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Background Approximately 30% of patients with Chronic Obstructive Pulmonary Disease (COPD) exhibit peripheral skeletal muscle dysfunction and a shift towards type II glycolytic fibres in the quadriceps compared to healthy controls (Nanatek et al , 2013). Previous work to elucidate the molecular mechanisms underlying these changes has relied on whole biopsy samples and may have missed fibre-specific pathways; thus a method to evaluate fibre specific signalling pathways would be useful. Objective To describe a novel laser capture micro-dissection (LCM) method to examine fibre-specific signalling in quadriceps biopsies. Methods First larger Intercostal muscle biopsies were used to validate the methodology since they yielded more RNA. Fibres were classified as type-2 positive or type-2 negative based on immunoreactivity with a type-2 fibre specific anti-myosin Heavy Chain Alexa FLUOR 488 antibody. The type-2 negative fibre population was hence assumed to contain type-1 fibres, which was confirmed by the type-2 negative fibre population exhibiting a higher myhc7/2 mRNA ratio and expressing higher levels of genes associated with type-1 fibres, e.g. TNNT-1 and STARS, and lower levels of genes associated with type-2 fibres, e.g. TGF-B, myostatin, GAPDH and HDAC-4 (n = 2). We then examined OCT-embedded vastus lateralis muscle biopsy specimens. 10micron cryosections underwent fixation with 4% paraformaldehyde before immunostaining. LCM (PALM Microbeam, Zeiss, UK) was used to capture type I and type II fibre populations, before RNA extraction with RNAeasy FFPE kit (Qiagen, USA) and rtPCR to obtain cDNA. Sybr-II qPCR was performed on fibre populations for target genes MHC I, MHC IIa, MHC IIx, HDAC-4 and RPLPO. Results Preliminary results from three 10micron slices indicate that this technique is feasible to study fibre-specific signalling in COPD. LCM following immunostaining captures distinct fibre populations (Figure 1) confirmed by a higher MHC I content in ‘type I fibres’, with ‘type II’ fibres containing more MHC IIa, MHC IIx and HDAC-4 as would be expected. Gene expression is normalised against RPLPO. Conclusion LCM can be used to study fibre specific inflammatory signalling in the skeletal muscle of COPD patients and immunostaining with MHC antibodies is a feasible way to distinguish between fibre types when capturing composite fibre populations.

Background There is evidence to suggest that Pulmonary Rehabilitation (PR) is beneficial for patients with chronic lung diseases other than COPD (AACP/AACVPR guidelines 2007, ILD consultation document 2013). However, there is little evidence to suggest that PR provides exercise tolerance benefits comparable to COPD patients who participate in the same PR programmes. Aim To determine whether walking distance improvements differ significantly between ILD and COPD patients following PR. Method Retrospective data of PR Endurance Shuttle Walk Test distances (ESWTD) pre- to post-PR were analysed and compared between 55 Interstitial Lung Disease (ILD) and 440 COPD patients from February 2005 to December 2012. Patients participated in a PR programme run by the same clinical team. Independent sample two-tailed t-tests were performed on data for pre-PR ESWTD, post-PR ESWTD and ESWTD change. Results There were no significant differences between group ESWTD prior to PR (t = -0.049, p = 0.961), following PR (t = -0.227, p = 0.820) or change in ESWTD (t = -0.228, p = 0.820). Conclusions These data indicate there is no significant difference between ILD and COPD patients’ walking distances. ILD patients with a reduced exercise tolerance should be included and referred to PR programmes.

Introduction Skeletal muscle impairment is a well recognised complication of COPD, predicting mortality in severe disease. 1 Evidence from animal models, genetic studies and observational cohorts suggest a role for the renin-angiotensin system in control of muscle phenotype. 2 We hypothesised that angiotensin-converting enzyme (ACE) inhibition would have a beneficial effect on quadriceps function in patients with COPD. Methods A single-centre, double-blind randomised controlled parallel-group trial investigating the effect of fosinopril versus placebo on quadriceps muscle dysfunction in COPD patients with quadriceps weakness. Muscle weakness was defined as a quadriceps maximum voluntary contraction (QMVC) less than 120% of the body mass index.1 Measurements The primary outcome was change in non-volitional quadriceps endurance at 3 months, measured using repetitive magnetic stimulation. QMVC, mid-thigh CT cross-sectional area (MT CSA ), incremental shuttle walk distance (ISWD) and serum inflammatory markers were secondary outcomes. Results 80 patients were enrolled (mean(SD), 65(8) years, FEV1 43(21)% predicted, 53% male). 67 patients (31 fosinopril and 36 placebo) completed the trial, with the treatment group demonstrating a significant reduction in systolic blood pressure (Δ-10.5mmHg, 95%CI –19.9 to –1.1, p=0.03) and serum ACE activity (Δ-20.4units/L, 95%CI –31.0 to –9.8, p Conclusion This randomised controlled trial found that ACE-inhibition did not improve quadriceps function in a COPD population with quadriceps weakness. Study funded by the Medical Research Council. Trial registration: NCT01014338. Swallow EB, et al . Thorax 2007; 62:115–20. Shrikrishna D, et al . Clin Sci 2012; 123:487–98.

Introduction Quadriceps muscle dysfunction is an important prognostic comorbidity in COPD. MicroRNAs (miRs) are small non-coding RNAs that regulate gene expression. Skeletal muscle expresses a number of tissue-specific microRNA including miR-1, which modulates muscle phenotype. MicroRNAs can be secreted from cells and maintained in blood within exosomes. Elevated levels of circulating miR-1 have been demonstrated in a number of human and animal models of muscle disease. We hypothesised that plasma levels of miR-1 would be elevated in COPD patients and would correlate with important physiological parameters. Methods 103 COPD patients and 25 controls were studied. MiR-1 was quantified in stored plasma samples using q-RT PCR.1 MiR-16 and miR-122 were quantified as negative controls. Results were normalised to an exogenous spiked-in control. Results Characteristics as mean (SD); COPD patients: M: 67, F: 36, age=66.47 (8.4), FEV 1 % pred= 43.5 (18.6), 6-minute walk (6MW) = 394 (120). Controls: M: 14, F: 11, age=67 (8.1), FEV 1 % pred=111.2 (13.1), 6MW=613 (83). Plasma miR-1 was significantly elevated in COPD patients, p=0.002. There was no difference in miR-16 and miR-122. MiR-1 was negatively associated with FEV 1 % predicted (r =−0.3, p Conclusion Our results show that stable COPD patients have elevated plasma levels of muscle-specific miR-1. The increase in miR-1 may be due to increased muscle degradation or turnover in the COPD patients studied. Our work raises the possibility of using other muscle-specific microRNAs in the future as potential biomarkers of muscle dysfunction in patients with COPD.