American Journal of Respiratory and Critical Care Medicine

Patients with chronic obstructive pulmonary disease (COPD) often lose weight during the course of their disease. We hypothesized that this may be due to skeletal muscle apoptosis. To investigate this possibility, we obtained quadriceps femoris biopsies in 15 patients with COPD (8 with normal body mass index [BMI] and 7 with low [< 20 kg/m2] BMI), 8 healthy volunteers, and 6 sedentary subjects undergoing orthopedic surgery (both groups with normal BMI). Skeletal muscle apoptosis was assessed by the transferase–mediated dUTP nick end labeling (TUNEL) technique and the immunodetection of poly-(ADP-ribose)-polymerase proteolytic fragments. Exercise tolerance on a cycloergometer was also determined in patients with COPD. We found that skeletal muscle apoptosis (by both techniques) was increased in patients with COPD and low BMI as compared with the other three groups (p < 0.005). In patients with COPD, BMI was inversely related to skeletal muscle apoptosis (TUNEL, p = 0.009), and it was better correlated with exercise capacity (p = 0.006) than with the degree of airflow obstruction present (p = 0.02). Markers of skeletal muscle apoptosis were not related to any of the measured lung function variables. This study shows that skeletal muscle apoptosis (1) is increased in patients with COPD having low BMI; and (2) is associated with a lower exercise tolerance despite a similar degree of lung function impairment.

Unexplained weight loss due to skeletal muscle atrophy occurs frequently in patients with chronic obstructive pulmonary disease (COPD) (1, 2). This is clinically relevant because it limits their exercise capacity and jeopardizes their quality of life (1). Besides, it is a negative and independent prognostic factor (3, 4). Yet, because the molecular mechanisms underlying skeletal muscle atrophy in COPD are unknown (5, 6), new therapeutic alternatives cannot be developed (7).

Apoptosis is a tightly regulated process in which cell death follows a programmed sequence of events (8). The results of this program, however, are different in mono- and multinucleated cells. In mononucleated cells, apoptosis leads directly to cell death (8, 9). In contrast, in multinucleated cells (like the myocyte), it causes cell atrophy (10). In fact, recent reports have shown that in experimental models of chronic heart failure (11), as well as in patients with cardiac cachexia (12), atrophy correlates with the magnitude of skeletal muscle apoptosis. Given that skeletal muscle atrophy is the main cause of unexplained weight loss in COPD (1, 2), we reasoned that increased apoptosis might occur in skeletal muscle of patients with COPD and low body weight. To investigate this possibility, we compared the level of skeletal muscle apoptosis seen in patients with COPD having low body weight with that determined in: (1) patients with COPD and normal body weight; (2) healthy volunteers of similar age; and (3) sedentary subjects who required orthopedic surgery. This last group provides some control for the direct effects of inactivity (which is frequent in patients with COPD) upon skeletal muscle structure (1). In the patients with COPD included in the study, we also investigated potential relationships between skeletal muscle apoptosis, lung function, and exercise tolerance.

Ethics

All participants gave written consent after being fully informed of the purpose, characteristics, and nature of the study, which had been approved by the Ethics Review Board of our institution.

Subjects

We recruited 15 patients with stable COPD from the outpatient clinic of our department. None of them had required hospital admission or treatment change during the preceding 3 months. Treatment included long-acting β2 agonists, ipratropium bromide, theophylline, and/or inhaled steroids. None were receiving oral steroids. Because previous studies have shown that a body mass index (BMI = weight [kg]/height [m2]) lower than 20 kg/m2 was associated with a poor prognosis in COPD (3), patients were grouped according to this threshold value. BMI was lower than 20 kg/m2 in seven patients (17.5 ± 0.5 kg/m2 [mean ± SEM]; range, 16.4–18.6 kg/m2) and normal in the remaining eight patients (24.8 ± 0.7 kg/m2; range, 23.2–26.3 kg/m2). Healthy volunteers (n = 8) were recruited from the general population by advertisement in local newspapers (BMI, 25.4 ± 0.6 kg/m2; range: 24.0–26.7 kg/m2). Patients undergoing hip or knee surgery (n = 6) were recruited from the corresponding wards in our institution (BMI, 26.4 ± 0.6 kg/m2; range, 24.8–27.9 kg/m2), and were otherwise healthy individuals receiving nonsteroidal antiinflammatory therapy only.

Lung Function and Exercise Tolerance

Spirometry (GS; Warren E. Collins, Braintree, MA) was obtained in all participants (13). In patients with COPD, the single-breath diffusion capacity for carbon monoxide (DlCO) (GS; Warren E. Collins) and arterial blood gases (by radial artery puncture under local anesthesia [IL BG3, Izasa, Spain]) were also determined. Spirometric and DlCO reference values were those of a Mediterranean population (14, 15). All participants (except orthopedic patients) performed an incremental, symptom-limited exercise test on a cycloergometer (CPXII; Warren E. Collins). The peak oxygen uptake (Vo2peak) was determined from analysis of the exhaled air. Reference values were those of Jones and coworkers (16).

Skeletal Muscle Biopsy

Needle biopsies were obtained, under local anesthesia, from the lateral portion of quadriceps femoris (at the mid-thigh level), as previously reported in our laboratory (17), except in orthopedic patients in whom they were obtained during the surgical procedure. Muscle samples were immediately frozen in liquid nitrogen and stored at −80°C until analyzed.

Assessment of Skeletal Muscle Apoptosis

The amount of skeletal muscle apoptosis was determined using two complementary techniques: the fluorescent transferase–mediated dUTP nick end labeling (TUNEL) assay (18) and the immunodetection and quantitation of poly-(ADP-ribose)-polymerase (PARP) proteolytic fragments by Western blot (19).

TUNEL assay. This method has been widely used to identify apoptosis in situ in different cell lines, including cardiac (8, 20) and skeletal muscle myocytes (12, 21). It detects the presence of 3′OH-free DNA ends associated with the DNA fragmentation that characterizes the late phase of the apoptotic pathway (8, 12, 20, 21). In our study, we used the APOPDETEK cell death assay system (Enzo Diagnostics, Inc., Farmingdale, NY), following the directions of the manufacturer. Briefly, skeletal muscle tissue cryosections were fixed overnight in 10% paraformaldehyde, dehydrated, and mounted in paraffin according to standard protocols. The slides were treated with proteinase K for 15 minutes at 37°C and, subsequently, with 3% hydrogen peroxide for 10 minutes at 37°C. The slides were then incubated for 30 minutes at 37°C in the presence of the buffer containing the enzyme and dUTP-biotin, followed by 30 minutes at 37°C with the solution containing alkaline phosphatase-conjugated streptavidin and, finally, 30 minutes at 37°C with the substrate solution. Apoptotic cells appeared with a brown nucleus, whereas negative nuclei were blue, due to the hematoxylin counterstaining. Two sections of each biopsy were screened for apoptotic nuclei by two independent observers. A myocyte was considered apoptotic if it contained at least two positively stained nuclei (21). To avoid counting endothelial cell or fibroblast nuclei as myocyte nuclei, the architecture of the muscle fiber boundary was assessed by hematoxylin staining, and only TUNEL positive nuclei clearly located within the muscle fiber boundary were counted as myonuclei (11, 12). The percentage of positive apoptotic myocytes was calculated for at least 200 myocytes. The mean value between the two observers was used for analysis.

Quantitation of PARP proteolytic fragments. PARP is a 117-kD DNA-binding protein cleaved by specific apoptotic proteases into 89- and 24-kD fragments during the execution phase of apoptosis (19). PARP proteolysis is associated with endonuclease activation and DNA fragmentation (22). Therefore, the presence of these two catalytic fragments (89 and 24 kD) indicates the activation of a late event in the apoptotic cascade (19). In our study, we used Western blotting to determine the level of apoptosis in each muscle biopsy by quantifying the ratio between the integrated optical density of one of these two proteolytic fragments (89 kD) to that of the native form of PARP (117 kD). To this end, muscle samples were homogenized (1:10 wt/vol) in cold 40 mM Tris buffer, pH 7.5, containing 1 mM ethylene diamine tetraacetic acid, and the protease inhibitors phenylmethylsulfonyl fluoride (1 mM) and leupeptin (40 μg/ml). Samples were centrifuged at 12,000 × g for 10 minutes at 4°C. One hundred microliters of the resulting supernatant was mixed with an equal volume of loading buffer (62.5 mM Tris, pH 6.8, 3% sodium dodecyl sulfate, 20% glycerol, 0.005% bromophenol blue), which was then boiled for 4 minutes. The protein concentration was determined by the method of Bradford (23). One hundred micrograms of the resulting suspension was loaded in a 10% polyacrylamide gel and submitted to electrophoresis (sodium dodecyl sulfate–polyacrylamide gel electrophoresis). Proteins were transferred to nitrocellulose membranes (Protran; Schleicher and Schuell, Germany), which were incubated in a phosphate-buffered saline containing 4% nonfat dry milk (blocking solution) for 1 hour at room temperature with gentle rocking. Then, membranes were incubated overnight at 4°C in blocking solution containing the primary antibody, anti-PARP (H-250) rabbit polyclonal antibody at 1:100 dilution (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). The specificity of the anti-PARP antibody in detecting the cleavage of the native PARP by apoptosis was determined by Western blot analysis of Jurkat cells cultured in the presence (10%) or absence of fetal calf serum (data not shown). The secondary antibody, a horseradish peroxidase-linked sheep anti-rabbit IgG (Amersham International, Buckinghamshire, UK) was incubated at 1:1000 dilution in blocking solution during 2 hours. Immunoreactivity was detected with a chemiluminescence Western blot detection system (Pierce, Rockford, IL). Films were scanned, and densitometric analysis (integrated optical density) of the immunoreactive bands (117 and 89 kD) was performed with the aid of the SigmaGel gel analysis software (Jandel Scientific Corporation, San Rafael, CA).

Statistical Analysis

Results are shown as mean ± SEM. The reproducibility of the immunohistochemical determinations between the two observers was tested using the intra-class correlation coefficient (24). A one-way analysis of variance, followed by post hoc contrast (LSD) if appropriate, was used to determine the statistical significance of differences between groups. Correlations between variables of interest were explored using the two-tailed Pearson test. A p value lower than 0.05 was considered significant.

Clinical and Functional Data

As shown in Table 1

TABLE 1. Clinical, lung function, and exercise data



Control Subjects

Patients with COPD

p Value*

Healthy
Inactive*
Normal BMI
Low BMI

Age, yrs45.6 ± 1.4 63.7 ± 2.5 62.0 ± 2.654.3 ± 3.50.0001
BMI, kg/m225.4 ± 0.6 26.4 ± 0.6 24.8 ± 0.717.5 ± 0.50.0001
Pack-year11.1 ± 4.521.7 ± 10.550.6 ± 6.752.1 ± 4.30.0001
FEV1/FVC, %82.1 ± 1.6 83.8 ± 1.644.4 ± 2.640.6 ± 5.00.0001
FEV1, L 3.9 ± 0.1 2.2 ± 0.2 0.9 ± 0.10.9 ± 0.10.0001
FEV1, % reference103.0 ± 4.498.3 ± 14.332.1 ± 3.128.4 ± 3.50.0001
DlCO, % referenceNANA 56.1 ± 11.4 42.1 ± 20.9NS
PaO2, mm HgNANA 69.3 ± 3.6 69.4 ± 5.2NS
PaCO2, mm HgNANA 44.8 ± 3.4 42.0 ± 2.9NS
Peak load, watts164.3 ± 10.9NA 55.0 ± 4.3 38.3 ± 6.50.0001
Vo2peak, L/min 1.9 ± 0.1NA 0.8 ± 0.1 0.6 ± 0.10.0001
Vo2peak, % reference
 74.4 ± 9.3
NA
 43.8 ± 4.2
 28.7 ± 4.5
 0.001

*Inactive control subjects refer to patients undergoing orthopedic surgery.

p < 0.05 versus healthy.

p < 0.05 versus inactive.

p < 0.05 versus COPD with normal BMI.

Definition of abbreviations: BMI = body mass index; COPD = chronic obstructive pulmonary disease; DlCO = carbon monoxide diffusing capacity of the lungs; FEV1 = forced expiratory volume in 1 second; FVC = forced vital capacity; NA = not available; NS = not significant; PaCO2 = arterial partial pressure of carbon dioxide; Po2 = arterial partial pressure of oxygen; Vo2 = oxygen uptake.

, healthy control subjects were younger than the rest of the groups; likewise, patients with COPD and low BMI were younger than patients with COPD and normal BMI, or patients undergoing orthopedic surgery. By definition, BMI was lower than 20 kg/m2 in the group of patients with COPD having low BMI and within the normal range in the remaining three groups (Table 1). The smoking history was not different between the two groups of patients with COPD. Forced spirometry was normal in healthy and inactive control subjects. By contrast, both groups of patients with COPD had similar levels of severe airflow obstruction, reduced DlCO, and arterial hypoxemia. Exercise tolerance (Vo2peak) was normal in healthy control subjects, but severely limited in patients with COPD, particularly among those with low BMI (p < 0.05).

Skeletal Muscle Apoptosis

The concordance between the two observers for the assessment of apoptosis by the TUNEL technique was excellent (intraclass correlation coefficient was 0.96, p < 0.001). Furthermore, the two techniques used to determine the level of apoptosis (TUNEL and PARP cleavage) were linearly related (Figure 1)

.

The percentage of apoptotic cells in muscle biopsies determined by the TUNEL technique increased progressively (p < 0.0001) from healthy subjects (3.8 ± 1.8%) to inactive control subjects (6.0 ± 2.3%), patients with COPD having normal BMI (17.1 ± 4.2%), and patients with COPD having low BMI (57.1 ± 12.4%). Figure 2

shows a representative example of a muscle biopsy from a healthy subject and a patient with COPD and low BMI. Likewise, the 89/117-kD PARP fragment ratio increased in these biopsies (p < 0.005) from healthy individuals (0.5 ± 0.2), to inactive controls (2.0 ± 0.9), and patients with COPD having normal BMI (1.1 ± 1.5), with the highest ratio occurring in patients with COPD and low BMI (5.3 ± 1.5) (Table 2

TABLE 2. Markers of skeletal muscle apoptosis



Control Subjects

Patients with COPD

p Value

Healthy
Inactive*
Normal BMI
Low BMI

TUNEL positive cells, %3.8 ± 1.86.0 ± 2.317.1 ± 4.257.1 ± 12.40.0001
PARP 89/117 kD ratio
0.5 ± 0.2
2.0 ± 0.9
1.1 ± 1.5
5.3 ± 1.5
 0.002

*Inactive control subjects refer to patients undergoing orthopedic surgery.

p < 0.05 versus healthy.

p < 0.05 versus inactive.

p < 0.05 versus COPD with normal BMI.

Definition of abbreviations: BMI = body mass index; COPD = chronic obstructive pulmonary disease; PARP = poly-(ADP-ribose) polymerase; TUNEL = transferance–mediated dUTP nick end labeling.

, Figure 2).

Physiologic Correlates

To investigate the potential mechanisms and consequences of skeletal muscle mass loss in COPD (and to avoid potential confounders), we looked for relationships between variables of interest only among patients with the disease (we excluded from this analysis the two groups of healthy control subjects). We observed in patients with COPD that BMI was inversely related to skeletal muscle apoptosis, determined either by the TUNEL (r = −0.65, p = 0.009) or PARP cleavage methods (r = −0.58, p = 0.04). Neither BMI nor skeletal muscle apoptosis was correlated significantly to any of the lung function variables determined in these patients (forced expiratory volume in 1 second, DlCO, arterial oxygen pressure, or arterial carbon dioxide pressure). Finally, BMI was better correlated with exercise capacity (Vo2peak [% reference]) (r = 0.74, p = 0.006) than the degree of airflow obstruction as measured by forced expiratory volume in 1 second (% predicted) (r = 0.66, p = 0.02).

The main observations of our study are, first, that increased apoptosis occurs in skeletal muscle of underweight patients with COPD and, second, that this is associated with lower exercise tolerance despite a similar degree of lung function impairment. To our knowledge, this has not been reported before in COPD. Yet, two recent studies have documented similar results in patients with chronic heart failure (12, 21), a disease characterized, like COPD, by the frequent occurrence of unexplained weight loss (2). Therefore, there is a growing body of evidence supporting a role for skeletal muscle apoptosis in chronic diseases characterized by muscle atrophy and unexplained weight loss.

Potential Mechanisms

Mechanistically, two different questions need to be addressed. First, what are the links between apoptosis and muscle fiber atrophy and, second, what are the potential triggers of apoptosis in these patients? The answer to the former question is unclear, but several experimental (10, 11) and clinical studies (12) indicate that apoptosis in multinucleated cells (like the myocytes) causes atrophy rather than cell death. The observation in our study of a high percentage (> 50%) of TUNEL positive fibers in underweight (i.e., atrophic) patients with COPD supports this view. Although this might seem somewhat surprising, previous studies in patients with end-stage heart failure (and cardiac cachexia) also reported values of TUNEL positive myocites that ranged from 20–30% (8, 12, 20) to 76% (9). As with these previous studies, we did our best to exclude the possibility that some of the TUNEL positive nuclei correspond to interstitial cells (fibroblasts, endothelial cells, or other recruited cells), and not to muscle nuclei (see Methods).

A different mechanistic question relates to the potential triggers of apoptosis in these patients. Our results lend some support to some potential candidate triggers but not others. For instance, although aging induces apoptosis in muscle cells (25), it is unlikely that aging can explain our findings because underweight patients with COPD were younger than those with normal BMI (Table 1). Inactivity is also unlikely to explain our findings because the levels of skeletal muscle apoptosis in underweight patients with COPD were higher than those seen in orthopedic patients, who were also inactive. Lung function impairment (including arterial hypoxemia) was similar in the two groups of patients with COPD studied, and is therefore unlikely to explain the observed differences in skeletal muscle apoptosis. Finally, smoking history was similar in both groups of patients with COPD (Table 1), and although oral steroids can cause skeletal myopathy (26), none of the patients had this treatment. On the other hand, we can postulate several, nonmutually exclusive, potential mechanisms that may trigger muscle apoptosis in these patients. Oxidative stress and systemic inflammation are obvious candidates because both occur in patients with COPD (1) and both have the potential to induce apoptosis in several cell systems (2729). Likewise, because the release of cytochrome c from the mitochondria is a primary regulator of apoptosis (30, 31), mitochondrial abnormalities may also play a pathogenic role. We have previously shown that the activity of cytochrome oxidase (which uses cytochrome c as substrate) is increased in the skeletal muscle of patients with COPD (17), and other authors have also demonstrated the presence of significant bioenergetic abnormalities in the muscles in these patients (1). Overall, these observations suggest a potential role of mitochondrial abnormalities in the genesis of skeletal muscle apoptosis in COPD. The precise role of these and other potential cellular pathways explaining the increased apoptosis observed the skeletal muscle of underweight patients with COPD need to be specifically addressed in future studies.

Limitations of the Study

Because of the well-known limitations in sensitivity and specificity of the TUNEL technique to quantitate apoptosis (32), many authors recommend its use in combination with other methods (33, 34). In our study, we complemented the TUNEL technique with quantitation by Western blot of two proteolytic fragments (89 and 24 kD) of PARP (19). PARP proteolysis is a late event in the apoptotic cascade and is associated with endonuclease activation and DNA fragmentation (22). Because the latter generates the 3′OH-free DNA ends that are detected by the TUNEL assay, both techniques should, in theory, be related. It was therefore reassuring to observe that they were linearly related (Figure 1), and yielded the same qualitative information (Table 2). We are therefore confident that our results support the presence of increased apoptosis in skeletal muscle of patients with COPD and low body weight.

Clinical Implications

It is notable that despite a similar degree of lung function impairment (Table 1), underweight patients with COPD showed poorer exercise tolerance (lower Vo2peak value) than did those with a normal BMI. Indeed, in these patients BMI was better correlated with exercise capacity than with the degree of airflow obstruction, which historically is believed to be the characteristic physiologic feature of COPD. These observations are in keeping with previous reports in patients with chronic heart failure (12, 21), and suggest that the level of skeletal muscle apoptosis in these patients may indeed be limiting their exercise capacity.

Conclusions

Our results show that increased apoptosis occurs in skeletal muscle of underweight COPD patients and that this may contribute to limit their exercise tolerance. Thus, a better understanding of the molecular mechanisms underlying this phenomenon may open the possibility of new therapeutic interventions in these patients (35) that contribute to improving their exercise tolerance, quality of life and, potentially, long-term prognosis.

:

The authors thank Carmen Santos, Maria Carmen Arroyo, Marga Bosch, Angels Noguera, and Francisca Bauzá for their technical help, and Prof. W. MacNee (University of Edinburgh) for his helpful comments and advice.

Supported in part by Fis (00/437), SEPAR, Fundación Barceló, and ABEMAR.

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Correspondence and requests for reprints should be addressed to Dr. Alvar Agustí, M.D., Servei Pneumología, Hospital Universitari Son Dureta, Andrea Doria 55, 07014 Palma Mallorca, Spain. E-mail:

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