Influence Marianne
of Sepsis in Rats on Muscle Protein Turnover In Vivo and in Tissue Incubated Under Different In Vitro Conditions Ulf Anger%,
Hall-Anger&,
Daniel von Allmen,
Per-Olof Hasselgren,
Takashi Higashiguchi,
Oded Zamir,
and Josef E. Fischer
We studied the influence of sepsis on muscle protein synthesis and degradation in vivo and in muscles, incubated flaccid or at resting length. Sepsis was induced in rats by cecal ligation and puncture (CLP). Control rats were sham-operated. A flooding dose of “C-phenylalanine was used to determine muscle protein synthesis rate in vivo, and protein breakdown was calculated from the difference between protein synthesis and growth rates. Protein synthesis rate in vitro was assessed by determining incorporation of “C-phenylalanine into protein in incubated extensor digitorum longus (EDL) and soleus (SOL) muscles. Total and myofibrillar protein breakdown rates were determined from release into incubation medium of tyrosine and 3-methylhistidine (3-MH), respectively. Muscle protein synthesis rate in vivo was reduced by 35%. similar to the reduction observed in muscles incubated flaccid or at resting length. The calculated protein breakdown rate in vivo was increased by 31% in septic rats. In incubated muscles, the increase in total protein breakdown (ie, tyrosine release) during sepsis was almost identical in muscles incubated flaccid or at resting length, ie, 83% to 88% in EDL and 47% to 49% in SOL. Myofibrillar protein degradation in vitro (ie, 3-MH release) was increased approximately lo-fold in EDL muscles incubated flaccid or at resting length, but was not significantly affected by sepsis in SOL. Results suggest that sepsis-induced changes in protein synthesis observed in muscles incubated either flaccid or at resting length reflect changes in vivo. Changes in protein breakdown were qualitatively similar in vivo and in vitro, but results in incubated muscles mav overestimate the increase in muscle proteolysis caused by sepsis. Copyright 0 19il by W.B. Saunders Company
I
NCUBATED rat skeletal muscles are frequently used to study the effects of different pathophysiologic conditions on protein turnover rates. The muscles are also responsive in vitro to hormones,‘,’ amino acids,3,4 and other substances.‘.’ In previous studies, muscles were often incubated unsupported, ie. in a flaccid state. Recent reports demonstrated that protein balance and energy levels are better maintained in muscles incubated at resting length, rather than faccid,‘,’ suggesting that these muscles represent a more physiologic in vitro preparation. More important, other studies suggested that not only are muscles at resting length less catabolic, but the response of protein syrlthesis and degradation to different stimuli may vary in muscles incubated at resting length or flaccid.’ In previous reports from our laboratory, the effect of sepsis
on protein
turnover
rates
was
determined
in incu-
rat extensor digitorum longus (EDL) and soleus (SOL) muscles, and results suggested that protein synthesis is reduced and degradation is increased during sepsis.2.4,“.‘o In most of those studies, muscles were incubated flaccid, and although one report indicated that the response to sepsis of protein synthesis is similar in flaccid muscles and between mxcles at resting length,‘” no direct comparisons the effect of sepsis on protein synthesis and breakdown rates in muscles incubated flaccid or at resting length have been reported. In the present study, therefore, protein turnover rates were determined in EDL and SOL muscles from control and septic rats, with the muscles unsupported or maintained at resting length during incubation. In addition, protein turnover rates were determined in vivo to compare the magnitude of sepsis-induced changes in protein metabolism in incubated muscles with calculated changes in vivo. bated
MATERIALS
AND METHODS
Sepsis was induced in male Sprague-Dawley rats (50 to 70 g) by cezal ligation and puncture (CLP) as previously described.’ Con-
Metabolism,
Vol40,
No 3 (March),
1991:
pp 247-251
trol animals underwent laparotomy with manipulation, but no ligation or puncture of the cecum. All animals were resuscitated with 5 mL of saline/l00 g body weight injected subcutaneously on the back immediately after the operative procedures. The animals were allowed water ad libitum, but were fasted after surgery. Sixteen hours after CLP or sham-operation, EDL and SOL muscles were dissected with intact tendons under ether anesthesia, and muscles were incubated for measurement of protein turnover rates (see below). In other animals, muscles were harvested at the same time point for determination of protein content.” In a separate series of experiments, bigger rats were used (120 to 135 g), and muscle protein synthesis and degradation rates were calculated in vivo in sham-operated and septic rats (see below).
Protein Synthesis and Degradation in Incubated Muscles For the study of protein synthesis, one EDL and one SOL muscle from each rat were mounted on a stainless steel support at resting length, and contralateral muscles were incubated in a flaccid state. The muscles were preincubated in a shaking water bath at 37°C for 30 minutes in 3 mL of a medium consisting of Krebs-Henseleit bicarbonate buffer (pH 7.4), glucose (10 mmol/L), and phenylalanine (0.5 mmol/L). The medium was gassed with 02:COL (95:5) for 15 to 20 minutes immediately before use, and the incubation flasks were flushed with 02:C02 (95:5) and sealed with rubber stoppers after addition of the muscles. Following preincubation, the muscles were transferred to 3 mL of fresh medium of the same composition as described above with the addition of “C-phenylalanine (0.05
From the Depatiment of Surgery. Universi~ of Cincinnati Medical Center, Cincinnati, OH. Supported in part by National Institutes of Health Grant No. 1ROl DK 37908. M.H.-A. and U.A. were also supported by grants from the Gothenburg Medical Society; the Medical Faculty. University of Gothenburg, Sweden: and the Swedish Socieg of Medical Sciences. Address reprint requests to Per-Olof Hasselgren, MD, Department of Surgery, University of Cincinnati, 231 Bethesda Ave. Cincinnati, OH 45267-0558. Copyright Q 1991 b.y W?B. Saunders Cornpun> 0026049519114003-0006$03.00l0
247
248
&i/mL). After incubation for 2 hours, the amount of phenylalanine incorporated into trichloroacetic acid precipitated protein was determined as described in detail previously.“’ For the study of protein degradation, release of tyrosine by the incubated muscles was used as a measure of total protein breakdown rate, and release of J-methylhistidine (3-MH) was used as a measure of myofibrillar protein degradation. In a recent study, we found that tissue levels of the amino acids, in particular 3-MH, may change during incubation.‘* In the present experiments, therefore, bilateral EDL and SOL muscles of each rat were dissected and individually preincubated in a shaking water bath at 37°C for 30 minutes in 3 mL of a medium consisting of Krebs-Henseleit bicarbonate buffer (pH 7.4) and glucose (10 mmol/L). Muscles were incubated flaccid or fixed on stainless steel supports at resting length. After preincubation, one EDL and one SOL muscle were transferred to ice-cold 3% (wt/vol) HCIO, for determination of tissue tyrosine and 3-MH, while the contralateral muscles were transferred to 3 mL of fresh medium containing cycloheximide (0.5 mmol/L) and incubated for 2 hours. After incubation, muscles were blotted and transferred to ice-cold 3% HCIO, and muscles and incubation media were stored at -20°C until analysis. The muscles were homogenized in 2 mL of ice-cold 3% HClO, and after centrifugation (2,000 x g, 20 minutes, 4”C), the supernatant was neutralized with 0.28 mL of 2 mol/L KOH containing 0.5 mol/L triethanolamine. The precipitated KCIO, was removed by centrifugation (2,000 x g, 5 minutes, 4°C). Tyrosine and 3-MH in HCIO, extracts of tissue and in incubation-medium samples were determined by high-performance liquid chromatography as described previously.“,” Thus, the experimental design was different when protein synthesis or degradation was studied in vitro. While paired muscles from the same rat were incubated flaccid or at resting length when protein synthesis rate was measured, muscles from different groups of rats were used when protein breakdown rates were measured in flaccid muscles or muscles at resting length. This was necessary, since tissue levels of tyrosine and 3-MH were determined both at the start (ie, after preincubation) and end of the 2-hour incubation in the degradation experiments, and changes in tissue levels were taken into account when net production of the amino acids was calculated.” Muscle Protein Synthesis and Degradation In Vivo Because there is no direct method available to determine muscle protein degradation in vivo, various indirect techniques have been used.14 In the present experiments, protein breakdown rate in vivo was calculated as the difference between protein synthesis and protein growth rate. Consequently, determination of the muscle protein growth rate over the 16-hour experimental period was an important component in the calculation of degradation rate. In order to increase the accuracy of the determinations of muscle protein content, bigger rats (120 to 135 g) were used here than in the experiments in which incubated muscles were studied. Rats were divided into five groups of five to six animals. For each rat allocated to one group, rats of the same body weight within a 5-g margin were allocated to the four other groups. This provided five groups of animals with almost identical initial body weight. Both EDL muscles from each rat were pooled and protein content was determined according to Lowry et al” before surgical procedure (O-hour protein content) and 16 hours after sham-operation or CLP (16-hour protein content). Animals were fasted, but had free access to water after the operative procedures. In two other groups of rats, protein synthesis rate was determined in vivo 16 hours after CLP or sham-operation using a flooding dose of 3H-phenylalanine
HALL-ANGER&
ET AL
as described in detail previously,“‘,” and protein degradation rate was calculated as the difference between protein synthesis and growth rate. ATP Concentration in Muscle Tissue EDL and SOL muscles from control or septic rats (50 to 70 g body weight) were freeze clamped in vivo with forceps that had been cooled in liquid nitrogen. The muscles were then immediately immersed in liquid nitrogen. In other experiments, paired muscles were incubated flaccid or at resting length as described above for protein synthesis, but without phenylalanine in the medium. After 2 hours’ incubation, muscles were immediately frozen in liquid nitrogen and stored at -70°C until determination of adenosine triphosphate (ATP), which was done fluorimetrically.‘6 Statistics Results are presented as means ? SEM. Student’s t test or ANOVA followed by Tukey’s test was used for statistical comparisons. RESULTS
Weight and protein content of EDL and SOL muscles from control and septic rats are shown in Fig 1. Protein content was reduced in EDL from septic rats, while the weights of both muscles and protein content in SOL were not significantly different between control and septic animals. The results shown in Fig 1 were not influenced by different body weights, since control and septic rats weighed 58 ? 1.6 g and 58 ? 1.8 g, respectively, when they were killed. Muscle ATP levels in vivo were unchanged by sepsis in EDL, but were slightly reduced (P < .05) in SOL (Fig 2). During incubation, tissue ATP levels were better maintained in muscles incubated at resting length than in unsupported muscles (Fig 2). ATP levels in vivo reported here and the decrease in energy levels during incubation of flaccid muscles are in line with previous reports.8.17 The response to sepsis of protein synthesis in vitro was similar in muscles incubated flaccid or at resting length, with an approximately 40% reduction of synthesis rate in EDL and an approximately 20% reduction in SOL (Table 1). The higher protein synthesis rate noted in SOL muscles incubated at resting length compared with muscles incubated flaccid (Table 1) is similar to a recent study from this laboratory,” and contrasts to protein synthesis in EDL, which was not significantly affected by incubating the muscle at resting length. The relative increase in tyrosine release during sepsis was almost identical in muscles incubated flaccid or at resting length, ie, 83% to 88% in EDL and 47% to 49% in SOL (Table 2). The increase in myofibrillar protein breakdown following CLP was similar in EDL muscles incubated flaccid or at resting length, but was not statistically significant in SOL (Table 2). Because one of the purposes of this report was to study the response to sepsis in muscles incubated under different in vitro conditions, sham-operated and septic rats within the different groups, ie, “flaccid” or “resting length,” were always studied on the same day to avoid day-to-day varia-
249
MUSCLE PROTEIN TURNOVER IN SEPSIS
EDL 8 I
6 4
EOL
I
I
Oh
2h
SOL
SOL 0 6 4
EDL
I SOL
Fig 1. Weight and protein content of EDL and SOL muscles from seven sham-operated (0) and seven septic rats(m). lf < .05.
Lions of protein turnover rates. Thus, in contrast to protein synthesis, when paired muscles were used, the experimental design does not allow for comparisons between protein breakdown rates in muscles incubated flaccid or at resting length. Because the present results and a recent studyI demonstrated that sepsis-induced changes in protein metabolism are more pronounced in EDL (a white, fast-twitch muscle) than in SOL (a red, slow-twitch muscle), EDL muscles were used for the in vivo experiments. Protein content in the pooled EDL muscles was 26.07 & 0.37 mg before shamoperation or CLP. Sixteen hours later, muscle protein content was 23.38 t 0.49 mg in control rats and 21.40 t 1.22 mg in septic rats (P < .OS). Thus, the protein “growth rate” was -2.69 mg in control animals and -4.67 mg in septic rats. The loss of muscle protein in the control animals probably reflects the combined effect of surgical trauma (sham-operation) and fasting. Protein synthesis rate was
2 b
I
I
Oh
2h
ATP in EDL and SOL muscles incubated for 2 hours at resting length or flaccid. ATP in freeze-clamped muscles in vivo is represented by Oh. O-O, Control muscles incubated at resting length; O---O, control muscles incubated flaccid; O-O, septic muscles incubated at resting length; O---O, septic muscles incubated flaccid. Results are from four to seven muscles in each group.
Table 1. Protein Synthesis Rates (nmol Phe/g
2 h) in EDL and SOL
Muscles From Control and Septic Rats: Muscles Incubated Flaccid or at Resting Length Resting
Resting Length
Flaccid
Length
v Flaccid
Sham
233 2 35
179 -i: 14
NS
CLP
138 t 7
110 2 7
NS
-41 %*
-39%X
SOL Sham
340 + 36
424 2 20
~25%~
CLP
257 2 13
345 t 29
+ 34% *
~ 24%X
-19%X
EDL
CLP Y sham
CLP v sham
NOTE. Results are from seven muscles in each group. *P < .05.
250
HALL-ANGER&
Table 2. Total and Myofibrillar Protein Degradation Rates (nmol/g
‘2
h) in Incubated EDL and SOL Muscles From Control and
Septic Rats: Muscles Incubated Flaccid or at Resting Length Tyrosine
3-MH Resting
Flaccid
Length
Resting Flaccid
Length
EDL Sham
283 + 17
188 -c 12
0.35 f 0.16
0.34 f 0.28
CLP
533 + 20
343 2 52
4.82 + 1.14
3.80 k 1.14
i-88%’
+83%*
+ 1.277%*
+1,017%x
CLP v sham SOL Sham
368 2 24
246 f 9
0.58 + 0.24
0.65 k 0.53
CLP
548 + 24
360 2 20
2.01 + 0.92
1.67 -t 0.71
+49%x
+47%*
NS
NS
CLP v sham
NOTE. Results are from six or seven muscles in each group. ‘P
< .05.
10.04 ? 0.77%/d and 6.48 f 058%/d in control and septic rats, respectively (P < .05). Thus, the relative change in protein synthesis in EDL in vivo during sepsis (-35%) was similar to the change noted in vitro (Table 1). Adjusting the in vivo synthesis rates to percent per 16 hours, and using the protein growth over the same period of time, the calculated protein breakdown rates were 4.43 and 5.80 mg protein/l6 h in the control and septic rats, respectively. This increase in calculated protein breakdown in vivo (+31%) during sepsis was less pronounced than that observed in incubated muscles from septic rats (Table 2). DISCUSSION
The present results show that the relative changes in protein synthesis and degradation rates induced by sepsis in rats are similar when measured in muscles incubated flaccid or at resting length. Thus, when incubated muscles are used to study the influence of sepsis on protein turnover rates, either flaccid or mounted muscles can be used. Flaccid muscles offer the advantage of shorter time required for preparation of the tissue before incubation. However, lower protein breakdown rates and better maintained energy levels in muscles incubated at resting length, suggest that these muscles are in a more physiologic state and should be used when experiments are performed to study absolute protein turnover rates and probably also when the effects of different substances in vitro are examined. One reason why flaccid muscles are more catabolic than muscles incubated at resting length is probably that they shorten during incubation. In a previous study .by Baracos and Goldberg,* flaccid EDL and SOL muscles shortened to 44% and 25% of their resting length, respectively, during incubation for 2 hours. The decrease in length was associated with an increase in diameter, most pronounced in SOL, the thickness of which increased more than threefold during incubation. The risk for development of a central, hypoxic core in the incubated tissue is greater in thicker muscles, which probably explains why energy levels are reduced in flaccid muscles. Recent studies suggest that hypoxia may even develop during incubation of very thin muscles from mice.‘8,‘9In a recent study from our labora-
ET AL
tory, the central, hypoxic core of incubated rat SOL muscles was somewhat larger in tissue incubated flaccid than at resting length.” While the central core of flaccid muscles increased progressively in size during incubation, it remained relatively constant in muscles incubated at resting length. More important was the finding that there were no obvious differences in the size of the central core between septic and control muscles, neither when incubated flaccid nor at resting length.‘” We also compared the response to sepsis of protein metabolism in vivo and in vitro. The relative changes in protein synthesis in EDL were similar in incubated muscles and in vivo. We recently found that the changes in protein synthesis in SOL were also similar in vivo and in vitro.‘” Hence, the current in vitro system reflects sepsis-induced changes in muscle protein synthesis in vivo, at least from a qualitative standpoint. In other studies using the same in vivo technique, muscle protein synthesis rates varied between 17%/d and 21%/d.‘5.‘8.“’The lower synthesis rate observed in control rats in the current study probably reflects the fact that control animals were sham-operated and fasted for 16 hours before protein synthesis rate was measured. Because there are no direct methods available for the measurement of muscle protein degradation in vivo, breakdown rates must be calculated from other measurements. The degradation rates in the present study were calculated from protein synthesis and “growth” rates and results suggested that the sepsis-induced increase in protein breakdown may be overestimated when measured in incubated muscles. This result may reflect the fact that incubated muscles are in a catabolic state, even when incubated at resting length, and it is possible that this aggravates the sepsis-induced changes in protein breakdown. However, it should be noted that other interpretations are possible as well. One important factor to take into account is that the incubated muscles reflected the situation at 16 hours after sham operation or CLP, whereas the in vivo data reflected the degradation over the entire experimental period. For the calculations it was assumed that the protein synthesis rates were unchanged over the 16-hour period. This, of course, is unlikely; if protein synthesis was inhibited later in the septic course than protein breakdown was increased, the contribution of protein breakdown to reduced muscle protein content would be greater than the current calculations indicate. In fact, previous studies suggest that muscle protein breakdown is increased earlier than protein synthesis is reduced following CLP in rats’ and consequently, it is likely that the current in vivo calculations underestimated the increase in protein breakdown. Therefore, the in vitro data may reflect the in vivo situation better than the in vivo calculations suggested. The present report did not address the question whether in vivo studies are superior to those in vitro when the influence of sepsis on muscle protein metabolism is examined. Both techniques probably have their roles in these types of experiments, provided results are interpreted with caution.
251
MUSCLE PROTEIN TURNOVER IN SEPSIS REFERENCES I. McGrath JA, Goldspink DF: Glucocorticoid action on protein synthesis and protein breakdown in isolated skeletal muscles. Biochem J 206:641-645,1982 2. Hasselgren PO. Warner BW. James JH, et al: Effect of insulin on amino acid uptake and protein turnover in skeletal muscle from septic rats: Evidence for insulin resistance of protein breakdown. Arch Surg 122122X-233.1987 3. Tischler ME, Desautels M, Goldberg AL: Does leucine, leucyl-tRNA, or some metabolite of leucine regulate protein synthesis and degradation in skeletal and cardiac muscle? J Biol Chem 257:1613-1621,1982 1. Hasselgren PO, James JH, Warner BW, et al: Protein synthesis and degradation in skeletal muscle from septic rats: Response to leucine and cy-ketoisocaproic acid. Arch Surg 123:640641.1988 i. Rodemann HP, Goldberg AL: Arachidonic acid, prostaglandin Ez and FZ, influence rates of protein turnover in skeletal and cardiac muscle. J Biol Chem 257:1632-1638. 1982 6. Goldberg AL, Baracos V. Rodemann P, et al: Control of pnrtein degradation in muscle by prostaglandins, Ca’+, and leukocytic pyrogen (interleukin-1). Fed Proc 43:1301-1306,1984 7. Etlinger JD, Kameyama T, Toner K, et al: Calcium and stretch-dependent regulation of protein turnover and myofibrillar disassembly in muscle. in Pette D (ed): Plasticity of Muscle. New York, NY. de Gruyter, 1980, p 541 3. Baracos VE, Goldberg AL: Maintenance of normal length improves protein balance and energy status in isolated rat skeletal muscle. Am J Physiol251:C588-C596, 1986 4. Hasselgren PO, Talamini MA, James JH, et al: Protein metabolism in different types of skeletal muscle during early and late sepsis in rats. Arch Surg 121:918-923. 1986 10. Hummel RP, Hasselgren PO, James JH, et al: 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. Metabolism 37:1120-1127, 1988 11. Lowry OH, Rosebrough NJ, Farr AL, et al: Protein measurement with the Folin phenol reagent. J Biol Chem 193:265-275, 1951 12. Hasselgren PO, Hall-Anger& M. Anger% LJ, et al: 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 13. Hasselgren PO, James JH, Benson DW, et al: Total and myofibrillar protein breakdown in different types of rat skeletal muscle: Effects of sepsis and regulation by insulin. Metabolism 38:634-640, 1989 14. Hasselgren PO, Pedersen P, Sax HC, et al: Methods for studying protein synthesis and degradation in liver and skeletal muscle. J Surg Res 45:389-415, 1988 15. Garlick PJ. McNurlan MA, Preedy VR: A rapid and convenient technique for measuring the rate of protein synthesis in tissues by injection of [‘HI-phenylalanine. Biochem J 192719-723. 1980 16. Lowty OH, Passonneau JV, Hasselberger FX, et al: Effect of ischemia on known substrates and cofactors of the glycolytic pathway in brain. J Biol Chem 239:18-30. 1964 17. Tullson PC, John-Alder HB, Hood DA, et al: De novo synthesis of adenine nucleotides in different skeletal muscle fiber types. Am J Physiol255:C271-C277,1988 18. Maltin CA. Harris Cl: Morphological observations and rates of protein synthesis in rat muscles incubated in vitro. Biochem J 232:927-930, 1985 19. Van Breda E, Keizer HA. Glatz JFC, et al: Use of the intact mouse skeletal-muscle preparation for metabolic studies. Evaluation of the model. Biochem J 267:257-260. 1990 20. McNurlan MA, Fern EB, Garlick PJ: Failure of leucine to stimulate protein synthesis in vivo. Biochem J 204:831-838. 1982
of Sepsis in Rats on Muscle Protein Turnover In Vivo and in Tissue Incubated Under Different In Vitro Conditions Ulf Anger%,
Hall-Anger&,
Daniel von Allmen,
Per-Olof Hasselgren,
Takashi Higashiguchi,
Oded Zamir,
and Josef E. Fischer
We studied the influence of sepsis on muscle protein synthesis and degradation in vivo and in muscles, incubated flaccid or at resting length. Sepsis was induced in rats by cecal ligation and puncture (CLP). Control rats were sham-operated. A flooding dose of “C-phenylalanine was used to determine muscle protein synthesis rate in vivo, and protein breakdown was calculated from the difference between protein synthesis and growth rates. Protein synthesis rate in vitro was assessed by determining incorporation of “C-phenylalanine into protein in incubated extensor digitorum longus (EDL) and soleus (SOL) muscles. Total and myofibrillar protein breakdown rates were determined from release into incubation medium of tyrosine and 3-methylhistidine (3-MH), respectively. Muscle protein synthesis rate in vivo was reduced by 35%. similar to the reduction observed in muscles incubated flaccid or at resting length. The calculated protein breakdown rate in vivo was increased by 31% in septic rats. In incubated muscles, the increase in total protein breakdown (ie, tyrosine release) during sepsis was almost identical in muscles incubated flaccid or at resting length, ie, 83% to 88% in EDL and 47% to 49% in SOL. Myofibrillar protein degradation in vitro (ie, 3-MH release) was increased approximately lo-fold in EDL muscles incubated flaccid or at resting length, but was not significantly affected by sepsis in SOL. Results suggest that sepsis-induced changes in protein synthesis observed in muscles incubated either flaccid or at resting length reflect changes in vivo. Changes in protein breakdown were qualitatively similar in vivo and in vitro, but results in incubated muscles mav overestimate the increase in muscle proteolysis caused by sepsis. Copyright 0 19il by W.B. Saunders Company
I
NCUBATED rat skeletal muscles are frequently used to study the effects of different pathophysiologic conditions on protein turnover rates. The muscles are also responsive in vitro to hormones,‘,’ amino acids,3,4 and other substances.‘.’ In previous studies, muscles were often incubated unsupported, ie. in a flaccid state. Recent reports demonstrated that protein balance and energy levels are better maintained in muscles incubated at resting length, rather than faccid,‘,’ suggesting that these muscles represent a more physiologic in vitro preparation. More important, other studies suggested that not only are muscles at resting length less catabolic, but the response of protein syrlthesis and degradation to different stimuli may vary in muscles incubated at resting length or flaccid.’ In previous reports from our laboratory, the effect of sepsis
on protein
turnover
rates
was
determined
in incu-
rat extensor digitorum longus (EDL) and soleus (SOL) muscles, and results suggested that protein synthesis is reduced and degradation is increased during sepsis.2.4,“.‘o In most of those studies, muscles were incubated flaccid, and although one report indicated that the response to sepsis of protein synthesis is similar in flaccid muscles and between mxcles at resting length,‘” no direct comparisons the effect of sepsis on protein synthesis and breakdown rates in muscles incubated flaccid or at resting length have been reported. In the present study, therefore, protein turnover rates were determined in EDL and SOL muscles from control and septic rats, with the muscles unsupported or maintained at resting length during incubation. In addition, protein turnover rates were determined in vivo to compare the magnitude of sepsis-induced changes in protein metabolism in incubated muscles with calculated changes in vivo. bated
MATERIALS
AND METHODS
Sepsis was induced in male Sprague-Dawley rats (50 to 70 g) by cezal ligation and puncture (CLP) as previously described.’ Con-
Metabolism,
Vol40,
No 3 (March),
1991:
pp 247-251
trol animals underwent laparotomy with manipulation, but no ligation or puncture of the cecum. All animals were resuscitated with 5 mL of saline/l00 g body weight injected subcutaneously on the back immediately after the operative procedures. The animals were allowed water ad libitum, but were fasted after surgery. Sixteen hours after CLP or sham-operation, EDL and SOL muscles were dissected with intact tendons under ether anesthesia, and muscles were incubated for measurement of protein turnover rates (see below). In other animals, muscles were harvested at the same time point for determination of protein content.” In a separate series of experiments, bigger rats were used (120 to 135 g), and muscle protein synthesis and degradation rates were calculated in vivo in sham-operated and septic rats (see below).
Protein Synthesis and Degradation in Incubated Muscles For the study of protein synthesis, one EDL and one SOL muscle from each rat were mounted on a stainless steel support at resting length, and contralateral muscles were incubated in a flaccid state. The muscles were preincubated in a shaking water bath at 37°C for 30 minutes in 3 mL of a medium consisting of Krebs-Henseleit bicarbonate buffer (pH 7.4), glucose (10 mmol/L), and phenylalanine (0.5 mmol/L). The medium was gassed with 02:COL (95:5) for 15 to 20 minutes immediately before use, and the incubation flasks were flushed with 02:C02 (95:5) and sealed with rubber stoppers after addition of the muscles. Following preincubation, the muscles were transferred to 3 mL of fresh medium of the same composition as described above with the addition of “C-phenylalanine (0.05
From the Depatiment of Surgery. Universi~ of Cincinnati Medical Center, Cincinnati, OH. Supported in part by National Institutes of Health Grant No. 1ROl DK 37908. M.H.-A. and U.A. were also supported by grants from the Gothenburg Medical Society; the Medical Faculty. University of Gothenburg, Sweden: and the Swedish Socieg of Medical Sciences. Address reprint requests to Per-Olof Hasselgren, MD, Department of Surgery, University of Cincinnati, 231 Bethesda Ave. Cincinnati, OH 45267-0558. Copyright Q 1991 b.y W?B. Saunders Cornpun> 0026049519114003-0006$03.00l0
247
248
&i/mL). After incubation for 2 hours, the amount of phenylalanine incorporated into trichloroacetic acid precipitated protein was determined as described in detail previously.“’ For the study of protein degradation, release of tyrosine by the incubated muscles was used as a measure of total protein breakdown rate, and release of J-methylhistidine (3-MH) was used as a measure of myofibrillar protein degradation. In a recent study, we found that tissue levels of the amino acids, in particular 3-MH, may change during incubation.‘* In the present experiments, therefore, bilateral EDL and SOL muscles of each rat were dissected and individually preincubated in a shaking water bath at 37°C for 30 minutes in 3 mL of a medium consisting of Krebs-Henseleit bicarbonate buffer (pH 7.4) and glucose (10 mmol/L). Muscles were incubated flaccid or fixed on stainless steel supports at resting length. After preincubation, one EDL and one SOL muscle were transferred to ice-cold 3% (wt/vol) HCIO, for determination of tissue tyrosine and 3-MH, while the contralateral muscles were transferred to 3 mL of fresh medium containing cycloheximide (0.5 mmol/L) and incubated for 2 hours. After incubation, muscles were blotted and transferred to ice-cold 3% HCIO, and muscles and incubation media were stored at -20°C until analysis. The muscles were homogenized in 2 mL of ice-cold 3% HClO, and after centrifugation (2,000 x g, 20 minutes, 4”C), the supernatant was neutralized with 0.28 mL of 2 mol/L KOH containing 0.5 mol/L triethanolamine. The precipitated KCIO, was removed by centrifugation (2,000 x g, 5 minutes, 4°C). Tyrosine and 3-MH in HCIO, extracts of tissue and in incubation-medium samples were determined by high-performance liquid chromatography as described previously.“,” Thus, the experimental design was different when protein synthesis or degradation was studied in vitro. While paired muscles from the same rat were incubated flaccid or at resting length when protein synthesis rate was measured, muscles from different groups of rats were used when protein breakdown rates were measured in flaccid muscles or muscles at resting length. This was necessary, since tissue levels of tyrosine and 3-MH were determined both at the start (ie, after preincubation) and end of the 2-hour incubation in the degradation experiments, and changes in tissue levels were taken into account when net production of the amino acids was calculated.” Muscle Protein Synthesis and Degradation In Vivo Because there is no direct method available to determine muscle protein degradation in vivo, various indirect techniques have been used.14 In the present experiments, protein breakdown rate in vivo was calculated as the difference between protein synthesis and protein growth rate. Consequently, determination of the muscle protein growth rate over the 16-hour experimental period was an important component in the calculation of degradation rate. In order to increase the accuracy of the determinations of muscle protein content, bigger rats (120 to 135 g) were used here than in the experiments in which incubated muscles were studied. Rats were divided into five groups of five to six animals. For each rat allocated to one group, rats of the same body weight within a 5-g margin were allocated to the four other groups. This provided five groups of animals with almost identical initial body weight. Both EDL muscles from each rat were pooled and protein content was determined according to Lowry et al” before surgical procedure (O-hour protein content) and 16 hours after sham-operation or CLP (16-hour protein content). Animals were fasted, but had free access to water after the operative procedures. In two other groups of rats, protein synthesis rate was determined in vivo 16 hours after CLP or sham-operation using a flooding dose of 3H-phenylalanine
HALL-ANGER&
ET AL
as described in detail previously,“‘,” and protein degradation rate was calculated as the difference between protein synthesis and growth rate. ATP Concentration in Muscle Tissue EDL and SOL muscles from control or septic rats (50 to 70 g body weight) were freeze clamped in vivo with forceps that had been cooled in liquid nitrogen. The muscles were then immediately immersed in liquid nitrogen. In other experiments, paired muscles were incubated flaccid or at resting length as described above for protein synthesis, but without phenylalanine in the medium. After 2 hours’ incubation, muscles were immediately frozen in liquid nitrogen and stored at -70°C until determination of adenosine triphosphate (ATP), which was done fluorimetrically.‘6 Statistics Results are presented as means ? SEM. Student’s t test or ANOVA followed by Tukey’s test was used for statistical comparisons. RESULTS
Weight and protein content of EDL and SOL muscles from control and septic rats are shown in Fig 1. Protein content was reduced in EDL from septic rats, while the weights of both muscles and protein content in SOL were not significantly different between control and septic animals. The results shown in Fig 1 were not influenced by different body weights, since control and septic rats weighed 58 ? 1.6 g and 58 ? 1.8 g, respectively, when they were killed. Muscle ATP levels in vivo were unchanged by sepsis in EDL, but were slightly reduced (P < .05) in SOL (Fig 2). During incubation, tissue ATP levels were better maintained in muscles incubated at resting length than in unsupported muscles (Fig 2). ATP levels in vivo reported here and the decrease in energy levels during incubation of flaccid muscles are in line with previous reports.8.17 The response to sepsis of protein synthesis in vitro was similar in muscles incubated flaccid or at resting length, with an approximately 40% reduction of synthesis rate in EDL and an approximately 20% reduction in SOL (Table 1). The higher protein synthesis rate noted in SOL muscles incubated at resting length compared with muscles incubated flaccid (Table 1) is similar to a recent study from this laboratory,” and contrasts to protein synthesis in EDL, which was not significantly affected by incubating the muscle at resting length. The relative increase in tyrosine release during sepsis was almost identical in muscles incubated flaccid or at resting length, ie, 83% to 88% in EDL and 47% to 49% in SOL (Table 2). The increase in myofibrillar protein breakdown following CLP was similar in EDL muscles incubated flaccid or at resting length, but was not statistically significant in SOL (Table 2). Because one of the purposes of this report was to study the response to sepsis in muscles incubated under different in vitro conditions, sham-operated and septic rats within the different groups, ie, “flaccid” or “resting length,” were always studied on the same day to avoid day-to-day varia-
249
MUSCLE PROTEIN TURNOVER IN SEPSIS
EDL 8 I
6 4
EOL
I
I
Oh
2h
SOL
SOL 0 6 4
EDL
I SOL
Fig 1. Weight and protein content of EDL and SOL muscles from seven sham-operated (0) and seven septic rats(m). lf < .05.
Lions of protein turnover rates. Thus, in contrast to protein synthesis, when paired muscles were used, the experimental design does not allow for comparisons between protein breakdown rates in muscles incubated flaccid or at resting length. Because the present results and a recent studyI demonstrated that sepsis-induced changes in protein metabolism are more pronounced in EDL (a white, fast-twitch muscle) than in SOL (a red, slow-twitch muscle), EDL muscles were used for the in vivo experiments. Protein content in the pooled EDL muscles was 26.07 & 0.37 mg before shamoperation or CLP. Sixteen hours later, muscle protein content was 23.38 t 0.49 mg in control rats and 21.40 t 1.22 mg in septic rats (P < .OS). Thus, the protein “growth rate” was -2.69 mg in control animals and -4.67 mg in septic rats. The loss of muscle protein in the control animals probably reflects the combined effect of surgical trauma (sham-operation) and fasting. Protein synthesis rate was
2 b
I
I
Oh
2h
ATP in EDL and SOL muscles incubated for 2 hours at resting length or flaccid. ATP in freeze-clamped muscles in vivo is represented by Oh. O-O, Control muscles incubated at resting length; O---O, control muscles incubated flaccid; O-O, septic muscles incubated at resting length; O---O, septic muscles incubated flaccid. Results are from four to seven muscles in each group.
Table 1. Protein Synthesis Rates (nmol Phe/g
2 h) in EDL and SOL
Muscles From Control and Septic Rats: Muscles Incubated Flaccid or at Resting Length Resting
Resting Length
Flaccid
Length
v Flaccid
Sham
233 2 35
179 -i: 14
NS
CLP
138 t 7
110 2 7
NS
-41 %*
-39%X
SOL Sham
340 + 36
424 2 20
~25%~
CLP
257 2 13
345 t 29
+ 34% *
~ 24%X
-19%X
EDL
CLP Y sham
CLP v sham
NOTE. Results are from seven muscles in each group. *P < .05.
250
HALL-ANGER&
Table 2. Total and Myofibrillar Protein Degradation Rates (nmol/g
‘2
h) in Incubated EDL and SOL Muscles From Control and
Septic Rats: Muscles Incubated Flaccid or at Resting Length Tyrosine
3-MH Resting
Flaccid
Length
Resting Flaccid
Length
EDL Sham
283 + 17
188 -c 12
0.35 f 0.16
0.34 f 0.28
CLP
533 + 20
343 2 52
4.82 + 1.14
3.80 k 1.14
i-88%’
+83%*
+ 1.277%*
+1,017%x
CLP v sham SOL Sham
368 2 24
246 f 9
0.58 + 0.24
0.65 k 0.53
CLP
548 + 24
360 2 20
2.01 + 0.92
1.67 -t 0.71
+49%x
+47%*
NS
NS
CLP v sham
NOTE. Results are from six or seven muscles in each group. ‘P
< .05.
10.04 ? 0.77%/d and 6.48 f 058%/d in control and septic rats, respectively (P < .05). Thus, the relative change in protein synthesis in EDL in vivo during sepsis (-35%) was similar to the change noted in vitro (Table 1). Adjusting the in vivo synthesis rates to percent per 16 hours, and using the protein growth over the same period of time, the calculated protein breakdown rates were 4.43 and 5.80 mg protein/l6 h in the control and septic rats, respectively. This increase in calculated protein breakdown in vivo (+31%) during sepsis was less pronounced than that observed in incubated muscles from septic rats (Table 2). DISCUSSION
The present results show that the relative changes in protein synthesis and degradation rates induced by sepsis in rats are similar when measured in muscles incubated flaccid or at resting length. Thus, when incubated muscles are used to study the influence of sepsis on protein turnover rates, either flaccid or mounted muscles can be used. Flaccid muscles offer the advantage of shorter time required for preparation of the tissue before incubation. However, lower protein breakdown rates and better maintained energy levels in muscles incubated at resting length, suggest that these muscles are in a more physiologic state and should be used when experiments are performed to study absolute protein turnover rates and probably also when the effects of different substances in vitro are examined. One reason why flaccid muscles are more catabolic than muscles incubated at resting length is probably that they shorten during incubation. In a previous study .by Baracos and Goldberg,* flaccid EDL and SOL muscles shortened to 44% and 25% of their resting length, respectively, during incubation for 2 hours. The decrease in length was associated with an increase in diameter, most pronounced in SOL, the thickness of which increased more than threefold during incubation. The risk for development of a central, hypoxic core in the incubated tissue is greater in thicker muscles, which probably explains why energy levels are reduced in flaccid muscles. Recent studies suggest that hypoxia may even develop during incubation of very thin muscles from mice.‘8,‘9In a recent study from our labora-
ET AL
tory, the central, hypoxic core of incubated rat SOL muscles was somewhat larger in tissue incubated flaccid than at resting length.” While the central core of flaccid muscles increased progressively in size during incubation, it remained relatively constant in muscles incubated at resting length. More important was the finding that there were no obvious differences in the size of the central core between septic and control muscles, neither when incubated flaccid nor at resting length.‘” We also compared the response to sepsis of protein metabolism in vivo and in vitro. The relative changes in protein synthesis in EDL were similar in incubated muscles and in vivo. We recently found that the changes in protein synthesis in SOL were also similar in vivo and in vitro.‘” Hence, the current in vitro system reflects sepsis-induced changes in muscle protein synthesis in vivo, at least from a qualitative standpoint. In other studies using the same in vivo technique, muscle protein synthesis rates varied between 17%/d and 21%/d.‘5.‘8.“’The lower synthesis rate observed in control rats in the current study probably reflects the fact that control animals were sham-operated and fasted for 16 hours before protein synthesis rate was measured. Because there are no direct methods available for the measurement of muscle protein degradation in vivo, breakdown rates must be calculated from other measurements. The degradation rates in the present study were calculated from protein synthesis and “growth” rates and results suggested that the sepsis-induced increase in protein breakdown may be overestimated when measured in incubated muscles. This result may reflect the fact that incubated muscles are in a catabolic state, even when incubated at resting length, and it is possible that this aggravates the sepsis-induced changes in protein breakdown. However, it should be noted that other interpretations are possible as well. One important factor to take into account is that the incubated muscles reflected the situation at 16 hours after sham operation or CLP, whereas the in vivo data reflected the degradation over the entire experimental period. For the calculations it was assumed that the protein synthesis rates were unchanged over the 16-hour period. This, of course, is unlikely; if protein synthesis was inhibited later in the septic course than protein breakdown was increased, the contribution of protein breakdown to reduced muscle protein content would be greater than the current calculations indicate. In fact, previous studies suggest that muscle protein breakdown is increased earlier than protein synthesis is reduced following CLP in rats’ and consequently, it is likely that the current in vivo calculations underestimated the increase in protein breakdown. Therefore, the in vitro data may reflect the in vivo situation better than the in vivo calculations suggested. The present report did not address the question whether in vivo studies are superior to those in vitro when the influence of sepsis on muscle protein metabolism is examined. Both techniques probably have their roles in these types of experiments, provided results are interpreted with caution.
251
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