EXPERIMENTAL
CELL
RESEARCH
195,
284-294
(19%)
The Biochemical and Structural Maturation of Human Skeletal Muscle Cells in Culture: The Effect of the Serum Substitute Ultroser G AD A.G.M.
BENDERS,TOIN H.M. Department
S. M. VANKUPPEVELT,ARIEOOSTERHOF,ANDJACQUESH.VEERKAMP'
of Biochemistry,
University
of Nijmegen, Nijmegen,
On the basis of the percentage creatine kinase-MM, human skeletal muscle cells cultured on growth and differentiation media containing the serum substitute Ultroser G reach a significantly higher maturation grade after 7 days of differentiation than cells cultured on serum-containing media. They also remain viable for longer periods. The myotubes are much longer, their nuclei are often localized in rows on the periphery, and they show cross-striation more frequently. The activities of creatine kinase, citrate synthase, cytochrome c oxidase, AMP deaminase, and phosphorylase are significantly higher. Extending the differentiation period to 3 weeks increases the maturation grade of the cultures and the activities of all the enzymes mentioned before, except phosphorylase. A correlation exists between the enzyme activities and the maturation grade of the muscle cells. The content of fatty acid-binding protein also increases significantly with the maturation grade in contrast to the palmitate oxidation rate. The AMP deaminase and creatine kinase activity and the percentage MM-type remain lower in cultured cells than in adult muscle and the hexokinase activity remains higher, but the other enzyme activities become comparable after 20 days of differentiation. The myotubes, derived from Ultroser G-containing culture media, show spontaneous contractions after 12 days and cross-striation after 20 days when immunostained for the M-subunit of creatine kinase. These cells possess clusters of acetylcholine receptors, but aggregation of desmin at the site of the clusters was never detectable. The possibility of cultivating muscle cells with a predictable maturation grade allows the study of muscle development and muscular diseases caused by differentiation defects or by deficiency of a maturation-dependent (iso)en8 1991 Academic Press, Inc. zyme.
INTRODUCTION Human muscle cell cultures obtained from biopsies are useful for studying various aspects of muscle cell 1 To whom correspondence and reprint requests should be addressed at: Department of Biochemistry, University of Nijmegen, P.O. Box 9101,650O HB Nijmegen, The Netherlands. 0014.4827/91 $3.00 Copyright 0 1991 by Academic Press, All rights of reproduction in any form
284 Inc. reserved.
The Netherlands
metabolism [l-9]. Many abnormalities of muscle can be reproduced in cell cultures derived from pathological muscle, but in other primary muscle defects, cultures do not show the expression of the disease [Z, 3, lo]. Compared to chicken and rat muscle cells, aneurally cultured human muscle cells remain relatively immature [ll, 121. They do not approach the degree of maturation achieved by adult human muscle with regard to creatine kinase (CK) activity and the CK isoenzyme pattern [3, 4, 9, 131. Some mitochondrial activities are, however, of the same order as in adult human muscle [4,9]. Spontaneous contractions are rarely observed and cells degenerate after 3 weeks [4, 5, 91. In contrast, muscle cells cultured in the presence of nerve cells can be maintained for several months, show spontaneous contractions, and are biochemically more mature [2, 13-161. Investigations with media containing serum and chicken embryo extract gave results consistent with the interpretation that mitogens block differentiation by forcing the cells to enter the S phase of the cell cycle and thus undergo another round of replication [14, 171. Therefore the need for more defined media increased and during the past decade the synergistic influence of trophic factors and polypeptide growth factors on cultured mammalian muscle cells became clear [17-261. In this study we compared the influence of serumcontaining media and the serum substitute, Ultroser G, on the growth and differentiation of cultured human skeletal muscle cells. Ultroser G has successfully been applied for the culture of a wide variety of cells such as human skin fibroblasts [27,28] and rabbit chondrocytes [29] and for cell lines such as MCDK [30] and HeLa [31]. W e also looked for the influence of rat brain extract, since this stimulates fusion of myoblasts and the subsequent differentiation and maintenance of chicken skeletal muscle cells in vitro [32]. W e studied the effect of the composition of the culture media and the duration of the differentiation period by biochemical and immunocytochemical methods. As parameters of metabolism we investigated the activities of CK and its muscle-specific isoenzyme CK-MM, the mitochondrial marker enzymes citrate synthase and cytochrome c oxidase, the glycolytic enzyme hexokinase, and the glycogenolytic enzyme phosphorylase. W e
MATURATION
TABLE
OF CULTURED
1
Composition of Growth and Differentiation Fetal calf serum Growth media A B C Differentiation I II III IV V VI
Ultroser G
Horse serum
20
Media
Chicken embryo extract
Rat brain
extract
2 2
4 4
10
media 10 2 2 2 0.4
2 2 10 10 10
Note. The growth and differentiation media contain DMEM and the above-mentioned additions. Volume percentages are given. Ultroser G contains a.o. insulin, epidermal growth factor, fibroblast growth factor, transforming growth factor, insulin-like growth factor I, thyroxine, dexamethasone, and bovine serum albumin.
also studied the activity of AMP deaminase, which plays a critical role in muscle energy metabolism; the content of fatty acid-binding protein (FABP); and the palmitate oxidation rate as indicators for fatty acid metabolism. In order to evaluate the sarcomeric organization of myotubes, immunostaining for myosin, the M subunit of CK, and desmin was carried out. The distribution of acetylcholine receptors (AChRs) was studied as well with a-bungarotoxin and specific antibodies.
METHODS
AND MATERIALS
Culture media and buffer. Dulbecco’s modified phosphate-buffered saline (DPBS) contains 145 mM NaCl, 5.4 mM KCl, 5 mM Na,HPO,, 25 mM glucose, and 25 mM sucrose (pH 7.3). The growth and differentiation media used were composed of Dulbecco’s modified Eagle’s medium (DMEM) with the additions indicated in Table 1. For preparation of rat brain extract, brains from lo- to 11-day-old Wistar albino rats were homogenized (100 mg tissue/ml DMEM) manually at 0°C in a PotterrElvehjem glass-Teflon homogenizer using a pestle with a clearance of 0.05 mm (5 strokes). The homogenate was centrifuged for 1 h at 100,OOOg and the supernatant was stored at -7O’C until used. Muscle cell cultures. Human muscle biopsies were obtained from individuals without any known metabolic muscular disorder. Biopsies from m. rectus abdominis and m. gluteus were obtained by open biopsy (100-500 mg) and biopsies from m. quadriceps by open (loo250 mg) or percutaneous needle biopsy (25-50 mg). The biopsies were dissociated according to the dispersion technique of Yasin et al. [33]. The obtained satellite cells were plated into 35.mm tissue culture dishes (lo5 cells per dish in 2 ml growth medium A). After 24 h cell debris and nonadherent cells were removed by washing with DPBS. From this moment on, the cells were cultured on the various growth media. At confluency (4 x 10’ cells per dish) the cells were subcultured as described previously [4]. When confluency was reached again
HUMAN
MUSCLE
CELLS
the growth media were replaced by the different differentiation dia. The cells were cultured in a humidified CO, atmosphere at and all media were refreshed every third day. All cultures were vested after 7 days of onset of myoblast fusion, unless indicated erwise.
285 me37°C haroth-
Biochemical assays. Homogenates of cultured muscle cells (l-5 mg protein/ml) and biopsies of m. quadriceps (5% w/v) were prepared in 250 mM sucrose/2 mM EDTA/lO mM Tris-HCl (pH 7.4) as described by Zuurveld et al. [4]. Creatine kinase (EC 2.7.3.2) activity was determined with the CK N-acetylcysteine-activated monotest at 37°C. Units of activity represent micromoles of NADP reduced per minute. The CK isoenzyme MM was separated from other CK isoenzymes by anion exchange chromatography essentially according to Jacobs et al. [9]. Citrate synthase (EC 4.1.3.7) activity was measured in sonicated homogenates as described earlier by Shepherd and Garland [34]. Cytochrome c oxidase (EC 1.9.3.1) activity was assayed according to Van Hinsbergh et al. [35]. Units of activity of these enzymes represent micromoles of coenzyme A liberated and of cytochrome c oxidized per minute at 25”C, respectively. AMP deaminase (EC 3.5.4.6) activity was radiochemically determined at 37°C following the conversion of 5 mM [8-i4C]AMP to IMP and inosine [36]. Hexokinase (EC 2.7.1.1) activity was measured as described by Henriksson et al. [37] and units of activity represent micromoles of NADP reduced per min at 37’C. Phosphorylase (EC 2.4.1.1) activity was determined as described by Martinuzzi et al. [13] with the modification that the reduction of NADP was directly spectrophotometrically measured. Units of activity represent micromoles of D-glucose-l-phosphate formed per minute at 37°C. The content of fatty acid-binding protein was assayed by an enzyme-linked immunosorbent assay according to Paulussen et al. [38]. The palmitate oxidation rate was measured with 120 &f [l-‘4C]palmitic acid bound to 24 FM albumin at 37°C and calculated from the production of 14C0, and 14C-labeled acid-soluble products [4]. Protein content was assayed according to Lowry et al. with bovine serum albumin as standard [39]. Statistics. Data represent means f SD. Statistical analysis was performed by means of the unpaired Student’s t test and significance was set at P
CELL
RESEARCH
195,
284-294
(19%)
The Biochemical and Structural Maturation of Human Skeletal Muscle Cells in Culture: The Effect of the Serum Substitute Ultroser G AD A.G.M.
BENDERS,TOIN H.M. Department
S. M. VANKUPPEVELT,ARIEOOSTERHOF,ANDJACQUESH.VEERKAMP'
of Biochemistry,
University
of Nijmegen, Nijmegen,
On the basis of the percentage creatine kinase-MM, human skeletal muscle cells cultured on growth and differentiation media containing the serum substitute Ultroser G reach a significantly higher maturation grade after 7 days of differentiation than cells cultured on serum-containing media. They also remain viable for longer periods. The myotubes are much longer, their nuclei are often localized in rows on the periphery, and they show cross-striation more frequently. The activities of creatine kinase, citrate synthase, cytochrome c oxidase, AMP deaminase, and phosphorylase are significantly higher. Extending the differentiation period to 3 weeks increases the maturation grade of the cultures and the activities of all the enzymes mentioned before, except phosphorylase. A correlation exists between the enzyme activities and the maturation grade of the muscle cells. The content of fatty acid-binding protein also increases significantly with the maturation grade in contrast to the palmitate oxidation rate. The AMP deaminase and creatine kinase activity and the percentage MM-type remain lower in cultured cells than in adult muscle and the hexokinase activity remains higher, but the other enzyme activities become comparable after 20 days of differentiation. The myotubes, derived from Ultroser G-containing culture media, show spontaneous contractions after 12 days and cross-striation after 20 days when immunostained for the M-subunit of creatine kinase. These cells possess clusters of acetylcholine receptors, but aggregation of desmin at the site of the clusters was never detectable. The possibility of cultivating muscle cells with a predictable maturation grade allows the study of muscle development and muscular diseases caused by differentiation defects or by deficiency of a maturation-dependent (iso)en8 1991 Academic Press, Inc. zyme.
INTRODUCTION Human muscle cell cultures obtained from biopsies are useful for studying various aspects of muscle cell 1 To whom correspondence and reprint requests should be addressed at: Department of Biochemistry, University of Nijmegen, P.O. Box 9101,650O HB Nijmegen, The Netherlands. 0014.4827/91 $3.00 Copyright 0 1991 by Academic Press, All rights of reproduction in any form
284 Inc. reserved.
The Netherlands
metabolism [l-9]. Many abnormalities of muscle can be reproduced in cell cultures derived from pathological muscle, but in other primary muscle defects, cultures do not show the expression of the disease [Z, 3, lo]. Compared to chicken and rat muscle cells, aneurally cultured human muscle cells remain relatively immature [ll, 121. They do not approach the degree of maturation achieved by adult human muscle with regard to creatine kinase (CK) activity and the CK isoenzyme pattern [3, 4, 9, 131. Some mitochondrial activities are, however, of the same order as in adult human muscle [4,9]. Spontaneous contractions are rarely observed and cells degenerate after 3 weeks [4, 5, 91. In contrast, muscle cells cultured in the presence of nerve cells can be maintained for several months, show spontaneous contractions, and are biochemically more mature [2, 13-161. Investigations with media containing serum and chicken embryo extract gave results consistent with the interpretation that mitogens block differentiation by forcing the cells to enter the S phase of the cell cycle and thus undergo another round of replication [14, 171. Therefore the need for more defined media increased and during the past decade the synergistic influence of trophic factors and polypeptide growth factors on cultured mammalian muscle cells became clear [17-261. In this study we compared the influence of serumcontaining media and the serum substitute, Ultroser G, on the growth and differentiation of cultured human skeletal muscle cells. Ultroser G has successfully been applied for the culture of a wide variety of cells such as human skin fibroblasts [27,28] and rabbit chondrocytes [29] and for cell lines such as MCDK [30] and HeLa [31]. W e also looked for the influence of rat brain extract, since this stimulates fusion of myoblasts and the subsequent differentiation and maintenance of chicken skeletal muscle cells in vitro [32]. W e studied the effect of the composition of the culture media and the duration of the differentiation period by biochemical and immunocytochemical methods. As parameters of metabolism we investigated the activities of CK and its muscle-specific isoenzyme CK-MM, the mitochondrial marker enzymes citrate synthase and cytochrome c oxidase, the glycolytic enzyme hexokinase, and the glycogenolytic enzyme phosphorylase. W e
MATURATION
TABLE
OF CULTURED
1
Composition of Growth and Differentiation Fetal calf serum Growth media A B C Differentiation I II III IV V VI
Ultroser G
Horse serum
20
Media
Chicken embryo extract
Rat brain
extract
2 2
4 4
10
media 10 2 2 2 0.4
2 2 10 10 10
Note. The growth and differentiation media contain DMEM and the above-mentioned additions. Volume percentages are given. Ultroser G contains a.o. insulin, epidermal growth factor, fibroblast growth factor, transforming growth factor, insulin-like growth factor I, thyroxine, dexamethasone, and bovine serum albumin.
also studied the activity of AMP deaminase, which plays a critical role in muscle energy metabolism; the content of fatty acid-binding protein (FABP); and the palmitate oxidation rate as indicators for fatty acid metabolism. In order to evaluate the sarcomeric organization of myotubes, immunostaining for myosin, the M subunit of CK, and desmin was carried out. The distribution of acetylcholine receptors (AChRs) was studied as well with a-bungarotoxin and specific antibodies.
METHODS
AND MATERIALS
Culture media and buffer. Dulbecco’s modified phosphate-buffered saline (DPBS) contains 145 mM NaCl, 5.4 mM KCl, 5 mM Na,HPO,, 25 mM glucose, and 25 mM sucrose (pH 7.3). The growth and differentiation media used were composed of Dulbecco’s modified Eagle’s medium (DMEM) with the additions indicated in Table 1. For preparation of rat brain extract, brains from lo- to 11-day-old Wistar albino rats were homogenized (100 mg tissue/ml DMEM) manually at 0°C in a PotterrElvehjem glass-Teflon homogenizer using a pestle with a clearance of 0.05 mm (5 strokes). The homogenate was centrifuged for 1 h at 100,OOOg and the supernatant was stored at -7O’C until used. Muscle cell cultures. Human muscle biopsies were obtained from individuals without any known metabolic muscular disorder. Biopsies from m. rectus abdominis and m. gluteus were obtained by open biopsy (100-500 mg) and biopsies from m. quadriceps by open (loo250 mg) or percutaneous needle biopsy (25-50 mg). The biopsies were dissociated according to the dispersion technique of Yasin et al. [33]. The obtained satellite cells were plated into 35.mm tissue culture dishes (lo5 cells per dish in 2 ml growth medium A). After 24 h cell debris and nonadherent cells were removed by washing with DPBS. From this moment on, the cells were cultured on the various growth media. At confluency (4 x 10’ cells per dish) the cells were subcultured as described previously [4]. When confluency was reached again
HUMAN
MUSCLE
CELLS
the growth media were replaced by the different differentiation dia. The cells were cultured in a humidified CO, atmosphere at and all media were refreshed every third day. All cultures were vested after 7 days of onset of myoblast fusion, unless indicated erwise.
285 me37°C haroth-
Biochemical assays. Homogenates of cultured muscle cells (l-5 mg protein/ml) and biopsies of m. quadriceps (5% w/v) were prepared in 250 mM sucrose/2 mM EDTA/lO mM Tris-HCl (pH 7.4) as described by Zuurveld et al. [4]. Creatine kinase (EC 2.7.3.2) activity was determined with the CK N-acetylcysteine-activated monotest at 37°C. Units of activity represent micromoles of NADP reduced per minute. The CK isoenzyme MM was separated from other CK isoenzymes by anion exchange chromatography essentially according to Jacobs et al. [9]. Citrate synthase (EC 4.1.3.7) activity was measured in sonicated homogenates as described earlier by Shepherd and Garland [34]. Cytochrome c oxidase (EC 1.9.3.1) activity was assayed according to Van Hinsbergh et al. [35]. Units of activity of these enzymes represent micromoles of coenzyme A liberated and of cytochrome c oxidized per minute at 25”C, respectively. AMP deaminase (EC 3.5.4.6) activity was radiochemically determined at 37°C following the conversion of 5 mM [8-i4C]AMP to IMP and inosine [36]. Hexokinase (EC 2.7.1.1) activity was measured as described by Henriksson et al. [37] and units of activity represent micromoles of NADP reduced per min at 37’C. Phosphorylase (EC 2.4.1.1) activity was determined as described by Martinuzzi et al. [13] with the modification that the reduction of NADP was directly spectrophotometrically measured. Units of activity represent micromoles of D-glucose-l-phosphate formed per minute at 37°C. The content of fatty acid-binding protein was assayed by an enzyme-linked immunosorbent assay according to Paulussen et al. [38]. The palmitate oxidation rate was measured with 120 &f [l-‘4C]palmitic acid bound to 24 FM albumin at 37°C and calculated from the production of 14C0, and 14C-labeled acid-soluble products [4]. Protein content was assayed according to Lowry et al. with bovine serum albumin as standard [39]. Statistics. Data represent means f SD. Statistical analysis was performed by means of the unpaired Student’s t test and significance was set at P
