American Journal of Respiratory and Critical Care Medicine

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-β 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-β 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-β 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.

Scientific Knowledge on the Subject

Muscle dysfunction associated with critical illness, intensive care unit–acquired weakness (ICUAW), is a common complication that has profound prognostic and economic implications. The mechanisms that result in muscle dysfunction are not fully understood, but a loss of mitochondrial function and increased atrophic signalling contribute to muscle dysfunction. MicroRNAs are critical regulators of cell phenotype, so are likely to be important contributors to muscle wasting.

What This Study Adds to the Field

MicroRNA-542-3p (miR-542-3p) and -5p were increased in the quadriceps of patients with ICUAW. Increased miR-542-3p targets mitochondrial ribosomal proteins, reducing mitochondrial translation and leading to mitochondrial dysfunction in vitro and in vivo. Increased miR-542-5p promoted SMAD signalling and reduced the expression of SMAD7, SMURF1, and SMAD phosphatases in vitro and in an animal model. This study therefore identifies miR-542-3p and -5p as mediators of mitochondrial and muscle dysfunction in ICUAW.

Admission to the intensive care unit (ICU) is often accompanied by a marked loss of skeletal muscle mass and function (1) that extends weaning time (2) and leads to a reduction in quality of life and an increase in mortality (3, 4). Recovery from muscle wasting can be very slow with many patients still having reduced function 5 years after critical illness (5). Muscle dysfunction also occurs in several chronic diseases including chronic obstructive pulmonary disease (COPD) contributing to increased morbidity and mortality (6). Skeletal muscle loss occurs as a result of an imbalance between protein synthesis and breakdown, with similar signaling pathways being implicated. For example, increased growth differentiation factor (GDF)-15 and reduced insulin-like growth factor-1 have been reported in both critical illness and COPD (710). In addition to occurring as a consequence of muscle loss, decreased function also results from a reduction in the muscles’ capacity to generate ATP, associated with a reduction in the activity of mitochondrial protein complexes, notably complexes I, III, and IV (1113). These complexes contain proteins encoded by mitochondrial DNA that require translation by mitochondrial ribosomes.

Although muscle mass and oxidative capacity are primarily thought to be controlled by separate processes (protein and mitochondrial turnover), they are to some extent interdependent, because increasing oxidative capacity requires protein synthesis and increasing protein synthesis requires energy. Similarly, reducing oxidative capacity requires protein breakdown and autophagy, processes that operate during muscle breakdown. It is perhaps therefore not surprising that the loss of muscle mass and oxidative capacity often occur together (14) raising the possibility that there are common regulatory mechanisms or factors. Understanding such factors is important in identifying novel therapeutic approaches to treating muscle dysfunction.

MicroRNAs (miRNAs) are small RNAs that control the translation and degradation of sets of mRNAs, modulating biologic responses and cell phenotype by regulating the levels of key proteins in multiple biologic pathways. For example, miRNA-1 (miR-1) is elevated during muscle differentiation and regeneration (15), contributing to myogenesis both by suppressing the expression of HDAC4 (an inhibitor of several myogenic transcription factors) and by entering mitochondria and increasing mitochondrial translation (16). Consistent with this, we and others have previously reported reduced expression of miR-1 is associated with muscle wasting in COPD (17), renal failure (18), and ICU-acquired weakness (ICUAW) (7), although there is some discrepancy in the COPD literature (10). We have also identified a miRNA pattern associated with muscle mass in patients with COPD that is distinct from that in healthy individuals (19). The miRNAs identified were associated with pluripotency and regeneration, indicating that patients who lost the most muscle were unable to respond sufficiently to the physiologic stress imposed by disease. This observation suggests that some patients are more susceptible to muscle atrophy in the presence of disease but does not identify factors associated with disease that drive the loss of muscle, nor did the miRNA pattern identified associate with the loss of oxidative capacity.

Consequently, we hypothesized that there would be changes in miRNA expression in the quadriceps of patients with COPD that promoted atrophy and a loss of oxidative capacity. In this study, we therefore reanalyzed our screen to identify miRNAs associated with COPD that were predicted to target pathways controlling protein turnover and energy balance; miR-542-3p and -5p fulfilled these criteria (mitochondrial translation and transforming growth factor [TGF]-β signaling, respectively). However analysis of the expression of these miRNAs in patients with established ICUAW showed that they were more markedly increased in those patients than in patients with COPD, consistent with the more rapid muscle atrophy that these patients undergo. We confirmed the ability of these miRNAs to target the appropriate biochemical pathways both in vitro and in vivo and showed that expression of the miRNA could cause muscle wasting in vivo. Finally, we analyzed the expression of these miRNAs in the response of patients to the insult of aortic surgery, which we have characterized as a human model of ICUAW (8). Some of the results of these studies have been previously reported in the form of an abstract (2022).

Subjects
COPD cohort

Stable patients with COPD according to the Global Initiative for Chronic Obstructive Lung Disease guidelines 2004 (23) were enrolled from clinics at the Royal Brompton Hospital. Exclusion criteria are described in the online supplement. Healthy age-matched control subjects were recruited by advertisement. COPD subjects in this study form part of a larger well-phenotyped cohort described by Natanek and coworkers (14). Samples from 14 patients and 7 control subjects were used in the initial miRNA screen. The validation cohort included samples from 24 patients and 12 control subjects, none of which formed part of the screen. Demographic data for these patients are shown in Tables E1 and E2 in the online supplement.

Physiologic measurements

Details of the methods used to assess physiologic parameters (body composition, lung function, physical performance, and strength) are given in the online supplement. Muscle biopsy was performed by percutaneous needle biopsy in the mid-thigh of the vastus lateralis, of the same leg in which strength had been tested, under local anesthesia using the Bergstrom technique (24).

ICUAW cohort

This cohort (17 patients with ICUAW and 7 control subjects) has been described previously (7) and recruitment details are given in the online supplement. The muscle biopsies were taken from the rectus femoris by the Bergstrom technique (24) or as open biopsies. There was no difference between patients and control subjects in age (62 ± 18 and 68 ± 11 yr) or in body mass index (27.8 ± 3.9 and 25.2 ± 3.8 kg/m2).

Aortic surgery cohort

Patients provided written informed consent and the study was approved by the National Research Ethics Committee (study 13/LO/0479). Patients undergoing elective cardiothoracic surgery at Royal Brompton Hospital were recruited. The principal inclusion criterion was a high-risk elective aortic surgical procedure requiring admission to the ICU. Presurgical exclusion criteria included preexisting muscular or neuromuscular disease, malignancy, and contraindication to muscle biopsy. Patients were also excluded if clinical instability precluded a biopsy after surgery. Rectus femoris cross-sectional area (RFCSA) was determined by ultrasound before surgery and 7 days after surgery, as previously described (25). An open biopsy was taken after the induction of anesthesia but before the start of surgery from the rectus femoris and a second biopsy was taken using the Bergstrom technique 24 hours after surgery. Blood samples were collected before surgery and regularly during the first 7 days after surgery. Demographic data for these patients are shown in Table E3.

Quantification of RNA and Protein in Biologic Samples

Details of the analysis methods for RNA and protein have been described before and are detailed in the online supplement.

In Vitro and Animal Experiments

Targets of miR-542-3p/5p were identified by Ago-2 pull down. The effects of the miRNAs on gene and protein expression, and on mitochondrial function and TGF-β signaling were determined in LHCN-M2 cells, a human skeletal muscle cell line. Overexpression of the miRNA in mouse muscle was achieved by electroporation of an enhanced green fluorescent protein (EGFP)-miRNA construct (pCAGGS-miR-542). Experimental procedures for the in vitro and animal experiments are detailed in the online supplement.

Statistical Analysis

Gene expression data were log transformed to produce a normal distribution and stabilize variance. Pearson correlations were performed for correlation analysis assuming linearity (Aabel). Differences between groups were calculated using Student’s t test for normally distributed data or by Mann-Whitney U test for nonparametric data (Aabel). In vitro data shown were produced in three independent experiments; for gene expression data and luciferase assays treatments were performed in sextuplet within each experiment.

Analyzing our original screen (19) of 14 male patients with Global Initiative for Chronic Obstructive Lung Disease 3–4 COPD and 7 age-matched healthy male control subjects to identify miRNAs associated with disease showed that 26 miRNAs were suppressed at P less than 0.01 (Table 1). The most statistically significantly down-regulated miRNAs were miR-144 and miR-499. Only six miRNAs were increased in the muscle of patients with COPD, five of which (miR-542-5p, miR-542-3p, miR-424, miR-424*, and miR-450a) came from a single locus on the X chromosome. The most up-regulated miRNA from this cluster was miR-542-5p and the miRNA showing the most significant increase was miR-542-3p. Analyzing the expression of miR-542-3p in a larger cohort of male control subjects and patients with the same inclusion criteria (n = 12 control subjects and n = 24 patients with COPD) confirmed the elevation of miR-542-3p twofold (P = 0.012) (see Figure E1) indicating that the finding was robust.

Table 1. miRNAs Differentially Expressed in the Quadriceps of Patients with COPD Compared with Control Subjects

miRNAChromosomal LocationMean Fold Change COPD/ControlP Value
hsa-miR-499-5p20q11.220.2110.006
hsa-miR-144#17q11.20.2500.006
hsa-miR-3674q250.3070.005
hsa-miR-302b4q250.3350.002
hsa-miR-31#9p21.30.3440.008
hsa-miR-126#9q34.30.3590.001
hsa-miR-374a#xq13.20.3740.005
hsa-miR-20a#13q31.30.3770.005
hsa-miR-651xp22.310.3830.006
hsa-miR-145#5q320.4400.006
hsa-miR-319p21.30.4490.001
hsa-miR-329q31.30.4560.002
hsa-miR-302a4q250.4770.006
hsa-miR-520c-3p19q13.420.5140.006
hsa-miR-545xq13.20.5300.001
hsa-miR-19015q22.20.5310.005
hsa-miR-5988p23.10.5710.003
hsa-miR-136#14q32.310.5850.001
hsa-miR-21011p15.50.6040.004
hsa-miR-15019q13.330.6280.008
hsa-miR-576-3p4q250.6490.008
hsa-miR-3405q35.30.6830.004
hsa-miR-590-5p7q11.230.7020.004
hsa-miR-652xq230.7230.007
hsa-miR-30c1p34.2, 6q130.7440.004
hsa-miR-140-5p16q22.10.7860.008
hsa-miR-1282q21.3, 3p21.31.7310.000
hsa-miR-542-3pxq26.34.0730.000
hsa-miR-424xq26.34.1240.000
hsa-miR-450axq26.34.7280.000
hsa-miR-424#xq26.36.7230.004
hsa-miR-542-5pxq26.39.6170.001

Definition of abbreviations: COPD = chronic obstructive pulmonary disease; miR = miRNA; miRNA = microRNA.

We had previously shown similar changes in miR-499 in patients with ICUAW (7), so we determined whether miR-542-3p and -5p were increased in samples from the same patients and found that they were markedly more elevated than in the patients with COPD (15-fold and 50-fold, respectively) (Figures 1A and 1B) and correlated with length of hospital stay (Figures 1C and 1D). Moreover miR-542-5p was higher in those with a Sequential Organ Failure Assessment score at the time of biopsy greater than 10 than in patients with a Sequential Organ Failure Assessment score less than 10 (Figures 1E and 1F).

miR-542-3p Promotes Mitochondrial Ribosomal Stress

For these miRNAs to be important regulators of muscle phenotype in disease they must target appropriate biochemical pathways. Bioinformatic analysis using miRwalk (26) showed that miR-542-3p may target components of the mitochondrial ribosome including mitochondrial ribosomal protein (MRP) S2, S10, S18C, S25, S26, and S27, so may induce mitochondrial ribosomal stress, thereby reducing the expression of mitochondrially encoded proteins, which would be expected to reduce mitochondrial function including energy production. Similar analysis showed that miR-542-5p would target inhibitors of the TGF-β signaling pathway, which is known to mediate muscle atrophy, suggesting that it may increase TGF-β signaling. Hence miR-542-3p/5p are predicted to target pathways that affect both muscle mass and function.

Ago-2 pull-down followed by quantitative polymerase chain reaction showed enrichment of all the MRPs tested (MRP S2, S27, S18C, and S10) in RNA isolated from miR-542-3p–transfected cells compared with cells transfected with control miRNA (see Figure E2A). To determine whether these interactions resulted in reduced MRP and thereby suppressed mitochondrial translation, we quantified the expression of one target (MRPS10) and one product of mitochondrial protein synthesis (Cytb5). Protein levels of MRPS10 were reduced in response to transfection with miR-542-3p by 50%. Cytb5 protein levels were also reduced but to a much smaller extent (Figures 2A–2C). Transfection with miR-542-3p also reduced the mitochondrial membrane potential measured using Mitotracker Red and JC1 (Figure 2E; see Figure E2B). Both effects were reversible by cotransfection with the antagomiR (Figure 2E; see Figure E2C). There was no effect of the miRNA on the amount of mitochondrial DNA relative to nuclear DNA, indicating that the results were not caused by a reduction in mitochondrial content.

Ribosome stress induced by reduced small subunit proteins is accompanied by a decrease in ribosomal RNA especially the small subunit RNA (27). Consistent with the induction of ribosome stress, miR-542-3p suppressed the expression of the 12S ribosomal RNA (rRNA) to a greater extent than the 16S rRNA (mitochondrial rRNAs from the small and large subunits, respectively) and these changes were reversible with an antagomiR (Figure 2D).

miR-542-5p Causes Ligand-Independent SMAD2/3 Phosphorylation and Suppresses SMAD7 In Vitro

Bioinformatic analysis predicted that miR-542-5p targets several components of the TGF-β family signaling pathway, including SMAD7 and SMURF1 (which inhibit the TGF-β type I receptor [28]), and the proteins that dephosphorylate and inactivate the SMAD2/3 complex (see PPP2CA in the online supplement Results and Figure E3). Ago2 pull-down and quantitative polymerase chain reaction confirmed enrichment for SMURF1 and PPP2CA (see Figure E3A) but SMAD7 mRNA expression was too low to prove targeting.

The effect of miR-542-5p on TGF-β signaling was determined using a SMAD-dependent luciferase reporter (29). In the absence of exogenous ligand, miR-542-5p increased luciferase activity twofold (analysis of variance, P < 0.001) (Figure 3B). Furthermore, transfection of cells with a dominant inhibitory form of the TGF-β type II receptor inhibited the effects of the miRNA suggesting that a functional receptor complex was required for the response (Figure 3C). After phosphorylation, SMAD2/3 translocates to the nucleus and, consistent with the increase in basal SMAD-dependent luciferase activity, immunofluorescence assay showed increased nuclear pSMAD2/3 in cells transfected with miR-542-5p compared with scrambled miRNA (analysis of variance, P < 0.001) (Figures 3D and 3E; see Figure E4). Western blotting confirmed increased pSMAD2/3 in the presence of miR-542-5p (see Figure E4).

To determine which predicted targets of miR-542-5p were suppressed, the mRNAs for SMAD7, SMURF1, and PPP2CA were quantified by polymerase chain reaction. miR-542-5p suppressed the expression of all components (Figure 4A). SMAD7 expression was not readily detectable by Western blotting in LHCN-M2 cells, but was detectable by immunoprecipitation, which demonstrated that miR-542-5p suppressed the expression of SMAD7 (Figures 4B and 4C) suggesting that SMAD7 is targeted by the miRNA. However, because we could not confirm an interaction between the miRNA and SMAD7 mRNA this reduction may be indirect.

Overexpression of miR-542 Promotes Mitochondrial Dysfunction, Suppresses SMAD7, and Causes Atrophy In Vivo

To determine whether the effects of miR-542-3p and -5p observed in vitro could also occur in vivo and to determine whether it could cause atrophy, miR-542 was overexpressed in the tibialis anterior of mice by electroporation with positively transfected fibers identified by coexpression of EGFP and compared with expression of EGFP alone. Three days after electroporation, 37% of the fibers expressed detectable EGFP (38% EGFP-only leg and 36% miR-542 leg) (see Figure E5). miR-542-3p levels were 10-fold higher than in the control leg (P = 0.04 paired Student’s t test). The miR-542–expressing muscle was smaller than the contralateral muscle expressing EGFP alone (Figure 5A). The average diameter of transfected, EGFP-expressing fibers in the miR-542–transfected leg were smaller than the untransfected fibers in the same leg (Figure 5B; see Figure E5A). There was no difference in size between the transfected and untransfected fibers in the muscle of the control-transfected leg, or between the untransfected fibers in the two muscles. Quantification of mitochondrial complex I activity (the first component of the electron transport chain) showed a marked reduction in the muscles expressing the miRNA compared with those expressing the EGFP alone (Figure 5C). Similarly, JC-1 staining of the mitochondria showed a reduction in mitochondrial membrane potential (Figure 5D).

Because the electroporation does not transfect all of the tissue, we enriched the samples by laser capture microdissection to quantify the expression of rRNAs in transfected fibers. This analysis showed a significant reduction in 12S rRNA in fibers expressing EGFP and miR-542 compared with those expressing EGFP alone that was greater than any reduction in 16S rRNA consistent with mitochondrial ribosomal stress (Figure 5E). Quantification of SMAD7 protein also showed a marked down-regulation of the protein in the miR-542–expressing leg (Figure 5F; see Figure E5).

Patients with ICUAW Show Reduced Expression of 12S rRNA and Inhibitors of TGF-β Signaling

Mitochondrial rRNAs and SMAD7, SMURF1, and PPP2CA were analyzed in patients with ICUAW to determine whether a similar pattern of changes occurred. Both the 12S and 16S rRNAs were reduced but the reduction in 12S rRNA was larger resulting in a significant reduction in the 12S:16S ratio (Figures 6A–6C) consistent with the in vitro and in vivo findings. Furthermore the expression of SMURF1 and PPP2CA were reduced at the mRNA level (Figure 7A), and both mRNA and protein levels of SMAD7 were reduced (Figures 7B and 7C). Our previous analysis of the ICUAW cohort samples analyzed here showed that patients with established ICUAW have higher nuclear pSMAD2/3 and increased expression of Cyr-61, a TGF-β–responsive gene (7). These data are consistent with mitochondrial ribosome stress and increased TGF-β signaling in the patients.

miR-542-3p/5p Is Associated with Muscle Loss after Cardiopulmonary Bypass Surgery

Half of patients admitted to the ICU after aortic surgery lose more than 10% of their RFCSA over the subsequent 7 days (8). We therefore quantified change in muscle size over 7 days in a separate cohort of 40 patients in whom we also took muscle biopsies immediately before surgery (see Table E3). Nineteen of these patients lost more than 10% RFCSA after surgery. Presurgery expression of miR-542-3p and -5p were weakly associated with presurgery left ventricular ejection fraction (LVEF%) (Figures 8A and 8B).

The preoperative expression of miR-542-3p and -5p was higher in patients who lost more than 10% RFCSA in the 7 days after surgery than in those who lost less than 10% RFCSA (Figures 8C and 8D). Furthermore, preoperative expression of these miRNAs correlated with the amount of muscle lost (Figures 8E and 8F) and length of hospital stay (see Figures E6A and E6B). Age and preoperative LVEF% were also associated with muscle loss after surgery (see Figure E7). However, in multiple regression analysis using LVEF%, age, and either miR-542-3p or miR-542-5p expression before surgery, only the miRNAs were retained as having a significant association.

ICUAW has a significant impact on patient mortality and morbidity, as well as on the economics of critical illness. A range of different factors (including oxidative stress, sepsis, and growth factor dysregulation [30]) mediate ICUAW by interfering with intracellular processes. Our data show that quadriceps expression of two miRNAs, miR-542-3p/5p, which are predicted to target processes essential for muscle maintenance, is markedly elevated in patients with established ICUAW suggesting that these miRNAs contribute to the wasting process. The in vitro studies confirm that miR-542-3p/5p regulate appropriate pathways (mitochondrial function and SMAD2/3 signaling) in a manner that would contribute to wasting. Furthermore, overexpression of miR-542, to a similar fold change as that seen in patients with ICUAW, caused muscle wasting and mitochondrial dysfunction in mice. Finally we show that in patients admitted to the ICU after aortic surgery, the level of expression of miR-542-3p/5p at the time of surgery is associated with muscle wasting in the 7 days after surgery. Consequently, we have demonstrated that the expression of specific miRNAs is associated with wasting in humans, identified mechanisms by which they may promote wasting, and demonstrated that they can cause wasting in mice.

miR-542-3p and Mitochondrial Dysfunction

Reduced mitochondrial number and mitochondrial dysfunction have been previously demonstrated in the quadriceps of patients in the ICU (12). Indeed, mitochondrial dysfunction has been suggested as a mechanism leading to the metabolic failure associated with ICUAW (31). One feature of the reported mitochondrial dysfunction in ICUAW is a reduction in the activity of electron transfer complexes that require mitochondrially translated proteins (notably complex I, III, and IV [11, 12]). Reduced activity of these complexes as a consequence of mitochondrial ribosomal stress is also found in individuals born with mutations in MRPS16 and MRPS22 (32, 33). The identification here of a miRNA that promotes mitochondrial ribosomal stress (as identified by the reduction in MRPS10, 12S rRNA, and mitochondrial membrane potential), together with the demonstration of a reduction in the 12S to 16S rRNA ratio in patients with established ICUAW suggest two novel findings: that mitochondrial ribosomal stress contributes to metabolic failure and wasting in ICUAW; and that elevated miR-542-3p is a contributor to this process.

At first glance, there is a discrepancy between our data and that of Fredriksson and coworkers (34), who showed no decrease in mitochondrial protein synthesis in patients with ICUAW. However, in that study they measured protein synthesis from whole mitochondria. Most proteins in mitochondria are encoded by nuclear genes, translated in the cytoplasm and imported into mitochondria with only 13 proteins translated by mitochondrial ribosomes. Consequently, although their experiment demonstrates no change in the synthesis of mitochondrial proteins it does not measure the rate of translation within mitochondria of key components of the complexes involved in oxidative phosphorylation because the bulk of the proteins assayed will have been synthesized in the cytoplasm.

miR-542-5p and TGF-β Signaling

Myostatin is an important regulator of muscle mass that promotes phosphorylation of SMAD2/3. Inhibiting the myostatin pathway and thereby suppressing SMAD2/3 phosphorylation has been suggested as a means of treating ICUAW (www.clinicaltrials.gov reference NCT01321320). However, circulating levels of myostatin did not increase in patients admitted to the ICU after aortic surgery (8). Our studies suggest that increased expression of miR-542-5p contributes to muscle wasting by promoting SMAD2/3 phosphorylation, a down-stream effect of myostatin binding to TGF-β receptors, thereby potentially bypassing the need for significantly elevated myostatin. The effect of the dominant inhibitory receptor, together with inability of miR-542-5p to increase luciferase activity in the presence of 5 ng/ml TGF-β, indicate that miR-542-5p is not acting via a separate pathway because the effects are not additive.

Our data indicate that miR-542-5p increases pSMAD2/3 by suppressing the inhibitory components of the TGF-β signaling pathway with a reduction in the expression of the SMAD7 and SMURF1, inhibitors of TGF-β type I receptors, and of the phosphatases that limit TGF-β signaling by dephosphorylating and inactivating SMADs. Our observation that the increase in luciferase activity in the absence of exogenous ligand was inhibited by the dominant inhibitory receptor suggests that it occurs as a result of either low-level receptor kinase activity in the absence of ligand or a sensitization of the signal to the low levels of TGF-β receptor ligands present in the serum used to culture the cells or produced by the cells themselves.

Our previous analysis of the samples from the ICUAW cohort analyzed here showed that patients with established ICUAW have higher nuclear pSMAD2/3 and increased expression of Cyr-61, a TGF-β–responsive gene (7). Here we show that these patients also have increased miR-542-5p, alongside reduced expression of SMAD7, SMURF1, and PPP2CA, suggesting that these factors contribute to the observed increases in TGF-β signaling.

miR-542-5p is not the only miRNA from this locus to target components of the TGF-β signaling pathway. For example, miRNAs from the miR-424-503 polycistron reduce SMAD7 and promote TGF-β signaling (35). Furthermore, because these miRNAs are also regulated by TGF-β signaling it is possible that they form part of a positive feedback loop (36). It seems likely therefore that increased expression of these miRNAs also contributes to elevated TGF-β signaling and atrophy in ICUAW.

miR-542 and Muscle Atrophy

The combined effects of miR-542 on mitochondrial function and TGF-β signaling suggest that it contributes to muscle wasting and dysfunction. We confirmed the potential of these miRNAs to promote wasting by overexpression in the muscle of mice. This overexpression caused marked muscle loss within 3 days that was accompanied by mitochondrial dysfunction and reduced SMAD7 expression, demonstrating the potential of miR-542 to drive atrophy. This loss of muscle is larger and more rapid than that achieved by overexpression of myostatin or GDF-15 using the same promoter as used here (9, 37).

Clinical Implications

The data presented here identify miR-542-3p/5p as potential therapeutic targets in patients with ICUAW. Therapy could take the form of antagomiRs for miR-542-3p/5p or the suppression of the mechanisms that drive the expression of the miRNAs. The data also show that some aspects of the propensity to waste are established in patients before they become critically ill because patients with the highest preaortic surgery miR-542 levels wasted more than those with the lowest, raising the possibility that a therapy could be given to vulnerable patients before major surgery. In addition, preoperative miR-542 levels could be used to stratify patients in high or lower risk of perioperative and postoperative muscle atrophy so that high-risk patients could have intensive nutritional and rehabilitation input. Because we identified miR-542 as being elevated in COPD and because the expression of this miRNA was associated with LVEF in our aortic surgery patients, it is possible that this miRNA locus contributes to muscle wasting in a wider range of conditions but that its effects are greatest with the insults imposed as a result of critical illness. Contributing to both chronic and acute wasting is not without precedent because many pathways including GDF-15 and insulin-like growth factor-1 have been associated with both chronic and acute wasting (810). Furthermore, the pathways leading to muscle loss are common with a suppression of protein synthesis relative to degradation and mitochondrial dysfunction being end components.

Critique of the Study

The study presents a cross-sectional analysis demonstrating elevation of miR-542-3p/5p in ICUAW and a longitudinal study showing an association of these miRNAs with muscle loss after surgery. We cannot therefore directly demonstrate that elevation of the miRNA causes either the reduction in oxidative capacity or promotes the loss of muscle mass in humans. However, the demonstration that the miRNAs cause appropriate biochemical changes in vitro, muscle wasting in mice, and the demonstration of appropriate phenotypic manifestations in humans strengthens our conclusions.

Conclusions

Our data indicate that expression of miR-542-3p/5p is elevated in patients with ICUAW. These miRNAs target the synthesis of MRPs and of inhibitors of TGF-β signaling. In mice their overexpression promotes mitochondrial dysfunction and muscle wasting. Elevation of these miRNAs in the muscle of patients with ICUAW is a very plausible mechanism contributing to the loss of oxidative capacity and muscle wasting observed in this condition.

1. Man WD, Kemp P, Moxham J, Polkey MI. Skeletal muscle dysfunction in COPD: clinical and laboratory observations. Clin Sci (Lond) 2009;117:251264.
2. De Jonghe B, Bastuji-Garin S, Sharshar T, Outin H, Brochard L. Does ICU-acquired paresis lengthen weaning from mechanical ventilation? Intensive Care Med 2004;30:11171121.
3. De Jonghe B, Sharshar T, Lefaucheur JP, Authier FJ, Durand-Zaleski I, Boussarsar M, Cerf C, Renaud E, Mesrati F, Carlet J, et al.; Groupe de Réflexion et d’Etude des Neuromyopathies en Réanimation. Paresis acquired in the intensive care unit: a prospective multicenter study. JAMA 2002;288:28592867.
4. Ali NA, O’Brien JM Jr, Hoffmann SP, Phillips G, Garland A, Finley JC, Almoosa K, Hejal R, Wolf KM, Lemeshow S, et al.; Midwest Critical Care Consortium. Acquired weakness, handgrip strength, and mortality in critically ill patients. Am J Respir Crit Care Med 2008;178:261268.
5. Fletcher SN, Kennedy DD, Ghosh IR, Misra VP, Kiff K, Coakley JH, Hinds CJ. Persistent neuromuscular and neurophysiologic abnormalities in long-term survivors of prolonged critical illness. Crit Care Med 2003;31:10121016.
6. Swallow EB, Reyes D, Hopkinson NS, Man WD, Porcher R, Cetti EJ, Moore AJ, Moxham J, Polkey MI. Quadriceps strength predicts mortality in patients with moderate to severe chronic obstructive pulmonary disease. Thorax 2007;62:115120.
7. Bloch SA, Lee JY, Syburra T, Rosendahl U, Griffiths MJ, Kemp PR, Polkey MI. Increased expression of GDF-15 may mediate ICU-acquired weakness by down-regulating muscle microRNAs. Thorax 2015;70:219228.
8. Bloch SA, Lee JY, Wort SJ, Polkey MI, Kemp PR, Griffiths MJ. Sustained elevation of circulating growth and differentiation factor-15 and a dynamic imbalance in mediators of muscle homeostasis are associated with the development of acute muscle wasting following cardiac surgery. Crit Care Med 2013;41:982989.
9. Patel MS, Lee J, Baz M, Wells CE, Bloch S, Lewis A, Donaldson AV, Garfield BE, Hopkinson NS, Natanek A, et al. Growth differentiation factor-15 is associated with muscle mass in chronic obstructive pulmonary disease and promotes muscle wasting in vivo. J Cachexia Sarcopenia Muscle 2016;7:436448.
10. Puig-Vilanova E, Martínez-Llorens J, Ausin P, Roca J, Gea J, Barreiro E. Quadriceps muscle weakness and atrophy are associated with a differential epigenetic profile in advanced COPD. Clin Sci (Lond) 2015;128:905921.
11. Brealey D, Brand M, Hargreaves I, Heales S, Land J, Smolenski R, Davies NA, Cooper CE, Singer M. Association between mitochondrial dysfunction and severity and outcome of septic shock. Lancet 2002;360:219223.
12. Jiroutková K, Krajčová A, Ziak J, Fric M, Waldauf P, Džupa V, Gojda J, Němcova-Fürstová V, Kovář J, Elkalaf M, et al. Mitochondrial function in skeletal muscle of patients with protracted critical illness and ICU-acquired weakness. Crit Care 2015;19:448.
13. van den Borst B, Slot IG, Hellwig VA, Vosse BA, Kelders MC, Barreiro E, Schols AM, Gosker HR. Loss of quadriceps muscle oxidative phenotype and decreased endurance in patients with mild-to-moderate COPD. J Appl Physiol 1985;2013:13191328.
14. Natanek SA, Gosker HR, Slot IG, Marsh GS, Hopkinson NS, Man WD-C, Tal-Singer R, Moxham J, Kemp PR, Schols AMWJ, et al. Heterogeneity of quadriceps muscle phenotype in chronic obstructive pulmonary disease (COPD); implications for stratified medicine? Muscle Nerve 2013;48:488497.
15. Chen JF, Mandel EM, Thomson JM, Wu Q, Callis TE, Hammond SM, Conlon FL, Wang DZ. The role of microRNA-1 and microRNA-133 in skeletal muscle proliferation and differentiation. Nat Genet 2006;38:228233.
16. Zhang X, Zuo X, Yang B, Li Z, Xue Y, Zhou Y, Huang J, Zhao X, Zhou J, Yan Y, et al. MicroRNA directly enhances mitochondrial translation during muscle differentiation. Cell 2014;158:607619.
17. Lewis A, Riddoch-Contreras J, Natanek SA, Donaldson A, Man WD, Moxham J, Hopkinson NS, Polkey MI, Kemp PR. Downregulation of the serum response factor/miR-1 axis in the quadriceps of patients with COPD. Thorax 2012;67:2634.
18. Watson E, Sylvius NB, Viana JLL, Greening N, Barratt J, Smith A. Differential microRNA expression in skeletal muscle of human CKD patients and healthy controls [abstract]. Nephrol Dial Transplant 2016;31:i473i474.
19. Lewis A, Lee JY, Donaldson AV, Natanek SA, Vaidyanathan S, Man WD, Hopkinson NS, Sayer AA, Patel HP, Cooper C, et al. Increased expression of H19/miR-675 is associated with a low fat-free mass index in patients with COPD. J Cachexia Sarcopenia Muscle 2016;7:330344.
20. Farre-Garros R, Natanek SA, Bloch S, Polkey MI, Kemp PR. miR-542: a novel regulator of muscle mass and function [abstract]. J Muscle Res Cell Motil 2015;36:593594.
21. Farre-Garros R, Paul R, Natanek SA, Griffiths M, Polkey MI, Kemp PR. A microRNA axis that regulates muscle mass and mitochondrial function in response to disease [abstract]. J Muscle Res Cell Motil 2017;S08:O05O253.
22. Paul R, Polkey M, Kemp P, Griffiths M. S116 GDF-15, the miR-542 cluster and miR-422a are associated with muscle wasting in intensive care unit acquired paresis [abstract]. Thorax 2015;70:A66A67.
23. Rabe KF, Hurd S, Anzueto A, Barnes PJ, Buist SA, Calverley P, Fukuchi Y, Jenkins C, Rodriguez-Roisin R, van Weel C, et al.; Global Initiative for Chronic Obstructive Lung Disease. Global strategy for the diagnosis, management, and prevention of chronic obstructive pulmonary disease: GOLD executive summary. Am J Respir Crit Care Med 2007;176:532555.
24. Bergstrom J. Percutaneous needle biopsy of skeletal muscle in physiological and clinical research. Scand J Clin Lab Invest 1975;35:609616.
25. Seymour JM, Ward K, Sidhu PS, Puthucheary Z, Steier J, Jolley CJ, Rafferty G, Polkey MI, Moxham J. Ultrasound measurement of rectus femoris cross-sectional area and the relationship with quadriceps strength in COPD. Thorax 2009;64:418423.
26. Dweep H, Sticht C, Pandey P, Gretz N. miRWalk--database: prediction of possible miRNA binding sites by “walking” the genes of three genomes. J Biomed Inform 2011;44:839847.
27. Wang Y, Huang JW, Castella M, Huntsman DG, Taniguchi T. p53 is positively regulated by miR-542-3p. Cancer Res 2014;74:32183227.
28. Yan X, Liu Z, Chen Y. Regulation of TGF-beta signaling by Smad7. Acta Biochim Biophys Sin (Shanghai) 2009;41:263272.
29. Dennler S, Itoh S, Vivien D, ten Dijke P, Huet S, Gauthier JM. Direct binding of Smad3 and Smad4 to critical TGF beta-inducible elements in the promoter of human plasminogen activator inhibitor-type 1 gene. EMBO J 1998;17:30913100.
30. Bloch S, Polkey MI, Griffiths M, Kemp P. Molecular mechanisms of intensive care unit-acquired weakness. Eur Respir J 2012;39:10001011.
31. Brealey D, Singer M. Mitochondrial dysfunction in sepsis. Curr Infect Dis Rep 2003;5:365371.
32. Saada A, Shaag A, Arnon S, Dolfin T, Miller C, Fuchs-Telem D, Lombes A, Elpeleg O. Antenatal mitochondrial disease caused by mitochondrial ribosomal protein (MRPS22) mutation. J Med Genet 2007;44:784786.
33. Miller C, Saada A, Shaul N, Shabtai N, Ben-Shalom E, Shaag A, Hershkovitz E, Elpeleg O. Defective mitochondrial translation caused by a ribosomal protein (MRPS16) mutation. Ann Neurol 2004;56:734738.
34. Fredriksson K, Tjäder I, Keller P, Petrovic N, Ahlman B, Schéele C, Wernerman J, Timmons JA, Rooyackers O. Dysregulation of mitochondrial dynamics and the muscle transcriptome in ICU patients suffering from sepsis induced multiple organ failure. PLoS One 2008;3:e3686.
35. Li Y, Li W, Ying Z, Tian H, Zhu X, Li J, Li M. Metastatic heterogeneity of breast cancer cells is associated with expression of a heterogeneous TGFβ-activating miR424-503 gene cluster. Cancer Res 2014;74:61076118.
36. Faraonio R, Salerno P, Passaro F, Sedia C, Iaccio A, Bellelli R, Nappi TC, Comegna M, Romano S, Salvatore G, et al. A set of miRNAs participates in the cellular senescence program in human diploid fibroblasts. Cell Death Differ 2012;19:713721.
37. Lee JY, Lori D, Wells DJ, Kemp PR. FHL1 activates myostatin signalling in skeletal muscle and promotes atrophy. FEBS Open Bio 2015;5:753762.
Correspondence and requests for reprints should be addressed to Paul R. Kemp, D.Phil., Molecular Medicine Section, National Heart and Lung Institute, Imperial College, South Kensington Campus, London SW7 2AZ, UK. E-mail:

Supported by the Medical Research Council COPDMAP, the British Heart Foundation, and the National Institute for Health Research Respiratory Disease Biomedical Research Unit at the Royal Brompton and Harefield NHS Foundation Trust and Imperial College London who part funded M.I.P.’s salary and wholly funded R.F.G. The views expressed in this publication are those of the authors and not necessarily those of the NHS, the National Institute for Health Research, or the Department of Health. A.L. received a Biotechnology and Biological Sciences Research Council funded studentship and was later funded by COPDMAP. S.B. was supported by a Medical Research Council Clinical Research Fellowship. M.C. is supported by a British Heart Foundation studentship. The LHCN-M2 cells were derived using the platform for immortalization of human cells from the Myology Institute in Paris.

Author Contributions: The overall study was designed by P.R.K. with contributions from M.J.G., M.I.P., and A.L. Patients were recruited and samples were collected by R.P., S.A.N., and S.B. The laboratory studies were performed by R.F.G., R.P., M.C., A.L., B.E.G., and P.R.K. V.M. isolated the human muscle cell line. P.R.K. wrote the first draft of the paper, and all authors provided critical appraisal and input into the manuscript.

Originally Published in Press as DOI: 10.1164/rccm.201701-0101OC on August 15, 2017

This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org

Author disclosures are available with the text of this article at www.atsjournals.org.

Related

No related items
Comments Post a Comment




New User Registration

Not Yet Registered?
Benefits of Registration Include:
 •  A Unique User Profile that will allow you to manage your current subscriptions (including online access)
 •  The ability to create favorites lists down to the article level
 •  The ability to customize email alerts to receive specific notifications about the topics you care most about and special offers
American Journal of Respiratory and Critical Care Medicine
196
11

Click to see any corrections or updates and to confirm this is the authentic version of record