RESUMEN

The primary function of dystrophin is to form a link between the cytoskeleton and the extracellular matrix. In addition to this crucial structural function, dystrophin also plays an essential role in clustering and organizing several signaling proteins, including ion channels. Proteomic analysis of the whole rodent brain has stressed the role of some components of the dystrophin-associated glycoprotein complex (DGC) as potential interacting proteins of the voltage-gated Ca2+ channels of the CaV2 subfamily. The interaction of CaV2 with signaling and scaffolding proteins, such as the DGC components, may influence their function, stability, and location in neurons. This work aims to study the interaction between dystrophin and CaV2.1. Our immunoprecipitation data showed the presence of a complex formed by CaV2.1, CaVα2δ-1, CaVß4e, Dp140, and α1-syntrophin in the brain. Furthermore, proximity ligation assays (PLA) showed that CaV2.1 and CaVα2δ-1 interact with dystrophin in the hippocampus and cerebellum. Notably, Dp140 and α1-syntrophin increase CaV2.1 protein stability, half-life, permanence in the plasma membrane, and current density through recombinant CaV2.1 channels. Therefore, we have identified the Dp140 and α1-syntrophin as novel interaction partners of CaV2.1 channels in the mammalian brain. Consistent with previous findings, our work provides evidence of the role of DGC in anchoring and clustering CaV channels in a macromolecular complex.

Asunto(s)

Distrofina , Proteómica , Animales , Distrofina/genética , Distrofina/metabolismo , Mamíferos/metabolismo , Neuronas/metabolismo

RESUMEN

The large DMD gene encodes a group of dystrophin proteins in brain and retina, produced from multiple promoters and alternative splicing events. Dystrophins are core components of different scaffolding complexes in distinct cell types. Their absence may thus alter several cellular pathways, which might explain the heterogeneous genotype-phenotype relationships underlying central comorbidities in Duchenne muscular dystrophy (DMD). However, the cell-specific expression of dystrophins and associated proteins (DAPs) is still largely unknown. The present study provides a first RNA-Seq-based reference showing tissue- and cell-specific differential expression of dystrophins, splice variants and DAPs in mouse brain and retina. We report that a cell type may express several dystrophin complexes, perhaps due to expression in separate cell subdomains and/or subpopulations, some of which with differential expression at different maturation stages. We also identified new splicing events in addition to the common exon-skipping events. These include a new exon within intron 51 (E51b) in frame with the flanking exons in retina, as well as inclusions of intronic sequences with stop codons leading to the presence of transcripts with elongated exons 40 and/or 41 (E40e, E41e) in both retina and brain. PCR validations revealed that the new exons may affect several dystrophins. Moreover, immunoblot experiments using a combination of specific antibodies and dystrophin-deficient mice unveiled that the transcripts with stop codons are translated into truncated proteins lacking their C-terminus, which we called N-Dp427 and N-Dp260. This study thus uncovers a range of new findings underlying the complex neurobiology of DMD.

Asunto(s)

Distrofina , Distrofia Muscular de Duchenne , Ratones , Animales , Distrofina/genética , Distrofina/metabolismo , Transcriptoma/genética , Codón de Terminación/metabolismo , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Retina/metabolismo , Encéfalo/metabolismo

RESUMEN

The shortest dystrophins, Dp71 and Dp40, are transcribed from the DMD gene through an internal promoter located in intron 62. These proteins are the main product of the DMD gene in the nervous system and have been involved in various functions related to cellular differentiation and proliferation as well as other cellular processes. Dp71 mRNA undergoes alternative splicing that results in different Dp71 protein isoforms. The subcellular localization of some of these isoforms in the PC12 cell line has been previously reported, and a differential subcellular distribution was observed, which suggests a particular role for each isoform. With the aim of obtaining information on their function, this study identified factors involved in the nuclear transport of Dp71 and Dp40 isoforms in the PC12 cell line. Cell cultures were treated with specific nuclear import/export inhibitors to determine the Dp71 isoform transport routes. The results showed that all isoforms of Dp71 and Dp40 included in the analysis have the ability to enter the cell nucleus through α/ß importin, and the main route of nuclear export for Dp71 isoforms is through the exportin CRM1, which is not the case for Dp40.

Asunto(s)

Distrofina , beta Carioferinas , Transporte Activo de Núcleo Celular , Animales , Distrofina/genética , Distrofina/metabolismo , Espacio Intracelular , Carioferinas/metabolismo , Células PC12 , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , ARN Mensajero/metabolismo , Ratas , beta Carioferinas/metabolismo

RESUMEN

Duchenne muscular dystrophy (DMD) is an X-linked genetic disorder induced by mutations in the dystrophin gene, leading to a degeneration of muscle fibers, triggering retrograde immunomodulatory, and degenerative events in the central nervous system. Thus, neuroprotective drugs such as pregabalin (PGB) can improve motor function by modulating plasticity, together with anti-inflammatory effects. The present work aimed to study the effects of PGB on axonal regeneration after axotomy in dystrophic and non-dystrophic mice. For that, MDX and C57BL/10 mouse strains were subjected to peripheral nerve damage and were treated with PGB (30 mg/kg/day, i.p.) for 28 consecutive days. The treatment was carried out in mice as soon as they completed 5 weeks of life, 1 week before the lesion, corresponding to the peak period of muscle degeneration in the MDX strain. Six-week-old mice were submitted to unilateral sciatic nerve crush and were sacrificed in the 9th week of age. The ipsi and contralateral sciatic nerves were processed for immunohistochemistry and qRT-PCR, evaluating the expression of proteins and gene transcripts related to neuronal and Schwann cell activity. Cranial tibial muscles were dissected for evaluation of neuromuscular junctions using α-bungarotoxin, and the myelinated axons of the sciatic nerve were analyzed by morphometry. The recovery of motor function was monitored throughout the treatment through tests of forced locomotion (rotarod) and spontaneous walking track test (Catwalk system). The results show that treatment with PGB reduced the retrograde cyclic effects of muscle degeneration/regeneration on the nervous system. This fact was confirmed after peripheral nerve injury, showing better adaptation and response of neurons and glia for rapid axonal regeneration, with efficient muscle targeting and regain of function. No side effects of PGB treatment were observed, and the expression of pro-regenerative proteins in neurons and Schwann cells was upregulated. Morphometry of the axons was in line with the preservation of motor endplates, resulting in enhanced performance of dystrophic animals. Overall, the present data indicate that pregabalin is protective and enhances regeneration of the SNP during the development of DMD, improving motor function, which can, in turn, be translated to the clinic.

Asunto(s)

Distrofia Muscular de Duchenne , Animales , Ratones , Distrofina/genética , Distrofina/metabolismo , Marcha , Ratones Endogámicos C57BL , Ratones Endogámicos mdx , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/metabolismo , Atrofia Muscular/metabolismo , Distrofia Muscular de Duchenne/metabolismo , Regeneración Nerviosa , Pregabalina/metabolismo

RESUMEN

Duchenne muscular dystrophy (DMD) is a muscle disease characterized by the absence of the protein dystrophin, which causes a loss of sarcolemma integrity, determining recurrent muscle injuries, decrease in muscle function, and progressive degeneration. Currently, there is a need for therapeutic treatments to improve the quality of life of DMD patients. Here, we investigated the effects of a low-intensity aerobic training (37 sessions) on satellite cells, peroxisome proliferator-activated receptor-gamma coactivator (PGC)-1α protein (PGC-1α), and different types of fibers of the psoas muscle from mdx mice (DMD experimental model). Wildtype and mdx mice were randomly divided into sedentary and trained groups (n = 24). Trained animals were subjected to 37 sessions of low-intensity running on a motorized treadmill. Subsequently, the psoas muscle was excised and analyzed by immunofluorescence for dystrophin, satellite cells, myosin heavy chain (MHC), and PGC-1α content. The minimal Feret's diameters of the fibers were measured, and light microscopy was applied to observe general morphological features of the muscles. The training (37 sessions) improved morphological features in muscles from mdx mice and caused an increase in the number of quiescent/activated satellite cells. It also increased the content of PGC-1α in the mdx group. We concluded that low-intensity aerobic exercise (37 sessions) was able to reverse deleterious changes determined by DMD.

Asunto(s)

Distrofia Muscular de Duchenne , Animales , Modelos Animales de Enfermedad , Distrofina/metabolismo , Humanos , Ratones , Ratones Endogámicos mdx , Músculo Esquelético/metabolismo , Distrofia Muscular de Duchenne/metabolismo , Músculos Psoas/metabolismo , Calidad de Vida

RESUMEN

Dp40 is ubiquitously expressed including the central nervous system. In addition to being present in the nucleus, membrane, and cytoplasm, Dp40 is detected in neurites and postsynaptic spines in hippocampal neurons. Although Dp40 is expressed from the same promoter as Dp71, its role in the cognitive impairment present in Duchenne muscular dystrophy patients is still unknown. Here, we studied the effects of overexpression of Dp40 and Dp40L170P during the neuronal differentiation of PC12 Tet-On cells. We found that Dp40 overexpression increased the percentage of PC12 cells with neurites and neurite length, while Dp40L170P overexpression decreased them compared to Dp40 overexpression. Two-dimensional gel electrophoresis analysis showed that the protein expression profile was modified in nerve growth factor-differentiated PC12-Dp40L170P cells compared to that of the control cells (PC12 Tet-On). The proteins α-internexin and S100a6, involved in cytoskeletal structure, were upregulated. The expression of vesicle-associated membrane proteins increased in differentiated PC12-Dp40 cells, in contrast to PC12-Dp40L170P cells, while neurofilament light-chain was decreased in both differentiated cells. These results suggest that Dp40 has an important role in the neuronal differentiation of PC12 cells through the regulation of proteins involved in neurofilaments and exocytosis of synaptic vesicles, functions that might be affected in PC12-Dp40L170P.

Asunto(s)

Sustitución de Aminoácidos , Distrofina/genética , Filamentos Intermedios/metabolismo , Proyección Neuronal/genética , Neuronas/metabolismo , Vesículas Sinápticas/metabolismo , Animales , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Diferenciación Celular , Citoesqueleto/metabolismo , Citoesqueleto/ultraestructura , Distrofina/metabolismo , Exocitosis , Regulación de la Expresión Génica , Proteínas de Filamentos Intermediarios/genética , Proteínas de Filamentos Intermediarios/metabolismo , Filamentos Intermedios/ultraestructura , Proteínas de Neurofilamentos/genética , Proteínas de Neurofilamentos/metabolismo , Neuronas/citología , Células PC12 , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , Ratas , Proteína A6 de Unión a Calcio de la Familia S100/genética , Proteína A6 de Unión a Calcio de la Familia S100/metabolismo , Transducción de Señal , Vesículas Sinápticas/ultraestructura

RESUMEN

PURPOSE: Cardiac transition from concentric (C-LVH) to eccentric left ventricle hypertrophy (E-LVH) is a maladaptive response of hypertension. Matrix metalloproteinases (MMPs), in particular MMP-2, may contribute to tissue remodeling by proteolyzing extra- and intracellular proteins. Troponin I and dystrophin are two potential targets of MMP-2 examined in this study and their proteolysis would impair cardiac contractile function. We hypothesized that MMP-2 contributes to the decrease in troponin I and dystrophin in the hypertensive heart and thereby controls the transition from C-LVH to E-LVH and cardiac dysfunction. METHODS: Male Wistar rats were divided into sham or two kidney-1 clip (2K-1C) hypertensive groups and treated with water (vehicle) or doxycycline (MMP inhibitor, 15 mg/kg/day) by gavage from the tenth to the sixteenth week post-surgery. Tail-cuff plethysmography, echocardiography, gelatin zymography, confocal microscopy, western blot, mass spectrometry, in silico protein analysis and immunofluorescence were performed. RESULTS: 6 out of 23 2K-1C rats (26%) had E-LVH followed by reduced ejection fraction. The remaining had C-LVH with preserved cardiac function. Doxycycline prevented the transition from C-LVH to E-LVH. MMP activity is increased in C-LVH and E-LVH hearts which was inhibited by doxycycline. This effect was associated with an increase in troponin I cleavage products and a decline in dystrophin in the left ventricle of E-LVH rats, which was prevented by doxycycline. CONCLUSION: Hypertension causes increased cardiac MMP-2 activity which proteolyzes troponin I and dystrophin, contributing to the transition from C-LVH to E-LVH and cardiac dysfunction.

Asunto(s)

Doxiciclina/farmacología , Distrofina/metabolismo , Hipertensión/complicaciones , Hipertrofia Ventricular Izquierda/etiología , Metaloproteinasa 2 de la Matriz/metabolismo , Troponina I/metabolismo , Animales , Antibacterianos/farmacología , Distrofina/genética , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Hipertrofia Ventricular Izquierda/metabolismo , Masculino , Metaloproteinasa 2 de la Matriz/genética , Inhibidores de la Metaloproteinasa de la Matriz/farmacología , Ratas , Ratas Wistar , Troponina I/genética

RESUMEN

Dp71 and Dp40 are the main products of the DMD gene in the central nervous system, and they are developmentally regulated from the early stages of embryonic development to adulthood. To further study the roles of Dp71 and Dp40 during cell proliferation and neural differentiation, we analyzed Dp71/Dp40 isoform expression at the mRNA level by RT-PCR assays to identify alternative splicing (AS) in the isoforms expressed in rat neural stem/progenitor cells (NSPCs) and in differentiated cells (neurons and glia). We found that proliferating NSPCs expressed Dp71d, Dp71dΔ71, Dp71f, Dp71fΔ71, Dp71dΔ74 and Dp40, as well as two Dp40 isoforms: Dp40Δ63,64 and Dp40Δ64-67. In differentiated cells we also found the expression of Dp71d, Dp71dΔ71, Dp71f, Dp71fΔ71 and Dp40. However, the expression frequencies were different in both stages. In addition, in differentiated cells, we found Dp71fΔ71-74, and interestingly, we did not find the expression of Dp71dΔ74 or the newly identified Dp40 isoforms. In this work we show that NSPC differentiation is accompanied by changes in Dp71/Dp40 isoform expression, suggesting different roles for these isoforms in NSPCs proliferation and neuronal differentiation, and we describe, for the first time, alternative splicing of Dp40.

Asunto(s)

Empalme Alternativo , Distrofina/genética , Células-Madre Neurales/metabolismo , Animales , Diferenciación Celular , Proliferación Celular , Distrofina/metabolismo , Células-Madre Neurales/citología , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Isoformas de ARN/metabolismo , Ratas Wistar

RESUMEN

Dystrophin deficiency makes the sarcolemma fragile and susceptible to degeneration in Duchenne muscular dystrophy. The proteasome is a multimeric protease complex and is central to the regulation of cellular proteins. Previous studies have shown that proteasome inhibition improved pathological changes in mdx mice. Ixazomib is the first oral proteasome inhibitor used as a therapy in multiple myeloma. This study investigated the effects of ixazomib on the dystrophic muscle of mdx mice. MDX mice were treated with ixazomib (7.5 mg/kg/wk by gavage) or 0.2 mL of saline for 12 weeks. The Kondziela test was performed to measure muscle strength. The tibialis anterior (TA) and diaphragm (DIA) muscles were used for morphological analysis, and blood samples were collected for biochemical measurement. We observed maintenance of the muscle strength in the animals treated with ixazomib. Treatment with ixazomib had no toxic effect on the mdx mouse. The morphological analysis showed a reduction in the inflammatory area and fibres with central nuclei in the TA and DIA muscles and an increase in the number of fibres with a diameter of 20 µm2 in the DIA muscle after treatment with ixazomib. There was an increase in the expression of dystrophin and utrophin in the TA and DIA muscles and a reduction in the expression of osteopontin and TGF-ß in the DIA muscle of mdx mice treated with ixazomib. Ixazomib was thus shown to increase the expression of dystrophin and utrophin associated with improved pathological and functional changes in the dystrophic muscles of mdx mice.

Asunto(s)

Compuestos de Boro/farmacología , Distrofina/efectos de los fármacos , Glicina/análogos & derivados , Músculo Esquelético/efectos de los fármacos , Distrofia Muscular de Duchenne , Inhibidores de Proteasas/farmacología , Animales , Distrofina/metabolismo , Glicina/farmacología , Ratones Endogámicos mdx , Músculo Esquelético/metabolismo , Músculo Esquelético/patología , Utrofina/efectos de los fármacos , Utrofina/metabolismo

RESUMEN

The mechanisms accounting for the loss of muscle function with obesity and type 2 diabetes are likely the result of a combination of neural and muscular factors. One muscular factor that is important, yet has received little attention, is the protein machinery involved in longitudinal and lateral force transmission. The purpose of this study was to compare the levels of force transfer and membrane integrity proteins before and after a 12-week endurance training program in lean, obese, and obese type 2 diabetic adults. Nineteen sedentary subjects (male = 8 and female = 11) were divided into three groups: Lean (n = 7; 50.3 ± 4.1 y; 69.1 ± 7.2 kg); Obese (n = 6; 49.8 ± 4.1 y; 92.9 ± 19.5 kg); and Obese with type 2 diabetes (n = 6; 51.5 ± 7.9 years; 88.9 ± 15.1 kg). Participants trained 150 min/week between 55% and 75% of VO2max for 12 weeks. Skeletal muscle biopsies were taken before and after the training intervention. Baseline dystrophin and muscle LIM protein levels were higher (~50% p < .01) in lean compared to obese and type 2 diabetic adults, while the protein levels of the remaining force transfer and membrane integrity proteins were similar between groups. After training, obese individuals decreased (-53%; p < .01) the levels of the muscle ankyrin repeat protein and lean individuals decreased dystrophin levels (-45%; p = .01), while the levels of the remaining force transfer and membrane integrity proteins were not affected by training. These results suggest that there are modest changes to force transfer and membrane integrity proteins in middle-aged individuals as a result of 12 weeks of lifestyle and training interventions.

Asunto(s)

Diabetes Mellitus Tipo 2/metabolismo , Entrenamiento Aeróbico/métodos , Terapia por Ejercicio/métodos , Proteínas de la Membrana/metabolismo , Músculo Esquelético/metabolismo , Obesidad/metabolismo , Delgadez/metabolismo , Ancirinas/metabolismo , Diabetes Mellitus Tipo 2/patología , Diabetes Mellitus Tipo 2/terapia , Distrofina/metabolismo , Femenino , Humanos , Masculino , Persona de Mediana Edad , Fuerza Muscular/fisiología , Obesidad/patología , Obesidad/terapia , Delgadez/patología , Delgadez/terapia

RESUMEN

In this review, we discuss the therapies used in the treatment of patients with Duchenne muscular dystrophy since the first description of the disease. A short description is given of the various theories based on disease pathogenesis, which give the substrates for the many therapeutic interventions. A brief review of the methods of evaluation used in therapeutic trials is made. Of all the treatments, the only drugs that are still considered able to modify the course of the disease are the corticosteroids (prednisone/prednisolone/deflazacort). Other drugs (coenzyme Q10 and creatine) have had a little effect in a few functions without adverse reactions. Idebenone seems to improve the respiratory function in the long term. The trials with mRNA transcription, through nonsense mutations or exon 51 skipping, show some beneficial results in a few functional tests, but they are limited to a small set of DMD patients.

Asunto(s)

Distrofia Muscular de Duchenne/tratamiento farmacológico , Corticoesteroides/uso terapéutico , Antiinflamatorios/uso terapéutico , Antioxidantes/uso terapéutico , Bloqueadores de los Canales de Calcio/uso terapéutico , Inhibidores de la Colinesterasa/uso terapéutico , Distrofina/efectos de los fármacos , Distrofina/metabolismo , Humanos , Distrofia Muscular de Duchenne/metabolismo

RESUMEN

ABSTRACT In this review, we discuss the therapies used in the treatment of patients with Duchenne muscular dystrophy since the first description of the disease. A short description is given of the various theories based on disease pathogenesis, which give the substrates for the many therapeutic interventions. A brief review of the methods of evaluation used in therapeutic trials is made. Of all the treatments, the only drugs that are still considered able to modify the course of the disease are the corticosteroids (prednisone/prednisolone/deflazacort). Other drugs (coenzyme Q10 and creatine) have had a little effect in a few functions without adverse reactions. Idebenone seems to improve the respiratory function in the long term. The trials with mRNA transcription, through nonsense mutations or exon 51 skipping, show some beneficial results in a few functional tests, but they are limited to a small set of DMD patients.

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RESUMO Nesta revisão são discutidas as terapêuticas empregadas no tratamento da distrofia muscular de Duchenne desde a descrição da doença. Apresentamos as diversas teorias que fundamentaram as intervenções terapêuticas, com uma breve descrição dos tipos de avaliação empregados nos ensaios terapêuticos. Dentre todos os tratamentos, a única medicação que até agora modificou o curso da doença foram os corticosteroides (prednisona/prednisolona/deflazacort). A coenzima Q10 e creatina tiveram algum efeito pequeno em algumas funções e evolução da doença sem efeitos colaterais. O idebenone mostrou efeito benéfico na função respiratória em longo prazo. As tentativas de intervir no gene da distrofina utilizando técnicas de transcrição do mRNA através das mutações sem sentido e técnicas que pulam o exon 51 mostram resultado muito discreto em algumas provas funcionais e limitado a uma parcela pequena de casos.

Asunto(s)

Humanos , Distrofia Muscular de Duchenne/tratamiento farmacológico , Bloqueadores de los Canales de Calcio/uso terapéutico , Inhibidores de la Colinesterasa/uso terapéutico , Distrofina/efectos de los fármacos , Distrofina/metabolismo , Corticoesteroides/uso terapéutico , Distrofia Muscular de Duchenne/metabolismo , Antiinflamatorios/uso terapéutico , Antioxidantes/uso terapéutico

RESUMEN

Dystrophin is a cytoskeleton-linked membrane protein that binds to a larger multiprotein assembly called the dystrophin-associated glycoprotein complex (DGC). The deficiency of dystrophin or the components of the DGC results in the loss of connection between the cytoskeleton and the extracellular matrix with significant pathophysiological implications in skeletal and cardiac muscle as well as in the nervous system. Although the DGC plays an important role in maintaining membrane stability, it can also be considered as a versatile and flexible molecular complex that contribute to the cellular organization and dynamics of a variety of proteins at specific locations in the plasma membrane. This review deals with the role of the DGC in transmembrane signaling by forming supramolecular assemblies for regulating ion channel localization and activity. These interactions are relevant for cell homeostasis, and its alterations may play a significant role in the etiology and pathogenesis of various disorders affecting muscle and nerve function.

Asunto(s)

Distrofina/metabolismo , Glicoproteínas/metabolismo , Canales Iónicos/metabolismo , Animales , Membrana Celular/metabolismo , Citoesqueleto/metabolismo , Humanos , Proteínas de la Membrana/metabolismo , Músculo Esquelético/metabolismo , Miocardio/metabolismo , Transducción de Señal

RESUMEN

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.

Asunto(s)

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/metabolismo

RESUMEN

Multiple dystrophin Dp71 isoforms have been identified in rats, mice, and humans and in several cell line models. These Dp71 isoforms are produced by the alternative splicing of exons 71 to 74 and 78 and intron 77. Three main groups of Dp71 proteins are defined based on their C-terminal specificities: Dp71d, Dp71f, and Dp71e. Dp71 is highly expressed in the brain and retina; however, the specific isoforms present in these tissues have not been determined to date. In this work, we explored the expression of Dp71 isoforms in the mouse brain and retina using RT-PCR assays followed by the cloning of PCR products into the pGEM-T Easy vector, which was used to transform DH5α cells. Dp71-positive colonies were later analyzed by PCR multiplex and DNA sequencing to determine the alternative splicing. We thus demonstrated the expression of Dp71 transcripts corresponding to Dp71, Dp71a, Dp71c, Dp71b, Dp71ab, Dp71 Δ110, and novel Dp71 isoforms spliced in exon 74; 71 and 74; 71, 73 and 74; and 74 and 78, which we named Dp71d Δ74 , Dp71d Δ71,74 , Dp71d Δ71,73-74 , and Dp71f Δ74 , respectively. Additionally, we demonstrated that the Dp71d group of isoforms is highly expressed in the brain, while the Dp71f group predominates in the retina, at both the cDNA and protein levels. These findings suggest that distinct Dp71 isoforms may play different roles in the brain and retina.

Asunto(s)

Encéfalo/metabolismo , Distrofina/metabolismo , Isoformas de Proteínas/metabolismo , Retina/metabolismo , Empalme Alternativo , Animales , Ratones , Fracciones Subcelulares/metabolismo

RESUMEN

Hypertension causes cardiac hypertrophy, one of the most important risk factors for heart failure (HF). Despite the importance of cardiac hypertrophy as a risk factor for the development of HF, not all hypertrophied hearts will ultimately fail. Alterations of cytoskeletal and sarcolemma-associated proteins are considered markers cardiac remodeling during HF. Dystrophin provides mechanical stability to the plasma membrane through its interactions with the actin cytoskeleton and, indirectly, to extracellular matrix proteins. This study was undertaken to evaluate dystrophin and calpain-1 in the transition from compensated cardiac hypertrophy to HF. Wistar rats were subjected to abdominal aorta constriction and killed at 30, 60 and 90 days post surgery (dps). Cardiac function and blood pressure were evaluated. The hearts were collected and Western blotting and immunofluorescence performed for dystrophin, calpain-1, alpha-fodrin and calpastatin. Statistical analyses were performed and considered significant when p<0.05. After 90 dps, 70% of the animals showed hypertrophic hearts (HH) and 30% hypertrophic+dilated hearts (HD). Systolic and diastolic functions were preserved at 30 and 60 dps, however, decreased in the HD group. Blood pressure, cardiomyocyte diameter and collagen content were increased at all time points. Dystrophin expression was lightly increased at 30 and 60 dps and HH group. HD group showed decreased expression of dystrophin and calpastatin and increased expression of calpain-1 and alpha-fodrin fragments. The first signals of dystrophin reduction were observed as early as 60 dps. In conclusion, some hearts present a distinct molecular pattern at an early stage of the disease; this pattern could provide an opportunity to identify these failure-prone hearts during the development of the cardiac disease. We showed that decreased expression of dystrophin and increased expression of calpains are coincident and could work as possible therapeutic targets to prevent heart failure as a consequence of cardiac hypertrophy.

Asunto(s)

Biomarcadores/metabolismo , Cardiomegalia/metabolismo , Distrofina/metabolismo , Animales , Presión Sanguínea , Western Blotting , Cardiomegalia/diagnóstico por imagen , Cardiomegalia/fisiopatología , Ecocardiografía , Técnica del Anticuerpo Fluorescente , Masculino , Ratas , Ratas Wistar

RESUMEN

INTRODUCTION: Multiple components of the dystrophin-associated protein complex (DAPC) are expressed in numerous tissues including the brain. Members of the DAPC and dysbindin are abnormally expressed in the brain of Duchenne Muscular Dystrophy (DMD) patients, which has been associated with cognitive impairments. However, little is known about the expression pattern of individual members of the DAPC in animal models of DMD and their relationship with dysbindin. METHODS: Ten mdx mice were randomly allocated into a control and intervention group [(-)-epicatechin (Epi) 1mg/kg/day for four weeks] and results compared to a wild-type mice. After sacrifice, brain pre-frontal cortices were collected for Western blotting and immunoprecipitation assays, and sagittal sections processed for immunohistochemistry. RESULTS: Epi promotes a partial recovery of DAPC members [α1-Syntrophin, sarcoglycans (SG), dystrophin 71 (Dp71)], dysbindin, and utrophin protein levels. Epi also appears to restore the association of DAPC between dysbindin, and utrophin with Dp71 and ε-SG. Co-immunostaining evidence increased protein levels of dysbindin, dystrophin, and ε-SG and their colocalization. CONCLUSIONS: Altogether, results suggest that Epi is capable of restoring pre-frontal cortex DAPC and dysbindin levels of mdx mice towards that of healthy brains. The functional implications of such studies warrant further investigation.

Asunto(s)

Catequina/genética , Disbindina/metabolismo , Complejo de Proteínas Asociado a la Distrofina/metabolismo , Lóbulo Frontal/metabolismo , Distrofia Muscular de Duchenne/metabolismo , Animales , Distrofina/metabolismo , Ratones Endogámicos mdx , Utrofina/metabolismo

RESUMEN

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 fármacos

RESUMEN

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/metabolismo

RESUMEN

Cardiac dysfunction caused by the impairment of myocardial contractility has been recognized as an important factor contributing to the high mortality in sepsis. Calpain activation in the heart takes place in response to increased intracellular calcium influx resulting in proteolysis of structural and contractile proteins with subsequent myocardial dysfunction. The purpose of the present study was to test the hypothesis that increased levels of calpain in the septic heart leads to disruption of structural and contractile proteins and that administration of calpain inhibitor-1 (N-acetyl-leucinyl-leucinyl-norleucinal (ALLN)) after sepsis induced by cecal ligation and puncture prevents cardiac protein degradation. We also tested the hypothesis that calpain plays a role in the modulation of protein synthesis/degradation through the activation of proteasome-dependent proteolysis and inhibition of the mTOR pathway. Severe sepsis significantly increased heart calpain-1 levels and promoted ubiquitin and Pa28ß over-expression with a reduction in the mTOR levels. In addition, sepsis reduced the expression of structural proteins dystrophin and ß-dystroglycan as well as the contractile proteins actin and myosin. ALLN administration prevented sepsis-induced increases in calpain and ubiquitin levels in the heart, which resulted in decreased of structural and contractile proteins degradation and basal mTOR expression levels were re-established. Our results support the concept that increased calpain concentrations may be part of an important mechanism of sepsis-induced cardiac muscle proteolysis.

Asunto(s)

Calpaína/metabolismo , Distrofina/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Sepsis/patología , Serina-Treonina Quinasas TOR/metabolismo , Ubiquitina/metabolismo , Actinas/metabolismo , Animales , Calpaína/antagonistas & inhibidores , Calpaína/genética , Cardiomiopatías/complicaciones , Cardiomiopatías/patología , Modelos Animales de Enfermedad , Expresión Génica/efectos de los fármacos , Leupeptinas/farmacología , Leupeptinas/uso terapéutico , Masculino , Ratones , Ratones Endogámicos C57BL , Microscopía Fluorescente , Miocardio/metabolismo , Miocardio/patología , Miosinas/metabolismo , Proteolisis/efectos de los fármacos , Sepsis/etiología , Sepsis/prevención & control , Serina-Treonina Quinasas TOR/antagonistas & inhibidores