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Effect of leupeptin of protein turnover in normal and dystrophic chicken skeletal muscle cells in culture
BIOCHEMICAL
MEDICINE
23, 316-323 (1980)
Effect of Leupeptin on Protein Turnover in Normal and Dystrophic Chicken Skeletal Muscle Cells in Culture’ JOHN
F.
RIEBOW
AND
RONALD
B.
YOUNG
Genetics Program. 28C Food Science Building, Michigan East Lansing, Michigan 48824
State University,
Received February 19, 1980
The primary defect in human genetic muscular dystrophy is not known, even though several secondary manifestations of the defect are well documented (l-7). Availability of several animal models for human dystrophy has encouraged a more detailed biochemical analysis of this inherited disease. The dystrophic chicken (line 307, University of California, Davis), for example, is an early-onset dystrophy that is characterized by atrophy, fatty infiltration, and other histopathological similarities to human muscular dystrophy (9, 18). Moreover, evidence from limb bud transplantation studies and cell culture analysis strongly suggest that the genetic lesion in chicken muscular dystrophy is myogenic in origin and is uniquely expressed in fully differentiated muscle cells (19-25). Muscle atrophy is one of the most obvious physiological characteristics of muscular dystrophy. Accordingly, dystrophic muscle contains elevated levels of several proteolytic enzymes (j-7,26-28). Treatment of muscular dystrophy with inhibitors of proteolytic activity may therefore be a promising avenue of therapy, since some of these inhibitors delay the degeneration of dystrophic muscle both in vivo ( 15, 16) and in vitro ( 14). One of these agents, leupeptin, inhibits the activity of at least two muscle proteases (17, 29). To more closely assess the effect of leupeptin on muscle protein turnover in muscle cells, the present study was conducted with the two following goals in mind: (i) to determine if leupeptin inhibits turnover of both the myofibrillar protein fraction and the soluble protein ’ Michigan Agricultural Experiment Station Article No. 9339. This was a cooperative effort between the Genetics Program, the Department of Food Science and Human Nutrition, and the Department of Animal Husbandry. Research was supported in part by Michigan Agricultural Experiment Station Project Nos. 1241 and 1265, and by research grants from the Muscular Dystrophy Association of America and the Upjohn Company. * To whom all correspondence should be addressed. 316 0006-2944/80/030316-08$02.00/O Copyright All rights
@ 1980 by Academic Press. Inc. of reproduction in any form reserved.
LEUPEPTIN
AND PROTEIN
fraction in dystrophic muscle cells in culture; and whether normal and dystrophic muscle cells exhibit sponse to leupeptin treatment. Our results suggest that soluble, but not myofibrillar, protein turnover and that trophic muscle cells respond identically to leupeptin. MATERIALS
317
TURNOVER
(ii) to determine a differential releupeptin inhibits normal and dys-
AND METHODS
Myogenic cells were isolated from the breast muscle of normal (white Leghorn, Reichardt’s Hatchery, St. Louis, MI) and genetically dystrophic (line 307, University of California, Davis) 12-day chick embryos as described elsewhere (10, 11). The cells were counted in a hemocytometer and plated at approximately 2 x 1oB cells per 60-mm, collagen-coated tissue culture plate. The cells were cultured in 85% Eagle’s minimum essential medium containing 10% horse serum, 5% chick embryo extract, 50 units/ml penicillin, 50 @g/ml streptomycin, and 2.5 pg/ml fungizone. Culture medium was changed every 24 hr, and the cells were incubated at 37°C in a 5% CO, atmosphere. The general experimental design for this investigation is summarized in Fig. 1. All cell cultures were fed on Day 3 and on each day thereafter with complete medium containing 10m7 M fluorodeoxyuridine (FdU) to inhibit fibroblast proliferation. On Day 7, all culture plates were labeled for 16 hr with complete medium containing FdU and 1 #X/ml [3H]Leu. At the end of the labeling period, the cells were rinsed twice with an isotonic saline solution at 37”C, refed complete medium, and reincubated for 6 hr with complete medium containing FdU. The purpose of this brief period was to permit sufficient time for residual, free intracellular [3H]Leu to be either incorporated into protein or released into the culture medium. Quadrupli-
DAY OF EXPERIMENT
0 i-2 3-6 7 6
9
MANIPULATIONS SET UP 16 MUSCLE CELL REFEED ALL CELLS
CULTURES
REFEED. ADO FLUOROOEOXYURIDINE tICi M) PULSE WITH 1 JJCI/MLh~ LEU FOR 16HR A. SACRIFICE WAORUPLICATE PLATES 6. RINSE OUT f3tll, START “CHASE” C. ADD LEUPEPTIN T O 12 PLATES As FOLLOW D,25,50, AND 75 UGlML (TRIPLICATE RAm FOR EACH TREATMENT) SACRIFICE ALL REMAINING PLATES
FIG. 1. Experimental design for evaluating the effect of leupeptin on protein turnover in normal and dystrophic chicken muscle cell cultures. Radioactivity was measured in the myofibrillar protein fraction, the soluble protein fraction, and the 200,000-dalton heavy chain of myosin.
318
RIEBOW
AND
YOUNG
cate plates were harvested at the end of this 6-hr period as described below, and the remaining culture plates were incubated for an additional 24 hr with complete medium and leupeptin at 0, 25, 50, and 75 ,+$ml. All subsequent measurements were in triplicate. At the end of the chase period, the plates were rinsed two times with a cold isotonic saline solution, and the cells were immediately scraped from the culture plates. The cells were homogenized in a 7-ml Dounce homogenizer (Wheaton Scientific, tightly fitting A pestle) using 1 ml of homogenization buffer (0.25 M NaCl, 10 mM Tris, 10 mM MgC&, pH 7.2). The volume of the homogenate was measured, and 25-~1 aliquots were taken for protein determination and trichloroacetic acid precipitation. The cell homogenate was then centrifuged at 3000g for 30 min to remove nuclei and mitochondria. Following the addition of 25 pg of carrier myosin to facilitate precipitation of myofibrillar proteins, the supernatant was diluted IO-fold with cold water. Samples were placed at 2°C for 16 hr, and the myofibrillar fraction was collected by centrifugation at 2000g for 45 min. The pellet was dissolved in 100 ~1 of a solution containing 1% NaDodSO,, 0.5% mercaptoethanol, 0.01% Pyronin Y, 20% glycerol, 0.5 M Tris, pH 7.2, and samples were then analyzed by polyacrylamide gel electrophoresis in the presence of NaDodSO, (13). The radioactivity present in total cellular protein was determined by precipitation of aliquots of the cell homogenate with 5% trichloroacetic acid. The precipitates were collected on Millipore Type HA filters. Filters were dissolved by heating at 100°C for 15 min in a OS-ml solution of 0.5 N HCl, followed by the addition of 1 ml of ethyl acetate. The radioactivity was analyzed using 10 ml of Formula 963 liquid scintillation cocktail from New England Nuclear. The radioactivity present in myosin heavy chain and the crude myofibrillar fraction was determined after electrophoresis of the crude myofibrillar fraction on polyacrylamide disc gels in the presence of NaDodSO,. The myosin heavy-chain band was removed from the gel and dissolved in 0.2 ml of 30% hydrogen peroxide. The remainder of the gel was dissolved in 2 ml of 30% hydrogen peroxide and then diluted to a final volume of 10 ml with HzO. The radioactivity in the myosin heavy-chain band and in an aliquot of the crude myofibrillar fraction was analyzed as described above. Once the amount of radioactivity in the myofibrillar fraction was known, this value was subtracted from the radioactivity present in total cellular protein. This difference was attributed to the soluble protein fraction. The total protein per culture plate was determined using the method of Lowry et al. (12). Total myotube nuclei and total nuclei per culture plate was counted using Giemsa-stained culture plates as previously described (13).
LEUPEPTIN
AND PROTEIN TURNOVER
319
RESULTS When normal and genetically dystrophic embryonic myogenic cells are grown in cell culture, they proliferate, fuse into multinucleated myotubes, and accumulate muscle-specific proteins. Dystrophic muscle cells consistently accumulated protein faster than normal cultures during the first several days in culture, but the maximum quantity of protein was the same in mature, steady-state cultures (Fig. 2). During the period of interest for this investigation (Days 8-9), total protein content (Fig. 2) and synthesis rate (23) are similar in normal and dystrophic muscle cells. Leupeptin at 75 &ml virtually eliminated the loss of [“H]Leu from the soluble protein fraction in muscle cultures, but release of radioactivity from the myofibrillar fraction and from myosin heavy chain was not affected by leupeptin (Fig. 3). Although contractile protein turnover in neither normal nor dystrophic cells was responsive to leupeptin treatment, the rate of [3H]Leu release at all levels of leupeptin was consistently higher from the dystrophic cells (Fig. 3B, 3C). Although O-75 pg/ml of leupeptin drastically inhibited soluble protein degradation (Fig. 3A), the actual protein content of leupeptin-treated cultures was only slightly higher than control cultures (Table 1). The number of muscle cells in each culture was not markedly affected by leupeptin (Table 1). DISCUSSION Turnover of soluble and myofibrillar proteins in skeletal muscle seems to be accomplished by different classes or groups of proteases, since leupeptin only inhibits soluble protein degradation. In the case of myosin heavy chain, it can be concluded only that leupeptin does not inhibit the
Eol&&--J 0
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CULTURE
6
8
IO
12
14
AGE, days
FIG. 2. Accumulation of total protein in normal and dystrophic muscle cell cultures. Protein content in each culture was divided between the number of nuclei per culture to normalize for possible differences in cell density. 0, normal; A, dystrophic. Each point represtnts the mean f. 1 SEM of five experiments in which all measurements were in duplicate.
320
RIEBOW AND YOUNG
J LEUFEPTIN
CCWENTRATDN,u~ml
FIG. 3. Effect of leupeptin on loss of [3H]Leu from the soluble protein fraction, the myofibrillar protein fraction, and myosin heavy chain. Cultures were prelabeled with [3H]Leu for 16 hr. Radioactivity was measured in one set of quadruplicate cultures 6 hr after the end of labeling period in order to determine the amount of radioactivity in each fraction at the start of the chase period. Cultures were then incubated for an additional 24 hr in culture medium without rSH]Leu but with O-75 &ml leupeptin. The quantity of radioactivity in each fraction at the end of the chase period was subtracted from the quantity at the start of the chase. This difference reflects the amount of radioactivity that was lost from each protein fraction during the 24-hr chase period. Experiments were conducted as shown in Fig. 1 and as detailed under Materials and Methods. (A) Soluble protein fraction, (B) myofibrillar protein fraction, and (C) myosin heavy chain. 0, normal; 0, dystrophic.
initial proteolytic step. The first break in the MHC molecule would produce two smaller breakdown products which would not have migrated with intact myosin heavy chains during electrophoresis under denaturing conditions. Therefore, inhibition of turnover of these smaller fragments would not have been detected. Leupeptin had no measurable effect on myofibtillar protein turnover in either normal or dystrophic cultures; however, the turnover rate of myofibrillar proteins was considerably higher in the dystrophic muscle cultures (Figs. 3B and C). In previous studies (23), we had tentatively reached this latter conclusion, based on the observation that the myofibtillar protein synthesis rate was approximately 50% higher in dystrophic muscle cultures under steady-state conditions (i.e.. no net change in
LEUPEPTIN
321
AND PROTEIN TURNOVER
TABLE 1 EFFECTOF LEUPEPTINTREATMENTON THEPROTEIN CONTENTOF MUSCLECULTURES ANDON THENUMBEROF MYOTUBE NUCLEI IN EACH CULTURE DISH
Cell type
Leupeptin concentration ( rglml)
Protein per dish (mfd
Myotube nuclei per dish (X 106)
Normal
0 25 50 75
1.33 2 1.34” 1.58 2 I.51 +
0.09 0.10 0.14 0.12
2.73 2.94 2.62 2.87
-c 0.10 2 0.17 +- 0.07 lr- 0.08
Dystrophic
0 25 50 75
1.32 1.37 1.43 1.50
0.10 0.11 0.12 0.14
3.04 3.07 2.91 2.90
-c 0.06 Ifr 0.20 rt 0.06 5 0.06
2 k + +
Note. Cells were analyzed at the end of the chase period on Day 9.
protein content). Data in Figs. 3B and C directly confirm that, under the conditions employed in our laboratory, contractile protein turnover is 50-100% higher in dystrophic muscle cultures than in normal cultures. Although leupeptin seems to inhibit only soluble protein turnover, an alternative explanation cannot be ruled out. The same results would be obtained if intracellular amino acid pools are highly compartmentalized for soluble and myofibrillar protein synthesis, and if leupeptin differentially inhibits reutilization of [3H]Leu in these pools during the chase period. Several previous studies have demonstrated that treatment with leupeptin of both normal and dystrophic muscle cells in vitro and in vivo delays the degenerative process (14- 16). The results of the present experiments suggest that this improvement resulted from diminished turnover of soluble, cytoplasmic protein species, rather than by effects on the contractile elements. However, the increase in quantity of cellular protein is relatively small (Table 1). The intracellular specificity of leupeptin on protein turnover is not yet clearly defined. Leupeptin has been reported to inhibit cathepsin B with a high degree of selectivity (17). Moreover, Azanza et al. (29) have shown that leupeptin completely inhibits the activity of a Ca*+-activated neutral protease that has been postulated to be a major enzyme in disassembly and degradation of myofibrillar proteins (30-32). Based on these observations, leupeptin would have been expected to inhibit turnover of both soluble and myofibrillar proteins. Identification of a unifying explanation for these observations is complicated further by the following: (i) the fact that Ca2+-activated protease purified in three separate laboratories exhibits differences in specificity for degradation of myofibrillar proteins
322
RIEBOW AND YOUNG
(29, 31-33), and (ii) contradictory evidence as to whether cathepsin B is actually involved in degradation of myofibrillar proteins (34-39). Development of a suitable therapy for genetic muscular dystrophy is the ultimate goal of research with protease inhibitors. Potential use of these agents requires that two major obstacles be overcome. First, the proper combination of inhibitors must be identified in order to achieve a balanced increase in the quantity of myofibrillar and soluble proteins. Second, a delivery system for specific targeting of the inhibitors to muscle tissue must be developed. In conclusion, leupeptin will inhibit soluble protein turnover and will somewhat increase the amount of total protein in muscle cell cultures. However, leupeptin neither inhibits myofibrillar protein turnover nor exerts a selective beneficial effect on dystrophic muscle cells. SUMMARY
The effect of leupeptin on turnover of the soluble protein fraction, the myofibrillar protein fraction, and myosin heavy chain was evaluated in muscle cell cultures. Cultures were prepared from the breast muscle of 12-day normal (white leghorn) and dystrophic (line 307) chick embryos. After 7 days in culture, cells were labeled for 16 hr with 1 &i/ml of [3H]Leu and then “chased” for an additional 24 hr in culture medium containing no E3H]Leu and O-75 pg/ml leupeptin. Cultures were analyzed for radioactivity in the soluble protein fraction, the myofibrillar protein fraction, and myosin heavy chain. Leupeptin (75 pgiml) virtually eliminated loss of radioactivity from the soluble protein fraction, but only minimally affected loss of radioactivity from the myofibrillar fraction or myosin heavy chain. Normal and dystrophic muscle cells responded identically to leupeptin treatment. Thus, the muscle proteases that are specifically inhibited by leupeptin seem to have no major role in initiating myofibrillar protein turnover. REFERENCES 1. Munsat. T. L., Balch. R., Pearson, C. M., and Farler, W., J. Amer. Med. Assoc. 226, 1536-1543 (1973). 2. Benedict, J. P., Kahnsey, J. J., Scarrone, L. A., Wertheim, A. R., and Stetten, D. E. W.. J. Clin. Invest. 34, 141-145 (1955). 3. Hughes, B. P., J. Neural. Neurosurg. Psychiar. 35, 658-663 (1972). 4. Lin, C. H., Hudson, A. J., and Strickland, K. P., Life Sci. 11, 355-362 (1972). 5. Pennington, R. J., and Robinson, J. F., Enzymol. Biol. C/in. 9, 175-182 (1968). 6. Toppel, A. L., Zalkin, H., Caldwell, K. A., Desai, I. D., and Shifko, S., Arch. Biochim. Biophys.
96, 340-346
(1962).
7. Iodice, A. A., Chin, J., Perker, S., and Weinstock, I. M., Arch. Eiochim. Biophys. 152, 166-174 (1972). 8. Appel. S. H.. and Roses, A. D., in “The Metabolic Basis of Inherited Disease” (John B. Stanburg. James B. Wyngarden. and Donald S. Fredrickson, Eds.). 4th ed., pp. 1260-1281. McGraw-Hill, New York. 1978.
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9. Wilson, B. W., Randall, W. R., Patterson, G. T.. and Entrikin, R. K., Ann. N. Y. Acad. Sci. 317, 224-246 (1979). 10. Tepperman, I., Morris, G., Essien, F., and Heywood, S. M., J. Cell. Physiol. 86, 561-565 (1975). II. Young, R. B., Goll, D. E., and Stromer, M. H., Develop. Biol. 47, 123-135 (1975). 12. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. J. Biof. Chem. 193, 265-275 ( 195 1). 13. Young, R. B., Bergen, W. G., and Blauwiekel, P. B.,J. Cell Viol. 81, 115-122 (1979). 14. McGowan, E. B., Shafiq, S. A., and Stracher, A., Exp. Neural. 50, 649-657 (1976). 15. Stracher, A., McGowan, E. B., and Shaftq, S. A., Science 200, 50-51 (1978). 16. Libby, P., and Goldberg, A. L., Science 199, 534-536 (1978). 17. Aoyagi, T., and Umezawa, U., (“Cold Spring Harbor Conference on Cell Proliferation (Proteases and Biological Control), pp. 429-450. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1975. 18. McMurtry, S. L., Kuhan, L. M., and Asmundson, U. S., Arch. Parhoi. 94, 217-224 (1972). 19. Cosmos, E., and Butter, J., in “Research in Muscle Development and the Muscle Spindle” (B. Q. Banker, R. J. Przybyski, J. P. van der Mevlen, and M. Victor, Eds.), pp. 149-162. Excerpta Medica, Amsterdam, 1972. 20. Linkhart, T. A., Yee, G. W., and Wilson, B. W., Science 187, 549-551 (1975). 21. Linkhart, T. A., Yee, G. W., Nieberg, P. S., and Wilson, B. W., Develop. Biol. 48, 447-457 (1976). 22. Askanas, V., Shahq, S. A., and Milhorat, A. T., Arch. Neural. 20, 259-265 (1971). 23. Young, R. B., McConnell, D. G., and Suelter, C. H., J. Cell Biol. 79,319a (1978). 24. Herman, B. A., and Femandez, S. M., Fed. Proc. 36, 1652a (1977). 25. Weinstock, 1. M., and Jones, K. B., Biochem. Med. 18, 245-255 (1977). 26. Mayer, M., Amin, R., Milholland, R. J., and Rosen, F., Exp. Mol. Pathol. 25, 9-19. (1976). 27. Sanada, Y., Yasogawa, N., and Katunuma, N., J. Biochem. 83, 27-33 (1978). 28. Katumuma, N., Yasogawa, N., Kito. K. Sanada, Y., Kawai, H.. and Miyoshi, K., J. Biochem.
83, 625-628
(1978).
29. Azanza, J., Raymond, J., Robin, J., Cottin, P., and Ducastaing, A.. B&hem. J. 183, 339-347 (1979). 30. Dayton, W. R., Gall. D. E., Zeece, M. G., Robson, R. M., and Revihe, W. J., Biochemistry 15, 2150-2158 (1976). 31. Dayton, W. R., Reville, W. J.. Gall, D. E.. and Stromer, M. H., Biochemistry 15, 2159-2167 (1976). 32. Dayton, W. R., Gall, D. E., Stromer, M. H., Reville, W. J., Zeece, M. G., and Robson, R. M., “Cold Spring Harbor Conference on Cell Proliferation (hoteases and Biological Control),” pp. 551-577. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1975. 33. Reddy, M. K., Etlinger, J. D., Rabinowitz, M.. Fischman, D. A., and Zak, R., J. Bid. Chem.
34. 35. 36. 37. 38. 39.
250, 4278-4284
(1975).
Schwartz, W. N., and Bird, J. W. C., Biochem. J. 167, 811-820 (1977). Bodwell, C. E., and Pearson, A. M., J. Food Sci. 29, 602-607 (1964). Friedman, I., Lamer, A., and Davis, A. M., Experentia 28, 1092-1093 (1979). Martins, C. B., and Whitaker, J. R., J. Food Sci. 33, 59-64 (1968). Park, D. C., and Pennington, R. J., Enzymol. Biol. Clin. 8, 149-160 (1%7). Sharp, J. G., J. Sci. Food Agr. 14, 468-479 (1%3).
MEDICINE
23, 316-323 (1980)
Effect of Leupeptin on Protein Turnover in Normal and Dystrophic Chicken Skeletal Muscle Cells in Culture’ JOHN
F.
RIEBOW
AND
RONALD
B.
YOUNG
Genetics Program. 28C Food Science Building, Michigan East Lansing, Michigan 48824
State University,
Received February 19, 1980
The primary defect in human genetic muscular dystrophy is not known, even though several secondary manifestations of the defect are well documented (l-7). Availability of several animal models for human dystrophy has encouraged a more detailed biochemical analysis of this inherited disease. The dystrophic chicken (line 307, University of California, Davis), for example, is an early-onset dystrophy that is characterized by atrophy, fatty infiltration, and other histopathological similarities to human muscular dystrophy (9, 18). Moreover, evidence from limb bud transplantation studies and cell culture analysis strongly suggest that the genetic lesion in chicken muscular dystrophy is myogenic in origin and is uniquely expressed in fully differentiated muscle cells (19-25). Muscle atrophy is one of the most obvious physiological characteristics of muscular dystrophy. Accordingly, dystrophic muscle contains elevated levels of several proteolytic enzymes (j-7,26-28). Treatment of muscular dystrophy with inhibitors of proteolytic activity may therefore be a promising avenue of therapy, since some of these inhibitors delay the degeneration of dystrophic muscle both in vivo ( 15, 16) and in vitro ( 14). One of these agents, leupeptin, inhibits the activity of at least two muscle proteases (17, 29). To more closely assess the effect of leupeptin on muscle protein turnover in muscle cells, the present study was conducted with the two following goals in mind: (i) to determine if leupeptin inhibits turnover of both the myofibrillar protein fraction and the soluble protein ’ Michigan Agricultural Experiment Station Article No. 9339. This was a cooperative effort between the Genetics Program, the Department of Food Science and Human Nutrition, and the Department of Animal Husbandry. Research was supported in part by Michigan Agricultural Experiment Station Project Nos. 1241 and 1265, and by research grants from the Muscular Dystrophy Association of America and the Upjohn Company. * To whom all correspondence should be addressed. 316 0006-2944/80/030316-08$02.00/O Copyright All rights
@ 1980 by Academic Press. Inc. of reproduction in any form reserved.
LEUPEPTIN
AND PROTEIN
fraction in dystrophic muscle cells in culture; and whether normal and dystrophic muscle cells exhibit sponse to leupeptin treatment. Our results suggest that soluble, but not myofibrillar, protein turnover and that trophic muscle cells respond identically to leupeptin. MATERIALS
317
TURNOVER
(ii) to determine a differential releupeptin inhibits normal and dys-
AND METHODS
Myogenic cells were isolated from the breast muscle of normal (white Leghorn, Reichardt’s Hatchery, St. Louis, MI) and genetically dystrophic (line 307, University of California, Davis) 12-day chick embryos as described elsewhere (10, 11). The cells were counted in a hemocytometer and plated at approximately 2 x 1oB cells per 60-mm, collagen-coated tissue culture plate. The cells were cultured in 85% Eagle’s minimum essential medium containing 10% horse serum, 5% chick embryo extract, 50 units/ml penicillin, 50 @g/ml streptomycin, and 2.5 pg/ml fungizone. Culture medium was changed every 24 hr, and the cells were incubated at 37°C in a 5% CO, atmosphere. The general experimental design for this investigation is summarized in Fig. 1. All cell cultures were fed on Day 3 and on each day thereafter with complete medium containing 10m7 M fluorodeoxyuridine (FdU) to inhibit fibroblast proliferation. On Day 7, all culture plates were labeled for 16 hr with complete medium containing FdU and 1 #X/ml [3H]Leu. At the end of the labeling period, the cells were rinsed twice with an isotonic saline solution at 37”C, refed complete medium, and reincubated for 6 hr with complete medium containing FdU. The purpose of this brief period was to permit sufficient time for residual, free intracellular [3H]Leu to be either incorporated into protein or released into the culture medium. Quadrupli-
DAY OF EXPERIMENT
0 i-2 3-6 7 6
9
MANIPULATIONS SET UP 16 MUSCLE CELL REFEED ALL CELLS
CULTURES
REFEED. ADO FLUOROOEOXYURIDINE tICi M) PULSE WITH 1 JJCI/MLh~ LEU FOR 16HR A. SACRIFICE WAORUPLICATE PLATES 6. RINSE OUT f3tll, START “CHASE” C. ADD LEUPEPTIN T O 12 PLATES As FOLLOW D,25,50, AND 75 UGlML (TRIPLICATE RAm FOR EACH TREATMENT) SACRIFICE ALL REMAINING PLATES
FIG. 1. Experimental design for evaluating the effect of leupeptin on protein turnover in normal and dystrophic chicken muscle cell cultures. Radioactivity was measured in the myofibrillar protein fraction, the soluble protein fraction, and the 200,000-dalton heavy chain of myosin.
318
RIEBOW
AND
YOUNG
cate plates were harvested at the end of this 6-hr period as described below, and the remaining culture plates were incubated for an additional 24 hr with complete medium and leupeptin at 0, 25, 50, and 75 ,+$ml. All subsequent measurements were in triplicate. At the end of the chase period, the plates were rinsed two times with a cold isotonic saline solution, and the cells were immediately scraped from the culture plates. The cells were homogenized in a 7-ml Dounce homogenizer (Wheaton Scientific, tightly fitting A pestle) using 1 ml of homogenization buffer (0.25 M NaCl, 10 mM Tris, 10 mM MgC&, pH 7.2). The volume of the homogenate was measured, and 25-~1 aliquots were taken for protein determination and trichloroacetic acid precipitation. The cell homogenate was then centrifuged at 3000g for 30 min to remove nuclei and mitochondria. Following the addition of 25 pg of carrier myosin to facilitate precipitation of myofibrillar proteins, the supernatant was diluted IO-fold with cold water. Samples were placed at 2°C for 16 hr, and the myofibrillar fraction was collected by centrifugation at 2000g for 45 min. The pellet was dissolved in 100 ~1 of a solution containing 1% NaDodSO,, 0.5% mercaptoethanol, 0.01% Pyronin Y, 20% glycerol, 0.5 M Tris, pH 7.2, and samples were then analyzed by polyacrylamide gel electrophoresis in the presence of NaDodSO, (13). The radioactivity present in total cellular protein was determined by precipitation of aliquots of the cell homogenate with 5% trichloroacetic acid. The precipitates were collected on Millipore Type HA filters. Filters were dissolved by heating at 100°C for 15 min in a OS-ml solution of 0.5 N HCl, followed by the addition of 1 ml of ethyl acetate. The radioactivity was analyzed using 10 ml of Formula 963 liquid scintillation cocktail from New England Nuclear. The radioactivity present in myosin heavy chain and the crude myofibrillar fraction was determined after electrophoresis of the crude myofibrillar fraction on polyacrylamide disc gels in the presence of NaDodSO,. The myosin heavy-chain band was removed from the gel and dissolved in 0.2 ml of 30% hydrogen peroxide. The remainder of the gel was dissolved in 2 ml of 30% hydrogen peroxide and then diluted to a final volume of 10 ml with HzO. The radioactivity in the myosin heavy-chain band and in an aliquot of the crude myofibrillar fraction was analyzed as described above. Once the amount of radioactivity in the myofibrillar fraction was known, this value was subtracted from the radioactivity present in total cellular protein. This difference was attributed to the soluble protein fraction. The total protein per culture plate was determined using the method of Lowry et al. (12). Total myotube nuclei and total nuclei per culture plate was counted using Giemsa-stained culture plates as previously described (13).
LEUPEPTIN
AND PROTEIN TURNOVER
319
RESULTS When normal and genetically dystrophic embryonic myogenic cells are grown in cell culture, they proliferate, fuse into multinucleated myotubes, and accumulate muscle-specific proteins. Dystrophic muscle cells consistently accumulated protein faster than normal cultures during the first several days in culture, but the maximum quantity of protein was the same in mature, steady-state cultures (Fig. 2). During the period of interest for this investigation (Days 8-9), total protein content (Fig. 2) and synthesis rate (23) are similar in normal and dystrophic muscle cells. Leupeptin at 75 &ml virtually eliminated the loss of [“H]Leu from the soluble protein fraction in muscle cultures, but release of radioactivity from the myofibrillar fraction and from myosin heavy chain was not affected by leupeptin (Fig. 3). Although contractile protein turnover in neither normal nor dystrophic cells was responsive to leupeptin treatment, the rate of [3H]Leu release at all levels of leupeptin was consistently higher from the dystrophic cells (Fig. 3B, 3C). Although O-75 pg/ml of leupeptin drastically inhibited soluble protein degradation (Fig. 3A), the actual protein content of leupeptin-treated cultures was only slightly higher than control cultures (Table 1). The number of muscle cells in each culture was not markedly affected by leupeptin (Table 1). DISCUSSION Turnover of soluble and myofibrillar proteins in skeletal muscle seems to be accomplished by different classes or groups of proteases, since leupeptin only inhibits soluble protein degradation. In the case of myosin heavy chain, it can be concluded only that leupeptin does not inhibit the
Eol&&--J 0
2
4
CULTURE
6
8
IO
12
14
AGE, days
FIG. 2. Accumulation of total protein in normal and dystrophic muscle cell cultures. Protein content in each culture was divided between the number of nuclei per culture to normalize for possible differences in cell density. 0, normal; A, dystrophic. Each point represtnts the mean f. 1 SEM of five experiments in which all measurements were in duplicate.
320
RIEBOW AND YOUNG
J LEUFEPTIN
CCWENTRATDN,u~ml
FIG. 3. Effect of leupeptin on loss of [3H]Leu from the soluble protein fraction, the myofibrillar protein fraction, and myosin heavy chain. Cultures were prelabeled with [3H]Leu for 16 hr. Radioactivity was measured in one set of quadruplicate cultures 6 hr after the end of labeling period in order to determine the amount of radioactivity in each fraction at the start of the chase period. Cultures were then incubated for an additional 24 hr in culture medium without rSH]Leu but with O-75 &ml leupeptin. The quantity of radioactivity in each fraction at the end of the chase period was subtracted from the quantity at the start of the chase. This difference reflects the amount of radioactivity that was lost from each protein fraction during the 24-hr chase period. Experiments were conducted as shown in Fig. 1 and as detailed under Materials and Methods. (A) Soluble protein fraction, (B) myofibrillar protein fraction, and (C) myosin heavy chain. 0, normal; 0, dystrophic.
initial proteolytic step. The first break in the MHC molecule would produce two smaller breakdown products which would not have migrated with intact myosin heavy chains during electrophoresis under denaturing conditions. Therefore, inhibition of turnover of these smaller fragments would not have been detected. Leupeptin had no measurable effect on myofibtillar protein turnover in either normal or dystrophic cultures; however, the turnover rate of myofibrillar proteins was considerably higher in the dystrophic muscle cultures (Figs. 3B and C). In previous studies (23), we had tentatively reached this latter conclusion, based on the observation that the myofibtillar protein synthesis rate was approximately 50% higher in dystrophic muscle cultures under steady-state conditions (i.e.. no net change in
LEUPEPTIN
321
AND PROTEIN TURNOVER
TABLE 1 EFFECTOF LEUPEPTINTREATMENTON THEPROTEIN CONTENTOF MUSCLECULTURES ANDON THENUMBEROF MYOTUBE NUCLEI IN EACH CULTURE DISH
Cell type
Leupeptin concentration ( rglml)
Protein per dish (mfd
Myotube nuclei per dish (X 106)
Normal
0 25 50 75
1.33 2 1.34” 1.58 2 I.51 +
0.09 0.10 0.14 0.12
2.73 2.94 2.62 2.87
-c 0.10 2 0.17 +- 0.07 lr- 0.08
Dystrophic
0 25 50 75
1.32 1.37 1.43 1.50
0.10 0.11 0.12 0.14
3.04 3.07 2.91 2.90
-c 0.06 Ifr 0.20 rt 0.06 5 0.06
2 k + +
Note. Cells were analyzed at the end of the chase period on Day 9.
protein content). Data in Figs. 3B and C directly confirm that, under the conditions employed in our laboratory, contractile protein turnover is 50-100% higher in dystrophic muscle cultures than in normal cultures. Although leupeptin seems to inhibit only soluble protein turnover, an alternative explanation cannot be ruled out. The same results would be obtained if intracellular amino acid pools are highly compartmentalized for soluble and myofibrillar protein synthesis, and if leupeptin differentially inhibits reutilization of [3H]Leu in these pools during the chase period. Several previous studies have demonstrated that treatment with leupeptin of both normal and dystrophic muscle cells in vitro and in vivo delays the degenerative process (14- 16). The results of the present experiments suggest that this improvement resulted from diminished turnover of soluble, cytoplasmic protein species, rather than by effects on the contractile elements. However, the increase in quantity of cellular protein is relatively small (Table 1). The intracellular specificity of leupeptin on protein turnover is not yet clearly defined. Leupeptin has been reported to inhibit cathepsin B with a high degree of selectivity (17). Moreover, Azanza et al. (29) have shown that leupeptin completely inhibits the activity of a Ca*+-activated neutral protease that has been postulated to be a major enzyme in disassembly and degradation of myofibrillar proteins (30-32). Based on these observations, leupeptin would have been expected to inhibit turnover of both soluble and myofibrillar proteins. Identification of a unifying explanation for these observations is complicated further by the following: (i) the fact that Ca2+-activated protease purified in three separate laboratories exhibits differences in specificity for degradation of myofibrillar proteins
322
RIEBOW AND YOUNG
(29, 31-33), and (ii) contradictory evidence as to whether cathepsin B is actually involved in degradation of myofibrillar proteins (34-39). Development of a suitable therapy for genetic muscular dystrophy is the ultimate goal of research with protease inhibitors. Potential use of these agents requires that two major obstacles be overcome. First, the proper combination of inhibitors must be identified in order to achieve a balanced increase in the quantity of myofibrillar and soluble proteins. Second, a delivery system for specific targeting of the inhibitors to muscle tissue must be developed. In conclusion, leupeptin will inhibit soluble protein turnover and will somewhat increase the amount of total protein in muscle cell cultures. However, leupeptin neither inhibits myofibrillar protein turnover nor exerts a selective beneficial effect on dystrophic muscle cells. SUMMARY
The effect of leupeptin on turnover of the soluble protein fraction, the myofibrillar protein fraction, and myosin heavy chain was evaluated in muscle cell cultures. Cultures were prepared from the breast muscle of 12-day normal (white leghorn) and dystrophic (line 307) chick embryos. After 7 days in culture, cells were labeled for 16 hr with 1 &i/ml of [3H]Leu and then “chased” for an additional 24 hr in culture medium containing no E3H]Leu and O-75 pg/ml leupeptin. Cultures were analyzed for radioactivity in the soluble protein fraction, the myofibrillar protein fraction, and myosin heavy chain. Leupeptin (75 pgiml) virtually eliminated loss of radioactivity from the soluble protein fraction, but only minimally affected loss of radioactivity from the myofibrillar fraction or myosin heavy chain. Normal and dystrophic muscle cells responded identically to leupeptin treatment. Thus, the muscle proteases that are specifically inhibited by leupeptin seem to have no major role in initiating myofibrillar protein turnover. REFERENCES 1. Munsat. T. L., Balch. R., Pearson, C. M., and Farler, W., J. Amer. Med. Assoc. 226, 1536-1543 (1973). 2. Benedict, J. P., Kahnsey, J. J., Scarrone, L. A., Wertheim, A. R., and Stetten, D. E. W.. J. Clin. Invest. 34, 141-145 (1955). 3. Hughes, B. P., J. Neural. Neurosurg. Psychiar. 35, 658-663 (1972). 4. Lin, C. H., Hudson, A. J., and Strickland, K. P., Life Sci. 11, 355-362 (1972). 5. Pennington, R. J., and Robinson, J. F., Enzymol. Biol. C/in. 9, 175-182 (1968). 6. Toppel, A. L., Zalkin, H., Caldwell, K. A., Desai, I. D., and Shifko, S., Arch. Biochim. Biophys.
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