Utrophin Expression Research Articles

PGC‐1alpha over‐expression alters the proteome of dystrophin deficient skeletal muscle

Duchenne muscular dystrophy is characterized by profound muscle injury and is caused by the production of an aberrant dystrophin gene product. In dystrophic muscle peroxisome proliferator-activated receptor-gamma coactivator (PGC)-1alpha appears to reduce muscle injury by increasing expression of the dystrophin-related protein, utrophin, as well as expression of oxidative proteins. The goal of the current study was to determine the extent to which PGC-1alpha over-expression altered the dystrophic proteome. Neonatal mdx mice (n=6) were injected in the right hind limb with adeno- associated virus (AAV) driving PGC-1alpha expression and the left hind limb was injected with an empty AAV vector. After 6 weeks the gastrocnemius was removed, total protein extracted, 2D- DIGE performed, and spots differing in abundance analyzed by mass spectroscopy. Forty-five spots in the 12% gel and 22 spots in the 8% gel were differentially expressed and 16 different proteins were identified. Limbs over-expressing PGC-1alpha had lower expression of serotransferrin indicating reduced pro-oxidant free iron. Additionally, expression of cytoskeletal components was decreased in PGC-1alpha over-expressing muscle suggesting that induced utrophin expression stabilized the muscle membrane. These data support the beneficial role of PGC-1alpha and provide additional insight regarding the underlying mechanism of protection.

Improved motor function in dko mice by intravenous transplantation of bone marrow-derived mesenchymal stromal cells

We explored the potential therapeutic value of transplanting bone marrow (BM)-derived mesenchymal stromal cells (MSC) into utrophin/dystrophin-deficient double knock-out (dko) mice, a murine model of Duchenne muscular dystrophy. MSC from male rats were isolated and transplanted into female dko mice via the caudal vein. Behavior and locomotor function were later evaluated, along with the expression of dystrophin and utrophin in the sarcolemma of myofiber tissues. The presence of grafted cells was confirmed via polymerase chain reaction for the sex-determining region of the Y-chromosome. Locomotor activity improved significantly (P < 0.05) from 5 to 15 weeks after cell transplantation, as measured by traction, rotating rod and running wheel tests. We also found that the expression of dystrophin and utrophin increased significantly (P < 0.05) and progressively in the sarcolemma from 5 to 15 weeks after transplantation. The median lifespan of mice in the normal group (74.1 weeks) was significantly (P < 0.001) higher than those in the control (22.0 weeks) and transplantation (35.0 weeks) groups, and the median lifespan of mice in the transplantation group was significantly (P < 0.001) higher than that in the control group. Results of this study demonstrate that BM MSC have potential value in xenogeneic transplantation therapy for muscular dystrophy.

Dynamics of dystroglycan complex proteins and laminin changes due to angiogenesis in rat cerebral hypoperfusion

Permanent bilateral carotid occlusion is a well known cerebral hypoperfusion model in rats. The aim of our study was to investigate the different stages of vascular reaction by detecting changes in the extracellular martix proteins and to examine their relationship to angiogenesis after occlusion. Experiments were performed on adult male rats. Brain samples were investigated from day 1 to day 30 post-surgery. Immunohistochemical analysis was performed on the whole hippocampus and on the adjacent cortex in order to investigate extracellular martix proteins, such as the markers of dystroglycan complex (β-dystroglycan, α-dystrobrevin and utrophin) and a marker of basal lamina (laminin). The levels of the proteins were estimated by western blot analysis. Vascular density as well as blood–brain barrier permeability were studied on brain slices from the same regions. Our results showed altered laminin and β-dystroglycan immunoreactivity beginning 2 days after the onset of occlusion followed by an increased utrophin immunoreactivity without blood–brain barrier disruption 5 days later. By day 30 of hypoperfusion, when increased vascular density was detected, all changes returned to baseline levels. Western blot analysis showed significant differences in β-dystroglycan and utrophin expression. Our results indicate that the different stages of neovascularisation resulting from cerebral hypoperfusion can be well defined by the markers laminin, β-dystroglycan, and utrophin and that these markers are more likely to correlate with glio-vascular decoupling than does altered blood–brain barrier function.

Biglycan recruits utrophin to the sarcolemma and counters dystrophic pathology in mdx mice

Duchenne muscular dystrophy (DMD) is caused by mutations in dystrophin and the subsequent disruption of the dystrophin-associated protein complex (DAPC). Utrophin is a dystrophin homolog expressed at high levels in developing muscle that is an attractive target for DMD therapy. Here we show that the extracellular matrix protein biglycan regulates utrophin expression in immature muscle and that recombinant human biglycan (rhBGN) increases utrophin expression in cultured myotubes. Systemically delivered rhBGN up-regulates utrophin at the sarcolemma and reduces muscle pathology in the mdx mouse model of DMD. RhBGN treatment also improves muscle function as judged by reduced susceptibility to eccentric contraction-induced injury. Utrophin is required for the rhBGN therapeutic effect. Several lines of evidence indicate that biglycan acts by recruiting utrophin protein to the muscle membrane. RhBGN is well tolerated in animals dosed for as long as 3 months. We propose that rhBGN could be a therapy for DMD.

Marginal Level Dystrophin Expression Improves Clinical Outcome in a Strain of Dystrophin/Utrophin Double Knockout Mice

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.

Expression of Utrophin at Dystrophin‐Deficient Neuromuscular Synapses of mdx Mice: A Study of Protected and Affected Muscles

In mdx mice, intrinsic laryngeal muscles are spared and sternomastoid muscles are affected, showing cycles of muscle regeneration. We observed that utrophin and acetylcholine receptors are fragmented only in affected muscles, providing further evidence that changes in the overall distribution of molecules at dystrophic neuromuscular junctions may be correlated with muscle regeneration.

Mechanism of action of somatic stem cell treatments: towards the concept of therapeutic plasticity

The initial perception that transplantation of stem cells could serve only to replace damaged cells has continued to be modifi ed as evidence for a protective multifaceted function of transplanted cells has emerged. Neural stem/precursor cells (NPC) were the fi rst to be shown to use this strategy to protect the central nervous system (CNS) from infl ammatory insults (1 – 5). Subsequently similar functions by other types of somatic stem cells (e.g. mesenchymal stromal cells; MSC) have been described (6). Currently the ability of transplanted stem cells to protect tissues from diverse injury using multifaceted ‘ bystander strategies ’ has been substantiated in several experiments. Somatic stem cells promote CNS repair following intralesional as well as systemic injection by producing in situ a milieu of protective molecules whose release is regulated, both temporally and spatially, by environmental demands. The milieu contains immunomodulatory substances, trophic growth factors, stem cell regulators, angiogenic factors, scavengers, etc.: all molecules constitutively expressed by somatic stem cells for maintaining tissue homeostasis during development and in adult life. The concept of ‘ therapeutic platicity ’ that is now emerging (7) also reconciles data showing that stem cells other than NPC, although endowed with negligible transdifferentiation capability, may have a benefi cial role in CNS repair. In four articles of this issue of Cytotherapy the concept of therapeutic plasticity of somatic stem cells is presented further. Although these reports do not completely clarify the mechanisms underlying this therapeutic plasticity, they demonstrate that transplantation of various somatic stem cells via different routes ameliorates diverse neurologic diseases without an overt terminal differentiation and functional integration of the transplanted cells. Alexanian et al. (8) observe that delayed intralesional transplantation of human glial-restricted NPC in rats affected by thoracic spinal cord injury ameliorates sensorimotor defi cits. He et al. (9) report that bone marrow (BM)-derived MSC and endothelial progenitor cells transplanted intravenously (i.v.) protect rats from focal ischemia via the secretion of different combinations of growth factors. Interestingly, an additive benefi cial effect was observed when the two different cell sources were co-transplanted. Li et al. (10) focus on a mouse model of muscular dystrophy where mean survival as well as loss of dystrophin and utrophin expression is delayed by i.v. transplantation of rat-derived MSC. Attar et al. (11) describe preliminary results of intralesional transplantation of BM-derived hematopoietic stem cells along with BM-derived mononuclear cells in four patients with spinal cord injury. Although the possibility of using bystander strategies as protection from tissue injury appears to be a very promising therapeutic use of somatic stem cells, we still need to resolve unsolved key questions before clinical translation. One crucial question is whether to transplant the cells heterotopically or homotopically. Transplantation is not without risks: co-transplantation of NPC and pancreatic islet cells under the kidney capsule of diabetic mice induced the generation, at the site of cell transplantation, of insulin-induced neuroblastoma-like neoplasms (12). Intracardiac transplantation of stem cell factor-overexpressing MSC into mice with myocardial infarction promoted the formation of a chest wall-invading soft tissue sarcoma (13). The unregulated transplantation Cytotherapy, 2011; 13: 6–7

Diaphragm rescue alone prevents heart dysfunction in dystrophic mice

Duchenne muscular dystrophy (DMD) is an X-linked recessive disease caused, in most cases, by the complete absence of the 427 kDa cytoskeletal protein, dystrophin. There is no effective treatment, and affected individuals die from respiratory failure and cardiomyopathy by age 30. Here, we investigated whether cardiomyopathy could be prevented in animal models of DMD by increasing diaphragm utrophin or dystrophin expression and thereby restoring diaphragm function. In a transgenic mdx mouse, where utrophin was over expressed in the skeletal muscle and the diaphragm, but not in the heart, we found cardiac function, specifically right and left ventricular ejection fraction as measured using in vivo magnetic resonance imaging, was restored to wild-type levels. In mdx mice treated with a peptide-conjugated phosphorodiamidate morpholino oligomer (PPMO) that resulted in high levels of dystrophin restoration in the skeletal muscle and the diaphragm only, cardiac function was also restored to wild-type levels. In dystrophin/utrophin-deficient double-knockout (dKO) mice, a more severely affected animal model of DMD, treatment with a PPMO again produced high levels of dystrophin only in the skeletal muscle and the diaphragm, and once more restored cardiac function to wild-type levels. In the dKO mouse, there was no difference in heart function between treatment of the diaphragm plus the heart and treatment of the diaphragm alone. Restoration of diaphragm and other respiratory muscle function, irrespective of the method used, was sufficient to prevent cardiomyopathy in dystrophic mice. This novel mechanism of treating respiratory muscles to prevent cardiomyopathy in dystrophic mice warrants further investigation for its implications on the need to directly treat the heart in DMD.

Utrophin Expression is Prevalent in Epidermis

Utrophin protein was discovered to localize to the neuromuscular junctions and is thought to provide a critical linkage between integral proteins bound to the extracellular matrix outside the cell and the actin cytoskeleton inside the cell. Clinical implications of this protein include the upregulation of utrophin as a treatment for Duchenne Muscular Dystrophy, a disease in which utrophin's homologue (dystrophin) is functionally absent. In addition, utrophin gene expression is reduced in a variety of cancers. This study set out to identify whether utrophin is expressed within the epidermis—the top layer of skin. To date, four unique mRNA transcripts have been characterized for the human UTRN gene, supporting translation of two full-length utrophin proteins (utrophin A and utrophin B) and two short isoforms (Up 140 and Up71). All four of these human utrophin transcripts were detected within human epidermis using reverse-transcriptase PCR performed on RNA purified from human epidermis. In addition to the detection of utrophin mRNA within human epidermis, the full-length utrophin protein was also detected within human epidermis using immunofluorescence labeling—a technique used to detect specific proteins. Utrophin protein localizes to the area of the epidermis closest to the dermis termed the basement membrane zone. These data demonstrate that utrophin expression is prevalent in epidermis. Utrophin has been characterized as a member of the dystroglycan complex and dystroglycan has previously been identified within epidermis and too localizes to the basement membrane zone. Therefore, this study provides support for existence of a dystroglycan complex within epidermis as utrophin is the second member of this complex to be found within the epidermis.

Rescue of a Dystrophin-like Protein by Exon Skipping In Vivo Restores GABAA-receptor Clustering in the Hippocampus of the mdx Mouse

Dystrophin, the cytoskeletal protein whose defect is responsible for Duchenne muscular dystrophy (DMD), is normally expressed in both muscles and brain. Genetic loss of brain dystrophin in the mdx mouse model of DMD reduces the capacity for type A gamma-aminobutyric acid (GABA(A))-receptor clustering in central inhibitory synapses, which is thought to be a main molecular defect leading to brain and cognitive alterations in this syndrome. U7 small nuclear RNAs modified to encode antisense sequences and expressed from recombinant adeno-associated viral (rAAV) vectors have proven efficient after intramuscular injection to induce skipping of the mutated exon 23 and rescue expression of a functional dystrophin-like product in muscle tissues of mdx mice in vivo. Here, we report that intrahippocampal injection of a single dose of rAAV2/1-U7 can rescue substantial levels of brain dystrophin expression (15-25%) in mdx mice for months. This is sufficient to completely restore GABA(A)-receptor clustering in pyramidal and dendritic layers of CA1 hippocampus, suggesting exon-skipping strategies offer the prospect to investigate and correct both brain and muscle alterations in DMD. This provides new evidence that in the adult brain dystrophin is critical for the control of GABA(A)-receptor clustering, which may have an important role in activity-dependent synaptic plasticity in hippocampal circuits.

CT-GalNAc transferase overexpression in adult mice is associated with extrasynaptic utrophin in skeletal muscle fibres

Duchenne muscular dystrophy is a genetic muscle disease characterized by the absence of sub-sarcolemmal dystrophin that results in muscle fibre necrosis, progressive muscle wasting and is fatal. Numerous experimental studies with dystrophin-deficient mdx mice, an animal model for the disease, have demonstrated that extrasynaptic upregulation of utrophin, an analogue of dystrophin, can prevent muscle fibre deterioration and reduce or negate the dystrophic phenotype. A different approach for ectopic expression of utrophin relies on augmentation of CT-GalNAc transferase in muscle fibre. We investigated whether CT-GalNAc transferase overexpression in adult mice influence appearance of utrophin in the extrasynaptic sarcolemma. After electrotransfer of plasmid DNA carrying an expression cassette of CT-GalNAc transferase into tibialis anterior muscle of wild type and dystrophic mice, muscle sections were examined by immunofluorescence. CT-GalNAc transgene expression augmented sarcolemmal carbohydrate glycosylation and was accompanied by extrasynaptic utrophin. A 6-week time course study showed that the highest efficiency of utrophin overexpression in a plasmid harboured muscle fibres was 32.2% in CD-1 and 52% in mdx mice, 2 and 4 weeks after CT-GalNAc gene transfer, respectively. The study provides evidence that postnatal CT-GalNAc transferase overexpression stimulates utrophin upregulation that is inherently beneficial for muscle structure and strength restoration. Thus CT-GalNAc may provide an important therapeutic molecule for treatment of dystrophin deficiency in Duchenne muscular dystrophy.

Arginine Metabolism by Macrophages Promotes Cardiac and Muscle Fibrosis in mdx Muscular Dystrophy

BackgroundDuchenne muscular dystrophy (DMD) is the most common, lethal disease of childhood. One of 3500 new-born males suffers from this universally-lethal disease. Other than the use of corticosteroids, little is available to affect the relentless progress of the disease, leading many families to use dietary supplements in hopes of reducing the progression or severity of muscle wasting. Arginine is commonly used as a dietary supplement and its use has been reported to have beneficial effects following short-term administration to mdx mice, a genetic model of DMD. However, the long-term effects of arginine supplementation are unknown. This lack of knowledge about the long-term effects of increased arginine metabolism is important because elevated arginine metabolism can increase tissue fibrosis, and increased fibrosis of skeletal muscles and the heart is an important and potentially life-threatening feature of DMD.MethodologyWe use both genetic and nutritional manipulations to test whether changes in arginase metabolism promote fibrosis and increase pathology in mdx mice. Our findings show that fibrotic lesions in mdx muscle are enriched with arginase-2-expressing macrophages and that muscle macrophages stimulated with cytokines that activate the M2 phenotype show elevated arginase activity and expression. We generated a line of arginase-2-null mutant mdx mice and found that the mutation reduced fibrosis in muscles of 18-month-old mdx mice, and reduced kyphosis that is attributable to muscle fibrosis. We also observed that dietary supplementation with arginine for 17-months increased mdx muscle fibrosis. In contrast, arginine-2 mutation did not reduce cardiac fibrosis or affect cardiac function assessed by echocardiography, although 17-months of dietary supplementation with arginine increased cardiac fibrosis. Long-term arginine treatments did not decrease matrix metalloproteinase-2 or -9 or increase the expression of utrophin, which have been reported as beneficial effects of short-term treatments.Conclusions/SignificanceOur findings demonstrate that arginine metabolism by arginase promotes fibrosis of muscle in muscular dystrophy and contributes to kyphosis. Our findings also show that long-term, dietary supplementation with arginine exacerbates fibrosis of dystrophic heart and muscles. Thus, commonly-practiced dietary supplementation with arginine by DMD patients has potential risk for increasing pathology when performed for long periods, despite reports of benefits acquired with short-term supplementation.

Sarcolemmal nNOS anchoring reveals a qualitative difference between dystrophin and utrophin.

Duchenne muscular dystrophy (DMD) is a lethal muscle disease caused by dystrophin deficiency. In normal muscle, dystrophin helps maintain sarcolemmal stability. Dystrophin also recruits neuronal nitric oxide synthase (nNOS) to the sarcolemma. Failure to anchor nNOS to the membrane leads to functional ischemia and aggravates muscle disease in DMD. Over the past two decades, a great variety of therapeutic modalities have been explored to treat DMD. A particularly attractive approach is to increase utrophin expression. Utrophin shares considerable sequence, structural and functional similarity with dystrophin. Here, we test the hypothesis that utrophin also brings nNOS to the sarcolemma. Full-length utrophin cDNA was expressed in dystrophin-deficient mdx mice by gutted adenovirus or via transgenic overexpression. Subcellular nNOS localization was determined by immunofluorescence staining, in situ nNOS activity staining and microsomal preparation western blot. Despite supra-physiological utrophin expression, we did not detect nNOS at the sarcolemma. Furthermore, transgenic utrophin overexpression failed to protect mdx muscle from exercise-associated injury. Our results suggest that full-length utrophin cannot anchor nNOS to the sarcolemma. This finding might have important implications for the development of utrophin-based DMD therapies.

The utrophin A 5'-UTR drives cap-independent translation exclusively in skeletal muscles of transgenic mice and interacts with eEF1A2

The molecular mechanisms regulating expression of utrophin A are of therapeutic interest since upregulating its expression at the sarcolemma can compensate for the lack of dystrophin in animal models of Duchenne Muscular Dystrophy (DMD). The 5'-UTR of utrophin A has been previously shown to drive cap-independent internal ribosome entry site (IRES)-mediated translation in response to muscle regeneration and glucocorticoid treatment. To determine whether the utrophin A IRES displays tissue specific activity, we generated transgenic mice harboring control (CMV/betaGAL/CAT) or utrophin A 5'-UTR (CMV/betaGAL/UtrA/CAT) bicistronic reporter transgenes. Examination of multiple tissues from two CMV/betaGAL/UtrA/CAT lines revealed that the utrophin A 5'-UTR drives cap-independent translation of the reporter gene exclusively in skeletal muscles and no other examined tissues. This expression pattern suggested that skeletal muscle-specific factors are involved in IRES-mediated translation of utrophin A. We performed RNA-affinity chromatography experiments combined with mass spectrometry to identify trans-factors that bind the utrophin A 5'-UTR and identified eukaryotic elongation factor 1A2 (eEF1A2). UV-crosslinking experiments confirmed the specificity of this interaction. Regions of the utrophin A 5'-UTR that bound eEF1A2 also mediated cap-independent translation in C2C12 muscle cells. Cultured cells lacking eEF1A2 had reduced IRES activity compared with cells overexpressing eEF1A2. Together, these results suggest an important role for eEF1A2 in driving cap-independent translation of utrophin A in skeletal muscle. The trans-factors and signaling pathways driving skeletal-muscle specific IRES-mediated translation of utrophin A could provide unique targets for developing pharmacological-based DMD therapies.

The artificial gene Jazz, a transcriptional regulator of utrophin, corrects the dystrophic pathology in mdx mice

The absence of the cytoskeletal protein dystrophin results in Duchenne muscular dystrophy (DMD). The utrophin protein is the best candidate for dystrophin replacement in DMD patients. To obtain therapeutic levels of utrophin expression in dystrophic muscle, we developed an alternative strategy based on the use of artificial zinc finger transcription factors (ZF ATFs). The ZF ATF 'Jazz' was recently engineered and tested in vivo by generating a transgenic mouse specifically expressing Jazz at the muscular level. To validate the ZF ATF technology for DMD treatment we generated a second mouse model by crossing Jazz-transgenic mice with dystrophin-deficient mdx mice. Here, we show that the artificial Jazz protein restores sarcolemmal integrity and prevents the development of the dystrophic disease in mdx mice. This exclusive animal model establishes the notion that utrophin-based therapy for DMD can be efficiently developed using ZF ATF technology and candidates Jazz as a novel therapeutic molecule for DMD therapy.

Myotendinous Junction Defects and Reduced Force Transmission in Mice that Lack α7 Integrin and Utrophin

The alpha7beta1 integrin, dystrophin, and utrophin glycoprotein complexes are the major laminin receptors in skeletal muscle. Loss of dystrophin causes Duchenne muscular dystrophy, a lethal muscle wasting disease. Duchenne muscular dystrophy-affected muscle exhibits increased expression of alpha7beta1 integrin and utrophin, which suggests that these laminin binding complexes may act as surrogates in the absence of dystrophin. Indeed, mice that lack dystrophin and alpha7 integrin (mdx/alpha7(-/-)), or dystrophin and utrophin (mdx/utr(-/-)), exhibit severe muscle pathology and die prematurely. To explore the contribution of the alpha7beta1 integrin and utrophin to muscle integrity and function, we generated mice lacking both alpha7 integrin and utrophin. Surprisingly, mice that lack both alpha7 integrin and utrophin (alpha7/utr(-/-)) were viable and fertile. However, these mice had partial embryonic lethality and mild muscle pathology, similar to alpha7 integrin-deficient mice. Dystrophin levels were increased 1.4-fold in alpha7/utr(-/-) skeletal muscle and were enriched at neuromuscular junctions. Ultrastructural analysis revealed abnormal myotendinous junctions, and functional tests showed a ninefold reduction in endurance and 1.6-fold decrease in muscle strength in these mice. The alpha7/utr(-/-) mouse, therefore, demonstrates the critical roles of alpha7 integrin and utrophin in maintaining myotendinous junction structure and enabling force transmission during muscle contraction. Together, these results indicate that the alpha7beta1 integrin, dystrophin, and utrophin complexes act in a concerted manner to maintain the structural and functional integrity of skeletal muscle.

Functional substitution by TAT-utrophin in dystrophin-deficient mice.

The loss of dystrophin compromises muscle cell membrane stability and causes Duchenne muscular dystrophy and/or various forms of cardiomyopathy. Increased expression of the dystrophin homolog utrophin by gene delivery or pharmacologic up-regulation has been demonstrated to restore membrane integrity and improve the phenotype in the dystrophin-deficient mdx mouse. However, the lack of a viable therapy in humans predicates the need to explore alternative methods to combat dystrophin deficiency. We investigated whether systemic administration of recombinant full-length utrophin (Utr) or DeltaR4-21 "micro" utrophin (muUtr) protein modified with the cell-penetrating TAT protein transduction domain could attenuate the phenotype of mdx mice. Recombinant TAT-Utr and TAT-muUtr proteins were expressed using the baculovirus system and purified using FLAG-affinity chromatography. Age-matched mdx mice received six twice-weekly intraperitoneal injections of either recombinant protein or PBS. Three days after the final injection, mice were analyzed for several phenotypic parameters of dystrophin deficiency. Injected TAT-muUtr transduced all tissues examined, integrated with members of the dystrophin complex, reduced serum levels of creatine kinase (11,290+/-920 U versus 5,950+/-1,120 U; PBS versus TAT), the prevalence of muscle degeneration/regeneration (54%+/-5% versus 37%+/-4% of centrally nucleated fibers; PBS versus TAT), the susceptibility to eccentric contraction-induced force drop (72%+/-5% versus 40%+/-8% drop; PBS versus TAT), and increased specific force production (9.7+/-1.1 N/cm(2) versus 12.8+/-0.9 N/cm(2); PBS versus TAT). These results are, to our knowledge, the first to establish the efficacy and feasibility of TAT-utrophin-based constructs as a novel direct protein-replacement therapy for the treatment of skeletal and cardiac muscle diseases caused by loss of dystrophin.

RhoA leads to up-regulation and relocalization of utrophin in muscle fibers

Up-regulation of utrophin, a homolog of dystrophin, is known to ameliorate the dystrophic phenotype in animal models of Duchenne muscular dystrophy. We have previously demonstrated that the active form of RhoA (RhoAV14) increases the expression of utrophin and its localization at the plasma membrane in cultured myoblasts. In this paper, we ask whether RhoAV14 can up-regulate utrophin also in mice. A plasmid encoding for RhoAV14 was injected into skeletal muscles followed by electroporation. Muscles expressing RhoAV14 were analyzed by Western-immunoblotting, real time PCR amplification and immunohistochemistry. We found that RhoAV14 increased utrophin protein expression and distribution specifically at the plasma membrane in muscle fibers without any effect on utrophin transcription. Utrophin up-regulation, uncoupled from that of its mRNA, has been previously observed in pathological processes and in normal regenerating conditions.

Sub-physiological sarcoglycan expression contributes to compensatory muscle protection in mdx mice.

Sarcoglycans are a group of single-pass transmembrane glycoproteins. In striated muscle, sarcoglycans interact with dystrophin and other dystrophin-associated proteins (DAPs) to form the dystrophin-associated glycoprotein complex (DGC). The DGC protects the sarcolemma from contraction-induced injury. Duchenne muscular dystrophy (DMD) is caused by dystrophin gene mutations. In the absence of dystrophin, the DGC is disassembled from the sarcolemma. This initiates a chain reaction of muscle degeneration, necrosis, inflammation and fibrosis. In contrast to human patients, dystrophin-null mdx mice are only mildly affected. Enhanced muscle regeneration and the up-regulation of utrophin and integrin are thought to protect mdx muscle. Interestingly, trace amounts of sarcoglycans and other DAPs can be detected at the mdx sarcolemma. It is currently unclear whether sub-physiological sarcoglycan expression also contributes to the mild phenotype in mdx mice. To answer this question, we generated delta-sarcoglycan/dystrophin double knockout mice (delta-Dko) in which residual sarcoglycans were completely eliminated from the sarcolemma. Interestingly, utrophin levels were further increased in these mice. However, enhanced utrophin expression did not mitigate disease. The clinical manifestation of delta-Dko mice was worse than that of mdx mice. They showed characteristic dystrophic signs, body emaciation and more macrophage infiltration. Their lifespan was reduced by 60%. Furthermore, delta-Dko muscle generated significantly less absolute muscle force and became more susceptible to contraction-induced injury. Our results suggest that sub-physiological sarcoglycan expression plays a critical role in ameliorating muscle disease in mdx mice. We speculate that low-level sarcoglycan expression may represent a useful strategy to palliate DMD.

Gene-mediated Restoration of Normal Myofiber Elasticity in Dystrophic Muscles

Dystrophin mediates a physical link between the cytoskeleton of muscle fibers and the extracellular matrix, and its absence leads to muscle degeneration and dystrophy. In this article, we show that the lack of dystrophin affects the elasticity of individual fibers within muscle tissue explants, as probed using atomic force microscopy (AFM), providing a sensitive and quantitative description of the properties of normal and dystrophic myofibers. The rescue of dystrophin expression by exon skipping or by the ectopic expression of the utrophin analogue normalized the elasticity of dystrophic muscles, and these effects were commensurate to the functional recovery of whole muscle strength. However, a more homogeneous and widespread restoration of normal elasticity was obtained by the exon-skipping approach when comparing individual myofibers. AFM may thus provide a quantification of the functional benefit of gene therapies from live tissues coupled to single-cell resolution.