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Oxidative phosphorylation and calcium transport by sarcotubular vesicles in myotonic dystrophy
BIOCHEMICAL
2,457-460
MEDICINE
Oxidative
( 1989)
Phosphorylation
Sarcotubular
Vesicles
J. B. PETER Departmmt
and
Calcium
in Myotonic
Transport
by
Dystrophy
M. WORSFOLD
AND
of Medicine, UCLA School of Medicine, Los Angeles, California 90024
Received January 24, 1969
Dystrophia myotonica (myotonic dystrophy) is a form of muscular dystrophy characterized by myotonia and progressive muscular weakness, which are generally apparent by late adolescence or early adulthood (1, 2). Clinically, myotonia is manifest as muscular stiffness or difficulty in relaxing the grip. Electrically, myotonia is characterized by repetitive discharge of muscle fibers in response to a variety of stimuli. This repetitive depolarization is not prevented by agents which block the nerve or neuromuscular junction ( 2). Several authors have suggested that the myotonia might be a manifestation of a defect in the production or utilization of ATP in affected muscle (3, 4). A defect in ATP production due to deficiency of oxidative phosphorylation would impair muscle relaxation because relaxation depends on ATP-dependent sequestration of calcium by the sarcoplasmic reticulum (9). Another possible mechanism of myotonia supposesthat ATP production is normal but that there is a primary abnormality of the sarcoplasmic reticulum which results in its failure to take up calcium properly. This latter possibility has been examined with negative results by standard techniques for studying calcium uptake by sarcotubular vesicles (fragmented sarcoplasmic reticulum) in the presence of excess calcium ( 5). However, recent studies in this laboratory (8) have shown that these techniques are relatively insensitive compared with the calcium affinity assay described below. METHODS
We have developed an efficient, reproducible method for isolation of skeletal muscle mitochondria from samples of human muscle weighing less than 1.5 gm (6, 7). From the same homogenates, sarcotubular vesicles may be isolated and assayed for their ATPase activity, their capacity to accumulate calcium from relatively concentrated (106 PM) solutions, and for their calcium affinity. In the calcium affinity assay the amount of added calcium is less than 457
458
PETER
AND
WORSFOLD
l/3 of that which would saturate the vesicles. Under these conditions (20 ,uM Ca’+; 0.25 pmoles Ca?+/mg protein) vesicles from normal muscle reduce the free calcium concentration to 0.3 JUM Ca”+ or less in 4 minutes at 26”. The free calcium concentration at 4 minutes is taken as a measure of the calcium affinity of the isolated vesicles and hence of the competence of the sarcoplasmic reticulum in viva to induce relaxation of myofibrils. The relaxation-contraction cycle of myofibrils occurs over the approximate range 0.1-1.0 PM Ca?+ (9). RESULTS
Figure 1 shows a polarographic tracing of the respiration of mitochondria isolated from the dystrophic quadriceps muscle of a Syear-old male with dystrophia myotonica. The rate of respiration ( Qo, ) and ADPIO ratios for both pyruvate-malate and succmate (-j- rotenone) are entirely normal. Also normal for both substrates are the acceptor ratios (ratio of Qo2 in the presence of substrate, oxygen, Pi, and ADP to Qoz in the same control ratios (ratio of system before addition of ADP) and respiratory or+:9 AR=76 I5 I5
mM mM
PYRUVATE MALATE
-
PYRUVATE-
00~
----
MALATE 02 /mg
I hr
SUCCINATE-ROTENONE Oo2=96@ Op/mg/hr RCR=
45 mM
= 66pi
2.6
SUCCINATE
0 9 rng PROTEIN STANDARD MEDIUM TOTAL VOLUME = 2ml 7~26. pH’7 4
ZO/.LLM o*
\\ \ lI In,”
\\ \
FIG. 1. Polarographic tracing of oxygen consumption cy skeletal muscle mitochondria (27M5OOg) isolated from a patient with dystrophia myotonica. The standard medium contains 30 mM PI, 25 mu TES, (N-tris [hydroxymethyl] methyl2-aminoethane sulfonic acid), 8 mu MgSO,, 0.5 mu EDTA, and 50 mu KC1 at pH 7.4. Values for Q% are avenges of those found in the presence of ADP. Respiratory control ratio (RCR) is defined in the text and is an average of the transitions shown.
MYOTONIC
DYS’lXOPHY
459
Qoz in the presence of substrate, oxygen, Pi and ADP to Qo, in the same system after added ADP has been converted to ATP by oxidative phosphorylation). The yield of mitochondria per gram of muscle was also normal. A similar study from another patient showed normal respiratory rates ( Qoz), acceptor ratios, respiratory control ratios and ADP/O ratios in fractions of mitochondria sedimented at 270-8SOOg, 8500-7OOOg, and 7000-15000g. Both electromyography and the histology of the muscles were consistent with dystrophia myotonica. We have also demonstrated normal oxidative phosphorylation in mitochondria isolated from patients with Duchenne muscular dystrophy ( 8).
I
TIME,
minutes
FIG.
2. Ca&m affinity of sarcotubular vesicles from the same patient (Fig. 1) with dystrophia myotonica ( 0). The hatched area is the normal range ( 8). Standard conditions for this assay are 26 O, 0.08 mg vesicle protein/ml, 5 mM ATP, 5 mu MgCL, 5 mu oxalate, 40 mM histidine, pH 7.2, 110 IIIM NaCl, 120 mu sucrose, and %I pM CaCL, labeled with %a”+. Samples are taken at the times indicated and free calcium remaining in solution is determined by Millipore filtration (8).
Figure 2 illustrates the calcium affinity of sarcotubular vesicles isolated from the same patient. Under these conditions the vesicles lowered the free calcium concentration in solution from 20 PM to 0.2 PM in 4 minutes. This is well within the normal range (8). Calcium accumulation from 100 pM solution (which indicates the total capacity) was also normal in this patient and in another with dystrophic myotonica, in confirmation of Samaha’s findings (5). The normal calcium affinity is, however, of partic-
460
PETER
AND
W~RSFOLD
ular significance because in contrast to these findings in myotonic dystrophy decreased calcium affinity is consistently demonstrable in vesicles from patients with the Duchenne form of muscular dystrophy, even when the total capacity for calcium accumulation from 100 pM solutions is normal (8). This illustrates the greater sensitivity of the calcium affinity assay. DISCUSSION
These studies show that contrary to some theories the oxidative phosphorylation of skeletal muscle mitochondria from patients with dystrophia myotonica is entirely normal. Very sensitive methods for studying calcium affinity of sarcotubular vesicles in one such patient revealed no abnormalities, Other data of Samaha (5) revealed no abnormality of actomyosin syneresis in dystrophia myotonica. The results as well as the eIectrica1 characteristics of myotonia are consistent with a primary abnormality of the sarcolemma in this disease. It is on purified sarcolemma that our biochemical studies of dystrophia myotonica are now focused. SUMMARY
Oxidative phosphorylation by muscle mitochondria and the competence of calcium sequestration by fragmented sarcoplasmic reticulum from the muscle of patients with myotonic dystrophy were studied. Both of these functions appeared to be entirely normal, lending no support to theories that abnormalities of mitochondrial respiration or calcium transport by the sarcotubular system are the primary cause of the disease. ACKNOWLEDGMENTS
These studies were supported by NIH grants HD 02584 and NB 07587. One the authors (M. Worsfold) was assisted by a Wellcome Research Travel Grant.
of
REFERENCES 1. WALTON, J. N., “Disorders of Voluntary Muscle,” p. 293. Little, Brown, Boston, Massachusetts, 1964. 2. ADAMS, R. D., DENNY-BROWN, D., AND PEARSON, C. M., “Diseases of Muscle,” p. 660. Harper, New York, 1962. 3. KUHN, E., Ard. Forsch. 15, 6 ( 1961). N. C., “Dystrophia Myotonica and 4. CAUGHEY, J. E., AND MYRIANTHOPOIJLOS, Related Disorders,” p. 238. Thomas, Springfield, Illinois, 1963. 5. SAMAHA, F. J., SCHROEDER, J. M., REBEIZ, J., AND ADAMS, R. D., Arch. Neural. 17, 22 (1967). 6. PETER, J. B., AND LEE, L. D., Biochem. Biophys. Res. Commun. 29, 430 (1967). 7. PETER, J. B., Biochem. Med. 2, 179 ( 1968). 8. PETER, J. B., AND WORSFOLD, M., Biochem. Med. 2, 364 (1969). 9. WEBER, A., in “Current Topics in Bioenergetics” (D. R. Sanadi, ed.), p. 263, Academic Press, New York, 1966.
2,457-460
MEDICINE
Oxidative
( 1989)
Phosphorylation
Sarcotubular
Vesicles
J. B. PETER Departmmt
and
Calcium
in Myotonic
Transport
by
Dystrophy
M. WORSFOLD
AND
of Medicine, UCLA School of Medicine, Los Angeles, California 90024
Received January 24, 1969
Dystrophia myotonica (myotonic dystrophy) is a form of muscular dystrophy characterized by myotonia and progressive muscular weakness, which are generally apparent by late adolescence or early adulthood (1, 2). Clinically, myotonia is manifest as muscular stiffness or difficulty in relaxing the grip. Electrically, myotonia is characterized by repetitive discharge of muscle fibers in response to a variety of stimuli. This repetitive depolarization is not prevented by agents which block the nerve or neuromuscular junction ( 2). Several authors have suggested that the myotonia might be a manifestation of a defect in the production or utilization of ATP in affected muscle (3, 4). A defect in ATP production due to deficiency of oxidative phosphorylation would impair muscle relaxation because relaxation depends on ATP-dependent sequestration of calcium by the sarcoplasmic reticulum (9). Another possible mechanism of myotonia supposesthat ATP production is normal but that there is a primary abnormality of the sarcoplasmic reticulum which results in its failure to take up calcium properly. This latter possibility has been examined with negative results by standard techniques for studying calcium uptake by sarcotubular vesicles (fragmented sarcoplasmic reticulum) in the presence of excess calcium ( 5). However, recent studies in this laboratory (8) have shown that these techniques are relatively insensitive compared with the calcium affinity assay described below. METHODS
We have developed an efficient, reproducible method for isolation of skeletal muscle mitochondria from samples of human muscle weighing less than 1.5 gm (6, 7). From the same homogenates, sarcotubular vesicles may be isolated and assayed for their ATPase activity, their capacity to accumulate calcium from relatively concentrated (106 PM) solutions, and for their calcium affinity. In the calcium affinity assay the amount of added calcium is less than 457
458
PETER
AND
WORSFOLD
l/3 of that which would saturate the vesicles. Under these conditions (20 ,uM Ca’+; 0.25 pmoles Ca?+/mg protein) vesicles from normal muscle reduce the free calcium concentration to 0.3 JUM Ca”+ or less in 4 minutes at 26”. The free calcium concentration at 4 minutes is taken as a measure of the calcium affinity of the isolated vesicles and hence of the competence of the sarcoplasmic reticulum in viva to induce relaxation of myofibrils. The relaxation-contraction cycle of myofibrils occurs over the approximate range 0.1-1.0 PM Ca?+ (9). RESULTS
Figure 1 shows a polarographic tracing of the respiration of mitochondria isolated from the dystrophic quadriceps muscle of a Syear-old male with dystrophia myotonica. The rate of respiration ( Qo, ) and ADPIO ratios for both pyruvate-malate and succmate (-j- rotenone) are entirely normal. Also normal for both substrates are the acceptor ratios (ratio of Qo2 in the presence of substrate, oxygen, Pi, and ADP to Qoz in the same control ratios (ratio of system before addition of ADP) and respiratory or+:9 AR=76 I5 I5
mM mM
PYRUVATE MALATE
-
PYRUVATE-
00~
----
MALATE 02 /mg
I hr
SUCCINATE-ROTENONE Oo2=96@ Op/mg/hr RCR=
45 mM
= 66pi
2.6
SUCCINATE
0 9 rng PROTEIN STANDARD MEDIUM TOTAL VOLUME = 2ml 7~26. pH’7 4
ZO/.LLM o*
\\ \ lI In,”
\\ \
FIG. 1. Polarographic tracing of oxygen consumption cy skeletal muscle mitochondria (27M5OOg) isolated from a patient with dystrophia myotonica. The standard medium contains 30 mM PI, 25 mu TES, (N-tris [hydroxymethyl] methyl2-aminoethane sulfonic acid), 8 mu MgSO,, 0.5 mu EDTA, and 50 mu KC1 at pH 7.4. Values for Q% are avenges of those found in the presence of ADP. Respiratory control ratio (RCR) is defined in the text and is an average of the transitions shown.
MYOTONIC
DYS’lXOPHY
459
Qoz in the presence of substrate, oxygen, Pi and ADP to Qo, in the same system after added ADP has been converted to ATP by oxidative phosphorylation). The yield of mitochondria per gram of muscle was also normal. A similar study from another patient showed normal respiratory rates ( Qoz), acceptor ratios, respiratory control ratios and ADP/O ratios in fractions of mitochondria sedimented at 270-8SOOg, 8500-7OOOg, and 7000-15000g. Both electromyography and the histology of the muscles were consistent with dystrophia myotonica. We have also demonstrated normal oxidative phosphorylation in mitochondria isolated from patients with Duchenne muscular dystrophy ( 8).
I
TIME,
minutes
FIG.
2. Ca&m affinity of sarcotubular vesicles from the same patient (Fig. 1) with dystrophia myotonica ( 0). The hatched area is the normal range ( 8). Standard conditions for this assay are 26 O, 0.08 mg vesicle protein/ml, 5 mM ATP, 5 mu MgCL, 5 mu oxalate, 40 mM histidine, pH 7.2, 110 IIIM NaCl, 120 mu sucrose, and %I pM CaCL, labeled with %a”+. Samples are taken at the times indicated and free calcium remaining in solution is determined by Millipore filtration (8).
Figure 2 illustrates the calcium affinity of sarcotubular vesicles isolated from the same patient. Under these conditions the vesicles lowered the free calcium concentration in solution from 20 PM to 0.2 PM in 4 minutes. This is well within the normal range (8). Calcium accumulation from 100 pM solution (which indicates the total capacity) was also normal in this patient and in another with dystrophic myotonica, in confirmation of Samaha’s findings (5). The normal calcium affinity is, however, of partic-
460
PETER
AND
W~RSFOLD
ular significance because in contrast to these findings in myotonic dystrophy decreased calcium affinity is consistently demonstrable in vesicles from patients with the Duchenne form of muscular dystrophy, even when the total capacity for calcium accumulation from 100 pM solutions is normal (8). This illustrates the greater sensitivity of the calcium affinity assay. DISCUSSION
These studies show that contrary to some theories the oxidative phosphorylation of skeletal muscle mitochondria from patients with dystrophia myotonica is entirely normal. Very sensitive methods for studying calcium affinity of sarcotubular vesicles in one such patient revealed no abnormalities, Other data of Samaha (5) revealed no abnormality of actomyosin syneresis in dystrophia myotonica. The results as well as the eIectrica1 characteristics of myotonia are consistent with a primary abnormality of the sarcolemma in this disease. It is on purified sarcolemma that our biochemical studies of dystrophia myotonica are now focused. SUMMARY
Oxidative phosphorylation by muscle mitochondria and the competence of calcium sequestration by fragmented sarcoplasmic reticulum from the muscle of patients with myotonic dystrophy were studied. Both of these functions appeared to be entirely normal, lending no support to theories that abnormalities of mitochondrial respiration or calcium transport by the sarcotubular system are the primary cause of the disease. ACKNOWLEDGMENTS
These studies were supported by NIH grants HD 02584 and NB 07587. One the authors (M. Worsfold) was assisted by a Wellcome Research Travel Grant.
of
REFERENCES 1. WALTON, J. N., “Disorders of Voluntary Muscle,” p. 293. Little, Brown, Boston, Massachusetts, 1964. 2. ADAMS, R. D., DENNY-BROWN, D., AND PEARSON, C. M., “Diseases of Muscle,” p. 660. Harper, New York, 1962. 3. KUHN, E., Ard. Forsch. 15, 6 ( 1961). N. C., “Dystrophia Myotonica and 4. CAUGHEY, J. E., AND MYRIANTHOPOIJLOS, Related Disorders,” p. 238. Thomas, Springfield, Illinois, 1963. 5. SAMAHA, F. J., SCHROEDER, J. M., REBEIZ, J., AND ADAMS, R. D., Arch. Neural. 17, 22 (1967). 6. PETER, J. B., AND LEE, L. D., Biochem. Biophys. Res. Commun. 29, 430 (1967). 7. PETER, J. B., Biochem. Med. 2, 179 ( 1968). 8. PETER, J. B., AND WORSFOLD, M., Biochem. Med. 2, 364 (1969). 9. WEBER, A., in “Current Topics in Bioenergetics” (D. R. Sanadi, ed.), p. 263, Academic Press, New York, 1966.