Mdx Myotubes Research Articles

Guanidino quinazolines and pyrimidines promote readthrough of premature termination codons in cells with native nonsense mutations

Using small molecules to induce readthrough of premature termination codons is a promising therapeutic approach to treating genetic diseases and cancers caused by nonsense mutations, as evidenced by the widespread use of ataluren to treat nonsense mutation Duchene muscular dystrophy. Herein we describe a series of novel guanidino quinazoline and pyrimidine scaffolds that induce readthrough in both HDQ-P1 mammary carcinoma cells and mdx myotubes. Linkage of basic, tertiary amines with aliphatic, hydrophobic substituents to the terminal guanidine nitrogen of these scaffolds led to significant potency increases. Further potency gains were achieved by flanking the pyrimidine ring with hydrophobic substituents, inducing readthrough at concentrations as low as 120 nM and demonstrating the potential of these compounds to be used either in combination with ataluren or as stand-alone therapeutics.

Cross-talk between TRPC-1, mTOR, PGC-1α and PPARδ in the dystrophic muscle cells treated with tempol

Background Ca2+ dysregulation and oxidative damage appear to have a central role in Duchenne muscular dystrophy (DMD) progression. The current study provides muscle cell-specific insights into the effect of Tempol on the TRPC 1 channel; on the positive and negative regulators of muscle cell differentiation; on the antioxidant enzymatic system; on the activators of mitochondrial biogenesis; and on the inflammatory process in the dystrophic primary muscle cells in culture. Methods: Mdx myotubes were treated with Tempol (5 mM) for 24 h. Untreated mdx myotubes and C57BL/10 myotubes were used as controls. Results: The Trypan Blue, MTT and Live/Dead Cell assays showed that Tempol (5 mM) presented no cytotoxic effect on the dystrophic muscle cells. The Tempol treated-mdx muscle cells showed significantly lower levels in the fluorescence intensity of intracellular calcium; TRPC-1 channel; MyoD; H2O2 and O2 •− production; 4-HNE levels; SOD2, CAT and GPx levels; and TNF levels. On the other hand, SOD, CAT and GR mRNA relative expression were significantly higher in Tempol treated-mdx muscle cells. In addition, higher levels of Myogenin, MHC-Slow, mTOR, PGC-1α and PPARδ were also observed in Tempol treated-mdx muscle cells. Conclusion: Our findings demonstrated that Tempol decreased intracellular calcium and oxidative stress in primary dystrophic muscle cells, promoting a cross-talk between TRPC-1, mTOR, PGC-1α and PPARδ.

Antisense oligonucleotide modified with serinol nucleic acid (SNA) induces exon skipping in mdx myotubes

We investigated the potential of SNA-modified antisense oligonucleotide (AO) for exon-skipping. We found that a 20-mer SNA-AO induced efficient exon-23 skipping in the mouse dystrophin gene transcript.

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.

Agents Which Inhibit NF-κB Signaling Block Spontaneous Contractile Activity and Negatively Influence Survival of Developing Myotubes.

Inhibiting the NF-κB signaling pathway provides morphological and functional benefits for the mdx mouse, a model for Duchenne muscular dystrophy characterized by chronic elevations in the nuclear expression of p65, the transactivating component of the NF-κB complex. The purpose of this study was to examine p65 expression in nondystrophic and mdx myotubes using confocal immunofluorescence, and determine whether inhibitors of the NF-κB pathway alter myotube development. Primary cultures of nondystrophic and mdx myotubes had identical levels of nuclear and cytosolic p65 expression and exhibited equivalent responses to TNF-α, thus excluding the hypothesis that the lack of dystrophin is sufficient to induce increases in NF-κB signaling. The NF-κB inhibitors pyrrolidine dithiocarbamate (PDTC) and sulfasalazine decreased spontaneous contractile activity and reduced myotube viability in a dose- and time-dependent manner. Similarly, a vivo-morpholino designed to block translation of murine p65 (m-p65tb-vivomorph1) rapidly abolished spontaneous contractile activity, reduced p65 expression measured by confocal immunofluorescence, and induced cell death in primary cultures of nondystrophic and mdx myotubes. Similar effects on p65 immunofluorescence and cell viability were observed following m-p65tb-vivomorph1 exposure to spontaneously inactive C2C12 myotubes, while exposure to a control scrambled vivo morpholino had no effect. These results indicate a direct role of the NF-κB pathway in myotube development and identify a potential therapeutic limitation to the use of NF-κB inhibitors in treating Duchenne and related muscular dystrophies. J. Cell. Physiol. 231: 788-797, 2016. © 2015 Wiley Periodicals, Inc.

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.

Elevation of NO Increases Ca2+ Entry and Resting Ca2+ and Na+ Concentrations in Skeletal Muscle Cells

Protein S-nitrosylation is a reversible post-translational modification and can have profound effects on protein function in skeletal muscle. S-nitrosylation has been proposed to exert regulatory effects on ion channels such as RyR1 and TRPCs, by modifying the redox state of critical thiols. Increased nitric oxide (NO) levels may play a crucial role in the alteration of intracellular resting [Ca2+]r and [Na+]r homeostasis in skeletal muscle. The present work aimed to characterize the contribution of NO using the nitric oxide donor S-nitroso-N-acetylpenicillamine (SNAP) on intracellular Ca2+ and Na+ homeostasis in Wt myotubes. In quiescent Wt myotubes 100 µM SNAP increased Ca2+ entry, [Ca2+]r and [Na+]r. These effects could be blocked with either Gd3+ or BTP-2 and partially reversed by DTT, a known inhibitor of S nitrosylation, suggesting that the effect of SNAP was through a reversible increase in the TRPC channel activity. Furthermore, we found that RyRs are not the principal target for S-nitrosylation because SNAP incubation increases [Ca2+]r and [Na+]r in RyR1/RYR3 null myotubes. It is known that NO production is elevated in mdx muscle. Here we found that DTT or BTP-2 significantly reduced [Ca2+]r in mdx myotubes and DTT was able to restore a normal Ca2+ entry. We then looked at the effects of SNAP on K+ induced Ca2+ release and SR Ca2+ content in WT and mdx myotubes. SNAP did not significantly decrease sarcoplasmic reticulum Ca2+ content in either WT or mdx myotubes but reduced the magnitude of the Ca2+ transient induced by K+ in Wt but not in mdx myotubes. These results suggest that nitrosative modifications play a key role regulating TRPC activity in muscular dystrophy and their role in the Ca2+ and Na+ overload seen in dystrophic muscles.

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 gp91phox/p47phox 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 iPLA2β and of stretch‐activated channels by doxorubicin alters dystrophic muscle function

Chronic elevation in intracellular Ca(2+) concentration participates in death of skeletal muscle from mdx mice, a model for Duchenne muscular dystrophy (DMD). Candidate pathways mediating this Ca(2+) overload involve store-operated channels (SOCs) and stretch-activated channels (SACs), which are modulated by the Ca(2+) -independent form of PL A2 (iPLA2 ). We investigated the effect of doxorubicin (Dox), a chemotherapeutic agent reported to inhibit iPLA2 in other systems, on the activity of this enzyme and on the consequences on Ca(2+) handling and muscle function in mdx mice. Effects of Dox on iPLA2 activity, reactive oxygen species production and on Ca(2+) influx were investigated in C2C12 and mdx myotubes. The mechanism of Dox-mediated iPLA2 inhibition was evaluated using purified 6x histidine-tagged enzyme. Aequorin technology was used to assess Ca(2+) concentrations underneath the plasma membrane. Isolated muscles were exposed to fatigue protocols and eccentric contractions to evaluate the effects of Dox on muscle function. Dox at 1-30 μM inhibited iPLA2 activity in cells and in the purified enzyme. Dox also inhibited SAC- but not SOC-mediated Ca(2+) influx in myotubes. Stimulated elevations of Ca(2+) concentrations below the plasmalemma were also blocked. Exposure of excised muscle to Dox was not deleterious to force production and promoted recovery from eccentric contractions. Dox showed efficacy against targets known to play a role in the pathology of DMD, namely iPLA2 and SAC. The potent SAC inhibitory effect of Dox is a novel finding that can explain partly the cardiomyopathy seen in chronic anthracycline treatment.

NF‐kB–dependent IL‐6 expression, induced by electrical stimulation, is reduced in dystrophic mdx skeletal muscle cells.

In skeletal muscle cells, membrane depolarization increases reactive oxygen species (ROS) due to NOX2 activation; this is related with changes in gene expression. Our aim was to study IL‐6 gene in wt and mdx muscle cells, and its relationship with NOX2 and NF‐kB activity. Differentiated wt and mdx myotubes were stimulated with 250p, 0.5 ms at 20 Hz. ROS production was assessed with CM‐DCFA. NOX2 was assessed using apocynin and DPI. NF‐kB transcriptional activity was studied with a luciferase reporter, p65 phosphorylation by western blot and gene expression by real time PCR.NOX2 subunits (gp91 and p47) were upregulated in mdx myotubes. After stimulation, ROS production was higher in mdx compared to wt. NF‐kB activity after both electrical and ROS stimulation increased in wt myotubes, but not in mdx. NOX2 inhibition reduced NF‐kB activation. IL‐6 gene expression induced by both electrical and ROS stimulation was higher in wt compared to mdx muscle cells.ROS production and NF‐kB activity are linked in normal cells. Altered NF‐kB, excess ROS production and differential gene expression could explain oxidative stress and progressive damage reported in DMD.Fondecyt 1110467 (EJ), AT‐24100066 (FA).

Dystrophin deficiency leads to disturbance of LAMP1-vesicle-associated protein secretion.

Duchenne muscular dystrophy results from loss of the protein dystrophin, which links the intracellular cytoskeletal network with the extracellular matrix, but deficiency in this function does not fully explain the onset or progression of the disease. While some intracellular events involved in the degeneration of dystrophin-deficient muscle fibers have been well characterized, changes in their secretory profile are undescribed. To analyze the secretome profile of mdx myotubes independently of myonecrosis, we labeled the proteins of mdx and wild-type myotubes with stable isotope-labeled amino acids (SILAC), finding marked enrichment of vesicular markers in the mdx secretome. These included the lysosomal-associated membrane protein, LAMP1, that co-localized in vesicles with an over-secreted cytoskeletal protein, myosin light chain 1. These LAMP1/MLC1-3-positive vesicles accumulated in the cytosol of mdx myotubes and were secreted into the culture medium in a range of abnormal densities. Restitution of dystrophin expression, by exon skipping, to some 30 % of the control value, partially normalized the secretome profile and the excess LAMP1 accumulation. Together, our results suggest that a lack of dystrophin leads to a general dysregulation of vesicle trafficking. We hypothesize that disturbance of the export of proteins through vesicles occurs before, and then concurrently with, the myonecrotic cascade and contributes chronically to the pathophysiology of DMD, thereby presenting us with a range of new potential therapeutic targets.

Altered mitogen‐activated protein kinase signaling in dystrophic (mdx) muscle

Duchenne muscular dystrophy (DMD) results from a deficiency in the protein, dystrophin. Dystrophic myotubes are susceptible to stressful stimuli. This may be partly due to altered regulation of pro-survival signaling pathways, but a role for mitogen-activated protein (MAP) kinases has not been investigated. We examined patterns of phosphorylation of key MAP kinase proteins in cultured myotubes responding to oxidative stress, and in muscle tissue in vivo. Dystrophic (mdx) myotubes have an increased susceptibility to oxidant-induced death compared with wild-type (C57Bl/10ScSn) myotubes. This correlates with late phosphorylation of c-Jun N-terminal kinase (JNK), and persistently high p38 MAP kinase phosphorylation in mdx myotubes. JNK and extracellular signal-regulated kinase 1/2 (ERK1/2) also showed altered phosphorylation levels in mdx muscle tissue. We show altered patterns of MAP kinase protein phosphorylation in dystrophic muscle in vitro and in vivo. These pathways may be novel pharmacological targets for treating DMD.

AMPK Activation Stimulates Autophagy and Ameliorates Muscular Dystrophy in the mdx Mouse Diaphragm

Duchenne muscular dystrophy (DMD) is characterized by myofiber death from apoptosis or necrosis, leading in many patients to fatal respiratory muscle weakness. Among other pathological features, DMD muscles show severely deranged metabolic gene regulation and mitochondrial dysfunction. Defective mitochondria not only cause energetic deficiency, but also play roles in promoting myofiber atrophy and injury via opening of the mitochondrial permeability transition pore. Autophagy is a bulk degradative mechanism that serves to augment energy production and eliminate defective mitochondria (mitophagy). We hypothesized that pharmacological activation of AMP-activated protein kinase (AMPK), a master metabolic sensor in cells and on-switch for the autophagy-mitophagy pathway, would be beneficial in the mdx mouse model of DMD. Treatment of mdx mice for 4 weeks with an established AMPK agonist, AICAR (5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside), potently triggered autophagy in the mdx diaphragm without inducing muscle fiber atrophy. In AICAR-treated mdx mice, the exaggerated sensitivity of mdx diaphragm mitochondria to calcium-induced permeability transition pore opening was restored to normal levels. There were associated improvements in mdx diaphragm histopathology and in maximal force-generating capacity, which were not linked to increased mitochondrial biogenesis or up-regulated utrophin expression. These findings suggest that agonists of AMPK and other inducers of the autophagy-mitophagy pathway can help to promote the elimination of defective mitochondria and may thus serve as useful therapeutic agents in DMD.

Increased Resting Intracellular Calcium Modulates NF-κB-dependent Inducible Nitric-oxide Synthase Gene Expression in Dystrophic mdx Skeletal Myotubes

Duchenne muscular dystrophy (DMD) is a genetic disorder caused by dystrophin mutations, characterized by chronic inflammation and severe muscle wasting. Dystrophic muscles exhibit activated immune cell infiltrates, up-regulated inflammatory gene expression, and increased NF-κB activity, but the contribution of the skeletal muscle cell to this process has been unclear. The aim of this work was to study the pathways that contribute to the increased resting calcium ([Ca(2+)](rest)) observed in mdx myotubes and its possible link with up-regulation of NF-κB and pro-inflammatory gene expression in dystrophic muscle cells. [Ca(2+)](rest) was higher in mdx than in WT myotubes (308 ± 6 versus 113 ± 2 nm, p < 0.001). In mdx myotubes, both the inhibition of Ca(2+) entry (low Ca(2+) solution, Ca(2+)-free solution, and Gd(3+)) and blockade of either ryanodine receptors or inositol 1,4,5-trisphosphate receptors reduced [Ca(2+)](rest). Basal activity of NF-κB was significantly up-regulated in mdx versus WT myotubes. There was an increased transcriptional activity and p65 nuclear localization, which could be reversed when [Ca(2+)](rest) was reduced. Levels of mRNA for TNFα, IL-1β, and IL-6 were similar in WT and mdx myotubes, whereas inducible nitric-oxide synthase (iNOS) expression was increased 5-fold. Reducing [Ca(2+)](rest) using different strategies reduced iNOS gene expression presumably as a result of decreased activation of NF-κB. We propose that NF-κB, modulated by increased [Ca(2+)](rest), is constitutively active in mdx myotubes, and this mechanism can account for iNOS overexpression and the increase in reactive nitrogen species that promote damage in dystrophic skeletal muscle cells.

Increased Resting Calcium Modulates NF-κB Activity and iNOS Expression in mdx Myotubes

Duchenne muscular dystrophy (DMD) is a genetic disorder characterized by severe muscle wasting. Dystrophic muscles exhibit activated immune cell infiltrates with up-regulated inflammatory gene expression and increased NF-κB activity, but the contribution of the skeletal muscle cell to this process has been unclear. The aim of this work was to study the resting calcium [Ca2+]rest deregulation and its possible link with NF-κB up-regulation and iNOS expression in mdx myotubes.[Ca2+]rest was measured with Ca2+-selective microelectrodes and NF-κB transcriptional activity was studied using luciferase reporter and immunofluorescence in wt and mdx myotubes. Gene expression was studied by real time PCR.[Ca2+]rest was higher in mdx than in wt myotubes (308±6 vs 113±2 nM, p<0.001). Both the inhibition of Ca2+ entry (Gd3+ and low Ca2+ solutions) and blockade of ryanodine (Ry) receptors or IP3 receptors (XeB), reduced [Ca2+]rest in mdx myotubes. Basal activity of NF-κB was significantly up-regulated in mdx myotubes. This was shown by an increased p65 nuclear localization and increased transcriptional activity, which could be reversed by inhibitors that reduced [Ca2+]rest. Levels of mRNA for TNFα, IL-1β and IL-6 were similar in wt and mdx myotubes, whereas iNOS expression was increased 5-fold in mdx myotubes. Moreover, both NF-κB inhibition and [Ca2+]rest inhibitors reduced iNOS gene expression.We propose that NF-κB is constitutively active in mdx myotubes, modulated by increased [Ca2+]rest and this condition can account for iNOS overexpression in dystrophic myotubes. We hypothesize that the differences in NF-κB activity may help to understand the mechanisms of muscle damage in DMD.Fondecyt 1110467, FONDAP15010006 and AFM14562 (EJ). NIH AR43140, AR052354 (PDA, JRL), AT-24100066 and VAA travel support, Universidad de Chile (FA).

Effect of Oxidative Stress on Intracellular Ca2+ and Na+ Concentration in Duchenne Muscular Dystrophy Myotubes

Duchenne muscular dystrophy (DMD) is an X-linked myopathy leading to progressive muscle weakness that is the most common and devastating type of muscular dystrophy in boys. Oxidative stress has been suggested as a potential upstream contributor to the skeletal muscle damage in DMD. Here we test this hypothesis by comparing the effect of ROS in intracellular concentrations of Ca2+ and Na+. Intracellular calcium concentration ([Ca2+]i) and intracellular Na+ concentration ([Na+]i) were measured using Ca2+ and Na+ selective microelectrodes in Wt and mdx myotubes. Mdx myotubes showed significantly elevated [Ca2+]i and [Na+]i in comparison to Wt cells. In mdx myotubes [Ca2+]i was 333±35 nM (mean±SD, n=35) and [Na+]i was 17.5±1.3 mM (n=15)versus115±10 nM (n=40) and 7.9±0.6 mM, (n=15) in Wt. Ca2+ influx at rest measured by Mn2+ quenching also reveled increased Ca2+ entry in mdx. The effect of ROS on cultured myotubes was tested by exposing myotubes to 10μM H2O2, a concentration that did not alter cell viability. H2O2 caused a significant elevation of [Ca2+]i and [Na+]i in both mdx and Wt. However, the relative increase of both ions was much greater in mdx than Wt. Whereas in mdx [Ca2+]i reached 1167±138 nM (n=14) and [Na+]i 23.6±2 mM (n=11) in Wt cells these concentration were 152±11 nM (n=15) and 10.5±0.5 mM (n=14), respectively. In addition, pre-treatment of mdx and Wt myotubes with nonselective TRPC blockers 5μM BTP2 or 20μM gadolinium prevented the increase in [Ca2+]i and [Na+]i induced by H2O2. These results suggest that mdx myotubes are more susceptible than Wt to elevation of [Ca2+]i and [Na+]I induced by oxidative stress. Moreover, H2O2-induced dysregulation of [Ca2+]i and [Na+]i seems primarily mediated by Ca2+ entry.Supported by Grants P01AR47605 (to PDA) and 5K01AR054818 (to CFP)

Ip3-Dependent Calcium Transients Differentially Modulate Nf-Kappa B Activity in Wild Type and Mdx Myotubes

In normal skeletal myotubes, tetanic electrical stimulation activates NFκB through intracellular calcium increase released by inositol 1,4,5 triphosphate receptor (IP3R) and by Ryanodine receptors (RyR). In this work, we studied the differences in calcium transients evoked by electrical stimuli and NFκB activation in wild type (C57) and in mdx (animal model of Duchenne muscular dystrophy, DMD) myotubes. Myoblasts were obtained from C57 or mdx neonatal mice (1-3 days). Myotubes were loaded with fluo-3AM and stimulated with 250 pulses of 0.5 ms duration at 20 Hz and the calcium signal was assessed by fluorescence microscopy. NFκB activation was determined by luciferase reporter assay, p65 immunofluorescence and p65-GFP translocation. Fast calcium signal (RyR dependent) was significantly reduced in electrically stimulated mdx compared to wild type C57 myotubes (0.62 vs 1.41 RLU, p<0.01). However, the amplitude and kinetics of (IP3 dependent) slow calcium signals were similar in mdx and C57 myotubes. Electrical stimulation increased the NFκB luciferase activity in normal but not in mdx myotubes. By immunofluorescence, p65-GFP translocation studies and reporter assays we determined that the basal activity of this transcription factor is up-regulated in mdx myotubes and this could be blocked by inhibitors of IP3-related pathways (100 µM suramine, 10 µM BAPTA-AM, 5 µM xestospongin B) but not by ryanodine (30 µM). We propose that NFκB up regulation in mdx myotubes is modulated by intracellular calcium release through IP3R. The differences in NFκB activation between normal and dystrophic muscle cells may help to understand the mechanisms of pro-inflamatory cytokines gene expression in Duchenne muscular dystrophy. FONDAP 15010006, AFM14562. CONICYT graduate student fellowship and doctoral support AT-24100066.

Halofuginone inhibits Smad3 phosphorylation via the PI3K/Akt and MAPK/ERK pathways in muscle cells: Effect on myotube fusion

Halofuginone, a novel inhibitor of Smad3 phosphorylation, has been shown to inhibit muscle fibrosis and to improve cardiac and skeletal muscle functions in the mdx mouse model of Duchenne muscular dystrophy. Here, we demonstrate that halofuginone promotes the phosphorylation of Akt and mitogen-activated protein kinase (MAPK) family members in a C2 muscle cell line and in primary myoblasts derived from wild-type and mdx mice diaphragms. Halofuginone enhanced the association of phosphorylated Akt and MAPK/extracellular signal-regulated protein kinase (ERK) with the non-phosphorylated form of Smad3, accompanied by a reduction in Smad3 phosphorylation levels. This reduction was reversed by inhibitors of the phosphoinositide 3′-kinase/Akt (PI3K/Akt) and MAPK/ERK pathways, suggesting their specific role in mediating halofuginone's inhibitory effect on Smad3 phosphorylation. Halofuginone enhanced Akt, MAPK/ERK and p38 MAPK phosphorylation and inhibited Smad3 phosphorylation in myotubes, all of which are crucial for myotube fusion. In addition, halofuginone increased the association Akt and MAPK/ERK with Smad3. As a consequence, halofuginone promoted myotube fusion, as reflected by an increased percentage of C2 and mdx myotubes containing high numbers of nuclei, and this was reversed by specific inhibitors of the PI3K and MAPK/ERK pathways. Together, the data suggest a role, either direct or via inhibition of Smad3 phosphorylation, for Akt or MAPK/ERK in halofuginone-enhanced myotube fusion, a feature which is crucial to improving muscle function in muscular dystrophies.

Nonsense suppression activity of PTC124 (ataluren)

Auld et al. (1) suggest that PTC124's nonsense suppression activity may be an indirect consequence of the compound's effects on firefly luciferase (FLuc) enzymatic activity. However, our initial characterization of potential nonsense-suppressing compounds in FLuc assays also utilized independent assays of nonsense suppression in disease-relevant systems, including assays measuring synthesis of full-length protein in mdx myotubes, mdx mice, and Cftr−/− transgenic mice (2, 3). These tests eliminated inactive chemical scaffolds and identified PTC124. Subsequently, PTC124 demonstrated nonsense suppression activity in cystic fibrosis and Duchenne muscular dystrophy patients (2, 4). These independent confirmations in validated suppression assays are to be contrasted with Auld et al.'s (1) use of a Renilla luciferase reporter that was an unsuitable and misinterpreted gauge of nonsense suppression because it failed to respond appreciably to the established nonsense-suppressing aminoglycoside, G418.

MECHANOSENSITIVE ION CHANNELS IN SKELETAL MUSCLE: A LINK IN THE MEMBRANE PATHOLOGY OF MUSCULAR DYSTROPHY

1. Mechanosensitive (MS) channels are expressed abundantly in skeletal muscle at all stages of development. In recordings from membrane patches, MS channels are constitutively active at the resting potential. The channels are selective for cations and have a large single-channel conductance (approximately 25 pS in physiological saline) and a high Ca2+ permeability (relative permeability of Ca2+ to K+ (PCa/PK) = 7). 2. Mechanosensitive channel activity recorded from the surface of myotubes from dystrophic mdx mice was substantially greater than the activity recorded from wild-type myotubes. Increased channel activity in the mutant results from the induction in a subpopulation of channels of a novel MS gating mode characterized by markedly prolonged channel openings and inactivation in response to membrane stretch. 3. Membrane stretch or a strong depolarization causes an irreversible switch to the stretch-inactivated gating mode in mdx myotubes. A stretch-induced shift in MS channel gating mode may contribute to stretch-induced elevations in [Ca2+]i during the early stages of disease pathogenesis. 4. Abnormalities of MS channel behaviour are also detected in recordings from patches on flexor digitorum brevis fibres acutely isolated from mdx mice. Mechanosensitive channel opening probability is higher in mdx fibres at all developmental stages. In addition, channel numbers are persistently elevated during postnatal development, failing to undergo a normal process of downregulation during the first 3 postnatal weeks. 5. Two distinct mechanisms may contribute to elevations of [Ca2+]i in dystrophin-deficient skeletal muscle: (i) a membrane stress-dependent switch of MS channels into to a prolonged opening mode; and (ii) a loss of developmental downregulation leading to persistent MS channel expression during postnatal muscle development.