Correlated NOS-Imu and myf5 expression by satellite cells in mdx mouse muscle regeneration during NOS manipulation and deflazacort treatment

Satellite cells, muscle precursor cells in skeletal muscle, are normally quiescent and become activated by disease or injury. A lack of dystrophin and changes in the expression or activity of neuronal nitric oxide synthase (NOS-I) affect the timing of activation in vivo. Nitric oxide synthase inhibition delays muscle repair in normal mice, and worsens muscular dystrophy in the mdx mouse, a genetic homologue of Duchenne muscular dystrophy. However, the potential role of activation and repair events mediated by nitric oxide in determining the outcome of steroid or other treatments for muscular dystrophy is not clear. We tested the hypothesis that the extent of repair in dystrophic muscles of mdx mice is partly dependent on NOS-Imu expression and activity. Myotube formation in regenerating muscle was promoted by deflazacort treatment of mdx dystrophic mice (P<0.05), and improved by combination with the nitric oxide synthase substrate, L-arginine, especially in the diaphragm. NOS-Imu mRNA expression and activity were present in satellite cells and very new myotubes of regenerating and dystrophic muscle. Deflazacort treatment resulted in increased NOS-Imu expression in regenerating muscles in a strong and specific correlation with myf5 expression (r=0.95, P<0.01), a marker for muscle repair. Nitric oxide synthase inhibition prevented the deflazacort-induced rise in NOS-Imu and myf5 expression in the diaphragm without affecting the diameter of non-regenerating fibres. These in vivo studies suggest that gains in NOS-Imu expression and nitric oxide synthase activity in satellite cells can increase the extent and speed of repair, even in the absence of dystrophin in muscle fibres. NOS-Imu may be a useful therapeutic target to augment the effects of steroidal or other treatments of muscular dystrophy.     

Microarray analysis of mdx mice expressing high levels of utrophin: therapeutic implications for dystrophin deficiency

Duchenne Muscular Dystrophy (DMD) is a fatal muscle wasting disorder caused by dystrophin deficiency. Previous work suggested that increased expression of the dystrophin-related protein utrophin in the mdx mouse can reduce the dystrophic pathophysiology. Physiological tests showed that the transgenic mouse muscle functioned in a way similar to normal muscle. More recently, it has become possible to analyse disease pathways using microarrays, a sensitive method to evaluate the efficacy of a therapeutic approach. We thus examined the gene expression profile of mdx mouse muscle compared to wild-type mouse muscle and compared the data with that obtained from the transgenic line overexpressing utrophin. The data confirm that the expression of utrophin in the mdx mouse muscle results in a global gene expression profile more similar to that seen for the wild-type mouse. This study confirms that a strategy to up-regulate utrophin is likely to be beneficial in dystrophin deficiency.     

重组腺相关病毒载体介导的小基因SMCKA3999治疗对Duchenne肌营养不良肌力的影响

目的研究重组腺相关病毒载体(rAAV)介导的人dystrophin小基因SMCKA3999对DMD病理、肌力改变的治疗作用.方法将dystrophin小基因SMCKA3999克隆至rAAV并包装成rAAVSMCKA3999病毒,以5×109病毒颗粒多点注射于DMD模型鼠mdx腓肠肌,基因治疗4月后免疫荧光法检测肌膜dystrophin基因表达,治疗5月后采用肌肉离体灌注电刺激测定腓肠肌肌力,观察rAAVSMCKA3999对mdx鼠肌力的疗效.结果rAAVSMCKA3999有效稳定表达并使肌膜缺失的dystrophin恢复,明显改善mdx鼠肌力.结论rAAVSMCKA3999对DMD治疗有效,能显著改善mdx鼠肌肉功能,应用重组腺相关病毒载体介导的dystrophin小基因SMCKA3999是治疗DMD有希望的方法.     

Muscle regeneration in mdx mouse, and a trial of normal myoblast transfer into regenerating dystrophic muscle

The most ideal therapeutic trial on Duchenne muscular dystrophy (DMD) is a transfer of normal myoblasts into dystrophic muscle which has been attempted on animal models in several institutes. In the process of muscle regeneration, the transferred normal myoblasts are expected to incorporate into the regenerating fibers in host dystrophic mouse. To know the capacity of muscle regeneration in dystrophic muscle, we compared the regenerating process of the normal muscle with that of the dystrophic muscle after myonecrosis induced by 0.25% bupivacaine hydrochloride (BPVC) chronologically. In the present study, C57BL/10ScSn-mdx (mdx) mouse was used as an animal model of DMD and C57BL/10ScSn (B10) mouse as a control. There was no definite difference in the behavior of muscle fiber regeneration between normal and dystrophic muscles. The dystrophic muscle regenerated rapidly at the similar tempo to the normal as to their size and fiber type differentiation. The variation in fiber size diameter of dystrophic muscle, however, was more obvious than that of normal. To promote successful myoblast transfer from B10 mouse into dystrophic mdx mouse at higher ratio, cultured normal myoblasts were transferred into the regenerating dystrophic muscle on the first and the second day after myonecrosis induced by BPVC. Two weeks after the myoblast injection, the muscles were examined with immunohistochemical stain using anti dystrophin antibody. Although dystrophin-positive fibers appeared in dystrophic muscle, the positive fibers were unexpectedly small in number (3.86 +/- 1.50%).(ABSTRACT TRUNCATED AT 250 WORDS)     

Expression and localization of protein inhibitor of neuronal nitric oxide synthase in Duchenne muscular dystrophy.

In skeletal muscle fibers, nitric oxide is synthesized by neuronal nitric oxide synthase (nNOS), which normally associates with the dystrophin complex in close proximity to the sarcolemma. Many reports have documented that very low levels of nNOS protein exist in muscle fibers of Duchenne muscular dystrophy (DMD) patients. In this study we investigated the functional significance of PIN (protein inhibitor of nNOS) in targeting of nNOS to the sarcolemma and the association between nNOS and the dystrophin complex in normal and dystrophic muscle fibers. Northern blotting for PIN mRNA in normal mouse muscles and muscles of mdx mice (an animal model of DMD) revealed a significant rise in PIN mRNA in dystrophic muscles compared with normal muscles. Immunohistochemical analysis showed that, in normal mouse muscle fibers, PIN expression was localized at the sarcolemma, peripheral nuclei, and the sarcoplasm. By comparison, PIN protein in muscles from mdx mice was more concentrated around the sarcolemma and central nuclei. The presence of PIN protein expression in muscles from mdx mice was evident despite the significant reduction in nNOS and dystrophin protein expressions in these fibers. In muscle sections of DMD patients, the absence of nNOS protein expression was accompanied by maintained PIN expression. Prominent PIN expression was also detectable in macrophages infiltrating dystrophic muscle fibers both in mdx mice and DMD patients. These results suggest that PIN expression in muscles from mdx mice and DMD patients is controlled by factors different from those involved in the regulation of nNOS and dystrophin. Moreover, our results indicate that PIN is not an integral component of the dystrophin complex inside skeletal muscle fibers.     

Expression profiling in stably regenerating skeletal muscle of dystrophin-deficient mdx mice

The mdx mouse is comparable to Duchenne muscular dystrophy in having an absence of dystrophin. While dystrophic human skeletal muscle undergoes progressive degeneration, in the mdx mouse regeneration and tissue remodeling substantially compensate for the lack of dystrophin. To better understand the molecular events leading to active muscle regeneration in mdx muscles, we have determined the gene expression profiles of wild-type and mdx hind limb muscles using oligonucleotide arrays. Compared to wild-type, 58 genes were found to be differentially expressed in mdx. The molecular signature of actively regenerating skeletal muscle in young adult mdx mice showed upregulation of muscle development genes and genes involved in immune response, proteolysis and extracellular matrix remodeling. Moreover, energy metabolism and mitochondrial function were not compromised. Insights into the processes activated in the mdx muscle to compensate for chronic degeneration may have important implications for therapy in patients with muscular dystrophy.     

DMD(mdx3Cv) and DMD(mdx4Cv) dystrophin mutations in mice: rapid polymerase chain reaction genotyping

We devised non-radioactive PCR assays for the DMD(mdx3Cv) and DMD(mdx4Cv) mouse dystrophin point mutations, in which mutant and wild type reactions electrophoresed separately diagnose whether the DNA carries the mutant, wild type, or both alleles. This simple and reliable assay facilitates the use of these mutant mouse models, which have an extended inflammatory phase (DMD(mdx3Cv)), less reversion to wild type (DMD(mdx4Cv)), and reduced expression of dystrophin mRNAs arising from internal promoter usage than the DMD(mdx) mouse. The PCR assays described facilitate the use of the DMD(mdx3Cv) and DMD(mdx4Cv) mutant mouse models, when maintaining the mutations as heterozygotes, backcrossing into different inbred genetic backgrounds, or when crossing targeted mutations into these dystrophic backgrounds.     

CD45 fraction bone marrow cells as potential delivery vehicles for genetically corrected dystrophin loci

Targeted correction of mutations in muscle can be delivered by direct i.m. injection of corrective DNA to the dystrophic muscle or by autologous injection of cells that have been genetically corrected after isolation from the individual with the dystrophic muscle. The successful application of chimeraplasty and short fragment homologous replacement to correct the exon 23 nonsense mdx transition at the mouse dys locus has opened up the possibility that with further development, targeted gene correction may have some future application for the treatment of muscular dystrophies. In vitro, application of targeted gene correction at the mdx dys locus results in better correction efficiencies than when applied directly to dystrophic muscle. This suggests that at least for the time being, a strategy involving ex vivo correction may be advantageous over a direct approach for delivery of gene correction to dystrophic muscle. This, particularly in view of recent developments indicating that bone-marrow-derived cells are able to systemically remodel dystrophic muscle, whilst penetration of DNA introduced to muscle is limited to individually injected muscles. Application of targeted gene correction to Duchenne dystrophy needs to account for the fact that about 65% of Duchenne muscular dystrophy cases involve large frame-shift deletion of gene sequence at the dys locus. Traditionally, whilst targeted gene correction is able to restore point mutations entirely, it remains to be seen as to whether a strategy for the 'correction' of frame shift deletions may be engineered successfully. This communication discusses the possibility of applying targeted gene correction to dystrophic muscle in Duchenne dystrophy.     

Tenascin-C expression in dystrophin-related muscular dystrophy

The mdx mouse has a mutated dystrophin gene and is used as a model for the study of Duchenne muscular dystrophy (DMD). We investigated whether regenerating mdx skeletal muscle contains the extracellular matrix protein tenascin-C (TN-C), which is expressed in wound healing and nerve regeneration. Prior to the initiation of muscle degeneration, both normal and mdx mice displayed similar weak staining for TN-C in skeletal muscle, but by 3 weeks of age the mice differed substantially. TN-C was undetectable in normal muscle except at the myotendinous junction, while in dystrophic muscle, TN-C was prominent in degenerating/regenerating areas but absent from undegenerated muscle. With increasing age, TN-C staining declined around stable regenerated mdx myofibers. TN-C was also observed in muscle from dogs with muscular dystrophy and in human boys with DMD. Therefore, in dystrophic muscle, TN-C expression may be stimulated by the degenerative process and remain upregulated unless the tissue undergoes successful regeneration. © 1996 John Wiley & Sons, Inc.     

Reduced necrosis of dystrophic muscle by depletion of host neutrophils, or blocking TNFalpha function with Etanercept in mdx mice

Necrosis of skeletal muscle fibres in the lethal childhood myopathy Duchenne Muscular Dystrophy results from deficiency of the cell membrane associated protein, dystrophin. We test the hypothesis in dystrophin-deficient mice, that the initial sarcolemmal breakdown resulting from dystrophin deficiency is exacerbated by inflammatory cells, specifically neutrophils, and that cytokines, specifically Tumour Necrosis Factor alpha (TNFalpha), contribute to myofibre necrosis. Antibody depletion of host neutrophils resulted in a delayed and significantly reduced amount of skeletal muscle breakdown in young dystrophic mdx mice. A more striking and prolonged protective effect was seen after pharmacological blockade of TNFalpha bioactivity using Etanercept. The extent of exercise induced myofibre necrosis in adult mdx mice after voluntarily wheel exercise was also reduced after Etanercept administration. These data show a clear role for neutrophils and TNFalpha in necrosis of dystrophic mdx muscle in vivo. Etanercept is a highly specific anti-inflammatory drug, widely used clinically, and potential application to muscular dystrophies is suggested by this reduced breakdown of mdx skeletal muscle.     

Cromolyn administration (to block mast cell degranulation) reduces necrosis of dystrophic muscle in mdx mice

Duchenne muscular dystrophy is a lethal muscle wasting disorder, resulting from mutations in the gene encoding for the skeletal muscle protein dystrophin. The absence of functional dystrophin leaves the muscle membrane vulnerable to damage during contraction. Damage initially occurs as 'tears' in the membrane, this damage can be exacerbated by the inflammatory response leading to myofibre necrosis rather than repair. Mast cells resident within skeletal muscle represent an immediate source of pro-inflammatory cytokines. We hypothesise that blockade of mast cell degranulation would reduce the extent of myofibre necrosis in the mdx mouse. Daily cromolyn injections were performed on young and exercised adult mdx mice and histological analysis confirmed that mast cell degranulation contributes to myofibre necrosis. This research identified high biological variation between individual mdx mice in the severity of the dystrophic pathology, and supported a relationship between extent of muscle damage in adult mdx mice and their individual enthusiasm for voluntary wheel running.     

重组腺相关病毒（rAAV）介导基因SMCKA3999对Duchenne肌营养不良病理和功能的影响

目的　 研究重组腺相关病毒 (rAAV)载体介导的dystrophin小基因SMCKA3999治疗DMD模型鼠mdx ,从病理和功能观察rAAVSMCKA3999治疗对DMD模型小鼠mdx的疗效。方法　以dystrophin小基因SMCK A3999为目的基因 ,将SMCKA3999克隆至rAAV并包装成rAAVSMCKA3999,以 5× 10 10 病毒颗粒单点注射于DMD模型鼠mdx腓肠肌 ,基因治疗后 4个月及 7个月 ,采用免疫荧光、光镜组织病理、肌电图等方法 ,从形态和功能观察rAAVSMCKA3999治疗对DMD模型小鼠mdx的疗效。 结果　rAAVSMCKA3999使肌膜缺失的dys trophin恢复并稳定表达持续 7个月以上 ,肌肉组织病理改变好转 ,肌病肌电图改变明显改善 ,疗效持续 4个月以上。 结论　rAAVSMCKA3999能改善mdx小鼠骨骼肌的病理及功能 ,采用rAAV介导的dystrophin小基因SMC KA3999对Duchenne肌营养不良基因治疗是有希望的治疗方法。     

Recombinant micro-genes and dystrophin viral vectors

An effective gene therapy for Duchenne muscular dystrophy ideally relies on the ability to provide long-term expression to muscle tissue of the missing protein, dystrophin. Early work in the mdx mouse using a 6.3 kb mini-dystrophin cDNA, carried out in either adenoviral or retroviral vectors was generally successful, however, expression was only transient. In an attempt to remedy this problem, two approaches are being investigated. The first of these is a hybrid vector system that combines the efficacy of gene transfer into skeletal muscle of adenoviral vectors with the long-term stability of retroviral vectors. The second utilises the inherently efficient transducing properties and stability of the adeno-associated viral delivery system. Using highly truncated micro-dystrophin cDNAs we have shown that both vector systems were able to restore dystrophin and dystrophin-associated protein expression at the plasma membrane of mdx mice for prolonged periods of time. Additionally, evaluation of central nucleation indicated a significant inhibition of degenerative dystrophic muscle pathology. These studies suggest that hybrid adenoviral-retroviral and adeno-associated viral vectors are capable of ameliorating dystrophic pathology at the cellular level and as such are useful tools in the development of a gene therapy for Duchenne muscular dystrophy.     

重组腺相关病毒载体介导的小基因SMCKA3999治疗Duchenne肌营养不良小鼠的研究

目的研究重组腺相关病毒载体(rAAV)介导的人抗肌萎缩蛋白(dystrophin)小基因SMCKA3999对Duchenne肌营养不良(DMD)在病理、肌电图、肌力方面的治疗作用。方法将dystrophin小基因SMCKA3999克隆至rAAV并包装成rAAV SMCKA3999病毒,以5×109病毒颗粒多点注射于DMD模型鼠(mdx鼠)腓肠肌,基因治疗4个月后,以免疫荧光双标法检测肌膜dystrophin基因表达,采用Nicolet肌电及诱发电位仪记录mdx鼠肌电图,基因治疗5个月后以肌肉离体灌注电刺激法测定腓肠肌肌力,观察rAAV SMCKA3999对DMD动物模型鼠的治疗作用。结果rAAVSMCKA3999能有效稳定表达,并使肌膜缺失的dystrophin恢复,明显改善DMD肌电图表现,肌力恢复。结论rAAV SMCKA3999对mdx鼠治疗有效,能显著改善其肌肉功能,应用rAAV介导的dystrophin小基因SMCKA3999基因治疗是临床治疗DMD有希望的方法。     

Analysis of dystrophin in fast- and slow-twitch skeletal muscles from mdx and dy2J mice at different ages

Muscles from mdx, control, and dy2J/dy2J mice at different ages were analyzed for dystrophin in an attempt to relate the chronology of the protein expression with the final phenotypes in regenerated, normal, and dystrophic muscle, respectively. Immunostaining and gold staining of electrophoresis gels were carried out in the investigation. At 5, 25, and 219 days of age, control muscles exhibited dystrophin bands in both the fast-twitch extensor digitorum longus (EDL) and the slow-twitch soleus (SOL) muscles. Muscles from the mdx mice at comparable ages (8, 28, and 217 days) never exhibited bands for dystrophin, although titin, nebulin, myosin, and other protein bands were present at intensities comparable to those in control muscles. The dystrophin band was present in both the EDL and SOL from dy2J/dy2J dystrophic mice. As indicated by the present study, the dystrophin deficiency from mdx tissue is not transient. This suggests that dystrophin is not necessary for the success of mdx muscle regeneration.     

GABAergic miniature spontaneous activity is increased in the CA1 hippocampal region of dystrophic mdx mice

Duchenne muscular dystrophy (DMD), a genetic disease due to dystrophin gene mutation and characterised by skeletal muscle failure, is associated with non-progressive cognitive deficits. In human and mouse brain, full-length dystrophin is localised postsynaptically in neocortical, hippocampal and cerebellar neurons. Evidence obtained in the CNS of dystrophic mice (mdx) suggested alterations of the GABAergic system. However, a direct functional evaluation of GABAergic synaptic transmission in mdx mice has not been conducted in the hippocampus, which is involved in cognitive processes and is rich in full-length dystrophin. Here, we investigated evoked and miniature inhibitory postsynaptic currents (IPSCs) in CA1 pyramidal neurons of mdx mice with patch clamp recording techniques. Results showed an increased frequency of miniature spontaneous IPSCs in mdx mice compared with controls, whereas evoked IPSCs did not show significant variations. Paired-pulse facilitation (PPF) analysis showed lack of facilitation at short intervals in mdx mice compared with that in wild-type mice. Analysis of density of synapses that innervate CA1 pyramidal cell bodies did not indicate significant differences between mdx mice and controls. Therefore, we suggest that increased miniature spontaneous IPSC frequency is due to altered pre-synaptic release probability. The present findings are discussed in the light of the accrued evidence for alterations of inhibitory synaptic transmission in the brain of dystrophic mice.     

Dystrophin expression in myotubes formed by the fusion of normal and dystrophic myoblasts

Mdx mouse dystrophy is characterized by the absence in the muscle cytoplasmic membrane of a high molecular weight protein called dystrophin. A possible avenue for treatment of muscular dystrophies is to inject normal myoblasts in a dystrophic muscle to form hybrid muscle fibers. Hybrid myotubes were formed in vitro by the fusion of normal rat and dystrophic mouse (mdx) myoblasts. Staining with Hoechst dye 33258 permitted the clear distinction of mouse and rat nuclei. Immunostaining demonstrated that dystrophin was present over the entire membrane of all hybrid myotubes even when nuclei ratio normal/dystrophic was low.