Sepsis-induced Muscle Research Articles

Dantrolene reduces serum TNFalpha and corticosterone levels and muscle calcium, calpain gene expression, and protein breakdown in septic rats.

The effects of dantrolene on serum TNFalpha and corticosterone levels and on muscle calcium, calpain gene expression, and protein breakdown were studied in rats with abdominal sepsis induced by cecal ligation and puncture. Treatment of rats with 10 mg/kg of dantrolene 2 h before and 8 h after induction of sepsis reduced serum TNFalpha and corticosterone, muscle calcium levels, mRNA levels for m- and mu-calpain, and the muscle specific calpain p94, as well as total and myofibrillar protein breakdown rates, determined as release of tyrosine and 3-methylhistidine, respectively, from incubated extensor digitorum longus muscles. The results support the concept that increased calcium concentrations may be an important mechanism of sepsis-induced muscle protein breakdown. The data also indicate that other mechanisms, in addition to reduced muscle calcium concentrations such as decreased levels of TNFalpha and glucocorticoids, may contribute to the anti-catabolic effects of dantrolene during sepsis. The observations are important from a clinical standpoint because they suggest that the catabolic response in skeletal muscle during sepsis may be prevented by treatment with a calcium antagonist.

Insulin-Like Growth Factor I Reduces Ubiquitin and Ubiquitin-Conjugating Enzyme Gene Expression but Does Not Inhibit Muscle Proteolysis in Septic Rats

We examined the effect of insulin-like growth factor I (IGF-I), administered in vivo, on protein turnover rates and gene expression of the ubiquitin-proteasome proteolytic pathway in skeletal muscle of septic rats. Sepsis was induced by cecal ligation and puncture. Other rats were sham-operated. Miniosmotic pumps were implanted sc, and groups of rats received IGF-I (7 mg/kg·24 h) or saline. Protein synthesis and breakdown rates were determined in incubated extensor digitorum longus muscles. Messenger RNA levels for ubiquitin and the ubiquitin-conjugating enzyme E214k were determined by Northern blot analysis. Sepsis resulted in an approximately 30% reduction of muscle protein synthesis, and this effect of sepsis was blunted in rats treated with IGF-I. In contrast, IGF-I did not prevent the sepsis-induced increase in total and myofibrillar muscle protein breakdown. Ubiquitin and E214k messenger RNA levels were increased several fold in muscle from septic rats, and this effect of sepsis was abolished in IGF-I treated rats. The results suggest that administration of IGF-I may improve sepsis-induced muscle cachexia by stimulating protein synthesis. However, because muscles were resistant to IGF-I, with regard to regulation of protein breakdown, the use of IGF-I to treat muscle cachexia during sepsis remains unclear. An additional important implication of the present study is that changes in messenger RNA levels for ubiquitin and the ubiquitin-conjugating enzyme E214k do not always reflect changes in muscle protein breakdown rates.

Insulin-like growth factor I reduces ubiquitin and ubiquitin-conjugating enzyme gene expression but does not inhibit muscle proteolysis in septic rats.

We examined the effect of insulin-like growth factor I (IGF-I), administered in vivo, on protein turnover rates and gene expression of the ubiquitin-proteasome proteolytic pathway in skeletal muscle of septic rats. Sepsis was induced by cecal ligation and puncture. Other rats were sham-operated. Miniosmotic pumps were implanted sc, and groups of rats received IGF-I (7 mg/kg x 24 h) or saline. Protein synthesis and breakdown rates were determined in incubated extensor digitorum longus muscles. Messenger RNA levels for ubiquitin and the ubiquitin-conjugating enzyme E2(14k) were determined by Northern blot analysis. Sepsis resulted in an approximately 30% reduction of muscle protein synthesis, and this effect of sepsis was blunted in rats treated with IGF-I. In contrast, IGF-I did not prevent the sepsis-induced increase in total and myofibrillar muscle protein breakdown. Ubiquitin and E2(14k) messenger RNA levels were increased several fold in muscle from septic rats, and this effect of sepsis was abolished in IGF-I treated rats. The results suggest that administration of IGF-I may improve sepsis-induced muscle cachexia by stimulating protein synthesis. However, because muscles were resistant to IGF-I, with regard to regulation of protein breakdown, the use of IGF-I to treat muscle cachexia during sepsis remains unclear. An additional important implication of the present study is that changes in messenger RNA levels for ubiquitin and the ubiquitin-conjugating enzyme E2(14k) do not always reflect changes in muscle protein breakdown rates.

Sepsis-Induced Muscle Proteolysis Is Prevented by a Proteasome Inhibitor in Vivo

Sepsis-induced muscle proteolysis mainly reflects ubiquitin-proteasome-dependent protein degradation. The effect of in vivo administration of a proteasome inhibitor on muscle protein breakdown during sepsis is not known. We treated rats with the proteasome inhibitor N-benzyloxycarbonyl-Ile-Glu-(O-t-butyl)-Ala-leucinal (PSI) or corresponding volume of vehicle i.p. 2 h before sham-operation or induction of sepsis by cecal ligation and puncture. The sepsis-induced increase in total and myofibrillar muscle protein breakdown was inhibited in rats treated in vivo with PSI and a maximal effect was seen following 15 mg/kg of the proteasome inhibitor. Results from in vitro experiments in which incubated muscles were treated with 100 μM PSI suggest that the drug has a direct effect on muscle and that the effect is specific for the proteasome. The results are important because they suggest that it may be possible to prevent or improve the cachectic response in skeletal muscle during sepsis by treatment with a proteasome inhibitor.

The Gene Expression of Ubiquitin Ligase E3α Is Upregulated in Skeletal Muscle during Sepsis in Rats—Potential Role of Glucocorticoids

Muscle protein breakdown during sepsis is associated with upregulated expression and activity of the ubiquitin-proteasome proteolytic pathway. Previous studies suggest that ubiquitination of proteins in skeletal muscle is regulated by the ubiquitin ligase E3α together with the 14 kDa ubiquitin-conjugating enzyme E214k. The E3α gene was cloned only recently. The influence of sepsis on the gene expression of E3α in skeletal muscle has not been reported. In the present study, induction of sepsis in rats by cecal ligation and puncture resulted in increased mRNA levels for E3α in white, fast-twitch but not in red slow-twitch muscle. Treatment with the glucocorticoid receptor antagonist RU38486 (10 mg/kg) prevented the sepsis-induced increase in E3α and E214k mRNA levels. The present study is the first report of increased E3α expression in skeletal muscle during sepsis. The results lend further support to the concept that glucocorticoid-mediated upregulation of the ubiquitin-proteasome proteolytic pathway is involved in sepsis-induced muscle cachexia. Increased expression of both E3α and E214k suggests that muscle proteins are degraded in the N-end rule pathway during sepsis.

Sepsis stimulates release of myofilaments in skeletal muscle by a calcium-dependent mechanism.

Sepsis is associated with a pronounced catabolic response in skeletal muscle, mainly reflecting degradation of the myofibrillar proteins actin and myosin. Recent studies suggest that sepsis-induced muscle proteolysis may reflect ubiquitin-proteasome-dependent protein breakdown. An apparently conflicting observation is that the ubiquitin-proteasome pathway does not degrade intact myofibrils. Thus, it is possible that actin and myosin need to be released from the myofibrils before they can be ubiquitinated and degraded by the proteasome. We tested the hypothesis that sepsis results in disruption of Z-bands, increased expression of calpains, and calcium-dependent release of myofilaments in skeletal muscle. Sepsis induced in rats by cecal ligation and puncture resulted in increased gene expression of micro-calpain, m-calpain, and p94 and in Z-band disintegration in the extensor digitorum longus muscle. The release of myofilaments from myofibrillar proteins was increased in septic muscle. This response to sepsis was blocked by treating the rats with dantrolene, a substance that inhibits the release of calcium from intracellular stores to the cytoplasm. The present results provide evidence that sepsis is associated with Z-band disintegration and a calcium-dependent release of myofilaments in skeletal muscle. Release of myofilaments may be an initial and perhaps rate-limiting component of sepsis-induced muscle breakdown.

Activity and expression of the 20S proteasome are increased in skeletal muscle during sepsis.

Recent studies suggest that sepsis stimulates ubiquitin-dependent protein breakdown in skeletal muscle. In this proteolytic pathway, ubiquitinated proteins are recognized, unfolded, and degraded by the multicatalytic 26S protease complex. The 20S proteasome is the catalytic core of the 26S protease complex. The role of the 20S proteasome in the regulation of sepsis-induced muscle proteolysis is not known. We tested the hypothesis that sepsis increases 20S proteasome activity and the expression of mRNA for various subunits of this complex. Proteolytic activity of isolated 20S proteasomes, assessed as activity against fluorogenic peptide substrates, was increased in extensor digitorum longus muscles from septic rats. The proteolytic activity was inhibited by specific proteasome blockers. Northern blot analysis revealed an approximately twofold increase in the relative abundance of mRNA for the 20S alpha-subunits RC3 and RC9 and the beta-subunit RC7. However, Western blot analysis did not show any difference in RC9 protein content between sham-operated and septic rats. The increased activity and expression of the 20S proteasome in muscles from septic rats lend further support for a role of the ubiquitin-proteasome-pathway in the regulation of sepsis-induced muscle proteolysis.

Pathways of muscle protein breakdown in injury and sepsis.

The purpose of this article is to review evidence that the ubiquitin-proteasome proteolytic pathway plays an important role in injury- and sepsis-induced muscle catabolism. Such evidence includes upregulated gene expression of several of the components of the ubiquitin-proteasome pathway as well as energy-dependency of the injury- and sepsis-induced muscle protein breakdown. Although the ubiquitin-proteasome pathway is the predominant mechanism of muscle breakdown in various catabolic conditions, other proteolytic mechanisms, in particular calcium-dependent, calpain-mediated protein degradation, probably participate as well.

Sepsis is associated with increased ubiquitinconjugating enzyme E214k mRNA in skeletal muscle.

Previous studies provided evidence that sepsis is associated with increased ubiquitin-proteasome-dependent protein breakdown in skeletal muscle. The 14-kDa ubiquitin-conjugating enzyme (E214k) has been proposed to be a key regulator of the ubiquitin proteolytic pathway. We tested the hypothesis that E214k message and protein levels are increased in skeletal muscle during sepsis. Sepsis was induced in rats by cecal ligation and puncture (CLP). Control rats were sham operated. E214k mRNA and protein levels were quantitated after Northern and Western blot analysis, respectively, 16 h after CLP or sham operation. Sepsis resulted in a 70% increase in the 1. 2-kb E214k transcript in the fast-twitch extensor digitorum longus muscle, whereas no changes were seen in the slow-twitch soleus muscle. E214k protein levels were not influenced by sepsis in any of the muscles studied. Although the changes in the expression of the E214k 1.2-kb transcript paralleled the differential effect of sepsis on protein breakdown in fast- and slow-twitch muscle, the potential role of E214k in the regulation of sepsis-induced muscle proteolysis needs to be interpreted with caution, because the results demonstrated that increased message levels were not associated with increased E214k protein levels.

Role of the ubiquitin-proteasome pathway in sepsis-induced muscle catabolism.

Several lines of evidence suggest that the ubiquitin-proteasome pathway is involved in sepsis-induced muscle catabolism. The gene expression of ubiquitin and several of the proteasome subunits was increased in muscle from both septic rats and patients. In other studies, the activity of isolated 20S proteasomes was stimulated in septic muscles. Sepsis-induced increase in muscle total and myofibrillar protein breakdown was inhibited with specific proteasome blockers. Although the ubiquitin-proteasome pathway is upregulated in septic muscle, it is still unclear how the myofibrillar proteins actin and myosin are ubiquitinated and become substrates for the 26S proteasome. Recent studies suggest that a calcium-dependent, calpain-mediated process releases myofilaments from the Z-disks during sepsis. It is possible that this process exposes destabilizing N-terminal residues on actin and myosin, making them suitable substrates for the N-end rule pathway involving the 14 kD ubiquitin-conjugating enzyme E214k and the ubiquitin-protein ligase E3alpha.

Sepsis in mice stimulates muscle proteolysis in the absence of IL-6.

We tested the role of interleukin-6 (IL-6) in sepsis-induced muscle proteolysis by determining ubiquitin mRNA levels and protein breakdown rates in incubated extensor digitorum longus muscles from septic and sham-operated IL-6 knockout and wild-type mice. In addition, the effect of treatment of mice with human recombinant IL-6 on muscle protein breakdown rates was determined. Finally, protein breakdown rates were measured in myotubes treated for up to 48 h with different concentrations of IL-6. Sepsis in wild-type mice resulted in an approximately ninefold increase in plasma IL-6 levels, whereas IL-6 was not detectable in plasma of sham-operated or septic IL-6 knockout mice. Total and myofibrillar muscle protein breakdown rates were increased by approximately 30% and threefold, respectively, in septic IL-6 wild-type mice with an almost identical response noted in septic IL-6 knockout mice. Ubiquitin mRNA levels determined by dot blot analysis were increased during sepsis in muscles from both IL-6 knockout and wild-type mice, although the increase was less pronounced in IL-6 knockout than in wild-type mice. Treatment of normal mice or of cultured L6 myotubes with IL-6 did not influence protein breakdown rates. The present results suggest that IL-6 does not regulate muscle proteolysis during sepsis.

Sepsis-induced increase in muscle proteolysis is blocked by specific proteasome inhibitors.

Recent studies suggest that sepsis stimulates ubiquitin-dependent protein breakdown in skeletal muscle. The 20S proteasome is the catalytic core of the ubiquitin-dependent proteolytic pathway. We tested the effects in vitro of the proteasome inhibitors N-acetyl-L-leucinyl-L-leucinal-L-norleucinal (LLnL) and lactacystin on protein breakdown in incubated muscles from septic rats. LLnL resulted in a dose- and time-dependent inhibition of protein breakdown in muscles from septic rats. Lactacystin blocked both total and myofibrillar muscle protein breakdown. In addition to inhibiting protein breakdown, LLnL reduced muscle protein synthesis and increased ubiquitin mRNA levels, probably reflecting inhibited proteasome-associated ribonuclease activity. Inhibited muscle protein breakdown caused by LLnL or lactacystin supports the concept that the ubiquitin-proteasome pathway plays a central role in sepsis-induced muscle proteolysis. The results suggest that muscle catabolism during sepsis may be inhibited by targeting specific molecular mechanisms of muscle proteolysis.

Sepsis is associated with increased mRNAs of the ubiquitin-proteasome proteolytic pathway in human skeletal muscle.

Previous studies provided evidence that sepsis-induced muscle proteolysis in experimental animals is caused by increased ubiquitin-proteasome-dependent protein breakdown. It is not known if a similar mechanism accounts for muscle proteolysis in patients with sepsis. We determined mRNA levels for ubiquitin and the 20 S proteasome subunit HC3 by Northern blot analysis in muscle tissue from septic (n = 7) and non-septic (n = 11) patients. Plasma and muscle amino acid concentrations and concentrations in urine of 3-methylhistidine (3-MH), creatinine, and cortisol were measured at the time of surgery to assess the catabolic state of the patients. A three- to fourfold increase in mRNA levels for ubiquitin and HC3 was noted in muscle tissue from the septic patients concomitant with increased muscle levels of phenylalanine and 3-MH and reduced levels of glutamine. Total plasma amino acids were decreased by approximately 30% in the septic patients. The 3-MH/creatinine ratio in urine was almost doubled in septic patients. The cortisol levels in urine were higher in septic than in control patients but this difference did not reach statistical significance. The results suggest that sepsis is associated with increased mRNAs of the ubiquitin-proteasome pathway in human skeletal muscle.

Sepsis stimulates nonlysosomal, energy-dependent proteolysis and increases ubiquitin mRNA levels in rat skeletal muscle.

We tested the role of different intracellular proteolytic pathways in sepsis-induced muscle proteolysis. Sepsis was induced in rats by cecal ligation and puncture; controls were sham operated. Total and myofibrillar proteolysis was determined in incubated extensor digitorum longus muscles as release of tyrosine and 3-methylhistidine, respectively. Lysosomal proteolysis was assessed by using the lysosomotropic agents NH4Cl, chloroquine, leupeptin, and methylamine. Ca(2+)-dependent proteolysis was determined in the absence or presence of Ca2+ or by blocking the Ca(2+)-dependent proteases calpain I and II. Energy-dependent proteolysis was determined in muscles depleted of ATP by 2-deoxyglucose and 2.4-dinitrophenol. Muscle ubiquitin mRNA and the concentrations of free and conjugated ubiquitin were determined by Northern and Western blots, respectively, to assess the role of the ATP-ubiquitin-dependent proteolytic pathway. Total and myofibrillar protein breakdown was increased during sepsis by 50 and 440%, respectively. Lysosomal and Ca(2+)-dependent proteolysis was similar in control and septic rats. In contrast, energy-dependent total and myofibrillar protein breakdown was increased by 172% and more than fourfold, respectively, in septic muscle. Ubiquitin mRNA was increased severalfold in septic muscle. The results suggest that the increase in muscle proteolysis during sepsis is due to an increase in nonlysosomal energy-dependent protein breakdown, which may involve the ubiquitin system.

Reduced muscle protein breakdown in septic rats following treatment with interleukin-1 receptor antagonist

1. 1. The role ofinterleukin-1 (IL-1) in sepsis-induced muscle proteolysis was assessed by treating septic rats with recombinant IL-1 receptor antagonist (rIL-Ira). 2. 2. In initial experiments, we tested the effectiveness of IL-Ira in preventing muscle proteolysis induced by administration of IL-1. 3. 3. When normal rats were treated with rIL-α (three intraperitoneal doses of 100 μ g/kg body weight each over 16 hr), total and myofibrillar muscle protein breakdown rates, measured as release oftyrosine and 3-methylhistidine, respectively, by incubated extensor digitorum longus muscles, were significantly increased. 4. 4. This metabolic response to IL-α was completely abolished by rIL-Ira, administered as three intraperitoneal doses of 3 mg/kg body weight each over 16hr. 5. 5. In subsequent experiments, sepsis was induced in rats by cecal ligation and puncture (CLP); non-septic rats were sham-operated. 6. 6. Treatment of septic rats over 16hr with a total dose of 25mg/kg body weight of rIL-Ira reduced, but did not normalize, the increased muscle protein breakdown rates seen during sepsis. 7. 7. When the dose of rIL-Ira was more than doubled and given as a constant infusion at a rate of 4.2 mg/kg body weight/hr for 16 hr, the increased rate of muscle proteolysis in septic rats was normalized. 8. 8. The present study offers the first direct evidence that IL-1 is involved in the regulation of muscle proteolysis during sepsis.

Evidence that tumor necrosis factor participates in the regulation of muscle proteolysis during sepsis.

The role of tumor necrosis factor (TNF) in the regulation of muscle protein turnover was studied in rats. Protein synthesis and total and myofibrillar protein breakdown rates were measured in incubated extensor digitorum longus muscles. Intraperitoneal administration of recombinant TNF-alpha (300 micrograms/kg of body weight) increased total and myofibrillar protein breakdown rates by 28% and threefold, respectively, with no effect on protein synthesis. In subsequent experiments, sepsis was induced by cecal ligation and puncture or a sham-operation was performed. Rats received TNF antiserum (1 mL/100 g of body weight) or control serum 2 hours before cecal ligation and puncture or sham-operation. Treatment with TNF antiserum reduced the mortality rate from 25% to 5% following cecal ligation and puncture. The treatment had no effect on protein synthesis but reduced total and myofibrillar protein breakdown rates by 26% and 39%, respectively, in septic animals. Results suggest TNF is involved in the regulation of sepsis-induced muscle proteolysis.