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Am J Physiol Regul Integr Comp Physiol 281: R1193-R1200, 2001;
0363-6119/01 $5.00
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Vol. 281, Issue 4, R1193-R1200, October 2001

Heat shock protects L6 myotubes from catabolic effects of dexamethasone and prevents downregulation of NF-kappa B

Guangju Luo, Xiaoyan Sun, Eric Hungness, and Per-Olof Hasselgren

Department of Surgery, University of Cincinnati, and Shriners Hospital for Children, Cincinnati, Ohio 45267-0558


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Glucocorticoids are the most important mediator of muscle cachexia in various catabolic conditions. Recent studies suggest that the transcription factor NF-kappa B acts as a suppressor of genes in the ubiquitin-proteasome proteolytic pathway and that glucocorticoids increase muscle proteolysis by downregulating NF-kappa B activity. The heat shock (stress) response, characterized by the induction of heat shock proteins, confers a protective effect against a variety of harmful stimuli. In the present study, we tested the hypothesis that the heat shock response protects muscle cells from the catabolic effects of dexamethasone and prevents downregulation of NF-kappa B. Cultured L6 myotubes were subjected to heat shock (43°C for 1 h) followed by recovery at 37°C for 1 h. Thereafter, cells were treated for 6 h with 1 µM dexamethasone, during which period protein degradation was measured as release of TCA-soluble radioactivity from proteins that had been prelabeled with [3H]tyrosine. Heat shock resulted in increased protein and mRNA levels for heat shock protein 70. The increase in protein degradation induced by dexamethasone was prevented in cells expressing the heat shock response. In the same cells, dexamethasone-induced downregulation of NF-kappa B DNA binding activity was blocked. The present results suggest that the heat shock response may protect muscle cells from the catabolic effects of dexamethasone and that this effect of heat shock may be related to inhibited downregulation of NF-kappa B activity.

heat shock protein 70; muscle cachexia; nuclear factor-kappa B; proteolysis


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

MUSCLE CACHEXIA IS A PROMINENT metabolic response to a number of different disease states, including cancer (32, 40), diabetes (19), uremia (1), injury (8), sepsis (13, 34, 35), and acquired immunodeficiency syndrome (21). Muscle breakdown in these conditions is mainly caused by increased protein breakdown; in particular, ubiquitin-proteasome-dependent myofibrillar protein breakdown (13). Although the pathogenesis of muscle protein degradation is probably multifactorial, there is evidence that glucocorticoids are the most important factor in regulating muscle protein breakdown in different catabolic conditions (14). For example, sepsis- and injury-induced muscle proteolysis can be blocked by a glucocorticoid receptor antagonist (7, 12), and muscle protein breakdown can be induced by treating normal animals (33) or humans (5) with glucocorticoids. We and others reported previously that treatment of cultured myotubes with dexamethasone resulted in increased protein degradation and that this effect of dexamethasone was associated with upregulated activity and expression of the ubiquitin-proteasome pathway (6, 17, 37), lending further support to the important role of glucocorticoids in the development of muscle cachexia.

A recent report by Du et al. (6) provided insight into the molecular regulation of glucocorticoid-induced muscle protein degradation. In that study, treatment of cultured L6 myotubes with dexamethasone stimulated proteolysis, increased proteasome C3 subunit gene transcription, and downregulated nuclear factor (NF)-kappa B DNA binding activity. Additional experiments in which myotubes were transfected with a proteasome subunit promoter plasmid suggested that NF-kappa B is a repressor of certain proteasome subunit genes in muscle cells and that glucocorticoids may stimulate protein degradation by opposing this suppressor activity.

Muscle cachexia is important from a clinical standpoint for a number of reasons. Continuous muscle protein breakdown results in muscle wasting and fatigue that prevent or delay ambulation of patients with sepsis or severe injury and increase the risk for thromboembolic complications. When respiratory muscles are affected (31), pulmonary complications may occur and there may be a need for prolonged ventilatory support. In cancer patients, almost one-third of deaths has been estimated to be related to muscle cachexia (38), and there is evidence that the response to chemotherapy is impaired in patients with cachexia (36). Thus means to prevent or reduce muscle cachexia may have important clinical implications.

In recent years, accumulating evidence suggests that the heat shock (stress) response confers a protective effect against a variety of harmful factors, including hyperthermia, oxidants, and inflammation (4, 41). Although the exact mechanisms by which the stress response exerts its protective effect are not completely understood, it is generally believed that heat shock proteins act as "chaperones" to damaged proteins and prevent aggregation of these proteins, thus preventing further cell injury (24). One of the most widely studied inducible heat shock proteins is the 70-kDa heat shock protein 70 (HSP70), the induction of which is commonly used to monitor the heat shock response.

The influence of the heat shock response on glucocorticoid-induced muscle cachexia is not known. In the present study, we tested the hypothesis that induction of the heat shock response would inhibit the catabolic effect of dexamethasone and prevent the downregulation of NF-kappa B activity in dexamethasone-treated muscle cells.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cell culture. L6 rat skeletal muscle cells were purchased from the American Type Culture Collection (Manassas, VA). Cells were grown and maintained as described in detail previously (37) and were used between passages 2 and 8. Cells were seeded in 24-well culture plates (2.5 × 104 cells/well) or 10-cm dishes (2.5 × 105 cells/dish). Experiments were performed when ~90% of the cells had formed myotubes.

The L6 muscle cell line was originally developed by Yaffe (44) from trypsin-suspended thigh muscle cells of newborn rats. During culture, the cells differentiate into multinucleated fibers that become cross-striated and develop contractility, thus resembling mature muscle cells. L6 muscle cells were used in previous studies examining the regulation of protein turnover, and results from those experiments suggest that the response in the myotubes to various treatments, including treatment with dexamethasone, calcium, and hormones, is similar to the response seen in vivo or in incubated intact muscles (6, 9-11, 17, 37). Thus, although results obtained in vitro in cultured cells always need to be interpreted with caution, regulatory mechanisms of protein turnover in L6 myotubes are likely to reflect mechanisms in muscle cells in vivo.

Protein degradation. Protein degradation was measured by determining the release of TCA-soluble radioactivity from proteins that had been labeled for 48 h with L-[3,5-3H]tyrosine (New England Nuclear, Boston, MA) as described previously (17, 37). Release of TCA-soluble radioactivity was measured over a 6-h period, and the rate of protein degradation (%/6 h) was equal to 100 times the TCA-soluble radioactivity in the medium divided by the TCA-soluble plus TCA-insoluble radioactivity in the medium plus the myotube radioactivity.

Myotubes were subjected to hyperthermia (41 or 43°C) or were kept at 37°C for 1 h followed by recovery at 37°C for 1 h. The cells were then rinsed twice in Hanks' balanced salt solution, whereafter nonradioactive Dulbecco's modified Eagle's medium containing 2 mM tyrosine was added to the cells. The cells were incubated in the absence of presence of 1 µM dexamethasone for 6 h at 37°C, during which period protein degradation was measured as described above. In additional experiments, cells were studied up to 7 days after induction of heat shock to examine the correlation between the heat shock response (assessed from HSP70 levels) and the protective effect.

Thermotolerance. Myotubes were incubated at 37 or 43°C for 1 h and were allowed to recover at 37°C for 1 h. The cells were then exposed to 50°C for 2 h, whereafter cell viability was assessed by determining the number of cells that excluded 0.4% trypan blue dye or by measuring release of lactate dehydrogenase (LDH) as described previously (22).

Western blot analysis. Western blot analysis was used to determine HSP70 and inhibitory kappa Balpha (Ikappa Balpha ) levels. Cells were lysed in ice-cold 50 mM Tris · HCl buffer (pH 8.0) containing 100 mM NaCl, 5 mM EDTA, 10% Triton X-100, and the protease inhibitors leupepsin and phenylmethylsulfonyl fluoride. The cell lysates were transferred to microcentrifuge tubes and were centrifuged at 3,000 g for 5 min. Protein concentration in the supernatant was determined by the Bradford method (BioRad, Hercules, CA). The cell lysates were boiled in Laemmli buffer for 5 min. Aliquots containing 50 µg protein were loaded on a 4-15% SDS-polyacrylamide gel and transblotted onto nitrocellulose membranes in a Trans-Blot semi-dry electrophoretic transfer cell (BioRad). The membranes were blocked with 10% nonfat dried milk in Tris-buffered saline (TBS; pH 7.6) containing 0.05% Tween 20 for 1 h. A rabbit polyclonal IgG-specific antibody against the human HSP70 family, including the inducible isoform HSP72 (StressGen Biotechnologies, Victoria, BC, Canada), or a polyclonal rabbit antibody against human Ikappa Balpha (Santa Cruz Biotechnology, Santa Cruz, CA), was used as primary antibody at a dilution of 1:1,000 for 2 h. After washing three times in TBS containing 0.1% Tween 20, a secondary antibody (peroxidase-conjugated goat anti-rabbit IgG) was applied at a dilution of 1:2,000 for 1 h. The blots were washed three times in TBS containing 0.1% Tween 20 and were then incubated in enhanced chemiluminescence reagent (Amersham, Buckinghamshire, UK) and exposed to radiographic film (Eastman Kodak, Rochester, NY) and quantitated by densitometry.

Dot blot hybridization. Dot blot hybridization was used to determine HSP70 mRNA levels. In these experiments, myotubes were cultured in 10-cm dishes to increase the amount of tissue. RNA was extracted and dot blot hybridization was carried out as described previously in detail from this laboratory (39, 40). cDNA probes for HSP70 and GAPDH were labeled with digoxigenin (DIG)-11 dUTP (Boehringer Mannheim, Indianapolis, IN) by random labeling. RNA was quantified by spectrophotometry and denatured by boiling for 5 min. Three amounts of RNA (20, 10, and 5 µg) from each sample were blotted on a nylon membrane (Boehringer Mannheim) using a minifold II slot-blot filtration manifold (BioRad) and fixed to the membrane by ultraviolet cross-linking for 5 min. Prehybridization was carried out at 56°C for 4 h in a buffer consisting of 50% formamide, 7% SDS, 50 mM sodium phosphate (pH 7.0), 2% blocking reagent (Boehringer Mannheim), 5× SSC (1× SSC = 0.15 M NaCl, 0.015 M sodium citrate), and 0.1% N-laurolysarcosine.

Hybridization was carried out overnight at 56°C in the same buffer with 25 ng/ml DIG-labeled cDNA probe. After hybridization, the membranes were washed twice in 2× SSC, 0.1% SDS for 5 min at room temperature and twice in 0.1 SSC, 0.1% SDS for 15 min at 68°C. Chemiluminescence was detected by using 4-DIG alkaline phosphatase-conjugated Fab fragment (37.5 mU/ml; Boehringer Mannheim) and the substrate CDP-Star (0.25 mM; Boehringer Mannheim). The membranes were then exposed to X-ray film (X-Omat, Eastman-Kodak, New Haven, CT) for 1-4 h, and the signal intensities were determined by densitometry. The ratio between the signal intensities for HSP70 and GAPDH mRNA was calculated for the 5-, 10-, and 20-µg RNA dots, and the mean of these calculations was used for each experiment.

Electrophoretic mobility shift assay. Electrophoretic mobility shift assay (EMSA) was used to determine NF-kappa B DNA binding activity as described in previous reports from this laboratory (29). In short, nuclear fractions were prepared by harvesting myotubes in ice-cold phosphate-buffered saline (pH 7.4) and centrifuging the samples at 3,000 g for 5 min. The cells were weighed and resuspended in lysis buffer. After incubation for 5 min on ice, the nuclear pellet was isolated by centrifugation (3,000 g for 5 min). The supernatant was saved as the cytoplasmic fraction (for determination of Ikappa Balpha levels by Western blotting). The nuclear pellet was resuspended in extract buffer and incubated for 15 min. The samples were then centrifuged at 16,000 g for 20 min, and the supernatants were saved as the nuclear fractions.

EMSA was performed using a 32P-gamma -ATP end-labeled NF-kappa B gel shift oligonucleotide (5'-AGT TGA GGG GAC TTT CCC AGG C-3') (Santa Cruz Biotechnology) as described previously (29). An excess (20 ng) of unlabeled consensus or mutant NF-kappa B oligonucleotide (5'-GT TGA GGC GAC TTT CCC AGG C-3'; mutation underlined) was added for competition reactions. For supershift analysis, 2 µl of an antibody against p50 or p65 (Biomol, Plymouth Meeting, PA) was added 30 min after the addition of the radiolabeled probe. Samples were subjected to electrophoretic separation on a nondenaturing 5% polyacrylamide gel at 100 V using Tris-borate EDTA buffer (0.45 M Tris-borate, 0.001 M EDTA, pH 8.3). Blots were dried overnight and analyzed by exposure on PhosphorImager screens (Molecular Dynamics, Sunnyvale, CA).

Statistics. Results are given as means ± SE. Analysis of variance followed by Tukey's test was used for statistical analysis.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In initial experiments, we characterized the heat shock response in cultured L6 myotubes. When cells were subjected to hyperthermia (41 or 43°C) for 1 h and then returned to 37°C, cellular concentrations of the inducible HSP70 increased three- to fourfold and remained elevated for at least 6 h (Fig. 1). The increase in HSP70 levels occurred earlier and was more pronounced after 43 than 41°C. In continued experiments, 43°C was used to induce the heat shock response in the myotubes.


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  		Fig. 1.   Heat shock protein (HSP) 70 levels in cultured L6 myotubes subjected to heat shock. Myotubes were subjected to heat shock (41 or 43°C for 1 h) and were then allowed to recover at 37°C for 1, 3, or 6 h, whereafter HSP70 levels were determined by Western blotting. Control cells were kept at 37°C. A: representative Western blots from cells that had recovered for 3 h after heat shock. B: quantitation by densitometry of HSP70 levels in myotubes that had been subjected to 41 or 43°C heat shock for 1 h and then allowed to recover at 37°C for 1, 3, or 6 h. HSP70 levels are expressed as percent of control values (HSP70 in cells cultured at 37°C throughout the experimental period) and are given as means ± SE with n = 6 for each group. *P < 0.05 vs. control.

In other cell types, the induction of HSP70 by hyperthermia was regulated at the transcriptional level (4, 41). To test if the heat shock response is regulated by a similar mechanism in muscle cells, HSP70 mRNA levels were determined after subjecting cultured L6 myotubes to 43°C for 1 h. One hour after the heat shock, mRNA levels for HSP70 were increased approximately threefold and, after 3 and 6 h, the HSP70 mRNA levels were increased >10-fold (Fig. 2). This result suggests that the induction of HSP70 by hyperthermia is at least, in part, regulated at the transcriptional level in muscle cells.


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  		Fig. 2.   HSP70 mRNA levels in L6 myotubes subjected to heat shock (43°C for 1 h) and allowed to recover at 37°C for 1, 3, or 6h. A: representative dot blots from cells that had recovered at 37°C for 1, 3, and 6 h after heat shock. Control cells were cultured at 37°C throughout the experimental period. Three dots are shown for each experimental condition and represent 20, 10, and 5 µg RNA as described in MATERIALS AND METHODS. B: quantitation by densitometry of HSP70 mRNA levels. The ratio between the signal intensities for HSP70 and GAPDH mRNA was calculated for the 20-, 10-, and 5-µg RNA dots, and the mean of these calculations was used for each experiment. Results are means ± SE with n = 6 for each group. *P < 0.05 vs. control (37°C).

The main purpose of the present study was to test the influence of the heat shock response on dexamethasone-induced protein degradation in the myotubes. Protein degradation was determined by measuring the release of TCA-soluble radioactivity from proteins that had been labeled with [3H]tyrosine for 48 h before the experiment as described previously (17, 37). When cells were treated with 1 µM dexamethasone, the degradation of proteins increased by ~20-30%, similar to previous reports (17, 37). This effect of dexamethasone was significantly reduced in myotubes that had been subjected to heat shock (43°C for 1 h) before the treatment with dexamethasone (Fig. 3). Although the degree of inhibition of dexamethasone-induced proteolysis by the heat shock varied somewhat from one experiment to another, the protective effect of the heat shock was a consistent finding in multiple experiments. Heat shock did not influence basal protein breakdown rates.


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  		Fig. 3.   Effects of dexamethasone (Dex) and heat shock on protein degradation in cultured L6 myotubes. Myotubes were subjected to heat shock (43°C for 1 h followed by 37°C for 1 h) or were cultured at 37°C throughout the experimental period (no heat shock). Cells were then treated for 6 h with 1 µM Dex during which time protein degradation was determined by measuring the release of TCA-soluble radioactivity from proteins that had been labeled with [3H]tyrosine for 48 h as described in MATERIALS AND METHODS. Results are means ± SE with n = 7 for each group. *P < 0.05 vs. all other groups. Ctr, control cells.

In other cell types, the heat shock response conferred protection against the noxious effects of high temperature (4, 41). To test whether heat shock can induce a similar thermotolerance in muscle cells, myotubes were subjected to hyperthermia of 50°C for 2 h, whereafter cell viability was assessed by determining trypan blue exclusion or release of LDH into the medium. High temperature (50°C for 2 h) resulted in a substantial loss of cell viability; this effect of high temperature was prevented in cells that had been subjected to heat shock (Fig. 4).


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  		Fig. 4.   Cell viability assessed by trypan blue exclusion (A) or lactate dehydrogenase (LDH) release (B). L6 myotubes were subjected to heat shock (43°C for 1 h followed by recovery at 37°C for 1 h) and were then treated at 50°C for 2 h. Ctr were kept at 37°C throughout the experimental period. Results are means ± SE with n = 6 in each group. * and dagger P < 0.05 vs. other groups.

Although the results reported here of upregulated HSP70 levels and protection of the myotubes against the catabolic effects of dexamethasone and the noxious effects of high temperature suggest that HSP70 may be involved in the mechanisms of cell protection, additional experiments were performed to further test that notion. In those experiments, we examined the correlation between HSP70 levels and the protective effects by testing the influence of dexamethasone and high temperature (50°C) at a time point when the HSP70 levels had declined. A time course with regards to HSP70 levels after subjecting the myotubes to 43°C for 1 h was first established. Results from that experiment showed that HSP70 levels were increased for a relatively long period of time after the 1-h treatment at 43°C. Thus HSP70 levels remained elevated 4 days after heat shock and did not approach control levels until after 7 days (Fig. 5).


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  		Fig. 5.   HSP70 levels in cultured L6 myotubes 4 and 7 days after subjecting the cells to hyperthermia (43°C for 1 h). Ctr were kept at 37°C.

Cells that had been subjected to hyperthermia 7 days earlier were then treated with 1 µM dexamethasone for 6 h and protein degradation was measured. No protective effect of the heat shock response on dexamethasone-induced proteolysis was seen in these cells (Fig. 6). Likewise, there was no protective effect of the heat shock response in these cells with regards to the noxious effects of high temperature (Fig. 7). These results suggest that there is a temporal relationship between HSP70 levels and the protective effects in the myotubes, lending support to the concept that HSP70 may at least, in part, account for the protective effects of the heat shock response noticed in this study.


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  		Fig. 6.   Effect of Dex (1 µM for 6 h) on protein degradation in cultured myotubes that had been subjected to heat shock (43°C for 1 h) 7 days earlier. Results are means ± SE with n = 6 in each group. *P < 0.05 vs. Ctr (cells not treated with Dex).



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  		Fig. 7.   Cell viability assessed by trypan blue exclusion (A) or LDH release (B). L6 myotubes were exposed to 50°C for 2 h 7 days after induction of the heat shock response. Ctr were kept at 37°C for the duration of the experiment. Results are means ± SE with n = 6 in each group. *P < 0.05 vs. control.

Because a recent study suggested that downregulated NF-kappa B activity may be an important mechanism by which dexamethasone stimulates protein degradation in cultured myotubes (6), we next examined the effect of the heat shock response on NF-kappa B activity in dexamethasone-treated myotubes. Treatment of the cells with 1 µM dexamethasone for 6 h resulted in reduced NF-kappa B DNA binding activity (Fig. 8), similar to a recent report by Du et al. (6). Addition of an excess amount of cold competitor to the reaction, but not of a mutant competitor, deleted the NF-kappa B band, confirming the specificity of the EMSA. The most common form of NF-kappa B in other cell types is a p50/p65 heterodimer (2). Supershift analysis suggested that NF-kappa B contained p50 and p65 subunits in the L6 myotubes as well (Fig. 8).


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  		Fig. 8.   Nuclear factor (NF)-kappa B DNA binding activity determined by electrophoretic mobility shift assay (EMSA) in control myotubes and myotubes treated with Dex (1 µM) for 6 h. Cold, excess of wild-type NF-kappa B gel shift oligonucleotide for competition reaction; mutant, excess of mutant NF-kappa B oligonucleotide. p50 And p65 indicates addition of antibody to these NF-kappa B subunits for supershift analysis.

When cells had been subjected to hyperthermia 1 h before treatment with dexamethasone, the downregulation of NF-kappa B activity was blocked (Fig. 9). Because NF-kappa B is retained in its inactive form in the cytoplasm by its inhibitory protein Ikappa Balpha , cytoplasmic levels of Ikappa Balpha were measured. Treatment of the myotubes with dexamethasone resulted in increased levels of Ikappa Balpha , providing a potential mechanism for the dexamethasone-induced downregulation of NF-kappa B activity. The effect of dexamethasone on Ikappa Balpha levels was blocked in cells expressing the heat shock response (Fig. 9).


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  		Fig. 9.   Effect of heat shock and Dex on inhibitory kappa Balpha (Ikappa Balpha ) levels and NF-kappa B DNA binding activity in cultured L6 myotubes. HT, heat shock (43°C for 1 h followed by 37°C for 1 h); Dex, treatment with 1 µM dexamethasone for 6 h; top, Ikappa Balpha levels determined by Western blotting; bottom, NF-kappa B DNA binding activity determined by EMSA.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In the present study, induction of the heat shock response protected cultured myotubes from the catabolic effect of dexamethasone and induced thermotolerance in the same cells. To our knowledge, this is the first report of inhibited glucocorticoid-induced muscle proteolysis by the heat shock response. The observation is significant because glucocorticoids are an important mediator of muscle protein breakdown in various catabolic conditions, including cancer (32, 40), sepsis (33), injury (7), and renal failure (1), and methods to reduce the catabolic effect of glucocorticoids may have important clinical implications.

The present finding of increased protein degradation in cultured muscle cells after treatment with dexamethasone is in line with previous reports from our (37) and other laboratories (6, 17). In a recent study, we found that treatment of cultured L6 myotubes with dexamethasone resulted in increased energy-proteasome-dependent protein degradation and upregulated gene expression of ubiquitin and the proteasome subunit C3 (37). These effects of dexamethasone are similar to the effects of sepsis, injury, and other catabolic conditions in skeletal muscle, providing support for the use of dexamethasone-treated myotubes as a model of muscle cachexia.

Although muscle cachexia during sepsis and other catabolic conditions is caused by a combination of increased protein degradation and reduced protein synthesis, there is evidence that the increase in protein breakdown is the most important component of muscle cachexia (3, 15, 16). In recent experiments, treatment of cultured myotubes with dexamethasone stimulated protein degradation but did not influence protein synthesis (37). Consequently, the present study focused on the effect of heat shock on protein breakdown in dexamethasone-treated myotubes.

In a recent elegant study, Du et al. (6) characterized some of the molecular mechanisms of dexamethasone-induced protein degradation in cultured myotubes. Results from those experiments provided evidence that the transcription factor NF-kappa B is a suppressor of the proteasome subunit C3 gene and that glucocorticoids stimulate C3 subunit expression (and muscle protein degradation) by downregulating NF-kappa B activity. Inhibition of the dexamethasone-induced downregulation of NF-kappa B may therefore be a potential mechanism by which the heat shock response protects the cells from the effects of dexamethasone. The finding in the present study that NF-kappa B binding activity was not reduced by dexamethasone in myotubes that had been subjected to heat shock lends support to this concept. In addition, the present results suggest that the effects of the heat shock response and dexamethasone on NF-kappa B binding activity were mediated by changes in Ikappa Balpha levels.

Although the exact mechanisms by which the heat shock response exerts a protective effect are not fully understood, production of heat shock proteins is central to the stress response (4, 24, 41). In the present study, we measured HSP70 levels to monitor the induction of the heat shock response. HSP70 is one of the best characterized inducible heat shock proteins, and its induction has been used as a "marker" of the heat shock response in different tissues and cell types. It is important to point out that elevated HSP70 levels do not necessarily mean that the protective effects of heat shock were conveyed by this specific protein under the present experimental conditions but could have been conferred by other heat shock protein(s) as well. The temporal relationship between HSP70 levels and the protective effects of the heat shock suggests (but does not prove) that HSP70 was at least, in part, responsible for the protective effects noticed here. More direct evidence for a protective effect of HSP70 was reported in other cell types including cardiomyocytes and enterocytes (18, 25, 27).

We and others reported previously that hyperthermia stimulates protein degradation in incubated muscles and cultured myotubes (23). The present result of downregulated protein degradation in dexamethasone-treated myotubes after induction of the heat shock response, therefore, may seem contradictory to earlier studies. It should be noted, however, that in the present study, cells that were subjected to heat shock for 1 h were allowed to recover at 37°C for 1 h before they were treated with dexamethasone. This model differs from previous experiments in which we found that protein degradation was increased in myotubes that were subjected to hyperthermia continuously for 6 h (23). In the present study, the heat shock inhibited the response to dexamethasone but did not alter basal protein degradation rates.

In several previous studies, the heat shock response downregulated NF-kappa B activity in different tissues during inflammation and in stimulated cells (30). This differs from the present study in which the heat shock prevented the inhibition of NF-kappa B activity induced by dexamethasone. Of interest was the finding that heat shock itself did not reduce NF-kappa B activity in the present study. One explanation for this may be that NF-kappa B activity was determined 7 h after the heat shock in the present study (1-h recovery at 37°C and 6-h incubation with or without dexamethasone at 37°C) and the influence of a limited period of hyperthermia on basal Ikappa Balpha levels and NF-kappa B activity may be of shorter duration.

Induction of the heat shock response in skeletal muscle was reported after physical exercise in several previous reports (20, 28), but the physiological significance of this response to exercise is somewhat unclear. Two recent reports examined the potential role of the heat shock response in the regulation of muscle protein turnover. In one of those studies, treatment of cultured L8 myotubes with glutamine potentiated the induction of HSP70 caused by hyperthermia, and it was speculated that the stimulation of protein synthesis and inhibition of protein degradation by glutamine were related to the stress response (45). In another study, induction of the heat shock response by subjecting rats to 60 min of hyperthermia resulted in increased expression of HSP72 (the inducible form of HSP70) in soleus muscles and blunted the catabolic effect of hindlimb unweighting (26). Potential mechanisms of stress response-induced prevention of muscle cachexia were not explored in that study, but it was speculated that the heat shock response blocked the inhibition of muscle protein synthesis and the increase in protein degradation caused by extremity unweighting.

Although the present results are novel and may have clinical implications, a number of limitations needs to be kept in mind when the results are interpreted. First, the observations were made in vitro and it will be important in future experiments to determine the influence of the stress response on glucocorticoid-induced muscle proteolysis in vivo. Second, cells were pretreated with heat shock before treatment with dexamethasone, and an important question from a clinical standpoint is whether subjecting muscle cells to the stress response after the induction of protein degradation by dexamethasone will reduce proteolysis. Finally, although a mechanism of heat shock-induced protection of the myotubes from the catabolic effect of glucocorticoids was provided in the present study (i.e., inhibition of the dexamethasone-induced increase in Ikappa Balpha levels and decrease in NF-kappa B activity), it remains to be determined which specific heat shock protein(s) is responsible for the protective effect. Despite the limitations, however, the present study is important because it provides the first support to the concept that the heat shock (stress) response may downregulate glucocorticoid-mediated muscle cachexia.


    ACKNOWLEDGEMENTS

This work was supported in part by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-37908 and by grants from the Shriners of North America, Tampa, FL. E. Hungness was supported by a Research Fellowship from the Surgical Infection Society and Dura Pharmaceuticals.


    FOOTNOTES

Address for reprint requests and other correspondence: P.-O. Hasselgren, Dept. of Surgery, Univ. of Cincinnati, 231 Bethesda Ave., Cincinnati, OH 45267-0558 (E-mail: hasselp{at}uc.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.

Received 30 January 2001; accepted in final form 22 June 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Bailey, JL, Wang X, England BK, Price SR, Ding X, and Mitch WE. The acidosis of chronic renal failure activates muscle proteolysis in rats by augmenting transcription of genes encoding proteins of the ATP-dependent ubiquitin-proteasome pathway. J Clin Invest 97: 1447-1453, 1996[Web of Science][Medline].

2.   Baldwin, AS. The NF-kappa B and Ikappa B proteins: new discoveries and insights. Annu Rev Immunol 14: 649-683, 1996[Web of Science][Medline].

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Am J Physiol Regul Integr Comp Physiol 281(4):R1193-R1200
0363-6119/01 $5.00 Copyright © 2001 the American Physiological Society



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