Oxidative stress and Nrf2 signaling in McArdle disease Yu Kitaoka, Daniel I. Ogborn, Mats I. Nilsson, Nicholas J. Mocellin, Lauren G. MacNeil, Mark A. Tarnopolsky ⁎ Departments of Pediatrics and Medicine, McMaster University, 1200 Main Street West, Hamilton, Ontario, L8N 3Z5 Canada abstract article info Article history: Received 25 June 2013 Accepted 25 June 2013 Available online 6 July 2013 Keywords: McArdle disease Oxidative stress Nuclear factor erythroid 2-related factor 2 McArdle disease (MD) is a metabolic myopathy due to myophosphorylase deficiency, which leads to a severe limitation in the rate of adenosine triphosphate (ATP) resynthesis. Compensatory flux through the myoadenylate deaminase NN xanthine oxidase pathway should result in higher oxidative stress in skeletal muscle; however, oxidative stress and nuclear factor erythroid 2-related factor 2 (Nrf2) mediated antioxidant response cascade in MD patients have not yet been examined. We show that MD patients have elevated muscle protein carbonyls and 4-hydroxynonenal (4-HNE) in comparison with healthy, age and activity matched controls (P b 0.05). Nuclear abundance of Nrf2 and Nrf2-antioxidant response element (ARE) binding was also higher in MD patients compared with controls (P b 0.05). The expressions of Nrf2 target genes were also higher in MD patients vs. controls. These observations suggest that MD patients experience elevated levels of oxidative stress, and that the Nrf2-mediated antioxidant response cascade is up-regulated in skeletal muscle to compensate. © 2013 Elsevier Inc. All rights reserved. 1. Introduction McArdle disease (Glycogen Storage Disease Type V; MD) (MIM #232600) is an inborn error of metabolism caused by a deficiency in muscle glycogen phosphorylase (EC 2.4.1.1) [1–3]. When the produc- tion of adenosine triphosphate (ATP) cannot match its consumption in working muscle, which is accentuated in MD patients due to the ab- sence of glycogenolysis, two adenosine diphosphate (ADP) molecules can combine to regenerate ATP by the adenylate kinase in an attempt to keep up with ATP demand. Adenosine monophosphate (AMP) is pro- duced in the reaction as a by-product, and removed by AMP deaminase, resulting in the production of inosine monophosphate (IMP) and am- monia (NH 3 ). IMP is metabolized to inosine and then to hypoxanthine, xanthine, and uric acid via xanthine oxidase, which generates reactive oxygen species (ROS). Previous studies have reported an excessive in- crease in muscle ADP [4], and plasma NH 3 and hypoxanthine [5,6] in exercising MD patients, which suggests an increased rate of this ade- nine nucleotide degradation. Consequently, MD patients may experi- ence elevated levels of oxidative stress in skeletal muscle. Nuclear factor erythroid 2-related factor 2 (Nrf2) has been regarded as a master regulator of antioxidant transcription [7,8]. Under oxidative stress conditions, Nrf2 dissociates from its cytosolic repressor, Kelch-like ECH-associated protein 1 (Keap1), and translocates into the nucleus to bind with the antioxidant response element (ARE) in the promoter region of target antioxidant genes including NAD(P)H quinone oxidore- ductase 1 (NQO1) and heme oxygenase 1 (HMOX1) [9,10]. These antiox- idants scavenge ROS and maintain intracellular reducing potential [11]. We hypothesized that Nrf2-Keap1 signaling would be up-regulated in skeletal muscle of MD patients to confer adaptive cytoprotection from el- evated oxidative stress. 2. Materials and methods 2.1. Subjects Thirteen patients with MD (10 males and 3 females; 53.6 ± 4.7 years) and thirteen age-matched controls (10 males and 3 fe- males; 57.4 ± 5.3 years) were recruited to participate in the study. All procedures were approved by the McMaster University Hamilton Health Sciences Human Research Ethics Board and conformed to the Declaration of Helsinki guidelines. All participants arrived at the clinic following an overnight (12 h) fast. Study personnel met with the participants and explained the study and the participants signed the consent form. A review of the patient's medical history, current med- ication and nutritional supplement use was performed. The diagnosis of MD was confirmed in each patient through biochemical and/or mo- lecular testing: Fasting plasma CK levels were elevated, histochemical phosphorylase activity was absent in muscle biopsy and plasma lactate levels did not increase with a 60 s non-ischemic forearm exercise test in all MD patients. Furthermore, all patients had at least one previously Molecular Genetics and Metabolism 110 (2013) 297–302 Abbreviations: ADP, adenosine diphosphate; AMP, adenosine monophosphate; ARE, antioxidant response element; ATP, adenosine triphosphate; GCLC, γ-glutamylcysteine ligase catalytic subunit; GCLM, γ-glutamylcysteine ligase regulatory subunit; GPX, glu- tathione peroxidase; HMOX1, heme oxygenase 1; Keap1, Kelch-like ECH-associated protein 1; MD, McArdle disease; NQO1, NAD(P)H quinone oxidoreductase 1; Nrf2, nu- clear factor erythroid 2-related factor 2; PYGM, muscle glycogen phosphorylase; TXNRD, thioredoxin reductase. ⁎ Corresponding author at: Departments of Pediatrics and Medicine, McMaster University, HSC-2H26, 1200 Main St. West, Hamilton, Ontario, Canada L8N 3Z5. Fax: +1 905 577 8380. E-mail addresses: kitaoka@mcmaster.ca (Y. Kitaoka), ogborndi@mcmaster.ca (D.I. Ogborn), mnilsson7714@gmail.com (M.I. Nilsson), nicmocellin@gmail.com (N.J. Mocellin), macneilg@mcmaster.ca (L.G. MacNeil), tarnopol@mcmaster.ca (M.A. Tarnopolsky). 1096-7192/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ymgme.2013.06.022 Contents lists available at ScienceDirect Molecular Genetics and Metabolism journal homepage: www.elsevier.com/locate/ymgme