Dystrophic Muscle Cells Research Articles

Can we talk about myoblast fusion?

Can we talk about myoblast fusion?

Vinculin Tension and YAP Activation are Altered in Dystrophic Muscle Cells

Duchenne muscular dystrophy is a lethal muscle wasting disease caused by the absence of dystrophin, a protein that links the actin cytoskeleton to the extracellular matrix. Cells generate, sense, and respond to forces via mechanosensitive protein complexes, and altered force transmission via these complexes such as focal adhesions (FAs) are known to contribute to the development and progression of muscular dystrophy. However, the interplay between loss of dystrophin and altered force transmission via FAs remains unknown. Here we show that FAs of dystrophin deficient muscle cells are under less mechanical load and have decreased activation of yes-associated protein 1 (YAP). Using a vinculin bioluminescent tension sensor, we measured FAs tension in transgenic muscle cells expressing wild type (WT) dystrophin, two patient derived dystrophin missense mutants (L172H and L54R), and a non-disease-causing mutation (I232M). We found that cells harboring WT dystrophin showed higher average tension across vinculin compared to the tension in dystrophic cells. I232M vinculin tension was the same as WT tension. YAP is a known FA mechano-target protein that functions in the nucleus as a transcriptional co-activator and regulates several cellular processes including cellular migration. Dystrophic cells had higher levels of cytoplasmic YAP when compared to nuclear YAP, increased YAP S127 inactivating phosphorylation and decreased migration rates, compared to WT and I23M. Our results suggest that the Hippo pathway may regulate YAP activation via LATS1/2 activity. LATS1/2 phosphorylates YAP in S172 sequestering it in the cytoplasm and preventing its translocation to the nucleus, decreasing the expression of YAP target genes in migration. Altogether, our results show that expression of disease-causing mutant dystrophins leads to decreased tension transmission and aberrant mechanotransduction via FAs. We propose that decreased vinculin tension leads to increased Hippo pathway signaling which causes decreased YAP activation that results in impaired migration. Therefore, our cell lines could be used as a platform to characterize the effectiveness of DMD therapies for patients with missense mutations by means of monitoring YAP and migration. Furthermore, they could also be used to explore novel therapeutic targets in the Hippo pathway.

ИСПОЛЬЗОВАНИЕ АНТИОКСИДАНТОВ В КАЧЕСТВЕ АДАПТОГЕНОВ для СВИНЕЙ (Sus scrofa domesticus Erxleben, 1777) (МЕТА-АНАЛИЗ)

The meat flash quality is basically dependent on muscle tissue characteristics. Feeding disorders and stresses can cause myopathy, a destabilizing factor of farm animal meat quality. The muscle tissue injury preventing is of particular interest as it can improve lifetime meat quality formation. Dietary enrichment of farm animal nutrition with natural adaptogens and antioxidants offers potential to reduce myopathies of various etiologies. This paper is an overview of nutrition factors as protective agents under stress loads and myopathies in intensively growing pigs. Dietary adaptogens, e.g. selenium, tocopherol, quercetins, etc., inhibit peroxidation of lipids, generation of reactive oxygen species and are important for the control of glycolysis and oxidative stress. Most adaptogens are antioxidants, they have a beneficial effect on the cardiovascular system, including blood capillaries, prevent damage to cell membranes caused by free radicals and apoptosis. The beneficial effects of vitamin E-enriched diets (from 10 to 1000 mg/kg feed, approximately 200 mg/kg mainly) on porcine meat quality characteristics have been well studied in pig breeds and breed combinations during various periods of growing. However, no effect of dietary vitamin E on the growth rate of animals has also been reported. Feed enrichment with dietary tocopherol leads to its deposition in all tissues and organs, primarily in the blood, liver, heart, and in muscle and fat tissues. Vitamin E has a membrane-stabilizing effect, reduces oxidation of membrane lipids, increases the total amount of fatty acids in mitochondria, antioxidant capacity and muscle glycogen content. It has been shown that in pigs fed diets supplemented with vitamin E during fattening phase the vitamin E deposition level in meat is higher. This, in turn, improves meat taste and flavor, reduces the smell characteristic of reheated dishes, does not change the aldehyde profile of meat volatiles and reduces the accumulation of nitrogenous volatiles resulted from the breakdown of meat proteins during storage, including in a vacuum. Less attention is paid to administration of selenium as an adaptogen. It was shown that selenium combined with higher vitamin E level can neutralize the adverse consequences of hyperthermia in growing pigs and increase free fatty acid content in fat. The organic form of dietary selenium improves the antioxidant status of muscles in pigs. However, selenium has different effects on the oxidation of proteins and lipids during meat storage. In some studies, selenium reduced oxidation; in others, on the contrary, it was proved to be unable to inhibit the accumulation of products of oxidative damage. Two flavonoids quercetin and dihydroquercetin (Taxifolin) are well known for their antioxidant properties. The research articles are mainly deal with quercetin and dihydroquercetin bioavailability and deposition, the impact on antioxidant status and reproductive functions of sows, leveling transportation stress, and pork quality. Quercetin supplements have a pronounced effect at 25-50 mg/kg live weight, dihydroquercetin supplements at 1-3.5 mg/kg live weight. The flavonoids are effective when administered both during the fattening period and before slaughter or transportation. Despite the encouraging reports, little research has focused on the role of these flavonoids in the pork meat quality formation, so further study requires. Quercetin when fed up to 6 months at 2 % of the diet reduced damage to dystrophic skeletal muscle fibers in laboratory animals due to a decrease in reduced production of hydrogen peroxide in mitochondria. Adaptogens and directed muscle tissue development regulators are proposed as potentially key supplements ensuring meat quality under intensive animal husbandry, therefore, further search for and study of bioactive substances which can protect muscle tissues from damaging factors are required.

Decreasing HepG2 Cytotoxicity by Lowering the Lipophilicity of Benzo[d]oxazolephosphinate Ester Utrophin Modulators.

Utrophin modulation is a disease-modifying therapeutic strategy for Duchenne muscular dystrophy that would be applicable to all patient populations. To improve the suboptimal profile of ezutromid, the first-in-class clinical candidate, a second generation of utrophin modulators bearing a phosphinate ester moiety was developed. This modification significantly improved the physicochemical and ADME properties, but one of the main lead molecules was found to have dose-limiting hepatotoxicity. In this work we describe how less lipophilic analogues retained utrophin modulatory activity in a reporter gene assay, upregulated utrophin protein in dystrophic mouse muscle cells, but also had improved physicochemical and ADME properties. Notably, ClogP was found to directly correlate with pIC50 in HepG2 cells, hence leading to a potentially safer toxicological profiles in this series. Compound 21 showed a balanced profile (H2K EC50: 4.17 μM, solubility: 477 μM, mouse hepatocyte T 1/2 > 240 min) and increased utrophin protein 1.6-fold in a Western blot assay.

Abstract 478: Connexin-43 Reduction Rescues Cardiac and Skeletal Muscle Pathology in a Novel Mouse Model of Duchenne Muscular Dystrophy Symptomatic Carriers

Duchenne muscular dystrophy (DMD) and its associated cardiomyopathy manifest in 8-10% of all female carriers however research remains male-centric. Although underrepresented, symptomatic females face the risk of cardiac, respiratory, and skeletal muscle problems. Basic research and clinical trials exclude female carriers therefore developments in treatment expose females to unknown safety and efficacy issues. The bottleneck is largely due to the absence of a faithful mouse model. To generate a mouse model, we injected mdx embryonic stem cells (ESCs) into wild-type (WT) blastocysts ( mdx /WT chimera). The cardiac and skeletal muscle phenotype recapitulates the same generated as a consequence of x-inactivation in human manifesting female patients. In the heart, mdx /WT chimeras develop fibrotic cardiomyopathy. In the skeletal muscle, we found evidence of fibrosis, inflammation and muscle weakness. We found that Connexin-43 (Cx43), the primary gap junctional protein in the heart, was pathologically enhanced and remodeled in mdx /WT chimeras. Cx43 was also enhanced in the dystrophic skeletal muscle. Genetic reduction of Cx43-copy number protected mdx /WT chimeras from cardiac and skeletal muscle fiber damage. The latter result was unexpected because Cx43 is not expressed in mature muscle fibers. Upon further investigation, Cx43 was localized to the mononuclear cells invading the interstitial space between dystrophic skeletal muscle fibers. Pathologically enhanced activity of Cx43 in mdx FACS-macrophages was observed via ethidium bromide uptake and the Cx43 hemichannel peptide mimetic, Gap19, inhibited Cx43 function in a dose-dependent manner. Because an excess of Cx43 has been associated with cell death, we believe that Cx43 reduction in invading mdx macrophages benefits the skeletal muscle of understudied DMD carriers, perhaps by a paracrine mechanism involving macrophage-skeletal muscle fiber communication.

Multiple poloxamers increase plasma membrane repair capacity in muscle and nonmuscle cells.

Various previous studies established that the amphiphilic tri-block copolymer known as poloxamer 188 (P188) or Pluronic-F68 can stabilize the plasma membrane following a variety of injuries to multiple mammalian cell types. This characteristic led to proposals for the use of P188 as a therapeutic treatment for various disease states, including muscular dystrophy. Previous studies suggest that P188 increases plasma membrane integrity by resealing plasma membrane disruptions through its affinity for the hydrophobic lipid chains on the lipid bilayer. P188 is one of a large family of copolymers that share the same basic tri-block structure consisting of a middle hydrophobic propylene oxide segment flanked by two hydrophilic ethylene oxide moieties [poly(ethylene oxide)80-poly(propylene oxide)27-poly(ethylene oxide)80]. Despite the similarities of P188 to the other poloxamers in this chemical family, there has been little investigation into the membrane-resealing properties of these other poloxamers. In this study we assessed the resealing properties of poloxamers P181, P124, P182, P234, P108, P407, and P338 on human embryonic kidney 293 (HEK293) cells and isolated muscle from the mdx mouse model of Duchenne muscular dystrophy. Cell membrane injuries from glass bead wounding and multiphoton laser injury show that the majority of poloxamers in our panel improved the plasma membrane resealing of both HEK293 cells and dystrophic muscle fibers. These findings indicate that many tri-block copolymers share characteristics that can increase plasma membrane resealing and that identification of these shared characteristics could help guide design of future therapeutic approaches.

Coenzyme Q10 supplementation acts as antioxidant on dystrophic muscle cells.

Increased oxidative stress is a frequent feature in Duchenne muscular dystrophy (DMD). High reactive oxygen species (ROS) levels, associated with altered enzyme antioxidant activity, have been reported in dystrophic patients and mdx mice, an experimental model of DMD. In this study, we investigated the effects of coenzyme Q10 (CoQ10) on oxidative stress marker levels and calcium concentration in primary cultures of dystrophic muscle cells from mdx mice. Primary cultures of skeletal muscle cells from C57BL/10 and mdx mice were treated with coenzyme Q10 (5 μM) for 24 h. The untreated mdx and C57BL/10 muscle cells were used as controls. The MTT and live/dead cell assays showed that CoQ10 presented no cytotoxic effect on normal and dystrophic muscle cells. Intracellular calcium concentration, H2O2 production, 4-HNE, and SOD-2 levels were higher in mdx muscle cells. No significant difference in the catalase, GPx, and Gr levels was found between experimental groups. This study demonstrated that CoQ10 treatment was able to reduce levels of oxidative stress markers, such as H2O2, acting as an antioxidant, as well as decreasing abnormal intracellular calcium influx in dystrophic muscles cells. This study demonstrated that CoQ10 treatment was able to reduce levels of oxidative stress markers, such as H2O2, acting as an antioxidant, as well as decreasing abnormal intracellular calcium influx in dystrophic muscles cells. Our findings also suggest that the decrease of oxidative stress reduces the need for upregulation of antioxidant pathways, such as SOD and GSH.

P.326Characterization of the ion channels involved in the increase of sarcolemmal calcium permeability in the dystrophin-deficient DMDmdx rat model

Duchenne duscular Dystrophy (DMD) is a lethal muscle disease due to genetic mutations leading to the lack of dystrophin expression. Even if the cascade of events leading to muscle necrosis and fibrosis observed in the disease remains partly unexplained, an increase of the sarcolemmal permeability to Ca2+ (SPCa) and a consequent intracellular Ca2+ overload seem to be crucial events. Although much evidence has accumulated to designate channels belonging to the TRP family, the exact nature of the channels involved in the SPCa increase remains subject to debate. This may be because most of the studies have been done in the mdx mouse model that lacks significant clinical phenotype. We recently generated the dystrophin-deficient DMDmdx rat model, which mimics the human disease at both the muscular and the cardiac levels. Herein, we show that skeletal muscle fibers and cardiomyocytes isolated from DMDmdx rats and loaded with the Fura-2 Ca2+ probe exhibit higher cytosolic Ca2+ concentration than in wild-type rats. Additionally, we found, using the Mn2+ quenching technique, that SPCa is increased in DMDmdx rat skeletal muscle fibres. We performed immunofluorescence and western-blot analysis to determine wether SPCa increase could be related to changes in the expression and/or in the localization of six TPR channels: TRPV2, TRPC1, TRPC3 and TRPML1-3. TRPV2 is sensitive to stretch and has been shown to accumulate at the sarcolemma of dystrophic fibres. TRPC1 and TRPC3 are involved in SPCa regulation in muscle cells differentiation. TRPML are lysosomal Ca2+ channels involved in plasma membrane repair process. Our results show that TRPV2, TRPC3, TRPML1 and TRPML2 channels accumulate at the sarcolemma and are overexpressed in striated muscle cells from DMDmdx rats, leading to suggest an involvement of these channels in the SPCa increase measured in the dystrophic muscle fibers. The complete results of our investigations will be presented as well as our current perspectives.

Polyrotaxane Nanocarriers Can Deliver CRISPR/Cas9 Plasmid to Dystrophic Muscle Cells to Successfully Edit the DMD Gene

AbstractGene editing with clustered regularly interspaced short palindromic repeats and CRISPR‐associated protein 9 (CRISPR/Cas9) has shown promise in models of Duchenne muscular dystrophy (DMD); however, nonviral strategies to deliver CRISPR to muscle have not been widely explored or optimized. Most studies have relied on viral vectors, which are likely limited to single dosing due to their immunogenicity, thus reducing their therapeutic potential. Therefore, there is a need to develop nonviral approaches that allow for delivery and repeat dosing of CRISPR/Cas9 therapies to skeletal muscle. Here, biocompatible multi‐arm polyrotaxane (PRX) nanocarriers, are iteratively optimized for packaging large plasmid DNA for delivery to muscle cells. The PRXs are optimized by addition of a disulfide‐responsive linker that enhances plasmid release. Furthermore, conjugation of peptides leads to quicker uptake and improved transfection efficiency in humanized dystrophic muscle cells in vitro. Finally, in vitro delivery of PRXs complexed with a CRISPR/Cas9 platform demonstrates effective deletion of DMD exons 45–55, a therapeutic strategy with potential to restore the reading frame for half of DMD patients. This work represents the first PRX platform that is optimized and designed for delivery of large plasmid DNA, such as CRISPR/Cas9, to dystrophic muscle cells.

Modeling the Cytotoxic Swelling of Dystrophic Muscle Fibers

Modeling the Cytotoxic Swelling of Dystrophic Muscle Fibers

Choline administration attenuates aspects of the dystrophic pathology in mdx mice

Background & aimsDystrophic muscle fibres are fragile and prone to breakage, leading to impaired Ca2+ homeostasis and excessive inflammation, resulting in muscle wasting and weakness. Choline, an essential water-soluble nutrient, is involved in multiple biological processes, including modulation of inflammation and oxidative stress. We tested the hypothesis that choline supplementation would ameliorate the dystrophic pathology in mdx mice.MethodsThree-week old male mdx mice (n = 40) were fed control purified laboratory chow (CON; n = 20) or a choline-enriched diet (5 g/kg choline; CHL, n = 20) for four weeks. Rotarod performance, grip strength and running (wheel) distance were assessed during treatment. Markers of Ca2+-handling, inflammation, oxidative stress and fibrosis were measured in the diaphragm, quadriceps muscle and the liver.ResultsCholine-treated mdx mice displayed less macrophage (CD68 -33%, P < 0.05) and collagen infiltration (−34%, P < 0.05) and reduced Tgfβ3 mRNA expression (P < 0.05) in the diaphragm compared to untreated mdx mice. Choline supplementation increased maximal SERCA activity (38%, P < 0.05) and reduced markers of inflammatory (Tnfα, F4/80 and Cd206 mRNA, P < 0.05) processes compared with untreated mdx mice. In the liver there was a reduction in Acta2 mRNA (P < 0.05) with choline treatment, as well as an improvement in serum ALT levels (P < 0.01). There were no differences between the groups for the whole-body functional analyses.ConclusionsCholine supplementation attenuated the progression of the dystrophic pathology. Although choline did not alter functional performance, the reduction in fibrosis is clinically relevant for increasing the efficacy of therapies for DMD. Dystrophic muscle fibres are fragile and prone to breakage, leading to impaired Ca2+ homeostasis and excessive inflammation, resulting in muscle wasting and weakness. Choline, an essential water-soluble nutrient, is involved in multiple biological processes, including modulation of inflammation and oxidative stress. We tested the hypothesis that choline supplementation would ameliorate the dystrophic pathology in mdx mice. Three-week old male mdx mice (n = 40) were fed control purified laboratory chow (CON; n = 20) or a choline-enriched diet (5 g/kg choline; CHL, n = 20) for four weeks. Rotarod performance, grip strength and running (wheel) distance were assessed during treatment. Markers of Ca2+-handling, inflammation, oxidative stress and fibrosis were measured in the diaphragm, quadriceps muscle and the liver. Choline-treated mdx mice displayed less macrophage (CD68 -33%, P < 0.05) and collagen infiltration (−34%, P < 0.05) and reduced Tgfβ3 mRNA expression (P < 0.05) in the diaphragm compared to untreated mdx mice. Choline supplementation increased maximal SERCA activity (38%, P < 0.05) and reduced markers of inflammatory (Tnfα, F4/80 and Cd206 mRNA, P < 0.05) processes compared with untreated mdx mice. In the liver there was a reduction in Acta2 mRNA (P < 0.05) with choline treatment, as well as an improvement in serum ALT levels (P < 0.01). There were no differences between the groups for the whole-body functional analyses. Choline supplementation attenuated the progression of the dystrophic pathology. Although choline did not alter functional performance, the reduction in fibrosis is clinically relevant for increasing the efficacy of therapies for DMD.

Cytotoxic Swelling of Sick Excitable Cells - Impaired Ion Homeostasis and Membrane Tension Homeostasis in Muscle and Neuron.

When they become simultaneously leaky to both Na+ and Cl-, excitable cells are vulnerable to potentially lethal cytotoxic swelling. Swelling ensues in spite of an isosmotic milieu because the entering ions add osmolytes to the cytoplasm's high concentration of impermeant anionic osmolytes. An influx of osmotically-obliged water is unavoidable. A cell that cannot stanch at least one the leaks will succumb to death by Donnan effect. "Sick excitable cells" are those injured through ischemia, trauma, inflammation, hyperactivity, genetically-impaired membrane skeletons and other insults, all of which foster bleb-damage to regions of the plasma membrane. Nav channels resident in damaged membrane exhibit left-shifted kinetics; the corresponding Nav window conductance constitutes a Na+-leak. In cortical neurons, sustained depolarization to ∼-20mV elicits a sustained lethal gCl. Underlying Vrest in skeletal muscle is a constitutively active gCl; not surprisingly therefore, dystrophic muscle fibers, which are prone to bleb damage and which exhibit Nav-leak and Na+-overload, are prone to cytotoxic swelling. To restore viability in cytotoxically swelling neurons and muscle, the imperative of fully functional ion homeostasis is well-recognized. However, as emphasized here, in a healthy excitable cell, fully functional membrane tension homeostasis is also imperative. ATPase-pumps keep plasma membrane batteries charged, and ATPase-motor proteins maintain membrane tone. In sick excitable cells, neither condition prevails.

Lectin-binding characterizes the healthy human skeletal muscle glycophenotype and identifies disease-specific changes in dystrophic muscle.

Our understanding of muscle glycosylation to date has derived from studies in mouse models and a limited number of human lectin histochemistry studies. As various therapeutic approaches aimed at treating patients with muscular dystrophies are being translated from rodent models to human, it is critical to better understand human muscle glycosylation and relevant disease-specific differences between healthy and dystrophic muscle. Here, we report the first quantitative characterization of human muscle glycosylation, and identify differentiation- and disease-specific differences in human muscle glycosylation. Utilizing a panel of 13 lectins with varying glycan specificities, we surveyed lectin binding to primary and immortalized myoblasts and myotubes from healthy and dystrophic sources. Following differentiation of primary and immortalized healthy human muscle cells, we observed increased binding of Narcissus pseudonarcissus agglutinin (NPA), PNA, MAA-II and WFA to myotubes compared to myoblasts. Following differentiation of immortalized healthy and dystrophic human muscle cells, we observed disease-specific differences in binding of NPA, Jac and Tricosanthes japonica agglutinin-I (TJA-I) to differentiated myotubes. We also observed differentiation- and disease-specific differences in binding of NPA, Jac, PNA, TJA-I and WFA to glycoprotein receptors in muscle cells. Additionally, Jac, PNA and WFA precipitated functionally glycosylated α-DG, that bound laminin, while NPA and TJA-I did not. Lectin histochemistry of healthy and dystrophic human muscle sections identified disease-specific differences in binding of O-glycan and sialic acid-specific lectins between healthy and dystrophic muscle. These results indicate that specific and discrete changes in glycosylation occur following differentiation, and identify specific lectins as potential biomarkers sensitive to changes in healthy human muscle glycosylation.

Interleukin-6 and neuregulin-1 as regulators of utrophin expression via the activation of NRG-1/ErbB signaling pathway in mdx cells

Duchenne muscular dystrophy (DMD) is a neuromuscular disease originated by mutations in the dystrophin gene. A promising therapeutic approach deals with functional substitution of dystrophin by utrophin, a structural homolog that might be able to compensate dystrophin absence in DMD muscle fibers.It has been described that both interleukin-6 (IL-6) and neuregulin-1 (NRG-1; Heregulin-HRG) induce utrophin expression in skeletal muscle. We investigated a possible functional link among IL-6, NRG-1 and utrophin, in normal (C57) and dystrophic (mdx) skeletal muscle cells. Western Blot analysis allowed us to demonstrate that IL-6 (100ng/mL) induces NRG-1 receptor phosphorylation (ErbB2/ErbB3) in both cell types, in a process that depends on intracellular Ca2+ and metalloproteinase activity; it also induces a transient increase of ERK1 and GABPα phosphorylation only in dystrophic myotubes. Semiquantitative PCR showed that IL-6 treatment increases utrophin mRNA levels just in mdx myotubes. We observed that utrophin mRNA induction was abolished by BAPTA-AM (an intracellular Ca2+ chelator), GM6001 (a general metalloproteinase inhibitor), genistein (a general protein tyrosine kinase inhibitor), PD-158780 (an ErbB receptor tyrosine kinase inhibitor) and PD-98059 (a MEK inhibitor), whereas Ly-294002 and wortmannin (PI3K inhibitors) did not affect utrophin induction evoked by IL-6 in dystrophic myotubes.Our results suggest that IL-6 induces utrophin expression in mdx myotubes through activation of a NRG-1/ErbBs signaling cascade. Soluble NRG-1 elicited by proteolytic processing of transmembrane NRG-1 might induce ErbBs phosphorylation and ERK1/2 pathway activation, leading to utrophin up-regulation.

Mitochondria mediate cell membrane repair and contribute to Duchenne muscular dystrophy

Dystrophin deficiency is the genetic basis for Duchenne muscular dystrophy (DMD), but the cellular basis of progressive myofiber death in DMD is not fully understood. Using two dystrophin-deficient mdx mouse models, we find that the mitochondrial dysfunction is among the earliest cellular deficits of mdx muscles. Mitochondria in dystrophic myofibers also respond poorly to sarcolemmal injury. These mitochondrial deficits reduce the ability of dystrophic muscle cell membranes to repair and are associated with a compensatory increase in dysferlin-mediated membrane repair proteins. Dysferlin deficit in mdx mice further compromises myofiber cell membrane repair and enhances the muscle pathology at an asymptomatic age for dysferlin-deficient mice. Restoring partial dystrophin expression by exon skipping improves mitochondrial function and offers potential to improve myofiber repair. These findings identify that mitochondrial deficit in muscular dystrophy compromises the repair of injured myofibers and show that this repair mechanism is distinct from and complimentary to the dysferlin-mediated repair of injured myofibers.

Dystrophin: The dead calm of a dogma.

ABSTRACTDuchenne muscular dystrophy (DMD) is the most common inherited muscle disease leading to severe disability and death of young men. Current interventions are palliative as no treatment improves the long-term outcome. Therefore, new therapeutic modalities with translational potential are urgently needed and abnormalities downstream from the absence of dystrophin are realistic targets. It has been shown that DMD mutations alter extracellular ATP (eATP) signaling via P2RX7 purinoceptor upregulation, which leads to autophagic death of dystrophic muscle cells. Furthermore, the eATP-P2RX7 axis contributes to DMD pathology by stimulating harmful inflammatory responses. We demonstrated recently that genetic ablation or pharmacological inhibition of P2RX7 in the mdx mouse model of DMD produced functional attenuation of both muscle and non-muscle symptoms, establishing this receptor as an attractive therapeutic target. Central to the argument presented here, this purinergic phenotype affects dystrophic myoblasts. Muscle cells were believed not to be affected at this stage of differentiation, as they do not produce detectable dystrophin protein. Our findings contradict the central hypothesis stating that aberrant dystrophin expression is inconsequential in myoblasts and the DMD pathology results from effects such as sarcolemma fragility, due to the absence of dystrophin, in differentiated myofibres. However, we discuss here the evidence that, already in myogenic cells, DMD mutations produce a plethora of abnormalities, including in cell proliferation, differentiation, energy metabolism, Ca2+ homeostasis and death, leading to impaired muscle regeneration. We hope that this discussion may bring to light further results that will help re-evaluating the established belief. Clearly, understanding how DMD mutations alter such a range of functions in myogenic cells is vital for developing effective therapies.

3D culture system of muscle precursor cell to reveal mechanosensing defects in nuclear envelope related disorders

Mutations in the LInker of the Nucleoskeleton and Cytoskeleton (LINC)-complex associated proteins, including lamins and nesprins cause human muscular dystrophies but disease mechanisms still remain to be elucidated. We aim to determine whether human muscular dystrophies resulting from mutations in A-type lamin and nesprin1 affected the capacity of myoblasts to sense the stiffness of the extracellular matrix. Human myoblasts with various mutations in the A-type lamins encoded by LMNA (LMNA), nesprin1 mutant (SYNE1), and control (WT) myoblasts were cultured in 3D soft matrix (1-10 kPa) or on 2D conventional glass (~ 106 kPa) surfaces. Focal adhesion (vinculin), actin cytoskeleton, and YAP signaling pathway, a particularly important regulator of the mechano-response, were investigated. On 2D hard surface, there was no obvious difference in actin cytoskeleton and focal adhesion between WT, LMNA and SYNE1 myoblasts. In contrast, LMNA and SYNE1 myoblasts cultured in soft matrix exhibited enlarged focal adhesions and stress fibers compared with WT. Cytoplasmic translocation of YAP observed in WT in response to reduced stiffness matrix was absent in LMNA and SYNE1 cells, suggesting a permanent activation of YAP in mutant cells. In conclusion, our data indicate that cell culture matrix stiffness is critical to reveal mechanosensing defects in dystrophic muscle cells.

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.

Altered ROS production, NF-κB activation and interleukin-6 gene expression induced by electrical stimulation in dystrophic mdx skeletal muscle cells

Duchenne muscular dystrophy is a fatal X-linked genetic disease, caused by mutations in the dystrophin gene, which cause functional loss of this protein. This pathology is associated with an increased production of reactive oxygen (ROS) and nitrogen species. The aim of this work was to study the alterations in NF-κB activation and interleukin-6 (IL-6) expression induced by membrane depolarization in dystrophic mdx myotubes. Membrane depolarization elicited by electrical stimulation increased p65 phosphorylation, NF-κB transcriptional activity and NF-κB-dependent IL-6 expression in wt myotubes, whereas in mdx myotubes it had the opposite effect. We have previously shown that depolarization-induced intracellular Ca2+ increases and ROS production are necessary for NF-κB activation and stimulation of gene expression in wt myotubes. Dystrophic myotubes showed a reduced amplitude and area under the curve of the Ca2+ transient elicited by electrical stimulation. On the other hand, electrical stimuli induced higher ROS production in mdx than wt myotubes, which were blocked by NOX2 inhibitors. Moreover, mRNA expression and protein levels of the NADPH oxidase subunits: p47phox and gp91phox were increased in mdx myotubes. Looking at ROS-dependence of NF-κB activation we found that in wt myotubes external administration of 50μM H2O2 increased NF-κB activity; after administration of 100 and 200μM H2O2 there was no effect. In mdx myotubes there was a dose-dependent reduction in NF-κB activity in response to external administration of H2O2, with a significant effect of 100μM and 200μM, suggesting that ROS levels are critical for NF-κB activity. Prior blockage with NOX2 inhibitors blunted the effects of electrical stimuli in both NF-κB activation and IL-6 expression. Finally, to ascertain whether stimulation of NF-κB and IL-6 gene expression by the inflammatory pathway is also impaired in mdx myotubes, we studied the effect of lipopolysaccharide on both NF-κB activation and IL-6 expression. Exposure to lipopolysaccharide induced a dramatic increase in both NF-κB activation and IL-6 expression in both wt and mdx myotubes, suggesting that the altered IL-6 gene expression after electrical stimulation in mdx muscle cells is due to dysregulation of Ca2+ release and ROS production, both of which impinge on NF-κB signaling. IL-6 is a key metabolic modulator that is released by the skeletal muscle to coordinate a multi-systemic response (liver, muscle, and adipocytes) during physical exercise; the alteration of this response in dystrophic muscles may contribute to an abnormal response to contraction and exercise.

The effect of an ActRIIB ligand trapping agent on the number of satellite cells and myonuclei in dystrophic (mdx) muscle fibers

Duchenne muscular dystrophy is characterized by repeated cycles of degeneration and regeneration ultimately leading to respiratory failure and death. Muscle growth is largely under the control of myostatin, which negatively influences fiber growth and regeneration by binding to the ActRIIB receptor. Ligand trapping agents for the ActRIIB receptor (RAP‐435) are therefore currently being tested as therapeutic agents using the mdx mouse model for Duchenne muscular dystrophy. The present study utilized confocal immunochemical techniques to examine the effects of in vivo RAP‐435 treatment on the number of satellite cells and myonuclei associated with isolated mdx costal diaphragm fibers. One month old mdx mice were treated with RAP435 or vehicle for one month. Following treatment, the isolated muscles were exposed to 0.2% collagenase. Satellite cells were identified with a PAX 7 primary and an AlexaFluor 488 secondary antibody. Myonuclei were identified by DAPI. Mdx costal diaphragm fibers exhibited approximately 700 myonuclei and 5 satellite cells per muscle fiber. Experiments comparing specific age‐matched nondystrophic and mdx respiratory muscles will determine if the regeneration characteristic of dystrophic muscle is associated with corresponding increases in the number of myonuclei per fiber. RAP‐435 treatment influenced neither the number of myonuclei nor satellite cells per muscle fiber. Morphometric analyses are being performed in conjunction with these confocal studies to determine if ActRIIB inhibition specifically influences myonuclear domain in promoting fiber growth and regeneration.