Proteins regulating cap-dependent translation are downregulated during total knee arthroplasty

Total knee arthroplasty (TKA) utilizes a tourniquet to reduce blood loss, maintain a clear surgical "bloodless" field, and to ensure proper bone-implant cementing. In 2007, over 600,000 TKAs were performed in the United States, and this number is projected to increase to 3.48 million procedures performed annually by 2030. The acute effects of tourniquet-induced ischemia-reperfusion (I/R) on human skeletal muscle cells are poorly understood and require critical investigation, as muscle atrophy following this surgery is rapid and represents the most significant clinical barrier to long-term normalization of physical function. To determine the acute effects of I/R on skeletal muscle cells, biopsies were obtained at baseline, maximal ischemia (prior to tourniquet release), and reperfusion (following tourniquet release). Quadriceps volume was determined before and 2 wk post-TKA by MRI. We measured a 36% decrease in phosphorylation of Akt Ser(473) during ischemia and 37% during reperfusion (P < 0.05). 4E-BP1 Thr(37/46) phosphorylation decreased 29% during ischemia and 22% during reperfusion (P < 0.05). eEF2 Thr(56) phosphorylation increased 25% during ischemia and 43% during reperfusion (P < 0.05). Quadriceps volume decreased 12% in the TKA leg (P < 0.05) and tended to decrease (6%) in the contralateral leg (P = 0.1). These data suggest cap-dependent translation initiation, and elongation may be inhibited during and after TKA surgery. We propose that cap-dependent translational events occurring during surgery may precipitate postoperative changes in muscle cells that contribute to the etiology of muscle atrophy following TKA.     

MAPK signaling in the quadriceps of patients with chronic obstructive pulmonary disease

Muscle atrophy in chronic obstructive pulmonary disease (COPD) is associated with reduced exercise tolerance, muscle strength, and survival. The molecular mechanisms leading to muscle atrophy in COPD remain elusive. The mitogen-activated protein kinases (MAPKs) such as p38 MAPK and ERK 1/2 can increase levels of MAFbx/Atrogin and MuRF1, which are specifically involved in muscle protein degradation and atrophy. Our aim was to investigate the level of activation of p38 MAPK, ERK 1/2, and JNK in the quadriceps of patients with COPD. A biopsy of the quadriceps was obtained in 18 patients with COPD as well as in 9 healthy controls. We evaluated the phosphorylated as well as total protein levels of p38 MAPK, ERK 1/2, and JNK as well as MAFbx/Atrogin and MuRF1 in these muscle samples. The corresponding mRNA expression was also assessed by RT-PCR. Ratios of phosphorylated to total level of p38 MAPK (P = 0.02) and ERK 1/2 (P = 0.01) were significantly elevated in patients with COPD compared with controls. Moreover, protein levels of MAFbx/Atrogin showed a tendency to be greater in patients with COPD (P = 0.08). mRNA expression of p38 MAPK (P = 0.03), ERK 1/2 (P = 0.02), and MAFbx/Atrogin (P = 0.04) were significantly elevated in patients with COPD. In addition, phosphorylated-to-total p38 MAPK ratio (Pearson's r = -0.45; P < 0.05) and phosphorylated-to-total ERK 1/2 ratio (Pearson's r = -0.47; P < 0.05) were negatively associated with the mid-thigh muscle cross-sectional area. These data support the hypothesis that the MAPKs might play a role in the development of muscle atrophy in COPD.     

Molecular mechanisms modulating muscle mass

Skeletal muscle atrophy occurs in multiple clinical settings, including cancer, AIDS and sepsis, and is caused in part by an increase in the rate of ATP-dependent ubiquitin-mediated proteolysis. The expression of two recently identified genes encoding ubiquitin-protein ligases, MAFbx/Atrogin-1 and MuRF1, has been shown to increase during muscle atrophy. Mouse knockout studies have demonstrated that MAFbx and MuRF1 are required for muscle atrophy, and thus might be targets for clinical intervention. A second strategy for blocking atrophy involves the stimulation of pathways leading to skeletal muscle hypertrophy. Insulin-like growth factor 1 (IGF-1) is a protein growth factor that can induce skeletal muscle hypertrophy by activating the phosphatidylinositol 3-kinase (PI3K)-Akt pathway. The pathways modulating hypertrophy and atrophy will be further discussed, to highlight potential targets for clinical intervention.     

Signalling pathways that mediate skeletal muscle hypertrophy and atrophy

Atrophy of skeletal muscle is a serious consequence of numerous diseases, including cancer and AIDS. Successful treatments for skeletal muscle atrophy could either block protein degradation pathways activated during atrophy or stimulate protein synthesis pathways induced during skeletal muscle hypertrophy. This perspective will focus on the signalling pathways that control skeletal muscle atrophy and hypertrophy, including the recently identified ubiquitin ligases muscle RING finger 1 (MuRF1) and muscle atrophy F-box (MAFbx), as a basis to develop targets for pharmacologic intervention in muscle disease.     

Curcumin prevents lipopolysaccharide-induced atrogin-1/MAFbx upregulation and muscle mass loss

Because elevated ubiquitin ligase atrogin-1/MAFbx and MuRF1 mediate skeletal muscle wasting associated with various catabolic conditions, the signaling pathways involved in the upregulation of these genes under pathological conditions are considered therapeutic targets. AKT and NF-kappaB have been previously shown to regulate the expression of atrogin-1/MAFbx or MuRF1, respectively. In addition, we recently found that p38 MAPK mediates TNF-alpha upregulation of atrogin-1/MAFbx expression, suggesting that multiple signaling pathways mediate muscle wasting in inflammatory diseases. To date, however, these advances have not resulted in a practical clinical intervention for disease-induced muscle wasting. In the present study, we tested the effect of curcumin--a non-toxic anti-inflammatory reagent that inhibits p38 and NF-kappaB--on lipopolysaccharide (LPS)-induced muscle wasting in mice. Daily intraperitoneal (i.p.) injection of curcumin (10-60 micro g/kg) for 4 days inhibited, in a dose-dependent manner, the LPS-stimulated (1 mg/kg, i.p.) increase of atrogin-1/MAFbx expression in gastrocnemius and extensor digitorum longus (EDL) muscles, resulting in the attenuation of muscle protein loss. It should also be noted that curcumin administration did not alter the basal expression of atrogin-1/MAFbx, nor did it affect LPS-stimulated MuRF1 and polyubiquitin expression. LPS activated p38 and NF-kappaB, while inhibiting AKT; whereas, curcumin administration inhibited LPS-stimulated p38 activation, without altering the effect of LPS on NF-kappaB and AKT. These results indicate that curcumin is effective in blocking LPS-induced loss of muscle mass through the inhibition of p38-mediated upregulation of atrogin-1/MAFbx.     

Dependence of dexamethasone-induced Akt/FOXO1 signaling,upregulation of MAFbx,and protein catabolism upon the glucocorticoid receptor

The muscle ubiquitin ligases MAFbx and MuRF1 are upregulated in and promote muscle atrophy. Upregulation of MAFbx and MuRF1 by glucocorticoids has been linked to activation of FOXO1 and FOXO3A resulting from reduced Akt activity. We determined the requirements for the glucocorticoid receptor (GR) in these biological responses in C2C12 cells in which GR expression was knocked down by stable expression of an shRNA. Loss of GR prevented dexamethasone-induced increases in protein catabolism. Loss of GR, or inhibition of ligand binding to GR with RU486, prevented upregulation of MAFbx and MuRF1 by dexamethasone. Loss of GR also prevented dexamethasone-induced decreases in Akt phosphorylation, and increases in the fraction of FOXO1 that was unphosphorylated. The findings establish a requirement for the GR in activating molecular signals that promote muscle protein catabolism.     

Modulation of autophagy and ubiquitin-proteasome pathways during ultra-endurance running

In this study, the coordinated activation of ubiquitin-proteasome pathway (UPP), autophagy-lysosomal pathway (ALP), and mitochondrial remodeling including mitophagy was assessed by measuring protein markers during ultra-endurance running exercise in human skeletal muscle. Eleven male, experienced ultra-endurance athletes ran for 24 h on a treadmill. Muscle biopsy samples were taken from the vastus lateralis muscle 2 h before starting and immediately after finishing exercise. Athletes ran 149.8 ± 16.3 km with an effective running time of 18 h 42 min ( ± 41 min). The phosphorylation state of Akt (-74 ± 5%; P < 0.001), FOXO3a (-49 ± 9%; P < 0.001), mTOR Ser2448 (-32 ± 14%; P = 0.028), and 4E-BP1 (-34 ± 7%; P < 0.001) was decreased, whereas AMPK phosphorylation state increased by 247 ± 170% (P = 0.042). Proteasome β2 subunit activity increased by 95 ± 44% (P = 0.028), whereas the activities associated with the β1 and β5 subunits remained unchanged. MuRF1 protein level increased by 55 ± 26% (P = 0.034), whereas MAFbx protein and ubiquitin-conjugated protein levels did not change. LC3bII increased by 554 ± 256% (P = 0.005), and the form of ATG12 conjugated to ATG5 increased by 36 ± 17% (P = 0.042). The mitochondrial fission marker phospho-DRP1 increased by 110 ± 47% (P = 0.003), whereas the fusion marker Mfn1 and the mitophagy markers Parkin and PINK1 remained unchanged. These results fit well with a coordinated regulation of ALP and UPP triggered by FOXO3 and AMPK during ultra-endurance exercise.     

Translational suppression of atrophic regulators by microRNA-23a integrates resistance to skeletal muscle atrophy

Muscle atrophy is caused by accelerated protein degradation and occurs in many pathological states. Two muscle-specific ubiquitin ligases, MAFbx/atrogin-1 and muscle RING-finger 1 (MuRF1), are prominently induced during muscle atrophy and mediate atrophy-associated protein degradation. Blocking the expression of these two ubiquitin ligases provides protection against muscle atrophy. Here we report that miR-23a suppresses the translation of both MAFbx/atrogin-1 and MuRF1 in a 3'-UTR-dependent manner. Ectopic expression of miR-23a is sufficient to protect muscles from atrophy in vitro and in vivo. Furthermore, miR-23a transgenic mice showed resistance against glucocorticoid-induced skeletal muscle atrophy. These data suggest that suppression of multiple regulators by a single miRNA can have significant consequences in adult tissues.     

Ghrelin receptor agonist GHRP-2 prevents arthritis-induced increase in E3 ubiquitin-ligating enzymes MuRF1 and MAFbx gene expression in skeletal muscle

Chronic arthritis is a catabolic state associated with an inhibition of the IGF system and a decrease in body weight. Cachexia and muscular wasting is secondary to protein degradation by the ubiquitin-proteasome pathway. The aim of this work was to analyze the effect of adjuvant-induced arthritis on the muscle-specific ubiquitin ligases muscle ring finger 1 (MuRF1) and muscle atrophy F-box (MAFbx) as well as on IGF-I and IGF-binding protein-5 (IGFBP-5) gene expression in the skeletal muscle. We also studied whether the synthetic ghrelin receptor agonist, growth hormone releasing peptide-2 (GHRP-2), was able to prevent arthritis-induced changes in the skeletal muscle. Arthritis induced an increase in MuRF1, MAFbx (P < 0.01), and tumor necrosis factor (TNF)-alpha mRNA (P < 0.05) in the skeletal muscle. Arthritis decreased the serum IGF-I and its gene expression in the liver (P < 0.01), whereas it increased IGF-I and IGFBP-5 gene expression in the skeletal muscle (P < 0.01). Administration of GHRP-2 for 8 days prevented the arthritis-induced increase in muscular MuRF1, MAFbx, and TNF-alpha gene expression. GHRP-2 treatment increased the serum concentrations of IGF-I and the IGF-I mRNA in the liver and in the cardiac muscle and decreased muscular IGFBP-5 mRNA both in control and in arthritic rats (P < 0.05). GHRP-2 treatment increased muscular IGF-I mRNA in control rats (P < 0.01), but it did not modify the muscular IGF-I gene expression in arthritic rats. These data indicate that arthritis induces an increase in the activity of the ubiquitin-proteasome proteolytic pathway that is prevented by GHRP-2 administration. The parallel changes in muscular IGFBP-5 and TNF-alpha gene expression with the ubiquitin ligases suggest that they can participate in skeletal muscle alterations during chronic arthritis.     

Activated protein synthesis and suppressed protein breakdown signaling in skeletal muscle of critically ill patients

Background

Skeletal muscle mass is controlled by myostatin and Akt-dependent signaling on mammalian target of rapamycin (mTOR), glycogen synthase kinase 3β (GSK3β) and forkhead box O (FoxO) pathways, but it is unknown how these pathways are regulated in critically ill human muscle. To describe factors involved in muscle mass regulation, we investigated the phosphorylation and expression of key factors in these protein synthesis and breakdown signaling pathways in thigh skeletal muscle of critically ill intensive care unit (ICU) patients compared with healthy controls.

Methodology/Principal Findings

ICU patients were systemically inflamed, moderately hyperglycemic, received insulin therapy, and showed a tendency to lower plasma branched chain amino acids compared with controls. Using Western blotting we measured Akt, GSK3β, mTOR, ribosomal protein S6 kinase (S6k), eukaryotic translation initiation factor 4E binding protein 1 (4E-BP1), and muscle ring finger protein 1 (MuRF1); and by RT-PCR we determined mRNA expression of, among others, insulin-like growth factor 1 (IGF-1), FoxO 1, 3 and 4, atrogin1, MuRF1, interleukin-6 (IL-6), tumor necrosis factor α (TNF-α) and myostatin. Unexpectedly, in critically ill ICU patients Akt-mTOR-S6k signaling was substantially higher compared with controls. FoxO1 mRNA was higher in patients, whereas FoxO3, atrogin1 and myostatin mRNAs and MuRF1 protein were lower compared with controls. A moderate correlation (r² = 0.36, p<0.05) between insulin infusion dose and phosphorylated Akt was demonstrated.

Conclusions/Significance

We present for the first time muscle protein turnover signaling in critically ill ICU patients, and we show signaling pathway activity towards a stimulation of muscle protein synthesis and a somewhat inhibited proteolysis.     

IFN-gamma does not mimic the catabolic effects of TNF-alpha

Cachexia is common in chronic inflammatory diseases and is attributed, in part, to an elevation of circulating proinflammatory cytokines. TNF-alpha is the prototype in this category. IFN-gamma is also thought to play a role, but the evidence supporting this model is primarily indirect. To determine the direct effects of IFN-gamma stimulation on muscle cells, we selected key components of the procatabolic signaling pathways by which TNF-alpha stimulates protein loss. We tested two hypotheses: 1) IFN-gamma mimics TNF-alpha signaling by increasing intracellular oxidant activity and activating MAPKs and NF-kappaB and 2) IFN-gamma increases the expression of the ubiquitin ligases atrogin1/MAFbx and muscle-specific ring finger protein 1 (MuRF1). Results showed that treatment with IFN-gamma at 60 ng/ml increased Stat1 phosphorylation after 15 min, indicating receptor activation. IFN-gamma had no effect on cytosolic oxidant activity, as measured by 2',7'-dichlorofluorescein oxidation. Nor did IFN-gamma activate JNK, ERK1/2, or p38 MAPK, as assessed by Western blot. Treatment for up to 60 min did not decrease IkappaB-alpha protein levels, as measured by Western blot analysis, or the DNA binding activity of NF-kappaB, as measured by EMSA. After 6 h, IFN-gamma decreased Akt phosphorylation and increased atrogin1/MAFbx and MuRF1 mRNA. Daily treatment for up to 72 h did not alter adult fast-type myosin heavy chain content or the total protein-to-DNA ratio. These data show that responses of myotubes to IFN-gamma and TNF-alpha differ markedly and provide little evidence for a direct catabolic effect of IFN-gamma on muscle.     

抗阻训练通过Akt-FoxO1通路抑制低氧诱导的骨骼肌萎缩

高原低氧环境会引起肌力下降和运动能力退化,而抗阻训练是刺激骨骼肌生长的重要手段,叉头转录因子1（fork head box protein O 1,FoxO1）在调控骨骼肌蛋白质分解通路中承担重要角色。为探究Akt-FoxO1通路是否参与抗阻训练抑制低氧诱导的骨骼肌萎缩,本研究构建低氧诱导骨骼肌萎缩的大鼠模型,并模拟海拔4 000 m低氧环境下（12.4% O2）进行抗阻训练,对比观察大鼠比目鱼肌和趾长伸肌湿重和横截面积,以及蛋白激酶B（protein kinase B,Akt）、叉头转录因子1、泛素蛋白连接酶1（muscle ring finger 1,MuRF1）的表达差异等。结果表明,低氧暴露导致大鼠趾长伸肌湿重显著下降,苏木精-伊红染色组织切片分析肌纤维横截面积、低氧环境下比目鱼肌横截面积明显下降,而低氧抗阻训练后趾长伸肌横截面积明显高于安静组。实时荧光定量PCR和蛋白质免疫印迹结果显示,低氧暴露后FoxO1和MuRF1基因表达明显上调,低氧下抗阻训练后发现,Akt基因表达明显上调而FoxO1、MuRF则明显下调。免疫荧光观察磷酸化FoxO1在细胞核内外表达情况,发现抗阻训练后FoxO1（S256）于细胞核外表达增强。上述结果表明,抗阻训练可以达到抑制低氧诱导骨骼肌萎缩的效果,Akt促进FoxO1磷酸化从而减缓骨骼肌蛋白质分解过程是抗阻训练能够抑制骨骼肌萎缩的分子机制之一。