Isolated Rat Heart Mitochondria Research Articles

Cytochrome c Oxidase Inhibition by ATP Decreases Mitochondrial ROS Production.

This study addresses the eventual consequence of cytochrome c oxidase (CytOx) inhibition by ATP at high ATP/ADP ratio in isolated rat heart mitochondria. Earlier, it has been demonstrated that the mechanism of allosteric ATP inhibition of CytOx is one of the key regulations of mitochondrial functions. It is relevant that aiming to maintain a high ATP/ADP ratio for the measurement of CytOx activity effectuating the enzymatic inhibition as well as mitochondrial respiration, optimal concentration of mitochondria is critically important. Likewise, only at this concentration, were the differences in ΔΨm and ROS concentrations measured under various conditions significant. Moreover, when CytOx activity was inhibited in the presence of ATP, mitochondrial respiration and ΔΨm both remained static, while the ROS production was markedly decreased. Consubstantial results were found when the electron transport chain was inhibited by antimycin A, letting only CytOx remain functional to support the energy production. This seems to corroborate that the decrease in mitochondrial ROS production is solely the effect of ATP binding to CytOx which results in static respiration as well as membrane potential.

Celecoxib decreases mitochondrial complex IV activity and induces oxidative stress in isolated rat heart mitochondria: An analysis for its cardiotoxic adverse effect.

In spite of the cardiotoxic effect of selective cyclooxygenase-2 inhibitors, they are most widely used as anti-inflammatory and analgesic drugs. Today, valdecoxib and rofecoxib have been withdrawn in the market but celecoxib remains. In this study, we focused on an analysis of celecoxib toxic effects on isolated mitochondria. Isolated rat heart mitochondria were obtained using differential centrifugation. Using flow cytometry and biochemical assays, we searched succinate dehydrogenases, mitochondrial membrane potential (MMP), reactive oxygen species (ROS) formation, mitochondrial swelling, ATP/ADP ratio, lipid peroxidation, and mitochondrial complexes activity in rat heart isolated mitochondria. Herein, our results indicated a significant decrease in the activity of complex IV after exposure with celecoxib (16 µg/ml). This decrease in the activity of complex IV is paralleled by the MMP collapse, ROS formation, mitochondrial swelling, depletion of ATP, and lipid peroxidation. For the first time, this introductory study has shown a significant decrease in the activity of complexIV and mitochondrial dysfunction after exposure with celecoxib in rat heart isolated mitochondria.

Antioxidant activity of calcitriol reduces direct methamphetamine-induced mitochondrial dysfunction in isolated rat heart mitochondria

We investigated this hypothesis that methamphetamine could directly cause mitochondrial dysfunction and calcitriol could reduce its adverse effects in rat heart isolated mitochondria. Results indicated that 250 µM methamphetamine caused a deleterious alteration in mitochondrial functions, reactive oxygen species (ROS) production, mitochondrial membrane potential (MMP) collapse, mitochondrial swelling, oxidative stress and lipid peroxidation. While our results indicated that calcitriol (5 µM) can reduce methamphetamine-induced toxicity in isolated cardiac mitochondria. Altogether, the results of the current study showed that methamphetamine directly induce mitochondrial dysfunction through oxidative stress in isolated cardiac mitochondria, which were ameliorated by calcitriol with its antioxidant potential.

Analysis of toxicity effects of delta-9-tetrahydrocannabinol on isolated rat heart mitochondria

Mitochondria have the main roles in myocardial tissue homeostasis, through providing ATP for the vital enzymes in intermediate metabolism, contractile apparatus and maintaining ion homeostasis. Mitochondria-related cardiotoxicity results from the exposure with illicit drugs have previously reported. These illicit drugs interference with processes of normal mitochondrial homeostasis and lead to mitochondrial dysfunction and mitochondrial-related oxidative stress. Cannabis consumption has been shown to cause ventricular tachycardia, to increase the risk of myocardial infarction (MI) and potentially sudden death. Here, we investigated this hypothesis that delta-9-tetrahydrocannabinol (Delta-9-THC) as a main cannabinoid found in cannabis could directly cause mitochondrial dysfunction. Cardiac mitochondria were isolated with mechanical lysis and differential centrifugation form rat heart. The isolated cardiac mitochondria were treated with different concentrations of THC (1, 5, 10, 50, 100 and 500 µM) for 1 hour at 37 °C. Then, succinate dehydrogenase (SDH) activity, mitochondrial swelling, reactive oxygen species (ROS) formation, mitochondrial membrane potential (MMP) collapse and lipid peroxidation were measured in the treated and nontreated isolated cardiac mitochondria. Our observation showed that THC did not cause a deleterious alteration in mitochondrial functions, ROS production, MMP collapse, mitochondrial swelling, oxidative stress and lipid peroxidation in used concentrations (5–100 µM), even in several tests, toxicity showed a decreasing trend. Altogether, the results of the current study showed that THC is not directly toxic in isolated cardiac mitochondria, and even may be helpful in reducing mitochondrial toxicity.

Impact of prolonged nicotine administration on myocardial function and susceptibility to ischaemia-reperfusion injury in rats.

This study investigated the impact of prolonged nicotine administration on myocardial susceptibility to ischaemia-reperfusion (I/R) injury in a rat model and determined whether nicotine affects mitochondrial reactive oxygen species (ROS) production and permeability transition in rat hearts. Sprague-Dawley rats were administered 0.6 or 1.2mg/kg nicotine for 28days, and their hearts were isolated at end-point for assessment of myocardial susceptibility to I/R injury ex vivo. Rat heart mitochondria were also isolated from a subset of rats for analysis of mitochondrial ROS production and permeability transition. Compared to the vehicle controls, rat hearts isolated from nicotine-administered rats exhibited poorer left ventricular function that worsened over the course of I/R. Coronary flow rate was also severely impaired in the nicotine groups at baseline and this worsened after I/R. Nicotine administration significantly increased mitochondrial ROS production and permeability transition relative to the vehicle controls. Interestingly, pre-incubation of isolated mitochondria with ROS scavengers (superoxide dismutase and mitoTEMPO) significantly abolished nicotine-induced increase in mitochondria permeability transition in isolated rat heart mitochondria. Overall, our data showed that prolonged nicotine administration enhances myocardial susceptibility to I/R injury in rats and this is associated with mitochondrial ROS-driven increase in mitochondrial permeability transition.

Mesalazine Induces Oxidative Stress and Cytochrome c Release in Isolated Rat Heart Mitochondria: An Analysis of Cardiotoxic Effects.

Mesalazine is widely used in the management of inflammatory bowel disease. Previous studies reported that mesalazine-induced cardiotoxicity is a rare, potentially fatal complication. Mitochondria play an important role in myocardial tissue homeostasis. Deterioration in mitochondrial function will eventually lead to cardiomyocyte death and consequently cardiovascular dysfunction. The aim of the current study was to investigate the effects of mesalazine on rat heart mitochondria. Rat heart mitochondria were isolated by mechanical lysis and differential centrifugation. Parameters of mitochondrial toxicity including succinate dehydrogenase (SDH) activity, reactive oxygen species (ROS) formation, mitochondrial membrane potential (MMP) collapse, mitochondrial swelling, and cytochrome c release were evaluated. Results revealed that mesalazine induced a concentration- and time-dependent rise in mitochondrial ROS formation, inhibition of SDH, MMP collapse, mitochondrial swelling, and cytochrome c release in rat heart mitochondria. These results indicate that the cardiotoxic effects of mesalazine are most likely associated with mitochondrial dysfunction and ROS formation, which finally ends in cytochrome c release signaling and induction of apoptosis.

Astaxanthin Prevents Mitochondrial Impairment Induced by Isoproterenol in Isolated Rat Heart Mitochondria.

Mitochondria are considered to be a power station of the cell. It is known that they play a major role in both normal and pathological heart function. Alterations in mitochondrial bioenergetics are one of the main causes of the origin and progression of heart failure since they have an inhibitory effect on the activity of respiratory complexes in the inner mitochondrial membrane. Astaxanthin (AST) is a xanthophyll carotenoid of mainly marine origin. It has both lipophilic and hydrophilic properties and may prevent mitochondrial dysfunction by permeating the cell membrane and co-localizing within mitochondria. The carotenoid suppresses oxidative stress-induced mitochondrial dysfunction and the development of diseases. In the present study, it was found that the preliminary oral administration of AST upregulated the activity of respiratory chain complexes and ATP synthase and the level of their main subunits, thereby improving the respiration of rat heart mitochondria (RHM) in the heart injured by isoproterenol (ISO). AST decreased the level of cyclophilin D (CyP-D) and increased the level of adenine nucleotide translocase (ANT) in this condition. It was concluded that AST could be considered as a potential mitochondrial-targeted agent in the therapy of pathological conditions associated with oxidative damage and mitochondrial dysfunction. AST, as a dietary supplement, has a potential in the prevention of cardiovascular diseases.

Curcumin attenuates bevacizumab-induced toxicity via suppressing oxidative stress and preventing mitochondrial dysfunction in heart mitochondria.

Heart failure was subsequently noted in 2-4% of patients on bevacizumab (BEV). Whereas mitochondria play an important role in myocardial tissue homeostasis, deterioration in mitochondrial function will eventually lead to cardiomyocyte cell death and consequently cardiovascular dysfunction. Therefore, the aim of our study is to search the effects of BEV on isolated rat heart mitochondria and cardiomyocytes, and survey the effect of curcumin as a mitochondrial protective and cardioprotective agent. Rat heart mitochondria and cardiomyocytes were isolated from adult rat heart ventricular. By using biochemical and flow cytometry evaluations, the parameters of mitochondrial toxicity including succinate dehydrogenase (SDH) activity, mitochondrial swelling, mitochondrial membrane potential (MMP) collapse, reactive oxygen species (ROS) formation and lipid peroxidation (LP), and cellular assays such as cytotoxicity and MMP collapse were evaluated. Results revealed that BEV (up to 50μg/ml) induced a concentration- and time-dependent rise in mitochondrial ROS formation, MMP collapse, mitochondrial swelling, LP, and inhibition of SDH in rat heart mitochondria. Our results showed that curcumin (10-100μM) significantly ameliorated BEV-induced mitochondrial toxicities. Also, our results in cellular assays confirmed amelioration effect of curcumin against BEV toxicity. These results indicate that the cardiotoxic effects of BEV are associated with mitochondrial dysfunction and ROS formation, which finally ends in MMP collapse and mitochondrial swelling as the "point of no return" in the cascade of events leading to apoptosis. Also, results of this study suggest that probably the combination therapy of BEV and curcumin could decrease mitochondrial effects of this drug.

Multi-walled carbon nanotubes induce oxidative stress, apoptosis, and dysfunction in isolated rat heart mitochondria: protective effect of naringin.

Multi-walled carbon nanotubes (MWCNTs) are material with exclusive features that can be applied in different fields including industrial and medicine. It has been determined that the accumulation of MWCNTs in the organs is along with genotoxic and cytotoxic injuries. Previous studies have shown mitochondrial dysfunction in MWCNTs exposure with cell lines, but their exact mechanisms with isolated mitochondria have remained unclear. The present study evaluated toxicity induced by MWCNTs in isolated rat heart mitochondria and protective effect of naringin. Our results showed that MWCNTs toxicity caused the prevention of heart mitochondrial complex II activity. Treatment of isolated heart mitochondria with MWCNTs led to an increase in mitochondrial reactive oxygen species (ROS) generation, mitochondrial membrane potential (MMP) collapse, and mitochondrial malondialdehyde (MDA) and a decrease in mitochondrial glutathione (GSH) level and mitochondrial catalase (CAT) activity. Pretreatment of isolated heart mitochondria with naringin decreased mitochondrial oxidative damage through decreasing lipid peroxidation, returned mitochondrial complex II changes, decreasing MMP collapse and ROS production, and restoration of GSH level and CAT activity. Our findings indicated that MWCNTs had toxic effects on isolated heart mitochondria by inducing oxidative stress and possibly apoptosis pathway. The protection effects of naringin may be accompanied by mitochondrial conservation by its antioxidant property or due to its free radical scavenging. Our findings indicated that naringin had a possible role in preventing the mitochondria complaints in the heart.

Bevacizumab as a monoclonal antibody inhibits mitochondrial complex II in isolated rat heart mitochondria: ameliorative effect of ellagic acid

Drug-induced cardiotoxicity usually manifests as heart failure or left ventricular systolic dysfunction. Left ventricular dysfunction is a rarely reported side effect of bevacizumab (BEV) with an incidence of 1.2%, and this occurs irrespective of the route of administration. In this study, we focused on an analysis of BEV effects on mitochondrial complexes activities and protective effect of ellagic acid (EA) against BEV-induced mitochondria toxicity. Rat heart mitochondria were isolated using differential centrifugation form wistar rats. Using biochemical and flowcytometry assays we evaluated mitochondrial complexes activity, succinate dehydrogenases (SDH), mitochondrial swelling, reactive oxygen species (ROS) formation and mitochondrial membrane potential (MMP) in isolated mitochondria. We observed only decreased activity of complexes II after exposure with BEV (50 and 100 µg/ml). The inhibition of complex II is paralleled by the decreased MMP, mitochondrial swelling, and ROS formation. Also, we showed that EA (10–100 µM) as an antioxidant and natural agent significantly decreases mitochondrial toxicity induced by BEV. Together, for the first time, this preliminary study has demonstrated a significant decrease in activity of complexes II after exposure with BEV and proved the protective effects of EA in alleviating BEV-mediated mitochondria toxicity.

Disturbance of bioenergetics and calcium homeostasis provoked by metabolites accumulating in propionic acidemia in heart mitochondria of developing rats

Propionic acidemia is caused by lack of propionyl-CoA carboxylase activity. It is biochemically characterized by accumulation of propionic (PA) and 3-hydroxypropionic (3OHPA) acids and clinically by severe encephalopathy and cardiomyopathy. High urinary excretion of maleic acid (MA) and 2-methylcitric acid (2MCA) is also found in the affected patients. Considering that the underlying mechanisms of cardiac disease in propionic acidemia are practically unknown, we investigated the effects of PA, 3OHPA, MA and 2MCA (0.05–5 mM) on important mitochondrial functions in isolated rat heart mitochondria, as well as in crude heart homogenates and cultured cardiomyocytes. MA markedly inhibited state 3 (ADP-stimulated), state 4 (non-phosphorylating) and uncoupled (CCCP-stimulated) respiration in mitochondria supported by pyruvate plus malate or α-ketoglutarate associated with reduced ATP production, whereas PA and 3OHPA provoked less intense inhibitory effects and 2MCA no alterations at all. MA-induced impaired respiration was attenuated by coenzyme A supplementation. In addition, MA significantly inhibited α-ketoglutarate dehydrogenase activity. Similar data were obtained in heart crude homogenates and permeabilized cardiomyocytes. MA, and PA to a lesser degree, also decreased mitochondrial membrane potential (ΔΨm), NAD(P)H content and Ca2+ retention capacity, and caused swelling in Ca2+-loaded mitochondria. Noteworthy, ΔΨm collapse and mitochondrial swelling were fully prevented or attenuated by cyclosporin A and ADP, indicating the involvement of mitochondrial permeability transition. It is therefore proposed that disturbance of mitochondrial energy and calcium homeostasis caused by MA, as well as by PA and 3OHPA to a lesser extent, may be involved in the cardiomyopathy commonly affecting propionic acidemic patients.

Astaxanthin Inhibits Mitochondrial Permeability Transition Pore Opening in Rat Heart Mitochondria

The mitochondrion is the main organelle of oxidative stress in cells. Increased permeability of the inner mitochondrial membrane is a key phenomenon in cell death. Changes in membrane permeability result from the opening of the mitochondrial permeability transition pore (mPTP), a large-conductance channel that forms after the overload of mitochondria with Ca2+ or in response to oxidative stress. The ketocarotenoid astaxanthin (AST) is a potent antioxidant that is capable of maintaining the integrity of mitochondria by preventing oxidative stress. In the present work, the effect of AST on the functioning of mPTP was studied. It was found that AST was able to inhibit the opening of mPTP, slowing down the swelling of mitochondria by both direct addition to mitochondria and administration. AST treatment changed the level of mPTP regulatory proteins in isolated rat heart mitochondria. Consequently, AST can protect mitochondria from changes in the induced permeability of the inner membrane. AST inhibited serine/threonine protein kinase B (Akt)/cAMP-responsive element-binding protein (CREB) signaling pathways in mitochondria, which led to the prevention of mPTP opening. Since AST improves the resistance of rat heart mitochondria to Ca2+-dependent stress, it can be assumed that after further studies, this antioxidant will be considered an effective tool for improving the functioning of the heart muscle in general under normal and medical conditions.

Mitochondrial Permeability Transition Pore Sealing Agents and Antioxidants Protect Oxidative Stress and Mitochondrial Dysfunction Induced by Naproxen, Diclofenac and Celecoxib

Nonsteroidal anti-inflammatory drugs (NSAIDs) like naproxen, diclofenac and celecoxib used to reduce pain. Many of these drugs have been associated with an increased risk of cardiovascular disease (CVD). The molecular mechanism(s) by which NSAIDs induce CVD up to now is unknown. We investigated the effects of naproxen, diclofenac and celecoxib with different structures and mechanism action on isolated rat heart mitochondria. All tested NSAIDs increased reactive oxygen species (ROS) formation, mitochondrial membrane collapse (MMP), mitochondrial swelling, lipid peroxidation, and glutathione and ATP depletion, which all of them play important roles in developing cardiotoxicity. We reported that mitochondrial permeability transition (MPT) pore sealing agents and antioxidants have the capacity to significantly prevent mitochondrial toxicity. Therefore, the inhibition of mitochondrial oxidative stress and mitochondrial dysfunction by MPT pore sealing agents and antioxidants can double confirm NSAID-induced cardiomyocytes toxicity is resulted from induction of apoptosis signaling trough ROS-mediated mitochondrial permeability transition.

An old medicine as a new drug to prevent mitochondrial complex I from producing oxygen radicals.

FindingsHere, we demonstrate that OP2113 (5-(4-Methoxyphenyl)-3H-1,2-dithiole-3-thione, CAS 532-11-6), synthesized and used as a drug since 1696, does not act as an unspecific antioxidant molecule (i.e., as a radical scavenger) but unexpectedly decreases mitochondrial reactive oxygen species (ROS/H2O2) production by acting as a specific inhibitor of ROS production at the IQ site of complex I of the mitochondrial respiratory chain. Studies performed on isolated rat heart mitochondria also showed that OP2113 does not affect oxidative phosphorylation driven by complex I or complex II substrates. We assessed the effect of OP2113 on an infarct model of ex vivo rat heart in which mitochondrial ROS production is highly involved and showed that OP2113 protects heart tissue as well as the recovery of heart contractile activity.Conclusion / SignificanceThis work represents the first demonstration of a drug authorized for use in humans that can prevent mitochondria from producing ROS/H2O2. OP2113 therefore appears to be a member of the new class of mitochondrial ROS blockers (S1QELs) and could protect mitochondrial function in numerous diseases in which ROS-induced mitochondrial dysfunction occurs. These applications include but are not limited to aging, Parkinson’s and Alzheimer's diseases, cardiac atrial fibrillation, and ischemia-reperfusion injury.

Effects of Nd3+ on Calcium-Dependent Processes in Isolated Rat Heart Mitochondria and Frog Heart Muscle

Effects of Nd3+ on Calcium-Dependent Processes in Isolated Rat Heart Mitochondria and Frog Heart Muscle

Effect of Melatonin on Rat Heart Mitochondria in Acute Heart Failure in Aged Rats.

Excessive generation of reactive oxygen species (ROS) in mitochondria and the opening of the nonselective mitochondrial permeability transition pore are important factors that promote cardiac pathologies and dysfunction. The hormone melatonin (MEL) is known to improve the functional state of mitochondria via an antioxidant effect. Here, the effect of MEL administration on heart mitochondria from aged rats with acute cardiac failure caused by isoprenaline hydrochloride (ISO) was studied. A histological analysis revealed that chronic intake of MEL diminished the age-dependent changes in the structure of muscle fibers of the left ventricle, muscle fiber swelling, and injury zones characteristic of acute cardiac failure caused by ISO. In acute heart failure, the respiratory control index (RCI) and the Ca2+ retention capacity in isolated rat heart mitochondria (RHM) were reduced by 30% and 40%, respectively, and mitochondrial swelling increased by 34%. MEL administration abolished the effect of ISO. MEL partially prevented ISO-induced changes at the subunit level of respiratory complexes III and V and drastically decreased the expression of complex I subunit NDUFB8 both in control RHM and in RHM treated with ISO, which led to the inhibition of ROS production. MEL prevents the mitochondrial dysfunction associated with heart failure caused by ISO. It was shown that the level of 2′,3′-cyclicnucleotide-3′-phosphodiasterase (CNPase), which is capable of protecting cells in aging, increased in acute heart failure. MEL also retained the CNPase content in RHM both in control experiments and after ISO-induced heart damage. We concluded that an increase in the CNPase level promotes cardioprotection.

Actions of Nitric Oxide Synthase Inhibitors on Mitochondrial Respiration in Isolated Rat Heart Mitochondria

ObjectiveThe existence of a nitric oxide (NO) producing enzyme, nitric oxide synthase (NOS), within cardiac mitochondria has been speculated in the most recent decades. There is conflicting data regarding the identity, physiological function, and the pathophysiological significance of mitochondrial NOS. Nevertheless, mitochondrial preparations have been shown to display an ability to produce NO that is sensitive to NOS inhibitors. Our objective was to evaluate the impact of pharmacological NOS inhibition on mitochondrial respiration. We hypothesized that pharmacological inhibition of NOS affects mitochondrial respiration.Methods and ResultsMitochondria were isolated from rats by homogenization and gradient separation. Isolated mitochondria were divided into three groups: untreated control, neuronal NOS inhibitor‐treated (nNOS; ARL dihydrochloride or ARL), and endothelial NOS inhibitor‐treated (eNOS; N5‐(1‐Iminoethyl)‐L‐ornithine or NIO). Mitochondrial function was assessed by measuring oxygen consumption rates (OCR) using Seahorse XFe Analyzer. First, we observed that isolated rat heart mitochondria displayed respiratory control ratios above 2 suggestive of the acceptable tightness of the coupling between respiration and phosphorylation indicative of the good quality of the preparation. Second, both NOS inhibitors decreased the OCR related to ATP production and increased proton leak in the mitochondria although the basal and maximal respiration were unaffected.ConclusionsPharmacological inhibition of NOS impairs ATP production in isolated rat heart mitochondria that is likely to be related to increased proton leak. Thus, the physiological function of mitochondrial NOS activity involves modulation of inner mitochondrial membrane permeability to promote efficiency of ATP productionSupport or Funding InformationAmerican Heart Association (PVG: 14SDG20490359 and VNS: 16PRE31450006); National Institute of Health: National Institute of Neurological Disorders and Stroke and National Institute of General Medical Sciences (PVK: R01NS094834); and National Institute of Diabetes and Digestive and Kidney Diseases (RS: DK107694).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Dronedarone-Induced Cardiac Mitochondrial Dysfunction and Its Mitigation by Epoxyeicosatrienoic Acids.

Dronedarone and amiodarone are structurally similar antiarrhythmic drugs. Dronedarone worsens cardiac adverse effects with unknown causes while amiodarone has no cardiac adversity. Dronedarone induces preclinical mitochondrial toxicity in rat liver and exhibits clinical hepatotoxicity. Here, we further investigated the relative potential of the antiarrhythmic drugs in causing mitochondrial injury in cardiomyocytes. Differentiated rat H9c2 cardiomyocytes were treated with dronedarone, amiodarone, and their respective metabolites namely N-desbutyldronedarone (NDBD) and N-desethylamiodarone (NDEA). Intracellular ATP content, mitochondrial membrane potential (Δψm), and inhibition of carnitine palmitoyltransferase I (CPT1) activity and arachidonic acid (AA) metabolism were measured in H9c2 cells. Inhibition of electron transport chain (ETC) activities and uncoupling of ETC were further studied in isolated rat heart mitochondria. Dronedarone, amiodarone, NDBD and NDEA decreased intracellular ATP content significantly (IC50 = 0.49, 1.84, 1.07, and 0.63 µM, respectively) and dissipated Δψm potently (IC50 = 0.5, 2.94, 12.8, and 7.38 µM, respectively). Dronedarone, NDBD, and NDEA weakly inhibited CPT1 activity while amiodarone (IC50 > 100 µM) yielded negligible inhibition. Only dronedarone inhibited AA metabolism to its regioisomeric epoxyeicosatrienoic acids (EETs) consistently and potently. NADH-supplemented ETC activity was inhibited by dronedarone, amiodarone, NDBD and NDEA (IC50 = 3.07, 5.24, 11.94, and 16.16 µM, respectively). Cytotoxicity, ATP decrease and Δψm disruption were ameliorated via exogenous pre-treatment of H9c2 cells with 11, 12-EET and 14, 15-EET. Our study confirmed that dronedarone causes mitochondrial injury in cardiomyocytes by perturbing Δψm, inhibiting mitochondrial complex I, uncoupling ETC and dysregulating AA-EET metabolism. We postulate that cardiac mitochondrial injury is one potential contributing factor to dronedarone-induced cardiac failure exacerbation.

Mitochondrial cytochrome c oxidase is inhibited by ATP only at very high ATP/ADP ratios.

In the past, divergent results have been reported based on different methods and conditions used for enzymatic activity measurements of cytochrome c oxidase (CytOx). Here, we analyze in detail and show comparable and reproducible polarographic activity measurements of ATP-dependent inhibition of CytOx kinetics in intact and non-intact rat heart mitochondria and mitoplasts. We found that this mechanism is always present in isolated rat heart mitochondria and mitoplasts; however, it is measurable only at high ATP/ADP ratios using optimal protein concentrations. In the kinetics assay, measurement of this mechanism is independent of presence or absence of Tween-20 and the composition of measuring buffer. Furthermore, the effect of atractyloside on intact rat heart mitochondria confirms that (i) ATP inhibition occurs under uncoupled conditions [in the presence of carbonly cyanide m-chlorophenyl hydrazone (CCCP)] when the classical respiratory control is absent and (ii) high ATP/ADP ratios in the matrix as well as in the cytosolic space are required for full ATP inhibition of CytOx. Additionally, ATP inhibition measured in intact mitochondria extends in the presence of oligomycin, thus indicating further that the problem to measure the inhibitory effect of ATP on CytOx is apparently due to the lack of very high ATP/ADP ratios in isolated mitochondria.

5 - Manganese Enhances Mitochondrial H2O2 Emission by Different Mechanisms

The mitochondrial respiratory chain is a major source of cellular reactive oxygen species (ROS). Recent data indicate that beside the established H 2 O 2 /O 2 ●– -generators NADH-ubiquinone- (complex I) and the ubiquinone-cytrochrome c -oxidoreductase (complex III), additional sources significantly contribute to the overall ROS production. These include the oxoglutarate dehydrogenase complex and the succinate dehydrogenase (complex II; SDH). The latter was shown to produce mainly H 2 O 2 from its FAD site. More recently manganese toxicity in the central nervous system has been linked to an elevated ROS production by complex II. However, the molecular mechanism as well as the distinct site of origin remained elusive. In this study with submitochondrial particles (SMP) and isolated rat heart mitochondria (RHM) we confirmed an inducing effect of manganese on ROS production by complex II. Furthermore, we tracked down H 2 O 2 /O 2 ●– -to be derived mainly from the ubiquinone binding site. In RHM, manganese additionally acted as a potent inducer of mitochondrial permeability transition, and hence potentiated the emission of H 2 O 2 from ‘substrate’ oxidoreductases of the Krebs cycle. Moreover, manganese generally led to an enhanced emission of hydrogen peroxide due to its accelerating effect on superoxide dismutation. These findings highlight the complex effects of manganese on mitochondrial H 2 O 2 emission. They also warrant further explorations aiming to understand/predict the functional consequences of H 2 O 2 /O 2 ●– formed at distinct sites of SDH.