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.     

On the role of mi-cK and VDAC in mitochondrial function of heart muscle cells

A two-compartment kinetic model was used to describe reconstituted systems in which mitochondria compete with pyruvate kinase for kinase-generated ADP. The modelling suggests that cytosolic CK deficiency results in a 50% increase in outer mitochondrial membrane permeability.     

Circumventing the Crabtree Effect: A method to induce lactate consumption and increase oxidative phosphorylation in cell culture

Most cells grown in glucose-containing medium generate almost all their ATP via glycolysis despite abundant oxygen supply and functional mitochondria, a phenomenon known as the Crabtree effect. By contrast, most cells within the body rely on mitochondrial oxidative phosphorylation (OXPHOS) to generate the bulk of their energy supply. Thus, when utilising the accessibility of cell culture to elucidate fundamental elements of mitochondria in health and disease, it is advantageous to adopt culture conditions under which the cells have greater reliance upon OXPHOS for the supply of their energy needs. Substituting galactose for glucose in the culture medium can provide these conditions, but additional benefit can be gained from alternate in vitro models. Herein we describe culture conditions in which complete autonomous depletion of medium glucose induces a lactate-consuming phase marked by increased MitoTracker Deep Red staining intensity, increased expression of Kreb’s cycle proteins, increased expression of electron transport chain subunits, and increased sensitivity to the OXPHOS inhibitor rotenone. We propose these culture conditions represent an alternate accessible model for the in vitro study of cellular processes and diseases involving the mitochondrion without limitations incurred via the Crabtree effect.     

Carbon monoxide exposure in rat heart: evidence for two modes of toxicity

Rat hearts were perfused for 30 min with buffer equilibrated with CO. Mean data for hearts exposed to 0.01% CO show a 15% decrease in heart rate (HR) during exposure followed by recovery, with a further 17% decrease post exposure. Examination of time courses from individual perfusions shows that in 10 hearts exposed to 0.01% CO HR responded in different ways: no response (5 hearts); decrease during exposure followed by recovery (3 hearts); and decrease post exposure (2 hearts). There was a strong association between CO-induced HR decrease and release of creatine kinase into the perfusate, both of which were not prevented by the antioxidants, ascorbate, and Trolox C. Perfusate flow rate declined post exposure (4.9% and 8.9% with 0.01% and 0.05% CO, respectively) and this was prevented by antioxidants. CO may have two, independent, cardiotoxic effects; these may be mediated by CO-induced elevation of oxidant production, H2O2 in one case and peroxynitrite in the other.     

Uncouple my heart: the benefits of inefficiency

Myocardial ischemia/reperfusion (IR) injury leads to structural changes in the heart muscle later followed by functional decline due to progressive fibrous replacement. Hence approaches to minimize IR injury are devised, including ischemic pre—and postconditioning. Mild uncoupling of oxidative phosphorylation is one of the mechanisms suggested to be cardioprotective as chemical uncoupling mimics ischemic preconditioning. Uncoupling protein 2 is proposed to be the physiological counterpart of chemical uncouplers and is thought to be a part of the protective machinery of cardiomyocytes. Morphological changes in the mitochondrial network likely accompany the uncoupling with mitochondrial fission dampening the signals leading to cardiomyocyte death. Here we review recent data on the role of uncoupling in cardioprotection and propose that low concentrations of dietary polyphenols may elicit the same cardioprotective effect as dinitrophenol and FCCP, perhaps accounting for the famed “French paradox”.     

Mitochondrial Glutathione Transport Is a Key Determinant of Neuronal Susceptibility to Oxidative and Nitrosative Stress

Mitochondrial oxidative stress significantly contributes to the underlying pathology of several devastating neurodegenerative disorders. Mitochondria are highly sensitive to the damaging effects of reactive oxygen and nitrogen species; therefore, these organelles are equipped with a number of free radical scavenging systems. In particular, the mitochondrial glutathione (GSH) pool is a critical antioxidant reserve that is derived entirely from the larger cytosolic pool via facilitated transport. The mechanism of mitochondrial GSH transport has not been extensively studied in the brain. However, the dicarboxylate (DIC) and 2-oxoglutarate (OGC) carriers localized to the inner mitochondrial membrane have been established as GSH transporters in liver and kidney. Here, we investigated the role of these carriers in protecting neurons from oxidative and nitrosative stress. Immunoblot analysis of DIC and OGC in primary cultures of rat cerebellar granule neurons (CGNs) and cerebellar astrocytes showed differential expression of these carriers, with CGNs expressing only DIC and astrocytes expressing both DIC and OGC. Consistent with these findings, butylmalonate specifically reduced mitochondrial GSH in CGNs, whereas both butylmalonate and phenylsuccinate diminished mitochondrial GSH in astrocytes. Moreover, preincubation with butylmalonate but not phenylsuccinate significantly enhanced susceptibility of CGNs to oxidative and nitrosative stressors. This increased vulnerability was largely prevented by incubation with cell-permeable GSH monoethylester but not malate. Finally, knockdown of DIC with adenoviral siRNA also rendered CGNs more susceptible to oxidative stress. These findings demonstrate that maintenance of the mitochondrial GSH pool via sustained mitochondrial GSH transport is essential to protect neurons from oxidative and nitrosative stress.     

Molecular Basis for the Resistance of Human Mitochondrial 2-Cys Peroxiredoxin 3 to Hyperoxidation

Peroxiredoxins (Prxs) detoxify peroxides and modulate H₂O₂-mediated cell signaling in normal and numerous pathophysiological contexts. The typical 2-Cys subclass of Prxs (human Prx1–4) utilizes a Cys sulfenic acid (Cys-SOH) intermediate and disulfide bond formation across two subunits during catalysis. During oxidative stress, however, the Cys-SOH moiety can react with H₂O₂ to form Cys sulfinic acid (Cys-SO₂H), resulting in inactivation. The propensity to hyperoxidize varies greatly among human Prxs. Mitochondrial Prx3 is the most resistant to inactivation, but the molecular basis for this property is unknown. A panel of chimeras and Cys variants of Prx2 and Prx3 were treated with H₂O₂ and analyzed by rapid chemical quench and time-resolved electrospray ionization-TOF mass spectrometry. The latter utilized an on-line rapid-mixing setup to collect data on the low seconds time scale. These approaches enabled the first direct observation of the Cys-SOH intermediate and a putative Cys sulfenamide (Cys-SN) for Prx2 and Prx3 during catalysis. The substitution of C-terminal residues in Prx3, residues adjacent to the resolving Cys residue, resulted in a Prx2-like protein with increased sensitivity to hyperoxidation and decreased ability to form the intermolecular disulfide bond between subunits. The corresponding Prx2 chimera became more resistant to hyperoxidation. Taken together, the results of this study support that the kinetics of the Cys-SOH intermediate is key to determine the probability of hyperoxidation or disulfide formation. Given the oxidizing environment of the mitochondrion, it makes sense that Prx3 would favor disulfide bond formation as a protection mechanism against hyperoxidation and inactivation.     

Functional dynamic compartmentalization of respiratory chain intermediate substrates: implications for the control of energy production and mitochondrial diseases

Activity defects in respiratory chain complexes are responsible for a large variety of pathological situations, including neuromuscular diseases and multisystemic disorders. Their impact on energy production is highly variable and disproportional. The same biochemical or genetic defect can lead to large differences in clinical symptoms and severity between tissues and patients, making the pathophysiological analysis of mitochondrial diseases difficult. The existence of compensatory mechanisms operating at the level of the respiratory chain might be an explanation for the biochemical complexity observed for respiratory defects. Here, we analyzed the role of cytochrome c and coenzyme Q in the attenuation of complex III and complex IV pharmacological inhibition on the respiratory flux. Spectrophotometry, HPLC–EC, polarography and enzymology permitted the calculation of molar ratios between respiratory chain components, giving values of 0.8:61:3:12:6.8 in muscle and 1:131:3:9:6.5 in liver, for CII:CoQ:CIII:Cyt c:CIV. The results demonstrate the dynamic functional compartmentalization of respiratory chain substrates, with the existence of a substrate pool that can be recruited to maintain energy production at normal levels when respiratory chain complexes are inhibited. The size of this reserve was different between muscle and liver, and in proportion to the magnitude of attenuation of each respiratory defect. Such functional compartmentalization could result from the recently observed physical compartmentalization of respiratory chain substrates. The dynamic nature of the mitochondrial network may modulate this compartmentalization and could play a new role in the control of mitochondrial respiration as well as apoptosis.     

The proximal N-terminal amino acid residues are required for the coupling activity of the bovine heart mitochondrial factor B

Treatment of the recombinant bovine factor B with trypsin yielded a fragment (amino acid residues 62-175) devoid of coupling activity. Removal of the N-terminal Trp2-Gly3-Trp4 peptide resulted in a significant loss of coupling activity in the FB_ΔW²_−W⁴ deletion mutant. Sucrose density gradient centrifugation demonstrated co-sedimentation of recombinant factor B with the ADP/ATP carrier, which is present in preparations of H⁺-translocating F₀F₁-ATPase, but not in preparations of complex V. The N-terminally truncated factor B mutant FB_ΔW²_−W⁴ did not co-sediment with the ADP/ATP carrier. Recombinant factor B co-sedimented with partially purified membrane sector F₀, extracted from F₁-stripped bovine submitochondrial particles with n-dodecyl-β-d-maltoside. Factor B inhibited the passive proton conductance catalyzed by F₀ reconstituted into asolectin liposomes. A factor B mutant, bearing a photoreactive unnatural amino acid pbenzoyl-l-phenylalanine (pBpa) substituted for Trp2, cross-linked with F₀ subunits e and g as well as the ADP/ATP carrier. These results suggest that the N-terminal domain and, in particular, the proximal N-terminal amino acids are important for the coupling activity and protein-protein interactions of bovine factor B.     

4-Methylene-2-octyl-5-oxotetrahydrofuran-3-carboxylic Acid (C75), an Inhibitor of Fatty-acid Synthase,Suppresses the Mitochondrial Fatty Acid Synthesis Pathway and Impairs Mitochondrial Function

4-Methylene-2-octyl-5-oxotetrahydrofuran-3-carboxylic acid (C75) is a synthetic fatty-acid synthase (FASN) inhibitor with potential therapeutic effects in several cancer models. Human mitochondrial β-ketoacyl-acyl carrier protein synthase (HsmtKAS) is a key enzyme in the newly discovered mitochondrial fatty acid synthesis pathway that can produce the substrate for lipoic acid (LA) synthesis. HsmtKAS shares conserved catalytic domains with FASN, which are responsible for binding to C75. In our study, we explored the possible effect of C75 on HsmtKAS and mitochondrial function. C75 treatment decreased LA content, impaired mitochondrial function, increased reactive oxygen species content, and reduced cell viability. HsmtKAS but not FASN knockdown had an effect that was similar to C75 treatment. In addition, an LA supplement efficiently inhibited C75-induced mitochondrial dysfunction and oxidative stress. Overexpression of HsmtKAS showed cellular protection against low dose C75 addition, whereas there was no protective effect upon high dose C75 addition. In summary, the mitochondrial fatty acid synthesis pathway has a vital role in mitochondrial function. Besides FASN, C75 might also inhibit HsmtKAS, thereby reducing LA production, impairing mitochondrial function, and potentially having toxic effects. LA supplements sufficiently ameliorated the toxicity of C75, showing that a combination of C75 and LA may be a reliable cancer treatment.     

Channel Formation by Yeast F-ATP Synthase and the Role of Dimerization in the Mitochondrial Permeability Transition

Purified F-ATP synthase dimers of yeast mitochondria display Ca²⁺-dependent channel activity with properties resembling those of the permeability transition pore (PTP) of mammals. After treatment with the Ca²⁺ ionophore ETH129, which allows electrophoretic Ca²⁺ uptake, isolated yeast mitochondria undergo inner membrane permeabilization due to PTP opening. Yeast mutant strains ΔTIM11 and ΔATP20 (lacking the e and g F-ATP synthase subunits, respectively, which are necessary for dimer formation) display a striking resistance to PTP opening. These results show that the yeast PTP originates from F-ATP synthase and indicate that dimerization is required for pore formation in situ.     

Targeting Dinitrophenol to Mitochondria: Limitations to the Development of a Self-limiting Mitochondrial Protonophore

The protonmotive force (Δp) across the mitochondrial inner membrane drives ATP synthesis. In addition, the energy stored in Δp can be dissipated by proton leak through the inner membrane, contributing to basal metabolic rate and thermogenesis. Increasing mitochondrial proton leak pharmacologically should decrease the efficiency of oxidative phosphorylation and counteract obesity by enabling fatty acids to be oxidised with decreased ATP production. While protonophores such as 2,4-dinitrophenol (DNP) increase mitochondrial proton leak and have been used to treat obesity, a slight increase in DNP concentration above the therapeutically effective dose disrupts mitochondrial function and leads to toxicity. Therefore we set out to develop a less toxic protonophore that would increase proton leak significantly at high Δp but not at low Δp. Our design concept for a potential self-limiting protonophore was to couple the DNP moiety to the lipophilic triphenylphosphonium (TPP) cation and this was achieved by the preparation of 3-(3,5-dinitro-4-hydroxyphenyl)propyltriphenylphosphonium methanesulfonate (MitoDNP). TPP cations accumulate within mitochondria driven by the membrane potential (Δψ), the predominant component of Δp. Our hypothesis was that MitoDNP would accumulate in mitochondria at high Δψ where it would act as a protonophore, but that at lower Δψ the accumulation and uncoupling would be far less. We found that MitoDNP was extensively taken into mitochondria driven by Δψ. However MitoDNP did not uncouple mitochondria as judged by its inability to either increase respiration rate or decrease Δψ. Therefore MitoDNP did not act as a protonophore, probably because the efflux of deprotonated MitoDNP was inhibited.     

Novel plasma phospholipid biomarkers of autism: Mitochondrial dysfunction as a putative causative mechanism

Autism is a neurological disorder that manifests as noticeable behavioral and developmental abnormalities predominantly in males between the ages of 2 and 10. Although the genetics, biochemistry and neuropathology of this disease have been extensively studied, underlying causal factors to this disease have remained elusive. Using a longitudinal trial design in which three plasma samples were collected from 15 autistic and 12 non-autistic age-matched controls over the course of 1 year, universal and unambiguous alterations in lipid metabolism were observed. Biomarkers of fatty acid elongation and desaturation (poly-unsaturated long chain fatty acids (PUFA) and/or saturated very long chain fatty acids (VLCFA)-containing ethanolamine phospholipids) were statistically elevated in all autistic subjects. In all 8 of the affected/non-affected sibling pairs, the affected sibling had higher levels of these biomarkers than the unaffected sibling. Exposure of neurons, astrocytes and hepatocytes in vitro to elevated extracellular glutamate levels resulted in lipid biomarker changes indistinguishable from those observed in autistic subjects. Glutamate stress also resulted in in vitro decreased levels of reduced glutathione (GSH), methionine and cysteine, in a similar way to the decreases we observed in autism plasma. Impaired mitochondrial fatty acid oxidation, elevated plasma VLCFAs, and glutamate toxicity as putative causal factors in the biochemistry, neuropathology, and gender bias in autism are discussed.     

Organization of the Mitochondrial Apoptotic BAK Pore: OLIGOMERIZATION OF THE BAK HOMODIMERS*

The multidomain pro-apoptotic Bcl-2 family proteins BAK and BAX are believed to form large oligomeric pores in the mitochondrial outer membrane during apoptosis. Formation of these pores results in the release of apoptotic factors including cytochrome c from the intermembrane space into the cytoplasm, where they initiate the cascade of events that lead to cell death. Using the site-directed spin labeling method of electron paramagnetic resonance (EPR) spectroscopy, we have determined the conformational changes that occur in BAK when the protein targets to the membrane and forms pores. The data showed that helices α1 and α6 disengage from the rest of the domain, leaving helices α2-α5 as a folded unit. Helices α2-α5 were shown to form a dimeric structure, which is structurally homologous to the recently reported BAX “BH3-in-groove homodimer.” Furthermore, the EPR data and a chemical cross-linking study demonstrated the existence of a hitherto unknown interface between BAK BH3-in-groove homodimers in the oligomeric BAK. This novel interface involves the C termini of α3 and α5 helices. The results provide further insights into the organization of the BAK oligomeric pores by the BAK homodimers during mitochondrial apoptosis, enabling the proposal of a BAK-induced lipidic pore with the topography of a “worm hole.”     

Role of mitochondria in the immune response to cancer: a central role for Ca2+

This study demonstrates that Ca²⁺ stimulates mitochondrial energy metabolism during spleen lymphocyte activation in response to the ascitic Walker 256 tumor in rats. Intracellular Ca²⁺ concentrations, phosphorylated protein kinase C (pPKC) levels, Bcl-2 protein contents, interleukin-2 (IL-2) levels, mitochondrial uncoupling protein-2 (UCP-2) contents and reactive oxygen species (ROS) were significantly elevated in these activated lymphocytes. Mitochondria of activated lymphocytes exhibited high free Ca²⁺ concentrations in the matrix and enhanced oligomycin-sensitive oxygen consumption, indicating an increased rate of oxidative phosphorylation. The production of ROS was largely decreased by diphenylene iodinium in the activated lymphocytes, suggesting that NADPH oxidase is the prevalent source of these species. Accumulation of UCP-2 and the anti-apoptotic protein Bcl-2 is probably important to prevent mitochondrial dysfunction and cell death elicited by the sustained high levels of intracellular Ca²⁺ and ROS and may explain the observed higher resistance from activated lymphocytes against the opening of the mitochondrial membrane permeability pore (MPT). All these changes were blocked by pretreatment of the rats with verapamil, an L-type Ca²⁺ channel antagonist. These data demonstrate a central role of Ca²⁺ in the control of mitochondrial bioenergetics in spleen lymphocytes during the immune response to cancer.     

Replication Intermediates of the Linear Mitochondrial DNA of Candida parapsilosis Suggest a Common Recombination Based Mechanism for Yeast Mitochondria

Variation in the topology of mitochondrial DNA (mtDNA) in eukaryotes evokes the question if differently structured DNAs are replicated by a common mechanism. RNA-primed DNA synthesis has been established as a mechanism for replicating the circular animal/mammalian mtDNA. In yeasts, circular mtDNA molecules were assumed to be templates for rolling circle DNA-replication. We recently showed that in Candida albicans, which has circular mapping mtDNA, recombination driven replication is a major mechanism for replicating a complex branched mtDNA network. Careful analyses of C. albicans-mtDNA did not reveal detectable amounts of circular DNA molecules. In the present study we addressed the question of how the unit sized linear mtDNA of Candida parapsilosis terminating at both ends with arrays of tandem repeats (mitochondrial telomeres) is replicated. Originally, we expected to find replication intermediates diagnostic of canonical bi-directional replication initiation at the centrally located bi-directional promoter region. However, we found that the linear mtDNA of Candida parapsilosis also employs recombination for replication initiation. The most striking findings were that the mitochondrial telomeres appear to be hot spots for recombination driven replication, and that stable RNA:DNA hybrids, with a potential role in mtDNA replication, are also present in the mtDNA preparations.