BackgroundA loss of muscle mass occurs as a consequence of a range of chronic and acute diseases as well as in older age. This wasting results from an imbalance of protein synthesis and degradation with a reduction in synthesis and resistance to anabolic stimulation often reported features. Ribosomes are required for protein synthesis, so changes in the control of ribosome synthesis are potential contributors to muscle wasting. MicroRNAs (miRNAs) are known regulators of muscle phenotype and have been shown to modulate components of the protein synthetic pathway. One miRNA that is predicted to target a number of components of protein synthetic pathway is miR‐424‐5p, which is elevated in the quadriceps of patients with chronic obstructive pulmonary disease (COPD).MethodsTargets of miR‐424‐5p were identified by Argonaute2 pull down, and the effects of the miRNA on RNA and protein expression were determined by quantitative polymerase chain reaction and western blotting in muscle cells in vitro. Protein synthesis was determined by puromycin incorporation in vitro. The miRNA was over‐expressed in the tibialis anterior muscle of mice by electroporation and the effects quantified. Finally, quadriceps expression of the miRNA was determined by quantitative polymerase chain reaction in patients with COPD and intensive care unit (ICU)‐acquired weakness and in patients undergoing aortic surgery as well as in individuals from the Hertfordshire Sarcopenia Study.ResultsPull‐down assays showed that miR‐424‐5p bound to messenger RNAs encoding proteins associated with muscle protein synthesis. The most highly enriched messenger RNAs encoded proteins required for the Pol I RNA pre‐initiation complex required for ribosomal RNA (rRNA) transcription, (PolR1A and upstream binding transcription factor). In vitro, miR‐424‐5p reduced the expression of these RNAs, reduced rRNA levels, and inhibited protein synthesis. In mice, over‐expression of miR‐322 (rodent miR‐424 orthologue) caused fibre atrophy and reduced upstream binding transcription factor expression and rRNA levels. In humans, elevated miR‐424‐5p associated with markers of disease severity in COPD (FEV1%), in patients undergoing aortic surgery (LVEF%), and in patients with ICU‐acquired weakness (days in ICU). In patients undergoing aortic surgery, preoperative miR‐424‐5p expression in skeletal muscle was associated with muscle loss over the following 7 days.ConclusionsThese data suggest that miR‐424‐5p regulates rRNA synthesis by inhibiting Pol I pre‐initiation complex formation. Increased miR‐424‐5p expression in patients with conditions associated with muscle wasting is likely to contribute to the inhibition of protein synthesis and loss of muscle mass.

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.

BackgroundLoss 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.MethodsThe 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).ResultsmiR‐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).ConclusionsThese 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.

Pulmonary arterial hypertension ( PAH ) is characterised by remodelling of the pulmonary vasculature leading to right ventricular hypertrophy. Here, we show that miR‐322‐5p (the rodent orthologue of miR‐424‐5p) expression is decreased in the right ventricle of monocrotaline‐treated rats, a model of PAH , whereas a putative target insulin‐like growth factor 1 ( IGF ‐1) is increased. IGF ‐1 mRNA was enriched 16‐fold in RNA immunoprecipitated with Ago2, indicating binding to miR‐322‐5p. In cell transfection experiments, miR‐322‐5p suppressed the activity of a luciferase reporter containing a section of the IGF ‐1 3′ untranslated region ( UTR ) as well as IGF ‐1 mRNA and protein levels. Taken together, these data suggest that miR‐322 targets IGF ‐1, a process downregulated in PAH ‐related RV hypertrophy.

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.

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.

While there have been significant advances in drug discovery for diabetes mellitus over the past couple of decades, there is an opportunity and need for improved therapies. While type 2 diabetic patients better manage their illness, many of the therapeutics in this area are peptide hormones with lengthy sequences and a molecular structure that makes them challenging and expensive to produce. Using machine learning, we present novel anti-diabetic peptides which are less than 16 amino acids in length, distinct from human signalling peptides. We validate the capacity of these peptides to stimulate glucose uptake and Glucose transporter type 4 (GLUT4) translocation in vitro. In obese insulin-resistant mice, predicted peptides significantly lower plasma glucose, reduce glycated haemoglobin and even improve hepatic steatosis when compared to treatments currently in use in a clinical setting. These unoptimised, linear peptides represent promising candidates for blood glucose regulation which require further evaluation. Further, this indicates that perhaps we have overlooked the class of natural short linear peptides, which usually come with an excellent safety profile, as therapeutic modalities.

The microRNA miR-1 is an important regulator of muscle phenotype including cardiac muscle. Down-regulation of miR-1 has been shown to occur in left ventricular hypertrophy but its contribution to right ventricular hypertrophy in pulmonary arterial hypertension are not known. Previous studies have suggested that miR-1 may suppress transforming growth factor-beta (TGF-β) signalling, an important pro-hypertrophic pathway but only indirect mechanisms of regulation have been identified. We identified the TGF-β type 1 receptor (TGF-βR1) as a putative miR-1 target. We therefore hypothesized that miR-1 and TGF-βR1 expression would be inversely correlated in hypertrophying right ventricle of rats with pulmonary arterial hypertension and that miR-1 would inhibit TGF-β signalling by targeting TGF-βR1 expression. Quantification of miR-1 and TGF-βR1 in rats treated with monocrotaline to induce pulmonary arterial hypertension showed appropriate changes in miR-1 and TGF-βR1 expression in the hypertrophying right ventricle. A miR-1-mimic reduced enhanced green fluorescent protein expression from a reporter vector containing the TGF-βR1 3'untranslated region and knocked down endogenous TGF-βR1. Lastly, miR-1 reduced TGFβ activation of a (mothers against decapentaplegic homolog) SMAD2/3-dependent reporter. Taken together, these data suggest that miR-1 targets TGF-βR1 and reduces TGF-β signalling, so a reduction in miR-1 expression may increase TGF-β signalling and contribute to cardiac hypertrophy.