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Improvement of cardiac contractile function by peptide-based inhibition of NF-κB in the utrophin/dystrophin-deficient murine model of muscular dystrophy
Dawn A Delfín, Ying Xu, Jennifer M Peterson, Denis C Guttridge, Jill A Rafael-Fortney, Paul ML Janssen
Journal of Translational Medicine , 2011, DOI: 10.1186/1479-5876-9-68
Abstract: To assess whether cardiac function in addition to diaphragm function can be improved, we investigated physiological and histological parameters of cardiac muscle in mice deficient for both dystrophin and its homolog utrophin (double knockout = dko) mice treated with NBD peptide. These dko mice show classic pathophysiological hallmarks of heart failure, including myocyte degeneration, an impaired force-frequency response and a severely blunted β-adrenergic response. Cardiac contractile function at baseline and frequencies and pre-loads throughout the in vivo range as well as β-adrenergic reserve was measured in isolated cardiac muscle preparations. In addition, we studied histopathological and inflammatory markers in these mice.At baseline conditions, active force development in cardiac muscles from NBD treated dko mice was more than double that of vehicle-treated dko mice. NBD treatment also significantly improved frequency-dependent behavior of the muscles. The increase in force in NBD-treated dko muscles to β-adrenergic stimulation was robustly restored compared to vehicle-treated mice. However, histological features, including collagen content and inflammatory markers were not significantly different between NBD-treated and vehicle-treated dko mice.We conclude that NBD can significantly improve cardiac contractile dysfunction in the dko mouse model of DMD and may thus provide a novel therapeutic treatment for heart failure.Duchenne muscular dystrophy (DMD) is a degenerating striated muscle disease caused by the absence of the dystrophin protein[1]. Although limb muscle weakness and the loss of ambulation are usually the initial clinical signs of the disease, patients with DMD die from respiratory failure or heart failure. Pertaining to the heart, ninety-five percent of DMD patients develop dilated cardiomyopathy, and over twenty-five percent die from heart failure [2]. These numbers are predicted to grow as prophylactic treatments targeted at maintaining respirat
Marginal Level Dystrophin Expression Improves Clinical Outcome in a Strain of Dystrophin/Utrophin Double Knockout Mice  [PDF]
Dejia Li,Yongping Yue,Dongsheng Duan
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0015286
Abstract: Inactivation of all utrophin isoforms in dystrophin-deficient mdx mice results in a strain of utrophin knockout mdx (uko/mdx) mice. Uko/mdx mice display severe clinical symptoms and die prematurely as in Duchenne muscular dystrophy (DMD) patients. Here we tested the hypothesis that marginal level dystrophin expression may improve the clinical outcome of uko/mdx mice. It is well established that mdx3cv (3cv) mice express a near-full length dystrophin protein at ~5% of the normal level. We crossed utrophin-null mutation to the 3cv background. The resulting uko/3cv mice expressed the same level of dystrophin as 3cv mice but utrophin expression was completely eliminated. Surprisingly, uko/3cv mice showed a much milder phenotype. Compared to uko/mdx mice, uko/3cv mice had significantly higher body weight and stronger specific muscle force. Most importantly, uko/3cv outlived uko/mdx mice by several folds. Our results suggest that a threshold level dystrophin expression may provide vital clinical support in a severely affected DMD mouse model. This finding may hold clinical implications in developing novel DMD therapies.
The Crystal Structures of Dystrophin and Utrophin Spectrin Repeats: Implications for Domain Boundaries  [PDF]
Muralidharan Muthu, Kylie A. Richardson, Andrew J. Sutherland-Smith
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0040066
Abstract: Dystrophin and utrophin link the F-actin cytoskeleton to the cell membrane via an associated glycoprotein complex. This functionality results from their domain organization having an N-terminal actin-binding domain followed by multiple spectrin-repeat domains and then C-terminal protein-binding motifs. Therapeutic strategies to replace defective dystrophin with utrophin in patients with Duchenne muscular dystrophy require full-characterization of both these proteins to assess their degree of structural and functional equivalence. Here the high resolution structures of the first spectrin repeats (N-terminal repeat 1) from both dystrophin and utrophin have been determined by x-ray crystallography. The repeat structures both display a three-helix bundle fold very similar to one another and to homologous domains from spectrin, α-actinin and plectin. The utrophin and dystrophin repeat structures reveal the relationship between the structural domain and the canonical spectrin repeat domain sequence motif, showing the compact structural domain of spectrin repeat one to be extended at the C-terminus relative to its previously defined sequence repeat. These structures explain previous in vitro biochemical studies in which extending dystrophin spectrin repeat domain length leads to increased protein stability. Furthermore we show that the first dystrophin and utrophin spectrin repeats have no affinity for F-actin in the absence of other domains.
Drug Discovery for Duchenne Muscular Dystrophy via Utrophin Promoter Activation Screening  [PDF]
Catherine Moorwood, Olga Lozynska, Neha Suri, Andrew D. Napper, Scott L. Diamond, Tejvir S. Khurana
PLOS ONE , 2011, DOI: 10.1371/journal.pone.0026169
Abstract: Background Duchenne muscular dystrophy (DMD) is a devastating muscle wasting disease caused by mutations in dystrophin, a muscle cytoskeletal protein. Utrophin is a homologue of dystrophin that can functionally compensate for its absence when expressed at increased levels in the myofibre, as shown by studies in dystrophin-deficient mice. Utrophin upregulation is therefore a promising therapeutic approach for DMD. The use of a small, drug-like molecule to achieve utrophin upregulation offers obvious advantages in terms of delivery and bioavailability. Furthermore, much of the time and expense involved in the development of a new drug can be eliminated by screening molecules that are already approved for clinical use. Methodology/Principal Findings We developed and validated a cell-based, high-throughput screening assay for utrophin promoter activation, and used it to screen the Prestwick Chemical Library of marketed drugs and natural compounds. Initial screening produced 20 hit molecules, 14 of which exhibited dose-dependent activation of the utrophin promoter and were confirmed as hits. Independent validation demonstrated that one of these compounds, nabumetone, is able to upregulate endogenous utrophin mRNA and protein, in C2C12 muscle cells. Conclusions/Significance We have developed a cell-based, high-throughput screening utrophin promoter assay. Using this assay, we identified and validated a utrophin promoter-activating drug, nabumetone, for which pharmacokinetics and safety in humans are already well described, and which represents a lead compound for utrophin upregulation as a therapy for DMD.
The dystrotelin, dystrophin and dystrobrevin superfamily: new paralogues and old isoforms
Hong Jin, Sipin Tan, Jane Hermanowski, Sabrina B?hm, Sabrina Pacheco, Joanna M McCauley, Marc J Greener, Yaniv Hinits, Simon M Hughes, Paul T Sharpe, Roland G Roberts
BMC Genomics , 2007, DOI: 10.1186/1471-2164-8-19
Abstract: Features of the superfamily revealed by our survey include: a) Dystrotelin, an entirely novel branch of the superfamily, present in most vertebrates examined. Dystrotelin is expressed in the central nervous system, and is a possible orthologue of Drosophila DAH. We describe the preliminary characterisation of its function, evolution and expression. b) A novel vertebrate member of the dystrobrevin family, γ-dystrobrevin, an ancient branch now extant only in fish, but probably present in our own ancestors. Like dystrophin, zebrafish γ-dystrobrevin mRNA is localised to myosepta. c) The extent of conservation of alternative splicing and alternative promoter use in the dystrophin and dystrobrevin genes; alternative splicing of dystrophin exons 73 and 78 and α-dystrobrevin exon 13 are conserved across vertebrates, as are the use of the Dp116, Dp71 and G-utrophin promoters; the Dp260 and Dp140 promoters are tetrapod innovations. d) The evolution of the unique N-terminus of DRP2 and its relationship to Dp116 and G-utrophin. e) A C-terminally truncated common ancestor of dystrophin and utrophin in cyclostomes. f) A severely restricted repertoire of dystrophin complex components in ascidians.We have refined our understanding of the evolutionary history and isoform diversity of the five previously reported vertebrate superfamily members and describe two novel members, dystrotelin and γ-dystrobrevin. Dystrotelins, dystrophins and dystrobrevins are roughly equally related to each other. Vertebrates therefore have a repertoire of seven superfamily members (three dystrophins, three dystrobevins, and one dystrotelin), with one lost in tetrapods. Most invertebrates studied have one member from each branch. Although the basic shared function which is implied by the common architecture of these distantly related proteins remains unclear, it clearly permeates metazoan biology.Dystrophin, identified nearly 20 years ago as the protein deficient in Duchenne muscular dystrophy (DMD)[1], is
Neuronal Nitric Oxide Synthase-Rescue of Dystrophin/Utrophin Double Knockout Mice does not Require nNOS Localization to the Cell Membrane  [PDF]
Michelle Wehling-Henricks, James G. Tidball
PLOS ONE , 2011, DOI: 10.1371/journal.pone.0025071
Abstract: Survival of dystrophin/utrophin double-knockout (dko) mice was increased by muscle-specific expression of a neuronal nitric oxide synthase (nNOS) transgene. Dko mice expressing the transgene (nNOS TG+/dko) experienced delayed onset of mortality and increased life-span. The nNOS TG+/dko mice demonstrated a significant decrease in the concentration of CD163+, M2c macrophages that can express arginase and promote fibrosis. The decrease in M2c macrophages was associated with a significant reduction in fibrosis of heart, diaphragm and hindlimb muscles of nNOS TG+/dko mice. The nNOS transgene had no effect on the concentration of cytolytic, CD68+, M1 macrophages. Accordingly, we did not observe any change in the extent of muscle fiber lysis in the nNOS TG+/dko mice. These findings show that nNOS/NO (nitric oxide)-mediated decreases in M2c macrophages lead to a reduction in the muscle fibrosis that is associated with increased mortality in mice lacking dystrophin and utrophin. Interestingly, the dramatic and beneficial effects of the nNOS transgene were not attributable to localization of nNOS protein at the cell membrane. We did not detect any nNOS protein at the sarcolemma in nNOS TG+/dko muscles. This important observation shows that sarcolemmal localization is not necessary for nNOS to have beneficial effects in dystrophic tissue and the presence of nNOS in the cytosol of dystrophic muscle fibers can ameliorate the pathology and most importantly, significantly increase life-span.
Differential Requirement for Utrophin in the Induced Pluripotent Stem Cell Correction of Muscle versus Fat in Muscular Dystrophy Mice  [PDF]
Amanda J. Beck,Joseph M. Vitale,Qingshi Zhao,Joel S. Schneider,Corey Chang,Aneela Altaf,Jennifer Michaels,Mantu Bhaumik,Robert Grange,Diego Fraidenraich
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0020065
Abstract: Duchenne muscular dystrophy (DMD) is an incurable degenerative muscle disorder. We injected WT mouse induced pluripotent stem cells (iPSCs) into mdx and mdx:utrophin mutant blastocysts, which are predisposed to develop DMD with an increasing degree of severity (mdx <<< mdx:utrophin). In mdx chimeras, iPSC-dystrophin was supplied to the muscle sarcolemma to effect corrections at morphological and functional levels. Dystrobrevin was observed in dystrophin-positive and, at a lesser extent, utrophin-positive areas. In the mdx:utrophin mutant chimeras, although iPSC-dystrophin was also supplied to the muscle sarcolemma, mice still displayed poor skeletal muscle histopathology, and negligible levels of dystrobrevin in dystrophin- and utrophin-negative areas. Not only dystrophin-expressing tissues are affected by iPSCs. Mdx and mdx:utrophin mice have reduced fat/body weight ratio, but iPSC injection normalized this parameter in both mdx and mdx:utrophin chimeras, despite the fact that utrophin was compromised in the mdx:utrophin chimeric fat. The results suggest that the presence of utrophin is required for the iPSC-corrections in skeletal muscle. Furthermore, the results highlight a potential (utrophin-independent) non-cell autonomous role for iPSC-dystrophin in the corrections of non-muscle tissue like fat, which is intimately related to the muscle.
Longitudinal ambulatory measurements of gait abnormality in dystrophin-deficient dogs
Inès Barthélémy, Eric Barrey, Pablo Aguilar, Ane Uriarte, Matthias Le Chevoir, Jean-Laurent Thibaud, Thomas Voit, Stéphane Blot, Jean-Yves Hogrel
BMC Musculoskeletal Disorders , 2011, DOI: 10.1186/1471-2474-12-75
Abstract: Six healthy and twelve GRMD dogs were evaluated twice monthly, from the age of two to nine months. The evolution of each gait variable previously shown to be modified in control and dystrophin-deficient adults was assessed using two-ways variance analysis (age, clinical status) with repeated measurements. A principal component analysis (PCA) was applied to perfect multivariate data interpretation.Speed, stride length, total power and force significantly already decreased (p < 0.01) at the age of 2 months. The other gait variables (stride frequency, relative power distributions along the three axes) became modified at later stages. Using the PCA analysis, a global gait index taking into account the main gait variables was calculated, and was also consistent to detect the early changes in the GRMD gait patterns, as well as the progressive degradation of gait quality.The gait variables measured by the accelerometers were sensitive to early detect and follow the gait disorders and mirrored the heterogeneity of clinical presentations, giving sense to monitor gait in GRMD dogs during progression of the disease and pre-clinical therapeutic trials.Duchenne muscular dystrophy (DMD), a still incurable severe and generalized muscle disease in man, represents a therapeutic challenge because of the numerous obstacles it presents for systemic gene, cell or pharmacological therapies. Several of these issues, such as efficacy of studied drugs, or locoregional or systemic medication pathways, can be assessed in the dystrophin-deficient dog, a large animal model which mimics the human disease in many points, better than other animal models [1].During pre-clinical trials, to assess a functional effect, these canine patients must undergo a clinical follow-up encompassing a qualitative and quantitative evaluation of their impaired functions, particularly the gait. Recently, two studies including ours have focused on gait analysis in adult dogs suffering from Golden retriever muscular dy
Translational Regulation of Utrophin by miRNAs  [PDF]
Utpal Basu, Olga Lozynska, Catherine Moorwood, Gopal Patel, Steve D. Wilton, Tejvir S. Khurana
PLOS ONE , 2011, DOI: 10.1371/journal.pone.0029376
Abstract: Background Utrophin is the autosomal homolog of dystrophin, the product of the Duchenne Muscular Dystrophy (DMD) locus. Its regulation is of therapeutic interest as its overexpression can compensate for dystrophin's absence in animal models of DMD. The tissue distribution and transcriptional regulation of utrophin have been characterized extensively, and more recently translational control mechanisms that may underlie its complex expression patterns have begun to be identified. Methodology/Principal Findings Using a variety of bioinformatic, molecular and cell biology techniques, we show that the muscle isoform utrophin-A is predominantly suppressed at the translational level in C2C12 myoblasts. The extent of translational inhibition is estimated to be ~99% in C2C12 cells and is mediated by both the 5′- and 3′-UTRs of the utrophin-A mRNA. In this study we identify five miRNAs (let-7c, miR-150, miR-196b, miR-296-5p, miR-133b) that mediate the repression, and confirm repression by the previously identified miR-206. We demonstrate that this translational repression can be overcome by blocking the actions of miRNAs, resulting in an increased level of utrophin protein in C2C12 cells. Conclusions/Significance The present study has identified key inhibitory mechanisms featuring miRNAs that regulate utrophin expression, and demonstrated that these mechanisms can be targeted to increase endogenous utrophin expression in cultured muscle cells. We suggest that miRNA-mediated inhibitory mechanisms could be targeted by methods similar to those described here as a novel strategy to increase utrophin expression as a therapy for DMD.
Serum cholinesterases are differentially regulated in normal and dystrophin-deficient mutant mice  [PDF]
Andrea R. Durrant,Liliya Tamayev,Lili Anglister
Frontiers in Molecular Neuroscience , 2012, DOI: 10.3389/fnmol.2012.00073
Abstract: The cholinesterases, acetylcholinesterase (AChE), and butyrylcholinesterase (BChE) (pseudocholinesterase), are abundant in the nervous system and in other tissues. The role of AChE in terminating transmitter action in the peripheral and central nervous system is well understood. However, both knowledge of the function(s) of the cholinesterases in serum, and of their metabolic and endocrine regulation under normal and pathological conditions, is limited. This study investigates AChE and BChE in sera of dystrophin-deficient mdx mutant mice, an animal model for the human Duchenne muscular dystrophy (DMD) and in control healthy mice. The data show systematic and differential variations in the concentrations of both enzymes in the sera, and specific changes dictated by alteration of hormonal balance in both healthy and dystrophic mice. While AChE in mdx-sera is elevated, BChE is markedly diminished, resulting in an overall cholinesterase decrease compared to sera of healthy controls. The androgen testosterone (T) is a negative modulator of BChE, but not of AChE, in male mouse sera. T-removal elevated both BChE activity and the BChE/AChE ratio in mdx male sera to values resembling those in healthy control male mice. Mechanisms of regulation of the circulating cholinesterases and their impairment in the dystrophic mice are suggested, and clinical implications for diagnosis and treatment are considered.
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