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Background Sepsis and inflammation can cause intensive care unit‐acquired weakness (ICUAW). Increased interleukin‐6 (IL‐6) plasma levels are a risk factor for ICUAW. IL‐6 signalling involves the glycoprotein 130 (gp130) receptor and the JAK/STAT‐pathway, but its role in sepsis‐induced muscle wasting is uncertain. In a clinical observational study, we found that the IL‐6 target gene, SOCS3, was increased in skeletal muscle of ICUAW patients indicative for JAK/STAT‐pathway activation. We tested the hypothesis that the IL‐6/gp130‐pathway mediates ICUAW muscle atrophy. Methods We sequenced RNA (RNAseq) from tibialis anterior (TA) muscle of cecal ligation and puncture‐operated (CLP) and sham‐operated wildtype (WT) mice. The effects of the IL‐6/gp130/JAK2/STAT3‐pathway were investigated by analysing the atrophy phenotype, gene expression, and protein contents of C2C12 myotubes. Mice lacking Il6st, encoding gp130, in myocytes (cKO) and WT controls, as well as mice treated with the JAK2 inhibitor AG490 or vehicle were exposed to CLP or sham surgery for 24 or 96 h. Results Analyses of differentially expressed genes in RNAseq (≥2‐log2‐fold change, P

Background Septic cardiomyopathy worsens the prognosis of critically ill patients. Clinical data suggest that interleukin‐1β (IL‐1β), activated by the NLRP3 inflammasome, compromises cardiac function. Whether or not deleting Nlrp3 would prevent cardiac atrophy and improve diastolic cardiac function in sepsis was unclear. Here, we investigated the role of NLRP3/IL‐1β in sepsis‐induced cardiomyopathy and cardiac atrophy. Methods Male Nlrp3 knockout (KO) and wild‐type (WT) mice were exposed to polymicrobial sepsis by caecal ligation and puncture (CLP) surgery (KO, n = 27; WT, n = 33) to induce septic cardiomyopathy. Sham‐treated mice served as controls (KO, n = 11; WT, n = 16). Heart weights and morphology, echocardiography and analyses of gene and protein expression were used to evaluate septic cardiomyopathy and cardiac atrophy. IL‐1β effects on primary and immortalized cardiomyocytes were investigated by morphological and molecular analyses. IonOptix and real‐time deformability cytometry (RT‐DC) analysis were used to investigate functional and mechanical effects of IL‐1β on cardiomyocytes. Results Heart morphology and echocardiography revealed preserved systolic (stroke volume: WT sham vs. WT CLP: 33.1 ± 7.2 μL vs. 24.6 ± 8.7 μL, P

Critically ill patients at the intensive care unit (ICU) often develop a generalized weakness, called ICU-acquired weakness (ICUAW). A major contributor to ICUAW is muscle atrophy, a loss of skeletal muscle mass and function. Skeletal muscle assures almost all of the vital functions of our body. It adapts rapidly in response to physiological as well as pathological stress, such as inactivity, immobilization, and inflammation. In response to a reduced workload or inflammation muscle atrophy develops. Recent work suggests that adaptive or maladaptive processes in the endoplasmic reticulum (ER), also known as sarcoplasmic reticulum, contributes to this process. In muscle cells, the ER is a highly specialized cellular organelle that assures calcium homeostasis and therefore muscle contraction. The ER also assures correct folding of proteins that are secreted or localized to the cell membrane. Protein folding is a highly error prone process and accumulation of misfolded or unfolded proteins can cause ER stress, which is counteracted by the activation of a signaling network known as the unfolded protein response (UPR). Three ER membrane residing molecules, protein kinase R-like endoplasmic reticulum kinase (PERK), inositol requiring protein 1a (IRE1a), and activating transcription factor 6 (ATF6) initiate the UPR. The UPR aims to restore ER homeostasis by reducing overall protein synthesis and increasing gene expression of various ER chaperone proteins. If ER stress persists or cannot be resolved cell death pathways are activated. Although, ER stress-induced UPR pathways are known to be important for regulation of skeletal muscle mass and function as well as for inflammation and immune response its function in ICUAW is still elusive. Given recent advances in the development of ER stress modifying molecules for neurodegenerative diseases and cancer, it is important to know whether or not therapeutic interventions in ER stress pathways have favorable effects and these compounds can be used to prevent or treat ICUAW. In this review, we focus on the role of ER stress-induced UPR in skeletal muscle during critical illness and in response to predisposing risk factors such as immobilization, starvation and inflammation as well as ICUAW treatment to foster research for this devastating clinical problem.

OBJECTIVE: In sepsis, the disease course of critically ill patients is often complicated by muscle failure leading to ICU-acquired weakness. The myokine transforming growth factor-{beta}1 increases during inflammation and mediates muscle atrophy in vivo. We observed that the transforming growth factor-{beta}1 inhibitor, secreted frizzled-related protein 2, was down-regulated in skeletal muscle of ICU-acquired weakness patients. We hypothesized that secreted frizzled-related protein 2 reduction enhances transforming growth factor-{beta}1-mediated effects and investigated the interrelationship between transforming growth factor-{beta}1 and secreted frizzled-related protein 2 in inflammation-induced atrophy. DESIGN: Observational study and prospective animal trial. SETTING: Two ICUs and research laboratory. PATIENTS/SUBJECTS: Twenty-six critically ill patients with Sequential Organ Failure Assessment scores greater than or equal to 8 underwent a skeletal muscle biopsy from the vastus lateralis at median day 5 in ICU. Four patients undergoing elective orthopedic surgery served as controls. To search for signaling pathways enriched in muscle of ICU-acquired weakness patients, a gene set enrichment analysis of our recently published gene expression profiles was performed. Quantitative reverse transcriptase-polymerase chain reaction, Western blot, and immunohistochemistry were used to analyze secreted frizzled-related protein 2 expression and protein content. A mouse model of inflammation-induced skeletal muscle atrophy due to polymicrobial sepsis and cultured myocytes were used for mechanistic analyses. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Gene set enrichment analysis uncovered transforming growth factor-{beta}1 signaling activation in vastus lateralis from ICU-acquired weakness patients. Muscular secreted frizzled-related protein 2 expression was reduced after 5 days in ICU. Likewise, muscular secreted frizzled-related protein 2 expression was decreased early and continuously in mice with inflammation-induced atrophy. In muscle, secreted frizzled-related protein 2 was predominantly contained in fast twitch/type II myofibers. Secreted frizzled-related protein 2 physically interacted and colocalized with transforming growth factor-{beta}1 through its cysteine-rich domain. Finally, secreted frizzled-related protein 2 prevented transforming growth factor-{beta}1-induced atrophy in C2C12 myotubes. CONCLUSIONS: Muscular secreted frizzled-related protein 2 is down-regulated in ICU-acquired weakness patients and mice with inflammation-induced muscle atrophy. Decreased secreted frizzled-related protein 2 possibly establishes a positive feedback loop enhancing transforming growth factor-{beta}1-mediated atrophic effects in inflammation-induced atrophy.

Chronic pressure overload due to aortic valve stenosis leads to pathological cardiac hypertrophy and heart failure. Hypertrophy is accompanied by an increase in myocyte surface area, which requires a proportional increase in the number of cell-cell and cell-matrix contacts to withstand enhanced workload. In a proteomic analysis we identified nerve injury-induced protein 1 (Ninjurin1), a 16kDa transmembrane cell-surface protein involved in cell adhesion and nerve repair, to be increased in hypertrophic hearts from patients with aortic stenosis. We hypothesised that Ninjurin1 is involved in myocyte hypertrophy. We analyzed cardiac biopsies from aortic-stenosis patients and control patients undergoing elective heart surgery. We studied cardiac hypertrophy in mice after transverse aortic constriction and angiotensin II infusions, and performed mechanistic analyses in cultured myocytes. We assessed the physiological role of ninjurin1 in zebrafish during heart and skeletal muscle development. Ninjurin1 was increased in hearts of aortic stenosis patients, compared to controls, as well as in hearts from mice with cardiac hypertrophy. Besides the 16kDa Ninjurin1 (Ninjurin1-16) we detected a 24kDa variant of Ninjurin1 (Ninjurin1-24), which was predominantly expressed during myocyte hypertrophy. We disclosed that the higher molecular weight of Ninjurin1-24 was caused by N-glycosylation. Ninjurin1-16 was contained in the cytoplasm of myocytes where it colocalized with stress-fibers. In contrast, Ninjurin1-24 was localized at myocyte membranes. Gain and loss-of-function experiments showed that Ninjurin1-24 plays a role in myocyte hypertrophy and myogenic differentiation in vitro. Reduced levels of ninjurin1 impaired cardiac and skeletal muscle development in zebrafish. We conclude that Ninjurin1 contributes to myocyte growth and differentiation, and that these effects are mainly mediated by N-glycosylated Ninjurin1-24.

The muscle-specific RING-finger protein MuRF1 (also known as TRIM63) constitutes a bona fide ubiquitin ligase that routes proteins like several different myosin heavy chain proteins (MyHC) to proteasomal degradation during muscle atrophy. In two unbiased screens, we identified DCAF8 as a new MuRF1-binding partner. MuRF1 physically interacts with DCAF8 and both proteins localize to overlapping structures in muscle cells. Importantly, similar to what is seen for MuRF1, DCAF8 levels increase during atrophy, and the downregulation of either protein substantially impedes muscle wasting and MyHC degradation in C2C12 myotubes, a model system for muscle differentiation and atrophy. DCAF proteins typically serve as substrate receptors for cullin 4-type (Cul4) ubiquitin ligases (CRL), and we demonstrate that DCAF8 and MuRF1 associate with the subunits of such a protein complex. Because genetic downregulation of DCAF8 and inhibition of cullin activity also impair myotube atrophy in C2C12 cells, our data imply that the DCAF8 promotes muscle wasting by targeting proteins like MyHC as an integral substrate receptor of a Cul4A-containing ring ubiquitin ligase complex (CRL4A).This article has an associated First Person interview with the first author of the paper.

Accumulation of aberrant proteins in the endoplasmic reticulum (ER) triggers the unfolded protein response pathway that helps the cell to survive under these stress conditions. Herp is a mammalian ubiquitin domain protein, which is strongly induced by the unfolded protein response. It is involved in ER-associated protein degradation (ERAD) and interacts directly with the ubiquitin ligase Hrd1, which is found in high molecular mass complexes of the ER membrane. Here we present the first evidence that Herp regulates Hrd1-mediated ubiquitylation in a ubiquitin-like (UBL) domain-dependent manner. We found that upon exposure of cells to ER stress, elevation of Herp steady state levels is accompanied by an enhanced association of Herp with pre-existing Hrd1. Hrd1-associated Herp is rapidly degraded and substituted by de novo synthesized Herp, suggesting a continuous turnover of the protein at Hrd1 complexes. Further analysis revealed the presence of multiple Hrd1 copies in a single complex enabling binding of a variable number of Herp molecules. Efficient ubiquitylation of the Hrd1-specific ERAD substrate α1-antitrypsin null Hong Kong (NHK) required the presence of the Herp UBL domain, which was also necessary for NHK degradation. In summary, we propose that binding of Herp to Hrd1-containing ERAD complexes positively regulates the ubiquitylation activity of these complexes, thus permitting survival of the cell during ER stress.

Rationale: Skeletal muscle wasting with accompanying cachexia is a life threatening complication in congestive heart failure. The molecular mechanisms are imperfectly understood, although an activated renin–angiotensin aldosterone system has been implicated. Angiotensin (Ang) II induces skeletal muscle atrophy in part by increased muscle-enriched E3 ubiquitin ligase muscle RING-finger-1 ( MuRF1 ) expression, which may involve protein kinase D1 (PKD1). Objective: To elucidate the molecular mechanism of Ang II–induced skeletal muscle wasting. Methods and Results: A cDNA expression screen identified the lysosomal hydrolase-coordinating transcription factor EB (TFEB) as novel regulator of the human MuRF1 promoter. TFEB played a key role in regulating Ang II–induced skeletal muscle atrophy by transcriptional control of MuRF1 via conserved E-box elements. Inhibiting TFEB with small interfering RNA prevented Ang II–induced MuRF1 expression and atrophy. The histone deacetylase-5 (HDAC5), which was directly bound to and colocalized with TFEB, inhibited TFEB-induced MuRF1 expression. The inhibition of TFEB by HDAC5 was reversed by PKD1, which was associated with HDAC5 and mediated its nuclear export. Mice lacking PKD1 in skeletal myocytes were resistant to Ang II–induced muscle wasting. Conclusion: We propose that elevated Ang II serum concentrations, as occur in patients with congestive heart failure, could activate the PKD1/HDAC5/TFEB/MuRF1 pathway to induce skeletal muscle wasting.

ObjectivesSystemic inflammation is a major risk factor for critical-illness myopathy (CIM) but its pathogenic role in muscle is uncertain. We observed that interleukin 6 (IL-6) and serum amyloid A1 (SAA1) expression was upregulated in muscle of critically ill patients. To test the relevance of these responses we assessed inflammation and acute-phase response at early and late time points in muscle of patients at risk for CIM.DesignProspective observational clinical study and prospective animal trial.SettingTwo intensive care units (ICU) and research laboratory.Patients/subjects33 patients with Sequential Organ Failure Assessment scores ≥ 8 on 3 consecutive days within 5 days in ICU were investigated. A subgroup analysis of 12 patients with, and 18 patients without CIM (non-CIM) was performed. Two consecutive biopsies from vastus lateralis were obtained at median days 5 and 15, early and late time points. Controls were 5 healthy subjects undergoing elective orthopedic surgery. A septic mouse model and cultured myoblasts were used for mechanistic analyses.Measurements and main resultsEarly SAA1 expression was significantly higher in skeletal muscle of CIM compared to non-CIM patients. Immunohistochemistry showed SAA1 accumulations in muscle of CIM patients at the early time point, which resolved later. SAA1 expression was induced by IL-6 and tumor necrosis factor-alpha in human and mouse myocytes in vitro. Inflammation-induced muscular SAA1 accumulation was reproduced in a sepsis mouse model.ConclusionsSkeletal muscle contributes to general inflammation and acute-phase response in CIM patients. Muscular SAA1 could be important for CIM pathogenesis.Trial registrationISRCTN77569430.

There is increasing evidence that reactive oxygen species (ROS) are mediators in growth factor and cytokine signaling pathways. Mechanisms by which ROS can interfere with signaling cascades may include regulation of protein activities by the modification of essential cysteines. Modification can be performed chemically or enzyme-catalyzed. Enzymes catalyzing a reversible thiol modification within proteins are to be able to react with both, ROS and protein thiols. If hydroperoxides are involved, promising candidates are peroxiredoxins and glutathione peroxidases (GPx), especially the phospholipid hydroperoxide GPx. Interleukin-1, one of the key players in inflammatory response, stimulates the production of ROS itself, but its signaling cascade can also be influenced by ROS and by thiol modifying agents. Targets are located in early, intermediate, and late events in the signaling cascade. We here summarize what is known about the effects of thiol modifying agents, selenium and glutathione peroxidases, on the assembly of the IL-1 receptor signaling complex as an early event, on the activation of NF-kappaB as an intermediate event, and on the expression of cell adhesion molecules as a late event in IL-1 signaling. (C) 2003 Elsevier Inc. All rights reserved

Introduction ICU-acquired weakness (ICUAW) complicates the disease course of critically ill patients. Inflammation and acute-phase response occur directly within myocytes and contribute to ICUAW. We observed that tripartite motif–containing 62 (TRIM62), an E3 ubiquitin ligase and modifier of inflammation, is increased in the skeletal muscle of ICUAW patients. We investigated the regulation and function of muscular TRIM62 in critical illness. Methods Twenty-six critically ill patients with Sequential Organ Failure Assessment scores ≥8 underwent two skeletal muscle biopsies from the vastus lateralis at median days 5 and 15 in the ICU. Four patients undergoing elective orthopedic surgery served as controls. TRIM62 expression and protein content were analyzed in these biopsies. The kinetics of Trim62, Atrogin1 and MuRF1 expression were determined in the gastrocnemius/plantaris and tibialis anterior muscles from mouse models of inflammation-, denervation- and starvation-induced muscle atrophy to differentiate between these contributors to ICUAW. Cultured myocytes were used for mechanistic analyses. Results TRIM62 expression and protein content were increased early and remained elevated in muscles from critically ill patients. In all three animal models, muscular Trim62 expression was early and continuously increased. Trim62 was expressed in myocytes, and its overexpression activated the atrophy-inducing activator protein 1 signal transduction pathway. Knockdown of Trim62 by small interfering RNA inhibited lipopolysaccharide-induced interleukin 6 expression. Conclusions TRIM62 is activated in the muscles of critically ill patients. It could play a role in the pathogenesis of ICUAW by activating and maintaining inflammation in myocytes. Trial registration Current Controlled Trials ID: ISRCTN77569430 (registered 13 February 2008) Electronic supplementary material The online version of this article (doi:10.1186/s13054-014-0545-6) contains supplementary material, which is available to authorized users.