Cellular Markers of Muscle Atrophy in Chronic Obstructive Pulmonary Disease

Cellular Markers of Muscle Atrophy in Chronic Obstructive Pulmonary Disease Pamela J. Plant 1, Dina Brooks 2, Marie Faughnan 1, Tanya Bayley 1, James Bain 3, Lianne Singer 4, Judy Correa 1, Dawn Pearce 5, Matthew Binnie 1, and Jane Batt 1 1 Department of Medicine and 5 Department of Radiology and St. Michael s Hospital, University of Toronto, Toronto, Ontario, Canada; 2 Department of Physical Therapy, University of Toronto and Toronto Rehab Institute, Toronto, Ontario, Canada; 3 Department of Surgery, McMaster University, Hamilton, Ontario, Canada; and 4 Department of Medicine, Toronto General Hospital, University Health Network, University of Toronto, Toronto, Ontario, Canada Skeletal muscle atrophy in individuals with advanced chronic obstructive pulmonary disease (COPD) is associated with diminished quality of life, increased health resource use, and worsened survival. Muscle wasting results from an imbalance between protein degradation and synthesis, and is enhanced by decreased regenerative repair. We investigated the activation of cellular signaling networks known to mediate muscle atrophy and regulate muscle regenerative capacity in rodent models, in individuals with COPD (FEV 1, 50% predicted). Nine patients with COPD and nine control individuals were studied. Quadriceps femoris muscle isometric contractile force and cross-sectional area were confirmed to be significantly smaller in the patients with COPD compared with control subjects. The vastus lateralis muscle was biopsied and muscle transcript and/or protein levels of key components of ubiquitin-mediated proteolytic systems (MuRF1, atrogin-1, Nedd4), inflammatory mediators (IkBa, NFkBp65/p50), AKT network (AKT, GSK3b, p70s6 kinase), mediators of autophagy (beclin-1, LC3), and myogenesis (myogenin, MyoD, Myf5, myostatin) were determined. Atrogin-1 and Nedd4, two ligases regulating ubiquitin-mediated protein degradation and myostatin, a negative regulator of muscle growth, were significantly increased in the muscle of patients with COPD. MuRF1, Myf5, myogenin, and MyoD were not differentially expressed. There were no differences in the level of phosphorylation of AKT, GSK3b, p70s6kinase, or IkBa, activation of NF-kBp65 or NF-kBp50, or level of expression of beclin-1 or LC3, suggesting that AKT signaling was not down-regulated and the NF-kB inflammatory pathway and autophagy were not activated in the COPD muscle. We conclude that muscle atrophy associated with COPD results from the recruitment of specific regulators of ubiquitin-mediated proteolytic pathways and inhibition of muscle growth. Keywords: vastus lateralis; ubiquitin ligase; myostatin; neural precursor cell expressed developmentally down-regulated 4; atrogin-1 Skeletal muscle atrophy is a critical phenomenon that occurs in individuals with chronic obstructive pulmonary disease (COPD). It is associated with diminished exercise capacity (1) and quality of life (2), increased health resource use and health care costs (3), and is a powerful negative predictor of survival (4, 5). Loss of skeletal muscle mass results from decreased muscle protein synthesis and increased proteolysis, and this may (Received in original form October 8, 2008 and in final form May 8, 2009) This work was supported by Canadian Institutes of Health Research New Investigator Awards (D.B. and J.B.), a Keenan Foundation Fellowship, St Michaels Hospital (P.P.), and by an Ontario Thoracic Society Grant-in-Aid Award ( J.B., D.B., and M.F.). Correspondence and requests for reprints should be addressed to Jane Batt, M.D., Ph.D., Room 7344, Medical Sciences Building, 1 Kings College Circle, University of Toronto, Toronto, ON, M5S 1A8 Canada. E-mail: jane.batt@ utoronto.ca Am J Respir Cell Mol Biol Vol 42. pp 461 471, 2010 Originally Published in Press as DOI: 10.1165/rcmb.2008-0382OC on June 11, 2009 Internet address: www.atsjournals.org be enhanced by decreased regenerative capacity of the muscle (reviewed in References 6 8). The molecular mechanisms underlying skeletal muscle atrophy have recently begun to be elucidated through in vitro experimentation in myotube cultures and in rodent models of disease. Although muscle proteolysis is mediated via the coordinated efforts of several cellular networks, including, for example, oxidative stress responses, activation of calpains, and nonlysosomal proteases, it is the ubiquitin proteasome pathway that predominates (6, 8 12). The ubiquitin ligases, atrogin-1, muscle-specific RING finger protein (MuRF) 1, and neural precursor cell expressed developmentally down-regulated (Nedd) 4, key enzymes regulating ubiquitin-mediated protein degradation, are variably up-regulated in multiple rodent models of skeletal muscle atrophy, ranging from traumatic denervation to chronic metabolic diseases, such as cancer and diabetes (13 17). More recently, activation of the autophagic/ lysosomal system has also been recognized to contribute to muscle atrophy and, at least in vitro, autophagy seems to play a substantial role in the loss of muscle protein (18, 19). In muscle atrophy associated with generalized inflammatory states (20, 21), the inflammatory cytokine TNF-a causes muscle proteolysis and inhibits muscle regenerative capacity by inducing oxidative stress, and destabilizing MyoD (20, 22) through NF-kB activation. MyoD is a muscle transcription factor essential to myogenesis and muscle regeneration. NF-kB activation also mediates expression of the ubiquitin ligase, MuRF1, thus enhancing proteasome-mediated protein degradation (23, 24). It is also known that the engagement of muscle atrophy signaling networks results in the concurrent down-regulation of the signaling pathways that induce muscle hypertrophy. Activation of the AKT (protein kinase B) pathway, and signaling through its downstream targets, such as glycogen synthase kinase (GSK)-3b and p70s6 kinase (70-kD ribosomal protein S6 kinase), results in muscle hypertrophy (8, 25). This pathway is down-regulated/deactivated in models of muscle wasting, demonstrating a key finding that there is a reciprocal link between the muscle hypertrophic and atrophic signaling networks (8, 25, 26). The muscle regulatory transcription factors, myogenin and myogenic factor (Myf) 5, which induce myoblast differentiation and muscle regeneration and myostatin, a negative regulator of muscle growth, can also influence muscle mass, as demonstrated in genetic rodent models (reviewed in References 7, 27). Few studies have assessed cellular signaling of muscle atrophy in humans, and thus the relevance of these networks and effectors of atrophy in human disease is not clear. In addition, in the myriad of signaling networks activated to induce muscle atrophy in rodent models, there is some specificity of pathway recruitment to the atrophy model assessed. Given this variability, we need to determine which cellular signaling

462 AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL 42 2010 networks are active in contributing to the clinically significant muscle atrophy of COPD. This will provide future targets for pharmacologic manipulation, with the goal of improving quality of life, functional status, and, potentially, survival in COPD. Thus, in this study, we sought to identify the cellular signaling pathways activated in atrophied skeletal muscle of patients with severe COPD by measuring mrna transcript and/or protein expression levels of key signaling network components in biopsies of the quadriceps muscles. MATERIALS AND METHODS Patient and Control Populations Patients with COPD were recruited from respirology clinics at St. Michael s Hospital (SMH) and the Toronto General Hospital, University of Toronto. Patients were deemed eligible if they had a physician diagnosis of COPD, as defined by the Canadian Thoracic Society guidelines (28), and severe disease (FEV 1, 50% predicted). Exclusion criteria included: (1) the use of systemic glucocorticoid therapy in the preceding 90 days or the chronic use of medication that causes muscle atrophy; (2) comorbidities associated with muscle atrophy (infection, diabetes, end-stage renal disease on dialysis, active cancer); (4) COPD exacerbation or hospitalization in the previous 3 months; (5) HIV, hepatitis B, or hepatitis C infection; (6) age greater than 75 years; and (8) institutionalized care or required assistance for activities of daily living. A healthy age-matched control population was drawn from volunteer participants registered with the SMH Healthy Lung Database and through advertisements that were placed throughout the hospitals. These individuals had no known lung disease and did not fulfill any of the study exclusion criteria. The study protocol was approved by the research ethics boards at Toronto General Hospital and SMH. All participating subjects provided informed consent. Function and Quality of Life Measurements Pulmonary disease severity and degree of impairment and disability were characterized using pulmonary function testing, BODE score, and 6-minute walk distance as outlined briefly here. Pulmonary function testing. Spirometry was performed in accordance with recommended techniques detailed by the American Thoracic Society guidelines (29). The primary parameters of assessment were FEV 1, FVC, and the FEV 1 :FVC ratio. The 6-minute walk test. The 6-minute walk test was used to assess functional exercise capacity, according to American Thoracic Society established criteria (30, 31). We applied standardized instruction and encouragement. At baseline, the patients and control subjects completed at least two training walks to account for learning effect. BODE score. The BODE score incorporates body mass index (BMI), FEV 1% predicted, score on the modified Medical Research Council dyspnea scale, and 6-minute walk distance to estimate survival in COPD (32). The BODE score was calculated for all participants. Assessment of Body Composition, Muscle Mass, and Strength Muscle mass and body composition were determined using clinical measures (BMI, sum of five skin folds, circumferential waist girth), and the midthigh quadriceps femoris muscle cross-sectional area (CSA) was determined as previously described (33). Briefly, computed tomography imaging of the right thigh, halfway between the pubic symphysis and the inferior condyle of the femur, using a Light Speed QXi 4 slice helical scanner (General Electric, Milwaukee, WI), was performed with the subject in the supine position. Each image was 10 to 20 mm thick, and the muscle identified as tissue with a density of 40 to 100 Hounsfield units. Images were analyzed and the CSA of muscle determined by a single investigator, blinded as to the categorization of each test subject. Muscle strength was determined using isometric contractile strength testing of the quadriceps femoris muscle. Isometric contraction of the quadriceps femoris was measured with the use of a strain gauge, with the subject seated on a chair extending the knee against a stationary metal bar, as previously described (34, 35). Isometric contraction is reflective of the amount of time a muscle group can perform and exercise. Vastus Lateralis Percutaneous Biopsy Biopsy of the vastus lateralis muscle was performed under local anesthetic using a modified Bergstrom needle, as previously described (36). Briefly, under sterile conditions, the skin and subcutaneous tissue were anesthetized, and a small incision was made in the outer fascial layer with a scalpel blade. The Bergstrom needle was advanced through the incision roughly 1 cm into the muscle, suction was applied as the trochar was advanced, and several pieces of muscle tissue were obtained. The needle was withdrawn under counter pressure, the skin closed with a single suture, and a pressure dressing applied. Extracted tissue was snap frozen in liquid nitrogen for RNA and protein extraction. RNA Extraction and Real-Time RT-PCR Real-time RT PCR was used to assess expression levels of transcripts for the ubiquitin ligases, atrogin-1, MuRF1, and the myogenic regulatory factors (MRFs), myogenin, Myf5, and myostatin (an inhibitor of muscle growth), and mediators of autophagy, beclin-1 and autophagyrelated protein LC (LC)-3. Transcript levels were assessed as opposed to protein levels, because good commercial antibodies were not available and/or the limited amount of protein available necessitated that mrna levels be determined. These genes do not produce proteins that require phosphorylation for activation, and determination of the level of expression of the transcripts was therefore deemed to be adequate. Tissue snap-frozen in liquid nitrogen was homogenized in Trizol (1 ml/mg tissue; Life Technologies, Burlington, ON, Canada) to isolate total muscle RNA, per the manufacturer s recommendations. RNA quality was assessed with agarose gel electrophoresis and the Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA), and quantified by absorption spectrophotometry at 260 and 280 nm. To remove any contaminating genomic DNA, the muscle total RNA samples were treated with DNA-free (Ambion, Austin, TX), per the manufacturer s instructions. cdna was then generated from the total RNA from each patient and control subject in a first-strand synthesis reaction using Superscript II Reverse Transcriptase (Invitrogen, Burlington, ON, Canada) at 428C for 2 hours, according to the manufacturer s instructions. Relative quantitative real-time RT-PCR was subsequently performed with an Applied Biosystems 7900 system and SYBR Green Master Mix (Applied Biosystems, Foster City, CA) for atrogin-1, MuRF1, myogenin, Myf5, myostatin, beclin-1, and microtubule-associated protein 1A/1B-light chain 3 (LC3). Primers were designed using Primer Express software (Applied Biosystems) (Table 1). For each gene, real-time PCR was performed in triplicate wells on cdna generated from the reverse transcription of 10 ng of total RNA. Negative controls for each gene included no template (water) and no reverse transcriptase (10 ng RNA). The PCR amplification consisted of 10-minute denaturation, followed by 40 cycles of amplification (15 s at 958C, 60 s at 608C). After amplification, amplicons were melted and the resulting dissociation curve assessed to ensure a single product. Aliquots (10 ml) of all products were run on DNA polyacrylamide gels to ensure the presence of a single amplicon of the expected size. The real-time experiments were repeated twice for all genes and all subjects. The expression level of each transcript for each individual (patient or control subject) was determined relative to a pooled RNA reference generated in the laboratory, using the relative quantification (DDCT) technique, according to Applied Biosystems instructions. Two housekeeping genes tyrosine 3 monooxygenase/tryptophan 5 monoxygenase (YWHAZ), or hydroxymethylbilane synthetase (HMBS) were used. Primer amplification efficiencies were equal for all genes tested and both housekeeping genes (data not shown). Protein Extraction and Western Blotting Proteins were extracted by homogenizing the frozen muscle in lysis buffer (5 mm Tris-HCL [ph 8.0], 1 mm EDTA, 1 mm EGTA, 1 mm b-mercaptoethanol, 1% glycerol with 1 mm PMSF, 10 mg/ml leupeptin, 10 mg/ml aprotinin, 1 mm orthovanadate) with a Polytron PTE 1200E homogenizer (Kinematica, Lucerne, Switzerland) in three 30-second pulses, and homogenates were cleared by centrifuging at 1,600 3 g for

Plant, Brooks, Faughnan, et al.: Markers of Muscle Atrophy in COPD 463 TABLE 1. REAL-TIME RT-PCR PRIMER SEQUENCES Gene Forward Primer Reverse Primer HMBS tgc aac ggc gga aga aaa agc tgg ctc ttg cgg gta c YWHAZ gca atg atg tac tgt ctc ttt tgg aa taa cgg tag taa tct cct ttc att ttc a Atrogin-1 gca cgt gct cag cga aga atc tgc cgc tcg gag aag t MuRF1 tcc agc aga cac tga acc aga a tcc att ttg cac caa tgt aga aa Myf5 agg tca acc agg ctt tcg aa gat gta gcg gat ggc att cc Myostatin ttg aga ccc gtc gag act cct a ttc aga gat cgg att cca gta tac c Myogenin gct gta tga gac atc ccc cta ctt cgt agc ctg gtg gtt cga a Beclin-1 agg aac tca cag ctc cat tac aat ggc tcc tct cct gag tt LC3 atg tca aca tga gcg agt tgg t ctg gtt cac cag cag gaa gaa Definition of abbreviations: HMBS, hydroxymethylbilane synthetase; LC3, microtubule-associated protein 1A/1B-light chain 3; MuRF, muscle-specific RING finger protein; Myf, myogenic factor; YWHAZ, tyrosine 3 monooxygenase/tryptophan 5 monoxygenase. 10 minutes at 48C. The supernatant (soluble fraction) was centrifuged further for 10 minutes at 48C and 10,000 3 g. The pellet (insoluble fraction) was washed with PBS, resuspended, dissolved by sonication in 200 ml buffer containing protease/phosphatase inhibitors (as described previously here), and further centrifuged (10,000 3 g, 48C) for 10 minutes, and the supernatant designated as the insoluble fraction. Protein lysate (7.5 mg) was separated by SDS-PAGE, immunoblotted, and bands detected using chemiluminescence generated by Super- Signal West Femto Chemiluminescent Substrate (Thermo Scientific, Waltham, MA) acquired with a charge-coupled device camera (Bio- Rad VersaDoc, Hercules, CA), and quantified using Quantity One software (Bio-Rad). Primary antibodies used included: actin (1:200 dilution; Sigma-Aldrich, St. Louis MO); AKT; phospho-akt (Ser 473); phospho-gsk3a/b (Ser 21/9); inhibitory kb (IkB)-a; phospho IkB-a (Ser 32/36); NF-kB p65; phospho-nf-kb p65 (Ser 536); p70 S6 kinase (1:1,000 dilution; all from Cell Signaling, Boston, MA); lamin A 1 C and MuRF1 (1:1,000 dilution; both from Abcam, Cambridge, MA); phospho p70s6 kinase (Thr 389, 1:1,000 dilution; Cell Signaling); MyoD (1:500 dilution; Santa Cruz, Santa Cruz, CA); GAPDH (1:100,000 dilution; Abcam); GSK3a/b (1:10,000 dilution; Invitrogen, Carlsbad, CA); and a-tubulin (1:50,000 dilution; Sigma). Secondary antibodies consisted of horseradish peroxidase (HRP) conjugated antimouse, anti-rabbit, and anti-goat at 1:20,000 dilution. Phosphorylated proteins were normalized in expression to their total unphosphorylated forms. MyoD, Nedd4, and MuRF1 were normalized to two housekeeping proteins, a-tubulin and GAPDH. Given the limited sample available from the muscle biopsies, the levels of some proteins of interest were not determined in all study participants. Total numbers assessed are detailed in the results section. Activated NF-kB(p65) and NF-kB(p50) Assays Quantitation of the expression of active NF-kB(p65) and active NFkB(p50) was completed using the Thermo Scientific Transcription Factor Kits for NF-kB(p65) and NF-kB(p50) (nos. 89858 and 89859; ThermoFisher Scientific, Waltham, MA), as directed by the manufacturer. Briefly, 7 mg of protein lysates were incubated with the NF-kB binding biotinylated consensus sequence DNA (p65 or p50) adherent to the streptavidin-coated 96-well plates provided. Only the active NFkB (p65 or p50) binds to the consensus sequence, and is subsequently detected with anti NF-kB(p65) or NF-kB(p50) primary antibody, an HRP-conjugated secondary antibody and a chemiluminescent substrate (all provided in the kits). Chemiluminescence was detected in an EnVision Multilabel Plate Reader Model 2102 (Perkin Elmer, Waltham, MA) and reported as relative chemiluminescence units. Specificity of the binding of the activated transcription factors to the consensus sequence DNA was ensured by using control wild-type and mutant NF-kB (p65 or p50) competitor duplexes supplied with the kit. Statistical Analysis Continuous data are reported as mean and SE, and were compared using unpaired Student s t test or Wilcoxon s rank-sum test after testing for normal distribution (Shapiro-Wilk). When comparing multiple independent means, a one-way ANOVA was first performed to confirm a difference across all groups prior to comparison of individuals means. Results were considered significant if P values were less than 0.05. RESULTS Characterization of the COPD Patient and Control Populations A total of 94 individuals with severe COPD were assessed for study inclusion. Of these, 37 were not eligible based on exclusion criteria. Of the remaining 57 eligible patients, 9 provided informed consent and took part in the study. Nine age-matched healthy control individuals provided informed consent and took part in the study. To characterize our study populations, body composition, pulmonary function, and functional capacity were assessed and data are provided in Table 2. As expected, individuals with severe COPD had significantly lower 6-minute walk distances and significantly higher BODE scores compared with control individuals. There were no differences between the COPD and control populations with respect to BMI, percent body fat, or sum of five skin folds. The distribution of males and females was not equal between groups, with more females in the control group. Two male patients with COPD were involved in a pre lung transplant rehabilitation program, and one female patient undertook regular aerobic and resistance training at least three times per week. The remaining patients with COPD were all fully independent and intermittently exercised. Four control participants (two male and two female) undertook regular aerobic training greater than three times per week. The remainder intermittently exercised. To confirm the presence of muscle atrophy in the patients with COPD, we measured CSA of the quadriceps femoris muscle. Patients with COPD demonstrated smaller quadriceps CSA (61.26 6 5.36 cm 2 ; n 5 9) compared with control individuals (89.88 6 4.59 cm 2 ; n 5 9) as determined by computed tomography imaging (P, 0.05; Figure 1A). Similarly, the strength of the quadriceps was lower in the patients with COPD than the control subjects (11.95 6 1.06 kg; n 5 8 versus TABLE 2. SUBJECT CHARACTERISTICS Patient Subjects Control Subjects Characteristics (n 5 9) (n 5 9) Significance Age, yr 64.0 6 2.1 59.6 6 1.3 N.S. Sex, male/female 5/4 3/6 Body mass index 24.4 6 1.1 25.9 6 1.8 N.S. Body fat, % 26.8 6 2.0 26.8 6 2.0 N.S. Sum of five skin folds, cm 58.7 6 7.5 65.3 6 5.0 N.S. Waist circumference, cm 92.7 6 3.0 99.8 6 11.9 N.S. FEV 1, L/s 0.88 6 0.09 2.87 6 0.22 P, 0.05 FEV 1% predicated 35.1 6 2.5 115.1 6 7.9 P, 0.05 BODE 4.78 6 0.45 0.11 6 0.11 P, 0.05 6-min walk, m 393 6 32 617 6 22 P, 0.05 Definition of abbreviations: N.S., not significant.

464 AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL 42 2010 Figure 1. Quadriceps cross-sectional area (CSA) and power. Patients with chronic obstructive pulmonary disease (COPD) demonstrate significantly decreased CSA of the quadriceps femoris muscle (A) and are weaker (B) than the agematched control subjects. Significant differences in muscle area between patients and control subjects are maintained even when data are assessed in sex-specific groups (C). Quadriceps of female patients are significantly weaker than in female control subjects (D), and male patients are significantly weaker than male control subjects, but male patients are not significantly weaker than female control subjects. F, female; M, male. 23.46 6 3.02 kg, n 5 9; P, 0.05; Figure 1B). One male patient declined strength testing. Due to the uneven distribution of males and females in the patient and control populations, we assessed differences on a sex-specific basis. Female patients muscle area (46.37 6 5.95 cm 2 ; n 5 4) was smaller than female control individuals (81.59 6 2.92 cm 2 ; n 5 6; P, 0.05; Figure 1C). Male patients muscle area (73.18 6 1.55 cm 2 ; n 5 5) was significantly smaller than both male control subjects (106.50 6 1.94 cm 2 ; n 5 3; P, 0.05) and female control subjects (P, 0.05; Figure 1C). Sexspecific comparisons were also performed for strength (Figure 1D). Female patients quadriceps strength was significantly weaker than female control subjects (11.4 6 1.9 kg [n 5 4] versus 20.1 6 2.6 kg [n 5 6]; P, 0.05). Male patients were weaker than male control subjects (12.5 6 1.1 kg [n 5 4] versus 30.1 6 6.5 kg [n 5 3]; P, 0.05). Male patients, however, were not significantly weaker than female control subjects (P 5 0.062). Ubiquitin Ligases Mediators of Protein Degradation by the Proteasome Good-quality RNA was extracted for all study participants. Patients with COPD demonstrated a significant increase in the level of atrogin-1 transcript expression (2.8 6 0.5 fold change; n 5 9) compared with the control individuals (1.4 6 0.2 fold change; n 5 9; P, 0.05) (Figure 2A). There was no difference in the expression level of MuRF1 transcripts between patients with COPD (10.7 6 2.1 fold change; n 5 9) and control individuals (8.8 6 2.2 fold change; n 5 9; P. 0.05) (Figure 2B), nor was there any difference in MuRF1 proteins levels in the soluble fraction of muscle protein lysates between patients with COPD (0.22 6 0.10; n 5 9) and control individuals (0.11 6 0.03; n 5 9; P. 0.05) (Figures 2D and 2F). In contrast, Nedd4 was increased in expression in the insoluble fraction of muscle protein lysates (insoluble myofibrils) in the patients with COPD (0.37 6 0.08; n 5 9) versus control subjects (0.09 6 0.03; n 5 9; P, 0.05) (Figures 2C and 2E). Nedd4 was also detectable in the soluble fraction of protein lysates, but no differences in expression were seen in this fraction between patients with COPD and control individuals (data not shown). Mediators of Muscle Regenerative Capacity Muscle Regulatory Transcription Factors and Myostatin Real-time RT-PCR was performed for myostatin, a negative regulator of muscle growth and the muscle regulatory transcription factors, myogenin and Myf5, which induce myoblast differentiation and muscle regeneration. Myostatin transcript levels (Figure 3A) were significantly higher in the patients with COPD (3.1 6 0.8 fold change; n 5 9) compared with the control group (0.9 6 0.1 fold change; n 5 9; P, 0.05). Neither Myf5 (Figure 3B) nor myogenin (Figure 3C) demonstrated significant differences in expression between patients with COPD (n 5 9) and the control population (n 5 9) (Myf5, 0.96 6 0.18 versus 0.88 6 0.15 fold change [P. 0.05], and myogenin, 1.32 6 0.27 versus 0.96 6 0.16 fold change [P. 0.05]). Similarly, there was no difference in the level of expression of the proregenerative MyoD in the soluble fraction of muscle protein lysates in patients with COPD (0.14 6 0.05; n 5 8) versus control individuals (0.06 6 0.03; n 5 8; P. 0.05; Figures 3D and 3E). NF-kB Signaling Network Inflammatory mediators Phosphorylation of IkBa leads to its proteolytic degradation, and subsequent release and activation of NF-kB transcription factors. Total IkBa and phosphorylated IkBa (pikba) protein levels (Figure 4) were determined in the soluble fraction of muscle protein lysates by Western blotting. There was no significant difference in the level of phosphorylation of IkBa in the patients with COPD compared with the control population (Figure 4), as determined by measuring the ratio of phosphorylated IkBa/total IkBa (patients with COPD, 0.34 6 0.11 [n 5 9], versus control subjects, 0.22 6 0.09 [n 5 8]; P. 0.05) or when normalizing phosphorylated IkBa levels to GAPDH (patients with COPD, 0.18 6 0.05 [n 5 9] versus control subjects, 0.20 6 0.04 [n 5 8]; P. 0.05).

Plant, Brooks, Faughnan, et al.: Markers of Muscle Atrophy in COPD 465 Figure 2. Ubiquitin ligases. Atrogin-1 mrna levels (A) were increased in patients with chronic obstructive pulmonary disease (COPD) versus control individuals, as determined by relative quantification with real-time RT-PCR. There was no difference in muscle-specific RING finger protein (MuRF) 1 transcript levels (B) between patients and control subjects. mrna levels in patients and control subjects were compared with a common reference standard. Tyrosine 3 monooxygenase/tryptophan 5 monoxygenase (YWHAZ) served as the housekeeping gene (HKG), and similar data were obtained using hydroxymethylbilane synthetase (HMBS) as the HKG (data not shown). Neural precursor cell expressed developmentally downregulated (Nedd) 4 protein levels (C) were increased in the insoluble fraction of patient muscle protein lysates compared with control subjects, as determined by SDS PAGE, Western blotting, and charge-coupled device camera image acquisition, and quantification of the chemiluminescent signal. Nedd4 expression was normalized to a-tubulin, and a representative Western blot is shown (E ). Similar results were obtained normalizing Nedd4 to GAPDH (data not shown). There was no difference in MuRF1 protein (D) expression in the soluble fraction of muscle protein lysates between patients and control subjects. MuRF1 protein levels were normalized to a-tubulin, and a representative Western blot is shown (F ). Similar results were obtained normalizing to GAPDH (data not shown). Co, control individual; N.S., not significant; Pt, patient; P. 0.05. Quantitation of activation of NF-kB(p65) and NF-kB(p50) was determined by measuring the chemiluminescence signal generated by binding of the activated transcription factors to their respective DNA consensus sequences fixed in a 96-well plate and detected with anti NF-kB(p65) and anti NF-kB(p50) primary antibodies and HRP-linked secondary antibodies. There was no significant difference in the level of expression of active NF-kB(p65) in patients with COPD (7.23 6 1.6 3 10 5 chemiluminescence units; n 5 6) versus control subjects (6.4 6 1.7 3 10 5 chemiluminescence units; n 5 6) (P. 0.05; Figure 5A). Similarly, there was no difference in the level of expression of active NF-kB(p50) between patients with COPD (1.9 6 0.8 3 10 6 chemiluminescence units; n 5 5) and control subjects (1.2 6 0.5 3 10 6 chemiluminescence units; n 5 6) (P. 0.05; Figure 5B). AKT Signaling Network Mediators of Muscle Hypertrophy Total AKT and phosphorylated AKT, total GSK3b and phosphorylated GSK3b, and total p70s6 kinase and phosphorylated p70s6 kinase protein levels were determined in the soluble fraction of the muscle protein lysates by Western blot analysis. There were no differences in the level of phosphorylation of AKT (Figure 6) in the patients with COPD (0.44 6 0.13; n 5 8) versus the control population (0.30 6 0.10; n 5 9) (P. 0.05), the level of phosphorylation of GSK3b (Figure 7) in the patients with COPD (0.58 6 0.16, n 5 8) versus the control population (0.74 6 0.17; n 5 9) (P. 0.05) or level of phosphorylation of p70s6 kinase (Figure 8) in the patients with COPD (0.34 6 0.10; n 5 8) versus the control population (0.29 6 0.08; n 5 9) (P. 0.05). Mediators of Autophagy Beclin-1 and LC3 There was no difference in the level of expression of beclin-1 transcripts in patients with COPD (6.7 6 1.2 fold change; n 5 9) compared with control individuals (7.4 6 1.1 fold change; n 5 9) (P. 0.05; Figure 9A). Similarly, there was no difference in the level of expression of LC3 transcripts between patients with COPD (52.4 6 16.5 fold change; n 5 9) and control individuals (54.3 6 8.5 fold change; n 5 9) (P. 0.05; Figure 9B). DISCUSSION Skeletal muscle atrophy and weakness occur in individuals with advanced COPD, and this reduction in muscle mass is associ-

466 AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL 42 2010 Figure 3. Muscle-regulatory transcription factors and myostatin. Myostatin mrna levels (A) were significantly increased in patients with chronic obstructive pulmonary disease (COPD) compared with control subjects, as determined by relative quantification with real-time RT-PCR. There were no differences in the level of expression of (B) myogenic factor (Myf) 5 transcripts or (C) myogenin transcripts between patients with COPD and control subjects. Transcript levels in patients and control subjects were compared with a common reference standard. YWHAZ served as the HKG. Results were similar using HMBS as the HKG (data not shown). MyoD (D) protein levels in the soluble fraction of muscle protein lysates were not significantly different between patients with COPD and control subjects, as determined by SDS PAGE, Western blotting, and charge-coupled device camera image acquisition, and quantification of the chemiluminescent signal. MyoD expression was normalized to a-tubulin. (E) A representative Western blot is shown. Similar results were obtained normalizing MyoD to GAPDH (data not shown). Co, control; N.S., not significant; Pt, patient; P. 0.05. ated with increased morbidity, mortality, health resource use, and cost (1, 3 5). The significant advances made in the past decade delineating the cellular signaling pathways and key mediators responsible for the development of skeletal muscle atrophy (reviewed in References 6 8) have been derived from experimentation conducted largely in rodent models of disease, and the clinical relevance of these findings is not known. In the present study, we sought to identify the cellular signaling Figure 4. Inhibitory kb (IkB)-a. (A) There was no significant difference in the level of IkBa phosphorylation in the patients with chronic obstructive pulmonary disease (COPD) versus control individuals. Total and phosphorylated IkBa (IkBa and pikba) protein levels were determined in the soluble cytoplasmic fraction of muscle protein lysates by SDS- PAGE, Western blotting, and charge-coupled device camera image acquisition, and quantification of the chemiluminescent signal. pikba expression was normalized to total IkBa and to GAPDH. (B) A representative Western blot is shown. Positive (Pos) and negative (Neg) controls for IkB phosphorylation consisted of protein lysates from HeLa cell cultures treated with TNF-a (20 ng/ml 3 5 min) and untreated, respectively. Co, control; N.S., not significant; Pt, patient; P. 0.05.

Plant, Brooks, Faughnan, et al.: Markers of Muscle Atrophy in COPD 467 Figure 5. NF-kB(p65) and NF-kB(p50) activation. Expression levels of the active forms of NF-kB(p65) and NF-kB(p50) proteins were determined in muscle protein lysates using the ThermoScientific Transcription Factor Kits for NF-kB(p65) and NF-kB(p50). Activated p65 and p50 bound to the NF-kB binding consensus sequence DNA immobilized in 96-well plates was detected using anti NF-kB(p65) and NF-kB(p50) antibodies, horseradish peroxidase linked secondary antibodies and a chemiluminescent substrate. The chemiluminescent signal was quantified in an EnVision multilabel plate reader. There was no significant difference in the level of expression of active NF-kB(p65) (A) or NF-kB(p50) (B) in the patients with chronic obstructive pulmonary disease (COPD) versus control individuals. N.S., not significant; P. 0.05 (59, 60). networks responsible for the development of muscle atrophy in individuals with COPD. Our patients with COPD were carefully selected to exclude comorbid illness and medical therapies that could affect muscle mass. Therefore, the significant atrophy and weakness of the quadriceps femoris muscle demonstrated most likely results from the advanced but stable COPD in our patient population. We report the novel observations of upregulation of myostatin and the ubiquitin ligase, Nedd4, and confirm up-regulation of a second ubiquitin ligase, atrogin-1 (which has been previously reported [37]), in the atrophied vastus lateralis muscle of individuals with severe COPD. Skeletal muscle protein loss is mediated by a series of intracellular signaling networks, but ubiquitin-dependent proteolytic mechanisms are the predominant mechanisms responsible for the development of muscle atrophy (reviewed in References 6, 8 12). Protein ubiquitination is a highly ordered process whereby proteins to be degraded are tagged with multiple ubiquitin moieties, which serve as recognition markers, targeting proteins for subsequent proteolytic cleavage by the 26S proteasome or lysosome. Ubiquitin ligases are the critical enzymes that both link ubiquitin moieties to the protein targeted for degradation and confer specificity to the system by interacting directly with the target protein through welldefined protein protein interaction domains (38). The ubiquitin ligases, atrogin-1 and MuRF1, are key regulators of muscle atrophy in multiple rodent models (14, 15, 17), and, more Figure 6. AKT. (A) There was no difference in the level of phosphorylation of AKT in the patients with chronic obstructive pulmonary disease (COPD) versus the control population. Total AKT and phosphorylated AKT (pakt) protein levels were determined by SDS-PAGE, Western blotting, charge-coupled device camera image acquisition, and quantitation of the chemiluminescent signal in the soluble fraction of muscle protein lysates. pakt protein levels were normalized to total AKT protein levels. (B) A representative Western blot is shown. Protein lysates of 293T cell cultures treated with insulin, and untreated, served as positive (Pos) and negative (Neg) controls, respectively, for AKT phosphorylation. Western blotting for GAPDH served as a loading control. Co, control; N.S., not significant; Pt, patient; P. 0.05. recently, a third ligase, Nedd4, has also been recognized (13, 16, 39) to contribute to this process in rodent models. The up-regulation of atrogin-1 and Nedd4 noted in our patients with COPD infers an increase in ubiquitin proteasome mediated proteolysis in the COPD muscle. Interestingly we did not find MuRF1 to be increased. Discordance in the upregulation of these three ubiquitin ligases has been previously reported, with the pattern and temporality of ligase recruitment dependent upon the etiology of the muscle atrophy. Ottenheijm and colleagues (40) reported enhanced proteasomal degradation in the diaphragms of patients with COPD, and found increased transcript levels of atrogin-1, but not MuRF1, in the diaphragmatic muscle. Nedd4 expression was not investigated in this population. Nedd4 is increased in atrophy associated with denervation or unloading (8, 13, 41), but is not increased in muscle atrophy resulting from diabetes, renal failure, or starvation in mice or rats (16, 17, 39). Atrophy resulting from denervation in rodents involves the up-regulation of atrogin-1, MuRF1, and Nedd4, but atrogin-1 and MuRF1 increases are short lived, with persistent increases seen only in the level of Nedd4 (13). Thus, there is specificity of these ubiquitin ligases to the systemic process responsible for the development of atrophy and in the temporality of its progression. This is not surprising, as the target substrates identified to date for each enzyme differ (13, 16, 42 44). Clearly, in the human population, further study is necessary to fully understand the implications of this specificity in muscle atrophy arising from different disease processes. Our novel finding that Nedd4 is up-regulated, and confirmation of atrogin-1 up-regulation in the COPD patient population, is significant, as it provides possible targets for future therapeutic intervention in the treatment of COPD. It is interesting that we note an increase in Nedd4 in the muscle of patients with COPD, whereas up-regulation of Nedd4 in rodent models of atrophy has been limited to unloaded or

468 AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL 42 2010 Figure 7. Glycogen synthase kinase (GSK) 3b. (A) There was no significant difference in the level of phosphorylation of GSK3b in the patients with chronic obstructive pulmonary disease (COPD) versus the control population. Total GSK3b and phosphorylated GSK3b (pgsk3b) protein levels in the soluble fractions of muscle protein lysates were determined by SDS-PAGE, Western blotting, charge-coupled device camera image acquisition, and quantification of the chemiluminescent signal. pgsk3b protein levels were normalized to total GSK3b levels. (B) A representative Western blot is shown. Protein lysates of 293T cell cultures treated with insulin, and untreated, served as positive (Pos) and negative (Neg) controls, respectively, for GSK3b phosphorylation. Western blotting for GAPDH served as a loading control. Co, control; N.S., not significant; Pt, patient; P. 0.05. denervated muscle. The unloading model in rats (hindlimb suspension) is an extreme muscle disuse model. It may be that there is a graded effect of muscle loading on Nedd4 expression, with relative muscle disuse resulting in only slightly increased Nedd4 expression. This would not have been noted in the rat models of diabetes, renal failure, or starvation, because these animals retain full activity. Our patients with COPD were ambulatory; their muscles are loaded and innervated, but they may experience relative disuse compared with control individuals, resulting in increased Nedd4 expression. An alternative explanation is that we noted increased Nedd4 expression only in the insoluble myofibrillar fraction of protein lysates, not in the soluble fraction. Koncarevic and colleagues (16) localized Nedd4 to the sarcolemma in healthy rodent muscle, and assessed levels only in cytoplasmic and crude membrane fractions. We have noted increased expression of Nedd4 in the insoluble myofibrillar fraction of protein lysates of rodent muscle atrophying from denervation (39). In other organs and cells, Nedd4 is expressed throughout the cytoplasm, with membrane localization occurring only upon target substrate binding (45, 46). Given the varying subcellular distribution, and the fact that the downstream target substrates of Nedd4 in muscle remain largely undefined, it is not possible to state in which fraction increased Nedd4 expression is necessary for the development of muscle atrophy. In addition to enhanced protein breakdown, alterations in the regeneration of muscle can influence muscle mass. Muscle satellite cells are the population of precursor cells that regenerate muscle and maintain muscle mass (47, 48). These cells are normally quiescent, but they are activated to proliferate after muscle injury, terminally differentiating to mature myocytes, and fusing to form new myofibers or repair damaged myofibers. The MRFs, MyoD, Myf5, and myogenin, are essential to the processes of muscle self-renewal, and specify satellite cell terminal differentiation into muscle cells. We found no differences in the level of expression of Myf5, myogenin, or MyoD between patients and control subjects, suggesting that myoblast differentiation in COPD muscles is unaffected. Myostatin is a member of the transforming growth factor b family, and is a negative regulator of skeletal muscle development and growth. Myostatin inhibits proliferation of satellite cells/myoblasts by up-regulating expression of p21 and decreasing Cdk2 and phosphorylated Rb, which results in cell cycle withdrawal (27). Myostatin also appears to inhibit satellite cell differentiation via the down-regulation of various MRFs, including MyoD, Myf5, and myogenin. Mice with a targeted deletion of the myostatin gene demonstrate a hypermuscular phenotype (27). Increased myostatin expression in both serum and muscle was associated with decreased muscle mass in HIVpositive patients with muscle wasting (49). Our finding of increased levels of myostatin transcripts in the patients with COPD implies that the loss of muscle mass in our COPD population is attributed to impaired muscle growth, in addition to active muscle degradation, reiterating the notion that atrophy is a multifactorial process rather than the result of a single biochemical pathway/signal. We cannot comment on the signaling pathways engaged downstream of myostatin, as we did not interrogate the cell cycle regulators. However, as we did not demonstrate any changes in the levels of MyoD, myogenin, Figure 8. p70s6 Kinase. (A) There was no significant difference in the level of phosphorylation of p70s6 kinase in the soluble fraction of muscle protein lysates of patients with chronic obstructive pulmonary disease (COPD) versus the control population. Total p70s6 kinase and phosphorylated p70s6 kinase (p-p70s6 kinase) protein levels were determined by SDS-PAGE, Western blotting, charge-coupled device camera image acquisition, and quantification of the chemiluminescent signal. p-p70s6kinase expression was normalized to total p70s6 kinase expression. (B) A representative Western blot is shown. Protein lysates of 293T cell cultures treated with insulin, and untreated, served as positive (Pos) and negative (Neg) controls, respectively, for p70s6 kinase phosphorylation. Western blotting for GAPDH served as a loading control. Co, control; N.S., not significant; Pt, patient; P. 0.05.

Plant, Brooks, Faughnan, et al.: Markers of Muscle Atrophy in COPD 469 Figure 9. Autophagy factors beclin-1 and microtubule-associated protein 1A/1B-light chain 3 (LC3). There was no difference in the level of expression of beclin-1 or LC3 transcripts between patients with chronic obstructive pulmonary disease (COPD) and control individuals, as determined by relative quantification with real-time RT-PCR. mrna levels in patients and control subjects were compared with a common reference standard. YWHAZ served as the HKG, and similar data were obtained using HMBS as HKG (data not shown). N.S., not significant; P. 0.05. or Myf5, it is probable that myostatin is affecting proliferation of the satellite cell population as opposed to differentiation. This would impair the regenerative capacity of COPD muscle, and potentially contribute to the loss of muscle mass. The NF-kB signaling pathway is known to mediate muscle atrophy in rodent models of muscle wasting associated with cachexia, systemic inflammation, denervation, and unloading (7, 8, 20, 23, 50) through the combined effects of stimulated proteolysis and impaired muscle growth. Of the five known transcription factors (p65, Rel B, C-rel, p52, and p50), p65 is the primary subunit known to regulate muscle biology. As with all NF-kB transcription factors, p65 heterodimers are held in the cytosol in an inactive complex with IkB (24, 51, 52). p65 activation occurs via phosphorylation of IkBa, release, and translocation to the nucleus. Phosphorylation of NF-kB(p65) in the nucleus enhances its transcriptional activity. It is there that the p65/p50 heterodimers bind the MuRF1 promotor, inducing MuRF1 transcription and proteasomal mediated muscle degradation (23). p65 is the sole subunit currently shown to regulate myogenesis (24, 53). There are reports suggesting that up-regulation of NF-kB signaling is involved in the development of muscle atrophy associated with COPD. Agusti and colleagues (50) demonstrated NF-kB(p65) activation in pooled muscle protein lysates from patients with COPD with low BMI, compared with patients with COPD with normal to high BMI. Inflammatory cytokines, such as TNF-a, induce muscle atrophy by engaging NF-kB(p65) signaling, which results in increased proteasomalmediated protein degradation and inhibition of muscle regenerative pathways via the induction of oxidative stress, and decreased MyoD expression (20, 22). Low-grade systemic inflammation (elevated circulating plasma levels of TNF-a, and IL-1 and -6) has been described in patients with COPD (54, 55). Despite these reports of up-regulation of systemic inflammation in patients with COPD at both a resting state and during acute exacerbations, and in contrast to Agusti s findings, we were unable to demonstrate engagement of IkBa or NFkB(p65) in the vastus lateralis muscles of our patients with COPD. The absence of engagement of NF-kB(p65) signaling in our patients is supported by normal expression levels of MuRF1 and MyoD, both downstream of p65. In contrast to Agusti s patient cohort, our patient population had a normal BMI, which may have contributed to the difference in findings between our studies. Other investigations have recently addressed the question of the presence of a proinflammatory milieu in the skeletal muscle of stable patients with COPD compared with healthy control subjects. Neither Barreiro and colleagues (56) nor Crul and colleagues (57) were able to demonstrate local inflammation in the vastus lateralis muscle of clinically and weightstable patients with COPD by assessing the level of expression of various cytokines. Barreiro did, however, demonstrate increased oxidative stress in the COPD muscle. Thus, studies performed in humans to date, including our results here, suggest that inflammatory mediators and signaling via NFkB(p65) are not active in the muscle of clinically stable patients with COPD, and do not contribute to the development of muscle atrophy in those with normal weight and BMI. This does not preclude the possibility that, during acute illness, this pathway may be recruited, but our study did not address this issue. Recently, NF-kB(p50) has been shown to mediate muscle atrophy associated with unloading, independent of activation of p65 (58). The downstream substrate targets of p50 that mediate this effect are unknown, and, to date, no other upstream stimulants that engage this atrophic pathway have been identified. We interrogated this pathway here, because muscle deconditioning and relative disuse can occur in the COPD population. However, it appears that, in the patient with stable COPD, p50 engagement does not contribute to muscle atrophy. Whether there is an incremental effect, with p50 recruitment during acute exacerbations of COPD in a comparable patient population, remains to be determined. Phosphorylation and activation of AKT is well known to induce skeletal muscle hypertrophy (6, 25). AKT activation results in the recruitment and subsequent phosphorylation of multiple direct and indirect downstream targets, including mammalian target of rapamycin, p70s6 kinase, PHAS-1, and/ or GSK3b, all of which contribute to the development of muscle hypertrophy, at least in part, via the induction of protein synthesis. It has also been recently appreciated that activation of AKT signaling suppresses the expression of atrogin-1 and MuRF1 via phosphorylation of the Forkhead O transcription factors, and thereby actively suppresses muscle proteolysis (25, 26). Conversely, down-regulation of AKT signaling occurs in muscle atrophied by denervation, disuse, and glucocorticoid administration, demonstrating reciprocal communication between pathways mediating muscle atrophy and hypertrophy (8). The amount of material available to us from the muscle biopsy limited the extent to which we were able to interrogate the AKT signaling network, and we focused our assessment on AKT, GSK3b, and p70s6 kinase. Surprisingly, despite the atrophy seen in the COPD population, we were unable to demonstrate down-regulation of AKT signaling in our cohort of patients with COPD. In contrast, Doucet and colleagues (37) demonstrated an increase in protein levels of phosphorylated AKT, GSK3b, and p70s6 kinase in patients with COPD, and hypothesized a compensatory mechanism of muscle mass recovery accounting for this activation (37). The discrepant findings between our study and that by Doucet and colleagues may be attributable to differences employed in the normalization of data, yet the critical observation is that neither study found AKT signaling to be down-regulated in the patients with COPD. This was unexpected and in contrast to data generated in rodent models of muscle atrophy. This finding suggests the possibility that a compensatory response is in place in the muscle of patients with COPD that attempts to counteract the atrophic stimuli. There is certainly precedent for this phenomenon. Attempts at muscle recovery after muscle atrophy are described in other