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

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

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 is a potential contributor 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 Ago2 pull-down and the effects of the miRNA on RNA and protein expression were determined by qPCR 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 qPCR in patients with COPD, intensive care unit acquired weakness (ICUAW), 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 mRNAs encoding proteins associated with muscle protein synthesis. The most highly enriched mRNAs encoded proteins required for the Pol I RNA pre-initiation complex (PIC) required for rRNA transcription, (PolR1A and Upstream binding transcription factor, UBTF). In vitro, miR-424-5p reduced 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 UBTF expression and rRNA levels. In humans elevated miR-424-5p associated with markers of disease severity in COPD (FEV1%), patients undergoing aortic surgery (LVEF%) and in patients with ICU acquired weakness (days in ICU). In patients undergoing aortic surgery, pre-operative 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 PIC formation. Increased miR-424-5p expression in patients with conditions associated with muscle wasting is likely to contribute to inhibition of protein synthesis and the loss of muscle mass

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 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 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 and -5p expression were elevated in patients with COPD (5-fold p<0.001) and the degree of elevation associated with impaired lung function (TLCO% and FEV1%) and physical performance (6-minute walk distance %). 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

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

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

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