Alteration of mitochondrial supercomplexes assembly in metabolic diseases.

Deregulation of nutrient, hormonal, or neuronal signaling produces metabolic alterations that result in increased mitochondrial reactive oxygen species (ROS) production. The associations of the mitochondrial respiratory chain components into supercomplexes could have pathophysiological relevance in metabolic diseases, as supramolecular arrangements, by sustaining a high electron transport rate, might prevent ROS generation. In this review, the relationship between mitochondrial dysfunction and supercomplex arrangement of the mitochondrial respiratory chain components in obesity, insulin resistance, hepatic steatosis and diabetes mellitus is summarized and discussed.

Sulforaphane protects from myocardial ischemia-reperfusion damage through the balanced activation of Nrf2/AhR.

The activation of the transcription factor Nrf2 and the consequent increment in the antioxidant response might be a powerful strategy to contend against reperfusion damage. In this study we compared the effectiveness between sulforaphane (SFN), a well known activator of Nrf2 and the mechanical maneuver of post-conditioning (PostC) to confer cardioprotection in an in vivo cardiac ischemia-reperfusion model. We also evaluated if additional mechanisms, besides Nrf2 activation contribute to cardioprotection. Our results showed that SFN exerts an enhanced protective response as compared to PostC. Bot, strategies preserved cardiac function, decreased infarct size, oxidative stress and inflammation, through common protective pathways; however, the aryl hydrocarbon receptor (AhR) also participated in the protection conferred by SFN. Our data suggest that SFN-mediated cardioprotection involves transient Nrf2 activation, followed by phase I enzymes upregulation at the end of reperfusion, as a long-term protection mechanism.

Cardioprotective strategies preserve the stability of respiratory chain supercomplexes and reduce oxidative stress in reperfused ischemic hearts.

Electron leakage from dysfunctional respiratory chain and consequent superoxide formation leads to mitochondrial and cell injury during ischemia and reperfusion (IR). In this work we evaluate if the supramolecular assembly of the respiratory complexes into supercomplexes (SCs) is associated with preserved energy efficiency and diminished oxidative stress in post-ischemic hearts treated with the antioxidant N-acetylcysteine (NAC) and the cardioprotective maneuver of Postconditioning (PostC). Hemodynamic variables, infarct size, oxidative stress markers, oxygen consumption and the activity/stability of SCs were compared between groups. We found that mitochondrial oxygen consumption and the activity of respiratory complexes are preserved in mitochondria from reperfused hearts treated with both NAC and PostC. Both treatments contribute to recover the activity of individual complexes. NAC reduced oxidative stress and maintained SCs assemblies containing Complex I, Complex III, Complex IV and the adapter protein SCAFI more effectively than PostC. On the other hand, the activities of CI, CIII and CIV associated to SCs assemblies were preserved by this maneuver, suggesting that the activation of other cardioprotective mechanisms besides oxidative stress contention might participate in maintaining the activity of the mitochondrial respiratory complexes in such superstructures. We conclude that both the monomeric and the SCs assembly of the respiratory chain contribute to the in vivo functionality of the mitochondria. However, although the ROS-induced damage and the consequent increased production of ROS affect the assembly of SCs, other levels of regulation as those induced by PostC, might participate in maintaining the activity of the respiratory complexes in such superstructures.

Nrf2: Molecular and epigenetic regulation during aging.

Increase in life-span is commonly related with age-related diseases and with gradual loss of genomic, proteomic and metabolic integrity. Nrf2 (Nuclear factor-erythroid 2-p45 derived factor 2) controls the expression of genes whose products include antioxidant proteins, detoxifying enzymes, drug transporters and numerous cytoprotective proteins. Several experimental approaches have evaluated the potential regulation of the transcription factor Nrf2 to enhance the expression of genes that contend against accumulative oxidative stress and promote healthy aging. Negative regulators of Nrf2 that act preventing it´s binding to DNA-responsive elements, have been identified in young and adult animal models. However, it is not clearly established if Nrf2 decreased activity in several models of aging results from disruption of that regulation. In this review, we present a compilation of evidences showing that changes in the levels or activity of Keap1 (Kelch-like ECH associated protein 1), GSK-3ß (glycogen synthase kinase-3), Bach1, p53, Hrd1 (E3 ubiquitin ligase) and miRNAs might impact on Nrf2 activity during elderly. We conclude that understanding Nrf2 regulatory mechanisms is essential to develop a rational strategy to prevent the loss of cellular protection response during aging.

3-NP-induced Huntington's-like disease impairs Nrf2 activation without loss of cardiac function in aged rats.

Cardiovascular diseases (CVDs) are one of the leading causes of death in patients over 60years with Huntington's disease (HD). Here, we investigated if age-related oxidative stress (OS) is a relevant factor to develop cardiac damage in an in vivo model of striatal neurodegeneration induced by 3-nitropropionic acid (3-NP). We also evaluated the potential effect of tert-butylhydroquinone (tBHQ) to increase the Nrf2-regulated antioxidant response in hearts from adult and aged rats intoxicated with 3-NP. Our results showed that 3-NP-treatment did not induce cardiac dysfunction, neither in adult nor in aged rats. However, at the cellular level, adult animals showed higher susceptibility to 3-NP-induced damage than aged rats, which suggest that chronic oxidative stress ongoing during aging might have induced an hormetic response that probably prevented from further 3-NP damage. We also found that the oxidative unbalance concurs with unresponsiveness of the Nrf2-mediated antioxidant response in old animals.

Role of sphingomyelinase in mitochondrial ceramide accumulation during reperfusion.

Ceramide accumulation in mitochondria has been associated with reperfusion damage, but the underlying mechanisms are not clearly elucidated. In this work we investigate the role of sphingomyelinases in mitochondrial ceramide accumulation, its effect on reactive oxygen species production, as well as on mitochondrial function by using the sphingomyelinase inhibitor, tricyclodecan-9-yl-xanthogenate (D609). Correlation between neutral sphingomyelinase (nSMase) activity and changes in ceramide content were performed in whole tissue and in isolated mitochondria from reperfused hearts. Overall results demonstrated that D609 treatment attenuates cardiac dysfuncion, mitochondrial injury and oxidative stress. Ceramide was accumulated in mitochondria, but not in the microsomal fraction of the ischemic-reperfused (I/R) group. In close association, the activity of nSMase increased, whereas glutathione (GSH) levels diminished in mitochondria after reperfusion. On the other hand, reduction of ceramide levels in mitochondria from I/R+D609 hearts correlated with diminished nSMase activity, coupling of oxidative phosphorylation and with mitochondrial integrity maintenance. These results suggest that mitochondrial nSMase activity contributes to compartmentation and further accumulation of ceramide in mitochondria, deregulating their function during reperfusion.

Curcumin prevents maleate-induced nephrotoxicity: relation to hemodynamic alterations, oxidative stress, mitochondrial oxygen consumption and activity of respiratory complex I.

The potential protective effect of the dietary antioxidant curcumin (120 mg/Kg/day for 6 days) against the renal injury induced by maleate was evaluated. Tubular proteinuria and oxidative stress were induced by a single injection of maleate (400 mg/kg) in rats. Maleate-induced renal injury included increase in renal vascular resistance and in the urinary excretion of total protein, glucose, sodium, neutrophil gelatinase-associated lipocalin (NGAL) and N-acetyl ß-D-glucosaminidase (NAG), upregulation of kidney injury molecule (KIM)-1, decrease in renal blood flow and claudin-2 expression besides of necrosis and apoptosis of tubular cells on 24 h. Oxidative stress was determined by measuring the oxidation of lipids and proteins and diminution in renal Nrf2 levels. Studies were also conducted in renal epithelial LLC-PK1 cells and in mitochondria isolated from kidneys of all the experimental groups. Maleate induced cell damage and reactive oxygen species (ROS) production in LLC-PK1 cells in culture. In addition, maleate treatment reduced oxygen consumption in ADP-stimulated mitochondria and diminished respiratory control index when using malate/glutamate as substrate. The activities of both complex I and aconitase were also diminished. All the above-described alterations were prevented by curcumin. It is concluded that curcumin is able to attenuate in vivo maleate-induced nephropathy and in vitro cell damage. The in vivo protection was associated to the prevention of oxidative stress and preservation of mitochondrial oxygen consumption and activity of respiratory complex I, and the in vitro protection was associated to the prevention of ROS production.

Cardiac responses to ß-adrenoceptor stimulation is partly dependent on mitochondrial calcium uniporter activity.

BACKGROUND AND PURPOSE: Despite the importance of mitochondrial Ca(2+) to metabolic regulation and cell physiology, little is known about the mechanisms that regulate Ca(2+) entry into the mitochondria. Accordingly, we established a system to determine the role of the mitochondrial Ca(2+) uniporter in an isolated heart model, at baseline and during increased workload following ß-adrenoceptor stimulation. EXPERIMENTAL APPROACH: Cardiac contractility, oxygen consumption and intracellular Ca(2+) transients were measured in ex vivo perfused murine hearts. Ru360 and spermine were used to modify mitochondrial Ca(2+) uniporter activity. Changes in mitochondrial Ca(2+) content and energetic phosphate metabolite levels were determined. KEY RESULTS: The addition of Ru360 , a selective inhibitor of the mitochondrial Ca(2+) uniporter, induced progressively and sustained negative inotropic effects that were dose-dependent with an EC50 of 7 µM. Treatment with spermine, a uniporter agonist, showed a positive inotropic effect that was blocked by Ru360 . Inotropic stimulation with isoprenaline elevated oxygen consumption (2.7-fold), Ca(2+) -dependent activation of pyruvate dehydrogenase (5-fold) and mitochondrial Ca(2+) content (2.5-fold). However, in Ru360 -treated hearts, this parameter was attenuated. In addition, ß-adrenoceptor stimulation in the presence of Ru360 did not affect intracellular Ca(2+) handling, PKA or Ca(2+) /calmodulin-dependent PK signalling. CONCLUSIONS AND IMPLICATIONS: Inhibition of the mitochondrial Ca(2+) uniporter decreases ß-adrenoceptor response, uncoupling between workload and production of energetic metabolites. Our results support the hypothesis that the coupling of workload and energy supply is partly dependent on mitochondrial Ca(2+) uniporter activity.

Pharmacological strategies to contend against myocardial reperfusion damage: diverse chemicals for multiple targets.

Acute myocardial infarction is a frequent and disabling disease. Paradoxically, reperfusion, the most effective treatment to reduce infarct size, can both protect and kill. Although reperfusion protects by preventing lesions occurring during prolonged ischemia, it causes damage because reflow is associated with an unbalance between oxygen availability and metabolic demand, altered ionic homeostasis, and reactive oxygen species (ROS) generation. Recently, more players in myocardial reperfusion injury have been described: protein kinase C (PKC) and members of the MAP kinase, which activate downstream cascades that may activate intricate processes compromising cardiac recovery after ischemia. All together, such mechanisms promote endothelial and vascular dysfunction, sequels of impaired blood flow, metabolic and contractile dysfunction, dysrhythmia, cellular necrosis and apoptosis. Different pharmacological agents, as well as mechanical strategies, have been used to challenge the outcome of the complex interactions among these mechanisms and with others. In this review, we focused on the potential of different compounds used in animal models and in the clinical practice to improve the prognosis after post-ischemic reperfusion. We also review mechanisms activated during reperfusion injury and the structure-activity relationship between some of the cardioprotective chemicals and their cellular targets.

Effects of alpha-mangostin on mitochondrial energetic metabolism.

Although alpha-mangostin prevents from toxicity associated to oxidative stress, it also promotes apoptotic cell death in cancer cells. Such effects have been associated with mitochondrial membrane depolarization and cytochrome c release. Therefore, the aim of this work was to analyze the potentially harmful effect of this natural compound on relevant parameters of mitochondrial function from normal tissue. Our results showed that alpha-mangostin protected mitochondria from peroxidative damage, but at high concentration, it acted as an uncoupler, reduced dramatically ADP-stimulated respiration and inhibited the activity of respiratory complex IV, making mitochondria prone to permeability transition, which is a mitochondrial player on cell fate.

Changes in specific lipids regulate BAX-induced mitochondrial permeability transition.

Recent evidence suggests the existence of lipid microdomains in mitochondria, apparently coexisting as structural elements with some of the mitochondrial permeability transition pore-forming proteins and members of the Bcl-2 family. The aim of this study was to investigate the relevance of the main components of membrane microdomains (e.g. cholesterol and sphingolipids) in activation of the mitochondrial permeability transition pore (mPTP) by recombinant BAX (rBAX). For this purpose, we used chemically modified renal cortex mitochondria and renal cortex mitochondria from hypothyroid rats that show a modified mitochondrial lipid composition in vivo. Oligomeric rBAX induced an enhanced permeability conformation in the mPTP of control mitochondria. rBAX failed to induce mPTP opening when the cholesterol and ganglioside content of mitochondria were modified with the chelator methyl-beta-cyclodextrin. Accordingly, hypothyroid mitochondria, with endogenously lower cholesterol and ganglioside content, showed resistance to mPTP opening induced by rBAX. These observations suggest that enriched cholesterol and ganglioside domains in the mitochondrial membranes may determine BAX interaction with the mPTP. An intriguing observation was that chemical extraction of cholesterol and ganglioside in control mitochondria did not have an effect on rBAX insertion. Conversely, in hypothyroid mitochondria, rBAX insertion was diminished dramatically compared with control mitochondria. The membrane and protein changes associated with thyroid status and their possible role in rBAX docking into the membranes are discussed.

Ru360, a specific mitochondrial calcium uptake inhibitor, improves cardiac post-ischaemic functional recovery in rats in vivo.

BACKGROUND AND PURPOSE: The mitochondrial permeability transition pore (mPTP), an energy-dissipating channel activated by calcium, contributes to reperfusion damage by depolarizing the mitochondrial inner membrane potential. As mitochondrial Ca(2+) overload is a main inductor of mPTP opening, we examined the effect of Ru(360), a selective inhibitor of the mitochondrial calcium uptake system against myocardial damage induced by reperfusion in a rat model. EXPERIMENTAL APPROACH: Myocardial reperfusion injury was induced by a 5-min occlusion of the left anterior descending coronary artery, followed by a 5-min reperfusion in anaesthetized open-chest rats. We measured reperfusion-induced arrhythmias and functions indicative of unimpaired mitochondrial integrity to evaluate the effect of Ru(360) treatment. KEY RESULTS: Reperfusion elicited a high incidence of arrhythmias, haemodynamic dysfunction and loss of mitochondrial integrity. A bolus intravenous injection of Ru(360) (15-50 nmol kg(-1)), given 30-min before ischaemia, significantly improved the above mentioned variables in the ischaemic/reperfused myocardium. Calcium uptake in isolated mitochondria from Ru(360)-treated ventricles was partially diminished, suggesting an interaction of this compound with the calcium uniporter. CONCLUSIONS AND IMPLICATIONS: We showed that Ru(360) treatment abolishes the incidence of arrhythmias and haemodynamic dysfunction elicited by reperfusion in a whole rat model. Ru(360) administration partially inhibits calcium uptake, preventing mitochondria from depolarization by the opening of the mPTP. We conclude that myocardial damage could be a consequence of failure of the mitochondrial network to maintain the membrane potential at reperfusion. Hence, it is plausible that Ru(360) could be used in reperfusion therapy to prevent the occurrence of arrhythmia.

Different subunit location of the inhibition and transport sites in the mitochondrial calcium uniporter.

The mitochondrial calcium uniporter behaves as a cooperative mechanism, where the velocity is dependent on [Ca2+]ex. Transport kinetics follows a sigmoidal behavior with a Hill coefficient near 2.0, indicating the binding of at least two calcium molecules. Calcium transport in mitochondria is dependent on a negative inner membrane potential and is inhibited by policationic ruthenium compounds. In this study, calcium uptake activity was reconstituted into cytochrome oxidase vesicles by incorporating solubilized mitochondrial proteins. Calcium accumulation plotted against increasing Ca2+ concentrations followed a sigmoidal behavior with a Hill coefficient of 1.53. The uptake was sensitive to ruthenium policationic inhibitors, e.g. ruthenium red and Ru360. After mitochondrial proteins were separated by preparative isoelectrofocusing and incorporated into cytochrome oxidase vesicles, two peaks of calcium uptake activity were recovered. One of the activities was inhibited by Ru360, while the second activity was insensitive to Ru360 and was associated with proteins focused at very acidic isoelectric points. By using a thiol-group crosslinker and radiolabeled Ru360, we proposed a scheme of partial dissociation of the uniporter inhibitor-binding subunit under acidic conditions.

Cardiolipin regulates the activity of the reconstituted mitochondrial calcium uniporter by modifying the structure of the liposome bilayer.

Reconstitution of mitochondrial calcium transport activity requires the incorporation of membrane proteins into a lipidic ambient. Calcium uptake has been measured previously using Cytochrome oxidase vesicles. The enrichment of these vesicles with cardiolipin, an acidic phospholipid that is found only in the inner mitochondrial membrane of eukaryotic cells, strongly inhibits calcium transport, in remarkable contrast with the activation effect that cardiolipin exerts upon other mitochondrial transporters and enzymes. The relation of the inactivation of calcium transport to the physical state of the bilayer was studied by following the polarization changes of 1,6-diphenyl-1,3,5-hexatriene (DPH) and by flow cytometry in the cardiolipin-enriched liposomes with incorporated mitochondrial solubilized proteins. Non-bilayer molecular arrangements in the cardiolipin-supplemented liposomes, detected by flow cytometry, may produce the fluidity changes observed by fluorescence polarization of DPH. Fluidity changes correlate with the abolition of calcium uptake, but have no effect on the establishment of a membrane potential in the vesicles required for calcium transport activity. Changes in the membrane structure and uniporter function are observed in the combined presence of cardiolipin and calcium leading to a modified lipid configuration.

Possible involvement of the adenine nucleotide translocase in the activation of the permeability transition pore induced by cadmium.

Low levels of cadmium induce a rapid calcium efflux in energized rat kidney mitochondria. This is accompanied by the collapse of the transmembrane gradient in a partial CSA-sensitive fashion. The binding of 109Cd2+ to mitochondria is a saturable function; in the presence of NEM, the binding of 2.5 nmol 109Cd2+/mg of protein suffices to induce the opening of the permeability transition pore. It was found that cadmium bound mainly to proteins of molecular weight between 30 and 50 kDa. In the presence of the monothiol reagent NEM, the label is concentrated in the 30 kDa protein. Following the addition of the reducing agent dithiothreitol, calcium is reaccumulated and the membrane potential restored. This correlates with a significant loss of label in the 30 kDa protein region. The 30 kDa protein was identified as the adenine nucleotide translocase by labelling experiments with eosin 5-maleimide and experiments of reconstitution.

Copper sensitizes the mitochondrial permeability transition to carboxytractyloside and oleate.

Addition of 5 microM copper to rat kidney mitochondria enhances the effect of carboxyatractyloside and oleate on pore opening, in a cyclosporin A-sensitive fashion. The effects of the pair copper-carboxyatractyloside were observed on matrix Ca2+ efflux, mitochondrial swelling and on the transmembrane electric gradient. The effect of Cu2+ emphasizes the importance of membrane thiol groups located, probably, in the ADP/ATP translocase (ANT), on permeability transition. It was also found that Cu2+ does not block the fluorescent label of ANT by eosin 5-maleimide, but abolishes the inhibition by CAT on the labeling. This suggests that the binding of Cu2+ to cysteine residues of ANT promotes a conformational change in the carrier, strengthening the effect of CAT and oleate on membrane leakage.

Inactivation of mitochondrial permeability transition pore by octylguanidine and octylamine.

Mitochondrial permeability transition occurs through a Ca2+-dependent opening of a transmembrane pore, whose identity has been attributed to that of the adenine nucleotide translocase (ANT). In this work, we induced permeability transition by adding 0.5 microM carboxyatractyloside. The process was evaluated analyzing Ca2+ efflux, a drop in transmembrane electric gradient, and swelling. We found that the amphiphyllic cations octylguanidine and octylamine, at the concentration of 100 microM, inhibited, almost completely, nonspecific membrane permeability. Hexylguanidine, hexylamine, as well as guanidine chloride and hydroxylamine failed to do so. The inhibition was reversed after the addition of 40 mM Li+, Na+ K+, Rb+, or Cs+; K+ was the most effective. We propose that the positive charge of the amines interact with negative charges of membrane proteins, more likely the ADP/ATP carrier, while the alkyl chain penetrates into the hydrophobic milieu of the inner membrane, fixing the reagent.

Carboxyatractyloside increases the effect of oleate on mitochondrial permeability transition.

Addition of a low concentration of carboxyatractyloside (0.075 microM) renders mitochondria susceptible to the opening of the non-specific pore by 5 microM oleate, in a cyclosporin A-sensitive fashion. Matrix Ca2+ efflux as well as collapse of the transmembrane potential reveal permeability transition. The effect of oleate is reached after the titration, by carboxyatractyloside, of 38 pmol of adenine nucleotide translocase per mg mitochondrial protein. We propose that permeability transition may result from an additive action of carboxyatractyloside plus oleate on the ADP/ATP carrier.

Inhibitory properties of ruthenium amine complexes on mitochondrial calcium uptake.

The recent finding that the inhibition of Ca2+-stimulated respiration by ruthenium red is mainly due to a binuclear ruthenium complex (Ru360) present in the commercial samples of the classical inhibitor ruthenium red (Ying et. al., 1991), showed that this complex is the more potent and specific inhibitor of the mitochondrial calcium uniporter. This work was aimed to provide insights into the mechanism by which Ru360 and other ruthenium-related compounds inhibits calcium uptake. Ruthenium red and a synthesized analog (Rrphen) were compared with Ru360. The inhibition by this binuclear complex was noncompetitive, with a Ki of 9.89 nM. The number of specific binding sites for Ru360 was 6.2 pmol/mg protein. Ruthenium red and Ru360 were mutually exclusive inhibitors. Bound La3+ was not displaced by Ru360. Rrphen was the least effective for inhibiting calcium uptake. The results support the notion of a specific binding site in the uniporter for the polycationic complexes and a negative charged region from the phospholipids in the membrane, closely associated with the uniporter inhibitor-binding site.

Oxygen free-radicals mediate the damaging effect of ultraviolet light on membrane mitochondria.

This paper reports evidence that exposure of mitochondria to near-ultraviolet light inhibits electron transport, collapses the electric gradient, and increases non-specific membrane permeability to matrix solutes such as Ca2+. Membrane energization, as well as superoxide dismutase and catalase avoid membrane leakiness. Increased permeability correlates with a diminution in the titrated thiol groups. Plausibly the pore is formed through the formation of sulfhydryl bridges by the action of UV light-derived oxygen-centered free- radicals on membrane proteins.