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doi:10.1378/chest.07-1592
(Chest. 2008; 133:13-18)
© 2008 American College of Chest Physicians
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Peripheral Muscle Alterations in Non-COPD Smokers*

Maria Montes de Oca, MD, PhD; Eduardo Loeb, MD; Sonia H. Torres, MD, PhD; Juan De Sanctis, PhD; Noelina Hernández, PhD and Carlos Tálamo, MD, FCCP

* From the Pulmonary Division (Drs. Montes de Oca, Loeb, and Tálamo), Hospital Universitario de Caracas; Instituto de Medicina Experimental (Drs. Torres and Hernández), Sección de Adaptación Muscular; and Instituto de Inmunología (Dr. De Sanctis), Universidad Central de Venezuela, Caracas, Venezuela.

Correspondence to: María Montes de Oca, MD, PhD, Servicio de Neumonología, Piso 8, Hospital Universitario de Caracas, Universidad Central de Venezuela, Los Chaguaramos, Caracas, Venezuela; e-mail: mmdeoca{at}cantv.net

Abstract

Background: Although tobacco smoke is the main cause of COPD, relatively little attention has been paid to its potential damage to skeletal muscle. This article addresses the effect of smoking on skeletal muscle.

Methods: The vastus lateralis muscle was studied in 14 non-COPD smokers (FEV1/FVC, 78 ± 5%) and 20 healthy control subjects (FEV1/FVC, 80 ± 3%). Muscular structure, enzyme activity, constitutive and inducible nitric oxide (NO) synthases (endothelial NO oxide synthase [eNOS], neuronal NO synthase [nNOS] and inducible NO synthase [iNOS]), nitrites, nitrates, nitrotyrosine, and the presence of macrophages were analyzed.

Results: In smokers, type I muscle fibers cross-sectional area was decreased, and a similar trend was found in type IIa fibers. Lactate dehydrogenase levels and the percentage of fibers with low oxidative and high glycolytic capacity were increased in smokers. nNOS (96.9 ± 11.7 vs 125.4 ± 31.9 ng/mg protein; p < 0.01) and eNOS (38.9 ± 11.0 vs 45.2 ± 7.7 ng/mg protein [± SD]; p < 0.05) were lower in smokers, while fiber type distribution, capillarity measures, β-hydroxy-acyl-CoA-dehydrogenase levels, iNOS, nitrite, nitrate, and nitrotyrosine levels, and macrophage number in the muscle tissue were similar to the nonsmoker subjects.

Conclusions: Smokers presented some alterations of skeletal muscle such as oxidative fiber atrophy, increased glycolytic capacity, and reduced expression of the constitutive NO synthases (eNOS and nNOS). The findings support some muscular structural and metabolic damage but not the presence of local inflammation in the smokers. In addition, they suggest a possible effect of tobacco smoke impairing the normal process of NO generation.

Key Words: inflammation • nitric oxide • skeletal muscle • smoking

Several studies12345 have shown that skeletal muscle function and structure are altered in COPD patients. Peripheral muscle dysfunction is probably the most studied systemic effect of COPD; however, the mechanisms responsible for its occurrence are still poorly understood. It is probably multifactorial in origin, and there is strong evidence suggesting a link with disuse, chronic hypoxia, nutritional abnormalities, and the development of systemic inflammation and oxidative stress.

Although it is accepted that tobacco smoke is the major cause of COPD, much less attention has been paid to its potential effect in the extrapulmonary manifestations of the disease. Tobacco smoke has > 4,700 compounds, including free radicals and other oxidants, which can be potentially harmful to skeletal muscle. In rats exposed to cigarette smoke, Nakatani et al67 showed a reduced proportion of type I fibers and cross-sectional area (CSA) of type I and IIa fibers in soleus muscle, and decreased CSA of IIa and IIb fibers in extensor digitorum longus muscles. Orlander et al8 studied the skeletal muscle characteristics in smokers and nonsmoking subjects. Their results indicated the presence of abnormalities in smokers characterized by a reduction of type I and increase in type IIb fibers, as well as reduced muscle oxidative capacity, findings similar to those reported in COPD patients. Unfortunately, pulmonary function was not performed in this study8; therefore, it is not clear whether the differences are an effect of smoking per se or if some of the smokers had COPD.

An oxidant/antioxidant imbalance has been reported in active and passive smokers.9101112 In addition, cigarette smoke exposure in experimental animals affects the antioxidative capacity of skeletal muscle.13 There is also evidence that the activity of endothelial nitric oxide (NO) may be impaired in smokers.14

Motivated by the lack of information about the potential damage to the skeletal muscle of cigarette smoke, the present study was designed to compare skeletal muscle enzyme activity and morphologic characteristics between smokers with normal spirometry results and normal subjects. In addition, we investigated the concentration of endothelial NO synthase (eNOS), neuronal NO synthase (nNOS), and inducible NO synthase (iNOS), and the products of NO: nitrite, nitrate, and nitrotyrosine. The possible presence of macrophages was evaluated with anti-CD68 in the muscle of some of the subjects.

Methods and Materials

Study Subjects
The study group consisted of 14 current smokers subjects (30 ± 10 pack-years), with normal spirometry results who were recruited from the smoking cessation clinic of the Hospital Universitario de Caracas. Twenty healthy sedentary nonsmoking subjects served as control subjects. The committee on human research approved the study, and all subjects signed the informed consent.

A non-COPD smoker was defined as a postbronchodilator FEV1/FVC ratio > 0.7, and a smoking history > 20 pack-years. Smokers and control subjects were excluded if they had reversibility of the FEV1 after 200 mg of inhaled salbutamol > 12% and 200 mL. Also excluded were subjects with congestive heart failure, hypertension, and neoplastic and metabolic diseases. Smoker and control subjects were asked about the frequency and duration of walking, climbing stairs, household activities, sporting activities, and physical activity during leisure time. All subjects in both groups led a similar sedentary lifestyle.

Pulmonary Function Tests
FEV1, FVC, and FEV1/FVC were calculated according to the recommendations of the American Thoracic Society.15 Normal values for pulmonary measurements were taken from a standard reference source.16

Skeletal Muscle Study
All subjects underwent vastus lateralis muscle biopsies.17 The muscle sample was divided in three parts. One was embedded with optimal cutting temperature compound (Tissue Tek II; Sakura Finetek U.S.A.; Torrance, CA) and frozen in isopentane cooled in liquid nitrogen for histochemical and immunohistochemical analysis. Two parts were frozen directly in liquid nitrogen for NO end products and enzymes determination.

Histochemical Analysis
Transverse 10-µm serial sections were cut in a cryostat at – 20°C and used for adenosine triphosphatase reaction after alkaline (pH 10.3) and acid (pH 4.37 and pH 4.6) preincubation.18 Sections were also stained for reduced nicotinamide adenine dinucleotide diaphorase (NADH-d) and {alpha}-glycerophosphate dehydrogenase ({alpha}-GPD)1920. The fibers were classified as high, medium, or low oxidative (NADH-d) and glycolytic ({alpha}-GPD) activity according to the intensity of the staining, evaluated blindly by two different observers. Capillaries were visualized by the {alpha}- amylase periodic acid Schiff reaction.21 Photomicrographs at a final magnification x 200 were made, and fibers were identified by comparison with the adenosine triphosphatase sections. An area of the photograph was delimited, measured with a planimeter, and fibers and capillaries were counted to calculate the mean fiber CSA, capillaries per square millimeter, and capillaries per fiber ratio. Capillaries around each fiber type were counted (capillary contacts), and all the fibers of one type were drawn together to measure area by planimetry to calculate the mean area of each fiber type.

Immunohistochemical Analysis
To evaluate the presence of macrophages in muscle sections, immunohistochemical analysis was performed using anti-CD68, clone MB11 (DAKO; Carpinteria, CA). Rapid staining was performed (Vecstain ABC Elite; Vector Laboratories; Burlingame, CA). Five fields per section were observed at x 100 with a 1 µm2 grid. For technical reasons, this study could only be performed in samples from eight smokers and seven control subjects.

Enzymes Determination
After weighing, the sample was homogenized in ice-cooled potassium phosphate buffer. The activity of β-hydroxy-acyl-CoA-dehydrogenase (HAD) and lactate dehydrogenase (LDH) was assayed using fluorometric techniques.22 Results are expressed in micromols per liter per minute per gram of wet tissue.

Assessment of Nitrite and Nitrate Levels
The muscle sample was weighed and homogenized with a glass pestle in 1 mL of Tris-HCl buffer, 10 mmol/L, pH 7.5; nNaCl, 150 mmol/L; ethylene-diamine tetra-acetic acid, 5 mmol/L; triton x-100, 1% (volume/volume); leupeptin, 10 µg/mL; aprotinin,10 µg/mL; and pepstatin, 2.5 g/mL. The homogenate was centrifuged at 90g for 5 min. Supernatant was used for the assays. Protein concentration was assessed by the BCA protein assay kit (Pierce Biochemicals; Rockfort IL).

NO levels were determined indirectly by quantification of their oxidized products of degradation, using nitrate reductase and the Griess reagent2324. A standard curve was obtained with sodium nitrate dissolved in water or in a pool of normal sera. Nitrite concentration was determined at 540 nm using enzyme-linked immunosorbent assay (ELISA) plate reader (Multiscan MCC/340; Labsystems; Helsinki, Finland).

Determination of NO Synthases, Nitrotyrosine by ELISA
All three NO synthases were assessed using sandwich ELISA assays. Constitutive eNOS was detected using a commercial kit (R&D Systems; Minneapolis, MN). Monoclonal and polyclonal antibodies for nNOS were from BD Biosciences (San Diego CA), and the recombinant enzyme for the standard curve was from Calbiochem (San Diego, CA). iNOS was assessed using a pair of antibodies (Serotec Corporation; Kidlington, Oxford, UK). The sensitivity of all assays was 25 pg/mL.

The total amount of nitrotyrosine was determined by ELISA.25 Antibodies (mouse IgG monoclonal, polyclonal against nitrotyrosine and polyclonal goat anti-rabbit IgG-peroxidase) were from Upstate Biotechnology (Lake Placid, NY). The quantification of nitrotyrosine was performed using a standard curve with known concentrations of nitrotyrosine from chemically modified bovine serum albumin. The sensitivity of the assay was 50 pg/mL.

Statistical Analysis
Data are presented as mean ± SD. Differences in skeletal muscle variables between smokers and control subjects were determined using nonparametric Mann-Whitney U test. A statistical software program was used (Statistica; Statsoft; Tulsa, OK). An acceptable level of statistical significance was at p ≤ 0.05.

Results

The clinical characteristics and spirometric data of the non-COPD smokers and controls subjects are detailed in Table 1 . There were no differences in age, weight, height, body mass index, and pulmonary function between the groups.


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  		Table 1.. Anthropometric and Spirometric Data in Smokers and Control Subjects*

 
The smokers and control subjects fiber type distribution, mean CSA of the fibers, CSA of the different fiber types, capillarity measured as capillaries per square millimeter, capillaries per fiber, and the number of capillary contacts with the different fiber types are shown in Table 2 . The CSA of type I fibers was significantly lower in the smoker subjects (p < 0.05), and a similar trend was observed in the type IIa fibers (p = 0.08). The rest of the parameters were similar in both groups. The percentage of fibers with low oxidative capacity was increased in smokers and also the percentage of fibers with high glycolytic capacity at the expense of a reduction in the percentage of medium glycolytic capacity fiber (Fig 1 ). These changes indicate more glycolytic and less oxidative activity of the muscle fibers.


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  		Table 2.. Skeletal Muscle Characteristics in Smokers and Control Subjects*

 

Figure 1
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  		Figure 1.. Top: Percentage of fibers with oxidative capacity (NADH-d) in smokers and control subjects. Bottom: Percentage of fibers with glycolytic capacity ({alpha}-GPD) in smokers and control subjects. *p < 0.001.

 
Figure 2 shows LDH activity that was increased in smokers compared with control subjects (253 ± 95 and 175 ± 50 µmol/L/min/g wet weight, respectively). The values in the HAD activity were similar in both groups (11.0 ± 3.4 and 9.8 ± 2.9 µmol/L/min/g wet weight, respectively).


Figure 2
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  		Figure 2.. LDH levels in smokers and control subjects. *p < 0.01.

 
The results of skeletal muscle nitrite, nitrate, and nitrotyrosine are presented in Table 3 . There were no differences in the level of these products between the groups. iNOS, eNOS, and nNOS levels of smokers and healthy subjects are shown in Figure 3 . Levels of eNOS and nNOS were significantly lower in smokers compared with control subjects. However, skeletal muscle iNOS expression was similar in both groups.


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  		Table 3.. Skeletal Muscle Levels of NO End Products in Smokers and Control Subjects*

 

Figure 3
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  		Figure 3.. eNOS, nNOS, and iNOS, levels in smokers and control subjects. *p < 0.005.

 
Immunohistochemical analysis of the muscle tissue showed that the number of macrophages was similar in smokers and control subjects (3.4 ± 1.1/mm2 vs 4.6 ± 2.9/mm2, respectively). A representative picture illustrating the muscle reaction with anti-CD68, clone MB11 in a smoker and a control subject is shown in Figure 4 .


Figure 4
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  		Figure 4.. Transversal section of vastus lateralis part of quadriceps muscle. Immunohistochemical reaction with anti-CD68, clone MB11. Arrows point positive reaction. Note that the CSA of some of the fibers is decreased in the smoker subject.

 
Discussion

There were several important findings in this study of non-COPD smoker skeletal muscle. Smoker subjects had reduced CSA of type I fibers and a similar trend in type IIa fibers compared with control subjects. LDH levels and the percentage of fibers with low oxidative and high glycolytic capacity were increased in smokers. Muscular eNOS and nNOS concentrations were decreased in smokers, and there was no evidence supporting the presence of local inflammation in the skeletal muscle of these subjects.

In subjects who smoke, several studies826 have reported decreased muscular strength, reduced oxidative enzymes activity and type I fiber proportion, as well as higher proportion of type II fibers, in particular IIb fibers. However, no difference has been observed in fiber areas. The results of this study contrast in part with those findings.8 We did not observe changes in fiber type proportion or in the HAD activity between smokers and control subjects, but in agreement with them we observed changes indicating more glycolytic activity in the smokers such as increased LDH levels and higher proportion of fibers with high glycolytic capacity. Although the HAD levels were not decreased in smokers, the proportion of fibers with low oxidative capacity was increased, indicating lower oxidative capacity of the fibers. In addition, reduced type I fibers CSA and a similar trend of type IIa fibers were found, indicating a preferential damage of oxidative fibers. These results are in agreement with those reported in the soleus muscle of rats exposed to cigarette smoke,7 but contrast with those reported by Orlander et al,8 probably due to the lack of pulmonary function assessment in their study that may include COPD patients in the smoker group. Fiber atrophy in smokers observed in the present study should not be attributed to the effect of physical activity because both groups of subjects had a similar sedentary lifestyle. The CSA of type I, IIa, and IIab fibers has been found reduced in skeletal muscle of COPD patients,1 which would lead to the question whether the atrophy in these fibers is due to the effect of tobacco smoke or to other factors.

Oxidant/antioxidant imbalance has been reported in COPD patients as well as in active and passive smokers.9101112 Although it is a matter of controversy,272829 there is evidence of local inflammation and nitrosative stress in COPD skeletal muscle. This is shown by increased iNOS activity, nitrotyrosine and cytokines levels, and the presence of macrophages. In addition, there are decreased eNOS, nNOS, nitrite, and nitrate levels in the muscles of these patients.29 It is important to know which of the mentioned alterations could be attributed to the effect of tobacco smoke per se. Cigarette smoke exposure induces transient decrease of skeletal muscle glutathione in the guinea pig.13 To our knowledge, no previous study has evaluated the levels of oxidative stress and inflammatory markers in the skeletal muscle of non-COPD smokers. The results of this study show that there were no differences in iNOS, nitrite, nitrate, and nitrotyrosine levels between smokers and healthy subjects. However, eNOS and nNOS levels were significantly lower in smokers, suggesting the presence of microvascular alteration leading to hypoxia that could explain the oxidative fiber atrophy observed in the smokers.

The number of macrophages present in the muscle sections was similar in both groups. This finding, in addition to the normal values of nitrotyrosine and iNOS, do not support the involvement of local inflammation in the skeletal muscle of non-COPD smokers, but the changes in the constitutive NO synthases levels suggest that tobacco smoke may produce an incipient alteration in the normal process of NO generation, probably not severe enough to reduce nitrite and nitrate levels.

Normally, NO is formed inside the skeletal muscle fibers by the constitutive NO synthase isoforms. The main source of NO synthesis is the nNOS isoform, which is localized in close proximity to the sarcolemma and acts as a neuromodulator or neuromediator.30 NO is also produced by eNOS, which is localized inside the skeletal muscle mitochondria.30 There is evidence indicating that constitutive NO production in the skeletal muscle promotes glucose uptake, plays a major role in regulating muscle glucose metabolism, enhances Ca++ release from the sarcoplasmic reticulum, improves muscle perfusion, and promotes muscle repair and sarcomere addition.30 Several studies3132 indicate that cigarette smoking appears to have a profound effect on glucose transport in skeletal muscle, reducing glucose uptake and causing impairment in the insulin-dependent portion of muscle recovery from glycogen-depleting exercise. In addition, there is evidence showing that cigarette smoke adversely affects the endothelial l-arginine NO synthase pathway, reduces NO production, and eNOS expression.33 The reduction in eNOS and nNOS levels, although not accompanied by changes in the NO end products assessed in the present study, argue in favor of tobacco smoke involvement in the muscular NO synthesis, which may eventually affect glucose transport, endothelial function, and muscle strength reported by other studies.8263132

Due to the invasive nature of the procedure used to obtain the muscle samples, a relative small number of subjects were studied. This probably limits the extrapolation of these results to the entire population of non-COPD smokers. However, the current report represents the first study that assesses the levels of nitrosative stress markers in their skeletal muscle.

In summary, this study shows that there are some incipient alterations in the skeletal muscle of non-COPD smokers characterized by type I fiber atrophy, increased glycolytic activity, and reduced expression of the constitutive NO synthases. The last finding supports a possible participation of tobacco smoke impairing the normal process of NO generation. However, there are no evidences indicating the presence of local inflammation in the skeletal muscle of non-COPD smokers that could eventually argue in favor to the potential contribution of tobacco smoke on the skeletal muscle inflammation in COPD patients.

Footnotes

Abbreviations: {alpha}-GPD = {alpha}-glycerophosphate dehydrogenase; CSA = cross-sectional area; ELISA = enzyme-linked immunosorbent assay; eNOS = endothelial nitric oxide synthase; HAD = β-hydroxy-acyl-coenzyme A-dehydrogenase; iNOS = inducible nitric oxide synthase; LDH = lactate dehydrogenase; NADH-d = nicotinamide adenine dinucleotide diaphorase; NO = nitric oxide; nNOS = neuronal nitric oxide synthase

Grant support was provided by Fondo Nacional de Ciencia, Tecnología e Innovación, S1-2005000149, G-2005000389, Consejo de Desarrollo Científico y Humanístico, Universidad Central de Venezuela, 09.33.4367.2005.

The authors have no conflicts of interest to disclose.

Received for publication June 25, 2007. Accepted for publication August 15, 2007.

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