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J Neurol (1982) 227:201-207 Journal of

Neurology © Springer-Verlag 1982

Cytochrome-C-oxidase Deficiency in Muscles of a Floppy Infant Without Mitochondrial Myopathy*

M. Rimoldi ~, E. Bottacchi ~, L. Rossi 3, F. Cornelio 2, G. Uziel 3, and S. Di Donato 3

1 Laboratory of Neurometabolic Disease and 2 Laboratory of Myopathology, Istituto Neurologico "C. Besta", Milan

~Pediatric Clinic, 1st Chair, Milan, Italy

Summary. A 4-month-old girl had hypotonia, muscle weakness and cardiomyopathy at birth and died at the age of 8 months from heart failure. Muscle biopsy showed normal muscle fibers, normal mitochondria and type II hypotrophy, in agreement with clinical and neurophysiological signs consistent with a suprasegmental lesion. Cytochrome-C-oxidase was the only respiratory chain enzyme that was profoundly decreased in the patient's muscle, but not in her brain, heart and cultured fibroblasts. We suggest that cytochrome-C- oxidase deficiency in skeletal muscle is not always associated with mitochondrial abnormalities and may not always indicate primary muscle pathology.

Key words: Cytochrome-C-oxidase deficiency - Mitochondrial myopathy - Floppy infant

Zusammenfassung. Ein 4 Monate altes M~idchen litt seit Geburt an einer Muskelhypotonie verbunden mit einer Kardiomyopathie, an der es im Alter von 8 Monaten starb. Eine Muskelbiopsy zeigte normale Muskelfasern und Mitochondrien und eine Muskelhypotrophie Typ II. Dies sprach in einer Obereinstimmung mit den klinischen und elektrophysiologischen Befunden fiJr eine suprasegmentale Lasion.

Cytochrome-C-Oxidase war das einzige Enzym der Atmungskette, welches im Muskel hochgradig vermindert war. Es war hingegen normal im Gehirn- gewebe, im Herz und in der Fibroblasten-Kultur. Es wird daraus gefolgert, dab ein Mangel an Cytochrome-C-Oxidase nicht immer mit morphologisch fafSbaren Anomalien der Mitochondrien vergesellschaftet ist und dab sic nicht immer eine prim~ire Muskelerkrankung darstellt.

* This work was carried out under a grant of the Muscular Dystrophy Association, USA and research grant CT 81.00146.04 from the Consiglio Nazionale delle Ricerche, Italy

Offprint requests to." Stefano Di Donato, MD -- Laboratory of Neurometabolic Diseases, Istituto Neurologico "C. Besta" via Celoria 11 -- 20133 Milan, Italy

0340-5354/82/0227/0201/$01.40

202 M. Rimoldi et al.

Introduction

The floppy baby syndrome is frequently diagnosed in children with poor skeletal muscle control and diminished muscle tone at birth.

Many of these children suffer from central or peripheral nervous system disorders, but some have muscle diseases [8], including congenital myopathies, mainly defined from distinct morphological features [2], and metabolic myopathies, mainly defined from distinct biochemical features [5]. The latter group of diseases includes many recognized disorders associated with the floppy infant syndrome, such as glycogenosis type II [5] and cytochrome-C-oxidase deficiency [6, 17]. In this last disease, in addi t ion to the enzyme deficiency in skeletal muscle the floppy babies have lactic acidosis, renal dysfunct ion resembling de Toni- Fancon i -Debr6 syndrome and muscle morphology consistent with the mi tochon- drial-l ipid-glycogen (MLG) myopathy described by Jerusalem et al. [ l l ] . We describe a f loppy baby with progressive lethal cardiomyopathy, moderate lactic acidosis, normal renal funct ion and central nervous system involvement, in whom a p ro found deficiency of cytochrome-C-oxidase (Cyt-C-Oxidase) was found in skeletal muscle, without any abnormali t ies of muscle mitochondria .

Case Report

The propositus, a 4-month-old girl, was born in an uncomplicated delivery after 37 weeks of pregnancy. The parents were first cousins. The 3-year-old sister of the patient was normal.

The patient's weight at birth was 2150 g. At age 4 months she weighed 4000 g, but her weight declined to 3600 g at age 6 months and to 3100 g at age 8 months. Mental development seemed normal: she smiled at age 2 months and could recognize parents and objects at 4 months.

Neurological examination at age 4 months showed marked generalized hypotonia and weakness of the skeletal muscles with difficulty in grasping objects and poor head control. Deep tendon reflexes were present. Visual function and ophthalmoscopic examination of the fundi were judged normal. The liver edges was palpable two fingers beneath the right costal margin. Precordial examination revealed a protomesodiastolic thrill: the second heart sound was variably split. Electrocardiograms revealed sinus tachycardia (175/min) and signs of left ventricular hypertrophy with moderate myocardial damage. Echocardiography showed a picture consistent with asymmetric ventricular hypertrophy, with marked septal hypertrophy. Chest radiographs showed heart enlargement. The patient was digitalized at age 4 months. EMG of both proximal and distal muscles and maximal motor conduction velocities and distal latencies on the left median and peroneal nerves were normal. Repeated EEG recordings showed clear signs of diffuse brain sufferance.

Routine laboratory investigations were normal except for increased levels of aminotrans- ferases (SGOT 120 and SGPT 63mU/1, normal values less than 24mU/1). There was no glycosuria or proteinuria and urinary aminoacids were in the normal range. Blood lactate and pyruvate were moderately increased (lactate 2.7, 2.8, 5.3 mM; pyruvate 0.20, 0.18, and 0.26 mM; normal values for lactate less than 2 mM; normal values for pyruvate less than 0.15 raM). The lactate to pyruvate ratio was normal. Overnight fasting acetoacetate and fl-hydroxybutyrate were 76 and 136~tM, and 391 and 7271aM, respectively, in two different determinations. The fi- hydroxybutyrate to acetoacetate ratio was high (normal value less than 3), indicating poor mitochondrial oxidation of nicotinamide adenine dinucleotide (NADH). At age 7 months neurological examination revealed marked generalized muscle weakness and wasting, diffuse muscle hypotonia and no deep tendon reflexes. Her general condition was poor and echocardiography revealed a worsening of the left ventricular hypertrophy. At age 8 months the patient died

Cytochrome-C-oxidase Deficiency in Muscles of a Floppy Infant 203

from heart failure. The main findings at autopsy were cardiomyopathy with asymmetric ventricular hypertrophy and diffuse fatty degeneration of the liver. Biopsies of the right biceps brachii and of skin for fibroblast culture were taken at age 4 months.

Methods

Carnitine and its esters were evaluated separately; carnitine was measured by the radiochemical method previously reported [3]. Carnitine acetyltransferase (CAT) was determined by the method previously reported [4]. Carnitine palmitoyltransferase (CPT) was measured by the "forward" reaction [10] and by the "backward" reaction, using labelled palmitoyl-1-3H-carnitine, synthesized according to the method of Stokke and Bremer [16]. Pyruvate-dehydrogenase complex (PDHC) was measured by a radiochemical method [4]. Succinate-cytochrome-C- reductase (Succ-Cyt-C-red) and NADH-cytochrome-C-reductase (NADH-Cyt-C-red) were measured spectrophotometrically [15]. Cyt-C-oxidase activity was determined spectrophotometrically by the decrease in absorbance at 550nm reduced cytochrome-C [18]. Oxidized cytochrome-C (Sigma), 1% in 10 mM phosphate buffer pH 7.0, was reduced by the addition of a few grains of NaBH4: the unreacted NaBH4 was eliminated by titration with 1 N HC1. Respiratory chain enzyme activities were determined in 10% skeletal muscle homogenates in TRIS-HC1- sucrose, pH 7.4 at 38°C, and in homogenates of fibroblasts in TRIS-buffered saline, pH 7.4 at 30 ° C. Rates of oxidation of pyruvate- 1 - 14C and palmitate- 1- ~4C (A mersham) by skeletal muscle homogenates were determined as previously described [1]. Proteins were measured by the Lowry method [13], non-collagen protein by the method of Lilienthal et al. [12]. Light and electron microscopic studies of skeletal muscle were performed as previously described [4]. The histochemical reaction for Cyt-C-oxidase was that of Seligman [14].

Results

Since the rates of pyruvate and palmitate oxidat ion by muscle homogenates were shown to be slow in the pat ient ' s muscle (Table 1), we measured some of the mi tochondr ia l enzymes involved in the oxidative pathways for pyruvate and fatty

acids. Table 2 shows that the activity of Cyt-C-oxidase was p rofoundly decreased in

the pat ient ' s muscle (less than 5% of the control mean) , while the activities of the other enzyme complexes of the respiratory chain were in the no rma l range. The activities of acyltransferases (CAT and CPT) and of citrate synthase were normal , while the activity of P D H C was reduced in the pat ient ' s muscle (25% of the mean of controls). Total carni t ine content in skeletal muscle was lower than the

Table 1. Oxidation rates in skeletal muscle homogenates

Substrate Controls Patient

- L-carnitine + L-carnitine - L-carnitine + L-carnitine

Pyruvate-124C (9) 1087 +_175 2187 +570 262 292

Palmitate-l-14C (10) 17.7+_ 3.7 35.5+_ 82 7.0 6.1

Values are given as picomoles labelled CO2 produced/min/mg noncollagen protein_+SD. L-carnitine, when added, was I mM. Number of controls in parentheses

204

Enzyme Controls Patient

CAT (8) 2.9 _+ 0.4 2.9

CPTfbrwar d (12) 0.14_+ 0.1 0.14

CPTbackward (12) 13.3 _+ 0.9 12.8

Citrate synthase (8) 77.1 _+ 15 86.1

PDHC (4) 2.75 + 0.90 0.68

NADH-Cyt-C-red (4) 20.1 + 0.6 17.9

Succ-Cyt-C-red (4) 5.2 _+ 1.7 4.6

Cyt-C-oxidase (7) 31.8 _+ 13.3 1.4

Values are given as nanomoles substrate catalyzed/min/ mg noncollagen protein_+SD. Number of controls in parentheses

NADH-Cyt-C-red = NADH-cytochrome-C-reductase Succ-Cyt-C-red = Succinate-cyt ochrome-C-reductase Cyt-C-oxidase = Cytochrome-C-oxidase

M. Rimoldi et al.

Table 2. Mitochondrial enzymes in muscle homogenates

Table 3. Carnitine and carnitine esters in plasma and skeletal muscle

Free Esterified Total

Plasma

Controls

Patient

Muscle

Controls

Patient

37.0_+ 2.5 5.7 42.4+ 2.5

(23.0 - 53.0) (3.0 - 10.0) (26.0 - 63.0)

27 23.5 50.5

19.5_+ 1.1 2.5_+ 0.2 22.0_+ 1.1

(11.0-28.0) (1 .1- 5.5) (12.1-33.5)

7.8 3.9 11.7

Values in plasma are given as nmol/ml + SE Values in muscle are given as nmol/mg noncollagen protein_+ SE The fange in controls in parentheses

lowes t c o n t r o l va lue (Tab le 3). T o t a l ca rn i t ine c o n t e n t in p l a s m a was n o r m a l , bu t

ca rn i t i ne d i s t r i bu t i on was not , s ince ca rn i t ine esters were inc reased f o u r f o l d o v e r the c o n t r o l m e a n value .

C y t - C - o x i d a s e , P D H C and c i t ra te syn thase were m e a s u r e d in c u l t u r e d f ib rob las t s and in a u t o p s y spec imens o f the p a t i e n t ' s b ra in and hear t a n d f o u n d to be n o r m a l (Tab le 4).

M o r p h o l o g i c a l s tudies o f the musc le b i o p s y s h o w e d a n o r m a l pa t t e rn o f

h i s t o c h e m i c a l r eac t i ons excep t for a se lect ive h y p o t r o p h y o f type II f ibers. E l e c t r o n

m i c r o g r a p h s s h o w e d n o r m a l musc le a r ch i t e c tu r e and m i t o c h o n d r i a o f n o r m a l


Table 4. Mitochondrial enzymes in brain, heart and cultured fibroblasts

Tissue Controls Patient

Heart

Cyt-C-oxidase (3) 11.9 + 2.0 I 1.5

Brain

Cyt-C-oxidase (3) 5.5 + 1.2 7.2

Fibroblasts

Cyt-C-oxidase (4) 6.6 + 1.5 5.5

PDHC (8) 3.13 + 0.65 3.26

CPTforwar d (4) 0.35 + 0.04 0.37 CPTbackward (4) 10.1 +2.1 10.2

Values are given as nmol substrate catalyzed/min/mg cell protein Number of controls in parentheses

Fig. lA, B. Cytochrome-C-oxidase stain of fresh frozen section of muscle biopsy. A Normal staining in muscle from a 5-month-old patient with suspected carnitine deficiency, showing punctate granules corresponding to mitochondria. B There is no staining of mitochondria in the muscle of the propositus

shape and size. The specific s ta in for Cy t -C-ox idase gave no s ta in ing in the muscle fibers f rom the pat ient , but s ta ining o f the muscle fibers f rom an age -ma tched cont ro l was n o r m a l (Fig. 1).

D i s c u s s i o n

F o u r pa t ien ts in two families with lethal f loppy infant synd rome and Cyt -C- oxidase deficiency in skeletal muscle have been descr ibed [6, 17]. These pa t ien ts had lactic acidosis , renal insuff iciency and clinical, morpho log ica l and n e u r o -

206 M. Rimoldi et al.

physiological signs of primary muscle pathology. Another patient with an M L G myopathy and Cyt-C-oxidase deficiency in skeletal muscle has been recently observed. However, in this patient the clinical course was benign and the Cyt-C- oxidase deficiency in muscle was reversible [7]. In addition to these myopathic patients, Cyt-C-oxidase deficiency has been found in muscles of patients with anatomically proved Leigh's syndrome [19] and of a patient with Menkes' trichopoliodystrophy [9]. In these patients the Cyt-C-oxidase defect in skeletal muscles was considered to be secondary [6]. Our patient had progressive cardiomyopathy, which caused the unfavorable clinical course and death. This pathology was not related to Cyt-C-oxidase activity, since this enzyme was normal in her heart muscle (Table 4). In addition to cardiomyopathy, our patient suffered from generalized muscle weakness and hypotonia. EMG and VCMM were normal at age 4 months and muscle morphology was inconsistent with a primary muscle pathology since it revealed only a selective type II hypotrophy of muscle fibers, a pattern consistent with a suprasegmental lesion [2]. This was in agreement with persistent EEG signs of diffuse brain sufference. Marked Cyt-C-oxidase deficiency was found both biochemically and histochemically in the skeletal muscle of this girl, but not in her brain, heart or cultured fibroblasts.

The defective function of the respiratory chain in muscle might well explain the slow rates of pyruvate and palmitate oxidation by muscle homogenates. The partial deficiency of P D H C in muscle might have contributed to lowering the pyruvate oxidation (Table 2). Carnitine content was low in muscle (Table 3), as observed by Di Mauro et al. [6] in a fatal case of Cyt-C-oxidase deficiency and by us in a patient with M L G myopathy and lactic acidosis [4]. Carnitine esters were increased in plasma, probably as the consequence of impaired acyl-CoA oxidation in muscles and consequent equilibration of acyl-CoAs with acylcarnitines through the action of carnitine acyltransferases. These enzymes activities were normal in the patient 's muscle (Table 2). In this patient, the Cyt-C-oxidase deficiency in muscle was quantitatively comparable to that in patients with primary genetic deficiencies of this enzyme in muscle [6, 17]. However, in our patient it could not be easily attributed to primary muscle mitochondrial pathology, since mitochondria were normal, muscle morphology and neurophysiology were not consistent with myopathy but were consistent with suprasegmental damage. Therefore, we suggest that Cyt-C-oxidase deficiency in muscles may result in heterogeneous clinical conditions. Alternatively CNS damage might have nega- tively affected the normal development of certain mitochondrial enzymes (Cyt-C- oxidase, PDHC), but not of others. Whatever the cause, Cyt-C°oxidase in muscle probably contributed to the progression of weakness and wasting in this patient.

Acknowledgement. We are gratefully indebted to Mr. Sergio Daniel for his skillful technical assistance.

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Received March 22, 1982