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Published ahead of print on December 18, 2002, doi:10.1164/rccm.200206-519OC
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American Journal of Respiratory and Critical Care Medicine Vol 167. pp. 873-879, (2003)
© 2003 American Thoracic Society

Original Article

Mitochondrial Electron Transport Chain Function Is Enhanced in Inspiratory Muscles of Patients with Chronic Obstructive Pulmonary Disease

Florence Ribera, Benoit N'Guessan, Joffrey Zoll, Dominique Fortin, Bernard Serrurier, Bertrand Mettauer, Xavier Bigard, Renée Ventura-Clapier and Eliane Lampert

Service des Explorations Fonctionnelles Respiratoires et de l'Exercice, Hôpitaux Universitaires de Strasbourg, Département de Physiologie, Equipe d'Accueil, Faculté de Médecine, Strasbourg; Faculté de Pharmacie, Cardiologie Cellulaire et Moléculaire U-446 Institut National de la Santé et de la Recherche Médicale, Université Paris-Sud, Châtenay-Malabry; and Unité de Bioénergétique, Centre de Recherche du Service de Santé des Armées, BP 87, La-Tronche Cedex, France

Correspondence and requests for reprints should be addressed to Eliane Lampert, Department of Physiology, Faculty of Medicine, 11 rue Humann, 67,000 Strasbourg, France. E-mail: Eliane.Lampert{at}physio-ulp.u-strasbg.fr


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In chronic obstructive pulmonary disease, inspiratory muscles face increased resistive and elastic workloads and therefore increased energy requirements. The adaptive response of these muscles to this higher energy demand includes increased oxidative enzymes and changes in contractile protein expression but the consequences on mitochondrial function and energy metabolism have not been assessed so far. We investigated the in situ properties of the mitochondria of costal diaphragm and external intercostal muscles using the skinned fiber technique in 9 emphysematous and 11 age-matched control patients. Biopsies obtained during thoracic surgery were placed in an oxygraphic chamber to measure maximal oxygen uptake. We observed that the maximal oxidative capacity of diaphragm and external intercostal muscles increased significantly in the emphysematous group compared with the control group (+135 and +37%, respectively). Significant correlations were found between the maximal oxidative capacity and patients' pulmonary indexes of obstruction (diaphragm: r = -0.637, intercostal: r = -0.667, p < 0.005) and hyperinflation (diaphragm: r = 0.639, p < 0.003, intercostal: r = 0.634, p < 0.01). Slow myosin heavy chain isoform increased in the diaphragm of the emphysematous group, with significant relationships between indexes of obstruction and hyperinflation and activities of biochemical mitochondrial markers. Thus, severe emphysema was associated with increased mitochondrial capacity and efficiency in the inspiratory muscles, supporting an endurance training-like effect.

Key Words: diaphragm • intercostal muscle • emphysema

In severe chronic obstructive pulmonary disease (COPD), there is an imbalance between inspiratory muscle function which is hampered and the load they face which is increased. This could make the inspiratory muscles vulnerable to the development of muscle fatigue (1). However, contractile fatigue of the diaphragm is an uncommon event after exhaustive exercise in patients with severe COPD (2, 3). Indeed, recent studies have shown that inspiratory muscles are able to respond to the chronically increased respiratory work by functional and structural adaptive processes. Changes in the contractile phenotype with a shift from fast to slow fiber type and myosin heavy chain isoforms in the diaphragm have been demonstrated in patients with severe emphysema (4–6). Moreover, an increased activity of oxidative enzymes such as citrate synthase or succinodehydrogenase (7, 8) consistent with an increase of the mitochondrial density (9), as well as a decrease in glycolytic enzyme activities (7, 10), have been described in the diaphragmatic fibers of these patients. On the other hand, the peripheral skeletal muscles of patients with COPD exhibit intrinsic biochemical and structural alterations, including atrophy and reduced oxidative capacity (11). Thus, the respiratory muscles of patients with COPD may be subjected to the opposite influence of increased load and systemic consequences of the disease as in congestive heart failure (12). Moreover, the contractile and metabolic phenotype of muscle fibers may also change in opposite direction (12, 13).

Importantly, increased resistance to fatigue relies not only on the capacity of the muscles to increase energy production by the oxidative pathways but also on energy transfer from sites of production to use. The creatine kinase, catalyzing the reversible transfer of a phosphate moiety between adenosine diphosphate (ADP) and creatine, plays a major role in this transfer through its specific mitochondrial and cytosolic isoforms (14–17). Neither the functional consequences on oxidative capacity and regulation of mitochondria electron transport chain nor the mechanisms of cellular energy transfer such as the creatine kinase system have ever been assessed in human respiratory muscles.

Mitochondrial respiration can be studied in situ using saponin-skinned muscle fibers. This technique allows the entire mitochondrial population of the sample to be studied in its natural structural environment without disturbing the connections between mitochondria and the cytoskeleton. Furthermore, it allows studying the ADP sensitivity of mitochondria, which has been shown to vary with the type of muscle in animal studies (15, 18).

The purpose of the present study was to investigate the mitochondrial function in situ and the muscle fiber phenotype and metabolic characteristics of inspiratory muscles (diaphragm and external intercostal muscle) in patients with severe COPD. We also searched for possible correlations between these cellular adaptations and pulmonary functional indexes of airflow obstruction and lung hyperinflation.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Patients
The twenty patients undergoing thoracic surgery included in this study were divided into two groups. The control group (n = 11, 7 males, 4 females) included nonsmoking patients undergoing limited resection of metastases of nonpulmonary cancer having pulmonary function within the normal range. The COPD group (n = 9, 8 males, 1 female) included patients undergoing lung volume reduction surgery (n = 7) or resection of large bullae (n = 2). Their characteristics are given in Table 1 . Our Institution's Ethics Committee approved the experimental protocol and patients gave their informed consent. None of the control group had been treated with agents likely to modify muscle metabolism (anabolic steroids, radiotherapy, and cytotoxic chemotherapy). Three patients with COPD received oral steroids. Spirometry, diffusion capacity, and plethysmographic lung volumes were determined with a spirometer and whole-body plethysmograph (MasterScreen; Jaeger, Würtzburg, Germany) and compared with predicted values (19).


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  		TABLE 1. Anthropomorphic and functional characteristics

 
Skeletal Muscle Biopsies
Anterior-costal diaphragm and external intercostal (anterior fourth intercostal space) muscle biopsies were obtained during thoracotomy by the Bergstrom technique (20). One part was immediately frozen in liquid nitrogen for enzymatic and myosin isoforms analysis, and another part served for respiration studies.

Enzyme Activities and Myosin Isoforms
Frozen tissue samples were weighed and placed into cold buffer (30 mg wet weight/ml) containing: N-2-hydroxyethylpiperazine-N'-ethane sulfonic acid (pH 8.7), ethyleneglycol-bis-(ß-aminoethyl ether)-N,N'-tetraacetic acid, dithiothreitol, magnesium chloride, and 0.1% Triton and incubated for 60 minutes at 0°C to ensure complete enzyme extraction. The citrate synthase, creatine kinase, and lactate dehydrogenase activities were assayed as described previously (13, 21). Myosin isoforms were determined as described previously (13).

Mitochondrial Respiration
Muscle biopsies were freed of connective tissue and muscle fibers were isolated. Respiratory parameters of the total mitochondrial population were studied in situ on fresh saponin-skinned fibers (50 µg/ml saponin for 30 minutes) and determined with a Clark electrode (Strathkelvin, Glasgow, Scotland) in an oxigraphic cell containing respiration solution with glutamate and malate as substrates at 22°C as described previously (17, 22). Respiration rates are expressed as µmol O2/minute g dry weight. A total of 5 to 10 mg fibers were placed in the chamber and basal oxygen consumption without ADP (Vo) was recorded. Then, increasing amounts of ADP were added to titrate ADP-stimulated respiration (VADP) until maximal respiration was reached. To check for the outer mitochondrial membrane integrity, cytochrome-c was added in all experiments (Figure 1A , see legend) (15). This technique allowed the determination of several parameters characterizing mitochondrial function (Figure 2 , see legend): basal respiration and the maximal respiration rate (Vmax = VADP + VO). The Km values, which represent the sensitivity of the mitochondrial oxidative phosphorylation to ADP, and VADP were calculated using a nonlinear fitting of the Michaelis–Menten equation. The efficiency of the coupling between oxidation and phosphorylation also called acceptor control ratio was calculated as the ratio between respiration with ADP and respiration without ADP (Vmax/Vo).



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  		Figure 1. An example of oxygen consumption of permeabilized diaphragm myofibers for increasing levels of the phosphate acceptor ADP. (A) Raw data obtained for diaphragm fiber respiration in oxigraphic chamber, from a representative patient of the severe chronic obstructive pulmonary disease (COPD) group. The tissue oxygen (O2) consumption increases with increasing doses of ADP up to the maximal activation of respiration, then cytochrome-c was added to a final concentration of 8 µM. Cytochrome C did not change the slope of O2 consumption (solid line), whereas it should increase the slope of O2 consumption if the mitochondrial outer membrane was altered (dotted line). (B) The ADP-related respiration characterizing the Michaelis–Menten kinetics of oxygen consumption for a patient of the control group and one of the severe COPD group. E = patient with severe COPD.

 


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  		Figure 2. Schematic representation of differences in respiratory features between glycolytic and oxidative muscle Schematic intracellular organization of mitochondria energy production and transfers (left side) and mitochondrial characteristics (right side) for glycolytic muscle (A) and oxidative muscle (B) (from References [14, 37]). Glycolytic muscle is characterized by (1) a low maximal oxidative capacity, represented by a low maximal respiration (Vmax) and effective glycolytic ATP production; (2) a high sensitivity of mitochondrial respiration for ADP (low Km), the signal for respiration is cytosolic ADP; (3) noncoupled energy transfers within the cell between mitochondria and ATPases. Oxidative muscle is characterized by (1) a high maximal oxidative capacity, represented by a high ADP-stimulated respiration; (2) a low sensitivity of mitochondrial respiration for cytosolic ADP (high Km), (lower left panel: solid line representing the mitochondrial outer membrane), the signal for respiration is creatine; (3) coupled energy transfers within the cell between mitochondria and ATPases by creatinekinase shuttles. Cr = creatine; PCr = phosphocreatine.

 
Statistical Analysis
Values are expressed as means ± SEM. If normality and equal variance tests were passed, we used an unpaired t test to compare groups. If the normality test or equal variance test failed, differences between groups were tested for with a Mann–Whitney rank sum test. Statistical significance was set at p values less than or equal to 0.05.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Patients and Ventilatory Function
Pulmonary function parameters for the two groups are given in Table 1. Although age and height were similar between the two groups, control patients were heavier than patients with COPD whose mean body mass index remained within the normal range, with three values less than 20 kg/m2. The COPD group was characterized by a greater residual volume (RV) and ratio of RV to total lung capacity than the control group and by significantly lower values of FEV1, FVC, FEV1/FVC, carbon monoxide transfer factor, and PaO2.

Myosin Heavy Chains
The distribution of the myosin isoforms is presented in Table 2 . Intercostal muscle contained a higher percentage of slow myosin isoform than diaphragm muscle. The diaphragm of patients with COPD had a significantly higher percentage of slow myosin isoform than control patients (p = 0.031), mainly at the expense of the fast myosin isoform. Myosin distribution in intercostal muscle was unaltered in patients with COPD.


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  		TABLE 2. Enzyme activities and myosin heavy chain isoforms

 
Enzyme Analysis
The activities of enzymes participating in the energy metabolism are presented in Table 2. The total creatine kinase activity (IU/g wet weight), as well the cytosolic isoforms MM-CK and MB-CK, expressed as percentages of total creatine kinase activity, remained almost identical in the two groups for both diaphragm and intercostal muscles. Mitochondrial creatine kinase isoform activity in intercostal muscle expressed the same way was significantly increased in patients with COPD in comparison with control patients (p = 0.024). Although near the limit of significance (p = 0.069), a 2.3-fold increase in percentage of mitochondrial creatine kinase isoform was found in the diaphragm of patients with COPD. What was highly significant, however, was the negative relationship between the percentage of mitochondrial creatine kinase isoform in the diaphragm and FEV1 (% predicted), which is an index of airflow obstruction, or the positive relationship with the ratio of RV to total lung capacity, an index of lung hyperinflation (Figure 3) . Although citrate synthase activity was 50% higher in patients with COPD than in control patients, this did not reach significance. This probably relates to the scattered values due to the variable content of connective tissue within respiratory muscles among patients. To correct for this sample heterogeneity, citrate synthase values were expressed as a percentage of creatine kinase, which represents the true muscular content of the sample, being a purely muscular enzyme. Relative citrate synthase activity was then significantly higher in the diaphragm of patients with COPD and strongly negatively correlated with FEV1 (% predicted) or positively with the ratio of RV to total lung capacity (Figure 3). Analysis of total lactate dehydrogenase activity and isoform profiles were similar in the two groups for the two muscles (data not shown).



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  		Figure 3. Correlations between oxidative enzymes of the diaphragm and indexes of airway obstruction (A) and lung hyperinflation (B). Diamond, patients with severe COPD; circle, control patients. CK = creatine kinase; CS = citrate synthase; mi-CK = mitochondrial isoenzyme of creatine kinase; RV/TLC = ratio of residual volume to total lung capacity.

 
Mitochondrial Respiration
An example of the oxygen consumption of saponin-skinned diaphragm fibers for increasing ADP concentrations is shown in Figure 1A. The addition of cytochrome-c did not increase the slope of oxygen consumption, showing that cytochrome-c had not leaked out and that the outer mitochondrial membrane was not damaged by the procedure. In Figure 1B, respiration rates are plotted as a function of ADP concentration for one representative control patient and one representative patient with COPD. As ADP concentration increased, the respiration rate reached a plateau (Vmax) that was significantly higher in the diaphragm of the patients with COPD. The Km values for ADP and Vmax are given in Table 3 for the diaphragm and intercostal muscles. Vo was similar in the diaphragm and intercostal muscles of both groups. For the two muscles, the maximal respiratory rates at saturating ADP concentrations (Vmax) were significantly higher in patients with COPD than in control patients. The degree of coupling between oxidation and phosphorylation (acceptor control ratio) was significantly higher in the COPD group for both muscles. The Km of mitochondrial respiration for ADP was 2.4-fold higher in diaphragm muscle of patients with COPD compared with control subjects, close to significance (p = 0.08). Moreover, for both muscles, Vmax was also negatively related to the degree of airflow obstruction or positively related to the degree of lung hyperinflation (Figure 4) . The relationship was stronger between Vmax and the ratio of RV to total lung capacity in the diaphragm than in intercostal muscles.


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  		TABLE 3. Mitochondrial respiration indexes

 


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  		Figure 4. Correlations between maximal oxidative capacity and indexes of obstruction (left) and hyperinflation (right) for diaphragm (A) and intercostal muscle (B). Diamond, patients with severe COPD; circle, control patients.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Using the saponin-skinned fiber oxigraphic technique, we provide the first direct evidence for an increase in mitochondrial oxidative capacity in inspiratory muscles of patients with severe COPD. The maximal respiration of mitochondria of both the diaphragm and the intercostal muscles was significantly increased in patients with COPD compared with patients with normal lung function. This increase was linearly correlated with pulmonary indexes of airflow obstruction and lung hyperinflation. Moreover, this was associated with a significant improvement in the coupling of oxidation to phosphorylation suggesting an increased efficiency of mitochondrial ATP production. These changes were combined with a significant increase in less direct indexes of oxidative capacity such as the citrate synthase activity and the slow myosin isoform in the diaphragm already reported by others (4, 5, 7).

Myosin Isoforms and Enzyme Activities
Our results on myosin isoforms in the diaphragm of patients with COPD are in agreement with previous studies on the diaphragm of patients with COPD having similar obstruction (4, 5, 8) and similar (5) or more severe inflation (4, 8). In accordance with Sanchez and coworkers (23), we found a higher proportion of slow myosin isoform in external intercostal muscle compared with diaphragm. However, the repartition of myosin isoforms within the external intercostal muscles was not altered by severe emphysema. This differs from observations made by Gea (24) that showed an increased expression of fast myosin isoform in external intercostal muscles of severely obstructed patients. This might be linked to different factors. First, as pointed out by Wilson and coworkers (25), the mechanical effect of the anterior part of the external intercostal muscles differs according to their localization within the chest wall. The maximal inspiratory action of external intercostals is observed in the upper part of the rib cage with a decreasing inspiratory action unto the sixth intercostal space, eventually becoming expiratory in the lower intercostal spaces (25). Because the biopsy of external intercostals were performed in the fourth intercostal space in the present study as opposed to the sixth in the study by Gea (24), this might explain some of the discrepancies. In addition, the lack of changes in the contractile phenotype and enzyme activity in the intercostal muscles could also result from the interaction between two opposite influences acting on these muscles: the increase in functional workload inducing more fatigue-resistant fibers would be counterbalanced by the detrimental influence of chronic hypoxia, nutritional depletion, medication, and oxidative stress as already described in the peripheral skeletal muscles of patients with severe COPD (11). Conversely, in the diaphragm, the effects of global workload increase may be more important than the negative consequences of the pulmonary disease, allowing an adaptation of myosin isoform expression and citrate synthase to occur, in agreement with the recently described increase in succinodehydrogenase activity (8). This also parallels the increase in the mitochondrial volume density in the diaphragm of patients with severe COPD (9). The observed correlations between citrate synthase and respiratory function parameters (Figure 3) did not allow us to affirm a cause to effect relationship. However, we believe that these and other correlations presented in Figures 3 and 4 suggest that the diaphragm adapts to a chronically increased workload induced by obstruction and hyperinflation by increasing oxidative enzymes and that the degree of adaptation correlates with the severity of COPD.

Our observation of a threefold to fivefold increase in mitochondrial creatine kinase activity in both inspiratory muscles of patients with COPD together with a significant relationship to functional indexes in the diaphragm suggests a response of the creatine kinase system of inspiratory muscles to a prolonged increased workload as already described in skeletal muscles of marathon runners after intense training (26) and approaching the characteristics of slow oxidative muscle in rats (16).

Mitochondrial Oxidative Capacity in Inspiratory Muscles
As discussed previously, earlier works by different groups have provided indirect evidences that have been interpreted as an increased oxidative capacity within the inspiratory muscles of patients with severe COPD, especially an increase in mitochondrial density (9). Human skeletal muscle is able to sustain more prolonged work not only because of a higher oxidative capacity but also owing to an increased efficiency of ATP production and changes in energy distribution (14). The skinned-fibers technique, used in the present study, offers the unique opportunity to study the maximal oxidative capacity of muscle cells and some of the mitochondrial regulatory properties. Applied to the rat diaphragm, this technique disclosed mitochondrial properties typical of an oxidative muscle (Figure 2), being intermediate between that of cardiac muscle and that of soleus, a slow-oxidative skeletal muscle (12). All these three muscles are characterized by a high oxidative capacity (Vmax ranged from 8 to 30 µmol O2/minute/g dry weight) and a low sensitivity to external ADP (high Km), reflecting a restricted diffusion of ADP through the outer mitochondrial membrane (12, 21, 27) and most likely highly coupled and compartimentalized energy transfers (28) (Figure 2).

However, the present results in human inspiratory muscles of the control patients showed striking differences with rodent diaphragm, being closer to those observed in the vastus lateralis of normal sedentary humans (14, 22). This was characterized by a low Vmax, a low acceptor control ratio, and an intermediate sensitivity toward external ADP as reflected by a relatively low Km in both the diaphragm and intercostal muscles of control patients, all these features being characteristics of an untrained skeletal muscle (14) and closer to the ones of rodent fast-glycolytic muscles (15). These differences between rodents and humans diaphragms may be explained by differences in breathing pattern and frequency, which have already been shown to be related to biochemical parameters of aerobic capacity such as citrate synthase activity and substrate oxidation rates in the diaphragm of different adult mammals (29).

However, in patients with COPD, the oxidative capacity of the diaphragm muscle, as expressed by Vmax, was significantly increased approaching values obtained for the vastus lateralis muscle after 6 weeks of endurance training (30) or in physically active subjects (14, 22). This demonstrates that in the diaphragm of patients with COPD, there is not only an increase in the mitochondrial density as shown by Orozco-Levi and coworkers (9), but also a higher maximal ADP-stimulated mitochondrial respiration. We observed strong correlations between maximal respiration and functional respiratory indexes (Figure 4), suggesting that the intensity of the response of mitochondrial respiration response was directly related to the severity of obstruction and hyperinflation. Moreover, the Km for ADP was threefold higher in the diaphragm of patients with COPD than in the diaphragm of control patients, suggesting a lower ADP sensibility of mitochondrial respiration and an increased role of creatine as stimulus for mitochondrial respiration in these patients. Although this value only approaches significance (p = 0.08) because of the limited number of patients included, this parallels the changes previously observed in the skeletal muscle of athletic as compared with sedentary subjects (14, 30) or in slow-oxidative as compared with fast-glycolytic muscles in animals (15). Accordingly, these changes in Km between patients with COPD and control patients may not only reflect differences in mitochondrial sensitivity to cytosolic ADP but also modifications in regulation of energy transfers toward a more coupled scheme and changes in cellular architecture and/or compartimentalization (28).

The basal respiration of inspiratory muscles, Vo, remained unchanged in patients with COPD. Because basal respiration results from the mitochondrial transmembrane proton leak and from the number of mitochondria, we expected the increase in mitochondrial density to result in an increase of basal respiration in the diaphragm of severe hyperinflated patients. Instead, maximal respiration increased in both muscles of the COPD group with no change in basal respiration, resulting in a significant increase of the coupling between oxidation and phosphorylation for both the diaphragm and the external intercostal muscles. A similar increase in maximal respiration without changes in basal respiration was reported by our group (14) in the m. vastus lateralis of normal subjects with a high level of physical activity. This suggests that not only oxidative capacity but also efficiency of the respiratory chain participate in the increased energy production for increased muscle activity. This efficiency may result from a change in the permeability of the inner mitochondrial membrane due to alterations in phospholipid fatty acid composition as shown after regular exercise training (31, 32), decreasing proton leak and thus contributing to the unchanged basal respiration found in highly active subjects despite increased mitochondrial density. It was suggested that long-term training was probably necessary to significantly improve mitochondrial efficiency (14). We believe that this explanation might also apply to inspiratory muscles of patients with COPD because the increased burden put on these muscles is long-lasting and increases with time.

Limitations of the Study
Thoracotomy allows us to obtain in vivo biopsies of the diaphragm and is the most used method in previous works concerning the structure of this muscle (4, 5, 9). However, in our study, it introduces possible bias such as comorbidity factors potentially associated with disseminate cancer; we tried to minimize these factors by carefully selecting our control patients. In the same way, underweight and oral steroid treatment in some of our patients with COPD might have affected the phenotype alterations of respiratory muscles (33, 34). It is noticeable, however, that patients with a low body mass index or those treated with steroids presented values of maximal respiration and oxidative enzymes above the normal range of the group mean value. These findings suggest that the oxidative capacity may not have been influenced by the weight loss or steroid treatment. Finally, we were not able to relate our results on mitochondrial function to respiratory muscle endurance indexes. Despite the recent description of a noninvasive method (35), inspiratory muscle endurance testing is not yet standardized or accepted on a broad basis (36).

In conclusion, the increase in functional demand on the inspiratory muscles of patients with severe COPD is associated with an increase in the maximal rate of mitochondrial respiration, increased efficiency of the electron transport chain for ATP production of these muscles, and increased transfer of energy from production to use through the creatine kinase pathway. These results expand previous findings demonstrating increases in indirect indexes and provide evidence for an increased oxidative capacity in the inspiratory muscles of patients with severe COPD. This response strongly correlates with functional parameters of obstruction and hyperinflation, suggesting a long-lasting training-like effect of these muscles.


   Acknowledgments
 
The authors thank Eric Marchand and Vladimir Veksler for extensive help and advice and Jean Lonsdorfer, Gilbert Massard, Xavier Ducrocq and Jean Marie Wihlm for their continuous support.


   FOOTNOTES
 
Supported by grants from Institut National de la Santé et de la Recherche Médicale; R.V. was supported by the Centre National de la Recherche Scientifique.

Received in original form June 6, 2002; accepted in final form December 13, 2002


   REFERENCES
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
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