Eccentric exercise training as a countermeasure to non-weight-bearing soleus muscle atrophy CHRISTOPHER Department
R. KIRBY,
MIRELLE
of Physiology and Cell Biology,
J. RYAN,
AND
FRANK
W. BOOTH
University of Texas Medical School, Houston, Texas 77225
eccentric resistance exercise (lengthening contractions) during non-weight bearing to be determined. Normal locomotion, posture, and activity incorporate eccentric contractions (14, 17, 29). Because loaded eccentric contracbeen tested, none has completely prevented muscle atrophy tions are absent during non-weight bearing (15), we hyduring non-weight bearing. Becauseloaded eccentric contracpothesized that the return of eccentric contractions tions occur during normal daily activity but are absent during during non-weight bearing might provide an effective non-weight bearing, this investigation tested whether eccentric countermeasure to muscle atrophy. The purpose of this resistancetraining could prevent soleusmuscleatrophy during non-weight bearing. Adult female rats were randomly assigned study was to determine whether an eccentric exercise to either weight bearing ztrintramuscular electrodes or non- training bout every other day during 10 days of nonweight bearing t intramuscular electrodesgroups. Electrically weight bearing would prevent soleus muscle atrophy. stimulated maximal eccentric contractions (4 sets of 6 repeti- This frequency of eccentric exercise training was setions at ~0.2 fiber lengths/s, 128” range of motion) were per- lected because a single bout of 24 eccentric contractions formed on anesthetized animals at 48-h intervals during the elevated total mixed protein synthesis rates for up to 41 h IO-day experiment. Non-weight bearing significantly reduced postexercise in normal rat tibialis anterior muscle (31). KIRBY, CHRISTOPHER R., MIRELL~ J. RYAN, ANDFRANKW. Eccentric exercise training as a countermeasure to nonweight-bearing soleus muscle atrophy. J. Appl. Physiol. 73(5): 1894-1899, 1992.-Although various exercise paradigms have
BOOTH.
soleusmusclewet weight (28-31%) and noncollagenousprotein content (30-31%) compared with controls. Eccentric exercise training during non-weight bearing attenuated but did not prevent the lossof soleusmusclewet weight and noncollagenous protein by 77 and 44%, respectively. The potential of eccentric exercise training as an effective and highly efficient countermeasureto non-weight-bearing atrophy is demonstrated in the 44% attenuation of soleusmusclenoncollagenousprotein loss by eccentric exercise during only 0.035% of the total nonweight-bearing time period. reduced use; muscleprotein content; hypokinesia; hypodynamia; musclewasting
SKELETALMUSCLEATROPHY resulting from non-weight bearing is one of the principal alterations associated with spaceflight deconditioning (4). The loss of muscle protein content and strength may result in reduced physical work capacity during long-duration spaceflight and/or difficulty in readaptation to the 1-G environment of Earth after spaceflight (4). Therefore several countermeasures have been tested for their ability to reverse or prevent muscle atrophy during non-weight bearing. The countermeasure most often employed has been exercise (5,6,9-12,22,24,26). However, no exercise countermeasure has completely prevented muscle atrophy during non-weight bearing. Of the exercise protocols utilized, resistance training is at least equal in its effectiveness (i.e., percentage of atrophy prevented) and superior in its efficiency (i.e., percentage of atrophy prevented/exercise time) as a countermeasure for attenuating muscle mass and protein content losses (1; see Table 3). To date, only the adequacy of concentric resistance exercise (shortening contractions) has been tested, leaving the efficacy of 1894
016L7567/92
$2.00
METHODS Animals. Thirty female retired-breeder rats (Harlan Sprague Dawley, 275 g body wt, 8-10 mo of age) were randomly assigned to either weight-bearing (WB), weight-bearing with bilateral intramuscular electrodes (WB-IM), non-weight-bearing (NWB), or non-weightbearing with bilateral intramuscular electrodes (NWBIM) groups. An additional 13 animals from this shipment were used for pilot studies. The contribution of growth inhibition of the soleus muscle during non-weight bearing was diminished by use of adult female rats. Animals had ad libitum access to standard rat chow and water throughout the experiment. All procedures using animals were approved by the Animal Welfare Committee of the University of Texas Medical School, Houston and conformed to the American Physiological Society “Guiding Principles for Research Involving Animals.” Experimental design. WB-IM and NWB-IM groups underwent surgery for bilateral electrode implantation into soleus muscles on day 0 of the experiment (Fig. 1). After a 7-day recovery (day 7), soleus muscles from the right leg of animals in WB-IM and NWB-IM underwent a single bout of eccentric exercise as a prophylactic measure to reduce eccentric exercise-induced muscle damage in the subsequent eccentric training program (8). On day 14 all animals began a lo-day period of tail casting with (WB and WB-IM groups) or without (NWB and NWBIM groups) hindlimb weight bearing. On day 14 WB-IM and NWB-IM groups also began eccentric exercise training of the right soleus muscle. The frequency of eccentric exercise training (every other day) was based on a previous report in which total mixed protein synthesis rates
Copyright 0 1992 the American Physiological Society
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ECCENTRIC Day:
Groups:
0
WB-IM NWB-IM
7
EXERCISE
14
16
18
ATTENUATES 20
Etc. Ex.
Ecc, Ex.
Tai I-cast
Etc. Ex.
WB-IM NWB-IM
WB-fM NWB-IM
WB WB-IM NWB NWB-IM
WB-IM NWB-IM
22
24
Kill all groups
FIG. 1. Experimental time line. Surgery, implantation of intramuscular electrodes; Ecc Ex, eccentric ex&&se training; Tail cast, tailcasted + non-weight bearing; WB, weight bearing; NWB, non-weight bearing; WB-IM, weight bearing with intramuscular electrodes; NWBIM, non-weight bearing with intramuscular electrodes.
were increased between 36 and 41 h after a single bout of 24 eccentric contractions in rat tibialis anterior muscle (31). Eccentric exercise training every other day continued through day 22, and all animals were killed on day 24 of the experiment. Surgical procedures. Animals in the WB-IM and NWB-IM groups were anesthetized for surgery with 60 pg ketamine, 2 pg xylazine, and 4 pg acepromazine per g of body weight (Aveco, Fort Dodge, IA). A small (3-cm) incision was made in the skin of the lateral aspect of each lower leg. A small opening (0.5 cm) was then made, parallel to the muscle fibers, in the biceps femoris. The distal and proximal ends of the soleus were then retracted with a curved forcep. Teflon-coated, braided, stainless steel wire electrodes (0.012-in. diam, Cooner Wire, Chatsworth, CA), to which a 30-gauge needle had been attached, were then passed through the soleus muscle and anchored by a piece of 6-O suture tied around each protruding end. Incisions in the biceps fern .oris and skin were then closed with 6 -0 suture and 4-O suture plus wound clips, respectively. Electrodes in the right soleus originated from a two-contact lamp socket (Augat, Attleboro, MA), which was mounted to the skull with stainless steel screws (O-80 X 3/16 in.) and dental cement (methyl methacrylate, Sybron, Romulus, MI). Electrodes in the left soleus extended from the muscle but were cut just below the skin. In addition, a hole was drilled, and blunted 27gauge needles were implanted into both calcanei for the purpose of securing the right foot to the pulley-bar apparatus during eccentric exercise training and as a sham treatment for the left foot. Animals received prophylactic antibiotic injections (gentamicin, 50 pg/g body wt, Smith & Nephew, Franklin Park, IL) on the day of and for 2 days after surgery. All surgical procedures were performed under aseptic conditions. Animals recovered from surgery for 1 wk, at which time body weights (Table 1) and food consu .mption (data not shown) were similar to control animals. Eccentric exercise training protocol. Eccentric exercise training was performed using modifications to a model of involuntary resistance exercise in rats (30,31). Anesthetized rats (80 pg ketamine and 8 ,ug acepromazine/g body wt; Aveco) were supported on a platform with their feet secured to a plate on a pulley-bar apparatus (Fig. 2). Eccentric contractions of the right soleus muscle were accomplished by manual advance of the right ankle through its full range of motion (from plantar flexion at 162 t 1” to dorsiflexion at 34 t lo) during electrical stim-
REDUCED-USE
MUSCLE
ATROPHY
1895
ulation. Right soleus muscles of WB-IM and NWB-IM groups, which underwent eccentric exercise training, are designated “exercise” (Table 2). Eccentric exercise training of the right soleus muscles utilized an electrical stimulation paradigm that produced maximal isometric muscle force in rats (n = 3) not used in the experiment (train rate, 0.2 trains/s; stimulation rate, 60 Hz; pulse duration, 0.5 ms; and voltage, 16 V). On the assigned days, rats in the WB-IM and NWB-IM groups performed four sets of six repetitions. The muscle was stimulated supramaximally for 2.0 s every 5 s, with 2-min rest periods between sets. Left soleus muscles of WB-IM and NWB-IM groups, designated “no exercise” (Table 2), received neither electrical stimulation nor manual movement of the ankle. Eccentric contractions were performed at ~0.2 fiber lengths per second (L,ls), a lengthening velocity that does not produce histological muscle injury in mouse extensor digitorum longus muscles (16). The duration of contraction used to produce a lengthening velocity of ~0.2 LJs was based on the following assumptions and data. On the basis of the measured optimal length [L,, for the soleus assumed to coincide with an ankle angle of 90* (21)], a calculated optimal fiber length (L,) of 12.9 mm [assuming that fiber-to-muscle length ratio = 0.51 (23) and L, occurs at L, (16)], and the measured change in muscle length over the 128’ ankle range of motion (10.3 t 0.2 mm), the movement speed of -0.2 L,ls required a 2-s contraction, Tail suspension procedure. Animals in all groups were tail-casted for 10 days as previously described (26). Tail casts of animals in WB and WB-IM gruups were attached to an overhead support but hindlimbs remained weight bearing. Tail casts of animals in NWB and NWBIM groups were attached to an overhead support such that their hind feet were unable to touch either the floor or the sides of the cage. Muscle wet weight und protein content determinations.
Animals were anesthetized with pentobarbital sodium (45 pg/g body wt, Abbott Laboratories, North Chicago, IL) and weighed to the nearest 0.1 g on a triple-beam balance (Ohaus, Florham, NJ). Soleus and extensor digitorum longus muscles were rapidly dissected, trimmed free of fat, tendons, and connective tissue, weighed to the nearest 0.1 mg on a Mettler balance (Hightstown, NJ), frozen with liquid nitrogen-cooled Wollenberger tongs, and stored at -8O*C until assayed for noncollagenous protein content. Soleus muscles were homogenized in 1 ml of 0.9% (wt/vol) NaCl solution, and a O.&ml aliquot of the homogenate was solubilized in 4.5 ml of 0.05 N NaOH. Samples were centrifuged for 10 min at 4,000 g. Triplicate 2O-~1 aliquots of the supernatant were taken for the determination of noncollagenous protein by the method of Bradford (2) by use of a commercial protein assay reagent (Bio-Rad Laboratories, Richmond, CA), with bovine serum albumin as the standard (Sigma Chemical, St. Louis, MO). Adrenals were dissected, trimmed free of fat, and weighed similarly to muscles. Data analysis. Body weights within each group were compared using a repeated-measures analysis of variance (ANOVA) with Fischer least significant difference (LSD) post hoc testing (28). Comparisons of body and
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1896 TABLE
ECCENTRIC
EXERCISE
ATTENUATES
REDUCED-USE
MUSCLE
ATROPHY
1. Animal body and adrenal weights Body Wt, g Adrenal
Group
n
WB NWB WB-IM NWB-IM
5 5 10 10
Day
277k8 275k5
0
Day
7
270~18 269k7
Day
14
Day
270tlO 28lt8 269t8 266+5t
24
Wt, mg
Day 24
27428 259+6$ 266k6-t 262+4t
Adrenal
WtlBody
Wt,
wk
6821 68t2 74t2 75t2*
0.248t0.028 0.2631tO.029 0.28OkO.030 0.288*0.027*
Values are means + SE; n, no. of animals. WB, weight bearing; NWB, non-weight bearing; WB-IM, weight bearing with intramuscular electrodes; NWB-IM, non-weight bearing with intramuscular electrodes. * P < 0.05 WB vs. WB-IM, NWB, or NWB-IM [factorial analysis of variance (ANOVA)]. t P < 0.05 day 0 vs. day 7, day 14, or day 24 (repeated-measures ANOVA). $ P < 0.05 NWB day 14 vs. NWB day 24 (repeated-measures ANOVA).
adrenal weights, muscle wet weights, and muscle noncollagenous protein contents and concentrations between groups were performed using a factorial ANOVA with Fischer LSD post hoc testing. No exercise muscles of all groups and exercise muscles of WB-IM and NWB-IM groups were considered as six equal treatments. Significant differences were at P < 0.05.
weights might be associated with stress because of surgery and/or the repeated administration of general anesthesia before exercise. In accord with previous studies, non-weight bearing alone altered neither adrenal wet weights nor adrenal-to-body weight ratios (9,11,12,26). Effect of non-weight bearing on muscle wet weight and noncollagenous protein content and concentration. Non-
weight bearing significantly reduced soleus muscle wet weight (28-31%) and noncollagenous protein content (30-31%; Table 2). Equivalent losses of wet-weight and Effects of non-weight bearing andlor eccentric exercise protein content produced similar protein concentrations training on body and adrenal weights. Slight (3-a%), but compared with weight-bearing sosignificant, reductions in body weight occurred within all in non-weight-bearing lei. These responses of soleus muscle are similar to pretreatment groups (NWB, WB-IM, and NWB-IM) during the final 10 days of the &k-day experiment (Table 1). vious studies of 7-10 days of non-weight bearing (5,6,11, However, on both day 14 and day 24, body weights were 12, 18). Also in agreement with earlier observations, extensor digitorum longus muscle wet weights were similar similar between groups. Although these results reflect in weight-bearing and non-weight-bearing animals (data the overall well-being of the animals used in the study, adrenal wet weights were increased in WB-IM (9%, NS) not shown; 13, 18). Because the exten~+m &$torum hmuscle not affected directly by nonand NWB-IM (lo%, P < 0.05) groups. This finding is gus is a hindlimb similar to previous reports of adrenal hypertrophy in re- weight bearing, this finding suggests that atrophy of the sponse to exercise during non-weight bearing (9, 26), in soleus muscle results from a direct effect of non-weight bearing and not from some systemic alteration. which this response was attributed to increased animal Analysis of eccentric exercise training effects during handling and exercise stress. Because in this study WBIM and NWB-IM animals were anesthetized during ex- weight bearing or non-weight bearing. To evaluate the efercise, adrenal hypertrophy may have been produced by feet of eccentric exercise training during non-weight the stress associated with post-eccentric exercise musbearing, this investigation utilized both internal controls cle soreness (8). Alternatively, increased adrenal wet (comparisons between contralateral muscles, exercise vs.
RESULTS
AND DISCUSSION
Start
Finish
/ Soleus muscle Soleus muscle lengthened during stimulation
---\ 0,
‘t, ‘l, ‘\, \
$
+ : ,. f ’ \\ \\ \c----d \\
FIG. 2. Model of involuntary eccentric exercise. Anesthetized rats were supported on a platform with their feet secured to a plate on a pulley-bar apparatus. Eccentric contractions of right soleus muscle were accomplished by manually advancing right ankle through its full range of motion (plantar flexion at 162 t 1” to dorsiflexion at 34 t 1”) during electrical stimulation. Left soleus muscle, into which electrodes were implanted, served as an internal control. Left soleus received neither electrical stimulation nor manual movement of ankle. For illustrative purposes, ankle range of motion is not depicted to scale.
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ECCENTRIC TABLE
EXERCISE
ATTENUATES
2. Soleus muscle wet weight and noncollagenous
REDUCED-USE
protein
Group
n
WB NWB WB-IM NWB-IM
10* 10*
Exercise
10
137.4+5.4$
10
112.6k6.33:
No exercise 121.6t3.7 84.1+3.5-f 120.5t5.2 86.8+3.7-t
1897
ATROPHY
content and concentration Noncollagenous Content,
Wet Wt, mg
MUSCLE
Exercise
Protein mg No exercise
Noncollagenous Protein Concn, % Exercise
17.6kO.6
12.4+0.4t 16.ML8 12.7+0.6$
15.4+0.6f
1 l&0.5$
10.6+0.5t
11.5t0.5
No exercise 14.5t0.3 14.9t0.4 12.9+0.4t 12.2*0.3tg
Values are means t SE; n, no. of muscles. Noncollagenous protein concentration = (mg noncollagenous protein/mg muscle wet wt) X 100. * Number of muscles = 2 muscles from each of 5 animals per group. t P < 0.05 No Exercise of WB vs, NWB, WB-IM, and NWB-IM (factorial ANOVA). $ P < 0.05 Exercise vs. No Exercise in same -group-. (factorial ANOVA). Q P < 0.05 No Exercise muscles of NWB vs. NWB-IM (factorial ANOVA).
no exercise, of WB-IM or NWB-IM groups) and external controls (comparisons between no exercise muscles of all groups). Although internal controls serve to minimize interanimal variability, external controls are necessary to evaluate the indirect effects of various experimental treatments associated with tail suspension and eccentric exercise training (e.g., tail casting, anesthesia, surgery, chronic electrode implantation, and animal handling). The presence of intramuscular electrodes decreased soleus muscle protein content in weight-bearing (12%) and non-weight-bearing (14%) hindlimbs similarly (Table 2). These results suggest that appropriate comparisons for eccentric exercise effects are between contralateral soleus muscles (exercise vs. no exercise) with intramuscular electrodes. In addition, non-weight bearing decreased soleus wet weight and protein content (28-31%), regardless of whether the comparison was made within animals that did or did not have bilateral intramuscular electrodes. These results suggest that the presence of intramuscular electrodes did not alter soleus muscle atrophy during non-weight bearing. Collectively, these results support comparisons using internal controls by demonstrating that the indirect effects of various experimental procedures neither alter nor are altered by the soleus muscle response to non-weight bearing. Thus the discussion that follows is based on comparisons between contralateral muscles (exercise vs. no exercise) of WB-IM and NWB-IM groups. Effects of eccentric exercise training on muscle wet weight and noncollagenous protein content and concentrution. Noncollagenous protein concentration was not altered by eccentric exercise training in non-weight-bearing hindlimbs (Table 2). Eccentric exercise training during non-weight bearing produced significantly greater wet weights (30%) and noncollagenous protein contents (20%) in NWB-IM exercise muscles than in contralatera1 no exercise muscles. However, soleus wet weights and noncollagenous protein contents of NWB-IM exercise muscles remained 7 and 18% lower, respectively, than WB-IM no exercise muscles. Thus these data support an attenuation but not prevention of soleus muscle atrophy by the eccentric exercise training protocol employed. In addition, only 8% of the increase in muscle wet weight can be accounted for by increased noncollagenous protein content (i.e., 2.1-mg increase of noncollagenous protein/25&mg increase of wet weight), compared with control muscle noncollagenous protein concentrations of X&-15%. These results suggest that muscle edema and/or increased collagenous protein content contributed to the
increased muscle wet weights in NWB-IM exercise muscles. Similarly, soleus muscle “spared” from non-weight bearing atrophy by the ladder-climbing countermeasure (primarily concentric exercise) had a noncollagenous protein concentration of 13% compared with 17% for controls (12). Although alternate days of eccentric exercise training did not completely prevent soleus muscle atrophy during 10 days of non-weight bearing, it is noteworthy that eccentric exercise during only 0.035% of the total non-weight-bearing time period attenuated 44% of the loss of soleus muscle noncollagenous protein content (Table 3). Eccentric exercise training significantly increased (14%) soleus wet weight in the WB-IM group (Table 2; exercise vs. no exercise muscles). However, because soleus noncollagenous protein contents were similar in these muscles, noncollagenous protein concentration was slightly reduced (8%; P < 0.05). These results imply that the increase of soleus wet weight resulted primarily from muscle edema and/or the accumulation of collagenous protein. A larger amount of scar tissue surrounding electrodes in exercised muscles was subjectively noted and supports a role for connective tissue accumulation (Kirby, unpublished observation). This increased scar tissue in exercised muscles was possibly due to the repeated delivery of supramaximal electrical stimuli (3,20)* The finding that eccentric exercise training did not alter extensor digitorum longus muscle wet weights during either weight bearing or non-weight bearing (data not shown) was expected, because the extensor digitorum longus is not actively recruited during eccentric training of the soleus. This result demonstrates that eccentric exercise training did not produce either changes within the exercised hindlimb or systemic alterations that could account for the effects of eccentric exercise on soleus muscle. Efficacy of eccentric exercise training us a countermeasure to soleus muscle atrophy during non-weight bearing. Because the soleus muscle exhibits the greatest degree of atrophy during non-weight bearing (26), it has been the focus of most exercise countermeasure studies. Table 3 summarizes the effects of various exercise countermeasures to soleus muscle atrophy during non-weight bearing tested to date. Virtually all exercise modalities attenuate soleus muscle atrophy. However, compared with other forms of exercise, resistance training is at least equal in its effectiveness (i.e., percentage of atrophy prevented) and superior in its efficiency (i.e., percentage of
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1898 TABLE
ECCENTRIC
3. Summary
EXERCISE
ATTENUATES
REDUCED-USE
MUSCLE
ofvarious exercisecountermea-suresto soleus muscle atrophy
ATROPHY
during non-weight bearing
% Atrophy Treatment
Stationary ground support 2 h/day x 4 wk 4 h/day X 4 wk 4 X 10 min/day X 1 wk 2 h/day x 1 wk 4 X 15 miniday X 1 wk Treadmill running 1.5 h/day last 4 wk of 8 wk (20 m/min, 30% grade) 1.5 h/day X 4 wk (20 m/min, 30% grade) 1.5 h/day X 4 wk (20 m/min, 30% grade) 1.5 h/day X 4 wk (20 m/min, 30% grade) 4 X 10 miniday X 1 wk (5 m/min, 19’ grade) 4 X 10 min/day X 1 wk (12 m/min, 19O grade) 4 X 10 min/day X 1 wk (20 m/min, 19” grade) 4 X 10 min/day X 1 wk (20 m/min, 19’ grade) Ten daily contractions upon impact from a fall of 58 cm X 4 wk Continuous hindlimb support Ladder climbing (primarily concentric resistance) 4 X 10 reps/day X 1 wk (50% load, 1 m at 85O grade) 4 X 8 reps/day X 1 wk (75% load, 1 m at 85’ grade) 2 X 10 reps/day X 1 wk (75% load, 1 m at 85’ grade) Eccentric exercise 4 X 6 reps/day X days (alternate days of maximal electrically stimulated eccentric contractions) Centrifugation 2 h/day x 1 wk (1.5 G) 4 X 15 min/day X wk (1.2 G)
Relative wet wt
MinIDay
120 240 40 120
31 42 70”
36
Prevented
Absolute wet wt
32 35
Protein content
33 493:
ND 43
ND w
Ref.
26 26 12
60
118
61
125-t
6 5
90 90 90 90 40 40 40 40
R. KIRBY,
MIRELLE
of Physiology and Cell Biology,
J. RYAN,
AND
FRANK
W. BOOTH
University of Texas Medical School, Houston, Texas 77225
eccentric resistance exercise (lengthening contractions) during non-weight bearing to be determined. Normal locomotion, posture, and activity incorporate eccentric contractions (14, 17, 29). Because loaded eccentric contracbeen tested, none has completely prevented muscle atrophy tions are absent during non-weight bearing (15), we hyduring non-weight bearing. Becauseloaded eccentric contracpothesized that the return of eccentric contractions tions occur during normal daily activity but are absent during during non-weight bearing might provide an effective non-weight bearing, this investigation tested whether eccentric countermeasure to muscle atrophy. The purpose of this resistancetraining could prevent soleusmuscleatrophy during non-weight bearing. Adult female rats were randomly assigned study was to determine whether an eccentric exercise to either weight bearing ztrintramuscular electrodes or non- training bout every other day during 10 days of nonweight bearing t intramuscular electrodesgroups. Electrically weight bearing would prevent soleus muscle atrophy. stimulated maximal eccentric contractions (4 sets of 6 repeti- This frequency of eccentric exercise training was setions at ~0.2 fiber lengths/s, 128” range of motion) were per- lected because a single bout of 24 eccentric contractions formed on anesthetized animals at 48-h intervals during the elevated total mixed protein synthesis rates for up to 41 h IO-day experiment. Non-weight bearing significantly reduced postexercise in normal rat tibialis anterior muscle (31). KIRBY, CHRISTOPHER R., MIRELL~ J. RYAN, ANDFRANKW. Eccentric exercise training as a countermeasure to nonweight-bearing soleus muscle atrophy. J. Appl. Physiol. 73(5): 1894-1899, 1992.-Although various exercise paradigms have
BOOTH.
soleusmusclewet weight (28-31%) and noncollagenousprotein content (30-31%) compared with controls. Eccentric exercise training during non-weight bearing attenuated but did not prevent the lossof soleusmusclewet weight and noncollagenous protein by 77 and 44%, respectively. The potential of eccentric exercise training as an effective and highly efficient countermeasureto non-weight-bearing atrophy is demonstrated in the 44% attenuation of soleusmusclenoncollagenousprotein loss by eccentric exercise during only 0.035% of the total nonweight-bearing time period. reduced use; muscleprotein content; hypokinesia; hypodynamia; musclewasting
SKELETALMUSCLEATROPHY resulting from non-weight bearing is one of the principal alterations associated with spaceflight deconditioning (4). The loss of muscle protein content and strength may result in reduced physical work capacity during long-duration spaceflight and/or difficulty in readaptation to the 1-G environment of Earth after spaceflight (4). Therefore several countermeasures have been tested for their ability to reverse or prevent muscle atrophy during non-weight bearing. The countermeasure most often employed has been exercise (5,6,9-12,22,24,26). However, no exercise countermeasure has completely prevented muscle atrophy during non-weight bearing. Of the exercise protocols utilized, resistance training is at least equal in its effectiveness (i.e., percentage of atrophy prevented) and superior in its efficiency (i.e., percentage of atrophy prevented/exercise time) as a countermeasure for attenuating muscle mass and protein content losses (1; see Table 3). To date, only the adequacy of concentric resistance exercise (shortening contractions) has been tested, leaving the efficacy of 1894
016L7567/92
$2.00
METHODS Animals. Thirty female retired-breeder rats (Harlan Sprague Dawley, 275 g body wt, 8-10 mo of age) were randomly assigned to either weight-bearing (WB), weight-bearing with bilateral intramuscular electrodes (WB-IM), non-weight-bearing (NWB), or non-weightbearing with bilateral intramuscular electrodes (NWBIM) groups. An additional 13 animals from this shipment were used for pilot studies. The contribution of growth inhibition of the soleus muscle during non-weight bearing was diminished by use of adult female rats. Animals had ad libitum access to standard rat chow and water throughout the experiment. All procedures using animals were approved by the Animal Welfare Committee of the University of Texas Medical School, Houston and conformed to the American Physiological Society “Guiding Principles for Research Involving Animals.” Experimental design. WB-IM and NWB-IM groups underwent surgery for bilateral electrode implantation into soleus muscles on day 0 of the experiment (Fig. 1). After a 7-day recovery (day 7), soleus muscles from the right leg of animals in WB-IM and NWB-IM underwent a single bout of eccentric exercise as a prophylactic measure to reduce eccentric exercise-induced muscle damage in the subsequent eccentric training program (8). On day 14 all animals began a lo-day period of tail casting with (WB and WB-IM groups) or without (NWB and NWBIM groups) hindlimb weight bearing. On day 14 WB-IM and NWB-IM groups also began eccentric exercise training of the right soleus muscle. The frequency of eccentric exercise training (every other day) was based on a previous report in which total mixed protein synthesis rates
Copyright 0 1992 the American Physiological Society
Downloaded from www.physiology.org/journal/jappl by ${individualUser.givenNames} ${individualUser.surname} (129.237.035.237) on January 19, 2019.
ECCENTRIC Day:
Groups:
0
WB-IM NWB-IM
7
EXERCISE
14
16
18
ATTENUATES 20
Etc. Ex.
Ecc, Ex.
Tai I-cast
Etc. Ex.
WB-IM NWB-IM
WB-fM NWB-IM
WB WB-IM NWB NWB-IM
WB-IM NWB-IM
22
24
Kill all groups
FIG. 1. Experimental time line. Surgery, implantation of intramuscular electrodes; Ecc Ex, eccentric ex&&se training; Tail cast, tailcasted + non-weight bearing; WB, weight bearing; NWB, non-weight bearing; WB-IM, weight bearing with intramuscular electrodes; NWBIM, non-weight bearing with intramuscular electrodes.
were increased between 36 and 41 h after a single bout of 24 eccentric contractions in rat tibialis anterior muscle (31). Eccentric exercise training every other day continued through day 22, and all animals were killed on day 24 of the experiment. Surgical procedures. Animals in the WB-IM and NWB-IM groups were anesthetized for surgery with 60 pg ketamine, 2 pg xylazine, and 4 pg acepromazine per g of body weight (Aveco, Fort Dodge, IA). A small (3-cm) incision was made in the skin of the lateral aspect of each lower leg. A small opening (0.5 cm) was then made, parallel to the muscle fibers, in the biceps femoris. The distal and proximal ends of the soleus were then retracted with a curved forcep. Teflon-coated, braided, stainless steel wire electrodes (0.012-in. diam, Cooner Wire, Chatsworth, CA), to which a 30-gauge needle had been attached, were then passed through the soleus muscle and anchored by a piece of 6-O suture tied around each protruding end. Incisions in the biceps fern .oris and skin were then closed with 6 -0 suture and 4-O suture plus wound clips, respectively. Electrodes in the right soleus originated from a two-contact lamp socket (Augat, Attleboro, MA), which was mounted to the skull with stainless steel screws (O-80 X 3/16 in.) and dental cement (methyl methacrylate, Sybron, Romulus, MI). Electrodes in the left soleus extended from the muscle but were cut just below the skin. In addition, a hole was drilled, and blunted 27gauge needles were implanted into both calcanei for the purpose of securing the right foot to the pulley-bar apparatus during eccentric exercise training and as a sham treatment for the left foot. Animals received prophylactic antibiotic injections (gentamicin, 50 pg/g body wt, Smith & Nephew, Franklin Park, IL) on the day of and for 2 days after surgery. All surgical procedures were performed under aseptic conditions. Animals recovered from surgery for 1 wk, at which time body weights (Table 1) and food consu .mption (data not shown) were similar to control animals. Eccentric exercise training protocol. Eccentric exercise training was performed using modifications to a model of involuntary resistance exercise in rats (30,31). Anesthetized rats (80 pg ketamine and 8 ,ug acepromazine/g body wt; Aveco) were supported on a platform with their feet secured to a plate on a pulley-bar apparatus (Fig. 2). Eccentric contractions of the right soleus muscle were accomplished by manual advance of the right ankle through its full range of motion (from plantar flexion at 162 t 1” to dorsiflexion at 34 t lo) during electrical stim-
REDUCED-USE
MUSCLE
ATROPHY
1895
ulation. Right soleus muscles of WB-IM and NWB-IM groups, which underwent eccentric exercise training, are designated “exercise” (Table 2). Eccentric exercise training of the right soleus muscles utilized an electrical stimulation paradigm that produced maximal isometric muscle force in rats (n = 3) not used in the experiment (train rate, 0.2 trains/s; stimulation rate, 60 Hz; pulse duration, 0.5 ms; and voltage, 16 V). On the assigned days, rats in the WB-IM and NWB-IM groups performed four sets of six repetitions. The muscle was stimulated supramaximally for 2.0 s every 5 s, with 2-min rest periods between sets. Left soleus muscles of WB-IM and NWB-IM groups, designated “no exercise” (Table 2), received neither electrical stimulation nor manual movement of the ankle. Eccentric contractions were performed at ~0.2 fiber lengths per second (L,ls), a lengthening velocity that does not produce histological muscle injury in mouse extensor digitorum longus muscles (16). The duration of contraction used to produce a lengthening velocity of ~0.2 LJs was based on the following assumptions and data. On the basis of the measured optimal length [L,, for the soleus assumed to coincide with an ankle angle of 90* (21)], a calculated optimal fiber length (L,) of 12.9 mm [assuming that fiber-to-muscle length ratio = 0.51 (23) and L, occurs at L, (16)], and the measured change in muscle length over the 128’ ankle range of motion (10.3 t 0.2 mm), the movement speed of -0.2 L,ls required a 2-s contraction, Tail suspension procedure. Animals in all groups were tail-casted for 10 days as previously described (26). Tail casts of animals in WB and WB-IM gruups were attached to an overhead support but hindlimbs remained weight bearing. Tail casts of animals in NWB and NWBIM groups were attached to an overhead support such that their hind feet were unable to touch either the floor or the sides of the cage. Muscle wet weight und protein content determinations.
Animals were anesthetized with pentobarbital sodium (45 pg/g body wt, Abbott Laboratories, North Chicago, IL) and weighed to the nearest 0.1 g on a triple-beam balance (Ohaus, Florham, NJ). Soleus and extensor digitorum longus muscles were rapidly dissected, trimmed free of fat, tendons, and connective tissue, weighed to the nearest 0.1 mg on a Mettler balance (Hightstown, NJ), frozen with liquid nitrogen-cooled Wollenberger tongs, and stored at -8O*C until assayed for noncollagenous protein content. Soleus muscles were homogenized in 1 ml of 0.9% (wt/vol) NaCl solution, and a O.&ml aliquot of the homogenate was solubilized in 4.5 ml of 0.05 N NaOH. Samples were centrifuged for 10 min at 4,000 g. Triplicate 2O-~1 aliquots of the supernatant were taken for the determination of noncollagenous protein by the method of Bradford (2) by use of a commercial protein assay reagent (Bio-Rad Laboratories, Richmond, CA), with bovine serum albumin as the standard (Sigma Chemical, St. Louis, MO). Adrenals were dissected, trimmed free of fat, and weighed similarly to muscles. Data analysis. Body weights within each group were compared using a repeated-measures analysis of variance (ANOVA) with Fischer least significant difference (LSD) post hoc testing (28). Comparisons of body and
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1896 TABLE
ECCENTRIC
EXERCISE
ATTENUATES
REDUCED-USE
MUSCLE
ATROPHY
1. Animal body and adrenal weights Body Wt, g Adrenal
Group
n
WB NWB WB-IM NWB-IM
5 5 10 10
Day
277k8 275k5
0
Day
7
270~18 269k7
Day
14
Day
270tlO 28lt8 269t8 266+5t
24
Wt, mg
Day 24
27428 259+6$ 266k6-t 262+4t
Adrenal
WtlBody
Wt,
wk
6821 68t2 74t2 75t2*
0.248t0.028 0.2631tO.029 0.28OkO.030 0.288*0.027*
Values are means + SE; n, no. of animals. WB, weight bearing; NWB, non-weight bearing; WB-IM, weight bearing with intramuscular electrodes; NWB-IM, non-weight bearing with intramuscular electrodes. * P < 0.05 WB vs. WB-IM, NWB, or NWB-IM [factorial analysis of variance (ANOVA)]. t P < 0.05 day 0 vs. day 7, day 14, or day 24 (repeated-measures ANOVA). $ P < 0.05 NWB day 14 vs. NWB day 24 (repeated-measures ANOVA).
adrenal weights, muscle wet weights, and muscle noncollagenous protein contents and concentrations between groups were performed using a factorial ANOVA with Fischer LSD post hoc testing. No exercise muscles of all groups and exercise muscles of WB-IM and NWB-IM groups were considered as six equal treatments. Significant differences were at P < 0.05.
weights might be associated with stress because of surgery and/or the repeated administration of general anesthesia before exercise. In accord with previous studies, non-weight bearing alone altered neither adrenal wet weights nor adrenal-to-body weight ratios (9,11,12,26). Effect of non-weight bearing on muscle wet weight and noncollagenous protein content and concentration. Non-
weight bearing significantly reduced soleus muscle wet weight (28-31%) and noncollagenous protein content (30-31%; Table 2). Equivalent losses of wet-weight and Effects of non-weight bearing andlor eccentric exercise protein content produced similar protein concentrations training on body and adrenal weights. Slight (3-a%), but compared with weight-bearing sosignificant, reductions in body weight occurred within all in non-weight-bearing lei. These responses of soleus muscle are similar to pretreatment groups (NWB, WB-IM, and NWB-IM) during the final 10 days of the &k-day experiment (Table 1). vious studies of 7-10 days of non-weight bearing (5,6,11, However, on both day 14 and day 24, body weights were 12, 18). Also in agreement with earlier observations, extensor digitorum longus muscle wet weights were similar similar between groups. Although these results reflect in weight-bearing and non-weight-bearing animals (data the overall well-being of the animals used in the study, adrenal wet weights were increased in WB-IM (9%, NS) not shown; 13, 18). Because the exten~+m &$torum hmuscle not affected directly by nonand NWB-IM (lo%, P < 0.05) groups. This finding is gus is a hindlimb similar to previous reports of adrenal hypertrophy in re- weight bearing, this finding suggests that atrophy of the sponse to exercise during non-weight bearing (9, 26), in soleus muscle results from a direct effect of non-weight bearing and not from some systemic alteration. which this response was attributed to increased animal Analysis of eccentric exercise training effects during handling and exercise stress. Because in this study WBIM and NWB-IM animals were anesthetized during ex- weight bearing or non-weight bearing. To evaluate the efercise, adrenal hypertrophy may have been produced by feet of eccentric exercise training during non-weight the stress associated with post-eccentric exercise musbearing, this investigation utilized both internal controls cle soreness (8). Alternatively, increased adrenal wet (comparisons between contralateral muscles, exercise vs.
RESULTS
AND DISCUSSION
Start
Finish
/ Soleus muscle Soleus muscle lengthened during stimulation
---\ 0,
‘t, ‘l, ‘\, \
$
+ : ,. f ’ \\ \\ \c----d \\
FIG. 2. Model of involuntary eccentric exercise. Anesthetized rats were supported on a platform with their feet secured to a plate on a pulley-bar apparatus. Eccentric contractions of right soleus muscle were accomplished by manually advancing right ankle through its full range of motion (plantar flexion at 162 t 1” to dorsiflexion at 34 t 1”) during electrical stimulation. Left soleus muscle, into which electrodes were implanted, served as an internal control. Left soleus received neither electrical stimulation nor manual movement of ankle. For illustrative purposes, ankle range of motion is not depicted to scale.
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ECCENTRIC TABLE
EXERCISE
ATTENUATES
2. Soleus muscle wet weight and noncollagenous
REDUCED-USE
protein
Group
n
WB NWB WB-IM NWB-IM
10* 10*
Exercise
10
137.4+5.4$
10
112.6k6.33:
No exercise 121.6t3.7 84.1+3.5-f 120.5t5.2 86.8+3.7-t
1897
ATROPHY
content and concentration Noncollagenous Content,
Wet Wt, mg
MUSCLE
Exercise
Protein mg No exercise
Noncollagenous Protein Concn, % Exercise
17.6kO.6
12.4+0.4t 16.ML8 12.7+0.6$
15.4+0.6f
1 l&0.5$
10.6+0.5t
11.5t0.5
No exercise 14.5t0.3 14.9t0.4 12.9+0.4t 12.2*0.3tg
Values are means t SE; n, no. of muscles. Noncollagenous protein concentration = (mg noncollagenous protein/mg muscle wet wt) X 100. * Number of muscles = 2 muscles from each of 5 animals per group. t P < 0.05 No Exercise of WB vs, NWB, WB-IM, and NWB-IM (factorial ANOVA). $ P < 0.05 Exercise vs. No Exercise in same -group-. (factorial ANOVA). Q P < 0.05 No Exercise muscles of NWB vs. NWB-IM (factorial ANOVA).
no exercise, of WB-IM or NWB-IM groups) and external controls (comparisons between no exercise muscles of all groups). Although internal controls serve to minimize interanimal variability, external controls are necessary to evaluate the indirect effects of various experimental treatments associated with tail suspension and eccentric exercise training (e.g., tail casting, anesthesia, surgery, chronic electrode implantation, and animal handling). The presence of intramuscular electrodes decreased soleus muscle protein content in weight-bearing (12%) and non-weight-bearing (14%) hindlimbs similarly (Table 2). These results suggest that appropriate comparisons for eccentric exercise effects are between contralateral soleus muscles (exercise vs. no exercise) with intramuscular electrodes. In addition, non-weight bearing decreased soleus wet weight and protein content (28-31%), regardless of whether the comparison was made within animals that did or did not have bilateral intramuscular electrodes. These results suggest that the presence of intramuscular electrodes did not alter soleus muscle atrophy during non-weight bearing. Collectively, these results support comparisons using internal controls by demonstrating that the indirect effects of various experimental procedures neither alter nor are altered by the soleus muscle response to non-weight bearing. Thus the discussion that follows is based on comparisons between contralateral muscles (exercise vs. no exercise) of WB-IM and NWB-IM groups. Effects of eccentric exercise training on muscle wet weight and noncollagenous protein content and concentrution. Noncollagenous protein concentration was not altered by eccentric exercise training in non-weight-bearing hindlimbs (Table 2). Eccentric exercise training during non-weight bearing produced significantly greater wet weights (30%) and noncollagenous protein contents (20%) in NWB-IM exercise muscles than in contralatera1 no exercise muscles. However, soleus wet weights and noncollagenous protein contents of NWB-IM exercise muscles remained 7 and 18% lower, respectively, than WB-IM no exercise muscles. Thus these data support an attenuation but not prevention of soleus muscle atrophy by the eccentric exercise training protocol employed. In addition, only 8% of the increase in muscle wet weight can be accounted for by increased noncollagenous protein content (i.e., 2.1-mg increase of noncollagenous protein/25&mg increase of wet weight), compared with control muscle noncollagenous protein concentrations of X&-15%. These results suggest that muscle edema and/or increased collagenous protein content contributed to the
increased muscle wet weights in NWB-IM exercise muscles. Similarly, soleus muscle “spared” from non-weight bearing atrophy by the ladder-climbing countermeasure (primarily concentric exercise) had a noncollagenous protein concentration of 13% compared with 17% for controls (12). Although alternate days of eccentric exercise training did not completely prevent soleus muscle atrophy during 10 days of non-weight bearing, it is noteworthy that eccentric exercise during only 0.035% of the total non-weight-bearing time period attenuated 44% of the loss of soleus muscle noncollagenous protein content (Table 3). Eccentric exercise training significantly increased (14%) soleus wet weight in the WB-IM group (Table 2; exercise vs. no exercise muscles). However, because soleus noncollagenous protein contents were similar in these muscles, noncollagenous protein concentration was slightly reduced (8%; P < 0.05). These results imply that the increase of soleus wet weight resulted primarily from muscle edema and/or the accumulation of collagenous protein. A larger amount of scar tissue surrounding electrodes in exercised muscles was subjectively noted and supports a role for connective tissue accumulation (Kirby, unpublished observation). This increased scar tissue in exercised muscles was possibly due to the repeated delivery of supramaximal electrical stimuli (3,20)* The finding that eccentric exercise training did not alter extensor digitorum longus muscle wet weights during either weight bearing or non-weight bearing (data not shown) was expected, because the extensor digitorum longus is not actively recruited during eccentric training of the soleus. This result demonstrates that eccentric exercise training did not produce either changes within the exercised hindlimb or systemic alterations that could account for the effects of eccentric exercise on soleus muscle. Efficacy of eccentric exercise training us a countermeasure to soleus muscle atrophy during non-weight bearing. Because the soleus muscle exhibits the greatest degree of atrophy during non-weight bearing (26), it has been the focus of most exercise countermeasure studies. Table 3 summarizes the effects of various exercise countermeasures to soleus muscle atrophy during non-weight bearing tested to date. Virtually all exercise modalities attenuate soleus muscle atrophy. However, compared with other forms of exercise, resistance training is at least equal in its effectiveness (i.e., percentage of atrophy prevented) and superior in its efficiency (i.e., percentage of
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1898 TABLE
ECCENTRIC
3. Summary
EXERCISE
ATTENUATES
REDUCED-USE
MUSCLE
ofvarious exercisecountermea-suresto soleus muscle atrophy
ATROPHY
during non-weight bearing
% Atrophy Treatment
Stationary ground support 2 h/day x 4 wk 4 h/day X 4 wk 4 X 10 min/day X 1 wk 2 h/day x 1 wk 4 X 15 miniday X 1 wk Treadmill running 1.5 h/day last 4 wk of 8 wk (20 m/min, 30% grade) 1.5 h/day X 4 wk (20 m/min, 30% grade) 1.5 h/day X 4 wk (20 m/min, 30% grade) 1.5 h/day X 4 wk (20 m/min, 30% grade) 4 X 10 miniday X 1 wk (5 m/min, 19’ grade) 4 X 10 min/day X 1 wk (12 m/min, 19O grade) 4 X 10 min/day X 1 wk (20 m/min, 19” grade) 4 X 10 min/day X 1 wk (20 m/min, 19’ grade) Ten daily contractions upon impact from a fall of 58 cm X 4 wk Continuous hindlimb support Ladder climbing (primarily concentric resistance) 4 X 10 reps/day X 1 wk (50% load, 1 m at 85O grade) 4 X 8 reps/day X 1 wk (75% load, 1 m at 85’ grade) 2 X 10 reps/day X 1 wk (75% load, 1 m at 85’ grade) Eccentric exercise 4 X 6 reps/day X days (alternate days of maximal electrically stimulated eccentric contractions) Centrifugation 2 h/day x 1 wk (1.5 G) 4 X 15 min/day X wk (1.2 G)
Relative wet wt
MinIDay
120 240 40 120
31 42 70”
36
Prevented
Absolute wet wt
32 35
Protein content
33 493:
ND 43
ND w
Ref.
26 26 12
60
118
61
125-t
6 5
90 90 90 90 40 40 40 40
