Permeability Transition Pore Formation Research Articles

What Makes You Can Also Break You, Part II: Mitochondrial Permeability Transition Pore Formation by Dimers of the F1FO ATP-Synthase?

What Makes You Can Also Break You, Part II: Mitochondrial Permeability Transition Pore Formation by Dimers of the F1FO ATP-Synthase?

3-Nitropropionic acid induces autophagy by forming mitochondrial permeability transition pores rather than activating the mitochondrial fission pathway.

Huntington's disease is a neurodegenerative process associated with mitochondrial alterations. Inhibitors of the electron-transport channel complex II, such as 3-nitropropionic acid (3NP), are used to study the molecular and cellular pathways involved in this disease. We studied the effect of 3NP on mitochondrial morphology and its involvement in macrophagy. Pharmacological and biochemical methods were used to characterize the effects of 3NP on autophagy and mitochondrial morphology. SH-SY5Y cells were transfected with GFP-LC3, GFP-Drp1 or GFP-Bax to ascertain their role and intracellular localization after 3NP treatment using confocal microscopy. Untreated SH-SY5Y cells presented a long, tubular and filamentous net of mitochondria. After 3NP (5 mM) treatment, mitochondria became shorter and rounder. 3NP induced formation of mitochondrial permeability transition pores, both in cell cultures and in isolated liver mitochondria, and this process was inhibited by cyclosporin A. Participation of the mitochondrial fission pathway was excluded because 3NP did not induce translocation of the dynamin-related protein 1 (Drp1) to the mitochondria. The Drp1 inhibitor Mdivi-1 did not affect the observed changes in mitochondrial morphology. Finally, scavengers of reactive oxygen species failed to prevent mitochondrial alterations, while cyclosporin A, but not Mdivi-1, prevented the generation of ROS. There was a direct correlation between formation of mitochondrial permeability transition pores and autophagy induced by 3NP treatment. Activation of autophagy preceded the apoptotic process and was mediated, at least partly, by formation of reactive oxygen species and mitochondrial permeability transition pores.

Is p53 the Long-Sought Molecular Trigger for Cyclophilin D–Regulated Mitochondrial Permeability Transition Pore Formation and Necrosis?

An article recently published in Cell concluded that p53 is necessary and sufficient to induce mitochondrial permeability transition pore (MPTP)–dependent necrosis through inducible p53 translocation to the matrix with cyclophilin D (CypD) binding. The results and implications are very provocative. The physiological significance of the proposed paradigm, however, is uncertain because calcium itself, which is a fundamental regulator of MPTP, is independent of p53, as shown by the authors. In addition, purified mitochondria from any unstimulated cell type or tissue, which presumably lacks p53 given the inducible mechanism proposed, have a fully functional MPTP to all the classic modes of stimulation as analyzed in vitro. Myocardial infarction produces an area of myocyte loss that is a mixture of apoptotic and necrotic cell death. Recent data from multiple laboratories have shown that necrosis can be a programmed event and that specific molecular inhibitors of this process can be potently cardioprotective. During ischemic injury, both reactive oxygen species (ROS) and calcium levels increase within cells, triggering a type of programmed necrosis through MPTP opening that is CypD-dependent.1–5 Indeed, treatment with cyclosporine A, a CypD inhibitor, as well as genetic deletion of CypD, reduces ischemia/reperfusion injury in both the brain and the heart.3–5 Thus, mitochondria are central regulators of ischemic cell death through a mechanism involving the MPTP, but until recently we have lacked an understanding of how CypD is activated and potentially induces MPTP directly. However, Moll et al6 recently published an interesting article in the journal Cell , which shows that p53 can open the MPTP leading to necrotic cell death (Figure). They propose that upon ROS stimulation, p53 translocates to the matrix of the mitochondria where it binds to CypD and somehow opens the MPTP (Figure). This function of p53 was independent of …

Mitochondrial Calcium and Reactive Oxygen Species Regulate Agonist-Initiated Platelet Phosphatidylserine Exposure

To study the interactions of cytoplasmic calcium elevation, mitochondrial permeability transition pore (mPTP) formation, and reactive oxygen species formation in the regulation of phosphatidylserine (PS) exposure in platelets. mPTP formation, but not the degree of cytoplasmic calcium elevation, was associated with PS exposure in wild-type, cyclophilin D-null, ionomycin-treated, and reactive oxygen species-treated platelets. In the absence of the mPTP regulator cyclophilin D, agonist-initiated mPTP formation and high-level PS exposure were markedly blunted, but cytoplasmic calcium transients were unchanged. Mitochondrial calcium (Ca(2+)(mit)) transients and reactive oxygen species, key regulators of mPTP formation, were examined in strongly stimulated platelets. Increased reactive oxygen species production occurred in strongly stimulated platelets and was dependent on extracellular calcium entry, but not the presence of cyclophilin D. Ca(2+)(mit) increased significantly in strongly stimulated platelets. Abrogation of Ca(2+)(mit) entry, either by inhibition of the Ca(2+)(mit) uniporter or mitochondrial depolarization, prevented mPTP formation and exposure but not platelet aggregation or granule release. Sustained cytoplasmic calcium levels are necessary, but not sufficient, for high-level PS exposure in response to agonists. Increased Ca(2+)(mit) levels are a key signal initiating mPTP formation and PS exposure. Blockade of Ca(2+)(mit) entry allows the specific inhibition of platelet procoagulant activity.

The Role of Cyclosporine in the Treatment of Myocardial Reperfusion Injury

Myocardial injury in adult, pediatric, and newborn patients is a leading cause of mortality and morbidity. Although the underlying etiologies are different among patient populations, the sequence of initial ischemic-hypoxic injury followed by secondary myocardial reperfusion injury is relatively consistent. Overall infarct size is important because it is believed to be a key determinant of mortality. The detrimental effects of myocardial reperfusion have been proposed to be at least partially related to the formation of mitochondrial permeability transition pore (MPTP). The MPTP is a nonspecific pore, which forms during myocardial reperfusion and allows the release of apoptotic signaling molecules and may also lead to cellular necrosis. Cyclosporine A has been shown to be a potent inhibitor of the MPTP, leading to its study as a potential treatment to limit myocardial reperfusion injury. Multiple adult animal models have demonstrated the protective effects of cyclosporine in ischemia-reperfusion. A recent human pilot clinical trial also reported reduced myocardial injury and infarct size in patients treated with cyclosporine intravenously before percutaneous coronary intervention for ST-elevation myocardial infarction. Despite the paucity of evidence of cyclosporine A demonstrating myocardial protection in pediatric and newborn patients, the existing animal experimental results are promising.

Regulation of Platelet Procoagulant Activity by Mitochondrial Calcium Influx

Abstract 2193Phosphatidylserine (PS) exposure is critical for blood coagulation. On platelets PS exposure provides a surface for the assembly of coagulation enzyme complexes. Two pathways of platelet PS exposure have been identified, an apoptotic Bax/Bak-mediated pathway and an agonist-initiated and cyclophilin D (CypD)-regulated pathway. In the agonist-initiated pathway sustained high levels of intracellular calcium that occur in strongly-stimulated platelets cause mitochondrial permeability transition pore (mPTP) formation and platelet PS exposure. Even in strongly-stimulating conditions PS exposure is limited to a subpopulation of the activated platelets. In this study we investigated how mitochondrial calcium (Ca2+mit) elevations link the intracellular processes of cytoplasmic calcium (Ca2+cyt) elevation, mPTP formation and platelet PS exposure, and identify Ca2+mit influx as a novel potential therapeutic target to manipulate platelet procoagulant activity independent of other platelet activation events.We have previously characterized the sequence of calcium and mitochondrial events that occur in procoagulant platelets and demonstrated that Ca2+cyt elevations occur prior to mPTP formation in platelets. Experiments were performed to investigate whether PS exposure could be modulated independently of elevations in Ca2+cyt through alterations in mPTP sensitivity. First we tested whether PS exposure initiated by calcium ionophore treatment was affected by the absence of CypD, a critical regulatory component of the mPTP that potentiates its calcium responsiveness. In the absence of CypD a two-threefold greater concentration of ionophore was required to initiate platelet PS exposure. Phenylarsine oxide (PAO) potentiates the calcium sensitivity of the mPTP. PAO-treated platelets required two-three fold less calcium ionophore to initiate PS exposure, and this increase was independent of Ca2+cyt elevation. Together, these experiments establish the concept that modulation of the mPTP response can affect platelet PS exposure independent of Ca2+cyt elevations.Ca2+mit influx regulates mPTP formation. Using the mitochondrial-specific and calcium-sensitive dye rhod-2, Ca2+mit was found to be rapidly elevated in strongly-stimulated platelets, and the degree of Ca2+mit elevation was closely associated with platelet PS exposure. Calcium influx in the mitochondria occurs as the result of passive flow of Ca2+cyt down its electrochemical gradient through the mitochondrial calcium uniporter (MCU), a recently identified inner-mitochondrial transmembrane channel. Treatment with the MCU-specific inhibitor Ru360 effectively prevented agonist-initiated Ca2+mit influx and platelet PS exposure. To further test the importance of Ca2+mit in the regulation of platelet PS exposure, the effects of altering prestimulatory mitochondrial transmembrane potential (ΔΨm) were examined using inhibitors of mitochondrial respiration and oxidative phosphorylation. Decreased prestimulatory ΔΨm should decrease the passive flow of calcium into the mitochondria down its electrical gradient. In these preconditioned platelets prestimulatory ΔΨm was strongly positively correlated with both agonist-initiated Ca2+mit elevation and PS exposure (R2=0.9153). Minimal correlation was observed between the degree of Ca2+cyt elevation and PS exposure (R2=0.4254). The therapeutic effects of the anti-diabetic agent metformin have been proposed to occur through inhibition of respiratory complex I, an effect that should decrease prestimulatory ΔΨm. Metformin treatment of platelets decreased prestimulatory ΔΨm, Ca2+mit elevation, and platelet PS exposure in strongly-stimulated platelets. Together, these data emphasize the importance of Ca2+mit elevation in the regulation of agonist-initiated PS exposure, and identify inhibition of Ca2+mit uptake either by MCU antagonists or by decreasing prestimulatory ΔΨm as a novel potential target in the prevention of platelet procoagulant activity. Disclosures:No relevant conflicts of interest to declare.

Mitochondrially-Mediated Platelet Integrin AlphaIIbbeta3 Inactivation Limits Platelet Recruitment and Thrombus Growth,

Abstract 3251Platelet stimulation with strong agonist(s) results in the formation of a phenotypically unique platelet subpopulation known as procoagulant platelets. All platelets stimulated in strongly-activating conditions undergo granule release, activation of integrin αIIbβ3, and spreading. However, minutes later a subpopulation of these activated platelets undergo additional phenotypic changes including phosphatidylserine (PS) exposure, adoption of a vesiculated and balloon-like morphology, and an altered integrin αIIbβ3 conformation that has a decreased binding affinity for activation-dependent antibodies such as PAC-1 or JON/A. Deletion of the mitochondrial protein cyclophilin D (CypD), a critical regulator of mitochondrial permeability transition pore (mPTP) formation, results in marked abrogation of procoagulant platelet formation. We have previously demonstrated remarkable impairment of dual-agonist-initiated PS exposure and procoagulant activity in CypD−/− platelets (Jobe et al, Blood 2008). In this study the functional importance of the morphological and adhesive changes that occur in procoagulant platelets were examined using CypD−/− platelets and mice with platelet-specific deficiency of CypD. (CypDplt−/−).The morphology and phenotype of fibrinogen-adherent CypD+/+ (WT) and CypD−/− platelets were examined following dual stimulation with thrombin and the GPVI agonist convulxin. In dual-stimulated WT platelets, platelets initially spread; then after three minutes, nearly half of the platelets demonstrated lamellopodial and filipodial retraction with platelet rounding and vesiculation, and these changes were associated with increased PS exposure and decreased binding of JON/A. These changes were delayed in CypD−/− platelets, but eventually started to be seen 30 minutes subsequent to stimulation. Adhesion and platelet recruitment to strongly-stimulated platelets were studied in flow conditions. Increased platelet accumulation was noted when unstimulated platelets were flowed over strongly-stimulated adherent CypD−/− platelets relative to WT platelets. Similarly, increased platelet accumulation of CypD−/− platelets was noted on a collagen surface compared to WT platelets. In platelet aggregation assays, dual stimulated WT platelets demonstrated a chaotic pattern of aggregation two to three minutes after activation consistent with aggregate disruption, a pattern that was not observed in CypD−/− platelets. Together these studies indicate that procoagulant platelet formation results in integrin inactivation. Since this process is impaired in the absence of CypD, CypD−/− platelets demonstrate increased platelet accumulation in flow assays and stable aggregate formation. Adhesive and cytoskeletal proteins were investigated in strongly-stimulated platelets. Western blot analysis demonstrated significant proteolytic cleavage of both talin and the cytoplasmic domain of integrin β3 in WT platelets stimulated with thrombin plus convulxin, and these events were only minimally observed in single-agonist stimulated platelets. Proteolytic cleavage of both talin and integrin β3 were markedly decreased in CypD−/− platelets and in WT platelets treated with calpain inhibitors.CypD is a ubiquitously expressed protein; therefore, in vivo thrombosis and hemostasis were tested in CypDplt−/− mice. Following photochemically mediated mesenteric endothelial injury, time to occlusion was shortened in CypDplt−/− mice (723 ± 111 sec in CypDplt+/− vs. 371 ± 106 sec in CypDplt−/− mice), and complete arterial occlusion was increased in CypDplt−/− mice (55% in CypDplt+/− vs. 86% in CypDplt−/−) (p=0.02). Our in vitro and in in vivo results are consistent with the hypothesis that in strongly-stimulated platelets mitochondrially-mediated events initiate proteolytic cleavage of talin and integrin β3 accompanied by platelet integrin αIIbβ3 inactivation, and that this process limits platelet recruitment and thrombus growth. Since these events are limited to platelets stimulated by strong agonist(s), these results suggest a novel negative feedback mechanism initiated by accumulation of multiple or strong agonist(s) that limits thrombotic occlusion in vivo. Disclosures:No relevant conflicts of interest to declare.

Do mitochondria play a role in remodelling lace plant leaves during programmed cell death?

BackgroundProgrammed cell death (PCD) is the regulated death of cells within an organism. The lace plant (Aponogeton madagascariensis) produces perforations in its leaves through PCD. The leaves of the plant consist of a latticework of longitudinal and transverse veins enclosing areoles. PCD occurs in the cells at the center of these areoles and progresses outwards, stopping approximately five cells from the vasculature. The role of mitochondria during PCD has been recognized in animals; however, it has been less studied during PCD in plants.ResultsThe following paper elucidates the role of mitochondrial dynamics during developmentally regulated PCD in vivo in A. madagascariensis. A single areole within a window stage leaf (PCD is occurring) was divided into three areas based on the progression of PCD; cells that will not undergo PCD (NPCD), cells in early stages of PCD (EPCD), and cells in late stages of PCD (LPCD). Window stage leaves were stained with the mitochondrial dye MitoTracker Red CMXRos and examined. Mitochondrial dynamics were delineated into four categories (M1-M4) based on characteristics including distribution, motility, and membrane potential (ΔΨm). A TUNEL assay showed fragmented nDNA in a gradient over these mitochondrial stages. Chloroplasts and transvacuolar strands were also examined using live cell imaging. The possible importance of mitochondrial permeability transition pore (PTP) formation during PCD was indirectly examined via in vivo cyclosporine A (CsA) treatment. This treatment resulted in lace plant leaves with a significantly lower number of perforations compared to controls, and that displayed mitochondrial dynamics similar to that of non-PCD cells.ConclusionsResults depicted mitochondrial dynamics in vivo as PCD progresses within the lace plant, and highlight the correlation of this organelle with other organelles during developmental PCD. To the best of our knowledge, this is the first report of mitochondria and chloroplasts moving on transvacuolar strands to form a ring structure surrounding the nucleus during developmental PCD. Also, for the first time, we have shown the feasibility for the use of CsA in a whole plant system. Overall, our findings implicate the mitochondria as playing a critical and early role in developmentally regulated PCD in the lace plant.

Reversible inhibition of hydrogen peroxide elimination by calcium in brain mitochondria

In the present work, the Ca(2+) dependence of mitochondrial H(2) O(2) elimination was investigated. Mitochondria isolated from guinea pig brain were energized by glutamate and malate and incubated with micromolar concentrations of Ca(2+) in the presence of ADP, preventing permeability transition pore formation. After the completion of Ca(2+) uptake, mitochondria were challenged with H(2) O(2) (5 μM), then at various time points residual H(2) O(2) was determined using the Amplex red method and compared with that in mitochondria incubated with H(2) O(2) without Ca(2+) addition. Dose-dependent inhibition of H(2) O(2) elimination by Ca(2+) was detected, which was prevented by the Ca(2+) -uptake inhibitor Ru 360. Stimulation of Ca(2+) release from Ca(2+) -loaded mitochondria by a combined addition of Ru 360 and Na(+) decreased the Ca(2+) -evoked inhibition of H(2) O(2) removal. After Ca(2+) uptake (50 μM), mitochondrial aconitase activity was found to be decreased, which was partially attributable to the impaired elimination of endogenously produced reactive oxygen species. We found that the effects of Ca(2+) and H(2) O(2) on the activity of aconitase were additive. These results confirm that Ca(2+) inhibits elimination of H(2) O(2) in mitochondria and demonstrate that this effect is concentration dependent and reversible. The phenomenon described here can play a role in the modulation of ROS handling under conditions involving excessive cellular Ca(2+) load.

Remote Ischemic Preconditioning

Circulatory arrest and stroke are among the leading causes of death and disability worldwide, and they are difficult to treat. Only a limited number of therapeutic approaches have reached the bedside (eg, hypothermia and recombinant tissue plasminogen activator)1 and, unfortunately, relatively few patients are eligible. Virtually all experimental compounds tested so far have failed at some stage of translational development. Preconditioning is an attractive strategy that makes the brain and other visceral organs relatively resistant to tissue injury. It develops when a noxious stimulus, below the damage threshold, leads to reduced tissue injury on subsequent challenge with a stimulus given above injury threshold. It is an appealing strategy to identify novel endogenous protective mechanisms and, as demonstrated by Jensen, Loukogeorgakis, and their colleagues in this issue of Circulation ,2 it may prove useful to prevent brain damage in the clinical context when there is a high probability of cerebral ischemia (eg, before high-risk surgery or in patients with subarachnoid hemorrhage). Article see p 714 Various types of preconditioning stimuli have been used experimentally to protect brain, heart, retina, liver, kidney, and other organs.3 Some use ischemic preconditioning, in which the blood supply to a target organ is temporarily interrupted before introducing a longer infarct-generating insult that would ordinarily produce infarction; other studies use hypoxic preconditioning, in which animals are exposed to oxygen levels around 8% to 9% for a few hours. But ischemia and hypoxia are just 2 examples in a larger list of strategies that induce tolerance to brain injury: hypoglycemia, hypoxia, kainate-induced seizures, and exposure to volatile anesthetics, to name a few.4 Preconditioning can protect the brain either rapidly after stimulation (known as early, first window, or classical preconditioning) or after a 24-hour delay to induce protein synthesis-dependent protection lasting up to 96 hours …

Focal cerebral ischemia and mitochondrial dysfunction in the TNFα-transgenic rat

Post-ischemic neurodegeneration may be accelerated by a cytokine-receptor mediated apoptotic pathway, as shown in a transgenic rat overexpressing tumor necrosis factor-alpha (TNFα) in brain. To further investigate the mechanism of ischemic cellular injury in this animal, we tested the hypothesis that increased synthesis of TNFα augments neuronal death by promoting mitochondrial dysfunction, calcium dysregulation, and oxidative stress. Adult male TNFα-transgenic (TNFα-Tg) and non-transgenic (non-Tg) littermates underwent reversible middle cerebral artery occlusion (MCAO) for 1hour followed by 1hour of reperfusion. Cortical mitochondria were isolated from injured (ipsilateral) and uninjured (contralateral) hemispheres of ischemic rats or from pooled hemispheres of control animals. ATP synthesis was attenuated in non-ischemic TNFα-Tg rats, demonstrated by reduction of state III and respiratory control ratio, increased production of reactive oxygen species, and earlier formation of the calcium-induced membrane permeability transition pore. After MCAO, mitochondrial dysfunction was augmented more significantly in ischemic TNFα-Tg brain mitochondria than in non-Tg rats. These results show that mitochondrial dysfunction may be caused by increased brain levels of TNFα without physiological stress but will be exacerbated after MCAO. We conclude that ischemic stress and synthesis of inflammatory cytokines synergistically augment mitochondrial dysfunction to promote neuronal death.

Role of MGST1 in reactive intermediate-induced injury

Microsomal glutathione transferase (MGST1, EC 2.5.1.18) is a membrane bound glutathione transferase extensively studied for its ability to detoxify reactive intermediates, including metabolic electrophile intermediates and lipophilic hydroperoxides through its glutathione dependent transferase and peroxidase activities. It is expressed in high amounts in the liver, located both in the endoplasmic reticulum and the inner and outer mitochondrial membranes. This enzyme is activated by oxidative stress. Binding of GSH and modification of cysteine 49 (the oxidative stress sensor) has been shown to increase activation and induce conformational changes in the enzyme. These changes have either been shown to enhance the protective effect ascribed to this enzyme or have been shown to contribute to cell death through mitochondrial permeability transition pore formation. The purpose of this review is to elucidate how one enzyme found in two places in the cell subjected to the same conditions of oxidative stress could both help protect against and contribute to reactive oxygen species-induced liver injury.

Changes In Mitochondrial Permeability Initiate Platelet Phosphatidylserine Exposure In Response to Elevated Cytoplasmic Calcium Levels

AbstractAbstract 2024Platelet phosphatidylserine (PS) exposure and procoagulant activity occur in a subset of activated platelets following stimulation with multiple or high levels of agonists. Key among the upstream regulators of agonist-initiated PS exposure is elevated cytoplasmic calcium levels. Additionally, the importance of mitochondrial permeability transition pore (mPTP) formation in regulating PS exposure has been highlighted by recent studies using cyclophilin D null (CypD -/-) platelets. CypD is a critical regulatory protein in the initiation of mPTP formation, and in the absence of CypD agonist-initiated PS exposure is markedly impaired. An alternative, slower pathway of PS exposure, initiated by the BH3 mimetic ABT-737, is regulated by members of the Bcl-2 protein pathway.It is currently unclear how mitochondrial and calcium-signaling pathways interact to regulate PS exposure in platelets. mPTP formation has been reported as both a consequence and a cause of elevated intracellular calcium levels in other cells. To clarify the relative roles of these two upstream signals, we examined the sequence of events that occur upon platelet activation using calcium and mitochondrial transmembrane potential (ΔΨm) sensitive fluorescent probes. In isolated platelets studied by flow cytometry and among adherent platelets evaluated by confocal microscopy, elevated intracellular calcium levels were observed prior to mPTP formation (as determined by loss of ΔΨm). A rise in mitochondrial calcium levels as measured by rhod-2 occurred coincidentally with elevations in cytoplasmic calcium levels. Similar increases in intracellular calcium levels were observed in CypD -/- and CypD +/+ platelets, despite the almost complete absence of mPTP formation and PS exposure in the CypD -/- platelets. These results are consistent with calcium acting as an upstream signal regulating mPTP formation.Ionomycin (3 μM) stimulation can overcome the defect in PS exposure in CypD -/- platelets. This result has been interpreted to indicate that calcium activates effector pathways downstream of the mPTP. However, an alternative explanation is that ionomycin raises cytoplasmic calcium to an elevated level that is sufficient to initiate mPTP formation even in CypD's absence. Dose response studies indicated that the calcium sensitivity of the mPTP was decreased in CypD -/- platelets, but ionomycin (3uM) caused mPTP opening and PS externalization in both CypD +/+ and CypD -/- platelets. Furthermore, in both CypD +/+ and CypD -/- platelets, mPTP formation and PS exposure were closely correlated at all doses of ionomycin utilized. These results indicate that high doses of ionomycin overcome the CypD -/- defect in PS exposure by initiating non-CypD-dependent mPTP formation.The roles of the mitochondrial and calcium pathways in regulating ABT-737-mediated PS exposure were examined. Extracellular calcium was not required for PS exposure initiated by ABT-737, distinct from its importance in agonist-initiated PS exposure. In contrast to previously reported results (Schoenwaelder et al, Blood 2009) we observed that ABT-737 stimulation causes a loss of ΔΨm coincident with PS exposure. Since disruptions in oxidative metabolism could also cause the observed effect on ΔΨm, ABT-737's effects on mitochondrial permeability were confirmed using a calcein-cobalt quenching assay. Unlike agonist-initiated PS exposure, both increased mitochondrial permeability and PS exposure were not dependent on CypD.Together these results indicate a central role for alterations in mitochondrial membrane permeability in mediating PS exposure. In agonist-stimulated platelets increases in cytoplasmic calcium result in mPTP formation and mitochondrial permeabilization, triggering downstream events that result in PS exposure. In ABT-737 stimulated platelets non-CypD-dependent mitochondrial permeability changes mediated by Bcl-2 proteins may also act as an important initiator of the events that result in PS exposure. Disclosures:No relevant conflicts of interest to declare.

Le postconditionnement au cours de l’infarctus myocardique aigu : l’angioplastie primaire revisitée ?

Early reperfusion of ischemic myocardium is the mean to improve prognosis of acute myocardial infarction. Nevertheless, reperfusion injury due to immediate acidosis correction and subsequent Ca 2+ overload results in formation of the mitochondrial permeability transition pore. The consequence is the death of viable myocardium due to onconecrosis and apoptosis. Mechanical (Stuttering reperfusion) or pharmacological postconditioning (cyclosporine A, adenosine…) is able to prevent reperfusion injury resulting in more myocardial salvage.

Augmented O-GlcNAc signaling attenuates oxidative stress and calcium overload in cardiomyocytes.

O-linked β-N-acetylglucosamine (O-GlcNAc) is an inducible, dynamically cycling and reversible post-translational modification of Ser/Thr residues of nucleocytoplasmic and mitochondrial proteins. We recently discovered that O-GlcNAcylation confers cytoprotection in the heart via attenuating the formation of mitochondrial permeability transition pore (mPTP) and the subsequent loss of mitochondrial membrane potential. Because Ca(2+) overload and reactive oxygen species (ROS) generation are prominent features of post-ischemic injury and favor mPTP formation, we ascertained whether O-GlcNAcylation mitigates mPTP formation via its effects on Ca(2+) overload and ROS generation. Subjecting neonatal rat cardiac myocytes (NRCMs, n≥6 per group) to hypoxia, or mice (n≥4 per group) to myocardial ischemia reduced O-GlcNAcylation, which later increased during reoxygenation/reperfusion. NRCMs (n≥4 per group) infected with an adenovirus carrying nothing (control), adenoviral O-GlcNAc transferase (adds O-GlcNAc to proteins, AdOGT), adenoviral O-GlcNAcase (removes O-GlcNAc to proteins, AdOGA), vehicle or PUGNAc (blocks OGA; increases O-GlcNAc levels) were subjected to hypoxia-reoxygenation or H(2)O(2), and changes in Ca(2+) levels (via Fluo-4AM and Rhod-2AM), ROS (via DCF) and mPTP formation (via calcein-MitoTracker Red colocalization) were assessed using time-lapse fluorescence microscopy. Both OGT and OGA overexpression did not significantly (P>0.05) alter baseline Ca(2+) or ROS levels. However, AdOGT significantly (P<0.05) attenuated both hypoxia and oxidative stress-induced Ca(2+) overload and ROS generation. Additionally, OGA inhibition mitigated both H(2)O(2)-induced Ca(2+) overload and ROS generation. Although AdOGA exacerbated both hypoxia and H(2)O(2)-induced ROS generation, it had no effect on H(2)O(2)-induced Ca(2+) overload. We conclude that inhibition of Ca(2+) overload and ROS generation (inducers of mPTP) might be one mechanism through which O-GlcNAcylation reduces ischemia/hypoxia-mediated mPTP formation.

Mitochondrial reprogramming through cardiac oxygen sensors in ischaemic heart disease

Under hypoxic conditions, mitochondria can represent a threat to the cell because of their capacity to generate toxic reactive oxygen species (ROS). However, cardiomyocytes are equipped with an oxygen-sensing pathway that involves prolyl hydroxylase oxygen sensors and hypoxia-inducible factors (HIFs), which induces a tightly regulated programme to keep ischaemic mitochondrial activity under control. The aim of this review is to provide an update on the pathways leading to mitochondrial reprogramming, which occurs in the myocardium during ischaemia, with particular emphasis on those induced by HIF activation. We start by studying the mechanisms of mitochondrial damage during ischaemia and upon reperfusion, highlighting the importance of the formation of the mitochondrial permeability transition pore during reperfusion and its consequences for cardiomyocyte survival. Next, we analyse hypoxia-induced metabolic reprogramming through HIF and its important consequences for mitochondrial bioenergetics, as well as the phenomenon known as the hibernating myocardium. Subsequently, we examine the mechanisms underlying ischaemic preconditioning, focusing, in particular, on those that involve the HIF pathway, such as adenosine signalling, sub-lethal ROS generation, and nitric oxide production. Finally, the role of the mitochondrial uncoupling proteins in ischaemia tolerance is discussed.

Recent Progress of Mitochondrial Dysfunction Induced by β-Amyloid Protein*

Alzheimer′ s disease (AD) is one of the most prevalent neurodegenerative disorders. However, the mechanisms of the disease are still unclear. Increasing evidences demonstrated that mitochondrial dysfunction and structural abnormalities induced by Aβ play important roles in this disease. β-Amyloid (Aβ) induced mitochondrial damage can be attributed to reduced activities of several key enzymes of mitochondrial energy metabolism, impaired balance of mitochondrial fission and fusion as well as mitochondrial membrane permeability transition pore (mPTP) formation. The recent progress in the mechanisms of Aβ induced mitochondrial damage was summarized.

Role of Carboxylic Groups in the Control of Nonspecific Permeability of Mitochondrial Membranes

Ionized COOH groups are present in molecular structures involved in the process of formation of mitochondrial permeability transition pores (MPTPs), in particular, in the ADP/ATP antiporter and/or voltage-dependent anion channels. In experiments on preparations of isolated mitochondria obtained from rat hepatocytes, we found that, in the case of induction of nonspecific permeability through mitochondrial membranes under the action of Cа2+ in a relatively low concentration (15 μM), modulation of the activity of COOH groups with the use of 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (1 mM) led to unidirectional effects, namely to acceleration of the processes of formation of MPTPs and transport of incubation solution and Са2+ through these megachannels, prolongation of the open state of the latter, as well as to increases in the final volume (swelling) of the mitochondria and to a rise in the amount of Са2+ released from these organelles. In contrast, when calcium was used in a high concentration (100 μM), the directions of the above processes were dissimilar. Slowing down of the flow of incubation solution through MPTPs and the process of their formation was observed; at the same time, Са2+ release from the mitochondria was accelerated. However, the final volume of the mitochondria and the amount of Са2+ released from these cellular structures increased. Differences between the effects of the used modulator of the activity of COOH groups on the nonspecific permeability of the mitochondria induced by calcium applied in low and high concentrations are perhaps determined by the following. The process of swelling of the mitochondria is saturable, while Са2+ release from these organelles shows an unlimited pattern. The latter process (Са2+ release) probably undergoes calcium-initiated inactivation. The mechanisms of induction of nonspecific permeability of the mitochondrial membranes under the action of low and high calcium concentrations differ from each other. The calcium uniporter in the mitochondria is sensitive to the modulator of the activity of COOH groups. Diffusion of water through the inner mitochondrial membrane and/or other systems provides some contribution to the studied processes; this can lead to changes in the transport of liquids in these organelles.

Role of Mitochondrial Amyloid-β in Alzheimer's Disease

Mitochondrial dysfunction is an early feature of Alzheimer's disease (AD). Abnormalities in mitochondrial properties include impaired energy metabolism, defects in key respiratory enzyme activity/function, accumulation/generation of mitochondrial reactive oxygen species, and formation of membrane permeability transition pore. While the mechanisms underlying mitochondrial dysfunction remain incompletely understood, recent studies provide substantial evidence for the progressive accumulation of mitochondrial Abeta, which directly links to mitochondria-mediated toxicity. In this review, we describe recent studies addressing the following key questions: 1) Does Abeta accumulate in mitochondria of AD brain and AD mouse models? 2) How does Abeta gain access to the mitochondria? 3) If mitochondria are loaded with Abeta, do they develop similar evidence of dysfunction? 4) What are the mechanisms underlying mitochondrial Abeta-induced neuronal toxicity? and 5) What is the impact of interaction of mitochondrial Abeta with its binding partners (cyclophilin D and ABAD) on mitochondrial and neuronal properties/function in an Abeta milieu? The answers to these questions provide new insights into mechanisms of mitochondrial stress related to the pathogenesis of AD and information necessary for developing therapeutic strategy for AD.

Mechanism of Cardioprotection by Early Ischemic Preconditioning

A series of brief ischemia/reperfusion cycles (termed ischemic preconditioning, IPC) limits myocardial injury produced by a subsequent prolonged period of coronary artery occlusion and reperfusion. Over the last 2 decades our understanding of IPC's mechanism has increased exponentially. Hearts exposed to IPC have a better metabolic and ionic status during prolonged ischemia compared to naïve hearts. However, this difference is not thought to be the main mechanism by which IPC protects against infarction. Signaling pathways that are activated by IPC distinguish IPC hearts from naïve hearts. During the trigger phase of IPC, adenosine, bradykinin and opioid receptors are occupied. Although these three receptors trigger signaling through divergent pathways, the signaling converges on protein kinase C. We have proposed that at the end of the index ischemia the activated PKC sensitizes the low-affinity A(2b) adenosine receptor (A(2b)AR) through phosphorylation of either the receptor or its coupling proteins so that A(2b)AR can be activated by endogenous adenosine released by the previously ischemic cardiomyocytes. The sensitized A(2b)AR would then be responsible for activation of the survival kinases including PI3 kinase, Akt and ERK which then act to inhibit lethal mitochondrial permeability transition pore formation which normally uncouples mitochondria and destroys many myocytes in the first minutes of reperfusion. Herein we review the evidence for the above mechanisms and their functional details.