miR-542: a novel regulator for muscle mass and function

Loss of skeletal muscle mass and function is a common co-morbidity of a number of chronic diseases including chronic obstructive pulmonary disease (COPD) and a range of critical illnesses as well as in ageing, affecting the quality of life of these individuals. However, the mechanisms by which this occurs have not been completely elucidated. Previous studies in the group identified changed in the levels of several microRNAs in the quadriceps of COPD patients. We focused on the microRNAs that showed the largest and most significant increased expression between patients and controls. One of the elevated microRNAs was miR-542-3p, which was chosen after performing a bioinformatics analysis and saw interesting predicted targets in the muscle wasting context. MiR-542-3p was also found to be elevated in the quadriceps muscle of sarcopenic patients. In this thesis, we aimed to determine if miR-542-5p was also elevated in those two cohorts and if a similar pattern for miR-542-3p/-5p was seen in critical illness such as patients with intensive care unit acquired weakness (ICUAW). We also aimed to identify the molecular pathways by which these miRNAs contributed to muscle impairment or dysfunction. miR-542-3p/5p levels were found elevated in COPD and sarcopenic patients but more markedly elevated in patients with ICUAW. In vitro, miR-542-3p decreased the expression of mitochondrial (MRPS10) and cytoplasmic (RPS23) ribosomal proteins and reduced 12S and 18S ribosomal RNA (rRNA) suggesting mitochondrial and cytoplasmic ribosomal stress. miR-542-3p/-5p promoted the nuclear accumulation of phospho SMAD2/3 and suppressed expression of SMURF1, SMAD7 and PPP2CA which are inhibitors of the system, indicative of increased TGF-β signalling. In vivo, miR-542 over expression caused muscle wasting in the targeted muscle, decreased mitochondrial function, 12S rRNA and 18S rRNA levels and SMAD7 expression, consistent with the effects of the miRNA in vitro. In patients with ICUAW similar results were observed, the expression of 12S and 18S rRNA and SMURF1, SMAD7 and PPP2CA were reduced, suggesting mitochondrial and cytoplasmic ribosomal stress and increased TGF-β signalling.Open Acces

miR‐424‐5p reduces ribosomal RNA and protein synthesis in muscle wasting

Abstract Background A 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). Methods Targets 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. Results Pull‐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. Conclusions These 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