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Myopathy caused by a deficiency of Ca2+-adenosine triphosphatase in sarcoplasmic reticulum (Brody's disease).

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Myopathy Caused by a Deficiency of Ca2+ Adenosine Triphosphatase in Sarcoplasmic
Reticulum (Brody’s Disease)
George Karpati, MD, Jeffrey Charuk, MSc, Stirling Carpenrer, MD, Charles Jablecki, MD,*
and Paul Holland, PhD
Four male patients from two families were first seen with impaired skeletal muscle relaxation that rapidly worsened
during exercise. Muscle biopsies from 2 patients were examined by appropriate biochemical and microscopic immunocytochemical techniques. The adenosine triphosphate (ATP)-dependent Ca” transport rate was extremely low
in a particulate membrane fraction of skeletal muscle, and there was also a marked reduction of the concentration o f
100-kDphosphoprotein, corresponding to Ca*+-ATPaseof sarcoplasmic reticulum, in muscle microsomes. The concentration of immunoreactive Ca2+-ATPaseof sarcoplasmic reticulum was markedly reduced on immunoblots. Evaluation
by microscopic immunocytochemical techniques, using one polyclonal and two monoclonal antibodies against sarcoplasmic reticulum CaZ+transport protein, revealed that the severe reduction of immunoreactive CaZ+-ATPaseWE,
limited to the histochemical type 2 fibers. The deficiency of the Ca2+transport protein in the sarcoplasmic reticulum of
type 2 fibers, which may be the primary expression of a presumed gene defect, can explain the impaired musclts
relaxation of the patients. This disease appears to be a clinically, electromyographically, and biochemically distinct
metabolic myopathy.
Karpati G, Charuk J, Carpenter S, Jablecki C, Holland P Myopathy caused by a deficiency of
Ca2+-adenostne triphosphatase in sarcoplasmic reticulum (Brody’s disease). Ann Neurol 20 38-40, 1086
An unusual disorder of skeletal muscle function, characterized by increasing impairment of relaxation during exercise, was observed by Brody in 1969 Cl]. Calcium ion (CaL+) uptake by isolated sarcoplasmic
reticulum (SR) was markedly reduced, and this was
believed to be the cause of the impaired muscle relaxation in vivo. Previously, Lambert and Goldstein 1161
had described a patient with “silent myotonia” who
exhibited impaired muscle relaxation and probably suffered from the same disease.
In this communication, we describe 4 similar patients from 2 families who had a striking syndrome of
impaired muscle relaxation caused by a specific defect
in SR function. SR has a major role in excitation contraction coupling in skeletal muscle fibers [ 2 2 ] . In response to the excitation signal of the T tubules, CaZ is
released from the lateral cisterns of the SR into the
myofibrillar compartment; this extra Ca2 triggers
molecular events that ultimately lead to myofilament
sliding and force generation. Subsequently, the Ca2
concentration of the myofibrillar compartment is rapidly restored to the resting level by uptake of Ca2+
into the SR lumen through the activity of a Ca2+-Mg2+
adenosine triphosphatase ( ATPase) located in the SR
membrane [lo, 2 1, 221. Thus, the normal functioning
of this ATP-dependent Ca2+ transport enzyme is critical for the normal relaxation of muscle fibers.
In 2 of our patients, a marked reduction of immunoreactive Ca” transport ATPase of the SR of
fast-twitch muscle fibers was demonstrated; this can
explain the clinical symptoms and signs.
From the Deparcrnent of Neurology and Neurosurgery, McGill
Universitv. and the Montreal Neurolorical Institute. Montreal.
Quebec, Canada, and the *Department oiNeuro10gy3 university Of
California at San Diego, San Diego, CA.
Received July 18, 1985, and in revised form Oct 11, 1985. Acceoted for Dublication Oct 28. 1985.
Case Reports
Patient 1
A 42-year-old man came to our attention in 1982 with exer-cise-induced stiffness and “cramping” in most arm and leg
muscles. The symptoms started during the first decade and
increased progressively so that by age 40 he could no longer
work as a plumber. There was no history of pigmenturia. His
parents were first cousins and had no neuromuscular symp-toms. Of his 4 brothers and 3 sisters, a brother (Patient 2 ) is
affected by a similar disorder.
Examination revealed no muscle wasting and only minor
proximal limb muscle weakness (MRC grade 4 + ). Tendon
reflexes were moderately active. Exercise such as opening
and closing the hand or deep knee bends induced progressive stiffening, some pain in the exercised muscles, and in.-
Aidress reLrint requests tO Dr Grpari, Montreal Neurologic, Institute, 3801 University St, Montreal, Quebec, Canada H3A 2B4.
creasing difficulty relaxing them. After about 30 to 40 seconds of continued exercise, the patient could no longer open
his fist or rise from a bent-knee position. After a 1-minute
rest period, he could exercise again but the time required for
relaxation of his muscles became longer. There was no grip
or percussion myotonia. Craniobulbar and trunk muscles
did not show overtly impaired relaxation. Immersing the
forearm in cold water did not influence the exercise-induced
muscle stiffness.
A normal routine biochemical and hematological profile
was obtained. Serum creatine kinase activity was 50 IU/liter
(normal, 5 to 140 IUlliter) and urinary myoglobin was absent
by spectrophotometry. The resting venous lactate level was
1.7 mM (normal control measured simultaneously was 1.28
mM). After ischemic forearm exercise, the maximum lactate
level reached at 5 minutes was 3.76 mM (control, 1.47 mM);
30 minutes after exercise, the lactate continued to be above
resting level at 2.16 mM when the control had dropped to
0.92 mM. Electromyography showed physiological recruitment of normal motor unit potentials. There were no myotonic discharges. Microscopic and biochemical findings in
three muscle biopsies are described under Results. Treatment of the patient with dantrolene sodium (25 mg) or
nifedipine (20 mg) four times a day for at least a month did
not improve muscular function.
Patient 2
Patient 2 came to our attention in 1975, at age 42. He is a
brother of Patient 1. Since childhood he had complained of
“cramping” and stiffness of his limb muscles after less than a
minute of exercise. Examination showed the same progressive failure of muscle relaxation, as that described for his
brother. Limited laboratory results were obtained, which included normal routine biochemical and hematological tests.
Serum creatine kinase activity was 62 IU/liter (normal, 5 to
140 IUAiter). Electromyograms of several upper and lower
extremity muscles were entirely normal. No myotonic discharges were observed even after exercise-induced muscle
stiffness developed. Two biopsies of the biceps muscle were
examined microscopically (see Results). No tissue was available for immunocytochemical or biochemical studies.
Patient 3
A 26-year-old medical technologist was first seen in 1982 at
the Muscle Clinic of the Children’s Hospital, San Diego,
California, for evaluation of exertional muscle stiffness dating
back to childhood. He first sought medical attention in college. At that time he was not able to run 50 yards in less than
11 seconds, whereas his friends could run the same distance
in 6 to 8 seconds. He had the same range of activities as his
peers, but could nor do them as quickly because he found
that with strenous effort his muscles “tightened up.” There
was no history of pigmenturia with exertion.
Neurological examination demonstrated slow relaxation of
the orbicularis oculi muscles after sustained forceful eyelid
closure for 10 seconds (Fig 1). Clenching and unclenching his
fist was accompanied by progressive slowing of the movements. After forcefully clenching his fists for 10 seconds, he
could only open the hand slowly, with initial flexion of the
wrist (Fig 1).There was no evidence of percussion myotonia
in the hands o r tongue. The strength of individual muscles
and the muscle stretch reflexes were normal, as were the
results of sensory examination. The patient has a 36-year-old
brother (Patient 4) and a 34-year-old unaffected sister. There
is no family history, in three generations, of similar neuromuscular disorders. There was no parental consanguinity.
Laboratory studies included a serum creatine kinase activity
of 246 IU/liter (upper limit of normal, 232 IU/liter). The
right median nerve conduction studies showed normal amplitude, distal latency, conduction velocity, and F wave latency. Repetitive stimulation at 2 Hz showed no decrement,
and the amplitude of the motor response immediately after 1
minute of sustained contraction was the same as that of the
rested muscle. There were no fasciculations, fibrillation potentials, or myotonic discharges noted in the rested muscles.
During the phase of delayed relaxation of strongly contracted muscles (orbicularis oculi, first dorsal interosseous,
flexor carpi ulnaris), there was electrical silence. Microscopic
and biochemical findings of a quadriceps biopsy are presented under Results.
Patient 4
A 36-year-old lawyer is the brother of Patient 3. He was not
available for neurological examination or laboratory investigation, but a description by his brother indicated that he, too,
suffers from exertional muscle stiffness, but to a lesser degree.
Materials and Methods
Muscle biopsies from Patients 1 (biceps) and 3 (quadriceps)
were used for microscopic and biochemical studies. A biceps
biopsy of Patient 2 was only studied by microscopy. Control
muscles for biochemical analysis included two biopsies that
showed no microscopic abnormalities (normal controls), 6
muscles that showed partial denervation due to chronic peripheral neuropathies, and l specimen from a patient with
latent malignant hyperthermia.
Microscopic Techniques
Transverse and longitudinal cryostat sections were prepared
and used for routine histochemistry and immunocytochemistry for the display of immunoreactive Ca*+-ATPase. For immunocytochemistry, 4-km-thick cryostat sections were fixed
with 2% glutaraldehyde in 0.1 M cacodylate buffer for 3
minutes, washed with water, then washed with phosphatebuffered saline (PBS). Sections were treated for 15 minutes
each with 0.05 M glycine in PBS to block reactive groups and
0.05% HrOr in methanol to block endogenous peroxidase
activity. After washings with HzO and PBS, either polyclonal
(1 :200 dilution) or monoclonal (1:50 dilution) antibody to
Ca*+-ATPase (in PBS containing 1% bovine serum albumin) was layered over the sections for 12 hours at 4°C in
a humidified chamber. A 1:10,000 dilution of antibody
yielded 50% maximal binding to 12.5 pg of chicken SR
membranes in a quantitative enzyme-linked immunosorbent
assay. Sections were washed with PBS, then covered for 2
hours with biotinylated antimouse or antirabbit IgG (1 : 100
dilution in PBS containing 1% bovine serum albumin). After
washings with PBS, sections were covered with a freshly
prepared 1 : 1 mixture of avidin-DH and biotinylated horse-
Karpati et al: Myoparhy Due to Ca’+-ATPase Deficiency
Annals of Neurology
Vol 20
No 1 July 1986
radish peroxidase H (Vector Laboratories, Burlingame, CA)
for 1 hour at 20°C. After washing with PBS, the GrahamKarnovsky reaction was used to display peroxidase activity.
The presence of immunoreactive calsequestrin was displayed with polyclonal affinity-purified antibodies produced
to purified rabbit calsequestrin f l11. (Calsequestrin antibodies were obtained through the courtesy of Drs David
MacLennan and A. 0.Jorgensen, University of Toronto.)
Controls for the immunocytochemical techniques included three muscles showing no microscopic abnormalities,
muscles from 2 patients with severe nonspecific type 2 fiber
atrophy, and two chronically denervated muscles. Technical
controls included sections exposed to nonimmune rabbit or
mouse IgG instead of antibodies; after this the same technique was followed as that for the experimental sections.
Monoclonal and polyclonal antibodies were mixed with
purified rabbit skeletal muscle Ca2+ -ATPase, previously
coupled to sepharose 4 B to absorb out immunoreactive
globulins. Absorptions were performed for 24-hour periods
at 4°C in PBS. The number of absorptions required to remove reactive immunoglobulins completely was determined
by testing the remaining antibody titer. Absorptions were
considered complete when less than a 1 : 10 dilution yielded
half maximal reactivity in an enzyme-linked immunosorbent
Portions of the biopsies were fixed in 2.5% glutaraldehyde
and subsequently embedded in epoxy resin. Semithin (9
blocks from Patient 1 and 8 blocks from Patient 2) and
ultrathin sections (4 blocks from Patient 1 and 1 block from
Patient 2) were examined by phase and electron microscopy,
Biochemical Techniques
MEMBRANE PREPARATIONS. A particulate membrane fraction was prepared from homogenates of muscle biopsies essentially according to the method of Charuk and Holland
141. Microsomes were isolated from muscle biopsy tissue
essentially by the method described by Brody [l}. Protein
determinations were performed on isolated membranes by
the method of Lowry and co-workers [ls}.
ENZYME ASSAYS. The rate of ATP-dependent Ca2+ transport by particulate membrane fractions was determined by
micropore filtration of vesicles incubated in 45CaC12,as described by Martonosi and associates [23]. A steady-state
phosphorylated intermediate of the Ca2 -ATPase was
formed by incubating microsomes with (y-’*P)-ATP, as described by Martonosi and co-workers 1231. Phosphoproteins
were separated by electrophoresis in the p H 2.4 gel system
described by Fairbanks and Auvuch [G}. Autoradiography of
Fig I . Patient 3 . Delayed relaxation of eyes closed andfist
clenched after a maximal effort sustained for I0 seconds. Each
sequence of pictures was taken over a period of 3 t o 5 seconds.
Note the furrowing of the brow as the eyes began to open, as the
frontalis muscle attempts to assist in opening the eyes. Also note
thejexion of the wrist as the patient attempts to extend the
fingers, a maneuver that may alro be seen in patients with myotonic dystrophy.
dried gels was performed by the method of Laskey and Mills
ANTIBODY PREPARATIONS. Polyclonal antibodies to the
Ca2+-ATPase were prepared by immunization of rabbits
with the purified chicken pectoralis major enzyme [19].
Monoclonal antibodies to the Ca2+-ATPasewere obtained
from cloned hybridomas El31 formed by the fusion of P3X63-Ag8.65 3 myeloma cells [ 12) to mouse lymphocytes using the method of Kohler and Milstein 1141. Spleen cells
were isolated from CD-1 Swiss white mice immunized with
chicken pectoralis major SR proteins. Selection of cloned
hybridomas was performed by a modified immunoreplica
technique E301. Secreted antibodies to SR proteins were detected by the method of Hsu and co-workers C91. Antibodies
were purified from rabbit sera and hybridoma serum-free
culture media [24} by salt fractionation 181. Monoclonal antibodies were radioiodinated by the chloramine T procedure
[ 7 ] . The specificities of antibodies for the 100-kD Caz+ATPase protein were determined by Western immunobiotting of chicken muscle SR proteins [5].
muscle biopsy extracts were prepared by homogenizing 1 to
2 gm of tissue with 20 strokes of a ground glass homogenizer
pestle in 1 ml of 0.6 M KCI, 10 mM imidazole (pH, 7.3), and
0.3 M sucrose. Homogenates were sonicated for 30 seconds
using a Model 150 sonic dismembranator (Artek Systems
Corp, Farmingdale, NY). Samples were precipitated overnight with 5% trichloroacetic acid at 4°C and were pelleted
in an Eppendorf centrifuge (Model 5412) for 5 minutes at
14,500 g. Pellets were washed twice with 0.01 M sodium
phosphate (pH, 7.5). The pellets were then solubilized overnight in 0.5 to 1.0ml of 2.0% sodium dodecyl sulfate (SDS),
10% glycerol, and 65.5 mM Tris (pH, 6.8) by vortexing in an
Eppendorf Model 5432 Mixer (Brinkman Instrument Co,
Rexdale, Ont.). Protein levels were determined by the
method of Peterson [27]. 2-Mercaptoethanol was added to
samples to a final concentration of 5%. Samples were boiled
for 4 minutes and aliquots were diluted with 2.0% SDS,
10% glycerol, 5% 2-mercaptoethanol, and 65.5 mM Tris
(pH, 6.8) to a protein concentration appropriate for gel electrophoresis. Gel electrophoresis of proteins was performed
by the method of Laemmli [15}. Gels of 0.8 mm thickness
were stained using the silver method described by Sammons
and co-workers 1291. Proteins were transferred overnight to
nitrocellulose paper by the method of Towbin and associates
1321. Nitrocellulose transfers were blocked by incubation for
24 hours at 4°C in Ca*+-freePBS and 0.01% NaN3 containing 3% bovine serum albumin, prior to incubation with antisera. Then 0.01 pCiiml of 1251-mousemonoclonal antibody
to chicken Ca2+-ATPase, in blocking solution, was allowed
to react for 24 hours at 4°C with transfers of muscle biopsy
proteins and purified rabbit Ca2+-ATPasestandards to form
immunoblots. These were then incubated in a solution of
0.25% gelatin, 5 mM EDTA, 0.15 M NaCI, 0.05% NP-40,
and 0.05 M Tris (pH, 7.5) [25} for 30 minutes at 4”C, and
washed five times over a period of 24 hours at 4°C in Ca2+free PBS and 0.01% NaN3. The immunoblots were airdried and exposed to Kodak X-OMAT film for autoradiography using intensifying screens 1171.
Karpati et al: Myopathy Due to Ca2+-ATPase Deficiency
Densitometry was performed using a PMQII spectrophotometer equipped with a motorized flatbed gel scanner
(Carl Zeiss Inc, New York, NY) and a logarithmic chart
recorder. The 100-kD band, corresponding to monomeric
Ca2+-ATPase, was scanned at 550inm and the peak absorption area was determined using a Zeiss MOP-3 image analyzer. Best-fit standard curves for antibody binding to
purified rabbit skeletal muscle Ca*+-ATPase were established, and the quantity of Ca*+-ATPase in human muscle
extracts was determined. The assay was linear over a very
wide range of Ca’+-ATPase concentrations and a limit of
detection was found to be less than 10 ng of previously
frozen purified enzyme. Optimal detection of Ca’ +-ATPase
in human muscle extracts occurred using concentration
greater than 10 pg of homogenate protein per 4-mm gel slot.
For comparative purposes, myosin was extracted from
muscle biopsy homogenates and quantitated by the method
of Paterson and Strohman [26].
I n Vitro Caffeine-Halotbane Contractwe Test
Two fresh slabs of the biopsy of Patient 1 (approximately 10
x 30 mm) were used to measure isometric tension in vitro at
20°C in the presence of increasing concentrations of caffeine
(0.25 to 16 mM) as described by Britt and co-workers 121.
The minimum concentration of caffeine that produced a
I-gm rise in basal tension was considered the “caffeinespecific concentration.” The concentration was also tested in
the presence of 2% halothane.
HISTOCHEMISTRY. Cryostat sections of the muscle
biopsies from Patient 1 showed that approximately
45% of the histochemical type 2 (A and B) muscle
fibers were significantly atrophic in relation to type 1
fibers (Fig 2C). The mean minimum diameter of type 2
fibers was 61 ? 20 pm (534 fibers measured) as compared to 81 k 12 pm for type 1 fibers (106 fibers
measured). Thirty percent of type 2 fibers were less
than 50 pm in diameter, whereas none of the type 1
fibers was in that size range. Type 2 fibers composed
71%, type 1 29% of the total fiber population. The
cross-sectional outline of most type 2 fibers was angular. About 50% of the type 2 fibers had one to five
internally placed myonuclei (Fig 3).
The atrophic type 2 fibers had noticeably reduced
glycogen content, but normal glycogen phosphorylase
activity was observed in the presence of adenosine
monophosphate; unlike normal controls, an appreciable level of phosphorylase activity was seen in the absence of adenosine monophosphate. There were scattered hypercontracted fibers. No muscle fiber necrosis
was noted.
Biopsies of Patients 2 and 3 showed similar features
(Fig 4C). In Patient 3 , the mean minimum type 2 fiber
diameter was 57
21 pm (401 fibers measured),
whereas that for type 1 fibers was 79 2 13 Fm (181
Annals of Neurology
Vol 20
N o 1 July 1986
Fig 2. Serial cryostat sections from biceps biopsy of Patient I .
(A) Four-micrometer-thick transzierse section displa.ying the
C d -adenosine triphosphatase (ATPase)protein with monoclonal antibody. The type 2fibers. many ofwhich are of abnormally small caliber, have practically no immunoreactive C a ” ATPase protein. whereas type 1 fibers show the normal amount.
(B and C) Ten-micrometer-thick sections reacted for the activity
of myofibrillar ATPase after p H 4.3 (B) and 9.4 (Cj inCuba.tion. In C, the marked atrophy of many type 2 fibers is consplcuozcs. (All x 220 before 30%, redzlcti0n.i
Fig 3. Patient 1. Numerous internally situated myonuclei are
present in the atrophic (type2)fibers. (H6E; x 350 b.efore 30%
fibers measured). Type 2 fibers constituted 67% and
type 1 fibers 31% of the total fiber population.
Minimum mean fiber diameter of an age- and sexmatched control was 67 ? 12 pm for type 1 and 67 t
7 pm for type 2 fibers. None of the control fibers was
less than 50 pm in minimum diameter.
(whose muscles were available for testing), there was
barely detectable immunoreactive Ca2+-ATPase by
polyclonal (Fig 5A) or monoclonal antibodies in type 2
A or B fibers (Figs 2A, 4A, 5B). In contrast, type 1
fibers had the normal amount of immunoreactive
Ca2+-ATPase. In normal controls, immunoreactive
Ca2'-ATPase is present in equal amounts in type 1
and 2 fibers (Fig 6). In nonspecific type 2 fiber atrophy,
type 2 fibers did not show a reduced amount of immunoreactive Ca2'-ATPase (Fig 7 ) . Absolutely no
staining was present in type 1 or 2 muscle fibers by
using solutions of antibodies that had been absorbed
by purified rabbit Ca2'-ATPase, or using nonspecific
I& instead of specific antibody. Immunoreactive
calsequestrin concentration in Patient 2 was comparable to that of normal controls.
ELECTRON MICROSCOPY. Sections from biopsies of
Patients 1 and 2 were examined. The general internal
structure of muscle fibers was normal. Special attention was paid to fibers with relatively few mitochondria, since they were assumed to be type 2 . Triads
were normal in size and distribution. Tubular elements
of the SR could be seen here and there between
myofibrils on longitudinal sections (Fig 8). On transverse sections they could be seen occasionally bordering the Z disc, although somewhat less often than in a
control with simple type 2 atrophy. Occasional fibers
showed a focal reduplication of their basal lamina.
F ig 4. Serial cryostat sections from quadriceps biopsy of Patient
3. (A) Four-micrometer-thick cryostat section displaying the
C d +-adenosine triphosphatase (ATPasej protein with monoclonal antibody. The type 2 fibers have barely detectable immunoreactive Cd +-ATPaseprotein, whereas type 1 fibers have
a normal amount. (€3 and C) Ten-micrometer-thicksections
reacted for the activity of myofibrillar ATPase after pH 4.3 (B)
and 9.4 (C) incubation. In C, marked atrophy of some type 2
fibers is evident. (All x 220 before 30% reducti0n.j
Karpati et al: Myopathy Due to Ca2+-ATPase Deficiency 43
Fig 5 . (A)Patient 1. Markedly reduced immunoreactioe (polyclonal antibody) C d ‘-adenosine triphosphatase (ATPasei content is present in the atrophic (type 2)fibers; the normal-caliber
(type 1) fibers display normal immunoreactii~ity.( B )A t high
magnification, a monoclonal antibody (differentfrom the one
used to produce the preparation illustrated in Figure 2) rezjeah a
trace of immunoreactitie Cd+-ATPase in the atrophic (type 21
fibers. (Both x 350 before 30% reduction.)
caz+ TRANSPORT. A consistently low rate of ATPdependent Ca2 transport was observed for particulate
membranes isolated from the muscle of Patient 1 as
compared to control muscle (Fig 9). These results implied that there was either a net reduction in the
amount of enzymatically active Ca*+-ATPase or a reduced efficiency of Ca2 transport.
A comparison of
the amount of high-energy phosphorylated intermediate of the Ca2+-ATPase found in muscle microsomes of a disease control (lane a) and Patient l
(lane b) is shown in Figure 10. A marked reduction in
the amount of Ca2+-dependent 100-kD phosphoprotein, corresponding to the Ca2+-ATPase of SR, was
observed for microsomes isolated from Patient 1 (Fig
10, lane b). The major band of radioactivity at 100 kD
showed Ca2 dependence for phosphoprotein formation, since in the absence of Ca2’ little radioactivity
was observed in this region of the gel (Fig 10, lanes c,
d). The band of radioactivity at 100 kD was completely
removed by hydroxylamine treatment of phosphory-
QUANTITATION OF THE Ca2+-ATPase. Both the reduced Ca2+ transport rate and amount of phosphorylated intermediate formed in membranes isolated from
Fig 6. Serial cvostat sections of a normal biceps muscle. (A)
Type 1 and type 2 fibers show similar amounts of immunoreactive C a ” -adenosine triphosphatase (ATPasei displayed by the
same monoclonal antibody as wed t o produce the preparations
illustrated in Figures 2 A and 4 A . (Bi Myofibrillar ATPase,
pH 9.4 preincubation. (Both x 350 before 8% reduction.)
44 Annals of Neurology
lated proteins before electrophoresis (Fig 10, lanes e,
h). This confirmed the presence of a high-energy acylphosphate bond in the 100-kD protein that is characteristic of the Ca2+-ATPase of SR.
Vol 20
No 1 July 1986
Fig 7 . Serial cyostat sections from a quadriceps biopsy of a patient with nonspeczflc type 2 fiber atrophy secondary to disuse.
(A)Four-micrometer-thick cryostat section reveals abundant immunoreactive C d '-adenosine triphosphatase (ATPasei protein
in the small type 2 fibers and low or intermediate amounts in the
type 1 fibers. The monoclonal antibody was the same as the one
for producing the preparations illustrated in Figures 2A and
4A. (B) Myofibrillar ATPase reaction afier pH 9.4 incubation
reveals conspicuous type 2 fiber atrophy. (Both X 100 before
30% reduction.)
Patients 1 and 3 could be accounted for by a decreased
amount of activity of the Ca2+-ATPaseenzyme. When
whole muscle biopsy extracts were prepared from patients, then electrophoresed and the gels stained with
silver, a protein band of 100 kD was noticeably reduced in Patients I and 3 (Fig 11).
A specific quantitative immunological technique was
used to confirm that the patients did indeed have a
deficiency of skeletal muscle Ca2 +-ATPase enzyme
protein. Monoclonal antibodies, prepared to the Ca2
ATPase isolated from chicken skeletal muscle SR,
were used to probe the amount of enzyme present in
muscle biopsy samples of Patients 1 and 3. As shown
in Figure 12, muscles of Patients 1 and 3 (lanes b, e)
lack the major immunoreactive form of the 100-kD
Ca2+-ATPase protein. Some antibody binding to proteins of slightly greater and lesser molecular weight
+ -
F i g 8. Electron micrographfrom the biopsy of Patient 1 shows
an apparently normal triad (TR) with adjacent tubular elements
of the sarcoplasmic reticulum (arrows). ( x 100,000 bejore 25%
Karpati et al: Myopathy Due to Ca'+-ATPase Deficiency 45
TIME (mmutes)
Fig 9. Cd' uptake by human muscle particulate niembrunes
l f r techniques see Materials and Methods). In Patient 1 (closed
squares), adenosine triphosphatase-dependent C h ' uptake is
practically absent. Open squares represent the uptakes (mean 2
SD)of3 diJease controls (2,denervation atrophy; 1 malignant
than the predominant immunoreactive Ca2+-ATPase
protein occurs in all of the extracts examined.
The amount of Ca2+-ATPase enzyme in whole muscle biopsy extracts was determined for Patients 1 and 3
and several disease control patients using radioiodinated monoclonal antibody to the chicken Ca2
ATPase. The Table shows that Patients 1 and 3 have
undetectable levels of Ca2+-ATPase enzyme, although
they have normal levels of myosin heavy chain.
Control patients, who clinically showed signs of a peripheral neuropathy, had variable amounts of Ca2' ATPase protein. This suggests that innervation of
muscle might regulate the accumulation of Ca2'ATPase, as previously suggested by others 131). One
patient with peripheral neuropathy appeared to have a
decreased level of Ca2+-ATPase protein associated
with an increased level of a higher-molecular-weight
cross-reactive peptide (Fig 12, lane 1). One of the
disease control patients with latent malignant hyperthermia showed both normal Ca2+ transport rates (Fig
9) and a high level of immunoreactive Ca2+-ATPase
(Fig 12, lane c, Table). All of the disease control
muscle biopsies we have studied to date have been
shown to contain detectable levels of immunoreactive
+ -
In Vitro Cufleeine-Halothane Contractwe Test
The caffeine-specific concentration of muscle from Patient l was 0.6 (normal is above 4.1). The halothanepotentiated concentration was 0.1 (normal is above
46 Annals of Neurology
Vol 20
No 1 July 1986
F i g 10. Gel electrophoresis of phosphoproteinsformed in microsomesfrom muscle biopsy specimens. k n e s a, c. e. and g are
from a disease control (Patient 5 of Tablet; 1ane.c b, A, and h
are from Patient I . Phosphoproteins were formed in the presence
of (lanes a, b, e, andfl or absence of ilanes c, d. g. and h) Ca' '.
Phosphoproteins were treated with hydroxylamine in hnes e
through h. Arrowhead indicates the position of a co-migrating
purified rabbit Ch'-adenosine triphosphatase (ATPase). The
C h -dependent phosphoprotein concentration is markedly reduced in the patient's microsomes (lane 61 in comparison t o control (lane a). Note the C d dependence ilanes a ZiersuJ ci and
hydroxylamine sensitivity (lane a versus e) of 100-kD phosphoprotein, which is characteristic of the sarcopkasmic reticulum
Ca" -ATPase.
The impaired muscle relaxation exhibited by our patients closely resembled the symptoms of a patient reported by Brody [l], who found an abnormally low
rate of Ca2+ uptake into fragmented SR from the patient's muscle biopsy. In one of our patients, we have
also demonstrated a very low rate of ATP-dependent
Ca2+-uptake by a particulate muscle membrane fraction. We have found that such preparations yield reliable rates of total SR Ca2+uptake [ 4 ) . A marked reduction of the 100-kD phosphoprotein in microsomes
prepared from muscle of one of our patients indicated
that the markedly reduced rate of CaL+ transport is
caused by reduction of enzymatically active SR Ca2 + ATPase. Immunoblots of muscle proteins separated by
SDS-polyacrylamide gel electrophoresis revealed a
marked reduction in the amount of immunoreactive
100-kD protein that corresponds to the SR Ca2 ATPase. By microscopic immunocytochemistry, we
have shown that the severe reduction of immunoreactive Ca2+-ATPasewas limited to the histochemical
type 2 fibers.
Since one polyclonal and two monoclonal antibodies
showed a barely detectable amount of immunoreactive
Ca2+-ATPase in type 2 fibers, we can surmise that a
major portion or all of the Ca’+-ATPase transport
protein, of which the molecular structure has been well
characterized [lo, 21, 22), is missing in these fibers.
The residual proteins detected on immunoblots presumably reflect other cross-reacting Ca2 -ATPase(s),
including those of the histochemical type 1 fibers. If
the deficiency of immunoreactive Ca’ -ATPase in
type 2 muscle fibers is a primary expression of a gene
defect, fiber type-specific isoforms of Ca’ +-ATPase of
SR must exist. This would be consistent with different
Ca2+ uptake kinetics of SR found in chemically
skinned, single slow and fast human muscle fibers [28).
The trace amounts of immunoreactive Ca2+ -ATPase
observed by microscopic immunocytochemistry in
type 2 fibers of our patients may be due to the expression of some putative “slow” isoform or other crossreacting Ca’ +-ATPase(s).
Our evidence suggests that the Ca2+-ATPase deficiency in type 2 fibers is not merely secondary to disuse or atrophy. None of our disease controls, most of
whom showed marked atrophy of type 2 muscle fibers,
had a comparable deficiency of the Ca”-ATPase
observed by microscopic immunocytochemistry or by
immunoblotting. Some type 1 fibers in disused control muscles showed considerable paucity of immunoreactive Ca2 -ATPase; the pathophysiological
basis for this is unclear. Electron microscopic examination revealed that the triads were normal in all
fibers. The tubular component of the SR was not absent or even grossly depleted in any fiber when compared to a control. Another major SR component,
calsequestrin, was not deficient. The relative myosin
content of the patients’ muscles was also normal.
The gene(s) that codes for the Ca2+ transport protein of SR is being isolated and cloned [20]. Determination of the molecular nature of the genetic defect in
Fig 1 I . Peptide projile of human muscle bzopsy extracts (for techniques see Material and Methods). Lane a = molecular weight
standards; lane c = Patient 1 ; lane g = Patient 3; lanes 6, d, e,
and f = disease controls. Arrowhead marks the position of the
co-migrating purrfied rabbit sarcoplasmic reticulum C d ’adenosine triphosphatase. Note the severe reduction of a 100-kD
protein band in Patient 1 (lane c) and Patient 3 (lane in
relation to controls (b. d, e, andf).
Fig 12. lmmunoblot of human C d ’-adenosine triphosphatase
(ATPase). Patient 1 = lane b; Patient 3 = lane e; normal
control = lanes d and g (autopsy);disease controls = a, c, f; h
through m. Lane n is purifed rabbit sarcoplasmic reticulum
C d ’-ATPase. Note the absence ofthe major immunoreactive
form of Cd+-ATPase in lane b (Patient I) and lane e (Patient
3). Two minor cross-reactive bands are still detected in lanes b
and e. Disease controls show variability in amount (but not an
absence) of the major immunoreactive band.
Karpati et al: Myopathy Due to Ca2+-ATPas?Deficiency 47
lmrnunoquantitation of Biopsied Human Muscle
SurcoplaImic Reticulum C d ’-ATPuse
Ca2 -ATPase
(&mg protein)”
Not detectable
Not detectable
Brody’s disease
(Patient 1)
Brody’s disease
(Patient 3 )
Normal control
Normal control
‘Total cellular protein.
adenosine triphosphatase.
these patients by use of complementary deoxyribonucleic acid probes may thus be possible in the future.
The deficiency of Ca2 transport ATPase in the SR
can explain the clinical presentation of the patients.
Progressive impairment of muscle relaxation during
exercise would occur because of a cumulative increase
of myofibrillar Ca2 . Since only fast-twitch muscle
fibers suffer from the enzyme deficiency, one can explain that impaired relaxation was only noted after
phasic exercise, when primarily fast-twitch motor units
are recruited. In contrast, during tonic activity, such as
maintaining posture, which requires slow-twitch motor
unit activation, no obvious muscular dysfunction was
noted. Since after rest the patients could relax their
muscles and start exercising again, it should be presumed that the free Ca2+ concentration in the
myofibrillar compartment is eventually restored to, or
at least near, normal levels. It is not clear how this is
achieved; perhaps compensatory T-tubular, sarcolemmal, or mitochondrial Ca2+ transport could account
for the removal of some of the extra Ca2+ from the
cytosol. Furthermore, some residual Ca2+ -ATPase in
type 2 fibers, as suggested by immunocytochemical
analysis, may just be sufficient to replenish Ca2 slowly
in SR of type 2 fibers.
The molecular mechanism of the enhanced caffeine
sensitivity of muscle in our patient is uncertain. If the
relevant target of caffeine action is the Ca2+-ATPase
[ 3 3 ] , it is possible that on account of the deficiency of
this enzyme in type 2 fibers, the excessive caffeine
Annals of Neurology
Vol 20
No 1 July 1986
sensitivity is attributable to type 1 fibers, which are
known to be more sensitive to caffeine [ 3 ] . Since an
abnormally low caffeine-specific concentration in an in
vitro contracture test usually correlates with predisposition to malignant hyperthermia reaction [2], appropriate anesthetic precautions appear to be advisable in
Brody’s disease.
High cytoplasmic Ca2+ content can explain some
other deleterious effects observed in our patients’ muscles. For example, a presumed increased ratio of active
to inactive glycogen phosphorylase, leading to presumed glycogen depletion and augmented lactate generation (as evidenced by persistently elevated arterial
lactate levels 30 minutes after ischemic forearm exercise), might well be due to increased activation of the
Ca2 -dependent phosphorylase kinase. As no muscle
fiber necrosis or significantly elevated serum creatine
kinase activity was noted, we presume that in the cytosol of muscle fibers, Ca2+ had not risen to a
sufficiently high level to induce muscle cell necrosis.
The pathophysiological basis of the atrophy, the prominent central nucleation of the type 2 fibers, and the
slight hypertrophy of type 1 fibers remains unclear.
The clinical presentation of these patients might lead
to considering myotonic dystrophy or myotonia congenita as the cause of the symptoms and signs. However, unlike those conditions, in our patients continued exercise aggravated the symptoms arid no
myotonic discharges were recorded electromyographically during the delayed muscle relaxation. Because of
the electrical silence recorded electromyographically
during the delayed relaxation after exercise, the possibility of McArdle’s disease or other disorders of glycolysis as the cause of the symptoms might arise. However, unlike those conditions, the delayed muscle
relaxation observed in our patients was transient and
The familial incidence indicates that the disease is a
genetic one. In the first family (Patients 1 and 2), parental consanguinity would be consistent with an autosomal recessive mode of inheritance. However, given
that all of our patients, as well as the 2 others reported
in the literature [I, 161, were males, X-linked reces-.
sive inheritance cannot be absolutely ruled out. Information concerning levels of Ca2’-ATPase in the p a
tients’ parents and unaffected siblings would be of
Supported by the Medical Research Council of Canada, The Muscular Dystrophy Association of Canada, and the Killam Fund of rhe
Montreal Neurological Institute.
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Karpati et al: Myopathy Due to Ca*+-ATPase Deficiency 49
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