From the respiratory department, 15 COPD patients were included for study (Global Initiative for Chronic Obstructive Lung Disease [GOLD] stages II [n=4], III [n=4] and IV [n=7]). The exclusion criteria were heart, kidney or liver failure or a systemic inflammatory, metabolic or neuromuscular disease or a moderate-severe exacerbation (meaning, with the need for antibiotics, oral steroids or hospitalisation) in the previous four weeks. Using an advertisement, 8 healthy control individuals were recruited. All individuals gave their written informed consent and the study was approved by the research committee of the Royal Brompton, Harefield NHS Trust and Ealing and West London Mental Health Trust.

In accordance with the guidelines of the American Thoracic/European Respiratory Society, we determined postbronchodilator spirometry,16 lung volumes with plethysmography17 and carbon monoxide diffusing capacity,18 while arterial blood gas was examined from an arterialised blood sample obtained from an earlobe. Lean body mass was determined with bioelectric impedance (Bodystat® 1500, Bodystat, United Kingdom),19 which was corrected for stature to derive the lean body mass index. Physical activity was determined by a triaxial Dynaport® ADL3 accelerometer (McRoberts BV, Netherlands) that the patients used for two days, 12h each day, during normal activity. The mean locomotion time was calculated as previously described.20 The strength of the quadriceps (right leg) was evaluated with the maximum voluntary isometric contraction, in supine decubitus, based on the method by Edwards.21 The percutaneous needle biopsy in the right vastus lateralis was done with local anaesthesia using the Bergstrom technique.22 The samples for the analysis of ribonucleic acid and proteins were immediately frozen in liquid nitrogen, while the histology samples were introduced in pre-cooled isopentane for 15s before being frozen in liquid nitrogen then stored at −80°C.

There was not enough tissue from each and every individual to complete all the analyses, therefore for each analysis a subgroup of samples was used.

The frozen 10μm slices from 10 patients and 8 control individuals were set in a solution of 10% formaldehyde, washed in Triton X-100 at 0.1% in a neutralised saline solution with phosphate buffered saline (PBS) and were blocked with 5% bovine serum albumin (BSA) in PBS, before incubation with rabbit anti-YY1 antibody (dilution 1:400, sc-281; Santa Cruz Biotechnology, United States) and murine anti-heavy chain myosin fast contraction antibody (MYSN02, dilution 1:200, Abcam, United Kingdom) in 3% BSA in PBS for the entire night at 4°C. After an incubation of 1h at room temperature in the dark with secondary antibodies marked with fluorescence (A11008 Invitrogen and A11005 Invitrogen, dilution 1:250 in PBS), the slices were treated with diamino-phenylindole (DAPI). The images (two fields per sample, two cuts per individual) were obtained using a wide-field Zeiss Axiovert microscope, with a 10× lense and Improvision Volocity software. Using a Leica SP2 microscope, we obtained confocal images with a 63× oil immersion lense and they were analysed with LCS Lite software (Leica Microsystems, Germany).

YY1 is not abundant in the skeletal muscle of adults and, consequently, before immunoblotting, immunoprecipitation was necessary. From nine patients and seven control individuals, for this analysis there were 3–6mg of protein.

Between 3 to 6 mg of homogenised protein were incubated in a solution with Nonidet P40, cocktails of protease inhibitor and phosphatase inhibitor (Sigma, Poole Dorset, United Kingdom) in ice for 30 min with 30μl de Protein G-Sephorose beads. After centrifuging for 2 min (8,000rpm), the supernatant was incubated for 1 h in ice with 1μg of rabbit anti-YY1 antibody (sc-281, Santa Cruz Biotechnologies, United States). For each sample, another 30μl Protein G-Sephorose beads were added and they were re-incubated in ice for 1h. The samples were centrifuged again and the supernatant was discarded. The beads were washed in Nonidet P40 and boiled with 30μl of the sample buffer (loading buffer and 2-mercaptoetanol) for 5 min at 100°C. The process was optimised to guarantee that the supernatant would not have a detectable quantity of YY1 in the Western blot.

The samples were analysed using electrophoresis in polyacrylamide gel with sodium dodecyl sulphate (SDS/PAGE) (10% gel) and a semidry immunoblotting technique (LKB). After blocking (5% BSA in PBS), the membranes were incubated with mouse anti-YY1 antibody (ab58066, dilution 1:200, Abcam, United Kingdom) in 3% BSA in PBS for the whole night at 4°C, and immediately followed with an IgG murine antibody bound with horseradish peroxidase (ab 6728, Abcam, United Kingdom, 1:5000 in 3% BSA in PBS) for 1 h at room temperature. The proteins were visualised with Supersignal (Pierce, Rockford, IL, United States), determining the density of the band with a densitometry 1D analysis (AIDA, Raytek, Sheffield, United Kingdom) and was normalised for the quantity of proteins used in the immunoprecipitation.

From all the subjects, the 10μm frozen transverse muscle slices were incubated with primary antibodies for type I myosin, type IIa myosin and laminin (A4.840 and N2.261 Developmental Studies Hybridoma Bank, University of Iowa, Unites States, and L-9393 Sigma, Zwijndrecht, Netherlands, respectively) followed by secondary antibodies marked with fluorescence (A-21121, A-21426 and A-11069, Invitrogen).23 The epifluorescence signal was registered using a Texas Red excitation filter (540-580nm) for type I myosin, an FITC excitation filter (465-495nm) for type IIa myosin and a UV DAPI excitation filter (340-380nm) for the laminin using a Nikon Eclipse 800 microscope. The fibres were classified into type I, IIa, IIx (without staining) and hybrid I/IIa (dual staining) and we used the laminin edge of the fibres to calculate the CSA of each fibre (and with this the mean CSA for each type of fibre) and the proportions of fibres using Lucia 4.81 statistical software (Nikon, Japan).24 For each individual, we analysed an average of 207 fibres, with a minimum of 103 fibres.

The TNF-α mRNA transcripts were determined by a chain reaction of the quantitative polymerase, in real time, using SYBR® Green PCR Master Mix (Applied Biosystems) in a 7900HT Fast Real-Time PCR System (Applied Biosystems). The transcripts normalised to a geNorm factor derived from two internal genes, acidic ribosomal phosphoprotein (RPLPO) and beta-2 microglobulin (β2M), as previously reported.25 The following are the primer sequences:

The data did not have normal distribution (according to the histogram and the test of symmetry), so they are reported as averages (25th percentile and 75th percentile). The group differences in the continuous variables were analysed with the Mann–Whitney U-test, while the Fisher's exact test was used to test the group differences in the categorical variables. Spearman's rank correlation coefficient (ρ) was calculated to determine the relationship among the variables (Statview 1.0, Abacus Instruments). To define the statistical significance, a two-tailed P value ≤.05 was used.

YY1 was absent from the nucleus but present in the cytoplasm in the quadriceps of the healthy control subjects (Figs. 3 and 4C, D, G, H). YY1 in the cytoplasm aligned with the sarcomeres, but it did not co-localise with myosin (Fig. 3).

Fig. 3.

Image from a wide-field microscope of a longitudinal slice of the quadriceps muscle of a healthy control subject demonstrating the localisation of YY1 regarding the muscle nuclei by means of immunohistochemistry. The muscle was stained for YY1 (green), the heavy chain of the fast myosin (red) and the nuclei using 4′,6-diamidino-2-phenylindole (blue). The YY1 staining is observed along the sarcomeres, not co-localised with the myosin, and the absence of nuclei is also observed. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

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Fig. 4.

Microscope images of quadriceps muscle slices from COPD patients and control subjects, demonstrating in the myotubules the localisation of YY1 with immunohistochemistry with regard to the muscle nuclei. (A–D) Wide-field microscope images. E-H: images from a confocal microscope with cross-sectional quadriceps muscle cuts that demonstrate the YY1 stain in green and for the nuclei with 4′,6-diamidino-2-phenylindole in blue (B, D, E, and G) and the same cuts that show staining for YY1 only in green (A, C, F, and H). (A, B, E, and F) The slices of a patient in whom the anomalous staining pattern of the factor was identified in the nucleus as well as in the cytoplasm. (C, D, G, and H) From a control subject showing exclusively cytoplasmic YY1 staining.

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YY1 staining was demonstrated in the nuclear region in none of the control individuals but in 5 of the 10 patients. This difference between groups was statistically significant (Fisher's exact test, P=.04). Compared with 6 of the 8 control subjects, only in 2 of the 10 patients was there a demonstrated exclusively cytoplasmic YY1 distribution that, once again, was statistically significant (Fisher's exact test, P=.05). No significant difference was identified between patients with or without nuclear YY1 localisation regarding muscle or lung function, quadriceps fibre CSA or YY1 protein levels.

Naturally, the present study presents limitations. First of all, the size of the sample was small and the associations detected had to stand out; therefore it is possible that no other associations or group differences were identified due to the lack of power. The control subjects included in the present study were characterised by a relatively low lean mass index and a wide variance in their degree of physical activity, for which there is no clear explanation, which increases the threshold for the statistical significance of the group differences. Although the study of patients in stage II, III, and IV of the GOLD initiative should have made it easier to find correlations between the muscle and lung function and the YY1 levels (because the data was not expected to gather), the exclusive use of patients in stage IV could have maximised the probability of finding a significant difference in YY1 expression between patients and control subjects. Second, the observational design does not allow for the extraction of conclusions about the effect of the differences in YY1 on muscle structure and function, which merely highlights an association between the increase in expression of the factor and the smaller size of the fibres and between the nuclear localisation of the factor and the presence of COPD and less strength in the quadriceps muscle than in control individuals. Lastly, we used the localisation and expression of the factor as an indirect variable of the activity because YY1 is activated by transport from the cytoplasm to the nucleus. Our findings could have been corroborated by the quantification of YY1 bound to DNA (immunoprecipitation of chromatin, a technique that has not been previously published as being used in human muscle) or the quantification of YY1 able to bind with DNA (for example, by means of electrophoretic analysis of the change in mobility).

It could also be argued that the YY1 factor detected in the nuclei is not present in the periphery of the myofibrils, but is instead in the latent, satellite cells. However, we consider that this is improbable as the satellite cells only represent 2%-5% of the muscle nuclei.28 Even supposing a massive increase in the population of satellite cells in the tissue of the patients, the number of nuclei that stained for the factor considerably surpasses the number that could be justified by the satellite cells. Furthermore, in the samples of this present study, the absence of centralised nuclei suggests a limited activation of the satellite cells in the period of time when the samples were taken.

The implication of a powerful inverse correlation between the levels of the YY1 factor and the size of the fibres is that YY1 could participate in the mechanism of fibre atrophy, particularly as the relationship is stronger with the CSA of the type IIx fibres and these fibres seem to be the type that atrophy the most in COPD.26 However, this hypothesis requires additional research, for example, examining the effect in the skeletal muscle of adult knockout or knockdown mice with emphysema induced by tobacco smoke or other means.

The finding that patients with a nuclear localisation of the factor do not necessarily have higher levels can be explained by the fact that its localisation and expression can be regulated independently. It is possible that the patients with a greater expression of YY1 and a smaller size of the fibres are characterised by greater levels of NF-κB activation in the muscle, while in others a greater reserve of depolymerised actin would give rise to a nuclear accumulation of the factor without an increase in its expression. However, we did not find a correlation between the local values of TNF-α and YY1 in the muscle of the patients, which supports the suggestion that the levels of the factor are mainly determined by local TNF-α that would activate the NF-κB factor.

In conclusion, we report the new finding that YY1, transcription factor for the repression of the specific gene expression of muscles and myogenesis, is expressed associated with a decrease in the CSA of type IIx and type I fibres of the quadriceps muscle in COPD. YY1 is also localised in the nuclei of the quadriceps of COPD patients in comparison with healthy control subjects, paired by age. We speculate that the greater activity of YY1 could be implicated in the deterioration of skeletal muscle regeneration and the atrophy of the muscle fibres in COPD.