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1.  Prednisolone Attenuates Improvement of Cardiac and Skeletal Contractile Function and Histopathology by Lisinopril and Spironolactone in the mdx Mouse Model of Duchenne Muscular Dystrophy 
PLoS ONE  2014;9(2):e88360.
Duchenne muscular dystrophy (DMD) is an inherited disease that causes striated muscle weakness. Recently, we showed therapeutic effects of the combination of lisinopril (L), an angiotensin converting enzyme (ACE) inhibitor, and spironolactone (S), an aldosterone antagonist, in mice lacking dystrophin and haploinsufficient for utrophin (utrn+/−;mdx, het mice); both cardiac and skeletal muscle function and histology were improved when these mice were treated early with LS. It was unknown to what extent LS treatment is effective in the most commonly used DMD murine model, the mdx mouse. In addition, current standard-of-care treatment for DMD is limited to corticosteroids. Therefore, potentially useful alternative or additive drugs need to be both compared directly to corticosteroids and tested in presence of corticosteroids. We evaluated the effectiveness of this LS combination in the mdx mouse model both compared with corticosteroid treatment (prednisolone, P) or in combination (LSP). We tested the additional combinatorial treatment containing the angiotensin II receptor blocker losartan (T), which is widely used to halt and treat the developing cardiac dysfunction in DMD patients as an alternative to an ACE inhibitor. Peak myocardial strain rate, assessed by magnetic resonance imaging, showed a negative impact of P, whereas in both diaphragm and extensor digitorum longus (EDL) muscle contractile function was not significantly impaired by P. Histologically, P generally increased cardiac damage, estimated by percentage area infiltrated by IgG as well as by collagen staining. In general, groups that only differed in the presence or absence of P (i.e. mdx vs. P, LS vs. LSP, and TS vs. TSP) demonstrated a significant detrimental impact of P on many assessed parameters, with the most profound impact on cardiac pathology.
PMCID: PMC3923790  PMID: 24551095
2.  Rapamycin Ameliorates Dystrophic Phenotype in mdx Mouse Skeletal Muscle 
Molecular Medicine  2011;17(9-10):917-924.
Duchenne muscular dystrophy (DMD) is an X-linked, lethal, degenerative disease that results from mutations in the dystrophin gene, causing necrosis and inflammation in skeletal muscle tissue. Treatments that reduce muscle fiber destruction and immune cell infiltration can ameliorate DMD pathology. We treated the mdx mouse, a model for DMD, with the immunosuppressant drug rapamycin (RAPA) both locally and systemically to examine its effects on dystrophic mdx muscles. We observed a significant reduction of muscle fiber necrosis in treated mdx mouse tibialis anterior (TA) and diaphragm (Dia) muscles 6 wks post-treatment. This effect was associated with a significant reduction in infiltration of effector CD4+ and CD8+ T cells in skeletal muscle tissue, while Foxp3+ regulatory T cells were preserved. Because RAPA exerts its effects through the mammalian target of RAPA (mTOR), we studied the activation of mTOR in mdx TA and Dia with and without RAPA treatment. Surprisingly, mTOR activation levels in mdx TA were not different from control C57BL/10 (B10). However, mTOR activation was different in Dia between mdx and B10; mTOR activation levels did not rise between 6 and 12 wks of age in mdx Dia muscle, whereas a rise in mTOR activation level was observed in B10 Dia muscle. Furthermore, mdx Dia, but not TA, muscle mTOR activation was responsive to RAPA treatment.
PMCID: PMC3188871  PMID: 21607286
3.  The HDAC Inhibitor TSA Ameliorates a Zebrafish Model of Duchenne Muscular Dystrophy 
PLoS Currents  2013;
Zebrafish are an excellent model for Duchenne muscular dystrophy. In particular, zebrafish provide a system for rapid, easy, and low-cost screening of small molecules that can ameliorate muscle damage in dystrophic larvae. Here we identify an optimal anti-sense morpholino cocktail that robustly knocks down zebrafish Dystrophin (dmd-MO). We use two approaches, muscle birefringence and muscle actin expression, to quantify muscle damage and show that the dmd-MO dystrophic phenotype closely resembles the zebrafish dmd mutant phenotype. We then show that the histone deacetylase (HDAC) inhibitor TSA, which has been shown to ameliorate the mdx mouse Duchenne model, can rescue muscle fiber damage in both dmd-MO and dmd mutant larvae. Our study identifies optimal morpholino and phenotypic scoring approaches for dystrophic zebrafish, further enhancing the zebrafish dmd model for rapid and cost-effective small molecule screening.
PMCID: PMC3870918  PMID: 24459606
4.  Histone Deacetylase Inhibitors in the Treatment of Muscular Dystrophies: Epigenetic Drugs for Genetic Diseases 
Molecular Medicine  2011;17(5-6):457-465.
Histone deacetylases inhibitors (HDACi) include a growing number of drugs that share the ability to inhibit the enzymatic activity of some or all the HDACs. Experimental and preclinical evidence indicates that these epigenetic drugs not only can be effective in the treatment of malignancies, inflammatory diseases and degenerative disorders, but also in the treatment of genetic diseases, such as muscular dystrophies. The ability of HDACi to counter the progression of muscular dystrophies points to HDACs as a crucial link between specific genetic mutations and downstream determinants of disease progression. It also suggests the contribution of epigenetic events to the pathogenesis of muscular dystrophies. Here we describe the experimental evidence supporting the key role of HDACs in the control of the transcriptional networks underlying the potential of dystrophic muscles either to activate compensatory regeneration or to undergo fibroadipogenic degeneration. Studies performed in mouse models of Duchenne muscular dystrophy (DMD) indicate that dystrophin deficiency leads to deregulated HDAC activity, which perturbs downstream networks and can be restored directly, by HDAC blockade, or indirectly, by reexpression of dystrophin. This evidence supports the current view that HDACi are emerging candidate drugs for pharmacological interventions in muscular dystrophies, and reveals unexpected common beneficial outcomes of pharmacological treatment or gene therapy.
PMCID: PMC3105131  PMID: 21308150
5.  Constitutively activated dystrophic muscle fibroblasts show a paradoxical response to TGF-β and CTGF/CCN2 
Transforming growth factor beta (TGF-β) and connective tissue growth factor (CTGF) have been described to induce the production of extracellular matrix (ECM) proteins and have been reported to be increased in different fibrotic disorders. Skeletal muscle fibrosis is a common feature of Duchenne muscular dystrophy (DMD). The mdx mouse diaphragm is a good model for DMD since it reproduces the muscle degenerative and fibrotic changes. Fibronectin (FN) and proteoglycans (PG) are some of the ECM proteins upregulated in dystrophic conditions. In view of understanding the fibrotic process involved in DMD we have isolated fibroblasts from dystrophic mdx diaphragms. Here we report that regardless of the absence of degenerative myofibers, adult mdx diaphragm fibroblasts show increased levels of FN and condroitin/dermatan sulfate PGs synthesis. Fibroblasts isolated from non fibrotic tissue, such as 1 week old mice diaphragms or skin, do not present elevated FN levels. Furthermore, mdx fibroblast conditioned media is able to stimulate FN synthesis in control fibroblasts. Autocrine TGF-β signaling was unaltered in mdx cells. When control fibroblasts are exposed to TGF-β and CTGF, FN increases as expected. Paradoxically, in mdx cells it decreases in a concentration dependent manner and this decrease is not due to a downregulation of FN synthesis. According to this data we hypothesize that a pathological environment is able to reprogram fibroblasts into an activated phenotype which can be maintained through generations.
PMCID: PMC2443238  PMID: 18600480
Muscular dystrophy; Fibronectin; Proteoglycans; Fibrosis; TGF-β; CTGF; Fibroblasts
6.  Molecular mechanism of sphingosine-1-phosphate action in Duchenne muscular dystrophy 
Disease Models & Mechanisms  2013;7(1):41-54.
Duchenne muscular dystrophy (DMD) is a lethal muscle-wasting disease. Studies in Drosophila showed that genetic increase of the levels of the bioactive sphingolipid sphingosine-1-phosphate (S1P) or delivery of 2-acetyl-5-tetrahydroxybutyl imidazole (THI), an S1P lyase inhibitor, suppresses dystrophic muscle degeneration. In the dystrophic mouse (mdx), upregulation of S1P by THI increases regeneration and muscle force. S1P can act as a ligand for S1P receptors and as a histone deacetylase (HDAC) inhibitor. Because Drosophila has no identified S1P receptors and DMD correlates with increased HDAC2 levels, we tested whether S1P action in muscle involves HDAC inhibition. Here we show that beneficial effects of THI treatment in mdx mice correlate with significantly increased nuclear S1P, decreased HDAC activity and increased acetylation of specific histone residues. Importantly, the HDAC2 target microRNA genes miR-29 and miR-1 are significantly upregulated, correlating with the downregulation of the miR-29 target Col1a1 in the diaphragm of THI-treated mdx mice. Further gene expression analysis revealed a significant THI-dependent decrease in inflammatory genes and increase in metabolic genes. Accordingly, S1P levels and functional mitochondrial activity are increased after THI treatment of differentiating C2C12 cells. S1P increases the capacity of the muscle cell to use fatty acids as an energy source, suggesting that THI treatment could be beneficial for the maintenance of energy metabolism in mdx muscles.
PMCID: PMC3882047  PMID: 24077965
HDAC; S1P; THI; dys; Dystrophin; mdx
7.  Effect of Nuclear Factor κB Inhibition on Serotype 9 Adeno-Associated Viral (AAV9) Minidystrophin Gene Transfer to the mdx Mouse 
Molecular Medicine  2012;18(1):466-476.
Gene therapy studies for Duchenne muscular dystrophy (DMD) have focused on viral vector-mediated gene transfer to provide therapeutic protein expression or treatment with drugs to limit dystrophic changes in muscle. The pathological activation of the nuclear factor (NF)-κB signaling pathway has emerged as an important cause of dystrophic muscle changes in muscular dystrophy. Furthermore, activation of NF-κB may inhibit gene transfer by promoting inflammation in response to the transgene or vector. Therefore, we hypothesized that inhibition of pathological NF-κB activation in muscle would complement the therapeutic benefits of dystrophin gene transfer in the mdx mouse model of DMD. Systemic gene transfer using serotype 9 adeno-associated viral (AAV9) vectors is promising for treatment of preclinical models of DMD because of vector tropism to cardiac and skeletal muscle. In quadriceps of C57BL/10ScSn-Dmdmdx/J (mdx) mice, the addition of octalysine (8K)–NF-κB essential modulator (NEMO)-binding domain (8K-NBD) peptide treatment to AAV9 minidystrophin gene delivery resulted in increased levels of recombinant dystrophin expression suggesting that 8K-NBD treatment promoted an environment in muscle tissue conducive to higher levels of expression. Indices of necrosis and regeneration were diminished with AAV9 gene delivery alone and to a greater degree with the addition of 8K-NBD treatment. In diaphragm muscle, high-level transgene expression was achieved with AAV9 minidystoophin gene delivery alone; therefore, improvements in histological and physiological indices were comparable in the two treatment groups. The data support benefit from 8K-NBD treatment to complement gene transfer therapy for DMD in muscle tissue that receives incomplete levels of transduction by gene transfer, which may be highly significant for clinical applications of muscle gene delivery.
PMCID: PMC3356420  PMID: 22231732
8.  Motor Physical Therapy Affects Muscle Collagen Type I and Decreases Gait Speed in Dystrophin-Deficient Dogs 
PLoS ONE  2014;9(4):e93500.
Golden Retriever Muscular Dystrophy (GRMD) is a dystrophin-deficient canine model genetically homologous to Duchenne Muscular Dystrophy (DMD) in humans. Muscular fibrosis secondary to cycles of degeneration/regeneration of dystrophic muscle tissue and muscular weakness leads to biomechanical adaptation that impairs the quality of gait. Physical therapy (PT) is one of the supportive therapies available for DMD, however, motor PT approaches have controversial recommendations and there is no consensus regarding the type and intensity of physical therapy. In this study we investigated the effect of physical therapy on gait biomechanics and muscular collagen deposition types I and III in dystrophin-deficient dogs. Two dystrophic dogs (treated dogs-TD) underwent a PT protocol of active walking exercise, 3×/week, 40 minutes/day, 12 weeks. Two dystrophic control dogs (CD) maintained their routine of activities of daily living. At t0 (pre) and t1 (post-physical therapy), collagen type I and III were assessed by immunohistochemistry and gait biomechanics were analyzed. Angular displacement of shoulder, elbow, carpal, hip, stifle and tarsal joint and vertical (Fy), mediolateral (Fz) and craniocaudal (Fx) ground reaction forces (GRF) were assessed. Wilcoxon test was used to verify the difference of biomechanical variables between t0 and t1, considering p<.05. Type I collagen of endomysium suffered the influence of PT, as well as gait speed that had decreased from t0 to t1 (p<.000). The PT protocol employed accelerates morphological alterations on dystrophic muscle and promotes a slower velocity of gait. Control dogs which maintained their routine of activities of daily living seem to have found a better balance between movement and preservation of motor function.
PMCID: PMC3979701  PMID: 24713872
9.  Levels of α7 integrin and laminin-α2 are increased following prednisone treatment in the mdx mouse and GRMD dog models of Duchenne muscular dystrophy 
Disease Models & Mechanisms  2013;6(5):1175-1184.
Duchenne muscular dystrophy (DMD) is a fatal neuromuscular disease for which there is no cure and limited treatment options. Prednisone is currently the first line treatment option for DMD and studies have demonstrated that it improves muscle strength. Although prednisone has been used for the treatment of DMD for decades, the mechanism of action of this drug remains unclear. Recent studies have shown that the α7β1 integrin is a major modifier of disease progression in mouse models of DMD and is therefore a target for drug-based therapies. In this study we examined whether prednisone increased α7β1 integrin levels in mdx mouse and GRMD dog models and myogenic cells from humans with DMD. Our results show that prednisone promotes an increase in α7 integrin protein in cultured myogenic cells and in the muscle of mdx and GRMD animal models of DMD. The prednisone-mediated increase in α7 integrin was associated with increased laminin-α2 in prednisone-treated dystrophin-deficient muscle. Together, our results suggest that prednisone acts in part through increased merosin in the muscle basal lamina and through sarcolemmal stabilization of α7β1 integrin in dystrophin-deficient muscle. These results indicate that therapies that target an increase in muscle α7β1 integrin, its signaling pathways and/or laminin could be therapeutic in DMD.
PMCID: PMC3759337  PMID: 23846963
10.  Efficient Whole-body Transduction with Trans-splicing Adeno-associated Viral Vectors 
Limited packaging capacity has hampered adeno-associated virus (AAV)-mediated gene therapy for many common genetic diseases such as cystic fibrosis (CF) and Duchenne muscular dystrophy (DMD). Trans-splicing AAV (tsAAV) vectors double AAV packaging capacity but their transduction efficiency has been too low to be useful. We have recently overcome this hurdle by rational vector design. We have shown that a pair of optimized mini-dystrophin tsAAV vectors can reach the same transduction efficiency as that of a single AAV vector after local injection in dystrophic muscle. However, global gene transfer is required to treat diseases like DMD. To test whether systemic delivery can be achieved with tsAAV vectors, we generated a set of optimized alkaline phosphatase (AP) tsAAV vectors. We delivered AAV serotype 9 pseudotyped AP tsAAV intravenously to newborn mice. Six weeks later, we observed high-level transduction in all body skeletal muscle and the heart, the tissues that are affected in DMD. We also detected efficient transduction in the lung, the primary organ affected in CF. Our results provide the first evidence of whole-body transduction with tsAAV vectors and further raise the hope of tsAAV gene therapy for DMD and CF.
PMCID: PMC2581720  PMID: 17264855
11.  Isobaric Tagging-Based Quantification for Proteomic Analysis: A Comparative Study of Spared and Affected Muscles from mdx Mice at the Early Phase of Dystrophy 
PLoS ONE  2013;8(6):e65831.
Duchenne muscular dystrophy (DMD) is the most common childhood myopathy, characterized by muscle loss and cardiorespiratory failure. While the genetic basis of DMD is well established, secondary mechanisms associated with dystrophic pathophysiology are not fully clarified yet. In order to obtain new insights into the molecular mechanisms of muscle dystrophy during earlier stages of the disease, we performed a comparative proteomic profile of the spared extraocular muscles (EOM) vs. affected diaphragm from the mdx mice, using a label based shotgun proteomic approach. Out of the 857 identified proteins, 42 to 62 proteins had differential abundance of peptide ions. The calcium-handling proteins sarcalumenin and calsequestrin-1 were increased in control EOM compared with control DIA, reinforcing the view that constitutional properties of EOM are important for their protection against myonecrosis. The finding that galectin-1 (muscle regeneration), annexin A1 (anti-inflammatory) and HSP 47 (fibrosis) were increased in dystrophic diaphragm provides novel insights into the mechanisms through which mdx affected muscles are able to counteract dystrophy, during the early stage of the disease. Overall, the shotgun technique proved to be suitable to perform quantitative comparisons between distinct dystrophic muscles and allowed the suggestion of new potential biomarkers and drug targets for dystrophinopaties.
PMCID: PMC3688818  PMID: 23823696
12.  Menstrual Blood-derived Cells Confer Human Dystrophin Expression in the Murine Model of Duchenne Muscular Dystrophy via Cell Fusion and Myogenic Transdifferentiation 
Molecular Biology of the Cell  2007;18(5):1586-1594.
Duchenne muscular dystrophy (DMD), the most common lethal genetic disorder in children, is an X-linked recessive muscle disease characterized by the absence of dystrophin at the sarcolemma of muscle fibers. We examined a putative endometrial progenitor obtained from endometrial tissue samples to determine whether these cells repair muscular degeneration in a murine mdx model of DMD. Implanted cells conferred human dystrophin in degenerated muscle of immunodeficient mdx mice. We then examined menstrual blood–derived cells to determine whether primarily cultured nontransformed cells also repair dystrophied muscle. In vivo transfer of menstrual blood–derived cells into dystrophic muscles of immunodeficient mdx mice restored sarcolemmal expression of dystrophin. Labeling of implanted cells with enhanced green fluorescent protein and differential staining of human and murine nuclei suggest that human dystrophin expression is due to cell fusion between host myocytes and implanted cells. In vitro analysis revealed that endometrial progenitor cells and menstrual blood–derived cells can efficiently transdifferentiate into myoblasts/myocytes, fuse to C2C12 murine myoblasts by in vitro coculturing, and start to express dystrophin after fusion. These results demonstrate that the endometrial progenitor cells and menstrual blood–derived cells can transfer dystrophin into dystrophied myocytes through cell fusion and transdifferentiation in vitro and in vivo.
PMCID: PMC1855042  PMID: 17314403
13.  BMP antagonists enhance myogenic differentiation and ameliorate the dystrophic phenotype in a DMD mouse model 
Neurobiology of disease  2010;41(2):353-360.
Duchenne Muscular Dystrophy (DMD) is an X-linked lethal muscle wasting disease characterized by muscle fiber degeneration and necrosis. The progressive pathology of DMD can be explained by an insufficient regenerative response resulting in fibrosis and adipose tissue formation. BMPs are known to inhibit myogenic differentiation and in a previous study we found increased expression of the BMP family member BMP4 in DMD myoblasts. The aim of the current study was therefore to investigate whether inhibition of BMP signaling could be beneficial for myoblast differentiation and muscle regeneration processes in a DMD context. All tested BMP inhibitors, Noggin, dorsomorphin and LDN-193189, were able to accelerate and enhance myogenic differentiation. However, dorsomorphin repressed both BMP and TGFβ signaling and was found to be toxic to primary myoblast cell cultures. In contrast, Noggin was found to be a potent and selective BMP inhibitor and was therefore tested in vivo in a DMD mouse model. Local adenoviral-mediated overexpression of Noggin in muscle resulted in increased expression of the myogenic regulatory genes Myog and Myod1 and improved muscle histology. In conclusion, our results suggest that repression of BMP signaling may constitute an attractive adjunctive therapy for DMD patients.
PMCID: PMC3674857  PMID: 20940052
DMD; BMP signaling; myogenic differentiation; BMP antagonists
14.  Dysfunctional Muscle and Liver Glycogen Metabolism in mdx Dystrophic Mice 
PLoS ONE  2014;9(3):e91514.
Duchenne muscular dystrophy (DMD) is a severe, genetic muscle wasting disorder characterised by progressive muscle weakness. DMD is caused by mutations in the dystrophin (dmd) gene resulting in very low levels or a complete absence of the dystrophin protein, a key structural element of muscle fibres which is responsible for the proper transmission of force. In the absence of dystrophin, muscle fibres become damaged easily during contraction resulting in their degeneration. DMD patients and mdx mice (an animal model of DMD) exhibit altered metabolic disturbances that cannot be attributed to the loss of dystrophin directly. We tested the hypothesis that glycogen metabolism is defective in mdx dystrophic mice.
Dystrophic mdx mice had increased skeletal muscle glycogen (79%, (P<0.01)). Skeletal muscle glycogen synthesis is initiated by glycogenin, the expression of which was increased by 50% in mdx mice (P<0.0001). Glycogen synthase activity was 12% higher (P<0.05) but glycogen branching enzyme activity was 70% lower (P<0.01) in mdx compared with wild-type mice. The rate-limiting enzyme for glycogen breakdown, glycogen phosphorylase, had 62% lower activity (P<0.01) in mdx mice resulting from a 24% reduction in PKA activity (P<0.01). In mdx mice glycogen debranching enzyme expression was 50% higher (P<0.001) together with starch-binding domain protein 1 (219% higher; P<0.01). In addition, mdx mice were glucose intolerant (P<0.01) and had 30% less liver glycogen (P<0.05) compared with control mice. Subsequent analysis of the enzymes dysregulated in skeletal muscle glycogen metabolism in mdx mice identified reduced glycogenin protein expression (46% less; P<0.05) as a possible cause of this phenotype.
We identified that mdx mice were glucose intolerant, and had increased skeletal muscle glycogen but reduced amounts of liver glycogen.
PMCID: PMC3953428  PMID: 24626262
15.  Increased sphingosine-1-phosphate improves muscle regeneration in acutely injured mdx mice 
Skeletal Muscle  2013;3:20.
Presently, there is no effective treatment for the lethal muscle wasting disease Duchenne muscular dystrophy (DMD). Here we show that increased sphingosine-1-phoshate (S1P) through direct injection or via the administration of the small molecule 2-acetyl-4(5)-tetrahydroxybutyl imidazole (THI), an S1P lyase inhibitor, has beneficial effects in acutely injured dystrophic muscles of mdx mice.
We treated mdx mice with and without acute injury and characterized the histopathological and functional effects of increasing S1P levels. We also tested exogenous and direct administration of S1P on mdx muscles to examine the molecular pathways under which S1P promotes regeneration in dystrophic muscles.
Short-term treatment with THI significantly increased muscle fiber size and extensor digitorum longus (EDL) muscle specific force in acutely injured mdx limb muscles. In addition, the accumulation of fibrosis and fat deposition, hallmarks of DMD pathology and impaired muscle regeneration, were lower in the injured muscles of THI-treated mdx mice. Furthermore, increased muscle force was observed in uninjured EDL muscles with a longer-term treatment of THI. Such regenerative effects were linked to the response of myogenic cells, since intramuscular injection of S1P increased the number of Myf5nlacz/+ positive myogenic cells and newly regenerated myofibers in injured mdx muscles. Intramuscular injection of biotinylated-S1P localized to muscle fibers, including newly regenerated fibers, which also stained positive for S1P receptor 1 (S1PR1). Importantly, plasma membrane and perinuclear localization of phosphorylated S1PR1 was observed in regenerating muscle fibers of mdx muscles. Intramuscular increases of S1P levels, S1PR1 and phosphorylated ribosomal protein S6 (P-rpS6), and elevated EDL muscle specific force, suggest S1P promoted the upregulation of anabolic pathways that mediate skeletal muscle mass and function.
These data show that S1P is beneficial for muscle regeneration and functional gain in dystrophic mice, and that THI, or other pharmacological agents that raise S1P levels systemically, may be developed into an effective treatment for improving muscle function and reducing the pathology of DMD.
PMCID: PMC3750760  PMID: 23915702
16.  Early Treatment with Lisinopril and Spironolactone Preserves Cardiac and Skeletal Muscle in Duchenne Muscular Dystrophy Mice 
Circulation  2011;124(5):582-588.
Nearly-universal cardiomyopathy in Duchenne muscular dystrophy (DMD) contributes to heart failure and death. As DMD patients show myocardial fibrosis well before functional impairment, we postulated that earlier treatment using drugs with anti-fibrotic effect may be beneficial.
Methods and Results
Three groups of 10 utrn+/−;mdx or “het” mice with skeletal myopathy and cardiomyopathy that closely mimics clinical DMD were studied. One het group received spironolactone and lisinopril starting at 8 weeks-of-life (het-treated-8), a second received the same starting at 4 weeks-of-life (het-treated-4), and the third het group was untreated. At 20 weeks, all mice had normal EFs though circumferential strain rate was abnormal (−0.21±0.08) in untreated hets. This improved to −0.40±0.07 in het-treated-8 mice (p=0.003), and further improved to −0.56±0.10 in het-treated-4 mice (p=0.014 for het-treated-4 vs. het-treated-8). Treated mice showed less cardiomyocyte damage, with a 44% reduction in intra-cardiomyocyte serum IgG localization in het-treated-8 mice (p<0.0001), and further 53% reduction in het-treated-4 mice (p=0.0003 vs. het-treated-8); matrix metalloproteinases were similarly reduced. Cardiac, limb and diaphragm function by ex vivo muscle testing remained at 80% of normal with early treatment compared to a decline to 40% of normal skeletal muscle function without treatment.
These findings offer clinically-available medications with proven anti-fibrotic effect as a new therapeutic strategy in DMD. Early initiation greatly attenuated myocardial disease and, for the first time with these drugs, improved skeletal myopathy. Thus, early initiation of such agents warrants further clinical evaluation to maintain ambulatory, respiratory and cardiac function for DMD and related myopathies.
PMCID: PMC3150227  PMID: 21768542
cardiomyopathy; muscles; aldosterone antagonist
17.  Inefficient dystrophin expression after cord blood transplantation in DMD 
Muscle & nerve  2010;41(6):746-750.
We report a boy who received two allogeneic stem cell transplantations from umbilical cord donors to treat chronic granulomatous disease (CGD). The CGD was cured after the second transplantation, but 2½ years later, he was diagnosed with Duchenne muscular dystrophy (DMD).
Examinations of his DNA, muscle tissue, and myoblast cultures derived from muscle tissue were performed to determine whether any donor dystrophin was being expressed.
The boy was found to have a large-scale deletion on the X chromosome that spanned the loci for CYBB and DMD. The absence of dystrophin led to muscle histology characteristic of DMD. Analysis of myofibers demonstrated no definite donor cell engraftment.
This case suggests that umbilical cord-derived hematopoietic stem cell transplantation will not be efficacious in the therapy of DMD without additional interventions that induce engraftment of donor cells in skeletal muscle.
PMCID: PMC2965738  PMID: 20513101
Duchenne muscular dystrophy; chronic granulomatous disease; umbilical cord blood transplantation; stem cell therapy
18.  Chronic Administration of a Leupeptin-Derived Calpain Inhibitor Fails to Ameliorate Severe Muscle Pathology in a Canine Model of Duchenne Muscular Dystrophy 
Calpains likely play a role in the pathogenesis of Duchenne muscular dystrophy (DMD). Accordingly, calpain inhibition may provide therapeutic benefit to DMD patients. In the present study, we sought to measure benefit from administration of a novel calpain inhibitor, C101, in a canine muscular dystrophy model. Specifically, we tested the hypothesis that treatment with C101 mitigates progressive weakness and severe muscle pathology observed in young dogs with golden retriever muscular dystrophy (GRMD). Young (6-week-old) GRMD dogs were treated daily with either C101 (17 mg/kg twice daily oral dose, n = 9) or placebo (vehicle only, n = 7) for 8 weeks. A battery of functional tests, including tibiotarsal joint angle, muscle/fat composition, and pelvic limb muscle strength were performed at baseline and every 2 weeks during the 8-week study. Results indicate that C101-treated GRMD dogs maintained strength in their cranial pelvic limb muscles (tibiotarsal flexors) while placebo-treated dogs progressively lost strength. However, concomitant improvement was not observed in posterior pelvic limb muscles (tibiotarsal extensors). C101 treatment did not mitigate force drop following repeated eccentric contractions and no improvement was seen in the development of joint contractures, lean muscle mass, or muscle histopathology. Taken together, these data do not support the hypothesis that treatment with C101 mitigates progressive weakness or ameliorates severe muscle pathology observed in young dogs with GRMD.
PMCID: PMC3253583  PMID: 22291646
muscular dystrophy; animal; calpain; skeletal muscle; protease; canine
19.  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.
PMCID: PMC2701330  PMID: 19401296
20.  Enalapril treatment discloses an early role of angiotensin II in inflammation- and oxidative stress-related muscle damage in dystrophic mdx mice☆ 
Pharmacological Research  2011;64(5):482-492.
Graphical abstract
► An early treatment with enalapril was performed in exercised mdx mice. ► In vivo, enalapril increased mouse fore limb strength dose-dependently. ► Ex vivo, enalapril reduced muscular markers of oxidative stress and inflammation. ► Results corroborate an early role of angiotensin II in muscular dystrophy. ► Pre-clinical evidences of therapeutic interest of ACE inhibitors for therapy of DMD.
Inhibitors of angiotensin converting enzymes (ACE) are clinically used to control cardiomyopathy in patients of Duchenne muscular dystrophy. Various evidences suggest potential usefulness of long-term treatment with ACE inhibitors to reduce advanced fibrosis of dystrophic muscle in the mdx mouse model. However, angiotensin II is known to exert pro-inflammatory and pro-oxidative actions that might contribute to early events of dystrophic muscle degeneration. The present study has been aimed at evaluating the effects of an early treatment with enalapril on the pathology signs of exercised mdx mouse model. The effects of 1 and 5 mg/kg enalapril i.p. for 4–8 weeks have been compared with those of 1 mg/kg α-methyl-prednisolone (PDN), as positive control. Enalapril caused a dose-dependent increase in fore limb strength, the highest dose leading to a recovery score similar to that observed with PDN. A dose-dependent reduction of superoxide anion production was observed by dihydroethidium staining in tibialis anterior muscle of enalapril-treated mice, approaching the effect observed with PND. In parallel, a significant reduction of the activated form of the pro-inflammatory Nuclear Factor-kB has been observed in gastrocnemious muscle. Histologically, 5 mg/kg enalapril reduced the area of muscle necrosis in both gastrocnemious muscle and diaphragm, without significant effect on non-muscle area. In parallel no significant changes have been observed in both muscle TGF-β1 and myonuclei positive to phosphorylated Smad2/3. Myofiber functional indices were also monitored by microelectrodes recordings. A dose-dependent recovery of macroscopic chloride conductance has been observed upon enalapril treatment in EDL muscle, with minor effects being exerted in diaphragm. However a modest effect, if any, was found on mechanical threshold, a functional index of calcium homeostasis. No recovery was observed in creatine kinase and lactate dehydrogenase. Finally the results suggest the ability of enalapril to blunt angiotensin-II dependent activation of pro-inflammatory and pro-oxidant pathways which may be earlier events with respect to the pro-fibrotic ones, and may in part account for both functional impairment and muscle necrosis. The PDN-like profile may corroborate the combined use of the two classes of drugs in DMD patients so to potentiate the beneficial effects at skeletal muscle level, while reducing both spontaneous and PDN-aggravated cardiomyopathy.
PMCID: PMC3184479  PMID: 21689754
DMD, Duchenne muscular dystrophy; ACE, angiotensin converting enzyme; RAS, renin-angiotensin system; Ang II, angiotensin II; PDN, α-methylprednisolone; NF-kB, nuclear factor-kB; TGF-β1, trasforming growth factor β1; EDL, extensor digitorum longus; CK, creatine kinase; LDH, lactate dehydrogenase; gCl, sarcolemmal chloride conductance; gK, sarcolemmal potassium conductance; MT, mechanical threshold; Muscular dystrophy; Pre-clinical pharmacological tests; Angiotensin-II; Inflammation; Oxidative stress
21.  Amelioration of Muscular Dystrophy by Transgenic Expression of Niemann-Pick C1 
Molecular Biology of the Cell  2009;20(1):146-152.
Duchenne muscular dystrophy (DMD) and other types of muscular dystrophies are caused by the loss or alteration of different members of the dystrophin protein complex. Understanding the molecular mechanisms by which dystrophin-associated protein abnormalities contribute to the onset of muscular dystrophy may identify new therapeutic approaches to these human disorders. By examining gene expression alterations in mouse skeletal muscle lacking α-dystrobrevin (Dtna−/−), we identified a highly significant reduction of the cholesterol trafficking protein, Niemann-Pick C1 (NPC1). Mutations in NPC1 cause a progressive neurodegenerative, lysosomal storage disorder. Transgenic expression of NPC1 in skeletal muscle ameliorates muscular dystrophy in the Dtna−/− mouse (which has a relatively mild dystrophic phenotype) and in the mdx mouse, a model for DMD. These results identify a new compensatory gene for muscular dystrophy and reveal a potential new therapeutic target for DMD.
PMCID: PMC2613093  PMID: 18946078
22.  Increased muscle expression of interleukin-17 in Duchenne muscular dystrophy 
Neurology  2012;78(17):1309-1314.
Duchenne muscular dystrophy (DMD) is a degenerative muscle wasting disease caused by mutations in the dystrophin gene. Dystrophic muscle is characterized by chronic inflammation, and inflammatory mediators could be promising targets for innovative therapeutic interventions. We analyzed muscle biopsy samples of DMD-affected children to characterize interleukin (IL)-17 and Forkhead box P3 (Foxp3) expression levels and to identify possible correlations with clinical status.
Expression levels of IL-17, Foxp3, tumor necrosis factor-α (TNF-α), monocyte chemoattractant protein-1 (MCP-1), IL-6, and transforming growth factor-β (TGF-β) were analyzed by real-time PCR in muscle biopsy samples from patients with DMD (n = 27) and juvenile dermatomyositis (JDM) (n = 8). Motor outcome of patients with DMD was evaluated by North Star Ambulatory Assessment score.
In DMD, we found higher levels of IL-17 and lower levels of Foxp3 mRNA compared with those for a typical inflammatory myopathy, JDM. Moreover, the IL-17/Foxp3 ratio was higher in DMD than in JDM biopsy samples. IL-17 mRNA levels appeared to be related to the expression levels of other proinflammatory cytokines (TNF-α and MCP-1) and significantly associated with clinical outcome of patients.
The association of IL-17 expression with levels of other inflammatory cytokines and with the clinical course of DMD suggests a possible pathogenic role of IL-17.
PMCID: PMC3335450  PMID: 22496194
23.  The Proton Pump Inhibitor Lansoprazole Improves the Skeletal Phenotype in Dystrophin Deficient mdx Mice 
PLoS ONE  2013;8(7):e66617.
In Duchenne muscular dystrophy (DMD), loss of the membrane stabilizing protein dystrophin results in myofiber damage. Microinjury to dystrophic myofibers also causes secondary imbalances in sarcolemmic ion permeability and resting membrane potential, which modifies excitation-contraction coupling and increases proinflammatory/apoptotic signaling cascades. Although glucocorticoids remain the standard of care for the treatment of DMD, there is a need to investigate the efficacy of other pharmacological agents targeting the involvement of imbalances in ion flux on dystrophic pathology.
Methodology/Principal Findings
We designed a preclinical trial to investigate the effects of lansoprazole (LANZO) administration, a proton pump inhibitor, on the dystrophic muscle phenotype in dystrophin deficient (mdx) mice. Eight to ten week-old female mice were assigned to one of four treatment groups (n = 12 per group): (1) vehicle control; (2) 5 mg/kg/day LANZO; (3) 5 mg/kg/day prednisolone; and (4) combined treatment of 5 mg/kg/day prednisolone (PRED) and 5 mg/kg/day LANZO. Treatment was administered orally 5 d/wk for 3 months. At the end of the study, behavioral (Digiscan) and functional outcomes (grip strength and Rotarod) were assessed prior to sacrifice. After sacrifice, body, tissue and organ masses, muscle histology, in vitro muscle force, and creatine kinase levels were measured. Mice in the combined treatment groups displayed significant reductions in the number of degenerating muscle fibers and number of inflammatory foci per muscle field relative to vehicle control. Additionally, mice in the combined treatment group displayed less of a decline in normalized forelimb and hindlimb grip strength and declines in in vitro EDL force after repeated eccentric contractions.
Together our findings suggest that combined treatment of LANZO and prednisolone attenuates some components of dystrophic pathology in mdx mice. Our findings warrant future investigation of the clinical efficacy of LANZO and prednisolone combined treatment regimens in dystrophic pathology.
PMCID: PMC3699610  PMID: 23843959
24.  Long-term persistence of donor nuclei in a Duchenne muscular dystrophy patient receiving bone marrow transplantation 
Duchenne muscular dystrophy (DMD) is a severe progressive muscle-wasting disorder caused by mutations in the dystrophin gene. Studies have shown that bone marrow cells transplanted into lethally irradiated mdx mice, the mouse model of DMD, can become part of skeletal muscle myofibers. Whether human marrow cells also have this ability is unknown. Here we report the analysis of muscle biopsies from a DMD patient (DMD-BMT1) who received bone marrow transplantation at age 1 year for X-linked severe combined immune deficiency and who was diagnosed with DMD at age 12 years. Analysis of muscle biopsies from DMD-BMT1 revealed the presence of donor nuclei within a small number of muscle myofibers (0.5-0.9%). The majority of the myofibers produce a truncated, in-frame isoform of dystrophin lacking exons 44 and 45 (not wild-type). The presence of bone marrow-derived donor nuclei in the muscle of this patient documents the ability of exogenous human bone marrow cells to fuse into skeletal muscle and persist up to 13 years after transplantation.
PMCID: PMC151133  PMID: 12235112
25.  Extensive and Prolonged Restoration of Dystrophin Expression with Vivo-Morpholino-Mediated Multiple Exon Skipping in Dystrophic Dogs 
Nucleic Acid Therapeutics  2012;22(5):306-315.
Duchenne muscular dystrophy (DMD) is a severe and the most prevalent form of muscular dystrophy, characterized by rapid progression of muscle degeneration. Antisense-mediated exon skipping is currently one of the most promising therapeutic options for DMD. However, unmodified antisense oligos such as morpholinos require frequent (weekly or bi-weekly) injections. Recently, new generation morpholinos such as vivo-morpholinos are reported to lead to extensive and prolonged dystrophin expression in the dystrophic mdx mouse, an animal model of DMD. The vivo-morpholino contains a cell-penetrating moiety, octa-guanidine dendrimer. Here, we sought to test the efficacy of multiple exon skipping of exons 6–8 with vivo-morpholinos in the canine X-linked muscular dystrophy, which harbors a splice site mutation at the boundary of intron 6 and exon 7. We designed and optimized novel antisense cocktail sequences and combinations for exon 8 skipping and demonstrated effective exon skipping in dystrophic dogs in vivo. Intramuscular injections with newly designed cocktail oligos led to high levels of dystrophin expression, with some samples similar to wild-type levels. This is the first report of successful rescue of dystrophin expression with morpholino conjugates in dystrophic dogs. Our results show the potential of phosphorodiamidate morpholino oligomer conjugates as therapeutic agents for DMD.
PMCID: PMC3464409  PMID: 22888777

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