C-X-C motif chemokine ligand 12: a potential therapeutic target in Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is an X-linked recessive disease caused by a mutant dystrophin protein. DMD patients undergo gradual progressive paralysis until death. Chronic glucocorticoid therapy remains one of the main treatments for DMD, despite the significant side effects. However, its mechanisms of action remain largely unknown. We used bioinformatics tools to identify pathogenic genes involved in DMD and glucocorticoid target genes. Two gene expression profiles containing data from DMD patients and healthy controls (GSE38417 and GSE109178) were downloaded for further analysis. Differentially expressed genes (DEGs) between DMD patients and controls were identified using GEO2R, and glucocorticoid target genes were predicted from the Pharmacogenetics and Pharmacogenomics Knowledge Base. Surprisingly, only one gene, CXCL12 (C-X-C motif chemokine ligand 12), was both a glucocorticoid target and a DEG. Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis, Gene Ontology term enrichment analysis, and gene set enrichment analysis were performed. A protein-protein interaction network was constructed and hub genes identified using the Search Tool for the Retrieval of Interacting Genes (STRING) database and Cytoscape. Enriched pathways involving the DEGs, including CXCL12, were associated with the immune response and inflammation. Levels of CXCL12 and its receptor CXCR4 (C-X-C motif chemokine receptor 4) were increased in X-linked muscular dystrophy (mdx) mice (DMD models) but became significantly reduced after prednisone treatment. Metformin also reduced the expression of CXCL12 and CXCR4 in mdx mice. In conclusion, the CXCL12-CXCR4 pathway may be a potential target for DMD therapy.

Dystrophin Deficiency Causes Progressive Depletion of Cardiovascular Progenitor Cells in the Heart.

Duchenne muscular dystrophy (DMD) is a devastating condition shortening the lifespan of young men. DMD patients suffer from age-related dilated cardiomyopathy (DCM) that leads to heart failure. Several molecular mechanisms leading to cardiomyocyte death in DMD have been described. However, the pathological progression of DMD-associated DCM remains unclear. In skeletal muscle, a dramatic decrease in stem cells, so-called satellite cells, has been shown in DMD patients. Whether similar dysfunction occurs with cardiac muscle cardiovascular progenitor cells (CVPCs) in DMD remains to be explored. We hypothesized that the number of CVPCs decreases in the dystrophin-deficient heart with age and disease state, contributing to DCM progression. We used the dystrophin-deficient mouse model (mdx) to investigate age-dependent CVPC properties. Using quantitative PCR, flow cytometry, speckle tracking echocardiography, and immunofluorescence, we revealed that young mdx mice exhibit elevated CVPCs. We observed a rapid age-related CVPC depletion, coinciding with the progressive onset of cardiac dysfunction. Moreover, mdx CVPCs displayed increased DNA damage, suggesting impaired cardiac muscle homeostasis. Overall, our results identify the early recruitment of CVPCs in dystrophic hearts and their fast depletion with ageing. This latter depletion may participate in the fibrosis development and the acceleration onset of the cardiomyopathy.

Twenty-one days of low-intensity eccentric training improve morphological characteristics and function of soleus muscles of mdx mice.

Duchene muscular dystrophy (DMD) is caused by the absence of the protein dystrophin, which leads to muscle weakness, progressive degeneration, and eventually death due to respiratory failure. Low-intensity eccentric training (LIET) has been used as a rehabilitation method in skeletal muscles after disuse. Recently, LIET has also been used for rehabilitating dystrophic muscles, but its effects are still unclear. The purpose of this study was to investigate the effects of 21 days of LIET in dystrophic soleus muscle. Thirty-six male mdx mice were randomized into six groups (n = 6/each): mdx sedentary group; mdx training group-3 days; mdx training group-21 days; wild-type sedentary group; wild-type training group-3 days and wild-type training group-21 days. After the training sessions, animals were euthanized, and fragments of soleus muscles were removed for immunofluorescence and histological analyses, and measurements of active force and Ca2+ sensitivity of the contractile apparatus. Muscles of the mdx training group-21 days showed an improvement in morphological characteristics and an increase of active force when compared to the sedentary mdx group. The results show that LIET can improve the functionality of dystrophic soleus muscle in mice.

Mineralocorticoid Receptor Antagonists in Muscular Dystrophy Mice During Aging and Exercise.

BACKGROUND: Mineralocorticoid receptor antagonists added to angiotensin converting enzyme inhibitors have shown preclinical efficacy for both skeletal and cardiac muscle outcomes in young sedentary dystrophin-deficient mdx mice also haploinsufficient for utrophin, a Duchenne muscular dystrophy (DMD) model. The mdx genotypic DMD model has mild pathology, making non-curative therapeutic effects difficult to distinguish at baseline. Since the cardiac benefit of mineralocorticoid receptor antagonists has been translated to DMD patients, it is important to optimize potential advantages for skeletal muscle by further defining efficacy parameters. OBJECTIVE: We aimed to test whether therapeutic effects of mineralocorticoid receptor antagonists added to angiotensin converting enzyme inhibitors are detectable using three different reported methods of exacerbating the mdx phenotype. METHODS: We tested treatment with lisinopril and the mineralocorticoid receptor antagonist spironolactone in: 10 week-old exercised, 1 year-old sedentary, and 5 month-old isoproterenol treated mdx mice and performed comprehensive functional and histological measurements. RESULTS: None of the protocols to exacerbate mdx phenotypes resulted in dramatically enhanced pathology and no significant benefit was observed with treatment. CONCLUSIONS: Since endogenous mineralocorticoid aldosterone production from immune cells in dystrophic muscle may explain antagonist efficacy, it is likely that these drugs work optimally during the narrow window of peak inflammation in mdx mice. Exercised and aged mdx mice do not display prolific damage and inflammation, likely explaining the absence of continued efficacy of these drugs. Since inflammation is more prevalent in DMD patients, the therapeutic window for mineralocorticoid receptor antagonists in patients may be longer.

Variable rescue of microtubule and physiological phenotypes in mdx muscle expressing different miniaturized dystrophins.

Delivery of miniaturized dystrophin genes via adeno-associated viral vectors is one leading approach in development to treat Duchenne muscular dystrophy. Here we directly compared the functionality of five mini- and micro-dystrophins via skeletal muscle-specific transgenic expression in dystrophin-deficient mdx mice. We evaluated their ability to rescue defects in the microtubule network, passive stiffness and contractility of skeletal muscle. Transgenic mdx mice expressing the short dystrophin isoform Dp116 served as a negative control. All mini- and micro-dystrophins restored elevated detyrosinated α-tubulin and microtubule density of mdx muscle to values not different from C57BL/10, however, only mini-dystrophins restored the transverse component of the microtubule lattice back to C57BL/10. Passive stiffness values in mdx muscles expressing mini- or micro-dystrophins were not different from C57BL/10. While all mini- and micro-dystrophins conferred significant protection from eccentric contraction-induced force loss in vivo and ex vivo compared to mdx, removal of repeats two and three resulted in less protection from force drop caused by eccentric contraction ex vivo. Our data reveal subtle yet significant differences in the relative functionalities for different therapeutic constructs of miniaturized dystrophin in terms of protection from ex vivo eccentric contraction-induced force loss and restoration of an organized microtubule lattice.

Proteomic Profiling of the Dystrophin-Deficient Brain.

Duchenne muscular dystrophy is a highly progressive neuromuscular disorder caused by primary abnormalities in the Dmd gene encoding the membrane cytoskeletal protein dystrophin. Dystrophinopathies are multi-systems disorders that are characterized by severe skeletal muscle wasting, with loss of independent ambulation in the early teenage years, followed by cardio-respiratory complications and premature death. Nonprogressive cognitive impairments are estimated to affect approximately one-third of dystrophic children. To identify the molecular mechanisms behind the impaired brain function in dystrophinopathy, liquid chromatography-based mass spectrometry offers an unbiased and technology-driven approach. In this chapter, we give a detailed description of a label-free mass spectrometric method to investigate proteome-wide changes in the dystrophin-deficient brain from a genetic mouse model of Duchenne muscular dystrophy.

Probing the Pathogenesis of Duchenne Muscular Dystrophy Using Mouse Models.

Investigations using mouse models have provided seminal insights into the pathogenesis of Duchenne muscular dystrophy and the development of novel therapeutics. Several important methods have been considered standard-in-the-field for analyses of skeletal muscle weakness, strength, endurance, and histopathology, as well as responses to therapeutics such as glucocorticoids, disease modifying drugs which are part of the current standard of care for patients with this disease. Here we describe optimized genetic, genomic, and physiologic assays to probe dystrophic pathobiology in the mdx mouse and related strains.

The Use of Antisense Oligonucleotides for the Treatment of Duchenne Muscular Dystrophy.

Antisense oligonucleotides (AONs) hold great promise for therapeutic splice-switching correction in many genetic diseases and in particular for Duchenne muscular dystrophy (DMD), where AONs can be used to reframe the dystrophin transcript and give rise to a partially deleted but yet functional dystrophin protein. Many different chemistries of AONs can be used for splice switching modulation, and some of them have been evaluated in clinical trials for DMD. However, despite advances in AON chemistry and design, systemic use of AONs is limited due to poor tissue uptake, and sufficient therapeutic efficacy is difficult to achieve. Therefore, there is still a critical need to develop efficient AONs able to restore the expression of dystrophin in all relevant tissues and international efforts are currently on going to develop new compounds or alternative chemistries with higher therapeutic potential. Here, we describe the methods to evaluate the potency of antisense oligonucleotides, and in particular of tricyclo-DNA (tcDNA)-AONs, a novel class of AONs which displays unique pharmacological properties and unprecedented uptake in many tissues after systemic administration. We focus on the most widely used mouse model for DMD, the mdx mouse and detail methods to analyze the skipping of the mouse exon 23 both in vitro in H2K mdx cells and in vivo in the mdx mouse model.

Whole genome sequencing reveals a 7 base-pair deletion in DMD exon 42 in a dog with muscular dystrophy.

Dystrophin is a key cytoskeletal protein coded by the Duchenne muscular dystrophy (DMD) gene located on the X-chromosome. Truncating mutations in the DMD gene cause loss of dystrophin and the classical DMD clinical syndrome. Spontaneous DMD gene mutations and associated phenotypes occur in several other species. The mdx mouse model and the golden retriever muscular dystrophy (GRMD) canine model have been used extensively to study DMD disease pathogenesis and show efficacy and side effects of putative treatments. Certain DMD gene mutations in high-risk, the so-called hot spot areas can be particularly helpful in modeling molecular therapies. Identification of specific mutations has been greatly enhanced by new genomic methods. Whole genome, next generation sequencing (WGS) has been recently used to define DMD patient mutations, but has not been used in dystrophic dogs. A dystrophin-deficient Cavalier King Charles Spaniel (CKCS) dog was evaluated at the functional, histopathological, biochemical, and molecular level. The affected dog's phenotype was compared to the previously reported canine dystrophinopathies. WGS was then used to detect a 7 base pair deletion in DMD exon 42 (c.6051-6057delTCTCAAT mRNA), predicting a frameshift in gene transcription and truncation of dystrophin protein translation. The deletion was confirmed with conventional PCR and Sanger sequencing. This mutation is in a secondary DMD gene hotspot area distinct from the one identified earlier at the 5' donor splice site of intron 50 in the CKCS breed.

[Research progress on disease models and gene therapy of Duchenne muscular dystrophy].

Duchenne muscular dystrophy (DMD) is an X-linked, recessive and lethal genetic disease, which usually caused by gene mutations and the underlying mechanisms are complicated and diverse. The causal gene of DMD is the largest one in human that locates in the region of Xp21.2, encoding dystrophin. Currently there is no effective treatment for DMD patients. The treatment of DMD depends on gene mutation and molecular mechanism study of the disease, which requires reliable disease models such as mdx mouse model. Recently, researchers have increasingly discovered gene therapy strategies for DMD, and the efficacy has been demonstrated in DMD animal models. In addition, induced pluripotent stem cell technology can provide patient-specific cell source, offering a new platform for mechanism and therapy study of DMD.

Recombinase-mediated reprogramming and dystrophin gene addition in mdx mouse induced pluripotent stem cells.

A cell therapy strategy utilizing genetically-corrected induced pluripotent stem cells (iPSC) may be an attractive approach for genetic disorders such as muscular dystrophies. Methods for genetic engineering of iPSC that emphasize precision and minimize random integration would be beneficial. We demonstrate here an approach in the mdx mouse model of Duchenne muscular dystrophy that focuses on the use of site-specific recombinases to achieve genetic engineering. We employed non-viral, plasmid-mediated methods to reprogram mdx fibroblasts, using phiC31 integrase to insert a single copy of the reprogramming genes at a safe location in the genome. We next used Bxb1 integrase to add the therapeutic full-length dystrophin cDNA to the iPSC in a site-specific manner. Unwanted DNA sequences, including the reprogramming genes, were then precisely deleted with Cre resolvase. Pluripotency of the iPSC was analyzed before and after gene addition, and ability of the genetically corrected iPSC to differentiate into myogenic precursors was evaluated by morphology, immunohistochemistry, qRT-PCR, FACS analysis, and intramuscular engraftment. These data demonstrate a non-viral, reprogramming-plus-gene addition genetic engineering strategy utilizing site-specific recombinases that can be applied easily to mouse cells. This work introduces a significant level of precision in the genetic engineering of iPSC that can be built upon in future studies.

Phenotypic differences between mdx black mice and mdx albino mice. Comparison of cytokine levels in the blood.

In mdx mice, mutation in the muscle protein dystrophin gene results in the development of chronic degeneration of the muscle tissue. We performed a comparative analysis of blood cytokine levels in mdx mice, classical black mice and mice with additional genetic defect responsible for the manifestations of oculocutaneous albinism. In mdx albino mice, the total pool of cytokines (IL-10, IL-6, IL-5, IL-2, IL-1α, IL-4, IL-17, granulocyte-macrophage growth factor, TNF-α, and IFN-γ) was increased. This increase was not associated with selective release of one of the above cytokines into the blood. The fraction of pro-inflammatory cytokines (IL-6, IL-1α, TNF-α) was increased in the total pool and the percentage of antiinflammatory cytokines (IL-4) was reduced. Changes in cytokine pool probably reflect the differences in the severity of the pathological process in the muscle tissue of both genetic variations of mdx mice.

The Rag2⁻Il2rb⁻Dmd⁻ mouse: a novel dystrophic and immunodeficient model to assess innovating therapeutic strategies for muscular dystrophies.

The development of innovative therapeutic strategies for muscular dystrophies, particularly cell-based approaches, is still a developing field. Although positive results have been obtained in animal models, they have rarely been confirmed in patients and resulted in very limited clinical improvements, suggesting some specificity in humans. These findings emphasized the need for an appropriate animal model (i.e., immunodeficient and dystrophic) to investigate in vivo the behavior of transplanted human myogenic stem cells. We report a new model, the Rag2(-)Il2rb(-)Dmd(-) mouse, which lacks T, B, and NK cells, and also carries a mutant Dmd allele that prevents the production of any dystrophin isoform. The dystrophic features of this new model are comparable with those of the classically used mdx mouse, but with the total absence of any revertant dystrophin positive fiber. We show that Rag2(-)Il2rb(-)Dmd(-) mice allow long-term xenografts of human myogenic cells. Altogether, our findings indicate that the Rag2(-)Il2rb(-)Dmd(-) mouse represents an ideal model to gain further insights into the behavior of human myogenic stem cells in a dystrophic context, and can be used to assess innovative therapeutic strategies for muscular dystrophies.

Sequencing protocols to genotype mdx, mdx(4cv), and mdx(5cv) mice.

Currently available polymerase chain reaction (PCR) genotyping methods for point mutations in the mouse dystrophin gene can lead to false positives and result in wasted time and money due to breeding or treating the wrong mice. Here we describe a simple and accurate method for sequencing the point mutations in mdx, mdx(4cv), and mdx(5cv) mice. This method clearly distinguishes between wildtype, heterozygous, and mutant transcripts, and thereby time and money can be saved by avoiding false positives.

A new genetic variant of mdx mice: study of the phenotype.

Genetic selection in a colony of mdx mice (suffering from X-chromosome-linked muscular dystrophy) resulted in generation of their new genetic variant. In this new variant, the genetic, biochemical, and histological markers of muscular dystrophy are combined with signs of oculocutaneous albinism (skin and fur depigmentation), transillumination of the iris, sharply reduced pigmentation of the retinal epithelium, and increase of the eyeball refraction). Two sensorimotor tests (negative geotaxis and wire back down hanging) detected other phenotypical characteristics of albino mdx mice carrying, in addition to the mutation in the dystrophin gene exon 23 (intrinsic of the "classical" black mdx mice), an extra mutation responsible for pigmentation disorders. Slow geotaxis, despite longer wire back down hanging capacity, was regarded as aggravation of the neurological dysfunction in albino mdx mice in comparison with black mdx mice.

Truncated dystrophins can influence neuromuscular synapse structure.

Duchenne muscular dystrophy (DMD) is characterized by muscle degeneration and structural defects in the neuromuscular synapse that are caused by mutations in dystrophin. Whether aberrant neuromuscular synapse structure is an indirect consequence of muscle degeneration or a direct result of loss of dystrophin function is not known. Rational design of truncated dystrophins has enabled the design of expression cassettes highly effective at preventing muscle degeneration in mouse models of DMD using gene therapy. Here we examined the functional capacity of a minidystrophin (minidysGFP) and a microdystrophin (microdystrophin(DeltaR4-R23)) transgene on the maturation and maintenance of neuromuscular junctions (NMJ) in mdx mice. We found that minidysGFP prevents fragmentation and the loss of postsynaptic folds at the NMJ. In contrast, microdystrophin (DeltaR4-R23) was unable to prevent synapse fragmentation in the limb muscles despite preventing muscle degeneration, although fragmentation was observed to temporally correlate with the formation of ringed fibers. Surprisingly, microdystrophin(DeltaR4-R23) increased the length of synaptic folds in the diaphragm muscles of mdx mice independent of muscle degeneration or the formation of ringed fibers. We also demonstrate that the number and depth of synaptic folds influences the density of voltage-gated sodium channels at the neuromuscular synapse in mdx, microdystrophin(DeltaR4-R23)/mdx and mdx:utrophin double knockout mice. Together, these data suggest that maintenance of the neuromuscular synapse is governed through its lateral association with the muscle cytoskeleton, and that dystrophin has a direct role in promoting the maturation of synaptic folds to allow more sodium channels into the junction.

Animal models for genetic neuromuscular diseases.

The neuromuscular disorders are a heterogeneous group of genetic diseases, caused by mutations in genes coding sarcolemmal, sarcomeric, and citosolic muscle proteins. Deficiencies or loss of function of these proteins leads to variable degree of progressive loss of motor ability. Several animal models, manifesting phenotypes observed in neuromuscular diseases, have been identified in nature or generated in laboratory. These models generally present physiological alterations observed in human patients and can be used as important tools for genetic, clinic, and histopathological studies. The mdx mouse is the most widely used animal model for Duchenne muscular dystrophy (DMD). Although it is a good genetic and biochemical model, presenting total deficiency of the protein dystrophin in the muscle, this mouse is not useful for clinical trials because of its very mild phenotype. The canine golden retriever MD model represents a more clinically similar model of DMD due to its larger size and significant muscle weakness. Autosomal recessive limb-girdle MD forms models include the SJL/J mice, which develop a spontaneous myopathy resulting from a mutation in the Dysferlin gene, being a model for LGMD2B. For the human sarcoglycanopahties (SG), the BIO14.6 hamster is the spontaneous animal model for delta-SG deficiency, whereas some canine models with deficiency of SG proteins have also been identified. More recently, using the homologous recombination technique in embryonic stem cell, several mouse models have been developed with null mutations in each one of the four SG genes. All sarcoglycan-null animals display a progressive muscular dystrophy of variable severity and share the property of a significant secondary reduction in the expression of the other members of the sarcoglycan subcomplex and other components of the Dystrophin-glycoprotein complex. Mouse models for congenital MD include the dy/dy (dystrophia-muscularis) mouse and the allelic mutant dy(2J)/dy(2J) mouse, both presenting significant reduction of alpha2-laminin in the muscle and a severe phenotype. The myodystrophy mouse (Large(myd)) harbors a mutation in the glycosyltransferase Large, which leads to altered glycosylation of alpha-DG, and also a severe phenotype. Other informative models for muscle proteins include the knockout mouse for myostatin, which demonstrated that this protein is a negative regulator of muscle growth. Additionally, the stress syndrome in pigs, caused by mutations in the porcine RYR1 gene, helped to localize the gene causing malignant hypertermia and Central Core myopathy in humans. The study of animal models for genetic diseases, in spite of the existence of differences in some phenotypes, can provide important clues to the understanding of the pathogenesis of these disorders and are also very valuable for testing strategies for therapeutic approaches.

Generation and characterization of transgenic mice with the full-length human DMD gene.

We report the generation of mice with an intact and functional copy of the 2.3-megabase human dystrophin gene (hDMD), the largest functional stretch of human DNA thus far integrated into a mouse chromosome. Yeast spheroplasts containing an artificial chromosome with the full-length hDMD gene were fused with mouse embryonic stem cells and were subsequently injected into mouse blastocysts to produce transgenic hDMD mice. Human-specific PCR, Southern blotting, and fluorescent in situ hybridization techniques demonstrated the intactness and stable chromosomal integration of the hDMD gene on mouse chromosome 5. Expression of the transgene was confirmed by RT-PCR and Western blotting. The tissue-specific expression pattern of the different DMD transcripts was maintained. However, the human Dp427p and Dp427m transcripts were expressed at 2-fold higher levels and human Dp427c and Dp260 transcripts were expressed at 2- and 4-fold lower levels than their endogenous counterparts. Ultimate functional proof of the hDMD transgene was obtained by crossing of hDMD mice with dystrophin-deficient mdx mice and dystrophin and utrophin-deficient mdx x Utrn-/- mice. The hDMD transgene rescued the lethal dystrophic phenotype of the mdx x Utrn-/- mice. All signs of muscular dystrophy disappeared in the rescued mice, as demonstrated by histological staining of muscle sections and gene expression profiling experiments. Currently, hDMD mice are extensively used for preclinical testing of sequence-specific therapeutics for the treatment of Duchenne muscular dystrophy. In addition, the hDMD mouse can be used to study the influence of the genomic context on deletion and recombination frequencies, genome stability, and gene expression regulation.

Dystrophin-deficient mdx mice display a reduced life span and are susceptible to spontaneous rhabdomyosarcoma.

Duchenne muscular dystrophy (DMD) is the most common, lethal genetic disorder of children. A number of animal models of muscular dystrophy exist, but the most effective model for characterizing the structural and functional properties of dystrophin and therapeutic interventions has been the mdx mouse. Despite the approximately 20 years of investigations of the mdx mouse, the impact of the disease on the life span of mdx mice and the cause of death remain unresolved. Consequently, a life span study of the mdx mouse was designed that included cohorts of male and female mdx and wild-type C57BL/10 mice housed under specific pathogen-free conditions with deaths restricted to natural causes and with examination of the carcasses for pathology. Compared with wild-type mice, both mdx male and female mice had reduced life spans and displayed a progressively dystrophic muscle histopathology. Surprisingly, old mdx mice were prone to develop muscle tumors that resembled the human form of alveolar rhabdomyosarcoma, a cancer associated with poor prognosis. Rhabdomyosarcomas have not been observed previously in nontransgenic mice. The results substantiate the mdx mouse as an important model system for studies of the pathogenesis of and potential remedies for DMD.