c-Jun N-terminal kinase (JNK)-mediated modulation of brain mitochondria function: new target proteins for JNK signalling in mitochondrion-dependent apoptosis

The molecular mechanisms underlying the initiation and control of the release of cytochrome c during mitochondrion-dependent apoptosis are thought to involve the phosphorylation of mitochondrial Bcl-2 and Bcl-x(L). Although the c-Jun N-terminal kinase (JNK) has been proposed to mediate the phosphorylation of Bcl-2/Bcl-x(L) the mechanisms linking the modification of these proteins and the release of cytochrome c remain to be elucidated. This study was aimed at establishing interdependency between JNK signalling and mitochondrial apoptosis. Using an experimental model consisting of isolated, bioenergetically competent rat brain mitochondria, these studies show that (i) JNK catalysed the phosphorylation of Bcl-2 and Bcl-x(L) as well as other mitochondrial proteins, as shown by two-dimensional isoelectric focusing/SDS/PAGE; (ii) JNK-induced cytochrome c release, in a process independent of the permeability transition of the inner mitochondrial membrane (imPT) and insensitive to cyclosporin A; (iii) JNK mediated a partial collapse of the mitochondrial inner-membrane potential (Deltapsim) in an imPT- and cyclosporin A-independent manner; and (iv) JNK was unable to induce imPT/swelling and did not act as a co-inducer, but as an inhibitor of Ca-induced imPT. The results are discussed with regard to the functional link between the Deltapsim and factors influencing the permeability transition of the inner and outer mitochondrial membranes. Taken together, JNK-dependent phosphorylation of mitochondrial proteins including, but not limited to, Bcl-2/Bcl-x(L) may represent a potential of the modulation of mitochondrial function during apoptosis.

Exercise Training Restores Cardiac Protein Quality Control in Heart Failure

Exercise training is a well-known coadjuvant in heart failure treatment; however, the molecular mechanisms underlying its beneficial effects remain elusive. Despite the primary cause, heart failure is often preceded by two distinct phenomena: mitochondria dysfunction and cytosolic protein quality control disruption. The objective of the study was to determine the contribution of exercise training in regulating cardiac mitochondria metabolism and cytosolic protein quality control in a post-myocardial infarction-induced heart failure (MI-HF) animal model. Our data demonstrated that isolated cardiac mitochondria from MI-HF rats displayed decreased oxygen consumption, reduced maximum calcium uptake and elevated H2O2 release. These changes were accompanied by exacerbated cardiac oxidative stress and proteasomal insufficiency. Declined proteasomal activity contributes to cardiac protein quality control disruption in our MI-HF model. Using cultured neonatal cardiomyocytes, we showed that either antimycin A or H2O2 resulted in inactivation of proteasomal peptidase activity, accumulation of oxidized proteins and cell death, recapitulating our in vivo model. Of interest, eight weeks of exercise training improved cardiac function, peak oxygen uptake and exercise tolerance in MI-HF rats. Moreover, exercise training restored mitochondrial oxygen consumption, increased Ca2+-induced permeability transition and reduced H2O2 release in MI-HF rats. These changes were followed by reduced oxidative stress and better cardiac protein quality control. Taken together, our findings uncover the potential contribution of mitochondrial dysfunction and cytosolic protein quality control disruption to heart failure and highlight the positive effects of exercise training in re-establishing cardiac mitochondrial physiology and protein quality control, reinforcing the importance of this intervention as a nonpharmacological tool for heart failure therapy.

Effet des acides gras polyinsaturés EPA et DHA dans un modèle d’infarctus du myocarde

Plusieurs études montrent que les acides gras (AG) oméga-3 sont bénéfiques pour la santé cardiovasculaire. Une étude antérieure dans notre laboratoire a montré que l’administration des acides gras oméga-3 réduit la taille de l'infarctus du myocarde (IM). Cependant, la question demeure de savoir si les deux principaux acides gras oméga-3 à longue chaîne, l'acide eicosapentaénoïque (EPA) et l'acide docosahexaénoïque (DHA) possèdent la même efficacité à réduire la taille de l'infarctus. Le but de ce projet sera de déterminer l’efficacité relative de chacun de ces acides gras oméga-3 à protéger le cœur dans un modèle d’ischémie/reperfusion et d’étudier certaines voies de cardioprotection. Des rats mâles adultes Sprague-Dawley ont été nourris pendant 14 jours avec une diète comprenant l'un: 1- aucun AG oméga-3; 2- 5 g d'EPA / kg de nourriture; 3- 5 g de DHA / kg de nourriture; 4- 2,5 g de chaque oméga-3 AG / kg de nourriture; 5- 5 g chaque AG oméga-3 / kg de nourriture. Par la suite, les animaux ont été soumis à une ischémie pendant 40 minutes, causée par l'occlusion de l'artère coronaire gauche descendante. Au bout de 24 heures de reperfusion, la taille de l'infarctus est déterminée. Dans un sous-groupe d'animaux, l'activité d’Akt et des caspase-3 sont mesurées dans la région ischémique après 30 minutes de reperfusion. Finalement, à 15 minutes de reperfusion, l'ouverture du pore de transition de perméabilité mitochondriale mPTP est déterminée dans un autre sous-groupe. Les résultats indiquent que les diètes EPA ou DHA réduisent de manière significative la taille de l'infarctus par rapport à la diète sans AG oméga-3, tandis que la combinaison de deux acides gras oméga-3 n'a pas montré de diminution de la taille de l'infarctus. L'activité de la caspase-3 est réduite pour le groupe DHA puis, l'activité d'Akt est augmentée avec les diètes EPA et DHA seules. Finalement, en présence d’une diète enrichie uniquement de DHA, l'ouverture des mPTP est retardée comparativement aux autres diètes.

Effet de l'hypertrophie cardiaque physiologique et pathologique sur la régulation du pore de perméabilité transitionnelle

Thèse numérisée par la Division de la gestion de documents et des archives de l'Université de Montréal

Implication de la cyclophiline-D et du pore de perméabilité transitionnelle dans la vulnérabilité mitochondriale du coeur hypertrophié

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal

Biological effects of anionic meso-tetrakis (para-sulfonatophenyl) porphyrins modulated by the metal center. Studies in rat liver mitochondria

In this paper, we present a study about the influence of the porphyrin metal center and mesa ligands on the biological effects of meso-tetrakis porphyrins. Different from the cationic meso-tetrakis 4-N-methyl pyridinium (Mn(III)TMPyP), the anionic Mn(III) meso-tetrakis (para-sulfonatophenyl) porphyrin (Mn(III)TPPS4) exhibited no protector effect against Fe(citrate)-induced lipid oxidation. Mn(III)TPPS4 did not protect mitochondria against endogenous hydrogen peroxide and only delayed the swelling caused by tert-BuOOH and Ca(2+). Fe(III)TPPS4 exacerbated the effect of the tert-BuOOH, and both porphyrins did not significantly affect Fe(II)citrate-induced swelling. Consistently, Fe(III)TPPS4 predominantly promotes the homolytic cleavage of peroxides and exhibits catalytic efficiency ten-fold higher than Mn(III)TPPS4. For Mn(III)TPPS4, the microenvironment of rat liver mitochondria favors the heterolytic cleavage of peroxides and increases the catalytic efficiency of the manganese porphyrin due to the availability of axial ligands for the metal center and reducing agents such as glutathione (GSH) and proteins necessary for Compound II (oxomanganese IV) recycling to the initial Mn(III) form. The use of thiol reducing agents for the recycling of Mn(III)TPPS4 leads to GSH depletion and protein oxidation and consequent damages in the organelle. (C) 2009 Elsevier Ireland Ltd. All rights reserved.

The targeting of plasmalemmal ceramide to mitochondria during apoptosis

Ceramide is a key lipid mediator of cellular processes such as differentiation, proliferation, growth arrest and apoptosis. During apoptosis, ceramide is produced within the plasma membrane. Although recent data suggest that the generation of intracellular ceramide increases mitochondrial permeability, the source of mitochondrial ceramide remains unknown. Here, we determine whether a stress-mediated plasmalemmal pool of ceramide might become available to the mitochondria of apoptotic cells. We have previously established annexin A1--a member of a family of Ca(2+) and membrane-binding proteins--to be a marker of ceramide platforms. Using fluorescently tagged annexin A1, we show that, upon its generation within the plasma membrane, ceramide self-associates into platforms that subsequently invaginate and fuse with mitochondria. An accumulation of ceramide within the mitochondria of apoptotic cells was also confirmed using a ceramide-specific antibody. Electron microscopic tomography confirmed that upon the formation of ceramide platforms, the invaginated regions of the plasma membrane extend deep into the cytoplasm forming direct physical contacts with mitochondrial outer membranes. Ceramide might thus be directly transferred from the plasma membrane to the mitochondrial outer membrane. It is conceivable that this "kiss-of-death" increases the permeability of the mitochondrial outer membrane thereby triggering apoptosis.

Identification of the mitochondrial ND3 subunit as a structural component involved in the active/deactive enzyme transition of respiratory complex I

Mitochondrial complex I (NADH: ubiquinone oxidoreductase) undergoes reversible deactivation upon incubation at 30-37 degrees C. The active/deactive transition could play an important role in the regulation of complex I activity. It has been suggested recently that complex I may become modified by S-nitrosation under pathological conditions during hypoxia or when the nitric oxide: oxygen ratio increases. Apparently, a specific cysteine becomes accessible to chemical modification only in the deactive form of the enzyme. By selective fluorescence labeling and proteomic analysis, we have identified this residue as cysteine-39 of the mitochondrially encoded ND3 subunit of bovine heart mitochondria. Cysteine-39 is located in a loop connecting the first and second transmembrane helix of this highly hydrophobic subunit. We propose that this loop connects the ND3 subunit of the membrane arm with the PSST subunit of the peripheral arm of complex I, placing it in a region that is known to be critical for the catalytic mechanism of complex I. In fact, mutations in three positions of the loop were previously reported to cause Leigh syndrome with and without dystonia or progressive mitochondrial disease.

Molecular mechanism and physiological role of active-deactive transition of mitochondrial complex I

The unique feature ofmitochondrial complex I is the so-called A/D transition (active-deactive transition). The A-form catalyses rapid oxidation of NADH by ubiquinone (k ~10 min) and spontaneously converts into the D-form if the enzyme is idle at physiological temperatures. Such deactivation occurs in vitro in the absence of substrates or in vivo during ischaemia, when the ubiquinone pool is reduced. The D-form can undergo reactivation given both NADH and ubiquinone availability during slow (k ~1-10 min) catalytic turnover(s). We examined known conformational differences between the two forms and suggested a mechanism exerting A/D transition of the enzyme. In addition, we discuss the physiological role of maintaining the enzyme in the D-form during the ischaemic period. Accumulation of the D-form of the enzyme would prevent reverse electron transfer from ubiquinol to FMN which could lead to superoxide anion generation. Deactivation would also decrease the initial burst of respiration after oxygen reintroduction. Therefore the A/D transition could be an intrinsic protective mechanism for lessening oxidative damage during the early phase of reoxygenation. Exposure of Cys of mitochondrially encoded subunit ND3 makes the Dform susceptible for modification by reactive oxygen species and nitric oxide metabolites which arrests the reactivation of the D-form and inhibits the enzyme. The nature of thiol modification defines deactivation reversibility, the reactivation timescale, the status of mitochondrial bioenergetics and therefore the degree of recovery of the ischaemic tissues after reoxygenation.

Characterisation of the active/de-active transition of mitochondrial complex I

Oxidation of NADH in the mitochondrial matrix of aerobic cells is catalysed by mitochondrial complex I. The regulation of this mitochondrial enzyme is not completely understood. An interesting characteristic of complex I from some organisms is the ability to adopt two distinct states: the so-called catalytically active (A) and the de-active, dormant state (D). The A-form in situ can undergo de-activation when the activity of the respiratory chain is limited (i.e. in the absence of oxygen). The mechanisms and driving force behind the A/D transition of the enzyme are currently unknown, but several subunits are most likely involved in the conformational rearrangements: the accessory subunit 39 kDa (NDUFA9) and the mitochondrially encoded subunits, ND3 and ND1. These three subunits are located in the region of the quinone binding site. The A/D transition could represent an intrinsic mechanism which provides a fast response of the mitochondrial respiratory chain to oxygen deprivation. The physiological role of the accumulation of the D-form in anoxia is most probably to protect mitochondria from ROS generation due to the rapid burst of respiration following reoxygenation. The de-activation rate varies in different tissues and can be modulated by the temperature, the presence of free fatty acids and divalent cations, the NAD/NADH ratio in the matrix, the presence of nitric oxide and oxygen availability. Cysteine-39 of the ND3 subunit, exposed in the D-form, is susceptible to covalent modification by nitrosothiols, ROS and RNS. The D-form in situ could react with natural effectors in mitochondria or with pharmacological agents. Therefore the modulation of the re-activation rate of complex I could be a way to ameliorate the ischaemia/reperfusion damage. This article is part of a Special Issue entitled: 18th European Bioenergetic Conference. Guest Editors: Manuela Pereira and Miguel Teixeira.

Ischemic A/D transition of mitochondrial complex I and its role in ROS generation

Mitochondrial complex I (NADH:ubiquinone oxidoreductase) is a key enzyme in cellular energy metabolism and provides approximately 40% of the proton-motive force that is utilized during mitochondrial ATP production. The dysregulation of complex I function – either genetically, pharmacologically, or metabolically induced – has severe pathophysiological consequences that often involve an imbalance in the production of reactive oxygen species (ROS). Slow transition of the active (A) enzyme to the deactive, dormant (D) form takes place during ischemia in metabolically active organs such as the heart and brain. The reactivation of complex I occurs upon reoxygenation of ischemic tissue, a process that is usually accompanied by an increase in cellular ROS production. Complex I in the D-form serves as a protective mechanism preventing the oxidative burst upon reperfusion. Conversely, however, the D-form is more vulnerable to oxidative/nitrosative damage. Understanding the so-called active/deactive (A/D) transition may contribute to the development of new therapeutic interventions for conditions like stroke, cardiac infarction, and other ischemia-associated pathologies. In this review, we summarize current knowledge on the mechanism of A/D transition of mitochondrial complex I considering recently available structural data and site-specific labeling experiments. In addition, this review discusses in detail the impact of the A/D transition on ROS production by complex I and the S-nitrosation of a critical cysteine residue of subunit ND3 as a strategy to prevent oxidative damage and tissue damage during ischemia–reperfusion injury.

Water absorption, permeability, and resistance to chloride-ion penetration of lightweight aggregate concrete

This paper presents an experimental study to evaluate effect of cumulative lightweight aggregate (LWA) content (including lightweight sand) in concrete [water/cement ratio (w/c) = 0.38] on its water absorption, water permeability, and resistance to chloride-ion penetration. Rapid chloride penetrability test (ASTM C 1202), rapid migration test (NT Build 492), and salt ponding test (AASHTO T 259) were conducted to evaluate the concrete resistance to chloride-ion penetration. The results were compared with those of a cement paste and a control normal weight aggregate concrete (NWAC) with the same w/c and a NWAC (w/c = 0.54) with 28-day compressive strength similar to some of the lightweight aggregate concrete (LWAC). Results indicate that although the total charge passed, migration coefficient, and diffusion coefficient of the LWAC were not significantly different from those of NWAC with the same w/c of 0.38, resistance of the LWAC to chloride penetration decreased with increase in the cumulative LWA content in the concretes. The water penetration depth under pressure and water sorptivity showed, in general, similar trends. The LWAC with only coarse LWA had similar water sorptivity, water permeability coefficient, and resistance to chloride-ion penetration compared to NWAC with similar w/c. The LWAC had lower water sorptivity, water permeability and higher resistance to chloride-ion penetration than the NWAC with similar 28-day strength but higher w/c. Both the NWAC and LWAC had lower sorptivity and higher resistance to chloride-ion penetration than the cement paste with similar w/c.

Assessment of gene expression of intracellular calcium channels, pumps and exchangers with epidermal growth factor-induced epithelial-mesenchymal transition in a breast cancer cell line

Background Epithelial-mesenchymal transition (EMT) is a process implicated in cancer metastasis that involves the conversion of epithelial cells to a more mesenchymal and invasive cell phenotype. In breast cancer cells EMT is associated with altered store-operated calcium influx and changes in calcium signalling mediated by activation of cell surface purinergic receptors. In this study, we investigated whether MDA-MB-468 breast cancer cells induced to undergo EMT exhibit changes in mRNA levels of calcium channels, pumps and exchangers located on intracellular calcium storing organelles, including the Golgi, mitochondria and endoplasmic reticulum (ER). Methods Epidermal growth factor (EGF) was used to induce EMT in MDA-MB-468 breast cancer cells. Serum-deprived cells were treated with EGF (50 ng/mL) for 12 h and gene expression was assessed using quantitative RT-PCR. Results and conclusions These data reveal no significant alterations in mRNA levels of the Golgi calcium pump secretory pathway calcium ATPases (SPCA1 and SPCA2), or the mitochondrial calcium uniporter (MCU) or Na+/Ca2+ exchanger (NCLX). However, EGF-induced EMT was associated with significant alterations in mRNA levels of specific ER calcium channels and pumps, including (sarco)-endoplasmic reticulum calcium ATPases (SERCAs), and inositol 1,4,5-trisphosphate receptor (IP3R) and ryanodine receptor (RYR) calcium channel isoforms. The most prominent change in gene expression between the epithelial and mesenchymal-like states was RYR2, which was enriched 45-fold in EGF-treated MDA-MB-468 cells. These findings indicate that EGF-induced EMT in breast cancer cells may be associated with major alterations in ER calcium homeostasis.

Arsenic trioxide promotes mitochondrial DNA mutation and cell apoptosis in primary APL cells and NB4 cell line

This study aimed to investigate the effects of arsenic trioxide (As2O3) on the mitochondrial DNA (mtDNA) of acute promyelocytic leukemia (APL) cells. The NB4 cell line was treated with 2.0 μmol/L As2O3in vitro, and the primary APL cells were treated with 2.0 μmol/L As2O3in vitro and 0.16 mg kg-1 d-1 As2O3in vivo. The mitochondrial DNA of all the cells above was amplified by PCR, directly sequenced and analyzed by Sequence Navigatore and Factura software. The apoptosis rates were assayed by flow cytometry. Mitochondrial DNA mutation in the D-loop region was found in NB4 and APL cells before As2O3 use, but the mutation spots were remarkably increased after As2O3 treatment, which was positively correlated to the rates of cellular apoptosis, the correlation coefficient: rNB4-As2O3=0.973818, and rAPL-As2O3=0.934703. The mutation types include transition, transversion, codon insertion or deletion, and the mutation spots in all samples were not constant and regular. It is revealed that As2O3 aggravates mtDNA mutation in the D-loop region of acute promyelocytic leukemia cells both in vitro and in vivo. Mitochondrial DNA might be one of the targets of As2O3 in APL treatment.

Fluidity, Permeability And Antioxidant Behavior Of Model Membranes Incorporated With Alpha-Tocopherol And Vitamin-E Acetate

Magnetic resonance studies reveal a marked difference between the binding of α-tocopherol and that of the corresponding acetate (vitamin E acetate) with dipalmitoylphosphatidylcholine (DPPC) vesicles. This is reflected in differences in the phase-transition curves of the DPPC vesicles incorporated with the two compounds, as well as in the 13C relaxation times and line widths. A model for the incorporation of these molecules in lipid bilayers has been suggested. α-Tocopherol binds strongly with the lipids, possibly through a hydrogen bond formation between the hydroxyl group of the former and one of the oxygen atoms of the latter. The possibility of such a hydrogen bond formation is excluded in vitamin E acetate, which binds loosely through the normal hydrophobic interaction. The model for lipid-vitamin interaction explains the in vitro decomposition of H2O2 by α-tocopherol. α-Tocopherol in conjuction with H2O2 can also act as a free-radical scavenger in the lipid phase. The incorporation of α-tocopherol and vitamin E acetate in DPPC vesicles enhances the permeability of lipid bilayers for small molecules such as sodium ascorbate.