Abstract
Duchenne muscular dystrophy arises from the loss of dystrophin and is characterized by calcium dysregulation, muscular atrophy, and metabolic dysfunction. The secondary reduction of neuronal nitric oxide synthase (nNOS) from the sarcolemma reduces NO production and bioavailability. As NO modulates glucose uptake, metabolism, and mitochondrial bioenergetics, we investigated whether an 8-week nitrate supplementation regimen could overcome metabolic dysfunction in the mdx mouse. Dystrophin-positive control (C57BL/10) and dystrophin-deficient mdx mice were supplemented with sodium nitrate (85 mg/l) in drinking water. Following the supplementation period, extensor digitorum longus and soleus were excised and radioactive glucose uptake was measured at rest (basal) and during contraction. Gastrocnemius was excised and mitochondrial respiration was measured using the Oroboros Oxygraph. Tibialis anterior was analyzed immunohistochemically for the presence of dystrophin, nNOS, nitrotyrosine, IgG and CD45+ cells, and histologically to assess areas of damage and regeneration. Glucose uptake in the basal and contracting states was normal in unsupplemented mdx muscles but was reduced following nitrate supplementation in mdx muscles only. The mitochondrial utilization of substrates was also impaired in mdx gastrocnemius during phosphorylating and maximal uncoupled respiration, and nitrate could not improve respiration in mdx muscle. Although nitrate supplementation reduced mitochondrial hydrogen peroxide emission, it induced mitochondrial uncoupling in red gastrocnemius, increased muscle fiber peroxynitrite (nitrotyrosine), and promoted skeletal muscle damage. Our novel data suggest that despite lower nNOS protein expression and likely lower NO production in mdx muscle, enhancing NO production with nitrate supplementation in these mice has detrimental effects on skeletal muscle. This may have important relevance for those with DMD.
Highlights
Electronic supplementary material The online version of this article contains supplementary material, which is available to authorized users.Duchenne muscular dystrophy (DMD) is a progressive Xlinked [1] neuromuscular disease affecting 1 in 3500 to 5000 live male births [2], which arises from the ablation of the cytoskeletal protein, dystrophin [3]
CON UNSUPP n = 11; CON NITR n = 12; mdx UNSUPP n = 11; mdx NITR n = 10. This is the first study to date to investigate NITR supplementation as a potential therapy for DMD, and we show that the metabolic perturbations in dystrophin-deficient skeletal muscle could not be overcome by enhancing neuronal nitric oxide synthase (nNOS)-independent nitric oxide (NO) production
Our data suggest that chronically increasing NO bioavailability without restoring nNOS protein expression and its regulatory role on metabolism, promotes pathological muscle damage, potentially via a ONOO–dependent mechanism
Summary
Electronic supplementary material The online version of this article (doi:10.1007/s13311-016-0494-7) contains supplementary material, which is available to authorized users.Duchenne muscular dystrophy (DMD) is a progressive Xlinked [1] neuromuscular disease affecting 1 in 3500 to 5000 live male births [2], which arises from the ablation of the cytoskeletal protein, dystrophin [3]. DMD is associated with impairments in glycolysis [20,21,22], and in β-fatty acid oxidation, the tricarboxylic acid cycle, and the electron transport system (ETS) (for detailed reviewed see [6]). These metabolic impairments result in reduced energy production [23], with reports of adenosine triphosphate (ATP) content being 50% lower under resting conditions [24, 25]. Increasing NO availability has the potential to be of therapeutic benefit
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