Increased skeletal muscle-specific microRNA in the blood of patients with COPD

Abstract

Background: Skeletal muscle weakness in chronic obstructive pulmonary disease (COPD) carries a poor prognosis, therefore a non-invasive marker of this process could be useful. Reduced expression of muscle-specific microRNA (myomiRs) in quadriceps muscle in patients with COPD is associated with skeletal muscle weakness and changes in muscle fibre composition. Circulating exosomal miRNAs can be measured in blood, making them candidate biomarkers of biopsy phenotype. To determine whether plasma myomiR levels were associated with fibre size or fibre proportion, we measured myomiRs in plasma from patients with COPD and healthy controls.

Methods and results: 103 patients with COPD and 25 age-matched controls were studied. Muscle-specific miRNA was elevated in the plasma of patients with COPD and showed distinct patterns. Specifically, miR-1 was inversely associated with fat-free mass in the cohort, whereas levels of miR-499 were more directly associated with strength and quadriceps type I fibre proportion. Two miRs not restricted to muscle in origin (miR-16 and miR-122) did not differ between patients and controls. Plasma miR-499 was also associated with muscle nuclear factor κB p50 but not p65 in patients with early COPD whereas plasma inflammatory cytokines were associated with miR-206 in patients with more advanced disease.

Conclusions: Plasma levels of individual myomiRs are altered in patients with COPD but alone do not predict muscle fibre size or proportion. Our findings are consistent with an increase in muscle wasting and turnover associated with the development of skeletal muscle dysfunction and fibre-type shift in patients with stable COPD.

Keywords: COPD Pathology; Exercise; Systemic disease and lungs.

Figure 1

Muscle-derived miRNAs are elevated in the plasma of patients with stable chronic obstructive pulmonary disease (COPD). Plasma miR levels were measured as described in ‘Methods’ and normalised to a spiked Caenorhabditis elegans control. Data are presented as log-normalised levels with the notched boxes showing median and IQR, error bars at the 10th and 90th percentile, outliers are also shown. Patients with Global Initiative for Obstructive Lung Disease (GOLD) stage I and II COPD are represented by filled grey circles, those with GOLD stage III and IV disease are represented by filled black circles and controls are shown as open grey circles. Statistical significance was calculated by t test or Mann–Whitney U test as the non-parametric alternative.

Figure 2

Association of plasma miRNAs with lung function. Plasma miRNAs were determined as described in ‘Methods’ and compared with forced expiratory volume in 1 s (FEV₁) % predicted. MiR-1 showed a negative association with FEV₁ across the whole cohort (A) whereas miR-499 (B) and miR-206 (C) were highest in patients with early stage chronic obstructive pulmonary disease (COPD) (Global Initiative for Obstructive Lung Disease (GOLD) I and II). Consequently although miR-499 and miR-206 levels were significantly higher in patients than controls (see figure 1) there was a negative association of these miRNAs with FEV₁ within the patients group alone (miR-499 r=0.29, p<0.003; miR-206 r=0.26, p<0.009). Patients with GOLD I and II COPD are represented by filled grey circles, those with GOLD III and IV COPD are represented by filled black circles and control subjects are represented by open grey circles.

Figure 3

Plasma miR-1 is weakly associated with fat-free mass and type I fibre cross-sectional area (CSA) in patients with chronic obstructive pulmonary disease (COPD). Physiological parameters and plasma miRNA levels were determined as described in ‘Methods’. (A) Plasma miR-1 levels negatively correlated with fat-free mass index when patients with COPD were analysed either with or without the controls (as shown in the figure), r=−0.21, p=0.036 and r=−0.25, p=0.013, respectively. (B) Plasma miR-1 levels were negatively correlated with type I fibre CSA in patients (r=–0.27, p=0.027). Patients with Global Initiative for Obstructive Lung Disease (GOLD) I and II COPD are represented by filled grey circles, those with GOLD III and IV COPD are represented by filled black circles.

Figure 4

Association of plasma miR-499 and fibre proportion in patients with Global Initiative for Obstructive Lung Disease (GOLD) III and IV chronic obstructive pulmonary disease (COPD). Plasma miR-499 was determined as described in ‘Methods’. In the entire patient group none of the miRNAs were associated with the percentage of type I fibre (TI%) in quadriceps muscle biopsy. (A) Comparison of fibre proportion and plasma miR-499 levels in patients with GOLD III and IV COPD showed a weak association of plasma miR-499 with TI% in quadriceps biopsies (r=0.26, p=0.033). (B) In the entire patient cohort, comparison of plasma miR-499 in patients with fibre shift (defined as a TI% below the 2.5th percentile for the control cohort) with those without fibre shift had higher plasma miR-499. Median (25th, 75th percentile) log AU=4.113 (3.781, 4.412) and 3.898 (3.594, 4.126) respectively (Mann–Whitney test). Error bars are 10th percentile and 90th percentile and outliers are shown. (C) Comparison of 6 min walk distance with plasma miR-499 showed a weak association in the entire patient cohort (r=0.22, p=0.028). Patients with GOLD I and II COPD are represented by filled grey circles and those with GOLD III and IV COPD are represented by black circles.

Figure 5

Receiver operating characteristic (ROC) analyses of physiological parameters alone or in combination with plasma miRNA levels to discriminate patients according to the presence of type I fibre shift. Patients with Global Initiative for Obstructive Lung Disease (GOLD) III and IV disease were defined as having type I fibre shift or not as described in ‘Results’. ROC analysis was then performed as described in ‘Methods’ using transfer factor of the lung for carbon monoxide (TL_CO) expressed as % predicted (A), 6 min walk distance (6MWD) as % predicted (B), combination score (generated by combining TL_CO % predicted, 6MWD % predicted, plasma miR-1 level, plasma miR-499 level, plasma miR-181 level and plasma miR-206 level, as described in ‘Methods’) (C). Area under the curve (AUC) is shown in each graph. (D) Comparison of the combined score in patients with or without fibre shift. Patients with GOLD III and IV disease with fibre shift have a significantly different score (p<0.001) than patients of the same disease severity without fibre shift. TPF, true positive fraction.

Figure 6

Plasma miR-499, miR-133 and miR-206 are correlated with the amount of nuclear factor κB (NFκB) in the quadriceps nuclei of patients with Global Initiative for Obstructive Lung Disease (GOLD) I and II chronic obstructive pulmonary disease (COPD). Log normalised plasma miR-499 (determined as described in ‘Methods’) was compared with NFκB p50 determined by transcription factor ELISA. In patients with GOLD I and II COPD there was a direct association between NFκB p50 (r=0.58, p<0.001) and miR-499 (A) that was not present when analysing patients with GOLD III and IV COPD (r=0.142, p=0.25 (B). In patients with GOLD I and II COPD NFκB p50 was also correlated with miR-206 (r=0.4 p=0.016 (C) and miR-133 (r=0.47, p=0.006 (D). Patients with GOLD I and II COPD are represented by filled grey circles, those with GOLD III and IV COPD are represented by filled black circles.

Figure 7

Plasma miR-499 and miR-206 are associated with plasma interleukin 2 (IL-2) in patients with chronic obstructive pulmonary disease (COPD). Log normalised plasma miR levels (determined as described in ‘Methods’) were compared with plasma IL-2 levels determined by ELISA. miR-206 was not associated with plasma IL-2 in patients with Global Initiative for Obstructive Lung Disease (GOLD) I and II COPD but was positively associated with plasma IL-2 in GOLD III and IV COPD (B) (r=0.39, p<0.001). miR-499 was not associated with IL-2 in patients with GOLD I and II COPD (C) or in patients with GOLD III and IV COPD (D). Patients with GOLD I and II COPD are represented by filled grey circles and those with GOLD III and IV COPD are represented by filled black circles.