225
Clinica Chimica Acta, 96 (1979) 225-231 @ Elsevier/North-Holland Biomedical Press
CCA 1082
DUCHENNE MUSCULAR DYSTROPHY: 45Ca EXCHANGE IN CULTURED SKIN FIBROBLASTS AND THE EFFECT OF CALCIUM IONOPHORE A23187
HELEN
E. STATHAM
* and VICTOR
DUBOWITZ
Jerry Lewis Muscle Research Centre and Department of Paediatrics and Neonatal Medicine (Institute of Child Health), Hammersmith Hospital, London, W12 OHS (U.K.) (Received
March 23rd, 1979)
Summary
Calcium exchange was studied in skin fibroblasts cultured from eight subjects with Duchenne muscular dystrophy, four with Limb Girdle dystrophy and eight normal controls using 45Ca. No difference was found in the time course of calcium exchange between the groups, nor in the level of 45Ca when maximal exchange had occurred. Treatment of the cultures with the calcium ionophore A23187 resulted in higher levels of calcium exchange over a 2-h period. The increase was similar in the cultures from the 3 patient groups studied.
Introduction
Duchenne muscular dystrophy (DMD) is an X-linked inherited disease, characterized by progressive degeneration of skeletal muscle and elevated serum levels of muscle-type creatine kinase. Although the basic defect in DMD is unknown, there is evidence to suggest that the plasma membrane of muscle, and of other tissues not obviously affected by the disease, may be abnormal. Structural and functional abnormalities have been reported for DMD muscle plasma membranes [ 1,2] and DMD erythrocytes [ 3-71. Platelets from DMD patients have reduced levels of [ 14C]serotonin incorporation [ 81 and interstitial cell nuclei in muscle biopsies have the same elevated calcium : phosphorus ratio found in muscle cell nuclei [ 91. Defects have also been observed in cells from DMD patients grown in tissue culture. Abnormalities in adenyl cyclase activity [lo] and in protein synthesis [ 111 have been described in DMD myotubes and unusual ‘clumping’ behaviour has been observed in mononuclear cells cultured from DMD muscle [ 121. Skin * To
whom
correspondence
should
be addressed.
226
fibroblasts from DMD patients have also been reported to have defects in ultrastructure [ 131 and in protein synthesis [ 141 but all of these reports are so far unconfirmed. Many of the abnormalities described for DMD patients and tissues are in some way connected with membrane calcium transport [6,15--171. In this study, we have examined some aspects of the calcium transport mechanisms of cultured skin fibroblasts from DMD patients, normal controls and patients with other neuromuscular disorders, to try to establish whether the reported defects in calcium transport systems might be part of the primary defect in DMD. Methods Fibroblast cultures were established from skin biopsies from patients with neuromuscular diseases, taken while subjects were undergoing muscle biopsy and from controls while undergoing surgery. Clinical details of the subjects are shown in Table I; neuro-muscular disorders were diagnosed by clinical assessment, serum enzyme levels and muscle biopsy. Primary fibroblast cultures were grown from 1 mm3 explants of skin in 25 cm* Falcon tissue culture flasks. The first subculture was usually made within 6-8 weeks of explanting the skin. Cells were maintained routinely in 75 cm* Lux flasks, sub-culturing as necessary with 0.25% trypsin in Hanks salt solution (HSS) [ 181 without calcium and magnesium. Tissue culture medium throughout was Eagles basal medium (BME), with Hank’s salts. Medium was supplemented with 10% new-born calf serum, 2 mmol/l glutamine, 50 I.U. ml-’ sodium benzylpenicillin, 50 pg ml-l streptomycin sulphate and 0.75 mg ml-l NaHC03. 10 mmol/l HEPES (N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid) was added to culture medium in flasks. For calcium exchange measurements, confluent monolayers of cells (passages 5-20) were trypsinized into suspension and plated at an initial density of 10” cells per 35-mm well of a 6-well tissue culture plate. Medium was additionally supplemented with 2.5 pg ml-l amphotericin-B (fungizone) and the cultures were maintained in a humidified incubator with 5% CO2 in air. Medium was changed after three days, and confluency was reached by five days. Calcium exchange was measured on the sixth day after plating. Prior to labelling with 45Ca, the cells were washed with Hank’s salt solution made up to contain (mg 1-l) KCl, 400; KH2P04, 60; MgC12(H20)6, 100; MgS04(H20)7, 100; NaCl, 8000; NaHC03, 350; Na2HP04(H20)2, 60; CaCl,, 140; glucose, 1000; pH = 7.2. Cells were preincubated in HSS for 120 min and were then incubated with HSS with 1.5-2.5 &i ml-l 45Ca, in the presence and absence of dimethylsulfoxide (DMSO), ethanol and calcium ionophore A23187. At the end of the labelling period, plates were washed five times with ice-cold HSS, without CaCl, and with 1 mmol/l EGTA (ethyleneglycolbis(P-aminoethyl-ether);N,N’-tetraacetic acid), air dried and the cells dissolved in 1 mmol/l NaOH. 1 mmol/l EGTA was found to displace extracellular calcium as effectively as 1 mmol/l lanthanum chloride in these conditions (Statham, unpublished observation). Aliquots were assayed for radioactivity in Bray’s scintillation fluid [ 191 in a Beckman LS230 liquid scintillation counter, and for protein by the method of Lowry et al. [20] using bovine serum albumin fraction V as standard.
227
Materials All media, sera, trypsin and 25 cm2 Falcon flasks were obtained from GibcoBiocult, 75 cm’ Lux flasks from Eurolab or Gateway International, Linbro multiwell tissue culture plates from Flow Laboratories, 45Ca from the Radiochenical Centre, Amersham, scintillation chemicals from Koch-Light Laboratories and inorganic chemicals (Analar grade) from B.D.H. A23187 was a gift from Eli-Lilley and Co. Results Cells were incubated in 45Ca in HSS for various times and the amount of 45Ca which had exchanged with cellular calcium assayed. Preliminary experiments show that 45Ca exchange was complete within 90 min and a representative experiment is shown in Fig. 1, in which cells from 3 patients were studied for times between 1 and 120 min. Although sometimes the amount of 45Ca which had exchanged with cellular calcium was significantly different between two cell types, this was not consistently observed and 2-way analysis of variance [21] showed that there was no significant variation in the time course of 45Ca exchange in cells from patients with different diseases (F = 0.331, p > 0.25). 4sCa exchange at ‘isotopic equilibrium’ (i.e. when cellular levels of 4sCa did not increase any further with prolonged incubation) was then assayed in cells from the patients shown in Table I. Cells were incubated in 4sCa for 120 min
60 -I
0.0
1
30
60
90
120
Mmutes
Fig. 1. A representative experiment to show the time course of 4 5 Ca exchange in cultured fibroblasts in Hanks salt solution at 37’C. Each point is the mean of six determinations. Mean f standard error of the mean is shown wherever the symbol is not greater than the S.E.M.
228 TABLE
1
EXCHANGE
OF
45Ca
WITH
CELLULAR
CALCIUM
IN
CULTURED
FIBROBLASTS
AFTER
120
MINUTES Patient
Diagnosis
Age
protein
gg Ca m-1 i- S.E.M.
X
(years)
RA
2
M
0.238
+ 0.046
(16)
WH
3
M
0.411
+ 0.072
(12)
PT
5
M
0.208
i 0.032
(34)
JO
6
M
0.275
i 0.033
(9)
DP
7
M
0.477
i 0.184
(7)
N
Clinica Chimica Acta, 96 (1979) 225-231 @ Elsevier/North-Holland Biomedical Press
CCA 1082
DUCHENNE MUSCULAR DYSTROPHY: 45Ca EXCHANGE IN CULTURED SKIN FIBROBLASTS AND THE EFFECT OF CALCIUM IONOPHORE A23187
HELEN
E. STATHAM
* and VICTOR
DUBOWITZ
Jerry Lewis Muscle Research Centre and Department of Paediatrics and Neonatal Medicine (Institute of Child Health), Hammersmith Hospital, London, W12 OHS (U.K.) (Received
March 23rd, 1979)
Summary
Calcium exchange was studied in skin fibroblasts cultured from eight subjects with Duchenne muscular dystrophy, four with Limb Girdle dystrophy and eight normal controls using 45Ca. No difference was found in the time course of calcium exchange between the groups, nor in the level of 45Ca when maximal exchange had occurred. Treatment of the cultures with the calcium ionophore A23187 resulted in higher levels of calcium exchange over a 2-h period. The increase was similar in the cultures from the 3 patient groups studied.
Introduction
Duchenne muscular dystrophy (DMD) is an X-linked inherited disease, characterized by progressive degeneration of skeletal muscle and elevated serum levels of muscle-type creatine kinase. Although the basic defect in DMD is unknown, there is evidence to suggest that the plasma membrane of muscle, and of other tissues not obviously affected by the disease, may be abnormal. Structural and functional abnormalities have been reported for DMD muscle plasma membranes [ 1,2] and DMD erythrocytes [ 3-71. Platelets from DMD patients have reduced levels of [ 14C]serotonin incorporation [ 81 and interstitial cell nuclei in muscle biopsies have the same elevated calcium : phosphorus ratio found in muscle cell nuclei [ 91. Defects have also been observed in cells from DMD patients grown in tissue culture. Abnormalities in adenyl cyclase activity [lo] and in protein synthesis [ 111 have been described in DMD myotubes and unusual ‘clumping’ behaviour has been observed in mononuclear cells cultured from DMD muscle [ 121. Skin * To
whom
correspondence
should
be addressed.
226
fibroblasts from DMD patients have also been reported to have defects in ultrastructure [ 131 and in protein synthesis [ 141 but all of these reports are so far unconfirmed. Many of the abnormalities described for DMD patients and tissues are in some way connected with membrane calcium transport [6,15--171. In this study, we have examined some aspects of the calcium transport mechanisms of cultured skin fibroblasts from DMD patients, normal controls and patients with other neuromuscular disorders, to try to establish whether the reported defects in calcium transport systems might be part of the primary defect in DMD. Methods Fibroblast cultures were established from skin biopsies from patients with neuromuscular diseases, taken while subjects were undergoing muscle biopsy and from controls while undergoing surgery. Clinical details of the subjects are shown in Table I; neuro-muscular disorders were diagnosed by clinical assessment, serum enzyme levels and muscle biopsy. Primary fibroblast cultures were grown from 1 mm3 explants of skin in 25 cm* Falcon tissue culture flasks. The first subculture was usually made within 6-8 weeks of explanting the skin. Cells were maintained routinely in 75 cm* Lux flasks, sub-culturing as necessary with 0.25% trypsin in Hanks salt solution (HSS) [ 181 without calcium and magnesium. Tissue culture medium throughout was Eagles basal medium (BME), with Hank’s salts. Medium was supplemented with 10% new-born calf serum, 2 mmol/l glutamine, 50 I.U. ml-’ sodium benzylpenicillin, 50 pg ml-l streptomycin sulphate and 0.75 mg ml-l NaHC03. 10 mmol/l HEPES (N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid) was added to culture medium in flasks. For calcium exchange measurements, confluent monolayers of cells (passages 5-20) were trypsinized into suspension and plated at an initial density of 10” cells per 35-mm well of a 6-well tissue culture plate. Medium was additionally supplemented with 2.5 pg ml-l amphotericin-B (fungizone) and the cultures were maintained in a humidified incubator with 5% CO2 in air. Medium was changed after three days, and confluency was reached by five days. Calcium exchange was measured on the sixth day after plating. Prior to labelling with 45Ca, the cells were washed with Hank’s salt solution made up to contain (mg 1-l) KCl, 400; KH2P04, 60; MgC12(H20)6, 100; MgS04(H20)7, 100; NaCl, 8000; NaHC03, 350; Na2HP04(H20)2, 60; CaCl,, 140; glucose, 1000; pH = 7.2. Cells were preincubated in HSS for 120 min and were then incubated with HSS with 1.5-2.5 &i ml-l 45Ca, in the presence and absence of dimethylsulfoxide (DMSO), ethanol and calcium ionophore A23187. At the end of the labelling period, plates were washed five times with ice-cold HSS, without CaCl, and with 1 mmol/l EGTA (ethyleneglycolbis(P-aminoethyl-ether);N,N’-tetraacetic acid), air dried and the cells dissolved in 1 mmol/l NaOH. 1 mmol/l EGTA was found to displace extracellular calcium as effectively as 1 mmol/l lanthanum chloride in these conditions (Statham, unpublished observation). Aliquots were assayed for radioactivity in Bray’s scintillation fluid [ 191 in a Beckman LS230 liquid scintillation counter, and for protein by the method of Lowry et al. [20] using bovine serum albumin fraction V as standard.
227
Materials All media, sera, trypsin and 25 cm2 Falcon flasks were obtained from GibcoBiocult, 75 cm’ Lux flasks from Eurolab or Gateway International, Linbro multiwell tissue culture plates from Flow Laboratories, 45Ca from the Radiochenical Centre, Amersham, scintillation chemicals from Koch-Light Laboratories and inorganic chemicals (Analar grade) from B.D.H. A23187 was a gift from Eli-Lilley and Co. Results Cells were incubated in 45Ca in HSS for various times and the amount of 45Ca which had exchanged with cellular calcium assayed. Preliminary experiments show that 45Ca exchange was complete within 90 min and a representative experiment is shown in Fig. 1, in which cells from 3 patients were studied for times between 1 and 120 min. Although sometimes the amount of 45Ca which had exchanged with cellular calcium was significantly different between two cell types, this was not consistently observed and 2-way analysis of variance [21] showed that there was no significant variation in the time course of 45Ca exchange in cells from patients with different diseases (F = 0.331, p > 0.25). 4sCa exchange at ‘isotopic equilibrium’ (i.e. when cellular levels of 4sCa did not increase any further with prolonged incubation) was then assayed in cells from the patients shown in Table I. Cells were incubated in 4sCa for 120 min
60 -I
0.0
1
30
60
90
120
Mmutes
Fig. 1. A representative experiment to show the time course of 4 5 Ca exchange in cultured fibroblasts in Hanks salt solution at 37’C. Each point is the mean of six determinations. Mean f standard error of the mean is shown wherever the symbol is not greater than the S.E.M.
228 TABLE
1
EXCHANGE
OF
45Ca
WITH
CELLULAR
CALCIUM
IN
CULTURED
FIBROBLASTS
AFTER
120
MINUTES Patient
Diagnosis
Age
protein
gg Ca m-1 i- S.E.M.
X
(years)
RA
2
M
0.238
+ 0.046
(16)
WH
3
M
0.411
+ 0.072
(12)
PT
5
M
0.208
i 0.032
(34)
JO
6
M
0.275
i 0.033
(9)
DP
7
M
0.477
i 0.184
(7)
N
