title: 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.
authors.affiliation: Centre for Chronic Disease, College of Health & Biomedicine, Victoria University, Melbourne, Victoria, 8001, Australia.
authors.forename: Cara A
authors.initials: CA
authors.lastname: Timpani
authors.affiliation: Institute of Sport, Exercise & Active Living (ISEAL), Victoria University, Melbourne, Victoria, 8001, Australia.
authors.forename: Adam J
authors.initials: AJ
authors.lastname: Trewin
authors.affiliation: Centre for Chronic Disease, College of Health & Biomedicine, Victoria University, Melbourne, Victoria, 8001, Australia.
authors.forename: Vanesa
authors.initials: V
authors.lastname: Stojanovska
authors.affiliation: Centre for Chronic Disease, College of Health & Biomedicine, Victoria University, Melbourne, Victoria, 8001, Australia.
authors.forename: Ainsley
authors.initials: A
authors.lastname: Robinson
authors.affiliation: Centre for Chronic Disease, College of Health & Biomedicine, Victoria University, Melbourne, Victoria, 8001, Australia.|Institute of Sport, Exercise & Active Living (ISEAL), Victoria University, Melbourne, Victoria, 8001, Australia.|Australian Institute of Musculoskeletal Science (AIMSS), Western Health, Melbourne, Victoria, 3021, Australia.
authors.forename: Craig A
authors.initials: CA
authors.lastname: Goodman
authors.affiliation: Centre for Chronic Disease, College of Health & Biomedicine, Victoria University, Melbourne, Victoria, 8001, Australia.
authors.forename: Kulmira
authors.initials: K
authors.lastname: Nurgali
authors.affiliation: Centre for Chronic Disease, College of Health & Biomedicine, Victoria University, Melbourne, Victoria, 8001, Australia.|Institute of Sport, Exercise & Active Living (ISEAL), Victoria University, Melbourne, Victoria, 8001, Australia.
authors.forename: Andrew C
authors.initials: AC
authors.lastname: Betik
authors.affiliation: Institute of Sport, Exercise & Active Living (ISEAL), Victoria University, Melbourne, Victoria, 8001, Australia.
authors.forename: Nigel
authors.initials: N
authors.lastname: Stepto
authors.affiliation: Centre for Chronic Disease, College of Health & Biomedicine, Victoria University, Melbourne, Victoria, 8001, Australia.|Institute of Sport, Exercise & Active Living (ISEAL), Victoria University, Melbourne, Victoria, 8001, Australia.|Australian Institute of Musculoskeletal Science (AIMSS), Western Health, Melbourne, Victoria, 3021, Australia.
authors.forename: Alan
authors.initials: A
authors.lastname: Hayes
authors.affiliation: Institute of Sport, Exercise & Active Living (ISEAL), Victoria University, Melbourne, Victoria, 8001, Australia.|Australian Institute of Musculoskeletal Science (AIMSS), Western Health, Melbourne, Victoria, 3021, Australia.
authors.forename: Glenn K
authors.initials: GK
authors.lastname: McConell
authors.affiliation: Centre for Chronic Disease, College of Health & Biomedicine, Victoria University, Melbourne, Victoria, 8001, Australia. emma.rybalka@vu.edu.au.|Institute of Sport, Exercise & Active Living (ISEAL), Victoria University, Melbourne, Victoria, 8001, Australia. emma.rybalka@vu.edu.au.|Australian Institute of Musculoskeletal Science (AIMSS), Western Health, Melbourne, Victoria, 3021, Australia. emma.rybalka@vu.edu.au.
authors.forename: Emma
authors.initials: E
authors.lastname: Rybalka
mesh_terms: D000818:Animals; D016189:Dystrophin / Q000172:deficiency; D004579:Electron Transport; D005947:Glucose / Q000378:metabolism; D008297:Male; D008810:Mice, Inbred C57BL; D018101:Mice, Inbred mdx; D008928:Mitochondria / Q000502:physiology; D018482:Muscle, Skeletal / Q000378:metabolism*; D020388:Muscular Dystrophy, Duchenne / Q000378:metabolism*; D009566:Nitrates / Q000008:administration & dosage*; D052248:Nitric Oxide Synthase Type I / Q000172:deficiency*; D017382:Reactive Oxygen Species
chemical_list: D016189:Dystrophin; D009566:Nitrates; D017382:Reactive Oxygen Species; C031618:sodium nitrate; D052248:Nitric Oxide Synthase Type I; D005947:Glucose
keywords: Duchenne muscular dystrophy; Glucose uptake; Metabolism; Mitochondria; Nitrate supplementation