Sepsis-induced muscle wasting has severe clinical consequences, including muscle weakness, need for prolonged ventilatory support and stay in the intensive care unit, and delayed ambulation with risk for pulmonary and thromboembolic complications. Understanding molecular mechanisms regulating loss of muscle mass in septic patients therefore has significant clinical implications. FOXO transcription factors have been implicated in muscle wasting, partly reflecting upregulation of the ubiquitin ligases atrogin-1 and MuRF1. The influence of sepsis on FOXO transcription factors in skeletal muscle is poorly understood. We tested the hypothesis that sepsis upregulates expression and activity of FOXO transcription factors in skeletal muscle by a glucocorticoid-dependent mechanism. Sepsis in rats increased muscle FOXO1 and 3a mRNA and protein levels but did not influence FOXO4 expression. Nuclear FOXO1 levels and DNA binding activity were increased in septic muscle whereas FOXO3a nuclear levels were not increased during sepsis. Sepsis-induced expression of FOXO1 was reduced by the glucocorticoid receptor antagonist RU38486 and treatment of rats with dexamethasone increased FOXO1 mRNA levels suggesting that the expression of FOXO1 is regulated by glucocorticoids. Reducing FOXO1, but not FOXO3a, expression by siRNA in cultured L6 myotubes inhibited dexamethasone-induced atrogin-1 and MuRF1 expression, further supporting a role of FOXO1 in glucocorticoid-regulated muscle wasting. Results suggest that sepsis increases FOXO1 expression and activity in skeletal muscle by a glucocorticoid-dependent mechanism and that glucocorticoid-dependent upregulation of atrogin-1 and MuRF1 in skeletal muscle is regulated by FOXO1. The study is significant because it provides novel information about molecular mechanisms involved in sepsis-induced muscle wasting.

The role of the calpain proteases in skeletal muscle atrophy is poorly understood. One goal of these experiments was to clarify whether calpains act upstream of the ubiquitin-proteasome pathway (UPP). Calpain activation may also inhibit the anabolic signalling of Akt, since a molecular chaperone previously shown to mediate Akt activity, heat shock protein 90 (HSP 90), is a calpain substrate. Thus, an additional objective was to determine whether calpain activation affects the Akt signalling pathway. Ex vivo experiments were conducted using isolated rat diaphragm muscle. Calpain activation increased total protein degradation by 65%. Proteasome inhibition prevented this large rise in proteolysis, demonstrating that the proteasome was necessary for calpain-activated protein degradation. In addition, calpain activation increased proteasome-dependent proteolysis by 144%, further supporting the idea of sequential proteolytic pathways. Calpain reduced Akt and mammalian target of rapamycin (mTOR) phosphorylation by 35 and 50%, respectively, and activated glycogen synthase kinase-3 beta (GSK-3β) by 40%. Additionally, calpain activation reduced HSP 90β and mTOR protein content by 33 and 50%, respectively. These data suggest that calpains play a dual role in protein metabolism by concomitantly activating proteasome-dependent proteolysis and inhibiting the Akt pathway of protein synthesis.

Smith IJ, Lecker SH, Hasselgren PO. Calpain activity and muscle wasting in sepsis. Am J Physiol Endocrinol Metab 295: E762-E771, 2008. First published May 20, 2008 doi:10.1152/ajpendo.90226.2008.-Muscle wasting in sepsis reflects activation of multiple proteolytic mechanisms, including lyosomal and ubiquitin-proteasome-dependent protein breakdown. Recent studies suggest that activation of the calpain system also plays an important role in sepsis-induced muscle wasting. Perhaps the most important consequence of calpain activation in skeletal muscle during sepsis is disruption of the sarcomere, allowing for the release of myofilaments (including actin and myosin) that are subsequently ubiquitinated and degraded by the 26S proteasome. Other important consequences of calpain activation that may contribute to muscle wasting during sepsis include degradation of certain transcription factors and nuclear cofactors, activation of the 26S proteasome, and inhibition of Akt activity, allowing for downstream activation of Foxo transcription factors and GSK-3␤. The role of calpain activation in sepsis-induced muscle wasting suggests that the calpain system may be a therapeutic target in the prevention and treatment of muscle wasting during sepsis. Furthermore, because calpain activation may also be involved in muscle wasting caused by other conditions, including different muscular dystrophies and cancer, calpain inhibitors may be beneficial not only in the treatment of sepsis-induced muscle wasting but in other conditions causing muscle atrophy as well. muscle proteolysis; calcium; atrophy; calpastatin LOSS OF MUSCLE MASS IS COMMONLY seen in patients with sepsis (44, 55). Several other catabolic conditions, such as burn injury, cancer, uremia, and AIDS, are also associated with muscle wasting (20,27,29,79). Muscle wasting has several significant clinical consequences. For example, loss of muscle mass results in weakness and fatigue, in turn resulting in delayed ambulation and prolonged rehabilitation. When patients are bedridden for long periods of time, the risks for thromboembolic events as well as for pneumonia and other pulmonary complications increase. Prolonged bed rest in itself causes loss of muscle mass, thus creating a vicious cycle (35). Patients treated in the intensive care unit may need ventilatory support for extended periods of time when respiratory muscles are atrophying (82).Under normal conditions, muscle mass is maintained by a balance between protein synthesis and degradation, and muscle wasting can occur in any situation when this equilibrium is perturbed. There is evidence that loss of muscle mass during sepsis to a great extent reflects activated breakdown of muscle proteins, in particular the contractile proteins actin and myosin (48), but reduced protein synthesis may also contribute to sepsis-induced muscle wasting (62).Although increased expression and activity of the ubiquitinproteasome proteolytic pathway, including a dramatic upregulation of the muscle-specific ubiquitin ligases atrogin-1 and M...

Alamdari N, Smith IJ, Aversa Z, Hasselgren PO. Sepsis and glucocorticoids upregulate p300 and downregulate HDAC6 expression and activity in skeletal muscle. Am J Physiol Regul Integr Comp Physiol 299: R509 -R520, 2010. First published June 10, 2010 doi:10.1152/ajpregu.00858.2009.-Muscle wasting during sepsis is in part regulated by glucocorticoids. In recent studies, treatment of cultured muscle cells in vitro with dexamethasone upregulated expression and activity of p300, a histone acetyl transferase (HAT), and reduced expression and activity of the histone deacetylases-3 (HDAC3) and -6, changes that favor hyperacetylation. Here, we tested the hypothesis that sepsis and glucocorticoids regulate p300 and HDAC3 and -6 in skeletal muscle in vivo. Because sepsis-induced metabolic changes are particularly pronounced in white, fast-twitch skeletal muscle, most experiments were performed in extensor digitorum longus muscles. Sepsis in rats upregulated p300 mRNA and protein levels, stimulated HAT activity, and reduced HDAC6 expression and HDAC activity. The sepsis-induced changes in p300 and HDAC expression were prevented by the glucocorticoid receptor antagonist RU38486. Treatment of rats with dexamethasone increased expression of p300 and HAT activity, reduced expression of HDAC3 and -6, and inhibited HDAC activity. Finally, treatment with the HDAC inhibitor trichostatin A resulted in increased muscle proteolysis and expression of the ubiquitin ligase atrogin-1. Taken together, our results suggest for the first time that sepsis-induced muscle wasting may be regulated by glucocorticoid-dependent hyperacetylation caused by increased p300 and reduced HDAC expression and activity. The recent development of pharmacological HDAC activators may provide a novel avenue to prevent and treat muscle wasting in sepsis and other catabolic conditions. acetylation; muscle wasting MUSCLE WASTING DURING SEPSIS is, at least in part, regulated by glucocorticoids (15, 50) and is mainly caused by increased degradation of myofibrillar proteins, although inhibited protein synthesis may contribute as well (16,25). Gene transcription is altered in atrophying muscle, and in recent studies in experimental animals, a common set of genes, so called atrogenes, was upregulated in different catabolic conditions (27). Among the atrogenes, the genes for the ubiquitin ligases muscle atrophy F-box, also known as atrogin-1 (MAFbx/atrogin-1) and muscle ring finger 1 (MuRF1) are particularly important (3,14). Because changes in gene transcription play an important role in loss of muscle mass it is likely that transcription factors are involved in muscle wasting. Indeed, recent reports from our and other laboratories suggest that the transcription factors NF-B (4, 44, 59), CCAAT/enhancer-binding protein (C/ EBP)␤ and -␦ (43, 62), AP-1 (41, 44), and forkhead box (FOXO)1 and -3a (11, 12, 21, 22, 52) regulate muscle-wastingrelated genes in sepsis and other catabolic conditions.In addition to transcription factors, gene activation is also regulated by other f...