Postconditioning by Delayed Administration of Ciclosporin A: Implication for Donation after Circulatory Death (DCD).

Heart transplantation is facing a shortage of grafts. Donation after Circulatory Death (DCD) would constitute a new potential of available organs. In the present work, we aimed to evaluate whether Postconditioning (ischemic or with ciclosporin-A (CsA)) could reduce ischemia-reperfusion injury in a cardiac arrest model when applied at the start of reperfusion or after a delay. An isolated rat heart model was used as a model of DCD. Hearts were submitted to a cardiac arrest of 40 min of global warm ischemia (37 °C) followed by 3 h of 4 °C-cold preservation, then 60 min reperfusion. Hearts were randomly allocated into the following groups: control, ischemic postconditioning (POST, consisting of two episodes each of 30 s ischemia and 30 s reperfusion at the onset of reperfusion), and CsA group (CsA was perfused at 250 nM for 10 min at reperfusion). In respective subgroups, POST and CsA were applied after a delay of 3, 10, and 20 min. Necrosis was lower in CsA and POST versus controls (p < 0.01) whereas heart functions were improved (p < 0.01). However, while the POST lost its efficacy if delayed beyond 3 min of reperfusion, CsA treatment surprisingly showed a reduction of necrosis even if applied after a delay of 3 and 10 min of reperfusion (p < 0.01). This cardioprotection by delayed CsA application correlated with better functional recovery and higher mitochondrial respiratory index. Furthermore, calcium overload necessary to induce mitochondrial permeability transition pore (MPTP) opening was similar in all cardioprotection groups, suggesting a crucial role of MPTP in this delayed protection of DCD hearts.

Preconditioning or Postconditioning with 8-Br-cAMP-AM Protects the Heart against Regional Ischemia and Reperfusion: A Role for Mitochondrial Permeability Transition.

The cAMP analogue 8-Br-cAMP-AM (8-Br) confers marked protection against global ischaemia/reperfusion of isolated perfused heart. We tested the hypothesis that 8-Br is also protective under clinically relevant conditions (regional ischaemia) when applied either before ischemia or at the beginning of reperfusion, and this effect is associated with the mitochondrial permeability transition pore (MPTP). 8-Br (10 µM) was administered to Langendorff-perfused rat hearts for 5 min either before or at the end of 30 min regional ischaemia. Ca2+-induced mitochondria swelling (a measure of MPTP opening) and binding of hexokinase II (HKII) to mitochondria were assessed following the drug treatment at preischaemia. Haemodynamic function and ventricular arrhythmias were monitored during ischaemia and 2 h reperfusion. Infarct size was evaluated at the end of reperfusion. 8-Br administered before ischaemia attenuated ventricular arrhythmias, improved haemodynamic function, and reduced infarct size during ischaemia/reperfusion. Application of 8-Br at the end of ischaemia protected the heart during reperfusion. 8-Br promoted binding of HKII to the mitochondria and reduced Ca2+-induced mitochondria swelling. Thus, 8-Br protects the heart when administered before regional ischaemia or at the beginning of reperfusion. This effect is associated with inhibition of MPTP via binding of HKII to mitochondria, which may underlie the protective mechanism.

Effects of plant alkaloids on mitochondrial bioenergetic parameters.

Mitochondria are among the first responders to various stress factors that challenge cell and tissue homeostasis. Various plant alkaloids have been investigated for their capacity to modulate mitochondrial activities. In this study, we used isolated mitochondria from mouse brain and liver tissues to assess nicotine, anatabine and anabasine, three alkaloids found in tobacco plant, for potential modulatory activity on mitochondrial bioenergetics parameters. All alkaloids decreased basal oxygen consumption of mouse brain mitochondria in a dose-dependent manner without any effect on the ADP-stimulated respiration. None of the alkaloids, at 1 nM or 1.25 µM concentrations, influenced the maximal rate of swelling of brain mitochondria. In contrast to brain mitochondria, 1.25 µM anatabine, anabasine and nicotine increased maximal rate of swelling of liver mitochondria suggesting a toxic effect. Only at 1 mM concentration, anatabine slowed down the maximal rate of Ca2+-induced swelling and increased the time needed to reach the maximal rate of swelling. The observed mitochondrial bioenergetic effects are probably mediated through a pathway independent of nicotinic acetylcholine receptors, as quantitative proteomic analysis could not confirm their expression in pure mitochondrial fractions isolated from mouse brain tissue.

ATP Synthase Subunit a Supports Permeability Transition in Yeast Lacking Dimerization Subunits and Modulates yPTP Conductance.

BACKGROUND/AIMS: Mitochondrial ATP synthase, in addition to being involved in ATP synthesis, is involved in permeability transition pore (PTP) formation, which precedes apoptosis in mammalian cells and programmed cell death in yeast. Mutations in genes encoding ATP synthase subunits cause neuromuscular disorders and have been identified in cancer samples. PTP is also involved in pathology. We previously found that in Saccharomyces cerevisiae, two mutations in ATP synthase subunit a (atp6-P163S and atp6-K90E, equivalent to those detected in prostate and thyroid cancer samples, respectively) in the OM45-GFP background affected ROS and calcium homeostasis and delayed yeast PTP (yPTP) induction upon calcium treatment by modulating the dynamics of ATP synthase dimer/oligomer formation. The Om45 protein is a component of the porin complex, which is equivalent to mammalian VDAC. We aimed to investigate yPTP function in atp6-P163S and atp6-K90E mutants lacking the e and g dimerization subunits of ATP synthase. METHODS: Triple mutants with the atp6-P163S or atp6-K90E mutation, the OM45-GFP gene and deletion of the TIM11 gene encoding subunit e were constructed by crossing and tetrad dissection. In spores capable of growing, the original atp6 mutations reverted to wild type, and two compensatory mutations, namely, atp6-C33S-T215C, were selected. The effects of these mutations on cellular physiology, mitochondrial morphology, bioenergetics and permeability transition (PT) were analyzed by fluorescence and electron microscopy, mitochondrial respiration, ATP synthase activity, calcium retention capacity and swelling assays. RESULTS: The atp6-C33S-T215C mutations in the OM45-GFP background led to delayed growth at elevated temperature on both fermentative and respiratory media and increased sensitivity to high calcium ions concentration or hydrogen peroxide in the medium. The ATP synthase activity was reduced by approximately 50% and mitochondrial network was hyperfused in these cells grown at elevated temperature. The atp6-C33S-T215C stabilized ATP synthase dimers and restored the yPTP properties in Tim11∆ cells. In OM45-GFP cells, in which Tim11 is present, these mutations increased the fraction of swollen mitochondria by up to 85% vs 60% in the wild type, although the time required for calcium release doubled. CONCLUSION: ATP synthase subunit e is essential in the S. cerevisiae atp6-P163S and atp6-K90E mutants. In addition to subunits e and g, subunit a is critical for yPTP induction and conduction. The increased yPTP conduction decrease the S. cerevisiae cell fitness.

Cyclosporin A Increases Mitochondrial Buffering of Calcium: An Additional Mechanism in Delaying Mitochondrial Permeability Transition Pore Opening.

Regulation of mitochondrial free Ca2+ is critically important for cellular homeostasis. An increase in mitochondrial matrix free Ca2+ concentration ([Ca2+]m) predisposes mitochondria to opening of the permeability transition pore (mPTP). Opening of the pore can be delayed by cyclosporin A (CsA), possibly by inhibiting cyclophilin D (Cyp D), a key regulator of mPTP. Here, we report on a novel mechanism by which CsA delays mPTP opening by enhanced sequestration of matrix free Ca2+. Cardiac-isolated mitochondria were challenged with repetitive CaCl2 boluses under Na+-free buffer conditions with and without CsA. CsA significantly delayed mPTP opening primarily by promoting matrix Ca2+ sequestration, leading to sustained basal [Ca2+]m levels for an extended period. The preservation of basal [Ca2+]m during the CaCl2 pulse challenge was associated with normalized NADH, matrix pH (pHm), and mitochondrial membrane potential (ΔΨm). Notably, we found that in PO43- (Pi)-free buffer condition, the CsA-mediated buffering of [Ca2+]m was abrogated, and mitochondrial bioenergetics variables were concurrently compromised. In the presence of CsA, addition of Pi just before pore opening in the Pi-depleted condition reinstated the Ca2+ buffering system and rescued mitochondria from mPTP opening. This study shows that CsA promotes Pi-dependent mitochondrial Ca2+ sequestration to delay mPTP opening and, concomitantly, maintains mitochondrial function.

Organelle dynamics and viral infections: at cross roads.

Viruses are obligate intracellular parasites of the host cells. A commonly accepted view is the requirement of internal membranous structures for various aspects of viral life cycle. Organelles enable favourable intracellular environment for several viruses. However, studies reporting organelle dynamics upon viral infections are scant. In this review, we aim to summarize and highlight modulations caused to various organelles upon viral infection or expression of its proteins.

Probing Mitochondrial Permeability Transition Pore Activity in Nucleated Cells and Platelets by High-Throughput Screening Assays Suggests Involvement of Protein Phosphatase 2B in Mitochondrial Dynamics.

Mitochondrial permeability transition pore (mPTP) formation is well documented in isolated mitochondria. However, convincing detection of mPTP in whole cells remains elusive. In this study, we describe a high-throughput assay for Ca2+-activated mPTP opening in platelets using HyperCyt flow cytometry. In addition, we demonstrate that in several nucleated cells, using multiple approaches, the detection of cyclophilin D-dependent mPTP opening is highly challenging. Results with the mitochondrial-targeted Ca2+-sensing green fluorescent protein (mito-Case12) suggest the involvement of protein phosphatase 2B (PP2B; calcineurin) in regulating mitochondrial dynamics. Our results highlight the danger of relying on cyclosporine A alone as a pharmacological tool, and the need for comprehensive studies of mPTP in the cell.

Mitochondrial drug targets in neurodegenerative diseases.

Growing evidence suggests that mitochondrial dysfunction is the main culprit in neurodegenerative diseases. Given the fact that mitochondria participate in diverse cellular processes, including energetics, metabolism, and death, the consequences of mitochondrial dysfunction in neuronal cells are inevitable. In fact, new strategies targeting mitochondrial dysfunction are emerging as potential alternatives to current treatment options for neurodegenerative diseases. In this review, we focus on mitochondrial proteins that are directly associated with mitochondrial dysfunction. We also examine recently identified small molecule modulators of these mitochondrial targets and assess their potential in research and therapeutic applications.

Identifying Compounds that Induce Opening of the Mitochondrial Permeability Transition Pore in Isolated Rat Liver Mitochondria.

The mitochondrial permeability transition pore (MPTP) is a protein pore that forms in the inner mitochondrial membrane and allows the membrane to be permeable to all molecules of less than 1500 Da. Ca(2+), numerous reactive chemicals, and oxidative stress induce MPTP opening, whereas cyclosporin A (CsA) or bongkrekic acid block it. In addition, several drugs have been shown to induce MPTP opening, leading to the loss of mitochondrial membrane potential, swelling of the matrix because of water accumulation, rupture of the outer mitochondrial membrane, and release of intermembrane space proteins into the cytosol. This ultimately leads to the rupture of the outer mitochondrial membrane and cell demise. Here, we describe an assay using isolated rat liver mitochondria that can detect Ca(2+)-dependent drug-induced opening of the MPTP, providing protocols for screening in both cuvette and 96-well format.

Neuroglobin and neuronal cell survival.

The balance between neuronal apoptosis and survival sculpts the developing brain and has an important role in neurodegenerative diseases. Thus, the individuation of signals that could modulate the cell death machinery as well as enhance survival in neurons promises to provide multiple points of therapeutic intervention in neurodegenerative diseases. Neuroglobin (NGB), the first nerve globin identified in neuronal tissues of humans, seems to possess a protective role in the brain only after up-regulation. Here, the NGB physiological role in the control of neuronal survival is reviewed. In vitro studies suggested that cytosolic NGB could react very rapidly with cytochrome c released from mitochondria, thus interfering with the intrinsic pathway of apoptosis. Although very suggestive, these data do not explain either the role of NGB up-regulation in neuroprotection or the recently reported NGB localization into mitochondria. Recently, we identified the steroid hormone 17ß-estradiol (E2) as an endogenous modulator of NGB levels in neuroblastoma SK-N-BE cell line. Upon E2 stimulation, NGB reallocates mainly into mitochondria where the association with the mitochondrial cytochrome c occurs. Remarkably, E2 treatment before an apoptotic stimulus strongly enhances the NGB:cytochrome c association reducing cytochrome c release into the cytosol. As a consequence, a decrease of caspase-3 activation and, in turn, of the apoptotic cascade activation take place. Besides E2, other compounds have been reported to up-regulate the NGB expression highlighting the possibility to develop NGB-mediated therapeutic strategies against stroke damage and neurodegenerative diseases. This article is part of a Special Issue entitled: Oxygen Binding and Sensing Proteins.