Preserved function and reduced angiogenesis potential of the quadriceps in patients with mild COPD

We studied 37 patients with GOLD 1 COPD [14] presenting a history of smoking (≥ 15 pack-years). Nineteen healthy age-matched subjects with normal spirometry and a history of smoking (≥ 15 pack-years) served as controls. None of them were involved in a previous study. Activity-related dyspnea was assessed by the Baseline Dyspnea Index (BDI) [15]. The research protocol was approved by the institutional ethics committee (Comité d’éthique, Institut Universitaire de cardiologie et de pneumologie de Québec, approval 20378) and all of the participants signed an informed consent prior to study enrolment. The sponsor (Boehringer Ingelheim and Pfizer) was not involved in the study design, data collection, analysis or interpretation. The sponsor had the opportunity to read and comment on the manuscript with no obligation for the authors to incorporate any suggestion into the final version.

Participants were characterized during a first visit with pulmonary function tests, and measurements of body composition, MTCSA, quadriceps muscle strength and endurance. Comorbidities were assessed using a structured questionnaire and by medical chart review. During this initial visit, they also performed an incremental shuttle walking test. Following ≥ 72-hrs, but within a week of the first visit, a needle biopsy of the quadriceps was performed and blood was sampled to measure systemic inflammatory biomarkers. Finally, participants had to wear a physical activity monitor for 6 to 8 days.

Standard pulmonary function tests, including lung volumes and diffusion capacity (DL_CO) were obtained according to previously described guidelines [16] and related to predicted normal values [17]. Similarly, spirometry was performed according to guidelines [16] and related to predicted normal values [18].

Computed tomography was performed in the supine position and MTCSA was determined halfway between the pubic symphysis and the inferior surface of the femoral condyle.

Needle biopsies of the quadriceps were performed after 30-min of immobilization as described by Bergström [22] and as routinely done in our laboratory [10]. Muscle specimens were immediately frozen in liquid nitrogen and stored at -80°C for future analysis. From these muscle samples, fiber typing and surface areas were determined. The cross-sectional area of each fiber-type was calculated based on 40 randomly selected fibres of each type [23]. Muscle capillarity and pro-angiogenesis factors (vascular endothelial growth factor A [VEGF-A], angiopoietin I [Ang-I], angiopoietin II [Ang-II]) were measured. Finally, quadriceps oxidative (citrate synthase [CS], hydroxyacyl-coenzyme A dehydrogenase [HADH]) and glycolytic (phosphofructokinase [PFK]) enzymatic activity was assessed as well as muscle oxidative stress. See Additional file 1 for more details. [Detailed methods used to analyze fiber typing, muscle surface area, muscle capillarity, enzymatic activity, real-time PCR and oxidative stress].

Plasma levels of systemic inflammatory markers (TNF-α, IL-6, CRP, Fibrinogen, SP-D) were measured from antecubital venous blood sample.

All variables are expressed as means ± SD. Comparisons between the two groups for baseline characteristics, muscle functional and morphometric properties, level of oxidative stress and systemic inflammation were performed using unpaired t-test. The homogeneity of variances was analysed with the Brown-Forsythe test. Multiple regression analyses were performed to identify potential determinants of quadriceps MVC, oxidative capacity (CS activity) and capillary to fiber ratio. Independent variables that were used in this model included age, sex, smoking history (pack-years), fat-free mass index (FFMI), forced expiratory flow in 1 s (FEV₁) % predicted, forced vital capacity (FVC) % predicted, total lung capacity (TLC) % predicted, functional residual capacity (FRC) % predicted, inspiratory capacity (IC) % predicted, residual volume (RV) % predicted, DL_CO % predicted, plasma IL-6 level, muscle oxidative stress indices, number of steps per day, energy expenditure > 3 METs and daily time > 3 METs. IL-6 was used in this model because it was the only inflammatory biomarker that was significantly increased in COPD. Beside this statistical consideration, IL-6 is the inflammatory biomarkers with the strongest association with important clinical outcomes such as mortality, in comparison with several other biomarkers, including TNF-α, CRP, fibrinogen and SP-D [24, 25]. A statistical level of significance of 0.05 was used for all analysis. The data were analyzed using the statistical package program JMP (Version 8.0.1, SAS Institute Inc., Cary, NC).

Characteristics of the participants are presented in Table 1. Patients with mild COPD displayed more activity-related dyspnea in addition to mildly increased lung volumes and decreased diffusion capacity. Total smoking exposure tended to be lower in controls but this did not reach statistical significance. Peak VO₂ was similar between the two groups. Comorbid conditions (hypertension, dyslipidemia, osteoporosis, anxiety) were similarly distributed between the two groups (Additional file 1) [Comorbidities for which participants were pharmacologically treated in patients with COPD and healthy controls].

No difference was found in MTCSA between patients with mild COPD and healthy controls (Figure 1A). Likewise, MVC, TwQ_pot and endurance were all similar between patients with mild COPD and controls (Figure 1B-D).

Plasmatic levels of IL-6 were significantly higher in patients with COPD and CRP levels tended to be higher (p = 0.08) when compared with healthy controls. On the opposite, plasmatic levels of fibrinogen were higher in healthy controls (Table 3).

Levels of physical activities in daily life are reported in Figure 3. Patients with COPD had similar daily step count when compared with healthy controls. However, when accounting for the intensity of daily life activities, patients with mild COPD showed a decreased amount of at least moderate intensity (> 3 METs) physical activities in daily life. Accordingly, mean daily time dedicated to at least moderate intensity daily activities was significantly reduced in patients with mild COPD.

In multiple regression analysis, quadriceps MVC was positively associated with male gender (p = 0.03) and FFMI (p = 0.003) and negatively with FRC (p = 0.002). Altogether the model explained 73% of the variance in MVC. CS activity was positively associated with energy expenditure > 3 METs (p < 0.001) and daily time > 3 METs (p = 0.007) with a total r² = 0.53. The capillary-to-fiber ratio was positively associated with number of steps per day (p = 0.045), energy expenditure > 3 METs (p < 0.001) and daily time > 3 METs (p = 0.007) with a total r² = 0.49. Plasma IL-6 levels and indices of muscle oxidative stress did not emerge as significant determinants of quadriceps MVC, oxidative capacity and capillarization.

Reduced muscle strength and endurance were previously reported in patients with GOLD 1 and 2 COPD [26]. One asset of the present study is that only patients with GOLD 1 COPD were enrolled, allowing to conclude about the preservation of muscle mass, strength and endurance in this specific patient population.

Our data suggests that fiber type shifting and metabolic alterations are not early events in the process leading to limb muscle dysfunction in COPD. This finding is consistent with the fact that the fiber type shifting of the quadriceps is associated with COPD severity in patients with GOLD 3 and 4 COPD [27, 28]. Furthermore, the proportion of type I fibers in our patients was well beyond the proposed pathological threshold of 27% [27], highlighting that muscle fiber type shifting occurs in more advanced disease. Lastly, evidence of reduced oxidative enzyme activities were not found in another study involving patients with mild to moderate COPD [29].

Our results about the preservation of muscle capillarization in mild COPD are consistent with a recent study reporting a similar number of capillaries in contact with type I and II muscle fibers between patients with mild COPD and healthy controls in the tibialis anterior[30]. Despite this, a decreased mRNA expression level of pro-angiogenesis factors was found in COPD. The reduced quadriceps VEGF-A mRNA expression is consistent with a previous report [31]. In contrast to our findings, elevated angiopoietin II mRNA expression level was previously reported in the quadriceps of patients with severe COPD [32]. A negative association between angiopoietin II mRNA expression and FEV₁ was also observed in this study [32] suggesting that the divergent conclusions reached about the quadriceps angiopoietin II expression could well be explained by differences in disease severity between study populations. Muscle angiogenesis is regulated by oxidative stress and inflammation [32], two features that are more likely to predominate in advanced COPD or during disease exacerbation. As such, the angiogenesis status of the quadriceps may differ according to disease severity.

Skeletal muscle angiogenesis is a highly regulated process involving several coordinated signaling pathways, including VEGF and the angiopoietin system whose activation precede and promote the formation of new capillaries [33]. Considering the influence of exercise on these key angiogenesis regulating pathways, reduced physical activity level emerges at a likely explanation for the reduction in angiogenic regulating pathways in patients with GOLD 1 COPD. In the absence of decreased capillarization, we could only speculate on the significance of the reduced pro-angiogenesis factors. One possibility is that biochemical regulation of the angiogenic process precedes the observable changes in capillarization [34]. This interpretation implies that patients with COPD could be vulnerable in situation where an increase capillarization would be required such as during hypoxemia. Alternatively, it can be submitted that muscle capillarization is fairly stable and relatively insensitive to the downregulation in angiogenic growth factors at this stage of the disease [35].

Similarly to other investigators [31, 36‐38], we found only equivoqual evidence of systemic inflammation in this population of mild COPD. Overall, our inflammatory biomarker data is consistent with the available literature in showing that IL-6 elevation is very consistent in COPD [38]. Our findings are also congruent with a previous study showing that TNF-α and surfactant-D protein (SP-D) levels are not increased in GOLD 2–4 COPD compared to smokers with normal lung function while fibrinogen and CRP levels are highly variable in COPD [38]. The finding of a reduced plasma fibrinogen level in GOLD 1 COPD was unexpected. Fibrinogen elevation was reported in a subset of the ECLIPSE cohort involving patients with GOLD 2–4 COPD, in comparison smokers with normal lung function [38]. However, significant overlap between COPD and controls exists in such a way most patients with COPD have normal fibrinogen levels [37]. Another point to consider in interpreting this finding is that our investigation involved patients with GOPD 1 COPD, for whom little information exists about the inflammatory status. Lastly, the relative small sample size of our investigation should be considered in interpreting the inflammatory biomarker data.

In the context that muscle oxidative stress was not present in patients with GOLD 1 COPD and because plasma IL-6 and indices of muscle oxidative stress were not associated with muscle function and capillarization, our results do not support the thesis that systemic inflammation or muscle oxidative stress initiate the establishment of limb muscle dysfunction associated with COPD.

Shrikrishna and colleagues reported reduced rectus femoris surface area and physical activity level in patients with GOLD 1 COPD in comparison to healthy controls [39]. Interestingly, and in contrast to the present study, the smoking exposure of their healthy controls was much lower than in COPD. This may suggest that smoking by itself may be associated with reduced muscle mass as previously suggested [40]. Altogether, these studies would support the idea that smoking exposure is important in the early development of limb muscle atrophy in COPD.

Our results provide further confirmation that the level of moderately intense physical activity is already low in GOLD I COPD [39, 41, 42]. The correlative findings between the level of moderately intense physical activity and quadriceps MVC and capillarity support a role for reduced physical activity in the initiation of limb muscle dysfunction in COPD. This is in line with recent evidences also pointing to the potential role of reduced physical activity level in the development of quadriceps weakness in COPD [43].

Although our results are based on a relatively limited sample size, they exclusively pertain to patients with mild (GOLD 1) COPD. Subjects were extensively characterized and the muscle profile was well defined. In fact, the levels of physical activity could be related to intrinsic and functional muscle properties, offering a unique opportunity to speculate on causal relationship in locomotors muscles alterations in early COPD. However, a definitive answer to this issue could only be answered by longitudinal studies evaluating the interactions between physical activity and limb muscle function over time.

Exercise training remains the most relevant strategy to reverse functional and metabolic impairments occurring in the skeletal muscle: it is known to partially reverse the loss of muscle cross-sectional area and improve muscle capillary density [44], irrespective of the disease severity when considering GOLD 1 to 4 patients [45]. Accordingly, our results provide support for the use of pulmonary rehabilitation in patients with GOLD 1 COPD as they already show subtle muscle abnormalities and reduced level of physical activity.

In conclusion the present study indicates that limb muscle function, fiber type distribution, enzymatic activities and capillarization was generally preserved in mild (GOLD 1) COPD. The level of muscle pro-angiogenesis factors was nevertheless reduced in these patients suggesting that pre-clinical muscle abnormalities were already present. Our findings also support a role for reduced physical activity in the initiation of limb muscle dysfunction in COPD.