Modification Of Mitochondrial Proteins Research Articles

C. elegans as Model for the Study of High Glucose– Mediated Life Span Reduction

OBJECTIVEEstablishing Caenorhabditis elegans as a model for glucose toxicity–mediated life span reduction.RESEARCH DESIGN AND METHODSC. elegans were maintained to achieve glucose concentrations resembling the hyperglycemic conditions in diabetic patients. The effects of high glucose on life span, glyoxalase-1 activity, advanced glycation end products (AGEs), and reactive oxygen species (ROS) formation and on mitochondrial function were studied.RESULTSHigh glucose conditions reduced mean life span from 18.5 ± 0.4 to 16.5 ± 0.6 days and maximum life span from 25.9 ± 0.4 to 23.2 ± 0.4 days, independent of glucose effects on cuticle or bacterial metabolization of glucose. The formation of methylglyoxal-modified mitochondrial proteins and ROS was significantly increased by high glucose conditions and reduced by mitochondrial uncoupling and complex IIIQo inhibition. Overexpression of the methylglyoxal–detoxifying enzyme glyoxalase-1 attenuated the life-shortening effect of glucose by reducing AGE accumulation (by 65%) and ROS formation (by 50%) and restored mean (16.5 ± 0.6 to 20.6 ± 0.4 days) and maximum life span (23.2 ± 0.4 to 27.7 ± 2.3 days). In contrast, inhibition of glyoxalase-1 by RNAi further reduced mean (16.5 ± 0.6 to 13.9 ± 0.7 days) and maximum life span (23.2 ± 0.4 to 20.3 ± 1.1 days). The life span reduction by glyoxalase-1 inhibition was independent from the insulin signaling pathway because high glucose conditions also affected daf-2 knockdown animals in a similar manner.CONCLUSIONSC. elegans is a suitable model organism to study glucose toxicity, in which high glucose conditions limit the life span by increasing ROS formation and AGE modification of mitochondrial proteins in a daf-2 independent manner. Most importantly, glucose toxicity can be prevented by improving glyoxalase-1–dependent methylglyoxal detoxification or preventing mitochondrial dysfunction.

A mitochondria-targeted S -nitrosothiol modulates respiration, nitrosates thiols, and protects against ischemia-reperfusion injury

Nitric oxide (NO(*)) competitively inhibits oxygen consumption by mitochondria at cytochrome c oxidase and S-nitrosates thiol proteins. We developed mitochondria-targeted S-nitrosothiols (MitoSNOs) that selectively modulate and protect mitochondrial function. The exemplar MitoSNO1, produced by covalently linking an S-nitrosothiol to the lipophilic triphenylphosphonium cation, was rapidly and extensively accumulated within mitochondria, driven by the membrane potential, where it generated NO(*) and S-nitrosated thiol proteins. MitoSNO1-induced NO(*) production reversibly inhibited respiration at cytochrome c oxidase and increased extracellular oxygen concentration under hypoxic conditions. MitoSNO1 also caused vasorelaxation due to its NO(*) generation. Infusion of MitoSNO1 during reperfusion was protective against heart ischemia-reperfusion injury, consistent with a functional modification of mitochondrial proteins, such as complex I, following S-nitrosation. These results support the idea that selectively targeting NO(*) donors to mitochondria is an effective strategy to reversibly modulate respiration and to protect mitochondria against ischemia-reperfusion injury.

Male‐female differences in post translational modifications of mitochondrial proteins

Premenopausal women have reduced cardiovascular disease compared to men. The mechanisms underlying such gender differences are unclear. Using a rat Langendorff model, we found that females (F) had smaller infarcts than males (M) after 30 minutes of ischemia. We hypothesized that this protection is mediated by altered protein levels or post‐translational modifications (PTM), and we used a proteomic approach to investigate these differences. Isolated mitochondria from M and F rats were analyzed by 2D fluorescence difference gel electrophoresis (2D‐DIGE) followed by mass spectrometry (MS). 2D‐DIGE analysis of M and F revealed differences in 20 proteins. PDH‐E1α and aldehyde dehydrogenase (ALDH) were both located at multiple pI values, suggesting PTM. Furthermore, for PDH E1α, F had higher levels at high pI values and lower levels at low pI values while the reverse was true for ALDH; these data suggest M‐F differences in PTM. Consistent with M‐F differences in PTM, using ProQ to identify phosphoprotein, we found increased PDH‐E1α phosphorylation in M compared to F. Quantitative MS identified 742 proteins, but no significant M‐F difference was found in any of these proteins. These data suggest that PTM plays a bigger role in M‐F differences than changes in expression of mitochondrial proteins.

The permissive role of mitochondria in the induction of haem oxygenase-1 in endothelial cells

HO-1 (haem oxygenase 1) is an essential antioxidant enzyme in the cell that exerts its effects through removal of pro-oxidant haem groups and the formation of antioxidant molecules and carbon monoxide. The electrophilic cyclopentenone 15d-PGJ2 (15-deoxy-Delta(12,14)-prostaglandin J2) induces the expression of HO-1 protein through the covalent modification of protein thiols. It has been shown that specific thiol residues of the redox-sensor Keap1 (Kelch-like ECH-associated protein 1) are modified by 15d-PGJ2, leading to activation of the transcription factor Nrf-2 (nuclear factor-erythroid 2 p45 subunit-related factor 2) and up-regulation of genes under control of the electrophile-response element, including HO-1. However, 15d-PGJ2 has also been shown to modify other proteins which comprise the electrophile-responsive proteome. Since 15d-PGJ2 has been shown to localize to the mitochondria in endothelial cells, we hypothesized that mitochondrial protein modification may also be important in Keap1/Nrf-2 signal transduction, leading to HO-1 up-regulation. In order to determine the role of mitochondrial protein thiol modification in HO-1 induction, we used the mitochondrial-targeted thiol-reactive compound IBTP [(4-iodobutyl)triphenylphosphonium]. IBTP had no effect on basal HO-1 levels, but effectively blocked HO-1 induction by a variety of reagents including haemin, iodoacetamide and 15d-PGJ2. Mechanistically, IBTP did not prevent the covalent modification of Keap1 by 15d-PGJ2. However, IBTP prevented the 15d-PGJ2-dependent increases in HO-1 mRNA and protein. Furthermore, IBTP prevented the nuclear accumulation of Nrf-2, suggesting cross-talk between mitochondria and antioxidant-response signal transduction. This effect was independent of reactive oxygen species formation or mitochondrial membrane potential. In addition, IBTP significantly enhanced the toxicity of high concentrations of 15d-PGJ2, suggesting that loss of mitochondrial control of HO-1 leads to increased susceptibility to electrophilic stress in endothelial cells. The implications for these studies in understanding the balance between cytoprotection and cytotoxicity in the context of diseases such as atherosclerosis is discussed.

Effects of the organochlorine pesticide methoxychlor on dopamine metabolites and transporters in the mouse brain

Pesticide exposure has been suggested as a risk factor in developing Parkinson's disease (PD). While the molecular mechanism underlying this association is not clear, several studies have demonstrated a role for mitochondrial dysfunction and oxidative damage in PD. Although data on specific pesticides associated with PD are often lacking, several lines of evidence point to the potential involvement of the organochlorine class of pesticides. Previously, we have found that the organochlorine pesticide methoxychlor (mxc) causes mitochondrial dysfunction and oxidative stress in isolated mitochondria. Here, we sought to determine whether mxc-induced mitochondrial dysfunction results in oxidative damage and dysfunction of the dopamine system. Adult female CD1 mice were dosed with either vehicle (sesame oil) or mxc (16, 32, or 64 mg/kg/day) for 20 consecutive days. Following treatment, we observed a dose-related increase in protein carbonyl levels in non-synaptic mitochondria, indicating oxidative modification of mitochondrial proteins which may lead to mitochondrial dysfunction. Mxc exposure also caused a dose-related decrease in striatal levels of dopamine (16–31%), which were accompanied by decreased levels of the dopamine transporter (DAT; 35–48%) and the vesicular monoamine transporter 2 (VMAT2; 21–44%). Because mitochondrial dysfunction, oxidative damage, and decreased levels of DAT and VMAT2 are found in PD patients, our data suggest that mxc should be investigated as a possible candidate involved in the association of pesticides with increased risk for PD, particularly in highly exposed populations.

High fat diet induces dysregulation of hepatic oxygen gradients and mitochondrial function in vivo

NAFLD (non-alcoholic fatty liver disease), associated with obesity and the cardiometabolic syndrome, is an important medical problem affecting up to 20% of western populations. Evidence indicates that mitochondrial dysfunction plays a critical role in NAFLD initiation and progression to the more serious condition of NASH (non-alcoholic steatohepatitis). Herein we hypothesize that mitochondrial defects induced by exposure to a HFD (high fat diet) contribute to a hypoxic state in liver and this is associated with increased protein modification by RNS (reactive nitrogen species). To test this concept, C57BL/6 mice were pair-fed a control diet and HFD containing 35% and 71% total calories (1 cal≈4.184 J) from fat respectively, for 8 or 16 weeks and liver hypoxia, mitochondrial bioenergetics, NO (nitric oxide)-dependent control of respiration, and 3-NT (3-nitrotyrosine), a marker of protein modification by RNS, were examined. Feeding a HFD for 16 weeks induced NASH-like pathology accompanied by elevated triacylglycerols, increased CYP2E1 (cytochrome P450 2E1) and iNOS (inducible nitric oxide synthase) protein, and significantly enhanced hypoxia in the pericentral region of the liver. Mitochondria from the HFD group showed increased sensitivity to NO-dependent inhibition of respiration compared with controls. In addition, accumulation of 3-NT paralleled the hypoxia gradient in vivo and 3-NT levels were increased in mitochondrial proteins. Liver mitochondria from mice fed the HFD for 16 weeks exhibited depressed state 3 respiration, uncoupled respiration, cytochrome c oxidase activity, and mitochondrial membrane potential. These findings indicate that chronic exposure to a HFD negatively affects the bioenergetics of liver mitochondria and this probably contributes to hypoxic stress and deleterious NO-dependent modification of mitochondrial proteins.

Altered Proteome Biology of Cardiac Mitochondria Under Stress Conditions

Myocardial ischemia-reperfusion induces mitochondrial dysfunction and, depending upon the degree of injury, may lead to cardiac cell death. However, our ability to understand mitochondrial dysfunction has been hindered by an absence of molecular markers defining the various degrees of injury. To address this paucity of knowledge, we sought to characterize the impact of ischemic damage on mitochondrial proteome biology. We hypothesized that ischemic injury induces differential alterations in various mitochondrial subcompartments, that these proteomic changes are specific to the severity of injury, and that they are important to subsequent cellular adaptations to myocardial ischemic injury. Accordingly, an in vitro model of cardiac mitochondria injury in mice was established to examine two stress conditions: reversible injury (induced by mild calcium overload) and irreversible injury (induced by hypotonic stimuli). Both forms of injury had a drastic impact on the proteome biology of cardiac mitochondria. Altered mitochondrial function was concomitant with significant protein loss/shedding from the injured organelles. In the setting of mild calcium overload, mitochondria retained functionality despite the release of numerous proteins, and the majority of mitochondria remained intact. In contrast, hypotonic stimuli caused severe damage to mitochondrial structure and function, induced increased oxidative modification of mitochondrial proteins, and brought about detrimental changes to the subproteomes of the inner mitochondrial membrane and matrix. Using an established in vivo murine model of regional myocardial ischemic injury, we validated key observations made by the in vitro model. This preclinical investigation provides function and suborganelle location information on a repertoire of cardiac mitochondrial proteins sensitive to ischemia reperfusion stress and highlights protein clusters potentially involved in mitochondrial dysfunction in the setting of ischemic injury.

Renal mitochondrial damage and protein modification in type-2 diabetes.

Although mitochondrial reduction-oxidation (redox) stress and increase in membrane permeability play an important role in diabetic-associated renal microvasculopathies, it is unclear whether the intra-renal mitochondrial oxidative stress induces mitochondrial protein modifications, leading to increase mitochondrial membrane permeability. The hypothesis is that mitochondrial oxidative stress induces mitochondrial protein modification and leakage in the mitochondrial membrane in type-2 diabetes. The present study was conducted to determine the involvement of intra-renal mitochondrial oxidative stress in mitochondrial protein modifications and modulation of membrane permeability in the setting of type-2 diabetes. Diabetes was induced by 6-week regimen of a high calorie and fat diet in C57BL/6J mice (Am J Physiol 291:F694-F701, 2006). Subcellular fractionation was carried out in kidney tissue from wild type and diabetic mice. All fractions were highly enriched in their corresponding marker enzyme. Subcellular protein modifications were determined by Western blot and 2-D proteomics. The results suggest that diabetes-induced oxidative stress parallels an increase in NADPH oxidase-4 (NOX-4) and decrease in superoxide dismutase-1, 2 (SOD-1, 2) expression, in mitochondrial compartment. We observed loss of mitochondrial membrane permeability as evidenced by leakage of mitochondrial cytochrome c and prohibitin to the cytosol. However, there was no loss in control tissue. The 2-D Western blots for mitochondrial post-translational modification showed an increase in nitrotyrosine generation in diabetes. We conclude that diabetes-induced intra-renal mitochondrial oxidative stress is reflected by an increase in mitochondrial membrane permeability and protein modifications by nitrotyrosine generation.

ANESTHETIC PRECONDITIONING CONFERS ACUTE CARDIOPROTECTION VIA UP-REGULATION OF MANGANESE SUPEROXIDE DISMUTASE AND PRESERVATION OF MITOCHONDRIAL RESPIRATORY ENZYME ACTIVITY

The cellular and molecular mechanisms that underlie cardioprotection against I/R by anesthetic-induced preconditioning (APC) require further elucidation. Using isoflurane as a representative anesthetic, we evaluated the hypothesis that APC induces myocardial protection against I/R by attenuation of excessive reactive oxygen species and restoration of mitochondrial bioenergetics through postischemic up-regulation of manganese superoxide dismutase (MnSOD) expression and preservation of respiratory enzyme activity. Pentobarbital anesthetized open-chest Sprague-Dawley rats were subject to 30-min left coronary artery occlusion, followed by 120-min reperfusion. Before ischemia, rats were randomly assigned to receive 0.9% saline, two cycles of brief coronary artery occlusion and reperfusion, or a 30-min exposure to 1.0 minimum alveolar concentration isoflurane in the absence or presence of a specific mitochondrial adenosine triphosphate-sensitive potassium (KATP) channel blocker, 5-hydroxydecanoate; a membrane-permeable superoxide scavenger, 4-hydroxy-2,2,6,6-tetramethyl piperidinoxyl; or a NOS inhibitor, N(G)-nitro-L-arginine methyl ester. Isoflurane exposure induced an initial increase in myocardial superoxide (O2-), but not NO level. It also significantly decreased infarct size and restored mitochondrial respiratory enzyme activity or ATP production in I/R rat hearts, along with suppression of the O2- surge at reperfusion and increase in MnSOD expression or enzyme activity. These protective effects were abrogated by 5-hydroxydecanoate or 4-hydroxy-2,2,6,6-tetramethyl piperidinoxyl, but not by N(G)-nitro-L-arginine methyl ester pretreatment. These results suggest that opening of mitochondrial KATP channel, followed by O2- signaling, induces postischemic augmentation of MnSOD and preservation of mitochondrial respiratory enzyme activities, leading to attenuated cardiac O2- surge and restored ATP production during reperfusion, and underlie APC-induced cardioprotection.

Glyoxalase‐1 prevents mitochondrial protein modification and enhances lifespan in Caenorhabditis elegans

Studies of mutations affecting lifespan in Caenorhabditis elegans show that mitochondrial generation of reactive oxygen species (ROS) plays a major causative role in organismal aging. Here, we describe a novel mechanism for regulating mitochondrial ROS production and lifespan in C. elegans: progressive mitochondrial protein modification by the glycolysis-derived dicarbonyl metabolite methylglyoxal (MG). We demonstrate that the activity of glyoxalase-1, an enzyme detoxifying MG, is markedly reduced with age despite unchanged levels of glyoxalase-1 mRNA. The decrease in enzymatic activity promotes accumulation of MG-derived adducts and oxidative stress markers, which cause further inhibition of glyoxalase-1 expression. Over-expression of the C. elegans glyoxalase-1 orthologue CeGly decreases MG modifications of mitochondrial proteins and mitochondrial ROS production, and prolongs C. elegans lifespan. In contrast, knock-down of CeGly increases MG modifications of mitochondrial proteins and mitochondrial ROS production, and decreases C. elegans lifespan.

Rapid characterization of covalent modifications to rat brain mitochondrial proteins after ex vivo exposure to 4‐hydroxy‐2‐nonenal by liquid chromatography–tandem mass spectrometry using data‐dependent and neutral loss‐driven MS3 acquisition

The modification of mitochondrial proteins enriched from rat forebrain by the major lipid peroxidation product 4-hydroxy-2-nonenal (HNE) was investigated using high performance liquid chromatography (HPLC) and tandem mass spectrometry. Subcellular fractionation in conjunction with a 'shotgun-based' approach that involved both conventional data-dependent and neutral loss (NL)-driven MS(3) data acquisition on a hybrid linear ion trap-Fourier transform ion cyclotron resonance mass spectrometer (LTQ-FT) was utilized. Using a relatively rapid linear HPLC gradient (1 h) for complex mixture analysis, 24 sites of HNE modification on 15 unique proteins were identified which corresponded exclusively to Michael adduct formation on histidine residues. Since a number of HNE-modified peptides produced a predominant HNE NL fragment-ion signal upon collision-induced dissociation (CID), NL-driven MS(3) data-dependent acquisition was a valuable method to enhance fragmentation information for these particular modified peptides. Of the 24 HNE modification sites identified, approximately 25% were determined from the MS(3) spectra alone. We envision the reported methodology as an efficient screening approach for HNE modification site selectivity that could ultimately provide a foundation for the development of targeted schemes for the characterization of in vivo HNE-protein adducts.

Free Radicals, Mitochondria, and Oxidized Lipids

Mitochondria have long been known to play a critical role in maintaining the bioenergetic status of cells under physiological conditions. It was also recognized early in mitochondrial research that the reduction of oxygen to generate the free radical superoxide occurs at various sites in the respiratory chain and was postulated that this could lead to mitochondrial dysfunction in a variety of disease states. Over recent years, this view has broadened substantially with the discovery that reactive oxygen, nitrogen, and lipid species can also modulate physiological cell function through a process known as redox cell signaling. These redox active second messengers are formed through regulated enzymatic pathways, including those in the mitochondrion, and result in the posttranslational modification of mitochondrial proteins and DNA. In some cases, the signaling pathways lead to cytotoxicity. Under physiological conditions, the same mediators at low concentrations activate the cytoprotective signaling pathways that increase cellular antioxidants. Thus, it is critical to understand the mechanisms by which these pathways are distinguished to develop strategies that will lead to the prevention of cardiovascular disease. In this review, we describe recent evidence that supports the hypothesis that mitochondria have an important role in cell signaling, and so contribute to both the adaptation to oxidative stress and the development of vascular diseases.

Mitochondrial Matrix Phosphoproteome: Effect of Extra Mitochondrial Calcium

Post-translational modification of mitochondrial proteins by phosphorylation or dephosphorylation plays an essential role in numerous cell signaling pathways involved in regulating energy metabolism and in mitochondrion-induced apoptosis. Here we present a phosphoproteomic screen of the mitochondrial matrix proteins and begin to establish the protein phosphorylations acutely associated with calcium ions (Ca(2+)) signaling in porcine heart mitochondria. Forty-five phosphorylated proteins were detected by gel electrophoresis-mass spectrometry of Pro-Q Diamond staining, while many more Pro-Q Diamond-stained proteins evaded mass spectrometry detection. Time-dependent (32)P incorporation in intact mitochondria confirmed the extensive matrix protein phosphoryation and revealed the dynamic nature of this process. Classes of proteins that were detected included all of the mitochondrial respiratory chain complexes, as well as enzymes involved in intermediary metabolism, such as pyruvate dehydrogenase (PDH), citrate synthase, and acyl-CoA dehydrogenases. These data demonstrate that the phosphoproteome of the mitochondrial matrix is extensive and dynamic. Ca(2+) has previously been shown to activate various dehydrogenases, promote the generation of reactive oxygen species (ROS), and initiate apoptosis via cytochrome c release. To evaluate the Ca(2+) signaling network, the effects of a Ca(2+) challenge sufficient to release cytochrome c were evaluated on the mitochondrial phosphoproteome. Novel Ca(2+)-induced dephosphorylation was observed in manganese superoxide dismutase (MnSOD) as well as the previously characterized PDH. A Ca(2+) dose-dependent dephosphorylation of MnSOD was associated with an approximately 2-fold maximum increase in activity; neither the dephosphorylation nor activity changes were induced by ROS production in the absence of Ca(2+). These data demonstrate the use of a phosphoproteome screen in determining mitochondrial signaling pathways and reveal new pathways for Ca(2+) modification of mitochondrial function at the level of MnSOD.

Efficient execution of cell death in non-glycolytic cells requires the generation of ROS controlled by the activity of mitochondrial H + -ATP synthase

There is a large body of clinical data documenting that most human carcinomas contain reduced levels of the catalytic subunit of the mitochondrial H+-ATP synthase. In colon and lung cancer this alteration correlates with a poor patient prognosis. Furthermore, recent findings in colon cancer cells indicate that downregulation of the H+-ATP synthase is linked to the resistance of the cells to chemotherapy. However, the mechanism by which the H+-ATP synthase participates in cancer progression is unknown. In this work, we show that inhibitors of the H+-ATP synthase delay staurosporine (STS)-induced cell death in liver cells that are dependent on oxidative phosphorylation for energy provision whereas it has no effect on glycolytic cells. Efficient execution of cell death requires the generation of reactive oxygen species (ROS) controlled by the activity of the H+-ATP synthase in a process that is concurrent with the rapid disorganization of the cellular mitochondrial network. The generation of ROS after STS treatment is highly dependent on the mitochondrial membrane potential and most likely caused by reverse electron flow to Complex I. The generated ROS promote the carbonylation and covalent modification of cellular and mitochondrial proteins. Inhibition of the activity of the H+-ATP synthase blunted ROS production prevented the oxidation of cellular proteins and the modification of mitochondrial proteins delaying the release of cytochrome c and the execution of cell death. The results in this work establish the downregulation of the H+-ATP synthase, and thus of oxidative phosphorylation, as part of the molecular strategy adapted by cancer cells to avoid ROS-mediated cell death. Furthermore, the results provide a mechanistic explanation to understand chemotherapeutic resistance of cancer cells that rely on glycolysis as the main energy provision pathway.

Disulphide formation on mitochondrial protein thiols

A large number of proteins contain free thiols that can be modified by the formation of internal disulphides or by mixed disulphides with low-molecular-mass thiols. The majority of these latter modifications result from the interaction of protein thiols with the endogenous glutathione pool. Protein glutathionylation and disulphide formation are of significance both for defence against oxidative damage and in redox signalling. As mitochondria are central to both oxidative damage and redox signalling within the cell, these modifications of mitochondrial proteins are of particular importance. In the present study, we review the mechanisms and physiological significance of these processes.

Disulphide formation on mitochondrial protein thiols.

A large number of proteins contain free thiols that can be modified by the formation of internal disulphides or by mixed disulphides with low-molecular-mass thiols. The majority of these latter modifications result from the interaction of protein thiols with the endogenous glutathione pool. Protein glutathionylation and disulphide formation are of significance both for defence against oxidative damage and in redox signalling. As mitochondria are central to both oxidative damage and redox signalling within the cell, these modifications of mitochondrial proteins are of particular importance. In the present study, we review the mechanisms and physiological significance of these processes.

Poly-ADP-ribosylation is a prevalent post-translational modification of mitochondrial proteins in the brain

Poly-ADP-ribosylation is a prevalent post-translational modification of mitochondrial proteins in the brain

Etiology of Primary Biliary Cirrhosis: The Search for the Culprit

Primary biliary cirrhosis (PBC) is an organ-specific autoimmune disease characterized by the presence of high titer antimitochondrial autoantibodies (AMAs) and destruction of intrahepatic small bile ducts. Despite vigorous efforts in the characterization of autoantibodies and bile duct histopathology, the etiology of this disease is unclear. Although there is no correlation between the titer of AMAs and disease severity, the presence of AMAs usually occurs before symptoms of liver abnormalities. We believe that the production of AMAs is not an epiphenomenon, and an understanding of the mechanism of AMA induction will shed light on the etiology of PBC. Recent studies have suggested that the induction of PBC is multifactorial, in which the primary player involves the xenobiotics modification of mitochondrial proteins or exposure to xenobiotic-modified bacterial mitochondrial protein homologs, leading to breaking of tolerance to the human mitochondrial autoantigens and eventually liver pathology in genetic susceptible individuals. We discuss the immunophysiological characteristics of biliary epithelial cells, biochemistry of the 2-oxo-acid dehydrogenase complex, and environmental and genetic factors relevant to PBC.

Endurance training limits the functional alterations of heart rat mitochondria submitted to in vitro anoxia-reoxygenation

Background Studies analysing the effect of endurance training on heart mitochondrial function submitted to in vitro anoxia-reoxygenation (A-R) are missing. The present study aimed to investigate the effect of moderate endurance treadmill training (14 weeks) against rat heart mitochondrial dysfunction induced by in vitro A-R. Methods Respiratory parameters (state 3, state 4, ADP/O and respiratory control ratio—RCR) and oxidative damage markers (carbonyl groups and malondialdehyde) were determined in isolated mitochondria before and after 1 min anoxia followed by 4 min reoxygenation. Levels of heat shock protein 60 kDa (HSP60) and 70 kDa (HSP70) were measured before A-R in mitochondria and whole muscle homogenate, respectively. Results A-R significantly impaired the rate of state 3 and state 4 respiration, as well as the RCR and ADP/O in the sedentary group. However, mitochondrial state 3 respiration was significantly higher in trained than in the sedentary group both before and after A-R. The impairments in RCR, ADP/O ratio and state 4 induced by A-R in sedentary group were significantly attenuated in endurance-trained group. The inhibition of state 4 induced by GDP was significantly higher in trained than in sedentary group. Oxidative modifications of mitochondrial proteins and phospholipids were found in sedentary group after A-R, although limited in trained group. Increased levels of mitochondrial HSP60 and tissue HSP70 accompanied the lower decrease in the respiratory function after A-R observed in trained group. Conclusion We therefore concluded that endurance training limited the impairments on rat heart mitochondria caused by the oxidant insult inflicted by in vitro A-R.

Manganese superoxide dismutase and inducible nitric oxide synthase modify early oxidative events in acute Adriamycin-induced mitochondrial toxicity

In the present study, we used genetically engineered B6C3 mice [mice overexpressing manganese superoxide dismutase (TgM(+/+)), mice in which inducible nitric oxide synthase had been inactivated (iNOSKO(-/-)), and crosses of these two genotypes] to study the role of manganese superoxide dismutase (MnSOD) and inducible nitric oxide synthase (iNOS) in the development of acute Adriamycin-induced cardiotoxicity. Both nontransgenic and genetically engineered mice were treated with 20 mg/kg Adriamycin and cardiac left ventricular tissues studied at 0, 3, 6, and 24 hours. Ultrastructural damage and levels of 4-hydroxy-2-nonenal (4HNE) protein adducts and 3-nitrotyrosine (3NT) were determined in cardiomyocytes using immunogold ultrastructural techniques. Our previous results showed that Adriamycin caused mitochondrial injury without significant nuclear or cytoplasmic damage at early time points. Interestingly, overexpression of MnSOD protected against acute mitochondrial injury, whereas deficiency in iNOS potentiated mitochondrial injury in comparison with levels of injury present in cardiomyocyte mitochondria of nontransgenic mice. In TgM(+/+) mice, there was a significant inverse correlation between mitochondrial injury and 4HNE/3NT levels at all time points analyzed, suggesting that reactive oxygen species/reactive nitrogen species damage products directly regulated acute Adriamycin-induced mitochondrial injury in these mice. The present studies are the first to directly quantify the effects of MnSOD and iNOS on mitochondrial injury during acute Adriamycin-induced cardiotoxicity and show extensive and specific patterns of posttranslational modifications of mitochondrial proteins following Adriamycin treatment.