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Increased calcium accumulation by brain mitochondria in dystrophic mice
Brain Research, 210 (1981) 437--440 © Elsevier/North-Holland Biomedical Press
437
Increased calcium accumulation by brain mitochondria in dystrophic mice
ADRIENNE FROSTHOLM, MICHEL BAUDRY and WILLIAM F. BENNETT Department of Psychobiology, School of Biological Sciences, University of California, Irvine, Calif. 92717 (U.S.A.)
(Accepted November 1lth, 1980) Key words: muscular dystrophy -- mitochondria -- Ca z+ accumulation
One of the major considerations in the etiology of the muscular dystrophies has been whether these maladies are of neural2,11 or muscular 5 origin. The latter view was predominant for many years since most of the deleterious symptoms occur in muscle. Recently, it has been suggested that the primary lesion in some types of dystrophy may be of a more general nature, such as a structural or enzymic abnormality of cellular membranes6,1a, 14. Evidence for this system-wide membrane defect has generally invoked abnormalities of the plasma membrane. However, literature reports of defective Ca ~+ transportg, 16-~8, and respiratory-chain linked functionsS, t0 suggest a mitochondrial membrane involvement. We have attempted to test this hypothesis more directly using crude and purified mitochondrial fractions from dystrophic mouse brain, a tissue not grossly affected by the disease. Six 129/ReJ dy/dy dystrophic mice and six littermate controls ( ? / + ) were used in the calcium accumulation studies. The average age on the day of sacrifice was 105 days. Each animal was lightly etherized and decapitated; the brain was quickly removed and homogenized in 4 ml of cold 0.32 M sucrose (700 rpm, teflon/glass). A crude synaptosomal/mitochondrial (P~) fraction was prepared by differential centrifugation as described by Browning et al. a. The pellet was resuspended in a hypotonic lysis buffer comprised of KCI (5 mM); MgSO4 (1.3 mM); NaH~PO4 (2.4 mM); HEPES (20 mM) and enough Tris base to bring the solution to a final p H of 6.6 at 22 °C. The suspension was allowed to equilibrate at 4 °C for 30-45 min before use. Aliquots of the lysate (approximately 30/zg protein/ml) were preincubated for 2 min at 37 °C in the presence and absence of 1 mM ATP. Reactions were initiated by the addition of 45Ca2+ (660 Ci/mmol, ICN, Irvine, Calif.), as previously described 1. After incubation periods of 1, 2.5, 5, 10, 15 and 20 min, the samples were filtered on Millipore cellulose filters (0.45 /~m pore), washed with 4 × 3 ml of homogenization buffer at 37 °C and counted in an aqueous scintillation cocktail (ACS, Amersham). Radioactivity was measured with an efficacy of 90 ~o. Blanks from which tissue had been omitted were subtracted from each sample value. Results are expressed as the
438
_0 E g2
-~'-CONTROL
In
+
[
I
2..5
5
[
I
I
1
g
0
1
INCUBATITIOMN(mi E n.) I0
15
20
Fig. 1. Time course of Ca .'+ accumulation in a mitochondrial preparation (P.') from control and dystrophic mouse brain. A typical experiment is shown in which each data point represents the mean of 3 samples measured after the indicated times of incubation in the presence of 45Ca2+ as described in the text.
accumulation of ng 45Ca~+/mg protein. Protein was assessed by the method of Lowry et al. 7. The synaptosomal-mitochondriat
(Pz)fraction
from brain tissue of 129/ReJ
dy/dy and littermate control mice exhibits two forms of calcium accumulation; ATP-independent
an
b i n d i n g w h i c h a p p r o a c h e s e q u i l i b r i u m w i t h i n less t h a n a m i n u t e ,
TABLE I
Rate of Ca 2+ accumulation in normal and dystrophic mouse brain mitochondrial (Pz) preparation Synaptosomal-mitochondrial (P2) fractions from brain of dystrophic and littermate control mice were prepared as described under Methods and the accumulation of 4~Ca.'+ was measured at various time intervals (i.e. 1', 2.5', 5', 10' and 20'). Rates of calcium accumulation were calculated from the slopes of the linear portion of the curves and are expressed in ng Ca .'+ rag/protein/rain. The free Ca .'+ concentration was calculated to be 2 # M by using a C a - E G T A buffer according to the formula Ca = K R / ( I - - R ) 1~ where R represents the ratio of total calcium over total E G T A (200 ktM) and K the dissociation constant for C a - E G T A which is equal to 1.28 × 10-n M at pH 6.6.
Experiment no.
129/ReJ dy/dy
Littermate controls ( ?/-~ )
1 2 3 4 5 6 Mean ± S.E.M.
272 290 247 366 241 268 281 ~ 19"
170 186 193 182 215 98 174 ± 16
* P < 0.01 (paired t-test).
439 and an ATP-dependent accumulation which proceeds linearly for about 5 min, reaches apparent equilibrium within 10-20 min (Fig. 1) and is sensitive to mitochondrial inhibitors. In contrast to ATP-independent Ca 2+ binding, which did not differ significantly from controls, the initial rate of ATP-dependent Ca 2+ accumulation in dystrophic tissue was markedly enhanced. The average rate of 45Ca2+ uptake in 6 experiments was 281 4- 19 and 174 4- 16 ng Ca2÷/mg protein/min for dystrophic and control tissue, respectively (Table I), representing an increased rate of 61 ~ in dystrophic tissue. Ca 2÷ accumulation at equilibrium was also 68% higher in dystrophic compared to control tissue. Additional experiments were conducted in order to determine more precisely the cellular location of the abnormalities observed in the Ca z+ transport mechanism of the P~ fraction; i.e. whether the defect originated in the plasma membrane or in the mitochondria of affected cells. A purified mitochondrial fraction, prepared according to Clark and Nicklas 4, exhibited a similar (although somewhat smaller) difference in Ca z+ uptake between dystrophic and control mice. After 10 min incubation, 45Ca z+ accumulation was measured at a free Ca 2÷ concentration of 2/~M in the presence of 1 mM ATP. In each of 3 experiments, the dystrophic values were from 15 ~ to 61% greater than controls (dy/dy ~ = 2390, control ~ = 1890 ng Ca2+/mg protein; average increase 2 6 ~ in dystrophic mitochondria). In each case, a mixture of mitochondrial poisons, (2,4 dinitrophenol, 0.I mM ; NAN3, 0.1 mM; oligomycin, 0.7 #g/ml) inhibited calcium accumulation by more than 9 0 ~ and eliminated the difference between dystrophic and control tissues. We have also obtained similar differences in Ca 2+ transport in mitochondrial fractions prepared from murine muscle tissue, brain and muscle from dystrophic hamsters, and brain and muscle from C57BL/6 dyZJ/dy2~ dystrophic mice. These data are in essential agreement with the observations of Wrogeman et al. is that dystrophic heart and skeletal muscle mitochondria have a greater calcium content than those from controls. The data presented here show that 129/ReJ dy/dy mice exhibit a marked increase in the rate of Ca 2+ accumulation by the synaptosomal-mitochondrial fraction from brain. In a purified mitochondrial preparation a similar increase was observed in dystrophic tissue. Mitochondrial poisons inhibited Ca z+ accumulation by more than 90 %, indicating that, in dystrophic mouse brain, a tissue not severely affected by the disease, there is a primary involvement of the mitochondrial membrane. Abnormalities in calcium metabolism have been observed in the more obviously affected tissues of a number of dystrophic animalsg,l~, 16-1s. While some biochemical abnormalities in mouse brain mitochondria have been reported 10, the calcium buffering capacity of that organelle has not been extensively studied. In view of the importance of intracellular calcium concentration on metabolic control, alterations of the calcium-buffering capacity of mitochondria would not be expected to pass unnoticed at the cellular, and even possibly at the behavioral levels. Both 129/ReJ dy/dy and C57BL/6 dystrophic mice have demonstrated behavioral abnormalities, including severe spontaneous seizures and learning deficits (Frostholm and Bennett, unpublished). The possible relationship between these observations and abnormalities in
440 calcium buffering, as well as the biochemical source of the calcium a c c u m u l a t i o n problem, are currently u n d e r study. This work was supported by a g r a n t f r o m the M u s c u l a r D y s t r o p h y Association to W.F.B.
1 Baudry, M., Browning, M. and Lynch, G., Phosphorylase kinase induced inhibition of Ca ~ ~ transport into rat brain mitochondria, Europ. J. Pharm., 58 (1979) 519-521. 2 Biscoe, T. J., Caddy, K. W. T., Pallor, D. J. and Pehrson, U. M. M., Investigation of cranial and other nerves in the mouse with muscular dystrophy, J. Neurol. Neurosurg. Psyehiat., 38 (1975) 391-403. 3 Browning, M., Dunwiddie, T., Bennett, W., Gispen, W. and Lynch, G., Synaptic phosphoproteins: specific changes after repetitive stimulation of the hippocampal slice, Science, 203 (1979) 60-62. 4 Clark, J. B. and Nicklas, W. J., The metabolism of rat brain mitochondria, J. biol. Chem., 245 (1970) 4724--4731. 5 Cosmos, E., Butler, J., Mazliah, J. and Allard, P., Animal models of muscle diseases II: murine dystrophy, Muscle and Nerve, 3 (1980) 350-359. 6 Kwok, C. T., Kuffer, A. D., Tang, B. Y. and Austin, L., Phospholipid metabolism in murine muscular dystrophy, Exp. NeuroL, 50 (1976) 362-375. 7 Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J., Protein measurements with the Folin phenol reagent, J. biol. Chem., 193 (1951) 265-275. 8 Martens, M. E., Jankulovska, M., Neymark, A. and Lee, C. P., Respiratory chain-linkedfunctions of skeletal muscle mitochondria from dystrophic mice, Fed. Proc., 36 (1977) 903. 9 Mendel, J. R., Higgins, R., Sahenk, Z. and Cosmos, E., Relevance of genetic animal models of muscular dystrophy to human muscular dystrophies. In John B. Harris (Ed.), Annals N. Y. Acad. Sci., New York, 317 (1979) 409-430. 10 Montalbo, R. G. and Kabara, J. J., Tricarboxylic acid cycle intermediates in muscular dystrophic mice (strain 129), Proc. Soc. exp. Biol. ( N. Y.), 145 (1974) 1225-1231. 11 Peterson, A. C., Frair, P. M., Rayburn, H. R. and Cross, D., Development and disease in the neuromuscular system of muscular dystrophic and normal mouse chimeras. In J. A. Ferrendelli (Ed.), Aspects of Developmental Neurobiology, Soc. Neurosci. Symp., Bethesda, 1979, pp. 258-273. 12 Plishker, G. A. Gitelman, H. J. and Appel, S. H., Myotonic muscular dystrophy: altered calcium transport in erythrocytes, Science, 200 (1978) 323-325. 13 Roses, A. D. and Appel, S. H., Muscle membrane protein kinase in mytonic muscular dystrophy, Nature (Lond.), 250 (1974) 245-247. 14 Roses, A. D. and Appel, S. H., Phosphorylation of component a of the human erythrocyte membrane in myotonic muscular dystrophy, J. Memb. Biol., 20 (1975) 51-58. 15 Schatzmann, H. J., Dependence on calcium concentrationsand stoichiometry of the calcium pump in human red cells, J. Physiol. (Lond.), 235 (1973) 551-569. 16 Seiler, D. und Kuhn, E., Kalziumtransport der isolierten Vesikel des sarkoplasmatischen Retikulums yon Patienten mit Myotonia congenita und Myotonia dystrophica, Schweiz med. Wschr., 100 (1970) 1374-1376. 17 Swift, L. L., Atkinson, J. V. and LeQuire, V. S., The composition and calcium transport activity of the sarcoplasmic reticulum from goats with and without heritable myotonia, Labl Invest., 40 (1979) 384-390. 18 Wrogeman, K., Hayward, W. A. K. and Blanchaer, M. C., Biochemical aspects of muscle necrosis in hamster dystrophy, Annals N. Y. Acad. Sci., 317 (1979) 30-45.
437
Increased calcium accumulation by brain mitochondria in dystrophic mice
ADRIENNE FROSTHOLM, MICHEL BAUDRY and WILLIAM F. BENNETT Department of Psychobiology, School of Biological Sciences, University of California, Irvine, Calif. 92717 (U.S.A.)
(Accepted November 1lth, 1980) Key words: muscular dystrophy -- mitochondria -- Ca z+ accumulation
One of the major considerations in the etiology of the muscular dystrophies has been whether these maladies are of neural2,11 or muscular 5 origin. The latter view was predominant for many years since most of the deleterious symptoms occur in muscle. Recently, it has been suggested that the primary lesion in some types of dystrophy may be of a more general nature, such as a structural or enzymic abnormality of cellular membranes6,1a, 14. Evidence for this system-wide membrane defect has generally invoked abnormalities of the plasma membrane. However, literature reports of defective Ca ~+ transportg, 16-~8, and respiratory-chain linked functionsS, t0 suggest a mitochondrial membrane involvement. We have attempted to test this hypothesis more directly using crude and purified mitochondrial fractions from dystrophic mouse brain, a tissue not grossly affected by the disease. Six 129/ReJ dy/dy dystrophic mice and six littermate controls ( ? / + ) were used in the calcium accumulation studies. The average age on the day of sacrifice was 105 days. Each animal was lightly etherized and decapitated; the brain was quickly removed and homogenized in 4 ml of cold 0.32 M sucrose (700 rpm, teflon/glass). A crude synaptosomal/mitochondrial (P~) fraction was prepared by differential centrifugation as described by Browning et al. a. The pellet was resuspended in a hypotonic lysis buffer comprised of KCI (5 mM); MgSO4 (1.3 mM); NaH~PO4 (2.4 mM); HEPES (20 mM) and enough Tris base to bring the solution to a final p H of 6.6 at 22 °C. The suspension was allowed to equilibrate at 4 °C for 30-45 min before use. Aliquots of the lysate (approximately 30/zg protein/ml) were preincubated for 2 min at 37 °C in the presence and absence of 1 mM ATP. Reactions were initiated by the addition of 45Ca2+ (660 Ci/mmol, ICN, Irvine, Calif.), as previously described 1. After incubation periods of 1, 2.5, 5, 10, 15 and 20 min, the samples were filtered on Millipore cellulose filters (0.45 /~m pore), washed with 4 × 3 ml of homogenization buffer at 37 °C and counted in an aqueous scintillation cocktail (ACS, Amersham). Radioactivity was measured with an efficacy of 90 ~o. Blanks from which tissue had been omitted were subtracted from each sample value. Results are expressed as the
438
_0 E g2
-~'-CONTROL
In
+
[
I
2..5
5
[
I
I
1
g
0
1
INCUBATITIOMN(mi E n.) I0
15
20
Fig. 1. Time course of Ca .'+ accumulation in a mitochondrial preparation (P.') from control and dystrophic mouse brain. A typical experiment is shown in which each data point represents the mean of 3 samples measured after the indicated times of incubation in the presence of 45Ca2+ as described in the text.
accumulation of ng 45Ca~+/mg protein. Protein was assessed by the method of Lowry et al. 7. The synaptosomal-mitochondriat
(Pz)fraction
from brain tissue of 129/ReJ
dy/dy and littermate control mice exhibits two forms of calcium accumulation; ATP-independent
an
b i n d i n g w h i c h a p p r o a c h e s e q u i l i b r i u m w i t h i n less t h a n a m i n u t e ,
TABLE I
Rate of Ca 2+ accumulation in normal and dystrophic mouse brain mitochondrial (Pz) preparation Synaptosomal-mitochondrial (P2) fractions from brain of dystrophic and littermate control mice were prepared as described under Methods and the accumulation of 4~Ca.'+ was measured at various time intervals (i.e. 1', 2.5', 5', 10' and 20'). Rates of calcium accumulation were calculated from the slopes of the linear portion of the curves and are expressed in ng Ca .'+ rag/protein/rain. The free Ca .'+ concentration was calculated to be 2 # M by using a C a - E G T A buffer according to the formula Ca = K R / ( I - - R ) 1~ where R represents the ratio of total calcium over total E G T A (200 ktM) and K the dissociation constant for C a - E G T A which is equal to 1.28 × 10-n M at pH 6.6.
Experiment no.
129/ReJ dy/dy
Littermate controls ( ?/-~ )
1 2 3 4 5 6 Mean ± S.E.M.
272 290 247 366 241 268 281 ~ 19"
170 186 193 182 215 98 174 ± 16
* P < 0.01 (paired t-test).
439 and an ATP-dependent accumulation which proceeds linearly for about 5 min, reaches apparent equilibrium within 10-20 min (Fig. 1) and is sensitive to mitochondrial inhibitors. In contrast to ATP-independent Ca 2+ binding, which did not differ significantly from controls, the initial rate of ATP-dependent Ca 2+ accumulation in dystrophic tissue was markedly enhanced. The average rate of 45Ca2+ uptake in 6 experiments was 281 4- 19 and 174 4- 16 ng Ca2÷/mg protein/min for dystrophic and control tissue, respectively (Table I), representing an increased rate of 61 ~ in dystrophic tissue. Ca 2÷ accumulation at equilibrium was also 68% higher in dystrophic compared to control tissue. Additional experiments were conducted in order to determine more precisely the cellular location of the abnormalities observed in the Ca z+ transport mechanism of the P~ fraction; i.e. whether the defect originated in the plasma membrane or in the mitochondria of affected cells. A purified mitochondrial fraction, prepared according to Clark and Nicklas 4, exhibited a similar (although somewhat smaller) difference in Ca z+ uptake between dystrophic and control mice. After 10 min incubation, 45Ca z+ accumulation was measured at a free Ca 2÷ concentration of 2/~M in the presence of 1 mM ATP. In each of 3 experiments, the dystrophic values were from 15 ~ to 61% greater than controls (dy/dy ~ = 2390, control ~ = 1890 ng Ca2+/mg protein; average increase 2 6 ~ in dystrophic mitochondria). In each case, a mixture of mitochondrial poisons, (2,4 dinitrophenol, 0.I mM ; NAN3, 0.1 mM; oligomycin, 0.7 #g/ml) inhibited calcium accumulation by more than 9 0 ~ and eliminated the difference between dystrophic and control tissues. We have also obtained similar differences in Ca 2+ transport in mitochondrial fractions prepared from murine muscle tissue, brain and muscle from dystrophic hamsters, and brain and muscle from C57BL/6 dyZJ/dy2~ dystrophic mice. These data are in essential agreement with the observations of Wrogeman et al. is that dystrophic heart and skeletal muscle mitochondria have a greater calcium content than those from controls. The data presented here show that 129/ReJ dy/dy mice exhibit a marked increase in the rate of Ca 2+ accumulation by the synaptosomal-mitochondrial fraction from brain. In a purified mitochondrial preparation a similar increase was observed in dystrophic tissue. Mitochondrial poisons inhibited Ca z+ accumulation by more than 90 %, indicating that, in dystrophic mouse brain, a tissue not severely affected by the disease, there is a primary involvement of the mitochondrial membrane. Abnormalities in calcium metabolism have been observed in the more obviously affected tissues of a number of dystrophic animalsg,l~, 16-1s. While some biochemical abnormalities in mouse brain mitochondria have been reported 10, the calcium buffering capacity of that organelle has not been extensively studied. In view of the importance of intracellular calcium concentration on metabolic control, alterations of the calcium-buffering capacity of mitochondria would not be expected to pass unnoticed at the cellular, and even possibly at the behavioral levels. Both 129/ReJ dy/dy and C57BL/6 dystrophic mice have demonstrated behavioral abnormalities, including severe spontaneous seizures and learning deficits (Frostholm and Bennett, unpublished). The possible relationship between these observations and abnormalities in
440 calcium buffering, as well as the biochemical source of the calcium a c c u m u l a t i o n problem, are currently u n d e r study. This work was supported by a g r a n t f r o m the M u s c u l a r D y s t r o p h y Association to W.F.B.
1 Baudry, M., Browning, M. and Lynch, G., Phosphorylase kinase induced inhibition of Ca ~ ~ transport into rat brain mitochondria, Europ. J. Pharm., 58 (1979) 519-521. 2 Biscoe, T. J., Caddy, K. W. T., Pallor, D. J. and Pehrson, U. M. M., Investigation of cranial and other nerves in the mouse with muscular dystrophy, J. Neurol. Neurosurg. Psyehiat., 38 (1975) 391-403. 3 Browning, M., Dunwiddie, T., Bennett, W., Gispen, W. and Lynch, G., Synaptic phosphoproteins: specific changes after repetitive stimulation of the hippocampal slice, Science, 203 (1979) 60-62. 4 Clark, J. B. and Nicklas, W. J., The metabolism of rat brain mitochondria, J. biol. Chem., 245 (1970) 4724--4731. 5 Cosmos, E., Butler, J., Mazliah, J. and Allard, P., Animal models of muscle diseases II: murine dystrophy, Muscle and Nerve, 3 (1980) 350-359. 6 Kwok, C. T., Kuffer, A. D., Tang, B. Y. and Austin, L., Phospholipid metabolism in murine muscular dystrophy, Exp. NeuroL, 50 (1976) 362-375. 7 Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J., Protein measurements with the Folin phenol reagent, J. biol. Chem., 193 (1951) 265-275. 8 Martens, M. E., Jankulovska, M., Neymark, A. and Lee, C. P., Respiratory chain-linkedfunctions of skeletal muscle mitochondria from dystrophic mice, Fed. Proc., 36 (1977) 903. 9 Mendel, J. R., Higgins, R., Sahenk, Z. and Cosmos, E., Relevance of genetic animal models of muscular dystrophy to human muscular dystrophies. In John B. Harris (Ed.), Annals N. Y. Acad. Sci., New York, 317 (1979) 409-430. 10 Montalbo, R. G. and Kabara, J. J., Tricarboxylic acid cycle intermediates in muscular dystrophic mice (strain 129), Proc. Soc. exp. Biol. ( N. Y.), 145 (1974) 1225-1231. 11 Peterson, A. C., Frair, P. M., Rayburn, H. R. and Cross, D., Development and disease in the neuromuscular system of muscular dystrophic and normal mouse chimeras. In J. A. Ferrendelli (Ed.), Aspects of Developmental Neurobiology, Soc. Neurosci. Symp., Bethesda, 1979, pp. 258-273. 12 Plishker, G. A. Gitelman, H. J. and Appel, S. H., Myotonic muscular dystrophy: altered calcium transport in erythrocytes, Science, 200 (1978) 323-325. 13 Roses, A. D. and Appel, S. H., Muscle membrane protein kinase in mytonic muscular dystrophy, Nature (Lond.), 250 (1974) 245-247. 14 Roses, A. D. and Appel, S. H., Phosphorylation of component a of the human erythrocyte membrane in myotonic muscular dystrophy, J. Memb. Biol., 20 (1975) 51-58. 15 Schatzmann, H. J., Dependence on calcium concentrationsand stoichiometry of the calcium pump in human red cells, J. Physiol. (Lond.), 235 (1973) 551-569. 16 Seiler, D. und Kuhn, E., Kalziumtransport der isolierten Vesikel des sarkoplasmatischen Retikulums yon Patienten mit Myotonia congenita und Myotonia dystrophica, Schweiz med. Wschr., 100 (1970) 1374-1376. 17 Swift, L. L., Atkinson, J. V. and LeQuire, V. S., The composition and calcium transport activity of the sarcoplasmic reticulum from goats with and without heritable myotonia, Labl Invest., 40 (1979) 384-390. 18 Wrogeman, K., Hayward, W. A. K. and Blanchaer, M. C., Biochemical aspects of muscle necrosis in hamster dystrophy, Annals N. Y. Acad. Sci., 317 (1979) 30-45.