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Effects of stretch and denervation on protease activities of normal and dystrophic chicken muscle
EXPERIMENTAL
NEUROLOGY
t?d,
420-427
(1984)
Effects of Stretch and Denervation on Protease Activities of Normal and Dystrophic Chicken Muscle Y. B. LEE, C. R. ASHMORE,
AND L. HITCHCOCK
Laboratory of Muscle Biology, Department of Animal Science, University of California, Davis, California 95616 Received September 19, 1983; revision received December 29, 1983 Changes of protease activities that follow passivestretch, denervation, and denervation plus stretch were followed in the patagiahs muscle of normal and dystrophic chicks between 6 and 7 weeks of age. The baseline activities of cathepsin C, cathepsin D, and leucine aminopeptidase in dystrophic muscle were 2 to 3.5 times higher than in normal muscle. Passive stretch and denervation induced increases in protease activities by 40 to 120% in normal muscle, whereas the same treatments did not significantly affect the activities of the enzymes in dystrophic muscle. We conclude that the level of protease activity in dystrophic chicken muscle at 6 weeks of age had already attained a maximum limit and could not be increased even by denervation. In spite of protease activities, which were not different from control dystrophic muscle, denervated dystrophic muscles lost muscle weight rapidly whether they were stretched or not. They weighed 60% less than the innervated control muscte after 7 days. Inherently high protease activities in dystrophic muscle do not vary at this age regardless of whether or not the muscle is gaining or losing weight. INTRODUCTION
Skeletal muscle growth induced by passive stretch has been well characterized in the patagialis (PAT) muscle of the normal chicken ( l-3,8). Muscles of 6-week-old chicks were increased in weight 60% more than the unstretched contralateral muscles after 1 week of passive stretch (3). This was preceded by increases of DNA and RNA, which subsequently resulted in increased protein content. When passive stretch was applied to l-week-old dystrophic birds, the PAT muscle at 7 weeks of age had doubled in weight and had a fiber cross-sectional area 80% greater than control muscles. This rapid growth of stretched muscles suggests that rates of protein synthesis may be elevated, Abbreviations: PAT-patagialis,
LAPase-leucine 420
0014-4886/84
$3.00
Copyright 0 1984 by Academic Press. Inc. All rights of reproduction in any form reserved.
aminopeptidase.
STRETCH
AND DENERVATION
ON PROTEASE
ACTIVITIES
421
or alternatively, that rates of degradation may be decreased. At present, no information is available to indicate whether activities of muscle proteolytic enzymes are affected by passive stretch in either normal or dystrophic muscle. Denervation is frequently used in the study of muscle atrophy (9, 12). In denervated normal muscle, increased rates of protein breakdown (9, 10, 14) and increased lysosomal enzyme activities (11, 15, 16) have both been observed. However, a recent study by Ashmore et al. (2) reported that denervation, with or without stretch, retarded the progress of pathology in dystrophic chickens. Therefore, it seemed possible that denervation of dystrophic muscles might actually result in a decline of proteolytic enzyme activities. Our objectives were to determine the effects of stretch, denervation, and denervation in combination with stretch on activities of cathepsins C and D and leucine aminopeptidase in normal and dystrophic muscles, and to relate the findings to changes in muscle weight and morphologic characteristics. MATERIALS
AND
METHODS
Normal chicks (line 03) and dystrophic chicks (line 433) were obtained from the Department of Avian Sciences, University of California, Davis, California. When the chicks were 6 weeks of age, 16 chicks of each genotype were assigned to each of the following experimental groups. Experiment 1. Passive stretch was applied to the PAT muscle of the right wing of each chick using a cardboard sleeve as described elsewhere (8). The contralateral muscle served as the control muscle. Four chicks of each genotype were killed at each of 1,3,5, and 7 days after stretching. The extent of passive stretch was such that after 24 h the sarcomeres of the experimental muscle were 40% longer than those in the control muscle as documented by Barnett et al. (3). The PAT muscle was removed, trimmed of gross connective tissue, weighed, and quick-frozen on dry ice. Experiment 2. One PAT muscle (right wing) of each chick was denervated as described elsewhere (2). The contralateral muscle served as the control muscle. Four chicks of each genotype were killed at each of 1, 3, 5, and 7 days after denervation and the PAT muscle was removed and handled as described in Experiment 1. Experiment 3. Both PAT muscles of each chick were denervated at 6 weeks of age. The PAT muscles on the right side were stretched for 1, 3, 5, and 7 days at which times four birds of each genotype were killed. The PAT muscles were removed and treated as described in Experiment 1. The PAT muscles on the left side served as the denervated control. For the determination of proteolytic enzyme activities, frozen muscle samples were homogenized 1 min in 10 vol 0.05 M Tris, 0.15 M KC1 buffer, pH 7.4, using a Polytron P- 10 homogenizer. The homogenate was centrifuged
422
LEE, ASHMORE,
AND HITCHCOCK
90 min at 100,000 g and the clear supernatant was used for the subsequent enzyme assays and protein determination. Cathepsin C (dipeptidylpeptidase I) activity was determined calorimetrically according to the method of Barrett and Heath (4) with the following modifications. Enzyme activity was determined in a total volume of 3 ml consisting of 2.8 ml buffered activator (3 mM cysteine, 1.33 mM EDTA, and 20 mM NaCl in 0.1 M phosphate buffer, pH 6.5), 0.1 ml supematant, and 0.1 ml gIycyl+phenylalanine 2-naphthylamide (30 mg/mI). After 30 min incubation at 37°C the reaction was stopped by adding 3 ml coupling reagent (4). After centrifugation 20 min at 20,000 g, the absorbance of the supematant was read at 520 nm. Enzyme activity was expressed as nmol 2naphthylamine/ mg protein/30 min. Cathepsin D assay was carried out according to Barrett and Heath (4) using hemoglobin as substrate. Enzyme activity was expressed as nmol tyrosine equivalent/mg protein/30 min. Leucine aminopeptidase (LAPase) activity described by Barrett and Heath (4) was assayed in the same procedure as described for cathepsin C except the substrate used was leucine-2-naphthylamide (20 mg/ml). Enzyme activity was expressed as nmol2-naphthlaminelmg protein/30 min. Protein content was determined by the biuret method as described by Layne (13). Bovine serum albumin was used as standard. RESULTS Patagialis Muscle Weight. Table 1 shows changes of PAT muscle weights. As reported (I), stretched muscles in both genotypes gained weight rapidly. After 7 days of stretching, the wet weight of the stretched muscle was 46.5% greater than the unstretched control muscle in the normal chick, whereas in the dystrophic chick the stretched muscles were 62% greater than the contralateral control muscles. The absolute weight difference between the stretched and the unstretched muscle was identical (17 1 mg) for both normal and dystrophic chicks after 7 days of stretching. However, the effect of denervation on muscle weight of the two genotypes was quite different. In normal chicks there was only a slight decrease in muscle weight after 7 days of denervation; whereas dystrophic PAT muscles lost 60% of their weight during the same period. When denervated muscles were stretched, muscle weights were slightly higher in both normal (14.3%) and dystrophic muscles (8%) compared with contralateral denervated unstretched muscles. The stretching effect on muscle weight gain was much smaller in denervated than in innervated muscle. Proteolytic Enzyme Activities. As shown in Table 2, proteolytic enzyme activities in PAT muscles were markedly higher in dystrophic chicks. The
STRETCH
AND
DENERVATION
ON
PROTEASE
ACTIVITIES
LEE, ASHMORE,
424
AND HITCHCOCK
TABLE 2 Proteolytic Enzyme Activities of Normal and Dystrophic Patagialis Muscles at 6 Weeks of Age” Enzymes
Normal chick
Dystrophic chick
Difference (W)
Cathepsin C b Leucine aminopeptidaseb Cathepsin DC
148 rt 15 174 * 18 163 + 12
512 f 32 418 + 41 353 t 29
246 140 116
LIMeans +- SD of eight animals. b Enzyme activity is expressed as nmol naphthylamine/mg protein/30 min. ’ Enzyme activity is expressed as nmol tyrosine equivalent/mg protein/30 min.
most significant difference was observed in cathepsin C activity, followed by LAPase and cathepsin D. The effect of stretching on proteolytic enzyme activities is illustrated in Fig. 1. In normal chicks, the activities of all three enzymes increased rapidly until 3 days of stretching, after which there was no significant change. In dystrophic chicks, passive stretching slightly lowered the activities of all three enzymes by 2 to 15%, although the decrease was not statistically significant. The effect of denervation on enzyme activities is illustrated in Fig. 2. Again in normal chicks, denervation induced rapid increases in enzyme activities. In spite of such increases, however, the enzyme activities of denervated normal muscles were still far below those of untreated dystrophic PAT muscles (Table 2). Again in dystrophic chicks, denervation did not result in significant increases in activities of all the enzymes. w
ae
DAYS AFTER
STRETCHING
PIG. 1. The effect of passive stretch on proteolytic enzyme activities in normal and dystrophic muscles. Cathespin C, - - - leucine aminopeptidase (LAPase), . . cathepsin D.
STRETCH
AND DENERVATION
ON PROTEASE
ACTIVITIES
425
Dyslrophic 3 ae
I
3
5
7
DAYS AFTER DENERVATION
FIG. 2. The effect of denervation on proteolytic enzyme activities in normal and dystrophic muscles. Cathepsin C, - - - LAPase, . . . cathepsin D.
The effect of stretching of denervated muscles on enzyme changes is summarized in Fig. 3. Compared with unstretched denervated PAT muscles there was no significant effect of stretching on normal chicks until 5 days, when all enzymes were slightly higher than denervated control muscles. In dystrophic chicks, there. were no significant differences in activities at any age examined. These results clearly indicated that in normal muscle, stretch and denervation induced marked increases (40 to 120%) in proteolytic enzyme activities. In dystrophic muscle, however, the baseline activities of proteolytic enzymes may have attained the biologically possible maximum limit and other treatments such as denervation and stretching could not change them to any significant extent.
0 ae
DAYS AFTER STRETCHING
FIG. 3. The eff&t of passive stretch on proteolytic enzyme activities in denervated normal and denervated dystrophic muscles. Cathepsin C, - - - LAPase, . . . cathepsin D.
426
LEE,
ASHMORE,
AND
HITCHCOCK
DISCUSSION Bamett et al. (3) demonstrated a significant increase of DNA, RNA, and protein content after 1 week of passive stretch, indicating that protein synthesis may be accelerated. We showed here that in the normal PAT muscle, stretching for 7 days enhanced proteolytic enzyme activities by 40 to 90%, whereas at the same time the muscle weight increased by 50%. Although the role of various proteolytic enzymes in muscle on the degradation of myofibrillar proteins is yet to be elucidated, a recent tissue culture study by Bird et al. (7) showed unusually high specific activities of lysosomal cathepsin B, D, II, and L in homogeneous myoblast populations. The authors suggested that the lysosomal enzymes play a significant role in the differentiation of muscle myotubes. Based on available evidence (5-7), the increase of catheptic enzyme activities in the stretched muscles indicates an accelerated rate of muscle protein tumover in stretch-induced rapid growth of PAT muscle. The most important observation in this study was the lack of effects of denervation or passive stretch on enzyme activities in dystrophic muscles whether or not the muscle was gaining or losing weight. The weight of denervated dystrophic muscles was only 60% of the weight of contralateral control muscles in spite of the fact that there was little difference in proteolytic enzyme activities. Similarly in stretched dystrophic muscle there was no alteration of proteolytic enzyme activities despite the fact that the muscle weight increased by 60%. These results strongly suggest that in the dystrophic muscle the baseline activities of cathepsins C and D, and leucine aminopeptidase have attained a biologic maximum limit and that these enzyme activities are not directly associated with changes in muscle weight. Further studies will be required to demonstrate the exact role of these proteolytic enzymes in the atrophy of dystrophic muscle. REFERENCES 1. ASHMORE, C. R. 1982. Stretch-induced growth in chicken wing muscles: effectson hereditary muscuku dystrophy. Am. I. Physiof. 242: Cl 78-C183. 2. ASHMORE, C. R., Y. B. LEE, P. SUMMERS, AND L. HITCHCOCK. 1984. Stretch-induced growth in chicken wing muscles: nerve muscle interaction in hereditary muscular dystrophy. Am. J. Physiol., 246 (Cell Physiol. 15). In press. 3. BARNETT, J. G., R. G. HOLLY, AND C. R. ASHMORE. 1980. Stretch-induced growth in chicken wing muscles: biochemical and morphological characterization. Am. J. Physiof. 239: C39-C46.
BARRETT, A. J., AND M. F. HEATH. 1977. Pages 19- 147 in J. T. DINGLE, Ed., Lysomes: A Laboratory Handbook, 2nd ed. North-Holland, Amsterdam. 5. BIRD, J. W. C. 1975. Skeletal muscle lysosomes. Pages 75-109 in J. DINGLE AND R. DEAN, Ed., Lysosomes in Biology and Pathology, Vol. 4. Elsevier, New York. 4.
STRETCH
AND
DENJZRVATION
ON
PROTEASE
ACTIVITIES
427
6. BIRD, J. W. C., J. CARTER, R. E. TRIEMER, AND A. M. SPANIER. 1980. Proteinases in cardiac and &&al muscle. Fed. Proc. 39: 20. 7. BIRD, J. W. C., F. J. ROISEN, G. YORKE, J. A. LEE, M. A. MCELLIGO’IT, D. F. TRIEMER, AND A. S. JOHN. 1981. Lysosomes and proteolytic enzyme activities in cultured striated muscle cells. J. Histochem. Cytochem. 29: 431-439. 8. FRANKENY, J., R. HOUY, AND C. R. ASHMORE. 1983. Effects of graded duration of stretch on normal and dystrophic skeletal muscle. Musck Nerve 6: 269-277. 9. GOLDBERG, A. L. 1969. Protein turnover in skeletal muscle. II. Effects of denervation and wrtisone on protein catabolism in skeletal muscle. J. Bier! &em. 2441 3223-3229. IO. GOLDSPINK, D. F. 1976. The effectsof denervatioo on protein turnover of tat skeletal muscle. B&hem. J. 156: 71-80. 11. GOLDSPINK, D. F., J. B. HARRIS, D. C. PARK, N. E. PARSONS,AND R. J. PENNINGTON. 1971. Quantitative enzyme studies in denervated extensor digitorum longus and soleus muscles of rats. J. Biochem. 2: 427-433. 12. GIJTMANN, E. 1962. The Denervated Muscle. Czechoslovakian Acad. of Sci., Prague. 13. LAYNE, E. 1957. Spectrophotometric and turbidimetric methods for measuring proteins. Page 447 in S. P. COWWKK AND N. 0. KAPLAN, Eds., Methods in Enzymology, Vol. 3. Academic Press, New York. 14. LI, J. B. 1980. Protein synthesis and degradation in skeletal muscle of normal and dystrophic hamsters. Am. J. Physiol. 239: E401-406. 15. PEARSON, C. M., AND’N. C. KAR. 1979. Muscle breakdown and lysosomal activation. Ann N.Y. Acad. Sci. 31’1: 465-477. 16. POLUCK, M. S., AND J. W. C. BIRD. 1968. Distribution and particle properties of acid hydrolases in denervated muscle. Am. J. Physiol. 215: 716-722.
NEUROLOGY
t?d,
420-427
(1984)
Effects of Stretch and Denervation on Protease Activities of Normal and Dystrophic Chicken Muscle Y. B. LEE, C. R. ASHMORE,
AND L. HITCHCOCK
Laboratory of Muscle Biology, Department of Animal Science, University of California, Davis, California 95616 Received September 19, 1983; revision received December 29, 1983 Changes of protease activities that follow passivestretch, denervation, and denervation plus stretch were followed in the patagiahs muscle of normal and dystrophic chicks between 6 and 7 weeks of age. The baseline activities of cathepsin C, cathepsin D, and leucine aminopeptidase in dystrophic muscle were 2 to 3.5 times higher than in normal muscle. Passive stretch and denervation induced increases in protease activities by 40 to 120% in normal muscle, whereas the same treatments did not significantly affect the activities of the enzymes in dystrophic muscle. We conclude that the level of protease activity in dystrophic chicken muscle at 6 weeks of age had already attained a maximum limit and could not be increased even by denervation. In spite of protease activities, which were not different from control dystrophic muscle, denervated dystrophic muscles lost muscle weight rapidly whether they were stretched or not. They weighed 60% less than the innervated control muscte after 7 days. Inherently high protease activities in dystrophic muscle do not vary at this age regardless of whether or not the muscle is gaining or losing weight. INTRODUCTION
Skeletal muscle growth induced by passive stretch has been well characterized in the patagialis (PAT) muscle of the normal chicken ( l-3,8). Muscles of 6-week-old chicks were increased in weight 60% more than the unstretched contralateral muscles after 1 week of passive stretch (3). This was preceded by increases of DNA and RNA, which subsequently resulted in increased protein content. When passive stretch was applied to l-week-old dystrophic birds, the PAT muscle at 7 weeks of age had doubled in weight and had a fiber cross-sectional area 80% greater than control muscles. This rapid growth of stretched muscles suggests that rates of protein synthesis may be elevated, Abbreviations: PAT-patagialis,
LAPase-leucine 420
0014-4886/84
$3.00
Copyright 0 1984 by Academic Press. Inc. All rights of reproduction in any form reserved.
aminopeptidase.
STRETCH
AND DENERVATION
ON PROTEASE
ACTIVITIES
421
or alternatively, that rates of degradation may be decreased. At present, no information is available to indicate whether activities of muscle proteolytic enzymes are affected by passive stretch in either normal or dystrophic muscle. Denervation is frequently used in the study of muscle atrophy (9, 12). In denervated normal muscle, increased rates of protein breakdown (9, 10, 14) and increased lysosomal enzyme activities (11, 15, 16) have both been observed. However, a recent study by Ashmore et al. (2) reported that denervation, with or without stretch, retarded the progress of pathology in dystrophic chickens. Therefore, it seemed possible that denervation of dystrophic muscles might actually result in a decline of proteolytic enzyme activities. Our objectives were to determine the effects of stretch, denervation, and denervation in combination with stretch on activities of cathepsins C and D and leucine aminopeptidase in normal and dystrophic muscles, and to relate the findings to changes in muscle weight and morphologic characteristics. MATERIALS
AND
METHODS
Normal chicks (line 03) and dystrophic chicks (line 433) were obtained from the Department of Avian Sciences, University of California, Davis, California. When the chicks were 6 weeks of age, 16 chicks of each genotype were assigned to each of the following experimental groups. Experiment 1. Passive stretch was applied to the PAT muscle of the right wing of each chick using a cardboard sleeve as described elsewhere (8). The contralateral muscle served as the control muscle. Four chicks of each genotype were killed at each of 1,3,5, and 7 days after stretching. The extent of passive stretch was such that after 24 h the sarcomeres of the experimental muscle were 40% longer than those in the control muscle as documented by Barnett et al. (3). The PAT muscle was removed, trimmed of gross connective tissue, weighed, and quick-frozen on dry ice. Experiment 2. One PAT muscle (right wing) of each chick was denervated as described elsewhere (2). The contralateral muscle served as the control muscle. Four chicks of each genotype were killed at each of 1, 3, 5, and 7 days after denervation and the PAT muscle was removed and handled as described in Experiment 1. Experiment 3. Both PAT muscles of each chick were denervated at 6 weeks of age. The PAT muscles on the right side were stretched for 1, 3, 5, and 7 days at which times four birds of each genotype were killed. The PAT muscles were removed and treated as described in Experiment 1. The PAT muscles on the left side served as the denervated control. For the determination of proteolytic enzyme activities, frozen muscle samples were homogenized 1 min in 10 vol 0.05 M Tris, 0.15 M KC1 buffer, pH 7.4, using a Polytron P- 10 homogenizer. The homogenate was centrifuged
422
LEE, ASHMORE,
AND HITCHCOCK
90 min at 100,000 g and the clear supernatant was used for the subsequent enzyme assays and protein determination. Cathepsin C (dipeptidylpeptidase I) activity was determined calorimetrically according to the method of Barrett and Heath (4) with the following modifications. Enzyme activity was determined in a total volume of 3 ml consisting of 2.8 ml buffered activator (3 mM cysteine, 1.33 mM EDTA, and 20 mM NaCl in 0.1 M phosphate buffer, pH 6.5), 0.1 ml supematant, and 0.1 ml gIycyl+phenylalanine 2-naphthylamide (30 mg/mI). After 30 min incubation at 37°C the reaction was stopped by adding 3 ml coupling reagent (4). After centrifugation 20 min at 20,000 g, the absorbance of the supematant was read at 520 nm. Enzyme activity was expressed as nmol 2naphthylamine/ mg protein/30 min. Cathepsin D assay was carried out according to Barrett and Heath (4) using hemoglobin as substrate. Enzyme activity was expressed as nmol tyrosine equivalent/mg protein/30 min. Leucine aminopeptidase (LAPase) activity described by Barrett and Heath (4) was assayed in the same procedure as described for cathepsin C except the substrate used was leucine-2-naphthylamide (20 mg/ml). Enzyme activity was expressed as nmol2-naphthlaminelmg protein/30 min. Protein content was determined by the biuret method as described by Layne (13). Bovine serum albumin was used as standard. RESULTS Patagialis Muscle Weight. Table 1 shows changes of PAT muscle weights. As reported (I), stretched muscles in both genotypes gained weight rapidly. After 7 days of stretching, the wet weight of the stretched muscle was 46.5% greater than the unstretched control muscle in the normal chick, whereas in the dystrophic chick the stretched muscles were 62% greater than the contralateral control muscles. The absolute weight difference between the stretched and the unstretched muscle was identical (17 1 mg) for both normal and dystrophic chicks after 7 days of stretching. However, the effect of denervation on muscle weight of the two genotypes was quite different. In normal chicks there was only a slight decrease in muscle weight after 7 days of denervation; whereas dystrophic PAT muscles lost 60% of their weight during the same period. When denervated muscles were stretched, muscle weights were slightly higher in both normal (14.3%) and dystrophic muscles (8%) compared with contralateral denervated unstretched muscles. The stretching effect on muscle weight gain was much smaller in denervated than in innervated muscle. Proteolytic Enzyme Activities. As shown in Table 2, proteolytic enzyme activities in PAT muscles were markedly higher in dystrophic chicks. The
STRETCH
AND
DENERVATION
ON
PROTEASE
ACTIVITIES
LEE, ASHMORE,
424
AND HITCHCOCK
TABLE 2 Proteolytic Enzyme Activities of Normal and Dystrophic Patagialis Muscles at 6 Weeks of Age” Enzymes
Normal chick
Dystrophic chick
Difference (W)
Cathepsin C b Leucine aminopeptidaseb Cathepsin DC
148 rt 15 174 * 18 163 + 12
512 f 32 418 + 41 353 t 29
246 140 116
LIMeans +- SD of eight animals. b Enzyme activity is expressed as nmol naphthylamine/mg protein/30 min. ’ Enzyme activity is expressed as nmol tyrosine equivalent/mg protein/30 min.
most significant difference was observed in cathepsin C activity, followed by LAPase and cathepsin D. The effect of stretching on proteolytic enzyme activities is illustrated in Fig. 1. In normal chicks, the activities of all three enzymes increased rapidly until 3 days of stretching, after which there was no significant change. In dystrophic chicks, passive stretching slightly lowered the activities of all three enzymes by 2 to 15%, although the decrease was not statistically significant. The effect of denervation on enzyme activities is illustrated in Fig. 2. Again in normal chicks, denervation induced rapid increases in enzyme activities. In spite of such increases, however, the enzyme activities of denervated normal muscles were still far below those of untreated dystrophic PAT muscles (Table 2). Again in dystrophic chicks, denervation did not result in significant increases in activities of all the enzymes. w
ae
DAYS AFTER
STRETCHING
PIG. 1. The effect of passive stretch on proteolytic enzyme activities in normal and dystrophic muscles. Cathespin C, - - - leucine aminopeptidase (LAPase), . . cathepsin D.
STRETCH
AND DENERVATION
ON PROTEASE
ACTIVITIES
425
Dyslrophic 3 ae
I
3
5
7
DAYS AFTER DENERVATION
FIG. 2. The effect of denervation on proteolytic enzyme activities in normal and dystrophic muscles. Cathepsin C, - - - LAPase, . . . cathepsin D.
The effect of stretching of denervated muscles on enzyme changes is summarized in Fig. 3. Compared with unstretched denervated PAT muscles there was no significant effect of stretching on normal chicks until 5 days, when all enzymes were slightly higher than denervated control muscles. In dystrophic chicks, there. were no significant differences in activities at any age examined. These results clearly indicated that in normal muscle, stretch and denervation induced marked increases (40 to 120%) in proteolytic enzyme activities. In dystrophic muscle, however, the baseline activities of proteolytic enzymes may have attained the biologically possible maximum limit and other treatments such as denervation and stretching could not change them to any significant extent.
0 ae
DAYS AFTER STRETCHING
FIG. 3. The eff&t of passive stretch on proteolytic enzyme activities in denervated normal and denervated dystrophic muscles. Cathepsin C, - - - LAPase, . . . cathepsin D.
426
LEE,
ASHMORE,
AND
HITCHCOCK
DISCUSSION Bamett et al. (3) demonstrated a significant increase of DNA, RNA, and protein content after 1 week of passive stretch, indicating that protein synthesis may be accelerated. We showed here that in the normal PAT muscle, stretching for 7 days enhanced proteolytic enzyme activities by 40 to 90%, whereas at the same time the muscle weight increased by 50%. Although the role of various proteolytic enzymes in muscle on the degradation of myofibrillar proteins is yet to be elucidated, a recent tissue culture study by Bird et al. (7) showed unusually high specific activities of lysosomal cathepsin B, D, II, and L in homogeneous myoblast populations. The authors suggested that the lysosomal enzymes play a significant role in the differentiation of muscle myotubes. Based on available evidence (5-7), the increase of catheptic enzyme activities in the stretched muscles indicates an accelerated rate of muscle protein tumover in stretch-induced rapid growth of PAT muscle. The most important observation in this study was the lack of effects of denervation or passive stretch on enzyme activities in dystrophic muscles whether or not the muscle was gaining or losing weight. The weight of denervated dystrophic muscles was only 60% of the weight of contralateral control muscles in spite of the fact that there was little difference in proteolytic enzyme activities. Similarly in stretched dystrophic muscle there was no alteration of proteolytic enzyme activities despite the fact that the muscle weight increased by 60%. These results strongly suggest that in the dystrophic muscle the baseline activities of cathepsins C and D, and leucine aminopeptidase have attained a biologic maximum limit and that these enzyme activities are not directly associated with changes in muscle weight. Further studies will be required to demonstrate the exact role of these proteolytic enzymes in the atrophy of dystrophic muscle. REFERENCES 1. ASHMORE, C. R. 1982. Stretch-induced growth in chicken wing muscles: effectson hereditary muscuku dystrophy. Am. I. Physiof. 242: Cl 78-C183. 2. ASHMORE, C. R., Y. B. LEE, P. SUMMERS, AND L. HITCHCOCK. 1984. Stretch-induced growth in chicken wing muscles: nerve muscle interaction in hereditary muscular dystrophy. Am. J. Physiol., 246 (Cell Physiol. 15). In press. 3. BARNETT, J. G., R. G. HOLLY, AND C. R. ASHMORE. 1980. Stretch-induced growth in chicken wing muscles: biochemical and morphological characterization. Am. J. Physiof. 239: C39-C46.
BARRETT, A. J., AND M. F. HEATH. 1977. Pages 19- 147 in J. T. DINGLE, Ed., Lysomes: A Laboratory Handbook, 2nd ed. North-Holland, Amsterdam. 5. BIRD, J. W. C. 1975. Skeletal muscle lysosomes. Pages 75-109 in J. DINGLE AND R. DEAN, Ed., Lysosomes in Biology and Pathology, Vol. 4. Elsevier, New York. 4.
STRETCH
AND
DENJZRVATION
ON
PROTEASE
ACTIVITIES
427
6. BIRD, J. W. C., J. CARTER, R. E. TRIEMER, AND A. M. SPANIER. 1980. Proteinases in cardiac and &&al muscle. Fed. Proc. 39: 20. 7. BIRD, J. W. C., F. J. ROISEN, G. YORKE, J. A. LEE, M. A. MCELLIGO’IT, D. F. TRIEMER, AND A. S. JOHN. 1981. Lysosomes and proteolytic enzyme activities in cultured striated muscle cells. J. Histochem. Cytochem. 29: 431-439. 8. FRANKENY, J., R. HOUY, AND C. R. ASHMORE. 1983. Effects of graded duration of stretch on normal and dystrophic skeletal muscle. Musck Nerve 6: 269-277. 9. GOLDBERG, A. L. 1969. Protein turnover in skeletal muscle. II. Effects of denervation and wrtisone on protein catabolism in skeletal muscle. J. Bier! &em. 2441 3223-3229. IO. GOLDSPINK, D. F. 1976. The effectsof denervatioo on protein turnover of tat skeletal muscle. B&hem. J. 156: 71-80. 11. GOLDSPINK, D. F., J. B. HARRIS, D. C. PARK, N. E. PARSONS,AND R. J. PENNINGTON. 1971. Quantitative enzyme studies in denervated extensor digitorum longus and soleus muscles of rats. J. Biochem. 2: 427-433. 12. GIJTMANN, E. 1962. The Denervated Muscle. Czechoslovakian Acad. of Sci., Prague. 13. LAYNE, E. 1957. Spectrophotometric and turbidimetric methods for measuring proteins. Page 447 in S. P. COWWKK AND N. 0. KAPLAN, Eds., Methods in Enzymology, Vol. 3. Academic Press, New York. 14. LI, J. B. 1980. Protein synthesis and degradation in skeletal muscle of normal and dystrophic hamsters. Am. J. Physiol. 239: E401-406. 15. PEARSON, C. M., AND’N. C. KAR. 1979. Muscle breakdown and lysosomal activation. Ann N.Y. Acad. Sci. 31’1: 465-477. 16. POLUCK, M. S., AND J. W. C. BIRD. 1968. Distribution and particle properties of acid hydrolases in denervated muscle. Am. J. Physiol. 215: 716-722.