EGF receptor (EGFR) inhibition promotes a slow-twitch oxidative, over a fast-twitch, muscle phenotype.

A low quadriceps slow-twitch (ST), oxidative (relative to fast-twitch) fiber proportion is prevalent in chronic diseases such Chronic Obstructive Pulmonary Disease (COPD) and is associated with exercise limitation and poor outcomes. Benefits of an increased ST fiber proportion are demonstrated in genetically modified animals. Pathway analysis of published data of differentially expressed genes in mouse ST and FT fibers, mining of our microarray data and a qPCR analysis of quadriceps specimens from COPD patients and controls were performed. ST markers were quantified in C2C12 myotubes with EGF-neutralizing antibody, EGFR inhibitor or an EGFR-silencing RNA added. A zebrafish egfra mutant was generated by genome editing and ST fibers counted. EGF signaling was (negatively) associated with the ST muscle phenotype in mice and humans, and muscle EGF transcript levels were raised in COPD. In C2C12 myotubes, EGFR inhibition/silencing increased ST, including mitochondrial, markers. In zebrafish, egfra depletion increased ST fibers and mitochondrial content. EGF is negatively associated with ST muscle phenotype in mice, healthy humans and COPD patients. EGFR blockade promotes the ST phenotype in myotubes and zebrafish embryos. EGF signaling suppresses the ST phenotype, therefore EGFR inhibitors may be potential treatments for COPD-related muscle ST fiber loss.

Quadriceps miR-542-3p and -5p are elevated in COPD and reduce function by inhibiting ribosomal and protein synthesis.

Reduced physical performance reduces quality of life in patients with chronic obstructive pulmonary disease (COPD). Impaired physical performance is, in part, a consequence of reduced muscle mass and function, which is accompanied by mitochondrial dysfunction. We recently showed that miR-542-3p and miR-542-5p were elevated in a small cohort of COPD patients and more markedly in critical care patients. In mice, these microRNAs (miRNAs) promoted mitochondrial dysfunction suggesting that they would affect physical performance in patients with COPD, but we did not explore the association of these miRNAs with disease severity or physical performance further. We therefore quantified miR-542-3p/5p and mitochondrial rRNA expression in RNA extracted from quadriceps muscle of patients with COPD and determined their association with physical performance. As miR-542-3p inhibits ribosomal protein synthesis its ability to inhibit protein synthesis was also determined in vitro. Both miR-542-3p expression and -5p expression were elevated in patients with COPD (5-fold P < 0.001) and the degree of elevation associated with impaired lung function (transfer capacity of the lung for CO in % and forced expiratory volume in 1 s in %) and physical performance (6-min walk distance in %). In COPD patients, the ratio of 12S rRNA to 16S rRNA was suppressed suggesting mitochondrial ribosomal stress and mitochondrial dysfunction and miR-542-3p/5p expression was inversely associated with mitochondrial gene expression and positively associated with p53 activity. miR-542-3p suppressed RPS23 expression and maximal protein synthesis in vitro. Our data show that miR-542-3p and -5p expression is elevated in COPD patients and may suppress physical performance at least in part by inhibiting mitochondrial and cytoplasmic ribosome synthesis and suppressing protein synthesis. NEW & NOTEWORTHY miR-542-3p and -5p are elevated in the quadriceps muscle of patients with chronic obstructive pulmonary disease (COPD) in proportion to the severity of their lung disease. These microRNAs inhibit mitochondrial and cytoplasmic protein synthesis suggesting that they contribute to impaired exercise performance in COPD.

MicroRNA-542 Promotes Mitochondrial Dysfunction and SMAD Activity and Is Elevated in Intensive Care Unit-acquired Weakness.

RATIONALE: Loss of skeletal muscle mass and function is a common consequence of critical illness and a range of chronic diseases, but the mechanisms by which this occurs are unclear. OBJECTIVES: To identify microRNAs (miRNAs) that were increased in the quadriceps of patients with muscle wasting and to determine the molecular pathways by which they contributed to muscle dysfunction. METHODS: miRNA-542-3p/5p (miR-542-3p/5p) were quantified in the quadriceps of patients with chronic obstructive pulmonary disease and intensive care unit-acquired weakness (ICUAW). The effect of miR-542-3p/5p was determined on mitochondrial function and transforming growth factor-ß signaling in vitro and in vivo. MEASUREMENTS AND MAIN RESULTS: miR-542-3p/5p were elevated in patients with chronic obstructive pulmonary disease but more markedly in patients with ICUAW. In vitro, miR-542-3p suppressed the expression of the mitochondrial ribosomal protein MRPS10 and reduced 12S ribosomal RNA (rRNA) expression, suggesting mitochondrial ribosomal stress. miR-542-5p increased nuclear phospho-SMAD2/3 and suppressed expression of SMAD7, SMURF1, and PPP2CA, proteins that inhibit or reduce SMAD2/3 phosphorylation, suggesting that miR-542-5p increased transforming growth factor-ß signaling. In mice, miR-542 overexpression caused muscle wasting, and reduced mitochondrial function, 12S rRNA expression, and SMAD7 expression, consistent with the effects of the miRNAs in vitro. Similarly, in patients with ICUAW, the expression of 12S rRNA and of the inhibitors of SMAD2/3 phosphorylation were reduced, indicative of mitochondrial ribosomal stress and increased transforming growth factor-ß signaling. In patients undergoing aortic surgery, preoperative levels of miR-542-3p/5p were positively correlated with muscle loss after surgery. CONCLUSIONS: Elevated miR-542-3p/5p may cause muscle atrophy in intensive care unit patients through the promotion of mitochondrial dysfunction and activation of SMAD2/3 phosphorylation.

Reduced HDAC2 in skeletal muscle of COPD patients.

BACKGROUND: Skeletal muscle weakness in chronic obstructive pulmonary disease (COPD) is an important predictor of poor prognosis, but the molecular mechanisms of muscle weakness in COPD have not been fully elucidated. The aim of this study was to investigate the role of histone deacetylases(HDAC) in skeletal muscle weakness in COPD. METHODS AND RESULTS: Twelve COPD patients, 8 smokers without COPD (SM) and 4 healthy non-smokers (NS) were recruited to the study. HDAC2 protein expression in quadriceps muscle biopsies of COPD patients (HDAC2/ß-actin: 0.59 ± 0.34) was significantly lower than that in SM (1.9 ± 1.1, p = 0.0007) and NS (1.2 ± 0.7, p = 0.029). HDAC2 protein in skeletal muscle was significantly correlated with forced expiratory volume in 1 s % predicted (FEV1 % pred) (rs = 0.53, p = 0.008) and quadriceps maximum voluntary contraction force (MVC) (rs = 0.42, p = 0.029). HDAC5 protein in muscle biopsies of COPD patients (HDAC5/ß-actin: 0.44 ± 0.26) was also significantly lower than that in SM (1.29 ± 0.39, p = 0.0001) and NS (0.98 ± 0.43, p = 0.020). HDAC5 protein in muscle was significantly correlated with FEV1 % pred (rs = 0.64, p = 0.0007) but not with MVC (rs = 0.30, p = 0.180). Nuclear factor-kappa B (NF-κB) DNA binding activity in muscle biopsies of COPD patients (10.1 ± 7.4) was significantly higher than that in SM (3.9 ± 7.3, p = 0.020) and NS (1.0 ± 1.2, p = 0.004and significantly correlated with HDAC2 decrease (rs = -0.59, p = 0.003) and HDAC5 (rs = 0.050, p = 0.012). HDAC2 knockdown by RNA interference in primary skeletal muscle cells caused an increase in NF-κB activity, NF-κB acetylation and basal tumour necrosis factor (TNF)-α production, as well as progressive cell death through apoptosis. CONCLUSION: Skeletal muscle weakness in COPD may result from HDAC2 down-regulation in skeletal muscle via acetylation and activation of NF-κB. The restoration of HDAC2 levels might be a therapeutic target for improving skeletal muscle weakness in COPD.

Increased expression of H19/miR-675 is associated with a low fat-free mass index in patients with COPD.

BACKGROUND: Loss of muscle mass and strength is a significant comorbidity in patients with chronic obstructive pulmonary disease (COPD) that limits their quality of life and has prognostic implications but does not affect everyone equally. To identify mechanisms that may contribute to the susceptibility to a low muscle mass, we investigated microRNA (miRNA) expression, methylation status, and regeneration in quadriceps muscle from COPD patients and the effect of miRNAs on myoblast proliferation in vitro. The relationships of miRNA expression with muscle mass and strength was also determined in a group of healthy older men. METHODS: We identified miRNAs associated with a low fat-free mass (FFM) phenotype in a small group of patients with COPD using a PCR screen of 750 miRNAs. The expression of two differentially expressed miRNAs (miR-675 and miR-519a) was determined in an expanded group of COPD patients and their associations with FFM and strength identified. The association of these miRNAs with FFM and strength was also explored in a group of healthy community-dwelling older men. As the expression of the miRNAs associated with FFM could be regulated by methylation, the relative methylation of the H19 ICR was determined. Furthermore, the proportion of myofibres with centralized nuclei, as a marker of muscle regeneration, in the muscle of COPD patients was identified by immunofluorescence. RESULTS: Imprinted miRNAs (miR-675 and from a cluster, C19MC which includes miR-519a) were differentially expressed in the quadriceps of patients with a low fat-free mass index (FFMI) compared to those with a normal FFMI. In larger cohorts, miR-675 and its host gene (H19) were higher in patients with a low FFMI and strength. The association of miR-519a expression with FFMI was present in male patients with severe COPD. Similar associations of miR expression with lean mass and strength were not observed in healthy community dwelling older men participating in the Hertfordshire Sarcopenia Study. Relative methylation of the H19 ICR was reduced in COPD patients with muscle weakness but was not associated with FFM. In vitro, miR-675 inhibited myoblast proliferation and patients with a low FFMI had fewer centralized nuclei suggesting miR-675 represses regeneration. CONCLUSIONS: The data suggest that increased expression of miR-675/H19 and altered methylation of the H19 imprinting control region are associated with a low FFMI in patients with COPD but not in healthy community dwelling older men suggesting that epigenetic control of this loci may contribute to a susceptibility to a low FFMI.