Jpn Circ J 1998; 62: 695 – 699

Cell Cycle of Myocytes of Cardiac and Skeletal
Muscle in Mitochondrial Myopathy
Atsushi Takeda, MD; Satoru Chiba, MD*; Iwai Takaaki, MD; Akira Tanamura, MD;
Yutaka Yamaguchi, MD*; Nobuakira Takeda, MD
Patients who have mitochondrial myopathy can present with specific pathological conditions (eg, diabetes mellitus and deafness). A 36-year-old woman presented with mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes (MELAS). An investigation was conducted into whether the abnormalitiy of mitochondrial DNA (a T to C transition at position 3271 in the mitochondrial tRNA [Leu(UUR)] gene) influences
nuclear DNA synthesis by cells in the heart, skeletal muscles, and brain. Myocardium, skeletal muscle, and brain
tissues were stained with hematoxylin-eosin, and Masson trichrome for histopathology. Target nuclei taken from
the myocardial and skeletal muscles and brain tissue were purified after removing debris by the modified Hedley
method. These nuclei were stained with propidium iodide (PI) for analysis by flow cytometry. The number of
nuclei in the G2M phase was bigger in myocytes of MELAS than in normal myocytes (Control) (MELAS
myocyte: Control myocyte=24.9±7.3: 6.1±1.6%, p<0.005), but there was no significant increase in the G2M
phase in brain tissue. The G1 phase was far more reduced in MELAS myocytes and skeletal muscle than in
Controls (MELAS myocyte: Control myocyte=65.8±9.1: 88.0±3.2%, p<0.005; MELAS skeletal muscle: Control
skeletal muscle=85.1±2.2: 90.1±3.2%, p<0.05), while there was no significant decrease of nuclei in the G1 phase
in brain tissue. Increased amount of nuclei in the G2M phase in cardiac myocytes and skeletal muscle cells
compared with that in neurons might depend on the capacity for proliferation and differentiation of these cells as
compared with brain tissue. It was concluded that the mitochondrial DNA mutation (3271T-to-C) of MELAS
may influence the nuclear DNA synthesis of cells in various tissues depending on their level of mitotic activity.
(Jpn Circ J 1998; 62: 695 – 699)
Key Words: Cell cycle; DNA; Gene; Mitochondria; Myopathy

M

itochondrial DNA (mtDNA) is located in the
matrix and is sometimes found attached to the
inner mitochondrial membrane. Human
mtDNA is a completely sequenced circular structure of
16,569 bp. Human mtDNA encodes 2 rRNAs and 22
tRNAs. Normal and abnormal mitochondrial DNA co-exist
in mammalian cells, which is called ‘heteroplasmy’, and
the ratio of the 2 types of mtDNA differs among tissues.1–3
Therefore, patients who have mtDNA mutations may
present with various symptoms and diseases; for example,.
mitochondrial myopathy (MELAS),4,5 diabetes mellitus,6,7
cardiomyopathy,4 deafness6 and others.8
Several recent reports on MELAS have suggested that
mutations of mtDNA, especially substitution of A to G at
position 3243 of the leucine tRNA (3243 mutation), may
cause diabetes and deafness.9–11 The 3243 point mutation
has been recognized in about 1–2% of patients with
diabetes mellitus.10,12–14
We report here on a 36-year-old woman who was finally
diagnosed as MELAS because she had epilepsy, lactic
acidosis, an mtDNA 3271 point mutation (a T to C transition at position 3271 in the mtDNA), and ragged-red fibers
(Received December 8, 1997; revised manuscript received April 20,
1998; accepted April 27, 1998)
Department of Internal Medicine, Aoto Hospital, and *Department of
Pathology, Kashiwa Hospital, Jikei University School of Medicine,
Tokyo, Japan
Mailing address: Atsushi Takeda, MD, Department of Internal
Medicine, Aoto Hospital, Jikei University School of Medicine, Aoto
6-41-2, Katsushika-Ku, Tokyo 125, Japan

Japanese Circulation Journal Vol.62, September 1998

in biopsy material of skeletal muscle. We investigated
whether this mtDNA point mutation influenced cell proliferation in several organs (heart, skeletal muscle, and brain).
Nuclear DNA synthesis in cardiac myocytes, skeletal
muscle cells, and brain tissue was investigated by flow
cytometry analysis.

Case Report
Patient Profile
The patient was a 36-year-old housewife with the chief
complaint of headache. There was nothing specific in her
past medical or family history.
Present Illness: She visited the Neurosurgery Department of our hospital because of the headache on October 1,
1994. Her neurological findings and brain computed
tomography (CT) scan were almost normal on that same
day, although she had sustained headache with muscle
cramps. 1 week later, she was hospitalized because of
disturbed consciousness, syncope, and left hemiparesis.
The diagnosis was cerebral infarction.
Physical Findings: Her height was 145 cm, body weight
was 31 kg, consciousness was drowsy, blood pressure was
110/60 mmHg, and heart rate was 100 beats/min (regular).
There was left hemiparesis and muscle cramping.
Laboratory Data: The blood lactate level was 47.5 mg/dl
(normal: 1–27 mg/dl) and the pyruvic acid level was 2.13
mg/dl (normal: <1.02 mg/dl). ECG showed sinus tachycardia (Fig 1). Brain CT showed multiple low density areas in
the cerebrum.

TAKEDA A et al.

696

Fig 1. Electrocardiogram of the 36-year-old MELAS patient.

Masson trichrome staining of the skeletal muscle biopsy
taken for the MELAS diagnosis revealed ragged-red fibers
and hypertrophic and atrophic muscle fibers th (Fig 2).
Mitochondrial DNA Analysis: A mtDNA (3271) point
mutation was identified in the skeletal muscle mitochondria
obtained from the biopsy specimen.
Progress after Admission: She was treated with steroids
and glycerol after diffuse patchy cerebral infarctions were
found on October 8, but her cramping and disorientation
persisted. The low density areas on brain CT showed a skip
lesion compared with the previous findings, so the patient
was diagnosed as MELAS syndrome based on the mtDNA
analysis, skeletal muscle biopsy, and biochemical data. She
was then treated with coenzyme Q10, but died of MRSA
infection on June 6, 1995.
Materials and Methods
At autopsy, heart muscle, skeletal muscle, and brain
tissue were obtained for analysis. Several pieces of tissue
were cut from each organ for flow cytometry analysis of
the cell cycle.
Pathological Study: Myocardium, skeletal muscle, and
brain tissue were stained with hematoxylin-eosin and
Masson trichrome after routine 10% formalin fixation,
paraffin embedding and sectioning. These tissues were also
examined by transmission electron microscopy.
Cell Cycle Analysis with Flow Cytometry: The percentage ratios of each phase were calculated automatically with
the integral calculus by FACS flow cytometry (Becton
Dickinson Co).15–17 With 4 different samples from each
organ (heart, skeletal muscle, brain tissue), the cell cycles
were measured by flow cytometry, and statistical analyzes
were performed comparing each with normal controls
(heart, skeletal muscle, brain tissue). Several 60-μm
sections were cut from each paraffin block of tissue18 and
placed in tubes with xylene for 20 min and then gradually

rehydrated with distilled water. The sample tubes were then
incubated for 1 h at 37°C in a water bath after adding 1 ml
of 0.5% pepsin solution (pH 1.5) in 0.9% NaCl. After pipetting off this solution, target nuclei were purified by filtering
through a 30–60μm teflon mesh to remove debris (modified
Hedley method).19,20 The nuclei were then stained with 50
μg/ml propidium iodide (PI) and 0.1% Triton X-100 in 4
mmol/l sodium citrate (Vindelov method). This solution
was centrifuged (4°C, 400×g, 10 min), the supernatant
was discarded, 0.5 ml of PI solution was added, and then a
FACSscan (Becton Dickinson Co) was used for flow
cytometry.15,16,21,22
In the fluoresceine DNA histogram, the first high peak
indicated the G0–1 phase of cell cycle, and also the second
peak is the G2M phase. The valley between the G0–1 and
G2M phases shows the S phase15,16 (Fig 7). The DNA
frequency histogram was deconvoluted and the results
were computer analyzed using the Multicycle software
(Phoenix Flow System, San Diego, CA, USA).15,16 Results
are presented as mean ± SD. Comparisons between the
values of MELAS and Controls were performed using
Student’s t-test. A p value of less than 0.05 was considered
statistically significant.

Results
Histopathological Study
In the myocardial tissue, myocardial degeneration and
perinuclear vacuolization of the cells were observed. The
fine perinuclear granules of the myocyte seen by light
microscopy could be seen to be mitochondria with the electron microscope (Fig 3). A variety of mitochondrial shapes
and mitochondrial proliferation could be also observed in
the myocardium (Figs 4,5). Myofibrilysis of skeletal
muscle cells was also detected and perinuclear fine granules were observed in the cytoplasm.
Japanese Circulation Journal Vol.62, September 1998

Cell Cycle of Myocytes in MELAS

Fig 2. Skeletal muscle biopsy from the MELAS patient (Masson
trichrome stain, ×400).

697

Fig 5. Left ventricular myocardial tissue from the MELAS patient
(Masson trichrome stain, ×400).

Fig 6. The naked nucleus after nuclear DNA analysis with hematoxylin-eosin stain (×600).

Fig 3. Transmission electron microscopy of
the left ventricularfree wall. Bar = 5μm.

Fig 7. The fluoresceine DNA histogram of a myocyte; the first high
peak is the G0–1 phase and the second one is the G2M phase.

Fig 4. Macroscopic view of the heart from the MELAS patient.

Cell Cycle Analysis With Flow Cytometry
The G2M phase was more prominent in MELAS
myocytes and skeletal muscles than in controls (MELAS
Japanese Circulation Journal Vol.62, September 1998

myocyte: Control myocyte=24.9±7.3: 6.1±1.6%, p<0.005;
MELAS skeletal muscle: Control skeletal muscle=6.4±1.0:
4.7±1.8, p<0.005), but there was no significant increase of
the G2 phase in brain tissue. The extent of the G1 phase
was significantly less in MELAS myocytes and skeletal
muscles than in controls (MELAS myocyte: Control
myocyte=65.8±9.1: 88.0±3.2%, p<0.005; MELAS skeletal
muscle: Control skeletal muscle=85.1±2.2: 90.0±3.2%,

TAKEDA A et al.

698
Table 1 Cell Cycle of Neurons and Cardiac and Skeletal Muscle Cells
Cardiac muscle

Skeletal muscle

Brain tissue

G1 phase (%)

65.8±9.1**
(88.0±3.2)

85.1±2.2*
(90.0±3.2)

90.8±1.5
(93.0±2.6)

S phase (%)

8.3±3.1
(6.0±1.8)

8.5±2.9
(5.2±1.4)

7.4±2.4
(5.4±2.2)

G2M phase (%)

24.9±7.3**
(6.1±1.6)

6.4±1.0**
(4.7±1.8)

1.8±0.9
(1.7±1.5)

n=4, mean ± SD. *p<0.05, **p<0.005, vs Control (Control data).

p<0.05), but there was no significant difference in the G1
phase of brain tissue (Table 1).
With regard to the selection of nuclei, the average
nuclear size in the myocardium was measured in hematoxylin-eosin stained tissue using the light microscope.
Only myocardial nuclei were filtrated through the optimum
Teflon mesh, whose hole size was a little bit smaller than
size of the nuclear myocyte, and then almost all the nuclei
of the interstitial cells were taken out (Fig 6). However, the
mixing ratio (%) of interstitial cell nuclei was technically
between about 2 and 8% (somewhere round 6%) after the
measurement.

Discussion
In mammals, cell differentiation and regeneration is
continuous. The varying lifespans of cells are dependent on
their ability to undergo mitosis modified by cell differentiation and regeneration.
Generally, mammalian cells are classified into 3 different mitotic categories: (i) vegetative intermitotics; (ii)
reverting postmitotics; and (iii) fixed postmitotics.
As reverting postmitotics, hepatocytes, secretory gland
cells, and renal tubular cells can undergo mitosis for regeneration after tissue damage. In other words, these cells are
normally postmitotic, but proliferative activity can be reactivated to allow regeneration.
On the other hand, fixed postmitotic cells like neurons
appear to lose the ability to regenerate and proliferate.
Thus, the various kinds of cells show different levels of
differentiation and regeneration. Our data suggested that
skeletal muscle cells and cardiac myocytes were reverting
postmitotic cells, whereas brain tissue cells were fixed
postmitotic cells. This is supported by the finding that the
G2M phase was lower in neurons than in the other cells. In
the case of skeletal muscle, each cell has many nuclei,
unlike other kinds of cells, so the amount of stimulation per
nucleus may be reduced and the cell cycle may not
progress to the G2 phase. In the present study, all the
filtered nuclei were not from myocytes, with about 6%
from interstitial cells. So the levels of the S and G2M
phases obtained must be a little higher than is the case
when all the nuclei are from myocytes. However, this influence is so little as to be somewhat ignored.
In conclusion, the difference in the extent of the G2M
phase between cardiac myocytes or skeletal muscle cells
and neurons might depend on the cell’s capability of proliferating and differentiating. Also the difference between
cardiac myocytes and skeletal muscle cells (myocytes:
skeletal muscle cells=24.9±7.3: 6.4±1.0%) may depend on
the severity of damage and the level of adaptation of the
cells. About 25% of cardiac myocytes were in the G2 phase
in MELAS syndrome.

We could not observe microscopically any double
nuclear myocytes in the heart tissue from the MELAS
patient and there were few polyploid cells by flow cytometric analysis, unlike in hypertrophic cardiomyopathy,
dilated cardiomyopathy and hypertensive heart disease.
Acknowledgments
The authors would like to thank Dr Yutaka Yamaguchi (Department of
Pathology, Kashiwa Hospital, Jikei University School of Medicine) for
providing the samples of MELAS syndrome.
This study was partly supported by the Research Committee for
Epidemiology and Etiology of Idiopathic Cardiomyopathy of the Ministry
of Health and Welfare of Japan, and a research grant from the Vehicle
Racing Commemorative Foundation.

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