Mitochondrial Permeability Transition Pore Formation Research Articles

Lysophosphatidylcholine induces oxidative stress and calcium-mediated cell death in human blood platelets.

Platelets are essential component of circulation that plays a major role in hemostasis and thrombosis. During activation and its demise, platelets release platelet-derived microvesicles, with lysophosphatidylcholine (LPC) being a prominent component in their lipid composition. LPC, an oxidized low-density lipoprotein, is involved in cellular metabolism, but its higher level is implicated in pathologies like atherosclerosis, diabetes, and inflammatory disorders. Despite this, its impact on platelet function remains relatively unexplored. To address this, we studied LPC's effects on washed human platelets. A multimode plate reader was employed to measure reactive oxygen species and intracellular calcium using H2DCF-DA and Fluo-4-AM, respectively. Flow cytometry was utilized to measure phosphatidylserine expression, mitochondrial membrane potential (ΔΨm), and mitochondrial permeability transition pore (mPTP) formation using FITC-Annexin V, JC-1, and CoCl2/calcein-AM, respectively. Additionally, platelet morphology and its ultrastructure were observed via phase contrast and electron microscopy. Sonoclot and light transmission aggregometry were employed to examine fibrin formation and platelet aggregation, respectively. The findings demonstrate that LPC induced oxidative stress and increased intracellular calcium in platelets, resulting in increased phosphatidylserine expression and reduced ΔΨm. LPC triggered caspase-independent platelet death and mPTP opening via cytosolic and mitochondrial calcium, along with microvesiculation and reduced platelet counts. LPC increased the platelet's size, adopting a balloon-shaped morphology, causing membrane fragmentation and releasing its cellular contents, while inducing a pro-coagulant phenotype with increased fibrin formation and reduced integrin αIIbβ3 activation. Conclusively, this study reveals LPC-induced oxidative stress and calcium-mediated platelet death, necrotic in nature with pro-coagulant properties, potentially impacting inflammation and repair mechanisms during vascular injury.

The effects of oxidative stress and intracellular calcium on mitochondrial permeability transition pore formation in equine spermatozoa

AbstractThe in vitro storage of stallion spermatozoa for use in artificial insemination leads to oxidative stress and imbalances in calcium homeostasis that trigger the formation of the mitochondrial permeability transition pore (mPTP), resulting in premature cell death. However, little is understood about the dynamics and the role of mPTP formation in mammalian spermatozoa. Here, we identify an important role for mPTP in stallion sperm Ca2+ homeostasis. We show that stallion spermatozoa do not exhibit “classical” features of mPTP; specifically, they are resistant to cyclosporin A‐mediated inhibition of mPTP formation, and they do not require exogenous Ca2+ to form the mPTP. However, chelation of endogenous Ca2+ prevented mPTP formation, indicating a role for intracellular Ca2+ in this process. Furthermore, our findings suggest that this cell type can mobilize intracellular Ca2+ stores to form the mPTP in response to low Ca2+ environments and that under oxidative stress conditions, mPTP formation preceded a measurable increase in intracellular Ca2+, and vice versa. Contrary to previous work that identified mitochondrial membrane potential (MMP) as a proxy for mPTP formation, here we show that a loss of MMP can occur independently of mPTP formation, and thus MMP is not an appropriate proxy for the detection of mPTP formation. In conclusion, the mPTP plays a crucial role in maintaining Ca2+ and reactive oxygen species homeostasis in stallion spermatozoa, serving as an important regulatory mechanism for normal sperm function, thereby contraindicating the in vitro pharmacological inhibition of mPTP formation to enhance sperm longevity.

ANT-dependent MPTP underlies necrotic myofiber death in muscular dystrophy.

Mitochondrial permeability transition pore (MPTP) formation contributes to ischemia-reperfusion injury in the heart and several degenerative diseases, including muscular dystrophy (MD). MD is a family of genetic disorders characterized by progressive muscle necrosis and premature death. It has been proposed that the MPTP has two molecular components, the adenine nucleotide translocase (ANT) family of proteins and an unknown component that requires the chaperone cyclophilin D (CypD) to activate. This model was examined in vivo by deleting the gene encoding ANT1 (Slc25a4) or CypD (Ppif) in a δ-sarcoglycan (Sgcd) gene-deleted mouse model of MD, revealing that dystrophic mice lacking Slc25a4 were partially protected from cell death and MD pathology. Dystrophic mice lacking both Slc25a4 and Ppif together were almost completely protected from necrotic cell death and MD disease. This study provides direct evidence that ANT1 and CypD are required MPTP components governing in vivo cell death, suggesting a previously unrecognized therapeutic approach in MD and other necrotic diseases.

Requirement for ER-mitochondria Ca2+ transfer, ROS production and mPTP formation in L-asparaginase-induced apoptosis of acute lymphoblastic leukemia cells.

Acute lymphoblastic leukemia (aLL) is a malignant cancer in the blood and bone marrow characterized by rapid expansion of lymphoblasts. It is a common pediatric cancer and the principal basis of cancer death in children. Previously, we reported that L-asparaginase, a key component of acute lymphoblastic leukemia chemotherapy, causes IP3R-mediated ER Ca2+ release, which contributes to a fatal rise in [Ca2+]cyt, eliciting aLL cell apoptosis via upregulation of the Ca2+-regulated caspase pathway (Blood, 133, 2222-2232). However, the cellular events leading to the rise in [Ca2+]cyt following L-asparaginase-induced ER Ca2+ release remain obscure. Here, we show that in acute lymphoblastic leukemia cells, L-asparaginase causes mitochondrial permeability transition pore (mPTP) formation that is dependent on IP3R-mediated ER Ca2+ release. This is substantiated by the lack of L-asparaginase-induced ER Ca2+ release and loss of mitochondrial permeability transition pore formation in cells depleted of HAP1, a key component of the functional IP3R/HAP1/Htt ER Ca2+ channel. L-asparaginase induces ER Ca2+ transfer into mitochondria, which evokes an increase in reactive oxygen species (ROS) level. L-asparaginase-induced rise in mitochondrial Ca2+ and reactive oxygen species production cause mitochondrial permeability transition pore formation that then leads to an increase in [Ca2+]cyt. Such rise in [Ca2+]cyt is inhibited by Ruthenium red (RuR), an inhibitor of the mitochondrial calcium uniporter (MCU) that is required for mitochondrial Ca2+ uptake, and cyclosporine A (CsA), an mitochondrial permeability transition pore inhibitor. Blocking ER-mitochondria Ca2+ transfer, mitochondrial ROS production, and/or mitochondrial permeability transition pore formation inhibit L-asparaginase-induced apoptosis. Taken together, these findings fill in the gaps in our understanding of the Ca2+-mediated mechanisms behind L-asparaginase-induced apoptosis in acute lymphoblastic leukemia cells.

ACTA1 H40Y mutant iPSC-derived skeletal myocytes display mitochondrial defects in an in vitro model of nemaline myopathy

Nemaline myopathies (NM) are a group of congenital myopathies that lead to muscle weakness and dysfunction. While 13 genes have been identified to cause NM, over 50% of these genetic defects are due to mutations in nebulin (NEB) and skeletal muscle actin (ACTA1), which are genes required for normal assembly and function of the thin filament. NM can be distinguished on muscle biopsies due to the presence of nemaline rods, which are thought to be aggregates of the dysfunctional protein. Mutations in ACTA1 have been associated with more severe clinical disease and muscle weakness. However, the cellular pathogenesis linking ACTA1 gene mutations to muscle weakness are unclear To evaluate cellular disease phenotypes, iPSC-derived skeletal myocytes (iSkM) harboring an ACTA1 H40Y point mutation were used to model NM in skeletal muscle. These were generated by Crispr-Cas9, and include one non-affected healthy control (C) and 2 NM iPSC clone lines, therefore representing isogenic controls. Fully differentiated iSkM were characterized to confirm myogenic status and subject to assays to evaluate nemaline rod formation, mitochondrial membrane potential, mitochondrial permeability transition pore (mPTP) formation, superoxide production, ATP/ADP/phosphate levels and lactate dehydrogenase release. C- and NM-iSkM demonstrated myogenic commitment as evidenced by mRNA expression of Pax3, Pax7, MyoD, Myf5 and Myogenin; and protein expression of Pax4, Pax7, MyoD and MF20. No nemaline rods were observed with immunofluorescent staining of NM-iSkM for ACTA1 or ACTN2, and these mRNA transcript and protein levels were comparable to C-iSkM. Mitochondrial function was altered in NM, as evidenced by decreased cellular ATP levels and altered mitochondrial membrane potential. Oxidative stress induction revealed the mitochondrial phenotype, as evidenced by collapsed mitochondrial membrane potential, early formation of the mPTP and increased superoxide production. Early mPTP formation was rescued with the addition of ATP to media. Together, these findings suggest that mitochondrial dysfunction and oxidative stress are disease phenotypes in the in vitro model of ACTA1 nemaline myopathy, and that modulation of ATP levels was sufficient to protect NM-iSkM mitochondria from stress-induced injury. Importantly, the nemaline rod phenotype was absent in our in vitro model of NM. We conclude that this in vitro model has the potential to recapitulate human NM disease phenotypes, and warrants further study.

Novel Regioselective Synthesis of 1,3,4,5-Tetrasubstituted Pyrazoles and Biochemical Valuation on F1FO-ATPase and Mitochondrial Permeability Transition Pore Formation.

An efficient, eco-compatible, and very cheap method for the construction of fully substituted pyrazoles (Pzs) via eliminative nitrilimine-alkene 1,3-dipolar cycloaddition (ENAC) reaction was developed in excellent yield and high regioselectivity. Enaminones and nitrilimines generated in situ were selected as dipolarophiles and dipoles, respectively. A deep screening of the employed base, solvent, and temperature was carried out to optimize reaction conditions. Recycling tests of ionic liquid were performed, furnishing efficient performance until six cycles. Finally, a plausible mechanism of cycloaddition was proposed. Then, the effect of three different structures of Pzs was evaluated on the F1FO-ATPase activity and mitochondrial permeability transition pore (mPTP) opening. The Pz derivatives' titration curves of 6a, 6h, and 6o on the F1FO-ATPase showed a reduced activity of 86%, 35%, and 31%, respectively. Enzyme inhibition analysis depicted an uncompetitive mechanism with the typical formation of the tertiary complex enzyme-substrate-inhibitor (ESI). The dissociation constant of the ESI complex (Ki') in the presence of the 6a had a lower order of magnitude than other Pzs. The pyrazole core might set the specific mechanism of inhibition with the F1FO-ATPase, whereas specific functional groups of Pzs might modulate the binding affinity. The mPTP opening decreased in Pz-treated mitochondria and the Pzs' inhibitory effect on the mPTP was concentration-dependent with 6a and 6o. Indeed, the mPTP was more efficiently blocked with 0.1 mM 6a than with 1 mM 6a. On the contrary, 1 mM 6o had stronger desensitization of mPTP formation than 0.1 mM 6o. The F1FO-ATPase is a target of Pzs blocking mPTP formation.

1,5-disubstituted-1,2,3-triazoles counteract mitochondrial dysfunction acting on F1FO-ATPase in models of cardiovascular diseases

The compromised viability and function of cardiovascular cells are rescued by small molecules of triazole derivatives (Tzs), identified as 3a and 3b, by preventing mitochondrial dysfunction. The oxidative phosphorylation improves the respiratory control rate in the presence of Tzs independently of the substrates that energize the mitochondria. The F1FO-ATPase, the main candidate in mitochondrial permeability transition pore (mPTP) formation, is the biological target of Tzs and hydrophilic F1 domain of the enzyme is depicted as the binding region of Tzs. The protective effect of Tz molecules on isolated mitochondria was corroborated by immortalized cardiomyocytes results. Indeed, mPTP opening was attenuated in response to ionomycin. Consequently, increased mitochondrial roundness and reduction of both length and interconnections between mitochondria. In in-vitro and ex-vivo models of cardiovascular pathologies (i.e., hypoxia-reoxygenation and hypertension) were used to evaluate the Tzs cardioprotective action. Key parameters of porcine aortic endothelial cells (pAECs) oxidative metabolism and cell viability were not affected by Tzs. However, in the presence of either 1 μM 3a or 0.5 μM 3b the impaired cell metabolism of pAECs injured by hypoxia-reoxygenation was restored to control respiratory profile. Moreover, endothelial cells isolated from SHRSP exposed to high-salt treatment rescued the Complex I activity and the endothelial capability to form vessel-like tubes and vascular function in presence of Tzs. As a result, the specific biochemical mechanism of Tzs to block Ca2+-activated F1FO-ATPase protected cell viability and preserved the pAECs bioenergetic metabolism upon hypoxia-reoxygenation injury. Moreover, SHRSP improved vascular dysfunction in response to a high-salt treatment.

Differences in mitochondrial efficiency and heart function after different modes of exercise training in hypertension

Abstract Physical exercise is often used for the cardioprotective purpose in hypertension. However, it's not clear whether the mitochondrial efficacy will depend on the intensity and mode of physical exercise. The aim of the work is to investigate the effect of different modes of exercise training on mitochondria function and cardyohemodynamic in hypertension. All experimental procedures were performed in accordance with the European Communities Council Directive of 24 November 1986 (86/609 /EEC). Six-month-old spontaneously hypertensive rats (SHR) and normotensive control–Wistar rats were randomly assigned to 2 different training modes: moderate (20 min) and enhanced (45 min). Swimming training was done five times per week for 5–6 weeks. The functional cardiohemodynamic indicators were registered via microcatheter and Millar Pressure-Volume System. Mitochondrial oxygen consumption registered by Oxygraph. Moderate exercise training mode in SHR had a positive effect on the respiratory chain of mitochondria, respiratory control increased by 25,3%, the mitochondrial membrane potential increased by 47,8%. Moderate exercise training mode protected mitochondrial permeability transition pore (MPTP) formation. The threshold of MPTP opening increased by 1.4 times and the sensitivity of MPTP to inductor Ca2+ decreased. It was shown that end-systolic and end-diastolic pressure of SHR was decreased by 23,8% and 32,7% respectively. Moderate exercise improves end-diastolic heart function in SHR by increasing dP/dtmin and reducing the end-diastolic myocardial stiffness in 8,3 times. Stroke volume increased in 3,3 times, stroke work decreased by 17.2%, which may indicate the heart work efficiency increased after moderate exercise training. The Frank-Starling mechanism efficiency after moderate exercise was increased by 3,7 times. After enhanced exercise mode we didn't observe a protected effect on heart mitochondria and heart function in SHR. The mitochondrial membrane potential didn't increase, mitochondrial respiration in states V2, V3, V4 were significantly decreased. In conclusion, we showed that a moderate mode of exercise in SHR rats increased mitochondrial efficiency and improved heart function. The enhanced mode of exercise was excessive for SHR which was confirmed by suppressing the mitochondrial respiration and end-diastolic function of the heart. Funding Acknowledgement Type of funding sources: None.

SS-31, a Mitochondria-Targeting Peptide, Ameliorates Kidney Disease.

Mitochondria are essential for eukaryotic cell activity and function, and their dysfunction is associated with the development and progression of renal diseases. In recent years, there has been a rapid development in mitochondria-targeting pharmacological strategies as mitochondrial biogenesis, morphology, and function, as well as dynamic changes in mitochondria, have been studied in disease states. Mitochondria-targeting drugs include nicotinamide mononucleotide, which supplements the NAD+ pool; mitochondria-targeted protective compounds, such as MitoQ; the antioxidant coenzyme, Q10; and cyclosporin A, an inhibitor of the mitochondrial permeability transition pore. However, traditional drugs targeting mitochondria have limited clinical applications due to their inability to be effectively absorbed by mitochondria in vivo and their high toxicity. Recently, SS-31, a mitochondria-targeting antioxidant, has received significant research attention as it decreases mitochondrial reactive oxygen species production and prevents mitochondrial depolarization, mitochondrial permeability transition pore formation, and Ca2+-induced mitochondrial swelling, and has no effects on normal mitochondria. At present, few studies have evaluated the effects of SS-31 against renal diseases, and the mechanism underlying its action is unclear. In this review, we first discuss the pharmacokinetics of SS-31 and the possible mechanisms underlying its protective effects against renal diseases. Then, we analyze its renal disease-improving effects in various experimental models, including animal and cell models, and summarize the clinical evidence of its benefits in renal disease treatment. Finally, the potential mechanism underlying the action of SS-31 against renal diseases is explored to lay a foundation for future preclinical studies and for the evaluation of its clinical applications.

Mitochondria Bioenergetic Functions and Cell Metabolism Are Modulated by the Bergamot Polyphenolic Fraction.

The bergamot polyphenolic fraction (BPF) was evaluated in the F1FO-ATPase activity of swine heart mitochondria. In the presence of a concentration higher than 50 µg/mL BPF, the ATPase activity of F1FO-ATPase, dependent on the natural cofactor Mg2+, increased by 15%, whereas the enzyme activity in the presence of Ca2+ was inhibited by 10%. By considering this opposite BPF effect, the F1FO-ATPase activity involved in providing ATP synthesis in oxidative phosphorylation and triggering mitochondrial permeability transition pore (mPTP) formation has been evaluated. The BPF improved the catalytic coupling of oxidative phosphorylation in the presence of a substrate at the first phosphorylation site, boosting the respiratory control ratios (state 3/state 4) by 25% and 85% with 50 µg/mL and 100 µg/mL BPF, respectively. Conversely, the substrate at the second phosphorylation site led to the improvement of the state 3/state 4 ratios by 15% only with 100 µg/mL BPF. Moreover, the BPF carried out its beneficial effect on the mPTP phenomenon by desensitizing the pore opening. The acute effect of the BPF on the metabolism of porcine aortica endothelial cells (pAECs) showed an ATP rate index greater than one, which points out a prevailing mitochondrial oxidative metabolism with respect to the glycolytic pathway, and this ratio rose by about three times with 100 µg/mL BPF. Consistently, the mitochondrial ATP turnover, in addition to the basal and maximal respiration, were higher in the presence of the BPF than in the controls, and the MTT test revealed an increase in cell viability with a BPF concentration above 200 µg/mL. Therefore, the molecule mixture of the BPF aims to ensure good performance of the mitochondrial bioenergetic parameters.

The mitochondrial F1FO-ATPase exploits the dithiol redox state to modulate the permeability transition pore

The dithiol reagents phenylarsine oxide (PAO) and dibromobimane (DBrB) have opposite effects on the F1FO-ATPase activity. PAO 20% increases ATP hydrolysis at 50 μM when the enzyme activity is activated by the natural cofactor Mg2+ and at 150 μM when it is activated by Ca2+. The PAO-driven F1FO-ATPase activation is reverted to the basal activity by 50 μM dithiothreitol (DTE). Conversely, 300 μM DBrB decreases the F1FO-ATPase activity by 25% when activated by Mg2+ and by 50% when activated by Ca2+. In both cases, the F1FO-ATPase inhibition by DBrB is insensitive to DTE. The mitochondrial permeability transition pore (mPTP) formation, related to the Ca2+-dependent F1FO-ATPase activity, is stimulated by PAO and desensitized by DBrB. Since PAO and DBrB apparently form adducts with different cysteine couples, the results highlight the crucial role of cross-linking of vicinal dithiols on the F1FO-ATPase, with (ir)reversible redox states, in the mPTP modulation.

Platelet Cyclophilin D-Dependent Events Limit Venous Thrombotic Occlusion and Platelet Accretion

The role of platelets in arterial thrombosis is well established. Recent findings have reinforced their importance in venous thrombosis as well. Which of the platelet's many functions mediate its effects in the venous setting remains unclear, and how procoagulant platelet formation regulates venous thrombosis has not been investigated. Cyclophilin D (CypD) is a mitochondrial peptidylprolylisomerase that has the key function of regulation of mitochondrial permeability transition pore (mPTP) formation. In the absence of CypD, mPTP and procoagulant platelet formation are markedly impaired in response to physiologic agonists. The functional consequence of CypD's absence in vivo has been examined in photochemical injury models in photochemical injury models of both the carotid and mesenteric arteries. In mice with platelet CypD-deficiency, in both of these high shear settings, thrombotic vasoocclusion was accelerated implicating procoagulant platelet fragility and loss of adhesiveness as a critical factor limiting thrombus growth. Whether CypD deficiency would similarly impact thrombus growth in a venous setting has not been previously examined.To investigate the role of CypD and procoagulant platelets in the low shear setting of venous thrombosis, we utilized the inferior vena cava (IVC) stenosis ligature murine model of deep vein thrombosis (DVT). This model was chosen as it utilizes mechanical narrowing to induce thrombosis in the setting of venous flow. 24 hours following IVC ligation, thrombi were significantly larger (1.83 fold) in CypD-deficient mice than wild type. (p<0.05). Similarly, in mice with platelet-specific CypD-deficiency, thrombi were 1.54 fold larger than those found in WT mice (p<0.05). To begin to investigate the mechanism by which CypD-deficiency resulted in increased thrombus size, the composition of the venous thrombi was analyzed. Western blots for markers of platelets (CD41), neutrophils (Ly6G), and fibrin (59D8) revealed that their relative levels in thrombi were unchanged, but indicated that red blood cell (spectrin α1) accumulation increased. Histologic appearance in both CypD-deficient and wild-type thrombi was similar with a consistent pattern observed. A white cap area dominated the thrombus closest to the site of ligation, while red thrombus was more common in the remaining thrombus area. To investigate whether the observed pattern is due to more rapid occlusion, ultrasound was utilized to investigate the time course of venous vasoocclusion following ligature placement. Preliminary ultrasound studies suggest that CypD-deficient mice develop vasoocclusion more rapidly, perhaps thus accounting for their larger size.Previous studies have delineated the role of CypD-dependent events in limiting platelet accretion in vitro in arterial, but not venous, flow conditions. In vitro studies utilizing whole blood from CypD-deficient mice showed increased accretion of platelets on collagen under venous flow conditions. Similarly, platelet accretion increased when human whole blood was treated with cyclosporin A, an inhibitor of CypD, and flowed over collagen at venous rates. Interestingly, washed platelets from CypD-deficient mice also showed increased accumulation on a von Willebrand factor (VWF) - coated surface under venous flow conditions, a condition in which neither collagen nor coagulation-mediated procoagulant platelet formation would be expected to occur. Thus, these studies have identified an important role for CypD-dependent platelet events in the regulation of platelet accumulation in vitro and thus slowing the early stages of thrombus growth in vivo . The results here, in venous settings, and previously in arterial settings are consistent with a limiting role for procoagulant platelet formation in thrombus growth. This regulatory feedback role should be taken into consideration prior to modulation of procoagulant platelet formation in the setting of thrombosis or inflammation. DisclosuresNo relevant conflicts of interest to declare.

Cytoplasmic Phospholipase A2 Is Essential in GPVI Signaling Initiated Procoagulant Platelet Formation

Procoagulant platelets (PP), a subpopulation of highly activated platelets, are essential in hemostasis by supporting active tenase and prothrombinase complexes on their phosphatidylserine (PSer) externalized surface. When platelets are stimulated with a GPVI agonist, such as convulxin (CVX) or collagen related peptide (CRP), both extracellular calcium entry and mitochondrial permeability transition pore (mPTP) formation are required to mediate the transition to the procoagulant phenotype. Activation of phospholipase A2 (PLA2) is an essential prerequisite for GPVI stimulated thromboxane A2 (TXA2) generation that induces paracrine platelet activation and aggregation. Active PLA2 cleaves the arachidonic acid from the second carbon group of glycerol within the phospholipid membrane. The resultant arachidonic acid, upon downstream enzymatic modification, is modified into TXA2, leukotrienes, and other metabolites. Inhibitors of cyclooxygenases (i.e. aspirin), which prevent arachidonic acid conversion to TXA2 are widely used clinically as antiplatelet agents. Here we investigate the role of PLA2 and its metabolites in inducing GPVI-induced PP formation.Human platelets (106/mL) were activated in the presence of three distinct PLA2 inhibitors: non-specific (PLA2; oleyloxyethyl phosphorylcholine, 1 μM), and specific to inducible (iPLA2; bromoenol lactone, 1 μM) and Ca2+-dependent cytosolic PLA2 (cPLA2; PACOCF3, 1 μM). Examination of platelets in these dilute conditions facilitates both the assessment of autocrine effects of PLA2 and its metabolites and flow cytometry analysis. Platelets were stimulated with different concentrations of GPVI agonists (CRP and CVX), stained (Annexin V for PSer, PAC1 for integrin activation and P-Selectin to assess granule release) and fixed. Additionally pharmacologic inhibitors of the TXA2 receptor (SQ29548, 0.5 μM) and lipoxygenases were utilized.As expected, PP formation in this setting was GPVI signaling and mPTP mediated, as dasatinib (SRC kinase inhibitor, 1 μM) and cyclosporin A (cyclophilin D inhibitor, 5 μM) completely inhibited PP formation. Analysis of platelet activation by flow cytometry demonstrated no effect of PLA2 inhibition on either platelet aggregatory ability or granule release. In contrast, procoagulant platelet formation was significantly decreased in the presence of the non-specific PLA2 inhibitor oleyloxyethyl phosphorylcholine (~59% decrease) and the specific cPLA2 inhibitor PACOCF3 (~51% decrease). Specific iPLA2 inhibition had no effect on any aspect of platelet activation. These results indicate that cPLA2-mediated events are required for autocrine induction of procoagulant platelet formation by GPVI. To begin to interrogate the downstream effectors mediating cPLA2's effects PP formation was examined in the presence of inhibitors of TXA2-mediated platelet activation (SQ29548) and lipoxygenase inhibitors, specifically zileuton, caffeic acid and 8,11,14-eicosatriynoate (all at 1 μM). Inhibition of either of these metabolite-mediated pathways had no effect on GPVI-induced PP formation. In contrast to TXA2's lack of effect on autocrine PP formation, platelet accretion on collagen, presumably mediated by paracrine stimulation of the TXA2 receptor, was decreased by 70% in the presence of SQ29548.These studies identify a novel pathway, mediated through activation of cPLA2, that is essential for GPVI induced PP formation. This cPLA2-mediated pathway is not mediated through the TXA2 receptor, and is distinct from that required for TXA2-mediated paracrine activation of platelet aggregation. Consistent with the findings presented here, previous studies have demonstrated minimal effect of cyclooxygenase inhibition on procoagulant platelet formation. Thus, our studies identify a novel aspirin-insensitive pathway mediating PP formation, and suggest a novel pharmacologic target that could be used for the treatment of procoagulant platelet related pathologic thromboses without affecting proaggregatory phenotype. DisclosuresNo relevant conflicts of interest to declare.

Autoinducer N-(3-oxododecanoyl)-l-homoserine lactone induces calcium and reactive oxygen species-mediated mitochondrial damage and apoptosis in blood platelets

Acylated homoserine lactones (AHL) such as N-(3-oxododecanoyl)-l-homoserine lactone (3-oxo-C12 HSL) and N-butyryl-l-homoserine lactone (C4 HSL) are the most common autoinducer molecules in Pseudomonas aeruginosa. These AHL molecules not only regulate the expression of virulence factors but also have been shown to interfere with the host cell and modulate its functions. Recently, we reported that 3-oxo-C12 HSL but not C4 HSL causes cytosolic Ca2+ rise and ROS production in platelets. In this study, we examined the potential of AHLs to induce apoptosis in the human blood platelet. Our result showed that 3-oxo-C12 HSL but not C4 HSL causes phosphatidylserine (PS) exposure, mitochondrial dysfunction (mitochondrial transmembrane potential loss, and mitochondrial permeability transition pore (mPTP) formation). Besides, 3-oxo-C12 HSL also inhibited thrombin-induced platelet aggregation and clot retraction. The pretreatment of an intracellular calcium chelator BAPTA-AM or ROS inhibitor (DPI) significantly attenuated the 3-oxo-C12 HSL induced apoptotic characters such as PS exposure and mitochondrial dysfunctions. These data, including our previous findings, confirmed that 3-oxo-C12 HSL induced intracellular Ca2+ mediated ROS production results in the activation and subsequent induction of apoptotic features in platelets. Our results demonstrated that the 3-oxo-C12 HSL modulates the functions of platelets that may cause severe thrombotic complications in P. aeruginosa infected individuals.

Вплив екзогенного глутатіону на кардіодинаміку і відкривання мітохондріальної пори при ішемії–реперфузії серця щурів

The effect of post-conditioning with reduced glutathione (GSH, hepaval Italy/Ukraine) on myocardial contractility, oxygen cost, and mitochondrial factor release as a marker of mitochondrial permeability transition pore (MPTP) opening was studied in ischemia–reperfusion model at Langendorffisolated rat heart. It was found that reperfusion with KrebsHenseleit solution containing GSH provided more complete restoration of the left ventricle developed pressure (70.2 and 56% at 5th and 40th min of reperfusion against 23.6 and 30.9% in control, P &lt; 0.05 for both), reduced oxygen cost of myocardial work (184 and 157% at 5th and 40th min of reperfusion against 413 and 216% in control, P &lt; 0.05 for both), and decreased the value of mitochondrial factor by 3 times, indicating inhibition of MPTP. It was shown that the level of GSH in cardiac tissues was significantly increased by 1.5 times 30 min after administration of hepaval (52 mg per kg) intraperitoneally, indicating accumulation of GSH from the bloodstream. Thus, we have shown that post-conditioning with GSH had cardioprotective effect, inhibited the formation of MPTP and can be used as a tool for correction of post-ischemic disturbances of heart function.

Mitochondria as a therapeutic target for cardiac ischemia‑reperfusion injury (Review)

Acute myocardial infarction is the leading cause of cardiovascular-related mortality and chronic heart failure worldwide. As regards treatment, the reperfusion of ischemic tissue generates irreversible damage to the myocardium, which is termed 'cardiac ischemia-reperfusion (IR) injury'. Due to the large number of mitochondria in cardiomyocytes, an increasing number of studies have focused on the roles of mitochondria in IR injury. The primary causes of IR injury are reduced oxidative phosphorylation during hypoxia and the increased production of reactive oxygen species (ROS), together with the insufficient elimination of these oxidative species following reperfusion. IR injury includes the oxidation of DNA, incorrect modifications of proteins, the disruption of the mitochondrial membrane and respiratory chain, the loss of mitochondrial membrane potential (∆Ψm), Ca2+ over-load, mitochondrial permeability transition pore formation, swelling of the mitochondria, and ultimately, cardiomyocyte necrosis. The present review article discusses the molecular mechanisms of IR injury, and summarizes the metabolic and dynamic changes occurring in the mitochondria in response to IR stress. The mitochondria are strongly recommended as a target for the development of therapeutic agents; however, the appropriate use of agents remains a challenge.

Time Dependent Ca2+ Induction Led to the Formation of Mitochondrial Permeability Transition Pore as a Function of Age

Ca2+ sequestration and its homeostasis is disrupted when mitochondrial membrane permeabilises to form a large opening - the mitochondrial permeability transition pore (MPTP). The MPTP formation is common during aging and age related pathologies, activating cell death pathways to avoid unhealthy consequences and malignancies in the brain tissue. Several studies have identified the participation of Cyclophilin-D (Cyp-D) and adenine nucleotide translocase(ANT) in forming MPTP. However, Ca2+ is known to participate significantly in MPTP induction, although its concentration and time dependent permeabilization mechanisms are still elusive. In this work, we have focused on the contribution of Ca2+ participation, its concentration and time taken to permeabilze the mitochondrial inner membrane as a measure of light scattering at 540 ηM. We have observed that MPTP formation is increased in mitochondria isolated from aged rats in comparison to young adult and neonatal rats. The cyclosporin A application blocks the cyclophilin interaction thus avoiding MPTP formation confirming the crucial role of Ca2+ inducted MPTP opening. A 100 µM Ca2+ incubation for 15 minutes allowed the 50% probability of formation of MPTP in mitochondria isolated from all age groups. Thus, alleviating its role in aging and neurodegeneration.

Melatonin Inhibits Formation of Mitochondrial Permeability Transition Pores and Improves Oxidative Phosphorylation of Frozen-Thawed Ram Sperm.

Structural and functional damages to mitochondria of frozen-thawed sperm are a typical cryoinjury, with mitochondrial permeability transition pore (MPTP) formation being the hallmark change. Mitochondria are both a primary synthesis site and principle target for melatonin; this compound can directly inhibit MPTP formation and therefore confer protection at a mitochondrial level. The objective was to determine effects of melatonin on MPTP opening, viability, motility, and oxidative phosphorylation (OXPHOS) of frozen-thawed ram sperm. Ram semen was diluted in glucose-egg yolk buffer with 0 or 10−7 M melatonin (frozen and frozen + melatonin groups, respectively) and slow frozen, with fresh semen as Control. In frozen-thawed sperm, melatonin inhibited MPTP opening and lactate concentrations and improved sperm viability, motility, acetyl-CoA concentration and adenosine triphosphate (ATP) production. With regard to the underlying physiological mechanism, melatonin suppressed movement of citrate synthase, isocitrate dehydrogenase, oxoglutarate dehydrogenase complex, and F0F1-ATP synthase permeability from mitochondrial to cytosolic fractions induced by MPTP opening; furthermore, it increased mRNA expressions of respiratory chain complex components and activities of complexes I, II, III, and IV and thereby improved oxygen consumption capacity in frozen-thawed sperm. In conclusion, melatonin improved OXPHOS of frozen-thawed ram sperm, attributed to inhibition of cryopreservation-induced MPTP opening.

Pseudomonas aeruginosa quorum-sensing molecule N-(3-oxo-dodecanoyl)-L-homoserine lactone triggers mitochondrial dysfunction and apoptosis in neutrophils through calcium signaling.

Pseudomonas aeruginosa is an opportunistic pathogen that utilizes the quorum-sensing (QS) process to regulate the production of different virulence factors and biofilm. N-3-oxo-dodecanoyl-L-homoserine lactone (C12) is a key QS molecule of P. aeruginosa which interacts with the mammalian immune cells and modulates their function. Here, we investigated the molecular mechanism of C12-induced apoptosis in neutrophils. Our data show that C12 causes apoptosis in neutrophils through an elevation in cytosolic and mitochondrial Ca2+ levels. Besides, C12 induces phosphatidylserine (PS) exposure,mitochondrial membrane potential (MMP) depolarization, mitochondrial permeability transition pore (MPTP) formation andmitochondrial reactive oxygen species (mROS) generation. C12-induced rise in intracellular Ca2+ level is majorly contributed by endoplasmic reticulum store through the activation of inositol 1, 4, 5-triphosphate receptor. Intracellular calcium chelation inhibited C12-induced mitochondrial dysfunction and apoptosis. Further, inhibition of mitochondrial Ca2+ uniporter by ruthenium red or Ru360 abrogated C12-induced mitochondrial Ca2+ uptake, MMP loss, MPTP opening, mROS production, and PS exposure. These mechanistic insights are expected to provide a better understanding of the role of C12 in P. aeruginosa pathogenesis.

Delayed Wound Healing in Mice with Impaired Procoagulant Platelet Formation

Platelets are well recognized as a key player in primary hemostasis and thrombosis, but their role in the subsequent initiation of tissue repair is less well characterized. Within the hemostatic thrombus, ballooned platelets reminiscent of the procoagulant or necrotic platelet are first observed in the periphery (extravascular and in contact with collagen) and subsequently throughout the platelet plug. The key role of cyclophilin D (CypD) in the regulation of platelet mitochondrial permeability transition pore (mPTP) and procoagulant platelet formation is well established. Furthermore, the cyclophilin inhibitor cyclosporine is known to both block procoagulant platelet formation in vitroand to result in delayed wound healing in patients. While the consequences of CypD's absence have been studied in vivo in models of venous and arterial thrombosis, the effects on wound healing and tissue repair have not been investigated.Here we investigated the role of CypD-dependent events in the setting of cutaneous wound healing. Punch biopsies (6 mm) were placed and wound diameter (days 0, 1,2, 5,7,10,14) was examined. In mice with a global absence of CypD and in mice with platelet/megakaryocyte-specific CypD deficiency, wound healing was substantially and similarly delayed (n=9 -11 for each group). This demonstrates that platelet CypD-mediated processes play a key role in the initiation of hemostatic thrombus resolution and subsequent wound healing. The initial resolution of wound healing was delayed by ~ 2-3 days. This resulted in a significant difference at days 4 and 7 post-injury in the size of the wounds of these mice (p<0.05). To elaborate possible mechanisms by which platelet CypD-deficiency resulted in delayed wound healing, skin from the area of the wound was harvested at key early time points (day 0,1,2,5) and histological examination was performed. Early in the initiation of wound resolution, polymorphonuclear cells (PMNs) successively infiltrate the platelet plug. In mice lacking CypD, either globally or platelet-specific, polymorphonuclear cell (PMN) infiltration of the resolving thrombus on days 1 and 2 was markedly impaired. However, in all mice, PMNs amassed at the edges of the wound at 2 days post-injury. Furthermore, subsequent wound resolution was dysregulated in the absence of platelet CypD. Wounds from these mice (day 5) demonstrated marked blebbing and structural abnormalities. Remarkably, these delays in wound healing and PMN succession occurred in the absence of either gross or microscopic bleeding consistent with the absence of a defect in initial hemostasis previously reported in platelet-CypD deficient mice.These results indicate that platelets, independent of their hemostatic role, play a key role in the initiation of wound resolution. And that, subsequent to hemostasis, a platelet CypD-mediated process, presumably the procoagulant platelet transition, is required for the timely and orchestrated initiation of wound resolution and successive wound PMN infiltration. These results are consistent with the established in vitro role of CypD in mediating the procoagulant platelet morphologic transition and the procoagulant platelet's known role in mediating neutrophil activation. Finally, our results identify a key platelet-dependent mechanism by which therapy with the immunosuppressant cyclosporine can result in delayed wound healing. DisclosuresJobe:CSL: Consultancy; Shire: Consultancy; Octapharma: Consultancy.