Attempting to Compensate for Reduced Neuronal Nitric Oxide Synthase Protein with Nitrate Supplementation Cannot Overcome Metabolic Dysfunction but Rather Has Detrimental Effects in Dystrophin-Deficient mdx Muscle

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.Electronic supplementary materialThe online version of this article (doi:10.1007/s13311-016-0494-7) contains supplementary material, which is available to authorized users.

BGP-15 Protects against Oxaliplatin-Induced Skeletal Myopathy and Mitochondrial Reactive Oxygen Species Production in Mice

“…While the mechanism behind this fiber size distribution shift remains unclear, the significant increase in fibers above 4,900 μm 2 in OXA treated mice (21x greater compared to VEH) suggests that OXA treatment induced pathological changes. Pseudohypertrophic fibers are a pronounced feature of myopathy, in which healthy fibers hypertrophy to compensate for the loss of strength induced by muscle atrophy, wasting and/or replacement with non-functional tissue (such as fat and connective tissue; Briguet et al, 2004; Timpani et al, 2016). Importantly, BGP-15 adjunct therapy ameliorated all of these OXA-induced pathologies by protecting against Ca 2+ , lipid and collagen accumulation and restoring a normal fiber distribution.…”

“…Slides were stained using a standard H&E staining protocol (30 s incubation in haematoxylin and 1 m 45 s incubation in eosin; Timpani et al, 2016) and mounted with DPx (BDH, Poole, UK). To determine fiber size frequency distributions, 200 fibers per section (except for those on the periphery that were cropped) were individually circled on three images taken from the top, middle and bottom along the midline from each TA cross section.…”

Exercise May Ameliorate the Detrimental Side Effects of High Vitamin D Supplementation on Muscle Function in Mice

“…Following the completion of the contractile protocol, the EDL and SOL muscles were coated in optimal cutting temperature (OCT) compound and snap frozen in liquid nitrogen‐chilled isopentane (Sigma Aldrich, Castle Hill NSW, Australia). The imbedded muscles were cryosectioned (12 μm) and stained using a standard hematoxylin and eosin (H&E) staining protocol (30 s incubation in hematoxylin and 1 m 45 s incubation in eosin) . Slides were mounted with distyrene‐plasticiser‐xylene (DPx) (BDH, Poole, UK), analyzed, and imaged at magnification ×20 using a Zeiss Axio Imager Z2 microscope (Carl Zeiss MicroImaging GmbH, Oberkochen, Germany) as described .…”

“…Slides were mounted with distyrene‐plasticiser‐xylene (DPx) (BDH, Poole, UK), analyzed, and imaged at magnification ×20 using a Zeiss Axio Imager Z2 microscope (Carl Zeiss MicroImaging GmbH, Oberkochen, Germany) as described . To assess the effects of vitamin D on mitochondrial content in the muscle, a succinate dehydrogenase (SDH) staining procedure was performed on cryosectioned muscle samples (prepared as per H&E staining), as described . After the mounting of slides in glycerol jelly, images were taken at magnification ×20 using the same Zeiss microscope and images were converted to 8‐bit.…”

“…All samples were analyzed for citrate synthase (CS) activity as a marker of mitochondrial density as described . Thawed tibialis anterior (TA) muscles were homogenized (TissueLyser; Qiagen, Hilden, Germany) for 20 s in 2 mL ice‐cold Western blot (WB) buffer (40mM Tris, pH 7.5; 1mM ethylenediaminetetraacetic acid; 5mM ethylene glycol tetraacetic acid [EGTA]; 0.5% Triton X‐100; 25mM β‐glycerophosphate; 25mM NaF; 1mM Na3VO4; 10 μg/mL leupeptin (LEU); and 1mM phenylmethylsulfonyl fluoride [PMSF]).…”