- Email: [email protected]
Effect of vitamin D and its metabolites on developing myogenic cultures
Experimental Cell Research 154 (1984) 485-491
Effect of Vitamin
D and Its Metabolites Myogenic Cultures
PETER STERN and LAWRENCE The Hospital
on Developing
J. KAGEN*
Division of Rheumatic Diseases, Department of Medicine, for Special Surgery and The New York Hospital- Cornell Medical New York, NY 10021, USA
Center,
Growth, protein synthesis and expression of creatine kinase (CK) by embryonic chick myogenic cells are inhibited by vitamin D and certain of its metabolites. 25OH cholecalciferol was most active in concentrations of 1O-‘-1O-6 M, with cholecalciferol and ergocalciferol less active in that order. Ergosterol had no activity of this sort. Inhibition of CK was most marked on the 4th and 5th day of culture and was due to suppression of the appearance of CK-MM and MB. CK-BB was not affected and CK-MB was more affected than CK-BB. Skin fibroblasts by comparison were slightly stimulated in growth at 10m6M and much less affected at lop5 M than the myogenic cells. It is suggested that vitamin D has a direct effect upon the muscle cell, to cause a selective diminution in the production of certain polypeptides.
Vitamin D is essential in the regulation of calcium and phosphorus homeostasis in higher animals. In addition, a relationship between vitamin D and muscle metabolism has been postulated, since vitamin D deficiency results in development of a myopathy which improves with administration of the vitamin [l-13]. The mechanism of the action of vitamin D on skeletal muscle might be direct, perhaps mediated by changes in calcium balance in intracellular and extracellular compartments, or its action might affect the secretion of other factors such as insulin which, in turn, might influence muscle metabolism [6]. Since vitamin D has been shown to bind to, and affect certain cells in tissue culture [14, 151, we attempted to investigate its effects directly on skeletal muscle cells developing in vitro. In this report we wish to present evidence of inhibition of growth and differentiation of skeletal muscle cells by vitamin D and its metabolites. Fibroblast cells in cultures were not affected in this way. MATERIALS
AND METHODS
Cell Cultures Cell cultures were prepared from muscle tissue and skin of 11-day-old white Leghorn chick embryos [17]. Skeletal muscle tissue samples from the pectoralis major and thigh extensor groups, as well as skin, were separately minced and disaggregated in 0.05% trypsin in Ca’+- and Mgzf-free * To whom offprint requests should be sent. Address: The Hospital for Special Surgery, 535 East 70th Street, New York, NY 10021, USA. 32-848340
Copyright 0 1984 by Academic Press, Inc. All rights of reproduction in any form reserved 0014-4827/84 $03.00
486 Stern and Kagen Table 1. Effect of cholecalciferol on pectoral and thigh muscle
Culture
Protein synthesis (cpm/culture)
Protein content O.&culture)
Thigh + ethanol diluent Thigh + cholecalciferol (lo-’ M) % inhibition Pectoral + ethanol diluent Pectoral + cholecalciferol (10T5 M) % inhibition
76 381+14 931 47 998+6 769” 37.2 67 391+9 057 40 81523 704” 39.4
341.2 266.5 21.9 311.3 232.5 25.0
Myotiber score 2 2 3 2
a Significantly different from control, pCO.01. Average of triplicate determinations + SD shown. Earle’s balanced salt solution (EBSS) at 37°C. After addition of chilled complete medium, the resulting suspensions were collected by centrifugation, filtered through lens paper to remove tissue clumps, and inoculated into plastic dishes (35x 10 mm, Falcon Plastics, Oxnard, Calif.) at 1x lo6 cells per dish in medium (2 ml Eagle’s medium supplemented with 10% fetal calf serum (FCS), 2 mM glutamine, 100 U/ml penicillin, 100 mg/ml streptomycin, and 0.25 mg/ml Fungizone). Pharmacologic agents, as indicated below, were added at the time of cell inoculation. Growth and development of the cultures was assessed as follows. (n) Semiquantitative myotiber score for muscle cell cultures based on phase-microscope evaluation of myotube-myofiber formation (0, no fibers seen; 1+, rare fibers seen in occasional fields; 2+, three to five fibers in most fields; 3+, many fibers in all fields). (6) Cell sap fluids were obtained from cultures on the indicated day of harvest by freezing and thawing x2 in 1 ml EBSS of the adherent cell layer after gentle but thorough rinsing with EBSS (x2) at 37°C. (c) Estimation of protein synthesis by incorporation of radioactive lysine into precipitates formed in 10% trichloroacetic acid (TCA). For these studies [U-‘4C]lysine (New England Nuclear Corp., Boston, Mass) was added, 0.5 @i/ml final concentration, after 2 days of culture and 24 h later the cell sap isolated from the rinsed adherent cell layer was assayed as described [16, 173. (d) Estimation of protein content of cell sap fluids was by the modified Folin method [18]. Creatine kinase (CK) activity was assessed by the modified method of Hughes [19] with reagents supplied by the Sigma Chemical Co., St. Louis, MO. CK isoenzymes were separated by electrophoresis for 20 min in agarose gel utilizing MOPS0 (3-(iV-morpholino) 2-hydroxypropane sulfonic acid) buffer (Corning, Medfield, Mass.), overlaid with a CK substrate solution at 37°C containing creatine phosphate, hexokinase, and glucose&phosphate dehydrogenase, and the reduction of NADP analysed fluorimetrically at 365 nm. For standardization of CK isoenzymes, homogenates of adult chicken brain, heart and thigh were prepared in 0.15 M saline and the resulting identified bands of CKMM, CK-MB, and CK-BB compared with those detected in tissue culture. (e) Ergosterol, cholecalciferol and ergocalciferol were obtained from the Sigma Chemical Co., St. Louis, MO. 25-Hydroxycholecalciferol was the kind gift of the Upjohn Company, Kalamazoo, Mich. These materials were dissolved in 95% ethanol and then further diluted in complete tissue culture medium before use. The final ethanol concentration ranged from 0X1954.95 %. Control cultures to which vitamin D preparations were not added contained 0.95 % ethanol, final concentration. (f) Statistical significance of data was determined by the paired r-test.
RESULTS Inhibition of Muscle Cell Protein Synthesis and Growth by Vitamin D When added to cultures of embryonic skeletal muscle cells, vitamin D inhibited myogenesis, growth and protein synthesis. As indicated in table 1, protein Erp Cell Res 154 (1984)
Effect of vitamin D on muscle cultures
487
14 no cholealclferol -
250H cholecalciferot
--
choledcileral
PROTEIN SYNTHESIS
PROTEIN CONTENT
0
0
10-6 10-7 Vit D analcq concentration Ml
10-s
Fig. I. Effect of vitamin D and its metabolites on protein synthesis of thigh muscle cultures. Average of triplicatedeterminations shown. At 10m5M all metabolites tested were significantly different from control; ~~0.01 at 10m6M, for protein synthesis; ~~0.01 for 2%OH cholecalciferol. Fig. 2. Effect of cholecalciferol on appearance of creatine kinase activity in cytoplasm of thigh muscle cells in culture. Average of triplicate determinations + SD shown. At 10m5M, treated group significantly different from controls, pCO.01 on days 3, 4, 5, 6, 7.
synthesis was depressed with addition of 10e5 M cholecalciferol equivalently in both cultured pectoral and thigh muscle, i.e., 37.2 and 39.4%, respectively. There was also a similar, although slightly less marked, inhibition of protein content in both cultures (21.9 and 25.0%). Myofiber score, or the semiquantitative assessment of myogenesis by microscopic appearance, was unchanged in the case of thigh muscle and slightly depressed in the cultures of pectoral muscle. Action of Vitamin D Metabolites The inhibitory activity of four vitamin D metabolites on protein synthesis in thigh muscle cultures is shown in fig. 1a. Ergosterol had no activity in doses tested from low7 to 10m5M. Ergocalciferol and cholecalciferol were active only at low5 M with 14% greater effect observed in cultures treated with cholecalciferol. The most potent of the agents tested was 25OH cholecalciferol which not only brought about 47% inhibition at 10F5 M but also had demonstrable, although lesser, inhibitory effects at 10V6 and 10m7 M. The effect on total protein content of the cultures was, however, less evident than on protein synthesis. Activity in this regard was demonstrable only at 10F5M with both 25OH cholecalciferol and cholecalciferol causing 22 % inhibition, ergocalciferol being less effective and ergosterol, again having no measurable effect. Exp Cell &s
154 (1984)
488 Stern and Kagen Table 2. Effect of cholecalciferol on creatine kinase and its isoenzymes Thigh muscle cultures treated with: Creatine kinase activity” BB MB MM Total activity
ethanol diluent Day Day Day Day Day Day Day Day
4 5 4 5 4 5 4 5
2.21 1.30 6.54 4.61 3.88 2.99 12.66 8.96
cholecalciferol (lo-’ M)
% Inhibition
2.06 1.41 3.316 1.976 1.36’ 1.316 6.83’ 4.65b
9.3 0 48.5 57.3 64.9 56.2 46.1 41.0
a U/mg prot. ’ Significantly different from control, p
Effect on CK Accumulation During myogenesis in vitro, skeletal muscle cells increase their content of CK rapidly, beginning near the time of fusion on day 2 and continuing for the next 2 days, after which CK activity/ug total protein in the cultures declines (fig. 2). Cholecalciferol-treated cultures were deficient in CK expression as shown, with maximum inhibition observed on days 4-6. This effect is shown in greater detail in table 2. Total CK content of the cells in culture was diminished by 46.1 and 47.0% on days 4 and 5, respectively, by cholecalciferol 10e5 M. However, this effect was not equivalently expressed on the three isoenzyme forms of CK examined. Both CK-MB and CK-MM were diminished (4g-64 %) in cell cultures, whereas CK-BB was essentially unchanged. As controls, the culture medium containing 10% fetal calf serum (FCS) contained essentially no CK activity after incubation for 2 days at 37°C. Cultures of skin fibroblasts produced small amounts of CK, which were too low for electrophoretic analysis using the techniques of collection of cell sap fluids employed for muscle cells. Effect on Protein Synthesis of Muscle Cells Compared with Fibroblasts The effect of cholecalciferol on cultured muscle cells was different from that on skin fibroblasts as shown in fig. 3. Increasing doses of cholecalciferol inhibited protein synthesis in muscle cells, whereas much less inhibition of fibroblast protein synthesis occurred at low5 M and a slight although reproducibly consistent stimulation of tibroblast protein synthesis was evident at 10e6 M. Exp Cell Res 154 (1984)
Effect of vitamin D on muscle cultures
Thigh muscle
Fibroblast
489
Fig. 3. Effect of cholecalciferol on protein synthesis of thigh muscle cell and skin fibroblast cultures. Both culture types established with inoculum size of 1x lo6 cells/dish. Average of triplicate determinations + SD shown. Inhibition by lo-’ M cholecalciferol on muscle cultures signiticant p
DISCUSSION Vitamin D is generally accepted as essential for life in higher animals. Vitamin D and its metabolites are recognized to form an endocrine system with receptorbinding proteins in many tissues, as well as in certain cell lines studied in vitro [20]. Deficiency of vitamin D results in myopathy [7]. Chicks raised on a diet deficient in vitamin D become weak, with diminished ability of the skeletal muscle to produce contractile force, and slowed rate of relaxation of tension after tetanic stimulation. In vitro transport of calcium by the sarcoplasmic reticulum is reduced, as is the calcium content of mitochondria. These effects are, however, not secondary to altered plasma concentrations of calcium and phosphate [8]. Weanling rats made rachitic by dietary deficiency of vitamin D had diminished uptake of calcium by the sarcoplasmic reticulum and reduced concentrations of troponin C [ 121. In rat muscle studied for single isometric contractions, the time to peak tension and recovery time were prolonged in vitamin D-deprived animals. Repletion of vitamin D returned these indices to normal, and neither dietary calcium deficiency nor thyroparathyroidectomy produced similar changes [lo]. In the fish, vitamin D deficiency also leads to myopathic change, with effects chiefly seen in white fibers. These changes are manifested by loss of definition of the Zline and M-band, increase in sarcomere length, shrinkage of the sarcoplasmic reticulum and T-tubules, and the presence of increased intracellular lipids [13]. Myofibrillar protein degradation in the rat was increased by vitamin D deficiency both in vivo and in vitro [6]. In these studies it was suggested that the effects of vitamin D on muscle protein turnover may be brought about by the prevention of hypocalcemia, as well as by stimulation of insulin secretion rather than by a direct effect on skeletal muscle. Muscle, although a target tissue for vitamin D action, does not contain specific Exp Cell Res IS4 (1984)
490 Stern and Kagen 1.25 (OH):! D receptor [14] and although phosphate accumulation in muscle is stimulated by vitamin D, a direct trophic action by the vitamin has not been demonstrated [22]. The concensus of these studies therefore points to an important relation between skeletal muscle metabolism and function and vitamin D; however, it is not clear to what extent vitamin D acts directly on the muscle cell, and to what degree its effects are secondary to changes in other systems. Therefore it was of interest to investigate possible effects directly on muscle tissue by exposing muscle cells to vitamin D and its metabolites under conditions of in vitro culture. When added to cell cultures of embryonic skeletal muscle, protein synthesis and expression of CK, a cellular differentiation product characteristic of muscle were inhibited. Vitamin D and its metabolites had demonstrable action of this sort in concentrations between 10e5 and 10e6 M. The most potent compound tested was 25OH cholecalciferol, with lesser activity apparent with cholecalciferol and ergocalciferol in that order. Ergosterol in doses tested was inactive. Appearance of CK was depressed due to inhibition of CK-MM and CK-MB. CK-BB was essentially unchanged. Since there was a 48% diminution of CK-MB and a greater inhibition of CK-MM (64%) which contains only the M subunit, it is suggested that the action of vitamin D on suppression of CK is directed mainly at the M subunit. This, in turn, suggests that the action of vitamin D on the suppression of protein synthesis may be selective, with some proteins or polypeptides inhibited, and others not. Under the conditions studied, skin fibroblasts were not affected by cholecalciferol in the same way as myogenic cells. They were less sensitive to its inhibitory action and were stimulated at doses of 10v6 M. On the cellular level, the action of vitamin D may also display selectivity. CK is produced in small amounts by fibroblasts [21]. Since the cell cultures studied here were not devoid of fibroblasts, it is possible that some of the resistance to the vitamin D effects on myogenic cultures, particularly of CK-BB, might be due to enzyme contributions of fibroblasts. This hypothesis remains possible, although in the culture system employed, the amount of CK-BB produced by skin fibroblasts analysed electrophoretically in 1 ml unconcentrated cytosol preparations was undetectably small and would not have been apparent in myogenic cell cultures. It is felt therefore that the CK isoenzyme changes observed in this study were the result of myogenie production. REFERENCES 1. 2. 3. 4. 5. 6. 7.
Ritz, E, Boland, R & Kreusser, W, Am j clin nutr 33 (1980) 1522. Floyd, M, Ayyar, D R, Bat-wick, D D, Hudgson, P 8c Weightman, D, Quart j med 43 (1974) 509. Schott, G D & Wills, M R, Lancet 1 (1976) 626. Stroder, J & Arensmeyer, E, Klin Wschr 43 (1965) 1201. Birge, S J & Haddad, J G, J clin invest 56 (1975) 1100. Wassner, S J, Li, J B, Sperduto, A & Norman, M E, J clin invest 72 (1983) 102. Curry, 0 B, Basten, J F, Francis, M J 0 & Smith, R, Nature 249 (1974) 83.
Exp Cell Res 154 (1984)
Effect of vitamin D on muscle cultures
491
8. Pleasure, D, Wyszynski, B, Sumner, A, Schotland, D, Feldman, B, Nugent, N, Hitz, K & Goodman, D B P, J clin invest 64 (1979) 1157. 9. Henderson, R G, Russell, R G G, Ledingham, J G G, Smith, R, Oliver, D 0, Walton, R J, Small, D G, Preston, C, Warner, G T & Norman, A W, Lancet i (1974) 379. 10. Rodnan, J S & Baker, T, Kidney int 13 (1978) 181. 11. Smith, R & Baker, T, Brain 90 (1967) 593. 12. Pointon, J J, Francis, M J 0 & Smith, R, Clin sci 57 (1979) 257. 13. George, J C, Bamett, B J, Cho, C Y & Singer, S J, Cytobios 31 (1981) 7. 14. Franceschi, R T, Simpson, R U & deluca, H F, Arch biochem biophys 210 (1981) 1. 15. Price, P A & Bankol, S A, J biol them 255 (1980) 11660. 16. Kagen, L J & Freedman, A, Exp cell res 88 (1974) 135. 17. Kagen, L J & Miller, S L, Exp neurol 58 (1978) 347. 18. Lowry, 0 H, Rosebrough, N J, Farr, A L & Randall, R J, J biol them 193 (1951) 265. 19. Hughes, B P, Clin chim acta 7 (1962) 597. 20. Norman, A W, Roth, J & Orci, L, Endocrin rev 3 (1982) 331. 21. Davis, M H, Cappel, R, Vester, J W, Samaha, F & Gruenstein, E, Muscle nerve 5 (1982) 1. 22. Bikle, D D, Hagler, L, Lollini, L 0, Hull, S F & Herman, R H, Am j clin nutr 32 (1979) 515. Received January 24, 1984 Revised version received April 10, 1984
Printed
in Sweden
Exp Cell Res 154 (1984)
Effect of Vitamin
D and Its Metabolites Myogenic Cultures
PETER STERN and LAWRENCE The Hospital
on Developing
J. KAGEN*
Division of Rheumatic Diseases, Department of Medicine, for Special Surgery and The New York Hospital- Cornell Medical New York, NY 10021, USA
Center,
Growth, protein synthesis and expression of creatine kinase (CK) by embryonic chick myogenic cells are inhibited by vitamin D and certain of its metabolites. 25OH cholecalciferol was most active in concentrations of 1O-‘-1O-6 M, with cholecalciferol and ergocalciferol less active in that order. Ergosterol had no activity of this sort. Inhibition of CK was most marked on the 4th and 5th day of culture and was due to suppression of the appearance of CK-MM and MB. CK-BB was not affected and CK-MB was more affected than CK-BB. Skin fibroblasts by comparison were slightly stimulated in growth at 10m6M and much less affected at lop5 M than the myogenic cells. It is suggested that vitamin D has a direct effect upon the muscle cell, to cause a selective diminution in the production of certain polypeptides.
Vitamin D is essential in the regulation of calcium and phosphorus homeostasis in higher animals. In addition, a relationship between vitamin D and muscle metabolism has been postulated, since vitamin D deficiency results in development of a myopathy which improves with administration of the vitamin [l-13]. The mechanism of the action of vitamin D on skeletal muscle might be direct, perhaps mediated by changes in calcium balance in intracellular and extracellular compartments, or its action might affect the secretion of other factors such as insulin which, in turn, might influence muscle metabolism [6]. Since vitamin D has been shown to bind to, and affect certain cells in tissue culture [14, 151, we attempted to investigate its effects directly on skeletal muscle cells developing in vitro. In this report we wish to present evidence of inhibition of growth and differentiation of skeletal muscle cells by vitamin D and its metabolites. Fibroblast cells in cultures were not affected in this way. MATERIALS
AND METHODS
Cell Cultures Cell cultures were prepared from muscle tissue and skin of 11-day-old white Leghorn chick embryos [17]. Skeletal muscle tissue samples from the pectoralis major and thigh extensor groups, as well as skin, were separately minced and disaggregated in 0.05% trypsin in Ca’+- and Mgzf-free * To whom offprint requests should be sent. Address: The Hospital for Special Surgery, 535 East 70th Street, New York, NY 10021, USA. 32-848340
Copyright 0 1984 by Academic Press, Inc. All rights of reproduction in any form reserved 0014-4827/84 $03.00
486 Stern and Kagen Table 1. Effect of cholecalciferol on pectoral and thigh muscle
Culture
Protein synthesis (cpm/culture)
Protein content O.&culture)
Thigh + ethanol diluent Thigh + cholecalciferol (lo-’ M) % inhibition Pectoral + ethanol diluent Pectoral + cholecalciferol (10T5 M) % inhibition
76 381+14 931 47 998+6 769” 37.2 67 391+9 057 40 81523 704” 39.4
341.2 266.5 21.9 311.3 232.5 25.0
Myotiber score 2 2 3 2
a Significantly different from control, pCO.01. Average of triplicate determinations + SD shown. Earle’s balanced salt solution (EBSS) at 37°C. After addition of chilled complete medium, the resulting suspensions were collected by centrifugation, filtered through lens paper to remove tissue clumps, and inoculated into plastic dishes (35x 10 mm, Falcon Plastics, Oxnard, Calif.) at 1x lo6 cells per dish in medium (2 ml Eagle’s medium supplemented with 10% fetal calf serum (FCS), 2 mM glutamine, 100 U/ml penicillin, 100 mg/ml streptomycin, and 0.25 mg/ml Fungizone). Pharmacologic agents, as indicated below, were added at the time of cell inoculation. Growth and development of the cultures was assessed as follows. (n) Semiquantitative myotiber score for muscle cell cultures based on phase-microscope evaluation of myotube-myofiber formation (0, no fibers seen; 1+, rare fibers seen in occasional fields; 2+, three to five fibers in most fields; 3+, many fibers in all fields). (6) Cell sap fluids were obtained from cultures on the indicated day of harvest by freezing and thawing x2 in 1 ml EBSS of the adherent cell layer after gentle but thorough rinsing with EBSS (x2) at 37°C. (c) Estimation of protein synthesis by incorporation of radioactive lysine into precipitates formed in 10% trichloroacetic acid (TCA). For these studies [U-‘4C]lysine (New England Nuclear Corp., Boston, Mass) was added, 0.5 @i/ml final concentration, after 2 days of culture and 24 h later the cell sap isolated from the rinsed adherent cell layer was assayed as described [16, 173. (d) Estimation of protein content of cell sap fluids was by the modified Folin method [18]. Creatine kinase (CK) activity was assessed by the modified method of Hughes [19] with reagents supplied by the Sigma Chemical Co., St. Louis, MO. CK isoenzymes were separated by electrophoresis for 20 min in agarose gel utilizing MOPS0 (3-(iV-morpholino) 2-hydroxypropane sulfonic acid) buffer (Corning, Medfield, Mass.), overlaid with a CK substrate solution at 37°C containing creatine phosphate, hexokinase, and glucose&phosphate dehydrogenase, and the reduction of NADP analysed fluorimetrically at 365 nm. For standardization of CK isoenzymes, homogenates of adult chicken brain, heart and thigh were prepared in 0.15 M saline and the resulting identified bands of CKMM, CK-MB, and CK-BB compared with those detected in tissue culture. (e) Ergosterol, cholecalciferol and ergocalciferol were obtained from the Sigma Chemical Co., St. Louis, MO. 25-Hydroxycholecalciferol was the kind gift of the Upjohn Company, Kalamazoo, Mich. These materials were dissolved in 95% ethanol and then further diluted in complete tissue culture medium before use. The final ethanol concentration ranged from 0X1954.95 %. Control cultures to which vitamin D preparations were not added contained 0.95 % ethanol, final concentration. (f) Statistical significance of data was determined by the paired r-test.
RESULTS Inhibition of Muscle Cell Protein Synthesis and Growth by Vitamin D When added to cultures of embryonic skeletal muscle cells, vitamin D inhibited myogenesis, growth and protein synthesis. As indicated in table 1, protein Erp Cell Res 154 (1984)
Effect of vitamin D on muscle cultures
487
14 no cholealclferol -
250H cholecalciferot
--
choledcileral
PROTEIN SYNTHESIS
PROTEIN CONTENT
0
0
10-6 10-7 Vit D analcq concentration Ml
10-s
Fig. I. Effect of vitamin D and its metabolites on protein synthesis of thigh muscle cultures. Average of triplicatedeterminations shown. At 10m5M all metabolites tested were significantly different from control; ~~0.01 at 10m6M, for protein synthesis; ~~0.01 for 2%OH cholecalciferol. Fig. 2. Effect of cholecalciferol on appearance of creatine kinase activity in cytoplasm of thigh muscle cells in culture. Average of triplicate determinations + SD shown. At 10m5M, treated group significantly different from controls, pCO.01 on days 3, 4, 5, 6, 7.
synthesis was depressed with addition of 10e5 M cholecalciferol equivalently in both cultured pectoral and thigh muscle, i.e., 37.2 and 39.4%, respectively. There was also a similar, although slightly less marked, inhibition of protein content in both cultures (21.9 and 25.0%). Myofiber score, or the semiquantitative assessment of myogenesis by microscopic appearance, was unchanged in the case of thigh muscle and slightly depressed in the cultures of pectoral muscle. Action of Vitamin D Metabolites The inhibitory activity of four vitamin D metabolites on protein synthesis in thigh muscle cultures is shown in fig. 1a. Ergosterol had no activity in doses tested from low7 to 10m5M. Ergocalciferol and cholecalciferol were active only at low5 M with 14% greater effect observed in cultures treated with cholecalciferol. The most potent of the agents tested was 25OH cholecalciferol which not only brought about 47% inhibition at 10F5 M but also had demonstrable, although lesser, inhibitory effects at 10V6 and 10m7 M. The effect on total protein content of the cultures was, however, less evident than on protein synthesis. Activity in this regard was demonstrable only at 10F5M with both 25OH cholecalciferol and cholecalciferol causing 22 % inhibition, ergocalciferol being less effective and ergosterol, again having no measurable effect. Exp Cell &s
154 (1984)
488 Stern and Kagen Table 2. Effect of cholecalciferol on creatine kinase and its isoenzymes Thigh muscle cultures treated with: Creatine kinase activity” BB MB MM Total activity
ethanol diluent Day Day Day Day Day Day Day Day
4 5 4 5 4 5 4 5
2.21 1.30 6.54 4.61 3.88 2.99 12.66 8.96
cholecalciferol (lo-’ M)
% Inhibition
2.06 1.41 3.316 1.976 1.36’ 1.316 6.83’ 4.65b
9.3 0 48.5 57.3 64.9 56.2 46.1 41.0
a U/mg prot. ’ Significantly different from control, p
Effect on CK Accumulation During myogenesis in vitro, skeletal muscle cells increase their content of CK rapidly, beginning near the time of fusion on day 2 and continuing for the next 2 days, after which CK activity/ug total protein in the cultures declines (fig. 2). Cholecalciferol-treated cultures were deficient in CK expression as shown, with maximum inhibition observed on days 4-6. This effect is shown in greater detail in table 2. Total CK content of the cells in culture was diminished by 46.1 and 47.0% on days 4 and 5, respectively, by cholecalciferol 10e5 M. However, this effect was not equivalently expressed on the three isoenzyme forms of CK examined. Both CK-MB and CK-MM were diminished (4g-64 %) in cell cultures, whereas CK-BB was essentially unchanged. As controls, the culture medium containing 10% fetal calf serum (FCS) contained essentially no CK activity after incubation for 2 days at 37°C. Cultures of skin fibroblasts produced small amounts of CK, which were too low for electrophoretic analysis using the techniques of collection of cell sap fluids employed for muscle cells. Effect on Protein Synthesis of Muscle Cells Compared with Fibroblasts The effect of cholecalciferol on cultured muscle cells was different from that on skin fibroblasts as shown in fig. 3. Increasing doses of cholecalciferol inhibited protein synthesis in muscle cells, whereas much less inhibition of fibroblast protein synthesis occurred at low5 M and a slight although reproducibly consistent stimulation of tibroblast protein synthesis was evident at 10e6 M. Exp Cell Res 154 (1984)
Effect of vitamin D on muscle cultures
Thigh muscle
Fibroblast
489
Fig. 3. Effect of cholecalciferol on protein synthesis of thigh muscle cell and skin fibroblast cultures. Both culture types established with inoculum size of 1x lo6 cells/dish. Average of triplicate determinations + SD shown. Inhibition by lo-’ M cholecalciferol on muscle cultures signiticant p
DISCUSSION Vitamin D is generally accepted as essential for life in higher animals. Vitamin D and its metabolites are recognized to form an endocrine system with receptorbinding proteins in many tissues, as well as in certain cell lines studied in vitro [20]. Deficiency of vitamin D results in myopathy [7]. Chicks raised on a diet deficient in vitamin D become weak, with diminished ability of the skeletal muscle to produce contractile force, and slowed rate of relaxation of tension after tetanic stimulation. In vitro transport of calcium by the sarcoplasmic reticulum is reduced, as is the calcium content of mitochondria. These effects are, however, not secondary to altered plasma concentrations of calcium and phosphate [8]. Weanling rats made rachitic by dietary deficiency of vitamin D had diminished uptake of calcium by the sarcoplasmic reticulum and reduced concentrations of troponin C [ 121. In rat muscle studied for single isometric contractions, the time to peak tension and recovery time were prolonged in vitamin D-deprived animals. Repletion of vitamin D returned these indices to normal, and neither dietary calcium deficiency nor thyroparathyroidectomy produced similar changes [lo]. In the fish, vitamin D deficiency also leads to myopathic change, with effects chiefly seen in white fibers. These changes are manifested by loss of definition of the Zline and M-band, increase in sarcomere length, shrinkage of the sarcoplasmic reticulum and T-tubules, and the presence of increased intracellular lipids [13]. Myofibrillar protein degradation in the rat was increased by vitamin D deficiency both in vivo and in vitro [6]. In these studies it was suggested that the effects of vitamin D on muscle protein turnover may be brought about by the prevention of hypocalcemia, as well as by stimulation of insulin secretion rather than by a direct effect on skeletal muscle. Muscle, although a target tissue for vitamin D action, does not contain specific Exp Cell Res IS4 (1984)
490 Stern and Kagen 1.25 (OH):! D receptor [14] and although phosphate accumulation in muscle is stimulated by vitamin D, a direct trophic action by the vitamin has not been demonstrated [22]. The concensus of these studies therefore points to an important relation between skeletal muscle metabolism and function and vitamin D; however, it is not clear to what extent vitamin D acts directly on the muscle cell, and to what degree its effects are secondary to changes in other systems. Therefore it was of interest to investigate possible effects directly on muscle tissue by exposing muscle cells to vitamin D and its metabolites under conditions of in vitro culture. When added to cell cultures of embryonic skeletal muscle, protein synthesis and expression of CK, a cellular differentiation product characteristic of muscle were inhibited. Vitamin D and its metabolites had demonstrable action of this sort in concentrations between 10e5 and 10e6 M. The most potent compound tested was 25OH cholecalciferol, with lesser activity apparent with cholecalciferol and ergocalciferol in that order. Ergosterol in doses tested was inactive. Appearance of CK was depressed due to inhibition of CK-MM and CK-MB. CK-BB was essentially unchanged. Since there was a 48% diminution of CK-MB and a greater inhibition of CK-MM (64%) which contains only the M subunit, it is suggested that the action of vitamin D on suppression of CK is directed mainly at the M subunit. This, in turn, suggests that the action of vitamin D on the suppression of protein synthesis may be selective, with some proteins or polypeptides inhibited, and others not. Under the conditions studied, skin fibroblasts were not affected by cholecalciferol in the same way as myogenic cells. They were less sensitive to its inhibitory action and were stimulated at doses of 10v6 M. On the cellular level, the action of vitamin D may also display selectivity. CK is produced in small amounts by fibroblasts [21]. Since the cell cultures studied here were not devoid of fibroblasts, it is possible that some of the resistance to the vitamin D effects on myogenic cultures, particularly of CK-BB, might be due to enzyme contributions of fibroblasts. This hypothesis remains possible, although in the culture system employed, the amount of CK-BB produced by skin fibroblasts analysed electrophoretically in 1 ml unconcentrated cytosol preparations was undetectably small and would not have been apparent in myogenic cell cultures. It is felt therefore that the CK isoenzyme changes observed in this study were the result of myogenie production. REFERENCES 1. 2. 3. 4. 5. 6. 7.
Ritz, E, Boland, R & Kreusser, W, Am j clin nutr 33 (1980) 1522. Floyd, M, Ayyar, D R, Bat-wick, D D, Hudgson, P 8c Weightman, D, Quart j med 43 (1974) 509. Schott, G D & Wills, M R, Lancet 1 (1976) 626. Stroder, J & Arensmeyer, E, Klin Wschr 43 (1965) 1201. Birge, S J & Haddad, J G, J clin invest 56 (1975) 1100. Wassner, S J, Li, J B, Sperduto, A & Norman, M E, J clin invest 72 (1983) 102. Curry, 0 B, Basten, J F, Francis, M J 0 & Smith, R, Nature 249 (1974) 83.
Exp Cell Res 154 (1984)
Effect of vitamin D on muscle cultures
491
8. Pleasure, D, Wyszynski, B, Sumner, A, Schotland, D, Feldman, B, Nugent, N, Hitz, K & Goodman, D B P, J clin invest 64 (1979) 1157. 9. Henderson, R G, Russell, R G G, Ledingham, J G G, Smith, R, Oliver, D 0, Walton, R J, Small, D G, Preston, C, Warner, G T & Norman, A W, Lancet i (1974) 379. 10. Rodnan, J S & Baker, T, Kidney int 13 (1978) 181. 11. Smith, R & Baker, T, Brain 90 (1967) 593. 12. Pointon, J J, Francis, M J 0 & Smith, R, Clin sci 57 (1979) 257. 13. George, J C, Bamett, B J, Cho, C Y & Singer, S J, Cytobios 31 (1981) 7. 14. Franceschi, R T, Simpson, R U & deluca, H F, Arch biochem biophys 210 (1981) 1. 15. Price, P A & Bankol, S A, J biol them 255 (1980) 11660. 16. Kagen, L J & Freedman, A, Exp cell res 88 (1974) 135. 17. Kagen, L J & Miller, S L, Exp neurol 58 (1978) 347. 18. Lowry, 0 H, Rosebrough, N J, Farr, A L & Randall, R J, J biol them 193 (1951) 265. 19. Hughes, B P, Clin chim acta 7 (1962) 597. 20. Norman, A W, Roth, J & Orci, L, Endocrin rev 3 (1982) 331. 21. Davis, M H, Cappel, R, Vester, J W, Samaha, F & Gruenstein, E, Muscle nerve 5 (1982) 1. 22. Bikle, D D, Hagler, L, Lollini, L 0, Hull, S F & Herman, R H, Am j clin nutr 32 (1979) 515. Received January 24, 1984 Revised version received April 10, 1984
Printed
in Sweden
Exp Cell Res 154 (1984)