Electron Transport Chain Enzyme Activities Research Articles

Contribution of mitochondrial function to exercise-induced attenuation of renal dysfunction in spontaneously hypertensive rats.

It is well known that exercise training exhibits renal protective effects in animal models of hypertension and chronic renal failure. However, the mechanisms regulating these effects of exercise training remain unclear. This study aimed to investigate the role of mitochondrial function in exercise-induced attenuation of renal injury in spontaneously hypertensive rats (SHR). The adult male SHR and age-matched normotensive Wistar-Kyoto rats (WKY) were given moderate-intensity exercise for 12 weeks or treated with MitoQ10 for 8 weeks. In this work, exercise training in SHR reduced blood pressure, and effectively attenuated renal dysfunction, marked by reduced creatinine excretion, albuminuria, blood urea nitrogen, and glomerular sclerosis. Exercise training in SHR reduced MDA levels in plasma and kidneys and suppressed formation of 3-nitrotyrosine in kidneys. Exercise training suppressed mitochondrial ROS and [Formula: see text] formation, enhanced ATP formation, reduced mitochondrial swelling, and restored electron transport chain enzyme activity in kidneys of SHR. Furthermore, exercise training upregulated protein expression of uncoupling protein 2 and manganese superoxide dismutase in kidneys of SHR. In addition, treatment with mitochondria-targeted antioxidant MitoQ10 exhibited similar renal protective effects in SHR. In conclusion, chronic aerobic exercise training preserved mitochondrial function and abated oxidative stress in the kidneys of SHR, which may in part explain the protective effect of exercise on renal function and structure in hypertensive individuals.

Mitochondrial Dihydrolipoamide Dehydrogenase is Upregulated in Response to Intermittent Hypoxic Preconditioning.

Intermittent hypoxia preconditioning (IHP) has been shown to protect neurons against ischemic stroke injury. Studying how proteins respond to IHP may identify targets that can help fight stroke. The objective of the present study was to investigate whether mitochondrial dihydrolipoamide dehydrogenase (DLDH) would respond to IHP and if so, whether such a response could be linked to neuroprotection in ischemic stroke injury. To do this, we subjected male rats to IHP for 20 days and measured the content and activity of DLDH as well as the three α-keto acid dehydrogenase complexes that contain DLDH. We also measured mitochondrial electron transport chain enzyme activities. Results show that DLDH content was indeed upregulated by IHP and this upregulation did not alter the activities of the three α-keto acid dehydrogenase complexes. Results also show that the activities of the five mitochondrial complexes (I-V) were not altered either by IHP. To investigate whether IHP-induced DLDH upregulation is linked to neuroprotection against ischemic stroke injury, we subjected both DLDH deficient mouse and DLDH transgenic mouse to stroke surgery followed by measurement of brain infarction volume. Results indicate that while mouse deficient in DLDH had exacerbated brain injury after stroke, mouse overexpressing human DLDH also showed increased brain injury after stroke. Therefore, the physiological significance of IHP-induced DLDH upregulation remains to be further investigated.

Fluoxetine-induced pancreatic beta cell dysfunction: New insight into the benefits of folic acid in the treatment of depression

BackgroundMajor depressive disorder is a common psychiatric illness with reported prevalence rates of 12–16% in persons aged 12 and over. Depression is also associated with a high risk of new onset of type 2 diabetes (T2D). This relationship between depression and diabetes may be related to depression itself and/or drugs prescribed. Importantly, the use of selective serotonin reuptake inhibitors (SSRIs), the most commonly prescribed class of antidepressants, increases the risk of developing T2D. However, the mechanism(s) underlying this association remains elusive. MethodsHere we examine the effects of the SSRI fluoxetine (Prozac®) on beta cell function utilizing INS-1E cells, a rat beta cell line, to elucidate the underlying molecular mechanisms. ResultsFluoxetine treatment significantly reduced glucose stimulated insulin secretion (GSIS). This decreased beta cell function was concomitant with an increased production of reactive oxygen species and oxidative damage which may contribute to decreased mitochondrial electron transport chain enzyme (ETC) activity. Importantly the fluoxetine-induced deficits in beta cell function were prevented by the addition of the antioxidant folic acid. LimitationsThese studies were conducted in vitro; the in vivo relevance remains to be determined. ConclusionsThese findings suggest that use of SSRI antidepressants may increase the risk of new-onset T2D by causing oxidative stress in pancreatic beta cells. However, folic acid supplementation in patients taking SSRIs may reduce the risk of new onset diabetes via protection of normal beta cell function.

Effect of Coenzyme Q10 supplementation on mitochondrial electron transport chain activity and mitochondrial oxidative stress in Coenzyme Q10 deficient human neuronal cells

Primary Coenzyme Q10 (CoQ10) deficiency is an autosomal recessive disorder with a heterogeneous clinical presentation. Common presenting features include both muscle and neurological dysfunction. Muscle abnormalities can improve, both clinically and biochemically following CoQ10 supplementation, however neurological symptoms are only partially ameliorated. At present, the reasons for the refractory nature of the neurological dysfunction remain unknown. In order to investigate this at the biochemical level we evaluated the effect of CoQ10 treatment upon a previously established neuronal cell model of CoQ10 deficiency. This model was established by treatment of human SH-SY5Y neuronal cells with 1mM para-aminobenzoic acid (PABA) which induced a 54% decrease in cellular CoQ10 status. CoQ10 treatment (2.5μM) for 5 days significantly (p<0.0005) decreased the level of mitochondrial superoxide in the CoQ10 deficient neurons. In addition, CoQ10 treatment (5μM) restored mitochondrial membrane potential to 90% of the control level. However, CoQ10 treatment (10μM) was only partially effective at restoring mitochondrial electron transport chain (ETC) enzyme activities. ETC complexes II/III activity was significantly (p<0.05) increased to 82.5% of control levels. ETC complexes I and IV activities were restored to 71.1% and 77.7%, respectively of control levels.In conclusion, the results of this study have indicated that although mitochondrial oxidative stress can be attenuated in CoQ10 deficient neurons following CoQ10 supplementation, ETC enzyme activities appear partially refractory to treatment. Accordingly, treatment with >10μM CoQ10 may be required to restore ETC enzyme activities to control level. Accordingly, these results have important implication for the treatment of the neurological presentations of CoQ10 deficiency and indicate that high doses of CoQ10 may be required to elicit therapeutic efficacy.

Beating oxygen: chronic anoxia exposure reduces mitochondrial F1FO-ATPase activity in turtle (Trachemys scripta) heart

The freshwater turtle Trachemys scripta can survive in the complete absence of O2 (anoxia) for periods lasting several months. In mammals, anoxia leads to mitochondrial dysfunction, which culminates in cellular necrosis and apoptosis. Despite the obvious clinical benefits of understanding anoxia tolerance, little is known about the effects of chronic oxygen deprivation on the function of turtle mitochondria. In this study, we compared mitochondrial function in hearts of T. scripta exposed to either normoxia or 2 weeks of complete anoxia at 5°C and during simulated acute anoxia/reoxygenation. Mitochondrial respiration, electron transport chain activities, enzyme activities, proton conductance and membrane potential were measured in permeabilised cardiac fibres and isolated mitochondria. Two weeks of anoxia exposure at 5°C resulted in an increase in lactate, and decreases in ATP, glycogen, pH and phosphocreatine in the heart. Mitochondrial proton conductance and membrane potential were similar between experimental groups, while aerobic capacity was dramatically reduced. The reduced aerobic capacity was the result of a severe downregulation of the F1FO-ATPase (Complex V), which we assessed as a decrease in enzyme activity. Furthermore, in stark contrast to mammalian paradigms, isolated turtle heart mitochondria endured 20 min of anoxia followed by reoxygenation without any impact on subsequent ADP-stimulated O2 consumption (State III respiration) or State IV respiration. Results from this study demonstrate that turtle mitochondria remodel in response to chronic anoxia exposure and a reduction in Complex V activity is a fundamental component of mitochondrial and cellular anoxia survival.

Assessment of mitochondrial electron transport chain function in a primary astrocyte cell model of hyperhomocystinaemia

Elevated plasma homocysteine (Hcy) has been detected in patients with various neurodegenerative conditions. Studies on neurones and cerebral tissue have revealed that hyperhomocystinaemia may inhibit mitochondrial electron transport chain (ETC) enzyme activity resulting in neuronal morbidity. As astrocytes convey a protective and supportive role towards neurones, we postulated that Hcy-induced astrocytic ETC inhibition may contribute to neurological dysfunction. In order to investigate this hypothesis, we established a cellular model of hyperhomocystinaemia using primary rat astrocytes. Which were incubated were incubated with 200 µM, 500 µM Hcy and the Hcy metabolite, thiolactone (10 µM). Following 96 h of incubation with 200 µM and 500 µM Hcy, an approximate two-fold (1.11 nmol/mg) and three-fold (1.45 nmol/mg) increase in mitochondrial levels of Hcy, respectively, were detected compared to control levels (0.54 nmol/mg). However, on exposure to Hcy (200 or 500 µM) and Hcy-thiolactone (10 µM), the activities of astrocytic ETC complex I, II–III and IV were found to be comparable to control levels. In addition, the extracellular lactate:pyruvate ratio and the intracellular glutathione status of primary rat astrocytes were not significantly different between Hcy (200 or 500 µM) treated and controls. In conclusion, the results of this study suggest that Hcy induced impairment of astrocytic ETC function may not contribute to the pathophysiology of hyperhomocystinaemia.

The influence of dietary lipid composition on liver mitochondria from mice following 1 month of calorie restriction

To investigate the role mitochondrial membrane lipids play in the actions of CR (calorie restriction), C57BL/6 mice were assigned to four groups (control and three 40% CR groups) and the CR groups were fed diets containing soya bean oil (also in the control diet), fish oil or lard. The fatty acid composition of the major mitochondrial phospholipid classes, proton leak and H2O2 production were measured in liver mitochondria following 1 month of CR. The results indicate that mitochondrial phospholipid fatty acids reflect the PUFA (polyunsaturated fatty acid) profile of the dietary lipid sources. CR significantly decreased the capacity of ROS (reactive oxygen species) production by Complex III but did not markedly alter proton leak and ETC (electron transport chain) enzyme activities. Within the CR regimens, the CR-fish group had decreased ROS production by both Complexes I and III, and increased proton leak when compared with the other CR groups. The CR-lard group showed the lowest proton leak compared with the other CR groups. The ETC enzyme activity measurements in the CR regimens showed that Complex I activity was decreased in both the CR-fish and CR-lard groups. Moreover, the CR-fish group also had lower Complex II activity compared with the other CR groups. These results indicate that dietary lipid composition does influence liver mitochondrial phospholipid composition, ROS production, proton leak and ETC enzyme activities in CR animals.

Impact of Cyanidin-3-Glucoside on Glycated LDL-Induced NADPH Oxidase Activation, Mitochondrial Dysfunction and Cell Viability in Cultured Vascular Endothelial Cells

Elevated levels of glycated low density lipoprotein (glyLDL) are frequently detected in diabetic patients. Previous studies demonstrated that glyLDL increased the production of reactive oxygen species (ROS), activated NADPH oxidase (NOX) and suppressed mitochondrial electron transport chain (mETC) enzyme activities in vascular endothelial cells (EC). The present study examined the effects of cyanidin-3-glucoside (C3G), a type of anthocyanin abundant in dark-skinned berries, on glyLDL-induced ROS production, NOX activation and mETC enzyme activity in porcine aortic EC (PAEC). Co-treatment of C3G prevented glyLDL-induced upregulation of NOX4 and intracellular superoxide production in EC. C3G normalized glyLDL-induced inhibition on the enzyme activities of mETC Complex I and III, as well as the abundances of NADH dehydrogenase 1 in Complex I and cytochrome b in Complex III in EC. Blocking antibody for the receptor of advanced glycation end products (RAGE) prevented glyLDL-induced changes in NOX and mETC enzymes. Combination of C3G and RAGE antibody did not significantly enhance glyLDL-induced inhibition of NOX or mETC enzymes. C3G reduced glyLDL-induced RAGE expression with the presence of RAGE antibody. C3G prevented prolonged incubation with the glyLDL-induced decrease in cell viability and the imbalance between key regulators for cell viability (cleaved caspase 3 and B cell Lyphoma-2) in EC. The findings suggest that RAGE plays an important role in glyLDL-induced oxidative stress in vascular EC. C3G may prevent glyLDL-induced NOX activation, the impairment of mETC enzymes and cell viability in cultured vascular EC.

The effect of smoking cessation pharmacotherapies on pancreatic beta cell function

The goal of our study was to evaluate whether drugs currently used for smoking cessation (i.e., nicotine replacement therapy, varenicline [a partial agonist at nicotinic acetylcholine receptors (nAChR)] and bupropion [which acts in part as a nAChR antagonist]) can affect beta cell function and determine the mechanism(s) of this effect. INS-1E cells, a rat beta cell line, were treated with nicotine, varenicline and bupropion to determine their effects on beta cell function, mitochondrial electron transport chain enzyme activity and cellular/oxidative stress. Treatment of INS-1E cells with equimolar concentrations (1μM) of three test compounds resulted in an ablation of normal glucose-stimulated insulin secretion by the cells. This disruption of normal beta cell function was associated with mitochondrial dysfunction since all three compounds tested significantly decreased the activity of mitochondrial electron transport chain enzyme activity. These results raise the possibility that the currently available smoking cessation pharmacotherapies may also have adverse effects on beta cell function and thus glycemic control in vivo. Therefore whether or not the use of nicotine replacement therapy, varenicline and bupropion can cause endocrine changes which are consistent with impaired pancreatic function warrants further investigation.

Beating anoxia: mitochondrial plasticity in the turtle heart

Freshwater turtles can survive without oxygen for several months. The cellular mechanisms that allow the turtle heart to survive without oxygen have remained almost totally unexplored. In particular, it is not known how the turtle heart avoids anoxic damage to the mitochondria. The objective of this study was to make a comprehensive analysis of mitochondrial function in chronically and acutely anoxic mitochondria from the heart of Trachemys scripta. Turtles were acclimated to 5°C under either normoxic or anoxic (2 weeks) conditions. Mitochondrial respiration, electron transport chain (ETC) activities, ADP affinity, enzyme activities, proton leak and membrane potential were measured in permeabilized cardiac fibres and isolated mitochondria. 2 weeks of anoxia led to a decrease in aerobic capacity and an increase in proton conductance (proton leak) of the mitochondrial membrane. The mechanism behind this plasticity was a downregulation of Complex V in the ETC. This strategy serves two purposes; to conserve ATP when energy is limited and to reduce the production of reactive oxygen species. Turtle heart mitochondria endured 20 min of simulated anoxia followed by reperfusion without any impact on subsequent ADP stimulated oxygen consumption, State IV respiration or respiratory control ratio. Thus, turtle mitochondria are robust and intrinsically tolerant to anoxia/reoxygenation.

Chronic hydrogen-rich saline treatment reduces oxidative stress and attenuates left ventricular hypertrophy in spontaneous hypertensive rats

In hypertensive animals and patients, oxidative stress represents the primary risk factor for progression of left ventricular hypertrophy. Recently, it has been demonstrated that hydrogen, as a novel antioxidant, can selectively reduce hydroxyl radicals and peroxynitrite anion to exert therapeutic antioxidant activity. In the current study, we explored the effect of chronic treatment with hydrogen-rich saline (HRS) on left ventricular hypertrophy in spontaneously hypertensive rats (SHR). The 8-week-old male SHR and age-matched Wistar-Kyoto rats (WKY) were randomized into HRS-treated (6 ml/kg/day for 3 months, i.p.) and vehicle-treated groups. HRS treatment had no significant effect on blood pressure, but it effectively attenuated left ventricular hypertrophy in SHR. HRS treatment abated oxidative stress, restored the activity of antioxidant enzymes including GPx, GST, catalase, and SOD, suppressed NADPH oxidase activity and downregulated Nox2 and Nox4 expression in left ventricles of SHR. HRS treatment suppressed pro-inflammatory cytokines including IL-1β, IL-6, TNF-α, and MCP-1, and inhibited NF-κB activation through preventing IκBα degradation in left ventricles of SHR. HRS treatment preserved mitochondrial function through restoring electron transport chain enzyme activity, repressing ROS formation, and enhancing ATP production in left ventricles of SHR. Moreover, HRS treatment suppressed ACE expression and locally reduced angiotensin II generation in left ventricles of SHR. In conclusion, HRS treatment attenuates left ventricular hypertrophy through abating oxidative stress, suppressing inflammatory process, preserving mitochondrial function, in which suppression of HRS on angiotensin II in left ventricles locally might be involved.

Rat Cardiac Mitochondrial Sub-populations Show Distinct Features of Oxidative Phosphorylation during Ischemia, Reperfusion and Ischemic Preconditioning

Background: Inter-fibrillar (IFM) and sub-sarcolemmal (SSM) mitochondria are two distinct mitochondrial sub-populations and are expected to behave differently during pathological conditions. This study was undertaken to compare functional oxidative phosphorylation (OXPHOS) in IFM and SSM during ischemia, reperfusion and ischemic preconditioning. Methods: Langendorff perfused Wistar rat hearts were subjected to 35minutes ischemia, 60minutes reperfusion and ischemic preconditioning (IPC) procedure (3cycles of 2-minutes ischemia followed by 3-minutes reperfusion).Subsequently IFM and SSM were isolated, and mitochondrial electron transport chain (ETC) enzyme activities and respiration were measured immediately. Results: Functional enzyme activities of ETC in IFM and SSM showed prominent differences especially in the proximal part of ETC enzymes during ischemia and reperfusion. SSM favor FADH₂while IFM prefer NADH as the main reducing equivalent for electron transport during ischemia and reperfusion. IPC preserved ETC enzyme activities in both IFM and SSM rendering cardio protection. Similarly IPC preserve ADP stimulated respiration with glutamate and malate as substrate in both sub populations, but not in IFM, with succinate as substrate. Apparently, the preconditioning imparts enhanced protection more to SSM than IFM during ischemia and reperfusion and especially to the proximal part of the ETC. Conclusion: We propose that mitochondrial dysfunction, one of the major targets of myocardial ischemia reperfusion injury needs to be evaluated by the synergic effect of both IFM and SSM.

Sirtuin 1 (SIRT1) Deacetylase Activity Is Not Required for Mitochondrial Biogenesis or Peroxisome Proliferator-activated Receptor-γ Coactivator-1α (PGC-1α) Deacetylation following Endurance Exercise

The protein deacetylase, sirtuin 1 (SIRT1), is a proposed master regulator of exercise-induced mitochondrial biogenesis in skeletal muscle, primarily via its ability to deacetylate and activate peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α). To investigate regulation of mitochondrial biogenesis by SIRT1 in vivo, we generated mice lacking SIRT1 deacetylase activity in skeletal muscle (mKO). We hypothesized that deacetylation of PGC-1α and mitochondrial biogenesis in sedentary mice and after endurance exercise would be impaired in mKO mice. Skeletal muscle contractile characteristics were determined in extensor digitorum longus muscle ex vivo. Mitochondrial biogenesis was assessed after 20 days of voluntary wheel running by measuring electron transport chain protein content, enzyme activity, and mitochondrial DNA expression. PGC-1α expression, nuclear localization, acetylation, and interacting protein association were determined following an acute bout of treadmill exercise (AEX) using co-immunoprecipitation and immunoblotting. Contrary to our hypothesis, skeletal muscle endurance, electron transport chain activity, and voluntary wheel running-induced mitochondrial biogenesis were not impaired in mKO versus wild-type (WT) mice. Moreover, PGC-1α expression, nuclear translocation, activity, and deacetylation after AEX were similar in mKO versus WT mice. Alternatively, we made the novel observation that deacetylation of PGC-1α after AEX occurs in parallel with reduced nuclear abundance of the acetyltransferase, general control of amino-acid synthesis 5 (GCN5), as well as reduced association between GCN5 and nuclear PGC-1α. These findings demonstrate that SIRT1 deacetylase activity is not required for exercise-induced deacetylation of PGC-1α or mitochondrial biogenesis in skeletal muscle and suggest that changes in GCN5 acetyltransferase activity may be an important regulator of PGC-1α activity after exercise.

SIRT1 Deacetylase Activity Is Not Required For Contraction-Induced Mitochondrial Biogenesis In Skeletal Muscle

The class III NAD+-dependent protein deacetylase, sirtuin1 (SIRT1), has been proposed as a key integrator/regulator of mitochondrial biogenesis in skeletal muscle due to its sensitivity to NAD+ and its ability to increase the activity of the transcriptional co-activator, PGC-1α. PURPOSE: To determine whether muscle-specific deletion of SIRT1 deacetylase activity (mKOSIRT1) blunts mitochondrial biogenesis following acute (AEX) or chronic (CEX) endurance exercise. METHODS: To generate mKOSIRT1, mice in which exon 4 of the SIRT1 gene was floxed, were crossed with MCK-Cre mice, resulting in offspring with muscle-specific "knockout" of deacetylase activity. For CEX studies, mKOSIRT1 and wildtype/floxed (WT) mice had free access to a running wheel for 21 days, after which skeletal muscle electron transport chain (ETC) enzyme activity and markers of mitochondrial biogenesis were measured. For AEX studies, mice ran for 1h on a motorized treadmill and skeletal muscle gene expression and PGC-1α acetylation status were measured 3h post-exercise. RESULTS: In WT mice, CEX increased mitochondrial and PGC-1α protein abundance, ETC enzyme activity and mtDNA content (P<0.05), and, importantly, this enhancement was not impaired in mKOSIRT1 mice. After AEX, PGC-1α expression was increased in both groups (∼4 fold; P<0.05), and increased to a greater extent in the mKOSIRT1 compared to WT mice (P<0.05). In addition, AEX increased the expression of a number of PGC-1α target genes including PPARα, CPT1, cytochrome-c and mitofusin-2, with the induction similar (2-4 fold) between mKOSIRT1 and WT mice (P<0.05). Interestingly, we also found that PGC-1α was deacetylated to the same extent in mKOSIRT1 and WT mice (P<0.05), suggesting that SIRT1 is not the primary regulator of PGC-1α acetylation status, in vivo. CONCLUSION: Altogether, our data suggest that, 1) SIRT1 activity is not required for endurance training-induced mitochondrial biogenesis in skeletal muscle and, 2) a protein other than SIRT1 mediates PGC-1α deacetylation after exercise.

Complex I-Associated Hydrogen Peroxide Production Is Decreased and Electron Transport Chain Enzyme Activities Are Altered in n-3 Enriched fat-1 Mice

The polyunsaturated nature of n-3 fatty acids makes them prone to oxidative damage. However, it is not clear if n-3 fatty acids are simply a passive site for oxidative attack or if they also modulate mitochondrial reactive oxygen species (ROS) production. The present study used fat-1 transgenic mice, that are capable of synthesizing n-3 fatty acids, to investigate the influence of increases in n-3 fatty acids and resultant decreases in the n-6∶n-3 ratio on liver mitochondrial H2O2 production and electron transport chain (ETC) activity. There was an increase in n-3 fatty acids and a decrease in the n-6∶n-3 ratio in liver mitochondria from the fat-1 compared to control mice. This change was largely due to alterations in the fatty acid composition of phosphatidylcholine and phosphatidylethanolamine, with only a small percentage of fatty acids in cardiolipin being altered in the fat-1 animals. The lipid changes in the fat-1 mice were associated with a decrease (p<0.05) in the activity of ETC complex I and increases (p<0.05) in the activities of complexes III and IV. Mitochondrial H2O2 production with either succinate or succinate/glutamate/malate substrates was also decreased (p<0.05) in the fat-1 mice. This change in H2O2 production was due to a decrease in ROS production from ETC complex I in the fat-1 animals. These results indicate that the fatty acid changes in fat-1 liver mitochondria may at least partially oppose oxidative stress by limiting ROS production from ETC complex I.

Omega‐3‐producing fat‐1 transgenic mice display reduced complex I‐associated hydrogen peroxide production and enzyme activity

The fat‐1 gene of C. elegans encodes a desaturase, which can convert n‐6 to n‐3 fatty acids. A transgenic mouse that constitutively expresses this gene was used as a mammalian model of increased tissue n‐3 fatty acids. The aim of the study was to evaluate the influence of changes in membrane fatty acid composition on the lipid‐protein interaction, specifically on the production of H2O2 and electron transport chain enzyme activities. Liver mitochondria were isolated by differential centrifugation and the activities of the electron transport chain enzymes determined spectrophotometrically, while H2O2 production was determined fluorimetrically. Fatty acid analyses of mitochondrial membranes were undertaken on Percoll‐purified mitochondria, and performed by Lipomics Technology, Inc. The results indicate that the transgenic mice show a decreased H2O2 production at complex I, as well as decreased complex I enzyme activity. Total H2O2 production was also decreased when mitochondria were respiring on succinate or succinate/glutamate/malate. Fatty acid analysis showed an increased n‐3/n‐6 ratio in mitochondrial PC, PE and LyPC lipid classes, with arachidonic acid (n‐6) decreased and eicosapentaenoic acid (n‐3) increased in all phospholipid classes. The results indicate that decreased complex I‐linked ROS production may provide some protection against oxidative stress in membranes with high n‐3 levels.

Mitochondrial cytochrome c release: a factor to consider in mitochondrial disease?

The pathogenesis of mitochondrial disorders has largely focused on the impairment of cellular energy metabolism. However, mitochondrial dysfunction has also been implicated as a factor in the initiation of apoptosis due to the translocation of cytochrome c, from mitochondria to the cytosol, and the subsequent cleavage of pro-caspase 3. In this study, we determined the cytochrome c content of cytosols (skeletal muscle) prepared from 22 patients with evidence of compromised mitochondrial electron transport chain enzyme activity and 26 disease controls. The cytochrome c content of the mitochondrial electron transport chain-deficient group was found to be significantly (p < 0.02) elevated when compared with the control group (63.7 +/- 15.5 versus 27.7 +/- 2.5 ng/mg protein). Furthermore, a relationship between the cytosolic cytochrome c content of skeletal muscle and complex I and complex IV activities was demonstrated. Such data raise the possibility that mitochondrial cytochrome c release may be a feature of mitochondrial disorders, particularly for those patients with marked deficiencies of respiratory chain enzymes. Whether initiation of apoptosis occurs as a direct consequence of this cytochrome c release has not been fully evaluated here. However, for one patient with the greatest documented cytosolic cytochrome c content, caspase 3 could be demonstrated in the cytosolic preparation. Further work is required in order to establish whether a relationship also exists between caspase 3 formation and the magnitude of respiratory chain deficiency.

Abstract 2609: Mitochondrial Oxidative Stress Promotes Failure of the Pressure-Loaded Infant Right Ventricle

Background: Right ventricular hypertrophy (RVH) and failure induced by chronic RV pressure overload is a major problem in congenital heart disease. We have previously shown that mitochondrial (Mito) damage accompanied load-induced infant RV failure. Here we assessed Mito electron transport chain(ETC) dysfunction and Mito oxidative stress in the transition of infant RVH to failure. Methods : Newborn rabbits underwent pulmonary artery banding (or sham) causing stable RV pressure load (~40 mmHg) and RVH by 2 weeks; RV contractile function (echocardiography) was preserved for ~4 weeks, followed by failure onset at ~8 weeks. Thus, RV free wall was assayed at 3, 4 and 8 weeks for ETC enzyme activities, high energy phosphates, Mito GSH/GSSH ratio (index of Mito ROS scavenging) , Mito 8-hydroxy-2-deoxyguanosine (8-OHdG, oxidative Mito DNA damage), Mito protein carbonyls (oxidative Mito protein damage) and Mito ultrastructure (electron microscopy). Results: By 4 weeks (RVH, preserved function), Mito 8-OHdG and protein carbonyl markedly increased, accompanied by decreased complex I and III activity. At 8 weeks (early failure), there were further increases in Mito 8-OHdG and protein carbonyls, significantly decreased GSH/GSSG ratio, further loss of complex I and III, and decreased activities of complexes IV and V; ATP content and ATP/ADP ratio were also significantly reduced. In parallel the number of abnormal disrupted RV mitochondria increased markedly. Conclusion: Pressure-load of the infant RV rapidly induces Mito oxidative stress. Due to the limited Mito scavenging and repair systems, this results in a cycle of Mito DNA and protein damage, progressive loss of ETC enzyme activity and further oxidative stress, as well as loss of functional mitochondria. This load-induced oxidative stress may be a therapeutic target to preserve RV function and oxidative metabolism and prevent the transition of RVH to RV failure. Changes in banded and sham-operated hearts after surgery

S10.28 Differences in H2O2 production, ΔΨm, JO2, electron transport chain enzyme activities and glutamate release in synaptosomes isolated from 6- and 18-month old rats

S10.28 Differences in H2O2 production, ΔΨm, JO2, electron transport chain enzyme activities and glutamate release in synaptosomes isolated from 6- and 18-month old rats

Age-related changes in H2O2 production and bioenergetics in rat brain synaptosomes

Detrimental changes to mitochondrial function have been shown to occur with age. In this study we examined the levels of H2O2 production, in situ mitochondrial membrane potential (Δψm), oxygen consumption (JO2) and electron transport chain (ETC) enzyme activities in synaptosomes isolated from rats of two age groups, 6 and 18 months. The rate of H2O2 production in synaptosomes was found to be higher in the 18-month old group compared to that of 6-month old. Δψm was found to be significantly lower in synaptosomes from the older rats, which also correlated with a reduction in JO2. Measurement of the individual electron transport chain enzyme activities revealed that reduced complex II/III and complex IV activities were the possible contributors to the reduced bioenergetic function in synaptosomes from the older rats. These data suggest that ageing may lead to increased nerve terminal H2O2 production while simultaneous deleterious effects on bioenergetic function occur in in situ synaptosomal mitochondria. In addition, Ca2+-independent glutamate release was found to be increased at lower levels of complex I inhibition in the synaptosomes from older rats, suggesting that reduction of mitochondrial function may potentiate excitotoxic conditions in the ageing brain.