The Structure of the Cardiac Mitochondria Respirasome Is Adapted for the ß-Oxidation of Fatty Acids.

It is well known that in the heart and kidney mitochondria, more than 95% of ATP production is supported by the ß-oxidation of long-chain fatty acids. However, the ß-oxidation of fatty acids by mitochondria has been studied much less than the substrates formed during the catabolism of carbohydrates and amino acids. In the last few decades, several discoveries have been made that are directly related to fatty acid oxidation. In this review, we made an attempt to re-evaluate the ß-oxidation of long-chain fatty acids from the perspectives of new discoveries. The single set of electron transporters of the cardiac mitochondrial respiratory chain is organized into three supercomplexes. Two of them contain complex I, a dimer of complex III, and two dimers of complex IV. The third, smaller supercomplex contains a dimer of complex III and two dimers of complex IV. We also considered other important discoveries. First, the enzymes of the ß-oxidation of fatty acids are physically associated with the respirasome. Second, the ß-oxidation of fatty acids creates the highest level of QH2 and reverses the flow of electrons from QH2 through complex II, reducing fumarate to succinate. Third, ß-oxidation is greatly stimulated in the presence of succinate. We argue that the respirasome is uniquely adapted for the ß-oxidation of fatty acids. The acyl-CoA dehydrogenase complex reduces the membrane's pool of ubiquinone to QH2, which is instantly oxidized by the smaller supercomplex, generating a high energization of mitochondria and reversing the electron flow through complex II, which reverses the electron flow through complex I, increasing the NADH/NAD+ ratio in the matrix. The mitochondrial nicotinamide nucleotide transhydrogenase catalyzes a hydride (H-, a proton plus two electrons) transfer across the inner mitochondrial membrane, reducing the cytosolic pool of NADP(H), thus providing the heart with ATP for muscle contraction and energy and reducing equivalents for the housekeeping processes.

Long-Chain and Medium-Chain Fatty Acids in Energy Metabolism of Murine Kidney Mitochondria.

Scientists have long established that fatty acids are the primary substrates for kidney mitochondria. However, to date we still do not know how long-chain and middle-chain fatty acids are oxidized at the mitochondrial level. Our previous research has shown that mitochondria from the heart, brain, and kidney oxidize palmitoylcarnitine at a high rate only in the presence of succinate, glutamate, or pyruvate. In this paper, we report properties of the isolated kidney mitochondria and how malate and succinate affect the oxidation of C16 and C8 acylcarnitines. The isolated kidney mitochondria contain very few endogenous substrates and require malate to oxidize pyruvate, glutamate, and C16 or C8 acylcarnitines. We discovered that with 10 µM of C16 or C8 acylcarnitines, low concentrations of malate (0.2 mM) or succinate (0.5 mM) enhance the States 4 and 3 respiratory rates several times. The highest respiration rates were observed with C16 or C8 acylcarnitines and 5 mM succinate mixtures. Results show that kidney mitochondria, unlike the heart and brain mitochondria, lack the intrinsic inhibition of succinate dehydrogenase. Additionally, results show that the oxidation of fatty acid by the small respirasome's supercomplex generates a high level of CoQH2, and this makes SDH in the presence of succinate reverse the flow of electrons from CoQH2 to reduce fumarate to succinate. Finally, we report evidence that succinate dehydrogenase is a key mitochondrial enzyme that allows fast oxidation of fatty acids and turns the TCA cycle function from the catabolic to the anabolic and anaplerotic metabolic pathways.

Cardiolipin, Perhydroxyl Radicals, and Lipid Peroxidation in Mitochondrial Dysfunctions and Aging.

Mitochondrial dysfunctions caused by oxidative stress are currently regarded as the main cause of aging. Accumulation of mutations and deletions of mtDNA is a hallmark of aging. So far, however, there is no evidence that most studied oxygen radicals are directly responsible for mutations of mtDNA. Oxidative damages to cardiolipin (CL) and phosphatidylethanolamine (PEA) are also hallmarks of oxidative stress, but the mechanisms of their damage remain obscure. CL is the only phospholipid present almost exclusively in the inner mitochondrial membrane (IMM) where it is responsible, together with PEA, for the maintenance of the superstructures of oxidative phosphorylation enzymes. CL has negative charges at the headgroups and due to specific localization at the negative curves of the IMM, it creates areas with the strong negative charge where local pH may be several units lower than in the surrounding bulk phases. At these sites with the higher acidity, the chance of protonation of the superoxide radical (O2 •), generated by the respiratory chain, is much higher with the formation of the highly reactive hydrophobic perhydroxyl radical (HO2 •). HO2 • specifically reacts with the double bonds of polyunsaturated fatty acids (PUFA) initiating the isoprostane pathway of lipid peroxidation. Because HO2 • is formed close to CL aggregates and PEA, it causes peroxidation of the linoleic acid in CL and also damages PEA. This causes disruption of the structural and functional integrity of the respirosomes and ATP synthase. We provide evidence that in elderly individuals with metabolic syndrome (MetS), fatty acids become the major substrates for production of ATP and this may increase several-fold generation of O2 • and thus HO2 •. We conclude that MetS accelerates aging and the mitochondrial dysfunctions are caused by the HO2 •-induced direct oxidation of CL and the isoprostane pathway of lipid peroxidation (IPLP). The toxic products of IPLP damage not only PEA, but also mtDNA and OXPHOS proteins. This results in gradual disruption of the structural and functional integrity of mitochondria and cells.

Targeting of reactive isolevuglandins in mitochondrial dysfunction and inflammation.

Inflammation is a major cause of morbidity and mortality in Western societies. Despite use of multiple drugs, both chronic and acute inflammation still represent major health burdens. Inflammation produces highly reactive dicarbonyl lipid peroxidation products such as isolevuglandins which covalently modify and cross-link proteins via lysine residues. Mitochondrial dysfunction has been associated with inflammation; however, its molecular mechanisms and pathophysiological role are still obscure. We hypothesized that inflammation-induced isolevuglandins contribute to mitochondrial dysfunction and mortality. To test this hypothesis, we have (a) investigated the mitochondrial dysfunction in response to synthetic 15-E2-isolevuglandin (IsoLG) and its adducts; (b) developed a new mitochondria-targeted scavenger of isolevuglandins by conjugating 2-hydroxybenzylamine to the lipophilic cation triphenylphosphonium, (4-(4-aminomethyl)-3-hydroxyphenoxy)butyl)-triphenylphosphonium (mito2HOBA); (c) tested if mito2HOBA protects from mitochondrial dysfunction and mortality using a lipopolysaccharide model of inflammation. Acute exposure to either IsoLG or IsoLG adducts with lysine, ethanolamine or phosphatidylethanolamine inhibits mitochondrial respiration and attenuates Complex I activity. Complex II function was much more resistant to IsoLG. We confirmed that mito2HOBA markedly accumulates in isolated mitochondria and it is highly reactive with IsoLGs. To test the role of mitochondrial IsoLGs, we studied the therapeutic potential of mito2HOBA in lipopolysaccharide mouse model of sepsis. Mito2HOBA supplementation in drinking water (0.1 g/L) to lipopolysaccharide treated mice increased survival by 3-fold, improved complex I-mediated respiration, and histopathological analyses supported mito2HOBA-mediated protection of renal cortex from cell injury. These data support the role of mitochondrial IsoLG in mitochondrial dysfunction and inflammation. We conclude that reducing mitochondrial IsoLGs may be a promising therapeutic target in inflammation and conditions associated with mitochondrial oxidative stress and dysfunction.

Physiological Levels of Nitric Oxide Diminish Mitochondrial Superoxide. Potential Role of Mitochondrial Dinitrosyl Iron Complexes and Nitrosothiols.

Mitochondria are the major source of superoxide radicals and superoxide overproduction contributes to cardiovascular diseases and metabolic disorders. Endothelial dysfunction and diminished nitric oxide levels are early steps in the development of these pathological conditions. It is known that physiological production of nitric oxide reduces oxidative stress and inflammation, however, the precise mechanism of "antioxidant" effect of nitric oxide is not clear. In this work we tested the hypothesis that physiological levels of nitric oxide diminish mitochondrial superoxide production without inhibition of mitochondrial respiration. In order to test this hypothesis we analyzed effect of low physiological fluxes of nitric oxide (20 nM/min) on superoxide and hydrogen peroxide production by ESR spin probes and Amplex Red in isolated rat brain mitochondria. Indeed, low levels of nitric oxide substantially attenuated both basal and antimycin A-stimulated production of reactive oxygen species in the presence of succinate or glutamate/malate as mitochondrial substrates. Furthermore, slow releasing NO donor DPTA-NONOate (100 µM) did not change oxygen consumption in State 4 and State 3. However, the NO-donor strongly inhibited oxygen consumption in the presence of uncoupling agent CCCP, which is likely associated with inhibition of the over-reduced complex IV in uncoupled mitochondria. We have examined accumulation of dinitrosyl iron complexes and nitrosothiols in mitochondria treated with fast-releasing NO donor MAHMA NONOate (10 µM) for 30 min until complete release of NO. Following treatment with NO donor, mitochondria were frozen for direct detection of dinitrosyl iron complexes using Electron Spin Resonance (ESR) while accumulation of nitrosothiols was measured by ferrous-N-Methyl-D-glucamine dithiocarbamate complex, Fe(MGD)2, in lysed mitochondria. Treatment of mitochondria with NO-donor gave rise to ESR signal of dinitrosyl iron complexes while ESR spectra of Fe(MGD)2 supplemented mitochondrial lysates showed presence of both dinitrosyl iron complexes and nitrosothiols. We suggest that nitric oxide attenuates production of mitochondrial superoxide by post-translational modifications by nitrosylation of protein cysteine residues and formation of protein dinitrosyl iron complexes with thiol-containing ligands and, therefore, nitric oxide reduction in pathological conditions associated with endothelial dysfunction may increase mitochondrial oxidative stress.

Metabolic and functional differences between brain and spinal cord mitochondria underlie different predisposition to pathology.

Mitochondrial dysfunctions contribute to neurodegeneration, the locations of which vary among neurodegenerative diseases. To begin to understand what mechanisms may underlie higher vulnerability of the spinal cord motor neurons in amyotrophic lateral sclerosis, compared with brain mitochondria, we studied three major functions of rat brain mitochondria (BM) and spinal cord mitochondria (SCM) mitochondria: oxidative phosphorylation, Ca(2+) sequestration, and production of reactive oxygen species (ROS), using a new metabolic paradigm (Panov et al., J. Biol. Chem. 284: 14448-14456, 2009). We present data that SCM share some unique metabolic properties of the BM. However, SCM also have several distinctions from the BM: 1) With the exception of succinate, SCM show significantly lower rates of respiration with all substrates studied; 2) immunoblotting analysis showed that this may be due to 30-40% lower contents of respiratory enzymes and porin; 3) compared with BM, SCM sequestered 40-50% less Ca(2+), and the total tissue calcium content was 8 times higher in the spinal cord; 4) normalization for mitochondria from 1 g of tissue showed that BM can sequester several times more Ca(2+) than was available in the brain tissue, whereas SCM had the capacity to sequester only 10-20% of the total tissue Ca(2+); and 5) with succinate and succinate-containing substrate mixtures, SCM showed significantly higher state 4 respiration than BM and generated more ROS associated with the reverse electron transport. We conclude that SCM have an intrinsically higher risk of oxidative damage and overload with calcium than BM, and thus spinal cord may be more vulnerable under some pathologic conditions. (250).

Mechanism of toxicity of pesticides acting at complex I: relevance to environmental etiologies of Parkinson's disease.

Parkinson's disease (PD) has been linked to mitochondrial dysfunction and pesticide exposure. The pesticide rotenone (ROT) inhibits complex I and reproduces features of PD in animal models, suggesting that environmental agents that inhibit complex I may contribute to PD. We have previously demonstrated that ROT toxicity is dependent upon complex I inhibition and that oxidative stress is the primary mechanism of toxicity. In this study, we examined the in vitro toxicity and mechanism of action of several putative complex I inhibitors that are commonly used as pesticides. The rank order of toxicity of pesticides to neuroblastoma cells was pyridaben > rotenone > fenpyroximate > fenazaquin > tebunfenpyrad. A similar order of potency was observed for reduction of ATP levels and competition for (3)H-dihydrorotenone (DHR) binding to complex I, with the exception of pyridaben (PYR). Neuroblastoma cells stably expressing the ROT-insensitive NADH dehydrogenase of Saccharomyces cerevisiae (NDI1) were resistant to these pesticides, demonstrating the requirement of complex I inhibition for toxicity. We further found that PYR was a more potent inhibitor of mitochondrial respiration and caused more oxidative damage than ROT. The oxidative damage could be attenuated by NDI1 or by the antioxidants alpha-tocopherol and coenzyme Q(10). PYR was also highly toxic to midbrain organotypic slices. These data demonstrate that, in addition to ROT, several commercially used pesticides directly inhibit complex I, cause oxidative damage, and suggest that further study is warranted into environmental agents that inhibit complex I for their potential role in PD.

Ca2+-induced permeability transition in human lymphoblastoid cell mitochondria from normal and Huntington's disease individuals.

Huntington's disease (HD) is associated with expansion of polyglutamine tract in a protein named huntingtin (htt) that is expressed in virtually all body tissues. Thus mutated htt (HD-htt) might affect all organs, although clinical manifestations of HD are associated with selective loss of corticostriatal neurons of the brain. In this work we studied how HD-htt affects mitochondria in human peripheral blood cells. We compared various functions of mitochondria isolated from cultured lymphoblastoid cells derived from three HD patients with juvenile onset of the disease (HD-LBM) and three age-matched control (C-LBM) individuals. Respiratory parameters in different metabolic states, with succinate and glutamate plus malate were the same for all control and HD cell lines. State 4 membrane potential in HD-LBM was slightly lower than in C-LBM. The calcium retention capacity (CRC) of mitochondria was estimated using simultaneously several methods to register permeability transition (PT). We found that LBM do not undergo swelling upon Ca2+-induced PT, and do not increase CRC in the presence of ADP + oligomycin. Although each cell line had different CRC values, qualitatively PT was different in C-LBM and HD-LBM. With C-LBM cyclosporin A (CsA) increased CRC significantly, while with HD-LBM CsA was ineffective. In C-LBM depolarization of mitochondria and a large pore opening (PT) always occurred simultaneously. In HD-LBM depolarization occurred at 20-50% lower Ca2+ loads than PT. We suggest that HD-htt promotes low H+ conductance of the mitochondria by interacting with proteins at the contacts sites without directly promoting PT or hampering mitochondrial oxidative phosphorylation.

Quantitative evaluation of the effects of mitochondrial permeability transition pore modifiers on accumulation of calcium phosphate: comparison of rat liver and brain mitochondria.

Mitochondria play a critical role in some forms of apoptosis, and the Ca(2+)-dependent permeability transition (PT) is a key initiator of this process. We quantitatively examined major control mechanisms of PT in rat brain (RBM) and liver (RLM) mitochondria. Compared with RLM, RBM were less sensitive to cyclosporin A (CsA), but the combined action of CsA+ADP was much more pronounced in RBM. Carboxyatractyloside abrogated the effects of all mPTP inhibitors in RBM but not in RLM, where the effects of CsA were not reduced. Estimated H(+)/Ca(2+) ratios were 0.81+/-0.01 for RLM and 0.84-0.93 for RBM, suggesting that Ca(2+) and Pi were sequestered in the matrix as CaHPO(4) and Ca(3)(PO(4))(2) salts, and that RBM sequester more CaPi as the least soluble salt. We conclude that: (1) RBM and RLM differ in their baseline behavior of the PT and in their responses to PT modifiers, and (2) PT modifiers can be functionally divided into those which directly affect the mitochondrial PT pore and are not energy-dependent (CsA, free Ca(2+), ADP(ex), and Mg(2+)), and those which affect the energy-dependent calcium phosphate sequestration process (ADP(mt), CATR, local anesthetics). We also conclude that ANT affects PT by changing mitochondrial capacity for energization.

In vitro effects of polyglutamine tracts on Ca2+-dependent depolarization of rat and human mitochondria: relevance to Huntington's disease.

The mechanisms by which neurons die in CAG triplet repeat (polyglutamine) disorders, such as Huntington's disease, are uncertain; however, mitochondrial dysfunction and disordered calcium homeostasis have been implicated. We previously demonstrated abnormal mitochondrial calcium handling in Huntington's disease cell lines and transgenic mice. To examine whether these abnormalities might arise in part from direct effects of the expanded polyglutamine tract contained in mutant huntingtin, we have exposed normal rat liver and human lymphoblast mitochondria to glutathione S-transferase fusion proteins containing polyglutamine tracts of 0, 19, or 62 residues. Similar to bovine serum albumin, each of the protein constructs nonspecifically inhibited succinate-supported respiration, independent of polyglutamine tract length. There was a small but significant reduction of mitochondrial membrane potential (state 4) only in the presence of the pathological-length polyglutamine tract. With successive addition of small Ca(2+) aliquots, mitochondria exposed to pathological-length polyglutamine tracts (approximately 5 microM) depolarized much earlier and to a greater extent than those exposed to the other protein constructs. These results suggest that the mitochondrial calcium handling defects seen in Huntington's disease cell lines and transgenic mice may be due, in part, to direct, deleterious effects of mutant huntingtin on mitochondria.

An in vitro model of Parkinson's disease: linking mitochondrial impairment to altered alpha-synuclein metabolism and oxidative damage.

Chronic systemic complex I inhibition caused by rotenone exposure induces features of Parkinson's disease (PD) in rats, including selective nigrostriatal dopaminergic degeneration and formation of ubiquitin- and alpha-synuclein-positive inclusions (Betarbet et al., 2000). To determine underlying mechanisms of rotenone-induced cell death, we developed a chronic in vitro model based on treating human neuroblastoma cells with 5 nm rotenone for 1-4 weeks. For up to 4 weeks, cells grown in the presence of rotenone had normal morphology and growth kinetics, but at this time point, approximately 5% of cells began to undergo apoptosis. Short-term rotenone treatment (1 week) elevated soluble alpha-synuclein protein levels without changing message levels, suggesting that alpha-synuclein degradation was retarded. Chronic rotenone exposure (4 weeks) increased levels of SDS-insoluble alpha-synuclein and ubiquitin. After a latency of >2 weeks, rotenone-treated cells showed evidence of oxidative stress, including loss of glutathione and increased oxidative DNA and protein damage. Chronic rotenone treatment (4 weeks) caused a slight elevation in basal apoptosis and markedly sensitized cells to further oxidative challenge. In response to H2O2, there was cytochrome c release from mitochondria, caspase-3 activation, and apoptosis, all of which occurred earlier and to a much greater extent in rotenone-treated cells; caspase inhibition provided substantial protection. These studies indicate that chronic low-grade complex I inhibition caused by rotenone exposure induces accumulation and aggregation of alpha-synuclein and ubiquitin, progressive oxidative damage, and caspase-dependent death, mechanisms that may be central to PD pathogenesis.

Early mitochondrial calcium defects in Huntington's disease are a direct effect of polyglutamines.

Huntington's disease (HD) is caused by an expansion of exonic CAG triplet repeats in the gene encoding huntingtin protein (Htt), but the mechanisms by which this mutant protein causes neurodegeneration remain unknown. Here we show that lymphoblast mitochondria from patients with HD have a lower membrane potential and depolarize at lower calcium loads than do mitochondria from controls. We found a similar defect in brain mitochondria from transgenic mice expressing full-length mutant huntingtin, and this defect preceded the onset of pathological or behavioral abnormalities by months. By electron microscopy, we identified N-terminal mutant huntingtin on neuronal mitochondrial membranes, and by incubating normal mitochondria with a fusion protein containing an abnormally long polyglutamine repeat, we reproduced the mitochondrial calcium defect seen in human patients and transgenic animals. Thus, mitochondrial calcium abnormalities occur early in HD pathogenesis and may be a direct effect of mutant huntingtin on the organelle.