Adeno-associated virus vector gene transfer and sarcolemmal expression of a 144 kDa micro-dystrophin effectively restores the dystrophin-associated protein complex and inhibits myofibre degeneration in nude/mdx mice

Duchenne muscular dystrophy is a severe life-threatening X-linked recessive disorder, caused by mutations in the dystrophin gene, for which currently there is no effective treatment. Because of the large size of the dystrophin cDNA (14 kb) this precluded it from being used in early adenovirus- or retrovirus-based gene therapy vectors. However, some therapeutic success has been achieved in mdx mice using adenovirus- and retrovirus-mediated transfer of a 6.3 kb recombinant mini-dystrophin cDNA. Despite this, problems with immunogenicity and inefficient transduction of mature myofibres make these vectors less than ideal for gene transfer to skeletal muscle. Adeno-associated viral (AAV) vectors overcome many of the problems associated with other vector systems. However, AAV vectors can only accommodate <5 kb of foreign DNA. For this reason we have produced a micro-dystrophin cDNA gene construct that is <3.8 kb. This construct, driven by a CMV promoter, was introduced into the skeletal muscle of 12-day-old nude/mdx mice using an AAV vector, resulting in specific sarcolemmal expression of micro-dystrophin in >50% of myofibres up to 20 weeks of age, and effective restoration of the dystrophin-associated protein (DAP) complex components. Additionally, evaluation of central nucleation indicated a significant inhibition of degenerative dystrophic muscle pathology. We have therefore shown that the current micro-dystrophin gene delivered in vivo using an AAV vector is not only capable of restoring sarcolemmal DAP complexes, but can also ameliorate dystrophic pathology at the cellular level.     

"mdx" mouse myopathy: histopathological, morphometric and histochemical observations on young mice

Histopathological examination of tibialis anterior (TA) and lateral gastrocnemius (LG) muscles from "mdx" mice sometimes revealed lesions of calcification and the close proximity of degenerative lesions with regenerative changes. Morphometric examinations showed that the TA was more severely affected than the LG in mice of all age groups. The soleus muscle was less severely affected than the TA in very young mice, but appeared to be affected almost as severely as the TA in older mice. Type 1 myofibres were more acid-labile in the mdx mice than in control mice and all three major fibre types degenerated and subsequently regenerated in young mdx mice. The aetiological significance of the above findings is unknown at present.     

转基因修饰后的自体骨髓间充质干细胞移植治疗mdx鼠后成肌调节因子的表达研究

目的探讨携带micro-dystrophin基因的自体骨髓间充质干细胞移植入mdx鼠后在移植鼠体内分化为肌细胞的可能机制。方法采用逆转录病毒介导micro-dystrophin基因转染mdx小鼠MSCs（mMSCs）,通过尾静脉注射移植治疗mdx鼠,在移植后免疫荧光检测micro-dystrophin的表达并在不同时间点检测MyoD的表达。结果移植后成功检测到micro-dystrophin,其表达随着移植时间的延长而增加;随移植时间延长MyoD阳性肌纤维比例增加,分别达到9%（4周时）、15%（8周时）、28%（12周时）。RT-PCR和Westernblot也发现,随着移植时间的延长,MyoD表达增加。结论自体mMSCs可携带外源性micro-dystrophin基因在受体鼠体内分化为micro-dystrophin阳性肌细胞,移植入的干细胞向肌细胞的分化是一个持久的、连续的过程,成肌调节因子在调节其分化过程中发挥了重大作用。     

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‐β 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.     

Electron microscopic and autoradiographic characterization of hindlimb muscle regeneration in the mdx mouse

The pattern of postnatal growth and development of skeletal muscle in mdx mice was studied by light and transmission electron microscopy and by autoradiography and was compared with that in their normal age-matched controls at 4 and 32 weeks of age. The muscle weights of both the extensor digitorum longus (EDL) and soleus muscles of mdx mice were significantly greater than those in control mice at both ages. Body weights of male and female mdx mice were also increased over controls up to 12 weeks of age. At 4 weeks, both the EDL and soleus muscles exhibited focal areas of degeneration, necrosis, and regeneration of centrally nucleated extrafusal fibers resulting in a wide range of fiber sizes. By 32 weeks, the majority of fibers in both muscles were centrally nucleated, and focal areas of recent regeneration were observed. By electron microscopy, the course of macrophage infiltration into areas of degenerating fibers and the ongoing regeneration of myofibers within redundant cylinders of external lamina were noted. This pattern was frequent in 4-week-old mdx muscles and was present to a lesser degree at 32 weeks. A notable lack of both adipose tissue infiltration and fibrotic change in the endomysium were observed in muscles at both ages. Autoradiograms of muscles from 4-week-old mdx mice injected with tritiated thymidine showed an increased proportion of labeled sublaminal nuclei at 24 and 48 hours after injection compared to controls. At 32 weeks of age, labeling of nuclei in muscles of mdx mice was also greater than in controls, but was reduced compared to muscle labeling in 4-week-old mdx mice. The observed features of mdx muscle tissue suggest that this animal model is more applicable to the study of regeneration dynamics than to Duchenne-type human muscular dystrophy.     

过量运动对肌营养不良模型鼠骨骼肌的损害作用

目的:观察过量运动对Duchenne型肌营养不良症模型鼠(mdx鼠)骨骼肌组织的影响。方法: 让成年和老年mdx小鼠、成年和老年C57小鼠连续进行游泳和提尾倒立运动试验(对照mdx不作运动), 每天1次, 每次13min, 连续3d后, 鼠尾静脉注谢Evans蓝, 次日杀小鼠, 剥去皮肤, 观察mdx鼠、C57鼠全身骨骼肌颜色改变, 对比照相后, 取腓肠肌、膈肌作冰冻切片, 荧光显微镜下, 观察Evans蓝的染色范围, 再作常规HE染色, 光学显微镜下, 观察骨骼肌细胞的组织学改变。结果:肉眼可见运动后的mdx鼠骨骼肌许多部位呈纵形条状蓝色, 而对照mdx鼠和C57鼠几乎无Evans蓝染色;荧光显微镜下, 运动后的mdx鼠骨骼肌可见Evans蓝荧光, 对照mdx鼠偶见此荧光, 而C57鼠无此荧光。光学显微镜下, 可见mdx鼠骨骼肌细胞变大、变圆, 伊红浓染, 胞核致密粗大, 坏死的肌细胞胞内结构降解, 呈无定型结构。膈肌中可见变性坏死的肌纤维居多, 而C57鼠无此改变。结论:过量运动造成了mdx鼠骨骼肌的损害;Evans蓝染色有助于鉴别变性坏死的肌纤维和研究dystrophin缺乏的肌肉的变性过程。     

成肌细胞移植阻止mdx鼠运动诱导的肌损害

目的:观察过量运动对肌营养不良症模型鼠(mdx鼠)骨骼肌的损害作用,以及成肌细胞移植对运动诱导损害肌纤维的保护作用。方法: 采用分离消化法对C57新生鼠的成肌细胞进行体外培养、纯化鉴定后,肌肉注射到mdx鼠左后肢,右后肢肌注DMEM作对照。成肌细胞移植后1个月,让mdx鼠作运动试验3 d后,静脉注射Evans蓝,次日取骨骼肌作冰冻切片,行dystrophin免疫荧光检测。荧光显微镜下,观察Evans蓝和dystrophin阳性纤维数,图像分析比较。 结果: 未移植肢体有26.82%±14.85%的肌纤维显示Evans蓝染色,而移植侧骨骼肌只有10.37%±2.87%的肌纤维显示Evans蓝染色。两者相比有显著差异(P＜0.05)。移植侧肢体有48.32%±6.54%的肌纤维dystrophin阳性,对照侧几乎没有dystrophin阳性肌纤维。Evans蓝染部分没有dystrophin表达。结论: 成肌细胞移植对运动诱导损害的肌纤维具有防护作用。     

Cell-lineage regulated myogenesis for dystrophin replacement: a novel therapeutic approach for treatment of muscular dystrophy

Duchenne muscular dystrophy (DMD) is characterized in skeletal muscle by cycles of myofiber necrosis and regeneration leading to loss of muscle fibers and replacement with fibrotic connective and adipose tissue. The ongoing activation and recruitment of muscle satellite cells for myofiber regeneration results in loss of regenerative capacity in part due to proliferative senescence. We explored a method whereby new myoblasts could be generated in dystrophic muscles by transplantation of primary fibroblasts engineered to express a micro-dystrophin/enhanced green fluorescent protein (muDys/eGFP) fusion gene together with a tamoxifen-inducible form of the myogenic regulator MyoD [MyoD-ER(T)]. Fibroblasts isolated from mdx(4cv) mice, a mouse model for DMD, were efficiently transduced with lentiviral vectors expressing muDys/eGFP and MyoD-ER(T) and underwent myogenic conversion when exposed to tamoxifen. These cells could also be induced to differentiate into muDys/eGFP-expressing myocytes and myotubes. Transplantation of transduced mdx(4cv) fibroblasts into mdx(4cv) muscles enabled tamoxifen-dependent regeneration of myofibers that express muDys. This lineage control method therefore allows replenishment of myogenic stem cells using autologous fibroblasts carrying an exogenous dystrophin gene. This strategy carries several potential advantages over conventional myoblast transplantation methods including: (i) the relative simplicity of culturing fibroblasts compared with myoblasts, (ii) a readily available cell source and ease of expansion and (iii) the ability to induce MyoD gene expression in vivo via administration of a medication. Our study provides a proof of concept for a novel gene/stem cell therapy technique and opens another potential therapeutic approach for degenerative muscle disorders.     

Stimulation of calcineurin signaling attenuates the dystrophic pathology in mdx mice

Utrophin has been studied extensively in recent years in an effort to find a cure for Duchenne muscular dystrophy. In this context, we previously showed that mice expressing enhanced muscle calcineurin activity (CnA*) displayed elevated levels of utrophin around their sarcolemma. In the present study, we therefore crossed CnA* mice with mdx mice to determine the suitability of elevating calcineurin activity in preventing the dystrophic pathology. Muscles from mdx/CnA* displayed increased nuclear localization of NFATc1 and a fiber type shift towards a slower phenotype. Measurements of utrophin levels in mdx/CnA* muscles revealed an approximately 2-fold induction in utrophin expression. Consistent with this induction, we also observed that members of the dystrophin-associated protein (DAP) complex were present at the sarcolemma of mdx/CnA* mouse muscle. This restoration of the utrophin-DAP complex was accompanied by significant reductions in the extent of central nucleation and fiber size variability. Importantly, assessment of myofiber sarcolemmal damage, as monitored by the intracellular presence of IgM and albumin as well as by Evans blue uptake in vivo, revealed a net amelioration of membrane integrity. Finally, immunofluorescence experiments using Mac-1 antibodies showed a reduction in the number of infiltrating immune cells in muscles from mdx/CnA* mice. These results show that elevated calcineurin activity attenuates the dystrophic pathology and thus provides an effective target for pharmacological intervention.     

The timing between skeletal muscle myoblast replication and fusion into myotubes, and the stability of regenerated dystrophic myofibres: an autoradiographic study in mdx mice

In mdx mice, a model for Duchenne muscular dystrophy, the timing between the replication of myoblasts and their incorporation into myotubes was determined autoradiographically. Thirty-eight mdx mice aged 23 d were injected with tritiated thymidine to label myoblasts replicating early in the dystrophic process. At intervals from 8 h to 30 d after injection the tibialis anterior muscles were removed, processed for autoradiography and analysed for labelled central myonuclei (derived from the progeny of myoblasts which had been labelled at 23 d). At 8 h after injection there were no labelled central myonuclei, showing that the labelled myoblasts had not fused within this time. At 1 d, 2% of central myonuclei were labelled, at 2 d, up to 32% were labelled, at 3 d ∼60% were labelled, and at 4 d the labelling peaked at 74%. In the 27 mice sampled from 5–30 d after injection, the levels of central myonuclear labelling varied enormously: from 1–63%. However, there was a consistent decrease in the numbers of labelled central myonuclei with time. This may have been due to dilution of the relative numbers of labelled myonuclei due to other, nonlabelled, myoblasts replicating after the availability of tritiated thymidine, and fusing. It was also possible that labelled myofibres underwent subsequent necrosis and were eliminated from the muscle. The proposal that a regenerated myofibre can undergo a subsequent cycle of necrosis and regeneration was supported by evidence of some necrotic myofibres with labelled and unlabelled central nuclei. These results have implications for understanding the cellular biology and pathology of dystrophic muscle, particularly in relation to myoblast transfer therapy as a potential treatment of Duchenne muscular dystrophy.     

Targeted inhibition of Ca2+ /calmodulin signaling exacerbates the dystrophic phenotype in mdx mouse muscle

In this study, we crossbred mdx mice with transgenic mice expressing a small peptide inhibitor for calmodulin (CaM), known as the CaM-binding protein (CaMBP), driven by the slow fiber-specific troponin I slow promoter. This strategy allowed us to determine the impact of interfering with Ca(2+)/CaM-based signaling in dystrophin-deficient slow myofibers. Consistent with impairments in the Ca(2+)/CaM-regulated enzymes calcineurin and Ca(2+)/CaM-dependent kinase, the nuclear accumulation of nuclear factor of activated T-cell c1 and myocyte enhancer factor 2C was reduced in slow fibers from mdx/CaMBP mice. We also detected significant reductions in the levels of peroxisome proliferator gamma co-activator 1alpha and GA-binding protein alpha mRNAs in slow fiber-rich soleus muscles of mdx/CaMBP mice. In parallel, we observed significantly lower expression of myosin heavy chain I mRNA in mdx/CaMBP soleus muscles. This correlated with fiber-type shifts towards a faster phenotype. Examination of mdx/CaMBP slow muscle fibers revealed significant reductions in A-utrophin, a therapeutically relevant protein that can compensate for the lack of dystrophin in skeletal muscle. In accordance with lower levels of A-utrophin, we noted a clear exacerbation of the dystrophic phenotype in mdx/CaMBP slow fibers as exemplified by several pathological indices. These results firmly establish Ca(2+)/CaM-based signaling as key to regulating expression of A-utrophin in muscle. Furthermore, this study illustrates the therapeutic potential of using targets of Ca(2+)/CaM-based signaling as a strategy for treating Duchenne muscular dystrophy (DMD). Finally, our results further support the concept that strategies aimed at promoting the slow oxidative myofiber program in muscle may be effective in altering the relentless progression of DMD.     

Aquaporin 4 Expression in the mdx Mouse Diaphragm

Expression of aquaporin (AQP) 4 in the surface membranes of skeletal myofibers is well established; however, its functional significance is still unknown. The alterations of AQP4 expressions in dystrophic muscles at RNA and protein levels have been reported in various dystrophic muscles such as dystrophinopathy, dysferlinopathy, and sarcoglycanopathy. We are interested in the relationship between the severity of dystrophic muscle degeneration and the expression of AQP4. Here we compared the AQP4 expression of the limb muscles with that of diaphragms in both mdx and control mice. The dystrophic muscle degeneration, such as rounding profile of cross sectional myofiber shape, dense eosin staining, central nuclei, and endomysial fibrosis in mdx mice, were more marked in diaphragms than in limb muscles. The decrease of AQP4 expression at protein level was more marked in diaphragms than in the limb muscles of mdx mice. However, the expression of AQP4 mRNA in the diaphragms of mdx mice was not reduced in comparison with limb muscles of mdx mice. The present study revealed that AQP4 expression at protein level was correlated with the severity of dystrophic changes in muscle tissues of mdx mice.     

Smooth muscle-specific dystrophin expression improves aberrant vasoregulation in mdx mice

Duchenne muscular dystrophy (DMD) is a fatal X-linked muscle-wasting disease caused by mutations of the gene encoding the cytoskeletal protein dystrophin. Therapeutic options for DMD are limited because the pathogenetic mechanism by which dystrophin deficiency produces the clinical phenotype remains obscure. Recent reports of abnormal alpha-adrenergic vasoregulation in the exercising muscles of DMD patients and in the mdx mouse, an animal model of DMD, prompted us to hypothesize that the dystrophin-deficient smooth muscle contributes to the vascular and dystrophic phenotypes of DMD. To test this, we generated transgenic mdx mice that express dystrophin only in smooth muscle (SMTg/mdx). We found that alpha-adrenergic vasoconstriction was markedly attenuated in the contracting hindlimbs of C57BL/10 wild-type mice, an effect that was mediated by nitric oxide (NO) and was severely impaired in the mdx mice. SMTg/mdx mice showed an intermediate phenotype, with partial restoration of the NO-dependent modulation of alpha-adrenergic vasoconstriction in active muscle. In addition, the elevated serum creatine kinase levels observed in mdx mice were significantly reduced in SMTg/mdx mice. This is the first report of a functional role of dystrophin in vascular smooth muscle.     

Increased susceptibility of EDL muscles from mdx mice to damage induced by contractions with stretch

Summary Absence of dystrophin in mdx muscles may render the muscle more susceptible to damage when submitted to high stress levels. To test this, typically slow (soleus) and fast (EDL) limb muscles of dystrophic (mdx) and normal (C57BL/10) mice were submitted (in vitro) to a series of isometric contractions, followed by a series of contractions with stretches. Muscle injury was assessed by monitoring the force signal. Membrane damage was evaluated by bathing the muscle in Procion Red, a dye that does not penetrate intact fibres, and subsequent analysis by light microscopy. After isometric contractions, only a very small force drop (<3% of maximal isometric force) was observed which indicated that no injury had occurred in soleus and EDL muscles in either mdx or C57 strains. After contractions with a stretch, a force drop of 10% was observed in soleus muscles from both strains and in EDL muscles from C57 mice. However, in mdx mice EDL muscles displayed an irreversible force drop of 40–60%. Histological analysis of the muscles indicates that force drop is associated with membrane damage. These results show that EDL muscles from mdx mice are more vulnerable than their controls, supporting the structural role hypothesis for dystrophin. Furthermore, they suggest that contractions with stretches may contribute to the muscle damage and degeneration observed in DMD-patients.     

Effect of cyclopiazonic acid, an inhibitor of the sarcoplasmic reticulum Ca-ATPase, on skeletal muscles from normal and mdx mice

AIM: In this study, we investigated Ca2+ loading by the sarcoplasmic reticulum in skeletal muscle from mdx mice, an animal model of human Duchenne's muscular dystrophy, at two stages of development: 4 and 11 weeks. METHOD: Experiments were conducted on fast- (extensor digitorum longus, EDL) and slow- (soleus) twitch muscles expressing different isoforms of Ca2+-ATPase, which is responsible for the uptake of Ca2+ by the sarcoplasmic reticulum. RESULTS: In sarcoplasmic reticulum vesicles, the ATP-dependent activity and sensitivity to cyclopiazonic acid (CPA), an inhibitor of the sarcoplasmic reticulum Ca2+-ATPase, were similar in mdx and normal EDL muscle. Furthermore, in chemically-skinned fibres from both normal and mdx muscles, the presence of CPA induced a decrease in Ca2+ uptake by the sarcoplasmic reticulum. However, the sensitivity to CPA was lower in mdx EDL muscle than in normal muscle. In addition, in EDL muscle from 4-week-old mdx mice, the expression of the slow Ca2+-pump isoform (SERCA2a) was significantly increased, without any accompanying change in slow myosin expression. In contrast, the expression and function of the Ca2+-ATPase in mdx soleus muscles at 4- and 11-weeks of development did not differ from those in age-matched controls. CONCLUSION: These findings show that in dystrophic muscle, where the Ca2+ homeostasis was perturbed, the Ca2+ handling by the sarcoplasmic reticulum was altered in fast-twitch muscle, and this was associated with the expression of the slow isoform of SERCA. In these muscles, reduced Ca2+ uptake could then contribute to an elevated concentration of Ca2+ in the cytosol, and also to Ca2+ depletion of the sarcoplasmic reticulum.     

骨髓干细胞移植治疗不同年龄mdx鼠的疗效研究

目的：研究不同年龄的Duchenne型肌营养不良鼠（mdx鼠）与骨髓干细胞移植后缺失蛋白表达的关系。 方法： 获取4-5周C57BL/6小鼠的骨髓干细胞，体外培养3 d，静脉移植到7Gy γ射线预处理的6周龄、8周龄两组各6只mdx鼠。移植12周后，对移植鼠骨骼肌dystrophin蛋白表达情况进行检测。 结果： 6周龄、8周龄两组mdx鼠，静脉移植1.2×107骨髓干细胞，3个月后，分别有16%和7%的骨骼肌纤维表达了dystrophin蛋白。 结论： 静脉移植同种、同系鼠骨髓干细胞的mdx鼠，3个月之后, 不同年龄mdx鼠骨骼肌细胞dystrophin蛋白表达的阳性率不同，幼年鼠骨髓干细胞移植有较高比率的缺失蛋白表达。     

Reduced density of intramembranous particle clusters: freeze-fracture study of mdx mouse muscle plasma membrane

Our previous freeze-fracture study demonstrated the decreased density of intramembranous particles (IMPs) on the protoplasmic (P) face of muscle plasma membranes in mdx mice. However, the molecular mechanism is unknown. In the present freeze-fracture study, we examined whether the reduced P-face IMP density in mdx mice would be caused by depletion of the rosette-like IMP clusters, which are IMP aggregations differing from crystal-like orthogonal arrays (OAs). By comparison with control mice, the P-face plasma membranes of extensor digitorum longus (EDL) and soleus (SOL) muscles of mdx mice demonstrated the following findings: (1) decreased IMP density with subunit particles of OAs and IMP clusters, (2) decreased IMP density without subunit particles of OAs, (3) normal IMP density without subunit particles of OAs and IMP clusters, (4) decreased OA density in EDL muscles and normal OA density in SOL muscles, and (5) decreased IMP cluster densities in both muscles. Thus, the reduced IMP density of P-face muscle plasma membranes in mdx mice may result from the decreased IMP clusters, suggesting the relationship between IMP clusters and the integral membrane proteins is influenced by dystrophin deficiency such as that of dystrophin-associated glycoproteins or other membrane proteins.     

mdx小鼠骨骼肌组织成肌、成脂、成骨基因的特异性表达

背景：干细胞移植是治疗肌营养不良症的有效方法之一,但移植的干细胞在病理骨骼肌中成肌表达较低。 目的：通过比较mdx小鼠和C57小鼠的骨骼肌形态及成肌、成脂、成骨基因表达的差异,探讨mdx小鼠骨骼肌病理改变的可能机制。 方法：取mdx小鼠与C57小鼠的骨骼肌组织行冰冻切片,苏木精-伊红染色和Vonkossa染色观察两种小鼠肌肉组织的形态特征;提取mdx小鼠和C57小鼠骨骼肌组织总RNA,real-time PCR检测成肌、成脂、成骨相关基因的表达。 结果与结论：mdx小鼠骨骼肌有肌纤维坏死和再生,伴有轻度脂肪、纤维结缔组织增生,Vonkossa染色可见钙结节沉积,而C57小鼠的骨骼肌细胞形态清晰,核位于细胞周边。与C57小鼠比较,mdx小鼠肌肉组织成骨、成脂基因表达有不同程度的上调(P < 0.05),而成肌基因表达下调(P< 0.05)。dystrophin基因缺失及成肌基因表达下调、成骨和成脂基因上调是造成mdx小鼠肌肉组织变性坏死的原因。     

Proteasome inhibitor (MG-132) treatment of mdx mice rescues the expression and membrane localization of dystrophin and dystrophin-associated proteins

Dystrophin, the protein product of the Duchenne muscular dystrophy (DMD) gene, is absent in the skeletal muscle of DMD patients and mdx mice. At the plasma membrane of skeletal muscle fibers, dystrophin associates with a multimeric protein complex, termed the dystrophin-glycoprotein complex (DGC). Protein members of this complex are normally absent or greatly reduced in dystrophin-deficient skeletal muscle fibers, and are thought to undergo degradation through an unknown pathway. As such, we reasoned that inhibition of the proteasomal degradation pathway might rescue the expression and subcellular localization of dystrophin-associated proteins. To test this hypothesis, we treated mdx mice with the well-characterized proteasomal inhibitor MG-132. First, we locally injected MG-132 into the gastrocnemius muscle, and observed the outcome after 24 hours. Next, we performed systemic treatment using an osmotic pump that allowed us to deliver different concentrations of the proteasomal inhibitor, over an 8-day period. By immunofluorescence and Western blot analysis, we show that administration of the proteasomal inhibitor MG-132 effectively rescues the expression levels and plasma membrane localization of dystrophin, beta-dystroglycan, alpha-dystroglycan, and alpha-sarcoglycan in skeletal muscle fibers from mdx mice. Furthermore, we show that systemic treatment with the proteasomal inhibitor 1) reduces muscle membrane damage, as revealed by vital staining (with Evans blue dye) of the diaphragm and gastrocnemius muscle isolated from treated mdx mice, and 2) ameliorates the histopathological signs of muscular dystrophy, as judged by hematoxylin and eosin staining of muscle biopsies taken from treated mdx mice. Thus, the current study opens new and important avenues in our understanding of the pathogenesis of DMD. Most importantly, these new findings may have clinical implications for the pharmacological treatment of patients with DMD.