MuRF-1 and Atrogin-1 Protein Expression and Quadriceps Fiber Size and Muscle Mass in Stable Patients with COPD

INTRODUCTION: Animal studies demonstrate the importance of the E3 ubiquitin ligases, Muscle RING-Finger Protein 1 (MuRF-1) and atrogin-1, in muscle protein degradation during acute muscle atrophy. Small clinical studies suggest MuRF-1 and atrogin-1 expression in the quadriceps muscle is also increased in stable patients with Chronic Obstructive Pulmonary Disease compared to controls. However, it remains unclear whether these ligases have a role in maintaining a muscle-wasted state in COPD patients. METHODS: 32 stable COPD patients (16 with a low fat-free mass index (FFMI), 16 with a normal FFMI) and 15 controls underwent lung function and quadriceps strength tests and a percutaneous quadriceps biopsy. Quadriceps MuRF-1 and atrogin-1 protein were quantified with western blotting. Quadriceps fiber cross-sectional area (CSA) and fiber proportions were determined by immunohistochemistry on muscle sections. MuRF-1 and atrogin-1 levels were compared between COPD patients with and without a low FFMI, and between patients and controls, and correlations between MuRF-1 and atrogin-1 levels and quadriceps fiber CSA in the patients were investigated. RESULTS: Atrogin-1 protein levels were lower in patients than controls, but similar in patients with a low and normal FFMI. MuRF-1 levels did not differ between any groups. MuRF-1 and atrogin-1 levels were not associated with quadriceps fiber CSA or quadriceps strength in patients. CONCLUSIONS: Chronic upregulation of ubiquitin ligases was not evident in the quadriceps muscle of stable COPD patients with a low muscle mass. This does not exclude the possibility of transient increases in ubiquitin ligases during acute catabolic episodes

miR-542 promotes mitochondrial dysfunction and SMAD activity and is raised in ICU 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: We aimed to identify 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: miR-542-3p/-5p were quantified in the quadriceps of patients with COPD and intensive care unit acquired weakness (ICUAW). The effect of miR-542-3p/5p was determined on mitochondrial function and TGF-β signaling in vitro and in vivo. Measurements and main results: miR-542-3p/5p were elevated in patients with COPD but more markedly in patients with ICUAW. In vitro, miR-542-3p suppressed the expression of the mitochondrial ribosomal protein MRPS10, and reduced 12S 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 TGF-β signaling. In mice, miR-542 over-expression caused muscle wasting, 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 TGF-β signaling. In patients undergoing aortic surgery, pre-operative levels of miR-542-3p/5p were positively correlated with muscle loss following surgery. Conclusion; Elevated miR-542-3p/5p may cause muscle atrophy in ICU patients through the promotion of mitochondrial dysfunction and activation of SMAD2/3 phosphorylation

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

© 2016 The Authors. Journal of Cachexia, Sarcopenia and Muscle published by John Wiley & Sons Ltd on behalf of the Society of Sarcopenia, Cachexia and Wasting DisordersBackground: 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

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/β-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/β-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

miR-422a suppresses SMAD4 protein expression and promotes resistance to muscle loss

Background Loss of muscle mass and strength are important sequelae of chronic disease, but the response of individuals is remarkably variable, suggesting important genetic and epigenetic modulators of muscle homeostasis. Such factors are likely to modify the activity of pathways that regulate wasting, but to date, few such factors have been identified. Methods The effect of miR-422a on SMAD4 expression and transforming growth factor (TGF)-β signalling were determined by western blotting and luciferase assay. miRNA expression was determined by qPCR in plasma and muscle biopsy samples from a cross-sectional study of patients with chronic obstructive pulmonary disease (COPD) and a longitudinal study of patients undergoing aortic surgery, who were subsequently admitted to the intensive care unit (ICU). Results miR-422a was identified, by a screen, as a microRNA that was present in the plasma of patients with COPD and negatively associated with muscle strength as well as being readily detectable in the muscle of patients. In vitro, miR-422a suppressed SMAD4 expression and inhibited TGF-beta and bone morphogenetic protein-dependent luciferase activity in muscle cells. In male patients with COPD and those undergoing aortic surgery and on the ICU, a model of ICU-associated muscle weakness, quadriceps expression of miR-422a was positively associated with muscle strength (maximal voluntary contraction r = 0.59, P < 0.001 and r = 0.51, P = 0.004, for COPD and aortic surgery, respectively). Furthermore, pre-surgery levels of miR-422a were inversely associated with the amount of muscle that would be lost in the first post-operative week (r = −0.57, P < 0.001). Conclusions These data suggest that differences in miR-422a expression contribute to the susceptibility to muscle wasting associated with chronic and acute disease and that at least part of this activity may be mediated by reduced TGF-beta signalling in skeletal muscle