AJP - Regu Ad Instruments
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Regul Integr Comp Physiol 288: R580-R590, 2005. First published November 24, 2004; doi:10.1152/ajpregu.00341.2004
0363-6119/05 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
288/3/R580    most recent
00341.2004v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (26)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Wei, W.
Right arrow Articles by Hasselgren, P.-O.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Wei, W.
Right arrow Articles by Hasselgren, P.-O.

INFLAMMATION AND CYTOKINES

Sepsis stimulates calpain activity in skeletal muscle by decreasing calpastatin activity but does not activate caspase-3

Wei Wei,1 Moin U. Fareed,1 Amy Evenson,1 Michael J. Menconi,1 Hongmei Yang,1 Victoria Petkova,2 and Per-Olof Hasselgren1

Departments of 1Surgery and 2Hematology/Oncology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts

Submitted 26 May 2004 ; accepted in final form 15 November 2004


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
We examined the influence of sepsis on the expression and activity of the calpain and caspase systems in skeletal muscle. Sepsis was induced in rats by cecal ligation and puncture (CLP). Control rats were sham operated. Calpain activity was determined by measuring the calcium-dependent hydrolysis of casein and by casein zymography. The activity of the endogenous calpain inhibitor calpastatin was measured by determining the inhibitory effect on calpain activity in muscle extracts. Protein levels of µ- and m-calpain and calpastatin were determined by Western blotting, and calpastatin mRNA was measured by real-time PCR. Caspase-3 activity was determined by measuring the hydrolysis of the fluorogenic caspase-3 substrate Ac-DEVD-AMC and by determining protein and mRNA expression for caspase-3 by Western blotting and real-time PCR, respectively. In addition, the role of calpains and caspase-3 in sepsis-induced muscle protein breakdown was determined by measuring protein breakdown rates in the presence of specific inhibitors. Sepsis resulted in increased muscle calpain activity caused by reduced calpastatin activity. In contrast, caspase-3 activity, mRNA levels, and activated caspase-3 29-kDa fragment were not altered in muscle from septic rats. Sepsis-induced muscle proteolysis was blocked by the calpain inhibitor calpeptin but was not influenced by the caspase-3 inhibitor Ac-DEVD-CHO. The results suggest that sepsis-induced muscle wasting is associated with increased calpain activity, secondary to reduced calpastatin activity, and that caspase-3 activity is not involved in the catabolic response to sepsis.

muscle wasting; sepsis; calpains; calpastatin; caspase-3; proteolysis

SEPSIS IS ASSOCIATED WITH a catabolic response in skeletal muscle, mainly reflecting increased breakdown of myofibrillar proteins (19, 21). Although ubiquitin- and proteasome-dependent proteolysis plays a prominent role in sepsis-induced muscle wasting, there is evidence that other mechanisms may be involved as well (22), including lysosomal protein degradation (13) and tripeptidyl peptidase II activity (53).

An additional proteolytic pathway that has been implicated in the development of muscle wasting is the calpain system. The calpains constitute a family consisting of at least 14 members, which may be ubiquitous enzymes, such as µ- and m-calpain, or tissue-specific proteins, such as the muscle-specific calpain 3, also called p94 (17). The regulation of calpain activity is complex. The most important activator of calpain is calcium, but calpain activity may also be influenced by other factors. For example, certain phospholipids, in particular, phosphatidylinositol, influence calpain activity by lowering the calcium concentration needed for autolysis of µ- and m-calpain (10, 38, 39). Other molecules as well have been proposed to regulate calpain activity by reducing the calcium requirements for activation, including isovalerylcarnitine (35) and an ~40- to 45-kDa endogenous "activator" present in skeletal muscle (36). Recent studies suggest that µ- and m-calpain can be phosphorylated at multiple sites (9), and, although the functional importance of calpain phosphorylation is not known at present, it is possible that µ- and m-calpain activity is influenced by phosphorylation of the enzymes (17).

An important additional regulator of calpain activity is the endogenous inhibitor calpastatin (17). Interestingly, calcium does not only activate calpains but also regulates the binding of calpastatin to calpain, resulting in inhibited calpain activity. In addition, calpain is autocatalyzed so that activated calpain degrades itself. Finally, activated calpain can degrade calpastatin, adding further complexity to the regulation of the calpain system.

Support for a role of calpains in muscle wasting comes from previous studies in which we found that the mRNA expression of calpains was increased in muscle from septic rats (16, 50). Reduced muscle calcium levels and inhibited protein breakdown after treatment with the calcium antagonist dantrolene provided further support for the concept that muscle protein breakdown in various catabolic conditions is at least in part regulated by a calcium-dependent proteolytic mechanism (16). On the basis of those and similar observations, we and others have proposed a model in which calpains provide an early, and perhaps rate-limiting, step in muscle wasting, accounting for degradation of Z-band-associated proteins, in particular titin and {alpha}-actinin and resulting in release of myosin and actin from the myofibrils (17, 19, 25, 41, 42, 50). In this model, the released actin and myosin are subsequently ubiquitinated and degraded by the 26S proteasome (19).

Although previous observations support a role of the calpain system in muscle wasting, the concept of calpain-mediated muscle proteolysis is controversial. One reason for this is that previous reports on calpain activity have been conflicting. For example, during sepsis, both increased (3) and unchanged (16) calpain activity in skeletal muscle have been reported. One reason why previous results were apparently conflicting may be differences in experimental models and methods used for the measurement of calpain activity, including differences in the substrate used in the assay and the method used for extraction of muscle proteins. Another reason why previous reports have been inconsistent may be that attention was not being paid to the potential role of calpastatin and its inhibitory effect on calpain. This is important because, when calpain activity is determined in tissue extracts by measuring the degradation of a substrate, results may be influenced by calpastatin bound to calpain. The importance of calpastatin was illustrated in a recent study in which muscle atrophy caused by unloading was reduced in transgenic mice overexpressing calpastatin in skeletal muscle (46). The influence of sepsis on calpastatin activity in skeletal muscle has not been reported.

Although previous studies suggest that calpain activity may be involved in the release of myofilaments from the sarcomer during muscle wasting (17, 19, 25, 41, 42, 50), a recent report by Du et al. (14) challenged that concept and instead proposed that activation of caspase-3 may be the mechanism by which myofilaments are released from the myofibrils, at least in muscle atrophy caused by acute diabetes or chronic uremia. The influence of sepsis on caspase-3 expression and activity in skeletal muscle and the role of caspase-3 in sepsis-induced muscle wasting have not been reported.

The purpose of the present study was to test the hypothesis that muscle calpain activity is increased during sepsis and that this effect of sepsis is at least in part regulated by changes in calpastatin activity. In addition, we examined the potential role of caspase-3 in sepsis-induced muscle wasting by determining caspase-3 expression and activity in septic muscle and the effect of caspase-3 inhibitors on protein breakdown rates in muscle from septic rats.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Experimental animals. Sepsis was induced in male Sprague-Dawley rats (50–60 g) by cecal ligation and puncture (CLP), as described in detail previously (16, 21, 44, 50). Control rats underwent sham operation, i.e., laparotomy and manipulation, but no ligation or puncture of the cecum. Small rats were used because rats of this size possess extremity muscles that are thin enough to allow for in vitro incubation with maintained tissue oxygenation, energy levels, and stable protein turnover rates (20). All rats were resuscitated with 10 ml/100 g body wt saline administered subcutaneously on the back at the time of surgery to prevent the development of hypovolemia and septic shock. The animals had free access to drinking water, but food was withheld after the surgical procedures to avoid the influence of differences in food intake between the septic and sham-operated rats on metabolic changes in muscle. After sham operation or CLP (16 h), extensor digitorum longus (EDL) and soleus muscles were harvested for determination of protein breakdown rates and calpain and caspase-3 expression and activity as described below. The septic model used here was used in several previous reports from our laboratory and results in a reproducible catabolic response in skeletal muscle and increased mRNA levels for multiple components of the ubiquitin-proteasome pathway and the calpain system (16, 21, 44, 50). The experiments were approved by the Institutional Animal Care and Utilization Committee at the Beth Israel Deaconess Medical Center (Boston, MA).

Muscle incubations. After sham operation or CLP (16 h), EDL and soleus muscles were dissected with intact tendons and incubated in vitro for 2 h under physiological conditions, as described previously (20, 21, 44). The tendons of the muscles were fixed to stainless steel supporters, and the muscles were maintained at resting length during incubation to provide optimal conditions with regard to energy levels and protein balance (1, 20). Bilateral muscles from the same rats were incubated pairwise in the absence or presence of calpain or caspase inhibitors, as described in RESULTS, followed by determination of protein breakdown rates and calpain activity. Protein breakdown rates were determined by measuring the net release of free tyrosine in the incubation medium using the fluorometric method described by Waalkes and Udenfriend (47). Cycloheximide (0.5 mM) was added to the incubation medium to prevent reincorporation of tyrosine into proteins. Because tyrosine is not synthesized or metabolized in skeletal muscle, its net release during incubation provides an accurate estimate of total protein breakdown.

Cell culture. L6 rat skeletal muscle cells (American Type Culture Collection, Manassas, VA) were thawed and maintained by repeated subculturing at low density in 162-cm2 culture flasks and were used between passages 2 and 8. Cells were grown in DMEM supplemented with 10% FBS, 100 U/ml penicillin, and 100 µg/ml streptomycin in 10% CO2 atmosphere at 37°C. When cells reached ~80% confluence, they were removed by trypsinization (0.25% trypsin in PBS) and seeded in 10-cm culture dishes or 12-well culture plates. The cells were grown in the presence of 10% FBS until they reached ~80% confluence, at which time the medium was replaced with DMEM containing 2% FBS for induction of differentiation into myotubes. Experiments were performed ~3 days later, when myotube formation was observed.

Rates of protein degradation were determined by measuring the release of TCA-soluble radioactivity from proteins labeled with [3H]tyrosine, as described previously (23, 49). After differentiation, myotubes were labeled with 1.0 µCi/ml L-[3,5-3H]tyrosine for 48 h in DMEM containing 2% FBS. Cells were washed two times with PBS and transferred to DMEM containing 2 mM unlabeled tyrosine. The myotubes were then treated for 24 h with 1 µM dexamethasone and different protease inhibitors, as indicated in RESULTS. After treatment, the culture medium was transferred to a microcentrifuge tube containing 100 µl BSA (10 mg/ml), and TCA was added to a final concentration of 10% (wt/vol). Samples were incubated at 4°C for 1 h followed by centrifugation for 5 min. The supernatant was used for determination of TCA-soluble radioactivity. The protein precipitates were dissolved with Tissue Solubilizer (Brinkman Instruments, Westburg, NY). Cell monolayers were washed with ice-cold PBS and solubilized with 0.5 M NaOH containing 0.1% Triton X-100. Radioactivity in the cell monolayer and TCA-soluble and -insoluble fractions were measured using a Packard TRI-CARB 1600 TR liquid scintillation analyzer. Protein degradation was expressed as the percentage of protein degraded over the 24-h period and was calculated as 100 times the TCA-soluble radioactivity in the medium divided by the TCA-soluble plus the TCA-insoluble radioactivity in the medium plus the cell layer (i.e., myotube) radioactivity.

Calpain activity. To test the effect of sepsis on muscle calpain activity, EDL and soleus muscles were harvested 16 h after CLP or sham operation and immediately frozen in liquid nitrogen. Muscles were then stored at –80°C until analysis. In an additional experiment, the effect of calpeptin on calpain activity was tested in incubated muscles. In that experiment, muscles were rinsed, blotted dry, and frozen at –80°C immediately after completion of the incubation. For determination of calpain activity, frozen muscles were pulverized and homogenized in a buffer consisting of 20 mM Tris·HCl, pH 8.0, 5 mM EDTA-Tris, pH 7.2, 0.1% {beta}-mercaptoethanol, 100 mg/l trypsin inhibitor, 2.5 µM E-64, and 2 mM serine protease inhibitor phenylmethylsulfonyl fluoride (PMSF). After centrifugation at 20,000 g for 30 min at 4°C, protein concentration in the supernatant was determined by using the Pierce Coomassie Blue R-250 method with BSA as standard.

Calpain activity in the supernatant was determined as described previously (43) with minor modifications. 4,4-Difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid labeled casein (BODIPY-FL-casein) was used as calpain substrate. Muscle extract (50 µg protein) was added to a microtiter plate well, and Dilution Buffer (20 mM Tris·HCl, pH 7.5, 1 mM EDTA, 100 mM KCl, and 0.1% mercaptoethanol) was added to bring the total volume to 100 µl. The reaction was initiated by adding 100 µl BODIPY-FL-casein (16 µg/ml) in 20 mM Tris·HCl, pH 7.5, 1 mM EDTA, 10 mM Ca2+, 100 mM KCl, and 0.1% mercaptoethanol to each well. The plate was incubated at 25°C for 60 min. The reaction was stopped by adding 25 µl of 100 mM EDTA, and fluorescence was read at 485-nm excitation and 530-nm emission wavelengths in a CytoFluor Multi-Well Plate Reader Series 4000 (Perseptive Biosystems, Framingham, MA). Duplicate assays were performed under identical conditions, except that calcium was omitted and 100 mM EDTA was added to the assay. Calpain activity was calculated as the difference between activity measured in the presence of 10 mM calcium and the activity measured in the absence of calcium and the presence of 100 mM EDTA. The assay, therefore, measured calcium-dependent degradation of casein. Calpain activity was expressed as fluorogenic units.

In additional experiments, calpain activity was assayed as described previously (16). The two major differences between that method and the method used here are 1) Suc-Leu-Tyr-7-amino-4-methylcoumarin (SLY) was used as substrate in our previous study (16), whereas BODIPY-FL-casein was used here; and 2) the techniques used for extraction of muscle proteins were different, with one major difference being that protease inhibitors and the reducing agent mercaptoethanol were not included in the extraction buffer in our previous report (16), whereas in the present study, multiple protease inhibitors and mercaptoethanol were included in the extraction buffer (see above). In one experiment, muscles were extracted as described previously (16) and homogenized in 5 vol of 100 mM Tris·HCl (pH 7.5), 10 mM EDTA, and 1 mM EGTA. The homogenates were centrifuged at 150,000 g for 90 min at 4°C. Aliquots (40 µg protein) of the supernatant were added to 160 µl of 50 µM SLY (dissolved in DMSO and buffer consisting of 100 mM Tris·HCl and 145 mM NaCl, pH 7.3). Incubations were performed at 30°C for 30 min in the absence or presence of 10 mM calcium and 400 nM of z-Leu-Leu-Tyr-CHN2, an inhibitor of calpains and cathepsin L. Amino-4-methyl coumarin release was measured by fluorometry using 360-nm excitation and 460-nm emission filters. Calpain activity was defined as the proteolytic activity at 10 mM calcium minus the activity in the presence of the calpain inhibitor and absence of calcium. Calpain activity was expressed as fluorogenic units. In one additional experiment, SLY was used as substrate after extraction was performed identical to the extraction used in the BODIPY-FL-casein method described above.

When calpain activity is measured in muscle extracts by determining the degradation of BODIPY-FL-casein or SLY, results may be influenced by the presence of calpastatin. To measure the activity of calpain independent of calpastatin, casein zymography was performed as described previously (12, 37). In this assay, µ- and m-calpain are separated by electrophoresis on a gel containing casein. Casein (0.02%, wt/vol) was copolymerized in a 10% (wt/vol) acrylamide gel (pH 8.8). The casein gel was subjected to 30 min of preelectrophoresis (125 volts) with Tris-glycine buffer (pH 8.3) containing 1 mM EDTA and 0.1% {beta}-mercaptoethanol. After protein loading (20 µg total cellular protein extracted as described above for the BODIPY-FL-casein method), electrophoresis was performed for 3 h at 150 volts and 4°C. The gel was then incubated for 16 h in activation buffer (pH 7.5) containing 50 mM Tris·HCl, 5 mM CaCl2, and 0.1% {beta}-mercaptoethanol at room temperature, typically with one or two changes of developing buffer. Finally, the casein gel was stained for 2 h with acid-based Coomassie blue. Calpain activity resulted in destained (clear) areas on the gel. The gels were scanned, and µ- and m-calpain activities were quantitated by densitometry.

Calpastatin activity. Calpastatin activity was measured by determining the inhibitory effect of muscle extracts on calpain-mediated degradation of BODIPY-FL-casein, as described in detail previously (28, 43). The muscle extracts were heated at 100°C for 5 min before the assay to destroy calpain activity (28). Purified µ-calpain (800 ng) from porcine erythrocytes (Calbiochem-Novabiochem, San Diego, CA) and aliquots (50 µg protein) of muscle extract were added to microtiter plate wells, and the volume was adjusted to 100 µl by adding Dilution Buffer (see above). The reaction was started by adding 100 µl BODIPY-FL-casein (16 µg casein/ml), and the calpastatin activity (expressed as %inhibition of calpain activity) was calculated from the difference in calpain activity measured in the absence or presence of muscle extract (43). To test the specificity of the calpastatin assay, a control experiment was performed in which an anti-calpastatin monoclonal antibody (Sigma, St. Louis, MO) was added to the reaction, as outlined in RESULTS.

Caspase-3 activity. Caspase-3 activity was determined as described in detail by Du et al. (14) with minor modifications. In short, frozen muscles were pulverized and homogenized on ice in a buffer consisting of 100 mM HEPES (pH 7.5), 10% sucrose, 0.1% Nonidet P-40, 10 mM dithiothreitol (DTT), and protease inhibitor cocktail (Sigma). Homogenates were subjected to three cycles of freeze-thaw before centrifugation at 18,000 g for 30 min. Aliquots (20 µg protein) were added to reaction buffer consisting of 100 mM HEPES (pH 7.5), 10 mM DTT, and 10% sucrose, and the mixture was preincubated for 30 min at 30°C with or without 50 µM of the cell-permeable caspase-3 inhibitor Ac-DEVD-FMK. The fluorogenic substrate Ac-DEVD-AMC (50 µM) was then added, and the reactions were performed at 30°C for 60 min. Fluorescence was measured using excitation and emission wavelengths of 360 nm and 460 nm, respectively. Results were expressed as fluorogenic units.

Western blotting. Aliquots (50 µg total cellular protein extracted as described above for the BODIPY-FL-casein method) of muscle extracts were loaded on 7 x 8 cm minigels (Millipore, Bedford, MA). SDS-PAGE was performed on 8% (for calpastatin) or 12.5% polyacrylamide gels (for all other proteins). The separated proteins were transferred electrophoretically using semi-dry transfer methodology to nitrocellulose membranes (Millipore). The membranes were blocked with blocking buffer (5% nonfat dry milk, 50 mM Tris·HCl, pH 7.5, 150 mM NaCl, and 1% Tween 20) for 1 h at room temperature. The membranes were then incubated overnight with one of the following primary antibodies: mouse anti-µ-calpain antibody that recognizes the large µ-calpain subunit (1:2,000; Sigma); rabbit anti-m-calpain antibody that recognizes the large m-calpain subunit (1:2,000; Sigma); goat anti-calpastatin antibody (1:200; Santa Cruz); rabbit anti-caspase-3 antibody (1:500; Santa Cruz); and mouse anti-{alpha}-tubulin antibody (1:1,000; Sigma). After incubation with the primary antibodies, the membranes were washed with TTBS (50 mM Tris·HCl, pH 7.5, 150 mM NaCl, and 1% Tween 20) three times and incubated for 1 h with the appropriate peroxidase-conjugated secondary antibody. Membranes were then washed exhaustively in TTBS. Immunoreactive protein bands were detected by using the Western Lightning kit for enhanced chemiluminescence (Perkin-Elmer Life Sciences, Boston, MA) and exposure on Kodak X-Omat blue film (Eastman Kodak, Rochester, NY). The identity of the bands on the Western blots was confirmed by using a molecular weight ladder when the Western blotting was performed. In addition, purified µ- and m-calpain was used as positive controls (data not shown). The molecular weight of the large subunits of µ- and m-calpain is 80 kDa, and of calpastatin 110 kDa. When the primary antibody was omitted, no bands corresponding to the calpain and calpastatin molecular weights were seen, providing further validation of the Western blots.

Real-time PCR. Caspase-3 and calpastatin mRNA levels were determined by real-time PCR performed as described in detail recently (34). Total RNA was extracted by the acid-guanidinium thiocyanate-phenol-chloroform method using Tri Reagent (MRC, Cincinnati, OH). The RNA was treated with DNase (DNA-free kit; Ambion). Multiplex real-time PCR was performed for quantitation of rat caspase-3 and calpastatin mRNA expression with amplification of 18S RNA as endogenous control; TaqMan analysis and subsequent calculations were performed with an ABI Prism 7700 Sequence Detection system (Perkin-Elmer, Foster City, CA). This system detects the increase in fluorescent signal released from an internal fluorogenic probe as the PCR proceeds. For each sample, 100 ng total RNA was subjected to real-time PCR according to the protocol provided by the manufacturer of the TaqMan One-Step PCR Master Mix Reagents Kit (part no. 4309169; ABI, Forster City, CA). A PCR reaction was also performed on total RNA that had not been reverse-transcribed to control for the absence of genomic DNA in the RNA preparation. The sequences of the forward, reverse, and double-labeled oligonucleotides for caspase-3 were as follows, respectively: 5'-CAC TGG AAT GTC AGC TCG CA-3', 5'- TCA GGG CCA TGA ATG TCT CTC-3', and 5'-TGG TAC CGA TGT CGA TGC AGC TAA CCT C-3'. The corresponding sequences for calpastatin were as follows: forward, 5'-GCT ATC ACA GGA CCT CTT CCA GA-3'; reverse, 5'-GGT GAA ATC AGA TGA CAA GGC A-3'; and double-labeled oligonucleotides, 5'-TCT CCT AAA CCT ATG GGA ATC GAC CAT GCT A-3'. Amplification of 18S RNA was performed in the same reaction tubes as an internal standard with an alternatively labeled probe (VIC-labeled probe) to distinguish its product from that derived from caspase-3 RNA. Caspase-3 and calpastatin mRNA concentrations were normalized to the 18S mRNA levels. Experiments were performed in triplicate for each standard and rat muscle sample.

Statistical analysis. Results are presented as means ± SE. Statistical analysis was performed by using Student’s t-test or ANOVA followed by Tukey’s test as appropriate. Most experiments were repeated at least three times to provide evidence of reproducibility.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Sepsis increases muscle calpain activity secondary to decreased calpastatin activity. When calpain activity was determined by measuring the degradation of BODIPY-FL-casein 16 h after sham operation or CLP in rats, results showed that calpain activity was increased by ~75% in EDL and 60% in soleus muscles of septic rats (Fig. 1A). This result differs from a previous report from our laboratory in which we found no evidence of increased muscle calpain activity in septic rats (16). In that study, the method used to measure calpain activity differed in two important aspects from the BODIPY-FL-casein method used here, i.e., the synthetic substrate SLY was used, and extraction of muscle proteins was performed in a buffer lacking protease inhibitors and mercaptoethanol. To test whether one or both of these differences may account for the different results observed here (increased calpain activity in septic muscle) and in our previous study (unchanged calpain activity), two experiments were performed. First, calpain activity was determined using an identical method described previously (16); calpain activity with this method did not show a difference between control and septic muscle (Fig. 1B), thus confirming our previous results (16). Second, SLY was used in muscle extracts that had been generated in an identical manner to that used here in the BODIPY-FL-casein method, i.e., using a buffer containing protease inhibitors and mercaptoethanol. When we used this approach, results showed that calpain activity increased in muscle from septic rats (Fig. 1C). Taken together, these results support the concept that sepsis increases calpain activity in skeletal muscle and suggest that the reason we were unable to demonstrate increased calpain activity in septic muscle in our previous report (16) reflected the fact that muscle proteins were extracted in buffer without protease inhibitors and mercaptoethanol (although the present experiments do not allow us to conclude whether the protease inhibitors or mercaptoethanol was most important for the present results, both groups of agents are important for maintaining the integrity and activity of the calpains).



View larger version (14K):
[in this window]
[in a new window]
  		Fig. 1. Effect of sepsis on calpain activity in skeletal muscle. A: calpain activity was determined by measuring the hydrolysis of the fluorogenic substrate BODIPY-FL-casein in extracts from extensor digitorum longus (EDL) and soleus (SOL) muscles. Sepsis was induced in rats by cecal ligation and puncture (CLP). Control rats were sham operated. Calpain activity is expressed as fluorogenic units (FU)/50 µg protein x 30 min. Results are means ± SE, with n = 8 rats in each group. *P < 0.05 vs. sham. B: calpain activity was determined in EDL and soleus muscles from sham-operated (sham) and septic (CLP) rats using SLY as substrate. Muscle proteins were extracted in a buffer without protease inhibitors and mercaptoethanol. Results are means ± SE, with n = 8 rats in each group. C: calpain activity was determined in EDL and soleus muscles from sham-operated and septic rats using SLY as substrate. Muscle proteins were extracted in a buffer containing protease inhibitors and mercaptoethanol. Results are means ± SE, with n = 8 rats in each group. *P < 0.05 vs. sham.

 
Because the methods used here to determine calpain activity do not give specific information about µ- and m-calpain activity, we next measured calpain activity by zymography. The basal activity of µ- and m-calpain was different in EDL and soleus muscles, with µ-calpain activity being higher in EDL and m-calpain activity being higher in soleus muscles (Fig. 2). Importantly, for the present study, µ- and m-calpain activities determined by zymography were not influenced by sepsis in either muscle when quantitations were performed by densitometry in multiple experiments (Fig. 2, B and C).



View larger version (16K):
[in this window]
[in a new window]
  		Fig. 2. Effect of sepsis (CLP) on µ- and m-calpain activity in EDL and soleus muscles determined by casein zymography. A: representative zymogram from extracts of EDL and soleus muscles from sham-operated and septic (CLP) rats. Quantification of zymograms by densitometry of µ- and m-calpain activity in EDL (B) and soleus (C) muscles from sham-operated and septic (CLP) rats. Results from densitometry are expressed as arbitrary units (AU) and are means ± SE, with n = 8 rats in each group.

 
Although it is possible that the apparently conflicting results from the BODIPY-FL-casein assay and zymography reflected insufficient sensitivity of the zymography method compared with the BODIPY-FL-casein method, a more likely explanation is that results in the BODIPY-FL-casein assay are influenced by the presence of calpastatin, whereas when zymography is performed, calpastatin is separated from the calpains (12, 37). Thus the increase in calpain activity noticed in the BODIPY-FL-casein assay may have been caused by reduced calpastatin activity. To test this mechanism, we next determined calpastatin activity in muscles from sham-operated and septic rats. Calpastatin activity was reduced by ~60% in EDL and by ~40% in soleus muscles from septic rats (Fig. 3), providing evidence for a potential mechanism of increased calpain activity determined in the BODIPY-FL-casein assay. In a control experiment, we tested the specificity of the calpastatin assay by adding an anticalpastatin monoclonal antibody to the reaction. Addition of 0.4 µl of the antibody to the reaction (n = 4) reduced the calpastatin activity by 100% in three assays and by 83% in one assay. In one experiment, two different amounts of anticalpastatin antibody were added to the reaction, and the calpastatin activity (expressed as %inhibition of calpain activity) was 18 (no antibody added), 6 (0.2 µl antibody added), and 3 (0.4 µl antibody added), suggesting a dose-dependent inhibition of calpastatin activity by the antibody, further validating the calpastatin assay.



View larger version (10K):
[in this window]
[in a new window]
  		Fig. 3. Effect of sepsis on calpastatin activity in EDL and soleus muscles from sham-operated and septic (CLP) rats. Results are means ± SE, with n = 8 rats in each group. *P < 0.05 vs. sham.

 
Calpastatin mRNA, but not protein, levels are reduced in septic muscle. To examine whether the reduced calpastatin activity in septic muscle was associated with changes in mRNA and protein expression of calpastatin, real-time PCR and Western blot analysis were performed. Calpastatin mRNA levels were reduced by 60–70% in both EDL and soleus muscles from septic rats (Fig. 4). Surprisingly, the decrease in calpastatin mRNA levels was not accompanied by reduced calpastatin protein levels (Fig. 5). When Western blots of proteins from 10 control and 10 septic muscle extracts were quantitated by densitometry, calpastatin protein levels were almost identical in muscles from sham-operated and septic rats (Fig. 5B). Similar to a previous report from this laboratory (16), muscle µ- and m-calpain levels were also unaffected by sepsis (Fig. 5 A, C, and D).



View larger version (9K):
[in this window]
[in a new window]
  		Fig. 4. Effect of sepsis on calpastatin mRNA levels in EDL and soleus muscles from sham-operated and septic (CLP) rats. mRNA levels were determined by real-time PCR and were normalized to 18S mRNA. Results are means ± SE, with n = 8 rats in each group. *P < 0.05 vs. sham.

 


View larger version (22K):
[in this window]
[in a new window]
  		Fig. 5. Protein expression of µ- and m-calpain and calpastatin in EDL and soleus muscles from sham-operated and septic (CLP) rats determined by Western blotting. {alpha}-Tubulin levels were determined as loading controls. A: representative Western blots of proteins from sham-operated and septic (CLP) rats. Quantification of calpastatin (B), µ-calpain (C), and m-calpain (D) protein levels was performed by densitometry. Results are means ± SE, with n = 10 rats in each group for calpastatin and n = 8 rats in each group for the calpains.

 
The calpain inhibitor calpeptin decreases muscle protein breakdown. To test whether calpain activity is involved in sepsis-induced muscle proteolysis, we treated muscles with the calpain inhibitor calpeptin (15). When EDL muscles from septic rats were incubated in the presence of 100 µM calpeptin, both protein degradation rates and calpain activity were reduced (Fig. 6). Interestingly, calpain activity and protein degradation were inhibited by calpeptin in muscles from sham-operated rats as well, suggesting that basal muscle protein breakdown rates are also regulated by calpains.



View larger version (12K):
[in this window]
[in a new window]
  		Fig. 6. Effect of calpeptin on protein breakdown (A) and calpain activity (B) in incubated EDL muscles from sham-operated and septic (CLP) rats. Protein breakdown rates were determined as net release of free tyrosine, as described in MATERIALS AND METHODS. Calpain activity was measured by using BODIPY-FL-casein as substrate. Muscles were incubated for 2 h in the absence or presence of 100 µM calpeptin, as indicated. Results are means ± SE, with n = 8 paired muscles in each group. *P < 0.05 vs. no calpeptin; {dagger}P < 0.05 vs. corresponding sham group by ANOVA.

 
Caspase-3 expression and activity are not increased in muscle from septic rats. Results in a recent study by Du et al. (14) suggested that caspase-3, rather than calpains, may be involved in the regulation of muscle protein breakdown in muscle-wasting conditions. To test whether caspase-3 plays a role in the present model of muscle catabolism, the expression and activity of caspase-3 were determined in muscles from sham-operated and septic rats. Caspase-3 mRNA levels were not influenced by sepsis in EDL or soleus muscles (Fig. 7A). The 32-kDa procaspase-3 is activated by caspase-9-regulated cleavage, resulting in 17-kDa and 12-kDa activated caspase-3 fragments (7, 14, 30, 32, 33). No activated caspase-3 17-kDa or 12-kDa fragments were detected in EDL or soleus muscles from sham-operated or septic rats (Fig. 7B). The expression of caspase-3 was somewhat different in EDL and soleus muscles; thus, in soleus muscles, a cleaved 29-kDa fragment was present. Although this fragment may represent activated caspase-3 (4), there were no differences in the 32-kDa and 29-kDa caspase-3 levels between soleus muscles from sham-operated and septic rats (Fig. 7B). Also, when caspase-3 activity was assessed by measuring hydrolysis of the fluorogenic substrate Ac-DEVD-AMC, there were no differences between muscles from sham-operated and septic rats (Fig. 7C).



View larger version (19K):
[in this window]
[in a new window]
  		Fig. 7. Effect of sepsis on caspase-3 expression and activity. A: caspase-3 mRNA levels determined by real-time PCR in EDL and soleus muscles from sham-operated and septic (CLP) rats. The mRNA levels were normalized to 18S mRNA and are expressed as arbitrary units (AU). Results are means ± SE, with n = 8 rats in each group. B: expression of procaspase-3 and cleaved caspase-3 fragments in EDL and soleus muscles from sham-operated and septic (CLP) rats determined by Western blotting. The location of molecular weight markers is indicated on right. {alpha}-Tubulin levels were measured as loading controls. Almost identical results were observed in three consecutive experiments. C: caspase-3 activity measured as hydrolysis of Ac-DEVD-AMC in extracts from EDL and soleus muscles of sham-operated and septic (CLP) rats. Results are means ± SE, with n = 8 rats in each group.

 
To further examine whether caspase-3 may be involved in sepsis-induced muscle proteolysis, EDL muscles from sham-operated and septic rats were incubated for 2 h in the absence or presence of the caspase-3 inhibitor Ac-DEVD-CHO. We used the same concentration (10 µM) of Ac-DEVD-CHO that was used in the study by Du et al. (14) in which the drug inhibited protein degradation in incubated epitrochlearis muscles from diabetic rats. Here, we found that Ac-DEVD-CHO did not reduce protein breakdown in EDL muscles from sham-operated or septic rats (Fig. 8).



View larger version (10K):
[in this window]
[in a new window]
  		Fig. 8. Protein breakdown rates in EDL muscles from sham-operated and septic (CLP) rats incubated in the absence (open bars) or presence (solid bars) of 10 µM of the caspase-3 inhibitor Ac-DEVD-CHO. Results are means ± SE, with n = 8 paired muscles in each group. *P < 0.05 vs. corresponding sham group.

 
Protein degradation in cultured myotubes is reduced by inhibitors of calpain, but not caspase, activity. Because it is possible that the lack of effect of the caspase-3 inhibitor on protein degradation in incubated EDL muscles reflected insufficient length of treatment (2 h), we next examined the effect of an extended period of treatment with the caspases-3 inhibitor Ac-DEVD-FMK. In this experiment, control and dexamethasone-treated L6 myotubes were exposed to Ac-DEVD-FMK for 24 h. Dexamethasone-treated myotubes were used because, in previous experiments, treatment of cultured L6 myotubes with dexamethasone resulted in a sepsis-like increase in proteasome- and calcium-dependent proteolysis (49). Ac-DEVD-FMK did not reduce basal or dexamethasone-induced protein degradation in the myotubes (Fig. 9A). Also, when the myotubes were treated for 24 h with the caspase-9 inhibitor LEHD-CHO, basal and dexamethasone-induced protein degradation was unaffected (Fig. 9B). In contrast, calpeptin blocked the dexamethasone-induced protein degradation in myotubes (Fig. 9C), lending further support to the concept that calpains regulate protein degradation in atrophying muscle.



View larger version (13K):
[in this window]
[in a new window]
  		Fig. 9. Effects of caspase and calpain inhibitors on dexamethasone-induced protein degradation. L6 myotubes were incubated for 24 h with or without dexamethasone (Dex; 1 µM) in the absence or presence of a caspase-3 inhibitor (Ac-DEVD-FMK, 20 µM; n = 6 wells/group; A), a caspase-9 inhibitor (LEHD-CHO, 20 µM; n = 6 wells/group; B), or the calpain inhibitor calpeptin (CP, 25 µM; n = 3 wells/group; C). Protein degradation was determined by measuring the release of TCA-soluble radioactivity from proteins prelabeled for 48 h with [3H]tyrosine. *P < 0.05 vs. control (Ctr; A and B); *P < 0.05 vs. all other groups (C).

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In the present study, sepsis in rats resulted in increased calpain activity in skeletal muscle, and this effect of sepsis reflected reduced calpastatin activity. The results are important because they reconcile, at least to some extent, previous apparently conflicting results from our laboratory with regard to muscle calpain activity during sepsis (16). Thus we found evidence that results may be influenced by the technique used to measure calpain activity. In particular, inclusion in the extraction buffer of agents that protect the calpains seems to be essential when calpain activity is measured. In addition, results are influenced by calpastatin activity when overall calpain activity is measured in muscle extracts, as suggested by zymography that determines µ- and m-calpain activity individually and independent of calpastatin activity.

The present results support the concept that increased calpain activity may be an important mechanism of muscle wasting during sepsis and perhaps other catabolic conditions as well (16, 17, 50). Additional support for a role of calpains in sepsis-induced muscle wasting was found in studies in which mRNA expression of µ- and m-calpain and p94 was increased in skeletal muscle of septic rats (16, 50). In other studies, muscle calcium levels were increased in various muscle-wasting conditions (26, 50), and treatment of rats with the calcium antagonist dantrolene prevented the sepsis-induced increase in muscle calcium levels and protein breakdown rates (16, 24, 50, 52). Taken together, those results suggest that sepsis-induced muscle proteolysis is at least in part regulated by calcium-activated proteases, possibly calpains. Because other reports provided evidence that the ubiquitin- and proteasome-dependent proteolytic pathway plays a central role in the development of muscle wasting (19, 44) despite the fact that the proteasome does not degrade intact myofibrils (27, 41, 42), a model has been proposed in which muscle protein breakdown during sepsis and other catabolic conditions is initiated by calcium- and calpain-mediated degradation of Z-band-associated proteins, in turn resulting in the release of the myofilaments actin and myosin or fragments of these proteins (17, 19). In this model, actin and myosin, released from the myofibrils, are subsequently ubiquitinated and degraded by the 26S proteasome. Recent studies suggest that the ubiquitin ligases atrogin-1 and MuRF1 may be particularly important for the regulation of ubiquitin- and proteasome-dependent protein breakdown in various catabolic conditions (5, 18, 51). Thus it is possible that calpain-dependent release of myofilaments is upstream of and at least in part may regulate the activation of atrogin-1 and MuRF1, although further studies are needed to determine the relationship between the activation of calpains and ubiquitin ligases in catabolic muscle.

It should be pointed out that, in addition to sepsis, other catabolic conditions as well have been associated with increased calpain expression and activity. For example, a role of calpain-mediated proteolysis was reported in patients with acute quadriplegic myopathy (40) and in various muscular dystrophies (45). Activation of calcium-dependent proteolysis (11) and increased mRNA expression of m-calpain (6) were observed in skeletal muscle of tumor-bearing rats. When cultured myocytes were transfected with a dominant negative m-calpain, protein degradation was reduced by 30%, and, when calpastatin was overexpressed, protein degradation was reduced by 63% (25). In the same study, inhibition of calpain activity also stabilized nebulin, and it was concluded that calpains play a key role in the disassembly of sarcomeric proteins.

The present results, indicating an important role of reduced calpastatin activity for the activation of calpains in skeletal muscle during sepsis, support a recent report by Tidball and Spencer (46). In that study, the authors generated transgenic mice that overexpressed calpastatin selectively in skeletal muscle. Overexpression of calpastatin reduced muscle atrophy by ~30% in their model of muscle wasting (unloading of hindlimb musculature for 10 days). Other reports as well support a role for changes in the balance between calpain and calpastatin in the regulation of muscle mass. For example, calcium-dependent muscle proteolysis in tumor-bearing rats was found to reflect reduced calpastatin activity in a recent study by Costelli et al. (11). In other studies, calpain-dependent protein degradation in differentiating myoblasts reflected reduced calpastatin expression and increased calpain-to-calpastatin ratio (2).

The present results of reduced calpastatin activity in muscle from septic rats despite unchanged calpastatin protein levels suggest that calpastatin activity may be regulated by mechanisms other than changes in the abundance of the peptide. Although it is well established that calpastatin-induced inhibition of calpain activity requires calcium-regulated binding of calpastatin to calpain, the exact mechanisms by which this binding is regulated and how calpain activity is inhibited are not fully understood at present. It is possible that binding of calcium to certain calpain domains results in conformational changes that increase the binding of calpastatin to calpain (31). It has also been speculated that phosphorylation of calpains and calpastatin may influence the ability of calpastatin to bind to and inhibit the calpains (9, 17). In a recent extensive review of the calpain/calpastatin system, Goll et al. (17) pointed out that there is little information available on the regulation of the calpain/calpastatin interaction and that this regulation will likely be an area of considerable attention in the coming years. The results in the present study are important because they provide evidence that changes in calpstatin activity can occur without changes in calpastatin protein expression.

The present results also suggest that the regulation of calpastatin protein and mRNA expression is complex, at least in septic muscle. Thus the unchanged calpastatin protein levels observed in muscles from septic rats, despite reduced calpastatin mRNA levels, suggest that the calpastatin protein levels were regulated by post-transcriptional mechanisms. The unchanged calpastatin protein levels in the presence of reduced calpastatin mRNA levels may be consistent with increased translational efficiency, reduced degradation of calpastatin protein, or a combination of these changes. The exact mechanisms of unchanged calpastatin protein levels in muscle from septic rats, despite reduced expression of calpastatin mRNA, remain to be determined.

In contrast to the present and other reports, a recent study by Du et al. (14) questioned the importance of calpains in the development of muscle atrophy. In that study, evidence was provided that increased caspase-3 activity may be the "upstream" mechanism by which myofilaments are released from the myofibrils followed by subsequent degradation by the ubiquitin-proteasome system. The conclusions in that study were based on experiments in which incubation of purified actomyosin or lysates from cultured L6 myotubes or rat psoas muscle with recombinant caspase-3 resulted in the production of a 14-kDa cleaved actin fragment. In subsequent experiments, the 14-kDa actin fragment was used as a marker of actin degradation in serum-deprived cultured L6 myotubes and in muscles from rats with acute diabetes or chronic uremia. When muscles from these rats were incubated in the presence of the caspase-3 inhibitor Ac-DEVD-CHO, the levels of the 14-kDa actin fragment were reduced, suggesting that caspase-3 was involved in the cleavage of actomyosin and actin. In addition, the authors reported data suggesting that caspase-3 activity was increased in gastrocnemius muscle of rats with acute diabetes. Unfortunately, calpain activity was not determined in the same muscles. In addition, the authors reported that tyrosine release from incubated epitrochlearis muscles from diabetic rats was reduced by ~13% by the caspase-3 inhibitor Ac-DEVD-CHO. This was a relatively modest reduction of protein degradation that did not prevent the diabetes-induced increase in muscle proteolysis. Thus protein degradation remained increased by ~35% compared with muscles from nondiabetic rats, even in the presence of Ac-DEVD-CHO. It would have been interesting to see the effect of a calpain inhibitor in the same experiment. Although data in the report by Du et al. (14) suggest that caspase-3 is involved in muscle protein degradation in catabolic (serum-starved) L6 myotubes and in muscle from diabetic and uremic rats, the results do not rule out the possibility that calpains played a role as well.

Although most reports are in agreement that the ubiquitin-proteasome system plays a central role in muscle wasting (19, 29), the present results support the concept that other mechanisms may also be important (22). The present study suggests that reduced calpastatin activity, resulting in increased calpain activity, may be a factor in sepsis-induced muscle wasting, similar to muscle wasting induced by unloading (46) and cancer (11). Our results do not rule out the possibility that additional mechanisms, including increased caspase activity, are involved in the development of muscle atrophy in certain muscle-wasting conditions, such as diabetes and chronic uremia (14).

It should also be noticed that recent studies provide evidence for a complex interaction between the calpain and caspase systems (32). For example, calpain-mediated truncation of caspase-3 may activate caspase-3 (4), whereas calpain-mediated cleavage of caspase-9 may result in loss of ability to activate caspase-3 (8). In addition, there is evidence that caspases may increase calpain activity by cleaving calpastatin (48). Thus it is possible that muscle wasting in various catabolic conditions is regulated by cross talk between calpains and caspases and that the relative importance of the two systems may vary in different conditions.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
The study was supported, in part, by National Institutes of Health Grants R01 DK-37908 and R01 NR-008545. During these studies, A. Evenson was a fellow under the Ruth L. Kirchstein National Research Service Award.


   FOOTNOTES
 

Address for reprint requests and other correspondence: P.-O. Hasselgren, Dept. of Surgery, Beth Israel Deaconess Medical Center, 330 Brookline Ave., Boston, MA 02215 (E-mail: phasselg{at}bidmc.harvard.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Baracos VE and Goldberg AL. Maintenance of normal length improves protein balance and energy status in isolated rat skeletal muscles. Am J Physiol Cell Physiol 251: C588–C596, 1986.[Abstract/Free Full Text]
  2. Barnoy S, Supino-Rosin L, and Kosower NS. Regulation of calpain and calpastatin in differentiating myoblasts: mRNA levels, protein synthesis and stability. Biochem J 351: 413–420, 2000.
  3. Bhattacharyya J, Thompson K, and Sayeed MM. Calcium-dependent and calcium-independent protease activities in skeletal muscle during sepsis. Circ Shock 35: 117–122, 1991.[Web of Science][Medline]
  4. Blomgren K, Zhu C, Wang X, Karlsson JO, Leverin AL, Bahr BA, Mallard C, and Hagberg H. Synergistic activation of caspase-3 by m-calpain after neonatal hypoxia-ischemia: a mechanism of "pathological apoptosis"? J Biol Chem 276: 10191–10198, 2001.[Abstract/Free Full Text]
  5. Bodine SC, Latres E, Baumheuter S, Lai VK, Nunez L, Clarke BA, Poueymirous WT, Panaro FJ, Na E, Dharmarajan K, Pan ZQ, Valenzuela DM, DeChiara TM, Stitt TN, Yancopoulos GD, and Glass DJ. Identification of ubiquitin ligases required for skeletal muscle atrophy. Science 294: 1704–1708, 2001.[Abstract/Free Full Text]
  6. Busquets S, Garcia-Martinez C, Alvarez B, Carbo N, Lopez-Soriano FJ, and Argiles JM. Calpain-3 gene expression is decreased during experimental cancer cachexia. Biochim Biophys Acta 1475: 5–9, 2000.[Medline]
  7. Carloni S, Mazzoni E, and Balduini W. Caspase-3 and calpain activities after acute and repeated ethanol administration during the rat brain growth spurt. J Neurochem 89: 197–203, 2004.[CrossRef][Web of Science][Medline]
  8. Chua BT, Guo K, and Li P. Direct cleavage by the calcium-activated protease calpain can lead to inactivation of caspases. J Biol Chem 275: 5131–5135, 2000.[Abstract/Free Full Text]
  9. Cong J, Thompson VF, and Goll DE. Phosphorylation of the calpains (Abstract). Mol Biol Cell 11: 386a, 2000.
  10. Coolican SA and Hathaway DR. Effect of L-alpha-phosphatidylinositol on a vascular smooth muscle Ca2+-dependent protease. J Biol Chem 259: 11627–11630, 1984.[Abstract/Free Full Text]
  11. Costelli P, Tullio RD, Baccino FM, and Melloni E. Activation of Ca(2+)-dependent proteolysis in skeletal muscle and heart in cancer cachexia. Br J Cancer 84: 946–950, 2001.[CrossRef][Web of Science][Medline]
  12. Croall DE, Moffett K, and Hatch H. Casein zymography of calpains using a 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid-imidazole buffer. Anal Biochem 304: 129–132, 2002.[CrossRef][Web of Science][Medline]
  13. Deval C, Mordier S, Obled C, Bechet D, Combaret L, Attaix D, and Ferrara M. Identification of cathepsin L as a differentially expressed message associated with skeletal muscle wasting. Biochem J 360: 143–150, 2001.[CrossRef][Web of Science][Medline]
  14. Du J, Wang X, Miereles C, Bailey JL, Debigare R, Zheng B, Price SR, and Mitch WE. Activation of caspase-3 is an initial step triggering accelerated muscle proteolysis in catabolic conditions. J Clin Invest 113: 115–123, 2004.[CrossRef][Web of Science][Medline]
  15. Ebisui C, Tsujinaka T, Kido Y, Iijima S, Yano M, Shibata H, Tanaka T, and Mori T. Role of intracellular proteases in differentiation of L6 myoblast cells. Biochem Mol Biol Int 32: 515–521, 1994.[Web of Science][Medline]
  16. Fischer DR, Sun X, Williams AB, Gang G, Pritts TA, James JH, Molloy M, Fischer JE, Paul RJ, and Hasselgren PO. Dantrolene reduces serum TNFalpha and corticosterone levels and muscle calcium, calpain gene expression, and protein breakdown in septic rats. Shock 15: 200–207, 2001.[Web of Science][Medline]
  17. Goll DE, Thompson VF, Li H, Wei W, and Cong J. The calpain system. Physiol Rev 83: 731–801, 2003.[Abstract/Free Full Text]
  18. Gomes MD, Lecker SH, Jagoe RT, Navon A, and Goldberg AL. Atrogin-1, a muscle specific F-box protein highly expressed during muscle atrophy. Proc Natl Acad Sci USA 98: 14440–14445, 2001.[Abstract/Free Full Text]
  19. Hasselgren PO and Fischer JE. Muscle cachexia: current concepts of intracellular mechanisms and molecular regulation. Ann Surg 233: 9–17, 2001.[CrossRef][Web of Science][Medline]
  20. Hasselgren PO, Hall-Angeras M, Angeras U, Benson D, James JH, and Fischer JE. Regulation of total and myofibrillar protein breakdown in rat extensor digitorum longus and soleus muscle incubated flaccid or at resting length. Biochem J 267: 37–44, 1990.[Web of Science][Medline]
  21. Hasselgren PO, James JH, Benson DW, Hall-Angeras M, Angeras U, Hiyama DT, Li S, and Fischer JE. Total and myofibrillar protein breakdown in different types of rat skeletal muscle: effects of sepsis and regulation by insulin. Metabolism 38: 634–640, 1989.[CrossRef][Web of Science][Medline]
  22. Hasselgren PO, Wray C, and Mammen J. Molecular regulation of muscle cachexia: it may be more than the proteasome. Biochem Biophys Res Commun 290: 1–10, 2002.[CrossRef][Web of Science][Medline]
  23. Hong DH and Forsberg NE. Effects of dexamethasone on protein degradation and protease gene expression in rat L8 myotube cultures. Mol Cell Endocrinol 108: 199–209, 1995.[CrossRef][Web of Science][Medline]
  24. Hotchkiss RS and Karl IE. Dantrolene ameliorates the metabolic hallmarks of sepsis in rats and improves survival in a mouse model of endotoxemia. Proc Natl Acad Sci USA 91: 3039–3043, 1994.[Abstract/Free Full Text]
  25. Huang J and Forsberg NE. Role of calpain in skeletal-muscle protein degradation. Proc Natl Acad Sci USA 95: 12100–12105, 1998.[Abstract/Free Full Text]
  26. Joffe M, Savage N, and Isaacs H. Increased muscle calcium. A possible cause of mitochondrial dysfunction and cellular necrosis in denervated rat skeletal muscle. Biochem J 196: 663–667, 1981.[Web of Science][Medline]
  27. Koohmaraie M. Ovine skeletal muscle multicatalytic proteinase complex (proteasome): purification, characterization, and comparison of its effects on myofibrils with mu-calpains. J Anim Sci 70: 3697–3708, 1992.[Abstract]
  28. Kosower NS and Barnoy S. Calpastatin (the endogenous calpain inhibitor) and membrane protein degradation in cell fusion. Methods Mol Biol 44: 181–194, 2000.
  29. Lecker SH, Jagoe RT, Gilbert A, Gomes M, Baracos V, Bailey J, Price SR, Mitch WE, and Goldberg AL. Multiple types of skeletal muscle atrophy involve a common program of changes in gene expression. FASEB J 18: 39–51, 2004.[Abstract/Free Full Text]
  30. McGinnis KM, Gnegy ME, Park YH, Mukerjee N, and Wang KK. Procaspase-3 and poly(ADP)ribose polymerase (PARP) are calpain substrates. Biochem Biophys Res Commun 263: 94–99, 1999.[CrossRef][Web of Science][Medline]
  31. Moldoveanu T, Hosfield CM, Lim D, Elce JS, Jia Z, and Davies PL. A Ca2+ switch aligns the active site of calpain. Cell 108: 649–660, 2002.[CrossRef][Web of Science][Medline]
  32. Neumar RW, Xu YA, Gada H, Guttmann RP, and Siman R. Cross-talk between calpain and caspase proteolytic systems during neuronal apoptosis. J Biol Chem 278: 14162–14167, 2003.[Abstract/Free Full Text]
  33. Nicholson DW and Thornberry NA. Caspases: killer proteases. Trends Biochem Sci 22: 299–306, 1997.[CrossRef][Web of Science][Medline]
  34. Okuno Y, Huettner CS, Radomska HS, Petkova V, Iwasaki H, Akashi K, and Tenen DG. Distal elements are critical for human CD34 expression in vivo. Blood 100: 4420–4426, 2002.[Abstract/Free Full Text]
  35. Pontremoli S, Melloni E, Viotti PL, Michetti M, DiLisa F, and Siliprandi N. Isovalerylcarnitine is a specific activator of the high calcium requiring forms of calpain. Biochem Biophys Res Commun 157: 373–380, 1990.[CrossRef]
  36. Pontremoli S, Viotti PL, Michetti M, Sparatore B, Salamino F, and Melloni E. Identification of an endogenous activator of calpain in rat skeletal muscle. Biochem Biophys Res Commun 171: 569–574, 1990.[CrossRef][Web of Science][Medline]
  37. Raser KJ, Posner A, and Wang KK. Casein zymography: a method to study µ-calpain, m-calpain, and their inhibitory agents. Arch Biochem Biophys 319: 211–216, 1995.[CrossRef][Web of Science][Medline]
  38. Saido TC, Mizuno K, and Suzuki K. Proteolysis of protein kinase C by calpain: effect of acidic phospholipids. Biomed Biochim Acta 50: 485–487, 1991.[Web of Science][Medline]
  39. Saido TC, Shibata M, Takenawa T, Murofushi H, and Suzuki K. Positive regulation of µ-calpain action by polyphosphoinositides. J Biol Chem 267: 24585–24590, 1992.[Abstract/Free Full Text]
  40. Showalter CJ and Engel AG. Acute quadriplegic myopathy: analysis of myosin isoforms and evidence for calpain-mediated proteolysis. Muscle Nerve 20: 316–322, 1997.[CrossRef][Web of Science][Medline]
  41. Solomon V, Baracos V, Sarraf P, and Goldberg AL. Rates of ubiquitin conjugation increase when muscles atrophy, largely through activation of the N-end rule pathway. Proc Natl Acad Sci USA 95: 12602–12607, 1998.[Abstract/Free Full Text]
  42. Solomon V, Lecker SH, and Goldberg AL. The N-end rule pathway catalyzes a major fraction of the protein degradation in skeletal muscle. J Biol Chem 273: 25216–25222, 1998.[Abstract/Free Full Text]
  43. Thompson VF, Saldana S, Cong J, and Goll DE. A BODIPY fluorescent microplate assay for measuring activity of calpains and other proteases. Anal Biochem 279: 170–178, 2000.[CrossRef][Web of Science][Medline]
  44. Tiao G, Fagan JM, Samuels N, James JH, Hudson K, Lieberman M, Fischer JE, and Hasselgren PO. Sepsis stimulates nonlysosomal, energy-dependent proteolysis and increases ubiquitin mRNA levels in rat skeletal muscle. J Clin Invest 94: 2255–2264, 1994.[Web of Science][Medline]
  45. Tidball JG and Spencer MJ. Calpains and muscular dystrophies. Int J Biochem Cell Biol 32: 1–5, 2000.[CrossRef][Web of Science][Medline]
  46. Tidball JG and Spencer MJ. Expression of a calpastatin transgene slows muscle wasting and obviates changes in myosin isoform expression during murine muscle disuse. J Physiol 545: 819–828, 2002.[Abstract/Free Full Text]
  47. Waalkes TP and Udenfriend S. A fluorometric method for the estimation of tyrosine in plasma and tissues. J Lab Clin Med 50: 733–736, 1957.[Web of Science][Medline]
  48. Wang KK, Posmantur R, Nadimpalli R, Nath R, Mohan P, Nixon RA, Talanian RV, Keegan M, Herzog L, and Allen H. Caspase-mediated fragmentation of calpain inhibitor protein calpastatin during apoptosis. Arch Biochem Biophys 356: 187–196, 1998.[CrossRef][Web of Science][Medline]
  49. Wang L, Luo GJ, Wang JJ, and Hasselgren PO. Dexamethasone stimulates proteasome- and calcium-dependent proteolysis in cultured L6 myotubes. Shock 10: 298–306, 1998.[Web of Science][Medline]
  50. Williams AB, Decourten-Myers GM, Fischer JE, Luo G, Sun X, and Hasselgren PO. Sepsis stimulates release of myofilaments in skeletal muscle by a calcium-dependent mechanism. FASEB J 13: 1435–1443, 1999.[Abstract/Free Full Text]
  51. Wray CJ, Mammen JMV, Hershko D, and Hasselgren PO. Sepsis upregulates the gene expression of multiple ubiquitin ligases in skeletal muscle. Int J Biochem Cell Biol 35: 698–705, 2003.[CrossRef][Web of Science][Medline]
  52. Wray CJ, Sun X, Gang GI, and Hasselgren PO. Dantrolene downregulates the gene expression and activity of the ubiquitin-proteasome proteolytic pathway in septic skeletal muscle. J Surg Res 104: 82–87, 2002.[CrossRef][Web of Science][Medline]
  53. Wray CJ, Tomkinson B, Robb BW, and Hasselgren PO. Tripeptidyl-peptidase II expression and activity are increased in skeletal muscle during sepsis. Biochem Biophys Res Commun 296: 41–47, 2002.[CrossRef][Web of Science][Medline]



This article has been cited by other articles:


Home page
 J. Appl. Physiol.Home page
S. M. Phillips, E. I. Glover, and M. J. Rennie
Alterations of protein turnover underlying disuse atrophy in human skeletal muscle
J Appl Physiol, September 1, 2009; 107(3): 645 - 654.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Cell Physiol.Home page
G. Neti, S. M. Novak, V. F. Thompson, and D. E. Goll
Properties of easily releasable myofilaments: are they the first step in myofibrillar protein turnover?
Am J Physiol Cell Physiol, June 1, 2009; 296(6): C1383 - C1390.
[Abstract] [Full Text] [PDF]

Home page
 Physiol. Rev.Home page
M. J. Tisdale
Mechanisms of Cancer Cachexia
Physiol Rev, April 1, 2009; 89(2): 381 - 410.
[Abstract] [Full Text] [PDF]

Home page
 J. Neurosci.Home page
M. V. Rao, P. S. Mohan, C. M. Peterhoff, D.-S. Yang, S. D. Schmidt, P. H. Stavrides, J. Campbell, Y. Chen, Y. Jiang, P. A. Paskevich, et al.
Marked Calpastatin (CAST) Depletion in Alzheimer's Disease Accelerates Cytoskeleton Disruption and Neurodegeneration: Neuroprotection by CAST Overexpression
J. Neurosci., November 19, 2008; 28(47): 12241 - 12254.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Endocrinol. Metab.Home page
I. J. Smith, S. H. Lecker, and P.-O. Hasselgren
Calpain activity and muscle wasting in sepsis
Am J Physiol Endocrinol Metab, October 1, 2008; 295(4): E762 - E771.
[Abstract] [Full Text] [PDF]

Home page
 J. Am. Soc. Nephrol.Home page
B. T. Workeneh, H. Rondon-Berrios, L. Zhang, Z. Hu, G. Ayehu, A. Ferrando, J. D. Kopple, H. Wang, T. Storer, M. Fournier, et al.
Development of a Diagnostic Method for Detecting Increased Muscle Protein Degradation in Patients with Catabolic Conditions
J. Am. Soc. Nephrol., November 1, 2006; 17(11): 3233 - 3239.
[Abstract] [Full Text] [PDF]

Home page
 J. Am. Soc. Nephrol.Home page
S. H. Lecker, A. L. Goldberg, and W. E. Mitch
Protein Degradation by the Ubiquitin-Proteasome Pathway in Normal and Disease States
J. Am. Soc. Nephrol., July 1, 2006; 17(7): 1807 - 1819.
[Full Text] [PDF]

Home page
 Am. J. Physiol. Regul. Integr. Comp. Physiol.Home page
M. U. Fareed, A. R. Evenson, W. Wei, M. Menconi, V. Poylin, V. Petkova, B. Pignol, and P.-O. Hasselgren
Treatment of rats with calpain inhibitors prevents sepsis-induced muscle proteolysis independent of atrogin-1/MAFbx and MuRF1 expression
Am J Physiol Regulatory Integrative Comp Physiol, June 1, 2006; 290(6): R1589 - R1597.
[Abstract] [Full Text] [PDF]

Home page
 Hum Mol GenetHome page
I. Kramerova, E. Kudryashova, G. Venkatraman, and M. J. Spencer
Calpain 3 participates in sarcomere remodeling by acting upstream of the ubiquitin-proteasome pathway
Hum. Mol. Genet., August 1, 2005; 14(15): 2125 - 2134.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
288/3/R580    most recent
00341.2004v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (26)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Wei, W.
Right arrow Articles by Hasselgren, P.-O.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Wei, W.
Right arrow Articles by Hasselgren, P.-O.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2005 by the American Physiological Society.