Interaction of NDPK-D with cardiolipin-containing membranes: Structural basis and implications for mitochondrial physiology

Nucleoside diphosphate kinases (NDPKs/Nm23), responsible for intracellular di- and tri-phosphonucleoside homeostasis, play multi-faceted roles in cellular energetic, signaling, proliferation, differentiation and tumor invasion. The mitochondrial NDPK-D, the NME4 gene product, is a peripheral protein of the inner membrane. Several new aspects of the interaction of NDPK-D with the inner mitochondrial membrane have been recently characterized. Surface plasmon resonance analysis using recombinant NDPK-D and different phospholipid liposomes showed that NDPK-D interacts electrostatically with anionic phospholipids, with highest affinity observed for cardiolipin, a phospholipid located mostly in the mitochondrial inner membrane. Mutation of the central arginine (R90) in a surface exposed cationic RRK motif unique to NDPK-D strongly reduced phospholipid interaction in vitro and in vivo. Stable expression of NDPK-D proteins in HeLa cells naturally almost devoid of this isoform revealed a tight functional coupling of NDPK-D with oxidative phosphorylation that depends on the membrane-bound state of the enzyme. Owing to its symmetrical hexameric structure exposing membrane binding motifs on two opposite sides, NDPK-D could bridge liposomes containing anionic phospholipids and promote lipid transfer between them. In vivo, NDPK-D could induce intermembrane contacts and facilitate lipid movements between mitochondrial membranes. Most of these properties are reminiscent to those of the mitochondrial creatine kinase. We review here the common properties of both kinases and we discuss their potential roles in mitochondrial functions such as energy production, apoptosis and mitochondrial dynamics.     

New aspects of impaired mitochondrial function in heart failure

This minireview focuses on the impairment of function in cardiac mitochondria in heart failure (HF). It is generally accepted that chronic energy starvation leads to cardiac mechanical dysfunction in HF. Mitochondria are the primary ATP generator for the heart. Current evidence suggests that the assembly of the electron transport chain (ETC) into respirasomes provides structural support for mitochondrial oxidative phosphorylation by facilitating electron channeling and perhaps by preventing electron leak and superoxide production. Defects have been purported to occur in the individual ETC complexes or components of the phosphorylation apparatus in HF, but these defects have not been linked to impaired mitochondrial function. Moreover, studies that reported decreased mitochondrial oxidative phosphorylation in HF did not identify the site of the defect. We propose a sequential mechanistic pathway in which the decrease in functional respirasomes in HF is the primary event causing decreased oxidative phosphorylation and increased reactive oxygen species production, leading to a progressive decrease in cardiac performance.     

Inhibition of cytosolic and mitochondrial creatine kinase by siRNA in HaCaT- and HeLaS3-cells affects cell viability and mitochondrial morphology

The creatine kinase (CK) system is essential for cellular energetics in tissues or cells with high and fluctuating energy requirements. Creatine itself is known to protect cells from stress-induced injury. By using an siRNA approach to silence the CK isoenzymes in human keratinocyte HaCaT cells, expressing low levels of cytoplasmic CK and high levels of mitochondrial CK, as well as HeLa cancer cells, expressing high levels of cytoplasmic CK and low levels of mitochondrial CK, we successfully lowered the respective CK expression levels and studied the effects of either abolishing cytosolic brain-type BB-CK or ubiquitous mitochondrial uMi-CK in these cells. In both cell lines, targeting the dominant CK isoform by the respective siRNAs had the strongest effect on overall CK activity. However, irrespective of the expression level in both cell lines, inhibition of the mitochondrial CK isoform generally caused the strongest decline in cell viability and cell proliferation. These findings are congruent with electron microscopic data showing substantial alteration of mitochondrial morphology as well as mitochondrial membrane topology after targeting uMi-CK in both cell lines. Only for the rate of apoptosis, it was the least expressed CK present in each of the cell lines whose inhibition led to the highest proportion of apoptotic cells, i.e., downregulation of uMi-CK in case of HeLaS3 and BB-CK in case of HaCaT cells. We conclude from these data that a major phenotype is linked to reduction of mitochondrial CK alone or in combination with cytosolic CK, and that this effect is independent of the relative expression levels of Mi-CK in the cell type considered. The mitochondrial CK isoform appears to play the most crucial role in maintaining cell viability by stabilizing contact sites between inner and outer mitochondrial membranes and maintaining local metabolite channeling, thus avoiding transition pore opening which eventually results in activation of caspase cell-death pathways.     

Regulation of respiration controlled by mitochondrial creatine kinase in permeabilized cardiac cells in situ: Importance of system level properties

The main focus of this investigation is steady state kinetics of regulation of mitochondrial respiration in permeabilized cardiomyocytes in situ. Complete kinetic analysis of the regulation of respiration by mitochondrial creatine kinase was performed in the presence of pyruvate kinase and phosphoenolpyruvate to simulate interaction of mitochondria with glycolytic enzymes. Such a system analysis revealed striking differences in kinetic behaviour of the MtCK-activated mitochondrial respiration in situ and in vitro. Apparent dissociation constants of MgATP from its binary and ternary complexes with MtCK, K_ia and K_a (1.94 ± 0.86 mM and 2.04 ± 0.14 mM, correspondingly) were increased by several orders of magnitude in situ in comparison with same constants in vitro (0.44 ± 0.08 mM and 0.016 ± 0.01 mM, respectively). Apparent dissociation constants of creatine, K_ib and K_b (2.12 ± 0.21 mM 2.17 ± 0.40 Mm, correspondingly) were significantly decreased in situ in comparison with in vitro mitochondria (28 ± 7 mM and 5 ± 1.2 mM, respectively). Dissociation constant for phosphocreatine was not changed. These data may indicate selective restriction of metabolites' diffusion at the level of mitochondrial outer membrane. It is concluded that mechanisms of the regulation of respiration and energy fluxes in vivo are system level properties which depend on intracellular interactions of mitochondria with cytoskeleton, intracellular MgATPases and cytoplasmic glycolytic system.     

Structural and functional changes in heart mitochondria from sucrose-fed hypertriglyceridemic rats

In the heart of sugar-induced hypertriglyceridemic (HTG) rats, cardiac performance is impaired with glucose as fuel, but not with fatty acids. Accordingly, the glycolytic flux and the transfer of energy diminish in the HTG heart, in comparison to control heart. To further explore the biochemical nature of such alteration in the HTG heart, the components of the non-glycolytic energy systems involved were evaluated. Total creatine kinase (CK) activity in the myocardial tissue was depressed by 30% in the HTG heart whereas the activity of the mitochondrial CK (mitCK) isoenzyme fraction that is functionally associated with oxidative phosphorylation decreased in isolated HTG heart mitochondria by 45%. Adenylate kinase (AK) was 20% lower in the HTG heart. In contrast, respiratory rates with 2-oxoglutarate (2-OG) and pyruvate/malate (pyr) were significantly higher in HTG heart mitochondria than in control mitochondria. 2-OG dehydrogenase activity was also higher in HTG mitochondria. Respiration with succinate was similar in both groups. Content of cytochromes b, c + c₁ and a + a₃, and cytochrome c oxidase activity, were also similar in the two kinds of mitochondria. A larger content of saturated and monounsaturated fatty acids was found in the HTG mitochondrial membranes with no changes in phospholipids composition or cholesterol content. Mitochondrial membranes from HTG hearts were more rigid, which correlated with the generation of higher membrane potentials. As the mitochondrial function was preserved or even enhanced in the HTG heart, these results indicated that deficiency in energy transfer was associated with impairment in mitCK and AK. This situation brought about uncoupling between the site of ATP production and the site of ATP consumption (contractile machinery), in spite of compensatory increase in mitochondrial oxidative capacity and membrane potential generation.     

Control of mitochondrial volume by mitochondrial metabolic water

It is well-known that mitochondrial volume largely controls mitochondrial functioning. We investigate whether metabolic water produced by oxidative phosphorylation could be involved in mitochondrial volume regulation. We modulated the generation of this water in liver mitochondria and assess their volume by two independent techniques.In liver mitochondria, the mitochondrial volume was specifically decreased when no water was produced independently of energetic parameters and uncoupling activity. In all other conditions associated with water generation, there was no significant change in mitochondrial metabolic volume.Altogether these data demonstrate that mitochondrial volume is regulated, independently of energetic status, by the mitochondrial metabolic water that acts as a signal.     

Fatty liver and insulin resistance in obese Zucker rats: no role for mitochondrial dysfunction

The relationship between insulin resistance and mitochondrial function is of increasing interest. Studies looking for such interactions are usually made in muscle and only a few studies have been done in liver, which is known to be a crucial partner in whole body insulin action. Recent studies have revealed a similar mechanism to that of muscle for fat-induced insulin resistance in liver. However, the exact mechanism of lipid metabolites accumulation in liver leading to insulin resistance is far from being elucidated. One of the hypothetical mechanisms for liver steatosis development is an impairment of mitochondrial function. We examined mitochondrial function in fatty liver and insulin resistance state using isolated mitochondria from obese Zucker rats. We determined the relationship between ATP synthesis and oxygen consumption as well as the relationship between mitochondrial membrane potential and oxygen consumption. In order to evaluate the quantity of mitochondria and the oxidative capacity we measured citrate synthase and cytochrome c oxidase activities. Results showed that despite significant fatty liver and hyperinsulinemia, isolated liver mitochondria from obese Zucker rats display no difference in oxygen consumption, ATP synthesis, and membrane potential compared with lean Zucker rats. There was no difference in citrate synthase and cytochrome c oxidase activities between obese and lean Zucker rats in isolated mitochondria as well as in liver homogenate, indicating a similar relative amount of hepatic mitochondria and a similar oxidative capacity. Adiponectin, which is involved in bioenergetic homeostasis, was increased two-fold in obese Zucker rats despite insulin resistance. In conclusion, isolated liver mitochondria from lean and obese insulin-resistant Zucker rats showed strictly the same mitochondrial function. It remains to be elucidated whether adiponectin increase is involved in these results.     

VDAC regulation: role of cytosolic proteins and mitochondrial lipids

It was recently asserted that the voltage-dependent anion channel (VDAC) serves as a global regulator, or governor, of mitochondrial function (Lemasters and Holmuhamedov, Biochim Biophys Acta 1762:181–190, 2006). Indeed, VDAC, positioned on the interface between mitochondria and the cytosol (Colombini, Mol Cell Biochem 256:107–115, 2004), is at the control point of mitochondria life and death. This large channel plays the role of a “switch” that defines in which direction mitochondria will go: to normal respiration or to suppression of mitochondria metabolism that leads to apoptosis and cell death. As the most abundant protein in the mitochondrial outer membrane (MOM), VDAC is known to be responsible for ATP/ADP exchange and for the fluxes of other metabolites across MOM. It controls them by switching between the open and “closed” states that are virtually impermeable to ATP and ADP. This control has dual importance: in maintaining normal mitochondria respiration and in triggering apoptosis when cytochrome c and other apoptogenic factors are released from the intermembrane space into the cytosol. Emerging evidence indicates that VDAC closure promotes apoptotic signals without direct involvement of VDAC in the permeability transition pore or hypothetical Bax-containing cytochrome c permeable pores. VDAC gating has been studied extensively for the last 30 years on reconstituted VDAC channels. In this review we focus exclusively on physiologically relevant regulators of VDAC gating such as endogenous cytosolic proteins and mitochondrial lipids. Closure of VDAC induced by such dissimilar cytosolic proteins as pro-apoptotic tBid and dimeric tubulin is compared to show that the involved mechanisms are rather distinct. While tBid mostly modulates VDAC voltage gating, tubulin blocks the channel with the efficiency of blockage controlled by voltage. We also discuss how characteristic mitochondrial lipids, phospatidylethanolamine and cardiolipin, could regulate VDAC gating. Overall, we demonstrate that VDAC gating is not just an observation made under artificial conditions of channel reconstitution but is a major mechanism of MOM permeability control.     

Dynamic changes in mitochondrial biogenesis and antioxidant enzymes during the spontaneous differentiation of human embryonic stem cells

Embryonic cells before implantation are exposed to a hypoxic condition and dependent on anaerobic metabolism. Human embryonic stem cells (HESCs) derived from pre-implantation blastocyst also grow well in hypoxic conditions. Expecting that the differentiating HESCs might mimic anaerobic-to-aerobic metabolic transition of the early human life, we examined the mitochondria-related changes in these cells. We observed that mitochondrial mass and mitochondrial DNA content were increased with differentiation, which was accompanied by the increase of the amount of ATP (4-fold) and its by-product reactive oxygen species (2.5-fold). The expression of various antioxidant enzymes including mitochondrial and cytoplasmic superoxide dismutases, catalase, and peroxiredoxins showed a dramatic change during the early differentiation. In conclusion, HESC differentiation was followed by dynamic changes in mitochondrial mass, ATP and ROS production, and antioxidant enzyme expressions. Therefore, the HESCs would serve as a good model to examine the mitochondrial biology during the early human differentiation.     

Protective effect of ursodeoxycholic acid on liver mitochondrial function in rats with alloxan-induced diabetes: link with oxidative stress

We investigated the effects of ursodeoxycholic acid (UDCA) on mitochondrial functions and oxidative stress and evaluated their relationships in the livers of rats with alloxan-induced diabetes. Diabetes was induced in male Wistar rats by a single alloxan injection (150 mg kg^− 1 b.w., i.p.). UDCA (40 mg kg^− 1 b.w., i.g., 30 days) was administered from the 5th day after the alloxan treatment. Mitochondrial functions were evaluated by oxygen consumption with Clark oxygen electrode using succinate, pyruvate + malate or palmitoyl carnitine as substrates and by determination of succinate dehydrogenase and NADH dehydrogenase activities. Liver mitochondria were used to measure chemiluminiscence enhanced by luminol and lucigenin, reduced liver glutathione and the end-products of lipid peroxidation. The activities of both NADH dehydrogenase and succinate dehydrogenase as well as the respiratory control (RC) value with all the substrates and the ADP/O ratio with pyruvate + malate and succinate as substrates were significantly decreased in diabetic rats. UDCA developed the beneficial effect on the mitochondrial respiration and oxidative phosphorylation parameters in alloxan-treated rats, whereas the activities of mitochondrial enzymes were increased insignificantly after the administration of UDCA. The contents of polar carbonyls and MDA as well as the chemiluminescence with luminol were elevated in liver mitochondria of diabetic rats. The treatment with UDCA normalized all the above parameters measured except the MDA content. UDCA administration prevents mitochondrial dysfunction in rats treated with alloxan and this process is closely connected with inhibition of oxidative stress by this compound.     

Direct measurement of energy fluxes from mitochondria into cytoplasm in permeabilized cardiac cells <Emphasis Type="Italic">in situ:</Emphasis> some evidence for mitochondrial interactosome

The aim of this study was to measure energy fluxes from mitochondria in isolated permeabilized cardiomyocytes. Respiration of permeabilized cardiomyocytes and mitochondrial membrane potential were measured in presence of MgATP, pyruvate kinase – phosphoenolpyruvate and creatine. ATP and phosphocreatine concentrations in medium surrounding cardiomyocytes were determined. While ATP concentration did not change in time, mitochondria effectively produced phosphocreatine (PCr) with PCr/O₂ ratio equal to 5.68 ± 0.14. Addition of heterodimeric tubulin to isolated mitochondria was found to increase apparent Km for exogenous ADP from 11 ± 2 μM to 330 ± 47 μM, but creatine again decreased it to 23 ± 6 μM. These results show directly that under physiological conditions the major energy carrier from mitochondria into cytoplasm is PCr, produced by mitochondrial creatine kinase (MtCK), which functional coupling to adenine nucleotide translocase is enhanced by selective limitation of permeability of mitochondrial outer membrane within supercomplex ATP Synthasome-MtCK-VDAC-tubulin, Mitochondrial Interactosome.     

Regulation of mitochondrial energy production in cardiac cells of rainbow trout (<Emphasis Type="Italic">Oncorhynchus mykiss</Emphasis>)

In skinned rat cardiac fibres, mitochondrial affinity for endogenous ADP generated by creatine kinase and Ca²⁺-activated ATPases is higher than for exogenous ADP added to the surrounding medium, suggesting that mitochondria are functionally coupled to creatine kinase and ATPases. Such a coupling may be weaker or absent in ectothermic vertebrate cardiac cells, because they typically have less elaborate intracellular membrane structures, higher glycolytic capacity and lower working temperature. Therefore, we examined skinned cardiac fibres from rainbow trout at 10 °C. The apparent mitochondrial affinity for endogenous ADP was obtained by stimulation with ATP and recording of the release of ADP into the surrounding medium. The apparent affinity for endogenous ADP was much higher than for exogenous ADP suggesting a functional coupling between mitochondria and ATPases. The apparent affinity for exogenous ADP and ATP was increased by creatine or an increase in Ca²⁺-activity, which should increase intrafibrillar turnover of ATP to ADP. In conclusion, ADP seems to be channelled from creatine kinase and ATPases to mitochondria without being released to the surrounding medium. Thus, despite difference in structure, temperature and metabolic capacity, trout myocardium resembles that of rat with regard to the regulation of mitochondrial respiration.Abbreviations ACR acceptor control ratio - ANT adenine nucleotide translocase - K_{M ADP} apparent mitochondrial affinity for ADP - K_{M ATP} apparent mitochondrial affinity for ATP - LDH lactate dehydrogenase - V_ADP ADP-stimulated respiration rate - V_{ADP max} maximal ADP-stimulated respiration rate - V_ATP ATP-stimulated respiration rate - V_{ATP max} maximal ATP-stimulated respiration rate - V₀ basal respiration rate in the absence of ADPCommunicated by G. Heldmaier     

Closure of VDAC causes oxidative stress and accelerates the Ca-induced mitochondrial permeability transition in rat liver mitochondria

The electron transport chain of mitochondria is a major source of reactive oxygen species (ROS), which play a critical role in augmenting the Ca²⁺-induced mitochondrial permeability transition (MPT). Mitochondrial release of superoxide anions (O₂⁻) from the intermembrane space (IMS) to the cytosol is mediated by voltage dependent anion channels (VDAC) in the outer membrane. Here, we examined whether closure of VDAC increases intramitochondrial oxidative stress by blocking efflux of O₂⁻ from the IMS and sensitizing to the Ca²⁺-induced MPT. Treatment of isolated rat liver mitochondria with 5 μM G3139, an 18-mer phosphorothioate blocker of VDAC, accelerated onset of the MPT by 6.8 ± 1.4 min within a range of 100-250 μM Ca²⁺. G3139-mediated acceleration of the MPT was reversed by 20 μM butylated hydroxytoluene, a water soluble antioxidant. Pre-treatment of mitochondria with G3139 also increased accumulation of O₂⁻ in mitochondria, as monitored by dihydroethidium fluorescence, and permeabilization of the mitochondrial outer membrane with digitonin reversed the effect of G3139 on O₂⁻ accumulation. Mathematical modeling of generation and turnover of O₂⁻ within the IMS indicated that closure of VDAC produces a 1.55-fold increase in the steady-state level of mitochondrial O₂⁻. In conclusion, closure of VDAC appears to impede the efflux of superoxide anions from the IMS, resulting in an increased steady-state level of O₂⁻, which causes an internal oxidative stress and sensitizes mitochondria toward the Ca²⁺-induced MPT.     

The voltage dependent anion channel affects mitochondrial cholesterol distribution and function

We have observed abnormally high membrane cholesterol levels and a subsequent deficiency of oxidative energy production in mitochondria from cultured Morris hepatoma cells (MH7777). Using cholesterol affinity chromatography and MALDI-TOF Mass Spectrometry, we have identified the voltage dependent anion channel (VDAC) as a necessary component of a protein complex involved in mitochondrial membrane cholesterol distribution. VDAC is known to associate strongly with hexokinase, particularly in glycolytic cancers. By constructing an E72Q mutant form of VDAC that inhibits its binding of hexokinase, we report an increase in oxidative phosphorylation activity of MH7777 cells, as well as reduced membrane cholesterol ratios to levels near that of normal liver mitochondria. This paper demonstrates that the ability of VDAC to influence mitochondrial membrane cholesterol distribution may have implications on mitochondrial characteristics such as oxidative phosphorylation and induction of apoptosis, as well as the propensity of cancer cells to exhibit a glycolytic phenotype.     

Peroxiredoxin 3 has a crucial role in the contractile function of skeletal muscle by regulating mitochondrial homeostasis

Antioxidant systems against reactive oxygen species (ROS) are important factors in regulating homeostasis in various cells, tissues, and organs. Although ROS are known to cause to muscular disorders, the effects of mitochondrial ROS in muscle physiology have not been fully understood. Here, we investigated the effects of ROS on muscle mass and function using mice deficient in peroxiredoxin 3 (Prx3), which is a mitochondrial antioxidant protein. Ablation of Prx3 deregulated the mitochondrial network and membrane potential of myotubes, in which ROS levels were increased. We showed that the DNA content of mitochondria and ATP production were also reduced in Prx3-KO muscle. Of note, the mitofusin 1 and 2 protein levels decreased in Prx3-KO muscle, a biochemical evidence of impaired mitochondrial fusion. Contractile dysfunction was examined by measuring isometric forces of isolated extensor digitorum longus (EDL) and soleus muscles. Maximum absolute forces in both the EDL and the soleus muscles were not significantly affected in Prx3-KO mice. However, fatigue trials revealed that the decrease in relative force was greater and more rapid in soleus from Prx3-KO compared to wild-type mice. Taken together, these results suggest that Prx3 plays a crucial role in mitochondrial homeostasis and thereby controls the contractile functions of skeletal muscle.     

The higher level of organization of the oxidative phosphorylation system: mitochondrial supercomplexes

The organization of the oxidative phosphorylation (OXPHOS) system within the inner mitochondrial membrane appears to be far more complicated than previously thought. In particular, the individual protein complexes of the OXPHOS system (complexes I to V) were found to specifically interact forming defined supramolecular structures. Blue-native polyacrylamide gel electrophoresis and single particle electron microscopy proved to be especially valuable in studying the so-called "respiratory supercomplexes". Based on these procedures, increasing evidence was presented supporting a "solid state" organization of the OXPHOS system. Here, we summarize results on the formation, organisation and function of the various types of mitochondrial OXPHOS supercomplexes.     

Structures of mitochondrial oxidative phosphorylation supercomplexes and mechanisms for their stabilisation

Oxidative phosphorylation (OXPHOS) is the main source of energy in eukaryotic cells. This process is performed by means of electron flow between four enzymes, of which three are proton pumps, in the inner mitochondrial membrane. The energy accumulated in the proton gradient over the inner membrane is utilized for ATP synthesis by a fifth OXPHOS complex, ATP synthase. Four of the OXPHOS protein complexes associate into stable entities called respiratory supercomplexes. This review summarises the current view on the arrangement of the electron transport chain in mitochondrial cristae. The functional role of the supramolecular organisation of the OXPHOS system and the factors that stabilise such organisation are highlighted. This article is part of a Special Issue entitled: Dynamic and ultrastructure of bioenergetic membranes and their components.     

Inhibition of mitochondrial respiratory chain in brain of rats subjected to an experimental model of depression

Depressive disorders, including major depression, are serious and disabling. However, the exact pathophysiology of depression is not clearly understood. Life stressors contribute in some fashion to depression and are an extension of what occurs normally. In this context, chronic stress has been used as an animal model of depression. Based on the hypothesis that metabolism impairment might be involved in the pathophysiology of depression, in the present work we evaluated the activities of mitochondrial respiratory chain complexes and creatine kinase in brain of rats subjected to chronic stress. After 40 days of mild stress, a reduction in sweet food ingestion was observed, as well as increased adrenal gland weight, when compared to control group. We also verified that control group gained weight after 40 days, but stressed group did not. Moreover, our findings showed that complex I, III and IV were inhibited in stress group only in cerebral cortex and cerebellum. On the other hand, complex II and creatine kinase were not affected in stressed group. Although it is difficult to extrapolate our findings to the human condition, the inhibition of mitochondrial respiratory chain by chronic stress may be one mechanism in the pathophysiology of depressive disorders.