Functional relevance of mitochondrial abnormalities in sporadic inclusion body myositis

Cytochrome c oxidase (COX)-deficient fibers and multiple mitochondrial DNA (mtDNA) deletions are frequent findings in sporadic inclusion body myositis (s-IBM). However, the functional impact of these defects is not known. We investigated oxygen desaturation during exercise using the forearm exercise test, accumulation of lactate during exercise using a cycle ergometry test and mitochondrial changes (COX-deficient fibers, biochemical activities of respiratory chain complexes, multiple mtDNA deletions by long-range polymerase chain reaction) in 10 patients with s-IBM and compared the findings with age and sex-matched normal and diseased controls (without mitochondrial disorders) as well as patients with mitochondrial disorder due to nuclear gene defects resulting in multiple mtDNA deletions (MITO group). The mean age of the s-IBM patients was 68.2 ± 5.7 years (range: 56–75). Patients with s-IBM had statistically significantly reduced oxygen desaturation (ΔsO₂) during the handgrip exercise (p < 0.05) and elevated peak serum lactate levels during cycle ergometry compared to normal controls (p < 0.05). The percentage of COX-deficient fibers in s-IBM and MITO patients was significantly increased compared to normal controls (p < 0.01). Five out of nine s-IBM patients had multiple mtDNA deletions. Thirty-three percent of s-IBM patients showed an increased citrate synthase content and decreased activities of complex IV (COX). The biochemical pattern of respiratory chain complexes in patients with s-IBM and MITO was similar. Histopathological analysis showed similar changes in s-IBM and MITO due to nuclear gene defects. Functional tests reflecting mitochondrial impairment suggest a contribution of mitochondrial defects to disease-related symptoms such as fatigue and exertion-induced symptoms.     

Genetic and biochemical findings in Chinese children with Leigh syndrome

This study investigated the genetic and enzymological features of Leigh syndrome due to respiratory chain complex deficiency in Chinese patients. The clinical features of 75 patients were recorded. Mitochondrial respiratory chain enzyme activities were determined via spectrophotometry. Mitochondrial gene sequence analysis was performed in 23 patients. Five core pedigrees were investigated via restriction fragment length polymorphism and gene sequencing. Psychomotor retardation (55%), motor regression (20%), weakness (29%), and epilepsy (25%) were the most frequent manifestations. Sixty-four patients (85.3%) had isolated respiratory complex deficiencies: complex I was seen in 28 patients (37.3%); complex II, seven (9.3%); complex III, six (8%); complex IV, ten (13.3%); and complex V, 13 patients (17.3%). Eleven patients (14.7%) had combined complex deficiencies. Mitochondrial DNA mutations were detected in 10 patients. Eight point mutations were found in mitochondrial structural genes: m.4833A > G in ND2, m.10191T > C in ND3, m.12338T > C and m.13513G > A in ND5, m.14502T > C and m.14487T > C in ND6, m.8108A > G in COXII, and m.8993T > G in ATPase6. Three mutations were found in tRNA genes: m.4395A > G in tRNA-Gln, m.10454T > C in tRNA-Arg, and m.5587T > C in tRNA-Ala. One patient and their mother both had the m.12338T > C and m.8993T > C mutations. In conclusion, mitochondrial respiratory chain complex I deficiency and structural gene mutations frequently occur in Chinese Leigh syndrome patients.     

Distal weakness with respiratory insufficiency caused by the m.8344A > G “MERRF” mutation

The m.8344A > G mutation in the mt-tRNA^Lys gene, first described in myoclonic epilepsy and ragged red fibers (MERRF), accounts for approximately 80% of mutations in individuals with MERRF syndrome. Although originally described in families with a classical syndrome of myoclonus, ataxia, epilepsy and ragged red fibers in muscle biopsy, the m.8344A > G mutation is increasingly recognised to exhibit marked phenotypic heterogeneity. This paper describes the clinical, morphological and laboratory features of an unusual phenotype in a patient harboring the m.8344A > G ‘MERRF’ mutation. We present the case of a middle-aged woman with distal weakness since childhood who also had ptosis and facial weakness and who developed mid-life respiratory insufficiency necessitating non-invasive nocturnal ventilator support. Neurophysiological and acetylcholine receptor antibody analyses excluded myasthenia gravis whilst molecular genetic testing excluded myotonic dystrophy, prompting a diagnostic needle muscle biopsy. Mitochondrial histochemical abnormalities including subsarcolemmal mitochondrial accumulation (ragged-red fibers) and in excess of 90% COX-deficient fibers, was seen leading to sequencing of the mitochondrial genome in muscle. This identified the m.8344A > G mutation commonly associated with the MERRF phenotype. This case extends the evolving phenotypic spectrum of the m.8344A > G mutation and emphasizes that it may cause indolent distal weakness with respiratory insufficiency, with marked histochemical defects in muscle. Our findings support consideration of screening of this gene in cases of indolent myopathy resembling distal limb-girdle muscular dystrophy in which screening of the common genes prove negative.     

Quercetin protects against aluminium induced oxidative stress and promotes mitochondrial biogenesis via activation of the PGC-1α signaling pathway

The present investigation was carried out to elucidate a possible molecular mechanism related to the protective effect of quercetin administration against aluminium-induced oxidative stress on various mitochondrial respiratory complex subunits with special emphasis on the role of PGC-1α and its downstream targets, i.e. NRF-1, NRF-2 and Tfam in mitochondrial biogenesis. Aluminium lactate (10 mg/kg b.wt./day) was administered intragastrically to rats, which were pre-treated with quercetin 6 h before aluminium (10 mg/kg b.wt./day, intragastrically) for 12 weeks. We found a decrease in ROS levels, mitochondrial DNA oxidation and citrate synthase activity in the hippocampus (HC) and corpus striatum (CS) regions of rat brain treated with quercetin. Besides this an increase in the mRNA levels of the mitochondrial encoded subunits – ND1, ND2, ND3, Cyt b, COX1, COX3 and ATPase6 along with increased expression of nuclear encoded subunits COX4, COX5A and COX5B of electron transport chain (ETC). In quercetin treated group an increase in the mitochondrial DNA copy number and mitochondrial content in both the regions of rat brain was observed. The PGC-1α was up regulated in quercetin treated rats along with NRF-1, NRF-2 and Tfam, which act downstream from PGC-1α. Electron microscopy results revealed a significant decrease in the mitochondrial cross-section area, mitochondrial perimeter length and increase in mitochondrial number in case of quercetin treated rats as compared to aluminium treated ones. Therefore it seems quercetin increases mitochondrial biogenesis and makes it an almost ideal flavanoid to control or limit the damage that has been associated with the defective mitochondrial function seen in many neurodegenerative diseases.     

Comparing clinical data and muscle imaging of DYSF and ANO5 related muscular dystrophies

In this retrospective cross-sectional study clinical and muscle imaging data of patients with Miyoshi distal myopathy phenotype (MMD1 and MMD3) and limb girdle muscular dystrophy 2L (LGMD2L) were described. MMD1 and MMD3 are genetically heterogenous diseases based on DYSF and ANO5 gene defects. MMD3 and LGMD2L are clinically different diseases caused by an ANO5 gene defect. All groups showed predominant fatty degeneration of the gluteus minimus muscle and of the posterior segments of the thigh and calf muscles with sparing of the gracilis muscle. Muscle atrophy, hypertrophy and asymmetric muscle involvement on muscle imaging did not differ between groups. The pattern of fatty degeneration of muscles and of muscle weakness shows only minor differences between MMD1 (n = 6) and MMD3 (n = 8) patients with more frequently fatty degeneration of the rectus femoris, anterior tibial, and extensor digitorum muscles and more frequently muscle weakness in the anterior tibial, peroneal and calf muscle in MMD1. In the ANO5 related phenotypes the lateral head of the gastrocnemius muscle was less frequently involved in LGMD2L (n = 13) and no differences in the incidence of muscle weakness was found. Therefore, MMD3 and LGMD2L should be considered as part of one spectrum of ANO5 related muscle disease.     

Inhibition of mitochondrial cytochrome c oxidase potentiates Aβ-induced ER stress and cell death in cortical neurons

Previously we reported that amyloid-β (Aβ) leads to endoplasmic reticulum (ER) stress in cultured cortical neurons and that ER-mitochondria Ca^2 + transfer is involved in Aβ-induced apoptotic neuronal cell death. In cybrid cells which recreate the defect in mitochondrial cytochrome c oxidase (COX) activity observed in platelets from Alzheimer's disease (AD) patients, we have shown that mitochondrial dysfunction affects the ER stress response triggered by Aβ. Here, we further investigated the impact of COX inhibition on Aβ-induced ER dysfunction using a neuronal model. Primary cultures of cortical neurons were challenged with toxic concentrations of Aβ upon chemical inhibition of COX with potassium cyanide (KCN). ER Ca^2 + homeostasis was evaluated under these conditions, together with the levels of ER stress markers, namely the chaperone GRP78 and XBP-1, a mediator of the ER unfolded protein response (UPR). We demonstrated that COX inhibition potentiates the Aβ-induced depletion of ER Ca^2 + content. KCN pre-treatment was also shown to enhance the rise of cytosolic Ca^2 + levels triggered by Aβ and thapsigargin, a widely used ER stressor. This effect was reverted in the presence of dantrolene, an inhibitor of ER Ca^2 + release through ryanodine receptors. Similarly, the increase in GRP78 and XBP-1 protein levels was shown to be higher in neurons treated with Aβ or thapsigargin in the presence of KCN in comparison with levels determined in neurons treated with the neurotoxins alone. Although the decrease in cell survival, the activation of caspase-9- and -3-mediated apoptotic cell death observed in Aβ- and thapsigargin-treated neurons were also potentiated by KCN, this effect is less pronounced than that observed in Ca^2 + signalling and UPR. Furthermore, in neurons treated with Aβ, the potentiating effect of the COX inhibitor in cell survival and death was not prevented by dantrolene. These results show that inhibition of mitochondrial COX activity potentiates Aβ-induced ER dysfunction and, to a less extent, neuronal cell death. Furthermore, data supports that the effect of impaired COX on Aβ-induced cell death occurs independently of Ca^2 + release through ER ryanodine receptors. Together, our data demonstrate that mitochondria dysfunction in AD enhances the neuronal susceptibility to toxic insults, namely to Aβ-induced ER stress, and strongly suggest that the close communication between ER and mitochondria can be a valuable future therapeutic target in AD.     

Mitofusin 2 mutations affect mitochondrial function by mitochondrial DNA depletion

Charcot–Marie–Tooth neuropathy type 2A (CMT2A) is associated with heterozygous mutations in the mitochondrial protein mitofusin 2 (Mfn2) that is intimately involved with the outer mitochondrial membrane fusion machinery. The precise consequences of these mutations on oxidative phosphorylation are still a matter of dispute. Here, we investigate the functional effects of MFN2 mutations in skeletal muscle and cultured fibroblasts of four CMT2A patients applying high-resolution respirometry. While maximal activities of respiration of saponin-permeabilized muscle fibers and digitonin-permeabilized fibroblasts were only slightly affected by the MFN2 mutations, the sensitivity of active state oxygen consumption to azide, a cytochrome c oxidase (COX) inhibitor, was increased. The observed dysfunction of the mitochondrial respiratory chain can be explained by a twofold decrease in mitochondrial DNA (mtDNA) copy numbers. The only patient without detectable alterations of respiratory chain in skeletal muscle also had a normal mtDNA copy number. We detected higher levels of mtDNA deletions in CMT2A patients, which were more pronounced in the patient without mtDNA depletion. Detailed analysis of mtDNA deletion breakpoints showed that many deleted molecules were lacking essential parts of mtDNA required for replication. This is in line with the lack of clonal expansion for the majority of observed mtDNA deletions. In contrast to the copy number reduction, deletions are unlikely to contribute to the detected respiratory impairment because of their minor overall amounts in the patients. Taken together, our findings corroborate the hypothesis that MFN2 mutations alter mitochondrial oxidative phosphorylation by affecting mtDNA replication.     

Power spectral analysis of surface electromyography (EMG) at matched contraction levels of the first dorsal interosseous muscle in stroke survivors

ObjectiveThe objective of this study was to help assess complex neural and muscular changes induced by stroke using power spectral analysis of surface electromyogram (EMG) signals.MethodsFourteen stroke subjects participated in the study. They were instructed to perform isometric voluntary contractions by abducting the index finger. Surface EMG signals were collected from the paretic and contralateral first dorsal interosseous (FDI) muscles with forces ranging from 30% to 70% maximum voluntary contraction (MVC) of the paretic muscle. Power spectral analysis was performed to characterize features of the surface EMG in paretic and contralateral muscles at matched forces. A Linear Mixed Model was applied to identify the spectral changes in the hemiparetic muscle and to examine the relation between spectral parameters and contraction levels. Regression analysis was performed to examine the correlations between spectral characteristics and clinical features.ResultsDifferences in power spectrum distribution patterns were observed in paretic muscles when compared with their contralateral pairs. Nine subjects showed increased mean power frequency (MPF) in the contralateral side (>15 Hz). No evident spectrum difference was observed in 3 subjects. Only 2 subjects had higher MPF in the paretic muscle than the contralateral muscle. Pooling all subjects’ data, there was a significant reduction of MPF in the paretic muscle compared with the contralateral muscle (paretic: 168.7 ± 7.6 Hz, contralateral: 186.1 ± 8.7 Hz, mean ± standard error, F = 36.56, p < 0.001). Examination of force factor on the surface EMG power spectrum did not confirm a significant correlation between the MPF and contraction force in either hand (F = 0.7, p > 0.5). There was no correlation between spectrum difference and Fugl–Meyer or Chedoke scores, or ratio of paretic and contralateral MVC (p > 0.2).ConclusionsThere appears to be complex muscular and neural processes at work post stroke that may impact the surface EMG power spectrum. The majority of the tested stroke subjects had lower MPF in the paretic muscle than in the contralateral muscle at matched isometric contraction force. The reduced MPF of paretic muscles can be attributed to different factors such as increased motor unit synchronization, impairments in motor unit control properties, loss of large motor units, and atrophy of muscle fibers.SignificanceSurface EMG power spectral analysis can serve as a useful tool to indicate complex neural and muscular changes after stroke.     

Frequency and phenotype of patients carrying TPM2 and TPM3 gene mutations in a cohort of 94 patients with congenital myopathy

Congenital myopathies are difficult to classify correctly through molecular testing due to the size and heterogeneity of the genes involved. Therefore, the prevalence of the various genetic causes of congenital myopathies is largely unknown. In our cohort of 94 patients with congenital myopathy, two related female patients and two sporadic, male patients were found to carry mutations in the tropomyosin 2 (TPM2) and tropomyosin 3 (TPM3) genes, respectively. This indicates a low (4.3%) frequency of TPM2 and TPM3 mutations as a cause of congenital myopathy. Compared to previously described patients carrying the same mutations as found in our study (c.503G > A, and c.502C > T in TPM3, and c.415_417delGAG in TPM2), clinical presentation and muscle morphological findings differed in our patients. Differences included variation in distribution of muscle weakness, presence of scoliosis and ptosis, physical performance and joint contractures. The variation in clinical profiles emphasizes the phenotypic heterogeneity. However, common features were also present, such as onset of symptoms in infancy or childhood, musculoskeletal deformities and normal or low plasma levels of creatine kinase.One patient had nemaline myopathy and fiber size disproportion, while three patients had congenital fiber type disproportion (CFTD) on muscle biopsies. TPM2-related CFTD has only been described in two cases, indicating that mutations in TPM2 are rare causes of CFTD.     

Multiple mitochondrial DNA deletions in hereditary inclusion body myopathy

We have recently described an autosomal dominant hereditary inclusion body myopathy (h-IBM). Clinically it is is characterized by congenital joint contractures and slowly progressive, proximal muscle weakness and ophthalmoplegia. There is deterioration of muscle function between 30 and 50 years of age. While young patients show minor pathological changes in muscle, the middle-aged and old patients show rimmed vacuoles and inclusions of filaments measuring 15–18 nm in diameter. Except for the absence of significant inflammation the histopathology is similar to that found in sporadic inclusion body myositis (s-IBM). In s-IBM mitochondrial alterations including cytochrome c oxidase (COX) -deficient muscle fibers are common. These are due to multiple mitochondrial DNA (mtDNA) deletions. In this study we investigated the occurrence of mitochondrial alterations in autosomal dominant h-IBM. Young affected individuals showed no mitochondrial changes but three patients aged 38, 51 and 59 years, respectively, showed ragged red fibers and COX-deficient muscle fibers. Polymerase chain reaction analysis showed multiple mtDNA deletions. By in situ hybridization clonal expansions of mtDNA with deletions were demonstrated in COX-deficient muscle fibers. Most of the analyzed deletion breakpoints showed nucleotide repeats flanking the deletions. The results show that COX-deficient muscle fibers and somatic mtDNA deletions are present in this family with h-IBM. The same factors may be involved in the development of mtDNA deletions in s-IBM and this family with h-IBM. Received: 13 July 1999 / Revised: 6 October 1999 · Accepted: 12 October 1999     

A further patient with parasitic myositis due to Haycocknema perplexum,a rare entity

A new genus of nematode, Haycocknema perplexum, causing polymyositis in humans, was first described in two Australian patients from Tasmania in 1998. Three patients with myositis due to the same nematode were reported from northern Queensland in 2008. We report the sixth case from Australia, a 50-year-old man, also from Tasmania. He had a 2-year history of progressive weakness, weight loss of 10 kg and dysphagia. Muscle biopsy was initially interpreted as polymyositis with eosinophils. Maximum creatine kinase (CK) level was 5700 U/L and full blood examination was normal. He deteriorated after several months of treatment with prednisolone and methotrexate and review of the muscle biopsy showed intramyofibre parasites of H. perplexum. After 3 months of treatment with albendazole therapy, he made a very good clinical recovery and his CK decreased to 470 U/L. This uniquely Australian parasite can mimic polymyositis and leads to significant irreversible morbidity (two of the previous patients still have weakness and elevated CK after years) and even mortality (one died), if diagnosed late or after corticosteroids. Diagnosis can only be made by histopathology of muscle biopsy.     

Ephedrine therapy in eight patients with congenital myasthenic syndrome due to DOK7 mutations

In congenital myasthenic syndrome with DOK7 mutations ephedrine was reported to be beneficial in single patients. We carried out a small, open and prospective cohort study in eight European patients manifesting from birth to 12 years. Five patients showed limb-girdle and facial weakness, three a floppy infant syndrome with bulbar symptoms and/or respiratory distress. Ephedrine was started with 25 mg/day and slowly increased to 75–100 mg/day. Within weeks after starting therapy an improvement was observed in all patients and clinical follow-up disclosed positive effects more pronounced on proximal muscle weakness and strength using MRC scale. Effects on facial weakness were less pronounced. Vital capacity measurements and repetitive stimulation tests did not improve in the same way as clinical symptoms did. These investigations are appropriate to confirm the diagnosis in case of pathological results, but they might not be appropriate means to monitor patients under ephedrine therapy.     

Objective evaluation of muscle strength in infants with hypotonia and muscle weakness

The clinical evaluation of an infant with motor delay, muscle weakness, and/or hypotonia would improve considerably if muscle strength could be measured objectively and normal reference values were available. The authors developed a method to measure muscle strength in infants and tested 81 typically developing infants, 6–36 months of age, and 17 infants with Prader–Willi Syndrome (PWS) aged 24 months. The inter-rater reliability of the measurement method was good (ICC = .84) and the convergent validity was confirmed by high Pearson's correlations between muscle strength, age, height, and weight (r = .79–.85). A multiple linear regression model was developed to predict muscle strength based on age, height, and weight, explaining 73% of the variance in muscle strength. In infants with PWS, muscle strength was significantly decreased. Pearson's correlations showed that infants with PWS in which muscle strength was more severely affected also had a larger motor developmental delay (r = .75).     

Skeletal muscle MRI of the lower limbs in congenital muscular dystrophy patients with novel POMT1 and POMT2 mutations

Alpha-dystroglycanopathies form a genetically heterogeneous group of congenital muscular dystrophies with a large variety of clinical phenotypes. Within this group mutations in the protein O-mannosyltransferase genes (POMT1 and POMT2) are known to cause a spectrum of CMD disorders including the Walker–Warburg Syndrome with severe brain and ocular malformations, and the limb girdle muscular dystrophy with and without mental retardation. In this case report the clinical phenotype and brain and muscle MRI findings of two siblings of 10 and 7 years (male/female) homozygous for a novel mutation in the POMT1 gene (c.2220G > C, p.Trp740Cys) and a 10 year old boy with two novel mutations in the POMT2 gene ((c.215G > A, p.Arg72His) and (c.713G > T, p.Gly238Val) are presented. Mutation detection was performed by direct sequencing of the FKRP, FKTN, POMT1 and POMT2 genes. T1-weighted axial muscle MRI of the lower limbs revealed diffuse fatty degeneration of thigh and calf muscles with predominance of gluteus maximus, adductor magnus, posterior thigh, medial gastrocnemius, and peroneus muscles, but no edematous changes. As a similar pattern of muscle involvement had been described in FKRP related α-dystroglycanopathy LGMD2I, we conclude that α-dystroglycanopathies may present with distinctive muscle MRI changes.     

An “inflammatory” mitochondrial myopathy. A case report

We describe a case of an adult male patient with progressive external ophthalmoplegia and upper limb weakness, who presented with an episode of sudden respiratory failure. Muscle biopsy showed ragged-red and COX-negative fibers associated with discrete inflammatory infiltrates and necrotizing features. Apart from artificial ventilator support, he was treated with intravenous immunoglobulins and carnitine, with excellent clinical outcome. Mitochondrial DNA analysis revealed the 3251A > G mutation, previously reported in association with rapidly progressive mitochondrial myopathy and respiratory failure. Our case expands the spectrum of this mutation and suggests a therapeutic attempt with immunoglobulins in mitochondrial patients with acute respiratory failure, at least when this mutation and/or muscle inflammation is present. Moreover, this case supports the idea of a pathologic inflammatory response induced by mitochondrial disease; such an abnormal response may be a contributory factor in disease progression or acute exacerbation typical of some mitochondrial diseases, but further studies are needed.     

Factors of importance for dynamic balance impairment and frequency of falls in individuals with myotonic dystrophy type 1 – A cross-sectional study – Including reference values of Timed Up & Go, 10 m walk and step test

Patients with myotonic dystrophy type 1 suffer from gait difficulties including stumbles and falls. To identify factors of importance for balance impairment and fall-risk a mapping of functional balance was performed, in a cross-sectional study of 51 adults. Walking, balance, falls and muscle force were self-assessed and measured. Reference values of balance were established through measurements of 220 healthy subjects. Falls were more frequently observed in the patients who were more severely affected of muscle weakness than in mildly affected patients, p = 0.014. The number of falls showed negative correlation with balance confidence (r_s = −0.516, p < 0.001). The ankle dorsiflexor force together with the time difference between comfortable and maximum speed in 10 m-walk proved to be significant factors for fall frequency. A ten Newton muscle force decrease showed 15% increase in odds ratio for frequent falls. One-second increase in time difference between comfortable and maximum walking speed showed 42% increase in odds ratio for frequent falls. In conclusion, assessing the ankle muscle force and the time difference in different walking speeds is important to detect risk of falling. The activities-specific balance confidence score reflects the consequences of the muscle force decrease. Certain patient strategies to diminish risk of falling could be due.     

Acute exposure to a Mn/Zn ethylene-bis-dithiocarbamate fungicide leads to mitochondrial dysfunction and increased reactive oxygen species production in Caenorhabditis elegans

Mn/Zn ethylene-bis-dithiocarbamate (Mn/Zn-EBDC) fungicides are among some the most widely-used fungicides in the world. Although they have been available for over 50 years, little is known about their mechanism of action in fungi, or their potentially toxic mechanisms in humans. To determine if exposure of Caenorhabditis elegans (C. elegans) to a representative fungicide (Manzate; MZ) from this group inhibits mitochondria or produces reactive oxygen species (ROS), we acutely (30 min) exposed worms to various MZ concentrations. Initial oxygen consumption studies showed an overall statistically significant decrease in oxygen consumption associated with addition of Complex I- and/or II-substrate in treatment groups compared to controls (*p < 0.05). In order to better characterize the individual complex activity, further studies were completed that specifically assessed Complex II or Complex IV. Data indicated that neither of these two complexes were targets of MZ treatment. Results from tetramethylrhodamine ethyl ester (proton gradient) and ATP assays showed statistically significant reductions in both endpoints (*p < 0.05, **p < 0.01, respectively). Additional studies were completed to determine if MZ treatment also resulted in increased ROS production. These assays provided evidence that hydrogen peroxide, but not superoxide or hydroxyl radical levels were statistically significantly increased (*p < 0.05). Taken together, these data indicate exposure of C. elegans to MZ concentrations to which humans are exposed leads to mitochondrial inhibition and concomitant hydrogen peroxide production. Since mitochondrial inhibition and increased ROS are associated with numerous neurodegenerative diseases, we suggest further studies to determine if MZ catalyzes similar toxic processes in mammals.     

Study of mitochondrial DNA depletion in muscle by single-fiber polymerase chain reaction

We studied muscle biopsies from 3 children with a mitochondrial myopathy characterized histochemically by the presence of ragged-red fibers (RRF) and various numbers of cytochrome c oxidase (COX)-negative fibers. We quantitated the absolute amounts of total mitochondrial DNA (mtDNA) in isolated normal COX-positive muscle fibers and in COX-negative RRF. There was severe mtDNA depletion in all fibers from the two most severe cases. In the third case mtDNA depletion could not be established with conventional diagnostic tools, but it was documented in single COX-negative fibers; COX-positive fibers showed the same amounts of mtDNA as fibers from aged-matched controls. Our observations indicate that mtDNA single-fiber PCR quantitation is a highly sensitive and specific method for diagnosing cases with focal mtDNA depletion. This method also allows one to correlate amounts of mtDNA with histochemical phenotypes in individual fibers from patients and age-matched controls, thereby providing important information about the functional role of residual mtDNA. © 1998 John Wiley & Sons, Inc. Muscle Nerve 21: 1374–1381, 1998     

Intérêt de la biopsie musculaire chez l’enfant en 2012

Muscle biopsy is a mainstay diagnostic tool for investigating neuromuscular disorders in children. We report the yield of pediatric muscle biopsy in a population of 415 children by a retrospective study of 419 biopsies performed between 1/01/2000 and 31/12/2009 in a neuropediatric department, including mitochondrial respiratory chain analysis for 87 children. Two hundred and fifty-five biopsies were from boys (61%) 164 from girls (39%). Their mean age at biopsy was 6.5 years; 155 (37%) biopsies were obtained before the child was 5 years old. Final histopathological diagnoses were: congenital myopathy (n = 193, including 15 structural congenital myopathies); progressive muscular dystrophy (n = 75 [18%] including 57 dystrophinopathies); congenital muscular dystrophy (n = 17, including six primary merosinopathies); dermatomyositis (n = 11); spinal muscular atrophy (n = 9, including six atypical spinal muscular atrophies); metabolic myopathy (n = 32, including 19 mitochondrial myopathies); encephalomyopathy (n = 53 [13%], including 27 with a mitochondrial respiratory chain defect). Pathological diagnosis remained undetermined in 16 cases. In 184 patients (44%), the muscle biopsy revealed specific histopathological anomalies (dystrophic process; specific ultrastructural abnormalities; perifascicular atrophy; neurogenic atrophy; metabolic anomalies) enabling a precise etiological diagnosis. For 85% of progressive muscular dystrophies, the biopsy resulted in a genetic diagnosis after identification of the protein defect. In 15% of the congenital myopathies, histopathological anomalies focused attention on one or several genes. Concerning dystrophinopathies, quantification of dystrophin deficiency on the biopsy specimen contributed to the definition of the clinical phenotype: Duchenne, or Becker. In children with a myopathy, muscle biopsy is often indispensable to establish the etiological diagnosis. Based on the results from this series, muscle biopsy can provide a precise orientation in 45% of patients, leading to a genetic hypothesis.     

Caenorhabditis elegans chronically exposed to a Mn/Zn ethylene-bis-dithiocarbamate fungicide show mitochondrial Complex I inhibition and increased reactive oxygen species

Reports have linked human exposure to Mn/Zn ethylene-bis-dithiocarbamate (Mn/Zn-EBDC) fungicides with multiple pathologies, from dermatitis to central nervous system dysfunction. Although members of this family of agrochemicals have been available for over 50 years, their mechanism of toxicity in humans is still unclear. Since mitochondrial inhibition and oxidative stress are implicated in a wide variety of diseases, we hypothesized that Caenorhabditis elegans (C. elegans) exposed to a commercially-available formulation of an Mn/Zn-EBDC-containing fungicide (Manzate; MZ) would also show these endpoints. Thus, worms were treated chronically (24 h) with various MZ concentrations and assayed for reduced mitochondrial function and increased levels of reactive oxygen species (ROS). Oxygen consumption studies suggested Complex I inhibition in all treatment groups compared to controls (^**p < 0.01). In order to verify these findings, assays specific for Complex II or Complex IV activity were also completed. Data analysis from these studies indicated that neither complex was adversely affected by MZ treatment. Additional data from ATP assays indicated a statistically significant decrease (^***p < 0.001) in ATP levels in all treatment groups when compared to control worms. Further studies were completed to determine if exposure of C. elegans to MZ also resulted in increased ROS concentrations. Studies demonstrated that hydrogen peroxide, but not superoxide or hydroxyl radical, levels were statistically significantly increased (*p < 0.05). Since hydrogen peroxide is known to up-regulate glutathione-S-transferase (GST), we used a GST:green fluorescent protein transgenic worm strain to test this hypothesis. Results from these studies indicated a statistically significant increase (^***p < 0.001) in green pixel number following MZ exposure. Taken together, these data indicate that C. elegans treated with MZ concentrations to which humans are exposed show mitochondrial Complex I inhibition with concomitant hydrogen peroxide production. Since these mechanisms are associated with numerous human diseases, we suggest further studies to determine if MZ exposure induces similar toxic mechanisms in mammals.