Differentially co-expressed myofibre transcripts associated with abnormal myofibre proportion in chronic obstructive pulmonary disease

Abstract

Background: Skeletal muscle dysfunction is a common extrapulmonary manifestation of chronic obstructive pulmonary disease (COPD). Alterations in skeletal muscle myosin heavy chain expression, with reduced type I and increased type II myosin heavy chain expression, are associated with COPD severity when studied in largely male cohorts. The objectives of this study were (1) to define an abnormal myofibre proportion phenotype in both males and females with COPD and (2) to identify transcripts and transcriptional networks associated with abnormal myofibre proportion in COPD.

Methods: Forty-six participants with COPD were assessed for body composition, strength, endurance and pulmonary function. Skeletal muscle biopsies from the vastus lateralis were assayed for fibre-type distribution and cross-sectional area via immunofluorescence microscopy and RNA-sequenced to generate transcriptome-wide gene expression data. Sex-stratified k-means clustering of type I and IIx/IIax fibre proportions was used to define abnormal myofibre proportion in participants with COPD and contrasted with previously defined criteria. Single transcripts and weighted co-expression network analysis modules were tested for correlation with the abnormal myofibre proportion phenotype.

Results: Abnormal myofibre proportion was defined in males with COPD (n = 29) as <18% type I and/or >22% type IIx/IIax fibres and in females with COPD (n = 17) as <36% type I and/or >12% type IIx/IIax fibres. Half of the participants with COPD were classified as having an abnormal myofibre proportion. Participants with COPD and an abnormal myofibre proportion had lower median handgrip strength (26.1 vs. 34.0 kg, P = 0.022), 6-min walk distance (300 vs. 353 m, P = 0.039) and forced expiratory volume in 1 s-to-forced vital capacity ratio (0.42 vs. 0.48, P = 0.041) compared with participants with COPD and normal myofibre proportions. Twenty-nine transcripts were associated with abnormal myofibre proportions in participants with COPD, with the upregulated NEB, TPM1 and TPM2 genes having the largest fold differences. Co-expression network analysis revealed that two transcript modules were significantly positively associated with the presence of abnormal myofibre proportions. One of these co-expression modules contained genes classically associated with muscle atrophy, as well as transcripts associated with both type I and type II myofibres, and was enriched for genetic loci associated with bone mineral density.

Conclusions: Our findings indicate that there are significant transcriptional alterations associated with abnormal myofibre proportions in participants with COPD. Transcripts canonically associated with both type I and type IIa fibres were enriched in a co-expression network associated with abnormal myofibre proportion, suggesting altered transcriptional regulation across multiple fibre types.

Keywords: COPD; fibre‐type shift; myofibre proportions; sex differences; skeletal muscle; transcriptomics.

Figure 1

Comparisons of muscle fibre‐type proportions (A, C) and cross‐sectional areas (CSAs [μm²]) (B, D) between female (A, B) and male (C, D) participants on the basis of disease status. Significant differences between groups are marked with horizontal brackets and P values; all other comparisons were non‐significant. COPD, chronic obstructive pulmonary disease.

Figure 2

Comparison of k‐means clustering before and after sex stratification. Results are presented for female (left) and male (right) participants separately. The grey vertical and horizontal dashed lines represent the criteria suggested by Gosker et al.; any point above or the left of these lines represents abnormal fibre type as defined by these criteria. (A) The results of k‐means clustering without sex stratification: Grey dots represent patients designated as having an abnormal myofibre proportion, and black dots represent patients with a normal myofibre proportion based on this clustering system. (B) The results of k‐means clustering after stratifying for sex.

Figure 3

Volcano plot of differentially expressed transcripts in participants with chronic obstructive pulmonary disease associated with the presence of our sex‐stratified abnormal myofibre proportion phenotype. Labelled dots are significant after false discovery rate (FDR) correction, and labels are their respective gene names. Red dots represent transcripts with an FDR‐corrected P value <0.05 and an absolute log fold difference (FD) ≥1. Blue dots represent transcripts with an FDR P value of <0.05. Green dots represent transcripts with an absolute log FD ≥ 1.

Figure 4

The blue and cyan modules from weighted gene co‐expression network analysis (WGCNA) are significantly positively correlated with our sex‐stratified abnormal myofibre proportion phenotype. (A) Transcriptomic module association with abnormal myofibre proportion. Values in each cell represent Pearson correlations and P values (in parentheses) between each module of co‐expressed transcripts and the presence of an abnormal myofibre proportion phenotype. Heatmap shading corresponds to the strength of association where darker red cells have higher upregulation and darker blue cells have higher downregulation based on correlation. Cells outlined in black are significantly associated (P < 0.05) with abnormal fibre type. (B, C) Network of hub transcriptomic modules significantly associated with abnormal myofibre proportion. Transcripts with a module membership (kME) >0.78 in the blue (B) and transcripts with a kME > 0.6 in the cyan (C) modules were selected for visualization, along with TRIM54 and FOXO4 in the blue module, due to their suspected role in skeletal muscle pathology in chronic obstructive pulmonary disease. The size of the circle in each network corresponds to increasing module membership, and the thickness of the edge corresponds to increasing topological overlap, a measure of the strength of correlation between transcript expression levels, which is Pearson's correlation obtained from the adjacency matrix. One hub transcript (TTN) in the blue module is significantly differentially expressed in the single‐transcript analysis and is coloured orange to reflect this.