Protein metabolism in rat gastrocnemius muscle after stimulated chronic concentric exercise THEODORE Department

AND FRANK W. BOOTH and Cell Biology, University of Texas h4edical School at Houston, Houston, Texas 77225




of Physiology







tabolism in rat gastrocnemiu muck after stimulated chronic concentric exercise.J. Appl. Physiol. 69(5): 1709-1717,1990.-

Previous results by use of a model of resistanceexercise consisting of nonvoluntary electrical contraction of rat skeletal musclehave shown that significant gastrocnemiusmuscleenlargement was produced after 16 wk of chronic concentric resistancetraining with progressively increasedweights but not after the same training program without weights (J. Appl. Physiol. 63: 950-934, 1988). In the present study we examined whether this differential effect on muscle massbetween highand low-resistanceexercise is mediated through differential actions on muscle protein synthesis rates. In addition, we determinedwhether accumulation of specific mRNA quantities had a primary role in the protein synthesis responseto this type of exercise. The data revealed that as little as 8 min of toti1 contractile duration increasedgastrocnemiusprotein synthesis rates by nearly 50%. Contrary to our hypothesis, postexercise protein synthesis rates do not appear to be differentially regulatedby the resistanceimposedon the muscleduring exercise but rather by the number of repetitions performed during the acute bout. This observation, the failure of highfrequency chronic training to produce gastrocnemiusenlargement, and the relatively minor effects on mRNA levels collectively suggestthat translational and posttranslational mechanisms, including protein degradation, may be the principal processesby which gastrocnemiusprotein expression is regulated in this model of stimulated concentric exercise. skeletal muscle;hypertrophy; protein synthesis;protein degradation; weight lifting; electrical stimulation; messengerribonucleic acid EFFECTS of increased contractile activity on the mechanisms that regulate adult skeletal muscle protein turnover are not fully understood. Furthermore, the exercise signal(s), their cellular level of regulation, and their influence on protein-regulating pathways are still unclear. Muscle activities that vary in frequency, duration, and intensity have been shown to decrease (28), increase (17, 21, 29), or have no effect on (15) muscle mass. Thus the regulation of muscle protein metabolism is likely a complex mechanism influenced by specific training stimuli. Chronic resistance training is a form of increased muscle activity that results in gross muscle enlargement (20). We have previously reported a model of resistance exercise that consists of nonvoluntary electrical contraction of rat skeletal muscle (29). Gastrocnemius muscle (GAST) enlargement was produced after 16 wk of


chronic concentric resistance training with progressively increased weights, whereas no increase in muscle mass was observed in animals completing the same training program without weights. Those results and others (17, 20) collectively suggested that high-intensity chronic resistance training is required to produce significant net protein synthesis by the muscle. One aim of the present study was to investigate whether the differential effect on protein accumulation of high- and low-resistance chronic weight training is mediated through differential actions on muscle protein synthesis rates after an acute resistance exercise bout. In addition, we wished to examine some of the biochemical changes that might support alterations in protein synthesis and muscle mass or indicate their cellular level of regulation (i.e., translational, transcriptional, etc.) after acute exercise and chronic training. We predicted that 1) GAST protein synthesis rates would be increased after acute exercise with weights but not without weights, 2) acute increases in synthesis rate are associatid with greater muscle size after chronic training, and 3) changes in synthesis would be partially mediated by increased levels of specific protein mRNAs. MATERIALS





Adult female Sprague-Dawley rats (Charles River Breeding Laboratories) were housed in animal quarters maintained at 21°C with a 12:12-h light-dark cycle. Animals were provided with water and rodent laboratory chow (23% protein; Purina Mills) ad libitum. Experimental protocols were approved by the Institutional Animal Care and Use Committee of the University of Texas Medical School at Houston. Electrically Stimulated MLwle Contraction

Animals were exercised by use of a model of nonvoluntary electrically contracted skeletal muscle (29). Briefly, Teflon-coated subcutaneous platinum electrodes (Medwire) were positioned bilaterally along the lower leg muscles of an anesthetized rat. In some experiments (chronic training), the contralateral muscles were used as a nonexercised internal control. The foot of the animal was secured to a pulley-bar apparatus with the rat supported on a platform above a foot lever. The muscle contraction of ether-anesthetized rats was induced with

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a Grass S48 electrical stimulator with 1-ms pulses at 100 Hz and 30 V with a 2.5-s train duration. The 15-V pulses

used to stimulate (submaximally) muscles in our earlier study (29) were increased to 30 V in the present study to increase the tension developed by the lower leg muscles. We have observed (data not shown) that tension output with 30-V stimulations produces 85400% of maximal tension, whereas 15-V stimulations produce only 50-85s of maximal tension (see below) during tibia1 nerve- or subcutaneous muscle-stimulated contractions at 15-90 V. Thus, by our estimates, muscles in the present study are near-maximally activated by stimulation. Muscle contraction is induced by activation of the stimulator, resulting in contraction of both posterior and anterior compartment muscles, with a net plantar flexion causing upward excursion of a weight by the pulley system. Electrically




We have previously reported a method by which estimates of the force output of isolated muscles are obtained during electrically stimulated muscle contractions (29) through intramuscular electrode stimulation. In addition to the reported results from those experiments, we found that the combined maximal force output of the GASTplantaris-soleus muscle complex (primary plantar flexors) was -1,100 g. In addition, the sum of the forces exerted by the tibialis anterior and extensor digitorum longus muscles (primary dorsiflexors) was -300 g. Thus in our model, in which both compartments are stimulated to contract, these values represent our closest approximation of the antagonistic tensions imposed on each respective muscle group. Exercise



Acute Exercise


assigned to one of four expergroup of rats received no electrically stimulated muscle contractions. The remaining three groups were classified as exercised groups, with each performing a defined regimen of electrically contracted muscle exercise. Each regimen varied in the number of repetitions (muscle contractions) per bout, the resistance (amount of weight added to the pulley), or both. High-frequency (repetition) groups were included in hopes of producing greater effects on protein synthesis rates and other parameters. These groups were designated and exercised as follows. Sedentary control (0 repetitions/O g). Control animals used in acute studies received no exercise but were anesthetized, catheterized, and infused in parallel with imental

were randomly


A control

exercised animals. Moderate frequency and moderate resistance (24 repetitions/500 g). Rats performed 24 total repetitions during the bout and were required to lift a 500-g weight attached to the pulley during each contraction. The adjusted resistance for the lever arm advantage (1.6) of this weight is -300 g. However, because of the cocontracting antag-

onistic dorsiflexor muscles, we have estimated that an additional resistance (-300 g) is imposed on the plantar flexor muscles. Thus rats must actually lift >600 g during each contraction or -55% of our estimated maximal leg



Repetitions were done in sets of six with 5-min rest periods between each set and 20-s rests between each 2.5-s muscle contraction. The protocol was completed in -30 min, during which rats underwent a total of 1 min of actual stimulated contractile activity. With very few exceptions, rats were able to lift the 500-g pulley weight through an -45” arc of the foot lever from the initial rest position during all repetitions of this paradigm. This protocol is similar to the regimens used in our previous tension.

study (29). High frequency

and low resistance

(192 repetitions/O


Animals completed 192 repetitions/bout divided into sets of six repetitions. Rest periods of 1 min were allowed between the first 16 sets, but only 30 s were allowed between each of’the last 16 sets with 10-s rests after each 2.5-s repetition. An additional 2.5.min rest was inserted after each group of four sets. No weights were placed onto the pulley, although muscles were required to overcome the resistance of the dorsiflexors (-300 g or 27% of maximal


This regimen required

-80 min to

complete, of which -8 min was actual stimulated contraction time. Because of the relatively lower resistance, rats performing this protocol were able to complete isotonic


of -90”

arc throughout

the exercise

bout. High frequency

and high resistance

(192 repetitions/

8004,100 g). A protocol identical to that described above for the high-frequency low-resistance group was performed, except weights were added to the pulley during contractions. Weights (8004,100 g) were regressively placed on the apparatus such that l,lOO-, l,OOO-, 900-, and 800-g weights were lifted in succession for one set of six repetitions. This cycle was completed eight times during the bout. We have calculated that when the mechanical advantage of the lever system is factored in, the weights (800-1,100 g) added to the pulley impose only 500-700 g of resistance or -4560% of the measured maximal tension (1,100 g) produced by the leg on the pulley during stimulation. Therefore, along with the -300 g of direct force imposed by the antagonistic dorsiflexors, the total resistance placed on the plantar flexors is -8OO-1,000 g or -TO-90% of our estimated maximal force output of the GAST-plantaris-soleus complex described above. This is almost three times the resistance lifted by the high-frequency low-resistance group. Depending on the pulley weight, rats were capable of moving the lever through an -20-30” arc. Exercise





groups of animals were trained by perform-

ing two acute bouts of exercise per week for 10 wk with 2 or 3 days of rest between bouts. The high-frequency

paradigms (192 repetitions/O g and 192 repetition@OO1,100 g) described above for acute exercise were examined in this part of the study.



a total of

only 160 min of actual stimulated contractile activity during the 10 wk (20 bouts) of training. The moderatefrequency moderate-resistance (24 repetitions/500 g) protocol was not included because the effects of chronic training were evaluated previously (29). A sedentary control group was maintained throughout the lo-wk

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training period, but rats were not subjected to repeated ether anesthesia. Ether anesthesia has no apparent effect

rats 3 days after the final exercise bout, which is the normal rest period between training days.

on muscle mass or body weight in this model (29). Strain gauge measurements of the net force output of the leg were recorded just before almost all training bouts and immediately after the bouts to estimate the degree of fatigue and functional adaptations of the muscle to

Cellular RNA was phenol extracted from frozen powdered muscle based on a method by Green (13) and after a procedure outlined previously (3) with the exception that RNA was quantified by ethidium bromide fluorimetry (18).



For RNA gel blot analysis, known amounts of extractable RNA (2-8 &lane) were denatured at 65°C in a 50%




Silastic (Dow Chemical) catheters were surgically placed into the right jugular vein of animals 2 days before the infusion of 5 mCi of L-[4,5-3H]leucine (45-130 Ci/ mmol; ICN Radiochemicals) at a rate of 1 mCi/h for 5 h in a lo-ml mixture of lactated Ringer solution and dextrose. Catheters were threaded subcutaneously through the nape of the neck to prevent access by the proximately 36 h before the start of infusion, leg of each animal was exercised according to nated exercise protocol. Later, 12 h before the the left leg of the rat was exercised with the

rat. Apthe right

its desiginfusion, identical regimen, This exercise schedule meant that synthesis rates could be measured during two 5-h intervals starting

at 12 (thus ending at 17 h) and 36 h (ending at 41. h) after exercise within the same animal. The 12- to 17-h interval was selected to avoid the expected transient

decline and delay (first 5 h postexercise) in the rise of protein synthesis postexercise (5) and because we speculated that the peak rise in synthesis would occur during this period. The 36- to 41-h period was chosen because this period coincided with the approximate midpoint of the rest period between exercise bouts used in chronic studies. It was expected that synthesis would be declining

or would be decreased back to control levels at this time point. Animals were ether anesthetized just before the completion of the [‘Hlleucine infusion, and the muscles were quickly dissected, frozen in liquid nitrogen-cooled Wollenberger tongs, and stored at -80°C. The exact time of infusion was noted as each muscle was frozen. Muscles were weighed and then powdered in liquid nitrogen. The aminoacyl tRNA was isolated, and total mixed protein acid hydrolysates were prepared as previously described (23). Myofibril protein was isolated (26)

and also hydrolyzed. The determination of the [3H]leutine specific radioactivities in the aminoacyl tRNA, mixed protein, and myofibril protein fractions was performed by the ultramicro method of Airhart et al. (1). The calculation of protein synthesis rates in total mixed and myofibril protein was done by the formula of Garlick et al. (11). Messenger and Ribosomal RNA Levels

Determinations after acute exercise were done on a separate set of rats from which protein synthesis measurements were made. Animals were exercised identically, and muscles were collected at time points comparable to 17 and 41 h postexercise. The moderate-frequency mod-

erate-resistance (24 repetitions/500 g) group was not included in this set of experiments. Measurements after chronic training were carried out in muscles taken from

formamide and 6% formaldehyde sample buffer containing 1 pg/ml ethidium bromide and electrophoresed onto 1.5% agarose gels containing 2.5% formaldehyde at 60 V for 4-5 h (10, 19). Gels were then checked for RNA degradation under ultraviolet light, and the RNA was electrohlotted (TransBlot Cell, Bio-Rad) onto a BioTrans (ICN Radiochemicals) nylon membrane, baked for 1 h at 80°C under vacuum, and hybridized. Dot blot analysis was carried out as described previously (3) with nylon instead of nitrocellulose filters baked for only 1 h. Probes used for hybridization to rat skeletal cw-actin and cytochrome c mRNAs and to 185 rRNA have been described previously (3, 23). In addition, a 28s rRNA probe, which is a 1.5-kb fragment from pSP64-28s subsequently subcloned into M13mp19 for hybridization, was also kindly given to us by L. Rothblum (24). Probe labeling with [32P]dATP, Northern and dot-blot filter hybridization, posthybridization washes, and autoradiography have also been outlined before (3). Radioactivity in dot blots was quantitated by liquid scintillation counting (Beckman LS3801). Protein, RNA, and DNA Muscle wet weights were determined on frozen muscles. Protein concentrations (mg/lOO mg muscle wet wt)

were obtained by the method of Bradford (6). RNA and DNA concentrations (mg/mg protein) were assayed according to the procedures of Munro and Fleck (24) and Burton (7), respectively. Detailed procedures used in this study were also reported previously (3). Statistics Protein synthesis rate and biochemical data are reported as means t SD unless otherwise noted and were analyzed by the general linear model analysis of variance (missing data) followed by the Scheffi! multiple range test to determine significance between individual means. Dot blots were examined by analysis of covariance for group differences between slopes constructed by plotting radioactivity vs. unit of extractable RNA. Significance level was defined as P < 0.05. RESULTS

Functional Effects of Resistance Exercise and Training

Muscles were significantly fatigued after completion of the initial exercise bout in rats used for chronic training studies. These values from the first bout were used to assess the acute effects of exercise. After highfrequency low-resistance exercise, the preexercise tension output of 1,084 t 110 g was significantly reduced by

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become insufficient for full force recovery. In animals performing the high-frequency high-resistance regimen, this progressive fatigue during the bout hindered the ability of rats to continue lifting the weights through the 20-30” arc at the beginning of exercise. During the initial bout, most rats were unable to maintain sufficient ten-

exercise. Mixed protein synthesis rates in the high-frequency low-resistance and high-frequency high-resistance groups were greater than moderate-frequency moderate-resistance rates, but myofibril synthesis was not statistically higher than that of this group during the interval 12-17 h after exercise. 36-41 h after acute exercise bout. Comparison of exercised groups indicated that synthesis rates 36 h after high-repetition high-intensity exercise (192 repetitions/ 800-1,100 g) had definitely begun to decrease back toward control levels because synthesis rates were now no longer greater than control or moderate-frequency moderate-resistance group values. In contrast, high repetitions of contraction without weights (192 repetitions/O

sion to lift the lever and performed

g) had a more prolonged effect on synthesis than higher-

41% (640 -+ 98 g) immediately postexercise. However, rats were able to perform isotonic contractions throughout the bout with a noticeably decreased excursion arc as exercise progressed. Fatigue was gradual such that animals underwent a fatigue-recovery cycle in which they become fatigued as each set (6 repetitions) was completed and then recovered during the subsequent rest period; however,

as the bout continued,

the shortened

rest times



contractions during the latter portions of the exercise. By the end of the bout, force output was decreased by 58% (457 k 134 g) compared




* 194 g). Decreases in preexercise force were not different between high-frequency regimens. Although (1,100

force measurements were not collected for the moderate-

frequency moderate-resistance group in this investigation, data not reported in our earlier study (29) showed that, compared with preexercise values, muscle knsion is significantly reduced by 17% at the end of the bout by

use of this general protocol. After 10 wk of chronic training, adaptational responses to training were evident because as muscles were less fatigued at the end of the last training bout compared with the frlrst bout. Preexercise force was decreased by only 28% (preexercise 1,243 $I 97 g, postexercise 807 t 170 g) at the end of bout 20 in rats performing the highfrequency low-resistance paradigm and by just 35%

(preexercise 1,280 t 190 g, postexercise 920 t 162 g) after high-frequency high-resistance training. This process occurred progressively over the training period. Preexercise tension after training was not significantly higher than pretraining values in either high-frequency training group.

intensity exercise (192 repetitions/800-1,100 g). Rates measured at 36-41 h after exercise were still elevated above controls, were at least maintained at levels observed 12-17 h after exercise, and were now greater than moderate-frequency moderate-resistance group synthesis rates for both mixed and myofibril protein. The reason for this apparent difference in transiency, how-

ever, is unknown comprehensive

but could be more fully answered by a time-course study. The moderate-fre-

quency moderate-resistance group again showed no significant differences from control GAST protein synthesis rates. The low inherent variability of synthesis rates was demonstrated by the similarity of l2- and 36-h values (left and right muscles) in control animals. Body and Muscle Weights The acute exercise values for the GAST from rats utilized for protein synthesis rate experiments are reported in Table 2. Comparison of preexercise body weights of rats used for the acute exercise studies revealed small but significant differences (S-13%) between experimental groups despite random group assignments. Acute exercise had no effect on body weights. Animals used for chronic concentric resistance-training

Protein Synthesis Rate The mixed and myofibril

protein synthesis rates de-

termined in rat GAST after a single acute concentric resistance exercise bout (acute exercise) and in nonexercised control animals are summarized in Table 1. 12-l 7 h after acute exercise bout. Rats that performed the moderate-frequency moderate-resistance regimen showed no significant change in either mixed or myofibril

protein synthesis rate compared with control (0 repetitions/O g) animals. However, in the high-frequency lowresistance group, mixed protein synthesis significantly increased 47% and myofibril protein synthesis rate was elevated by 38% compared with control muscles. Increasing the resistance by placing additional weights onto the


had similar body weights between groups before and after training. All groups showed significant and similar weight gains during the IO-wk training period. After acute exercise, there were not significant differences in GAST wet weight between or within any of the groups or time points. Interestingly,

chronic training did

not result in any significant compared with nontrained

change in GAST wet weight contralateral control muscle or nontrained sedentary control muscles or between chronic training groups. Protein

GAST total protein concentrations (mg/lOO mg wet wt) and contents (mg/muscle; Table 2) were statistically

pulley did not further increase synthesis rates because

unchanged after the acute exercise bout. Three days after


chronic training, protein concentration was reduced by 10 and 11% in high-frequency high-resistance and highfrequency low-resistance group muscles, respectively. This suggested a possible inflammatory response due to the exercise protocols. Protein content per muscle, how-



showed percent increases in fractional



synthesis rates

similar to those observed after lower-intensity exercise (192 repetitions/O g). Comparisons between exercised groups suggested that smaller percent increases in myofibril vs. mixed protein synthesis were induced by the

ever, was not different between any of the groups.


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1. Fractional protein synthesis rates in CAST muscle after acute concentric resistance exercise .__~ .......-~-... . --.... n

Control Exercised 24 rep/lSOO




Time Yostexercit;e,


- -_h


12-17 X.41

X0+0.6 X0+0.6


x4-+0.7 3.1 to.4 4.4*0.6*-f


.%Change from con troi




.. .-- -

-.-Y&Change from control

2e4kO.5 2.4k0.7

NS 2.720.5 NS NS 2.5&0,4 NS 192 rep/O g 7 +47 3.3*0.4* +38 +57 4.7+ 1.0*+ 3*4&l ,o*t +42 192 rep/ML1,100 8 g 4.4*1.:3*-f +47 3.3*0.9* +38 3.9kO.9 NS 2.9kO.9 _ . . --NS Total mixed and myofibril protein synthesis rates were measured in GAST muscles from Values are means t SD; n, no. of muscles. exercise bout using various paradigms of a nonvoluntary resistance nonexercised control rats and from animals after 1 acute concen tric resistance with control. t P < 0.05 compared with 24 rep/500 g group. exercise model. * I’ < 0.05 compared 36 41 12-17 .36- 41 12-17 36-4. --.-1

though GAST wet weights were not correspondingly increased nor protein contents decreased, this is probably due to normal variation. RNA and DNA

after the high-resistance exercise paradigm (192 repetitions/800-1,100 g). After chronic training, mRNA concentrations per unit of extractable RNA appeared to show a trend toward a decrease in trained animals. Although skeletal cu-actin mRNA per unit of extractable RNA was not significantly

After only a single acute exercise bout, RNA concentration (mg/mg protein) had increased with all exercise paradigms at 17 h postexercise and remained elevated at 41 h postexercise (Table 3). Because of differences in body size, RNA contents per muscle were significantly different from control only for the moderate-frequency

altered by either high-frequency regimen, the slopes of trained rats tended to be decreased compared with sedentary control animals (Table 5) and contralateral nontrained muscles (data not shown). Similar to skeletal cyactin mRNA, cytochrome c mRNA per unit of extractable RNA tended to be lower than sedentary control

moderate-resistance group (24 repetitions/500 g). RNA concentrations per milligram protein did not differ significantly between exercise regimens or time points. High-frequency chronic training resulted in a 45 (highfrequency low-resistance) and a 36% (high-frequency high-resistance) increase in RNA concentration. RNA content was 33% greater than sedentary control level in both chronic trained groups. Changes in DNA concentration per milligram protein and content per muscle were directionally similar to the changes in total RNA after acute exercise and c hronic training (Table 3).

animal levels (Table 5) and was significantly decreased in the high-frequency low-resistance (36%) and the highfrequency high-resistance (27%) groups compared with respective nontrained contralateral muscle levels. However, although the proportion of these mRNAs per unit of extractable RNA may be decreased, skeletal cr-actin and cytochrome c mRNA content per whole muscle may not be actually decreased if the 33% increase in total RNA content per muscle is considered. That is, if the proportion of an mRNA in the total RNA pool (mRNA/ unit extractable RNA) remains the same but the total pool of RNA is increased (extractable RNA/unit protein), then the absolute content of the mRNA in the whole muscle could be increased. To analyze this possiblity, we approximated the percent changes in skeletal cu-actin and cytochrome c mRNA per whole GAST (Table 4) by taking into account the increases in the total RNA concentration (Table 3) in the muscle. These estimates suggest that the absolute levels of these mRNAs are not

Messenger and Ribosomal RNA

Skeletal a-actin and cytochrome c mRNAs and 18s and 28s rRNA subunit levels were measured after acute exercise and chronic training. The levels of skeletal cu-actin and cytochrome c mRNA per unit of extractable RNA were not changed significantly from sedentary control rat or contralateral control muscle levels by either exercise paradigm 17 and 41 h after acute exercise (Table 4). These results support the conclusion from total RNA measurements (Table 3) that increases in RNA do not have a primary role in increasing protein synthesis rates after a single bout of resist-

ance exercise. Estimates of MS and 28s rRNA (Table 4) indicate that the level of these components may be elevated 41 h after low-resistance exercise (192 repetitions/O g), which is consistent with the observed increase in total RNA per milligram protein (Table 3). However, in light of the increased RNA in the muscle, it is not clear why 18s and 2% subunits were not also increased

decreased but that their relative proportion per unit of extractable RNA is diluted due to a greater proportional increase in rRNA than in mRNA levels during training. This is supported by the relatively unchanged 18s and 28s rRNA subunit levels per unit of extractable RNA in the muscles of trained rats. However, these calculations

are equivocal because of the nature of these estimates, which prohibits statistical analysis (Table 5). DISCUSSION

A number of observations in the present study were unexpected and contrary to our initial hypotheses. For example, it was not predicted that protein synthesis rates

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286 *36

Protein content, mg/muscle

17.6 (NS) 287d kll (NW

18.0 t0.8

1 .58d kO.08 WS) 0.50


*27 (W

16.9 k1.0 WS) 259

mg protein day-’ - mg










. -...


9 0




l.O**_---- l.O+O.ld Values a~ means k SD; n, no. of observations observations, respectively. TR and UNT, trained control.

RNA activity, synthesized RNA-’ DNA, mg/mg






Total d:ly bout-a: Time postexercise, h:



1.53 to.13 WS)


._-. 7 1

283 *41 (NS)








1.69 kO.16


8 1

192 rep/800-1,100

1.52 t0.09


192 rep/o g


18.0 *a9 WS) 293 k30

(NS) 0.53

1.62 f0.15



291 k23




1.66 io.09

(+15) 1.2+0.1’


(+W 3.9k0.5a (+l5)


I+311 4.1AO.3'





1.5i0.1 (NS) 7.4k2.3'






7 1

-192 rep/O




._ .-

(+lO) 1.1~0.1

(+45) 3.8H.3'

1.5kO.2 (NS) 8.3k2.0'






(NS) l.lkO.1



1.6kO.l (NS) 7.lt1.4'











6 0


0.9&O. 1






10 wk

changes control.

264 *28

16.4 kO.8


1.60 kO.13


20 72

TR rep/0

1 .3+0.2”b




7.7+0.7mmb (+45) 2.0*0.2*b (+33)




from sedentary f P < 0.05 from

15.6'~~ MI.5 (-11) 267 k27 (W

(NS) 0.52

1.71 *0.14

20 72


(192 rep/o g)


are directional percent changes from sedentary mP < 0.05 from sedentary ND, not determined.

FI 1

192 rep/MU-1,100

--1.1iO.l unless otherwise denoted. Numbers in and untrained Iegs, respectively, of the same rat;



5.720.3' (+14) 1.6kO.l' (+13) 5.4kO.8







(+W 1.8kO.3"


6 1

24 rep/500




after a single acute bout, and after

6 0


unless otherwise denoted. Numbers in parentheses are directional percent and untrained legs, respectively, of the same rat; ’ P < 0.05 fro m sedentary

tQ.5 (NS) 302' *31 (NW

6 1


3, Nucleic acid levels in CAST muscle from control, of twice-weekly chronic concentric resistance exercise ---__-

Values are means k SD; n, no. of observations observations, respectively. TR and UNT, trained

279" *37


WS) 0.54




1.72" kO.18


1.5gb *o. 19


w-h gmQ



1.57. *o. 19


wet wt/



9 0

Probin concn, mg/ 100 mg wet wt

wet wt, g


Time postexercise,





2. Body weights artd CAST wet weights and protein levels after a single acute bout resistance exercke

after 10 wk of twice-weekly chronic concentric





15.7'*' iO.8 (-10) 287 *37 (NS)

1.82 *0.14 WS) 0.53

20 72


TR (192 rep/

d 5

WS) 297 *12 WS)

16.8 kO.6


1.77 io. 12 WS)

0 72

UNT (0 rePI


control control.

(NS) 0.9*0.2


5.5+0.3 (NS) 1.6k0.1 WS)

0 72

UNT (0 rep)



(NW 1.0*0.1



1.5iO.l WS)






(0 rep)

values, C*bd*f ft = 8, 7,4, and 5 b P < 0.05 from contralateral


4.7f0.4'b (+38)


2.0i0.1~b I+W



20 72

TR (192 rep/ 8004,100 g)

control valuea. 8*b-cmdn = 8, 7, 4, and 5 contralateral control.


kO.8 (NS) 287 *21



kO.11 (NS)


0 72

UNT (0 rep)






TABLE 4. Percent changes in mRNA and rRNA subun it levels determined in GAST muscles after a single acute bout of concentric resistance exercise mRNA Acute

or rRNA/Unit RNA,





% 41 h

17 h



cu-Actin mRNA 192 rep/o g 192 rep/NO-1,100


Cytochrome c mRNA 192 rep/O g 192 rep/8OO-1,100





by both low- (192 repetitions/O g) and high-resistance

+36* NS

( 192 repetitions/800-1,100 g) exercise, Furthermore, synthesis rates were not different between these two groups at 12-17 h after exercise and even tended to be higher in the low-resistance group at 36-41 h postexer-

TABLE 5. Percent changes in mRNA and rRNA subunit levels determined in GAST muscles after 10 wk of chronic concentric resistance training Total RNA/W hole GAST, %

Estimated mRNA or rRNA/Whote GAST, %

a-Actin mHNA 192 rep/O g

192rep/8uo-1,100 g

-14 -16

+33 +33


+33 +33

+18 +10

+33 +33

+42 +42

+33 +33

+37 +29


Cytochrume c mRNA 192 rep/O g 192 rep/W-1,100


18s tRNA +7

192 rep/O g 192 rep/800-1,100



28s rRNA +3

192 rep/O g 192 rep/800-1,100

-11 -17



Values were taken 3 days after final exercise bout. Total RNA/ whole GAST values are from Table 3. mRNA and rRNA concentrations were determined by analysis of RNA dot blots (see MATERIALS AND METHODS); IZ = 6/g~oup. Indicated changes for mRNA or rRNA/unit of extractable RNA are not statistically significant and were estimated from differences between mean slope values determined for each exercised group and nonexercised control group. Changes in mRNA and rRNA contents per whole muscle were approximated from product of percent change in mRNA or rRNA/unit of extractable RNA and percent change in total RNA/whole GAST. Statistical analysis of mRNA or rRNA/whole CAST could not be performed because of nature of estimation.

would be increased similarly by both high-

and lowresistance protocols during the interval 12-17 h after 192,repetitions/bout acute concentric resistance exer-

cise. In light of the positive effects on synthesis rate, we were also surprised by the lack of hypertrophy in the GAST of both groups after 10 wk of performing 192 concentric contractions every 3rd or 4th day. In addition,

our hypothesis that GAST protein

of 192 contractions) can stimulate the acute increases in synthesis rate observed in the study. The implications of


Mean percent changes in mRNA or rRNA concentrations were determined by analysis of RNA dot blots (see MATP:RIALS AND METHODS); n = G/group. Indicated changes are in comparison to nonexercised control rats. * P < 0.05 from control.

mRNA or rRNA/Unit of Extractable RNA, o/c

after only 24 concentric contractions (24 g) did not hold, This was especially surprising because our previous chronic training study showed an 18% increase in GAST muscle mass after 16 wk of training by use of similar paradigms (29). Nevertheless, we considered it remarkable that a total of as few as 8 min of contractile activity (a single exercise bout repetitions/500


28s rRNA 192 rep/O g 192 rep/800-1,100

be increased

loads do not result in a greater effect on GAST protein synthesis rates or muscle mass, respectively, using highfrequency concentric contraction protocols. GAST protein synthesis rates after acute exercise were increased

18s rRNA 192 rep/o g 192 rep/800-1,100


dicate that exercising and training with higher resistance NS NS




the results from this study are discussed below. In contrast to our hypotheses, the following data in-





synthesis rates would

cise. Likewise, GAST muscle weights after chronic training were not different between these two groups. These results are in contrast to studies that have shown a significant correlation between acutely increased protein synthesis rates and subsequent muscle growth (17, 21).

Furthermore, they are contrary to data showing that lowresistance types of muscle activity such as daily running exercise (15) and chronic electrical stimulation of muscle (28) do not cause muscle enlargement, whereas highresistance types of exercise such as chronic resistance training (20) and experimental models of constant muscle loading (2, 4, 17) do result in greater muscle mass, One reason why protein synthesis rates were increased by both high- and low-resistance regimens may be that

the antagonistic dorsiflexor muscles are cocontracted with plantar-flexor muscles in this model, which results in the loading of the GAST during the so-called “zeroresistance” regimen (192 repetition@ g). In addition, it could be speculated that the source of the increased

protein synthesis is not within muscle fibers but is due to nonmuscle cell activity. However, although this is a reasonable argument for changes in mixed protein syn-

thesis, results also indicate that myofibril protein, which is virtually absent in inflammatory and connective tissue cell types, also showed a significant increase in synthesis rate. To explain the lack of GAST muscle growth with either

paradigm, it is possible that additional protein regulatory responses are activated by high frequencies of concentric resistance exercise, which may override the ability of high-resistance exercise to stimulate a net increase in protein synthesis. Hickson (14) has shown that simul-

taneous strength (high intensity) duration) training may hinder

and endurance (highthe development of

strength. This implies that gains in muscle mass may be overridden by longer durations of increased contractile

activity. Moreover, the finding that muscles atrophy after weeks of continuous (24 h/day) indirect stimulation (28) demonstrates that long durations of muscle activity can result in negative protein balance in the muscle. One explanation could be an increased protein degra-

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rate or some other posttranslational






changes that may mediate or indicate the cellular level

that could offset enhanced protein synthesis mechanisms during chronic training. It is clear that protein degradation can have an important role in protein expression.

of control of protein metabolism during concentric resistance exercise. The rise in the amount of RNA per protein after acute exercise has been observed in other



models of muscle weight bearing (12,17). A consequence

weighting report increases in both synthesis and degradation rates, but in contrast to findings in this study they show a net increase in protein mass (17). The finding that the muscles in the study above undergo



of greater RNA is an increase in the capacity of the muscle to synthesize protein. Thus RNA synthesis may be stimulated during acute exercise (27) and may be partially responsible for the increases in protein synthe-


sis. However, the smaller percent increases in RNA per





of constant



and perform eccentric muscle training may contribute to the divergence from our results. Other evidence for a major role for degradation comes from speculation that increased protein degradation activity may be necessary to clear tissue debris for deposition

of newly synthesized

protein during muscle remodeling (22). Another attractive hypothesis that could explain why the GAST did not enlarge in this study is that the resistance


on the GAST

was not high enough

to induce hypertrophy. Low resistance appears to be a feasible explanation for results from the high-frequency low-resistance groups because the 300 g of resistance produced by the dorsiflexors is relatively small compared with

the 1,100 g exerted

by the plantar-flexors.

On the

other hand, our estimates (see MATERIALS AND METHODS) indicate that for the high-frequency high-resistance regimen, the combined resistance of the dorsiflexors plus the adjusted pulley weight is -7O-90% of’ the maximum force output of the major plantar flexors. This resistance is thought to be sufficient to produce muscle hypertrophy (20). However, because our approximations of maximal force were based only on measurements of the GASTplantaris-soleus muscle complex, our values could be low. Although sufficient resistance is certainly

an impor-

tant factor, we believe that regulation may be more complex, because the above hypothesis cannot fully explain why large acute increases in protein synthesis are produced without a net protein increase after chronic training. It has been suggested that protein synthesis mechanisms may become less responsive as training


because synthesis rates were only determined

protein compared with the percent increases in protein synthesis rates support the idea that other mechanisms (i.e., translational or posttranscriptional controls) are concurrently activated (17). This was deduced from increased RNA activities (i.e., the amount of protein synthesized per day per unit of RNA; Table 3), indicating that mechanisms such as peptide initiation or elongation rates might be faster and could account for a major portion of the acute increases in synthesis rates. The expansion of RNA content per muscle (increased

protein-synthesizing capacity) after chronic training (Table 3) without a corresponding increase in muscle size (Table 2) supports the involvement of posttranslational

mechanisms in the determination

of muscle

growth during high-repetition concentric resistance training. The increase in RNA seems to be the result of accretion during the training period and does not appear to be a residual effect from the final acute bout because increases of RNA after chronic training were consider-

ably greater than after acute exercise. Thus muscles appear to undergo gradual biochemical as well as functional (less fatigue) adaptations as a consequence of chronic training. On the other hand, the origin of the increased RNA in the muscle after chronic training is

not known. It is feasible that the RNA could be associated with nonmuscle cells (16) or exogenous cells that migrate to the muscle as part of a postexercise inflammatory

response (8). This is supported by decreased

muscle protein concentrations without changes in protein content and the finding that DNA profiles were in general altered in parallel with changes in RNA after

after the first bout. However, the finding that total RNA

training. In either case, the increase of RNA content per

accumulates throughout the training period (see below) implies that the protein-synthesizing capacity of the muscle is progressively challenged during chronic training.

muscle without an increase in muscle size implies a dissociation between RNA and protein accumulation during chronic training. A similar finding has been previously observed in muscles that were chronically stim-

The finding that other resistance-training


ulated 24 h/day (28).

result in significant enlargement (29) by use of a range of resistances similar to that in this study except for lower-frequency repetitions per bout (24 repetitions/ bout) also cannot be explained by insufficient muscle

Unlike RNA, the source of the greater DNA levels cannot be mature muscle contractile fibers. Increased DNA in adult skeletal muscle must be from satellite cell activation, connective tissue cell proliferation, or infil-

loading. This implies that a low frequency of concentric


repetitions per day may be important to produce muscle hypertrophy. Thus, based on the collective data above, we speculate that low frequencies (24 contractions) of

distinguish between these mechanisms from the present data, all three cell types may contribute to this observation.

cells (8, 9, 16). Although

it is not possible to

high-resistance concentric exercise and training stimu-

The unchanged levels of skeletal n-actin mRNA per

late a small but preferential effect on protein synthesis over degradation mechanisms that result in muscle enlargement (29) but that at greater frequencies (192 contractions) increases in protein synthesis are offset by

unit of extractable RNA and per whole muscle after acute exercise suggest that mRNA accumulation is not a major mediator of the acute increase in myofibril protein synthesis rates. This provides further evidence that the

large and equivalent increases in protein degradation.

acute changes in protein synthesis principally



study also examined

the biochemical


or posttranslational




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these data do not exclude the possibility that changes in mRNA levels may occur at other time points not measured in the present experiments. Furthermore, the apparent accumulation of total RNA in the muscle after chronic training suggests that transcriptional or pretranslational pathways are probably implemented for at least rRNA synthesis. In summary, the regulation of protein metabolism during concentric resistance exercise in the GAST muscle is exerted at several cellular levels but is hypothesized to be primarily through translational or posttranslational pathways. The production of increased muscle protein after chronic training is apparently dependent on a delicate balance between protein synthesis and degradative mechanisms that can be biased by the number of repetitions and/or by the relative resistances placed on the muscle during the concentric exercise and training regimen. It is speculated that the do-5096 increases in protein synthesis rate caused by acute concentric resistance exercise may be masked by equivalent increases in degrad&ion during high repetitions of exercise with this model of nonvoluntary resistance exercise. This model predicts that multiple shorter bouts of this type of exercise may be more productive. The authors thank Marjorie Tucker for typing the tables, Dr. Kenneth RO for technical assistance, and Chris Kirby for assistance in editing the manuscript. This study was supported by National Institute of Arthritis and Musculoskeletal and Skin Disease Grant AR-19393 (F. W. Booth). This work was performed in partial fulfillment of doctoral dissertation requirements (T. S. Wong). Address for reprint requests: F. W. Booth, Dept. of Physiology and Cell Biology, University of Texas Medical School at Houston, P. 0. Box 20708, Houston, TX 77225. Received 25 January 1990; accepted in final form 6 June 1990. REFERENCES R. B. Low, AND W. S. amino acid analysis: applimetabolism in cultured cells. Anal.

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Protein metabolism in rat gastrocnemius muscle after stimulated chronic concentric exercise.

Previous results by use of a model of resistance exercise consisting of nonvoluntary electrical contraction of rat skeletal muscle have shown that sig...
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