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Reactive Oxygen Species (ROS) have been profusely studied as agents of potential damage to living cells and they have been related to a number of pathological processes. Increasing evidence points to a more positive role of ROS in cell signaling and the detailed mechanism that regulates the precise amount of ROS needed for cell functioning without the deleterious effects of excess ROS still needs to be resolved in detail. In skeletal muscle the main source of ROS during normal functioning appears to be NADPH oxidase 2 (NOX2), which is activated by electrical stimuli (or exercise) through a cascade of events that include ATP release through pannexin1 channels. NOX2 is a protein complex that assembles in the T-tubule membrane before activation and ROS production by NOX2 appears to be important for muscle adaptation through gene expression and mitochondrial biogenesis as well as for improving glucose transport after insulin action. Excess ROS production (or diminished antioxidant defenses) plays a role in a number of pathological processes in skeletal muscle. Together with increased reactive nitrogen species, an increase in ROS appears to have a deleterious role in a model of Duchenne muscular dystrophy as well as muscle wasting in other diseases such as aging sarcopenia and cancer cachexia. In addition, ROS is involved in obesity and muscle insulin resistance, both of which are causally related to type 2 diabetes. A detailed description of the fine-tuning of ROS (including all sources of ROS) in skeletal muscle in health and disease will significantly contribute to our knowledge of both muscle adaptation and muscle related pathologies. [ABSTRACT FROM AUTHOR]

Abstract: Mitochondria sense cytoplasmic Ca2+ signals in many cell types. In mammalian skeletal myotubes, depolarizing stimuli induce two independent cytoplasmic Ca2+ signals: a fast signal associated with contraction and a slow signal that propagates to the nucleus and regulates gene expression. How mitochondria sense and possibly affect these cytoplasmic Ca2+ signals has not been reported. We investigated here (a) the emergence of mitochondrial Ca2+ signals in response to electrical stimulation of myotubes, (b) the contribution of mitochondrial Ca2+ transients to ATP generation and (c) the influence of mitochondria as modulators of cytoplasmic and nuclear Ca2+ signals. Rhod2 and Fluo3 fluorescence determinations revealed composite Ca2+ signals associated to the mitochondrial compartment in electrically stimulated (400 pulses, 45Hz) skeletal myotubes. Similar Ca2+ signals were detected when using a mitochondria-targeted pericam. The fast mitochondrial Ca2+ rise induced by stimulation was inhibited by pre-incubation with ryanodine, whereas the phospholipase C inhibitor U73122 blocked the slow mitochondrial Ca2+ signal, showing that mitochondria sense the two cytoplasmic Ca2+ signal components. The fast but not the slow Ca2+ transient enhanced mitochondrial ATP production. Inhibition of the mitochondrial Ca2+ uniporter prevented the emergence of mitochondrial Ca2+ transients and significantly increased the magnitude of slow cytoplasmic Ca2+ signals after stimulation. Precluding mitochondrial Ca2+ extrusion with the Na+/Ca2+ exchanger inhibitor CGP37157 decreased mitochondrial potential, increased the magnitude of the slow cytoplasmic Ca2+ signal and decreased the rate of Ca2+ signal propagation from one nucleus to the next. Over expression of the mitochondrial fission protein Drp-1 decreased mitochondrial size and the slow Ca2+ transient in mitochondria, but enhanced cytoplasmic and nuclear slow transients. The present results indicate that mitochondria play a central role in the regulation of Ca2+ signals involved in gene expression in myotubes. [Copyright &y& Elsevier]

Summary: Brevetoxin-3 (PbTx-3), described to increase the open probability of voltage-dependent sodium channels, caused trains of action potentials and fast oscillatory changes in fluorescence intensity of fluo-3-loaded rat skeletal muscle cells in primary culture, indicating that the toxin increased intracellular Ca2+ levels. PbTx-3 did not elicit calcium transients in dysgenic myotubes (GLT cell line), lacking the α1 subunit of the dihydropyridine receptor (DHPR), but after transfection of the α1DHPR cDNA to GLT cells, PbTx-3 induced slow calcium transients that were similar to those of normal cells. Ca2+ signals evoked by PbTx-3 were inhibited by blocking either IP3 receptors, with 2-aminoethoxydiphenyl borate, or phospholipase C with U73122. PbTx-3 caused a tetrodotoxin-sensitive increase in intracellular IP3 mass levels, dependent on extra-cellular Na+. A similar increase in IP3 mass was induced by high K+ depolarization but no action potential trains (nor calcium signals) were elicited by prolonged depolarization under current clamp conditions. The increase in IP3 mass induced by either PbTx-3 or K+ was also detected in Ca2+-free medium. These results establish that the effect of the toxin on both intracellular Ca2+ and IP3 levels occurs via a membrane potential sensor instead of directly by Na+ flux and supports the notion of a train of action potentials being more efficient as a stimulus than sustained depolarization, suggesting that tetanus is the physiological stimulus for the IP3-dependent calcium signal involved in regulation of gene expression. [Copyright &y& Elsevier]

Membrane depolarization of skeletal muscle cells induces slow inositol trisphosphate-mediated calcium signals that regulate the activity of transcription factors such as the cAMP-response element-binding protein (CREB), jun, and fos. Here we investigated whether such signals regulate CREB phosphorylation via protein kinase C (PKC)-dependent pathways. Western blot analysis revealed the presence of seven isoforms (PKCα, -βI, -βII, -δ, -∈, -θ, and -ζ) in rat primary myotubes. The PKC inhibitors bisindolymaleimide I and Gö6976, blocked CREB phosphorylation. Chronic exposure to phorbol ester triggered complete down-regulation of several isoforms, but reduced PKCα levels to only 40%, and did not prevent CREB phosphorylation upon myotube depolarization. Immunocytochemical analysis revealed selective and rapid PKCα translocation to the nucleus following depolarization, which was blocked by 2amino-ethoxydiphenyl borate, an inositol trisphosphate receptor inhibitor, and by the phospholipase C inhibitor U73122. In C2Cl2 cells, which expressed PKCα, -∈, and -ζ, CREB phosphorylation also depended on PKCα. These results strongly implicate nuclear PKCα translocation in CREB phosphorylation induced by skeletal muscle membrane depolarization. [ABSTRACT FROM AUTHOR]

Skeletal muscle plays a central role in insulin-controlled glucose homeostasis. The molecular mechanisms related to insulin resistance in this tissue are incompletely understood. Herpud1 is an endoplasmic reticulum membrane protein that maintains intracellular Ca 2+ homeostasis under stress conditions. It has recently been reported that Herpud1 -knockout mice display intolerance to a glucose load without showing altered insulin secretion. The functions of Herpud1 in skeletal muscle also remain unknown. Based on these findings, we propose that Herpud1 is necessary for insulin-dependent glucose disposal in skeletal muscle. Here we show that Herpud1 silencing decreased insulin-dependent glucose uptake, GLUT4 translocation to the plasma membrane, and Akt Ser 473 phosphorylation in cultured L6 myotubes. A decrease in insulin-induced Akt Ser 473 phosphorylation was observed in soleus but not in extensor digitorum longus muscle samples from Herpud1 -knockout mice. Herpud1 knockdown increased the IP 3 R-dependent cytosolic Ca 2+ response and the activity of Ca 2+ -dependent serine/threonine phosphatase calcineurin in L6 cells. Calcineurin decreased insulin-dependent Akt phosphorylation and glucose uptake. Moreover, calcineurin inhibition restored the insulin response in Herpud1-depleted L6 cells. Based on these findings, we conclude that Herpud1 is necessary for adequate insulin-induced glucose uptake due to its role in Ca 2+ /calcineurin regulation in L6 myotubes. [ABSTRACT FROM AUTHOR]

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 (100 ng/mL) induces NRG-1 receptor phosphorylation (ErbB2/ErbB3) in both cell types, in a process that depends on intracellular Ca 2 + 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 Ca 2 + 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. [ABSTRACT FROM AUTHOR]

Homocysteine-inducible, endoplasmic reticulum (ER) stress-inducible, ubiquitin-like domain member 1 (HERPUD1), an ER resident protein, is upregulated in response to ER stress and Ca 2+ homeostasis deregulation. HERPUD1 exerts cytoprotective effects in various models, but its role during oxidative insult remains unknown. The aim of this study was to investigate whether HERPUD1 contributes to cytoprotection in response to redox stress and participates in mediating stress-dependent signaling pathways. Our data showed that HERPUD1 protein levels increased in HeLa cells treated for 30 min with H 2 O 2 or angiotensin II and in aortic tissue isolated from mice treated with angiotensin II for 3 weeks. Cell death was higher in HERPUD1 knockdown (sh- HERPUD1 ) HeLa cells treated with H 2 O 2 in comparison with control (sh- Luc ) HeLa cells. This effect was abolished by the intracellular Ca 2+ chelating agent BAPTA-AM or the inositol 1,4,5-trisphosphate receptor (ITPR) antagonist xestospongin B, suggesting that the response to H 2 O 2 was dependent on intracellular Ca 2+ stores and the ITPR. Ca 2+ kinetics showed that sh- HERPUD1 HeLa cells exhibited greater and more sustained cytosolic and mitochondrial Ca 2+ increases than sh- Luc HeLa cells. This higher sensitivity of sh- HERPUD1 HeLa cells to H 2 O 2 was prevented with the mitochondrial permeability transition pore inhibitor cyclosporine A. We concluded that the HERPUD1-mediated cytoprotective effect against oxidative stress depends on the ITPR and Ca 2+ transfer from the ER to mitochondria. [ABSTRACT FROM AUTHOR]

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 Ca 2 + 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 Ca 2 + 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: p47 phox and gp91 phox were increased in mdx myotubes. Looking at ROS-dependence of NF-κB activation we found that in wt myotubes external administration of 50 μM H 2 O 2 increased NF-κB activity; after administration of 100 and 200 μM H 2 O 2 there was no effect. In mdx myotubes there was a dose-dependent reduction in NF-κB activity in response to external administration of H 2 O 2 , with a significant effect of 100 μM and 200 μM, 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 Ca 2 + 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. [ABSTRACT FROM AUTHOR]

Highlights: [•] Insulin-like growth factor 1 (IGF-1) has pleotropic actions in the heart. [•] Deficiency in IGF-1 may drive cardiovascular disease. [•] IGF-1 activates canonical and noncanonical signaling pathways in the heart. [•] Local IGF-1 and progenitor cell therapies prevent heart injuries in experimental models. [ABSTRACT FROM AUTHOR]

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 ([Ca2+]rest) observed in mdx myotubes and its possible link with up-regulation of NF-κB and pro-inflammatory gene expression in dystrophic muscle cells. [Ca2+]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 Ca2+ entry (low Ca2+ solution, Ca2+-free solution, and Gd3+) and blockade of either ryanodine receptors or inositol 1,4,5-trisphosphate receptors reduced [Ca2+]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 [Ca2+]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 [Ca2+]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 [Ca2+]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. [ABSTRACT FROM AUTHOR]

ATP released from cells is known to activate plasma membrane P2X (ionotropic) or P2Y (metabotropic) receptors. In skeletal muscle cells, depolarizing stimuli induce both a fast calcium signal associated with contraction and a slow signal that regulates gene expression. Here we show that nucleotides released to the extracellular medium by electrical stimulation are partly involved in the fast component and are largely responsible for the slow signals. In rat skeletal myotubes, a tetanic stimulus (45 Hz, 400 1-ms pulses) rapidly increased extracellular levels of ATP, ADP, and AMP after 15 s to 3 mm. Exogenous ATP induced an increase in intracellular free Ca2+ concentration, with an EC50 value of 7.8 ± 3.1 μM. Exogenous ADP, UTP, and UDP also promoted calcium transients. Both fast and slow calcium signals evoked by tetanic stimulation were inhibited by either 100 μM suramin or 2 units/ml apyrase. Apyrase also reduced fast and slow calcium signals evoked by tetanus (45 Hz, 400 0.3-ms pulses) in isolated mouse adult skeletal fibers. A likely candidate for the ATP release pathway is the pannexin-1 hemichannel; its blockers inhibited both calcium transients and ATP release. The dihydropyridine receptor co-precipitated with both the P2Y2 receptor and pannexin-1. As reported previously for electrical stimulation, 500 μM ATP significantly increased mRNA expression for both c-fos and interleukin 6. Our results suggest that nucleotides released during skeletal muscle activity through pannexin-1 hemichannels act through P2X and P2Y receptors to modulate both Ca2+ homeostasis and muscle physiology. [ABSTRACT FROM AUTHOR]

Skeletal muscle is one of the main physiological targets of insulin, a hormone that triggers a complex signaling cascade and that enhances the production of reactive oxygen species (ROS) in different cell types. ROS, currently considered second messengers, produce redox modifications in proteins such as ion channels that induce changes in their functional properties. In myotubes, insulin also enhances calcium release from intracellular stores. In this work, we studied in myotubes whether insulin stimulated ROS production and investigated the mechanisms underlying the insulin-dependent calcium increase: in particular, whether the late phase of the Ca[sup2+] increase induced by insulin required ROS. We found that insulin stimulated ROS production, as detected with the probe 2', 7'-dichlorofluorescein diacetate (CM-H2DCFDA). We used the translocation of p47[sup[supphox]] from the cytoplasm to the plasma membrane as a marker of the activation of NADPH oxidase. Insulin-stimulated ROS generation was suppressed by the NADPH oxidase inhibitor apocynin and by small interfering RNA against p47[sup[supphox]], a regulatory NADPH oxidase subunit. Additionally, both protein kinase C and phosphatidylinositol 3-kinase are presumably involved in insulin-induced ROS generation because bisindolylmaleimide, a nonspecific protein kinase C inhibitor, and LY290042, an inhibitor of phosphatidylinositol 3-kinase, inhibited this increase. Bisindolylmaleimide, LY290042, apocynin, small interfering RNA against p47[sup[supphox]], and two drugs that interfere with inositol 1,4,5-trisphosphate-mediated Ca[sup2+] release, xestospongin C and U73122, inhibited the intracellular Ca[sup2+] increase produced by insulin. These combined results strongly suggest that insulin induces ROS generation trough NADPH activation and that this ROS increase is required for the intracellular Ca[sup2+] rise mediated by inositol 1,4,5-trisphosphate receptors. [ABSTRACT FROM AUTHOR]

Tetanic electrical stimulation of myotubes evokes a ryanodine receptor-related fast calcium signal, during the stimulation, followed by a phospholipase C/inositol 1,4,5-trisphosphate-dependent slow calcium signal few seconds after stimulus end. L-type calcium channels (Cay 1.1, dihydropyridine receptors) acting as voltage sensors activate an unknown signaling pathway involved in phospholipase C activation, We demonstrated that both G protein and phosphatidylinositol 3-kinase were activated by electrical stimulation, and both the inositol 1,4,5-trisphosphate rise and slow calcium signal induced by electrical stimulation were blocked by pertussis toxin, by a Gβγ scavenger peptide, and by phosphatidylinositol 3-kinase inhibitors. Immunofluorescence using anti-phosphatidylinositol 3-kinase γ antibodies showed a clear location in striations within the cytoplasm, consistent with a position near the I band region of the sarcomere. The time course of phosphatidylinositol 3-kinase activation, monitored in single living cells using a pleckstrin homology domain fused to green fluorescent protein, was compatible with sequential phospholipase Cγ1 activation as confirmed by phosphorylation assays for the enzyme. Co-transfection of a dominant negative form of phosphatidylinositol 3-kinase γ inhibited the phosphatidylinositol 3-kinase activity as well as the slow calcium signal. We conclude that Gβγ/phosphatidylinositol 3-kinase γ signaling pathway is involved in phospholipase C activation and the generation of the slow calcium signal induced by tetanic stimulation. We postulate that membrane potential fluctuations in skeletal muscle cells can activate a pertussis toxin-sensitive G protein, phosphatidylinositol 3-kinase, phospholipase C pathway toward modulation of long term, activity-dependent plastic changes. [ABSTRACT FROM AUTHOR]

In the heart, insulin-like growth factor-1 (IGF-1) is a pro-hypertrophic and anti-apoptotic peptide. In cultured rat cardiomyocytes, IGF-1 induced a fast and transient increase in Cai2+ levels apparent both in the nucleus and cytosol, releasing this ion from intracellular stores through an inositol 1,4,5-trisphosphate (IP3)-dependent signaling pathway. Intracellular IP3 levels increased after IGF-1 stimulation in both the presence and absence of extracellular Ca2+. A different spatial distribution of IP3 receptor isoforms in cardiomyocytes was found. Ryanodine did not prevent the IGF-1-induced increase of Cai2+ levels but inhibited the basal and spontaneous Cai2+ oscillations observed when cardiac myocytes were incubated in Ca2+-containing resting media. Spatial analysis of fluorescence images of IGF-1-stimulated cardiomyocytes incubated in Ca2+-containing resting media showed an early increase in Cai2+, initially localized in the nucleus. Calcium imaging suggested that part of the Ca2+ released by stimulation with IGF-1 was initially contained in the perinuclear region. The IGF-1-induced increase on Cai2+ levels was prevented by 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-AM, thapsigargin, xestospongin C, 2-aminoethoxy diphenyl borate, U-73122, pertussis toxin, and βARKct (a peptide inhibitor of Gβγ, signaling). Pertussis toxin also prevented the IGF-1-dependent IP3 mass increase. Genistein treatment largely decreased the IGF1-induced changes in both Cai2+ and IP3. LY29402 (but not PD98059) also prevented the IGF-1-dependent Cai2+ increase. Both pertussis toxin and U73122 prevented the IGF-1-dependent induction of both ERKs and protein kinase B. We conclude that IGF-1 increases Cai2+ levels in cultured cardiac myocytes through a Gβγ subunit of a pertussis toxin-sensitive G protein-PI3K-phospholipase C signaling pathway that involves participation of IP3. [ABSTRACT FROM AUTHOR]

Reactive oxygen species (ROS) are increasingly recognized as key regulators of cellular metabolism. In skeletal muscle, ROS has been linked to glucose transport in isolated electrically stimulated muscles but the exact source of ROS in the context of in vivo exercise and whether it controls in vivo glucose uptake by glucose transporter 4 (GLUT4) is unknown. Here, we utilized a combination of genetically-encoded biosensors, fluorescent dyes and mouse models lacking the NADPH oxidase 2 (NOX2) regulatory subunits p47phox and Rac1 to demonstrate that NOX2 is the predominant source of ROS during in vivo treadmill exercise in skeletal muscle. Furthermore, p47phox-deficient mice phenocopy key aspects of metabolic dysregulation in Rac1 mKO mice, including decreased in vivo glucose uptake and GLUT4 translocation. These findings provide strong evidence that NOX2 is a major ROS source during in vivo exercise and its activity is crucial to the translocation of GLUT4 to increase glucose uptake. [ABSTRACT FROM AUTHOR]