Zaira Aversa

Sepsis increases the expression and activity of the transcription factor Forkhead Box O 1 (FOXO1) in skeletal muscle by a glucocorticoid-dependent mechanism

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. Forkhead Box O (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.

CORTICOSTEROIDS AND MUSCLE WASTING ROLE OF TRANSCRIPTION FACTORS, NUCLEAR COFACTORS, AND HYPERACETYLATION

Purpose of reviewThe purpose of this review is to discuss novel insight into mechanisms of glucocorticoid-regulated muscle wasting, in particular the role of transcription factors and nuclear cofactors. In addition, novel strategies that may become useful in the treatment or prevention of glucocorticoid-induced muscle wasting are reviewed. Recent findingsStudies suggest that glucocorticoid-induced upregulation of the transcription factors Forkhead box O 1 and CCAAT/enhancer-binding protein β and downregulation of MyoD and myogenin are involved in glucocorticoid-induced muscle wasting. In addition, glucocorticoid-induced hyperacetylation caused by increased expression of the nuclear cofactor p300 and its histone acetyl transferase activity and decreased expression and activity of histone deacetylases plays an important role in glucocorticoid-induced muscle proteolysis and wasting. Other mechanisms may also be involved in glucocorticoid-induced muscle wasting, including insulin resistance and store-operated calcium entry. Novel potential strategies to prevent or treat glucocorticoid-induced muscle wasting include the use of small molecule histone deacetylase activators, dissociated glucocorticoid receptor agonists, and 11β-hydroxysteroid dehydrogenase type 1 inhibitors. SummaryAn increased understanding of molecular mechanisms regulating glucocorticoid-induced muscle wasting will help develop new strategies to prevent and treat this debilitating condition.

Acetylation and deacetylation—novel factors in muscle wasting

We review recent evidence that acetylation and deacetylation of cellular proteins, including transcription factors and nuclear cofactors, may be involved in the regulation of muscle mass. The level of protein acetylation is balanced by histone acetyltransferases (HATs) and histone deacetylases (HDACs) and studies suggest that this balance is perturbed in muscle wasting. Hyperacetylation of transcription factors and nuclear cofactors regulating gene transcription in muscle wasting may influence muscle mass. In addition, hyperacetylation may render proteins susceptible to degradation by different mechanisms, including intrinsic ubiquitin ligase activity exerted by HATs and by dissociation of proteins from cellular chaperones. In recent studies, inhibition of p300/HAT expression and activity and stimulation of SIRT1-dependent HDAC activity reduced glucocorticoid-induced catabolic response in skeletal muscle, providing further evidence that hyperacetylation plays a role in muscle wasting. It should be noted, however, that although several studies advocate a role of hyperacetylation in muscle wasting, apparently contradictory results have also been reported. For example, muscle atrophy caused by denervation or immobilization may be associated with reduced, rather than increased, protein acetylation. In addition, whereas hyperacetylation results in increased degradation of certain proteins, other proteins may be stabilized by increased acetylation. Thus, the role of acetylation and deacetylation in the regulation of muscle mass may be both condition- and protein-specific. The influence of HATs and HDACs on the regulation of muscle mass, as well as methods to modulate protein acetylation, is an important area for continued research aimed at preventing and treating muscle wasting.

Sepsis and glucocorticoids upregulate p300 and downregulate HDAC6 expression and activity in skeletal muscle

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.

β-Hydroxy-β-methylbutyrate (HMB) prevents dexamethasone-induced myotube atrophy.

High levels of glucocorticoids result in muscle wasting and weakness. β-hydroxy-β-methylbutyrate (HMB) attenuates the loss of muscle mass in various catabolic conditions but the influence of HMB on glucocorticoid-induced muscle atrophy is not known. We tested the hypothesis that HMB prevents dexamethasone-induced atrophy in cultured myotubes. Treatment of cultured L6 myotubes with dexamethasone resulted in increased protein degradation and expression of atrogin-1 and MuRF1, decreased protein synthesis and reduced myotube size. All of these effects of dexamethasone were attenuated by HMB. Additional experiments provided evidence that the inhibitory effects of HMB on dexamethasone-induced increase in protein degradation and decrease in protein synthesis were regulated by p38/MAPK- and PI3K/Akt-dependent cell signaling, respectively. The present results suggest that glucocorticoid-induced muscle wasting can be prevented by HMB.

Sepsis downregulates myostatin mRNA levels without altering myostatin protein levels in skeletal muscle.

Myostatin is a negative regulator of muscle mass and has been reported to be upregulated in several conditions characterized by muscle atrophy. The influence of sepsis on myostatin expression and activity is poorly understood. Here, we tested the hypothesis that sepsis upregulates the expression and downstream signaling of myostatin in skeletal muscle. Because sepsis‐induced muscle wasting is at least in part regulated by glucocorticoids, we also determined the influence of glucocorticoids on myostatin expression. Sepsis was induced in rats by cecal ligation and puncture and control rats were sham‐operated. In other experiments, rats were injected intraperitoneally with dexamethasone (10 mg/kg) or corresponding volume of vehicle. Surprisingly, myostatin mRNA levels were reduced and myostatin protein levels were unchanged in muscles from septic rats. Muscle levels of activin A, follistatin, and total and phosphorylated Smad2 (p‐Smad2) were not influenced by sepsis, suggesting that myostatin downstream signaling was not altered during sepsis. Interestingly, total and p‐Smad3 levels were increased in septic muscle, possibly reflecting altered signaling through pathways other than myostatin. Similar to sepsis, treatment of rats with dexamethasone reduced myostatin mRNA levels and did not alter myostatin protein levels. Fasting, an additional condition characterized by muscle wasting, reduced myostatin mRNA and activin A protein levels, increased myostatin protein, and did not influence follistatin and p‐Smad2 levels. Of note, total and p‐Smad3 levels were reduced in muscle during fasting. The results suggest that sepsis and glucocorticoids do not upregulate the expression and activity of myostatin in skeletal muscle. The role of myostatin may vary between different conditions characterized by muscle wasting. Downstream signaling through Smad2 and 3 is probably regulated not only by myostatin but by other mechanisms as well. J. Cell. Biochem. 111: 1059–1073, 2010.

Sepsis and glucocorticoids downregulate the expression of the nuclear cofactor PGC-1β in skeletal muscle

Muscle wasting during sepsis is at least in part regulated by glucocorticoids and is associated with increased transcription of genes encoding the ubiquitin ligases atrogin-1 and muscle-specific RING-finger protein-1 (MuRF1). Recent studies suggest that muscle atrophy caused by denervation is associated with reduced expression of the nuclear cofactor peroxisome proliferator-activated receptor-γ coactivator (PGC)-1β and that PGC-1β may be a repressor of the atrogin-1 and MuRF1 genes. The influence of other muscle-wasting conditions on the expression of PGC-1β is not known. We tested the influence of sepsis and glucocorticoids on PGC-1β and examined the potential link between downregulated PGC-1β expression and upregulated atrogin-1 and MuRF1 expression in skeletal muscle. Sepsis in rats and mice and treatment with dexamethasone resulted in downregulated expression of PGC-1β and increased expression of atrogin-1 and MuRF1 in the fast-twitch extensor digitorum longus muscle, with less pronounced changes in the slow-twitch soleus muscle. In additional experiments, adenoviral gene transfer of PGC-1β into cultured C2C12 myotubes resulted in a dose-dependent decrease in atrogin-1 and MuRF1 mRNA levels. Treatment of cultured C2C12 myotubes with dexamethasone or PGC-1β small interfering RNA (siRNA) resulted in downregulated PGC-1β expression and increased protein degradation. Taken together, our results suggest that sepsis- and glucocorticoid-induced muscle wasting may, at least in part, be regulated by decreased expression of the nuclear cofactor PGC-1β.

CALPAIN activity is increased in skeletal muscle from gastric cancer patients with no or minimal weight loss

The influence of cancer on skeletal muscle calpain expression and activity in humans is poorly understood. We tested the hypothesis that calpain activity is increased in skeletal muscle from gastric cancer patients with no or <5% weight loss. Muscle biopsies were obtained from rectus abdominis muscle in 15 patients who underwent surgery for gastric cancer and had <5% weight loss and also in 15 control patients. Calpain activity was determined using a calpain‐specific substrate in the absence or presence of calcium. The expression of μ‐ and m‐calpain, calpastatin, atrogin‐1, and MuRF1 was determined by real‐time polymerase chain reaction. Calpain activity was increased by 70% in cancer patients compared with controls. There were no differences in mRNA levels for μ‐ and m‐calpain, calpastatin, atrogin‐1, or MuRF1 between control and cancer patients. Calpain activity may be increased in muscle from gastric cancer patients even before changes in molecular markers of muscle wasting and significant weight loss occur. Muscle Nerve, 2011

C/EBPβ regulates dexamethasone‐induced muscle cell atrophy and expression of atrogin‐1 and MuRF1

Muscle wasting in catabolic patients is in part mediated by glucocorticoids and is associated with increased expression and activity of the transcription factor C/EBPβ. It is not known, however, if C/EBPβ is causally linked to glucocorticoid‐induced muscle atrophy. We used dexamethasone‐treated L6 myoblasts and myotubes to test the role of C/EBPβ in glucocorticoid‐induced expression of the muscle‐specific ubiquitin ligases atrogin‐1 and MuRF1, protein degradation, and muscle atrophy by transfecting cells with C/EBPβ siRNA. In myoblasts, silencing C/EBPβ expression with siRNA inhibited dexamethasone‐induced increase in protein degradation, atrogin‐1 and MuRF1 expression, and muscle cell atrophy. Similar effects of C/EBPβ siRNA were seen in myotubes except that the dexamethasone‐induced increase in MuRF1 expression was not affected by C/EBPβ siRNA in myotubes. In additional experiments, overexpressing C/EBPβ did not influence atrogin‐1 or MuRF1 expression in myoblasts or myotubes. Taken together, our observations suggest that glucocorticoid‐induced muscle wasting is at least in part regulated by C/EBPβ. Increased C/EBPβ expression alone, however, is not sufficient to upregulate atrogin‐1 and MuRF1 expression. J. Cell. Biochem. 112: 1737–1748, 2011.

PPARβ/δ regulates glucocorticoid- and sepsis-induced FOXO1 activation and muscle wasting.

FOXO1 is involved in glucocorticoid- and sepsis-induced muscle wasting, in part reflecting regulation of atrogin-1 and MuRF1. Mechanisms influencing FOXO1 expression in muscle wasting are poorly understood. We hypothesized that the transcription factor peroxisome proliferator-activated receptor β/δ (PPARβ/δ) upregulates muscle FOXO1 expression and activity with a downstream upregulation of atrogin-1 and MuRF1 expression during sepsis and glucocorticoid treatment and that inhibition of PPARβ/δ activity can prevent muscle wasting. We found that activation of PPARβ/δ in cultured myotubes increased FOXO1 activity, atrogin-1 and MuRF1 expression, protein degradation and myotube atrophy. Treatment of myotubes with dexamethasone increased PPARβ/δ expression and activity. Dexamethasone-induced FOXO1 activation and atrogin-1 and MuRF1 expression, protein degradation, and myotube atrophy were inhibited by PPARβ/δ blocker or siRNA. Importantly, muscle wasting induced in rats by dexamethasone or sepsis was prevented by treatment with a PPARβ/δ inhibitor. The present results suggest that PPARβ/δ regulates FOXO1 activation in glucocorticoid- and sepsis-induced muscle wasting and that treatment with a PPARβ/δ inhibitor may ameliorate loss of muscle mass in these conditions.