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1.  P2RX7 Purinoceptor: A Therapeutic Target for Ameliorating the Symptoms of Duchenne Muscular Dystrophy 
PLoS Medicine  2015;12(10):e1001888.
Background
Duchenne muscular dystrophy (DMD) is the most common inherited muscle disease, leading to severe disability and death in young men. Death is caused by the progressive degeneration of striated muscles aggravated by sterile inflammation. The pleiotropic effects of the mutant gene also include cognitive and behavioral impairments and low bone density.
Current interventions in DMD are palliative only as no treatment improves the long-term outcome. Therefore, approaches with a translational potential should be investigated, and key abnormalities downstream from the absence of the DMD product, dystrophin, appear to be strong therapeutic targets. We and others have demonstrated that DMD mutations alter ATP signaling and have identified P2RX7 purinoceptor up-regulation as being responsible for the death of muscles in the mdx mouse model of DMD and human DMD lymphoblasts. Moreover, the ATP–P2RX7 axis, being a crucial activator of innate immune responses, can contribute to DMD pathology by stimulating chronic inflammation. We investigated whether ablation of P2RX7 attenuates the DMD model mouse phenotype to assess receptor suitability as a therapeutic target.
Methods and Findings
Using a combination of molecular, histological, and biochemical methods and behavioral analyses in vivo we demonstrate, to our knowledge for the first time, that genetic ablation of P2RX7 in the DMD model mouse produces a widespread functional attenuation of both muscle and non-muscle symptoms. In dystrophic muscles at 4 wk there was an evident recovery in key functional and molecular parameters such as improved muscle structure (minimum Feret diameter, p < 0.001), increased muscle strength in vitro (p < 0.001) and in vivo (p = 0.012), and pro-fibrotic molecular signatures. Serum creatine kinase (CK) levels were lower (p = 0.025), and reduced cognitive impairment (p = 0.006) and bone structure alterations (p < 0.001) were also apparent. Reduction of inflammation and fibrosis persisted at 20 mo in leg (p = 0.038), diaphragm (p = 0.042), and heart muscles (p < 0.001). We show that the amelioration of symptoms was proportional to the extent of receptor depletion and that improvements were observed following administration of two P2RX7 antagonists (CK, p = 0.030 and p = 0.050) without any detectable side effects. However, approaches successful in animal models still need to be proved effective in clinical practice.
Conclusions
These results are, to our knowledge, the first to establish that a single treatment can improve muscle function both short and long term and also correct cognitive impairment and bone loss in DMD model mice. The wide-ranging improvements reflect the convergence of P2RX7 ablation on multiple disease mechanisms affecting skeletal and cardiac muscles, inflammatory cells, brain, and bone. Given the impact of P2RX7 blockade in the DMD mouse model, this receptor is an attractive target for translational research: existing drugs with established safety records could potentially be repurposed for treatment of this lethal disease.
Dariusz Gorecki and colleagues investigate the effect of P2RX7 ablation on muscle, brain, and bone pathology in a DMD model mouse.
Editors' Summary
Background
Muscular dystrophies are inherited diseases in which the body’s muscles gradually weaken and waste away. The most common and severe muscular dystrophy—Duchenne muscular dystrophy (DMD)—also includes cognitive (thinking) and behavioral impairments and low bone density as well as chronic inflammation. DMD affects about 1 in 3,500 boys; girls can be carriers of DMD but rarely have any symptoms. At birth, boys who carry a mutation (genetic change) in the gene that makes the protein dystrophin seem normal, but the symptoms of DMD soon begin to appear. Affected children may initially have difficulty walking or may find it hard to sit or stand independently. As they age, their muscle strength progressively declines, a process that is aggravated by sterile inflammation (an immune system response to tissue damage that occurs in the absence of an infectious agent), and most affected boys are confined to a wheelchair by the time they are 12 years old. The diaphragm and other muscles involved in breathing also weaken, and the heart muscle becomes enlarged. Consequently, few boys with DMD live beyond their early 20s, usually dying from breathing or heart problems. At present, there is no cure for DMD. However, physical therapy and treatment with steroids (intended to reduce sterile inflammation) can prolong the ability of patients to walk, and assisted ventilation can help with their breathing.
Why Was This Study Done?
One way to treat DMD under investigation is replacement of the defective dystrophin in muscles using gene therapy. Dystrophin normally forms structural scaffolds that sit in the membranes that surround muscle fibers and protect the fibers from damage during muscle contraction. In DMD, the loss of dystrophin, dystrophin-associated proteins, and specific signaling processes causes progressive muscle loss. Although gene therapy approaches that target dystrophin hold some promise, achieving sufficient dystrophin expression in all the crucial muscle groups to prevent progressive muscle damage is hard. Moreover, gene therapy targeted at muscles will not treat the non-muscle-related characteristics of DMD. Targeting an abnormality downstream of dystrophin might therefore be a better approach to the treatment of DMD. One such target is P2RX7. This purinoceptor was originally identified as a sensor of ATP released from damaged cells and is an activator of innate immune responses. Because upregulation of P2RX7 is responsible for muscle death in the mdx mouse model of DMD and for the death of human DMD lymphoblasts, in this study, the researchers investigate whether genetic ablation of P2RX7 can attenuate the DMD symptoms of the mdx mouse model.
What Did the Researchers Do and Find?
The researchers mated mdx mice and mice that lack the gene for P2RX7 to obtain Pf-mdx/P2RX7−/− mice, which make no functional dystrophin or P2RX7. They then compared the structure and function of the muscles (dystrophic pathology) in these mice with those in mdx mice. They also examined specific aspects of the behavior of the mice. At four weeks, there was improved muscle structure and strength, decreased inflammation, and decreased fibrosis (thickening and scarring of the connective tissue covering the muscles) in the Pf-mdx/P2RX7−/− mice compared to the mdx mice. P2RX7 ablation also reduced blood levels of creatinine kinase (a marker of muscle, heart, and brain injury), cognitive impairment, and bone structure alterations. Importantly, the reduction in inflammation and fibrosis was still evident at 20 months in the leg, diaphragm, and heart muscles of the Pf-mdx/P2RX7−/− mice compared to the mdx mice. Finally, the dystrophic pathology in mdx mice could also be reduced by treating these mice with P2RX7 antagonists (molecules that bind to P2RX7 and prevent its function).
What Do These Findings Mean?
These findings show that genetic ablation of P2RX7 can improve muscle function in the short and long term and can also correct cognitive impairment and bone loss in a mouse model of DMD. Thus, in mdx mice, P2RX7 ablation affects multiple disease mechanisms that affect skeletal and heart muscles, inflammatory cells, brain, and bone. Other preliminary findings suggest that P2RX7 blockade in mdx mice also improves DMD symptoms. These are promising results, but results in animals do not necessarily translate into effective clinical treatments. Nevertheless, these findings identify P2RX7 as an attractive target for the treatment of DMD, particularly since it might be possible to repurpose P2RX7 antagonists originally developed for the treatment of chronic pain for the treatment of DMD.
Additional Information
This list of resources contains links that can be accessed when viewing the PDF on a device or via the online version of the article at http://dx.doi.org/10.1371/journal.pmed.1001888.
The US National Institute of Neurological Disorders and Stroke provides information on muscular dystrophy (in English and Spanish)
The US National Human Genome Research Institute also provides basic information on Duchenne muscular dystrophy and links to additional resources
The US Centers for Disease Control and Prevention has information about muscular dystrophy
The not-for-profit Nemours Foundation provides information about muscular dystrophy for parents, children, and teenagers (in English and Spanish)
The US not-for-profit organization Parent Project Muscular Dystrophy provides detailed information about all aspects of Duchenne muscular dystrophy and parents’ stories about Duchenne muscular dystrophy
MedlinePlus provides links to further resources on muscular dystrophy and an encyclopedia page on Duchenne muscular dystrophy (in English and Spanish)
Wikipedia has pages about Duchenne muscular dystrophy and P2RX7 (note that Wikipedia is a free online encyclopedia that anyone can edit; available in several languages)
TREAT-NMD is a network for the neuromuscular field that provides an infrastructure to ensure that the most promising new therapies reach patients as quickly as possible
doi:10.1371/journal.pmed.1001888
PMCID: PMC4604078  PMID: 26461208
2.  Functional Substitution by TAT-Utrophin in Dystrophin-Deficient Mice 
PLoS Medicine  2009;6(5):e1000083.
James Ervasti and colleagues show that injection of a truncated form of utrophin transduced all tissues examined, integrated with members of the dystrophin complex, and reduced serum levels of creatine kinase in a mouse model of muscular dystrophy.
Background
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 ΔR4-21 “micro” utrophin (μUtr) protein modified with the cell-penetrating TAT protein transduction domain could attenuate the phenotype of mdx mice.
Methods and Findings
Recombinant TAT-Utr and TAT-μUtr 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-μUtr 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/cm2 versus 12.8±0.9 N/cm2; PBS versus TAT).
Conclusions
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.
Editors' Summary
Background
Muscular dystrophies are genetic (inherited) diseases in which the body's muscles gradually weaken and degenerate. The commonest and most severe muscular dystrophy—Duchenne muscular dystrophy—affects 1 in 3,500 boys (girls can be carriers of the disease but rarely have any symptoms). At birth, these boys seem normal but the symptoms of their disease begin to appear in early childhood. Affected children may initially have difficulty walking or find it to hard to sit or stand independently. As they age, their muscle strength progressively declines and most affected boys are confined to a wheelchair by the time they are 12 years old. The muscles involved in breathing also weaken and the heart muscle becomes enlarged. Few boys with Duchenne muscular dystrophy live beyond their early 20 s, usually dying from breathing or heart problems. At present there is no cure for Duchenne muscular dystrophy. However, physical therapy and treatment with steroids can prolong the ability of patients to walk, and assisted ventilation can help with their breathing.
Why Was This Study Done?
In all muscular dystrophies, one of the proteins needed to build and maintain healthy muscles is missing or nonfunctional because of a genetic change (mutation). In Duchenne muscular dystrophy the mutation is in dystrophin, a protein that is involved in the formation of the dystrophin–glycoprotein complex. This complex normally sits in the membranes that surround muscle fibers and protects these membranes from damage during muscle contraction. Consequently, in Duchenne muscular dystrophy, the muscle fiber membranes become damaged and eventually the muscle fibers die. Thus, if functional dystrophin could be introduced into the muscles of patients with Duchenne muscular dystrophy, it might be possible to reduce their symptoms and prolong their lives. Indeed, the effects of dystrophin deficiency in the dystrophin-deficient mdx mouse can be reduced by the introduction of an artificial gene that expresses dystrophin or the closely related protein utrophin. Unfortunately, this gene therapy approach has not yet been effectively demonstrated in humans. In this study, therefore, the researchers investigate whether utrophin protein can be introduced directly into dystrophin-deficient mouse muscles by exposing the muscle cells to utrophin fused to the protein transduction domain of the HIV-1 TAT protein. Most proteins will not cross cell membranes, but proteins fused to this cell-penetrating domain readily enter many cell types, including muscle cells.
What Did the Researchers Do and Find?
The researchers injected full-length utrophin fused to the TAT protein transduction domain (TAT-Utr) and a short, “micro” version of utrophin fused to the same domain (TAT-μUtr) into the abdomens of mdx mice and looked to see where the proteins ended up. After two injections, both proteins were present in a wide range of tissues and organs, including several types of muscle. However, the levels of TAT-Utr were much lower than those of TAT-μUtr. Next, the researchers injected another group of mdx mice with TAT-μUtr six times over three weeks. Again, TAT-μUtr was present in all the tissues that the researchers examined. Furthermore, μUtr–glycoprotein complexes formed in the TAT-μUtr injected mdx mice and the membrane integrity and overall health of the dystrophin-deficient muscles of the mdx mice improved compared to mdx mice treated with saline. Finally, the researchers report, TAT-μUtr injections greatly improved the contractile performance of the muscles of the mdx mice.
What Do These Findings Mean?
These findings provide the first demonstration that injection of TAT-utrophin protein fusions may provide a way to treat muscular dystrophies caused by the loss of dystrophin. However, although this direct protein-replacement therapy looks hopeful, approaches that work in animals do not necessarily work in people. In particular, for this approach to work in patients with muscular dystrophy, it would be necessary to give frequent, high-dose injections of the TAT-μUtr fusion protein, a process that could eventually trigger a deleterious immune response. Nevertheless, the researchers suggest that by combining this novel approach with other approaches that also increase utrophin expression, it might be possible to prevent or delay the development of the symptoms of Duchenne muscular dystrophy.
Additional Information
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.1000085.
The US National Institute of Neurological Disorders and Stroke provides information on muscular dystrophy and ongoing research into possible treatments (in English and Spanish)
The US National Human Genome Research Institute also provides basic information on Duchenne muscular dystrophy and links to additional resources
The UK National Health Service Choices Web site has pages for patients and caregivers on muscular dystrophy
The Nemours Foundation provides information about muscular dystrophy for parents, children, and teenagers
For links to further resources on muscular dystrophy, see also MedlinePlus
doi:10.1371/journal.pmed.1000083
PMCID: PMC2680620  PMID: 19478831
3.  γ-Sarcoglycan Deficiency Leads to Muscle Membrane Defects and Apoptosis Independent of Dystrophin  
The Journal of Cell Biology  1998;142(5):1279-1287.
γ-Sarcoglycan is a transmembrane, dystrophin-associated protein expressed in skeletal and cardiac muscle. The murine γ-sarcoglycan gene was disrupted using homologous recombination. Mice lacking γ-sarcoglycan showed pronounced dystrophic muscle changes in early life. By 20 wk of age, these mice developed cardiomyopathy and died prematurely. The loss of γ-sarcoglycan produced secondary reduction of β- and δ-sarcoglycan with partial retention of α- and ε-sarcoglycan, suggesting that β-, γ-, and δ-sarcoglycan function as a unit. Importantly, mice lacking γ-sarco- glycan showed normal dystrophin content and local- ization, demonstrating that myofiber degeneration occurred independently of dystrophin alteration. Furthermore, β-dystroglycan and laminin were left intact, implying that the dystrophin–dystroglycan–laminin mechanical link was unaffected by sarcoglycan deficiency. Apoptotic myonuclei were abundant in skeletal muscle lacking γ-sarcoglycan, suggesting that programmed cell death contributes to myofiber degeneration. Vital staining with Evans blue dye revealed that muscle lacking γ-sarcoglycan developed membrane disruptions like those seen in dystrophin-deficient muscle. Our data demonstrate that sarcoglycan loss was sufficient, and that dystrophin loss was not necessary to cause membrane defects and apoptosis. As a common molecular feature in a variety of muscular dystrophies, sarcoglycan loss is a likely mediator of pathology.
PMCID: PMC2149352  PMID: 9732288
muscular dystrophy; sarcolemma; dystrophin; extracellular matrix; apoptosis
4.  Thrombospondin expression in myofibers stabilizes muscle membranes 
eLife  null;5:e17589.
Skeletal muscle is highly sensitive to mutations in genes that participate in membrane stability and cellular attachment, which often leads to muscular dystrophy. Here we show that Thrombospondin-4 (Thbs4) regulates skeletal muscle integrity and its susceptibility to muscular dystrophy through organization of membrane attachment complexes. Loss of the Thbs4 gene causes spontaneous dystrophic changes with aging and accelerates disease in 2 mouse models of muscular dystrophy, while overexpression of mouse Thbs4 is protective and mitigates dystrophic disease. In the myofiber, Thbs4 selectively enhances vesicular trafficking of dystrophin-glycoprotein and integrin attachment complexes to stabilize the sarcolemma. In agreement, muscle-specific overexpression of Drosophila Tsp or mouse Thbs4 rescues a Drosophila model of muscular dystrophy with augmented membrane residence of βPS integrin. This functional conservation emphasizes the fundamental importance of Thbs’ as regulators of cellular attachment and membrane stability and identifies Thbs4 as a potential therapeutic target for muscular dystrophy.
DOI: http://dx.doi.org/10.7554/eLife.17589.001
eLife digest
Muscle cells, also known as myofibers, need to be robust in order to withstand the physical stresses of contracting and relaxing. As a result, the cell surface membrane that surrounds myofibers is more strongly anchored to its surroundings than that of other cells. Muscular dystrophies are a group of muscle-wasting disorders that usually arise when this surface membrane becomes less stable. For example, mutations that affect a protein called dystrophin-glycoprotein or integrin protein complexes can cause muscular dystrophy since these proteins normally keep the membrane anchored and stable when the muscle contracts and relaxes.
When myofibers in mammals become injured, as is the case during muscular dystrophy, they produce more proteins called thrombospondins – with thrombospondin-4 being the most common. However, until now it was not clear what these proteins did in muscle cells.
Vanhoutte et al. hypothesized that thrombospondin-4 may protect injured myofibers and tested their theory by first deleting the gene for thrombospondin-4 from mutant mice that were predisposed to develop muscular dystrophy. This worsened the muscle wasting in the mutant mice, and furthermore, deleting the gene for thrombospondin-4 also caused otherwise normal mice to develop muscular dystrophy in their old age. Conversely, when Vanhoutte et al. artificially increased the levels of thrombospondin-4 in the myofibers, it protected the mice against muscular dystrophy. Additional experiments conducted in fruit flies demonstrated that the protective effects of thrombospondin are conserved or similar in insects too. Lastly, biochemical experiments in mouse and rat cells showed that thrombospondin-4 aids dystrophin-glycoproteins and integrins in getting to the cell surface membrane, increasing its stability.
Overall these findings provide a clearer picture of the molecular underpinnings of muscular dystrophies. In the future, more experiments will have to focus on exactly how thrombospondins stabilize and direct dystrophin-glycoproteins and integrins to the cell surface membrane.
DOI: http://dx.doi.org/10.7554/eLife.17589.002
doi:10.7554/eLife.17589
PMCID: PMC5063588  PMID: 27669143
intracellular trafficking; thrombospondin; Muscular Dystrophy; D. melanogaster; Mouse
5.  Linking cytoarchitecture to metabolism: sarcolemma-associated plectin affects glucose uptake by destabilizing microtubule networks in mdx myofibers 
Skeletal Muscle  2013;3:14.
Background
Duchenne muscular dystrophy (DMD) is one of the most frequent forms of muscular disorders. It is caused by the absence of dystrophin, a core component of the sarcolemma-associated junctional complex that links the cytoskeleton to the extracellular matrix. We showed previously that plectin 1f (P1f), one of the major muscle-expressed isoforms of the cytoskeletal linker protein plectin, accumulates at the sarcolemma of DMD patients as well as of mdx mice, a widely studied animal model for DMD.
Based on plectin’s dual role as structural protein and scaffolding platform for signaling molecules, we speculated that the dystrophic phenotype observed after loss of dystrophin was caused, at least to some extent, by excess plectin. Thus, we hypothesized that elimination of plectin expression in mdx skeletal muscle, while probably resulting in an overall more severe phenotype, may lead to a partial phenotype rescue. In particular, we wanted to assess whether excess sarcolemmal plectin contributes to the dysregulation of sugar metabolism in mdx myofibers.
Methods
We generated plectin/dystrophin double deficient (dKO) mice by breeding mdx with conditional striated muscle-restricted plectin knockout (cKO) mice. The phenotype of these mice was comparatively analyzed with that of mdx, cKO, and wild-type mice, focusing on structural integrity and dysregulation of glucose metabolism.
Results
We show that the accumulation of plectin at the sarcolemma of mdx muscle fibers hardly compensated for their loss of structural integrity. Instead, it led to an additional metabolic deficit by impairing glucose uptake. While dKO mice suffered from an overall more severe form of muscular dystrophy compared to mdx or plectin-deficient mice, sarcolemmal integrity as well as glucose uptake of their myofibers were restored to normal levels upon ablation of plectin. Furthermore, microtubule (MT) networks in intact dKO myofibers, including subsarcolemmal areas, were found to be more robust than those in mdx mice. Finally, myotubes differentiated from P1f-overexpressing myoblasts showed an impairment of glucose transporter 4 translocation and a destabilization of MT networks.
Conclusions
Based on these results we propose that sarcolemma-associated plectin acts as an antagonist of MT network formation in myofibers, thereby hindering vesicle-mediated (MT-dependent) transport of glucose transporter 4. This novel role of plectin throws a bridge between extra-sarcomeric cytoarchitecture and metabolism of muscle fibers. Our study thus provides new insights into pathomechanisms of plectinopathies and muscular dystrophies in general.
doi:10.1186/2044-5040-3-14
PMCID: PMC3695810  PMID: 23758845
Plectin; Dystrophin; Sarcolemmal integrity; Glucose metabolism; Microtubules
6.  Transcriptomic analysis of dystrophin RNAi knockdown reveals a central role for dystrophin in muscle differentiation and contractile apparatus organization 
BMC Genomics  2010;11:345.
Background
Duchenne muscular dystrophy (DMD) is a fatal muscle wasting disorder caused by mutations in the dystrophin gene. DMD has a complex and as yet incompletely defined molecular pathophysiology hindering development of effective ameliorative approaches. Transcriptomic studies so far conducted on dystrophic cells and tissues suffer from non-specific changes and background noise due to heterogeneous comparisons and secondary pathologies. A study design in which a perfectly matched control cell population is used as reference for transcriptomic studies will give a much more specific insight into the effects of dystrophin deficiency and DMD pathophysiology.
Results
Using RNA interference (RNAi) to knock down dystrophin in myotubes from C57BL10 mice, we created a homogenous model to study the transcriptome of dystrophin-deficient myotubes. We noted significant differences in the global gene expression pattern between these myotubes and their matched control cultures. In particular, categorical analyses of the dysregulated genes demonstrated significant enrichment of molecules associated with the components of muscle cell contractile unit, ion channels, metabolic pathways and kinases. Additionally, some of the dysregulated genes could potentially explain conditions and endophenotypes associated with dystrophin deficiency, such as dysregulation of calcium homeostasis (Pvalb and Casq1), or cardiomyopathy (Obscurin, Tcap). In addition to be validated by qPCR, our data gains another level of validity by affirmatively reproducing several independent studies conducted previously at genes and/or protein levels in vivo and in vitro.
Conclusion
Our results suggest that in striated muscles, dystrophin is involved in orchestrating proper development and organization of myofibers as contractile units, depicting a novel pathophysiology for DMD where the absence of dystrophin results in maldeveloped myofibers prone to physical stress and damage. Therefore, it becomes apparent that any gene therapy approaches for DMD should target early stages in muscle development to attain a maximum clinical benefit. With a clear and specific definition of the transcriptome of dystrophin deficiency, manipulation of identified dysregulated molecules downstream of dystrophin may lead to novel ameliorative approaches for DMD.
doi:10.1186/1471-2164-11-345
PMCID: PMC2890566  PMID: 20515474
7.  Leaky ryanodine receptors in β-sarcoglycan deficient mice: a potential common defect in muscular dystrophy 
Skeletal Muscle  2012;2:9.
Background
Disruption of the sarcolemma-associated dystrophin-glycoprotein complex underlies multiple forms of muscular dystrophy, including Duchenne muscular dystrophy and sarcoglycanopathies. A hallmark of these disorders is muscle weakness. In a murine model of Duchenne muscular dystrophy, mdx mice, cysteine-nitrosylation of the calcium release channel/ryanodine receptor type 1 (RyR1) on the skeletal muscle sarcoplasmic reticulum causes depletion of the stabilizing subunit calstabin1 (FKBP12) from the RyR1 macromolecular complex. This results in a sarcoplasmic reticular calcium leak via defective RyR1 channels. This pathological intracellular calcium leak contributes to reduced calcium release and decreased muscle force production. It is unknown whether RyR1 dysfunction occurs also in other muscular dystrophies.
Methods
To test this we used a murine model of Limb-Girdle muscular dystrophy, deficient in β-sarcoglycan (Sgcb−/−).
Results
Skeletal muscle RyR1 from Sgcb−/− deficient mice were oxidized, nitrosylated, and depleted of the stabilizing subunit calstabin1, which was associated with increased open probability of the RyR1 channels. Sgcb−/− deficient mice exhibited decreased muscle specific force and calcium transients, and displayed reduced exercise capacity. Treating Sgcb−/− mice with the RyR stabilizing compound S107 improved muscle specific force, calcium transients, and exercise capacity. We have previously reported similar findings in mdx mice, a murine model of Duchenne muscular dystrophy.
Conclusions
Our data suggest that leaky RyR1 channels may underlie multiple forms of muscular dystrophy linked to mutations in genes encoding components of the dystrophin-glycoprotein complex. A common underlying abnormality in calcium handling indicates that pharmacological targeting of dysfunctional RyR1 could be a novel therapeutic approach to improve muscle function in Limb-Girdle and Duchenne muscular dystrophies.
doi:10.1186/2044-5040-2-9
PMCID: PMC3605002  PMID: 22640601
Muscular dystrophy; Ryanodine receptor; Calstabin1; Calcium
8.  Characterization of a DmdEGFP reporter mouse as a tool to investigate dystrophin expression 
Skeletal Muscle  2016;6:25.
Background
Dystrophin is a rod-shaped cytoplasmic protein that provides sarcolemmal stability as a structural link between the cytoskeleton and the extracellular matrix via the dystrophin-associated protein complex (DAPC). Mutations in the dystrophin-encoding DMD gene cause X-linked dystrophinopathies with variable phenotypes, the most severe being Duchenne muscular dystrophy (DMD) characterized by progressive muscle wasting and fibrosis. However, dystrophin deficiency does not only impair the function of skeletal and heart muscle but may also affect other organ systems such as the brain, eye, and gastrointestinal tract. The generation of a dystrophin reporter mouse would facilitate research into dystrophin muscular and extramuscular pathophysiology without the need for immunostaining.
Results
We generated a DmdEGFP reporter mouse through the in-frame insertion of the EGFP coding sequence behind the last Dmd exon 79, which is known to be expressed in all major dystrophin isoforms. We analyzed EGFP and dystrophin expression in various tissues and at the single muscle fiber level. Immunostaining of various members of the DAPC was done to confirm the correct subsarcolemmal location of dystrophin-binding partners. We found strong natural EGFP fluorescence at all expected sites of dystrophin expression in the skeletal and smooth muscle, heart, brain, and retina. EGFP fluorescence exactly colocalized with dystrophin immunostaining. In the skeletal muscle, dystrophin and other proteins of the DAPC were expressed at their correct sarcolemmal/subsarcolemmal localization. Skeletal muscle maintained normal tissue architecture, suggesting the correct function of the dystrophin-EGFP fusion protein. EGFP expression could be easily verified in isolated myofibers as well as in satellite cell-derived myotubes.
Conclusions
The novel dystrophin reporter mouse provides a valuable tool for direct visualization of dystrophin expression and will allow the study of dystrophin expression in vivo and in vitro in various tissues by live cell imaging.
Electronic supplementary material
The online version of this article (doi:10.1186/s13395-016-0095-5) contains supplementary material, which is available to authorized users.
doi:10.1186/s13395-016-0095-5
PMCID: PMC4932663  PMID: 27382459
Dystrophin; Dmd; EGFP; Duchenne muscular dystrophy; Reporter mouse
9.  Cell-lineage regulated myogenesis for dystrophin replacement: a novel therapeutic approach for treatment of muscular dystrophy 
Human Molecular Genetics  2008;17(16):2507-2517.
Duchenne muscular dystrophy (DMD) is characterized in skeletal muscle by cycles of myofiber necrosis and regeneration leading to loss of muscle fibers and replacement with fibrotic connective and adipose tissue. The ongoing activation and recruitment of muscle satellite cells for myofiber regeneration results in loss of regenerative capacity in part due to proliferative senescence. We explored a method whereby new myoblasts could be generated in dystrophic muscles by transplantation of primary fibroblasts engineered to express a micro-dystrophin/enhanced green fluorescent protein (µDys/eGFP) fusion gene together with a tamoxifen-inducible form of the myogenic regulator MyoD [MyoD-ER(T)]. Fibroblasts isolated from mdx4cv mice, a mouse model for DMD, were efficiently transduced with lentiviral vectors expressing µDys/eGFP and MyoD-ER(T) and underwent myogenic conversion when exposed to tamoxifen. These cells could also be induced to differentiate into µDys/eGFP-expressing myocytes and myotubes. Transplantation of transduced mdx4cv fibroblasts into mdx4cv muscles enabled tamoxifen-dependent regeneration of myofibers that express µDys. This lineage control method therefore allows replenishment of myogenic stem cells using autologous fibroblasts carrying an exogenous dystrophin gene. This strategy carries several potential advantages over conventional myoblast transplantation methods including: (i) the relative simplicity of culturing fibroblasts compared with myoblasts, (ii) a readily available cell source and ease of expansion and (iii) the ability to induce MyoD gene expression in vivo via administration of a medication. Our study provides a proof of concept for a novel gene/stem cell therapy technique and opens another potential therapeutic approach for degenerative muscle disorders.
doi:10.1093/hmg/ddn151
PMCID: PMC2574879  PMID: 18511457
10.  Dystrophin deficiency in canine X-linked muscular dystrophy in Japan (CXMDJ) alters myosin heavy chain expression profiles in the diaphragm more markedly than in the tibialis cranialis muscle 
Background
Skeletal muscles are composed of heterogeneous collections of muscle fiber types, the arrangement of which contributes to a variety of functional capabilities in many muscle types. Furthermore, skeletal muscles can adapt individual myofibers under various circumstances, such as disease and exercise, by changing fiber types. This study was performed to examine the influence of dystrophin deficiency on fiber type composition of skeletal muscles in canine X-linked muscular dystrophy in Japan (CXMDJ), a large animal model for Duchenne muscular dystrophy.
Methods
We used tibialis cranialis (TC) muscles and diaphragms of normal dogs and those with CXMDJ at various ages from 1 month to 3 years old. For classification of fiber types, muscle sections were immunostained with antibodies against fast, slow, or developmental myosin heavy chain (MHC), and the number and size of these fibers were analyzed. In addition, MHC isoforms were detected by gel electrophoresis.
Results
In comparison with TC muscles of CXMDJ, the number of fibers expressing slow MHC increased markedly and the number of fibers expressing fast MHC decreased with growth in the affected diaphragm. In populations of muscle fibers expressing fast and/or slow MHC(s) but not developmental MHC of CXMDJ muscles, slow MHC fibers were predominant in number and showed selective enlargement. Especially, in CXMDJ diaphragms, the proportions of slow MHC fibers were significantly larger in populations of myofibers with non-expression of developmental MHC. Analyses of MHC isoforms also indicated a marked increase of type I and decrease of type IIA isoforms in the affected diaphragm at ages over 6 months. In addition, expression of developmental (embryonic and/or neonatal) MHC decreased in the CXMDJ diaphragm in adults, in contrast to continuous high-level expression in affected TC muscle.
Conclusion
The CXMDJ diaphragm showed marked changes in fiber type composition unlike TC muscles, suggesting that the affected diaphragm may be effectively adapted toward dystrophic stress by switching to predominantly slow fibers. Furthermore, the MHC expression profile in the CXMDJ diaphragm was markedly different from that in mdx mice, indicating that the dystrophic dog is a more appropriate model than a murine one, to investigate the mechanisms of respiratory failure in DMD.
doi:10.1186/1471-2474-9-1
PMCID: PMC2257929  PMID: 18182116
11.  The Dystrophin Complex Controls BK Channel Localization and Muscle Activity in Caenorhabditis elegans 
PLoS Genetics  2009;5(12):e1000780.
Genetic defects in the dystrophin-associated protein complex (DAPC) are responsible for a variety of pathological conditions including muscular dystrophy, cardiomyopathy, and vasospasm. Conserved DAPC components from humans to Caenorhabditis elegans suggest a similar molecular function. C. elegans DAPC mutants exhibit a unique locomotory deficit resulting from prolonged muscle excitation and contraction. Here we show that the C. elegans DAPC is essential for proper localization of SLO-1, the large conductance, voltage-, and calcium-dependent potassium (BK) channel, which conducts a major outward rectifying current in muscle under the normal physiological condition. Through analysis of mutants with the same phenotype as the DAPC mutants, we identified the novel islo-1 gene that encodes a protein with two predicted transmembrane domains. We demonstrate that ISLO-1 acts as a novel adapter molecule that links the DAPC to SLO-1 in muscle. We show that a defect in either the DAPC or ISLO-1 disrupts normal SLO-1 localization in muscle. Consistent with observations that SLO-1 requires a high calcium concentration for full activation, we find that SLO-1 is localized near L-type calcium channels in muscle, thereby providing a mechanism coupling calcium influx with the outward rectifying current. Our results indicate that the DAPC modulates muscle excitability by localizing the SLO-1 channel to calcium-rich regions of C. elegans muscle.
Author Summary
Dystrophin is a long rod-shaped protein that forms a complex with several membrane and cytoplasmic proteins in muscle. Genetic defects in components of this dystrophin complex are responsible for many forms of muscular dystrophy, including Duchenne muscular dystrophy. C. elegans possesses the dystrophin complex and mutations in its components cause muscular defects, indicating that the dystrophin complex has an evolutionary conserved role in muscle. Accumulating evidence in mammals indicates that dystrophic muscle exhibits an abnormal calcium homeostasis. It is not clear how defects in the dystrophin complex are linked to calcium homeostasis, however. In a C. elegans genetic study we found that a novel adaptor protein links the dystrophin complex to a calcium-sensitive potassium channel that mediates muscle inactivation. We further demonstrated that both the dystrophin complex and the adaptor protein localize the potassium channel in a close proximity to a muscle-activating calcium channel. This arrangement ensures that calcium increases accompanied by muscle activation are coupled to muscle inactivation. Defects in the dystrophin complex or the adaptor disrupt the localization of the potassium channel, thereby resulting in prolonged muscle activation and calcium ion increases. Our study provides a mechanism by which the dystrophin complex regulates cellular signaling and muscle excitability.
doi:10.1371/journal.pgen.1000780
PMCID: PMC2788698  PMID: 20019812
12.  Sub-physiological sarcoglycan expression contributes to compensatory muscle protection in mdx mice 
Human Molecular Genetics  2009;18(7):1209-1220.
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 δ-sarcoglycan/dystrophin double knockout mice (δ-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 δ-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, δ-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.
doi:10.1093/hmg/ddp015
PMCID: PMC2655768  PMID: 19131360
13.  Mitigation of muscular dystrophy in mice by SERCA overexpression in skeletal muscle 
The Journal of Clinical Investigation  2011;121(3):1044-1052.
Muscular dystrophies (MDs) comprise a group of degenerative muscle disorders characterized by progressive muscle wasting and often premature death. The primary defect common to most MDs involves disruption of the dystrophin-glycoprotein complex (DGC). This leads to sarcolemmal instability and Ca2+ influx, inducing cellular necrosis. Here we have shown that the dystrophic phenotype observed in δ-sarcoglycan–null (Sgcd–/–) mice and dystrophin mutant mdx mice is dramatically improved by skeletal muscle–specific overexpression of sarcoplasmic reticulum Ca2+ ATPase 1 (SERCA1). Rates of myofiber central nucleation, tissue fibrosis, and serum creatine kinase levels were dramatically reduced in Sgcd–/– and mdx mice with the SERCA1 transgene, which also rescued the loss of exercise capacity in Sgcd–/– mice. Adeno-associated virus–SERCA2a (AAV-SERCA2a) gene therapy in the gastrocnemius muscle of Sgcd–/– mice mitigated dystrophic disease. SERCA1 overexpression reversed a defect in sarcoplasmic reticulum Ca2+ reuptake that characterizes dystrophic myofibers and reduced total cytosolic Ca2+. Further, SERCA1 overexpression almost completely rescued the dystrophic phenotype in a mouse model of MD driven solely by Ca2+ influx. Mitochondria isolated from the muscle of SERCA1-Sgcd–/– mice were no longer swollen and calpain activation was reduced, suggesting protection from Ca2+-driven necrosis. Our results suggest a novel therapeutic approach using SERCA1 to abrogate the altered intracellular Ca2+ levels that underlie most forms of MD.
doi:10.1172/JCI43844
PMCID: PMC3049367  PMID: 21285509
14.  Diagnosis and cell-based therapy for Duchenne muscular dystrophy in humans, mice, and zebrafish 
Journal of human genetics  2006;51(5):397-406.
The muscular dystrophies are a heterogeneous group of genetically caused muscle degenerative disorders. The Kunkel laboratory has had a longstanding research program into the pathogenesis and treatment of these diseases. Starting with our identification of dystrophin as the defective protein in Duchenne muscular dystrophy (DMD), we have continued our work on normal dystrophin function and how it is altered in muscular dystrophy. Our work has led to the identification of the defective genes in three forms of limb girdle muscular dystrophy (LGMD) and a better understanding of how muscle degenerates in many of the different dystrophies. The identification of mutations causing human forms of dystrophy has lead to improved diagnosis for patients with the disease. We are continuing to improve the molecular diagnosis of the dystrophies and have developed a high-throughput sequencing approach for the low-cost rapid diagnosis of all known forms of dystrophy. In addition, we are continuing to work on therapies using available animal models. Currently, there are a number of mouse models of the human dystrophies, the most notable being the mdx mouse with dystrophin deficiency. These mice are being used to test possible therapies, including stem-cell-based approaches. We have been able to systemically deliver human dystrophin to these mice via the arterial circulation and convert 8% of dystrophin-deficient fibers to fibers expressing human dystrophin. We are now expanding our research to identify new forms of LGMD by analyzing zebrafish models of muscular dystrophy. Currently, we have 14 different zebrafish mutants exhibiting various phenotypes of muscular dystrophy, including muscle weakness and inactivity. One of these mutants carries a stop codon mutation in dystrophin, and we have recently identified another carrying a mutation in titin. We are currently positionally cloning the disease-causative mutation in the remaining 12 mutant strains. We hope that one of these new mutant strains of fish will have a mutation in a gene not previously implicated in human muscular dystrophy. This gene would become a candidate gene to be analyzed in patients which do not carry a mutation in any of the known dystrophy-associated genes. By studying both disease pathology and investigating potential therapies, we hope to make a positive difference in the lives of people living with muscular dystrophy.
doi:10.1007/s10038-006-0374-9
PMCID: PMC3518425  PMID: 16583129
DNA sequencing; Muscle; Muscular dystrophy; Stem cells; Zebrafish
15.  Matrix Metalloproteinase-9 Inhibition Improves Proliferation and Engraftment of Myogenic Cells in Dystrophic Muscle of mdx Mice 
PLoS ONE  2013;8(8):e72121.
Duchenne muscular dystrophy (DMD) caused by loss of cytoskeletal protein dystrophin is a devastating disorder of skeletal muscle. Primary deficiency of dystrophin leads to several secondary pathological changes including fiber degeneration and regeneration, extracellular matrix breakdown, inflammation, and fibrosis. Matrix metalloproteinases (MMPs) are a group of extracellular proteases that are involved in tissue remodeling, inflammation, and development of interstitial fibrosis in many disease states. We have recently reported that the inhibition of MMP-9 improves myopathy and augments myofiber regeneration in mdx mice (a mouse model of DMD). However, the mechanisms by which MMP-9 regulates disease progression in mdx mice remain less understood. In this report, we demonstrate that the inhibition of MMP-9 augments the proliferation of satellite cells in dystrophic muscle. MMP-9 inhibition also causes significant reduction in percentage of M1 macrophages with concomitant increase in the proportion of promyogenic M2 macrophages in mdx mice. Moreover, inhibition of MMP-9 increases the expression of Notch ligands and receptors, and Notch target genes in skeletal muscle of mdx mice. Furthermore, our results show that while MMP-9 inhibition augments the expression of components of canonical Wnt signaling, it reduces the expression of genes whose products are involved in activation of non-canonical Wnt signaling in mdx mice. Finally, the inhibition of MMP-9 was found to dramatically improve the engraftment of transplanted myoblasts in skeletal muscle of mdx mice. Collectively, our study suggests that the inhibition of MMP-9 is a promising approach to stimulate myofiber regeneration and improving engraftment of muscle progenitor cells in dystrophic muscle.
doi:10.1371/journal.pone.0072121
PMCID: PMC3744489  PMID: 23977226
16.  Identification of New Dystroglycan Complexes in Skeletal Muscle 
PLoS ONE  2013;8(8):e73224.
The dystroglycan complex contains the transmembrane protein β-dystroglycan and its interacting extracellular mucin-like protein α-dystroglycan. In skeletal muscle fibers, the dystroglycan complex plays an important structural role by linking the cytoskeletal protein dystrophin to laminin in the extracellular matrix. Mutations that affect any of the proteins involved in this structural axis lead to myofiber degeneration and are associated with muscular dystrophies and congenital myopathies. Because loss of dystrophin in Duchenne muscular dystrophy (DMD) leads to an almost complete loss of dystroglycan complexes at the myofiber membrane, it is generally assumed that the vast majority of dystroglycan complexes within skeletal muscle fibers interact with dystrophin. The residual dystroglycan present in dystrophin-deficient muscle is thought to be preserved by utrophin, a structural homolog of dystrophin that is up-regulated in dystrophic muscles. However, we found that dystroglycan complexes are still present at the myofiber membrane in the absence of both dystrophin and utrophin. Our data show that only a minority of dystroglycan complexes associate with dystrophin in wild type muscle. Furthermore, we provide evidence for at least three separate pools of dystroglycan complexes within myofibers that differ in composition and are differentially affected by loss of dystrophin. Our findings indicate a more complex role of dystroglycan in muscle than currently recognized and may help explain differences in disease pathology and severity among myopathies linked to mutations in DAPC members.
doi:10.1371/journal.pone.0073224
PMCID: PMC3738564  PMID: 23951345
17.  Dystrophin expression in muscle stem cells regulates their polarity and asymmetric division 
Nature medicine  2015;21(12):1455-1463.
Dystrophin is expressed in differentiated myofibers where it is required for sarcolemmal integrity, and loss-of-function mutations in its gene result in Duchenne Muscular Dystrophy (DMD), a disease characterized by progressive and severe skeletal muscle degeneration. Here we found that dystrophin is also highly expressed in activated muscle stem cells (also known as satellite cells) where it associates with the Ser/Thr kinase Mark2 (also known as Par1b), an important regulator of cell polarity. In the absence of dystrophin, expression of Mark2 protein is downregulated, resulting in the inability to polarize Pard3 to the opposite side of the cell. Consequently, the number of asymmetric divisions is strikingly reduced in dystrophin-deficient satellite cells, while also displaying a loss of polarity, abnormal division patterns including centrosome amplification, impaired mitotic spindle orientation, and prolonged cell divisions. Altogether, these intrinsic defects strongly reduce the generation of myogenic progenitors needed for proper muscle regeneration. Therefore, we conclude that dystrophin has an essential role in the regulation of satellite cell polarity and asymmetric division. Our findings indicate that muscle wasting in DMD is not only caused by myofiber fragility, but is also exacerbated by impaired regeneration due to intrinsic satellite cell dysfunction.
doi:10.1038/nm.3990
PMCID: PMC4839960  PMID: 26569381
Duchenne Muscular Dystrophy; dystrophin; Skeletal muscle; Satellite cell; Stem cell; Asymmetric cell division; Polarity; PAR-complex; Mark2; Mitotic defect
18.  Enhanced Ca2+ influx from STIM1–Orai1 induces muscle pathology in mouse models of muscular dystrophy 
Human Molecular Genetics  2014;23(14):3706-3715.
Muscular dystrophy is a progressive muscle wasting disease that is thought to be initiated by unregulated Ca2+ influx into myofibers leading to their death. Store-operated Ca2+ entry (SOCE) through sarcolemmal Ca2+ selective Orai1 channels in complex with STIM1 in the sarcoplasmic reticulum is one such potential disease mechanism for pathologic Ca2+ entry. Here, we generated a mouse model of STIM1 overexpression in skeletal muscle to determine whether this type of Ca2+ entry could induce muscular dystrophy. Myofibers from muscle-specific STIM1 transgenic mice showed a significant increase in SOCE in skeletal muscle, modeling an observed increase in the same current in dystrophic myofibers. Histological and biochemical analysis of STIM1 transgenic mice showed fulminant muscle disease characterized by myofiber necrosis, swollen mitochondria, infiltration of inflammatory cells, enhanced interstitial fibrosis and elevated serum creatine kinase levels. This dystrophic-like disease in STIM1 transgenic mice was abrogated by crossing in a transgene expressing a dominant-negative Orai1 (dnOrai1) mutant. The dnOrai1 transgene also significantly reduced the severity of muscular dystrophy in both mdx (dystrophin mutant mice) and δ-sarcoglycan-deficient (Sgcd−/−) mouse models of disease. Hence, Ca2+ influx across an unstable sarcolemma due to increased activity of a STIM1–Orai1 complex is a disease determinant in muscular dystrophy, and hence, SOCE represents a potential therapeutic target.
doi:10.1093/hmg/ddu079
PMCID: PMC4065147  PMID: 24556214
19.  Cell-lineage Regulated Myogenesis for Dystrophin Replacement: a Novel Therapeutic Approach for Treatment of Muscular Dystrophy 
Human molecular genetics  2008;17(16):2507-2517.
Duchenne muscular dystrophy (DMD) is characterized in skeletal muscle by cycles of myofiber necrosis and regeneration leading to loss of muscle fibers and replacement with fibrotic connective and adipose tissue. The ongoing activation and recruitment of muscle satellite cells for myofiber regeneration results in loss of regenerative capacity in part due to proliferative senescence. We explored a method whereby new myoblasts could be generated in dystrophic muscles by transplantation of primary fibroblasts engineered to express a micro-dystrophin/eGFP (μDys/eGFP) fusion gene together with a tamoxifen-inducible form of the myogenic regulator MyoD [MyoD-ER(T)]. Fibroblasts isolated from mdx4cv mice, a mouse model for DMD, were efficiently transduced with lentiviral vectors expressing μDys/eGFP and MyoD-ER(T) and underwent myogenic conversion when exposed to tamoxifen. These cells could also be induced to differentiate into μDys/eGFP-expressing myocytes and myotubes. Transplantation of transduced mdx4cv fibroblasts into mdx4cv muscles enabled tamoxifen-dependent regeneration of myofibers that express micro-dystrophin. This lineage control method therefore allows replenishment of myogenic stem cells using autologous fibroblasts carrying an exogenous dystrophin gene. This strategy carries several potential advantages over conventional myoblast transplantation methods including: 1) the relative simplicity of culturing fibroblasts compared with myoblasts, 2) a readily available cell source and ease of expansion, and 3) the ability to induce MyoD gene expression in vivo via administration of a medication. Our study provides a proof of concept for a novel gene/stem cell therapy technique and opens another potential therapeutic approach for degenerative muscle disorders.
doi:10.1093/hmg/ddn151
PMCID: PMC2574879  PMID: 18511457
20.  MicroRNA-199a is induced in dystrophic muscle and affects WNT signaling, cell proliferation, and myogenic differentiation 
Cell Death and Differentiation  2013;20(9):1194-1208.
In patients with Duchenne muscular dystrophy (DMD), the absence of a functional dystrophin protein results in sarcolemmal instability, abnormal calcium signaling, cardiomyopathy, and skeletal muscle degeneration. Using the dystrophin-deficient sapje zebrafish model, we have identified microRNAs (miRNAs) that, in comparison to our previous findings in human DMD muscle biopsies, are uniquely dysregulated in dystrophic muscle across vertebrate species. MiR-199a-5p is dysregulated in dystrophin-deficient zebrafish, mdx5cv mice, and human muscle biopsies. MiR-199a-5p mature miRNA sequences are transcribed from stem loop precursor miRNAs that are found within the introns of the dynamin-2 and dynamin-3 loci. The miR-199a-2 stem loop precursor transcript that gives rise to the miR-199a-5p mature transcript was found to be elevated in human dystrophic muscle. The levels of expression of miR-199a-5p are regulated in a serum response factor (SRF)-dependent manner along with myocardin-related transcription factors. Inhibition of SRF-signaling reduces miR-199a-5p transcript levels during myogenic differentiation. Manipulation of miR-199a-5p expression in human primary myoblasts and myotubes resulted in dramatic changes in cellular size, proliferation, and differentiation. MiR-199a-5p targets several myogenic cell proliferation and differentiation regulatory factors within the WNT signaling pathway, including FZD4, JAG1, and WNT2. Overexpression of miR-199a-5p in the muscles of transgenic zebrafish resulted in abnormal myofiber disruption and sarcolemmal membrane detachment, pericardial edema, and lethality. Together, these studies identify miR-199a-5p as a potential regulator of myogenesis through suppression of WNT-signaling factors that act to balance myogenic cell proliferation and differentiation.
doi:10.1038/cdd.2013.62
PMCID: PMC3741500  PMID: 23764775
microRNA; zebrafish; miR-199a; WNT signaling; dystrophin; skeletal muscle
21.  Genetic epidemiology of muscular dystrophies resulting from sarcoglycan gene mutations. 
Journal of Medical Genetics  1997;34(12):973-977.
BACKGROUND: The autosomal recessive limb-girdle muscular dystrophies (LGMDs) are a group of genetically heterogeneous muscle diseases characterised by progressive proximal limb muscle weakness. Six different loci have been mapped and pathogenetic mutations in the genes encoding the sarcoglycan complex components (alpha-, beta-, gamma-, and delta-sarcoglycan) have been documented. LGMD patients affected with primary "sarcoglycanopathies" are classified as LGMD2D, 2E, 2C, and 2F, respectively. METHODS: A geographical area in north east Italy (2,319,147 inhabitants) was selected for a genetic epidemiological study on primary sarcoglycanopathies. Within the period 1982 to 1996, all patients living in this region and diagnosed with muscular dystrophy were seen at our centre. Immunohistochemical and immunoblot screening for alpha-sarcoglycan protein deficiency was performed on all muscle biopsies from patients with a progressive muscular dystrophy of unknown aetiology and normal dystrophin. Sarcoglycan mutation analyses were conducted on all patient muscle biopsies shown to have complete or partial absence of alpha-sarcoglycan immunostaining or a decreased quantity of alpha-sarcoglycan protein on immunoblotting. RESULTS: Two hundred and four patient muscle biopsies were screened for alpha-sarcoglycan protein deficiency and 18 biopsies showed a deficiency. Pathogenetic mutations involving one gene for sarcoglycan complex components were identified in 13 patients: alpha-sarcoglycan in seven, beta-sarcoglycan in two, gamma-sarcoglycan in four, and none in the delta-sarcoglycan gene. The overall prevalence of primary sarcoglycanopathies, as of 31 December 1996, was estimated to be 5.6 x 10(-6) inhabitants. CONCLUSION: The prevalence rate estimated in this study is the first to be obtained after biochemical and molecular genetic screening for sarcoglycan defects.
PMCID: PMC1051145  PMID: 9429136
22.  Label-free mass spectrometric analysis reveals complex changes in the brain proteome from the mdx-4cv mouse model of Duchenne muscular dystrophy 
Clinical Proteomics  2015;12:27.
Background
X-linked muscular dystrophy is a primary disease of the neuromuscular system. Primary abnormalities in the Dmd gene result in the absence of the full-length isoform of the membrane cytoskeletal protein dystrophin. Besides progressive skeletal muscle wasting and cardio-respiratory complications, developmental cognitive deficits and behavioural abnormalities are clinical features of Duchenne muscular dystrophy. In order to better understand the mechanisms that underlie impaired brain functions in Duchenne patients, we have carried out a proteomic analysis of total brain extracts from the mdx-4cv mouse model of dystrophinopathy.
Results
The comparative proteomic profiling of the mdx-4cv brain revealed a significant increase in 39 proteins and a decrease in 7 proteins. Interesting brain tissue-associated proteins with an increased concentration in the mdx-4cv animal model were represented by the glial fibrillary acidic protein GFAP, the neuronal Ca2+-binding protein calretinin, annexin AnxA5, vimentin, the neuron-specific enzyme ubiquitin carboxyl-terminal hydrolase isozyme L1, the dendritic spine protein drebrin, the cytomatrix protein bassoon of the nerve terminal active zone, and the synapse-associated protein SAP97. Decreased proteins were identified as the nervous system-specific proteins syntaxin-1B and syntaxin-binding protein 1, as well as the plasma membrane Ca2+-transporting ATPase PMCA2 that is mostly found in the brain cortex. The differential expression patterns of GFAP, vimentin, PMCA2 and AnxA5 were confirmed by immunoblotting. Increased GFAP levels were also verified by immunofluorescence microscopy.
Conclusions
The large number of mass spectrometrically identified proteins with an altered abundance suggests complex changes in the mdx-4cv brain proteome. Increased levels of the glial fibrillary acidic protein, an intermediate filament component that is uniquely associated with astrocytes in the central nervous system, imply neurodegeneration-associated astrogliosis. The up-regulation of annexin and vimentin probably represent compensatory mechanisms involved in membrane repair and cytoskeletal stabilization in the absence of brain dystrophin. Differential alterations in the Ca2+-binding protein calretinin and the Ca2+-pumping protein PMCA2 suggest altered Ca2+-handling mechanisms in the Dp427-deficient brain. In addition, the proteomic findings demonstrated metabolic adaptations and functional changes in the central nervous system from the dystrophic phenotype. Candidate proteins can now be evaluated for their suitability as proteomic biomarkers and their potential in predictive, diagnostic, prognostic and/or therapy-monitoring approaches to treat brain abnormalities in dystrophinopathies.
doi:10.1186/s12014-015-9099-0
PMCID: PMC4657206  PMID: 26604869
Annexin; Ca2+-ATPase PMCA2; Dystrophinopathy; Glial fibrillary acidic protein; Glyosis; Intermediate filament; Mental retardation; Vimentin; von Willebrand factor
23.  Decrease of myofiber branching via muscle-specific expression of the olfactory receptor mOR23 in dystrophic muscle leads to protection against mechanical stress 
Skeletal Muscle  2016;6:2.
Background
Abnormal branched myofibers within skeletal muscles are commonly found in diverse animal models of muscular dystrophy as well as in patients. Branched myofibers from dystrophic mice are more susceptible to break than unbranched myofibers suggesting that muscles containing a high percentage of these myofibers are more prone to injury. Previous studies showed ubiquitous over-expression of mouse olfactory receptor 23 (mOR23), a G protein-coupled receptor, in wild type mice decreased myofiber branching. Whether mOR23 over-expression specifically in skeletal muscle cells is sufficient to mitigate myofiber branching in dystrophic muscle is unknown.
Methods
We created a novel transgenic mouse over-expressing mOR23 specifically in muscle cells and then bred with dystrophic (mdx) mice. Myofiber branching was analyzed in these two transgenic mice and membrane integrity was assessed by Evans blue dye fluorescence.
Results
mOR23 over-expression in muscle led to a decrease of myofiber branching after muscle regeneration in non-dystrophic mouse muscles and reduced the severity of myofiber branching in mdx mouse muscles. Muscles from mdx mouse over-expressing mOR23 significantly exhibited less damage to eccentric contractions than control mdx muscles.
Conclusions
The decrease of myofiber branching in mdx mouse muscles over-expressing mOR23 reduced the amount of membrane damage induced by mechanical stress. These results suggest that modifying myofiber branching in dystrophic patients, while not preventing degeneration, could be beneficial for mitigating some of the effects of the disease process.
Electronic supplementary material
The online version of this article (doi:10.1186/s13395-016-0077-7) contains supplementary material, which is available to authorized users.
doi:10.1186/s13395-016-0077-7
PMCID: PMC4721043  PMID: 26798450
mOR23; Myofiber branching; Mechanical stress; Muscular dystrophy; Muscle regeneration
24.  Animal Models for Muscular Dystrophy Show Different Patterns of Sarcolemmal Disruption  
The Journal of Cell Biology  1997;139(2):375-385.
Genetic defects in a number of components of the dystrophin–glycoprotein complex (DGC) lead to distinct forms of muscular dystrophy. However, little is known about how alterations in the DGC are manifested in the pathophysiology present in dystrophic muscle tissue. One hypothesis is that the DGC protects the sarcolemma from contraction-induced damage. Using tracer molecules, we compared sarcolemmal integrity in animal models for muscular dystrophy and in muscular dystrophy patient samples. Evans blue, a low molecular weight diazo dye, does not cross into skeletal muscle fibers in normal mice. In contrast, mdx mice, a dystrophin-deficient animal model for Duchenne muscular dystrophy, showed significant Evans blue accumulation in skeletal muscle fibers. We also studied Evans blue dispersion in transgenic mice bearing different dystrophin mutations, and we demonstrated that cytoskeletal and sarcolemmal attachment of dystrophin might be a necessary requirement to prevent serious fiber damage. The extent of dye incorporation in transgenic mice correlated with the phenotypic severity of similar dystrophin mutations in humans. We furthermore assessed Evans blue incorporation in skeletal muscle of the dystrophia muscularis (dy/dy) mouse and its milder allelic variant, the dy2J/dy2J mouse, animal models for congenital muscular dystrophy. Surprisingly, these mice, which have defects in the laminin α2-chain, an extracellular ligand of the DGC, showed little Evans blue accumulation in their skeletal muscles. Taken together, these results suggest that the pathogenic mechanisms in congenital muscular dystrophy are different from those in Duchenne muscular dystrophy, although the primary defects originate in two components associated with the same protein complex.
PMCID: PMC2139791  PMID: 9334342
25.  Matrix metalloproteinase-9 inhibition ameliorates pathogenesis and improves skeletal muscle regeneration in muscular dystrophy 
Human Molecular Genetics  2009;18(14):2584-2598.
Duchenne muscular dystrophy (DMD) is a fatal X-linked genetic disorder of skeletal muscle caused by mutation in dystrophin gene. Although the degradation of skeletal muscle extracellular matrix, inflammation and fibrosis are the common pathological features in DMD, the underlying mechanisms remain poorly understood. In this study, we have investigated the role and the mechanisms by which increased levels of matrix metalloproteinase-9 (MMP-9) protein causes myopathy in dystrophin-deficient mdx mice. The levels of MMP-9 but not tissue inhibitor of MMPs were drastically increased in skeletal muscle of mdx mice. Besides skeletal muscle, infiltrating macrophages were found to contribute significantly to the elevated levels of MMP-9 in dystrophic muscle. In vivo administration of a nuclear factor-kappa B inhibitory peptide, NBD, blocked the expression of MMP-9 in dystrophic muscle of mdx mice. Deletion of Mmp9 gene in mdx mice improved skeletal muscle structure and functions and reduced muscle injury, inflammation and fiber necrosis. Inhibition of MMP-9 increased the levels of cytoskeletal protein β-dystroglycan and neural nitric oxide synthase and reduced the amounts of caveolin-3 and transforming growth factor-β in myofibers of mdx mice. Genetic ablation of MMP-9 significantly augmented the skeletal muscle regeneration in mdx mice. Finally, pharmacological inhibition of MMP-9 activity also ameliorated skeletal muscle pathogenesis and enhanced myofiber regeneration in mdx mice. Collectively, our study suggests that the increased production of MMP-9 exacerbates dystrophinopathy and MMP-9 represents as one of the most promising therapeutic targets for the prevention of disease progression in DMD.
doi:10.1093/hmg/ddp191
PMCID: PMC2701330  PMID: 19401296

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