Mitochondrial Permeability Transition Opening Research Articles

Annona muricata Linn Stem Bark Protects against Uterine Proliferative Disorder Induced by Estradiol Benzoate in Female Rat

Aim: The opening of mitochondrial permeability transition (mPT) pore is an important event in the execution of mitochondrial-mediated apoptosis. Some bioactive compounds elicit their chemotherapeutic effects against tumor/cancer cells via the induction of mitochondrial-mediated apoptosis. Annona muricata, a medicinal plant, is folklorically used in the treatment of tumors and cancers. This study therefore aimed at investigating the effect of methanol stem bark extract of Annona muricata (MEAM) on apoptosis via mPT pore and estradiol benzoate (EB)-induced proliferative disorder using female Wistar rats.
 Methodology: Mitochondria were isolated using differential centrifugation. The mPT pore opening, cytochrome c release and mitochondrial ATPase activity were determined spectrophotometrically. The levels of estrogen (E2), luteinizing hormone (LH), follicle stimulating hormone (FSH), malondialdehyde (MDA) and activities of superoxide dismutase (SOD), glutathione peroxidase (GSH), were determined using ELISA technique. Histological and histochemical assessments of the uterine sections were carried out using standard methods. Phytochemical constituents of MEAM were determined using Gas Chromatography-Mass Spectroscopy (GC-MS).
 Results: The in vitro results showed a significant induction of mPT pore opening, release of cytochrome c and enhancement of mitochondrial ATPase (mATPase) activity in a concentration-dependent manner. However, oral administration of MEAM did not induce rat uterine mPT pore opening, neither any significant release of cytochrome c nor enhancement of mATPase activity at the dosages used. Interestingly, MEAM reversed the EB-induced increase in E2, LH and FSH. The MEAM also improved the antioxidant milleu by reducing MDA level and increasing the SOD and GSH-Px activities in the treatment groups. Administration of EB induced endometrial hyperplasia in the model group which was mitigated by MEAM in the treatment group. The GC-MS analysis of MEAM revealed the presence of some important phytochemicals that are pharmacological relevant in cancer treatment.
 Conclusions: This study suggests that the methanol stem bark extract of Annona muricata contains bioactive compounds that protect against EB-induced uterine proliferative disorder in female Wistar rats.

Mitochondrial stress response in drug-induced liver injury.

Drug-induced liver injury (DILI) caused by the ingestion of medications, herbs, chemicals or dietary supplements, is a clinically widespread health problem. The underlying mechanism of DILI is the formation of reactive metabolites, which trigger mitochondrial oxidative stress and the opening of mitochondrial permeability transition (MPT) pores through direct toxicity or immune response, leading to cell inflammation, apoptosis, and necrosis. Traditionally,mitochondria play an indispensable role in maintaining the physiological and biochemical functions of cells by producing ATP and mediating intracellular signal transduction; drugs can typically stimulate the mitochondria and, in the case of sustained stress, can eventually cause impairment of mitochondrial function and metabolic activity. Meanwhile,the mitochondrial stress response, as an adaptive protective mechanism, occurs when mitochondrial homeostasis is threatened. In this review, we summarize the relevant frontier researches of the protective effects of mitochondrial stress response in DILI as well as the potential related mechanisms, thus providing some thoughts for the clinical treatment of DILI.

Stigmasterol isolated from the chloroform fraction of Adenopus breviflorus Benth fruit induces mitochondrial-dependent apoptosis in rat liver.

Decoction of Adenopus breviflorus fruit is used in folkloric medicine for treating dysmenorrhea and gonorrhea. Phytochemicals from A.breviflorus may be potent in inducing mitochondrial-dependent apoptosis via the opening of the mitochondrial permeability transition (MPT) pore. Therefore, this study investigated the in vitro effects of stigmasterol isolated from the chloroform fraction of A.breviflorus (CFAB) and also the increasing concentration of CFAB on the opening of rat liver mitochondrial permeability transition (MPT) pore. Fractionation of CFAB on column chromatography yielded a needle-like crystal which structure was elucidated by standard spectroscopic techniques. The effects of stigmasterol and CFAB on MPT pore opening were assayed spectrophotometrically. Also, the effect of CFAB on mitochondrial ATPase (mATPase) activity and cytochrome c (Cyt c) release were determined. Stigmasterol isolated from CFAB induced MPT pore opening significantly (p<0.05) when compared withthe control. Similarly, CFAB significantly (p<0.05) induced MPT pore opening in rat liver mitochondria in a concentration-dependent manner in the presence and absence of the triggering agent- calcium ion. Furthermore, the increasing concentration of CFAB significantly (p<0.05) stimulated mitochondrial ATPase (mATPase) activity and Cyt c release in a concentration-dependent manner. The study showed that stigmasterol isolated from the chloroform fraction of A.breviflorus is a potent inducer of mitochondrial-dependent apoptosis. Also, the study further revealed that CFAB possesses potent bioactive compounds which can induce the mitochondrial-dependent apoptosis through the opening of the mitochondrial permeability transition pore, activation of mitochondrial ATPase (mATPase) activity and cytochrome c release.

Cardioprotective effect of H2S and glutathione synthesis modulation is mediated by inhibition mitochondrial permeability transition pore opening

Hydrogen sulfide (H2S) was recently classified as the third gaseous transmitter produces by two cytosolic cystathionine γ-lyase (CSE) and cystathionine β-synthase, and one mitochondrial enzyme – 3-mercaptopyruvate sulfurtransferase. It was clearly shown that H2S protects against cardiac ischemia/reperfusion (I/R) injury in a wide range of exogenously applied doses of H2S donors. Cell damage within I/R injury is caused by extensive reactive oxygen species (ROS) mainly produced by mitochondria. ROS fleshes are associated with massive opening of mitochondrial permeability transition (MPT) pores and contribute to deterioration of heart function. However, it may be prevented in case of pharmacological inhibition of MPT pores opening. Mixture of mitochondrial metabolites released through the opened MPT pores can be detected in situ as increased optical density of outflow solutions at a wavelength of 245-250 nm and was called mitochondrial factor (MF). One of the most powerful antioxidant agents that preserve redox status in tissues is tripeptide glutathione. It forms in two ATP-depended reactions and exists in two forms reduced and oxidized one. Glutathione and H2S have a common precursor – amino acid L-cysteine. In this study, we used Langendorff isolated rat heart model to investigate the effect of H2S and glutathione synthesis modulation on MPT pores opening in I/R injury. Rats were pretreated intraperitoneally with D,L-propargylglycine (11,3 mg/kg), an inhibitor of H2S-producing enzyme CSE, L-cysteine (121 mg/kg) and buthionine sulfoximine (BSO, 22,2 mg/kg) an inhibitor of first step of glutathione synthesis. Cardiac function, oxygen metabolism and MPT pores opening in situ were measured. We clearly showed that treatment with PAG and L-cysteine provided pharmacological precondition and exerted cardioprotective effect inhibiting MPT pores opening as greatly decreased MF release from isolated heart. Pretreatment with BSO abolished cardioprotective effect of PAG+L-cysteine combination. Absorbance spectra in L-cysteine pretreated group did not differ from the control. Thus, we demonstrate that PAG+L-cysteine induced cardioprotection mediated via inhibition of MPT pores opening.

Mitochondria as a promising target for developing novel agents for treating Alzheimer's disease.

The mitochondria-targeting drugs can be conventionally divided into the following groups: those compensating for the energy deficit involved in neurodegeneration, including stimulants of mitochondrial bioenergetics and activators of mitochondrial biogenesis; and neuroprotectors, that are compounds increasing the resistance of mitochondria to opening of mitochondrial permeability transition (MPT) pores. Although compensating for the energy deficit and inhibition of MPT are obvious targets for drugs used in the very early stages of Alzheimer-like pathology, but their use as the monotherapy for patients with severe symptoms is unlikely to be sufficiently effective. It would be optimal to combine targets that would provide the cognitive-stimulating, the neuroprotective effects and the ability to affect specific disease-forming mechanisms. In the design of such drugs, assessment of their potential mitochondrial-targeted effects is of particular importance. The possibility of targeted drug design for simultaneous action on mitochondrial and neurotransmitter's receptors targets is, in particularly, based on the known interplay of various cellular pathways and the presence of common structural components. Of particular interest is directed search for multitarget drugs that would act simultaneously on mitochondrial calcium-dependent functions, the targets (receptors, enzymes, etc.) facilitating neurotransmission, and the molecular targets related to the action of so-called disease-modifying factors, in particular, the formation and overcoming of the toxicity of β-amyloid or hyperphosphorylated tau protein. The examples of such approaches realized on the level of preclinical and clinical trials are presented below.

In vitro and in vivo effect of 3-Para-fluorobenzoyl-propionic acid on rat liver mitochondrial permeability transition pore opening and lipid peroxidation

Abstract The opening of mitochondrial permeability transition (mPT) pore is a well recognized important event in the execution of mitochondrial-mediated apoptosis. Some bioactive compounds induce apoptosis in tumour cells via the induction of mPT pore opening. This study therefore investigated the effect of 3-Para-fluorobenzoyl-propionic acid (3PFBPA), a metabolite of haloperidol on mPT pore, mitochondrial ATPase activity (mATPase), mitochondrial lipid peroxidation (mLPO) and cytochrome c release (CCR). Thirty-two male Wistar rats, were acclimatized for 14 days in clean cages. After 30 days of treatment, they were sacrificed and the liver mitochondria isolated using differential centrifugation. The mPT pore, mATPase, mLPO and CCR were determined by standard methods using a spectrophotometer. The mPT pore opening was induced by 3PFBPA by 1.4, 3.6, 5.6, 6.6 and 7.4 folds, when compared with the control. Also, there was release of cytochrome c and enhancement of mATPase activity by 3PFBPA. The results also show that 3PFBPA reduced lipid peroxidation. However, oral administration of 3PFBPA at 50, 100 and 200 mg/kg did not have any effect on mPT pore opening and mATPase activity when compared with the control but there was inhibition of mLPO. These findings suggested the pharmacological potential of 3PFBPA against the pathological processes related to insufficient apoptosis (based on the in vitro data) and oxidative stress due to its anti-lipidperoxidative effect.

Mitochondrial Ca2+ concentrations in live cells: quantification methods and discrepancies.

Intracellular Ca2+ signaling controls numerous cellular functions. Mitochondria respond to cytosolic Ca2+ changes by adapting mitochondrial functions and, in some cell types, shaping the spatiotemporal properties of the cytosolic Ca2+ signal. Numerous methods have been developed to specifically and quantitatively measure the mitochondrial-free Ca2+ concentrations ([Ca2+ ]m ), but there are still significant discrepancies in the calculated absolute values of [Ca2+ ]m in stimulated live cells. These discrepancies may be due to the distinct properties of the methods used to measure [Ca2+ ]m , the calcium-free/bound ratio, and the cell-type and stimulus-dependent Ca2+ dynamics. Critical processes happening in the mitochondria, such as ATP generation, ROS homeostasis, and mitochondrial permeability transition opening, depend directly on the [Ca2+ ]m values. Thus, precise determination of absolute [Ca2+ ]m values is imperative for understanding Ca2+ signaling. This review summarizes the reported calibrated [Ca2+ ]m values in many cell types and discusses the discrepancies among these values. Areas for future research are also proposed.

High doses of sodium tungstate can promote mitochondrial dysfunction and oxidative stress in isolated mitochondria

Tungstate (W) is recognized as an agent of environmental pollution and a substitute to depleted uranium. According to some preliminary studies, tungstate toxicity is related to the formation of reactive oxygen species (ROS) under abnormal pathological conditions. The kidneys and liver are the main tungstate accumulation sites and important targets of tungstate toxicity. Since the mitochondrion is the main ROS production site, we evaluated the mechanistic toxicity of tungstate in isolated mitochondria for the first time, following a two-step ultracentrifugation method. Our findings demonstrated that tungstate-induced mitochondrial dysfunction is related to the increased formation of ROS, lipid peroxidation, and potential membrane collapse, correlated with the amelioration of adenosine triphosphate and glutathione contents. The present study indicated that mitochondrial dysfunction was associated with disruptive effects on the mitochondrial respiratory chain and opening of mitochondrial permeability transition (MPT) pores, which is correlated with cytochrome c release. Our findings suggest that high concentrations of tungstate (2 mM)-favored MPT pore opening in the inner membranes of liver and kidney mitochondria of rats. Besides, the results indicated higher tungstate susceptibility in the kidneys, compared with the liver.

Sphingolipids in Genetic and Acquired Forms of Chronic Kidney Diseases.

Sphingolipids (SLs) regulate apoptosis, proliferation, and stress response. SLs, including ceramide, glycosphingolipids (glucosylceramide, lactosylceramide, and gangliosides) and sphingosine-1-phosphate (S1P), play a role in the pathogenesis and progression of genetic (lysosomal storage disease, congenital nephrotic syndrome and polycystic kidney disease) and non-genetic forms of chronic kidney diseases (CKDs). SLs metabolism defects promote complications (cardiovascular events, etc.) via oxidant stress in CKDs. A balancing role of apoptotic SLs and anti-apoptotic S1P is crucial in the regulation of glomerular injury and complications associated with CKDs. Interaction between SLs, endothelial function and reninangiotensin- aldosterone system (RAAS) plays an important role in the regulation of glomerular injury. SLs affect mitochondrial function that regulate the opening of mitochondrial permeability transition (MPT) pore, mitochondrial outer membrane permeability (MOMP), generation of reactive oxygen species (ROS), and expression of BcL-2 family proteins, which result in cytochrome c release and caspase activation, leading to apoptosis, and regulate glomerular cell proliferation or renal fibrosis. This review article summarizes the current evidence supporting a role of SLs metabolism defects in the pathogenesis and progression of glomerular injury and discusses a role of mitochondria, including MPT pore, MOMP, ROS generation, BcL-2 family proteins, interaction between SLs, endothelial function and RAAS, and SLs-induced downstream signaling events in CKDs. Crosstalk between these factors plays a role in the pathogenesis and progression of CKDs. Therapeutic strategy of targeting SLs metabolism defects for CKDs through modulation of the enzymes responsible for SLs metabolism defects is also discussed.

Calcium-Induced Mitochondrial Permeability Transitions: Parameters of Ca2+ Ion Interactions with Mitochondria and Effects of Oxidative Agents.

We evaluated the parameters of Ca2+-induced mitochondrial permeability transition (MPT) pore formations, Ca2+ binding constants, stoichiometry, energy of activation, and the effect of oxidative agents, tert-butyl hydroperoxide (tBHP), and hypochlorous acid (HOCl), on Ca2+ -mediated process in rat liver mitochondria. From the Hill plot of the dependence of MPT rate on Ca2+ concentration, we determined the order of interaction of Ca2+ ions with the mitochondrial sites, n = 3, and the apparent Kd = 60 ± 12µM. We also found the apparent Michaelis-Menten constant, Km, for Ca2+ interactions with mitochondria to be equal to 75 ± 20µM, whereas that in the presence of 300µM tBHP was 120 ± 20µM. Using the Arrhenius plots of the temperature dependences of apparent mitochondrial swelling rate at various Ca2+ concentrations, we calculated the activation energy of the MPT process. ΔEa was 130 ± 20kJ/mol at temperatures below the break point of the Arrhenius plot (30-34 °C) and 50 ± 9kJ/mol at higher temperatures. Ca2+ ions induced rapid mitochondrial NADH depletion and membrane depolarization. Prevention of the pore formation by cyclosporin A inhibited Ca2+-dependent mitochondrial depolarization and Mg2+ ions attenuated the potential dissipation. tBHP (10-150µM) dose-dependently enhanced the rate of MPT opening, whereas the effect of HOCl on MPT depended on the ratio of HOCl/Ca2+. The apparent Km of tBHP interaction with mitochondria in the swelling reaction was found to be Km = 11 ± 3µM. The present study provides evidence that three calcium ions interact with mitochondrial site with high affinity during MPT. Ca2+-induced MPT pore formations due to mitochondrial membrane protein denaturation resulted in membrane potential dissipation. Oxidants with different mechanisms, tBHP and HOCl, reduced mitochondrial membrane potential and oxidized mitochondrial NADH in EDTA-free medium and had an effect on Ca2+-induced MPT onset.

Morphine Postconditioning Protects against Reperfusion Injury via Inhibiting JNK/p38 MAPK and Mitochondrial Permeability Transition Pores Signaling Pathways

Background: The purpose of this study was to determine whether c-jun NH₂ amino-terminal kinases (JNK) and p38 mitogen-activated protein kinases (MAPK) were involved in morphine postconditioning (MpostC). Methods: The isolated rat hearts were randomly assigned into one of the following groups. Hearts in the time control (TC) group were constantly perfused for 105min. Hearts in the ischemia-reperfusion (I/R) group were subjected to 45 min of ischemia followed by 1 h of reperfusion. MpostC was induced by 10 min of morphine administration at the onset of reperfusion. Anisomycin (an activator of JNK/p38 kinases) was administered with or without morphine during the first 10 min of reperfusion following the 45 min of ischemia. Mitochondria and cytosolic proteins were prepared to detect mitochondrial permeability transition (MPT) and cytochrome C (Cyt-c) respectively. Results: MpostC markedly reduced infarct size (IS/AAR), CK-MB release, and improved cardiac function recovery. However, these protective effects were partly abolished in the presence of anisomycin. I/R significantly increased the phosphorylation of JNK and p38 kinases, mitochondrial permeability transition (MPT) opening and Cyt-c release, while these effects were partly abolished by MpostC. The inhibitory effects of MpostC on the phosphorylation of JNK/p38 kinases, MPT opening and Cyt-c release were totally reversed by Anisocycin, which, used individually, did not show any influence on perfusion injury. Conclusions: These findings suggest that MpostC protects isolated rat hearts against reperfusion injury via inhibiting JNK/p38 MAPKs and mitochondrial permeability transition pores signaling pathways.

Glucose fluctuation increased hepatocyte apoptosis under lipotoxicity and the involvement of mitochondrial permeability transition opening

Oxidative stress is considered to be an important factor in producing lethal hepatocyte injury associated with nonalcoholic fatty liver disease (NAFLD). Glucose fluctuation, more pronounced in patients with diabetes, has been recognized as an even stronger oxidative stress inducer than the sustained hyperglycemia. Here, we investigated the role of glucose variability in the development of the NAFLD based on hepatocyte apoptosis and possible mechanisms. To achieve this goal we studied C57BL/6J mice that were maintained on a high fat diet (HFD) and injected with glucose (3 g/kg) twice daily to induce intermittent high glucose (IHG). We also studied hepatic L02 cells incubated with palmitic acid (PA) to induce steatosis. The following experimental groups were compared: normal glucose (NG), sustained high glucose (SHG) and IHG with or without PA. We found that, although hepatic enzyme levels and liver lipid deposition were comparable between HFD mice injected with glucose or saline, the glucose injected mice displayed marked hepatocyte apoptosis and inflammation, accompanied by increased lipid peroxide in liver. In vitro, in the presence of PA, IHG increased L02 cell apoptosis and oxidative stress and produced pronounced mitochondrial dysfunction relative to the NG and SHG groups. Furthermore, treatment with the mitochondrial permeability transition (MPT) inhibitor, cyclosporin A (1.5 μmol/l), prevented mitochondrial dysfunction, oxidative stress and hepatocyte apoptosis. Our data suggests that IHG under lipotoxicity might contribute to the development of NAFLD by increasing oxidative stress and hepatocyte apoptosis via MPT and its related mitochondrial dysfunction.

Methylene blue attenuates acute liver injury induced by paraquat in rats.

Paraquat (PQ) poisoning often leads to severe oxidative liver injury. Recent studies have reported that methylene blue (MB) can prevent oxidative stress-induced diseases. This study tested the hypothesis that MB treatment reduced acute liver injury induced by PQ in rats. Adult male Sprague-Dawley (SD) rats were randomly divided into four groups: (1) normal group, (2) MB group (2mg/kg i.p.), (3) PQ group (35 mg/kg i.p.) and (4) PQ+MB group (MB 2mg/kg i.p. administrated 2h after PQ). We evaluated the changes of liver histopathology, serum liver enzymatic activities, oxidative stress, heme oxygenase-1 expression, and mitochondrial permeability transition. The rats were injected with PQ produced liver injury, evidenced by histological changes and elevated serum alkaline phosphatase and alanine transaminase levels; PQ also led to oxidative stress, an increase of malondialdehyde content and mitochondrial permeability transition pore opening. Pathological damage and all of the above mentioned markers were reversed in the animals treated with MB than in those who received PQ alone. Meanwhile, MB significantly increased the contents of superoxide dismutase, adenosine triphosphate and the expression of heme oxygenase-1. In conclusion, MB had a protective effect against PQ-induced hepatic damage in rats. The mechanisms of the protection seem to be the inhibition of mitochondrial permeability transition opening and the increase of heme oxygenase-1 expression.

Mitochondrial Neuroprotection in Traumatic Brain Injury: Rationale and Therapeutic Strategies

Traumatic brain injury (TBI) is still the worldwide, leading cause of mortality and morbidity in young adults. The prognosis of TBI patients is strongly affected by secondary brain damage including mitochondrial dysfunctions. In many basic and clinical studies, mitochondrial dysfunctions, including the opening of mitochondrial permeability transition (mPT) pore, and treatments including cyclosporine A (CsA) have been studied. These evidences suggest an important role for mitochondria as therapeutic targets for neuroprotection after TBI. This review summarizes the data about normal and pathological mitochondrial function after TBI, TBI pathobiology relating to mitochondrial dysfunction and therapeutic strategies including drug treatment. This review also mentioned about glucose, lactate, and pyruvate metabolisms in TBI, including the "astrocyte-neuron lactate shuttle (ANLS)" hypothesis. Mitochondrial pathophysiology in TBI is still unclear. Thus, the pharmacological treatment in TBI patient is still challenging. This review could help further understanding of this topic. Hopefully, this could help further development and innovation for drug therapies in TBI.

Protective role of amantadine in mitochondrial dysfunction and oxidative stress mediated by hepatitis C virus protein expression

Amantadine is an antiviral and antiparkinsonian drug that has been evaluated in combination therapies against hepatitis C virus (HCV) infection. Controversial results have been reported concerning its efficacy, and its mechanism of action remains unclear. Data obtained in vitro suggested a role of amantadine in inhibiting HCV p7-mediated cation conductance. In keeping with the fact that mitochondria are responsible to ionic fluxes and that HCV infection impairs mitochondrial function, we investigated a potential role of amantadine in modulating mitochondrial function. Using a well-characterized inducible cell line expressing the full-length HCV polyprotein, we found that amantadine not only prevented but also rescued HCV protein-mediated mitochondrial dysfunction. Specifically, amantadine corrected (i) overload of mitochondrial Ca2+; (ii) inhibition of respiratory chain activity and oxidative phosphorylation; (iii) reduction of membrane potential; and (iv) overproduction of reactive oxygen species. The effects of amantadine were observed within 15min following drug administration and confirmed in Huh-7.5 cells transfected with an infectious HCV genome. These effects were also observed in cells expressing subgenomic HCV constructs, indicating that they are not mediated or only in part mediated by p7. Single organelle analyzes carried out on isolated mouse liver mitochondria demonstrated that amantadine induces hyperpolarization of the membrane potential. Moreover, amantadine treatment increased the calcium threshold required to trigger mitochondrial permeability transition opening. In conclusion, these results support a role of amantadine in preserving cellular bioenergetics and redox homeostasis in HCV-infected cells and unveil an effect of the drug which might be exploited for a broader therapeutic utilization.

Translocation of iron from lysosomes to mitochondria during ischemia predisposes to injury after reperfusion in rat hepatocytes

The mitochondrial permeability transition (MPT) initiated by reactive oxygen species (ROS) plays an essential role in ischemia–reperfusion (IR) injury. Iron is a critical catalyst for ROS formation, and intracellular chelatable iron promotes oxidative injury-induced and MPT-dependent cell death in hepatocytes. Accordingly, our aim was to investigate the role of chelatable iron in IR-induced ROS generation, MPT formation, and cell death in primary rat hepatocytes. To simulate IR, overnight-cultured hepatocytes were incubated anoxically at pH 6.2 for 4h and reoxygenated at pH 7.4. Chelatable Fe2+, ROS, and mitochondrial membrane potential were monitored by confocal fluorescence microscopy of calcein, chloromethyldichlorofluorescein, and tetramethylrhodamine methyl ester, respectively. Cell killing was assessed by propidium iodide fluorimetry. Ischemia caused progressive quenching of cytosolic calcein by more than 90%, signifying increased chelatable Fe2+. Desferal and starch–desferal 1h before ischemia suppressed calcein quenching. Ischemia also induced quenching and dequenching of calcein loaded into mitochondria and lysosomes, respectively. Desferal, starch–desferal, and the inhibitor of the mitochondrial Ca2+ uniporter (MCU), Ru360, suppressed mitochondrial calcein quenching during ischemia. Desferal, starch–desferal, and Ru360 before ischemia also decreased mitochondrial ROS formation, MPT opening, and cell killing after reperfusion. These results indicate that lysosomes release chelatable Fe2+ during ischemia, which is taken up into mitochondria by MCU. Increased mitochondrial iron then predisposes to ROS-dependent MPT opening and cell killing after reperfusion.

Exploiting the Role of Resveratrol in Rat Mitochondrial Permeability Transition

Resveratrol (RSV), a natural polyphenolic antioxidant, has been considered an anticarcinogenic agent as it triggers tumor cell apoptosis through activation of the mitochondrial pathway. In our study, the effects of RSV on mitochondria, especially on the mitochondrial permeability transition (MPT) process, were investigated by multiple methods. We found that RSV induced a collapse of membrane potential and matrix swelling related to MPT. We further demonstrated that Ca²⁺ was necessary for this RSV-induced MPT opening. In addition, RSV induced the inner membrane permeabilization to H⁺ and K⁺, the depression of respiration and changes in membrane fluidity. The results suggested that RSV-induced MPT was accompanied by mitochondrial dysfunction. But the prohibition on lipid peroxidation and different effects of low- and high-dose RSV on membrane fluidity and respiration showed that the interaction of RSV and the mitochondria could not be the result of a single simple mechanism.

Ischemia‐induced mobilization of lysosomal iron predisposes rat hepatocytes to ischemia‐reperfusion (IR) injury

Reactive oxygen species (ROS) initiate onset of the mitochondrial permeability transition (MPT) and play a key role in IR injury. Iron is a critical catalyst for ROS generation. Accordingly, our aim was to investigate the role of chelatable iron in ROS‐dependent MPT formation and cell death during IR to rat hepatocytes. Cells were incubated anoxically at pH 6.2 for 4 h and re‐oxygenated at pH 7.4 to simulate IR. Chelatable iron, ROS, mitochondrial membrane potential and cell death were monitored by confocal imaging of calcein, chloromethyl dichlorofluorescein, tetramethylrhodamine methyl ester and propidium iodide, respectively. Ischemia caused progressive quenching of cytosolic calcein, indicating an increase of chelatable Fe2+, which was suppressed by desferal and starch‐desferal pretreatment. Quenching of calcein was also blocked by ebselen and ferristatin, inhibitors of lysosomal divalent metal transporter‐1. Ischemia induced quenching of calcein loaded into mitochondria, which was blocked by desferal, starch‐desferal and Ru360. These agents also decreased mitochondrial ROS formation, MPT onset and cell killing after reperfusion. In conclusion, lysosomal iron is mobilized into mitochondria during ischemia. Increased mitochondrial iron then predisposes cells for ROS‐mediated MPT opening and cell killing after reperfusion. (Supported by NIH grants DK073336 and DK37034)

Disruption of Hexokinase II–Mitochondrial Binding Blocks Ischemic Preconditioning and Causes Rapid Cardiac Necrosis

Isoforms I and II of the glycolytic enzyme hexokinase (HKI and HKII) are known to associate with mitochondria. It is unknown whether mitochondria-bound hexokinase is mandatory for ischemic preconditioning and normal functioning of the intact, beating heart. We hypothesized that reducing mitochondrial hexokinase would abrogate ischemic preconditioning and disrupt myocardial function. Ex vivo perfused HKII(+/-) hearts exhibited increased cell death after ischemia and reperfusion injury compared with wild-type hearts; however, ischemic preconditioning was unaffected. To investigate acute reductions in mitochondrial HKII levels, wild-type hearts were treated with a TAT control peptide or a TAT-HK peptide that contained the binding motif of HKII to mitochondria, thereby disrupting the mitochondrial HKII association. Mitochondrial hexokinase was determined by HKI and HKII immunogold labeling and electron microscopy analysis. Low-dose (200 nmol/L) TAT-HK treatment significantly decreased mitochondrial HKII levels without affecting baseline cardiac function but dramatically increased ischemia-reperfusion injury and prevented the protective effects of ischemic preconditioning. Treatment for 15 minutes with high-dose (10 μmol/L) TAT-HK resulted in acute mitochondrial depolarization, mitochondrial swelling, profound contractile impairment, and severe cardiac disintegration. The detrimental effects of TAT-HK treatment were mimicked by mitochondrial membrane depolarization after mild mitochondrial uncoupling that did not cause direct mitochondrial permeability transition opening. Acute low-dose dissociation of HKII from mitochondria in heart prevented ischemic preconditioning, whereas high-dose HKII dissociation caused cessation of cardiac contraction and tissue disruption, likely through an acute mitochondrial membrane depolarization mechanism. The results suggest that the association of HKII with mitochondria is essential for the protective effects of ischemic preconditioning and normal cardiac function through maintenance of mitochondrial potential.

Blockade of electron transport during ischemia preserves bcl-2 and inhibits opening of the mitochondrial permeability transition pore

Myocardial ischemia damages the electron transport chain and augments cardiomyocyte death during reperfusion. To understand the relationship between ischemic mitochondrial damage and mitochondrial-driven cell death, the isolated perfused heart underwent global stop-flow ischemia with and without mitochondrial protection by reversible blockade of electron transport. Ischemic damage to electron transport depleted bcl-2 content and favored mitochondrial permeability transition (MPT). Reversible blockade of electron transport preserved bcl-2 content and attenuated calcium-stimulated mitochondrial swelling. Thus, the damaged electron transport chain leads to bcl-2 depletion and MPT opening. Chemical inhibition of bcl-2 with HA14-1 also dramatically increased mitochondrial swelling, augmented by exogenous H2O2 stress, indicating that bcl-2 depleted mitochondria are poised to undergo MPT during the enhanced oxidative stress of reperfusion.