We here used fluorescence imaging to explore the effect of co-overexpression of Mcl-1 and Bak/BH3-only proteins on mitochondrial morphology. The cells co-expressing CFP-Mcl-1 and YFP-Bak/BimL/Puma/tBid showed co-localization of Mcl-1 with Bak/Puma/BimL/tBid and also showed the inhibitory action of Mcl-1 on the Bak-, BimL-, Puma- or tBid-mediated cell death. Co-expression of Mcl-1 and Bak but not BH3-only proteins induced time-dependent mitochondrial swelling. Fluorescence resonance energy transfer (FRET) imaging proved the direct binding of Mcl-1 to Bak, BimL, Puma and tBid, respectively. In addition, Mcl-1 prevented Bak oligomerization by retrotranslocating Bak from mitochondria into cytoplasm. Moreover, Mcl-1-Bak complex exhibited a good co-localization with mitochondria, and co-expression of Mcl-1 and Bak for more than 24 h not only induced mitochondrial swelling but also impaired mitochondrial membrane potential. Collectively, co-expression of Mcl-1 and Bak but not BH3-only proteins significantly induced mitochondrial swelling and subsequent loss of mitochondrial membrane potential.

To demonstrate the causal role in disease of the MT-TP m.15992A>T mutation observed in patients from 5 independent families.

Outer mitochondrial membrane (OMM) rupture was first noted in isolated mitochondria in which the inner mitochondrial membrane (IMM) had lost its selective permeability. This phenomenon referred to as mitochondrial permeability transition (MPT) refers to a permeabilized inner membrane that originates a large swelling in the mitochondrial matrix, which distends the outer membrane until it ruptures. Here, we have expanded previous electron microscopic observations that in apoptotic cells, OMM rupture is not caused by a membrane stretching promoted by a markedly swollen matrix. It is shown that the widths of the ruptured regions of the OMM vary from 6 to 250 nm. Independent of the perforation size, herniation of the mitochondrial matrix appeared to have resulted in pushing the IMM through the perforation. A large, long focal herniation of the mitochondrial matrix, covered with the IMM, was associated with a rupture of the OMM that was as small as 6 nm. Contextually, the collapse of the selective permeability of the IMM may precede or follow the release of the mitochondrial proteins of the intermembrane space into the cytoplasm. When the MPT is a late event, exit of the intermembrane space proteins to the cytoplasm is unimpeded and occurs through channels that transverse the outer membrane, because so far, the inner membrane is impermeable. No channel within the outer membrane can expose to the cytoplasm a permeable inner membrane, because it would serve as a conduit for local herniation of the mitochondrial matrix.

Mitochondrial swelling is a hallmark of mitochondrial dysfunction, and is an indicator of the opening of the mitochondrial permeability transition pore. We introduce here a novel quantitative in situ single-cell assay of mitochondrial swelling based on standard wide-field or confocal fluorescence microscopy. This morphometric technique quantifies the relative diameter of mitochondria labeled by targeted fluorescent proteins. Fluorescence micrographs are spatial bandpass filtered transmitting either high or low spatial frequencies. Mitochondrial swelling is measured by the fluorescence intensity ratio of the high- to low-frequency filtered copy of the same image. We have termed this fraction the "thinness ratio". The filters are designed by numeric optimization for sensitivity. We characterized the thinness ratio technique by modeling microscopic image formation and by experimentation in cultured cortical neurons and astrocytes. The frequency domain image processing endows robustness and subresolution sensitivity to the thinness ratio technique, overcoming the limitations of shape measurement approaches. The thinness ratio proved to be highly sensitive to mitochondrial swelling, but insensitive to fission or fusion of mitochondria. We found that in situ astrocytic mitochondria swell upon short-term uncoupling or inhibition of oxidative phosphorylation, whereas such responses are absent in cultured cortical neurons.

Parkin gene mutations have been implicated in autosomal-recessive early-onset parkinsonism and lead to specific degeneration of dopaminergic neurons in midbrain. To investigate the role of Parkin in neuronal cell death, we overproduced this protein in PC12 cells in an inducible manner. In this cell line, neuronally differentiated by nerve growth factor, Parkin overproduction protected against cell death mediated by ceramide, but not by a variety of other cell death inducers (H(2)O(2), 4-hydroxynonenal, rotenone, 6-OHDA, tunicamycin, 2-mercaptoethanol and staurosporine). Protection was abrogated by the proteasome inhibitor epoxomicin and disease-causing variants, indicating that it was mediated by the E3 ubiquitin ligase activity of Parkin. Interestingly, Parkin acted by delaying mitochondrial swelling and subsequent cytochrome c release and caspase-3 activation observed in ceramide-mediated cell death. Subcellular fractionation demonstrated enrichment of Parkin in the mitochondrial fraction and its association with the outer mitochondrial membrane. Together, these results suggest that Parkin may promote the degradation of substrates localized in mitochondria and involved in the late mitochondrial phase of ceramide-mediated cell death. Loss of this function may underlie the degeneration of nigral dopaminergic neurons in patients with Parkin mutations.

Kidney proximal tubules (PTs) contain a high density of mitochondria, which are required to generate ATP to power solute transport. Mitochondrial dysfunction is implicated in the pathogenesis of numerous kidney diseases. Damaged mitochondria are thought to produce excess reactive oxygen species (ROS), which can lead to oxidative stress and activation of cell death pathways. MitoQ is a mitochondrial targeted anti-oxidant that has shown promise in preclinical models of renal diseases. However, recent studies in nonkidney cells have suggested that MitoQ might also have adverse effects. Here, using a live imaging approach, and both in vitro and ex vivo models, we show that MitoQ induces rapid swelling and depolarization of mitochondria in PT cells, but these effects were not observed with SS-31, another targeted anti-oxidant. MitoQ consists of a lipophilic cation (Tetraphenylphosphonium [TPP]) joined to an anti-oxidant component (quinone) by a 10-carbon alkyl chain, which is thought to insert into the inner mitochondrial membrane (IMM). We found that mitochondrial swelling and depolarization was also induced by dodecyltriphenylphosphomium (DTPP), which consists of TPP and the alkyl chain, but not by TPP alone. Surprisingly, MitoQ-induced mitochondrial swelling occurred in the absence of a decrease in oxygen consumption rate. We also found that DTPP directly increased the permeability of artificial liposomes with a cardiolipin content similar to that of the IMM. In summary, MitoQ causes mitochondrial swelling and depolarization in PT cells by a mechanism unrelated to anti-oxidant activity, most likely because of increased IMM permeability due to insertion of the alkyl chain.

Receptor-interacting protein kinase 3 (RIP3) is a convergence point of multiple signalling pathways, including necroptosis, inflammation and oxidative stress; however, it is completely unknown whether it underlies acute myocardial ischemia/reperfusion (I/R) injury. Langendorff-perfused rat hearts subjected to 30 min ischemia followed by 10 min reperfusion exhibited compromised cardiac function which was not abrogated by pharmacological intervention of RIP3 inhibition. An immunoblotting analysis revealed that the detrimental effects of I/R were unlikely mediated by necroptotic cell death, since neither the canonical RIP3-MLKL pathway (mixed lineage kinase-like pseudokinase) nor the proposed non-canonical molecular axes involving CaMKIIδ-mPTP (calcium/calmodulin-dependent protein kinase IIδ-mitochondrial permeability transition pore), PGAM5-Drp1 (phosphoglycerate mutase 5-dynamin-related protein 1) and JNK-BNIP3 (c-Jun N-terminal kinase-BCL2-interacting protein 3) were activated. Similarly, we found no evidence of the involvement of NLRP3 inflammasome signalling (NOD-, LRR- and pyrin domain-containing protein 3) in such injury. RIP3 inhibition prevented the plasma membrane rupture and delayed mPTP opening which was associated with the modulation of xanthin oxidase (XO) and manganese superoxide dismutase (MnSOD). Taken together, this is the first study indicating that RIP3 regulates early reperfusion injury via oxidative stress- and mitochondrial activity-related effects, rather than cell loss due to necroptosis.

Mechanical ventilation is a life-saving therapy for critically ill patients, providing rest to the respiratory muscles and facilitating gas exchange in the lungs. Ventilator-induced lung injury (VILI) is an unfortunate side effect of mechanical ventilation that may lead to serious consequences for the patient and increase mortality. The four main injury mechanisms associated with VILI are: baro/volutrauma caused by overstretching the lung tissues; atelectrauma, caused by repeated opening and closing of the alveoli resulting in shear stress; oxygen toxicity due to use of high ratio of oxygen in inspired air, causing formation of free radicals; and biotrauma, the resulting biological response to tissue injury, that leads to a cascade of events due to excessive inflammatory reactions and may cause multi-organ failure. An often-overlooked part of the inflammatory reaction is oxidative stress. In this research, a mouse model of VILI was set up with three tidal volume settings (10, 20 and 30 mL/kg) at atmospheric oxygen level. Airway pressures and heart rate were monitored and bronchoalveolar lavage fluid (BALF) and lung tissue samples were taken.

Optic atrophy-1 (OPA1) plays a crucial role in the regulation of mitochondria fusion and participates in maintaining the structural integrity of mitochondrial cristae. Here we elucidate the role of OPA1 cleavage induced by calcium swelling in the presence of Myls22 (an OPA1 GTPase activity inhibitor) and TPEN (an OMA1 inhibitor). The rate of ADP-stimulated respiration was found diminished by both inhibitors, and they did not prevent Ca2+-induced mitochondrial respiratory dysfunction, membrane depolarization, or swelling. L-OPA1 cleavage was stimulated at state 3 respiration; therefore, our data suggest that L-OPA1 cleavage produces S-OPA1 to maintain mitochondrial bioenergetics in response to stress.

The reduction in cholesterol in mitochondria, observed after exercise, is related to the inhibition of mitochondrial swelling. Caveolin-1 (Cav-1) plays an essential role in the regulation of cellular cholesterol metabolism and is required by various signalling pathways. Therefore, the aim of this study was to investigate the effect of prolonged swimming on the mitochondrial Cav-1 concentration; additionally, we identified the results of these changes as they relate to the induction of changes in the mitochondrial swelling and cholesterol in rat skeletal muscle and liver. Male Wistar rats were divided into a sedentary control group and an exercise group. The exercised rats swam for 3 hours and were burdened with an additional 3% of their body weight. After the cessation of exercise, their quadriceps femoris muscles and livers were immediately removed for experimentation. The exercise protocol caused an increase in the Cav-1 concentration in crude muscle mitochondria; this was related to a reduction in the cholesterol level and an inhibition of mitochondrial swelling. There were no changes in rat livers, with the exception of increased markers of oxidative stress in mitochondria. These data indicate the possible role of Cav-1 in the adaptive change in the rat muscle mitochondria following exercise.

Osmotic swelling of retinal glial (Müller) cells may contribute to the development of edema in diabetic retinopathy. Here, we tested whether oxidative stress and mitochondrial dysfunction are pathogenic factors involved in the osmotic swelling of Müller cells in retinal slices from control and streptozotocin-injected hyperglycemic rats. Hypotonic challenge did not change the size of Müller cell somata from control animals but induced soma swelling in Müller cells of diabetic animals. Administration of a reducing agent blocked the osmotic swelling of Müller cell somata. In retinal tissues from control animals, administration of the reducing agent blocked also the swelling-inducing effects of antagonists of P2Y₁ and adenosine A₁ receptors. In tissues from diabetic animals, inhibition of xanthine oxidase decreased the soma swelling by approximately 50% while inhibition of NADPH oxidase and nitric oxide synthase had no effects. Blockade of mitochondrial oxidative stress by perindopril, as well as of mitochondrial permeability transition by cyclosporin A or minocycline, attenuated the swelling. In addition, activation of mitochondrial K(ATP) channels by pinacidil fully prevented the swelling. The data suggest that oxidative stress produced by xanthine oxidase, as well as the mitochondria, are implicated in the induction of osmotic swelling of Müller cells from diabetic rats.

The iron chelator Deferasirox (DFX) causes severe toxicity in patients for reasons that were previously unexplained. Here, using the kidney as a clinically relevant in vivo model for toxicity together with a broad range of experimental techniques, including live cell imaging and in vitro biophysical models, we show that DFX causes partial uncoupling and dramatic swelling of mitochondria, but without depolarization or opening of the mitochondrial permeability transition pore. This effect is explained by an increase in inner mitochondrial membrane (IMM) permeability to protons, but not small molecules. The movement of water into mitochondria is prevented by altering intracellular osmotic gradients. Other clinically used iron chelators do not produce mitochondrial swelling. Thus, DFX causes organ toxicity due to an off-target effect on the IMM, which has major adverse consequences for mitochondrial volume regulation.

Naphthoquinones (NQs) are privileged structures in medicinal chemistry due to the biological effects associated with the induction of oxidative stress. The present study evaluated the activities of sixteen NQs derivatives on Trypanosoma cruzi.

Pregnancy complicated by preeclampsia (PE) and intrauterine growth restriction (IUGR) promotes endothelial cell (EC) dysfunction. Our in vitro study aimed to evaluate the endothelial cell morphology after acute and chronic exposition to medium supplemented with serum taken from healthy pregnant women and women with IUGR and IUGR with PE. In the same condition, ECs viability, proliferation, reactive oxygen species (ROS) production, and serum concentration of vascular endothelial growth factor (VEGF) were also measured. Pregnant women with IUGR and IUGR with PE-delivered babies with reduced body mass and were characterized in elevated blood pressure, urine protein loss, and reduced level of VEGF. The 24 hours of exposition did not exert any morphological changes in ECs, except the reduction in cell viability, but prolonged exposition resulted in significant morphological changes concerning mostly the swelling of mitochondria with accompanying ROS production, cell autophagy, reduced cell viability, and proliferation only in complicated pregnancies. In conclusion, the sera taken from women with IUGR and IUGR with PE show a detrimental effect on ECs, reducing their viability, proliferation, and generating oxidative stress due to dysfunctional mitochondria. This multidirectional effect might have an adverse impact on the cardiovascular system in women with IUGR and PE.

Human subcutaneous dirofilariasis is an emerging mosquitoborne zoonosis. A traveler returning to Germany from India experienced Dirofilaria infection with concomitant microfilaremia. Molecular analysis indicated Dirofilaria repens nematodes of an Asian genotype. Microfilaremia showed no clear periodicity. Presence of Wolbachia endosymbionts enabled successful treatment with doxycycline.

Bcl-2 family members Bak and Bax constitute a mitochondrial gateway for multiple death pathways. Both proteins are also present in the endoplasmic reticulum where they control apoptosis through the regulation of calcium levels. We show here that reticular Bak has the additional capacity of modulating the structure of this organelle. Coexpression of Bak and Bcl-X(L) provokes extensive swelling and vacuolization of reticular cisternae. A Bak version lacking the BH3 domain suffices to induce this phenotype, and reticular targeting of this mutant retains the activity. Expression of upstream BH3-only activators in similar conditions recapitulates ER swelling and vacuolization if ryanodine receptor calcium channel activity is inhibited. Experiments with Bak and Bax-deficient mouse embryonic fibroblasts show that endogenous Bak mediates the effect, whereas Bax is mainly irrelevant. These results reveal a previously unidentified role of Bak in regulating reticular conformation. Because this activity is absent in Bax, it constitutes one of the first examples of functional divergence between the two multidomain homologues.

Volume-regulated anion channel (VRAC) is a glutamate-permeable channel that is activated by physiological and pathological cell swelling and promotes ischemic brain damage. However, because VRAC opening requires cytosolic ATP, it is not clear if and how its activity is sustained in the metabolically compromised CNS. In the present study, we used cultured astrocytes - the cell type which shows prominent swelling in stroke - to model how metabolic stress and changes in gene expression may impact VRAC function in the ischemic and post-ischemic brain. The metabolic state of primary rat astrocytes was modified with chemical inhibitors and examined using luciferin-luciferase ATP assays and a Seahorse analyzer. Swelling-activated glutamate release was quantified with the radiotracer D-[3 H]aspartate. The specific contribution of VRAC to swelling-activated glutamate efflux was validated by RNAi knockdown of the essential subunit, leucine-rich repeat-containing 8A (LRRC8A); expression levels of VRAC components were measured with qRT-PCR. Using this methodology, we found that complete metabolic inhibition with the glycolysis blocker 2-deoxy-D-glucose and the mitochondrial poison sodium cyanide reduced astrocytic ATP levels by > 90% and abolished glutamate release from swollen cells (via VRAC). When only mitochondrial respiration was inhibited by cyanide or rotenone, the intracellular ATP levels and VRAC activity were largely preserved. Bypassing glycolysis by providing the mitochondrial substrates pyruvate and/or glutamine led to partial recovery of ATP levels and VRAC activity. Unexpectedly, the metabolic block of VRAC was overridden when ATP-depleted cells were exposed to extreme cell swelling (≥ 50% reduction in medium osmolarity). Twenty-four hour anoxic adaptation caused a moderate reduction in the expression levels of the VRAC component LRRC8A, but no significant changes in VRAC activity. Overall, our findings suggest that (i) astrocytic VRAC activity and metabolism can be sustained by low levels of glucose and (ii) the inhibitory influence of diminishing ATP levels and the stimulatory effect of cellular swelling are the two major factors that govern VRAC activity in the ischemic brain.

Noxa, a Bcl-2 homology 3 (BH3)-only protein of the Bcl-2 family, is responsive to cell stresses and triggers apoptosis by binding the prosurvival Bcl-2-like proteins Mcl1, BclXL, and Bcl2A1. Although the Noxa BH3 domain is necessary to induce apoptosis, the mitochondrial targeting domain (MTD) of Noxa functions as a pronecrotic domain, an inducer of mitochondrial fragmentation, and delivery to mitochondria. In this study, we demonstrate that the extended MTD (eMTD) peptide induces necrotic cell death by interaction with the VDAC2 protein. The eMTD peptide penetrates the cell membrane, causing cell membrane blebbing, cytosolic calcium influx, and mitochondrial swelling, fragmentation, and ROS generation. The MTD domain binds VDACs and opens the mitochondrial permeability transition pore (mPTP) in a CypD-independent manner. The opening of mPTP induced by eMTD is inhibited either by down-regulation of VDAC2 or by the VDACs inhibitor DIDS. These results indicate that the MTD domain of Noxa causes mitochondrial damage by opening mPTP through VDACs, especially VDAC2, during necrotic cell death.

Anoxia/reoxygenation injury of isolated rat liver mitochondria was investigated. During anoxia of up to 60 min, the membrane potential was largely preserved and mitochondrial swelling was not observed. Reoxygenation of anoxic mitochondria rapidly caused swelling, cyclosporin A-sensitive Ca2+ efflux, [14C]sucrose trapping, and loss of the membrane potential along with increased generation of reactive oxygen intermediates (ROI). Although pretreatment with catalase and superoxide dismutase completely abolished reoxygenation-induced generation of ROI, mitochondrial damage was not prevented, as indicated by swelling, loss of the membrane potential, a decrease of the ATP content, and cyclosporin A-sensitive Ca2+ efflux. However, addition of the immunosuppressant cyclosporin A or addition of ADP completely prevented the mitochondrial damage induced by reoxygenation. The same protective effect was noted when Ca2+ cycling was prevented, either by chelating Ca2+ with EGTA or by inhibiting Ca2+ reuptake with ruthenium red. These findings indicate that mitochondrial anoxia/reoxygenation injury is caused by the cyclosporin A-sensitive and Ca2+-dependent membrane permeability transition. In contrast, reoxygenation injury does not appear to be triggered by the enhanced production of ROI.

Amino acid sequence from 65th to 76th residue of the N-terminus of Chromogranin A (CGA-N12) is an antimicrobial peptide (AMP). Our previous studies showed that CGA-N12 reduces Candida tropicalis mitochondrial membrane potential. Here, we explored the mechanism that CGA-N12 collapsed the mitochondrial membrane potential by investigations of its action on the mitochondrial permeability transition pore (mPTP) complex of C. tropicalis. The results showed that CGA-N12 induced cytochrome c (Cyt c) leakage, mitochondria swelling and led to polyethylene glycol (PEG) of molecular weight 1000 Da penetrate mitochondria. mPTP opening inhibitors bongkrekic acid (BA) could contract the mitochondrial swelling induced by CGA-N12, but cyclosporin A (CsA) could not. Therefore, we speculated that CGA-N12 could induce C. tropicolis mPTP opening by preventing the matrix-facing (m) conformation of adenine nucleotide transporter (ANT), thereby increasing the permeability of the mitochondrial membrane and resulted in the mitochondrial potential dissipation.