A mitochondrial perspective on striated muscle physiopathology: insights from sepsis, denervation, and dystrophinopathies.

La mitochondrie est de plus en plus reconnue pour sa contribution à la dégénerescence musculaire. Les dysfonctions mitochondriales, en plus de causer une défaillance énergétique, contribuent à la signalisation apoptotique, stimule la production de ROS et peuvent induire une surcharge calcique. Ces caractéristiques sont tous reliées à certains types de myopathies. Cette thèse met en lumières comment certaines dysfonctions mitochondriales peuvent intervenir dans la pathogenèse de diverses myopathies. Nous démontrons que les dysfonctions mitochondriales sont impliqués dans l’atrophie dû à la perte d’innervation. Par contre, la désensabilisation de l’ouverture du pore mitochondrial de transition de perméabilité, via ablation génétique de cyclophiline-D, ne prévient ni la signalisation apoptotique mitochondrial ni l’atrophie. Nous avons aussi observé des dysfonctions mitochondriales dans le muscle atteint de dystrophie musculaire de Duchenne qui furent améliorés suite à une transfection de PGC1-α, laquelle résulta aussi en une amélioration de la pathologie. Finalement, nous démontrons que le recyclage de mitochondrie par les voies de mitophagies et de contrôles de la qualité impliquant Parkin et possiblement d’autres voies de signalisation inconnues sont cruciales au recouvrement cardiaqe lors d’un choc septique.

Role of E3-ligase parkin in mitochondrial quality control in a cardiotoxicity model to anthracyclines

Dues à leur importance croissante dans la dégénérescence musculaire, les mitochondries sont de plus en plus étudiées en relation à diverses myopathies. Leurs mécanismes de contrôle de qualité sont reconnus pour leur rôle important dans la santé mitochondrial. Dans cette étude, nous tentons de déterminer si le déficit de mitophagie chez les souris déficiente du gène Parkin causera une exacerbation des dysfonctions mitochondriales normalement induite par la doxorubicine. Nous avons analysé l’impact de l’ablation de Parkin en réponse à un traitement à la doxorubicine au niveau du fonctionnement cardiaque, des fonctions mitochondriales et de l’enzymologie mitochondriale. Nos résultats démontrent qu’à l’état basal, l’absence de Parkin n’induit pas de pathologie cardiaque mais est associé à des dysfonctions mitochondriales multiples. La doxorubicine induit des dysfonctions respiratoires, du stress oxydant mitochondrial et une susceptibilité à l’ouverture du pore de transition de perméabilité (PTP). Finalement, contrairement à notre hypothèse, l’absence de Parkin n’accentue pas les dysfonctions mitochondriales induites par la doxorubicine et semble même exercer un effet protecteur.

Mitochondrial energy metabolism in neurodegeneration associated with methylmalonic acidemia

Methylmalonic acidemia is one of the most prevalent inherited metabolic disorders involving neurological deficits. In vitro experiments, animal model studies and tissue analyses from human patients suggest extensive impairment of mitochondrial energy metabolism in this disease. This review summarizes changes in mitochondrial energy metabolism occurring in methylmalonic acidemia, focusing mainly on the effects of accumulated methylmalonic acid, and gives an overview of the results found in different experimental models. Overall, experiments to date suggest that mitochondrial impairment in this disease occurs through a combination of the inhibition of specific enzymes and transporters, limitation in the availability of substrates for mitochondrial metabolic pathways and oxidative damage.

Evidence that OGG1 glycosylase protects neurons against oxidative DNA damage and cell death under ischemic conditions

7,8-Dihydro-8-oxoguanine DNA glycosylase (OGG1) is a major DNA glycosylase involved in base-excision repair (BER) of oxidative DNA damage to nuclear and mitochondrial DNA (mtDNA). We used OGG1-deficient (OGG1(-/-)) mice to examine the possible roles of OGG1 in the vulnerability of neurons to ischemic and oxidative stress. After exposure of cultured neurons to oxidative and metabolic stress levels of OGG1 in the nucleus were elevated and mitochondria exhibited fragmentation and increased levels of the mitochondrial fission protein dynamin-related protein 1 (Drp1) and reduced membrane potential. Cortical neurons isolated from OGG1(-/-) mice were more vulnerable to oxidative insults than were OGG1(+/+) neurons, and OGG1(-/-) mice developed larger cortical infarcts and behavioral deficits after permanent middle cerebral artery occlusion compared with OGG1(+/+) mice. Accumulations of oxidative DNA base lesions (8-oxoG, FapyAde, and FapyGua) were elevated in response to ischemia in both the ipsilateral and contralateral hemispheres, and to a greater extent in the contralateral cortex of OGG1(-/-) mice compared with OGG1(+/+) mice. Ischemia-induced elevation of 8-oxoG incision activity involved increased levels of a nuclear isoform OGG1, suggesting an adaptive response to oxidative nuclear DNA damage. Thus, OGG1 has a pivotal role in repairing oxidative damage to nuclear DNA under ischemic conditions, thereby reducing brain damage and improving functional outcome. Journal of Cerebral Blood Flow & Metabolism (2011) 31, 680-692; doi:10.1038/jcbfm.2010.147; published online 25 August 2010

The mitochondrial transcription factor A functions in mitochondrial base excision repair

Mitochondrial transcription factor A (TFAM) is an essential component of mitochondrial nucleoids TFAM plays an important role in mitochondrial transcription and replication TFAM has been previously reported to inhibit nucleotide excision repair (NER) in vitro but NER has not yet been detected in mitochondria, whereas base excision repair (BER) has been comprehensively characterized in these organelles The BER proteins are associated with the inner membrane in mitochondria and thus with the mitochondrial nucleoid, where TFAM is also situated However, a function for TFAM in BER has not yet been investigated This study examines the role of TFAM in BER In vitro studies with purified recombinant TFAM indicate that it preferentially binds to DNA containing 8-oxoguanines, but not to abasic sites, uracils, or a gap in the sequence TFAM inhibited the in vitro incision activity of 8-oxoguanine DNA glycosylase (OGG1), uracil-DNA glycosylase (UDG), apurinic endonuclease 1 (APE1), and nucleotide incorporation by DNA polymerase gamma (pol gamma) On the other hand, a DNA binding-defective TFAM mutant, L58A, showed less inhibition of BER in vitro Characterization of TFAM knockdown (KD) cells revealed that these lysates had higher 8oxoG incision activity without changes in alpha OGG1 protein levels TFAM KD cells had mild resistance to menadione and increased damage accumulation in the mtDNA when compared to the control cells In addition, we found that the tumor suppressor p53, which has been shown to interact with and alter the DNA binding activity of TFAM, alleviates TFAM-Induced inhibition of BER proteins Together, the results suggest that TFAM modulates BER in mitochondria by virtue of its DNA binding activity and protein interactions Published by Elsevier B V

Mitochondrial ATP-sensitive K(+) channels as redox signals to liver mitochondria in response to hypertriglyceridemia

We have recently demonstrated that hypertriglyceridemic (HTG) mice present both elevated body metabolic rates and mild mitochondrial uncoupling in the liver owing to stimulated activity of the ATP-sensitive potassium channel (mitoK(ATP)). Because lipid excess normally leads to cell redox imbalance, we examined the hepatic oxidative status in this model. Cell redox imbalance was evidenced by increased total levels of carbonylated proteins, malondialdehydes, and GSSG/GSH ratios in HTG livers compared to wild type. In addition, the activities of the extramitochondrial enzymes NADPH oxidase and xanthine oxidase were elevated in HTG livers. In contrast, Mn-superoxide dismutase activity and content, a mitochondrial matrix marker, were significantly decreased in HTG livers. isolated HTG liver mitochondria presented lower rates of H(2)O(2) production, which were reversed by mitoK(ATP) antagonists. In vivo antioxidant treatment with N-acetylcysteine decreased both mitoKATP activity and metabolic rates in HTG mice. These data indicate that high levels of triglycerides increase reactive oxygen generation by extramitochondrial enzymes that promote MitoK(ATP) activation. The mild uncoupling mediated by mitoK(ATP) increases metabolic rates and protects mitochondria against oxidative damage. Therefore, a biological role for mitoK(ATP) is a redox sensor is shown here for the first time in an in vivo model of systemic and cellular lipid excess, (C) 2009 Elsevier Inc. All rights reserved.

Trypanocidal action of eupomatenoid-5 is related to mitochondrion dysfunction and oxidative damage in Trypanosoma cruzi

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Overexpression of plant uncoupling mitochondrial protein in transgenic tobacco increases tolerance to oxidative stress

An Arabidopsis thaliana cDNA clone encoding a plant uncoupling mitochondrial protein (AtPUMP1) was overexpressed in transgenic tobacco plants. Analysis of the AtPUMP1 mRNA content in the transgenic lines, determined by Northern blot, revealed variable levels of transgene expression. Antibody probing of Western blots of mitochondrial proteins from three independent transgenic lines showed significant accumulation of AtPUMP1 in this organelle. Overproduction of AtPUMP1 in transgenic tobacco plants led to a significant increase in tolerance to oxidative stress promoted by exogenous hydrogen peroxide as compared to wild-type control plants. These results provide the first biological evidence for a role of PUMP in protection of plant cells against oxidative stress damage.

Can trifluoperazine protect mitochondria against reactive oxygen species-induced damage?

Trifluoperazine (TFP) (35 μM) prevents mitochondrial transmembrane potential (ΔΨ) collapse and swelling induced by 10 μM Ca2+ plus oxyradicals generated from δ-aminolevulinic acid autoxidation. In contrast with EGTA, TFP cannot restore the totally collapsed ΔΨ. So, TFP might not remove Ca2+ from its 'harmful site', but could impair the ROS-driven cross-linking between membrane -SH proteins. Our data are correlated with the protective uses of TFP against oxidative processes promoted by oxyradicals plus Ca2+.

Impaired protamination and sperm DNA damage in a Nellore bull with high percentages of morphological sperm defects in comparison to normospermic bulls

The routine semen evaluation assessing sperm concentration, motility and morphology, does not identify subtle defects in sperm chromatin architecture. Bulls appear to have stable chromatin, with low levels of DNA fragmentation. However, the nature of fragmentation and its impact on fertility remain unclear and there are no detailed reports characterizing the DNA organization and damage in this species. The intensive genetic selection, the use of artificial insemination and in vitro embryo production associated to the cryopreservation process can contribute to the chromatin damage and highlights the importance of sperm DNA integrity for the success of these technologies. Frozen-thawed semen samples from three ejaculates from a Nellore bull showed high levels of morphological sperm abnormalities (55.8±5.1%), and were selected for complementary tests. Damage of acrosomal (76.9±8.9%) and plasma membranes (75.7±9.3%) as well as sperm DNA strand breaks (13.8±9.5%) and protamination deficiency (3.7±0.6%) were significantly higher compared to the values measured in the semen of five Nellore bulls with normospermia (24.3±3.3%; 24.5±6.1%; 0.6±0.5%; 0.4±0.6% for acrosome, plasma membrane, DNA breaks and protamine deficiency, respectively) (P<0.05). Motility and percentage of spermatozoa with low mitochondrial potential showed no differences between groups. This study shows how routine semen analyses (in this case morphology) may point to the length and complexity of sperm cell damage emphasizing the importance of sperm function testing.

Mechanisms involved in reproductive damage caused by gossypol in rats and protective effects of vitamin E

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Effects of bovine sperm cryopreservation using different freezing techniques and cryoprotective agents on plasma, acrosomal and mitochondrial membranes

The success of semen cryopreservation is influenced by several factors, such as freezing curves and cryoprotectants. These two factors are of special interest once they may lead to many important physical-chemical changes resulting in different degrees of damage in spermatozoa structure. This experiment was designed to compare the effect of bull semen cryopreservation using two freezing techniques: conventional (CT cooling rate of -0.55 degrees C min-1 and freezing rate of -19.1 degrees C min-1) and automated (AT cooling rate of -0.23 degrees C min-1 and freezing rate of -15 degrees C min-1), performed with different curves, and with three cryoprotectants (glycerol, ethylene glycol and dimethyl formamide) on bovine sperm motility and integrity of plasma, acrosomal and mitochondrial membranes. These variables were simultaneously evaluated using the fluorescence probes propidium iodide, fluorescein-conjugated Pisum sativum agglutinin and MitoTracker Green FM. The effects of freezing techniques, as well as of different cryoprotectants were analysed by the analysis of variance. The means were compared by Fishers test. There were no significant differences between freezing techniques (P > 0.05). Glycerol showed higher percentages of motility, vigour and integrity of plasma, acrosomal and mitochondrial membranes than other two cryoprotectants (P < 0.05). Ethylene glycol preserved higher motility and integrity of plasma and mitochondrial membranes than dimethyl formamide (P < 0.05). Sperm motility with glycerol was 30.67 +/- 1.41% and 30.50 +/- 1.06%, with ethylene glycol was 21.17 +/- 1.66% and 21.67 +/- 1.13% and with dimethyl formamide was 8.33 +/- 0.65% and 9.17 +/- 0.72% to CT and AT curves, respectively. The percentage of spermatozoa with simultaneously intact plasma membrane, intact acrosome and mitochondrial function (IPIAH) was 14.82 +/- 1.49% (CT) and 15.83 +/- 1.26% (AT) to glycerol, 9.20 +/- 1.31% (CT) and 9.92 +/- 1.29% (AT) to ethylene glycol 4.65 +/- 0.93% (CT) and 5.17 +/- 0.87% (AT) to dimethyl formamide. Glycerol provided the best results, although nearly 85% of spermatozoa showed some degree of injury in their membranes, suggesting that further studies are required to improve the results of cryopreservation of bovine semen.

Activation of the mitochondrial ATP-sensitive K+ channel reduces apoptosis of spleen mononuclear cells induced by hyperlipidemia

Background We have previously demonstrated that increased rates of superoxide generation by extra-mitochondrial enzymes induce the activation of the mitochondrial ATP-sensitive potassium channel (mitoKATP) in the livers of hypertriglyceridemic (HTG) mice. The resulting mild uncoupling mediated by mitoKATP protects mitochondria against oxidative damage. In this study, we investigate whether immune cells from HTG mice also present increased mitoKATP activity and evaluate the influence of this trait on cell redox state and viability. Methods Oxygen consumption (Clark-type electrode), reactive oxygen species production (dihydroethidium and H2-DCF-DA probes) and cell death (annexin V, cytocrome c release and Trypan blue exclusion) were determined in spleen mononuclear cells. Results HTG mice mononuclear cells displayed increased mitoKATP activity, as evidenced by higher resting respiration rates that were sensitive to mitoKATP antagonists. Whole cell superoxide production and apoptosis rates were increased in HTG cells. Inhibition of mitoKATP further increased the production of reactive oxygen species and apoptosis in these cells. Incubation with HTG serum induced apoptosis more strongly in WT cells than in HTG mononuclear cells. Cytochrome c release into the cytosol and caspase 8 activity were both increased in HTG cells, indicating that cell death signaling starts upstream of the mitochondria but does involve this organelle. Accordingly, a reduced number of blood circulating lymphocytes was found in HTG mice. Conclusions These results demonstrate that spleen mononuclear cells from hyperlipidemic mice have more active mitoKATP channels, which downregulate mitochondrial superoxide generation. The increased apoptosis rate observed in these cells is exacerbated by closing the mitoKATP channels. Thus, mitoKATP opening acts as a protective mechanism that reduces cell death induced by hyperlipidemia.

Mitochondrial dysfunction in a cell model of thyroid oncocytoma

The role of mitochondrial dysfunction in cancer has long been a subject of great interest. In this study, such dysfunction has been examined with regards to thyroid oncocytoma, a rare form of cancer, accounting for less than 5% of all thyroid cancers. A peculiar characteristic of thyroid oncocytic cells is the presence of an abnormally large number of mitochondria in the cytoplasm. Such mitochondrial hyperplasia has also been observed in cells derived from patients suffering from mitochondrial encephalomyopathies, where mutations in the mitochondrial DNA(mtDNA) encoding the respiratory complexes result in oxidative phosphorylation dysfunction. An increase in the number of mitochondria occurs in the latter in order to compensate for the respiratory deficiency. This fact spurred the investigation into the presence of analogous mutations in thyroid oncocytic cells. In this study, the only available cell model of thyroid oncocytoma was utilised, the XTC-1 cell line, established from an oncocytic thyroid metastasis to the breast. In order to assess the energetic efficiency of these cells, they were incubated in a medium lacking glucose and supplemented instead with galactose. When subjected to such conditions, glycolysis is effectively inhibited and the cells are forced to use the mitochondria for energy production. Cell viability experiments revealed that XTC-1 cells were unable to survive in galactose medium. This was in marked contrast to the TPC-1 control cell line, a thyroid tumour cell line which does not display the oncocytic phenotype. In agreement with these findings, subsequent experiments assessing the levels of cellular ATP over incubation time in galactose medium, showed a drastic and continual decrease in ATP levels only in the XTC-1 cell line. Furthermore, experiments on digitonin-permeabilised cells revealed that the respiratory dysfunction in the latter was due to a defect in complex I of the respiratory chain. Subsequent experiments using cybrids demonstrated that this defect could be attributed to the mitochondrially-encoded subunits of complex I as opposed to the nuclearencoded subunits. Confirmation came with mtDNA sequencing, which detected the presence of a novel mutation in the ND1 subunit of complex I. In addition, a mutation in the cytochrome b subunit of complex III of the respiratory chain was detected. The fact that XTC-1 cells are unable to survive when incubated in galactose medium is consistent with the fact that many cancers are largely dependent on glycolysis for energy production. Indeed, numerous studies have shown that glycolytic inhibitors are able to induce apoptosis in various cancer cell lines. Subsequent experiments were therefore performed in order to identify the mode of XTC-1 cell death when subjected to the metabolic stress imposed by the forced use of the mitochondria for energy production. Cell shrinkage and mitochondrial fragmentation were observed in the dying cells, which would indicate an apoptotic type of cell death. Analysis of additional parameters however revealed a lack of both DNA fragmentation and caspase activation, thus excluding a classical apoptotic type of cell death. Interestingly, cleavage of the actin component of the cytoskeleton was observed, implicating the action of proteases in this mode of cell demise. However, experiments employing protease inhibitors failed to identify the specific protease involved. It has been reported in the literature that overexpression of Bcl-2 is able to rescue cells presenting a respiratory deficiency. As the XTC-1 cell line is not only respiration-deficient but also exhibits a marked decrease in Bcl-2 expression, it is a perfect model with which to study the relationship between Bcl-2 and oxidative phosphorylation in respiratory-deficient cells. Contrary to the reported literature studies on various cell lines harbouring defects in the respiratory chain, Bcl-2 overexpression was not shown to increase cell survival or rescue the energetic dysfunction in XTC-1 cells. Interestingly however, it had a noticeable impact on cell adhesion and morphology. Whereas XTC-1 cells shrank and detached from the growth surface under conditions of metabolic stress, Bcl-2-overexpressing XTC-1 cells appeared much healthier and were up to 45% more adherent. The target of Bcl-2 in this setting appeared to be the actin cytoskeleton, as the cleavage observed in XTC-1 cells expressing only endogenous levels of Bcl-2, was inhibited in Bcl-2-overexpressing cells. Thus, although unable to rescue XTC-1 cells in terms of cell viability, Bcl-2 is somehow able to stabilise the cytoskeleton, resulting in modifications in cell morphology and adhesion. The mitochondrial respiratory deficiency observed in cancer cells is thought not only to cause an increased dependency on glycolysis but it is also thought to blunt cellular responses to anticancer agents. The effects of several therapeutic agents were thus assessed for their death-inducing ability in XTC-1 cells. Cell viability experiments clearly showed that the cells were more resistant to stimuli which generate reactive oxygen species (tert-butylhydroperoxide) and to mitochondrial calcium-mediated apoptotic stimuli (C6-ceramide), as opposed to stimuli inflicting DNA damage (cisplatin) and damage to protein kinases(staurosporine). Various studies in the literature have reported that the peroxisome proliferator-activated receptor-coactivator 1(PGC-1α), which plays a fundamental role in mitochondrial biogenesis, is also involved in protecting cells against apoptosis caused by the former two types of stimuli. In accordance with these observations, real-time PCR experiments showed that XTC-1 cells express higher mRNA levels of this coactivator than do the control cells, implicating its importance in drug resistance. In conclusion, this study has revealed that XTC-1 cells, like many cancer cell lines, are characterised by a reduced energetic efficiency due to mitochondrial dysfunction. Said dysfunction has been attributed to mutations in respiratory genes encoded by the mitochondrial genome. Although the mechanism of cell demise in conditions of metabolic stress is unclear, the potential of targeting thyroid oncocytic cancers using glycolytic inhibitors has been illustrated. In addition, the discovery of mtDNA mutations in XTC-1 cells has enabled the use of this cell line as a model with which to study the relationship between Bcl-2 overexpression and oxidative phosphorylation in cells harbouring mtDNA mutations and also to investigate the significance of such mutations in establishing resistance to apoptotic stimuli.

Pathogenic mechanisms in mitochondrial optic neuropathies

Leber’s hereditary optic neuropathy (LHON) and Autosomal Dominant Optic Atrophy (ADOA) are the two most common inherited optic neuropathies and both are the result of mitochondrial dysfunctions. Despite the primary mutations causing these disorders are different, being an mtDNA mutation in subunits of complex I in LHON and defects in the nuclear gene encoding the mitochondrial protein OPA1 in ADOA, both pathologies share some peculiar features, such a variable penetrance and tissue-specificity of the pathological processes. Probably, one of the most interesting and unclear aspect of LHON is the variable penetrance. This phenomenon is common in LHON families, most of them being homoplasmic mutant. Inter-family variability of penetrance may be caused by nuclear or mitochondrial ‘secondary’ genetic determinants or other predisposing triggering factors. We identified a compensatory mechanism in LHON patients, able to distinguish affected individuals from unaffected mutation carriers. In fact, carrier individuals resulted more efficient than affected subjects in increasing the mitochondrial biogenesis to compensate for the energetic defect. Thus, the activation of the mitochondrial biogenesis may be a crucial factor in modulating penetrance, determining the fate of subjects harbouring LHON mutations. Furthermore, mtDNA content can be used as a molecular biomarker which, for the first time, clearly differentiates LHON affected from LHON carrier individuals, providing a valid mechanism that may be exploited for development of therapeutic strategies. Although the mitochondrial biogenesis gained a relevant role in LHON pathogenesis, we failed to identify a genetic modifying factor for the variable penetrance in a set of candidate genes involved in the regulation of this process. A more systematic high-throughput approach will be necessary to select the genetic variants responsible for the different efficiency in activating mitochondrial biogenesis. A genetic modifying factor was instead identified in the MnSOD gene. The SNP Ala16Val in this gene seems to modulate LHON penetrance, since the Ala allele in this position significantly predisposes to be affected. Thus, we propose that high MnSOD activity in mitochondria of LHON subjects may produce an overload of H2O2 for the antioxidant machinery, leading to release from mitochondria of this radical and promoting a severe cell damage and death ADOA is due to mutation in the OPA1 gene in the large majority of cases. The causative nuclear defects in the remaining families with DOA have not been identified yet, but a small number of families have been mapped to other chromosomal loci (OPA3, OPA4, OPA5, OPA7, OPA8). Recently, a form of DOA and premature cataract (ADOAC) has been associated to pathogenic mutations of the OPA3 gene, encoding a mitochondrial protein. In the last year OPA3 has been investigated by two different groups, but a clear function for this protein and the pathogenic mechanism leading to ADOAC are still unclear. Our study on OPA3 provides new information about the pattern of expression of the two isoforms OPA3V1 and OPA3V2, and, moreover, suggests that OPA3 may have a different function in mitochondria from OPA1, the major site for ADOA mutations. In fact, based on our results, we propose that OPA3 is not involved in the mitochondrial fusion process, but, on the contrary, it may regulate mitochondrial fission. Furthermore, at difference from OPA1, we excluded a role for OPA3 in mtDNA maintenance and we failed to identify a direct interaction between OPA3 and OPA1. Considering the results from overexpression and silencing of OPA3, we can conclude that the overexpression has more drastic consequences on the cells than silencing, suggesting that OPA3 may cause optic atrophy via a gain-of-function mechanism. These data provide a new starting point for future investigations aimed at identifying the exact function of OPA3 and the pathogenic mechanism causing ADOAC.