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Myofibrillar creatine kinase in Duchenne and avian muscular dystrophy
BIGCHEMICAL
MEDICINE
29, 355-359
(1983)
Myofibrillar Creatine Kinase in Duchenne and Avian Muscular Dystrophy HOWARD FEIT,’
JOHN FUSELER,AND JAY D. COOK
Departments of Neurology and Cell Biology, University at Dallas, and Texas Scottish Rite Hospital for Texas 75235
of Texas Health Crippled Children,
Sciences Dallas,
Center
ReceivedJuly 2, 1982
Dysfunction of sarcomeric relaxation has been suggested by some workers to be important in the pathogenesis of both Duchenne muscular dystrophy (1) and muscular dystrophy in the hamster (2) and chicken (3). Sarcomeric relaxation is a complex event related to both the control of calcium concentration and reversal of the cross-bridge between actin and myosin. Two general theories have been proposed which attempt to account on a molecular basis for the final events in sarcomeric relaxation which result in cessation of the interaction of the thick and thin filaments. In both theories, the physical separation between actin and myosin occurs when the nucleotide binding site on myosin is occupied by ATP (4,5,6). In one theory, based on proposed reaction mechanisms for the actomyosin ATPase (6), the ADP associated with myosin is simply replaced by ATP. In the other theory, known as the phosphocreatine shuttle hypothesis, Bessman and Geiger (7) have suggested that myosin-ADP may be converted to myosin-ATP by the action of creatine kinase (CK) located within the myofibril itself. A small fraction (about 3-5%) of the total CK in skeletal muscle has been shown to be associated with the myofibril and is located in the M lines and to a lesser extent in the Z lines (8). In this report we examine the possibility that a failure of integration of CK into the myofibril might be associated with muscular dystrophy of the Duchenne or avian forms. MATERIALS
AND METHODS
Normal (line 412) and dystrophic (line 413) chickens were obtained from the Avian Science Department, University of California, Davis, ’ To whomcorrespondence shouldbe addressed. 355 0006-2944/83
$3.00
Copyright 0 1983 by Academic Press, Inc. All rights of reproduction in any form rexwed.
California. The pectoralis major muscle was removed from animals that were 90-days P.Yoi’o. Dystrophic human muscle was obtained from the quadriceps of three male patients between the ages of 5 to 7 years. The patients fulfilled the research criteria for the diagnosis of Duchenne dystrophy (9). Normal human pectoral muscle was obtained at autopsy within 6 hr of death from two men. age 20 and 41 years. who were the victims of gunshot wounds. Myofibrils were isolated by a modification of the methods of Wallimann et al. (8). The muscle was homogenized for 15 set in 20 vol of Solution A (0.1 M KCI, 1 mM EGTA. 1 mM dithiothreitol, and 5 mM EDTA, pH 7.0) using the PT35-10 generator of a Polytron homogenizer (Brinkman Instruments, Westbury, N.J.) at a power setting of 3.5. The homogenate was centrifuged at 8OOg for 10 min. The supernatant was discarded and the pellet was rehomogenized as described above and centrifuged at 15OOg x 10 min. Thereafter, the pellet was washed with Solution A a total of nine times. With each wash, the pellet was suspended in 7 ml of Solution A by homogenization with five strokes of a loose-fitting pestle in a Dounce homogenizer. CK activity of the washed myofibrils was determined with phosphocreatine and exogerzous ADP as substrates by measuring spectrophotometrically the production of ATP using a commercially available CK assay kit (Boehringer Mannheim, Indianapolis, Ind., uv method activated 158160). The final reaction mixture contained 100 mM triethanolamine buffer, pH 7.0, 20 mM o-glucose, 10 mM magnesium acetate, I.0 mM ADP, 10 mM AMP. 35 mM creatine phosphate, 0.6 mM NADP, 9 mM glutathione (reduced), 2 1.2 units/ml hexokinase, 2 1.2 units/ml glucose6-phosphate dehydrogenase, and an aliquot of myofibrils ~0.005 enzyme units. The reaction was monitored in triplicate or quadruplicate in a spectrophotometer at 25°C. Protein was determined by the method of Lowry et al. (10) after overnight digestion of the myofibrils in I N NaOH. The antibody to chicken pectoral muscle MM-CPK was a generous gift from Dr. Barry Eckert. The specificity of this antibody has been reported previously (11). Immunofluorescent staining of the washed myofibril preparations were performed according to the procedures of Walliman et al. (8). The anti-MM-CPK was used at a concentration of 50 pg/ml and visualized by fluorescein-conjugated rat anti-rabbit IgG. The antibody absorbed with an excess of antigen gave no staining of the myofibrils. RESULTS
Using an antibody to the MM-isoenzyme of chicken skeletal muscle CK, this protein was detected in the M line by immunofluorescent techniques applied to 9 x-washed myofibrils isolated from both normal and
MYOFIBRILLAR
CREATINE
KINASE
357
dystrophic human and avian muscle (Fig. 1). There was no visual evidence by these indirect immunofluorescent methods that dystrophic muscle had a subpopulation of myofibrils which lacked CK. There were no differences in the localization of the CK in myofibrils from normal compared with dystrophic muscle. As reported previously (8), the CK was predominantly localized to the M-line region of the sarcomeres.
FIG. 1. Immunofluorescent localization of creatine kinase in 9 x -washed myofibrils. (A) Normal human; (B) Duchenne muscular dystrophy. Identical results were obtained with myofibrils from normal and dystrophic avian muscle. Scale bar equals 10 pm.
358
FEIT. FUSELEK.
AND (‘OOK
The creatine kinase activity of human 9x -washed myofibrils using phosphocreatine and exogenous ADP as substrates is shown in Fig. 2. There were no statistically significant differences (Student’s t test) in the activity of myofibrillar CK in myofibrils from normal human pectoral muscle when compared with myofibrils prepared from dystrophic human quadriceps muscle. For avian pectoral muscle myofibrils, the CK activity for the washed myofibrils from the normal muscle was 0.33 + 0.03 unit/mg and for the washed myofibrils from dystrophic muscle was 0.29 t 0.03 unit/mg based on quadruplicate assays. DISCUSSION Myofibrils prepared from dystrophic muscle contain CK in the M and Z lines and this CK is enzymatically active with exogenous ADP as substrate. It is, therefore, unlikely that dysfunction of dystrophic sarcomeres results from the lack of integration of CK into the myofibril. In a previous study using cryostat cross-sections of muscle and immunocytochemistry, dystrophic muscle was noted to contain CK in the intermyofibrillary space (12). Our results do not exclude the possibility that the CK in myofibrils from dystrophic muscle cannot use myosinbound ADP as substrate, but this feature of the phosphocreatine shuttle hypothesis remains unproven in normal muscle. Myofibrillar CK, which may have a unique and important role in maintaining the concentration of ATP in the local environment of the sarcomere (71, is present and active in the forms of muscular dystrophy that were studied. In addition, the total concentrations of ATP and creatine phosphate in both human 15-
r 3
..
DMD-I MD-2
DMD-3
NORM-1
NORM-:
FIG. 2. Creatine kinase activity in units/mg total protein at 2s”C in 9 x-washed myofibrils prepared from quadriceps muscle from three patients with Duchenne muscular dystrophy and from pectoral muscle from two normal males. Individual data points are shown with an adjacent bar showing the mean and standard error.
MYOFIBRILLAR
CREATlNE
KINASE
359
(13) and avian (14) dystrophic muscle are in the normal range. It is also unlikely that the elevated serum concentration of CK in dystrophic humans or chickens results from failure of integration of CK into the sarcomere. Creatine kinase is also a constituent of muscle mitochondria. Our studies do not eliminate possible defects in the mitochondrially bound creatine kinase or in the transport of adenine nucleotides, creatine, and creatine phosphate between the mitochondrial and the myofibrillar compartments (15, 16). These processes could possibly be disturbed in dystrophic muscle as a nonspecific consequence of the disease state with alteration in the cytoarchitecture of the muscle or as the result of an effect of proteases on mitochondrial membranes ( 15, 16). SUMMARY The presence and activity of the fraction of creatine kinase (CK) which was associated with myofibrils and located in the M line of the sarcomeres was determined in normal and dystrophic avian muscle and in normal and dystrophic (Duchenne) human muscle. Myofibrils were isolated from homogenates of muscle and washed nine times so as to remove nonmyofibrillar CK. In myofibrils from dystrophic muscle the enzyme CK was localized to the M line using immunofluorescent techniques and was enzymatically active. These results suggest that in both avian and Duchenne muscular dystrophy, there is not a myofibrillar disorder of the phosphocreatine shuttle. REFERENCES 1. Cullen, M. J., and Fulthorpe. J. J., J. Neural. Sci. 24, 179 (1975). 2. Wrogemann, K.. Hayward, W. A. K.. and Blanchaer. M. C., Ann. N.Y. Acad. Sci. 317, 30 (1979). 3. Feit. H., Stauver, M. G., Domke. R., and Shay, J. W., Mu.scle Nerve 5, 373 (1982). 4. Bremen, R. D.. and Weber, A., Nature Neu, Biol. 238, 97 (1972). 5. Lymm. R. W., and Taylor, E. W., Biochemisfry 10, 4617 (1971). 6. Taylor, E. W., CRC Crit. Rev. Biochem. 6, 103 (1979). 7. Bessman, S. P., and Geiger, P. J.. Science 211, 448 (1981). 8. Wallimann, T., Turner, D. C., and Eppenberger, H. M., J. Cell Biol. 75, 297 (1977). 9. Brooke, M. H.. Griggs, R. C.. Mendell, J. R.. Fenichel. G. M.. Shumate, J. B.. and Pellegrino, R. J.. Muscle Nerve 3, 186 (1981). IO. Lowry, 0. H., Rosebrough. N. J., Farr, A. L., and Randall, R. J., J. Biol. Chem. 193, 265 (1951). 1 I. Eckert. B. S.. Koons, S. J.. Schontz. A. W.. and Zobel. C. R.. J. Cell Biol. 86. I (1980). 12. Karpati, G., and Sherwin. A. L., J. Neural. Sci. 14, 153 (1971). 13. Samaha, F. J., Davis, B., and Nagy, B., Neurology 31, 916 (1981). 14. Chalovich, J. M., Burt, C. T., Donnon, M. J.. Glonek, K. T.. and Barany, M.. Ann. N.Y. Acad. Sri. 317, 649 (1979). IS. Erickson-Viitanen, S.. Viitanen, P., Geiger, P. J., Yang, W. C. T., and Bessman, S. P., J. Biol. Chem. 257, 14395 (1982). 16. Erickson-Viitanen, S., Geiger, P. J., Viitanen, P., and Bessman, S. P.. J. Bid. Chem. 257, 14405 (1982).
MEDICINE
29, 355-359
(1983)
Myofibrillar Creatine Kinase in Duchenne and Avian Muscular Dystrophy HOWARD FEIT,’
JOHN FUSELER,AND JAY D. COOK
Departments of Neurology and Cell Biology, University at Dallas, and Texas Scottish Rite Hospital for Texas 75235
of Texas Health Crippled Children,
Sciences Dallas,
Center
ReceivedJuly 2, 1982
Dysfunction of sarcomeric relaxation has been suggested by some workers to be important in the pathogenesis of both Duchenne muscular dystrophy (1) and muscular dystrophy in the hamster (2) and chicken (3). Sarcomeric relaxation is a complex event related to both the control of calcium concentration and reversal of the cross-bridge between actin and myosin. Two general theories have been proposed which attempt to account on a molecular basis for the final events in sarcomeric relaxation which result in cessation of the interaction of the thick and thin filaments. In both theories, the physical separation between actin and myosin occurs when the nucleotide binding site on myosin is occupied by ATP (4,5,6). In one theory, based on proposed reaction mechanisms for the actomyosin ATPase (6), the ADP associated with myosin is simply replaced by ATP. In the other theory, known as the phosphocreatine shuttle hypothesis, Bessman and Geiger (7) have suggested that myosin-ADP may be converted to myosin-ATP by the action of creatine kinase (CK) located within the myofibril itself. A small fraction (about 3-5%) of the total CK in skeletal muscle has been shown to be associated with the myofibril and is located in the M lines and to a lesser extent in the Z lines (8). In this report we examine the possibility that a failure of integration of CK into the myofibril might be associated with muscular dystrophy of the Duchenne or avian forms. MATERIALS
AND METHODS
Normal (line 412) and dystrophic (line 413) chickens were obtained from the Avian Science Department, University of California, Davis, ’ To whomcorrespondence shouldbe addressed. 355 0006-2944/83
$3.00
Copyright 0 1983 by Academic Press, Inc. All rights of reproduction in any form rexwed.
California. The pectoralis major muscle was removed from animals that were 90-days P.Yoi’o. Dystrophic human muscle was obtained from the quadriceps of three male patients between the ages of 5 to 7 years. The patients fulfilled the research criteria for the diagnosis of Duchenne dystrophy (9). Normal human pectoral muscle was obtained at autopsy within 6 hr of death from two men. age 20 and 41 years. who were the victims of gunshot wounds. Myofibrils were isolated by a modification of the methods of Wallimann et al. (8). The muscle was homogenized for 15 set in 20 vol of Solution A (0.1 M KCI, 1 mM EGTA. 1 mM dithiothreitol, and 5 mM EDTA, pH 7.0) using the PT35-10 generator of a Polytron homogenizer (Brinkman Instruments, Westbury, N.J.) at a power setting of 3.5. The homogenate was centrifuged at 8OOg for 10 min. The supernatant was discarded and the pellet was rehomogenized as described above and centrifuged at 15OOg x 10 min. Thereafter, the pellet was washed with Solution A a total of nine times. With each wash, the pellet was suspended in 7 ml of Solution A by homogenization with five strokes of a loose-fitting pestle in a Dounce homogenizer. CK activity of the washed myofibrils was determined with phosphocreatine and exogerzous ADP as substrates by measuring spectrophotometrically the production of ATP using a commercially available CK assay kit (Boehringer Mannheim, Indianapolis, Ind., uv method activated 158160). The final reaction mixture contained 100 mM triethanolamine buffer, pH 7.0, 20 mM o-glucose, 10 mM magnesium acetate, I.0 mM ADP, 10 mM AMP. 35 mM creatine phosphate, 0.6 mM NADP, 9 mM glutathione (reduced), 2 1.2 units/ml hexokinase, 2 1.2 units/ml glucose6-phosphate dehydrogenase, and an aliquot of myofibrils ~0.005 enzyme units. The reaction was monitored in triplicate or quadruplicate in a spectrophotometer at 25°C. Protein was determined by the method of Lowry et al. (10) after overnight digestion of the myofibrils in I N NaOH. The antibody to chicken pectoral muscle MM-CPK was a generous gift from Dr. Barry Eckert. The specificity of this antibody has been reported previously (11). Immunofluorescent staining of the washed myofibril preparations were performed according to the procedures of Walliman et al. (8). The anti-MM-CPK was used at a concentration of 50 pg/ml and visualized by fluorescein-conjugated rat anti-rabbit IgG. The antibody absorbed with an excess of antigen gave no staining of the myofibrils. RESULTS
Using an antibody to the MM-isoenzyme of chicken skeletal muscle CK, this protein was detected in the M line by immunofluorescent techniques applied to 9 x-washed myofibrils isolated from both normal and
MYOFIBRILLAR
CREATINE
KINASE
357
dystrophic human and avian muscle (Fig. 1). There was no visual evidence by these indirect immunofluorescent methods that dystrophic muscle had a subpopulation of myofibrils which lacked CK. There were no differences in the localization of the CK in myofibrils from normal compared with dystrophic muscle. As reported previously (8), the CK was predominantly localized to the M-line region of the sarcomeres.
FIG. 1. Immunofluorescent localization of creatine kinase in 9 x -washed myofibrils. (A) Normal human; (B) Duchenne muscular dystrophy. Identical results were obtained with myofibrils from normal and dystrophic avian muscle. Scale bar equals 10 pm.
358
FEIT. FUSELEK.
AND (‘OOK
The creatine kinase activity of human 9x -washed myofibrils using phosphocreatine and exogenous ADP as substrates is shown in Fig. 2. There were no statistically significant differences (Student’s t test) in the activity of myofibrillar CK in myofibrils from normal human pectoral muscle when compared with myofibrils prepared from dystrophic human quadriceps muscle. For avian pectoral muscle myofibrils, the CK activity for the washed myofibrils from the normal muscle was 0.33 + 0.03 unit/mg and for the washed myofibrils from dystrophic muscle was 0.29 t 0.03 unit/mg based on quadruplicate assays. DISCUSSION Myofibrils prepared from dystrophic muscle contain CK in the M and Z lines and this CK is enzymatically active with exogenous ADP as substrate. It is, therefore, unlikely that dysfunction of dystrophic sarcomeres results from the lack of integration of CK into the myofibril. In a previous study using cryostat cross-sections of muscle and immunocytochemistry, dystrophic muscle was noted to contain CK in the intermyofibrillary space (12). Our results do not exclude the possibility that the CK in myofibrils from dystrophic muscle cannot use myosinbound ADP as substrate, but this feature of the phosphocreatine shuttle hypothesis remains unproven in normal muscle. Myofibrillar CK, which may have a unique and important role in maintaining the concentration of ATP in the local environment of the sarcomere (71, is present and active in the forms of muscular dystrophy that were studied. In addition, the total concentrations of ATP and creatine phosphate in both human 15-
r 3
..
DMD-I MD-2
DMD-3
NORM-1
NORM-:
FIG. 2. Creatine kinase activity in units/mg total protein at 2s”C in 9 x-washed myofibrils prepared from quadriceps muscle from three patients with Duchenne muscular dystrophy and from pectoral muscle from two normal males. Individual data points are shown with an adjacent bar showing the mean and standard error.
MYOFIBRILLAR
CREATlNE
KINASE
359
(13) and avian (14) dystrophic muscle are in the normal range. It is also unlikely that the elevated serum concentration of CK in dystrophic humans or chickens results from failure of integration of CK into the sarcomere. Creatine kinase is also a constituent of muscle mitochondria. Our studies do not eliminate possible defects in the mitochondrially bound creatine kinase or in the transport of adenine nucleotides, creatine, and creatine phosphate between the mitochondrial and the myofibrillar compartments (15, 16). These processes could possibly be disturbed in dystrophic muscle as a nonspecific consequence of the disease state with alteration in the cytoarchitecture of the muscle or as the result of an effect of proteases on mitochondrial membranes ( 15, 16). SUMMARY The presence and activity of the fraction of creatine kinase (CK) which was associated with myofibrils and located in the M line of the sarcomeres was determined in normal and dystrophic avian muscle and in normal and dystrophic (Duchenne) human muscle. Myofibrils were isolated from homogenates of muscle and washed nine times so as to remove nonmyofibrillar CK. In myofibrils from dystrophic muscle the enzyme CK was localized to the M line using immunofluorescent techniques and was enzymatically active. These results suggest that in both avian and Duchenne muscular dystrophy, there is not a myofibrillar disorder of the phosphocreatine shuttle. REFERENCES 1. Cullen, M. J., and Fulthorpe. J. J., J. Neural. Sci. 24, 179 (1975). 2. Wrogemann, K.. Hayward, W. A. K.. and Blanchaer. M. C., Ann. N.Y. Acad. Sci. 317, 30 (1979). 3. Feit. H., Stauver, M. G., Domke. R., and Shay, J. W., Mu.scle Nerve 5, 373 (1982). 4. Bremen, R. D.. and Weber, A., Nature Neu, Biol. 238, 97 (1972). 5. Lymm. R. W., and Taylor, E. W., Biochemisfry 10, 4617 (1971). 6. Taylor, E. W., CRC Crit. Rev. Biochem. 6, 103 (1979). 7. Bessman, S. P., and Geiger, P. J.. Science 211, 448 (1981). 8. Wallimann, T., Turner, D. C., and Eppenberger, H. M., J. Cell Biol. 75, 297 (1977). 9. Brooke, M. H.. Griggs, R. C.. Mendell, J. R.. Fenichel. G. M.. Shumate, J. B.. and Pellegrino, R. J.. Muscle Nerve 3, 186 (1981). IO. Lowry, 0. H., Rosebrough. N. J., Farr, A. L., and Randall, R. J., J. Biol. Chem. 193, 265 (1951). 1 I. Eckert. B. S.. Koons, S. J.. Schontz. A. W.. and Zobel. C. R.. J. Cell Biol. 86. I (1980). 12. Karpati, G., and Sherwin. A. L., J. Neural. Sci. 14, 153 (1971). 13. Samaha, F. J., Davis, B., and Nagy, B., Neurology 31, 916 (1981). 14. Chalovich, J. M., Burt, C. T., Donnon, M. J.. Glonek, K. T.. and Barany, M.. Ann. N.Y. Acad. Sri. 317, 649 (1979). IS. Erickson-Viitanen, S.. Viitanen, P., Geiger, P. J., Yang, W. C. T., and Bessman, S. P., J. Biol. Chem. 257, 14395 (1982). 16. Erickson-Viitanen, S., Geiger, P. J., Viitanen, P., and Bessman, S. P.. J. Bid. Chem. 257, 14405 (1982).