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Cytochrome C OxidaseЧdeficient myogenic cell lines in mitochondrial myopathy.

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Cytochrome c Omdase-Deficient Myogenic
Cell Lines in Mitochondrial Myopathy
Hideo Shimoizumi, MD," htiariko Yoshida Momoi, MD, PhD," Shigeo Ohta, PhD,t
Yasuo Kagawa, MD, PhD,t Takashi Momoi, PhD,S and Masayoshi Yanagisawa, MD"
In a patient with mitochondrial myopathy, the defect of cytochrome c oxidase activity was restricted to some muscle
fibers. To isolate cell lines with or without oxidase activity from a single muscle sample, primary cultured cells were
transformed by replication origin-defective simian virus 40, and then cloned. The clones were examined by cytochemical staining for cytochrome c oxidase activity. Eight myogenic clones were completely devoid of activity, while the
other myogenic and nonmyogenic clones were not. Deficiency of cytochrome c oxidase was stable in culture for at least
a year after serial passaging. The amount of mitochondrial DNA in cytochrome c oxidase-deficient cells was the same
as in control cells, and no deletion in the mitochondrial DNA was detected. Protein synthesis in mitochondria of the
subunits of cytochrome c oxidase and subunit 6 of the ATP synthase complex was markedly decreased, whereas
synthesis of the other subunits encoded by mitochondrial DNA was normal. These cloned cell lines provide an
excellent system for clarifying the cause of mitochondrial myopathy and for investigating nuclear-mitochondrial
genetic interaction.
Shimoizumi H, Momoi MY, Ohta S, Kagawa Y, Momoi T, Yanagisawa M. Cytochrome c oxidase-deficient
myogenic cell lines in mitochondrial myopathy. Ann Neurol 1989;25:615-621
A variety of functional defects in the mitochondrial
respiratory chain have been identified in patients with
mitochondrial myopathy El]. The diagnosis for a considerable number of patients has been described as
mitochondrial myopathy or mitochondrial encephalomyopathy. These patients had diverse clinical characteristics, including epileptic seizures, psychomotor deteriorations, stroke-like episodes, muscle weakness,
retinal degeneration, abnormal cardiac conduction, and
episodic vomiting. In some the disorder was fatal and
in others it was chronic or episodic. The observed clinical diversity indicated multisystem involvement, and
biochemical investigations have led to the identification of various specific errors in the mitochondrial metabolism in certain tissues [2-41. Tissue-specific errors
in the expression of the affected enzymes, the presence of a partial or mosaic enzyme defect in muscle
tissue, and deficiencies in multiple enzymes or subunits reported in some patients [5-81 make mitochondrial myopathy unique. These complex pathological
findings of this disorder may be a consequence of the
dual genetic control of some of the mitochondrial enzymes. Enzymes of the respiratory chain are composed
of multiple subunits; 5 to 6 subunits of complex I, one
subunit of complex 111, and 3 subunits of complex IV
(cytochrome c oxidase [COX]) are encoded by mito-
chondrial genes, and others are encoded by nuclear
genes [9, lo].
Investigations on the molecular and cellular mechanisms of these complex pathological entities, especially
in the partial or mosaic enzyme defect in muscle tissue,
require a homogenous experimental system with
which to study the expression and disorders of mitochondrial enzymes. Biopsied muscle specimens have
often been used for such pathological and molecular
investigations; however, there are some critical disadvantages such as limited availability, cellular heterogeneity, and difficulty in use for the study of gene
expression. To solve these problems, we established
clonal cell lines that retained the mitochondrial abnormality, by transforming primary muscle cells with simian virus 40 (SV40) DNA defective in a replication
origin (ori- SV40). Since the development of oriSV40 by Gluzman and colleagues [ll], human macrophages {12], embryonic kidney cells [13], and
enzyme-deficient fibroblasts [14] have been successively transformed and cloned. In our previous study,
ori- SV40 DNA was transfected into a primary culture of human muscle cells. The transformed myogenic cells exhibited an enhanced growth rate and an
unlimited life span and retained certain differentiated
traits [lS].
From the Departments of *Pediatrics and ?Biochemistry, Jichi Medical School, Minamikawachi-machi, Tochigi-ken, and the $National
Institute of Neuroscience, NCNP, Kodaira, Tokyo, Japan.
Address correspondence to Dr Momoi, Department of Pediatrics,
Jichi Medical School, Minamikawachi-machi, Kawachi-gun, Tochigi,
329-04 Japan.
Received Aug 29, 1988, and in revised form Dec 4. Accepted for
publication Dec 15, 1988.
Copyright 0 1989 by the American Neurological Association 615
In the present study, we report the establishment of
two kinds of myogenic cell lines, one with and one
without defective enzymatic activity, from a patient
with mitochondrial encephalomyopathy who showed a
partial defect in COX activity of the muscle. This indicates that a partial enzyme defect is the result of the
cellular mosaicism in the tissue. In addition, we describe an unusual pattern of protein synthesis in the
mitochondria of the cells deficient in COX activity,
and molecular mechanisms for this affected protein
synthesis are discussed.
into the cells by the calcium-phosphate precipitation method
of Wigler and associates 1201. In brief, cells were fed 24
hours before the transfection, and then transfected with 10
pg D N A per dish (60 mm in diameter) for 8 hours. The cells
were washed with DNA-free medium, cultured for a further
24 hours, treated with trypsin, and then allowed to grow
until foci were observed. About 18 foci appeared in a 60mm dish. Each focus was transferred to a 24-well microplate
by applying a small piece of filter paper soaked with 0.125%
trypsin onto each focus, and cells were recloned using the
limiting dilution method.
Identification of Myogenic Cells
Materials and Methods
Clinical Features
The detailed clinical features of the patient will be published
elsewhere. Briefly, his psychomotor deterioration started at
the age of 5 years, when generalized convulsive seizures developed. Fatigability, epileptic seizures, vomiting episodes,
and ataxia slowly progressed in severity, and by the age of
15, he was mute and bedridden. His parents and a sister
were healthy. His older brother had similar neurological
symptoms and died at the age of 19 years. Results of laboratory studies indicated elevated levels of lactic acid in the
serum (17.1-41.9 mg/dl) and cerebrospinal fluid (45.8 mg/
dl) and elevated levels of pyruvic acid in the serum (1.102.14 mg/dl) and cerebrospinal fluid (1.69mg/dl). The clinical
diagnosis of his condition was mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes.
Muscle from the Patient
Muscle was obtained by biopsy of the biceps brachii, with the
informed consent of his parents. The mitochondrial enzyme
activities of the reduced form of nicotinamide-adenine dinucleotide (NADH)-cytochrome c reductase (complex I and
III), succinate-cytochrome c reductase (complex 11 and 111),
and COX (complex IV) were measured by the methods of
Sottocasa and associates [ 163 and Cooperstein and Lazarow
f17). However, no apparent differences from normal values
were detected. Histochemical staining was performed by
modified Gomori-trichrome staining 118) and staining for
COX activity, as described by Seligman and co-workers t19).
Ragged-red fibers and partial deficiency of COX activity
were detected using these methods.
Primary Culture ofthe Patient's Muscle Cells
For the regular culture conditions, a complete medium containing 10% fetal calf serum (FCS), penicillin, streptomycin,
and glutamine in Ham F12 was used. Cells were cultured at
37°C under an atmosphere of carbon dioxide and 95% air.
'The muscle tissues obtained by biopsy were processed as
described previously { 15). Myoblast-rich fractions were obtained by leaving the harvested cells for 40 minutes, to allow
the fibroblasts to adhere to the polystyrene culture dishes.
The cells were fed every third day with the complete medium and subjected to transfection after the second passage.
Trans-ction of Cells with Ori- SV40 DNA
An ori- SV40 D N A was supplied in plasmid pMKl6 as an
insert at the BamHI site 2113. The DNA was transfected
616 Annals of Neurology Vol 25 No 6 June 1989
Identification of the origin of the myogenic cells was performed by examining the expression of the muscle type
isomer of creatine kinase and the ability to form multinucleated cells in the serum-free medium as previously described fl5).
COX Staining o f Cultured Cells
The cultured cells were stained for COX activity according
to the method of Seligman and colleagues [I93 with some
modifications. Cloned cells were cultured on cover-glasses
(22 x 22 mm) at the concentration of 1 to 2 x lo3 cells/
glass. O n day 7, each cover-glass was washed, fixed with 3%
glutaraldehyde in 0.1 M cacodylate buffer (pH 7.3) at 4°C
and incubated with 2 mg/ml of 3,3-diaminobenzidine tetrachloride, 1 mg/ml of cytochrome c, and 1% hydrogen
peroxide in 0.05 M sodium phosphate buffer (pH 7.4) at
37°C for 2 hours. The cover-glass was washed, reacted with
1% copper sulfate, and then dehydrated in ethanol.
Growth Rate of the Cells
Cells from the transformed clones were plated in a 60-mm
dish at the concentration of 1 X lo5 cellsldish. They were
cultured in the complete medium, fed with the fresh medium
every third day, and then counted after trypsinization on
days 2, 4, and 8. Triplicate dishes were plated for each day
and for each medium, and all the counts were performed in
Southern Blotting
Total D N A was prepared from the cultured cells using the
method described by Maniatis and associates f2 1). The
D N A (1 kg) was digested with BamHI and then subjected
to Southern blotting. The HindIII-Sac1 fragment (3.4 kilobase-pairs) of the mitochondrial DNA from HeLa cells was
cloned into pBR322, and the fragment obtained from a large
scale preparation of the plasmid was used as a hybridization
probe. The fragment covers most of part of COX subunits I,
11, and I11 and subunit 6 of ATP synthase {5].
Protein Synthesis in Mitochondria
Protein synthesis in mitochondria was examined by labeling
proteins with 35S-methionine in the presence of emetine, a
potent inhibitor for cytoplasmic protein synthesis. Confluent
cells in a 9-cm dish were cultured in methionine-free Dulbecco's modified Eagle medium supplemented with 2% FCS
for 30 minutes at 37"C, and then treated with 150 pg/ml
emetine for 5 minutes. The medium was changed into the
Fig I . Photomicrograph of stained,fixed muscle section for histochemicalstudy of cytochmme c oxidase activity. Thepartial
defect in cytochrome c oxidase activity is restricted to some muscle
fibers from the patient with mitochondrial myopathy. The dark
spots show mitochondria with normal oxidase activity. The arrows indicate muscle fibers without cytochrome c oxidase activity.
( x 100 before 54% reduction.)
methionine-free medium containing 2% FCS and 150 p,g/ml
emetine; 100 p,Ci/ml 35S-methioninewas added, and culturing was continued for 2 hours. The cells were harvested by
scraping with a rubber policeman, followed by centrifugation. The mitochondrial fraction was isolated using the
method of Whitfield and associates [227, and it was dissolved
in 7 M urea, 2% sodium dodecyl sulfate (SDS), 1% 2mercaptoethanol at room temperature. The samples were
applied to 12.5% polyacrylamide gel containing 7 M urea,
followed by electrophoresis. The labeled proteins were visualized by fluorography.
Partial Ddect of COX in Muscle
Although no decreased total COX activity was detected in the isolated mitochondria of the biopsy muscle specimen (data not shown), histochemical staining
of the fixed muscle sections revealed a partial deficiency of COX activity, as shown in Figure 1.
Transformation of Myogenic Cells and Their Cloning
The muscle tissue from the patient was subjected to
primary culture. After removal of most of the fibroblasts by the differential adhesion procedure at the
second passage, the transfection of ori- SV40 DNA
was performed. The efficiency of the transfection was
similar to that of normal muscle or of muscle from a
patient with another form of myopathy, as reported
previously 1153. After the first cloning, as described in
the Materials and Methods section, the myogenic origins of the clones were confirmed by examining the
expression of muscle type isomers of creatine kinase
and the ability to form multinucleated cells in the
serum-free medium, because some transformed cells
Fig 2. Transformed myogenic cells from the patient. The muscle
was subjected to primary culture and transjicted with origindefective simian virus 40 (0s- SV40) DNA as described in the
Materials and Methods section. (A) Afier the culture was in the
serum-free medium for 7 days, primary myotubes were observed
(arrows). ( x 200 before 32% reduction.) (B) Higher magnifcation of a multinuclear cell cultured under similar conditions is
shown. ( x 400 before 32% reduction.)
still retained their ability to form primary myotubes,
as shown in Figure 2. Nonmyogenic clones might have
originated from fibroblasts or they might be myogenic
cells that lost the ability to express the differentiated
traits during transformation. Clones were examined after cytochemical staining for COX activity, and more
COX-negative myogenic clones were selectively recloned. Eight myogenic clones were negative and 3
myogenic and all nonmyogenic clones were positive
for COX activity, as shown in Figure 3. Perinuclear
brown spots in the COX-positive clones were considered mitochondria with normal COX activity. As
shown in Figure 3, there were no COX-positive cells
in the COX-negative clones and no COX-negative
cells in the COX-positive clones. To examine the
specificity and reliability of this cytochemical staining,
one of the COX-positive clones was stained, using the
same method, in the presence of 0.02% sodium aide,
a potent inhibitor of oxidase activity. As shown in Figure 3C, no perinuclear brown staining was observed,
which was similar to the COX-negative clones. Each
Shimoizumi et al: Cytochrome c Oxidase-Deficient Muscle 617
P 1o.oj
Time (day)
F i g 4. Growth rates of cytochrome c oxidase (COX)-deficient
cell lines. Growth rates of one COX-positive (a) and 2 negative
clones (b, c) wtw mamined afer plating. Celh were plated in triplicate dishes and counts were performed in duplicate under the
conditions described in the Materials and Methods section. Cell
counts were plotted on semilogarithmic coordinates a g h s t time.
Fig 3. Photomicrograph of stained section for cytochemical study.
The cloned cells were stainedfor cytochrome c oxidase (COX)
activity, as described in the Materials and Methods section. (A,
B) A COX-positive clone shows the dark spots (arrow) indicating the presence of mitochondria with normal COX activity. (D,
E) A COX-negative one does not. (F) In thefibroblasts, the dark
spots (arrow)are also seen. (C) In the presence of sodium azi&
(0.02%),the dark spots are abolished i n the COX-positive clone.
(A, D: x 200; and B, C, E, F: x 400 before 32% reduction.)
clone was examined for COX activity after 5 passages
(approximately 2 5 generations), to confirm the reproducibility of the staining and the stability of the phenotype. The presence or the absence of COX activity
remained stable after the prolonged culture for a year.
Clones were stained either positive or negative, with
no midway type of staining observed.
Properties of the COX-Negative Cell Line
The cell lines were maintained with occasional passages with trypsinization. Both COX-positive cells and
COX-negative cells reached the growing state at 48
hours. However, the doubling times of the COXnegative cells were longer (33 and 36 hours) than that
of control one (21 hours) (Fig 4).
Amount of Mitochondriul D N A
The amount of mitochondrial DNA was measured by
total Southern blotting using a cloned mitochondrial
DNA fragment as the hybridization probe. N o differences were observed between the COX-negative and
COX-positive cells in the amount and length of mitochondrial DNA (Fig 5). This suggests that the observed deficiency in COX activity is not due to a de618 Annals of Neurology Vol 25 No 6 June 1989
creased number of mitochondria but to a defect in the
enzymatic activity itself.
Protein Synthesis in Mitochondria
Subunits I, 11, and 111 of COX are encoded by mitochondrial DNA. Protein synthesis in mitochondria was
examined using pulse-labeling with 35S-methionine in
the presence of emetine, which is a potent inhibitor of
cytoplasmic protein synthesis. Figure 6 shows the
bands of synthesized proteins; as judged by the molecular mass, each band was identified as shown in the
figure. N o differences between the apparent molecular
masses were detected by SDS-polyacrylamide gel electrophoresis. However, the bands corresponding to
COX subunits I, 11, and I11 and subunit 6 of the ATP
synthase complex were decreased markedly, while the
bands representing synthesis of the other proteins
looked normal.
Mitochondrial myopathy has unusual characteristics as
a genetic disease. In some patients, the defects are
profound and widely distributed; in others, they are
often partial and limited to some tissues, for example,
in skeletal muscle [2, 7). Furthermore, the partial defect of the enzyme activity is often mosaic, as observed
in muscle tissue 123-251. To analyze the molecular
and cellular mechanisms of these heterogeneous
pathological changes, we established cultured myogenic cell lines that were defective in a mitochondrial
enzyme from a patient with mitochondrial encephalomyopathy. The primary cultured muscle cells
were transformed by ori - SV40 DNA. Transformed
cells were then selected for myogenic origin [l51 and
Fig 5. Amount of mitochondrial DNA. One microgram of total
D N A from the cells was digested with BamHI. The D N A was
subjected to Southern blotting. The cloned Hindlll-Sac1 fragment
of HeLa mitochondria1D N A (encoding cytochrome c oxidase
subunit I, 11, and 111 and subunit 6 of ATP synthase) was used
as the hybridization probe. Lane a: the cloned HindIIl-Sac1
fragment of HeLa mitochondria1DNA; lanes b and c: the total
D N A from the COX-negativeclone and the COX-positive one,
Fig 6. Protein synthesis i n mitochondria. The cells were labeled
with 100 pCilml of "S-mthionine for 2 hours in methioninefree Dulbecco's modijied Eagle medium supplemented with 2%
fetal carfserum and in the presence of150 pglml emetine, as
described in the Materials and Methods Section. After the labeling, mitochondria were isolated and dissolved in 7 M urea, 2%
sodium and dodeql sulfate, and 2% 2-mercaptoethanol at room
temperature and applied to a 12.5% polyacvylamide gel containing 7 M urea. After electrophoresis, the gel was dried, followed Ly
fEuorography. Lane a: the COX-positiveclone; lane 6: the COXnegative clone. The synthesized polypeptides were identified as
follows, according t o Chomyn and colleagues (10). N D 1, 2, 3,
and 5 = subunits ofthe reducedform of nicotinamide-adenine,
dinucleotzde-ubiquinone oxidoreductase (complex I). CO I , 11,
and 111 = the subunits of cytochrome c oxidase (complex IV).
ATP 6 and 8 = the subunits of A T P synthase (complex V).
Shimoizumi et
Cytochrome c Oxidase-Deficient Muscle 619
cloned. Cytochemical staining of the myogenic clones
revealed that some of them were defective in COX
activity, while others stained positive for COX activity.
This revealed that the mosaic defect in the enzymatic
activity in the muscle tissue was the consequence of
the presence of both COX-positive and COX-negative
cells in the tissue.
This heterogeneity and mosaicism are supposed to
reflect the complex control mechanism of mitochondrial traits. Three subunits of COX (complex VI) are
encoded by the mitochondrial genome 15, 61, and
the rest of the subunits are encoded by the nuclear
genome. A genetic regulation of the two genomes
must exist so that equimolar amounts of subunits are
synthesized, which are then assembled to form complex VI. Thus, the gene responsible for cellular mosaicism must reside in either the nucleus or the
mitochondria. Nuclear-coded regulations have been
reported for the tissue-dependent expression of the
nuclear-coded subunits of COX, ATP synthase and
ADP/ATP translocator 126-281. In contrast, recent
analysis of mitochondrial DNA from patients with mitochondrial myopathy revealed the presence of an abnormal mitochondrial DNA, with a long deletion in
the muscle but not in the leukocytes {29, 30).
Our clones were examined for the affected sites of
the enzyme defect. Because the amount of mitochondria and mitochondrial DNA is dependent on the cell
type, and it is often changed by stimulation 1311, the
possibility of a decrease of mitochondrial DNA in a
COX-negative clone was examined. The amount of
mitochondrial DNA in a COX-negative clone was
comparable with that in a COX-positive clone. In addition, no large deletion was detected after BamHI digestion. Although no further experiments were done
with other restriction endonucleases to detect possible
deletions, our data on the protein synthesis in mitochondria should give us the answer. Subunits I, 11, and
I11 are encoded by a mitochondrial genome, and they
can be synthesized in the presence of emetine, an inhibitor of cytoplasmic ribosomes. These proteins were
synthesized in markedly decreased amounts in a COXnegative clone, as shown in Figure 6. The subunits
synthesized by a COX-negative clone had apparent
molecular weights similar to those of a control clone,
indicating that no deletions were present in the coding
region for each subunit. The marked decrease in synthesis might be caused by a rapid degradation, as has
been reported in a yeast mutant 1321. In the yeast
mutant, the primary abnormahty was a rapid destruction of a nuclear-coded subunit, and as a result, the
mitochondrial-coded subunits were easily degraded in
a short time 1327. Such an explanation is not likely in
our case, however, because the apparent protein synthesis of subunit 6 of ATP synthase was also decreased. The apparent syntheses of the affected pro620 Annals of Neurology Vol 25 No 6 June 1989
teins are nearly one-half of the normal level, while the
loss of the COX activity, as judged by cytochemical
staining, is almost complete. Unbalanced syntheses
might induce accelerated degradation or they might
interfere with complex formation, which should result
in the loss of the activity.
The messenger RNAs in the mitochondria are first
synthesized as polycistronic RNA and then cleaved
into the individual mRNA molecules 1331. The gene
for subunit 6 is located between subunit I1 and subunit
I11 of COX 151. Thus, the coding regions for the 4
proteins whose syntheses were decreased are adjacent
to each other on the mitochondrial genome. The decreased synthesis of more than 2 proteins can be explained by two possible mechanisms. The first possibility is that there is defective processing on the level of
mRNA synthesis in the enzyme-deficient cells. An alternative possibility is that there are common regulatory regions for the 4 affected subunits. If this is the
case, there still remains the question of whether a
molecule that would work on the regulatory region is
encoded by the nuclear genome or the mitochondrial
genome. To investigate these ideas, more detailed
analyses of the transcription and processing of RNA in
mitochondria are required.
The cell lines that we have described in this report
will provide an excellent system for investigating the
molecular pathological changes of mitochondrial encephalomyopathy and the genetic interactions between
nuclear and mitochondrial genomes.
This work was supported in part by grant No. 87-02 from the National Center of Neurology and Psychiatry of the Ministry of Health
and Welfare ofJapan, and grant No. 62570435 from the Ministry of
Education, Science and Culture of Japan.
We thank Ms Sachiko Kojima and Ms Kakuko Matsuda for their
excellent technical assistance.
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