The present study was aimed at evaluating the effects of a specific inspiratory muscle training protocol on the structure of inspiratory muscles in patients with chronic obstructive pulmonary disease. Fourteen patients (males, FEV1, 24 ± 7% predicted) were randomized to either inspiratory muscle or sham training groups. Supervised breathing using a threshold inspiratory device was performed 30 minutes per day, five times a week, for 5 consecutive weeks. The inspiratory training group was subjected to inspiratory loading equivalent to 40 to 50% of their maximal inspiratory pressure. Biopsies from external intercostal muscles and vastus lateralis (control muscle) were taken before and after the training period. Muscle samples were processed for morphometric analyses using monoclonal antibodies against myosin heavy chain isoforms I and II. Increases in both the strength and endurance of the inspiratory muscles were observed in the inspiratory training group. This improvement was associated with increases in the proportion of type I fibers (by approximately 38%, p < 0.05) and in the size of type II fibers (by approximately 21%, p < 0.05) in the external intercostal muscles. No changes were observed in the control muscle. The study demonstrates that inspiratory training induces a specific functional improvement of the inspiratory muscles and adaptive changes in the structure of external intercostal muscles.
Chronic obstructive pulmonary disease (COPD) is characterized by a long natural history and elevated costs for health care services (1). Depending on the individual, patients with COPD present diverse degrees of dyspnea and deterioration in exercise capacity in association with impaired pulmonary and cardiovascular functions (2). Weakness and deconditioning of respiratory and peripheral muscles are currently recognized in these patients as additional factors implicated in the reduction of exercise capacity as well as in the quality of life (3). The function of inspiratory muscles is frequently found to be impaired (decreased strength and/or endurance) in patients with COPD (4). Inspiratory muscle dysfunction appears to be the result of geometric changes of the thorax, systemic factors, and/or potential structural changes of the inspiratory muscles (5, 6). It is probable that inspiratory muscle dysfunction does not limit minimal ventilatory needs at rest, but it does appear to contribute to dyspnea, decreased exercise capacity, and ventilatory failure during exacerbations (7). For these reasons, specific inspiratory muscle training could be justified as a strategy with potential clinical benefits in patients with COPD who remain symptomatic, despite optimal therapy (8). Although controversy still exists, several studies in healthy individuals and patients with COPD have demonstrated that inspiratory muscle training can increase the strength and endurance of inspiratory muscles (9, 10). This functional improvement is observed only when specific inspiratory muscle training is performed but not when general exercise programs are applied (8). On the basis of previous experimental studies (11, 12), the authors hypothesized that inspiratory muscle training could be associated with adaptive changes within the structure of inspiratory muscles. Consequently, the present study was aimed at evaluating the effects of a specific, short-term inspiratory muscle training protocol on the structural characteristics of the external intercostal muscles in patients with severe COPD. Using an outpatient biopsy model, samples from the external intercostal muscles were taken before and after 5 weeks of a supervised training period.
This was a randomized, placebo-controlled trial conducted in accordance with World Medical Association guidelines for research on humans. Our institutional ethics board approved all protocols, and all the patients gave informed consent before participating in the study. Fourteen community-based patients with COPD (males, 66 ± 5 years) were selected for the study from a hospital respiratory clinic. The COPD diagnosis was determined from a clinical history consistent with chronic bronchitis and/or emphysema, a long history of cigarette smoking, and pulmonary function tests revealing fixed airflow obstruction (FEV1 < 75% predicted, and FEV1/FVC ratio < 65%) (13). Subjects were sedentary and were observed during a 4-week period while their regular treatment was maintained to evaluate functional status and to verify clinical stability. Patients showing severe hypoxemia (i.e., PaO2 lower than 60 mm Hg breathing room air); asthma; coronary disease; undernourishment (body mass index < 20 kg/m2); chronic metabolic diseases (e.g., diabetes, hypo- or hyperthyroidism); orthopedic diseases; previous abdominal or thoracic surgery; or treatment with steroids, hormones, or cancer chemotherapy were not considered eligible for the study. The patient's characteristics are summarized in Table 1
Sham Training Group
Inspiratory Training Group
|Age, yr||66 ± 6||65 ± 5|
|Body mass index, kg/m2||26 ± 4||29 ± 4|
|Cholesterol, mg%||229 ± 37||235 ± 50|
|Serum proteins, gr%||7.5 ± 3||7 ± 0.8|
|Albumin, gr%||4.5 ± 0.2||4.3 ± 0.4|
|Prothrombin consumption time, s||105 ± 12||104 ± 7|
Nutritional assessment, pulmonary function tests, inspiratory and expiratory muscle strength, and transdiaphragmatic pressure (14) during both quiet breathing and maximal sniff maneuvers were measured in each patient and compared with reference values (15–19), as described elsewhere (20). The inspiratory muscle pressure–time index (PTI) while the patient was seated during tidal breathing was calculated according to the equation PTI = (Pes/Pesmax) · (TI/TTOT), where Pes is the esophageal pressure, Pesmax is the esophageal pressure during sniff maneuvers, and TI/TTOT is the duty cycle.
Exercise capacity was evaluated using two tests. First, a submaximal exercise test was performed to assess the distance the patient was able to walk in 6 minutes along a measured flat corridor as described by Butland and coworkers (21). Second, maximal exercise capacity was evaluated during an incremental exercise test to volitional exhaustion on a cyclergometer referenced to values from Jones (22)
Specific inspiratory muscle endurance was assessed during two different threshold inspiratory loading tests performed using a device that was similar to the one described by Nickerson and Keens (23). In the first loading test, the volunteers breathed against incremental loads (∼ 8 cm H2O every 2 minutes) until maximal sustainable threshold pressure was reached (Pthmax) (24). In the second loading test, subjects breathed against a submaximal constant load (equivalent to 80% of maximal threshold pressure) until exhaustion. The elapsed time was defined as the inspiratory sustainable threshold endurance time (Tth80, in minutes).
Biopsies from the external intercostal (inspiratory muscle) and vastus lateralis (control) muscles were taken before and after the inspiratory muscle training period (25). The pretraining biopsies were taken from the nondominant or dominant side, as previously randomized. Biopsies from the external intercostal muscles were taken along the anterior axillary line at the sixth intercostal space. Biopsies from the vastus lateralis were taken from the middle portion of the thigh. The post-training biopsies were obtained from the same anatomical site but from the contralateral side. The size of the muscle samples was approximately 5 × 5 × 5 mm. Each biopsy was quick-frozen in isopentane cooled in liquid nitrogen and stored at −70°C. Ten-micrometer thick sections were cut, varying the inclination of the holder by 5° increments until the minimum cross-sectional area was obtained, which was defined as truly transverse (26, 27). Consecutive cross-sections were processed by immunohistochemical techniques using monoclonal antibodies directed against myosin heavy chain (MyHC) isoforms type I and type II (Figure 1)(MHCs and MHCf clones; Biogenesis, New Fields, Poole, UK). The cross-sectional area, mean least diameter, and proportions of type I and -II fibers were assessed using a light microscope (OLYMPUS, Series BX50F3; Olympus Optical Co., Hamburg, Germany) coupled with an image-digitizing camera (PIXERA STUDIO, Version 1.2; Pixera Corporation, Los Gatos, CA) and a morphometry program (NIH IMAGE, Version 1.60). At least 100 fibers were measured from each biopsy (26). Fiber diameters between 40 and 80 μm were considered normal (28, 29).
The subjects received either inspiratory muscle training or sham training for 30 minutes, breathing through a threshold inspiratory device 5 days a week for 5 consecutive weeks. Appropriate personnel supervised training sessions. Loaded breathing was intermittent for 3-minute periods, with a 2-minute rest period in between. The patients breathed against a load equivalent to 60% of their maximal sustainable inspiratory pressure (which represented approximately 40–50% of the initial PImax). The load could be increased depending on patient tolerance. Patients who received sham training breathed through the same inspiratory muscle training device with no additional load.
Values throughout the text and tables are expressed as mean ± SD. Baseline and post-training data were compared within groups using the nonparametric (Wilcoxon) tests for paired samples. The Mann–Whitney U-test was used to compare data between groups. An α (p) value less than or equal to 0.05 was considered statistically significant.
Sixteen patients were initially recruited for the study. Two of them were excluded, one due to pulmonary infection at the beginning of the training period and the other due to noncompliance with the training dates. Complications derived from either the functional evaluations or muscle sampling were not detected in any patient. The general characteristics of the study population are shown in Table 1. Changes in conventional pulmonary function tests (Table 2)
Sham Training Group
Inspiratory Training Group
|FEV1, l||836 ± 184||913 ± 185||974 ± 312||997 ± 341|
|FEV1, % pred||27 ± 7||29 ± 7||33 ± 8||34 ± 11|
|TLC, l||6.8 ± 7.8||6.7 ± 8.9||6.3 ± 1.5||6.2 ± 1.3|
|TLC, % pred||115 ± 7||115 ± 17||112 ± 22||111 ± 19|
|RV, % pred||190 ± 18||179 ± 32||177 ± 46||179 ± 52|
|PaO2, torr||68 ± 5||66 ± 5||68 ± 7||72 ± 10|
|PaCO2, torr||47 ± 5||46 ± 4||43 ± 7||44 ± 4|
Sham Training Group
Inspiratory Training Group
|Inspiratory muscle strength|
|PImax, cm H2O||77 ± 9||79 ± 10||77 ± 22||99 ± 22*|
|PImax, % pred||74 ± 7||76 ± 7||69 ± 19||90 ± 20*|
|Pesmax, cm H2O||−55 ± 17||−58 ± 16||−49 ± 16||−74 ± 19*|
|Pdimax, cm H2O||91 ± 24||90 ± 13||74 ± 19||110 ± 23*|
|PTI, index (at rest)||0.06 ± 0.03||0.04 ± 0.04||0.07 ± 0.02||0.04 ± 0.01|
|Inspiratory muscle endurance|
|Pthmax, cm H2O||−39 ± 21||−41 ± 20||−30 ± 19||−39 ± 22*|
|Tth80, min||9.3 ± 4||9.2 ± 2||11 ± 6||22 ± 6*|
|Expiratory muscle strength|
|PEmax, cm H2O||147 ± 31||145 ± 28||146 ± 24||144 ± 30|
|PEmax, % pred||77 ± 8||76 ± 12||82 ± 13||81 ± 16|
|Six minute walking test|
|Distance, m||429 ± 115||407 ± 114||445 ± 63||433 ± 81|
|Incremental cycle test|
|Work rate, Wattmax||91 ± 25||82 ± 23||79 ± 17||86 ± 23|
|VO2max, ml/kg/min||14 ± 2||12 ± 2||16 ± 3||15 ± 5|
The overall function of the diaphragm and accessory inspiratory muscles showed significant changes after training in the inspiratory muscle training group (Figure 2) as expressed by an increase, first, in the inspiratory muscle strength (PImax, Pesmax, and Pdimax), and second, in the inspiratory muscle endurance (Table 3). Neither the breathing pattern nor the strength of the expiratory muscles (PEmax) showed differences after training (Table 3).
Before training, the size of the external intercostal fibers was found to be 47 ± 8 μm, and a total of 46 ± 18% of the fibers expressed type I MyHC. The inspiratory muscle training clearly associated with structural changes in the muscle, as assessed by both fiber type distributions and fiber size. Specifically, both the proportion of type I fibers (p < 0.05) and the size of type II fibers (p < 0.05) increased after training (Table 4
Sham Training Group
Inspiratory Training Group
|Global fiber size|
|Least diameter, μm||47 ± 9||51 ± 10||47 ± 8||55 ± 6 p=0.09|
|CSA, μm2||3.08 ± 1.25||3.27 ± 1.28||2.73 ± 0.81||3.88 ± 0.48*|
|Type I fibers|
|Proportion, %||50 ± 14||47 ± 16||42 ± 20||58 ± 14*|
|CSA, μm2 × 103||2.60 ± 0.94||2.94 ± 1.24||2.92 ± 1.39||3.72 ± 0.68 p=0.06|
|Least diameter, μm||44 ± 7||49 ± 9||49 ± 10||55 ± 10|
|SD, μm||6 ± 1||7 ± 1||8 ± 1||10 ± 3|
|Type II fibers|
|Proportion, %||50 ± 14||53 ± 16||57 ± 20||42 ± 14*|
|CSA, μm2 × 103||3.67 ± 1.47||3.58 ± 1.32||2.82 ± 0.91||4.06 ± 0.86*|
|Least diameter, μm||50 ± 12||53 ± 11||47 ± 10||57 ± 8*|
|SD, μm||7 ± 2||8 ± 1||9 ± 2||11 ± 2 |
Before training, the mean size of vastus lateralis fibers was found to be 57 ± 10 μm. A total of 30 ± 11% of the fibers expressed type I MyHC. The inspiratory muscle training did not promote structural changes within the vastus lateralis, as assessed by fiber size or fiber type distributions (see Table E1 in the online data supplement).
The present study is the first to evaluate structural changes in the respiratory muscles of patients with COPD after a specific program of respiratory muscle training. Significant increases were observed both in the proportion of type I fibers (by about 38%) and in the size of type II fibers (by ≅ 21%) of the external intercostal muscles after the training period. These findings demonstrate that the external intercostal muscles of patients with severe COPD have the capacity to express structural remodeling. Functional improvement induced by the inspiratory muscle training (in terms of both inspiratory muscle strength and endurance) could be explained in part by structural adaptation within the inspiratory muscles.
The present study was aimed at evaluating the effects of a specific inspiratory muscle training on the structure of inspiratory muscles in community-based patients with COPD. This could be controversial regarding the current state of respiratory muscle adaptations. In fact, the diaphragm of nontrained patients with COPD exhibit structural changes that presumably represent “adaptive effects.” Studies of Levine and coworkers (30), Mercadier and coworkers (31), and one study of our group (32) demonstrate that the diaphragm shows an increase of type I fibers, MyHC-I, and mitochondria (supporting a fiber type transformation), whereas the length of the sarcomere decreases (supporting adaptation to chronic diaphragm flattening) (32). A reasonable presumption is that other primary respiratory muscles (such as the external intercostals) could exhibit similar adaptations. From a clinical point of view, however, such structural adaptations only appear to partially restore their functional capacity. In fact, inspiratory muscles of patients with COPD show a lower capacity to generate maximal pressures (strength) or tolerate submaximal inspiratory loads (endurance) (4). This muscle dysfunction could be explained by the coexistence of other factors (33–35) with deleterious effects such as (1) geometric changes of the thorax due to increased lung volume and shortening of the diaphragm fibers; (2) intrinsic changes within the muscles due to malnutrition, ionic, or arterial gas disorders; or (3) the effect of drugs or concomitant diseases or conditions (e.g., aging). Altogether, these arguments have prompted several investigators to hypothesize that inspiratory muscle training could offer clinical benefits to patients with severe COPD. However, the results of previous studies have been controversial.
Some authors have demonstrated that inspiratory muscle training may have a more general impact when tolerance is evaluated in terms of exercise capacity, endurance time on a treadmill, or dyspnea (10, 36–39). However, other studies (40–43), including the present one, were unable to show any changes in either walking distance or maximal oxygen uptake.
Whereas some studies have reported that inspiratory muscle strength and endurance can improve with specific training (10, 43), others have not found significant changes in inspiratory muscle function (44). The present results are consistent with the former because inspiratory muscle strength as well as inspiratory endurance were significantly increased after inspiratory muscle training. The differences in previous studies regarding the effects of inspiratory training could be related to the differences either in the magnitude or in the duration of inspiratory muscle loading (45). Taking this into consideration, specific inspiratory muscle training has been found to be capable of improving inspiratory muscle function when intensity is monitored and exceeds 20% of PImax (46, 47). In addition, some studies have simultaneously included multidimensional intervention as a part of the rehabilitation of patients with COPD. From a methodologic point of view, such an assessment could make it difficult to independently analyze the specific effect of inspiratory muscle training.
The size of external intercostal fibers was found to be within the normal range before specific training (48). The most important finding of the present study is that inspiratory muscle training induced structural changes within the trained muscles of stable patients with COPD. A few experimental studies have shown that the diaphragm and other inspiratory muscles show structural changes in animals submitted to inspiratory loads (11). However, the authors have been unable to find any study evaluating structural changes in human inspiratory muscles after specific training.
The findings of the present study highlight three main concepts. First, muscle response; the study demonstrates that external intercostal muscles of patients with COPD retain the capacity to be remodeled (conditioned) after a short-term loading period. The muscles exhibited a classical response to training that would be predictable in limb muscles. Similar findings have been described in the peripheral muscles of patients with COPD after general muscle training (49). The novelty of the present report is that this response is demonstrated in an inspiratory muscle group from patients with severe airflow obstruction. Second, functional and structural changes; the results allow us to hypothesize that the increase in inspiratory muscle endurance and strength after specific training could be related to the observed switching of MyHC isoforms (as assessed by the increase in fibers expressing MyHC-I) and to the increase in fiber size (mainly in type II fibers). Other factors such as perceptual adaptation to additional inspiratory loading (e.g., dyspnea desensitization), learning of specific maneuvers, or even a placebo effect could also have participated in improving inspiratory muscle strength and endurance. And third, specificity; we found that inspiratory muscle training had specific functional and structural effects only on the trained (inspiratory) muscles. The study included data from an unaffected (limb) muscle as a negative control. No changes were observed either in the fiber size or in fiber type proportions of the vastus lateralis (control muscle) when results before and after training were compared. Similarly, a transfer effect to other respiratory muscle groups (e.g., changes in function of the expiratory muscles) was not found. These facts support the conclusion that inspiratory training has a specific effect only on the trained muscles and allow us to hypothesize that structural adaptation occurred only in the inspiratory muscles. However, the design of the study does not permit total confidence in the structural changes within the external intercostals are representative of inspiratory muscles in general. The diaphragm is the most important of the inspiratory muscles, but there are obvious ethical and practical difficulties in repeatedly accessing the diaphragm either from healthy subjects or from stable patients with COPD, even if thoracotomy was performed for other reasons (e.g., lung volume reduction, lung cancer, or transplant).
The principal limitations of the present study are the relatively small number of subjects studied and the possibility that the results could be related to regional differences in the external intercostal muscles within a given volunteer. The authors believed that a greater number of volunteers was not essential because the results reached statistical significance from both structural and functional points of view, and the study that used relatively high invasive procedures. The results do not appear to be related to regional differences within the external intercostal muscles. A previous study demonstrated that biopsies from the external intercostal muscles taken from different anatomic regions are comparable in patients with COPD, even when the samples were taken either from the dominant or from the nondominant side (50). In addition, we believe that anatomical knowledge and experience allow us to ensure that muscle samples came from the external intercostals in all cases. The experience is the result of both anatomical reviews and practical evidence. At least nine arguments should be highlighted: (1) the technique is an “open” biopsy, in contrast to “blind” needle techniques; (2) we have reviewed and we have enough knowledge of the specific body zone; (3) we have practiced in cadaver models; (4) we normalized the body location for the muscle biopsies; (5) we performed a sequential and careful dissection technique with identification of all tissue plans (fascia, muscle); (6) we discarded a potential error by taking biopsies from the pectoralis because the “anatomical window” to access the external intercostals is below and relatively far from the lower insertions of the pectoralis muscles; (7) the biopsy was taken from the intercostal space; (8) we identified the orientation of the muscle fibers that is clearly different for external and internal intercostals; and (9) due to intimate contact between internal intercostals and the pleura, the absence of yatrogenic pneumo-thorax confirms that such muscles were not sampled.
This is the first report demonstrating that structural changes occur in respiratory muscles of patients with COPD after specific training, in association with improvement in the inspiratory muscle function. The authors believe that this evidence offers additional information, improves the knowledge of the physiologic basis of inspiratory muscle training, and reinforces the inclusion of inspiratory muscle training as a part of pulmonary rehabilitation. The results are innovative and therefore could not have been directly extrapolated either from previous animal studies (because many factors present in COPD do not exist in animal models) or from previous studies assessing the effects of peripheral muscle training (because pretraining phenotype is completely different from that reported for the respiratory muscles). Our selection criteria excluded patients showing malnutrition, respiratory failure, hypercapnia, or systemic steroid treatment, which are features usually found in patients with COPD. However, these conditions would introduce confounding factors to the study. Further studies evaluating the potential effect of inspiratory muscle training in patients with these associated conditions, the time course of the muscle remodeling process, and the minimal external loading capable of muscle adaptation seem to be warranted.
The present study demonstrates that the external intercostal muscles of patients with COPD have the capacity to express structural remodeling after specific inspiratory training. Both the proportion of type I fibers and the size of type II fibers were found to increase after training. These structural adaptations could explain, in part, the functional improvements observed in the trained muscles (increased inspiratory muscle strength and endurance) after specific training.
The authors gratefully acknowledge Professor J. Sanchís for contributing leadership and valuable suggestions; Drs. I. Solanes and M. Brufal for participating in some functional studies; R. Méndez and J. Palacio for technical assistance; and H. Lock for editing aid.
|1.||Speizer FE. The rise in chronic obstructive pulmonary disease: overview and summary. Am Rev Respir Dis 1989;140:S106–S107.|
|2.||Killian KJ, Leblanc P, Martin DH, Summers E, Jones NL, Campbell EJM. Exercise capacity and ventilatory, circulatory, and symptom limitation in patients with chronic airflow limitation. Am Rec Respir Dis 1992;146:935–940.|
|3.||Hamilton N, Killian KJ, Summers E, Jones NL. Muscle strength, symptom intensity, and exercise capacity in patients with cardiorespiratory disorders. Am J Respir Crit Care Med 1995;152:2021–2031.|
|4.||Rochester DF. The respiratory muscles in COPD: state of the art. Chest 1984;85:47–50.|
|5.||American Thoracic Society–European Respiratory Society. Skeletal muscle dysfunction in chronic obstructive pulmonary disease. Am J Respir Crit Care Med 1999;159:S1–S28.|
|6.||Arora NS, Rochester DF. Effect of chronic obstructive pulmonary disease in diaphragm muscle dimensions. Am Rev Respir Dis 1981;123:176–184.|
|7.||Begin P, Grassino A. Inspiratory muscle dysfunction and chronic hypercapnia in chronic obstructive pulmonary disease. Am Rev Respir Dis 1991;143:905–912.|
|8.||ACCP/AACVPR Pulmonary Rehabilitation Guidelines Panel. Pulmonary rehabilitation: joint ACCP/AACVPR evidence-based guidelines. Chest 1997;112:1363–1396.|
|9.||Leith DE, Bradley M. Ventilatory muscle strength and endurance training. J Appl Physiol 1976;41:508–516.|
|10.||Weiner P, Magadle Rasmi, Berar-Yanay R, Berar-Yanay N, Davidovich A, Weiner M. The cumulative effect of long-acting broncodilators, exercise, and inspiratory muscle training in patients with advanced COPD. Chest 2000;118:672–678.|
|11.||Gea J, Hamid Q, Czaika G, Zhu E, Mohan-Ram V, Goldspink G, Grassino A. Expression of myosin heavy-chain isoforms in the respiratory muscles following inspiratory resistive breathing. Am J Respir Crit Care Med 2000;161:1274–1278.|
|12.||Bisschop A, Gayan-Ramírez G, Rollier H, Gooselink R, Dom R, de Bock V, Decramer M. Intermittent inspiratory muscle training induces fiber hypertrophy in rat diaphragm. Am J Respir Crit Care Med 1997;155:1583–1589.|
|13.||American Thoracic Society. Standard for the diagnosis and care of patients with COPD. Am J Respir Crit Care Med 1995;152:s78–s121.|
|14.||Aran X, Gea J, Guiu R, Aguar MC, Sauleda J, Broquetas JM. Comparison between three different maneouvres for achieving maximal transdiapragmatic pressures. Arch Bronconeumol 1992;28:112–115.|
|15.||Feliffe DB. The assessment of the nutritional status in the community. Geneva, Switzerland: WHO; 1996.|
|16.||Roca J, Sanchís J, Agustí-Vidal A, Rodriguez-Roisin R. Spirometric reference values for a Mediterranean population. Bull Eur Physiopathol Respir 1986;22:217–224.|
|17.||Roca J, Burgos F, Barbera JA, Sunyer J, Rodriguez-Roisin R, Castellsague J, Sanchís J, Anto JM, Casan P, Clausen JL. Prediction equations for plethysmographic lung volumes. Respir Med 1998;92:454–460.|
|18.||Morales P, Sanchís J, Cordero PJ, Diez JL. Maximum static respiratory pressures in adults: the reference values for a Mediterranean Caucasian population. Arch Bronconeumol 1997;33:213–219.|
|19.||Roca J, Rodríguez-Roisin R, Cobo E, Burgos F, Pérez J, Clausen JL. Single breath carbon monoxide diffusing capacity (DLCO) prediction equations for a Mediterranean population. Am Rev Respir Dis 1990;141:1026–1032.|
|20.||Orozco-Levi M, Lloreta J, Minguella J, Serrano S, Broquetas JM, Gea J. Injury of the human diaphragm associated with exertion and chronic obstructive pulmonary disease. Am J Respir Crit Care Med 2001;164:1734–1739.|
|21.||Butland RJA, Pang J, Gross ER, Woodcock AA, Geddes DM. Two, six and 12 minute walking tests in respiratory disease. BMJ 1982;284:1607–1608.|
|22.||Jones NL. Clinical exercise testing, 3rd ed. Philadelphia: W.B. Saunders; 1988.|
|23.||Nickerson BG, Keens TG. Measuring ventilatory muscle endurance in humans as sustainable inspiratory pressure. J Appl Physiol 1982;52:768–772.|
|24.||Martyn JB, Brown RH, Pare PD, Pardy RL. Measurement of inspiratory muscle performance with incremental threshold loading. Am Rev Respir Dis 1987;135:919–923.|
|25.||Gea J, Pastó M, Carmona MA, Orozco-Levi M, Palomeque J, Broquetas J. Metabolic characteristics of the deltoid muscle in patients with chronic obstructive pulmonary disease. Eur Respir J 2001;17:1–7.|
|26.||Dubowitz V, Brooke MH. Muscle biopsy: a modern approach. In: Dubowitz V, Brooke MH, editors. Major problems in neurology. London: Saunders; 1973. p. 74–85.|
|27.||Poole DC, Lieber RL, Mathieu-Costello O. Myosin and actin filaments length in diaphragms from emphysematous hamsters. J Appl Physiol 1994;76:1220–1225.|
|28.||Brooke MH, Engel WK. The hystographic analysis of human biopsies with regard to fiber types. 1: adult male and female. Neurology 1969;19:221–233.|
|29.||Hards JM, Reid WD, Pardy RL, Pare PD. Respiratory muscle morphometry: correlation with lung function and nutrition. Chest 1990;97:1037–1044.|
|30.||Levine F, Kaiser L, Leferovich J, Tikunov B. Cellular adaptation in the diaphragm in chronic obstructive pulmonary disease. N Engl J Med 1997;337:1799–1806.|
|31.||Mercadier JJ, Schwartz K, Schiaffino S, Wisnewsky C, Ausoni S, Heimburger M, Marrash R, Pariente R, Aubier M. Myosin heavy chain gene expression changes in the diaphragm of patients with chronic lung hyperinflation. Am J Physiol 1998;274(4 Pt 1):L527–L534.|
|32.||Orozco-Levi M, Gea J, Lloreta JL, Felez M, Minguella J, Serrano S, Broquetas JM. Subcellular adaptation of the human diaphragm in chronic obstructive pulmonary disease. Eur Respir J 1999;13:371–378.|
|33.||Arora NS, Rochester DF. Effect of chronic obstructive pulmonary disease in diaphragm muscle dimensions. Am Rev Respir Dis 1981;123:176–184.|
|34.||American Thoracic Society–European Respiratory Society. Skeletal muscle dysfunction in chronic obstructive pulmonary disease. Am J Respir Crit Care Med 1999;159:S1–S28.|
|35.||Hards JM, Reid WD, Pardy RL, Pare PD. Respiratory muscle fiber morphometry: correlation with pulmonary function and nutrition. Chest 1990;97:1037–1044.|
|36.||Dekuitjzen PR, Folgering HT, Van Herwaarden CL. Target-flow inspiratory muscle training during pulmonary rehabilitation in patients with COPD. Chest 1991;99:128–133.|
|37.||Larson JL, Kim MJ, Sharp JT, Larson DA. Inspiratory muscle training with a pressure threshold breathing device in patients with COPD. Am Rev Respir Dis 1988;138:689–696.|
|38.||Weiner P, Azgad Y, Ganam R. Inspiratory muscle training combined with general exercise in patients with COPD. Chest 1992;102:1351–1356.|
|39.||Lisboa C, Muñoz V, Beroiza T, Leiva A, Cruz E. Inspiratory muscle training in chronic airflow limitation: comparison of two different training loads with a threshold device. Eur Respir J 1994;7:1266–1274.|
|40.||Noseda A, Carpieux JP, Vandeput W, Prigogine T, Schmerber J. Resistive inspiratory muscle training and exercise performance in COPD patients: a comparative study with conventional breathing training. Bull Eur Respir Physiopathol Respir 1987;23:457–463.|
|41.||Flynn MG, Barter CE, Nosworthy JC, Pretto JJ, Rockford PD, Pierce RJ. Threshold pressure training, breathing pattern, and exercise performance in chronic airflow obstruction. Chest 1989;95:535–540.|
|42.||Madsen F, Secher NH, Kay L, Kok-Jensen A, Rube N. Inspiratory resistance versus general physical training in patients with chronic obstructive pulmonary disease. Am Rev Respir Dis 1985;67:167–176.|
|43.||Belman MJ, Shadmehr R. Targeted resistive ventilatory muscle training in chronic obstructive pulmonary disease. J Appl Physiol 1988;65:2726–2735.|
|44.||Guyatt G, Keller J, Singer J, Halcrow S, Newhouse M. Controlled trial of respiratory muscle training in chronic airflow limitation. Thorax 1992;47:598–602.|
|45.||Donner CF, Muir JF. Selection criteria and programmes for pulmonary rehabilitation in COPD patients rehabilitation and chronic care scientific group of the European Respiratory Society. Eur Respir J 1997;10:744–757.|
|46.||Belman MJ, Shadmehr R. Targeted resistive ventilatory muscle training in chronic pulmonary disease. J Appl Physiol 1988;65:2726–2735.|
|47.||Heijdra YF, Dekhuijzen PNR, Van Herwaarden CLA, Folgering HTM. Nocturnal saturation improves by target-flow inspiratory muscle training in patients with COPD. Am J Respir Crit Care Med 1996;153:260–265.|
|48.||Sauleda J, Gea J, Orozco-Levi M, Corominas J, Minguella J, Aguar C, Broquetas J, Agusti AGN. Structure and function relationships of the respiratory muscles. Eur Respir J 1998;11:906–911.|
|49.||Maltais F, Leblanc P, Simard C, Jobin J, Berube C, Bruneau J, Carrier L. Belleau: skeletal muscle adaptation to endurance training in patients with chronic obstructive pulmonary disease. Am J Respir Crit Care Med 1996;154:442–447.|
|50.||Jimenez-Fuentes MA, Gea J, Pallas O, Gallego F, Felez MA, Broquetas JM. Fiber morphometry of the external intercostal muscle: comparison of dominant and nondominat sides in patients with severe COPD. Arch Bronconeumol 1998;34:189–193.|