Aim: We hypothesize that both type-1 ryanodine receptor (RyR1) and IP3-receptor (IP3R) calcium channels are necessary for the mitochondrial Ca2+ increase caused by membrane depolarization induced by potassium (or by electrical stimulation) of single skeletal muscle fibers; this calcium increase would couple muscle fiber excitation to an increase in metabolic output from mitochondria (excitation-metabolism coupling).Methods: Mitochondria matrix and cytoplasmic Ca2+ levels were evaluated in fibers isolated from flexor digitorium brevis muscle using plasmids for the expression of a mitochondrial Ca2+ sensor (CEPIA3mt) or a cytoplasmic Ca2+ sensor (RCaMP). The role of intracellular Ca2+ channels was evaluated using both specific pharmacological inhibitors (xestospongin B for IP3R and Dantrolene for RyR1) and a genetic approach (shIP3R1-RFP). O2 consumption was detected using Seahorse Extracellular Flux Analyzer.Results: In isolated muscle fibers cell membrane depolarization increased both cytoplasmic and mitochondrial Ca2+ levels. Mitochondrial Ca2+ uptake required functional inositol IP3R and RyR1 channels. Inhibition of either channel decreased basal O2 consumption rate but only RyR1 inhibition decreased ATP-linked O2 consumption. Cell membrane depolarization-induced Ca2+ signals in sub-sarcolemmal mitochondria were accompanied by a reduction in mitochondrial membrane potential; Ca2+ signals propagated toward intermyofibrillar mitochondria, which displayed increased membrane potential. These results are compatible with slow, Ca2+-dependent propagation of mitochondrial membrane potential from the surface toward the center of the fiber.Conclusion: Ca2+-dependent changes in mitochondrial membrane potential have different kinetics in the surface vs. the center of the fiber; these differences are likely to play a critical role in the control of mitochondrial metabolism, both at rest and after membrane depolarization as part of an “excitation-metabolism” coupling process in skeletal muscle fibers.

Reactive oxygen species (ROS) participate as signaling molecules in response to exercise in skeletal muscle. However, the source of ROS and the molecular mechanisms involved in these phenomena are still not completely understood. The aim of this work was to study the role of skeletal muscle NADPH oxidase isoform 2 (NOX2) in the molecular response to physical exercise in skeletal muscle. BALB/c mice, pre-treated with a NOX2 inhibitor, apocynin, (3 mg/kg) or vehicle for 3 days, were swim-exercised for 60 min. Phospho–p47phox levels were significantly upregulated by exercise in flexor digitorum brevis (FDB). Moreover, exercise significantly increased NOX2 complex assembly (p47phox–gp91phox interaction) demonstrated by both proximity ligation assay and co-immunoprecipitation. Exercise-induced NOX2 activation was completely inhibited by apocynin treatment. As expected, exercise increased the mRNA levels of manganese superoxide dismutase (MnSOD), glutathione peroxidase (GPx), citrate synthase (CS), mitochondrial transcription factor A (tfam) and interleukin-6 (IL-I6) in FDB muscles. Moreover, the apocynin treatment was associated to a reduced activation of p38 MAP kinase, ERK 1/2, and NF-κB signaling pathways after a single bout of exercise. Additionally, the increase in plasma IL-6 elicited by exercise was decreased in apocynin-treated mice compared with the exercised vehicle-group (p < 0.001). These results were corroborated using gp91-dstat in an in vitro exercise model. In conclusion, NOX2 inhibition by both apocynin and gp91dstat, alters the intracellular signaling to exercise and electrical stimuli in skeletal muscle, suggesting that NOX2 plays a critical role in molecular response to an acute exercise.

Duchenne Muscular Dystrophy (DMD) is a fatal X-linked genetic disease, caused by mutations in the dystrophin gene, which cause functional loss of this protein. DMD pathology is associated with an increased production of reactive oxygen and nitrogen species (ROS and RNS). 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 (ES) 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 ES. On the other hand, ES 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 200 µM 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 100 µM and 200 µM, suggesting that ROS levels are critical for NF-κB activity. Prior blockage with NOX2 inhibitors blunted the effects of ES 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 (LPS) on both NF-κB activation and IL-6 expression. Exposure to LPS induced a dramatic increase in both NF-κB and IL-6 expression in both wt and mdx myotubes, suggesting that the altered IL-6 gene expression after ES 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 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.

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.