Life Sciences, Vol. Printed in the USA
51, pp. 1691-1698
Pergamon Press
ADAPTATION IN SYNERGISTIC MUSCLES TO SOLEUS AND PLANTARIS MUSCLE REMOVAL IN THE RAT HINDLIMB S.C. Kandarian, J.C. Young, and E.E. Gomez Department of Health Sciences, Boston University, Boston, MA 02215 (Received in final form September 18, 1992)
Summary Although the soleus muscle comprises only 6% of the ankle plantar flexor mass in the rat, a major role in stance and walking has been ascribed to it. The purpose of this study was to determine if removal of the soleus muscle would result in adaptations in the remaining gastrocnemius and plantaris muscles due to the new demands for force production imposed on them during stance or walking. A second purpose was to determine whether the mass or the fiber type of the muscle(s) removed was a more important determinant of compensatory adaptations. Male Sprague-Dawley rats underwent bilateral removal of soleus muscle, plantaris muscle, or both muscles. For comparison, compensatory hypertrophy was induced in soleus and plantaris muscles by gastrocnemius muscle ablation. After forty days, synergist muscles remaining intact were removed. Mass, and oxidative, glycolytic, and contractile enzyme activities were determined. Despite its role in stance and slow walking, removal of the soleus muscle did not elicit a measurable alteration in muscle mass, or in citrate synthase, lactate dehydrogenase, or myofibrillar ATPase activity in gastrocnemius or plantaris muscles. Similarly, removal of the plantaris muscle, or soleus and plantaris muscles, had no effect on the gastrocnemius muscle, suggesting that this muscle was able to easily meet the new demands placed on it. These results suggest that amount of muscle mass removed, rather than fiber type, is the most important stimulus for compensatory hypertrophy. They also suggest that slow-twitch motor units in the gastrocnemius muscle play an important role during stance and locomotion in the intact animal. Of the ankle plantar flexor muscles, the soleus has been thought to have the largest role in maintaining force in stance and walking (1,13,17,19,25). The soleus muscle, which is comprised almost exclusively of slow-twitch (type I) fibers (2), is chronically activated but has a low power output in vivo (17,25). The other plantar flexor muscles, the plantaris and gastrocnemius, are predominantly mixed fast-twitch (type IIa and lib) muscles (2), Please send correspondence to: John C. Young, PhD, College of Human Performance and Development, University of Nevada, Las Vegas, 4505 Maryland Parkway, Las Vegas, NV 89154 0024-3205/92 $5.00 + .00 Copyright © 1992 Pergamon Press Ltd All rights reserved.
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Muscle Response to Synergist Ablation
Vol. 51, No. 21, 1992
intermittently activated with a high power output in vivo (13,25). If the soleus muscle has the primary responsibility for maintaining plantar flexion during stance and walking (17,19,25), removal of the soleus should result in its role as a chronically recruited postural muscle being assumed by the remaining plantar flexor muscles. However, the soleus muscle represents only 6% of the plantar flexor muscle mass, whereas the plantaris muscle (16%) and gastrocnemius muscle (78%) (2,3,21) comprise 94% of the mass. Thus, the slow-twitch motor units in plantaris and gastrocnemius muscles, which together are approximately equal in mass to the soleus muscle (3), may provide sufficient force during standing and walking to act in place of the soleus muscle when it is removed. In light of its relatively high level of activity and role in postural maintenance and walking despite its small contribution to total plantar flexor muscle mass, the purpose of this study was to determine the morphological and/or metabolic consequences of soleus muscle ablation on the remaining synergist muscles. In addition, we determined whether adaptations in synergist muscles to ablation were a function of muscle fiber type or of the mass of muscle removed by studying the consequences of plantaris muscle removal, and of plantaris and soleus muscle removal. The parameters selected to assess metabolic, contractile, and/or morphologic alterations are known markers of adaptation to changes in load and recruitment pattern, as seen with run training or increased functional load (4,5,6,8,20). Methods Animals, surgery, and design. Male Sprague-Dawley rats (205 + 11 g) were obtained from Charles River Breeding Laboratories (Kingston, NY). Rats were housed in metal cages on a 12:12 hr light/dark cycle. Food (Purina Lab Chow 5008) and water were provide ad-libitum. Rats were randomly divided into four groups. The soleus muscle was removed in the first group (n = 8), the plantaris muscle in the second group (n= 8), and both the soleus and plantaris muscles in the third group (n= 8). The fourth group served as controls (n = 10). All surgery for a given group was performed on the same day. Rats were anesthetized with pentobarbital sodium (5mg/100g body wt, ip). Bilateral ablations were performed. The lateral aspect of each limb was shaved and a longitudinal incision was made through the skin and fascia along the lateral aspect of the tibia. To ablate soleus muscle, the proximal and distal tendons were carefully isolated using blunt dissection and severed. The muscle was then freed from its neurovascular connections and removed from the animal. To ablate plantaris muscle, the distal tendon was isolated and severed. The distal 50% of the muscle was then removed. The partial removal of the plantaris muscle was necessary in order to maintain patency of the neurovasculature of the muscles left intact. The fascia and skin were sutured, distal to proximal, with 5-0 silk. The duration of time for the entire procedure for both hindlimbs was 40 min. Animals were returned to their cages and were observed walking immediately after recovery from anesthesia (=3 hr). Forty days following ablation, control and experimental rats were anesthetized as before, and the remaining plantar flexor muscles were excised. Immediately after removal, muscles were trimmed of visible fat and tendon projections, washed in saline, blotted, and weighed on a Mettler analytical balance. Muscles were frozen in liquid nitrogen and stored at -70°C until enzyme assays were performed.
Vol. 51, NO. 21, 1992
Muscle Response to Synergist Ablation
1693
For comparison purposes, a secondary study was performed in which mass of intact synergist muscles was determined 40 days following ablation of the gastrocnemius muscle only, or the gastrocnemius muscle and the soleus muscle or plantaris muscle. Gastrocnemius muscle ablation has been commonly used to induce compensatory hypertrophy in plantaris and/or soleus muscles. The surgeries were performed as described previously (16).
Enzyme assays. For the determination of citrate synthase (CS) and lactate dehydrogenase (LDH) activity, muscle samples were homogenized in 175 mM KC1, containing 10 mM glutathione, 2 mM EDTA (pH 7.4). CS activity was determined by the method of Srere (22) using 5,5-dithiobis-(2-nitrobenzoate). Homogenates were frozen and thawed four times to disrupt mitochondria, and centrifuged at 700 x g. Enzyme activity was measured spectrophotometrically on the 700 x g supernatant. LDH activity was determined on whole muscle homogenates by the method of Bergmeyer (7). Enzyme activity was measured by the rate of NADH oxidation. The amount of enzyme added was adjusted until the reaction was linear for four minutes. Skeletal muscle myofibrils were isolated for the measurement of Mg2÷-activated myofibrillar ATPase activity as described by Baldwin et al. (4). Protein concentration of the total myofibrillar suspension was determined by the method of Lowry (18), and adjusted to 6 mg/ml. ATPase activity was determined by measuring the release of inorganic phosphate (9). Statistical analysis. Statistical differences between means for each variable were determined by one-way ANOVA. Data are reported as mean _+ S.E. Results Body weight and muscle mass. Body weight did not differ between control (447 + 7 g) and experimental groups (451 _+5, 438 _+4, 447 + 6 g for soleus, plantaris, and soleus + plantaris muscle ablation, respectively) at the time of sacrifice (40 days post ablation). Mass of intact soleus, plantaris, and gastrocnemius muscles did not differ between any experimental groups and the control group 40 days after synergist removal (Table 1 and 2). Hypertrophy of soleus and/or plantaris muscles occurred only when the gastrocnemius muscle was ablated (Figure 1).
Enzyme activity. Citrate synthase activity (~mol/g wet wt/min) in intact plantaris or soleus muscles did not differ from control muscles after ablation of soleus or plantaris muscles, respectively (Table 1). The activity of citrate synthase in red and white gastrocnemius following ablation of soleus, plantaris, or both muscles was not different from that in control gastrocnemius muscles (Table 2). LDH (/~mol/g wet wt/min) activity did not differ from control levels in remaining plantaris, soleus, or gastrocnemius muscles following soleus and/or plantaris muscle ablation (Table 1 and 2). MgZ+-activated myofibrillar ATPase (/~mol Pi/mg myofibrillar protein/min) did not differ between control and experimental animals for any muscle studied (Table 1 and 2). Changes in CS, LDH, and myofibrillar ATPase activity in soleus and plantaris muscles following gastrocnemius muscle ablation were commensurate with previously reported results (4,5,6,21,24) (data not shown). There were no differences in myofibrillar yield between groups (mg myofibril/g wet wt); the values for control muscles were as follows: soleus = 107 + 3; plantaris = 106 + 2; gastrocnemius medial head red = 113 _+4, white = 123 _+5; gastrocnemius lateral head red = 103 + 3, white = 117 +_4.
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Muscle Response to Synergist Ablation
Vol.
51, No. 21, 1992
TABLE 1 Plantaris and Soleus Muscle Response to Soleus and Plantaris Ablation, Respectively
Mass
Plantaris Control Soleus Ablation Soleus Control Plantaris Ablation
(mg)
ATPase (/~mol Pi/mg prot./min)
Citrate Synthase (~mol/g/min)
LDH
468 _+ 10
0.497 _+ 0.017
25 + 1
438 + 21
497 + 15
0.530 _+ 0.025
27 _+ 3
420 _+ 31
177 _+ 10
0.149 + 0.014
23 + 3
109 +
12
198_+ 5
0.157_+0.008
22_+3
98+
7
(~mol/g/min)
Values are m e a n _+ SE for n = 10 for control and n= 8 for experimental groups. *Significantly different from control, P
51, pp. 1691-1698
Pergamon Press
ADAPTATION IN SYNERGISTIC MUSCLES TO SOLEUS AND PLANTARIS MUSCLE REMOVAL IN THE RAT HINDLIMB S.C. Kandarian, J.C. Young, and E.E. Gomez Department of Health Sciences, Boston University, Boston, MA 02215 (Received in final form September 18, 1992)
Summary Although the soleus muscle comprises only 6% of the ankle plantar flexor mass in the rat, a major role in stance and walking has been ascribed to it. The purpose of this study was to determine if removal of the soleus muscle would result in adaptations in the remaining gastrocnemius and plantaris muscles due to the new demands for force production imposed on them during stance or walking. A second purpose was to determine whether the mass or the fiber type of the muscle(s) removed was a more important determinant of compensatory adaptations. Male Sprague-Dawley rats underwent bilateral removal of soleus muscle, plantaris muscle, or both muscles. For comparison, compensatory hypertrophy was induced in soleus and plantaris muscles by gastrocnemius muscle ablation. After forty days, synergist muscles remaining intact were removed. Mass, and oxidative, glycolytic, and contractile enzyme activities were determined. Despite its role in stance and slow walking, removal of the soleus muscle did not elicit a measurable alteration in muscle mass, or in citrate synthase, lactate dehydrogenase, or myofibrillar ATPase activity in gastrocnemius or plantaris muscles. Similarly, removal of the plantaris muscle, or soleus and plantaris muscles, had no effect on the gastrocnemius muscle, suggesting that this muscle was able to easily meet the new demands placed on it. These results suggest that amount of muscle mass removed, rather than fiber type, is the most important stimulus for compensatory hypertrophy. They also suggest that slow-twitch motor units in the gastrocnemius muscle play an important role during stance and locomotion in the intact animal. Of the ankle plantar flexor muscles, the soleus has been thought to have the largest role in maintaining force in stance and walking (1,13,17,19,25). The soleus muscle, which is comprised almost exclusively of slow-twitch (type I) fibers (2), is chronically activated but has a low power output in vivo (17,25). The other plantar flexor muscles, the plantaris and gastrocnemius, are predominantly mixed fast-twitch (type IIa and lib) muscles (2), Please send correspondence to: John C. Young, PhD, College of Human Performance and Development, University of Nevada, Las Vegas, 4505 Maryland Parkway, Las Vegas, NV 89154 0024-3205/92 $5.00 + .00 Copyright © 1992 Pergamon Press Ltd All rights reserved.
1692
Muscle Response to Synergist Ablation
Vol. 51, No. 21, 1992
intermittently activated with a high power output in vivo (13,25). If the soleus muscle has the primary responsibility for maintaining plantar flexion during stance and walking (17,19,25), removal of the soleus should result in its role as a chronically recruited postural muscle being assumed by the remaining plantar flexor muscles. However, the soleus muscle represents only 6% of the plantar flexor muscle mass, whereas the plantaris muscle (16%) and gastrocnemius muscle (78%) (2,3,21) comprise 94% of the mass. Thus, the slow-twitch motor units in plantaris and gastrocnemius muscles, which together are approximately equal in mass to the soleus muscle (3), may provide sufficient force during standing and walking to act in place of the soleus muscle when it is removed. In light of its relatively high level of activity and role in postural maintenance and walking despite its small contribution to total plantar flexor muscle mass, the purpose of this study was to determine the morphological and/or metabolic consequences of soleus muscle ablation on the remaining synergist muscles. In addition, we determined whether adaptations in synergist muscles to ablation were a function of muscle fiber type or of the mass of muscle removed by studying the consequences of plantaris muscle removal, and of plantaris and soleus muscle removal. The parameters selected to assess metabolic, contractile, and/or morphologic alterations are known markers of adaptation to changes in load and recruitment pattern, as seen with run training or increased functional load (4,5,6,8,20). Methods Animals, surgery, and design. Male Sprague-Dawley rats (205 + 11 g) were obtained from Charles River Breeding Laboratories (Kingston, NY). Rats were housed in metal cages on a 12:12 hr light/dark cycle. Food (Purina Lab Chow 5008) and water were provide ad-libitum. Rats were randomly divided into four groups. The soleus muscle was removed in the first group (n = 8), the plantaris muscle in the second group (n= 8), and both the soleus and plantaris muscles in the third group (n= 8). The fourth group served as controls (n = 10). All surgery for a given group was performed on the same day. Rats were anesthetized with pentobarbital sodium (5mg/100g body wt, ip). Bilateral ablations were performed. The lateral aspect of each limb was shaved and a longitudinal incision was made through the skin and fascia along the lateral aspect of the tibia. To ablate soleus muscle, the proximal and distal tendons were carefully isolated using blunt dissection and severed. The muscle was then freed from its neurovascular connections and removed from the animal. To ablate plantaris muscle, the distal tendon was isolated and severed. The distal 50% of the muscle was then removed. The partial removal of the plantaris muscle was necessary in order to maintain patency of the neurovasculature of the muscles left intact. The fascia and skin were sutured, distal to proximal, with 5-0 silk. The duration of time for the entire procedure for both hindlimbs was 40 min. Animals were returned to their cages and were observed walking immediately after recovery from anesthesia (=3 hr). Forty days following ablation, control and experimental rats were anesthetized as before, and the remaining plantar flexor muscles were excised. Immediately after removal, muscles were trimmed of visible fat and tendon projections, washed in saline, blotted, and weighed on a Mettler analytical balance. Muscles were frozen in liquid nitrogen and stored at -70°C until enzyme assays were performed.
Vol. 51, NO. 21, 1992
Muscle Response to Synergist Ablation
1693
For comparison purposes, a secondary study was performed in which mass of intact synergist muscles was determined 40 days following ablation of the gastrocnemius muscle only, or the gastrocnemius muscle and the soleus muscle or plantaris muscle. Gastrocnemius muscle ablation has been commonly used to induce compensatory hypertrophy in plantaris and/or soleus muscles. The surgeries were performed as described previously (16).
Enzyme assays. For the determination of citrate synthase (CS) and lactate dehydrogenase (LDH) activity, muscle samples were homogenized in 175 mM KC1, containing 10 mM glutathione, 2 mM EDTA (pH 7.4). CS activity was determined by the method of Srere (22) using 5,5-dithiobis-(2-nitrobenzoate). Homogenates were frozen and thawed four times to disrupt mitochondria, and centrifuged at 700 x g. Enzyme activity was measured spectrophotometrically on the 700 x g supernatant. LDH activity was determined on whole muscle homogenates by the method of Bergmeyer (7). Enzyme activity was measured by the rate of NADH oxidation. The amount of enzyme added was adjusted until the reaction was linear for four minutes. Skeletal muscle myofibrils were isolated for the measurement of Mg2÷-activated myofibrillar ATPase activity as described by Baldwin et al. (4). Protein concentration of the total myofibrillar suspension was determined by the method of Lowry (18), and adjusted to 6 mg/ml. ATPase activity was determined by measuring the release of inorganic phosphate (9). Statistical analysis. Statistical differences between means for each variable were determined by one-way ANOVA. Data are reported as mean _+ S.E. Results Body weight and muscle mass. Body weight did not differ between control (447 + 7 g) and experimental groups (451 _+5, 438 _+4, 447 + 6 g for soleus, plantaris, and soleus + plantaris muscle ablation, respectively) at the time of sacrifice (40 days post ablation). Mass of intact soleus, plantaris, and gastrocnemius muscles did not differ between any experimental groups and the control group 40 days after synergist removal (Table 1 and 2). Hypertrophy of soleus and/or plantaris muscles occurred only when the gastrocnemius muscle was ablated (Figure 1).
Enzyme activity. Citrate synthase activity (~mol/g wet wt/min) in intact plantaris or soleus muscles did not differ from control muscles after ablation of soleus or plantaris muscles, respectively (Table 1). The activity of citrate synthase in red and white gastrocnemius following ablation of soleus, plantaris, or both muscles was not different from that in control gastrocnemius muscles (Table 2). LDH (/~mol/g wet wt/min) activity did not differ from control levels in remaining plantaris, soleus, or gastrocnemius muscles following soleus and/or plantaris muscle ablation (Table 1 and 2). MgZ+-activated myofibrillar ATPase (/~mol Pi/mg myofibrillar protein/min) did not differ between control and experimental animals for any muscle studied (Table 1 and 2). Changes in CS, LDH, and myofibrillar ATPase activity in soleus and plantaris muscles following gastrocnemius muscle ablation were commensurate with previously reported results (4,5,6,21,24) (data not shown). There were no differences in myofibrillar yield between groups (mg myofibril/g wet wt); the values for control muscles were as follows: soleus = 107 + 3; plantaris = 106 + 2; gastrocnemius medial head red = 113 _+4, white = 123 _+5; gastrocnemius lateral head red = 103 + 3, white = 117 +_4.
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Muscle Response to Synergist Ablation
Vol.
51, No. 21, 1992
TABLE 1 Plantaris and Soleus Muscle Response to Soleus and Plantaris Ablation, Respectively
Mass
Plantaris Control Soleus Ablation Soleus Control Plantaris Ablation
(mg)
ATPase (/~mol Pi/mg prot./min)
Citrate Synthase (~mol/g/min)
LDH
468 _+ 10
0.497 _+ 0.017
25 + 1
438 + 21
497 + 15
0.530 _+ 0.025
27 _+ 3
420 _+ 31
177 _+ 10
0.149 + 0.014
23 + 3
109 +
12
198_+ 5
0.157_+0.008
22_+3
98+
7
(~mol/g/min)
Values are m e a n _+ SE for n = 10 for control and n= 8 for experimental groups. *Significantly different from control, P
