Calcium-induced Mitochondrial Permeability Transition Pore Opening Research Articles

Age-Dependent Changes in the Function of Mitochondrial Membrane Permeability Transition Pore in Rat Liver Mitochondria

Mitochondria play an important role in the cell aging process. Changes in calcium homeostasis and/or increased reactive oxygen species (ROS) production lead to the opening of mitochondrial permeability transition pore (MPTP), depolarization of the inner mitochondrial membrane, and decrease of ATP production. Our work aimed to monitor age-related changes in the Ca2+ ion effect on MPTP and the ability of isolated rat liver mitochondria to accumulate calcium. The mitochondrial calcium retention capacity (CRC) was found to be significantly affected by the age of rats. Measurement of CRC values of the rat liver mitochondria showed two periods when 3 to17-week old rats were tested. 3-week and 17-week old rats showed lower CRC values than 7-week old animals. Similar changes were observed while testing calcium-induced swelling of rat liver mitochondria. These findings indicate that the mitochondrial energy production system is more resistant to calcium-induced MPTP opening accompanied by the damaging effect of ROS in adult rats than in young and aged animals.

Modulation of cardiac mitochondrial permeability transition and apoptotic signaling by endurance training and intermittent hypobaric hypoxia

BackgroundModulation of the mitochondrial permeability transition pore (MPTP) and inhibition of the apoptotic signaling are critically associated with the cardioprotective phenotypes afforded by both intermittent hypobaric-hypoxia (IHH) and endurance-training (ET). We recently proposed that IHH and ET improve cardiac function and basic mitochondrial capacity, although without showing addictive effects. Here we investigate whether a combination of IHH and ET alters cardiac mitochondrial vulnerability to MPTP and related apoptotic signaling. MethodsMale Wistar rats were divided into normoxic-sedentary (NS), normoxic-exercised (NE, 1h/day/5week treadmill-running), hypoxic-sedentary (HS, 6000m, 5h/day/5weeks) and hypoxic-exercised (HE) to study susceptibility to calcium-induced cardiac MPTP opening. Mitochondrial cyclophilin D (CypD), adenine nucleotide translocator (ANT), Bax and Bcl-2 protein contents were semi-quantified by Western blotting. Cardiac caspase 3-, 8- and 9-like activities were measured. Mitochondrial aconitase and superoxide dismutase (MnSOD) activity and malondialdehyde (MDA) and sulphydryl group (–SH) content were determined. ResultsSusceptibility to MPTP decreased in NE and HS vs. NS and even further in HE. The ANT content increased in HE vs. NS. Bcl-2/Bax ratio increased in NE and HS compared to NS. Decreased activities in tissue caspase 3-like (HE vs. NS) and caspase 9-like (HS and HE vs. NS) were observed. Mitochondrial aconitase increased in NE and HS vs. NS. No alterations between groups were observed for caspase 8-like activity, MnSOD, CypD, MDA and –SH. ConclusionsData confirm that IHH and ET modulate cardiac mitochondria to a protective phenotype characterized by decreased MPTP induction and apoptotic signaling, although without visible addictive effects as initially hypothesized.

DJ-1 protects against cell death following acute cardiac ischemia–reperfusion injury

Novel therapeutic targets are required to protect the heart against cell death from acute ischemia–reperfusion injury (IRI). Mutations in the DJ-1 (PARK7) gene in dopaminergic neurons induce mitochondrial dysfunction and a genetic form of Parkinson's disease. Genetic ablation of DJ-1 renders the brain more susceptible to cell death following ischemia–reperfusion in a model of stroke. Although DJ-1 is present in the heart, its role there is currently unclear. We sought to investigate whether mitochondrial DJ-1 may protect the heart against cell death from acute IRI by preventing mitochondrial dysfunction. Overexpression of DJ-1 in HL-1 cardiac cells conferred the following beneficial effects: reduced cell death following simulated IRI (30.4±4.7% with DJ-1 versus 52.9±4.7% in control; n=5, P<0.05); delayed mitochondrial permeability transition pore (MPTP) opening (a critical mediator of cell death) (260±33 s with DJ-1 versus 121±12 s in control; n=6, P<0.05); and induction of mitochondrial elongation (81.3±2.5% with DJ-1 versus 62.0±2.8% in control; n=6 cells, P<0.05). These beneficial effects of DJ-1 were absent in cells expressing the non-functional DJ-1L166P and DJ-1Cys106A mutants. Adult mice devoid of DJ-1 (KO) were found to be more susceptible to cell death from in vivo IRI with larger myocardial infarct sizes (50.9±3.5% DJ-1 KO versus 41.1±2.5% in DJ-1 WT; n≥7, P<0.05) and resistant to cardioprotection by ischemic preconditioning. DJ-1 KO hearts showed increased mitochondrial fragmentation on electron microscopy, although there were no differences in calcium-induced MPTP opening, mitochondrial respiratory function or myocardial ATP levels. We demonstrate that loss of DJ-1 protects the heart from acute IRI cell death by preventing mitochondrial dysfunction. We propose that DJ-1 may represent a novel therapeutic target for cardioprotection.

Induction of autophagy restores the loss of sevoflurane cardiac preconditioning seen with prolonged ischemic insult

Sevoflurane preconditioning against myocardial ischemia-reperfusion injury is lost if the ischemic insult is too long. Emerging evidence suggests that induction of autophagy may also confer cardioprotection against ischemia-reperfusion injury. We examined whether induction of autophagy prolongs sevoflurane preconditioning protection during a longer ischemic insult. Isolated guinea pigs hearts were subjected to 30 or 45min ischemia, followed by 120min reperfusion (control). Anesthetic preconditioning was elicited with 2% sevoflurane for 10min prior to ischemia (SEVO-30, SEVO-45). Chloramphenicol (autophagy upregulator, 300µM) was administered starting 20min before ischemia and throughout reperfusion in SEVO-45 (SEVO-45+CAP). To inhibit autophagy, 3-methyladenine (10μM) was administered during sevoflurane administration in SEVO-45+CAP. Infarct size was determined by triphenyltetrazolium chloride stain. Tissue samples were obtained before ischemia to determine autophagy-related protein (microtubule-associated protein light chain I and II: LC3-I, II), Akt and glycogen synthase kinase 3β (GSK3β) expression using Western blot analysis. The effect of autophagy on calcium-induced mitochondrial permeability transition pore (MPTP) opening in isolated calcein-loaded mitochondria was assessed. Electron microscopy was used to detect autophagosomes. Infarct size was significantly reduced in SEVO-30, but not in SEVO-45. Chloramphenicol restored sevoflurane preconditioning lost by 45min ischemia. There were more abundant autophagozomes and LC3-II expression was significantly increased in SEVO-45+CAP. Induction of autophagy before ischemia enhanced GSK3β phosphorylation and inhibition of calcium-induced MPTP opening. These effects were abolished by 3-methyladenine. Pre-ischemic induction of autophagy restores sevoflurane preconditioning lost by longer ischemic insult. This effect is associated with enhanced inhibition of MPTP by autophagy.

The STAT3 Inhibitor Stattic Impairs Cardiomyocyte Mitochondrial Function Through Increased Reactive Oxygen Species Formation

The signal transducer and activator of transcription 3 (STAT3) transduces stress signals from the plasma membrane to the nucleus but has recently also been identified in mitochondria. Inhibition of cardiomyocyte mitochondrial STAT3 with the STAT3-specific inhibitor Stattic decreases ADP-stimulated respiration and enhances calcium-induced mitochondrial permeability transition pore (MPTP) opening. The aim of the present study was to analyze whether or not these effects of STAT3 inhibition by Stattic are mediated by the formation of reactive oxygen species (ROS). The H2O2 formation from isolated rat left ventricular mitochondria was measured continuously in the presence of the complex 1 substrates glutamate and malate using the H2O2 indicator Amplex UltraRed. Stattic dose-dependently increased mitochondrial ROS formation (slope of Amplex UltraRed fluorescence/time; DMSO: 0.39±0.01; 1 µM Stattic: 0.40±0.03; 10 µM Stattic: 0.71±0.04; 25 µM Stattic: 1.43±0.05; 50 µM Stattic: 3.53±0.23; 100 µM Stattic: 9.23±0.69, n=5 mitochondrial preparations, p<0.05 for 10-100 µM Stattic). The increase in the ROS signal by 100 µM Stattic was abolished in the presence of the ROS scavenger N-acetylcysteine (Nac, 0.46±0.02, n=7, p<0.05). Mitochondria treated with 100 µM Stattic produced less ATP than control mitochondria (86±3 arbitrary units (a.u.) vs. 128±7 a.u., n=9, p<0.05). Again, in the presence of Nac ATP production was similar between Stattic-treated and control mitochondria (142±4 a.u. vs. 147±12 a.u., n=5, p=ns). MPTP opening was induced by lower amounts of calcium in Stattic-treated than in control mitochondria (in nmol CaCl2/mg protein, Stattic: 507±57, n=7; control: 857±70, n=7, p<0.05). There was no difference in calcium-induced MPTP opening between Stattic-treated (833±57, n=6) and control mitochondria (921±75, n=7, p=ns) in the presence of Nac. Taken together, our data show that inhibition of mitochondrial STAT3 by Stattic impacts on mitochondrial ATP production and MPTP opening through enhanced ROS formation.

Eccentric exercise transiently affects mice skeletal muscle mitochondrial function

Eccentric exercise (EE) is known to induce damage and dysfunction in skeletal muscle. However, the possible role of mitochondrial (dys)function, including the vulnerability to mitochondrial permeability transition pore (MPTP) opening, is unclear. Therefore, this study aimed to analyze the impact of a single acute bout of downhill running on skeletal muscle mitochondrial function. Thirty 12-week-old Charles River CD1 male mice were randomly assigned into control (C) or exercised groups. EE consisted of 120 min of downhill treadmill running at a -16° gradient. Exercised animals were sacrificed immediately (Ecc0h) and 48 h (Ecc48h) after the end of the running bout. Plasma and skeletal muscles were then obtained. Muscle mitochondrial function, including oxygen consumption prior to and after anoxia and reoxygenation, membrane potential, and MPTP opening, were evaluated. Respiratory chain complexI, II, and V activities were determined. EE significantly increased plasma creatine kinase activity (119.4 ± 5.6 vs. 1061.3 ± 46.3 vs. 256.8 ± 15.3 U·L(-1), C, Ecc0h and Ecc48h, respectively) and myoglobin and interleukin-6 content. Impaired state 3 and respiratory control ratio (8.4 ± 0.4 vs. 5.6 ± 0.9 vs. 8.4 ± 0.5, C, Ecc0h and Ecc48h, respectively), as well as increased susceptibility to MPTP opening, seen by cyclosporin A-sensitive high swelling amplitude, lower time to maximal swelling velocity (313.8 ± 17.7 vs. 244.5 ± 19.4 vs. 298.5 ± 8.7 s, C, Ecc0h and Ecc48h, respectively), and calcium release immediately after the end of exercise (C vs. Ecc0h) were observed. EE induced a transient impairment in the activity of complex V (C vs. Ecc0h). No significant changes from the C group were observed 48 h after the end of EE (C vs. Ecc48h) in any analyzed parameters. In conclusion, prolonged EE transiently impaired mice skeletal muscle mitochondrial function and increased susceptibility to calcium-induced MPTP opening.

Sevoflurane Confers Additive Cardioprotection to Ethanol Preconditioning Associated With Enhanced Phosphorylation of Glycogen Synthase Kinase-3β and Inhibition of Mitochondrial Permeability Transition Pore Opening

The purposes of this study were to investigate whether sevoflurane (SEVO) enhances moderate-dose ethanol (EtOH) preconditioning and whether this additional cardioprotection is associated with glycogen synthase kinase-3β (GSK-3β), protein kinase B (Akt), mammalian target of rapamycin (mTOR), 70-kDa ribosomal s6 kinase-1 (p70s6K), and/or mitochondrial permeability transition pore (MPTP) opening. In vitro study using an isolated heart Langendorff preparation. University research laboratory. Male guinea pigs (n = 170). Isolated perfused guinea pig hearts underwent 30-minute ischemia and 120-minute reperfusion (control). The EtOH group received 5% EtOH in the drinking water for 8 weeks. Anesthetic preconditioning was elicited by a 10-minute exposure to 2% SEVO in EtOH (EtOH + SEVO group) or non-EtOH (SEVO group) hearts. The inhibition of GSK-3β phosphorylation and mTOR was achieved with LY294002 and rapamycin, respectively. GSK-3β, Akt, mTOR, and p70s6K expressions were determined by western blot. Calcium-induced MPTP opening was assessed in isolated calcein-loaded mitochondria. After ischemia-reperfusion, the EtOH, SEVO, and EtOH + SEVO groups had higher left ventricular developed pressure recovery and lower end-diastolic pressure versus the control group. Infarct size was smaller in the EtOH and SEVO groups versus control and even smaller in the EtOH + SEVO group. Phosphorylation of GSK-3β and Akt, but not mTOR and p70s6K, was increased in the EtOH and SEVO groups. Phosphorylation of GSK-3β, but not mTOR and p70s6K, was further increased in the EtOH + SEVO group. The EtOH and SEVO groups exhibited a smaller calcium-induced MPTP opening, and the EtOH + SEVO presented an even smaller MPTP opening. SEVO and chronic EtOH preconditioning offer additive cardioprotection. This effect is associated with an increased GSK-3β phosphorylation and an inhibition of MPTP opening.

Direct evidence for inhibition of mitochondrial permeability transition pore opening by sevoflurane preconditioning in cardiomyocytes: Comparison with cyclosporine A

To assess whether sevoflurane preconditioning is associated with inhibition of mitochondrial permeability transition pore (MPTP), the effects of sevoflurane were compared with those of cyclosporine A, a known inhibitor of MPTP opening. Isolated perfused guinea pig hearts underwent 30 min global ischemia and 120 min reperfusion (control). Sevoflurane preconditioning was elicited by administration of 2% sevoflurane for 10 min with 10 min washout before ischemia (sevoflurane). A preconditioning-like cardioprotection was also induced by administering cyclosporine A (0.2 μM) for 15 min, starting 5 min before ischemia and for 10 min after the onset of reperfusion (cyclosporine A). Left ventricular developed and end-diastolic pressures, coronary flow and infarct size were measured. Expressions of Akt and glycogen synthase kinase 3β (GSK3β), known mediators of inhibition of MPTP opening, were determined by Western blot analysis. GSK3β inhibition was achieved with LY294002. The effects of sevoflurane and cyclosporine A on calcium-induced MPTP opening in isolated calcein-loaded mitochondria were assessed. After ischemia–reperfusion, sevoflurane and cyclosporine A had higher left ventricular developed pressure. Infarct size was significantly reduced in sevoflurane and cyclosporine A vs. control. This was abolished by LY294002 in sevoflurane, but not in cyclosporine A. Akt and GSK3β phosphorylation after reperfusion were significantly increased in sevoflurane and cyclosporine A. Ca 2 + -induced reduction in calcein fluorescence was significantly attenuated in sevoflurane and cyclosporine A. Preconditioning agents, sevoflurane and cyclosporine A increase the threshold of calcium-induced MPTP opening to a similar extent. This effect by sevoflurane, but not cyclosporine A is at least partially mediated by GSK3β inactivation.

Acute exercise protects against calcium-induced cardiac mitochondrial permeability transition pore opening in doxorubicin-treated rats

The use of DOX (doxorubicin), an antibiotic used in oncological treatments, is limited by a dose-related cardiotoxicity against which acute exercise is protective. However, the mitochondrial-related mechanisms of this protection remain unknown. Therefore the present study aimed to determine the effects of an acute endurance exercise bout performed 24 h before DOX treatment on heart and liver mitochondrial function. A total of 20 adult male Wistar rats were divided into groups as follows: non-exercised with saline (NE + SAL), non-exercised DOX-treated (NE + DOX), exercised with saline (EX + SAL) and exercised DOX-treated (EX + DOX). The animals performed a 60 min exercise bout on a treadmill or remained sedentary 24 h before receiving either a DOX bolus (20 mg/kg of body weight) or saline. Heart and liver mitochondrial function [oxygen consumption, membrane potential (DeltaPsi) and cyclosporin-A-sensitive calcium-induced MPTP (mitochondrial permeability transition pore) opening] were evaluated. The activities of the respiratory complex, Mn-SOD (superoxide dismutase), caspases 3 and 9, as well as the levels of ANT (adenine nucleotide translocase), VDAC (voltage-dependent anion channel), CypD (cyclophilin D), Bax and Bcl-2, were measured. Acute exercise prevented the decreased cardiac mitochondrial function (state 3, phosphorylative lagphase; maximal DeltaPsi generated both with complex I- and II-linked substrates and calcium-induced MPTP opening) induced by DOX treatment. Exercise also prevented the DOX-induced decreased activity of cardiac mitochondrial chain complexes I and V, and increased caspase 3 and 9 activities. DOX administration and exercise caused increased cardiac mitochondrial SOD activity. Exercise ameliorated liver mitochondrial complex activities. No alterations were observed in the measured MPTP and apoptosis-related proteins in heart and liver mitochondria. The results demonstrate that acute exercise protects against cardiac mitochondrial dysfunction, preserving mitochondrial phosphorylation capacity and attenuating DOX-induced decreased tolerance to MPTP opening.

Endurance training improves gastrocnemius mitochondrial function despite increased susceptibility to permeability transition

The aim of the present work was to test the hypothesis that moderate endurance treadmill training ameliorates gastrocnemius mitochondrial bioenergetics and increases the tolerance to the calcium-induced mitochondrial permeability transition pore (MPTP) opening. Twelve adult (6–8 week old) male Wistar rats were randomly divided into two groups ( n = 6 ⧹ per group): sedentary and trained (14 week of endurance treadmill running, 60 min/day). Several end-points for in vitro gastrocnemius mitochondrial function including oxygen consumption, transmembrane electric potential and susceptibility to calcium-induced MPTP opening were evaluated. Caspase-9 activity was measured in the intact tissue. Endurance training induced significant increases in state 3 and in respiratory control ratio both with complex I and II-linked substrates (malate + pyruvate and succinate, respectively). Increased CCCP-induced uncoupled respiration with succinate as substrate was also observed ( p < 0.05). No differences were found regarding state 4 and ADP/O ratio with both substrates. In addition, training significantly decreased the phosphorylative lag phase, whereas no changes were observed on maximal transmembrane electric potential, ADP-induced depolarization and repolarization potential ( p < 0.05). Interestingly and as opposed to our hypothesis, muscle mitochondria isolated from trained rats were more susceptible to MPTP induction by calcium, although in an initial phase muscle mitochondria isolated from trained rats had an increased calcium uptake. Interestingly, we also verified that endurance training increased the activity of caspase 9. The data obtained confirms that endurance training results in a general improvement in the gastrocnemius mitochondrial respiratory function, although mitochondrial and cellular alterations during training also result in increased calcium-induced MPTP opening.

Urocortin prevents mitochondrial permeability transition in response to reperfusion injury indirectly by reducing oxidative stress

Urocortin (UCN) protects hearts against ischemia and reperfusion injury whether given before ischemia or at reperfusion. Here we investigate the roles of PKC, reactive oxygen species, and the mitochondrial permeability transition pore (MPTP) in mediating these effects. In Langendorff-perfused rat hearts, acute UCN treatment improved hemodynamic recovery during reperfusion after 30 min of global ischemia; this was accompanied by less necrosis (lactate dehydrogenase release) and MPTP opening (mitochondrial entrapment of 2-[(3)H]deoxyglucose). UCN pretreatment protected mitochondria against calcium-induced MPTP opening, but only if the mitochondria had been isolated from hearts after reperfusion. These mitochondria also exhibited less protein carbonylation, suggesting that UCN decreases levels of oxidative stress. In isolated adult and neonatal rat cardiac myocytes, both acute (60 min) and chronic (16 h) treatment with UCN reduced cell death following simulated ischemia and re-oxygenation. This was accompanied by less MPTP opening as measured using tetramethylrhodamine methyl ester. The level of oxidative stress during reperfusion was reduced in cells that had been pretreated with UCN, suggesting that this is the mechanism by which UCN desensitizes the MPTP to reperfusion injury. Despite the fact that we could find no evidence that either PKC-epsilon or PKC-alpha translocate to the mitochondria following acute UCN treatment, inhibition of PKC with chelerythrine eliminated the effect of UCN on oxidative stress. Our data suggest that acute UCN treatment protects the heart by inhibiting MPTP opening. However, the mechanism appears to be indirect, involving a PKC-mediated reduction in oxidative stress.

Desflurane-induced preconditioning alters calcium-induced mitochondrial permeability transition.

Recent investigations have focused on the pivotal role of the mitochondria in the underlying mechanisms volatile anesthetic-induced myocardial preconditioning. This study aimed at examining the effect of anesthetic preconditioning on mitochondrial permeability transition (MPT) pore opening. Anesthetized open chest rabbits were randomized to one of four groups and underwent 10 min of ischemia, except for the sham 1 group (n = 12). Before this, they underwent a treatment period consisting of (1) no intervention (ischemic group; n = 12), (2) 30 min of desflurane inhalation (8.9% end-tidal concentration) followed by a 15-min washout period (desflurane group; n = 12), or (3) ischemic preconditioning (IPC group; n = 12). A second set of experiments was performed to evaluate the effect of a putative mitochondrial adenosine triphosphate-sensitive potassium channel antagonist, 5-hydroxydecanoate (5-HD). The animals underwent the same protocol as previously, plus pretreatment with 5 mg/kg 5-HD. They were randomized to one of five groups: the sham 2 group, receiving no 5-HD (n = 12); the sham 5-HD group (n = 12); the ischemic 5-HD group (n = 12), the desflurane 5-HD group (n = 12), and the IPC 5-HD group (n = 12). At the end of the protocol, the hearts were excised, and mitochondria were isolated. MPT pore opening was assessed by measuring the amount of calcium required to trigger a massive calcium release indicative of MPT pore opening. Desflurane and IPC group mitochondria needed a higher calcium load than ischemic group mitochondria (362 +/- 84, 372 +/- 74, and 268 +/- 110 microM calcium, respectively; P < 0.05) to induce MPT pore opening. The sham 1 and sham 2 groups needed a similar amount of calcium to trigger mitochondrial calcium release (472 +/- 70 and 458 +/- 90 microM calcium, respectively). 5-HD preadministration had no effect on sham animals (458 +/- 90 and 440 +/- 128 microM calcium without and with 5-HD, respectively) and ischemic group animals (268 +/- 110 and 292 +/- 102 microM calcium without and with 5-HD, respectively) but abolished the effects of desflurane on calcium-induced MPT pore opening (362 +/- 84 microM calcium without 5-HD vs. 238 +/- 96 microM calcium with 5-HD; P < 0.05) and IPC (372 +/- 74 microM calcium without 5-HD vs. 270 +/- 104 microM calcium with 5-HD; P < 0.05). Like ischemic preconditioning, desflurane improved the resistance of the transition pore to calcium-induced opening. This effect was inhibited by 5-HD, suggesting a link between mitochondrial adenosine triphosphate-sensitive potassium and MPT.