Dysfunctional Nrf2–Keap1 redox signaling in skeletal muscle of the sedentary old

Free Radical Biology & Medicine 49 (2010) 1487–1493

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Free Radical Biology & Medicine j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / f r e e r a d b i o m e d

Original Contribution

Dysfunctional Nrf2–Keap1 redox signaling in skeletal muscle of the sedentary old Adeel Safdar a,b,c, Justin deBeer d, Mark A. Tarnopolsky b,c,⁎ a

Department of Kinesiology, McMaster University, Hamilton, ON L8N 3Z5, Canada Department of Pediatrics, McMaster University, Hamilton, ON L8N 3Z5, Canada c Department of Medicine, McMaster University, Hamilton, ON L8N 3Z5, Canada d Department of Surgery, McMaster University, Hamilton, ON L8N 3Z5, Canada b

a r t i c l e

i n f o

Article history: Received 28 January 2010 Revised 15 June 2010 Accepted 4 August 2010 Available online 11 August 2010 Keywords: Sarcopenia Aging Skeletal muscle Physical inactivity Nrf2 Keap1 Oxidative stress Free radicals

a b s t r a c t The role of nuclear factor-erythroid 2 p45-related factor 2 (Nrf2) and Kelch-like ECH-associated protein 1 (Keap1) redox signaling has not been characterized in human skeletal muscle despite an extensive delineation of oxidative stress in the etiology of aging and sarcopenia. We assessed whether the ageassociated decline in antioxidant response is due, at least in part, to dysfunction in Nrf2–Keap1 redox signaling. We also evaluated whether an active lifestyle can conserve skeletal muscle cellular redox status via activation of Nrf2–Keap1 signaling. Here we show that a recreationally active lifestyle is associated with the activation of upstream modulators that induce the Nrf2-mediated antioxidant response cascade in skeletal muscle of the elderly. Conversely, a sedentary lifestyle is negatively associated with these adaptations mainly because of dysregulation of Nrf2–Keap1 redox signaling that renders the intracellular environment prone to reactive oxygen species-mediated toxicity. Our results indicate that an active lifestyle is an important determinant of cellular redox status. We propose that the metabolic induction of Nrf2–Keap1 redox signaling promises to be a viable therapy for attenuating oxidative stress-mediated damage in skeletal muscle associated with physical inactivity. © 2010 Elsevier Inc. All rights reserved.

Introduction Individuals over the age of 69 years represent one of the fastest growing segments of the North American population [1]. Although an important public health outcome continues to be an increase in life expectancy, of even greater importance is that extended life span is accompanied by an improved capacity to function independently and a better quality of life. One of the most striking and debilitating ageassociated alterations is the progressive loss of fat-free skeletal muscle mass, a phenomenon known as sarcopenia [2]. This loss of muscle mass and strength and the increase in body fat with aging are physiological phenomena that occur in part as a consequence of metabolic changes associated with a sedentary lifestyle in older adults [3,4]. A chronic sedentary lifestyle (hypodynamia) can lead to a decreased resistance to stressors resulting in cumulative systemic declines [5]. Clinical trials have shown that sedentary older adults significantly improved their physical performance and health after a physical activity intervention, compared to controls [6,7]. LongitudiAbbreviations: AO, Active Old; SO, Sedentary Old; Nrf2, nuclear factor-erythroid 2 p45-related factor 2; Keap1, kelch-like ECH-associated protein 1; ARE, Antioxidant Response Element; 8-OHdG, 8-hydroxy 2-deoxyguanosine; PC, protein carbonyls; γGCLC, γ-glutamylcysteine ligase; HMOX1, heme oxygenase 1. ⁎ Corresponding author. Department of Pediatrics and Medicine, HSC-2H26, McMaster University Medical Center 1200 Main St. W., Hamilton, Ontorio, Canada, L8N 3Z5. Fax: +1 905 577 8380. E-mail address: [email protected] (M.A. Tarnopolsky). 0891-5849/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.freeradbiomed.2010.08.010

nal studies have shown that regular physical activity may extend life expectancy, reduce morbidity (cancer, neurological disease, etc.), and reduce physical disability in later life [8,9]. These findings suggest that preserving mobility and an active lifestyle are central to maintaining a high quality of life in the elderly. The mitochondrial theory of aging has received support in recent years from studies in model organisms in which overexpression of antioxidant genes augmented oxidative stress tolerance and promoted longevity [10–12]. By extension, signaling pathways that regulate antioxidant responses are plausible regulators of the aging process. The Cap ‘n’ Collar transcription factor nuclear factor-erythroid 2 p45related factor 2 (Nrf2) and its cytosolic repressor, Kelch-like ECHassociated protein 1 (Keap1), have been identified as indispensable mediators of cellular responses to oxidative stresses and electrophilic xenobiotics [13]. Under oxidative stress conditions, Nrf2 dissociates from Keap1 and translocates into the nucleus and coordinately induces the transcription of several phase II antioxidants through a 5′ cis-acting regulatory enhancer known as the antioxidant response element (ARE) [13]. The phase II antioxidants work synergistically to mount a pleiotropic cellular defense that scavenges reactive oxygen species (ROS), detoxifies electrophiles and xenobiotics, and maintains intracellular reducing potential [14]. The striking similarities between older mice and nrf2 knockout mice [13] suggest that disruptions in Nrf2–Keap1 redox signaling may occur during aging. The role of Nrf2– Keap1 redox signaling has not yet been established within the context of human aging and sarcopenia. In this study, our principal aim was to

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determine whether the age-associated decline in oxidative stressmediated antioxidant response in human skeletal muscle is due, at least in part, to dysfunction in Nrf2–Keap1 redox signaling and if a recreationally active lifestyle can conserve skeletal muscle redox status.

lateralis at the time of their first total knee-joint arthroplasty procedure immediately after the first incision and without vascular occlusion 10 min after induction. Biopsies from SO subjects were collected by a single surgeon and the wet muscle tissue was stored as aforementioned.

Experimental procedures

Skeletal muscle tissue homogenization

Subjects and experimental protocol

The total protein was extracted from the frozen skeletal muscle biopsy as described previously in detail by our group [17]. Briefly, ~30 mg of skeletal muscle was homogenized on ice in a 2-ml Wheaton glass homogenizer (Fisher Scientific, Ottawa, ON, Canada) with 25 volumes of phosphate homogenization buffer [50 mM KPi, 5 mM EDTA, 0.5 mM dithiothreitol, 1.15% KCl supplemented with a Complete Mini, EDTA-free protease inhibitor cocktail tablet and a PhosSTOP, phosphatase inhibitor cocktail, tablet (Roche Applied Science, Mannheim, Germany) per 10 ml of buffer]. The lysate was centrifuged at 600 g for 15 min at 4 °C to pellet cellular debris. The supernatant was aliquotted, snap-frozen in liquid nitrogen, and stored at −86 °C until further analysis. The Lowry assay was used to quantify the total protein content of samples.

We recruited recreationally active university young students and recreationally active old (AO) and sedentary old (SO) men and women from the Hamilton area for this cross-sectional study (Table 1). The detailed description of the subjects’ characteristics has been previously reported [15]. Both young and active old subjects carried out activities of daily living (walking, grocery shopping, gardening, etc.) and participated in modest recreational activities (golfing, gardening, tennis, and/or cycling) three or more times a week, but were not competitive athletes, and were healthy. Consequently, the young and AO subjects had similar volumes and intensities of physical activity. The SO subjects were patients with a primary diagnosis of osteoarthritis (a nonpathological condition secondary to age-related wear and tear) that rendered them with a sedentary lifestyle. The SO subjects were recruited through the total knee-joint arthroplasty program at the Hamilton Health Sciences Corp. and had no previous knee surgeries. The inclusion criteria for this study included evidence of severe knee-joint osteoarthritis by radiography, age 50–75 years, and no previous joint arthroplasty in the knee to be operated upon. The exclusion criteria for this study included evidence of coronary artery disease, congestive heart failure, renal failure (creatinine N120 μmol/L, potassium N5.00 mmol/L), diabetes requiring insulin or glyburide dosage of 5 mg or more, previous stroke with motor loss, rheumatoid or other known inflammatory arthritis, uncontrolled hypertension or hypertension requiring more than monopharmacotherapy, inability to give consent because of cognitive difficulties, chronic obstructive pulmonary disease (forced vital capacity or forced expiratory volume in 1 s b85% of age-predicted mean value or requiring any medication other than an inhaler as needed), and smoking. The elderly women (both AO and SO) were postmenopausal and were not taking hormone replacement therapy, and the young women were not taking oral contraceptives. All subjects provided written consent before their participation. The study was approved by the Hamilton Health Sciences Human Research Ethics Board and conformed to the guidelines outlined in the Declaration of Helsinki. Muscle biopsy All subjects were instructed to abstain from physical activity for 48 h before the muscle biopsy. Young and AO subjects arrived between 0800 and 0930 h in the postabsorptive state after an overnight fast. We collected a muscle biopsy of the vastus lateralis of the dominant leg, 10 cm proximal or distal to the knee joint using a modified Bergström needle (5 mm diameter) with suction modification [16]. After quickly dissecting the biopsied muscle tissue of fat and connective tissue, we placed ~ 100 mg of wet muscle in an RNase-free cryovial, immediately snap-froze it in liquid nitrogen, and stored it at −86 °C until analysis. SO subjects were biopsied from the vastus Table 1 Subject characteristics. Young Sample size (N) Age (years) Weight (kg) Height (cm) BMI (kg/m2)

a

10 22 ± 2 70 ± 10 176 ± 11 23 ± 2

Values are means ± SD. a♀ = ♂.

AO

SO a

10 70 ± 5 77 ± 10 165 ± 7 28 ± 3

10a 63 ± 10 75 ± 9 161 ± 5 29 ± 3

Protein carbonyl content Muscle lysates were also analyzed for total protein carbonyls (Zenith Technology Corp., Dunedin, New Zealand) content using an enzyme immunoassay, as per the manufacturer's instructions. All samples, standards, and controls were run in duplicate and results were expressed as nmol∙mg protein− 1 for PC (total protein carbonyls). C2C12 cell culture and H2O2-mediated redox stress The C2C12 mouse skeletal myoblast cell line was obtained commercially (CRL-1722; American Type Culture Collection, Manassas, VA, USA) and maintained at subconfluent densities in Dulbecco's modified Eagle's medium (Invitrogen, Burlington, ON, Canada) supplemented with 10% fetal bovine serum, 4 mM glutamine, 1 mM pyruvate, and 1% penicillin and streptomycin under an atmosphere of 5% CO2 in air at 37 °C. Cells were treated with 100 mM H2O2 for 30 min or 3 h, whereas control C2C12 cells were treated with phosphate-buffered saline (PBS). H2O2 was diluted in the culture medium from a freshly prepared stock solution immediately before addition to the cells. Cells were washed twice with cold PBS after H2O2 treatment and were resuspended in 400 μl of ice-cold SDS lysis buffer (10 mM Tris–HCl, pH 7.5, 10 mM NaCl, 1% SDS, supplemented with a Complete Mini, EDTAfree protease inhibitor cocktail tablet and a PhosSTOP phosphatase inhibitor cocktail tablet per 10 ml of buffer) for 15 min on ice and passed through a 26-gauge needle 10 times. Cellular debris was removed by centrifugation at 10,000 g for 15 min at 4 °C. The supernatant was separated from the pellet, aliquotted in separate tubes, snap-frozen in liquid nitrogen, and stored at −86 °C until further analysis. The Lowry assay was used to quantify the total protein content of cell lysate. Immunoblotting Proteins were resolved on 7.5, 10, or 12.5% SDS–PAGE gels depending on the molecular weight of the protein of interest. The gels were transferred onto Hybond ECL nitrocellulose membranes (Amersham, Piscataway, NJ, USA) and immunoblotted using the following commercially available primary antibodies: anti-Nrf2 (sc-13032), anti-Keap1 (sc-15246), and γ-GCLC (γ-glutamylcysteine ligase; sc-22755; Santa Cruz Biotechnology, Santa Cruz, CA, USA); anti-4-HNE (4-hydroxy-2nonenal; ab48506), anti-catalase (ab1877), anti-GPx1 (glutathione peroxidase 1; ab22604), anti-HMOX1 (heme oxygenase 1; ab13248; Abcam, Cambridge, MA, USA); anti-phospho-p38 MAPK (Thr180/Tyr182; 9228), anti-phospho-p44/42 (Thr202/Tyr204; 9101), anti-phospho-Akt

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(Ser473; 4058), and anti-phospho-GSK-3β (Ser9, 9323; Cell Signaling Technology, Danvers, MA, USA). Anti-actin (Novus Biologicals, Littleton, CO, USA) was used as a housekeeping loading control and to normalize the expression of proteins of interest. Healthy human heart (ab29431) and lung (ab43320; Abcam) and healthy C57Bl/6 J mouse liver lysate were used as positive controls for skeletal muscle Nrf2–Keap1 discovery blots. All antibodies were used at 1:1000 dilution, except for anti-actin (1:10,000 dilution). Membranes were then incubated with the appropriate anti-mouse or anti-rabbit (depending on the primary antibody source) horseradish peroxidase-linked secondary antibody (1:5000 dilution) and visualized by enhanced chemiluminescence detection reagent (Amersham). Relative intensities of the protein bands were digitally quantified by using NIH ImageJ, version 1.37, analysis software (Scion Image; National Institutes of Health, Bethesda, MD, USA).

regulator of Nrf2, was significantly lower in the skeletal muscle of both the AO and the SO groups compared to the young group (50 and 53%, respectively; Fig. 2B). In the AO group, the Nrf2/Keap1 ratio was also significantly different from both the young and the SO groups (Fig. 2C). Despite the increase in oxidative damage, the protective increase in Nrf2 content was ablated in SO subjects. This suggests that in AO subjects, aging-associated accretion of oxidative stress results in a concomitant increase in Nrf2 content and a decrease in Keap1 content. This induction of Nrf2–Keap1 redox signaling failed in the skeletal muscle of SO subjects despite the occurrence of oxidative damage.

Statistical analysis

In vitro and in vivo studies have implicated numerous upstream regulatory signal transduction pathways, including PI3K/Akt, p38 MAPK, and p44/42 MAPK, that are involved in cellular ROS sensing, nuclear translocation and stabilization of Nrf2, and activation of phase II antioxidants [21–24]. Because an aberrant Nrf2–Keap1 response was observed in SO vs AO adults, we next examined whether the upstream signaling-mediated activation of Nrf2–Keap1 redox signaling is dysfunctional. Phospho-p44/42 (Thr202/Tyr204) and phospho-Akt (Ser473) levels were significantly lower in the SO group in comparison to both the young (43 and 41%, respectively) and the AO (74 and 42%, respectively) groups (Fig. 3A). In addition, the SO group had 63% higher levels of phosphorylated GSK-3β (Ser9) relative to the AO group (Fig. 3A). Based on these findings, we speculate that dysfunctional Nrf2 signaling in the skeletal muscle of SO subjects is due to impaired upstream p44/42 MAPK and PI3K/Akt signal transduction. To further confirm our findings, we then analyzed downstream phase II antioxidant protein content in the skeletal muscle of young, AO, and SO subjects. The AO group had significantly higher Nrf2mediated contents of the antioxidant enzymes HMOX1 and γ-GCLC (59 and 67%, respectively) compared to the young group, whereas the SO group had significantly lower HMOX1 and γ-GCLC protein content (65 and 47%, respectively) compared to the young group (Fig. 3B). Interestingly, we observed that SO subjects had 147% higher catalase protein content than both the young and the AO groups (Fig. 3B). There were no significant differences in total and phospho-p38 MAPK (Thr180/Tyr182), total p44/42 MAPK and Akt, or GPx1 protein content between the three groups (Fig. 3A and data not shown). Collectively, the differential phase II antioxidant content in AO vs SO further strengthens our speculation that the skeletal muscle of AO subjects is primed to coordinately activate multiple signal transduction pathways to induce Nrf2-mediated antioxidant enzymes.

A one-way analysis of variance was used to test differences between groups using Statistica 5.0 software (Statsoft, Tulsa, OK, USA). Tukey's HSD post hoc test was used to identify individual differences when statistical significance was observed. We used a one-tailed test with PC content analyses, because we a priori hypothesized that the skeletal muscle of AO and SO individuals would have higher PC content compared with the young group based upon earlier work by our group [18]. For all other analyses, a two-tailed test was employed. Statistical significance was established at P ≤0.05. Data are presented as means ±standard deviation. Asterisks denote significant changes vs young, and daggers denote significant changes vs AO. Results Nrf2-Keap1 signaling cascade is conserved in human skeletal muscle The Nrf2–Keap1 signaling cascade is a putative regulator of the basal and inducible expression of antioxidant and detoxifying genes [13]. Consequently, to test whether Nrf2 and Keap1 proteins are expressed in human skeletal muscle, we analyzed the expression of these proteins using Western blot and compared it with the known homologs of Nrf2–Keap1 previously reported in C2C12 cells in vitro, in human lung and heart tissue, and in Mus musculus (house mouse) liver (Fig. 1) [19,20]. For Nrf2–Keap1 signaling positive control, C2C12 cells were treated with hydrogen peroxide. We observed an increase in Nrf2 and a decrease in Keap1 content as previously shown [19]. Both Nrf2 and Keap1 protein homologs are conserved and stably expressed in human skeletal muscle.

Altered upstream signal transduction dysregulates Nrf2-mediated antioxidant response in sedentary older adults

Nrf2–Keap1 redox signaling is dysfunctional in sedentary, but not active, older adults

Physical inactivity is associated with increased skeletal lipid peroxidation

Next we evaluated Nrf2 and Keap1 protein content to determine their differential expression in response to age-associated oxidative stress and physical inactivity. Nrf2 protein content was significantly higher in the AO group compared to both the SO and the young groups (91 and 69%, respectively; Fig. 2A). In addition, Keap1, a negative

Protein carbonyl content, a marker of oxidative damage to proteins, was significantly elevated in the skeletal muscle of both the AO and the SO groups in comparison with the young group (39 and 53%, respectively; Fig. 4A). We also measured the skeletal muscle 4-HNE content, a marker of lipid peroxidation. There were no differences in

Fig. 1. Nrf2 and Keap1 proteins are expressed in human skeletal muscle. Nrf2 and Keap1 protein expression in (lane 1) C2C12 cells + PBS (control), C2C12 cells + 100 mM H2O2 for (lane 2) 30 min and (lane 3) 3 h, (lane 4) human lung, (lane 5) human heart, (lane 6) human skeletal muscle, and (lane 7) M. musculus liver. The molecular weights of both Nrf2 and Keap1 are estimated using Precision Plus Protein dual color standards (Bio-Rad Life Sciences, Mississauga, ON, Canada).

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Fig. 2. Nrf2–Keap1 redox signaling is impaired in the sedentary old. (A) Nrf2 and (B) Keap1 protein content and (C) Nrf2/Keap1 content ratio in the vastus lateralis of young, AO, and SO subjects (N = 10/group; ♀ = ♂). *Significant change vs young. † Significant change vs SO (P ≤ 0.05).

Fig. 3. Upstream activators of Nrf2 redox switch are altered in the sedentary old thus preventing cellular antioxidant response. (A) Phospho-p44/42 (Thr202/Tyr204), phospho-Akt (Ser473), and phospho-GSK-3β (Ser9) and (B) HMOX1, γ-GCLC, and catalase protein content in the vastus lateralis of young, AO, and SO subjects (N = 10/group; ♀ = ♂). *Significant change vs young. † Significant change vs AO (P ≤ 0.05).

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Fig. 4. Physical inactivity is associated with an increase in skeletal muscle protein oxidation and lipid peroxidation. (A) Protein carbonyl content (nmol × mg protein− 1) and (B) 4HNE-positive protein content in the vastus lateralis of young, AO, and SO subjects (N = 10/group; ♀ = ♂). *Significant change vs young. † Significant change vs SO (P ≤ 0.05).

total 4-HNE content between the groups. We observed that the 4-HNEpositive proteins were distributed over a large range of molecular weights. Individual lanes were divided into two groups (proteins weighing more or less than 25 kDa). When the 4-HNE signal from each group was requantified for 4-HNE-positive proteins that were less than 25 kDa, a substantially significant increase in 4-HNE content was detected in SO compared to the young and AO groups (284 and 276%, respectively; Fig. 4B). We suggest that an active lifestyle may confer resistance to higher levels of certain types of oxidative damage (such as lipid peroxidation) via induction of antioxidant responses that would maintain cellular redox status. Discussion The role of Nrf2–Keap1 signaling has not been previously characterized in human skeletal muscle. In this study, we have shown that the Nrf2–Keap1 redox signaling is conserved, and Nrf2–Keap1 proteins are stably expressed, in human skeletal muscle. In sedentary old subjects, a physically inactive lifestyle (hypodynamia) promoted oxidative damage to skeletal muscle. However, in recreationally active older adults, agingmediated accretion of oxidative stress is associated with the activation of signaling pathways that regulate cell survival, including PI3K/Akt and p44/42 MAPK signal transducers and a concomitant induction of Nrf2– Keap1 redox signaling. The activation of Nrf2 is consistent with the induction of phase II antioxidants in physically active older individuals. Conversely, the skeletal muscle of sedentary older adults fails to activate upstream survival pathways and shows dysregulation of Nrf2–Keap1 redox signaling that renders the intracellular environment prone to ROS-mediated toxicity and aberrant redox homeostasis. A recently elucidated Cap ‘n’ Collar transcription factor, Nrf2, and its repressor, Keap1, play indispensable roles in protecting a variety of tissues (e.g., lung, liver, kidney, central nervous system, etc.) from a wide array of toxic insults including carcinogens, electrophiles, reactive

oxygen species, inflammation, calcium disturbance, UV light, and cigarette smoke [13,14]. Studies using keap1 and nrf2 knockout mice have demonstrated a crucial role for Nrf2–Keap1 as a “multiorgan protector” in vivo [14]. nrf2 knockout mice exhibit reduced phase II antioxidant expression, enhanced susceptibility to toxicological insults, and increased oxidative stress-induced cell death [25–27]. Conversely, nrf2 overexpression in vitro and in vivo defends against oxidative stress implicated in neurodegeneration, chronic inflammation, and cancer [28–30]. Interestingly, aged animals exhibit losses in cellular redox capacity similar to those observed in nrf2 knockout mice [31,32]. To our knowledge, Nrf2–Keap1 redox signaling has not been characterized in human skeletal muscle, especially in the context of aging-associated oxidative stress, sarcopenia, and hypodynamia. In this study, we have shown that both Nrf2 and Keap1 proteins are conserved and stably expressed in human skeletal muscle (Fig. 1). Phase II antioxidants scavenge reactive oxygen/nitrogen species, detoxify electrophiles and xenobiotics, and maintain intracellular reducing potential [13]. Thus, a proper regulation of Nrf2–Keap1 redox signaling provides an important mechanistic link between oxidative stress (leading to cell death) and antioxidant gene expression (supporting cell survival). In young subjects, we observed low levels of total Nrf2 and high levels of total Keap1 in skeletal muscle (Figs. 2A and B). This is characteristic of basal physiologic levels of Nrf2–Keap1 redox signaling [13], showing that under normal redox homeostatic conditions both Nrf2 and Keap1 protein contents are tightly regulated in skeletal muscle (Fig. 2C). Conversely, we observed a significant increase in total Nrf2 content along with a decrease in Keap1 content in the skeletal muscle of AO adults, indicating that the Nrf2 signaling pathway is activated in this group and hence the redox homeostatic response is functioning in response to increased oxidative stress (Figs. 2A and B). The induction of the Nrf2–Keap1 redox signaling was muted in the SO adults, who showed total Nrf2 content similar to that in the young despite a decrease in Keap1 content (Figs. 2A and B). Whereas oxidative stress in AO adults

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is met with an appropriate induction of the Nrf2–Keap1-mediated survival response, this response is altered in hypodynamia-induced sedentary adults (Fig. 2C). The paradoxical failure to increase total Nrf2 content in the SO adults despite an increased age-associated pro-oxidant cellular milieu suggests that there may be alterations in the upstream signaling pathways that are involved in activation and stabilization of Nrf2. Also, Nrf2 gene expression is itself regulated by an ARE; therefore, the intriguing possibility exists that the dysregulation that alters AREmediated gene transcription also detrimentally represses Nrf2 transcription [13]. The upstream signaling pathways that regulate Nrf2 activation are not fully elucidated although several upstream regulatory pathways, including PI3K, Akt, p38 MAPK, p44/42 MAPK, protein kinase C, and JNK1, have been investigated [21–24]. The PI3K/Akt and MAPK signaling pathways, well-known regulators of cell survival, proliferation, and death, are activated by ROS-generating agents and H2O2 [33,34]. Loss of oxidative stress tolerance and increased muscle atrophy with aging is linked to the reduced Akt activity and increased GSK-3β activity [35– 37]. Activation of PI3K by oxidative stress resulted in activation of Nrf2 [38]. Interestingly, nrf2 knockout animals show impaired activation of PI3K/Akt, which resulted in enhanced cell death and delayed proliferation of hepatocytes [39,40]. We also observed that AO had significantly higher levels of active Akt along with inhibition of GSK-3β in comparison with SO adults (Fig. 3A). We suggest that physical activity may promote the activation of the PI3K/Akt signaling and thereby promote cell survival, counteracting the effects of age-associated oxidative stress. Alternatively, some studies show that long-term exposure to H2O2 inhibits Akt and activates GSK-3β [41]. We did not directly measure the endogenous H2O2 production; however we observed higher levels of catalase, an antioxidant enzyme induced in response to elevated cellular H2O2, in sedentary elderly subjects compared to both young and active elderly subjects (Fig. 3B). The higher levels of catalase in sedentary elderly subjects are indicative of elevated H2O2 levels, which in turn could inhibit Akt and cell survival pathways. We postulate that the increase in catalase in sedentary elderly subjects is a compensatory, albeit incomplete, response to the much greater levels of oxidative stress. Another possibility is that an alternative redox signaling pathway might be regulating the gene expression of catalase in the skeletal muscle of SO adults. The p44/42 MAPK signaling pathway has also been linked to the redox response of Nrf2–Keap1. Shen et al. [42] reported that both p44/42 MAPK and JNK signaling pathways enhance the transcriptional activity of Nrf2 in hepatocytes, whereas p38 MAPK negatively regulates Nrf2 activation. We observed that AO had significantly higher levels of active p44/42 MAPK in comparison with SO adults (Fig. 3A). We propose that the reduced activity of the PI3K/Akt and p44/42 MAPK signaling pathways contributes to the dysregulation of Nrf2–Keap1 redox signaling in SO adults and compromises their ability to respond to the age-associated increase in oxidative stress. Furthermore, physical activity effectively mitigates the decline in the function of these upstream regulatory pathways, thus preserving Nrf2 redox signaling and hence allowing muscle to better cope with the ageassociated increase in oxidative stress by maintaining cellular antioxidant capacity. Because we observed a dysregulation of Nrf2–Keap1 redox signaling in the skeletal muscle of SO adults, we sought to examine whether the protein content of Nrf2–Keap1 target genes was affected in these subjects. The phase II antioxidants HMOX1 (involved in the oxidative degradation of free heme to prevent hydroxyl radical production via the Fenton reaction) and γ-GCLC (the catalytic subunit of the rate-limiting enzyme γglutamylcysteine ligase in glutathione synthesis) are directly regulated by Nrf2 and were thus chosen for further study [13]. The content of HMOX1 and γ-GCLC was expectedly low in the skeletal muscle of young subjects (Fig. 3B). In contrast, the skeletal muscle of AO adults had significantly elevated HMOX1 and γ-GCLC content in comparison with the young adults, which further confirms the increased Nrf2 signaling observed in AO (Fig. 3B). Finally, skeletal muscle of SO adults failed to show an increase

in γ-GCLC and HMOX1 content compared to the young group. HMOX1 transcription is responsive to “recreational” low levels of physical activity [43]. Ding and colleagues [19] recently noted that catalase negatively regulates the expression of γ-GCLC, along with myogenin and myosin heavy chain content, hence suppressing myogenesis. This observation provides an alternate reasoning to the lack of increase in γ-GCLC content in the skeletal muscle of SO adults because they had significantly higher levels of catalase relative to both the AO and the young adults (Fig. 3B). Collectively, the differential phase II antioxidant content in our older adult populations strengthens our speculation that a life-long recreationally active lifestyle may provide an adequate and constant stressor to induce physiological levels of ROS that coordinately activate multiple signal transduction pathways and Nrf2-mediated phase II antioxidant response in an attempt to maintain skeletal muscle redox status. In contrast, a sedentary lifestyle may not only promote, but even accelerate, the aging phenotype and associated comorbidities via a dysregulation in the cellular antioxidant status and energy homeostasis. The role of oxidative stress and macromolecular damage in the etiology of sarcopenia has been extensively characterized [44–49]. We have previously shown that recreational activity in old adults confers higher levels of oxidative damage in skeletal muscle relative to young adults [18]. Indeed, we observed a significant increase in protein carbonyl content in the skeletal muscle of the older adults in comparison to the young (Fig. 1C). In addition, low-molecular-weight 4-HNEpositive protein content was significantly higher in SO in comparison to both the young and the AO groups (Fig. 1D). These finding suggests that there will be an increase in oxidative damage to muscle with aging (such as protein carbonyl content in both AO and SO groups) and that a certain type of oxidative damage is progressive depending on the physical activity status of the elderly (such as lipid peroxidation content in SO vs AO groups). Because the recreationally active older adults exhibited significantly higher skeletal muscle strength and lower levels of chronic muscle inflammation compared to the sedentary old group, we predicted that the skeletal muscle of physically active older adults would show an increase in cellular antioxidant response to preserve tissue function. In future, we will be utilizing mass spectrometry to identify the proteins that are oxidized as a function of aging and/or physical activity status of the elderly. The novel findings of this study highlight the complexity of coordinated regulation of Nrf2–Keap1 signaling in maintaining cellular redox homeostasis in the skeletal muscle of older adults. Because a physically active lifestyle is suggested to have therapeutic potential against cancer, atherosclerosis, obesity, type II diabetes, metabolic syndrome, and associated comorbidities [9,50], it is intriguing to speculate that maintenance of the Nrf2–Keap1 redox signaling via regular exercise training may combat these pathologies. In addition, we propose that the pharmaceutical/nutraceutical induction of Nrf2–Keap1 redox signaling [51–54] promises to be a viable therapy for attenuating oxidative stressmediated damage in skeletal muscle associated with physical inactivity. Author contribution MAT and AS conceived and designed the experiments. MAT and JdB collected muscle biopsies for anlayses. AS performed the experiments. AS analysed the data. MAT contributed reagens/materials/analysis tools and all funding. AS and MAT interpreted the data. AS wrote the manuscirpt. MAT critcically reviewed the manuscript. Competing interests The authors have declared no competing interests exist. Acknowledgments We acknowledge the invaluable time, patience, and dedication of the participants in the study. We also acknowledge Mr. Jonathan P. Little for

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his gracious assistance in critically reviewing the manuscript. Funding for this work was primarily from grants to M.A.T. from the Canadian Institutes of Health Research (CIHR), from Physician Services Incorporated, and from a donation from Mr. Warren Lammert and family. A.S. was funded by a CIHR Institute of Aging Doctoral Research Award. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. References [1] Manton, K. G.; Vaupel, J. W. Survival after the age of 80 in the United States, Sweden, France, England, and Japan. N. Engl. J. Med. 333:1232–1235; 1995. [2] Tarnopolsky, M. A.; Safdar, A. The potential benefits of creatine and conjugated linoleic acid as adjuncts to resistance training in older adults. Appl. Physiol. Nutr. Metab. 33:213–227; 2008. [3] Hollmann, W.; Struder, H. K.; Tagarakis, C. V.; King, G. Physical activity and the elderly. Eur. J. Cardiovasc. Prev. Rehabil. 14:730–739; 2007. 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