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Muscular dystrophy and other myopathies: sarcotubular vesicles in early disease
BIOCHEMICAL
MEDICINE
Muscular
2, 364-371 ( 1969)
Dystrophy
Sarcotubular
and Vesicles
J. B. PETER Department
AND
Other
Myopathies:
in Early
Disease
M. WORSFOLD
of Medicine, UCLA School of Medicine, Los Angeles, California 90024
ReceivedNovember 1, 1968 Vesicles derived from the sarcoplasmic reticulum of muscle from patients with Duchenne muscular dystrophy exhibit a decreased capacity for accumulating calcium with a concomitant increase in ATPase activity (1, 2). Sarcotubular vesicles from dystropbic mice also possess a reduced capacity for calcium accumulation (3). These observations with subcellular particles are an interesting corollary to the finding of delayed relaxation of muscIe in Duchenne muscular dystrophy (4). We have investigated several aspects of calcium uptake by vesicles from muscle obtained at biopsy from patients with no evidence of myopathy and from patients with a variety of muscle and neuromuscular diseases. Oxidative phosphorylation of intact mitochondria prepared from the same muscle samples was assayed concurrently. Cytochrome oxidase and sue&rate oxidase activities of dystrophic muscle are said to be normal (S), but a comparative study of the oxidative phosphorylation of mitochondria and calcium accumulation by sarcotubular vesicles in muscular dystrophy has not, to our knowledge, been published. By measuring calcium affinity of vesicles we have demonstrated biochemical abnormalities of vesicles from dystrophic and myositic muscle which were detected by the usual techniques for studying calcium accumulation. Oxidative phosphorylation of skeletal muscle mitochondria isolated from these same patients was normal. METHODS Mitochondria and vesicles were isolated by differential centrifugation of a homogenate prepared by shaking minced muscle with glass beads in a CO,-cooled shaker, as previously described in detail (6, 7 ). Respfmtory rates (Qo? ), acceptor ratios (AR), respiratory control ratios ( RCR) and ADP/O ratios were determined immediately after the isolation of the mitochondria (6, 7). The ZO-minute, 15,000-g supernatant fraction from the high-speed 364
MUSCULAR
DYSTROPHY
AND
OTHER
365
MYOPATHIES
mitochondrial pellet was centrifuged at 40,000g for 90 minutes to obtain sarcotubular vesicles. This pellet was rinsed with lO!Z sucrose and resuspended by gentle homogenization in 46% sucrose (0.8 mg protein/ ml). Calcium accumulation at intervals over a 1Sminute period was measured by the Millipore filtration method at 26” (8). The standard incubation medium contained 0.08 mg of vesicle protein/ml, 5 mM ATP, 5 mM MgCl,, 5 mM oxalate, 40 mM histidine, pH 7.2, 110 mM NaCl, 10 mM KCl, 120 mM sucrose, and either 100 PM CaCI, (calcium accumulation assay) or 20 PM CaCl, (calcium affinity assay), labeled with ““Ca. ATPase activity was related to inorganic phosphate (9) found in a portion of the Millipore filtrate, which contained no ATPase activity. Contaminating calcium was removed from ATP by passage through a column of Dowex-50 (H’). Sucrose solutions were passed through Dowex-50 (Na+) prior to use. Samples of dystrophic muscle were taken from thigh muscles (quadriceps or biceps femoris) and simultaneously in three patients from the pseudohypertrophied gastrocnemius muscles which were much stronger clinically and showed much less damage histologically than the thigh muscles. RESULTS
Thigh muscle from patients with far-advanced Duchenne dystrophy gave very low yields of mitochondria with low QO,, poor respiratory oo2:40 AVERAGE -GASTROCNEMlUS !!gc2‘J
-GASTROCNEMIUS
I I 0 rnq PROTEIN 15 0 mM PYRuvQTE 15 0 mM MALATE STANDARI TOTAL VOLUME = 2ml T:26*.pH:74
‘\
‘\
l_
015mf.4 -.
_-__
ADP
‘. ‘-.
BICEPS
FEMORIS
I
“‘.---T
..\\,___ OlSmM ADP
FIG. 1. Oxidative phosphorylation of skeletal muscle mitochondria isolated from two muscles of a 4-year-old boy with Ducherme dystrophy (patient 5). Conditions were exactly as described previously; note the high acceptor (AR) and respiratory control ratios ( RCR) and the near-“theoretical” ADP/O ratios (see 5).
A 0
NORMAL
I AND AlRoPHlES
MYOPATHY
x
DUCWENIIE: YUSCULAR
BECKER
l
l *
l
l * l
l
x x xx $xXxX
xx
x
x
[XX
xx
1
DYSTROPHY
DYSTROPHY
** l
x xxx x xxx xxx X
l PoLYYYoslTls
9l
l *m
x”
ALCOllOLIC
X
I?
.
YlSCLLLAWLOUS YYOPATnlts WEUnosLWIC
l .
0
l
x
.
X
1
r - - -i-------__- - - - - - - - - - - - - - - - - --, 30 om b l bo 1 x
+
F FX _-- ,e1 - - - - -- - - - - - - - -
1
-1
fACIO-SCAPULOHUYLRAL DYSTROPHY LIMO-SIROLE DYSTROPHY
.
I
t I
I
0
0.5
r_-_
0
l
X
I
x
x
X
I
I I .o
I I.5
B MISCELLANEOUS MYOPATHIES AND NEUROGENIC ATROPHIES ALCOHOLIC
MYOPATHY
DUCHENNE MUSCULAR
l ***
:
l **
l
t’t .
I
.
I
POLYMYOSlTlS
BECKER
. .
I
NORMAL
-
0
.
0 .
I I
1
DYSTROPHY
DYSTROPHY
FACIO- SCAPULOHUMERAL DYSTROPHY LIMB-GIRDLE UYSTROPHY
I IT----I-001
i
. -7-l
01
IO
L 1 &Xl++
/LLM
01 FOUR
IO
MINUTES
FIG. 2A. Calcium-accumulating capacity and ATPase activity of human vesicles. Maximum calcium uptake is expressed as amoles/mg vesicle protein ( l , normals
20
MUSCULAR
DYSTTIOPHY
AND
OTHER
MYOPATHIES
367
control and low ADP/ratios. The less-affected gastrocnemius muscles from the same patients, and the biceps femoris from one, were essentially normal in these respects (Fig. 1). One of the four patients with polymyositis and the first specimen from the patient with alcoholic myopathy also gave low yields of mitochondria, with low Qoz and poor respiratory control (Fig. 2B). The calcium-accumulation capacity with 100 PM calcium (1.25 pmoles/mg protein) was generally lower in the vesicles from severely affected muscle than in normal vesicles or in vesicles from muscle less affected by Duchenne dystrophy (Fig. 2A). Initial rates of calcium uptake also tended to be lower than normal in the severely affected group as did the initial Mg’+-Ca”-stimulated ATPase activity. Abnormalities of mitochondria were likewise more common in severely affected muscle (Fig. 2A). In an attempt to approach physiological conditions more closely, the removal of calcium from more dilute solutions of calcium (20 PM) was studied with the same concentration of vesicles (0.03 mg protein/ml). We shall refer to this as an assay of the calcium aflinity of vesicles as opposed to the total capacity for calcium accumulation. On incubation with only 20 PM calcium (0.25 pmoles/mg protein) normal vesicles quickly reduce the calcium concentration of the medium to less than 0.3 p~hr which is in the range required to induce relaxation of muscle or syneresis of myofibrils ( 10). The calcium af&ity assay therefore may be a more physiologically signiiicant test of the capacity of vesicles to bind calcium than is their rate of calcium uptake or total capacity for calcium aoxunulation from concentrated (100 PM) solutions of Caz+. Some of the vesicle preparations from Duchenne dystrophic muscle showed normal capacity for calcium accumulation but none had normal calcium affinity under the conditions of the assay (Fig. 2B). Vesicles from all three samples of dystrophic gastrocnemius showed normal and alfected subjects, normal mitochondria; 0, normals and affected subjects, ‘abnormal’ mttochondria; n , symptomless female heterozygotes). ATPase activity (~moles/min/mg protein) over the trst two minutes was measured simultaneously (x, normals and affected subjects; +, symptomless female heterozygotes). ‘Abnormal’ mitochondria are considered to be those with Qe, < 25 ~1 OJmg protein/ hour, in the presence of pyruvate, malate, PI and ADP, or RCR < 2.0. Symbols joined by tie-lines (- - - - ) represent samples obtained at one operation from different sites on the same patient, e.g., quadriceps and gastrocnemius. B. Calcium affinity of human vesicles. See Fig. 2A for explanation of symbols. Vesicles were incubated with 20 pM Ca*+ as described in the text. The differences between the various groups were most striking after 4 minutes’ incubation, but a similar pattern was evident at 2 and 8 minutes.
368
PETER
AND
WORSFOLD
capacity for calcium accumulation but all had impaired calcium affinity. This was also true of one vesicle preparation each from the biceps femoris and quadriceps muscles of patients with Duchemre dystrophy. Vesicles from the gastrocnemius of a female carrier of Duchenne dystrophy had normal calcium-accumulating capacity (Fig. 2A) but exhibited slightly impaired calcium affinity (Fig. 2B). Vesicles from a patient with the Becker form of pseudohypertrophic muscular dystrophy and from his daughter (a known carrier of this disease) showed grossly and slightly decreased calcium affinity respectively, whereas calcium accumulation capacity was normal in both. We have studied two biopsies from one patient with facie-scapula-humeral dystrophy. Calciumaccumulation was normal in vesicles from the quadriceps and deltoid muscles, but calcium affinity was abnormal in the quadriceps and normal in the deltoid. Three patients with limb-girdle dystrophy have shown normal capacities for calcium-accumulation and, in the only one of these tested, the assay for calcium affinity was also normal. Vesicles from three of four patients with polymyositis and from one with severe alcoholic myopathy also demonstrated impaired calcium affinity. A biopsy from the latter patient taken three months later (after disappearance of the myopathy) yielded vesicles with slightly abnormal calcium affinity and normal mitochondria. Mitochondria from three of the patients with polymyositis were normal, but the fourth patient yielded mitochondria with lower than normal Qoz and respiratory control ratios. DISCUSSION
Our finding of impaired calcium accumulation by vesicles from the affected muscles of patients with Duchenne dystrophy is in agreement with the report by Sugita et al. (1, 2). However, our measurement of the ATPase of the vesicle fraction is not strictly comparable to theirs, because our assay was conducted under conditions of optimal [Ca2+], whereas Sugita et al. ( 1, 2), using the method of Ebashi and Lipmann ( 11)) measured ATPase in the nominal absence of calcium, although no mention was made of any precautions which were taken to remove contaminating traces of calcium. Seidel and Gergely (12) have shown that contamination of commercial ATP might amount to a 6nal calcium concentration of about 10 PM in the medium used by Ebashi and Lipmann ( 11)) and contamination from other sources might also contribute calcium. The Mg-ATPase of skeletal muscle vesicles is optimally stimulated by calcium at concentrations anywhere between about 1 PM and 100 ,uM (cf. 10). At the start of the reaction, then, the medium of Sugita et al. (1, 2) would support optimal ATPase activity, if contaminating
MUSCULAR
DYSTROPHY
AND
OTHER
MYOPATHIES
369
Ca”+ had not been removed. Under these conditions with normal vesicles, uptake of calcium by the vesicles would quickly reduce [Caz+] to 0.3 PM or less, and the ATPase assay of Sugita et al. (2) would be conducted essentially in the absence of calcium. “Dystrophic” vesicles would not remove calcium so effectively as the normals, so that the calcium-stimulated ATPase is measured, leading to an apparent increase over the normal. This does not necessarily reflect an increased enzyme level. The analogy drawn by Sugita et al. between “dystrophic” vesicles and deoxycholate-treated, phospholipase C-treated, or aged vesicles which also show low capacity for calcium accumulation and high ATPase is not, therefore, necessarily valid. Rather the apparent increase in the MgATPase of such vesicles, like that of “dystrophic” vesicles, is probably due to impairment of their calcium-removing ability. The calcium dependence of vesicle ATPase is illustrated in Fig. 3. In this experiment far
MINUTES
FIG. 3. ATPase activity and calcium accumulation by vesicles from normal human skeletal muscle. Initial
calcium concentration
was 100 PM ( 1.25 ~mole8/mg
protein).
This amount of calcium is normally sufficient to saturatethe vesicles,leaving an excessof more than 10 FM. In tbis experimentreduction of the caldum concentration to 1 pM resuks in a simultaneous decrease of the calcium-stimulated activity. Composition of the mediumis described in the text.
ATPase
more calcium was added initially than might be present as contaminant in the ATPase assay of Sugita et al. (1, 2) and this resulted in a large initial burst of ATPase followed by a striking fall in ATPase activity as the free calcium level of the medium was lowered. This particular vesicle preparation was somewhat more active than the average but is shown to emphasize the striking stimulation of vesicle ATPase by calcium.
370
PETER
AND
WORSFOLD
In most of our samples of quadriceps muscle from patients with Duchenne dystrophy poor yields of mitochondria and vesicles and the abnormal properties of both these fractions indicate that gross, nonspecific degeneration had taken place. On the other hand, the less degenerate gastrocnemius muscles of our patients and the quadriceps and biceps femoris of two additional patients showed abnormality only in the ability of vesicles to reduce calcium concentration of a 20 pM solution, whereas calcium-accumulation capacity and rate of calcium uptake from 100 PM [Ca2+] were essentially normal. Of additional significance are our observations that with these less-degenerated muscles the respiratory rates, acceptor ratios, ADP/O ratios, and respiratory control ratios of isolated mitochondia were normal. Thus the calcium affinity assay has demonstrated a biochemical defect in vesicles from muscles in which the damage is early or at least is not general to all subcellular components and has detected an abnormality of vesicles which is not apparent with previously employed methods of studying calcium accumulation. Our current studies are directed to the possibility that an increased efflux of Ca2+ (increased leakiness of the vesicles) may contribute to this decrease in calcium affinity. The abnormal calcium affinity of vesicles seen in the Duchenne and Becker types of muscular dystrophy and in the diseases called polymyositis was not detected in a wide variety of other muscle and neuromuscular diseases, including myotonic dystrophy, periodic paralysis, thyrotoxic myopathy, and neurogenic muscular atrophies. A patient with acute alcoholic rhabdomyolysis in whom the disease was so marked at the time of initial biopsy that the mitochondria were also abnormal showed slightly abnormal calcium affinity on repeat biopsy, at which time oxidative phosphorylation had returned to normal. The decreased calcium affinity of vesicles with preservation of normal oxidative phosphorylation of isolated mitochondria seems to be relatively specific for muscular dystrophy and polymyositis. Its detection in the early stages of these diseases and also in carriers of Duchenne dystrophy and Becker dystrophy, whiIe mitochondrial function is unaffected, may indicate that the involvement of the sarcoplasmic reticulum takes place early in these disease processes, even though their etiologies are undoubtedly different. SUMMARY
Sarcotubular vesicles from patients with certain types of muscular dystrophy or polymyositis show a diminished ability to remove calcium from very dilute solutions (decreased calcium aflinity), whereas in some of these patients the capacity of vesicles to accumulate calcium, in the
MUSCULAR
DYSTROPHY
AND
OTHER
MYOPATHDZS
371
presence of excess calcium, is normal. Decreased calcium aflinity is detectable in these disorders when oxidative phosphorylation of concomitantly isolated mitochondria is normal. It is not found in a wide variety of other muscular or neuromuscular diseases. ACKNOWLEDGMENTS One Grant. by the Mrs. J.
1. 2.
3. 4. 5. 6. 7. 8. 9. 10. 11. 12.
of the authors (M. Worsfold) was assisted by a Wellcome Research Travel Supported by NIH grants NB 97587, HD 02584; CRC, NIH FR 00238, and Muscular Dystrophy Associations of America. The technical assistance of Payne is acknowledged.
REFERENCES SUGITA, H., OKIMOTO, K., AND EBAS~, S., Proc. Japan Aced. 42, 295 (1986). SUGITA, H., 0rrr~crr-q K., EBASHI, S., AND OIUNAKA, S., in “Exploratory Concepts in Muscular Dystrophy and Related Disorders” (A. T. Milhorat, ea.), Excerptu Med. Found., Zntern. Congr. Ser. No. 147, p. 321 (1967). MA~TONO~I, A., Proc. Sot. Exptl. Biol. Med. 127, 824 (1988). ROE, R. D., YAMAJI, K., AND S-w, A., in “Exploratory Concepts in Muscular Dystrophy and Related Disorders” (A. T. Milhorat, ea.), Excerpta Med. Found., Intern. Ccmgr. Ser. No. 147, p. 299 (1987). D~EYFUS, J. C., SHAPIRA, G., SCHAPIRA, F., ANY DEMOS, J., C&n. Chim. Acta 1, 434 (1958). PETER, J. B., B&&em. Med. 2, 179 (1988). PETER, J. B., ANI) LEE, L. D., Biochem. Biophys. Res. Commun. 29,430 ( 1967). MARTONO~U, A., AND Fmmn>s, R., J. Bfol. Chem. 239, 848 (1984). Rocrrs~~~m, M., AND HERRON, P. W., An& Chemistq 23, 1500 ( 1951). WFBER, A., in “Current Topics in Bioeneregtics” (D. R. Sanadi, ea.), Vol. 1, p. 293. Academic Press, New York ( 1988). Es~rq S., AND LIPMANN, F., J. CeE Btol. 14, 389 ( 1982). SEIDEL, J. C., AND GERGELY, J., J. Biol. Chem. 238, 3848 (1983).
MEDICINE
Muscular
2, 364-371 ( 1969)
Dystrophy
Sarcotubular
and Vesicles
J. B. PETER Department
AND
Other
Myopathies:
in Early
Disease
M. WORSFOLD
of Medicine, UCLA School of Medicine, Los Angeles, California 90024
ReceivedNovember 1, 1968 Vesicles derived from the sarcoplasmic reticulum of muscle from patients with Duchenne muscular dystrophy exhibit a decreased capacity for accumulating calcium with a concomitant increase in ATPase activity (1, 2). Sarcotubular vesicles from dystropbic mice also possess a reduced capacity for calcium accumulation (3). These observations with subcellular particles are an interesting corollary to the finding of delayed relaxation of muscIe in Duchenne muscular dystrophy (4). We have investigated several aspects of calcium uptake by vesicles from muscle obtained at biopsy from patients with no evidence of myopathy and from patients with a variety of muscle and neuromuscular diseases. Oxidative phosphorylation of intact mitochondria prepared from the same muscle samples was assayed concurrently. Cytochrome oxidase and sue&rate oxidase activities of dystrophic muscle are said to be normal (S), but a comparative study of the oxidative phosphorylation of mitochondria and calcium accumulation by sarcotubular vesicles in muscular dystrophy has not, to our knowledge, been published. By measuring calcium affinity of vesicles we have demonstrated biochemical abnormalities of vesicles from dystrophic and myositic muscle which were detected by the usual techniques for studying calcium accumulation. Oxidative phosphorylation of skeletal muscle mitochondria isolated from these same patients was normal. METHODS Mitochondria and vesicles were isolated by differential centrifugation of a homogenate prepared by shaking minced muscle with glass beads in a CO,-cooled shaker, as previously described in detail (6, 7 ). Respfmtory rates (Qo? ), acceptor ratios (AR), respiratory control ratios ( RCR) and ADP/O ratios were determined immediately after the isolation of the mitochondria (6, 7). The ZO-minute, 15,000-g supernatant fraction from the high-speed 364
MUSCULAR
DYSTROPHY
AND
OTHER
365
MYOPATHIES
mitochondrial pellet was centrifuged at 40,000g for 90 minutes to obtain sarcotubular vesicles. This pellet was rinsed with lO!Z sucrose and resuspended by gentle homogenization in 46% sucrose (0.8 mg protein/ ml). Calcium accumulation at intervals over a 1Sminute period was measured by the Millipore filtration method at 26” (8). The standard incubation medium contained 0.08 mg of vesicle protein/ml, 5 mM ATP, 5 mM MgCl,, 5 mM oxalate, 40 mM histidine, pH 7.2, 110 mM NaCl, 10 mM KCl, 120 mM sucrose, and either 100 PM CaCI, (calcium accumulation assay) or 20 PM CaCl, (calcium affinity assay), labeled with ““Ca. ATPase activity was related to inorganic phosphate (9) found in a portion of the Millipore filtrate, which contained no ATPase activity. Contaminating calcium was removed from ATP by passage through a column of Dowex-50 (H’). Sucrose solutions were passed through Dowex-50 (Na+) prior to use. Samples of dystrophic muscle were taken from thigh muscles (quadriceps or biceps femoris) and simultaneously in three patients from the pseudohypertrophied gastrocnemius muscles which were much stronger clinically and showed much less damage histologically than the thigh muscles. RESULTS
Thigh muscle from patients with far-advanced Duchenne dystrophy gave very low yields of mitochondria with low QO,, poor respiratory oo2:40 AVERAGE -GASTROCNEMlUS !!gc2‘J
-GASTROCNEMIUS
I I 0 rnq PROTEIN 15 0 mM PYRuvQTE 15 0 mM MALATE STANDARI TOTAL VOLUME = 2ml T:26*.pH:74
‘\
‘\
l_
015mf.4 -.
_-__
ADP
‘. ‘-.
BICEPS
FEMORIS
I
“‘.---T
..\\,___ OlSmM ADP
FIG. 1. Oxidative phosphorylation of skeletal muscle mitochondria isolated from two muscles of a 4-year-old boy with Ducherme dystrophy (patient 5). Conditions were exactly as described previously; note the high acceptor (AR) and respiratory control ratios ( RCR) and the near-“theoretical” ADP/O ratios (see 5).
A 0
NORMAL
I AND AlRoPHlES
MYOPATHY
x
DUCWENIIE: YUSCULAR
BECKER
l
l *
l
l * l
l
x x xx $xXxX
xx
x
x
[XX
xx
1
DYSTROPHY
DYSTROPHY
** l
x xxx x xxx xxx X
l PoLYYYoslTls
9l
l *m
x”
ALCOllOLIC
X
I?
.
YlSCLLLAWLOUS YYOPATnlts WEUnosLWIC
l .
0
l
x
.
X
1
r - - -i-------__- - - - - - - - - - - - - - - - - --, 30 om b l bo 1 x
+
F FX _-- ,e1 - - - - -- - - - - - - - -
1
-1
fACIO-SCAPULOHUYLRAL DYSTROPHY LIMO-SIROLE DYSTROPHY
.
I
t I
I
0
0.5
r_-_
0
l
X
I
x
x
X
I
I I .o
I I.5
B MISCELLANEOUS MYOPATHIES AND NEUROGENIC ATROPHIES ALCOHOLIC
MYOPATHY
DUCHENNE MUSCULAR
l ***
:
l **
l
t’t .
I
.
I
POLYMYOSlTlS
BECKER
. .
I
NORMAL
-
0
.
0 .
I I
1
DYSTROPHY
DYSTROPHY
FACIO- SCAPULOHUMERAL DYSTROPHY LIMB-GIRDLE UYSTROPHY
I IT----I-001
i
. -7-l
01
IO
L 1 &Xl++
/LLM
01 FOUR
IO
MINUTES
FIG. 2A. Calcium-accumulating capacity and ATPase activity of human vesicles. Maximum calcium uptake is expressed as amoles/mg vesicle protein ( l , normals
20
MUSCULAR
DYSTTIOPHY
AND
OTHER
MYOPATHIES
367
control and low ADP/ratios. The less-affected gastrocnemius muscles from the same patients, and the biceps femoris from one, were essentially normal in these respects (Fig. 1). One of the four patients with polymyositis and the first specimen from the patient with alcoholic myopathy also gave low yields of mitochondria, with low Qoz and poor respiratory control (Fig. 2B). The calcium-accumulation capacity with 100 PM calcium (1.25 pmoles/mg protein) was generally lower in the vesicles from severely affected muscle than in normal vesicles or in vesicles from muscle less affected by Duchenne dystrophy (Fig. 2A). Initial rates of calcium uptake also tended to be lower than normal in the severely affected group as did the initial Mg’+-Ca”-stimulated ATPase activity. Abnormalities of mitochondria were likewise more common in severely affected muscle (Fig. 2A). In an attempt to approach physiological conditions more closely, the removal of calcium from more dilute solutions of calcium (20 PM) was studied with the same concentration of vesicles (0.03 mg protein/ml). We shall refer to this as an assay of the calcium aflinity of vesicles as opposed to the total capacity for calcium accumulation. On incubation with only 20 PM calcium (0.25 pmoles/mg protein) normal vesicles quickly reduce the calcium concentration of the medium to less than 0.3 p~hr which is in the range required to induce relaxation of muscle or syneresis of myofibrils ( 10). The calcium af&ity assay therefore may be a more physiologically signiiicant test of the capacity of vesicles to bind calcium than is their rate of calcium uptake or total capacity for calcium aoxunulation from concentrated (100 PM) solutions of Caz+. Some of the vesicle preparations from Duchenne dystrophic muscle showed normal capacity for calcium accumulation but none had normal calcium affinity under the conditions of the assay (Fig. 2B). Vesicles from all three samples of dystrophic gastrocnemius showed normal and alfected subjects, normal mitochondria; 0, normals and affected subjects, ‘abnormal’ mttochondria; n , symptomless female heterozygotes). ATPase activity (~moles/min/mg protein) over the trst two minutes was measured simultaneously (x, normals and affected subjects; +, symptomless female heterozygotes). ‘Abnormal’ mitochondria are considered to be those with Qe, < 25 ~1 OJmg protein/ hour, in the presence of pyruvate, malate, PI and ADP, or RCR < 2.0. Symbols joined by tie-lines (- - - - ) represent samples obtained at one operation from different sites on the same patient, e.g., quadriceps and gastrocnemius. B. Calcium affinity of human vesicles. See Fig. 2A for explanation of symbols. Vesicles were incubated with 20 pM Ca*+ as described in the text. The differences between the various groups were most striking after 4 minutes’ incubation, but a similar pattern was evident at 2 and 8 minutes.
368
PETER
AND
WORSFOLD
capacity for calcium accumulation but all had impaired calcium affinity. This was also true of one vesicle preparation each from the biceps femoris and quadriceps muscles of patients with Duchemre dystrophy. Vesicles from the gastrocnemius of a female carrier of Duchenne dystrophy had normal calcium-accumulating capacity (Fig. 2A) but exhibited slightly impaired calcium affinity (Fig. 2B). Vesicles from a patient with the Becker form of pseudohypertrophic muscular dystrophy and from his daughter (a known carrier of this disease) showed grossly and slightly decreased calcium affinity respectively, whereas calcium accumulation capacity was normal in both. We have studied two biopsies from one patient with facie-scapula-humeral dystrophy. Calciumaccumulation was normal in vesicles from the quadriceps and deltoid muscles, but calcium affinity was abnormal in the quadriceps and normal in the deltoid. Three patients with limb-girdle dystrophy have shown normal capacities for calcium-accumulation and, in the only one of these tested, the assay for calcium affinity was also normal. Vesicles from three of four patients with polymyositis and from one with severe alcoholic myopathy also demonstrated impaired calcium affinity. A biopsy from the latter patient taken three months later (after disappearance of the myopathy) yielded vesicles with slightly abnormal calcium affinity and normal mitochondria. Mitochondria from three of the patients with polymyositis were normal, but the fourth patient yielded mitochondria with lower than normal Qoz and respiratory control ratios. DISCUSSION
Our finding of impaired calcium accumulation by vesicles from the affected muscles of patients with Duchenne dystrophy is in agreement with the report by Sugita et al. (1, 2). However, our measurement of the ATPase of the vesicle fraction is not strictly comparable to theirs, because our assay was conducted under conditions of optimal [Ca2+], whereas Sugita et al. ( 1, 2), using the method of Ebashi and Lipmann ( 11)) measured ATPase in the nominal absence of calcium, although no mention was made of any precautions which were taken to remove contaminating traces of calcium. Seidel and Gergely (12) have shown that contamination of commercial ATP might amount to a 6nal calcium concentration of about 10 PM in the medium used by Ebashi and Lipmann ( 11)) and contamination from other sources might also contribute calcium. The Mg-ATPase of skeletal muscle vesicles is optimally stimulated by calcium at concentrations anywhere between about 1 PM and 100 ,uM (cf. 10). At the start of the reaction, then, the medium of Sugita et al. (1, 2) would support optimal ATPase activity, if contaminating
MUSCULAR
DYSTROPHY
AND
OTHER
MYOPATHIES
369
Ca”+ had not been removed. Under these conditions with normal vesicles, uptake of calcium by the vesicles would quickly reduce [Caz+] to 0.3 PM or less, and the ATPase assay of Sugita et al. (2) would be conducted essentially in the absence of calcium. “Dystrophic” vesicles would not remove calcium so effectively as the normals, so that the calcium-stimulated ATPase is measured, leading to an apparent increase over the normal. This does not necessarily reflect an increased enzyme level. The analogy drawn by Sugita et al. between “dystrophic” vesicles and deoxycholate-treated, phospholipase C-treated, or aged vesicles which also show low capacity for calcium accumulation and high ATPase is not, therefore, necessarily valid. Rather the apparent increase in the MgATPase of such vesicles, like that of “dystrophic” vesicles, is probably due to impairment of their calcium-removing ability. The calcium dependence of vesicle ATPase is illustrated in Fig. 3. In this experiment far
MINUTES
FIG. 3. ATPase activity and calcium accumulation by vesicles from normal human skeletal muscle. Initial
calcium concentration
was 100 PM ( 1.25 ~mole8/mg
protein).
This amount of calcium is normally sufficient to saturatethe vesicles,leaving an excessof more than 10 FM. In tbis experimentreduction of the caldum concentration to 1 pM resuks in a simultaneous decrease of the calcium-stimulated activity. Composition of the mediumis described in the text.
ATPase
more calcium was added initially than might be present as contaminant in the ATPase assay of Sugita et al. (1, 2) and this resulted in a large initial burst of ATPase followed by a striking fall in ATPase activity as the free calcium level of the medium was lowered. This particular vesicle preparation was somewhat more active than the average but is shown to emphasize the striking stimulation of vesicle ATPase by calcium.
370
PETER
AND
WORSFOLD
In most of our samples of quadriceps muscle from patients with Duchenne dystrophy poor yields of mitochondria and vesicles and the abnormal properties of both these fractions indicate that gross, nonspecific degeneration had taken place. On the other hand, the less degenerate gastrocnemius muscles of our patients and the quadriceps and biceps femoris of two additional patients showed abnormality only in the ability of vesicles to reduce calcium concentration of a 20 pM solution, whereas calcium-accumulation capacity and rate of calcium uptake from 100 PM [Ca2+] were essentially normal. Of additional significance are our observations that with these less-degenerated muscles the respiratory rates, acceptor ratios, ADP/O ratios, and respiratory control ratios of isolated mitochondia were normal. Thus the calcium affinity assay has demonstrated a biochemical defect in vesicles from muscles in which the damage is early or at least is not general to all subcellular components and has detected an abnormality of vesicles which is not apparent with previously employed methods of studying calcium accumulation. Our current studies are directed to the possibility that an increased efflux of Ca2+ (increased leakiness of the vesicles) may contribute to this decrease in calcium affinity. The abnormal calcium affinity of vesicles seen in the Duchenne and Becker types of muscular dystrophy and in the diseases called polymyositis was not detected in a wide variety of other muscle and neuromuscular diseases, including myotonic dystrophy, periodic paralysis, thyrotoxic myopathy, and neurogenic muscular atrophies. A patient with acute alcoholic rhabdomyolysis in whom the disease was so marked at the time of initial biopsy that the mitochondria were also abnormal showed slightly abnormal calcium affinity on repeat biopsy, at which time oxidative phosphorylation had returned to normal. The decreased calcium affinity of vesicles with preservation of normal oxidative phosphorylation of isolated mitochondria seems to be relatively specific for muscular dystrophy and polymyositis. Its detection in the early stages of these diseases and also in carriers of Duchenne dystrophy and Becker dystrophy, whiIe mitochondrial function is unaffected, may indicate that the involvement of the sarcoplasmic reticulum takes place early in these disease processes, even though their etiologies are undoubtedly different. SUMMARY
Sarcotubular vesicles from patients with certain types of muscular dystrophy or polymyositis show a diminished ability to remove calcium from very dilute solutions (decreased calcium aflinity), whereas in some of these patients the capacity of vesicles to accumulate calcium, in the
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DYSTROPHY
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presence of excess calcium, is normal. Decreased calcium aflinity is detectable in these disorders when oxidative phosphorylation of concomitantly isolated mitochondria is normal. It is not found in a wide variety of other muscular or neuromuscular diseases. ACKNOWLEDGMENTS One Grant. by the Mrs. J.
1. 2.
3. 4. 5. 6. 7. 8. 9. 10. 11. 12.
of the authors (M. Worsfold) was assisted by a Wellcome Research Travel Supported by NIH grants NB 97587, HD 02584; CRC, NIH FR 00238, and Muscular Dystrophy Associations of America. The technical assistance of Payne is acknowledged.
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