Sex differences in COPD-related quadriceps muscle dysfunction and fibre abnormalities

In chronic obstructive pulmonary disease (COPD), lower limb dysfunction is associated with reduced exercise capacity, increased hospitalizations and mortality. We investigated sex differences in the prevalence of quadriceps dysfunction and fibre abnormalities in a large COPD cohort, controlling for the normal sex differences in health. We compared existing data from 76 male and 38 female COPD patients where each variable was expressed as a function of gender-specific normal values (obtained from 16 male and 14 female controls). Female COPD patients had lower quadriceps muscle strength and peak workload on a maximal incremental cycle ergometry protocol compared to male patients. Female patients had a smaller type II fibre cross-sectional area (CSA) compared to male patients, suggesting a greater female preponderance to fibre atrophy, although this result was largely driven by a few male patients with a large type II fibre CSA. Female patients had significantly higher concentrations of a number of plasma pro-inflammatory cytokines including tumour necrosis factor alpha and interleukin 8 (IL8), but not lower levels of physical activity or arterial oxygenation, compared to males. Our data confirm results from a previous small study and suggest that female COPD patients have a greater prevalence of muscle wasting and weakness. Larger studies investigating sex differences in COPD-related muscle atrophy and weakness are needed, as the results will have implications for monitoring in clinical practice and for design of clinical trials evaluating novel muscle anabolic agents.


Introduction
Chronic obstructive pulmonary disease (COPD) is a systemic disease. 1 Lower limb muscle atrophy (wasting), weakness and loss of oxidative muscle fibres are associated with reduced exercise capacity 2,3 and increased mortality in COPD patients. [4][5][6] Sex differences in the risk of developing COPD are well described. Females are more likely to develop COPD as a result of tobacco exposure, 7 plasma levels of inflammatory biomarkers in COPD patients differ between sexes 8 and sex hormones play an important part in the pathogenesis of COPD. 9 Only one small study has investigated sex differences in the prevalence of skeletal muscle dysfunction and abnormalities in COPD. 10 Female patients had more evidence of muscle damage, less evidence of muscle regeneration and more muscle dysfunction than male patients. Sex differences in health, seen in the control group, were not accounted for, thus the reported differences between female and male COPD patients could simply have reflected normal gender differences.
We have previously collected clinical and quadriceps biopsy data from the largest single centre cohort of COPD patients. 2 We reanalysed these data to determine whether there were sex differences in the prevalence of quadriceps muscle dysfunction and fibre abnormalities in COPD patients, over and above sex differences in our healthy age-matched controls, and if so, whether differences could be attributed to sex differences in potential aetiological factors.

Methods
Data from our prior study 2 were reanalysed. The original study was approved by the Royal Brompton and Harefield NHS Trust and Ealing and West London Mental Health Trust Research Ethics Committees (06/Q0404/35 and 06/Q0410/54), and participants gave written informed consent. The cohort consisted of 114 COPD patients (76 male and 38 female) and 30 healthy matched controls (16 male and 14 female). Exclusion criteria were diagnoses of heart, renal or liver failure; systemic inflammatory, metabolic or neuromuscular disorders; use of warfarin; or a moderate/severe exacerbation (i.e. requiring intervention) within the preceding 4 weeks. Post-bronchodilator spirometry, lung volumes (plethysmography), carbon monoxide diffusion capacity and resting arterialized capillary earlobe blood gas tensions were measured using previously described techniques. [11][12][13] Fat-free mass index (FFMI) was calculated from bioelectrical impedance measurements (using the Bodystat 1500, UK) taken after the subject had rested for 20 minutes lying supine. 14 A low FFMI was defined as an FFMI less than 15 kg/m 2 in women and less than 16 kg/m 2 in men according to previously published cut-offs in the work by Schols et al. 15 Physical activity, that is, locomotion, standing, sitting and lying time over 12 hours on 2 days was measured using the DynaPort accelerometer (McRoberts BV, Netherlands), as previously described. 16 Quadriceps strength was measured as supine isometric maximal voluntary contraction (MVC) 17 in the dominant leg. Quadriceps endurance (T 80 ) was assessed by time for force decline to 80% of initial force during repetitive magnetic femoral nerve stimulation in the dominant leg as described in the work by Swallow et al. 18 Whole body exercise performance was assessed by a 6-minute walk test according to American Thoracic Society (ATS) guidelines 19 and by symptom-limited incremental cycle ergometry with metabolic testing, as we have done previously. 20 Percutaneous biopsy of the vastus lateralis of quadriceps was performed using the Bergstrom technique 21 on an occasion separate from strength and cycle tests. Muscle samples were stored as previously described 2 until cryosectioning. Detection of type I, I/IIa, IIa and IIx fibres in a sample of at least 100 fibres (to calculate fibre type proportions and median fibre cross-sectional area (CSA)) was done with immunohistochemistry as described in the work by Natanek et al. 2 Variables analysed Patient demographics and COPD disease severity measures. Includes smoking history (pack-years), current medication, lung function (including FEV 1, TL CO ), arterial partial pressures of oxygen and carbon dioxide, health-related quality of life (SF36 and SGRQ scores).
Quadriceps function. Quadriceps strength measured as MVC and T 80 (time in seconds for muscle force to drop from initial force (30% of MVC) to 80% of the initial force with repetitive magnetic stimulation of the femoral nerve).
Quadriceps fibre characteristics. Proportion of slowtwitch, oxidative type I; fast-twitch, oxidative/glycolytic type IIa; and fast-twitch, glycolytic type IIx fibres and median CSA per fibre type (mm 2 ) as assessed by immunohistochemistry of frozen transverse muscle sections.
Whole body exercise performance and daily physical activity. Six-minute walk distance (6 MW, m) and peak oxygen consumption (VO 2 , ml/kg/min) from an incremental cycle ergometry protocol, expressed as a percentage predicted 23 which corrects for sex. Time spent in locomotion and standing (minutes over 12 hours) and movement intensity in m/s 2 from accelerometer readings over 2 days.

Selected markers of systemic and muscle inflammation.
Plasma concentrations of a number of proinflammatory cytokines and the anti-inflammatory cytokine IL-10 and soluble tumour necrosis factor alpha receptors I and II (TNF-aRI and II) were available from luminex quantifications (performed by GSK laboratories, UK). Muscle NF-kB p65 and p50 and AP-1 c-Jun subunit bound to DNA were quantified by transcription factor assays run on nuclear extracts (Panomics lab, Fremont, California, USA). Muscle TNF-a transcript levels were quantified by qPCR.

Normalization
Male and female controls were matched in terms of demographic data as previously reported. 2 However, some measurements, for example height, were expected to differ by gender in normal subjects (see Online Supplemental Table E1). In those cases, raw data for each patient (for variables not already subjected to any normalization) were divided by the mean or median value (depending on distribution) for the same variable from controls of the same sex, to give 'normalized values'. Where there were wellvalidated existing prediction equations that correct for sex, for example, differing equations that convert 6minute walk distance, 24 peak oxygen consumption on incremental cycle ergometry 23 and quadriceps MVC 25 to a % predicted for females and for males, these were used as a method of normalization, allowing % predicted for female and male COPD patients to be compared directly. This was because we considered this a more accurate and sophisticated correction than dividing by the mean or median value of the same sex control group. Certain variables, such as physical activity levels, would not be expected to have a systematic bias between healthy females and males, but variation between individuals as a result of other factors. For these variables, it was logical to compare absolute (not normalized) values between patients of different sexes.

Statistical analysis
This was carried out using Microsoft ® Excel 2011 and GraphPad Prism version 7.00 for Mac, GraphPad Software, La Jolla California USA, http://www.graph pad.com. Data distribution was determined through visual inspection of graphed data, D'Agostino and Pearson, Shapiro-Wilk and KS normality tests. Group differences were tested using unpaired t-tests or Mann-Whitney for normally and non-normally distributed data, respectively. Pearson's correlation coefficient and w 2 statistics were calculated to determine significance of linear correlations and differences in proportion. Level of significance was determined to 5% (a ¼ 0.05). Comparisons of proportions were performed using Fisher's exact test.

Results
Demographics and clinical parameters in COPD patients and controls are shown in Table 1.

Healthy controls
As expected, healthy males were taller and heavier and had greater muscle mass and quadriceps strength (MVC) than healthy females (Table 1). Minor fibre type proportion differences existed between the two sexes ( Table 2), consistent with previous reports. 26 Fibre CSA did not differ between sexes in the controls (Figure 2(c) and Table 2). Female controls had a 15% higher 6 MW % predicted (p ¼ 0.02; Table 1) and spent significantly more time standing than the male controls (298 (82) minutes/12 hours vs. 219 (126) minutes/12 hours, p ¼ 0.02; Figure 3(b)). Plasma interferon gamma (IFNg) and TNF-a were the only markers of inflammation different in female than male controls, where levels were higher in females (p ¼ 0.01 and 0.047, respectively).

COPD patients
Lung function, smoking history and exacerbations. Male and female patients were matched in their lung disease severity, yet female patients reported less smoke exposure than male patients (37.5 vs. 50 pack-years, p ¼ <0.0001). Three percent of both female and male patients had GOLD I COPD, 37% of females and 25% of males had GOLD II COPD, 37% of females and 34% of males had GOLD III COPD and 24% of females and 37% of males had GOLD IV COPD. There were no statistically significant differences between the percentages of females and males at each GOLD stage. The proportion of female and male patients in chronic respiratory failure (PaO 2 <8kPa) were identical at 16% and of female and male patients using long term oxygen therapy (LTOT) were not significantly different (55% and 8%, respectively, p  Figure 1(a)), and normalized quadriceps MVC/BMI and MVC/FFM values (muscle strength per unit body/muscle mass) were both significantly lower in COPD females than COPD males (Figure 1(a)). Quadriceps MVC as % predicted, calculated using existing prediction equations from As described previously in this cohort, the COPD patients had a reduced type I fibre and an increased type II fibre proportion, compared to healthy agematched controls 2 (Figure 2(a) and Table 2). Prior to normalization, the only sex difference in fibre type proportions was that female patients had a slightly lower type IIx fibre proportion than male patients (3 (5) % vs. 5 (9) %, p ¼ 0.037; Table 2), which is unlikely to be clinically significant. The normalized value comparison revealed that female patients had a slightly higher proportion of hybrid type I/IIa fibres compared to male patients (2 (3) vs. 1 (2), respectively, p ¼ 0.04; Figure 2 Table 2).
Factors that may contribute to skeletal muscle dysfunction C1 Resting blood gas tensions. There were no significant differences in resting arterial partial pressures of oxygen and carbon dioxide between male and female patients (Online Supplemental Figure E2A).
C3 Systemic levels of inflammation. There were no significant differences in plasma pro-inflammatory cytokine levels between male and female COPD patients (data not shown). Since there were differences between healthy male and female in levels of systemic inflammation and activation of muscle inflammatory cascades (either statistically significant or trends), levels of inflammation in patients normalized for sex-specific values were compared. Female patients had significantly higher levels of normalized pro-inflammatory cytokines granulocyte monocyte colony stimulating factor, TNF-a, IFNg and interleukins (IL) 2, 4, 5, 8, and anti-inflammatory IL-10 while there were no significant differences in IL-1b, IL6, C-reactive protein (CRP) levels or soluble TNF-aRI or RII (Figure 3(a) and (b) and Table 3). Fat mass and fat mass index were positively correlated with plasma CRP concentration in both females and males (females r ¼ 0.46, p ¼ 0.004, males r ¼ 0.38, p ¼ 0.01, and females r ¼ 0.50, p ¼ 0.01, males r ¼ 0.40, p ¼ 0.0004, respectively). There were no significant correlations between levels of proinflammatory cytokines and either MVC/BMI, MVC/FFM or type II fibre CSA in female patients. In male patients, there was a modest positive correlation between plasma CRP and type IIx fibre CSA (r ¼ 0.38, p ¼ 0.002; Online Supplemental Figure E3B).

C4 Muscle inflammation/inflammatory cascades.
Absolute levels of muscle NF-kB p50, p65 or AP-1 DNA binding were not significantly different between female and male patients. However, COPD females had a significantly lower normalized muscle NF-kB p50 level than male patients (0.6 vs. 1.1, p ¼ 0.003), but there were no significant differences in normalized muscle NF-kB p65 or AP-1 DNA binding (Figure 3(c)) nor muscle TNF-a mRNA levels between groups (Figure 3(d)). There was no correlation between muscle NF-kB p50 binding and fibre CSA in male patients.

Summary of main findings
Female COPD patients have a greater prevalence of signs of type II fibre atrophy and loss of quadriceps strength compared to their male counterparts, above and beyond the normal sex differences in health. The finding of smaller type II fibres in female compared to male patients was, however, largely driven by the effect of three male COPD patients who had large type II fibre CSAs, even relative to male controls. Consistent with greater evidence of muscle fibre wasting and weakness, female patients achieved a lower peak workload on cycle ergometry than male patients, and a larger proportion of female patients cited leg muscle discomfort as the reason for stopping cycling compared with male patients. However, interestingly female patients had a greater percentage predicted 6 MW performance and peak oxygen consumption on cycle ergometry than their male counterparts. The reductions in muscle fibre size, muscle strength and peak workload occurred in female patients despite them having a lower packyear smoking history and longer periods spent standing, which when carrying a load requires quadriceps activity, than male patients. However, this sex difference occurred in the presence of increased circulating pro-inflammatory cytokines in female compared to male patients (once normalized for sex differences in cytokines seen in the healthy controls). Although COPD females had evidence of a slightly increased early type I to type II fibre shift, there was no difference in the pure fibre type proportions nor T 80 . Muscle levels of NF-kB p50, relative to levels in their same sex counterparts, were lower in female than male patients.

Comparison with existing literature on the subject
Our findings of female COPD patients demonstrating lower muscle strength and greater muscle atrophy are consistent with those of Ausin et al. 10 However, with our larger sample size, we did not find that male patients had a greater type II fibre proportion as the previous study reports 10 but data supporting a subtle increase in fibre type switch in females with COPD. Like the previous study, we found that female COPD patients have relative higher systemic inflammatory markers than males but we did not find direct correlations between increased markers of systemic inflammation and fibre atrophy, nor indeed between physical activity levels and fibre atrophy, in either sex. This suggests that any contribution of systemic inflammation or physical activity to fibre atrophy is moderated by other influences so that simple linear associations are not evident.
The reduction in peak workload on a maximal incremental cycle protocol in female compared to male patients is consistent with a previous report from Yquel et al. 27 However, it is notable that we did not see the differences between male and female COPD patients in muscle fibre size and muscle strength translating to greater reductions in % predicted 6 MW and peak VO 2 on cycle ergometry in the female patients. Published predictive equations have a high variability in their predictive power, suggesting that other factors not included in the calculations play an important role in the distance walked. It may be that using % predicted values introduces a sex difference not dependent on muscle function and mass but on other factors. However, with height (hence leg length), weight and lung function having such an effect on exercise performance using uncorrected values to compare male and female patients would have been less satisfactory. Furthermore, there are advantages of smaller fibre size to performance measured as peak oxygen consumption, namely smaller diffusion distance for oxygen, 28 which may explain our results.
The canonical NF-kB pathway that signals in response to inflammation is dependent on heterodimers of p65 and p50 units translocating to the nucleus to induce transcription promoting skeletal muscle protein degradation 29 and inhibiting myogenesis, 30 at least with acute pathway activation. The fact that there were no differences in p60 DNA binding in males and females suggests that activation of the canonical NF-kB pathway in muscle is similar in males and females, despite the increased plasma levels of pro-inflammatory cytokines in female patients. The non-canonical NF-kB pathway associated with muscle disuse atrophy is dependent on p50 (and Bcl3) subunits not p65 subunits. 31 There was evidence of increased p50 DNA binding, despite the fact there was less evidence of fibre atrophy, in male, compared to female, patients. Increased activation of the non-canonical NF-kB pathway is therefore unlikely to account for the greater prevalence of fibre atrophy seen in females. This also suggests that there are differences in activation of signalling mechanisms in the locomotor muscle of male and female COPD patients. The influence of oestrogens and menopause on skeletal muscle inflammation, damage and regeneration 32 and differences in muscle protein synthesis/protein metabolism between sexes 33 are well described and are good examples of how the biology of muscle damage and repair differs between the sexes. Sex hormones also modulate systemic inflammation; for example, androgens have been shown to suppress the immune system. 34 Therefore, the sex differences we report could reflect differences in sex hormone levels between female and male COPD patients, which would support the use of androgenic agents, such as selective androgen receptor modulators, in the treatment of COPD-related muscle wasting, particularly in females.

Study limitations
This is a cross-sectional study and therefore we are unable to comment on sex differences in rate of muscle fibre atrophy over time. It is possible, therefore, that the finding of more fibre atrophy and weakness in female patients was not a genuine sex difference, but that it was related to another factor, for example, that diffusion abnormalities develop earlier in female than male patients, that causes muscle atrophy and weakness. Although our sample size was large for a single cohort including biopsy data, there was still a relatively small number of patients, particularly females, and therefore it cannot be discounted that the sex differences relate to sampling bias of females who had greater evidence of fibre atrophy and weakness than the average female COPD population. Our control sample is also small and therefore may not be representative, although it is larger than the previous similar study and many other COPD muscle studies. The female controls had less cigarette exposure than the male controls which, if it affected the physiological variables we measured, may also have confounded genuine sex differences. For these reasons, we were careful only to normalize to control values where there was previous literature supporting the observed sex differences we found in our controls. Had there been measurements of thigh muscle CSA or another measurement of lower limb muscle mass, it would have been preferable to correct our strength data to one of these measurements, particularly as musculature versus adipose tissue distribution in the lower limb is likely to be different in females and males. We did replicate our findings of greater quadriceps weakness in female patients when MVC was converted to a % predicted value, using validated equations for expected values in females and males, although these equations again use global rather than lower limb muscle mass. Analyses of the minority fibre types in quadriceps, for example, hybrid I/IIa fibres and type IIx fibres were likely to be underpowered because of the small number of these fibres per subject. The multiple comparisons we made increased the possibility of false positive results; however, on the whole, our significant results were consistent using a number of similar parameters, for example, consistent between MVC, MVC/BMI, MVC/FFM and MVC % predicted giving us confidence that they were not random positive results. Had we measured circulating sex hormone levels in female and male patients, this would have been valuable to examine whether these could account for the observed sex differences and which, if any, physiological measurements correlated with androgen and oestrogen levels. From the age of our female patients, it is likely that the majority were postmenopausal; therefore our results cannot be assumed to generalize to comparisons of premenopausal female and age-matched male patients. It would have been useful to have had data on menopausal status and hormone replacement therapy (HRT) use from our female patients and controls.

Implications for clinical practice
We have confirmed a previous report that female COPD patients appear more prone to quadriceps fibre atrophy and weakness than their male counterparts and identified that, in COPD, there are sex differences in aetiological factors and downstream signalling pathways that influence skeletal muscle structure and function. Further studies confirming sex differences in a larger patient cohort with a large comparative control population are required to see if these results hold, and whether the wasting and weakness in females is associated with androgen levels. These studies are necessary as the results would have implications for monitoring of patients in clinical practice, for stratification of participants in clinical trials of new treatments for COPD-related muscle atrophy, including new androgenic agents.

Declaration of conflicting interests
The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.