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.     

Ubiquitin-proteasome-dependent proteolytic activity remains elevated after zymosan-induced sepsis in rats while muscle mass recovers

We studied the role of the ubiquitin-proteasome system in rat skeletal muscle during sepsis and subsequent recovery. Sepsis was induced with intraperitoneal zymosan injections. This model allows one to study a sustained and reversible catabolic phase and mimics the events that prevail in septic and subsequently recovering patients. In addition, the role of the ubiquitin-proteasome system during muscle recovery is poorly documented. There was a trend for increased ubiquitin-conjugate formation in the muscle wasting phase, which was abolished during the recovery phase. The trypsin- and chymotrypsin-like peptidase activities of the 20S proteasome peaked at day 6 following zymosan injection (i.e. when both muscle mass and muscle fiber cross-sectional area were reduced the most), but remained elevated when muscle mass and muscle fiber cross-sectional area were recovering (11 days). This clearly suggests a role for the ubiquitin-proteasome pathway in the muscle remodeling and/or recovery process. Protein levels of 19S complex and 20S proteasome subunits did not increase throughout the study, pointing to alternative mechanisms regulating proteasome activities. Overall these data support a role for ubiquitin-proteasome dependent proteolysis in the zymosan septic model, in both the catabolic and muscle recovery phases.     

Downregulation of ubiquitin-dependent proteolysis by eicosapentaenoic acid in acute starvation.

A number of acute wasting conditions are associated with an upregulation of the ubiquitin-proteasome system in skeletal muscle. Eicosapentaenoic acid (EPA) is effective in attenuating the increased protein catabolism in muscle in cancer cachexia, possibly due to inhibition of 15-hydroxyeicosatetraenoic acid (15-HETE) formation. To determine if a similar pathway is involved in other catabolic conditions, the effect of EPA on muscle protein degradation and activation of the ubiquitin-proteasome pathway has been determined during acute fasting in mice. When compared with a vehicle control group (olive oil) there was a significant decrease in proteolysis of the soleus muscles of mice treated with EPA after starvation for 24 h, together with an attenuation of the proteasome "chymotryptic-like" enzyme activity and the induction of the expression of the 20S proteasome alpha-subunits, the 19S regulator and p42, an ATPase subunit of the 19S regulator in gastrocnemius muscle, and the ubiquitin-conjugating enzyme E2(14k). The effect was not shown with the related (n-3) fatty acid docosahexaenoic acid (DHA) or with linoleic acid. However, 2,3,5-trimethyl-6-(3-pyridylmethyl)1,4-benzoquinone (CV-6504), an inhibitor of 5-, 12- and 15-lipoxygenases also attenuated muscle protein catabolism, proteasome "chymotryptic-like" enzyme activity and expression of proteasome 20S alpha-subunits in soleus muscles from acute fasted mice. These results suggest that protein catabolism in starvation and cancer cachexia is mediated through a common pathway, which is inhibited by EPA and is likely to involve a lipoxygenase metabolite as a signal transducer.     

Clenbuterol induces muscle-specific attenuation of atrophy through effects on the ubiquitin-proteasome pathway.

The ubiquitin-proteasome pathway is primarily responsible for myofibrillar protein degradation during hindlimb unweighting (HU). Beta-adrenergic agonists such as clenbuterol (CB) induce muscle hypertrophy and attenuate muscle atrophy due to disuse or inactivity. However, the molecular mechanism by which CB exerts these effects remains poorly understood. The aims of this study were to investigate whether CB attenuates HU-induced muscle atrophy through an inhibition of the ubiquitin-proteasome pathway and whether insulin-like growth factor I (IGF-I) mediates this inhibition. Rats were randomized to the following groups: weight-bearing control, 14-day CB-treated, 14-day HU, and CB + HU. HU-induced atrophy was associated with increased proteolysis and upregulation of components of the ubiquitin-proteasome pathway (ubiquitin conjugates, ubiquitin conjugating enzyme E2-14 kDa, and 20S proteasome activity). Upregulation of the ubiquitin proteasome occurred in all muscles tested but was more pronounced in muscles composed primarily of slow-twitch fibers (soleus) than in fast-twitch muscles (plantaris and tibialis anterior). Although CB induced hypertrophy in all muscles, CB attenuated the HU-induced atrophy and reduced ubiquitin conjugates only in the fast plantaris and tibialis anterior and not in the slow soleus muscle. CB did not elevate IGF-I protein content in either of the muscles examined. These results suggest that CB induces hypertrophy and alleviates HU-induced atrophy, particularly in the fast muscles, at least in part through a muscle-specific inhibition of the ubiquitin-proteasome pathway and that these effects are not mediated by the local production of IGF-I in skeletal muscle.     

Molt cycle-associated changes in calcium-dependent proteinase activity that degrades actin and myosin in crustacean muscle

The role of calcium-dependent proteinase (CDP) in the proecdysial atrophy of crustacean claw muscle has been investigated. During atrophy the molar ratio of actin to myosin heavy chain decreased 31%, confirming earlier ultrastructural observations that the ratio of thin:thick myofilaments declined from 9:1 to 6:1 (D. L. Mykles and D. M. Skinner, 1981, J. Ultrastruct. Res., 75, 314–325). The release of TCA-soluble material in muscle homogenates at neutral pH was stimulated by Ca²⁺ and completely inhibited by EGTA. The specific degradation of the major myofibrillar proteins (actin, myosin heavy and light chains, paramyosin, tropomyosin, troponin-T, and troponin-I) was demonstrated by SDS-polyacrylamide gel electrophoresis. Proteolytic activity was more than twofold greater in proecdysial muscle homogenates. Degradation of myofibrillar proteins was inhibited by EGTA, and the two inhibitors of cysteine proteinases, leupeptin and antipain, but not pepstatin, an inhibitor of aspartic proteinases. Unlike CDPs from vertebrate muscle, the CDP(s) in crab claw muscle degrades actin and myosin in addition to other myofibrillar proteins.     

Downregulation of muscle protein degradation in sepsis by eicosapentaenoic acid (EPA)

Eicosapentaenoic acid (EPA) has been shown to attenuate muscle atrophy in cancer, starvation and hyperthermia by downregulating the increased expression of the ubiquitin-proteasome proteolytic pathway leading to a reduction in protein degradation. In the current study EPA (0.5 g/kg) administered to septic mice completely attenuated the increased protein degradation in skeletal muscle by preventing the increase in both gene expression and protein concentration of the α- and β-subunits of the 20S proteasome, as well as functional activity of the proteasome, as measured by the ‘chymotrypsin-like’ enzyme activity. These results suggest that muscle protein catabolism in sepsis is mediated by the same intracellular signalling pathways as found in other catabolic conditions.     

The gene expression of ubiquitin ligase E3alpha 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 E3alpha together with the 14 kDa ubiquitin-conjugating enzyme E2(14k). The E3alpha gene was cloned only recently. The influence of sepsis on the gene expression of E3alpha 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 E3alpha 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 E3alpha and E2(14k) mRNA levels. The present study is the first report of increased E3alpha 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 E3alpha and E2(14k) suggests that muscle proteins are degraded in the N-end rule pathway during sepsis.     

Circulating 20S Proteasome Is Independently Associated with Abdominal Muscle Mass in Hemodialysis Patients

Protein-energy wasting is highly prevalent in hemodialysis patients, and it contributes to patient morbidity and mortality. The ubiquitin-proteasome system is the major pathway for intracellular protein degradation and it is involved in the regulation of basic cellular processes. However, the role of this system in the determination of nutritional status is largely unknown. To examine a relationship between protein-energy wasting and the ubiquitin-proteasome system, a cross-sectional study of 76 hemodialysis patients was performed. Plasma concentrations of 20S proteasome were studied to evaluate its association with muscle and fat mass, which were investigated by abdominal muscle and fat areas measured using computed tomography and by creatinine production estimated using the creatinine kinetic model. Plasma 20S proteasome concentrations significantly and negatively correlated with abdominal muscle areas and creatinine production (rho = -0.263, P < 0.05 and rho = -0.241, P < 0.05, respectively), but not abdominal subcutaneous and visceral fat areas. Multiple regression analyses showed that 20S proteasome was a significant independent predictor of abdominal muscle area (P < 0.05). In conclusion, plasma 20S proteasome concentrations were independently associated with abdominal muscle mass in hemodialysis patients. Our findings indicate a relationship between circulating 20S proteasomes and muscle metabolism in these patients.

Trial Registration

UMIN Clinical Trials Registry UMIN000012341     

The effects of the interaction of myosin essential light chain isoforms with actin in skeletal muscles

In order to compare the ability of different isoforms of myosin essential light chain to interact with actin, the effect of the latter protein on the proteolytic susceptibility of myosin light chains (MLC-1S and MLC-1V - slow specific and same as ventricular isoform) from slow skeletal muscle was examined. Actin protects both slow muscle essential light chain isoforms from papain digestion, similarly as observed for fast skeletal muscle myosin (Nieznanska et al., 1998, Biochim. Biophys. Acta 1383: 71). The effect of actin decreases as ionic strength rises above physiological values for both fast and slow skeletal myosin, confirming the ionic character of the actin-essential light chain interaction. To better understand the role of this interaction, we examined the effect of synthetic peptides spanning the 10-amino-acid N-terminal sequences of myosin light chain 1 from fast skeletal muscle (MLC-1F) (MLCFpep: KKDVKKPAAA), MLC-1S (MLCSpep: KKDVPVKKPA) and MLC-1V (MLCVpep: KPEPKKDDAK) on the myofibrillar ATPase of fast and slow skeletal muscle. In the presence of MLCFpep, we observed an about 19% increase, and in the presence of MLCSpep about 36% increase, in the myofibrillar ATPase activity of fast muscle. On the other hand, in myofibrillar preparations from slow skeletal muscle, MLCSpep as well as MLCVpep caused a lowering of the ATPase activity by about 36%. The above results suggest that MLCSpep induces opposite effects on ATPase activity, depending on the type of myofibrils, but not through its specific N-terminal sequence - which differs from other MLC N-terminal peptides. Our observations lead to the conclusion that the action of different isoforms of long essential light chain is similar in slow and fast skeletal muscle. However the interaction of essential light chains with actin leads to different physiological effects probably depending on the isoforms of other myofibrillar proteins.     

Myofibril-bound serine protease and its endogenous inhibitor in mouse: extraction, partial characterization and effect on myofibrils

The protein content of muscle is determined by the relative rates of synthesis and degradation. The balance between this process determines the number of functional contractile units within each muscle cell. Myofibril-bound protease, protease M previously reported in mouse skeletal muscle could be solubilized from the myofibrillar fraction by salt and acid treatment and partially purified by Mono Q and Superose 12 chromotagraphy. Isolated protease M activity in vitro on whole myofibrils resulted in myosin, actin, troponin T, α-actinin and tropomyosin degradation. Protease M is serine type and was able to hydrolyze trypsin-type synthetic substrates but not those of chymotrypsin type. In gel filtration chromatography, protease M showed Mr 120.0 kDa. The endogenous inhibitor (MHPI) is a glycoprotein (110.0 kDa) that efficiently blocks the protease M-dependent proteolysis of myofibrillar proteins in a dose-dependent way, as shown by electrophoretic analysis and synthetic substrates assays. Protease M-Inhibitor system would be implicated in myofibrillar proteins turnover.     

Proteolytic activity of proteasome on myofibrillar structures

The physiologic function of proteasome remains unclear. Evidence suggests a role in degradation of ubiquitin-protein conjugates, MHC antigen presentation, and some specificity of substrate within certain cell types. To explore further the properties of proteasome we have examined its effect on a well defined structure, the myofibril. We find that despite its large size (20S) proteasome is able to degrade myofibrils and intact, permeabilized muscle fibrils. The proteins degraded showed some specificity because actin, myosin and desmin were degraded faster than -actinin, troponin T and tropomyosin. Changes in ultrastructure were slow and included a general loss of structure with Z and I bands effected before the M band and costameres.     

IGF-I has no effect on postexercise suppression of the ubiquitin-proteasome system in rat skeletal muscle.

Both exercise and insulin-like growth factor I (IGF-I) are known to have major hypertrophic effects in skeletal muscle; however, the interactive effect of exogenous IGF-I and exercise on muscle protein turnover or the ubiquitin-proteasome pathway has not been reported. In the present study, we have examined the interaction between endurance exercise training and IGF-I treatment on muscle protein turnover and the ubiquitin-proteasome pathway in the postexercise period. Adult male rats (270-280 g) were randomized to receive 5 consecutive days of progressive treadmill exercise and/or IGF-I treatment (1 mg. kg body wt(-1). day(-1)). Twenty-four hours after the last bout of exercise, the rate of protein breakdown in incubated muscles was significantly reduced compared with that in unexercised rats. This was associated with a significant reduction in the chymotrypsin-like activity of the proteasome and the rate of ubiquitin-proteasome-dependent casein hydrolysis in muscle extracts from exercised compared with unexercised rats. In contrast, the muscle expression of the 20S proteasome subunit beta-1, ubiquitin, and the 14-kDa E2 ubiquitin-conjugating enzyme was not altered by exercise or IGF-I treatment 24 h postexercise. Exercise had no effect on the rates of total mixed muscle protein synthesis in incubated muscles 24 h postexercise. IGF-I treatment had no effect on muscle weights or the rates of protein turnover 24 h after endurance exercise. These results suggest that a suppression of the ubiquitin-proteasome proteolytic pathway after endurance exercise may contribute to the acute postexercise net protein gain.     

AMP and IMP Dissociate Actomyosin into Actin and Myosin

We investigated to determine why heating of squid muscle at 60 °C induced the liberation of actin from myofibrils. When a mixture of a myofibrillar fraction and a low-molecular sarcoplasmic fraction prepared from squid muscle was heated at 60 °C, actin liberation occurred. When a myofibrillar fraction was heated with ATP, AMP, or IMP, actin liberation occurred. Hence, AMP is perhaps one of the factors causing actin liberation in postmortem squid muscle. It was found that AMP and IMP reversibly dissociated actomyosin of chicken, bovine, and porcine skeletal muscles into actin and myosin on incubation at 0 °C at pH 7.2 in 0.2 M KCl. These results led us to conclude that AMP and IMP were the most responsible factors causing actin liberation from myofibrils in the heated muscle and causing reversible dissociation of actomyosin on storage of skeletal muscle at a low temperature. Hence, AMP and IMP are possible factors causing the resolution of rigor mortis in muscles.     

Redistribution of myosin heavy chain mRNA in the midregion of stretched muscle fibers

Summary Myosin mRNA distribution was compared to the distribution of striations, nuclei, and cytoskeletal components in normal fibers and in fibers undergoing growth and repair processes in response to stretch. Plantarflexion of rabbit lower hindlimb for 4 or 6 days resulted in a 35% increase in weight of the tibialis anterior muscle. Slow myosin expression in stretched fibers increased such that the proportion of fibers shifted from the fast type towards an intermediate type. Semi-quantitative in situ hybridization revealed a large increase in concentration of slow myosin mRNA in stretched fibers. Polysomes translating myosin heavy chain were excluded from the intact myofibrillar lattice. Significant increases of myosin mRNA concentration occurred only in the outer 8 m subsarcolemmal annulus of these stretched fibers (P<0.001) where myofibril formation also was evident. In some fibers, stretch caused myofibrillar disorder where nuclei became centrally located, and focal concentrations of myosin mRNA also occurred. We discuss mechanisms for mRNA accumulation and favor free diffusion to loosely packed cytoplasmic regions where myosin is needed for myofibrillar growth and repair.     

Adaptation of the ubiquitin-proteasome proteolytic pathway in cancer cachexia

The ubiquitin-proteasome proteolytic pathway is of major importance in the breakdown of skeletal muscle proteins. The first step in this pathway is the covalent attachment of polyubiquitin chains to the targeted protein. Polyubiquitinylated proteins are then recognized and degraded by the 26S proteasome complex. In this review, we critically analyze recent findings in the regulation of ubiquitinylation of protein substrates and of their subsequent proteasome-dependent degradation in animal models of cancer cachexia. In particular, we discuss the influence of various mediators (anorexia, hormones, prostaglandins, cytokines, and proteolysis-inducing factor) in signaling the activation of ubiquitin-proteasome proteolysis in skeletal muscle. These findings have lead to new concepts that are starting to be used for preventing cachexia in cancer and other wasting diseases.     

Localization to the proteasome is sufficient for degradation

The majority of unstable proteins in eukaryotic cells are targeted for degradation through the ubiquitin-proteasome pathway. Substrates for degradation are recognized by the E1, E2, and E3 ubiquitin conjugation machinery and tagged with polyubiquitin chains, which are thought to promote the proteolytic process through their binding with the proteasome. We describe a method to bypass the ubiquitination step artificially both in vivo and in a purified in vitro system. Seven proteasome subunits were tagged with Fpr1, and fusion reporter constructs were created with the Fpr1-rapamycin binding domain of Tor1. Reporter proteins were localized to the proteasome by the addition of rapamycin, a drug that heterodimerizes Fpr1 and Tor1. Degradation of reporter proteins was observed with proteasomes that had either Rpn10 or Pre10 subunits tagged with Fpr1. Our experiments resolved a simple but central problem concerning the design of the ubiquitin-proteasome pathway. We conclude that localization to the proteasome is sufficient for degradation and, therefore, any added functions polyubiquitin chains possess beyond tethering substrates to the proteasome are not strictly necessary for proteolysis.