Ixazomib, an oral proteasome inhibitor, exhibits potential effect in dystrophin-deficient mdx mice.

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.

Utrophin, MHC and M1/M2 macrophages in GRMD dogs / Utrofina, MHC e macrófagos M1 e M2 em cães DMGR

Muscular dystrophies are hereditary diseases that lead to progressive degeneration of the skeletal musculature. Golden Retriever dogs are used as animal models because they show a hereditary muscle disease similar to muscular dystrophy in humans. Aims: To evaluate the immunostaining of M1 (CD68) and M2 (CD163) macrophages, MHC I, MHC II and, utrophin in muscles of Golden Retriever dogs affected by muscular dystrophy (GRMD). Methods: Samples from 17 male dogs affected by GRMD were divided into GI - dystrophic dogs up to one year of age; and GII - dystrophic dogs over one-year-old. Results: Immunostaining of CD163 was higher than CD68 in both GI and GII. CD68 showed no variation between groups of dystrophic animals. MHC class I immunostaining was most evident in the biceps femoris and triceps brachialis. MHC class II was expressed mildly in four dystrophic muscle types in GI and GII. Utrophin immunostaining was higher in GII. Conclusion: M2 macrophages were one of the main mononuclear inflammatory cells found in dystrophic muscles. The number of M2 in muscles of dogs with GRMD increases with age, linking this cell subtype to permanent muscle damage.(AU)

As distrofias musculares são doenças hereditárias que levam à degeneração progressiva da musculatura esquelética. Cães Golden Retriever são usados como modelos animais, pois desenvolvem uma doença muscular hereditária semelhante à distrofia muscular em humanos. Objetivos: Avaliar a imunomarcação dos macrófagos M1 (CD68) e M2 (CD163), MHC I, MHC II e utrofina nos músculos de cães Golden Retriever afetados pela distrofia muscular (DMGR). Métodos: Amostras de 17 cães machos afetados por DMGR foram divididas em cães distróficos GI - até um ano de idade; e GII - cães distróficos com mais de um ano de idade. Resultados: a imunomarcação de CD163 foi maior que CD68 nos grupos GI e GII. CD68 não mostrou variação entre os grupos de animais distróficos. A imunomarcação de MHC classe I foi mais evidente no bíceps femoral e tríceps braquial. O MHC classe II foi expresso discretamente nos quatro tipos de músculo distrófico no GI e GII. A imunomarcação de utrofina foi maior no GII. Conclusão: Os macrófagos M2 foram uma das principais células inflamatórias mononucleares encontradas nos músculos distróficos. O número de M2 nos músculos de cães com DMGR aumentou com a idade, ligando esse subtipo de célula a danos musculares permanentes.(AU)

Utrophin, MHC and M1/M2 macrophages in GRMD dogs / Utrofina, MHC e macrófagos M1 e M2 em cães DMGR

Muscular dystrophies are hereditary diseases that lead to progressive degeneration of the skeletal musculature. Golden Retriever dogs are used as animal models because they show a hereditary muscle disease similar to muscular dystrophy in humans. Aims: To evaluate the immunostaining of M1 (CD68) and M2 (CD163) macrophages, MHC I, MHC II and, utrophin in muscles of Golden Retriever dogs affected by muscular dystrophy (GRMD). Methods: Samples from 17 male dogs affected by GRMD were divided into GI - dystrophic dogs up to one year of age; and GII - dystrophic dogs over one-year-old. Results: Immunostaining of CD163 was higher than CD68 in both GI and GII. CD68 showed no variation between groups of dystrophic animals. MHC class I immunostaining was most evident in the biceps femoris and triceps brachialis. MHC class II was expressed mildly in four dystrophic muscle types in GI and GII. Utrophin immunostaining was higher in GII. Conclusion: M2 macrophages were one of the main mononuclear inflammatory cells found in dystrophic muscles. The number of M2 in muscles of dogs with GRMD increases with age, linking this cell subtype to permanent muscle damage.

As distrofias musculares são doenças hereditárias que levam à degeneração progressiva da musculatura esquelética. Cães Golden Retriever são usados como modelos animais, pois desenvolvem uma doença muscular hereditária semelhante à distrofia muscular em humanos. Objetivos: Avaliar a imunomarcação dos macrófagos M1 (CD68) e M2 (CD163), MHC I, MHC II e utrofina nos músculos de cães Golden Retriever afetados pela distrofia muscular (DMGR). Métodos: Amostras de 17 cães machos afetados por DMGR foram divididas em cães distróficos GI - até um ano de idade; e GII - cães distróficos com mais de um ano de idade. Resultados: a imunomarcação de CD163 foi maior que CD68 nos grupos GI e GII. CD68 não mostrou variação entre os grupos de animais distróficos. A imunomarcação de MHC classe I foi mais evidente no bíceps femoral e tríceps braquial. O MHC classe II foi expresso discretamente nos quatro tipos de músculo distrófico no GI e GII. A imunomarcação de utrofina foi maior no GII. Conclusão: Os macrófagos M2 foram uma das principais células inflamatórias mononucleares encontradas nos músculos distróficos. O número de M2 nos músculos de cães com DMGR aumentou com a idade, ligando esse subtipo de célula a danos musculares permanentes.

Immunoglobulin therapy ameliorates the phenotype and increases lifespan in the severely affected dystrophin-utrophin double knockout mice.

Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder, caused by mutations in the dystrophin gene, affecting 1:3500-5000 boys worldwide. The lack of dystrophin induces degeneration of muscle cells and elicits an immune response characterized by an intensive secretion of pro-inflammatory cytokines. Immunoglobulins modulate the inflammatory response through several mechanisms and have been widely used as an adjuvant therapy for autoimmune diseases. Here we evaluated the effect of immunoglobulin G (IG) injected intraperitoneally in a severely affected double knockout (dko) mouse model for Duchenne muscular dystrophy. The IG dko treated mice were compared regarding activity rates, survival and histopathology with a control untreated group. Additionally, dendritic cells and naïve lymphocytes from these two groups and WT mice were obtained to study in vitro the role of the immune system associated to DMD pathophysiology. We show that IG therapy significantly enhances activity rate and lifespan of dko mice. It diminishes muscle tissue inflammation by decreasing the expression of costimulatory molecules MHC, CD86 and CD40 and reducing Th1-related cytokines IFN-γ, IL-1ß and TNF-α release. IG therapy dampens the effector immune responses supporting the hypothesis according to which the immune response accelerates DMD progression. As IG therapy is already approved by FDA for treating autoimmune disorders, with less side-effects than currently used glucocorticoids, our results may open a new therapeutic option aiming to improve life quality and lifespan of DMD patients.

Effects of (-)-epicatechin on frontal cortex DAPC and dysbindin of the mdx mice.

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.

Sarcolemmal targeting of nNOSµ improves contractile function of mdx muscle.

Nitric oxide (NO) is a key regulator of skeletal muscle function and metabolism, including vasoregulation, mitochondrial function, glucose uptake, fatigue and excitation-contraction coupling. The main generator of NO in skeletal muscle is the muscle-specific form of neuronal nitric oxide synthase (nNOSµ) produced by the NOS1 gene. Skeletal muscle nNOSµ is predominantly localized at the sarcolemma by interaction with the dystrophin protein complex (DPC). In Duchenne muscular dystrophy (DMD), loss of dystrophin leads to the mislocalization of nNOSµ from the sarcolemma to the cytosol. This perturbation has been shown to impair contractile function and cause muscle fatigue in dystrophic (mdx) mice. Here, we investigated the effect of restoring sarcolemmal nNOSµ on muscle contractile function in mdx mice. To achieve this, we designed a modified form of nNOSµ (NOS-M) that is targeted to the sarcolemma by palmitoylation, even in the absence of the DPC. When expressed specifically in mdx skeletal muscle, NOS-M significantly attenuates force loss owing to damaging eccentric contractions and repetitive isometric contractions (fatigue), while also improving force recovery after fatigue. Expression of unmodified nNOSµ at similar levels does not lead to sarcolemmal association and fails to improve muscle function. Aside from the benefits of sarcolemmal-localized NO production, NOS-M also increased the surface membrane levels of utrophin and other DPC proteins, including ß-dystroglycan, α-syntrophin and α-dystrobrevin in mdx muscle. These results suggest that the expression of NOS-M in skeletal muscle may be therapeutically beneficial in DMD and other muscle diseases characterized by the loss of nNOSµ from the sarcolemma.

Description of a utrophin associated protein complex in lipid raft domains of human artery smooth muscle cells.

The dystrophin-associated protein complex (DAPC) is a multimeric complex that links the extracellular matrix to the actin cytoskeleton, and in some cases dystrophin can be substituted by its autosomal homologue utrophin to form the utrophin-associated protein complex (UAPC). Both complexes maintain the stability of plasma membrane during contraction process and play an important role in transmembrane signaling. Mutations in members of the DAPC are associated with muscular dystrophy and dilated cardiomyopathy. In a previous study with human umbilical cord vessels, we observed that utrophin colocalize with caveolin-1 (Cav-1) which proposed the presence of UAPC in the plasma membrane of vascular smooth muscle (VSM). In the current study, we demonstrated by immunofluorescence analysis, co-immunoprecipitation assays, and subcellular fractionation by sucrose gradients, the existence of an UAPC in lipid raft domains of human umbilical artery smooth muscle cells (HUASMC). This complex is constituted by utrophin, ß-DG, ε-SG, α-smooth muscle actin, Cav-1, endothelial nitric oxide synthase (eNOS) and cavin-1. It was also observed the presence of dystrophin, utrophin Dp71, ß-SG, δ-SG, δ-SG3 and sarcospan in non-lipid raft fractions. Furthermore, the knockdown of α/ß-DG was associated with the decrease in both the synthesis of nitric oxide (NO) and the presence of the phosphorylated (active) form of eNOS; and with a reduction in the downstream activation of some cGMP signaling transduction pathway components. Together these results show the presence of an UAPC complex in HUASMC that may participate in the activity regulation of eNOS and in the vascular function.

Caveolae and non-caveolae lipid raft microdomains of human umbilical vein endothelial cells contain utrophin-associated protein complexes.

Several studies have shown the importance of dystrophin-associated protein complex in the development of muscular dystrophies and dilated cardiomyopathy associated to vascular dysfunction. In vascular endothelium, dystrophin is substituted for utrophin (autosomal homolog of dystrophin); however, its role in this tissue is unknown. Therefore, it is important to obtain a more extensive knowledge of utrophin and its associated proteins in endothelial cells. In a previous study, we demonstrated the presence of utrophin-associated protein complex (UAPC) in human umbilical vein endothelial cells HUVEC, which interacts with caveolin-1 (Cav-1) and endothelial nitric oxide synthase (eNOS). Also, some of our observations suggested the presence of this complex in distinct membrane domains. Therefore, the aim of this study was to analyze the presence of the UAPC in caveolae and non-caveolae lipid rafts domains of HUVEC at baseline and with a mechanical stimulus. It was demonstrated, by subcellular fractionation and co-immunoprecipitation assays, the association of UAPC with Cav-1 and eNOS in caveolae domains, as well as its interaction with eNOS in non-caveolae lipid raft domains. Additionally, it was also observed that mechanical stress on endothelial cells induced activation and release of eNOS from both caveolae and non-caveolae lipid raft associated to UAPC. Together these results suggest that UAPC located in caveolae and non-caveolae lipid raft domains of HUVECs may have a mechanosensory function that could participate in the control of eNOS activity.

Age-related changes in dystrophin-glycoprotein complex and in utrophin are not correlated with intrinsic laryngeal muscles protection in mdx mice.

In this study we investigate whether dystrophic intrinsic laryngeal muscles (ILM) from aged mdx mice show alterations in dystrophin-glycoprotein complex (DGC) components.Immunofluorescence and immunoblotting analyses of beta-sarcoglycan, beta-dystroglycan, and utrophin showed that aged ILM had a similar pattern of changes in aged affected muscles (diaphragm and limb), suggesting that aging leads to changes in utrophin and DGC proteins in dystrophic ILM that cannot be correlated with their protection from dystrophic change.

Expression of utrophin at dystrophin-deficient neuromuscular synapses of mdx mice: a study of protected and affected muscles.

In mdx mice, intrinsic laryngeal muscles are spared and sternomastoid muscles are affected, showing cycles of muscle regeneration. We observed that utrophin and acetylcholine receptors are fragmented only in affected muscles, providing further evidence that changes in the overall distribution of molecules at dystrophic neuromuscular junctions may be correlated with muscle regeneration.

Dystrophins and DAPs are expressed in adipose tissue and are regulated by adipogenesis and extracellular matrix.

The dystrophin-associated protein complex (DAPC), consisting of dystrophin, dystroglycans, sarcoglycans, dystrobrevins and syntrophins, provides a linkage between the cytoskeleton and the extracellular matrix. The disruption of DAPC leads to Duchenne/Becker muscular dystrophy and other neuromuscular diseases. Although adipose-derived stem cells had been used for the experimental treatment of Duchenne/Becker disease with promising results, little is known on the expression and function of DAPC in adipose tissue. Here we show that visceral and subcutaneous rat adipose depots express mRNAs for all known dystrophin isoforms, utrophin, α- and ß-dystrobrevins, and α-, ßI-, ßII-, and γII-syntrophins. Visceral and subcutaneous rat preadipocytes express Dp116 and Dp71 mRNAs and proteins, and this expression is differentially regulated during adipogenesis. Rat preadipocytes also express ß-dystrobrevin, α-, ßI-, ßII- and γII-syntrophins, ß-dystroglycan and ß-, δ-, and ε-sarcoglycans with no changes during adipogenesis. We also show that α-dystrobrevin increases their expression during adipose differentiation and extracellular matrix differentially regulates the expression of dystrophin isoforms mRNAs during adipogenesis. Our results show that DAPC components are expressed in adipose tissues and suggest that this complex has a role on the adipose biology.

Ringo: discordance between the molecular and clinical manifestation in a golden retriever muscular dystrophy dog.

Of the various genetic homologues to Duchenne Muscular Dystrophy (DMD), the Golden Retriever Muscular Dystrophy (GRMD) dog, which presents a variable but usually severe and progressive muscle weakness, has the closest relevance to DMD in both clinical severity and histopathological change. Among 77 GRMD dogs born in our colony in Brazil, we have identified a very mildly affected dog, Ringo, born July 2003. Among his descendants, at least one male, Suflair, is also showing a mild course. In an attempt to better characterize these two dogs, we studied the pattern of muscle proteins expression in Ringo and Suflair, as compared to severely affected and normal control dogs. Dystrophin was absent in both and utrophin was overexpressed in a pattern similar to the observed in severely affected dogs. Understanding the mechanism that is protecting Ringo and Suflair from the deleterious effect of the dystrophin gene mutation is of utmost interest. In addition it points out that the clinical impact of therapeutic trials should be interpreted with caution.

Dp71, utrophin and beta-dystroglycan expression and distribution in PC12/L6 cell cocultures.

Function of dystrophin Dp71 isoforms is unknown but seems related to neurite outgrowth and synapse formation. To evaluate Dp71 role in myoneural synapses, we established a coculture model using PC12 cells and L6 myotubes and analyzed expression and localization of Dp71 and related proteins, utrophin and beta-dystroglycan, in PC12 cells. Confocal microscopy showed Dp71d isoform in PC12 nuclei, golgi-complex-like and endoplasmic reticulum-like structures, whereas Dp71ab concentrates at neurite tips and cytoplasm, colocalizing with beta-dystroglycan, utrophin, synaptophysin and acetylcholine receptors. Evidences suggest that Dp71ab isoform, unlike Dp71d, may take part in neurite-related processes. This is the first work on Dp and members of Dp-associated protein complex roles in a cell-line based coculturing system, which may be useful in determining Dp71 isoforms associations.

Expression and function of utrophin associated protein complex in stretched endothelial cells: dissociation and activation of eNOS.

Several studies have emphasized the relevance of dystrophin-associated protein complex (DAPC) to maintain the vascular function. Previously we postulated the presence of an utrophin associated protein complex (UAPC) in endothelium from umbilical cord vessels. In the present work, we demonstrate that utrophin (UTR) indeed forms a complex, with beta-dystroglycan (DG), epsilon-sarcoglycan (SG), caveolin-1 (cav-1), and endothelial nitric oxide synthase (eNOS) in human umbilical vein endothelial cells (HUVEC) by co-immunoprecipitation analysis. Additionally, we observed an increment in the protein levels of epsilon-SG, beta-DG, UTR and cav-1 after mechanical stretching. Interestingly, this stimulus also induced eNOS up-regulation, activation and release from the UAPC, and led to a significant increase in nitric oxide (NO) production. Finally, we propose that UAPC in HUVECs may play an important role in the regulation of vascular tone.

Absence of correlation between utrophin localization and quantity and the clinical severity in Duchenne/Becker dystrophies.

While present in the surface membrane of embryonic muscle fibers, in adult normal muscle fibers, utrophin is restricted to the motor endplate and cells of blood vessel walls. However, the observation that utrophin is maintained in the extrajunctional plasma membrane in Duchenne (DMD) and in mdx muscle fibers has led to the suggestion that excess utrophin might compensate for dystrophin deficiency in the Xp21 muscular dystrophies. In order to detect an inverse correlation of utrophin presence and clinical severity, we have assessed utrophin distribution and quantity in DMD and Becker (BMD) patients of different ages and stages of clinical severity. All patients showed a positive discontinuous immunolabeling of utrophin on the sarcolemma, staining equally small and large muscle fibers, indicating that immature characteristics are maintained in such fibers. On Western blot, utrophin bands with concentrations 2- to 10-fold greater than in normal controls were detected in all DMD/BMD patients. However, no negative correlation was found between the amount of utrophin and the severity of clinical course, implying that the detectable utrophin levels in these patients did not compensate for dystrophin deficiency. In a DMD patient with growth hormone (GH) deficiency and a BMD-like clinical course, utrophin levels were comparable to the other typical DMD cases, which reinforces the hypothesis that the observed increase in utrophin is apparently not responsible for a milder clinical course in some patients with Xp21 muscular dystrophies.

Intrafamilial variability in dystrophin abundance correlated with difference in the severity of the phenotype.

In Duchenne muscular dystrophy, the progression of the disease is always severe and predictable, while in Becker dystrophy there is a wide variability (intra and inter familial) in the severity of the phenotype. We report here a family in which the proband, who is currently 15 years old, is showing a severe DMD progression, while his affected maternal uncle, aged 29, has a more benign course, compatible with BMD. No DNA deletion was detected in both patients. Dystrophin analysis through immunofluorescence and western blotting showed a negative pattern in the youngest patient and a positive one in the oldest. Apparently, this is the first report on intrafamilial variability in dystrophin abundance correlated with a difference in the severity of the phenotype.