Up-regulation of mitogen activated protein kinases in mdx skeletal muscle following chronic treadmill exercise

Dystrophin, a product of the Duchenne muscular dystrophy gene, is a cytoskeletal protein of skeletal and cardiac muscle fibers. Dystrophin-deficient muscle fibers are abnormally vulnerable to mechanical stress including physical exercise, which is a powerful stimulator of mitogen-activated protein kinases (MAPKs). To examine how treadmill exercise affects MAPK family members in dystrophin-deficient skeletal muscle, we subjected both mdx mice, an animal model for Duchenne muscular dystrophy, and C57BL/10 mice to treadmill exercise and examined the phosphorylated protein levels of extracellular-signal regulated kinase (ERK1/2), p38 MAPK and c-Jun N terminal kinase 1 and 2 (JNK1 and JNK2) in the gastrocnemius muscle. Phosphorylation of ERK1/2, p38 MAPK and JNK2, but not JNK1, increased more in the muscles of exercise trained mdx mice than in muscles of trained C57BL/10 or untrained mdx mice. These results show that physical exercise aberrantly up-regulates the phosphorylated form of ERK1/2, p38 MAPK and JNK2 in dystrophin-deficient skeletal muscle and that their up-regulation might play a role in the degeneration and regeneration process of dystrophic features.     

Activation of AKT signaling promotes cell growth and survival in α7β1 integrin-mediated alleviation of muscular dystrophy

Transgenic expression of the α7 integrin can ameliorate muscle pathology in a mouse model of Duchenne muscular dystrophy (mdx/utr^−/−) and thus can compensate for the loss of dystrophin in diseased mice. In spite of the beneficial effects of the α7 integrin in protecting mice from dystrophy, identification of molecular signaling events responsible for these changes remains to be established. The purpose of this study was to determine a role for signaling in the amelioration of muscular dystrophy by α7 integrin. Activation of PI3K, ILK, AKT, mTOR, p70S6K, BAD, ERK, and p38 was measured in the muscle from wild type (WT), mdx/utr^−/− and α7BX2-mdx/utr^−/− mice using in vitro activity assays or phosphospecific antibodies and western blotting. Significant increases in PI3K activity (47%), ILK activity (2.0-fold), mTOR (Ser2448) (57%), p70S6K (Thr389) (11.7-fold), and ERK (Thr202/Tyr204) (66%) were demonstrated in dystrophic mdx/utr^−/− muscle compared to WT. A significant decrease in p38 phosphorylation (2.9-fold) was also observed. Although most of these signaling events were similar in dystrophic mdx/utr^−/− mice overexpressing the α7 integrin, the AKT (Ser473):AKT ratio (2-fold vs. WT) and p70S6K phosphorylation (18-fold vs. WT) were higher in α7BX2-mdx/utr^−/− compared to mdx/utr^−/− mice. In addition, increased phosphorylation of BAD Serine 112 may contribute to the significant reduction in TUNEL⁺ cells observed in α7BX2-mdx/utr^−/− mice. We conclude that the α7β1 integrin confers a protective effect in dystrophic muscle through the activation of the ILK, AKT, p70S6K and BAD signaling to promote muscle cell survival.     

Chronic AMPK stimulation attenuates adaptive signaling in dystrophic skeletal muscle

In the present study, we evaluated how a pharmacologically induced phenotype shift in dystrophic skeletal muscle would affect subsequent intracellular signaling in response to a complementary, adaptive physiological stimulus. mdx mice were treated with the AMP-activated protein kinase (AMPK) activator 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR; 500 mg·kg(-1)·day(-1)) for 30 days, and then one-half of the animals were subjected to a bout of treadmill running to induce acute AMPK and p38 MAPK signaling. The mRNA levels of phenotypic modifiers, including peroxisome proliferator-activated receptor-δ (PPARδ), PPARγ coactivator-1α (PGC-1α), receptor interacting protein 140 (RIP 140), and silent information regulator two ortholog 1 (SIRT1) were assessed in skeletal muscle, as well as the expression of the protein arginine methyltransferase genes PRMT1 and CARM1. We found unique AMPK and p38 phosphorylation and expression signatures between dystrophic and healthy muscle. In dystrophic skeletal muscle, treadmill running induced PPARδ, PGC-1α, and SIRT1 mRNAs, three molecules that promote the slow, oxidative myogenic program. In the mdx animals that received the chronic AICAR treatment, running-elicited AMPK and p38 phosphorylation was attenuated compared with vehicle-treated mice. Similarly, acute stress-evoked expression of PPARδ, PGC-1α, and SIRT1 was also blunted by chronic pharmacological AMPK stimulation. Skeletal muscle PRMT1 and CARM1 protein contents were higher in mdx mice compared with wild-type littermates. The acute running-evoked induction of PRMT1 and CARM1 mRNAs was also attenuated by the AICAR treatment. Our data demonstrate that prior pharmacological conditioning is a salient determinant in how dystrophic muscle adapts to subsequent complementary, acute physiological stress stimuli. These results provide insight into possible therapeutic applications of synthetic agonists in neuromuscular diseases, such as during chronic administration to Duchenne muscular dystrophy patients.     

Inspiratory loading does not accelerate dystrophy in mdx mouse diaphragm: implications for regenerative therapy.

Since the finding that the mdx mouse diaphragm, in contrast to limb muscles, undergoes progressive degeneration analogous to that seen in Duchenne muscular dystrophy, the relationship between the workload on a muscle and the pathogenesis of dystrophy has remained controversial. We increased the work performed by the mdx mouse diaphragm in vivo by tracheal banding and evaluated the progression of dystrophic changes in that muscle. Despite the establishment of dramatically increased respiratory workload and accelerated myofiber damage documented by Evans blue dye, no change in the pace of progression of dystrophy was seen in banded animals vs. unbanded, sham-operated controls. At the completion of the study, more centrally nucleated fibers were evident in the diaphragms of banded mdx mice than in sham-operated mdx controls, indicating that myofiber regeneration increases to meet the demands of the work-induced damage. These data suggest that there is untapped regenerative capacity in dystrophin-deficient muscle and validates experimental efforts aimed at augmenting regeneration within skeletal muscle as a therapeutic strategy in the treatment of dystrophinopathies.     

Stretch-activated calcium channel protein TRPC1 is correlated with the different degrees of the dystrophic phenotype in mdx mice

In Duchenne muscular dystrophy (DMD) and in the mdx mouse model of DMD, the lack of dystrophin is related to enhanced calcium influx and muscle degeneration. Stretch-activated channels (SACs) might be directly involved in the pathology of DMD, and transient receptor potential cation channels have been proposed as likely candidates of SACs. We investigated the levels of transient receptor potential canonical channel 1 (TRPC1) and the effects of streptomycin, a SAC blocker, in muscles showing different degrees of the dystrophic phenotype. Mdx mice (18 days old, n = 16) received daily intraperitoneal injections of streptomycin (182 mg/kg body wt) for 18 days, followed by removal of the diaphragm, sternomastoid (STN), biceps brachii, and tibialis anterior muscles. Control mdx mice (n = 37) were injected with saline. Western blot analysis showed higher levels of TRPC1 in diaphragm muscle compared with STN and limb muscles. Streptomycin reduced creatine kinase and prevented exercise-induced increases of total calcium and Evans blue dye uptake in diaphragm and in STN muscles. It is suggested that different levels of the stretch-activated calcium channel protein TRPC1 may contribute to the different degrees of the dystrophic phenotype seen in mdx mice. Early treatment designed to regulate the activity of these channels may ameliorate the progression of dystrophy in the most affected muscle, the diaphragm.     

P2X7 purinoceptor alterations in dystrophic mdx mouse muscles: relationship to pathology and potential target for treatment

Duchenne muscular dystrophy (DMD) is a lethal inherited muscle disorder. Pathological characteristics of DMD skeletal muscles include, among others, abnormal Ca(2+) homeostasis and cell signalling. Here, in the mdx mouse model of DMD, we demonstrate significant P2X7 receptor abnormalities in isolated primary muscle cells and cell lines and in dystrophic muscles in vivo. P2X7 mRNA expression in dystrophic muscles was significantly up-regulated but without alterations of specific splice variant patterns. P2X7 protein was also up-regulated and this was associated with altered function of P2X7 receptors producing increased responsiveness of cytoplasmic Ca(2+) and extracellular signal-regulated kinase (ERK) phosphorylation to purinergic stimulation and altered sensitivity to NAD. Ca(2+) influx and ERK signalling were stimulated by ATP and BzATP, inhibited by specific P2X7 antagonists and insensitive to ivermectin, confirming P2X7 receptor involvement. Despite the presence of pannexin-1, prolonged P2X7 activation did not trigger cell permeabilization to propidium iodide or Lucifer yellow. In dystrophic mice, in vivo treatment with the P2X7 antagonist Coomassie Brilliant Blue reduced the number of degeneration-regeneration cycles in mdx skeletal muscles. Altered P2X7 expression and function is thus an important feature in dystrophic mdx muscle and treatments aiming to inhibit P2X7 receptor might slow the progression of this disease.     

Reduced expression of regucalcin in young and aged mdx diaphragm indicates abnormal cytosolic calcium handling in dystrophin-deficient muscle

The cytosolic Ca2+ -binding protein regucalcin is involved in intracellular signaling and present in high abundance in the liver. Here, we could show by comparative mass spectrometry-based proteomics screening of normal versus dystrophic fibres that regucalcin of 33.9 kDa and pI5.2 also exists in diaphragm muscle. Since the expression of sarcolemmal Ca2+ -leak channels and luminal Ca2+ -binding elements is altered in dystrophin-deficient muscle, we initiated this study in order to determine whether additional soluble muscle proteins involved in Ca2+ -handling are affected in muscular dystrophy. Following separation by two-dimensional gel electrophoresis, the spot pattern of the normal versus the mdx diaphragm muscle proteome was evaluated by densitometry. The expression levels of 20 major protein spots were shown to change and their identity determined by mass spectrometry. A 2-fold reduction of regucalcin in mdx diaphragm, as well as in dystrophic limb muscle and heart, was confirmed by immunoblotting in both young and aged mdx mice. The results from our proteomics analysis of dystrophic diaphragm support the concept that abnormal Ca2+ -handling is involved in x-linked muscular dystrophy. The reduction in key Ca2+ -handling proteins may result in an insufficient maintenance of Ca2+ -homeostasis and an abnormal regulation of Ca2+ -dependent enzymes resulting in disturbed intracellular signaling mechanisms in dystrophinopathies.     

Uncoupling protein-2 polymorphisms in type 2 diabetes,obesity, and insulin secretion

The aim of the study was to investigate the effect of resistance exercise alone or in combination with oral intake of branched-chain amino acids (BCAA) on phosphorylation of the 70-kDa S6 protein kinase (p70(S6k)) and mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase (ERK1/2), and p38 MAPK in skeletal muscle. Seven male subjects performed one session of quadriceps muscle resistance training (4 x 10 repetitions at 80% of one repetition maximum) on two occasions. In a randomized order, double-blind, crossover test, subjects ingested a solution of BCAA or placebo during and after exercise. Ingestion of BCAA increased plasma concentrations of isoleucine, leucine, and valine during exercise and throughout recovery after exercise (2 h postexercise), whereas no change was noted after the placebo trial. Resistance exercise led to a robust increase in p70(S6k) phosphorylation at Ser(424) and/or Thr(421), which persisted 1 and 2 h after exercise. BCAA ingestion further enhanced p70(S6k) phosphorylation 3.5-fold during recovery. p70(S6k) phosphorylation at Thr(389) was unaltered directly after resistance exercise. However, during recovery, Thr(389) phosphorylation was profoundly increased, but only during the BCAA trial. Furthermore, phosphorylation of the ribosomal protein S6 was also increased in the recovery period only during the BCAA trial. Exercise led to a marked increase in ERK1/2 and p38 MAPK phosphorylation, which was completely suppressed upon recovery and unaltered by BCAA. In conclusion, BCAA, ingested during and after resistance exercise, mediate signal transduction through p70(S6k) in skeletal muscle.     

Amplification of proinflammatory phenotype, damage, and weakness by oxidative stress in the diaphragm muscle of mdx mice

Duchenne muscular dystrophy (DMD) is a common and devastating type of childhood-onset muscular dystrophy, attributed to an X-linked defect in the gene that encodes dystrophin. Myopathy with DMD is most pronounced in the diaphragm muscle and fast-twitch limb muscles and is dependent upon susceptibility to damage, inflammatory cell infiltration, and proinflammatory signaling (nuclear factor-κB; NF-κB). Although recent papers have reawakened the notion that oxidative stress links inflammatory signaling with pathology in DMD in limb muscle, the importance of redox mechanisms had been clouded by inconsistent results from indirect scavenger approaches, including in the diaphragm muscle. Therefore, we used a novel catalytic mimetic of superoxide dismutase and catalase (EUK-134) as a direct scavenger of oxidative stress in myopathy in the diaphragm of the mdx mouse model. EUK-134 reduced 4-hydroxynonenal and total hydroperoxides, markers of oxidative stress in the mdx diaphragm. EUK-134 also attenuated positive staining of macrophages and T-cells as well as activation of NF-κB and p65 protein abundance. Moreover, EUK-134 ameliorated markers of muscle damage including internalized nuclei, variability of cross-sectional area, and type IIc fibers. Finally, impairment of contractile force was partially rescued by EUK-134 in the diaphragm of mdx mice. We conclude that oxidative stress amplifies DMD pathology in the diaphragm muscle.     

PKC theta ablation improves healing in a mouse model of muscular dystrophy

Inflammation is a key pathological characteristic of dystrophic muscle lesion formation, limiting muscle regeneration and resulting in fibrotic and fatty tissue replacement of muscle, which exacerbates the wasting process in dystrophic muscles. Limiting immune response is thus one of the therapeutic options to improve healing, as well as to improve the efficacy of gene- or cell-mediated strategies to restore dystrophin expression. Protein kinase C θ (PKCθ) is a member of the PKCs family highly expressed in both immune cells and skeletal muscle; given its crucial role in adaptive, but also innate, immunity, it is being proposed as a valuable pharmacological target for immune disorders. In our study we asked whether targeting PKCθ could represent a valuable approach to efficiently prevent inflammatory response and disease progression in a mouse model of muscular dystrophy. We generated the bi-genetic mouse model mdx/θ(-/-), where PKCθ expression is lacking in mdx mice, the mouse model of Duchenne muscular dystrophy. We found that muscle wasting in mdx/θ(-/-) mice was greatly prevented, while muscle regeneration, maintenance and performance was significantly improved, as compared to mdx mice. This phenotype was associated to reduction in inflammatory infiltrate, pro-inflammatory gene expression and pro-fibrotic markers activity, as compared to mdx mice. Moreover, BM transplantation experiments demonstrated that the phenotype observed was primarily dependent on lack of PKCθ expression in hematopoietic cells.These results demonstrate a hitherto unrecognized role of immune-cell intrinsic PKCθ activity in the development of DMD. Although the immune cell population(s) involved remain unidentified, our findings reveal that PKCθ can be proposed as a new pharmacological target to counteract the disease, as well as to improve the efficacy of gene- or cell- therapy approaches.     

Large-scale analysis of differential gene expression in the hindlimb muscles and diaphragm of mdx mouse

The mdx mouse is an animal model for Duchenne muscular dystrophy (DMD), which is caused by the absence of dystrophin. Mdx limb muscles substantially compensate for the lack of dystrophin while the diaphragm is affected like DMD skeletal muscles. To understand better the complex cascade of molecular events leading to muscle degeneration and compensatory processes in mdx muscles, we analyzed alterations of gene expression in mdx hindlimb and diaphragm muscles as compared to their normal counterparts. The strategy was based on suppression subtractive hybridization followed by reverse Northern quantitative hybridization. Four subtracted/normalized libraries, containing cDNA clones up- or downregulated in mdx hindlimb muscles or diaphragm, were constructed and a total of 1536 cDNA clones were analyzed. Ninety-three cDNAs were found to be differentially expressed in mdx hindlimb muscles and/or diaphragm. They corresponded to 54 known genes and 39 novel cDNAs. The potential role of the known genes is discussed in the context of the mdx phenotype.     

Circadian rhythm of intracellular protein synthesis signaling in rat cardiac and skeletal muscles

Intracellular signaling exhibits circadian variation in the suprachiasmatic nucleus and liver. However, it is unclear whether circadian regulation also extends to intracellular signaling pathways in the cardiac and skeletal muscles. Here, we examined circadian variation in the intracellular mammalian target of rapamycin (mTOR)/70 kDa ribosomal protein S6 kinase 1 (p70S6K) and extracellular signal-regulated kinase (ERK) pathways, which regulate protein synthesis in rat cardiac and skeletal muscles. Seven-week-old male Wistar rats were assigned to six groups: Zeitgeber time (ZT) 2, ZT6, ZT10, ZT14, ZT18, and ZT22 (ZT0, lights on; ZT12, lights off). The cardiac, plantaris, and soleus muscles were removed after a 12-h fasting period, and signal transducers involved in protein synthesis (mTOR, p70S6K, and ERK) were analyzed by western blotting. Circadian rhythms of signal transducers were observed in both cardiac (mTOR, p70S6K, and ERK) and plantaris (p70S6K and ERK) muscles (p<0.05), but not in the soleus muscle. In the cardiac muscle, the phosphorylation rate of mTOR was significantly higher at ZT6 (peak) than at ZT18 (bottom), and the phosphorylation rate of p70S6K was significantly higher at ZT2 (peak) than at ZT18 (bottom). In contrast, in the plantaris muscle, the phosphorylation rate of ERK was significantly lower at ZT2 (bottom) than at ZT18 (peak). Our data suggested that protein synthesis via mTOR/p70S6K and ERK signaling molecules exhibits circadian variation in rat cardiac and fast-type plantaris muscles.     

Stimulation of calcineurin Aalpha activity attenuates muscle pathophysiology in mdx dystrophic mice

Calcineurin activation ameliorates the dystrophic pathology of hindlimb muscles in mdx mice and decreases their susceptibility to contraction damage. In mdx mice, the diaphragm is more severely affected than hindlimb muscles and more representative of Duchenne muscular dystrophy. The constitutively active calcineurin Aalpha transgene (CnAalpha) was overexpressed in skeletal muscles of mdx (mdx CnAalpha*) mice to test whether muscle morphology and function would be improved. Contractile function of diaphragm strips and extensor digitorum longus and soleus muscles from adult mdx CnAalpha* and mdx mice was examined in vitro. Hindlimb muscles from mdx CnAalpha* mice had a prolonged twitch time course and were more resistant to fatigue. Because of a slower phenotype and a decrease in fiber cross-sectional area, normalized force was lower in fast- and slow-twitch muscles of mdx CnAalpha* than mdx mice. In the diaphragm, despite a slower phenotype and a approximately 35% reduction in fiber size, normalized force was preserved. This was likely mediated by the reduction in the area of the diaphragm undergoing degeneration (i.e., mononuclear cell and connective and adipose tissue infiltration). The proportion of centrally nucleated fibers was reduced in mdx CnAalpha* compared with mdx mice, indicative of improved myofiber viability. In hindlimb muscles of mdx mice, calcineurin activation increased expression of markers of regeneration, particularly developmental myosin heavy chain isoform and myocyte enhancer factor 2A. Thus activation of the calcineurin signal transduction pathway has potential to ameliorate the mdx pathophysiology, especially in the diaphragm, through its effects on muscle degeneration and regeneration and endurance capacity.     

PAI-1-regulated miR-21 defines a novel age-associated fibrogenic pathway in muscular dystrophy

Disruption of skeletal muscle homeostasis by substitution with fibrotic tissue constitutes the principal cause of death in Duchenne muscular dystrophy (DMD) patients, yet the implicated fibrogenic mechanisms remain poorly understood. This study identifies the extracellular PAI-1/urokinase-type plasminogen activator (uPA) balance as an important regulator of microribonucleic acid (miR)-21 biogenesis, controlling age-associated muscle fibrosis and dystrophy progression. Genetic loss of PAI-1 in mdx dystrophic mice anticipated muscle fibrosis through these sequential mechanisms: the alteration of collagen metabolism by uPA-mediated proteolytic processing of transforming growth factor (TGF)-β in muscle fibroblasts and the activation of miR-21 expression, which inhibited phosphatase and tensin homologue and enhanced AKT signaling, thus endowing TGF-β with a remarkable cell proliferation-promoting potential. Age-associated fibrogenesis and muscle deterioration in mdx mice, as well as exacerbated dystrophy in young PAI-1(-/-) mdx mice, could be reversed by miR-21 or uPA-selective interference, whereas forced miR-21 overexpression aggravated disease severity. The PAI-1-miR-21 fibrogenic axis also appeared dysregulated in muscle of DMD patients, providing a basis for effectively targeting fibrosis and muscular dystrophies in currently untreatable individuals.     

IGF-II ameliorates the dystrophic phenotype and coordinately down-regulates programmed cell death

Duchenne muscular dystrophy (DMD) is a fatal and crippling disease of skeletal muscle which displays increased fibre turnover and elevated levels of programmed cell death (PCD) in muscle stem cells. Previously we showed that this cell death is inhibited by the growth factor IGF-II. To determine the functional significance of PCD to the dystrophic phenotype, we used a transgene to over-express IGF-II in the mdx mouse. We found that ectopic expression of IGF-II inhibited the elevated PCD observed in skeletal muscles in the absence of functional dystrophin and significantly ameliorates the early gross histopathological changes in skeletal muscles characteristic of the dystrophic phenotype. Replacement of the dystrophin gene abolished abnormal skeletal muscle cell PCD levels in vivo in a dose-dependent manner and in dystrophic SMS cell lines cultured in vitro. Thus elevation of stem cell PCD in dystrophic skeletal muscle is a direct consequence of the loss of functional dystrophin. Together these data demonstrate that elevated skeletal muscle cell PCD is a critical component of dystrophic pathology and is inversely correlated with both dystrophin gene dosage and with muscle fibre pathology. Targeting PCD in dystrophic muscles reduces both PCD and the classical features of dystrophic pathology in the mdx mouse suggesting that IGF-II is a strong candidate for therapeutic intervention in the dystrophinopathies.     

Aging does not alter the mechanosensitivity of the p38, p70S6k, and JNK2 signaling pathways in skeletal muscle.

The capacity for skeletal muscle to recover its mass following periods of unloading (regrowth) has been reported to decline with age. Although the mechanisms responsible for the impaired regrowth are not known, it has been suggested that aged muscles have a diminished capacity to sense and subsequently respond to a given amount of mechanical stimuli (mechanosensitivity). To test this hypothesis, extensor digitorum longus muscles from young (2-3 mo) and old (26-27 mo) mice were subjected to intermittent 15% passive stretch (ex vivo) as a source of mechanical stimulation and analyzed for alterations in the phosphorylation of stress-activated protein kinase (p38), ribosomal S6 kinase (p70S6k), and the p54 jun N-terminal kinase (JNK2). The results indicated that the average magnitude of specific tension (mechanical stimuli) induced by 15% stretch was similar in muscles from young and old mice. Young and old muscles also revealed similar increases in the magnitude of mechanically induced p38, p70S6k (threonine/serine 421/424 and threonine 389), and JNK2 phosphorylation. In addition, coincubation experiments demonstrated that the release of locally acting growth factors was not sufficient for the induction of JNK2 phosphorylation, suggesting that JNK2 was activated by a mechanical rather than a mechanical/growth factor-dependent mechanism. Taken together, the results of this study demonstrate that aging does not alter the mechanosensitivity of the p38, p70S6k, and JNK2 signaling pathways in skeletal muscle.     

Matrix Metalloproteinase-9 Inhibition Improves Proliferation and Engraftment of Myogenic Cells in Dystrophic Muscle of mdx Mice

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.