Cytochromec oxidase and coenzyme Q in neuromuscular diseases: a histochemical study

Summary Cytochromec oxidase (CCO) has been histochemically studied in 250 muscle biopsies from patients with different neuromuscular diseases. The results were compared with those obtained on serial sections stained with Gomori's trichrome and with the methods for NADH tetrazolium reductase, succinate dehydrogenase and lactate dehydrogenase. In 58 selected cases serial sections were also stained with a method demonstrating coenzyme Q (CoQ) activity. Demonstration of structural alterations was as good with CCO as with the methods for other oxidative enzymes: particularly evident were alterations of the distribution of mitochondria, such as core areas in central core and multiminicore diseases. Unstained fibers were observed in mitochondrial myopathies, in Becker, Emery-Dreifuss, limb-girdle, facio-scapulo-humeral muscular dystrophies, muscle infarction, polymyositis, motor neuron diseases and neuropathies. The histochemical method for CoQ showed only low specificity, since partial staining was also present in areas devoid of mitochondria, such as cores. CoQ deficiency was not observed in any of the 19 mitochondrial myopathies examined.     

Vascular pathology in chronic progressive external ophthalmoplegia with ragged-red fibers]

Vascular involvement in biopsied muscle specimens from 11 patients with chronic progressive external ophthalmoplegia (CPEO) with ragged-red fibers (RRF) was studied. Almost none of 69 intramuscular arteries examined were strongly stained with succinate dehydrogenase (SDH) except one patient who had 2 SSV (strongly SDH-reactive blood vessels) in his muscle biopsy. Although RRF and focal cytochrome c oxidase (CCO) deficiency in muscle fibers were the common histochemical changes in muscle biopsy specimens from CPEO patients, all mitochondria in both endothelial and smooth muscle cells of the arteries had normal morphology except for the two SSV and all mitochondria in the blood vessels had normal CCO activity by electron cytochemistry. The findings obtained from the present study were quite different from those in mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS), and myoclonus epilepsy associated with ragged-red fibers (MERRF) in which the striking vascular involvement with SSV is the most common and major abnormality in muscle biopsy specimens. To study vascular involvement in mitochondrial encephalomyopathies is the one of very important clues to understand the pathophysiology of phenotypic expressions in mitochondrial encephalomyopathies.     

"Ragged-red" fibres in myotonic dystrophy

Twenty-five muscle biopsies (18 from the left biceps and 7 from the left quadriceps) of 25 patients suffering from myotonic dystrophy (MyD) were studied, 13 of which showed "ragged-red" fibres (RRFs); all the RRFs, which were type I fibres, were found in biceps muscles, while none of the quadriceps muscles showed RRFs. The incidence of RRFs varied from 0.5% to 20.0% (average 4.2%). On electron microscopy, RRFs contained enlarged mitochondria, usually in subsarcolemmal clusters, including dense granular matrix materials, concentrically whired membranous cristae, and paracrystalline inclusions, consistent with those of previously reported cases of mitochondrial myopathy, suggesting that RRFs observed in biopsies from patients with MyD are due to abnormal mitochondria. The biopsy findings indicative of MyD including pyknotic nuclear clumps, moth-eaten fibres, ring fibres, type I fibre atrophy, and type I fibre predominance, were much more common findings in biceps muscles than quadriceps muscles, and in biopsies with RRFs than those without RRFs. From our observations, it is possible that RRFs in biopsied muscles from patients with MyD are not incidental observations but are intimately associated with the pathogenesis of this disorder, and that RRFs may be a special form of pathological reaction in which accumulation of abnormal mitochondria occurs.     

Cytochrome c oxidase activity is deficient in blood vessels of patients with myoclonus epilepsy with ragged-red fibers

Summary More than half of the intramuscular blood vessels in muscle biopsies from five patients with myoclonus epilepsy with ragged-fibers (MERRF) who had a point mutation in mitochondrial DNA at the tRNA^Lys region were darkly stained with succinate dehydrogenase (SDH) stain, showing the morphologic characteristics of strongly SDH-reactive blood vessels (SSV), but they had no cytochrome c oxidase (CCO) activity. By electron cytochemistry, the mitochondria in the smooth muscle cells of SSV had no CCO activity. On the other hand, SSV in muscle biopsies from patients with mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) had normal CCO activity as shown by light and electron microscopy. The defect in CCO activity in the arteriolar smooth muscle cells and in muscle fibers suggests that CCO deficiency is related to the pathophysiology of MERRF.     

Quantitative skeletal muscle pathology of aging regarding ragged-red fibers and cytochrome c oxidase-negative fibers]

A statistical analysis of mitochondrial abnormality of aging in human skeletal muscle fibers was performed. Sixty one muscle samples were obtained from patients with acute medical illness autopsied strictly within 2 hours after death, or with orthopedic or surgical diseases biopsied with informed consents. The patients aged from 16 to 89, averaging 58 +/- 21 years in males and 21 to 92, averaging 55 +/- 20 years in females. Sections were stained by modified Gomori's trichrome, succinate dehydrogenase and cytochrome c oxidase-negative [CCO(-)] fibers approximately in 10,000 fibers in each muscle were evaluated. Both RRF and CCO (-) fibers were not observed below the fourth decade, but sequentially increased with age, especially after the seventh decade. The incidence of CCO (-) fibers was higher than that of RRF. RRF did not necessarily correspond to CCO (-) fibers. The present quantitative pathological result is a useful tool to evaluate the mitochondrial function in fresh human skeletal muscles by the age.     

Mitochondrial myopathy, encephalopathy, lactic acidosis, and strokelike episodes with recurrent abdominal symptoms and coenzyme Q10 administration.

A male with mitochondrial myopathy, encephalopathy, lactic acidemia, and strokelike episodes is reported. He had also recurrent episodes of ileus. Muscle biopsy revealed ragged-red fibres. The cytochemistry of cytochrome c oxidase (CCO) showed scattered nonstained fibres, while all muscle fibres were heavily stained by immunocytochemistry using CCO antibody. These findings suggest that partical CCO deficiency may be present in the skeletal muscles of the patient. NADH cytochrome c reductase in the patient's muscle mitochondria was low compared with normal controls (about 26%), although succinate cytochrome c reductase was normal. Coenzyme Q10 administration (90 mg/day) did not improve CSF lactate levels, but did decrease plasma lactate levels. His muscle weakness slightly improved.     

Congenital nemaline myopathy with mitochondrial abnormalities. An adult case report]

We report a 42-year-old male suffering from congenital nemaline myopathy accompanied with mitochondrial abnormalities in his muscle biopsy. He had a dysmorphic face with a high-arched and narrow palate and slowly progressive generalized muscle weakness. He was still able to walk with a cane. CT showed symmetrical muscle atrophy and low densities in the thigh muscles, especially in the posterior compartment, and in the soleus muscles. Preferential posterior thigh involvement was unusual in congenital nemaline myopathy. The lumbar quadrate and paravertebral muscles were relatively well preserved; these muscles were reported to be severely involved in adult-onset nemaline myopathy patients. Muscle biopsy findings were consistent with nemaline myopathy; nemaline rods in approximately 10% of fibers, type 1 fiber atrophy, and type 2B fiber deficiency. In addition, ragged-red fibers were scattered and focal cytochrome c oxidase (CCO) deficiency was present. Formazan granules were large on succinate dehydrogenase stain. Many fibers with nemaline rods showed focal CCO deficiency. On electron microscopy, large (megaconial) mitochondria were lined regularly between Z lines. PCR and Southern blot analysis of muscle mitochondrial DNA revealed multiple deletions. It remains to be clarified whether mitochondrial abnormalities are primarily related to nemaline myopathy or secondarily induced phenomenon after a long-standing disease process.     

线粒体脑肌病的临床与病理

目的探讨线粒体脑肌病的临床与肌肉病理特点。方法对16例肌活检证实的线粒体脑肌病病例的临床表现、肌肉组织化学及超微结构进行分析。结果16例患者破碎红纤维(RRF)的平均比例为5.9%,11例有中央核增多,13例的SDH/CCO双染示12例有蓝纤维,且与RRF的分布一致。超微结构观察有4例找到典型晶格状包涵体。结论SDH/CCO双染有蓝纤维为线粒体肌病的诊断提供了依据,借此可与其他肌病鉴别。     

Mitochondrial cytopathy with lactic acidosis, carnitine deficiency and DeToni-Fanconi-Debré syndrome

We reported a 6-year-old girl with mitochondrial cytopathy with lactic acidosis. The patient developed hypotonia, hearing loss, mental retardation, short stature, cataracta, hypoparathyroidism, DeToni-Fanconi-Debré syndrome and carnitine deficiency. Histological examination disclosed ragged red fibers and moderate lipid storage in skeletal muscle tissue and several structural abnormalities of mitochondria both in muscle tissue and proximal renal tubules. Biochemical examination of muscle tissue revealed a partial deficiency of pyruvate dehydrogenase complex and normal activities of cytochrome c oxidase, succinate cytochrome c reductase and NADH cytochrome c reductase. This is the first report of mitochondrial cytopathy representing DeToni-Fanconi-Debré syndrome associated with partial deficiency of pyruvate dehydrogenase complex and normal cytochrome c oxidase activity.     

Familial mitochondrial encephalomyopathy with deaf-mutism, ophthalmoplegia and leukodvstrophy.

We report two sisters (32 and 36 years old) with familial deaf-mutism, progressive external ophthalmoplegia, leukodystrophy and mitochondrial myopathy. T2-weighted brain MRI demonstrated diffuse symmetrical high intensity areas in the white matter. Their muscle biopsies showed ragged-red fibers and cytochrome c oxidase (CCO)-negative fibers. CCO activity in biopsied muscle decreased to about 20% of normal control. They had no deletions of the mitochondrial DNA and no point mutations in mitochondrial tRNA. Their brother was diagnosed as having Kugelberg-Welander disease, grand mal seizures and urinary dysfunction. Their parents and grandparents had consanguinity. Three relatives were found to have deaf-mutism without accompanying ophthalmoplegia. This rare combination of mitochondrial encephalomyopathy and familial deaf-mutism might be caused by a nuclear DNA mutation in these sisters.     

Findings in muscle in complex I (NADH coenzyme Q reductase) deficiency

Thirteen of 15 patients with complex I deficiency had the multisystemic form, with strokelike episodes and other symptoms that fulfilled the diagnostic requirements for MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis, and strokelike episodes), and 2 had only muscle fatigability and weakness, having the purely myopathic form. In the multisystemic form, 12 patients had ragged-red fibers. All multisystemic patients had myopathic histochemical abnormalities that consisted of mild to moderate variation in fiber size, disorganized intermyofibrillar networks, type 2 fiber atrophy, and an increased number of type 2C fibers. Five of 13 multisystemic patients had decreased cytochrome c oxidase (CCO) activity in extrafusal fibers, with sparing of intrafusal muscle fibers. In the myopathic form, pathological findings were similar to those in the multisystemic form. In addition to complex I and NADH dehydrogenase activities being decreased, the CCO activity was significantly decreased (less than 50% of control value) in 8 patients, especially when the disease was in its advanced stages, suggesting that CCO enzyme might be secondarily affected as the disease progresses.     

Mitochondrial encephalomyopathy (focal cytochrome c oxidase deficiency) with transient episodes of muscle weakness and elevation of serum creatine kinase activity

A 17-year-old boy who had mitochondrial encephalomyopathy with focal deficiency of cytochrome c oxidase (CCO) activity is described. He experienced 3 episodes of muscle weakness, fatigability, nausea, vomiting and concomitant increase of serum creatine kinase activity, at the age of 13, 15 and 17 years. During interval there was no muscle weakness and the serum creatine kinase activity was within normal range. Increased levels of lactic acid and pyruvic acid were observed in the blood and cerebrospinal fluid. After an aerobic exercise test, lactic acid and pyruvic acid in the blood increased to an abnormally high level, and the arterial blood became acidic (pH 7.297). On EEG, occasional intermittent irregular theta activities were observed in the anterior region, but there were no abnormalities on CT and MRI in the central nervous system. In the biopsied muscle, ragged-red fibers comprised 20% on modified Gomori-trichrome staining and a number of fibers with no CCO activity were scattered throughout. The CCO activity in the mitochondria isolated from the biopsied muscle was reduced to 49.2 nmol/min/mg protein (normal range 144.7-355.8), while other mitochondrial enzyme activities in the electron transport system were normal. From these data, the patient was considered to have a unique form of mitochondrial encephalomyopathy. By the administration of a large amount of coenzyme Q10, episodes of muscle weakness and nausea, and an increase of lactic acid and pyruvic acid in the blood after aerobic exercise test were no longer observed.     

肢带型线粒体肌病一个家系的临床和病理特点研究

目的研究肢带型线粒体肌病一个家系中患者的临床和骨骼肌病理特点。方法对一组以进行性肌肉无力为主要特点的家系中的3名患者进行临床资料收集及肌肉活检病理分析,对家系成员行线粒体基因检测。结果该家系患者发病年龄为30~47岁,主要表现为躯干肌及四肢近端肌无力和运动耐力下降,晚期出现呼吸肌受累。血肌酸激酶水平轻度升高,血乳酸升高,电生理检查结果示肌源性损害。骨骼肌病理检查显示3名患者均存在典型的破碎红纤维(RRF),且患者病程越长(分别为2年、4年和14年),肌无力严重程度越重,RRF数量越多(分别为5%、10%和30%)。细胞色素c氧化酶(COX)染色中RRF以深染为主,也可见阴性的RRF。琥珀酸脱氢酶(SDH)染色均未发现琥珀酸脱氢酶高反应性血管(SSVs)。基因检测发现该家系存在mt DNA A3243G突变。结论肢带型线粒体肌病患者选择性累及躯干和四肢近端肌群,骨骼肌中的RRF数量与患者病情进展程度呈正相关。     

Defect in succinate oxidation by isolated muscle mitochondria in a patient with symmetrical lesions in the basal ganglia

A 3-year-old boy was referred for evaluation of psychomotor retardation. He had a waddling gait with proximal hypotonia and paresis. Computed tomography (CT scan) and magnetic resonance imaging (MRI) of the brain demonstrated symmetrical lesions in the basal ganglia suggesting bilateral necrosis. Lactate and pyruvate levels in blood and cerebrospinal fluid were persistently elevated. A biopsy of the quadriceps muscle showed normal light microscopic findings except for a slightly raised number of lipid droplets. Electron microscopy confirmed this and also showed a rather large number of subsarcolemmal mitochondria without crystalline inclusions. Biochemical studies showed a normal carnitine level and normal mitochondrial enzyme activities in muscle homogenate, including succinate-cytochrome c reductase. However, intact isolated mitochondria failed to oxidize succinate. An explanation for this paradoxical finding is a deficiency in that part of the coenzyme Q (CoQ) that is reduced by the succinate dehydrogenase complex. The differential diagnosis between Leigh's syndrome and infantile bilateral striatal necrosis (IBSN) is discussed. The role of neuroradiology in prompting complementary investigations is stressed.     

Diagnosis of mitochondrial diseases: clinical and histological study of sixty patients with ragged red fibers

BACKGROUND: Mitochondrial diseases are caused by mutations in mitochondrial or nuclear genes, or both and most patients do not present with easily recognizable disorders. The characteristic morphologic change in muscle biopsy, ragged-red fibers (RRFs) provides an important clue to the diagnosis. MATERIALS AND METHODS: Demographic data, presenting symptoms, neurological features, and investigative findings in 60 patients with ragged-red fibers (RRFs) on muscle biopsy, seen between January 1990 and December 2002, were analyzed. The authors applied the modified respiratory chain (RC) diagnostic criteria retrospectively to determine the number of cases fulfilling the diagnostic criteria of mitochondrial disease. RESULTS: The most common clinical syndrome associated with RRFs on muscle biopsy was progressive external ophthalmoplegia (PEO) with or without other signs, in 38 (63%) patients. Twenty-six patients (43%) had only external ophthalmoplegia, 5 (8%) patients presented with encephalomyopathy. Specific syndromes were the presenting feature in 8 (13%), Kearns-Sayre syndrome (KSS) in 4 and myoclonus epilepsy with ragged-red fibers (MERRF) in 4. Myopathy was the presenting feature in 5 (8%) and 4 presented with infantile myopathy. Of the 60 patients, 18 (30%) had proximal muscle weakness. Two patients with KSS and one patient with myopathy had complete heart block necessitating pace making. When the modified RC diagnostic criteria were applied, only 26 (43%) patients had one other major criterion in addition to RRFs for the diagnosis of mitochondrial diseases. The remaining 34 (57%) patients with RRFs on muscle biopsy had only some clinical features suggestive of RC disorder but did not fulfill the clinical criteria (of the modified diagnostic criteria) for the diagnosis of mitochondrial diseases. CONCLUSION: In patients with clinical features suggestive of RC disorder, demonstration of RRFs on muscle biopsy helps in confirming the diagnosis of mitochondrial disease in only a subgroup of patients.     

Cytoplasmic body and mitochondrial DNA deletion

A patient with chronic progressive external ophthalmoplegia (CPEO) who had abundant cytoplasmic bodies in muscle fibers and a deletion of mitochondrial DNA is reported. The patient was a 26-year-old male suffering from ophthalmoplegia from age 21. He had a marfanoid skeletal abnormality and perceptive hearing loss, but had neither retinopathy, ataxia, nor dementia. In the mitochondria isolated from the biopsied skeletal muscle, NADH-ubiquinone oxidoreductase activity was slightly decreased, succinate-cytochrome c reductase activity was slightly increased, and cytochrome c oxidase activity remained normal. Southern blot analysis of the muscle DNA identified heteroplasmy composed of a normal-sized mitochondrial DNA and a mutant mitochondrial DNA with a 4.2-kilobase deletion. The PCR plus S1 analysis showed that the deletion extended from nucleotide position 7860 +/- 60 to 12,090 +/- 70. The histological studies of the biopsied muscle revealed ragged-red fibers and cytochrome c oxidase-negative fibers in 15.7% and 18.6% of the muscle fibers, respectively. Other conspicuous histological change was abundant cytoplasmic bodies surrounded by clusters of abnormal mitochondria. The cytoplasmic bodies were found preferentially in type 1 fibers, and exclusively in cytochrome c oxidase-negative fibers and in ragged-red fibers. Focal existence of cytoplasmic bodies in muscle fibers with abnormal mitochondria suggests that segregated distribution of the abnormal mitochondria with deleted mitochondrial DNA is involved in the pathogenesis of cytoplasmic bodies.     

Autopsied case with MERRF/MELAS overlap syndrome accompanied by stroke‐like episodes localized to the precentral gyrus

We present an autopsied case with A8344G‐mutated myoclonus epilepsy with ragged red fibers (MERRF)/mitochondrial encephalomyopathy with lactic acidosis and stroke‐like episodes (MELAS) overlap syndrome accompanied by stroke‐like episodes localized to the precentral gyrus. A 16‐year‐old Japanese woman suddenly experienced repetitive consciousness disturbances with increased serum lactate and creatine kinase levels. Magnetic resonance imaging showed abnormal intensity of bilateral precentral gyrus. She was clinically diagnosed as having a mitochondrial disorder and the A8344G mutation was detected in mitochondrial DNA. At 17 years of age, she died from congestive heart failure secondary to a third episode of lactic acidosis. Neuropatho‐logically, multifocal laminar necrosis, which is responsible for stroke‐like episodes in MELAS, was seen in the frontal cortex including the precentral gyrus, but there was no neuronal loss and gliosis in the basal ganglia, cerebellum, and brainstem, which were compatible with MERRF. Hypertrophy of the vascular smooth muscle and choroidal epithelium were seen, and were strongly visualized by an anti‐mitochondrial antibody. Skeletal muscles showed uneven muscular diameters, increased central nuclei, and ragged red fibers (RRFs). Decreased cytochrome c oxidase (COX) activity and strongly succinate dehydrogenase (SDH)‐reactive blood vessels were also noted. Stroke‐like episodes in MERRF/MELAS overlap syndrome are thought to be rare in the frontal cortex including the precentral gyrus. Only two cases of MERRF/MELAS overlap syndrome with A8344G mutation, including this case, have shown stroke‐like episodes in the frontal lobes. Other than the A8344G mutation and frontal lobe involvement, they had a high degree of similarity in terms of presence of RRFs, gastrointestinal dysfunction, and lack of typical MERRF neuropathology. In conclusion, this is an important case describing the clinical spectrum associated with A8344G‐mutated MERRF/MELAS overlap syndrome.     

Monozygotic twins with MELAS-like syndrome lacking ragged red fibers and lactacidaemia

Typical cases of MELAS present a combination of clinical and neuroradiological features, lactacidaemia, and ragged red fibers (RRFs) in striated muscle. We have observed a MELAS-like syndrome in monozygotic twins. They developed seizures typically in conjunction with physical exertion, sleep deprivation or febrile episodes. Stroke-like episodes occurred usually during seizures. In twin 2 the course was fatal at age 20 years. Neuroradiological findings were typical of MELAS. Plasma lactate was normal in both. CSF lactate was normal in twin 1 and normal/elevated in twin 2. RRFs were not seen in muscle biopsies of the twins. Complex I activity was reduced in muscle in twin 1. Brain tissue removed at epilepsy surgery in twin 2 showed the presence of mitochondrial angiopathy. The commonest mitochondrial DNA mutation in MELAS, at base pair 3243, was absent. Lactacidaemia and mitochondrial myopathy with RRFs constitute part of the diagnostic criteria of MELAS. However, the absence of these features does not exclude mitochondrial disorder with the serious manifestations of MELAS (seizures and stroke-like episodes) as seen in these twins.     

Leigh encephalopathy: histologic and biochemical analyses of muscle biopsies.

To elucidate the pathogenesis of Leigh encephalopathy, histologic, biochemical, and mitochondrial DNA analyses were performed on biopsied muscles from 33 patients with the clinical characteristics of this disorder. On muscle histochemistry, cytochrome c oxidase activity was decreased or absent in 7 patients (21%), although none had ragged-red fibers. In 2 patients with cytochrome c oxidase deficiency, staining for this enzyme was poor in the muscle fibers and fibroblasts but was normal in the arterial wall, indicating tissue-specific involvement. Ten patients (30%) had biochemical defects, including 2 with pyruvate dehydrogenase complex, 4 with cytochrome c oxidase, 1 with NADH-cytochrome c reductase (complex I), and 3 with multiple complex deficiencies. None of the 28 patients in whom muscle mitochondrial (mt)DNA was analyzed had DNA deletions or point mutation at nucleotide positions 3,243 or 8,344. These results indicate that the underlying defect in Leigh encephalopathy is heterogeneous because only 30% of patients had enzyme defects demonstrable in muscle biopsy material.     

Exogenous coenzyme Q (coq) fails to increase coq in skeletal muscle of two patients with mitochondrial myopathies

Recently, several studies were published on therapy with coenzyme Q (CoQ) in patients with mitochondrial myopathies without biochemically established muscular deficiency of CoQ. Two patients with mitochondrial myopathies presenting as oculocraniosomatic syndromes were treated with coenzyme Q (CoQ). The muscle biopsy of both patients showed ragged-red fibers and single muscle fibers without histochemical reaction for cytochrome c oxidase. Biochemical analysis revealed normal activities of the respiratory chain complexes in muscle and normal levels of CoQ in serum and muscle. After one year of treatment CoQ in serum of both patients had increased 1.4-fold and 2.0-fold, respectively. In muscle, however, there was no increase of CoQ in either patient. In both patients the activities of citrate synthase and of the respiratory chain complexes I + III and IV, and in 1 patient also of complex II + III, were lower in the second biopsy compared with the first biopsy. In both patients there was no improvement of maximal isometric muscle strength assessed by a quantitative electronic strain gauge. The exercise-induced pathological rise of lactate in 1 patient remained essentially unchanged during therapy. The data indicate that orally administered CoQ fails to increase total CoQ in muscle of patients with mitochondrial myopathies but without muscular CoQ deficiency.