Proteomic assessment of the acute phase of dystrophin deficiency in mdx mice

Duchenne muscular dystrophy (DMD) is caused by the absence of a functional dystrophin protein and is modeled by the mdx mouse. The mdx mouse suffers an early necrotic bout in the hind limb muscles lasting from approximately 4 to 7 weeks. The purpose of this investigation was to determine the extent to which dystrophin deficiency changed the proteome very early in the disease process. In order to accomplish this, proteins from gastrocnemius from 6-week-old C57 (n = 6) and mdx (n = 6) mice were labeled with fluorescent dye and subjected to two-dimensional differential in-gel electrophoresis (2D-DIGE). Resulting differentially expressed spots were excised and protein identity determined via MALDI-TOF followed by database searching using MASCOT. Proteins of the immediate energy system and glycolysis were generally down-regulated in mdx mice compared to C57 mice. Conversely, expression of proteins involved in the Kreb’s cycle and electron transport chain were increased in dystrophin-deficient muscle compared to control. Expression of cytoskeletal components, including tubulins, vimentin, and collagen, were increased in mdx mice compared to C57 mice. Importantly, these changes are occurring at only 6 weeks of age and are caused by acute dystrophin deficiency rather than more chronic injury. These data may provide insight regarding early pathologic changes occurring in dystrophin-deficient skeletal muscle.     

Prolonged dystrophin expression and functional correction of mdx mouse muscle following gene transfer with a helper-dependent (gutted) adenovirus-encoding murine dystrophin

Dystrophin gene transfer using helper-dependent adenoviruses (HDAd), which are deleted of all viral genes, is a promising option to treat muscles in Duchenne muscular dystrophy. We investigated the benefits of this approach by injecting the tibialis anterior (TA) muscle of neonatal and juvenile (4-6-week-old) dystrophin-deficient (mdx) mice with a fully deleted HDAd (HDCBDysM). This vector encoded two full-length murine dystrophin cDNAs regulated by the powerful cytomegalovirus enhancer/beta-actin promoter. At 10 days post-injection of neonatal muscles, 712 fibers (42% of the total number of TA fibers) were dystrophin-positive (dys+), a value that did not decrease for 6 months (the study duration). In treated juveniles, maximal transduction occurred at 30 days post-injection (414 dys+ fibers, 24% of the total number of TA fibers), but decreased by 51% after 6 months. All studied aspects of the pathology were improved in neonatally treated muscles: the percentage of dys+ fibers with centrally localized myonuclei remained low, localization of the dystrophin associated protein complex was restored at the plasma membrane, muscle hypertrophy was reduced, and maximal force-generating capacity and resistance to contraction-induced injuries were increased. The same pathological aspects were improved in the treated juveniles, except for reduction of muscle hypertrophy and maximal force-generating capacity. We demonstrated a strong humoral response against murine dystrophin in both animal groups, but mild inflammatory response occurred only in the treated juveniles. HDCBDysM is thus one of the most promising and efficient vectors for treating DMD by gene therapy.     

Effective detection of corrected dystrophin loci in mdx mouse myogenic precursors

Targeted corrective gene conversion (TCGC) holds much promise as a future therapy for many hereditary diseases in humans. Mutation correction frequencies varying between 0.0001% and 40% have been reported using chimeraplasty, oligoplasty, triplex-forming oligonucleotides, and small corrective PCR amplicons (CPA). However, PCR technologies used to detect correction events risk either falsely indicating or greatly exaggerating the presence of corrected loci. This is a problem that is considerably exacerbated by attempted improvement of the TCGC system using high corrective nucleic acid (CNA) to nuclear ratios. Small fragment homologous replacement (SFHR)-mediated correction of the exon 23 dystrophin (DMD) gene mutation in the mdx mouse model of DMD has been used in this study to evaluate the effect of increasing CPA amounts. In these experiments, we detected extremely high levels of apparently corrected loci and determined that at higher CNA to nuclear ratios the extent of locus correction was highly exaggerated by residual CNA species in the nucleic acids extracted from the treated cells. This study describes a generic locus-specific detection protocol designed to eradicate residual CNA species and avoid the artifactual or exaggerated detection of gene correction.     

Direct retroviral-mediated transfer of a dystrophin minigene into mdx mouse muscle in vivo

At the cellular level, the primary pathology in Duchenne musculardystrophy (DMD) is caused by deficiency of the sarcolemmal-associatedprotein, dystrophin, in the striated musculature. Here we describethe somatic transfer and longterm expression of a human dystrophinminigene corresponding to a mild Becker muscular dystrophy (BMD)phenotype in skeletal muscle tissues of the dystrophin-deficientmdx mouse by direct retroviral transduction. Following a singleintramuscular injection of recombinant retrovirus, sarcolemmalexpression of dystrophin was observed in an average of     

Full-length dystrophin expression in half of the heart cells ameliorates beta-isoproterenol-induced cardiomyopathy in mdx mice

Gene therapy holds great promise for curing Duchenne muscular dystrophy (DMD), the most common fatal inherited childhood muscle disease. Success of DMD gene therapy depends upon functional improvement in both skeletal and cardiac muscle. Numerous gene transfer studies have been performed to correct skeletal muscle pathology, yet little is known about cardiomyopathy gene therapy. Since complete transduction of the entire heart is an impractical goal, it becomes critical to determine the minimal level of correction needed for successful DMD cardiomyopathy gene therapy. To address this question, we generated heterozygous mice that persistently expressed the full-length dystrophin gene in 50% of the cardiomyocytes of mdx mice, a model for DMD. We questioned whether dystrophin expression in half of the heart cells was sufficient to prevent stress-induced cardiomyopathy. Heart function of mdx mouse is normal in the absence of external stress. To determine the therapeutic effect, we challenged 3-month-old mice with beta-isoproterenol. Cardiomyocyte sarcolemma integrity was significantly impaired in mdx but not in heterozygous and C57Bl/10 mice. Importantly, in vivo closed-chest hemodynamic assays revealed normal left ventricular function in beta-isoproterenol-stimulated heterozygous mice. Since the expression profile in the heterozygous mice mimicked viral transduction, we conclude that gene therapy correction in 50% of the heart cells may be sufficient to treat cardiomyopathy in mdx mice. This finding may also apply to the gene therapy of other inherited cardiomyopathies.     

Read-through compound 13 restores dystrophin expression and improves muscle function in the mdx mouse model for Duchenne muscular dystrophy

Molecules that induce ribosomal read-through of nonsense mutations in mRNA and allow production of a full-length functional protein hold great therapeutic potential for the treatment of many genetic disorders. Two such read-through compounds, RTC13 and RTC14, were recently identified by a luciferase-independent high-throughput screening assay and were shown to have potential therapeutic functions in the treatment of nonsense mutations in the ATM and the dystrophin genes. We have now tested the ability of RTC13 and RTC14 to restore dystrophin expression into skeletal muscles of the mdx mouse model for Duchenne muscular dystrophy (DMD). Direct intramuscular injection of compound RTC14 did not result in significant read-through activity in vivo and demonstrated the levels of dystrophin protein similar to those detected using gentamicin. In contrast, significant higher amounts of dystrophin were detected after intramuscular injection of RTC13. When administered systemically, RTC13 was shown to partially restore dystrophin protein in different muscle groups, including diaphragm and heart, and improved muscle function. An increase in muscle strength was detected in all treated animals and was accompanied by a significant decrease in creatine kinase levels. These studies establish the therapeutic potential of RTC13 in vivo and advance this newly identified compound into preclinical application for DMD.     

Properties of branchiomeric and somite-derived muscle development in Tbx1 mutant embryos

Vertebrate craniofacial and trunk myogenesis are regulated by distinct genetic programs. Tbx1, homologue of the del22q11.2 syndrome candidate gene TBX1, controls branchiomeric craniofacial muscle development. Here, we demonstrate using immunohistochemistry that myogenic regulatory factors are activated in Tbx1-positive cells within pharyngeal mesoderm. These cells are also Islet1 and Capsulin-positive and in the absence of Tbx1 persist in the core of the first arch. Sporadic hypoplastic mandibular muscles in Tbx1-/- embryos contain Pax7-positive myocytes with indistinguishable differentiation properties from wild-type muscles and have normal tendon attachments and fiber-type patterning. In contrast to TBX1 haploinsufficient del22q11.2 syndrome patients, no alteration in fiber-type distribution was detected in Tbx1+/- adult masseter and pharyngeal constrictor muscles. Furthermore, Tbx1-expressing limb muscles display normal patterning, differentiation, fiber-type growth, fiber-type distribution and fetal maturation in the absence of Tbx1. The critical requirement for Tbx1 during muscle development is thus in the robust onset of myogenic specification in pharyngeal mesoderm.     

Human dystrophin expression corrects the myopathic phenotype in transgenic mdx mice.

Duchenne and the less severe Becker form of muscular dystrophy (DMD,BMD) result from genetic deficiency in the level and/or activity of the protein dystrophin. The recent availability of cDNA based minigenes encoding recombinant dystrophin polypeptides has raised the possibility of somatic gene transfer as a therapeutic approach to treat dystrophin deficiency. In this respect, the mdx mouse provides a useful model of DMD exhibiting features characteristic of both the early myopathic and later fibrotic phases of the human disease. Using a mutated human cDNA, compatible in size with virus-based somatic gene transfer vectors, the pathophysiological consequences of restoring dystrophin expression have been examined in transgenic mdx mice. Transgene expression was correlated with a marked reduction of the skeletal myofibre necrosis and regeneration which is a major feature of the dystrophin-deficient phenotype in young mdx mice. The cDNA construct which is based on a very mild BMD phenotype thus encodes a highly functional dystrophin molecule whose reduced size renders it an attractive candidate for development as a therapeutic gene transfer reagent.     

Expression of full-length and truncated dystrophin mini-genes in transgenic mdx mice

Duchenne and Becker muscular dystrophy are caused by defectsin the dystrophin gene, and are candidates for treatment bygene therapy. We have shown previously that overexpression ofa full-length dystrophin cDNA prevents the development of dystrophicsymptoms in mdx mice. We show here that this functional correctioncan be achieved by expressing the full-length muscle isoformat a lower level than is present in control animals. Gene therapyfor DMD may necessitate the use of truncated dystrophin mini-genesto accommodate the limited cloning capacity of current-generationviral delivery vectors. We have constructed both murine andhuman mini-genes deleted for exons 17–48, and have demonstratedthat expression of either mini-gene can almost completely preventthe development of dystrophic symptoms in transgenic mdx mice.These results suggest that viral-mediated expression of moderatelevels of a truncated dystrophin could be an effective treatmentfor DMD.     

Understanding dystrophinopathies: an inventory of the structural and functional consequences of the absence of dystrophin in muscles of the mdx mouse

Journal of Muscle Research and Cell Motility -     

An improved procedure for the efficient injection of radioactive steroids into mouse embryos

Retention of 3H-moxestrol in mouse fetuses after transmaternal, intrafetal, and intraplacental injection were compared. Direct injection into the allantoic placenta resulted in greater retention of radioactivity by the fetus than the other modes of administration between 12 and 16 days of gestation. By this same criterion, intrafetal injection was best for older fetuses. Maternal injection was the least efficient way to transfer 3H-moxestrol to the fetus.     

T-cell-dependent fibrosis in the mdx dystrophic mouse

In Duchenne muscular dystrophy patients, the pathological hallmark of the disease, namely, the chronic accumulation of sclerotic scar tissue in the interstitial space of skeletal muscle is attributed to manifestation of secondary pathological processes. Such anomalous generation of matrix protein is thought to be driven by the continuous degeneration and regeneration of muscle both in Duchenne Muscular Dystrophy and in the mdx mouse homolog. We examined mdx and the control strain C57bl/10 mice over a range of ages with respect to the amounts of collagen present in muscles and other organs, finding that the mdx have significantly higher collagen content at later time points in their kidney and lung as well as their muscles. Surprisingly, when we bred the mdx mice on the nu/nu background, the time course of fibrogenesis was modified depending on the tissue and the collagen content was significantly different in age-matched mice. Transplantation of normal thymic tissue into the mdx-nu/nu mice replenished their T-cells and concomitantly altered the collagen content in their tissues to levels comparable with those in immunocompetent mdx mice. This suggests that T-cells play a role in the onset of the fibrotic events that undermines the ability of dystrophic muscle to regenerate.     

Failure to infect embryos after virus injection in mouse zygotes

BACKGROUND: The intracytoplasmic injection of sperm raises the problem that viral elements may be transported into the oocyte by the spermatozoon or the surrounding medium. It also raises questions about how the developing zygote will behave. METHODS: We used the murine model to microinject murine cytomegalovirus (MCMV) into the zygote ooplasm and followed the changes in these microinjected zygotes in vivo and in vitro over time. RESULTS: 80% of zygotes microinjected with viral suspension, and 80% injected with medium alone, survived. Although MCMV DNA was detected in 56% of injected embryos, up until the blastocyst stage, the mice born from these injected zygotes developed normally and did not contain MCMV DNA. When embryonic stem cells were co-incubated with MCMV and then transferred into healthy blastocysts, the offspring were normal and did not contain any MCMV DNA. CONCLUSIONS: Our observations suggest that even if MCMV DNA persists from the zygote to the blastocyst stage, its presence has no detrimental effect on pre-implantation or post-implantation development.     

Expression of dystrophin in the mouse myenteric neurones

Dystrophin, a membrane-associated protein, plays relevant roles in cell functions. Its lack or trunkated expression results in Duchenne muscular dystrophy (DMD), a pathology associated with alterations in gastrointestinal motility considered to be neural in origin. No data are available on the presence of dystrophin in myenteric neurones. We labelled mouse myenteric neurones with DYS1-, DYS2-, DYS3-antibodies; staining was located on the perikarya and processes, with no differences in distribution or intensity among the antibodies; the western immunoblot analysis indicated that myenteric neurones express several dystrophin isoforms; anti-dystrophins/anti-neuronal specific enolase double-labeling confirmed that all neurones express dystrophin. Dystrophin in myenteric neurones might play a role in cytoskeletal organization, axonal transport and signal pathways; its lack might cause the intestinal motor abnormalities reported in DMD patients.     

Suppression of revertant fibers in mdx mice by expression of a functional dystrophin.

Duchenne muscular dystrophy (DMD) is characterized by progressive muscle degeneration that results from the absence of dystrophin. Despite null mutations in the dystrophin gene, many DMD patients display a low percentage of dystrophin-positive fibers. These "revertant fibers" are also present in the dystrophin-deficient mdx mouse and are believed to result from alternative splicing or second mutation events that bypass the mutation and restore an open reading frame. However, it is unclear what role dystrophin and the dystrophic pathology might play in revertant fiber formation and accumulation. We have analyzed the role of dystrophin expression and the dystrophic pathology in this process by monitoring revertant fibers in transgenic mdx mice that express truncated dystrophins. We found that newborn transgenic mice displayed approximately the same number of revertant fibers as newborn mdx mice, indicating that expression of a functional dystrophin does not suppress the initiation of revertant fiber formation. Surprisingly, when the transgene encoded a functional dystrophin, revertant fibers were not detected in adult or old mdx mice. In contrast, adult transgenic mice expressing a non-functional dystrophin accumulated increasing numbers of revertant fibers, similar to mdx mice, suggesting that positive selection is required for the persistence of revertant fibers. Finally, we provide evidence that the loss of revertant dystrophin in transgenic mdx muscle fibers overexpressing a functional dystrophin results from displacement of the revertant protein by the transgene-encoded dystrophin.