4 research outputs found
Electrical stimulation induces calcium-dependent up-regulation of neuregulin-1β in dystrophic skeletal muscle cell lines
Duchenne muscular dystrophy (DMD) is a
neuromuscular disease originated by reduced or no
expression of dystrophin, a cytoskeletal protein that
provides structural integrity to muscle fibres. A
promising pharmacological treatment for DMD aims
to increase the level of a structural dystrophin
homolog called utrophin. Neuregulin-1 (NRG-1), a
growth factor that potentiates myogenesis, induces
utrophin expression in skeletal muscle cells.
Microarray analysis of total gene expression allowed
us to determine that neuregulin-1β (NRG-1β) is one
of 150 differentially expressed genes in electrically
stimulated (400 pulses, 1 ms, 45 Hz) dystrophic
human skeletal muscle cells (RCDMD). We
investigated the effect of depolarization, and the
involvement of intracellular Ca2+ and PKC isoforms
on NRG-1β expression in dystrophic myotubes.
Electrical stimulation of RCDMD increased NRG-1β mRNA and protein levels, and mRNA enhancement
was abolished by actinomycin D. NRG-1β transcription
was inhibited by BAPTA-AM, an intracellular Ca2+
chelator, and by inhibitors of IP3-dependent slow Ca2+
transients, like 2-APB, Ly 294002 and Xestospongin
B. Ryanodine, a fast Ca2+ signal inhibitor, had no effect
on electrical stimulation-induced expression. BIM VI
(general inhibitor of PKC isoforms) and Gö 6976
(specific inhibitor of Ca2+-dependent PKC isoforms)
abolished NRG-1β mRNA induction. Our results
suggest that depolarization induced slow Ca2+ signals
stimulate NRG-1β transcription in RCDMD cells, and
that Ca2+-dependent PKC isoforms are involved in this
process. Based on utrophin´s ability to partially
compensate dystrophin disfunction, knowledge on the
mechanism involved on NRG-1 up-regulation could
be important for new therapeutic strategies design
Insulin-Dependent H2O2 Production Is Higher in Muscle Fibers of Mice Fed with a High-Fat Diet
Insulin resistance is defined as a reduced ability of insulin to stimulate glucose utilization. C57BL/6 mice fed with a high-fat diet (HFD) are a model of insulin resistance. In skeletal muscle, hydrogen peroxide (H2O2) produced by NADPH oxidase 2 (NOX2) is involved in signaling pathways triggered by insulin. We evaluated oxidative status in skeletal muscle fibers from insulin-resistant and control mice by determining H2O2 generation (HyPer probe), reduced-to-oxidized glutathione ratio and NOX2 expression. After eight weeks of HFD, insulin-dependent glucose uptake was impaired in skeletal muscle fibers when compared with control muscle fibers. Insulin-resistant mice showed increased insulin-stimulated H2O2 release and decreased reduced-to-oxidized glutathione ratio (GSH/GSSG). In addition, p47phox and gp91phox (NOX2 subunits) mRNA levels were also high (~3-fold in HFD mice compared to controls), while protein levels were 6.8- and 1.6-fold higher, respectively. Using apocynin (NOX2 inhibitor) during the HFD feeding period, the oxidative intracellular environment was diminished and skeletal muscle insulin-dependent glucose uptake restored. Our results indicate that insulin-resistant mice have increased H2O2 release upon insulin stimulation when compared with control animals, which appears to be mediated by an increase in NOX2 expression