American Journal of Respiratory and Critical Care Medicine

Rationale: The molecular mechanisms of muscle atrophy in chronic obstructive pulmonary disease (COPD) are poorly understood. In wasted animals, muscle mass is regulated by several AKT-related signaling pathways.

Objectives: To measure the protein expression of AKT, forkhead box class O (FoxO)-1 and -3, atrogin-1, the phosphophrylated form of AKT, p70S6K glycogen synthase kinase-3β (GSK-3β), eukaryotic translation initiation factor 4E binding protein-1 (4E-BP1), and the mRNA expression of atrogin-1, muscle ring finger (MuRF) protein 1, and FoxO-1 and -3 in the quadriceps of 12 patients with COPD with muscle atrophy and 10 healthy control subjects. Five patients with COPD with preserved muscle mass were subsequently recruited and were compared with six patients with low muscle mass.

Methods: Protein contents and mRNA expression were measured by Western blot and quantitative polymerase chain reaction, respectively.

Measurements and Main Results: The levels of atrogin-1 and MuRF1 mRNA, and of phosphorylated AKT and 4E-BP1 and FoxO-1 proteins, were increased in patients with COPD with muscle atrophy compared with healthy control subjects, whereas atrogin-1, p70S6K, GSK-3β, and FoxO-3 protein levels were similar. Patients with COPD with muscle atrophy showed an increased expression of p70S6K, GSK-3β, and 4E-BP1 compared with patients with COPD with preserved muscle mass.

Conclusions: An increase in atrogin-1 and MuRF1 mRNA and FoxO-1 protein content was observed in the quadriceps of patients with COPD. The transcriptional regulation of atrogin-1 and MuRF1 may occur via FoxO-1, but independently of AKT. The overexpression of the muscle hypertrophic signaling pathways found in patients with COPD with muscle atrophy could represent an attempt to restore muscle mass.

Scientific Knowledge on the Subject

Muscle atrophy is common in patients with chronic obstructive pulmonary disease (COPD). It is associated with increased mortality. However, the mechanisms leading to muscle wasting remain to be elucidated.

What This Study Adds to the Field

An increase in atrogin-1 and MuRF1 mRNA and FoxO-1 protein content was observed in the quadriceps of patients with COPD. The overexpression of the muscle hypertrophic signaling pathways found in patients with COPD with muscle atrophy could represent an attempt to restore muscle mass.

Skeletal muscle atrophy is an important systemic consequence of chronic obstructive pulmonary disease (COPD) (1, 2). In patients with severe COPD, reduced mid-thigh muscle cross-sectional area (MTCSA) has a strong impact on mortality (3). Although the biochemical pathways engaged in the development of muscle atrophy are poorly understood, an imbalance between protein breakdown and synthesis, in favor of the former, is believed to play a role in this process (4, 5). Within this tightly regulated balance, AKT, also called protein kinase B, appears to be a central mediator involved in the regulation of both atrophy and hypertrophy signaling pathways (6). In its phosphorylated active form, AKT is able to block muscle protein breakdown by downregulating two muscle-specific E3-ligases (7), atrogin-1 (muscle atrophy F-box) and muscle ring finger (MuRF), the roles of which are to ensure specific protein degradation by the ubiquitin–proteasome pathway (8). This action of AKT on both E3-ligases is mediated by the inactivation of the forkhead box class O (FoxO) of transcription factors (9, 10). AKT-driven phosphorylation of FoxO-1 and -3 sequesters them to the nucleus, where they are unable to activate gene transcription of the muscle-specific E3 ubiquitin ligases (7, 9). Phosphorylated AKT also stimulates a variety of hypertrophic pathways, including mammalian target of rapamycin. In turn, mammalian target of rapamycin can promote protein synthesis by the activation of 70-kD ribosomal S6 protein kinase (p70S6K) (6, 11) and by the inhibition of eukaryotic translation initiation factor 4E binding protein-1 (4E-BP1/PHAS-I) (12). Upon phosphorylation, 4E-BP1 is released from eIF (eukaryotic translation initiation factor)-4E, allowing it to exert its protein synthesis activity (13). Another hypertrophy mediator downstream of AKT is glycogen synthase kinase-3β (GSK-3β) (6, 11), a repressor of protein synthesis. The active form of AKT induces GSK-3β phosphorylation, which leads to the release of eIF-2B (14) and to an increase in protein synthesis, independently of the p70S6K and 4E-BP1 pathways. These different pathways are summarized in Figure 1.

Although major strides have been made in understanding the regulation of skeletal muscle mass in several experimental models, the relevance of these pathways remains to be confirmed in humans with COPD or other chronic diseases associated with muscle wasting. The aim of the present study was, therefore, to measure the mRNA and protein level of AKT and its downstream regulated atrophic (FoxO1 and -3, atrogin-1, MuRF1) and hypertrophic (phosphorylated forms of GSK-3β, p70S6K, and 4E-BP1) signaling pathways to provide a thorough evaluation of the molecular mechanisms potentially involved in muscle atrophy in patients with stable COPD in comparison to healthy control subjects with preserved muscle mass. A complementary substudy was also conducted with the objective of addressing whether these muscle-regulating pathways were differentially activated in patients with COPD with and without muscle atrophy.

This work has been published in part in the form of an abstract (15).

Study Design

This study was conducted in two phases. The main study objective was to study the muscle mass–regulating pathways in patients with COPD with muscle atrophy in comparison to healthy control subjects with preserved muscle mass. A secondary question was to address whether the muscle-regulating pathways of interest were differentially activated in patients with COPD with and without muscle atrophy. To do so, five additional patients with COPD with preserved muscle mass were subsequently recruited with the objective of comparing their muscle data to those of patients with COPD with low muscle mass. As sufficient muscle tissue was available in 6 out of the 12 patients with muscle atrophy, it was possible to reanalyze their muscle tissue simultaneously with those of patients with COPD and preserved muscle mass. The two phases of this study are thus presented and analyzed separately.


A total of 22 ex-smoker (> 6 mo) males were recruited for this study. Of these, 10 subjects had normal lung function and 12 had COPD. COPD diagnosis was based on a previous smoking history (> 10 pack-years) and pulmonary function testing showing irreversible airflow obstruction (post-bronchodilatator FEV1 < 80% predicted and FEV1/FVC < 70%). All patients with COPD were in a stable condition at the time of the study without any acute exacerbation of their disease or exposure to systemic corticosteroids within 2 mo of their participation in the study. None was receiving long-term oxygen therapy. Patients with comorbid conditions that could be associated with muscle wasting, such as active inflammatory illnesses, heart failure, or diabetes, were excluded. In recruiting study participants, we ensured that there was no overlap between patients with COPD and healthy control subjects for MTCSA and fat-free mass. All participants were considered as sedentary (physical activity scores < 9) according to a physical activity questionnaire adapted for older and retired subjects and used in COPD (16). After this main part of the study, five additional patients with severe COPD (FEV1 < 50% predicted) without muscle atrophy (MTCSA and fat-free mass similar to normal control subjects) were recruited. The Hôpital Laval ethics committee approved the study protocol, and all patients gave written informed consent.

Pulmonary function tests.

Standard pulmonary function tests, including spirometry, lung volumes with body plethysmography, and diffusion capacity, were obtained according to previously described guidelines (17) and related to the normal values of Quanjer and colleagues (18).

Anthropometric measurements and body composition.

Height and weight were measured according to standardized methods (19). Dual-energy X-ray absorptiometry provided regional assessment of fat and fat-free mass (General Electric Healthcare, Chicago, IL). Mid-thigh muscle cross-sectional area was determined using computed tomography of the right thigh halfway between the pubic symphysis and the inferior condyle of the femur, and was obtained by a fourth-generation Toshiba scanner (900S; Toshiba Inc., Tokyo, Japan), as previously described (3).

Blood sampling and analysis.

Arterial blood was drawn from a radial artery and PaO2 was then analyzed with a blood gas analyser (AVL 995; AVL Scientific, Roswell, GA). The antecubital venous blood was sampled between 8:00 a.m. and 10:00 a.m. in overnight-fasted subjects. Blood was centrifuged for 15 minutes, aliquoted, and stored at −80°C until further analysis. Commercial ELISA kits (R&D Systems, Minneapolis, MN) were used to measure the plasma levels of systemic inflammatory markers and anabolic hormones (5).

Muscle biopsies.

Needle biopsies of the quadriceps were performed as described by Bergström (20), and as routinely done in our laboratory (21).

Skeletal Muscle and Data Analysis
Real-time quantitative polymerase chain reaction.

Quantitative polymerase chain reaction was performed using an MX3000p thermal cycler system (Stratagene, La Jolla, CA), as previously reported (2224). Polymerase chain reaction primer and probe sequences are provided in Table E1 in the online supplement.

Protein extraction and Western blotting.

Nuclear and cytoplasmic proteins were extracted using an NE-PER kit (Pierce Biotechnology, Rockford, IL). Western blotting techniques were used (as detailed in the online supplement) for the measurement of AKT, phosphorylated AKT, and its related downstream markers, atrophic (FoxOs and E3-ligases) and hypertrophic (phosphorylated form of GSK-3β, p70S6K and 4E-BP1) pathways. The antibodies and dilutions used in the present study are shown Table E2.

Statistical Analysis

Results are expressed as mean (± SEM). For analytic purposes, continuous variables that were not normally distributed (total AKT and phosphorylated AKT) were log transformed to achieve normality and to meet variance assumptions. Between-group comparisons were done using unpaired Student's t tests, whereas possible relationships were evaluated using Pearson's correlations. Using the same statistical approaches, subsequent analyses were conducted comparing muscle data of six patients with COPD with low muscle mass already involved in the study to that of five additional patients with COPD with preserved muscle mass. Results were considered significant if p values were less than 0.05.

Main Study: Comparison of Patients with COPD and Muscle Atrophy and Healthy Control Subjects
Subject characteristics.

Anthropometric characteristics, smoking history, and pulmonary function data are provided in Table 1. On average, patients with COPD had severe airflow obstruction, resting hyperinflation, reduced diffusion capacity, and a slightly reduced resting PaO2. Body mass index, MTCSA, total fat-free mass index, and fat mass were significantly reduced in patients with COPD compared with control subjects.



Control Subjects (n = 10)

Patients with COPD (n = 12)
Age, yr65 ± 265 ± 2
Smoking history, pack-years74 ± 2163 ± 9
Weight, kg84 ± 364 ± 3*
BMI, kg/m229 ± 123 ± 1*
FEV1, L2.98 ± 0.120.86 ± 0.09*
FEV1% predicted102 ± 431 ± 3*
FEV1/FVC, %72 ± 235 ± 2*
TLC % predicted105 ± 4119 ± 2
RV % predicted94 ± 4157 ± 16
RV/TLC, %34 ± 157 ± 2*
DlCO % predicted100 ± 557 ± 4*
PaO2, mm Hg76 ± 2
PaCO2, mm Hg42 ± 1
MTCSA, cm298 ± 472 ± 3*
Fat-free mass, kg59 ± 247 ± 1*
FFMI, kg/m220 ± 217 ± 1*
Fat mass, kg
22 ± 2
14 ± 2*

Definition of abbreviations: BMI = body mass index; TLC = total lung capacity; RV = residual volume; DlCO = diffusing capacity of carbon monoxide; MTCSA = mid-thigh muscle cross-sectional area; FFMI = fat-free mass index.

Values are means ± SEM.

*p < 0.001.

p < 0.01.

Systemic inflammatory markers and anabolic hormones.

As depicted in Table 2, tumor necrosis factor-α levels were slightly lower in patients with COPD compared with healthy control subjects. All other inflammatory marker and anabolic hormone levels were similar in both groups.



Control Subjects (n = 10)

Patients with COPD (n = 12)
Inflammatory markers
 CRP, mg/L2.58 ± 0.792.64 ± 0.68
 TNF-α, pg/ml0.87 ± 0.060.64 ± 0.07*
 TNF-R55, pg/ml1180 ± 1361173 ± 107
 TNF-R75, pg/ml2424 ± 2712566 ± 250
 IL-6, pg/ml2.51 ± 0.352.77 ± 0.26
Anabolic hormones
 Insulin, pmol/L35.30 ± 4.7033.92 ± 9.85
 IGF-I, ng/ml
67.47 ± 4.51
72.77 ± 8.84

Definition of abbreviations: CRP = C-reactive protein; TNF = tumor necrosis factor; TNF-R = TNF receptor; IGF-I = insulin-like growth factor-I.

Values are mean ± SEM.

*p < 0.05.

Phosphorylated-AKT protein levels and phosphorylated-AKT/total AKT ratio.

A typical AKT Western blot is provided in Figure 2A. Group mean values for phosphorylated AKT protein levels and the phosphorylated AKT:total AKT ratio within the quadriceps are illustrated in Figures 2B and 2C. Increased AKT activation in COPD was supported by the elevation in cytoplasmic protein content of phosphorylated AKT (171%; p < 0.05). The phosphorylated AKT:total AKT ratio, also reflecting the AKT activation state, was higher in patients with COPD (490%) compared with control subjects.

Muscle atrophy signaling.

FoxO-1 mRNA expression (435%) and its nuclear protein content (169%) were significantly increased in the quadriceps of patients with COPD compared with control subjects (Figures 3A and 3C). A representative Western blot for FoxO-1 is provided in Figure 3B. There was no difference in FoxO-3 mRNA and nuclear protein level between the groups (data not shown).

The mRNA expression of atrogin-1 (232%) and MuRF1 (517%) were significantly increased in the quadriceps of patients with COPD compared with control subjects (Figures 4A and 4B). A representative Western blot for atrogin-1 is provided in Figure 4C. Atrogin-1 protein levels tended to be increased in patients with COPD (p = 0.14) (Figure 4D). Atrogin-1 and MuRF1 mRNA expression correlated significantly (Figure 4E).

Muscle hypertrophy signaling.

A representative Western blot for the phosphorylated form of 4E-BP1 is provided in Figure 5A. A downstream target of AKT, 4E-BP1, was significantly up-regulated in its phosphorylated state in the quadriceps of patients with COPD when compared with healthy control subjects (Figure 5B). This finding would argue in favor of a net release in eIF-4E which is known to increase protein synthesis (13). Representative Western blots for phosphorylated p70S6K and GSK-3β are provided in Figures 5C and 5E. There was no significant difference in phosphorylated p70S6K and GSK-3β between groups (Figures 5D and 5F, respectively).

Substudy: Comparison of Patients with COPD with and without Muscle Atrophy

Subjects' characteristics are provided in Table 3. Patients of both groups were well matched for age and severity of airflow obstruction. Patients with muscle atrophy (n = 6) all had an MTCSA less than 70 cm2; they also showed lower body mass index, total fat mass, and fat-free mass index than patients with normal muscle mass (n = 5; MTCSA > 90 cm2). Patients with COPD without muscle atrophy had an MTCSA and a fat-free mass index that was similar to the healthy control group.



COPD with Preserved Muscle Mass (n = 5)

COPD with Low Muscle Mass (n = 6)
Age, yr68 ± 370 ± 1
Smoking history, pack-years69 ± 870 ± 17
Weight, kg93 ± 360 ± 2*
BMI, kg/m233 ± 121 ± 1*
FEV1, L0.94 ± 0.070.72 ± 0.13
FEV1, % predicted34 ± 328 ± 4
FEV1/FVC, %37 ± 232 ± 3
TLC, % predicted118 ± 6121 ± 3
RV, % predicted182 ± 8168 ± 22
RV/TLC, %57 ± 160 ± 3
DlCO % predicted61 ± 555 ± 5
PaO2, mm Hg71 ± 376 ± 4
PaCO2, mm Hg48 ± 243 ± 1
MTCSA, cm295 ± 362 ± 3*
Fat-free mass, kg60 ± 246 ± 1*
FFMI, kg/m221 ± 116 ± 1*
Fat mass, kg
27 ± 3
10 ± 2*

For definition of abbreviations see Table 1.

Values are mean ± SEM.

*p < 0.001.

p < 0.05.

Muscle atrophy/hypertrophy signaling.

The protein levels of phosphorylated AKT and phosphorylated AKT:total AKT were not statistically different between the two groups of patients with COPD. The same was true for FoxO-1 and FoxO-3 mRNA and nuclear protein levels, MuRF1 and atrogin-1 (data not shown).

Representative Western blots for phosphorylated 4E-BP1, p70S6K and GSK-3β are provided in Figures 6A, 6C, and 6E, respectively. In their phosphorylated forms, these hypertrophy-related signals were significantly overexpressed in the quadriceps of patients with low muscle mass in comparison to patients with preserved muscle mass (Figures 6B, 6D, and 6F, respectively).

This study provides a thorough investigation of several plausible biochemical mechanisms involved in muscle atrophy in patients with COPD. Evidence was found supporting the activation of the ubiquitin–proteasome pathway in patients with COPD and muscle atrophy in comparison with healthy control subjects, as indicated by the increased atrogin-1 and MuRF1 mRNA expression. Our data also suggest a role of FoxO-1 in the transcriptional up-regulation of these two muscle-specific E3-ligases. The increase in active AKT protein content suggests that FoxO-1 upregulation was AKT independent. Comparing patients with COPD with and without muscle atrophy provided further insights about the muscle mass–regulating pathways in this disease. The striking finding in this substudy was that patients with COPD with low muscle mass could be differentiated from patients with COPD with preserved muscle mass by a clear, and apparently paradoxical, increase in phosphorylation status of three key signaling proteins involved in muscle hypertrophy. Our observation that a similar activation of the atrophying factors found in patients with COPD with and without muscle atrophy raises the possibility that the activation of the muscle E3 ligases may occur early in the wasting process. The increase in hypertrophy signaling pathways could be interpreted as a failed attempt to maintain or restore muscle mass in patients with muscle atrophy.

Activation of the Ubiquitin–Proteasome Pathway

Several animal models of cachexia clearly demonstrated a consistent increase in the activation in the atrogin-1 (25) and MuRF1 (8) genes. These observations suggest that these conditions of muscle atrophy share a common transcriptional program that induces the up-regulation of one or both of these two muscle-specific E3 ligases (26). The up-regulation of atrogin-1 and MuRF1 mRNA found in patients with COPD suggests an increased muscle protein proteolysis, which could contribute to a reduction in muscle mass reported in this population. Although muscle protein breakdown was not directly assessed in the present study, previous investigations have reported an increased in protein breakdown in muscle-wasted patients with COPD (27).

In humans, the initiation of muscle protein breakdown can be triggered by several factors, such as the frequent use of systemic glucocorticoids, low levels of circulating anabolic hormones, muscle inactivity, or starvation (28). These conditions are known to modulate the expression of some proteasome components, including muscle-specific E3 ligases. In the present study, the impact of these factors has been minimized by including patients with COPD during periods of disease stability without recent use of systemic corticosteroids. These patients were compared with sedentary control subjects of a similar age in an attempt to diminish the influence of aging and physical activity on the modulation of signaling proteins involved in muscle atrophy and hypertrophy.

Regulation of Muscle Atrophy via FoxO-1/Atrogin-1/MuRF1

The upstream signals mediating the activation of the ubiquitin–proteasome pathway in muscle atrophy is not fully understood. However, recent rodent in vitro studies have demonstrated that expression of atrogin-1 and MuRF1 is controlled by a complex signaling network that comprises the FoxO transcription factors and their regulation via the IGF-1/AKT (7, 9) and nuclear factor-κB (29, 30) signaling pathways.

FoxO transcription factors, in their unphosphorylated form, are predominantly located in the nuclear compartment, where they are active and DNA bound (10). It is known that, upon AKT-induced phosporylation, FoxO family members translocate from the nucleus to the cytosol, where they become inactive (10). Phosporylation of specific sites on FoxO protein is believed to be essential for its nuclear exclusion, although these mechanisms are complex and not fully understood (10, 31). Increased expression of FoxO-1 mRNA (26) is a common feature in several models of animal suffering from muscle atrophy (26). Moreover, transgenic mice with muscle-specific overexpression of FoxO-1 are characterized by reduced body weight and muscle mass (32). Taken together, these studies suggest that FoxO-1 plays a key role in the development of muscle wasting.

The up-regulation of FoxO-1 mRNA and protein levels found in patients with COPD is an important finding of the present study, as it supports the concept that FoxO-1 could be involved in the atrophy process in this disease. Our findings also suggest that FoxO-1 up-regulation could not be explained by a reduction in phosphorylated AKT as observed in rodent models of muscle wasting. Our observation that the AKT/FoxOs regulatory relationship did not follow its usual pattern is not without precedent. It has previously been observed that, in human primary breast tumors lacking a detectable phosphorylated AKT protein, FoxO-3 was not increased in the nucleus, but was instead predominantly confined in the cytoplasm (33). Additionally, decreased levels of active AKT protein in atrophied muscle of patients with amyotrophic lateral sclerosis did not reveal an expected increase in nuclear FoxO content (24). Therefore, it appears that, under certain conditions, regulation of AKT and FoxO-1 seems to be independent of each other (24, 33, 34).

Regulation of Muscle Hypertrophy Pathways

One striking and apparently paradoxical finding in this study was the increase in phosphorylation status of three muscle proteins involved in muscle hypertrophy in patients with COPD with low muscle mass when compared with those with preserved muscle mass. Up-regulation of p70S6K, GSK-3β, and 4E-BP1, three mediators involved in protein translation, is tightly associated with muscle regrowth during recovery from atrophy (35). The reason that overexpression of these pathways was associated with atrophy in the present investigation is unclear. One possible interpretation is that overexpression of p70S6K, GSK-3β, and 4E-BP1 may represent a failed attempt to compensate for the loss of muscle mass. The molecular mechanisms responsible for signaling muscle regrowth are poorly understood. However, muscle regrowth may be attenuated in aged rodent skeletal muscles in comparison to those of younger animals (36). Alternatively, the increase in protein synthesis, through compensatory hypertrophy, is reported to occur in two steps: an increase of translational efficiency, followed by changes in translational capacity through the addition of myonuclei (37). In stable COPD, a dysfunction located at either of these two steps may be, in part, involved in muscle atrophy. Also, factors other than AKT-regulated protein translation may be rate limiting for protein synthesis (37, 38), and it can be speculated that a defective hypertrophy response may be present in patients with COPD. Testing this hypothesis was beyond the scope of the present investigation. These results may nevertheless have potential clinical relevance; if confirmed, these data suggest that AKT would not be a proper therapeutic target to reduce the rate of muscle atrophy or restore muscle mass in COPD, as AKT and several of its downstream protein synthesis targets were already overexpressed in patients with COPD and muscle atrophy.

Systemic Factors versus Local Factors in Muscle Wasting

The previously reported absence of inflammation within the quadriceps of muscle-wasted patients with COPD argues against the inflammatory hypothesis of muscle cachexia in COPD (39). One study reported increased tumor necrosis factor-α in the quadriceps of patients with COPD, but the significance of this finding remains unclear (40). Furthermore, inflammation in COPD is of low intensity, and there are inconsistencies between studies about its presence in this disease (2, 5, 41, 42). Evidence of heightened systemic inflammation in patients with low muscle mass was also lacking in the present study. The current information is therefore insufficient to confirm (or refute) the inflammatory hypothesis of muscle cachexia in COPD. To address this question appropriately, larger trials that include a thorough evaluation of the inflammatory mediators at the muscle level, in addition to the measurement of circulating levels of inflammatory mediators, will be necessary. It will also be important to consider the possibility that bursts of systemic inflammation occurring during a period of acute exacerbation (43, 44) could trigger the atrophying cascade within the skeletal muscle. In this regard, studying patients during the time frame of a stable disease state may not be appropriate to detect the presence of inflammation.

Limitations of the Study

This investigation has the merit of reporting on several molecular mechanisms that have been convincingly associated with the development of muscle wasting in animals (26). However, a number of methodologic considerations and potential limitations need to be taken into account. As this study was exploratory, with no previous data available in this study population, we decided to focus the main study on the comparison of patients with COPD with muscle atrophy with healthy control subjects to maximize the potential differences between the two groups. This study design has the limitation of not allowing the determination of whether the reported modifications in intracellular muscle signaling are related to the presence of COPD itself, or to muscle atrophy. To begin to investigate which biochemical process could be specific to muscle atrophy in COPD, a substudy was subsequently undertaken to compare patients with low muscle mass to those with preserved muscle mass. Although preliminary, this substudy provides novel and challenging observations about the molecular pathways involved in muscle atrophy that should help generate new hypotheses in the continued effort to unravel the mechanisms underlying the wasting process in this chronic condition. It is also debatable whether our patients with COPD were indeed undergoing a cachectic process, as clear evidence of body weight loss was lacking in most of them, and no evidence of systemic inflammation was found. It is still unclear how cachexia should be defined and diagnosed clinically; this topic is a matter of intense debate (45, 46). To circumvent this difficulty, an operational definition of cachexia, based on the reduction in MTCSA and fat-free mass index, was used in the present investigation. This decision was justified given that a reduction in these two indices of muscle mass is a strong predictor of mortality in COPD (3, 47). We also realize that a causal relationship between FoxO-1, atrogin-1, and MuRF1 up-regulation and muscle wasting cannot be inferred from our investigation.

Also, studies in humans are limited by the quantity of the muscle tissue available for analysis. We chose to measure several AKT-regulated signaling proteins involved in skeletal muscle atrophy and hypertrophy processes, downstream targets of the atrophic and hypertrophic signaling pathways. Finally, in the absence of a specific antibody for MuRF1 protein, only mRNA levels were determined. From a methodologic point of view, the determination of protein content by Western blotting techniques is semiquantitative rather than quantitative. It should be emphasized, however, that the methods and antibodies used in this study have been previously validated in other human models of muscle atrophy (24, 48).


The current study highlights potentially relevant molecular mechanisms involved in muscle atrophy in COPD. Evidence of an increase in the activation of muscle atrophy pathways in patients with COPD with muscle atrophy is demonstrated by the up-regulation of two muscle-specific E3-ligases: atrogin-1 and MuRF1. This up-regulation is likely to be mediated by FoxO-1 that was also increased in nuclear protein extracts, despite increased levels of phosphorylated AKT. On the other hand, up-regulation of p70S6K, GSK-3β, and 4E-BP1 in patients with COPD with low muscle mass, in comparison with those with preserved muscle mass, would appear to be a failed attempt to restore muscle mass in the former individuals. The present findings suggest that research should be directed toward the understanding of persisting muscle atrophy despite the overexpression of the muscle hypertrophy pathways in patients with COPD and muscle atrophy.

The authors acknowledge the contribution of Annie Michaud and Marie-Ève Paré for their technical assistance, Serge Simard for his statistical assistance, and Marthe Bélanger, Marie-Josée Breton, Brigitte Jean, Josée Picard, and Hélène Villeneuve for their help in accomplishing this study.

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Correspondence and requests for reprints should be addressed to François Maltais, M.D., Centre de Pneumologie, 2725 Chemin Ste-Foy, PQ, G1V 4G5 Canada. E-mail:


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