Original Articles

Cytochrome C Oxidase-deficient Mitochondria in Mitochondrial Myopathy K a z u h i r o H a g i n o y a , M D * , Shigeaki M i y a b a y a s h i , M D * , K a z u i e Iinuma, M D * , Eizo O k i n o , MD*, H a t a e M a e s a k a , MD*, and Keiya Tada, M D *

Electron microscopic cytochemistry was used to evaluate the behavior of cytochrome c oxidase (COX) in cultured skin fibroblasts from 4 patients with decreased COX activity (Leigh encephalopathy, fatal infantile COX deficiency). In patients with Leigh encephalopathy, all mitochondria reacted to COX staining either equivocally or negatively, indicating that all mitochondria were abnormal in these patients. In 1 patient with fatal infantile COX deficiency, intercellular heterogeneity of mitochondria was observed by COX staining. In another patient with fatal infantile COX deficiency, intracellular heterogeneity of mitochondria was observed. Patients with Leigh encephalopathy appeared to have a different type of mitochondrial COX deficiency than those with fatal infantile COX deficiency. Our results suggest that these 2 diseases may result from different genetic mechanisms. Haginoya K, Miyabayashi S, Iinuma K, Okino E, Maesaka H, Tada K. Cytochrome c oxidase-deficient mitochondria in mitochondrial myopathy. Pediatr Neurol 1992;8:13-8.

Introduction Cytochrome c oxidase (COX) deficiency appears in patients with various types of encephalomyopathy [1]. Recent studies revealed that genetic defects in COX, an enzyme that is composed of 13 subunits and is coded by both nuclear and mitochondrial DNA, may manifest clinically in several different ways [2-6]. Large deletions of mitochondrial DNA cause chronic, progressive external ophthalmoplegia (CPEO), including Kearns-Sayre syndrome [3,4]. Transition mutations of the mitochondrial transfer RNA gene give rise to myoclonus epilepsy associated with ragged-red fibers (MERRF) [6] and mitochondrial myopathy, encephalopathy, lactic acidosis, and strokelike episodes (MELAS) [2]. Mutant and wild-type mitochondrial DNA coexisted in these patients (heteroplasmy); these 2 types of mitochondrial DNA were distributed in tissues in varying proportions. Heteroplasmy of mitochondrial DNA

From the *Department of Pediatrics; Tohoku University School of Medicine; Sendai, Japan; tDepartment of Pediatrics; Kanazawa Medical College; Kanazawa, Japan; *Department of Pediatrics; Kanagawa Children's Medical Center; Yokohama, Japan.

could occur as a mosaicism of mitochondrial function intercellularly, intracellularly, or even intramitochondrially. Electron microscopic histochemistry has been used to study the heterogenous expression of COX by mitochondria, resulting in tissue specificity [7] and the mosaicism of COX deficiency in muscle tissues [8]; however, electron microscopic cytochemistry has not been utilized previously to study COX in cultured fibroblasts from patients with mitochondrial encephalomyopathy. There is little molecular genetic information concerning the pathogenesis of Leigh encephalopathy and fatal infantile COX deficiency. To elucidate the manner in which details of the pathogenesis of fatal infantile COX deficiency differ from that of Leigh encephalopathy at the level of the mitochondrion, we studied COX activity in cultured skin fibroblasts from 4 patients with mitochondrial encephalomyopathy, including 2 with Leigh encephalopathy and 2 with fatal infantile COX deficiency, using electron microscopic cytochemistry.

Case Reports Patient 1. This 8-year-old boy, the first son of healthy, nonconsanguineous parents, had unsteady gait and poor coordination since late infancy. Although his delivery and early development had been normal, he developed truncal ataxia, nystagmus, and incoordination of the extremities which worsened during an acute febrile illness at 5 years of age. At 8 years of age, he was admitted to the Tohoku University Hospital. Cerebrospinal fluid (CSF) lactate and pyruvate levels were 28 mg/dl (normal: < 20 mg/dl) and 1.64 mg/dl (normal: < 0.9 mg/dl), respectively. Mental deterioration and cerebeUar ataxia gradually progressed. Cranial computed tomographic (CT) scans demonstrated lowdensity areas in the tegmentum. Magnetic resonance imaging (MRI) revealed symmetric prolongation of T2 relaxation time bilaterally in the putamen [9]. He was diagnosed as having Leigh encephalopathy. Skin and muscle biopsies were performed; muscle biopsy revealed diffuse, weak activity with COX staining. Biochemical study of the biopsied muscle disclosed COX activity to be only 30% of that in normal controis [10]. His younger brother exhibited psychomotor deterioration beginning at 10 months of age. While admitted to our hospital, the brother had lactic acidosis and bilateral areas of low density in the putamen on cranial CT. He died from respiratory insufficiency at 21 months of age. The

Communications should be addressed to: Dr. Haginoya; Department of Pediatrics; Tohoku University School of Medicine; l-1 Seiryo-machi, Aoba-ku, Sendai 980; Miyagi, Japan. Received February 27, 1991; accepted August 19, 1991.

Haginoya et al: COX-deficient Mitochondria

13

A

B

('

Figure l. Patterns of COX staining: (A) positive, (B) equivocal, and (C) negative. Reaction products are observed in imracristal Sl~m'c,~ and o~1 the outer surface of the inner membrane. Original magn~[ications: (A,C) x16,300 and (B) x30,O00. autopsy revealed bilateral neuronal loss, astrocytosis, and blood vessel proliferation in the putamen, caudate nuclei, and mid-brain; therefore, a diagnosis of Leigh encephalopathy was made. Postmortem biochemical study disclosed decreased COX activity in all tissues examined [10]. Patient 2. This 4-year-old boy, the offspring of healthy, nonconsanguineous parents, had intermittent episodes of vomiting and severe dehydration during infancy. The first episodes of metabolic acidosis (pH 7.4, Pao2 79.3 mm Hg, Paco2 29.3 m m Hg, base excess 7.2 mEq/L in arterial blood) and hyperlactic acidemia occurred at 18 months of age. The levels of lactate and pyruvate ranged 20-34 mg/dl (normal: 4-16 mg/dl) and 0.78-2.1 mg/dl (normal: 0.3-0.9 mg/dl), respectively, in blood and were 39.7 mg/dl (normal: < 20 mg/dl) and 1.8 mg/dl (normal: < 0.9 mg/dl), respectively, in CSE A skin biopsy was performed at that time. His psychomotor development was profoundly retarded. Beginning at age 2 years, he had seizures, nystagmus, intention tremor, and ataxic gait. Growth retardation and respiratory disturbance also became apparent at this age. Cranial CT disclosed diffuse, low-density areas in the brainstem and cerebellum. Ophthalmologic examination revealed bilateral optic atrophy. At age 4 years, respiratory failure developed and he required assisted ventilation. Biochemical analysis of a muscle biopsy specimen and peripheral leukocytes obtained at that time revealed that COX activity was decreased to 5% and 4% of normal levels, respectively [1 l]. Immunoblot studies in Patients 1 and 2 [11] disclosed uniformly decreased amounts of all subunits of COX in all tissues examined. He was suspected of having Leigh encephalopathy. Patient 3. A female neonate, the second child of nonconsanguineous parents, was born after an uneventful, term pregnancy, and uncomplicated labor and delivery. Her older brother had died at birth from an unknown cause. She developed cyanosis, muscular hypotonia, and apneic episodes shortly after birth. Blood gas analysis revealed severe metabolic acidosis (pH 7.0, Paco2 20.6 m m Hg, Pao2 96.9 rnm Hg, base excess -27.3 mEq/L). She was admitted while cyanotic and in a deep coma to the Tohoku University Hospital at 4 days of age. Lactate level was 88 mg/dl in blood (normal: 4-16 mg/dl) and 140 mg/dl in CSF (normal: < 20 mg/dl). Urinalysis demonstrated lactic aciduria, but no other unusual findings. Deep coma, severe metabolic acidosis, and hypotension persisted in spite of peritoneal dialysis and intensive care. Heart and renal failure developed soon after admission; she died at 10 days of age. Muscle and skin biopsies were performed on the eighth day of life. The muscle specimens stained poorly for COX activity, yet both COX-negative fibers and COX-positive fibers were found at high

14 PEDIATRIC NEUROLOGY

Vol. 8 No. 1

magnification [8]. A biochemical study of biopsied muscle revealed decreased levels of both complex I and COX [8]. Postmortem studies demonstrated COX activity to be decreased in specific tissues, yet normal in others. COX activities were within normal limits in the liver and kidney, but were decrettsed in muscle and brain to 18% and 30% of normal levels, respectively. Patient 4. This 5-month-old female was born to healthy, nonconsanguineous parents after a term. uneventful pregnancy and delivery. Birth

Table 1. COX activity in the fibroblasts from the controls and 4 patients* COX Deficiency

Leigh Encephalopathy Patient 1

17.0

(30);

Patient 2

9.0

(15)

Fatal Infantile COX Deficiency Patient 3

13.5

(23)

Patient 4

32.6

(56)

Control Group Control I

54.0

Control 2

95.8

Control 3

73.6

Control 4

66.7

Control 5

61.1

* Normal (mean + S.D.; 58.5 + 13.2; N = 28; nmol/min/mg protein). t Percentage of mean control values are listed in parentheses.

Table 2. Mitochondrial COX staining patterns expressed as percentage of total mitocbondrial area

Cell Line

No. of Examined Cells

Mitochondrial COX Activity Staining Patterns Equivocal Negative Positive

Leigh Encephalopathy Patient 1

20

1.6 _+ 1.9

68.3±23.2

31.1±23.6

Patient 2

20

0

56.5±25.9

43.5±25.9

Fatal Infantile COX Deficiency Patient 3

20

3.7 ± 12.6

19.7±35.9

76.6±41.4

Patient 4

20

31.5 :t: 25.3

45.3±25.1

23.2±31.5

Control 1

20

91.6 + 8.8

8.3±8.5

0.1±0.5

Control 2

20

86.2 _+ 14.7

13.5±13.9

0.3±1.3

Control 3

17

79.4 ± 17.0

19.3±15.0

1.3±3.4

Control 4

19

82.1 ± 10.1

16.1±9.6

1.0±1.5

Control 5

20

83.6 +_ 10.8

14.5±9.9

2.2±3.6

84.7 ± 12.9

14.2±11.9

1.0±2.4

Control Group

Mean ± S.D.

weight was 3.2 kg and the neonatal period was uneventful. At 1 month of age, hypotonia and generalized weakness were observed. On admission, she had profound hypotonia, absent tendon reflexes, and generalized weakness with few spontaneous movements. At 3 months of age, she suddenly developed apnea and required assisted ventilation. Serum lactate level was 57.5 mg/dl (normal: 3-16 mg/dl) and pyruvate level was 3.6 mg/dl (normal: 0.3-0.9 mg/dl). Urinalysis suggested de-ToniFanconi syndrome. She died at 8 months of age. A biochemical analysis of biopsied muscle obtained at 5 months of age revealed multiple deficiencies of mitochondrial respiratory chain enzymes, including COX [ 12].

Methods A skin biopsy was performed in each patient. Biochemical study was performed on the 4 patients and in the control group without mitochondrial encephalomyopathy, including 3 patients with epilepsy and 2 with mental retardation. These results were compared with those of 28 normal patients from whom tissue was obtained for diagnostic biopsy. Electron microscopic cytochemistry was performed on fibroblast cell lines from the 4 patients and 5 controls. Biochemical Study. The fibroblasts were harvested by trypsinization, disrupted by freeze-thawing, and suspended in a buffer containing 0.2 M sucrose, 0.13 M NaCl, and 1 mM Tris-HCl (pH 7.4). The COX activity was measured [13] in supematants after centrifugation at 600 × g for 10 min. Molecular Genetic Analysis. Mitochondrial DNA was extracted from muscle mitochondria isolated from Patients 1 and 3. After digestion with BamHI, electrophoresis on 0.8% agarose gel, and transfer to nylon filter (Gene Screen Plus, NGN Research Products), hybridization was performed using human linearized mitochondrial DNA probe radiolabeled with a random-primed DNA labeling kit (Boehringer Manheim, Indianapolis, IN). Electron Microscopic Cytochemistry. Skin fibroblasts were cultured for 3 days in Eagle's MEM supplemented with 10% FCS. The following procedures all were performed within the culture dishes to preserve the form of the fibroblasts in sire. Cultured fihroblasts were fixed with freshly prepared 2% glutaraldehyde for 15 min. COX staining was performed by Seligman's method [14] for 3 hours at 37°C. For negative control staining, other dishes from the same patient were incubated in a

medium containing 1 mM potassium cyanide. After incubation, fibroblasts were post-fixed in 1% osmium tetraoxide and dehydrated in graded ethanols. The monolayer of cultured fibroblasts which was a few micrometers thick on the bottom of the dish, was embedded in epoxy resin. After polymerization, the embedded fibroblasts were removed from culture dishes by heating the bottom of the dishes on a hot plate. Ultrathin sections were examined without counterstaining using an H-600 electron microscope (Hitachi). Micrographs of about 20 cells which contained nuclei were obtained randomly at magnification x2,000 in each case. To confirm that COX activity decreases with increased fixation time, various fixation times (15, 30, 45, 60, 120 min) were applied to control fibroblasts. COX activity decreased at fixation times of 60 min or more, disappearing completely after 120 min of fixation. To estimate the penetration of reaction substrate into the inner mitochondrial membranes of cultured fibroblasts, small pieces of muscle from control patients were incubated in the same medium. Reaction products were visible 30 ~tm beneath the surface of muscle within three hours of incubation. The percentage of mitochondrial area relative to cytoplasmic area was calculated for each fibroblast using the following formula: % Mitochondrial area =

Total mitochondrial area Whole cell area - nuclear area

× 100

The whole cell area and the nuclear area were measured for each cell from 1l/2-fold photographs of micrographs magnified x2,000. The total mitochondrial area was measured from 5-fold photographs of micrographs magnified x2,000 (total: xl0,000). Each mitochondrion was classified into 1 of 3 grades according to the intensity of staining (Figs IA-1C): positive staining (i.e., when all intracristal spaces were stained); equivocal staining (i.e., at least 1 intracristal space was stained weakly but some spaces were not stained at all); and negative staining (i.e., no intracristal space was stained). The percentage areas of positively-, equivocally-, and negatively-stained mitochondria relative to total mitochondrial area were measured in each cell from 5-fold photographs of micrographs magnified ×2,000. In each cell line, the mean area for each mitochondrial type was calculated from the areas in about 20 fibroblasts. Statistical analyses were performed using the Student t test.

Haginoya et al: COX-deficient Mitochondria

15

D Figure 2. COX staining in controls and 4 patienta. (A) Control 5. Among positively-stained mitochondria, rare negatively-stained mito~:hondria are observed in control fibroblasts. (B) Patient 1. All mitochondria react equivocally or negatively in Patients 1 and 2. (C) Patient 3. Inter