ACE2 Is Augmented in Dystrophic Skeletal Muscle and Plays a Role in Decreasing Associated Fibrosis

Duchenne muscular dystrophy (DMD) is the most common inherited neuromuscular disease and is characterized by absence of the cytoskeletal protein dystrophin, muscle wasting, and fibrosis. We previously demonstrated that systemic infusion or oral administration of angiotensin-(1-7) (Ang-(1-7)), a peptide with opposing effects to angiotensin II, normalized skeletal muscle architecture, decreased local fibrosis, and improved muscle function in mdx mice, a dystrophic model for DMD. In this study, we investigated the presence, activity, and localization of ACE2, the enzyme responsible for Ang-(1-7) production, in wild type (wt) and mdx skeletal muscle and in a model of induced chronic damage in wt mice. All dystrophic muscles studied showed higher ACE2 activity than wt muscle. Immunolocalization studies indicated that ACE2 was localized mainly at the sarcolemma and, to a lesser extent, associated with interstitial cells. Similar results were observed in the model of chronic damage in the tibialis anterior (TA) muscle. Furthermore, we evaluated the effect of ACE2 overexpression in mdx TA muscle using an adenovirus containing human ACE2 sequence and showed that expression of ACE2 reduced the fibrosis associated with TA dystrophic muscles. Moreover, we observed fewer inflammatory cells infiltrating the mdx muscle. Finally, mdx gastrocnemius muscles from mice infused with Ang-(1-7), which decreases fibrosis, contain less ACE2 associated with the muscle. This is the first evidence supporting ACE2 as an important therapeutic target to improve the dystrophic skeletal muscle phenotype.     

Quantitative T2 Combined with Texture Analysis of Nuclear Magnetic Resonance Images Identify Different Degrees of Muscle Involvement in Three Mouse Models of Muscle Dystrophy: mdx,Largemyd and mdx/Largemyd

Quantitative nuclear magnetic resonance imaging (MRI) has been considered a promising non-invasive tool for monitoring therapeutic essays in small size mouse models of muscular dystrophies. Here, we combined MRI (anatomical images and transverse relaxation time constant—T2—measurements) to texture analyses in the study of four mouse strains covering a wide range of dystrophic phenotypes. Two still unexplored mouse models of muscular dystrophies were analyzed: The severely affected Large^myd mouse and the recently generated and worst double mutant mdx/Large^myd mouse, as compared to the mildly affected mdx and normal mice. The results were compared to histopathological findings. MRI showed increased intermuscular fat and higher muscle T2 in the three dystrophic mouse models when compared to the wild-type mice (T2: mdx/Large^myd: 37.6±2.8 ms; mdx: 35.2±4.5 ms; Large^myd: 36.6±4.0 ms; wild-type: 29.1±1.8 ms, p<0.05), in addition to higher muscle T2 in the mdx/Large^myd mice when compared to mdx (p<0.05). The areas with increased muscle T2 in the MRI correlated spatially with the identified histopathological alterations such as necrosis, inflammation, degeneration and regeneration foci. Nevertheless, muscle T2 values were not correlated with the severity of the phenotype in the 3 dystrophic mouse strains, since the severely affected Large^myd showed similar values than both the mild mdx and worst mdx/Large^myd lineages. On the other hand, all studied mouse strains could be unambiguously identified with texture analysis, which reflected the observed differences in the distribution of signals in muscle MRI. Thus, combined T2 intensity maps and texture analysis is a powerful approach for the characterization and differentiation of dystrophic muscles with diverse genotypes and phenotypes. These new findings provide important noninvasive tools in the evaluation of the efficacy of new therapies, and most importantly, can be directly applied in human translational research.     

Nifedipine Treatment Reduces Resting Calcium Concentration,Oxidative and Apoptotic Gene Expression,and Improves Muscle Function in Dystrophic mdx Mice

Duchenne Muscular Dystrophy (DMD) is a recessive X-linked genetic disease, caused by mutations in the gene encoding dystrophin. DMD is characterized in humans and in mdx mice by a severe and progressive destruction of muscle fibers, inflammation, oxidative/nitrosative stress, and cell death. In mdx muscle fibers, we have shown that basal ATP release is increased and that extracellular ATP stimulation is pro-apoptotic. In normal fibers, depolarization-induced ATP release is blocked by nifedipine, leading us to study the potential therapeutic effect of nifedipine in mdx muscles and its relation with extracellular ATP signaling. Acute exposure to nifedipine (10 µM) decreased [Ca²⁺]_r, NF-κB activity and iNOS expression in mdx myotubes. In addition, 6-week-old mdx mice were treated with daily intraperitoneal injections of nifedipine, 1 mg/Kg for 1 week. This treatment lowered the [Ca²⁺]_r measured in vivo in the mdx vastus lateralis. We demonstrated that extracellular ATP levels were higher in adult mdx flexor digitorum brevis (FDB) fibers and can be significantly reduced after 1 week of treatment with nifedipine. Interestingly, acute treatment of mdx FDB fibers with apyrase, an enzyme that completely degrades extracellular ATP to AMP, reduced [Ca²⁺]_r to a similar extent as was seen in FDB fibers after 1-week of nifedipine treatment. Moreover, we demonstrated that nifedipine treatment reduced mRNA levels of pro-oxidative/nitrosative (iNOS and gp91^phox/p47^phox NOX2 subunits) and pro-apoptotic (Bax) genes in mdx diaphragm muscles and lowered serum creatine kinase (CK) levels. In addition, nifedipine treatment increased muscle strength assessed by the inverted grip-hanging test and exercise tolerance measured with forced swimming test in mdx mice. We hypothesize that nifedipine reduces basal ATP release, thereby decreasing purinergic receptor activation, which in turn reduces [Ca²⁺]_r in mdx skeletal muscle cells. The results in this work open new perspectives towards possible targets for pharmacological approaches to treat DMD.     

Inhibition of the angiotensin-converting enzyme decreases skeletal muscle fibrosis in dystrophic mice by a diminution in the expression and activity of connective tissue growth factor (CTGF/CCN-2)

The renin-angiotensin system (RAS), through angiotensin II and the angiotensin-converting enzyme (ACE), is involved in the genesis and progression of fibrotic diseases characterized by the replacement of normal tissue by an accumulation of an extracellular matrix (ECM). Duchenne muscular dystrophy (DMD) presents fibrosis and a decrease in muscle strength produced by chronic damage. The mdx mouse is a murine model of DMD and develops the same characteristics as dystrophic patients when subjected to chronic exercise. The connective tissue growth factor (CTGF/CCN2) and transforming growth factor type beta (TGF-β), which are overexpressed in muscular dystrophies, play a major role in many progressive scarring conditions. We have tested the hypothesis that ACE inhibition decreases fibrosis in dystrophic skeletal muscle by treatment of mdx mice with the ACE inhibitor enalapril. Both sedentary and exercised mdx mice treated with enalapril showed improvement in gastrocnemius muscle strength explained by a reduction in both muscle damage and ECM accumulation. ACE inhibition decreased CTGF expression in sedentary or exercised mdx mice and diminished CTGF-induced pro-fibrotic activity in a model of CTGF overexpression by adenoviral infection. Enalapril did not have an effect on TGF-β1 expression or its signaling activity in sedentary or exercised dystrophic mice. Thus, ACE inhibition might improve muscle strength and decrease fibrosis by diminishing specifically CTGF expression and activity without affecting TGF-β1 signaling. Our data provide insights into the pathogenic events in dystrophic muscle. We propose ACE as a target for developing therapies for DMD and related diseases.     

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.     

Mutation Types and Aging Differently Affect Revertant Fiber Expansion in Dystrophic Mdx and Mdx52 Mice

Duchenne muscular dystrophy (DMD), one of the most common and lethal genetic disorders, and the mdx mouse myopathies are caused by a lack of dystrophin protein. These dystrophic muscles contain sporadic clusters of dystrophin-expressing revertant fibers (RFs), as detected by immunohistochemistry. RFs are known to arise from muscle precursor cells with spontaneous exon skipping (alternative splicing) and clonally expand in size with increasing age through the process of muscle degeneration/regeneration. The expansion of revertant clusters is thought to represent the cumulative history of muscle regeneration and proliferation of such precursor cells. However, the precise mechanisms by which RFs arise and expand are poorly understood. Here, to test the effects of mutation types and aging on RF expansion and muscle regeneration, we examined the number of RFs in mdx mice (containing a nonsense mutation in exon 23) and mdx52 mice (containing deletion mutation of exon 52) with the same C57BL/6 background at 2, 6, 12, and 18months of age. Mdx mice displayed a significantly higher number of RFs compared to mdx52 mice in all age groups, suggesting that revertant fiber expansion largely depends on the type of mutation and/or location in the gene. A significant increase in the expression and clustering levels of RFs was found beginning at 6months of age in mdx mice compared with mdx52 mice. In contrast to the significant expansion of RFs with increasing age, the number of centrally nucleated fibers and embryonic myosin heavy chain-positive fibers (indicative of cumulative and current muscle regeneration, respectively) decreased with age in both mouse strains. These results suggest that mutation types and aging differently affect revertant fiber expansion in mdx and mdx52 mice.     

Orai1 Mediates Exacerbated Ca2+ Entry in Dystrophic Skeletal Muscle

There is substantial evidence indicating that disruption of Ca²⁺ homeostasis and activation of cytosolic proteases play a key role in the pathogenesis and progression of Duchenne Muscular Dystrophy (DMD). However, the exact nature of the Ca²⁺ deregulation and the Ca²⁺ signaling pathways that are altered in dystrophic muscles have not yet been resolved. Here we examined the contribution of the store-operated Ca²⁺ entry (SOCE) for the pathogenesis of DMD. RT-PCR and Western blot found that the expression level of Orai1, the pore-forming unit of SOCE, was significantly elevated in the dystrophic muscles, while parallel increases in SOCE activity and SR Ca²⁺ storage were detected in adult mdx muscles using Fura-2 fluorescence measurements. High-efficient shRNA probes against Orai1 were delivered into the flexor digitorum brevis muscle in live mice and knockdown of Orai1 eliminated the differences in SOCE activity and SR Ca²⁺ storage between the mdx and wild type muscle fibers. SOCE activity was repressed by intraperitoneal injection of BTP-2, an Orai1 inhibitor, and cytosolic calpain1 activity in single muscle fibers was measured by a membrane-permeable calpain substrate. We found that BTP-2 injection for 2 weeks significantly reduced the cytosolic calpain1 activity in mdx muscle fibers. Additionally, ultrastructural changes were observed by EM as an increase in the number of triad junctions was identified in dystrophic muscles. Compensatory changes in protein levels of SERCA1, TRP and NCX3 appeared in the mdx muscles, suggesting that comprehensive adaptations occur following altered Ca²⁺ homeostasis in mdx muscles. Our data indicates that upregulation of the Orai1-mediated SOCE pathway and an overloaded SR Ca²⁺ store contributes to the disrupted Ca²⁺ homeostasis in mdx muscles and is linked to elevated proteolytic activity, suggesting that targeting Orai1 activity may be a promising therapeutic approach for the prevention and treatment of muscular dystrophy.     

Amelioration of muscular dystrophy by transgenic expression of Niemann-Pick C1

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.     

Wild-Type Mouse Models to Screen Antisense Oligonucleotides for Exon-Skipping Efficacy in Duchenne Muscular Dystrophy

A readily available animal model is essential for rapidly identifying effective treatments for Duchenne muscular dystrophy (DMD), a devastating neuromuscular disorder caused by the lack of dystrophin protein, which results from frame-disrupting mutations in the DMD gene. Currently, the mdx mouse is the most commonly used model for antisense oligonucleotide (AO)-mediated exon skipping pre-clinical studies, with a mild phenotype. However, the accessibility of mdx mouse colonies particularly in developing countries can constrain research. Therefore in this study we explore the feasibility of using wild-type mice as models to establish exon-skipping efficiency of various DMD AO chemistries and their conjugates. Four different strains of wild-type mice and six different AO chemistries were investigated intramuscularly and the results indicated that the same exon-skipping efficiency was achieved for all tested AOs as that from mdx mice. Notably, levels of exon-skipping obtained in C57BL6 and C3H and mdx mice were most closely matched, followed by ICR and BALB/C mice. Systemic validation revealed that wild-type mice are less responsive to AO-mediated exon skipping than mdx mice. Our study provides evidence for the first time that wild-type mice can be appropriate models for assessing DMD AO exon-skipping efficiency with similar sensitivity to that of mdx mice and this finding can further accelerate the development of effective DMD AOs.     

Energy status of cells lacking dystrophin: an in vivo/in vitro study of mdx mouse skeletal muscle

Although great strides have been made in understanding the genetics of Duchenne muscular dystrophy (DMD), uncertainty still remains as to the metabolic changes which are associated with the disease. We have used the recently discovered animal model of DMD, the mdx mouse, to study aspects of high energy phosphate metabolism and metabiolic control indices in dystrophic muscle. This model of DMD has the dual advantage of having a genetic defect which is homologous to that in human DMD, and it lacks the fatty infiltration and ncecrosis which makes biochemical analysis of DMD so difficult. We have used nuclear magnetic resonance sperctroscopy (NMR) to monitor developmental changes in high energy phosphates and pH. No differences were observed between young (< 40–50 days old) control and mdx mice. The pH increase and alterations in phosphate ratios (i.e., decline in PCr/ATP) observed in adult mdx vs. control mice are quantilatively similar to those observed in humans. Biochemical analysis showed a small decline in ATP and PCr content and a decline in some indices of energy status in adult mdx mice. As young mdx mice appeared to be normal, the lack of dystrophin does not correlate with metabolic changes. The changes which were observed were small enough that alterations in fibre composition could be the major contributory factor.     

Non-Invasive MRI and Spectroscopy of mdx Mice Reveal Temporal Changes in Dystrophic Muscle Imaging and in Energy Deficits

In Duchenne muscular dystrophy (DMD), a genetic disruption of dystrophin protein expression results in repeated muscle injury and chronic inflammation. Magnetic resonance imaging shows promise as a surrogate outcome measure in both DMD and rehabilitation medicine that is capable of predicting clinical benefit years in advance of functional outcome measures. The mdx mouse reproduces the dystrophin deficiency that causes DMD and is routinely used for preclinical drug testing. There is a need to develop sensitive, non-invasive outcome measures in the mdx model that can be readily translatable to human clinical trials. Here we report the use of magnetic resonance imaging and spectroscopy techniques for the non-invasive monitoring of muscle damage in mdx mice. Using these techniques, we studied dystrophic mdx muscle in mice from 6 to 12 weeks of age, examining both the peak disease phase and natural recovery phase of the mdx disease course. T2 and fat-suppressed imaging revealed significant levels of tissue with elevated signal intensity in mdx hindlimb muscles at all ages; spectroscopy revealed a significant deficiency of energy metabolites in 6-week-old mdx mice. As the mdx mice progressed from the peak disease stage to the recovery stage of disease, each of these phenotypes was either eliminated or reduced, and the cross-sectional area of the mdx muscle was significantly increased when compared to that of wild-type mice. Histology indicates that hyper-intense MRI foci correspond to areas of dystrophic lesions containing inflammation as well as regenerating, degenerating and hypertrophied myofibers. Statistical sample size calculations provide several robust measures with the ability to detect intervention effects using small numbers of animals. These data establish a framework for further imaging or preclinical studies, and they support the development of MRI as a sensitive, non-invasive outcome measure for muscular dystrophy.     

The heart in Duchenne muscular dystrophy: early detection of contractile performance alteration

Progressive cardiomyopathy is a major cause of death in Duchenne muscular dystrophy (DMD) patients. Coupling between Ca²⁺ handling and contractile properties in dystrophic hearts is poorly understood. It is also not clear whether developing cardiac failure is dominated by alterations in Ca²⁺ pathways or more related to the contractile apparatus. We simultaneously recorded force and Ca²⁺ transients in field‐stimulated papillary muscles from young (10–14 weeks) wild‐type (wt) and dystrophic mdx mice. Force amplitudes were fivefold reduced in mdx muscles despite only 30 % reduction in fura‐2 ratio amplitudes. This indicated mechanisms other than systolic Ca²⁺ to additionally account for force decrements in mdx muscles. pCa‐force relations revealed decreased mdx myofibrillar Ca²⁺ sensitivity. ‘In vitro’ motility assays, studied in mdx hearts here for the first time, showed significantly slower sliding velocities. mdx MLC/MHC isoforms were not grossly altered. Dystrophic hearts showed echocardiography signs of early ventricular wall hypertrophy with a significantly enlarged end‐diastolic diameter ‘in vivo’. However, fractional shortening was still comparable to wt mice. Changes in the contractile apparatus satisfactorily explained force drop in mdx hearts. We give first evidence of early hypertrophy in mdx mice and possible mechanisms for already functional impairment of cardiac muscle in DMD.     

Muscle Structure Influences Utrophin Expression in mdx Mice

Duchenne muscular dystrophy (DMD) is a severe muscle wasting disorder caused by mutations in the dystrophin gene. To examine the influence of muscle structure on the pathogenesis of DMD we generated mdx^4cv:desmin double knockout (dko) mice. The dko male mice died of apparent cardiorespiratory failure at a median age of 76 days compared to 609 days for the desmin^−/− mice. An ∼2.5 fold increase in utrophin expression in the dko skeletal muscles prevented necrosis in ∼91% of 1a, 2a and 2d/x fiber-types. In contrast, utrophin expression was reduced in the extrasynaptic sarcolemma of the dko fast 2b fibers leading to increased membrane fragility and dystrophic pathology. Despite lacking extrasynaptic utrophin, the dko fast 2b fibers were less dystrophic than the mdx^4cv fast 2b fibers suggesting utrophin-independent mechanisms were also contributing to the reduced dystrophic pathology. We found no overt change in the regenerative capacity of muscle stem cells when comparing the wild-type, desmin^−/−, mdx^4cv and dko gastrocnemius muscles injured with notexin. Utrophin could form costameric striations with α-sarcomeric actin in the dko to maintain the integrity of the membrane, but the lack of restoration of the NODS (nNOS, α-dystrobrevin 1 and 2, α1-syntrophin) complex and desmin coincided with profound changes to the sarcomere alignment in the diaphragm, deposition of collagen between the myofibers, and impaired diaphragm function. We conclude that the dko mice may provide new insights into the structural mechanisms that influence endogenous utrophin expression that are pertinent for developing a therapy for DMD.     

Molecular mechanism of sphingosine-1-phosphate action in Duchenne muscular dystrophy

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.KEY WORDS: HDAC, S1P, THI, dys, Dystrophin, mdx     

A Highlights from MBoC Selection: Calpain-mediated proteolysis of tropomodulin isoforms leads to thin filament elongation in dystrophic skeletal muscle

Duchenne muscular dystrophy (DMD) induces sarcolemmal mechanical instability and rupture, hyperactivity of intracellular calpains, and proteolytic breakdown of muscle structural proteins. Here we identify the two sarcomeric tropomodulin (Tmod) isoforms, Tmod1 and Tmod4, as novel proteolytic targets of m-calpain, with Tmod1 exhibiting ∼10-fold greater sensitivity to calpain-mediated cleavage than Tmod4 in situ. In mdx mice, increased m-calpain levels in dystrophic soleus muscle are associated with loss of Tmod1 from the thin filament pointed ends, resulting in ∼11% increase in thin filament lengths. In mdx/mTR mice, a more severe model of DMD, Tmod1 disappears from the thin filament pointed ends in both tibialis anterior (TA) and soleus muscles, whereas Tmod4 additionally disappears from soleus muscle, resulting in thin filament length increases of ∼10 and ∼12% in TA and soleus muscles, respectively. In both mdx and mdx/mTR mice, both TA and soleus muscles exhibit normal localization of α-actinin, the nebulin M1M2M3 domain, Tmod3, and cytoplasmic γ-actin, indicating that m-calpain does not cause wholesale proteolysis of other sarcomeric and actin cytoskeletal proteins in dystrophic skeletal muscle. These results implicate Tmod proteolysis and resultant thin filament length misspecification as novel mechanisms that may contribute to DMD pathology, affecting muscles in a use- and disease severity–dependent manner.     

Low-Level Laser Therapy (LLLT) in Dystrophin-Deficient Muscle Cells: Effects on Regeneration Capacity,Inflammation Response and Oxidative Stress

The present study evaluated low-level laser therapy (LLLT) effects on some physiological pathways that may lead to muscle damage or regeneration capacity in dystrophin-deficient muscle cells of mdx mice, the experimental model of Duchenne muscular dystrophy (DMD). Primary cultures of mdx skeletal muscle cells were irradiated only one time with laser and analyzed after 24 and 48 hours. The LLLT parameter used was 830 nm wavelengths at 5 J/cm² fluence. The following groups were set up: Ctrl (untreated C57BL/10 primary muscle cells), mdx (untreated mdx primary muscle cells), mdx LA 24 (mdx primary muscle cells - LLLT irradiated and analyzed after 24 h), and mdx LA 48 (mdx primary muscle cells - LLLT irradiated and analyzed after 48 h). The mdx LA 24 and mdx LA 48 groups showed significant increase in cell proliferation, higher diameter in muscle cells and decreased MyoD levels compared to the mdx group. The mdx LA 48 group showed significant increase in Myosin Heavy Chain levels compared to the untreated mdx and mdx LA 24 groups. The mdx LA 24 and mdx LA 48 groups showed significant increase in [Ca²⁺]i. The mdx group showed significant increase in H₂O₂ production and 4-HNE levels compared to the Ctrl group and LLLT treatment reduced this increase. GSH levels and GPx, GR and SOD activities increased in the mdx group. Laser treatment reduced the GSH levels and GR and SOD activities in dystrophic muscle cells. The mdx group showed significant increase in the TNF-α and NF-κB levels, which in turn was reduced by the LLLT treatment. Together, these results suggest that the laser treatment improved regenerative capacity and decreased inflammatory response and oxidative stress in dystrophic muscle cells, indicating that LLLT could be a helpful alternative therapy to be associated with other treatment for dystrophinopathies.     

Characterization of Dystrophin Deficient Rats: A New Model for Duchenne Muscular Dystrophy

A few animal models of Duchenne muscular dystrophy (DMD) are available, large ones such as pigs or dogs being expensive and difficult to handle. Mdx (X-linked muscular dystrophy) mice only partially mimic the human disease, with limited chronic muscular lesions and muscle weakness. Their small size also imposes limitations on analyses. A rat model could represent a useful alternative since rats are small animals but 10 times bigger than mice and could better reflect the lesions and functional abnormalities observed in DMD patients. Two lines of Dmd mutated-rats (Dmd^mdx) were generated using TALENs targeting exon 23. Muscles of animals of both lines showed undetectable levels of dystrophin by western blot and less than 5% of dystrophin positive fibers by immunohistochemistry. At 3 months, limb and diaphragm muscles from Dmd^mdx rats displayed severe necrosis and regeneration. At 7 months, these muscles also showed severe fibrosis and some adipose tissue infiltration. Dmd^mdx rats showed significant reduction in muscle strength and a decrease in spontaneous motor activity. Furthermore, heart morphology was indicative of dilated cardiomyopathy associated histologically with necrotic and fibrotic changes. Echocardiography showed significant concentric remodeling and alteration of diastolic function. In conclusion, Dmd^mdx rats represent a new faithful small animal model of DMD.     

Notch signaling deficiency underlies age-dependent depletion of satellite cells in muscular dystrophy

Duchenne muscular dystrophy (DMD) is a devastating disease characterized by muscle wasting, loss of mobility and death in early adulthood. Satellite cells are muscle-resident stem cells responsible for the repair and regeneration of damaged muscles. One pathological feature of DMD is the progressive depletion of satellite cells, leading to the failure of muscle repair. Here, we attempted to explore the molecular mechanisms underlying satellite cell ablation in the dystrophin mutant mdx mouse, a well-established model for DMD. Initial muscle degeneration activates satellite cells, resulting in increased satellite cell number in young mdx mice. This is followed by rapid loss of satellite cells with age due to the reduced self-renewal ability of mdx satellite cells. In addition, satellite cell composition is altered even in young mdx mice, with significant reductions in the abundance of non-committed (Pax7⁺ and Myf5⁻) satellite cells. Using a Notch-reporter mouse, we found that the mdx satellite cells have reduced activation of Notch signaling, which has been shown to be necessary to maintain satellite cell quiescence and self-renewal. Concomitantly, the expression of Notch1, Notch3, Jag1, Hey1 and HeyL are reduced in the mdx primary myoblast. Finally, we established a mouse model to constitutively activate Notch signaling in satellite cells, and show that Notch activation is sufficient to rescue the self-renewal deficiencies of mdx satellite cells. These results demonstrate that Notch signaling is essential for maintaining the satellite cell pool and that its deficiency leads to depletion of satellite cells in DMD.KEY WORDS: Muscular dystrophy, Notch signaling, Stem cell     

Transport of Ca2+ and Ca2+-dependent permeability transition in heart mitochondria in the early stages of Duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD) is a progressive skeletal muscle disease that is associated with severe cardiac complications in the late stages. Significant mitochondrial dysfunction is reportedly responsible for the development of cardiomyopathy with age. At the same time, adaptive changes in mitochondrial metabolism in cardiomyocytes were identified in the early stages of DMD. In this work, we evaluate the functioning of calcium transport systems (MCU and NCLX), and MPT pore in the heart mitochondria of young dystrophin-deficient mice. As compared to wild-type animals, heart mitochondria of mdx mice have been found to be more efficient both in respect to Ca²⁺ uniport and Na⁺-dependent Ca²⁺ efflux. The data obtained indicate that the increased rate of Ca²⁺ uptake by heart mitochondria of mdx mice may be due to an increase in the ratio of MCU and MCUb subunits. In turn, an increase in the rate of Ca²⁺ efflux from organelles in DMD may be the result of a significant increase in the level of NCLX. Moreover, the heart mitochondria of mdx mice were more resistant to MPT pore opening, which may be due to an increase in the microviscosity of mitochondrial membranes of DMD mice. At the same time, the level of putative MPT pore proteins did not change. The paper discusses the effect of rearrangements of the mitochondrial proteome involved in the transport and accumulation of calcium on the adaptation of this organ to DMD.