Effects of elevated temperature in muscles from septic rats MARIANNE PER-OLOF Department
HALL-ANGER&, HASSELGREN,
AND
on protein
HALL-ANGER&, ULF ANGER&, HASSELGREN, AND JOSEF E. FISCHER of Surgery, University of Cincinnati Medical Center, Cincinnati, Ohio 45267-0558
MARIANNE, ULF ANGER& PER-OLOF JOSEF E. FISHER. Effects of elevated tem-
perature on protein breakdown in muscles from septic rats. Am. J. Physiol. 258 (Cell Physiol. 27): C589-C592, 1990.-Elevated temperature has been proposed to contribute to accelerated muscle protein degradation during fever and sepsis. The present study examined the effect of increased temperature in vitro on protein turnover in skeletal muscles from septic and control rats. Sepsis was induced by cecal ligation and puncture (CLP); control rats were sham operated. After 16 h, the extensor digitorum longus (EDL) and soleus (SOL) muscles were incubated at 37 or 40°C. Protein synthesis was determined by measuring incorporation of [ 14C]phenylalanine into protein. Total and myofibrillar protein breakdown was assessed from release of tyrosine and 3-methylhistidine (3-MH), respectively. Total protein breakdown was increased at 40°C by 15% in EDL and by 29% in SOL from control rats, whereas 3-MH release was not affected. In muscles from septic rats, total and myofibrillar protein breakdown was increased by 22 and 30%, respectively, at 40°C in EDL but was not altered in SOL. Protein synthesis was unaffected by high temperature both in septic and nonseptic muscles. The present results suggest that high temperature is not the primary mechanism of increased muscle protein breakdown in sepsis because the typical response to sepsis, i.e., a predominant increase in myofibrillar protein breakdown, was not induced by elevated temperature in normal muscle. It is possible, however, that increased temperature may potentiate protein breakdown that is already stimulated by sepsis because elevated temperature increased both total and myofibrillar protein breakdown in EDL from septic rats. skeletal
breakdown
muscle; sepsis; proteolysis
INCREASEDMUSCLE PROTEIN degradationisacharacteristic response to injury and sepsis (6, 10, 11). The mediator(s) and mechanism(s) of this metabolic response remain unknown, but different monokines, especially interleukin 1 (IL-l) (1, 5), prostaglandin E2 (PGE2) (19), and a circulating proteolysis inducing factor (PIF) (6) have been implicated. An additional factor that has been suggested to contribute to increased muscle protein breakdown is the elevated body temperature, which typically accompanies infection and sepsis. A recent study by Baracos et al. (2) demonstrated that protein degradation in isolated rat skeletal muscle incubated at different temperatures between 33 and 42°C increased by about ll%/OC. In contrast, protein synthesis was relatively insensitive to changes in temperature. 0363-6143/90
$1.50
Copyright
0 1990
In the study by Baracos et al. (2), protein degradation was assessedby measuring release of tyrosine from incubated muscle. Although tyrosine release adequately reflects total proteolysis, it does not distinguish between the breakdown of myofibrillar and nonmyofibrillar proteins. The recent development of a sensitive high-performance liquid chromatography (HPLC) method to determine 3-methylhistidine (3-MH) at low concentrations has made it possible to separately measure total (i.e., tyrosine release) and myofibrillar (i.e., 3-MH release) protein breakdown in incubated skeletal muscles (9). 3MH is a minor amino acid present only in actin and myosin and is not degraded or reutilized for protein synthesis after its release during proteolysis (20). Consequently, the release of 3-MH from muscle reflects myofibrillar protein breakdown. Studies in which release of both tyrosine and 3-MH was measured suggested that the degradation of myofibrillar and nonmyofibrillar proteins is regulated independently and by different pathways (9, 13, 15, 16). A recent study from our laboratory suggested that sepsis mainly stimulates the breakdown of myofibrillar protein, in particular in white fast-twitch muscle (10). Thus determination of both total and myofibrillar proteolysis is important to achieve a more complete understanding of muscle protein breakdown and its regulation. The effect of high temperature on myofibrillar protein breakdown has not been reported. The purpose of the present study was to determine the effect of elevated temperature in vitro on total and myofibrillar protein degradation in muscles from nonseptic and septic rats. MATERIALS
AND
METHODS
Animals and operative procedures. Male Sprague-Dawley rats weighing 40-60 g were used because they possess thin extensor digitorum longus (EDL) and soleus (SOL) muscles, facilitating adequate diffusion of oxygen and substrate into tissue under in vitro conditions (8). Sepsis was induced by cecal ligation and puncture (CLP) as previously described (11). Sham-operated animals underwent laparotomy and manipulation of the cecum. Saline (5 ml/100 g body wt) was injected subcutaneously on the back for hydration in both groups of rats. The animals were fasted but allowed water after the operative procedure. Protein synthesis and degradation in incubated muscles. Sixteen hours after CLP or sham operation, the EDL C589 the American Physiological Society
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c590
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and SOL muscles from both sides were dissected with intact tendons and mounted on stainless steel supports at resting length. The muscles were weighed and individually preincubated at 37°C in 3 ml of Krebs-Henseleit bicarbonate buffer (pH 7.4) with glucose (10 mM) in a shaking water bath for 30 min. The incubation medium was vigorously gassed with Oa-COe (95:5) for 15 min immediately before use, and the incubation flasks were flushed with OZ-COZ (95:5) after addition of muscle and were then sealed with a rubber stopper. After preincubation, the muscles were gently blotted and transferred to fresh medium containing cycloheximide (0.5 mM) and were incubated for an additional 2 h. The Z-h incubation was performed at 37°C for one EDL and one SOL muscle while the contralateral muscles were incubated at 40°C. Because two shaking water baths were used, muscles could be incubated pairwise at the different temperatures. At the end of the Z-h incubation, tyrosine and 3MH were determined in tissue and medium by HPLC as described previously (lo), and any difference in tissue and medium free tyrosine or 3-MH between the paired muscles was used to calculate differences in the protein breakdown rate. Paired muscles were used to increase the sensit!ivity of the system, since interindividual variations in protein breakdown rates were avoided. It is important to recognize that this experimental design does not allow for determinations of actual rates of protein breakdown, because changes in tissue levels in each individual muscle during incubation were not measured; the experimental design only allows for determination of differences in protein breakdown rates between the paired muscles. For the study of protein synthesis, muscles were preincubated and incubated as described above, but the incubation medium contained [14C]phenylalanine (0.05 &i/ ml; 0.5 mM) and no cycloheximide. At the end of Z-h incubation, the amount of amino acid incorporated into trichloroacetic acid-precipitated proteins was determined as described in detail previously (11). Because a high precursor amino acid concentration was used in the incubation medium, equilibration between extracellular, intracellular, and aminoacyl-tRNA specific radioactivities rapidly occurred (4,17). Consequently, actual protein synthesis rates could be assessedby using the extracellular specific radioactivity for calculation of amount of phenylalanine incorporated into protein. Statistics. Results are presented as means t SE. Student’s t test for paired or unpaired observations was used for statistical comparisons.
IN
SEPTIC
MUSCLE
septic SOL compared with nonseptic muscle (Figs. I and 2). These data are consistent with previous reports from this laboratory (10, 11) in suggesting that muscle proteolysis is increased during sepsis, especially in the white fast EDL muscle. It should be noted, however, that, as explained in MATERIALS AND METHODS, the amounts of amino acids released into incubation medium did not reflect actual proteolytic rates, because changes in tissue levels during incubation were not measured in each individual muscle. The main purpose of this study was not to study the effect of sepsison muscle protein breakdown but to study the influence in vitro of elevated temperature on proteolysis in nonseptic and septic muscle, and this is why paired muscles were used in these experiments. In nonseptic muscle, total protein breakdown was increased at 40°C by 15 and 29% in EDL and SOL, respectively, while myofibrillar protein breakdown was not affected (Fig. 1). In septic EDL muscle, total and myofibrillar protein breakdown rates were increased at 40°C by 22 and 30%, respectively, whereas proteolysis in septic SOL was not affected by elevated temperature (Fig. 2). Protein synthesis rate in EDL muscles from nonseptic rats (n = 7) was 224 t 20 nmol phenylalanine (Phe) . g-l.2 h-l under basal conditions, i.e., incubation at 37°C. The corresponding value in EDL from septic rats (n = 7) was 149 t 10 nmol Phe-g-l.2 h-l (-33%, P < 0.01). Although these results suggest that muscle protein synthesis is reduced during sepsis, similar to previous reports (12), the data should be interpreted with caution because the two groups of rats were not studied on the same day, and results could have been influenced by day-to-day variation. Protein synthesis rates were not significantly affected by high temperature in vitro, in either nonseptic or septic muscles (Fig. 3). -
500
EDL
is
*
RESULTS
Tissue levels of tyrosine and 3-MH were similar in paired muscles incubated at 37 or 40°C both in muscles from nonseptic and septic rats (data not shown). The effect of elevated temperature on total and myofibrillar protein breakdown rates could therefore be assessedby determining differences in release into incubation medium of tyrosine and 3-MH, respectively. Tyrosine and 3-MH release into incubation medium under basal conditions (i.e., incubation at 37°C) was increased bv 80-100% in septic EDL and bv ZO-35% in
FIG. 1. Tyrosine and 3-methylhistidine (3-MH) release by nonseptic extensor digitorum long-us (EDL) and soleus (SOL) muscles incubated pairwise at 37 (open bars) or 40°C (hatched bars). Results are from 20 naired EDL and 7 naired SOL muscles. * P < 0.05: ** P < 0.01.
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PROTEIN EDL
BREAKDOWN
EDL
1u ti -
X
8
[
FIG. 2. Tyrosine and 3-MH release by septic EDL and SOL muscles incubated pairwise at 37 (open bars) or 40°C (hatched bars). Results are from 17 paired EDL and 7 paired SOL muscles. Definitions are as in Fig. 1. * P < 0.05; ** P < 0.01. Non-septic muscle
n r N
300
X
-s it 0E
Septic
muscle
-
+ 200
-
100
-
T
25 -2 e m
.-: 5 ii .-c 3 P a
0,
FIG. 3. Protein synthesis (open bars) or 40°C (hatched and 7 paired septic muscles.
in EDL muscles incubated pairwise at 37 bars). Results are from 7 paired nonseptic Definitions are as in Fig. 1.
DISCUSSION
In a recent study, protein breakdown in incubated rat skeletal muscle was stimulated by increased temperature, and fever was proposed to be a mechanism of accelerated muscle proteolysis in sepsis (2). However, because only tyrosine release was measured in that study, it was not known if myofibrillar protein breakdown was increased by elevated temperature as well. This would be important because sepsis mainly stimulates the degradation of myofibrillar proteins, with only a modest increase in sarcoplasmic protein breakdown (lo), similar to other catabolic conditions, such as burn injury (7), glucocorticoid treatment (13), and fasting (16). The purpose of the present report therefore was to study the influence of high temperature in vitro on both total and myofibrillar protein degradation in skeletal muscle. Our results confirmed the study of Baracos et al. (2) in demonstrating increased tvrosine release bv nonseetic
IN
SEPTIC
MUSCLE
a91
rat EDL and SOL muscles incubated at high temperature, although the response per degree of increased temperature noted here, i.e., 5%/“C in EDL and lO%/OC in SOL, was somewhat smaller than the ll%/"C reported earlier (2). The finding that protein synthesis rate was not affected by high temperature suggests that the increased release of tyrosine was not the result of a general increase in protein turnover but reflected a specific effect on protein breakdown. Also, in the study of Baracos et al. (Z), protein synthesis was relatively insensitive to elevated temperature, and at 42”C, protein synthesis was even inhibited. In contrast to total protein breakdown, myofibrillar protein degradation (i.e., 3-MH release) in nonseptic muscle was unaffected by high temperature in vitro. This result suggests that increased temperature is not the primary mechanism of myofibrillar protein degradation in sepsis. It should be noted, however, that muscles were incubated for only 2 h, and it is possible that myofibrillar proteins are sensitive to high temperature over a longer period of time. The slower turnover rate of myofibrillar than nonmyofibrillar proteins (3) could explain why a longer time span may be required to detect alteration of myofibrillar protein breakdown rate. Although elevated temperature did not increase myofibrillar protein degradation in nonseptic muscle, high temperature may be a potentiating factor, further stimulating sepsis-induced proteolysis. To test this hypothesis, paired muscles from septic rats were incubated at different temperatures. Results from these experiments demonstrated that both total and myofibrillar protein degradation was further stimulated by high temperature in EDL muscle from septic animals, whereas septic SOL did not respond to elevated temperature. This suggests that the present in vitro findings may have in vivo implications because the effect of sepsis on both total and myofibrillar protein breakdown were more pronounced in white than in red muscle (10). The present findings that high temperature regulated total and myofibrillar protein breakdown individually and that the response was different in the white EDL and red SOL muscles illustrates the importance of measuring both tyrosine and 3-MH release in different types of skeletal muscle when possible mediators and mechanisms of muscle proteolysis are elucidated. Because the increased tyrosine release in nonseptic muscle was not associated with increased 3-MH release, the high temperature must primarily have stimulated breakdown of nonmyofibrillar proteins. Thus, in this respect, the effect of high temperature in vitro was different from the effect of sepsis (10). The mechanisms of temperature-induced changes in muscle proteolysis remain to be determined. Previous studies by Baracos et al. (2) found no evidence that lysosomal proteases or prostaglandins were involved in the response to elevated temperature. The mechanism of the specific effect of high temperature on protein breakdown in septic EDL muscle is also unknown, but it may be speculated that elevated temperature made the muscle more sensitive to one or several mediators of the septic
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c592
PROTEIN
BREAKDOWN
response, provided the mediators were retained in the muscle in vitro. Although the present study suggests that elevated temperature is not a primary factor for increased muscle proteolysis in sepsis, it must be remembered that the effects of high temperature in vitro may be quite different from the effects of fever in vivo. Thus conclusions from the present experiments must be drawn with great caution. However, the interpretation of the present results that high temperature alone does not stimulate muscle proteolysis in sepsis was supported by a recent study in which administration of tumor necrosis factor induced fever in rats without activating muscle protein breakdown (14). When the cyclooxygenase inhibitor ibuprofen was administered to endotoxin-treated human volunteers, body temperature was almost normalized while the hypoferremia and elevation of plasma C reactive protein level in .duced by endotoxin were unaffected (18), suggesting that some metabolic alterations in endotoxemia, and perhaps sepsis as well, can occur in the absence of elevated body temperature. This work was supported in part by the National Institute of Diabetes and Digestive and Kidney Diseases Grant lROl-DK-3790801. M. Hall-Anger&s and U. Angeras were also supported by grants from the Gothenborg Medical Society, The Medical Faculty, University of Gothenborg, and the Swedish Society of Medical Sciences. Address reprint requests to J. E. Fisher. Received
15 May
1989; accepted
in final
form
21 November
IN
6.
7.
8.
9.
10.
11.
12.
13.
14.
1989.
REFERENCES 1. BARACOS, V. E., H. P. RODEMANN, C. A. DINARELLO, AND A. L. GOLDBERG. Stimulation of muscle protein degradation and prostaglandin E2 release by leukocytic pyrogen (interleukin 1). N. Engl. J. Med. 308: 553-558,1983. 2. BARACOS, V. E., E. J. WILSON, AND A. L. GOLDBERG. Effects of temperature on protein turnover in isolated rat skeletal muscle. Am. J. Physiol. 246 (Cell Physiol. 15): C125-C130, 1984. 3. BATES, P. C., AND D. J. MILLWARD. Myofibrillar protein turnover: synthesis rates of myofibrillar and sarcoplasmic protein fractions in different muscles and the changes observed during postnatal development and in response to feeding and starvation. Biochem. J. 214: 587-592,1983. 4. CLARK, A. S., AND W. E. MITCH. Comparison of protein synthesis and degradation in incubated and perfused muscle. Biochem. J. 212: 649-653, 1983. 5. CLOWES, G. H. A., B. C. GEORGE, S. BOSARI, AND W. LOVE. Induction of muscle protein degradation by recombinant interleu-
15.
16.
17.
18.
19.
20.
SEPTIC
MUSCLE
kin 1 (rIL-1) and its spontaneously occurring fragments (Abstract). J. Leukocyte Biol. 42: 547-548, 1987. CLOWES, G. H. A., B. C. GEORGE, C. A. VILLEE, AND C. A. SARAVIS. Muscle proteolysis induced by a circulating peptide in patients with sepsis or trauma. N. Engl. J. Med. 308: 545-552, 1983. DOWNEY, R. S., W. W. MONATO, I. E. KARL, D. E. MATTHEWS, AND D. M. BIER. Protein dynamics in skeletal muscle after trauma: local and systemic effects. Surgery 99: 265-273, 1986. GOLDBERG, A. L., S. B. MARTEL, AND M. J. KUSHMERICK. In vitro preparations of the diaphragm and other skeletal muscles. In: Methods in Enzymology, edited by J. G. Hardman and B. W. O’Malley. New York: Academic, 1975, vol. 39, p. 82-94. GOODMAN, M. N. Differential effects of acute changes in cell Ca2+ concentration on myofibrillar and non-myofibrillar protein breakdown in the rat extensor digitorum long-us muscle in vitro. Assessment by production of tyrosine and NT-methylhistidine. Biochem. J. 241: 121-127, 1987. HASSELGREN, P. O., J. H. JAMES, D. W. BENSON, M. HALLANGER&, U. ANGERAS, D. T. HIYAMA, S. LI, AND J. E. FISCHER. Total and myofibrillar protein breakdown in different types of rat skeletal muscle: effects of sepsis and regulation by insulin. Metabolism 38: 634-640, 1989. HASSELGREN, P. O., M. A. TALAMINI, J. H. JAMES, AND J. E. FISCHER. Protein metabolism in different types of skeletal muscle during early and late sepsis in rats. Arch. Surg. 121: 918-923, 1986. HUMMEL, R. P., P. 0. HASSELGREN, J. H. JAMES, B. W. WARNER, AND J. E. FISCHER. The effect of sepsis in rats on skeletal muscle protein synthesis in vivo and in periphery and central core of incubated muscle preparations in vitro. Metabohsm 37: 1120-1127, 1988. KAYALI, A. G., V. R. YOUNG, AND M. N. GOODMAN. Sensitivity of myofibrillar proteins to glucocorticoid-induced muscle proteolysis. Am. J. PhysioZ. 252 (Endocrinol. Metab. 15): E62GE626, 1987. KETTELHUT, I. C., AND A. L. GOLDBERG. Tumor necrosis factor can induce fever in rats without activating protein breakdown in muscle or lipolysis in adipose tissue. J. Clin. Inuest. 81: 1384-1389, 1988. LOWELL, B. B., N. B. RUDERMAN, AND M. N. GOODMAN. Evidence that lysosomes are not involved in the degradation of myofibrillar proteins in rat skeletal muscle. Biochem. J. 234: 237-240, 1986. LOWELL, B. B., N. B. RUDERMAN, AND M. N. GOODMAN. Regulation of myofibrillar protein degradation in rat skeletal muscle during brief and prolonged starvation. Metabolism 35: 1121-1127, 1986. RANNELS, D. E., S. A. WARTELL, AND C. A. WATKINS. The measurement of protein synthesis in biological systems. Life Sci. 30: 1679-1690, 1982. REVHAUG, A., H. R. MICHIE, J. McK. MANSON, J. M. WATTERS, C. A. DINARELLO, S. M. WOLFF, AND D. W. WILMORE. Inhibition of cycle-oxygenase attenuates the metabolic response to endotoxin in humans. Arch. Surg. 123: 162-170, 1988. RODEMANN, H. P., AND A. L. GOLDBERG. Arachidonic acid, prostaglandin E2 and Fzol influence rates of protein turnover in skeletal and cardiac muscle. J. Biol. Chem. 257: 1632-1638, 1982. YOUNG, V. R., AND H. N. MUNRO. NT-Methylhistidine (3-methylhistidine) and muscle protein turnover: an overview. Federation Proc. 37: 2291-2300, 1978.
Downloaded from www.physiology.org/journal/ajpcell by ${individualUser.givenNames} ${individualUser.surname} (130.070.008.131) on November 6, 2018. Copyright © 1990 American Physiological Society. All rights reserved.
HALL-ANGER&, HASSELGREN,
AND
on protein
HALL-ANGER&, ULF ANGER&, HASSELGREN, AND JOSEF E. FISCHER of Surgery, University of Cincinnati Medical Center, Cincinnati, Ohio 45267-0558
MARIANNE, ULF ANGER& PER-OLOF JOSEF E. FISHER. Effects of elevated tem-
perature on protein breakdown in muscles from septic rats. Am. J. Physiol. 258 (Cell Physiol. 27): C589-C592, 1990.-Elevated temperature has been proposed to contribute to accelerated muscle protein degradation during fever and sepsis. The present study examined the effect of increased temperature in vitro on protein turnover in skeletal muscles from septic and control rats. Sepsis was induced by cecal ligation and puncture (CLP); control rats were sham operated. After 16 h, the extensor digitorum longus (EDL) and soleus (SOL) muscles were incubated at 37 or 40°C. Protein synthesis was determined by measuring incorporation of [ 14C]phenylalanine into protein. Total and myofibrillar protein breakdown was assessed from release of tyrosine and 3-methylhistidine (3-MH), respectively. Total protein breakdown was increased at 40°C by 15% in EDL and by 29% in SOL from control rats, whereas 3-MH release was not affected. In muscles from septic rats, total and myofibrillar protein breakdown was increased by 22 and 30%, respectively, at 40°C in EDL but was not altered in SOL. Protein synthesis was unaffected by high temperature both in septic and nonseptic muscles. The present results suggest that high temperature is not the primary mechanism of increased muscle protein breakdown in sepsis because the typical response to sepsis, i.e., a predominant increase in myofibrillar protein breakdown, was not induced by elevated temperature in normal muscle. It is possible, however, that increased temperature may potentiate protein breakdown that is already stimulated by sepsis because elevated temperature increased both total and myofibrillar protein breakdown in EDL from septic rats. skeletal
breakdown
muscle; sepsis; proteolysis
INCREASEDMUSCLE PROTEIN degradationisacharacteristic response to injury and sepsis (6, 10, 11). The mediator(s) and mechanism(s) of this metabolic response remain unknown, but different monokines, especially interleukin 1 (IL-l) (1, 5), prostaglandin E2 (PGE2) (19), and a circulating proteolysis inducing factor (PIF) (6) have been implicated. An additional factor that has been suggested to contribute to increased muscle protein breakdown is the elevated body temperature, which typically accompanies infection and sepsis. A recent study by Baracos et al. (2) demonstrated that protein degradation in isolated rat skeletal muscle incubated at different temperatures between 33 and 42°C increased by about ll%/OC. In contrast, protein synthesis was relatively insensitive to changes in temperature. 0363-6143/90
$1.50
Copyright
0 1990
In the study by Baracos et al. (2), protein degradation was assessedby measuring release of tyrosine from incubated muscle. Although tyrosine release adequately reflects total proteolysis, it does not distinguish between the breakdown of myofibrillar and nonmyofibrillar proteins. The recent development of a sensitive high-performance liquid chromatography (HPLC) method to determine 3-methylhistidine (3-MH) at low concentrations has made it possible to separately measure total (i.e., tyrosine release) and myofibrillar (i.e., 3-MH release) protein breakdown in incubated skeletal muscles (9). 3MH is a minor amino acid present only in actin and myosin and is not degraded or reutilized for protein synthesis after its release during proteolysis (20). Consequently, the release of 3-MH from muscle reflects myofibrillar protein breakdown. Studies in which release of both tyrosine and 3-MH was measured suggested that the degradation of myofibrillar and nonmyofibrillar proteins is regulated independently and by different pathways (9, 13, 15, 16). A recent study from our laboratory suggested that sepsis mainly stimulates the breakdown of myofibrillar protein, in particular in white fast-twitch muscle (10). Thus determination of both total and myofibrillar proteolysis is important to achieve a more complete understanding of muscle protein breakdown and its regulation. The effect of high temperature on myofibrillar protein breakdown has not been reported. The purpose of the present study was to determine the effect of elevated temperature in vitro on total and myofibrillar protein degradation in muscles from nonseptic and septic rats. MATERIALS
AND
METHODS
Animals and operative procedures. Male Sprague-Dawley rats weighing 40-60 g were used because they possess thin extensor digitorum longus (EDL) and soleus (SOL) muscles, facilitating adequate diffusion of oxygen and substrate into tissue under in vitro conditions (8). Sepsis was induced by cecal ligation and puncture (CLP) as previously described (11). Sham-operated animals underwent laparotomy and manipulation of the cecum. Saline (5 ml/100 g body wt) was injected subcutaneously on the back for hydration in both groups of rats. The animals were fasted but allowed water after the operative procedure. Protein synthesis and degradation in incubated muscles. Sixteen hours after CLP or sham operation, the EDL C589 the American Physiological Society
Downloaded from www.physiology.org/journal/ajpcell by ${individualUser.givenNames} ${individualUser.surname} (130.070.008.131) on November 6, 2018. Copyright © 1990 American Physiological Society. All rights reserved.
c590
PROTEIN
BREAKDOWN
and SOL muscles from both sides were dissected with intact tendons and mounted on stainless steel supports at resting length. The muscles were weighed and individually preincubated at 37°C in 3 ml of Krebs-Henseleit bicarbonate buffer (pH 7.4) with glucose (10 mM) in a shaking water bath for 30 min. The incubation medium was vigorously gassed with Oa-COe (95:5) for 15 min immediately before use, and the incubation flasks were flushed with OZ-COZ (95:5) after addition of muscle and were then sealed with a rubber stopper. After preincubation, the muscles were gently blotted and transferred to fresh medium containing cycloheximide (0.5 mM) and were incubated for an additional 2 h. The Z-h incubation was performed at 37°C for one EDL and one SOL muscle while the contralateral muscles were incubated at 40°C. Because two shaking water baths were used, muscles could be incubated pairwise at the different temperatures. At the end of the Z-h incubation, tyrosine and 3MH were determined in tissue and medium by HPLC as described previously (lo), and any difference in tissue and medium free tyrosine or 3-MH between the paired muscles was used to calculate differences in the protein breakdown rate. Paired muscles were used to increase the sensit!ivity of the system, since interindividual variations in protein breakdown rates were avoided. It is important to recognize that this experimental design does not allow for determinations of actual rates of protein breakdown, because changes in tissue levels in each individual muscle during incubation were not measured; the experimental design only allows for determination of differences in protein breakdown rates between the paired muscles. For the study of protein synthesis, muscles were preincubated and incubated as described above, but the incubation medium contained [14C]phenylalanine (0.05 &i/ ml; 0.5 mM) and no cycloheximide. At the end of Z-h incubation, the amount of amino acid incorporated into trichloroacetic acid-precipitated proteins was determined as described in detail previously (11). Because a high precursor amino acid concentration was used in the incubation medium, equilibration between extracellular, intracellular, and aminoacyl-tRNA specific radioactivities rapidly occurred (4,17). Consequently, actual protein synthesis rates could be assessedby using the extracellular specific radioactivity for calculation of amount of phenylalanine incorporated into protein. Statistics. Results are presented as means t SE. Student’s t test for paired or unpaired observations was used for statistical comparisons.
IN
SEPTIC
MUSCLE
septic SOL compared with nonseptic muscle (Figs. I and 2). These data are consistent with previous reports from this laboratory (10, 11) in suggesting that muscle proteolysis is increased during sepsis, especially in the white fast EDL muscle. It should be noted, however, that, as explained in MATERIALS AND METHODS, the amounts of amino acids released into incubation medium did not reflect actual proteolytic rates, because changes in tissue levels during incubation were not measured in each individual muscle. The main purpose of this study was not to study the effect of sepsison muscle protein breakdown but to study the influence in vitro of elevated temperature on proteolysis in nonseptic and septic muscle, and this is why paired muscles were used in these experiments. In nonseptic muscle, total protein breakdown was increased at 40°C by 15 and 29% in EDL and SOL, respectively, while myofibrillar protein breakdown was not affected (Fig. 1). In septic EDL muscle, total and myofibrillar protein breakdown rates were increased at 40°C by 22 and 30%, respectively, whereas proteolysis in septic SOL was not affected by elevated temperature (Fig. 2). Protein synthesis rate in EDL muscles from nonseptic rats (n = 7) was 224 t 20 nmol phenylalanine (Phe) . g-l.2 h-l under basal conditions, i.e., incubation at 37°C. The corresponding value in EDL from septic rats (n = 7) was 149 t 10 nmol Phe-g-l.2 h-l (-33%, P < 0.01). Although these results suggest that muscle protein synthesis is reduced during sepsis, similar to previous reports (12), the data should be interpreted with caution because the two groups of rats were not studied on the same day, and results could have been influenced by day-to-day variation. Protein synthesis rates were not significantly affected by high temperature in vitro, in either nonseptic or septic muscles (Fig. 3). -
500
EDL
is
*
RESULTS
Tissue levels of tyrosine and 3-MH were similar in paired muscles incubated at 37 or 40°C both in muscles from nonseptic and septic rats (data not shown). The effect of elevated temperature on total and myofibrillar protein breakdown rates could therefore be assessedby determining differences in release into incubation medium of tyrosine and 3-MH, respectively. Tyrosine and 3-MH release into incubation medium under basal conditions (i.e., incubation at 37°C) was increased bv 80-100% in septic EDL and bv ZO-35% in
FIG. 1. Tyrosine and 3-methylhistidine (3-MH) release by nonseptic extensor digitorum long-us (EDL) and soleus (SOL) muscles incubated pairwise at 37 (open bars) or 40°C (hatched bars). Results are from 20 naired EDL and 7 naired SOL muscles. * P < 0.05: ** P < 0.01.
Downloaded from www.physiology.org/journal/ajpcell by ${individualUser.givenNames} ${individualUser.surname} (130.070.008.131) on November 6, 2018. Copyright © 1990 American Physiological Society. All rights reserved.
PROTEIN EDL
BREAKDOWN
EDL
1u ti -
X
8
[
FIG. 2. Tyrosine and 3-MH release by septic EDL and SOL muscles incubated pairwise at 37 (open bars) or 40°C (hatched bars). Results are from 17 paired EDL and 7 paired SOL muscles. Definitions are as in Fig. 1. * P < 0.05; ** P < 0.01. Non-septic muscle
n r N
300
X
-s it 0E
Septic
muscle
-
+ 200
-
100
-
T
25 -2 e m
.-: 5 ii .-c 3 P a
0,
FIG. 3. Protein synthesis (open bars) or 40°C (hatched and 7 paired septic muscles.
in EDL muscles incubated pairwise at 37 bars). Results are from 7 paired nonseptic Definitions are as in Fig. 1.
DISCUSSION
In a recent study, protein breakdown in incubated rat skeletal muscle was stimulated by increased temperature, and fever was proposed to be a mechanism of accelerated muscle proteolysis in sepsis (2). However, because only tyrosine release was measured in that study, it was not known if myofibrillar protein breakdown was increased by elevated temperature as well. This would be important because sepsis mainly stimulates the degradation of myofibrillar proteins, with only a modest increase in sarcoplasmic protein breakdown (lo), similar to other catabolic conditions, such as burn injury (7), glucocorticoid treatment (13), and fasting (16). The purpose of the present report therefore was to study the influence of high temperature in vitro on both total and myofibrillar protein degradation in skeletal muscle. Our results confirmed the study of Baracos et al. (2) in demonstrating increased tvrosine release bv nonseetic
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rat EDL and SOL muscles incubated at high temperature, although the response per degree of increased temperature noted here, i.e., 5%/“C in EDL and lO%/OC in SOL, was somewhat smaller than the ll%/"C reported earlier (2). The finding that protein synthesis rate was not affected by high temperature suggests that the increased release of tyrosine was not the result of a general increase in protein turnover but reflected a specific effect on protein breakdown. Also, in the study of Baracos et al. (Z), protein synthesis was relatively insensitive to elevated temperature, and at 42”C, protein synthesis was even inhibited. In contrast to total protein breakdown, myofibrillar protein degradation (i.e., 3-MH release) in nonseptic muscle was unaffected by high temperature in vitro. This result suggests that increased temperature is not the primary mechanism of myofibrillar protein degradation in sepsis. It should be noted, however, that muscles were incubated for only 2 h, and it is possible that myofibrillar proteins are sensitive to high temperature over a longer period of time. The slower turnover rate of myofibrillar than nonmyofibrillar proteins (3) could explain why a longer time span may be required to detect alteration of myofibrillar protein breakdown rate. Although elevated temperature did not increase myofibrillar protein degradation in nonseptic muscle, high temperature may be a potentiating factor, further stimulating sepsis-induced proteolysis. To test this hypothesis, paired muscles from septic rats were incubated at different temperatures. Results from these experiments demonstrated that both total and myofibrillar protein degradation was further stimulated by high temperature in EDL muscle from septic animals, whereas septic SOL did not respond to elevated temperature. This suggests that the present in vitro findings may have in vivo implications because the effect of sepsis on both total and myofibrillar protein breakdown were more pronounced in white than in red muscle (10). The present findings that high temperature regulated total and myofibrillar protein breakdown individually and that the response was different in the white EDL and red SOL muscles illustrates the importance of measuring both tyrosine and 3-MH release in different types of skeletal muscle when possible mediators and mechanisms of muscle proteolysis are elucidated. Because the increased tyrosine release in nonseptic muscle was not associated with increased 3-MH release, the high temperature must primarily have stimulated breakdown of nonmyofibrillar proteins. Thus, in this respect, the effect of high temperature in vitro was different from the effect of sepsis (10). The mechanisms of temperature-induced changes in muscle proteolysis remain to be determined. Previous studies by Baracos et al. (2) found no evidence that lysosomal proteases or prostaglandins were involved in the response to elevated temperature. The mechanism of the specific effect of high temperature on protein breakdown in septic EDL muscle is also unknown, but it may be speculated that elevated temperature made the muscle more sensitive to one or several mediators of the septic
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response, provided the mediators were retained in the muscle in vitro. Although the present study suggests that elevated temperature is not a primary factor for increased muscle proteolysis in sepsis, it must be remembered that the effects of high temperature in vitro may be quite different from the effects of fever in vivo. Thus conclusions from the present experiments must be drawn with great caution. However, the interpretation of the present results that high temperature alone does not stimulate muscle proteolysis in sepsis was supported by a recent study in which administration of tumor necrosis factor induced fever in rats without activating muscle protein breakdown (14). When the cyclooxygenase inhibitor ibuprofen was administered to endotoxin-treated human volunteers, body temperature was almost normalized while the hypoferremia and elevation of plasma C reactive protein level in .duced by endotoxin were unaffected (18), suggesting that some metabolic alterations in endotoxemia, and perhaps sepsis as well, can occur in the absence of elevated body temperature. This work was supported in part by the National Institute of Diabetes and Digestive and Kidney Diseases Grant lROl-DK-3790801. M. Hall-Anger&s and U. Angeras were also supported by grants from the Gothenborg Medical Society, The Medical Faculty, University of Gothenborg, and the Swedish Society of Medical Sciences. Address reprint requests to J. E. Fisher. Received
15 May
1989; accepted
in final
form
21 November
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