Abstract
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.
Highlights
Duchenne muscular dystrophy (DMD) is a severe neuromuscular disorder characterized by the absence of the dystrophin protein and is the most common form of muscular dystrophy with a frequency of 1 in 3500 male births [1]
After incubation with nifedipine (10 mM, 6 h) NF-kB activity was reduced by 33% (P,0.05) and inducible nitric oxide synthase (iNOS) mRNA levels were diminished by 61% (P,0.01) in mdx myotubes, without any significant effect in wt myotubes (P.0.05) (Figure 2)
Our data show that [Ca2+]r was elevated in mdx myotubes and to a similar extent in adult skeletal muscle fibers from mdx mice
Summary
Duchenne muscular dystrophy (DMD) is a severe neuromuscular disorder characterized by the absence of the dystrophin protein and is the most common form of muscular dystrophy with a frequency of 1 in 3500 male births [1]. Extracellular ATP stimulation was pro-apoptotic, inducing the transcription of Bax, BIM and PUMA and increasing the levels of activated Bax and cytosolic cytochrome C [12]. These data suggest the potential for involvement of the ATP pathway in the activation of mechanisms related with cell death in muscular dystrophy, opening new perspectives towards possible targets for pharmacological therapies. In FDB muscle fibers isolated from the same nifedipine treated mdx mice, basal ATP release was reduced and the gene expression of pro-oxidative and pro-apoptotic genes in diaphragm (the most seriously affected muscle) were down-regulated.
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