Although arterial limb tourniquet is one of the first-line treatments to prevent exsanguinating hemorrhage in both civilian pre-hospital and battlefield casualty care, prolonged application of a limb tourniquet can lead to serious ischemia-reperfusion injury. However, the underlying pathomechanisms of tourniquet-induced ischemia-reperfusion injury are still poorly understood. Using a murine model of acute limb ischemia-reperfusion, we investigated if acute limb ischemia-reperfusion injury is mediated by superoxide overproduction and mitochondrial dysfunction. Hind limbs of C57/BL6 mice were subjected to 3 h ischemia and 4 h reperfusion via placement and release of a rubber tourniquet at the greater trochanter. Approximately 40% gastrocnemius muscle suffered infarction in this model. Activities of mitochondrial electron transport chain complexes including complex I, II, III, and IV in gastrocnemius muscle were decreased in the ischemia-reperfusion group compared to sham. Superoxide production was increased while activity of manganese superoxide dismutase (MnSOD, the mitochondria-targeted SOD isoform) was decreased in the ischemia-reperfusion group compared to sham group. Pretreatment with tempol (a SOD mimetic, 50 mg/kg) or co-enzyme Q10 (50 mg/kg) not only decreased the superoxide production, but also reduced the infarct size and normalized mitochondrial dysfunction in the gastrocnemius muscle. Our results suggest that tourniquet-induced skeletal muscle ischemia-reperfusion injuries including infarct size and mitochondrial dysfunction may be mediated via the superoxide over-production and reduced antioxidant activity. In the future, this murine ischemia-reperfusion model can be adapted to mechanistically evaluate anti-ischemic molecules in tourniquet-induced skeletal muscle injury.
Infarct size; Ischemia-reperfusion injury; Mitochondria; Superoxide; Tourniquet
Although the mitochondrial permeability transition pore (mPTP) was first discovered almost 30 years ago , it did not attract significant research attention until the 1990's when several studies implicated mPTP in apoptosis . Today, the dogma suggests that opening of mPTP is detrimental to the cell and mPTP activation is widely thought to contribute to disease in cancer, neurodegenerative diseases, stroke, muscular dystrophy, and cardiac reperfusion injury . Multiple factors including Ca2+, OH−, Pi, cyclophilin D, reactive oxygen and nitrogen species (ROS and RNS) trigger mPTP opening . However, whether mPTP activation feeds back to alter mitochondrial ROS generation remains unclear. We recently demonstrated that under normal conditions, individual mitochondria undergo spontaneous transient bursts of quantal superoxide generation, termed “superoxide flashes” . Superoxide flashes are observed in all cell types investigated to date and are triggered by a surprising functional coupling between mPTP activation and electron transport chain (ETC) dependent superoxide production. Additionally, reoxgenation following anoxia leads to uncontrolled superoxide flash genesis in cardiomyocytes. This positive feedback mechanism for mPTP/ETC-dependent ROS generation may drive localized redox signaling in individual mitochondria under physiological conditions, and when left unchecked, contribute to global cellular oxidative stress under pathological conditions in cardiac disease. The mPTP activity-dependent cell life and death determination imposes new challenges and opportunities in the pursuit of therapeutic agents for treating diseases in which oxidative stress has been implicated such as cardiac ischemia-reperfusion injury.
Ablation of mouse occipital cortex induces precisely timed and uniform p53-modulated and Bax-dependent apoptosis of thalamocortical projection neurons in the dorsal lateral geniculate nucleus (LGN) by 7 days postlesion. We tested the hypothesis that this neuronal apoptosis is initiated by oxidative stress and the mitochondrial permeability transition pore (mPTP). Pre-apoptotic LGN neurons accumulate mitochondria, Zn2+ and Ca2+, and generate higher levels of reactive oxygen species (ROS), including superoxide, nitric oxide (NO) and peroxynitrite, than LGN neurons with an intact cortical target. Pre-apoptosis of LGN neurons is associated with increased formation of protein carbonyls, protein nitration, and protein S-nitrosylation. Genetic deletion of nitric oxide synthase 1 (nos1) and inhibition of NOS1 with nitroindazole protected LGN neurons from apoptosis, revealing NO as a mediator. Putative components of the mPTP are expressed in mouse LGN, including the voltage-dependent anion channel (VDAC), adenine nucleotide translocator (ANT), and cyclophilin D (CyPD). Nitration of CyPD and ANT in LGN mitochondria occurs by 2 days after cortical injury. Chemical cross-linking showed that LGN neuron pre-apoptosis is associated with formation of CyPD and VDAC oligomers, consistent with mPTP formation. Mice without CyPD are rescued from neuron apoptosis as are mice treated with the mPTP inhibitors TRO-19622 and TAT-Bcl-XL-BH4. Manipulation of the mPTP markedly attenuated the early pre-apoptotic production of reactive oxygen/nitrogen species in target-deprived neurons. Our results demonstrate in adult mouse brain neurons that the mPTP functions to enhance ROS production and the mPTP and NO trigger apoptosis; thus, the mPTP is a target for neuroprotection in vivo.
Alzheimer’s disease; amyotrophic lateral sclerosis; axotomy; cell death; DNA damage; Parkinson’s disease; target deprivation; traumatic brain injury
Insulin resistance is associated with mitochondrial dysfunction, but the mechanism by which mitochondria inhibit insulin-stimulated glucose uptake into the cytoplasm is unclear. The mitochondrial permeability transition pore (mPTP) is a protein complex that facilitates the exchange of molecules between the mitochondrial matrix and cytoplasm, and opening of the mPTP occurs in response to physiological stressors that are associated with insulin resistance. In this study, we investigated whether mPTP opening provides a link between mitochondrial dysfunction and insulin resistance by inhibiting the mPTP gatekeeper protein cyclophilin D (CypD) in vivo and in vitro. Mice lacking CypD were protected from high fat diet-induced glucose intolerance due to increased glucose uptake in skeletal muscle. The mitochondria in CypD knockout muscle were resistant to diet-induced swelling and had improved calcium retention capacity compared to controls; however, no changes were observed in muscle oxidative damage, insulin signaling, lipotoxic lipid accumulation or mitochondrial bioenergetics. In vitro, we tested 4 models of insulin resistance that are linked to mitochondrial dysfunction in cultured skeletal muscle cells including antimycin A, C2-ceramide, ferutinin, and palmitate. In all models, we observed that pharmacological inhibition of mPTP opening with the CypD inhibitor cyclosporin A was sufficient to prevent insulin resistance at the level of insulin-stimulated GLUT4 translocation to the plasma membrane. The protective effects of mPTP inhibition on insulin sensitivity were associated with improved mitochondrial calcium retention capacity but did not involve changes in insulin signaling both in vitro and in vivo. In sum, these data place the mPTP at a critical intersection between alterations in mitochondrial function and insulin resistance in skeletal muscle.
MPTP, mitochondrial permeability transition pore; CYPD, cyclophilin D; HFD, high fat diet; LFD, low fat diet; WT, wild type; KO, knockout; CSA, cyclosporin A; BKA, bongkrekic acid; O2•, superoxide; [3H]-2-DOG, [3H]-2-deoxyglucose; Rg′, rate of glucose transport; FFA, free fatty acid; DAG, diacylglycerol; TEM, transmission electron microscopy; PDH, pyruvate dehydrogenase; PDHa, active PDH; PDHt, total PDH; MCAD, medium chain acyl-CoA dehydrogenase; β-HAD, β-hydroxyacyl-CoA dehydrogenase; PM, plasma membrane; ANT, adenine nucleotide translocator; VDAC, voltage-dependent anion channel; HK2, hexokinase 2; ETC, electron transport chain; OXPHOS, oxidative phosphorylation; MnSOD, mitochondrial manganese superoxide dismutase; MIRKO, muscle insulin receptor knockout; MHC, myosin heavy chain; TBARS, thiobarbituric acid reactive substances; Glucose; Insulin resistance; Mitochondrial dysfunction; Mitochondrial permeability transition pore; Cyclophilin D; Skeletal muscle
To clarify the relationship between reactive oxygen species (ROS) and cell death during ischemia-reperfusion (I/R), we studied cell death mechanisms in a cellular model of I/R. Oxidant stress during simulated ischemia was detected in the mitochondrial matrix using mito-roGFP, a ratiometric redox sensor, and by Mito-Sox Red oxidation. Reperfusion-induced death was attenuated by over-expression of Mn-superoxide dismutase (Mn-SOD) or mitochondrial phospholipid hydroperoxide glutathione peroxidase (mito-PHGPx), but not by catalase, mitochondria-targeted catalase, or Cu,Zn-SOD. Protection was also conferred by chemically distinct antioxidant compounds, and mito-roGFP oxidation was attenuated by NAC, or by scavenging of residual O2 during the ischemia (anoxic ischemia). Mitochondrial permeability transition pore (mPTP) oscillation/opening was monitored by real-time imaging of mitochondrial calcein fluorescence. Oxidant stress caused release of calcein to the cytosol during ischemia, a response that was inhibited by chemically diverse antioxidants, anoxia, or over-expression of Mn-SOD or mito-PHGPx. These findings suggest that mitochondrial oxidant stress causes oscillation of the mPTP prior to reperfusion. Cytochrome c release from mitochondria to the cytosol was not detected until after reperfusion, and was inhibited by anoxic ischemia or antioxidant administration during ischemia. Although DNA fragmentation was detected after I/R, no evidence of Bax activation was detected. Over-expression of the anti-apoptotic protein Bcl-XL in cardiomyocytes did not confer protection against I/R-induced cell death. Moreover, murine embryonic fibroblasts with genetic depletion of Bax and Bak, or over-expression of Bcl-XL, failed to show protection against I/R. These findings indicate that mitochondrial ROS during ischemia triggers mPTP activation, mitochondrial depolarization, and cell death during reperfusion through a Bax/Bak-independent cell death pathway. Therefore, mitochondrial apoptosis appears to represent a redundant death pathway in this model of simulated I/R.
Mitochondrial dysfunction caused by excessive Ca2+ accumulation is a major contributor to cardiac cell and tissue damage during myocardial infarction and ischemia–reperfusion injury (IRI). At the molecular level, mitochondrial dysfunction is induced by Ca2+-dependent opening of the mitochondrial permeability transition pore (mPTP) in the inner mitochondrial membrane, which leads to the dissipation of mitochondrial membrane potential (ΔΨm), disruption of adenosine triphosphate production, and ultimately cell death. Although the role of Ca2+ for induction of mPTP opening is established, the exact molecular mechanism of this process is not understood. The aim of the present study was to test the hypothesis that the adverse effect of mitochondrial Ca2+ accumulation is mediated by its interaction with inorganic polyphosphate (polyP), a polymer of orthophosphates linked by phosphoanhydride bonds. We found that cardiac mitochondria contained significant amounts (280 ± 60 pmol/mg of protein) of short-chain polyP with an average length of 25 orthophosphates. To test the role of polyP for mPTP activity, we investigated kinetics of Ca2+ uptake and release, ΔΨm and Ca2+-induced mPTP opening in polyP-depleted mitochondria. polyP depletion was achieved by mitochondria-targeted expression of a polyP-hydrolyzing enzyme. Depletion of polyP in mitochondria of rabbit ventricular myocytes led to significant inhibition of mPTP opening without affecting mitochondrial Ca2+ concentration by itself. This effect was observed when mitochondrial Ca2+ uptake was stimulated by increasing cytosolic [Ca2+] in permeabilized myocytes mimicking mitochondrial Ca2+ overload observed during IRI. Our findings suggest that inorganic polyP is a previously unrecognized major activator of mPTP. We propose that the adverse effect of polyphosphate might be caused by its ability to form stable complexes with Ca2+ and directly contribute to inner mitochondrial membrane permeabilization.
The inhalation anesthetic isoflurane has been shown to open the mitochondrial permeability transition pore (mPTP) and induce caspase activation and apoptosis, which may lead to learning and memory impairment. Cyclosporine A, a blocker of mPTP opening might attenuate the isoflurane-induced mPTP opening, lessening its ripple effects. Magnesium and anesthetic propofol are also mPTP blockers. We therefore set out to determine whether propofol and magnesium can attenuate the isoflurane-induced caspase activation and mPTP opening.
We investigated the effects of magnesium sulfate (Mg2+), propofol, and isoflurane on the opening of mPTP and caspase activation in H4 human neuroglioma cells stably transfected to express full-length human amyloid precursor protein (APP) (H4 APP cells) and in six day-old wild-type mice, employing Western blot analysis and flowcytometry.
Here we show that Mg2+ and propofol attenuated the isoflurane-induced caspase-3 activation in H4-APP cells and mouse brain tissue. Moreover, Mg2+ and propofol, the blockers of mPTP opening, mitigated the isoflurane-induced mPTP opening in the H4-APP cells.
These data illustrate that Mg2+ and propofol may ameliorate the isoflurane-induced neurotoxicity by inhibiting its mitochondrial dysfunction. Pending further studies, these findings may suggest the use of Mg2+ and propofol in preventing and treating anesthesia neurotoxicity.
Objective: To investigate the beneficial effect of bicyclol on rat hearts subjected to ischemia-reperfusion (IR) injuries and its possible mechanism. Methods: Male Sprague-Dawley rats were intragastrically administered with bicyclol (25, 50 or 100 mg/(kg∙d)) for 3 d. Myocardial IR was produced by occlusion of the coronary artery for 1 h and reperfusion for 3 h. Left ventricular hemodynamics was continuously monitored. At the end of reperfusion, myocardial infarct was measured by 2,3,5-triphenyltetrazolium chloride (TTC) staining, and serum lactate dehydrogenase (LDH) level and myocardial superoxide dismutase (SOD) activity were determined by spectrophotometry. Isolated ventricular myocytes from adult rats were exposed to 60 min anoxia and 30 min reoxygenation to simulate IR injuries. After reperfusion, cell viability was determined with trypan blue; reactive oxygen species (ROS) and mitochondrial membrane potential of the cardiomyocytes were measured with the fluorescent probe. The mitochondrial permeability transition pore (mPTP) opening induced by Ca2+ (200 μmol/L) was measured with the absorbance at 520 nm in the isolated myocardial mitochondria. Results: Low dose of bicyclol (25 mg/(kg∙d)) had no significant improving effect on all cardiac parameters, whereas pretreatment with high bicyclol markedly reduced the myocardial infarct and improved the left ventricular contractility in the myocardium exposed to IR (P<0.05). Medium dose of bicyclol (50 mg/(kg∙d)) markedly improved the myocardial contractility, left ventricular myocyte viability, and SOD activity, as well decreased infarct size, serum LDH level, ROS production, and mitochondrial membrane potential in rat myocardium exposed to IR. The reduction of ventricular myocyte viability in IR group was inhibited by pretreatment with 50 and 100 mg/(kg∙d) bicyclol (P<0.05 vs. IR), but not by 25 mg/(kg∙d) bicyclol. The opening of mPTP evoked by Ca2+ was significantly inhibited by medium bicyclol. Conclusions: Bicyclol exerts cardioprotection against IR injury, at least, via reducing oxidative stress and its subsequent mPTP opening.
Ischemia-reperfusion injury; Cardioprotection; Oxidative stress; Mitochondrial permeability transition pore; Bicyclol
Multidrug resistance (MDR) is a critical problem in the chemotherapy of cancers. Human hepatocellular carcinoma (HCC) responds poorly to chemotherapy owing to its potent MDR. Chemotherapeutic drugs primarily act by inducing apoptosis of cancer cells, and defects in apoptosis may result in MDR. Mitochondrial permeability transition (mPT) is implicated as an important event in the control of cell death or survival and mPT represents a target for the development of cytotoxic drugs. This study aimed to investigate the effects of selective opener (Atractyloside glycoside, ATR) and inhibitor (Cyclosporine A, CsA) of mitochondrial permeability transition pore (mPTP) on a CDDP-resistant HCC cell line (SK-Hep1 cells). In this study, a stable MDR phenotype characterization of SK-Hep1 cell line (SK-Hep1/CDDP cells) was established and used to investigate the role of mPTP in MDR. Results suggested that ATR accelerated the decrease of mitochondrial membrane potential (ΔΨm), reduced the Bax activity, and increased the apoptosis of SK-Hep1/CDDP cells; while CsA inhibited mPTP opening, reduced and delayed the decline of mitochondrial membrane potential, and increased the Bax activity, leading to increased tolerance of SK-Hep1/CDDP cells to apoptosis induction. However, mPTP activity had no effect on the expression of MDR1 in cells,meanwhile the P-gp translocation to mitochondria was increased, and functionally activated. In conclusion, selective modulation of mPTP can affect MDR in human HCC cells. Therefore, activation of mPTP may provide a new strategy to sensitize cancer cells to chemotherapeutic drugs and to reverse the MDR in cancer cells.
hepatocellular carcinoma; mitochondrial permeability transition pore; multidrug resistance; mitochondrial membrane potential.
We have recently shown that post-ischemic administration of intralipid protects the heart against ischemia/reperfusion injury. Here we compared the cardioprotective effects of intralipid with cyclosporine-A, a potent inhibitor of the mitochondrial permeability transition pore opening.
In-vivo rat hearts or isolated Langendorff-perfused mouse hearts were subjected to ischemia followed by reperfusion with Intralipid (0.5%, 1% and 2% ex-vivo and 20% in-vivo), cyclosporine-A (0.2μM, 0.8μM and 1.5μM ex-vivo and 10mg/kg in-vivo) or vehicle. The hemodynamic function, infarct size, calcium retention capacity, mitochodrial superoxide production and phosphorylation levels of Akt/GSK-3β were measured. The values are mean±SEM.
Administration of intralipid at reperfusion significantly reduced myocardial infarct size compared with cyclosporine-A in-vivo ((infarct size/area at risk)%: 22.9±2.5% vs. 35.2±3.5%; p=0.030, n=7/group). Postischemic administration of intralipid at its optimal dose (1%) was more effective than cyclosporine-A (0.8μM) in protecting the ex-vivo heart against ischemia/reperfusion injury as the rate pressure product at the end of reperfusion was significantly higher (mmHg*beats/min:12740±675(n=7) vs. 9203±10781(n=5), p=0.024), and the infarct size was markedly smaller (17.3±2.9(n=7) vs. 29.2±2.7(n=5), p=0.014). Intralipid was as efficient as cyclosporine-A in inhibiting the mPTP opening (calcium retention capacity=280±8.2 vs. 260.3±2.9nmol/mg-mitochondria-protein in cyclosporine-A, p=0.454, n=6) and in reducing cardiac mitochondrial superoxide production. Unlike intralipid, which increased phosphorlyation of Akt (6-fold) and GSK-3β (5-fold), cyclosporine-A had no effect on the activation of these pro-survival kinases.
Although intralipid inhibits the opening of the mitochondrial permeability transition pore as efficiently as cyclosporine-A, intralipid is more effective in reducing the infarct size and improving the cardiac functional recovery.
A critical event in ischemia-based cell death is the opening of the mitochondrial permeability transition pore (MPTP). However, the molecular identity of the components of the MPTP remains unknown. Here, we determined that the Bcl-2 family members Bax and Bak, which are central regulators of apoptotic cell death, are also required for mitochondrial pore-dependent necrotic cell death by facilitating outer membrane permeability of the MPTP. Loss of Bax/Bak reduced outer mitochondrial membrane permeability and conductance without altering inner membrane MPTP function, resulting in resistance to mitochondrial calcium overload and necrotic cell death. Reconstitution with mutants of Bax that cannot oligomerize and form apoptotic pores, but still enhance outer membrane permeability, permitted MPTP-dependent mitochondrial swelling and restored necrotic cell death. Our data predict that the MPTP is an inner membrane regulated process, although in the absence of Bax/Bak the outer membrane resists swelling and prevents organelle rupture to prevent cell death.
In all multicellular plants and animals, cells are continuously dying and being replaced. There are a number of different types of cell death, but two of the best studied are apoptosis and necrosis. Apoptosis, sometimes referred to as ‘cell suicide’, is a form of programmed cell death that is generally beneficial to the organism. Necrosis, however, occurs whenever cells are damaged—for example, due to a lack of oxygen—and can trigger harmful inflammation in surrounding tissue. Although the processes leading up to apoptosis and necrosis are very different, they both involve regulated changes in mitochondria—the organelles that supply cells with chemical energy.
Mitochondria have a distinctive appearance, being enclosed by two membranes, the innermost of which is highly folded. During apoptosis, large pores form in the outer membranes of mitochondria. These pores are generated by two proteins—Bax and Bak—and they enable the mitochondrion to release proteins that activate processes involved in apoptosis. Pores also form in the mitochondrial membrane during necrosis. However, these mitochondrial permeability transition pores (MPTPs) occur simultaneously in both the inner and outer membranes and are thought to lead to swelling and rupture of mitochondria.
Now, Karch et al. have shown that Bax and Bak are also involved in the formation of these permeability pores that underlie necrosis. When mouse cells that had been genetically modified to lack Bak and Bax were grown in cell culture, they were found to be resistant to substances that normally induce necrosis. Instead, their mitochondria continued to function normally, suggesting that MPTPs cannot form in the absence of Bak and Bax.
Karch et al. then generated mice with heart cells that lack Bax and Bak, and deprived their hearts of oxygen to simulate a heart attack. Compared to normal mice, the genetically modified animals experienced less damage to their heart muscle, suggesting that the absence of Bax and Bak prevents cell death due to necrosis. If Bax and Bak are involved in both apoptosis and necrosis, inhibiting them could be a powerful therapeutic approach for preventing all forms of cell death during heart attacks or in certain degenerative diseases.
cell death; mitochondria; necrosis; Mouse
Mitochondrial dysfunction caused by amyloid β-peptide (Aβ) plays an important role in the pathogenesis of Alzheimer disease (AD). Substantial evidence has indicated that the mitochondrial permeability transition pore (mPTP) opening is involved in Aβ-induced neuronal death and reactive oxygen species (ROS) generation. Astragaloside IV (AS-IV), one of the major active constituents of Astragalus membranaceus, has been reported as an effective anti-oxidant for treating neurodegenerative diseases. However, the molecular mechanisms still need to be clarified. In this study, we investigated whether AS-IV could prevent Aβ1-42-induced neurotoxicity in SK-N-SH cells via inhibiting the mPTP opening. The results showed that pretreatment of AS-IV significantly increased the viability of neuronal cells, reduced apoptosis, decreased the generation of intracellular reactive oxygen species (ROS) and decreased mitochondrial superoxide in the presence of Aβ1-42. In addition, pretreatment of AS-IV inhibited the mPTP opening, rescued mitochondrial membrane potential (ΔΨm), enhanced ATP generation, improved the activity of cytochrome c oxidase and blocked cytochrome c release from mitochondria in Aβ1-42 rich milieu. Moreover, pretreatment of AS-IV reduced the expression of Bax and cleaved caspase-3 and increased the expression of Bcl-2 in an Aβ1-42 rich environment. These data indicate that AS-IV prevents Aβ1-42-induced SK-N-SH cell apoptosis via inhibiting the mPTP opening and ROS generation. These results provide novel insights of AS-IV for the prevention and treatment of neurodegenerative disorders such as AD.
Oxidized cytochrome c is a powerful superoxide scavenger within the mitochondrial IMS (intermembrane space), but the importance of this role in situ has not been well explored. In the present study, we investigated this with particular emphasis on whether loss of cytochrome c from mitochondria during heart ischaemia may mediate the increased production of ROS (reactive oxygen species) during subsequent reperfusion that induces mPTP (mitochondrial permeability transition pore) opening. Mitochondrial cytochrome c depletion was induced in vitro with digitonin or by 30 min ischaemia of the perfused rat heart. Control and cytochrome c-deficient mitochondria were incubated with mixed respiratory substrates and an ADP-regenerating system (State 3.5) to mimic physiological conditions. This contrasts with most published studies performed with a single substrate and without significant ATP turnover. Cytochrome c-deficient mitochondria produced more H2O2 than control mitochondria, and exogenous cytochrome c addition reversed this increase. In the presence of increasing [KCN] rates of H2O2 production by both pre-ischaemic and end-ischaemic mitochondria correlated with the oxidized cytochrome c content, but not with rates of respiration or NAD(P)H autofluorescence. Cytochrome c loss during ischaemia was not mediated by mPTP opening (cyclosporine-A insensitive), neither was it associated with changes in mitochondrial Bax, Bad, Bak or Bid. However, bound HK2 (hexokinase 2) and Bcl-xL were decreased in end-ischaemic mitochondria. We conclude that cytochrome c loss during ischaemia, caused by outer membrane permeabilization, is a major determinant of H2O2 production by mitochondria under pathophysiological conditions. We further suggest that in hypoxia, production of H2O2 to activate signalling pathways may be also mediated by decreased oxidized cytochrome c and less superoxide scavenging.
Bcl-xL; cytochrome c; hexokinase (HK); mitochon-drial permeability transition pore (mPTP); superoxide; ANT, adenine nucleotide translocase; CHO, Chinese-hamster ovary; CsA, cyclosporine A; DEA, diethylamine NONOate diethylammonium salt; DTT, dithiothreitol; FCCP, carbonyl cyanide p-trifluoromethoxyphenylhydrazone; GMS, 5 mM L-glutamate+2 mM L-malate+5 mM succinate; HK, hexokinase; IMS, intermembrane space; IP, ischaemic pre-conditioning; mPTP, mitochondrial permeability transition pore; OMM, outer mitochondrial membrane; ROS, reactive oxygen species; SOD, superoxide dismutase; VDAC, voltage-dependent anion channel
Reperfusion therapy must be applied as soon as possible to attenuate the ischemic insult of acute myocardial infarction (AMI). However reperfusion is responsible for additional myocardial damage, which likely involves opening of the mitochondrial permeability transition pore (mPTP). In reperfusion injury, mitochondrial damage is a determining factor in causing loss of cardiomyocyte function and viability. Major mechanisms of mitochondrial dysfunction include the long lasting opening of mPTPs and the oxidative stress resulting from formation of reactive oxygen species (ROS). Several signaling cardioprotective pathways are activated by stimuli such as preconditioning and postconditioning, obtained with brief intermittent ischemia or with pharmacological agents. These pathways converge on a common target, the mitochondria, to preserve their function after ischemia/reperfusion. The present review discusses the role of mitochondria in cardioprotection, especially the involvement of adenosine triphosphate-dependent potassium channels, ROS signaling, and the mPTP. Ischemic postconditioning has emerged as a new way to target the mitochondria, and to drastically reduce lethal reperfusion injury. Several clinical studies using ischemic postconditioning during angioplasty now support its protective effects, and an interesting alternative is pharmacological postconditioning. In fact ischemic postconditioning and the mPTP desensitizer, cyclosporine A, have been shown to induce comparable protection in AMI patients.
Adenosine triphosphate-dependent potassium channels; Cardioprotection; Ischemia-reperfusion injury; Mitochondrial permeability transition pore; Reactive oxygen species
There are approximately 8.5 million Alzheimer disease (AD) patients who need anesthesia and surgery care every year. The inhalation anesthetic isoflurane, but not desflurane, has been shown to induce caspase activation and apoptosis, which are part of AD neuropathogenesis, through the mitochondria-dependent apoptosis pathway. However, the in vivo relevance, underlying mechanisms, and functional consequences of these findings remain largely to be determined.
We therefore set out to assess the effects of isoflurane and desflurane on mitochondrial function, cytotoxicity, learning, and memory using flow cytometry, confocal microscopy, Western blot analysis, immunocytochemistry, and the fear conditioning test.
Here we show that isoflurane, but not desflurane, induces opening of mitochondrial permeability transition pore (mPTP), increase in levels of reactive oxygen species, reduction in levels of mitochondrial membrane potential and adenosine-5′-triphosphate, activation of caspase 3, and impairment of learning and memory in cultured cells, mouse hippocampus neurons, mouse hippocampus, and mice. Moreover, cyclosporine A, a blocker of mPTP opening, attenuates isoflurane-induced mPTP opening, caspase 3 activation, and impairment of learning and memory. Finally, isoflurane may induce the opening of mPTP via increasing levels of reactive oxygen species.
These findings suggest that desflurane could be a safer anesthetic for AD patients as compared to isoflurane, and elucidate the potential mitochondria-associated underlying mechanisms, and therefore have implications for use of anesthetics in AD patients, pending human study confirmation.
Mitochondria in motor nerve terminals temporarily sequester large Ca2+ loads during repetitive stimulation. In wild-type mice this Ca2+ uptake produces a small (<5 mV), transient depolarization of the mitochondrial membrane potential (Ψm, motor nerve stimulated with at 100 Hz for 5 s). We demonstrate that this stimulation-induced Ψm depolarization attains much higher amplitudes in motor terminals of symptomatic mice expressing the G93A or G85R mutation of human superoxide dismutase 1 (SOD1), models of familial amyotrophic lateral sclerosis (fALS). These large Ψm depolarizations decayed slowly and incremented with successive stimulus trains. Additional Ψm depolarizations occurred that were not synchronized with stimulation. These large Ψm depolarizations were reduced (a) by cyclosporin A (CsA, 1-2 uM), which inhibits opening of the mitochondrial permeability transition pore (mPTP), or (b) by replacing bath Ca2+ with Sr2+, which enters motor terminals and mitochondria but does not support mPTP opening. These results are consistent with the hypothesis that the large Ψm depolarizations evoked by repetitive stimulation in motor terminals of symptomatic fALS mice result from mitochondrial dysfunction that increases the likelihood of transient mPTP opening during Ca2+ influx. Such mPTP openings, a sign of mitochondrial stress, would disrupt motor terminal handling of Ca2+ loads and might thereby contribute to motor terminal degeneration in fALS mice. Ψm depolarizations resembling those in symptomatic fALS mice could be elicited in wild-type mice following 0.5-1 hr exposure to diamide (200 μM), which produces an oxidative stress, but these depolarizations were not reduced by CsA.
motor nerve terminal; superoxide dismutase 1 G93A; superoxide dismutase 1 G85R; mouse models of familial amyotrophic lateral sclerosis; mitochondria; mitochondrial permeability transition pore; mitochondrial calcium uptake; oxidative stress; mitochondrial membrane potential; motor neuron
Successful treatment of myocardial infarction related to early reperfusion therapy has caused growing interest in not only ischemic but also myocardial reperfusion injury. Most experimentally confirmed preservation myocardial reperfusion injury methods have failed in clinical practice. Probably one reason for their ineffectiveness was the very narrow “time window” necessitating application of protective methods before obtaining reperfusion. Reducing the myocardial necrosis and preservation of the left ventricular function are the main goals of the therapy. Experimental data suggest that up to 50% of the infarct size may be related to reperfusion injury. Function of the mitochondrial permeability transition pore (mPTP) in the inner mitochondrial membrane, being closed during myocardial ischemia and opening at the beginning of reperfusion, is the common element linking protective methods. Their opening gives rise to metabolic alterations and may lead to cardiomyocyte death (lethal reperfusion injury). That is why successful intervention, very difficult to achieve, has to take precedence over coronary blood flow restoration. Cyclosporin A, an mPTP blocker, was effective in the first small clinical trial in preservation of myocardial reperfusion injury in acute coronary syndrome intervention. Second mitochondrial injury action is related to generation of reactive oxygen species (ROS) including superoxide anions. Reactive oxygen species accumulation results in mitochondrial pH increase leading to mPTP opening. Discovery of a small molecule cationic peptide, readily penetrating cell membranes and concentrating in mitochondria, may give new therapy perspectives. Combining therapy may be possible as well.
myocardial infarction; reperfusion; cyclosporin A; Bendavia; reactive oxygen species
During the aging process, an accumulation of non-heme iron disrupts cellular homeostasis and contributes to the mitochondrial dysfunction typical of various neuromuscular degenerative diseases. Few studies have investigated the effects of iron accumulation on mitochondrial integrity and function in skeletal muscle and liver tissue. Thus, we isolated liver mitochondria (LM), as well as quadriceps-derived subsarcolemmal mitochondria (SSM) and interfibrillar mitochondria (IFM), from male Fischer 344× Brown Norway rats at 8, 18, 29 and 37 months of age. Non-heme iron content in SSM, IFM and LM was significantly higher with age, reaching a maximum at 37 months of age. The mitochondrial permeability transition pore (mPTP) was more susceptible to the opening in aged mitochondria containing high levels of iron (i.e. SSM and LM) compared to IFM. Furthermore, mitochondrial RNA oxidation increased significantly with age in SSM and LM, but not in IFM. Levels of mitochondrial RNA oxidation in SSM and LM correlated positively with levels of mitochondrial iron, whereas a significant negative correlation was observed between the maximum Ca2+ amounts needed to induce mPTP opening and iron contents in SSM, IFM and LM. Overall, our data suggest that age-dependent accumulation of mitochondrial iron may increase mitochondrial dysfunction and oxidative damage, thereby enhancing the susceptibility to apoptosis.
mitochondrial aging; mitochondrial iron homeostasis; mitochondrial permeability transition pore; mitochondrial RNA; oxidative stress; skeletal muscle subsarcolemmal and interfibrillar mitochondria
Mitochondria serve as a “powerhouse” which provides near 90% of ATP necessary for cell life. However, recent studies provide strong evidence that mitochondria also play a central role in cell death. Mitochondrial permeability transition (mPT) at high conductance in response to oxidative or other cellular stresses is accompanied by pathological and non-specific mPT pore (mPTP) opening in the inner membrane of mitochondria. Mitochondrial PTP can serve as a target to prevent cell death under pathological conditions such as cardiac and brain ischemia/reperfusion injury and diabetes. On the other hand, mPTP can be used as an executioner to specifically induce cell death thus blocking tumorigenesis in cancer diseases. Despite many studies, the molecular identity of the mPTP remains unclear. Cyclophilin D (CyP-D) plays an essential regulatory role in pore opening. This review will discuss direct and indirect mechanisms underlying CyP-D interaction with a target protein of the mPTP complex. Understanding of the mechanisms of mPTP opening will be helpful to further develop new pharmacological agents targeting mitochondria-mediated cell death.
mitochondria; permeability transition pore; cyclophilin D; cell death
Ischemic postconditioning (PoCo) and cyclosporine A (CysA) given prior to reperfusion reduce myocardial infarct size after ischemia/reperfusion. Ischemic postconditioning's protection is characterized by better preservation of mitochondrial respiration and calcium retention capacity. Protection by CysA is not entirely clear. Cyclosporine A inhibits not only mitochondrial permeability transition pore (mPTP) opening but also the phosphatase calcineurin. We have investigated whether CysA mediates protection not only by mPTP inhibition but also through a more upstream inhibition of calcineurin with subsequently better preserved mitochondrial respiration.
Material and methods
Anesthetized pigs were subjected to 90 min ischemia and 10 min reperfusion initiated with either PoCo (6 × 20 s reperfusion/re-occlusion; n = 9), CysA infusion (5 mg/kg i.v.; 5 min before reperfusion; n = 4), or immediate full reperfusion (IFR; n = 8). Mitochondria were isolated from myocardial tissue for measurement of respiration and calcium retention capacity.
In mitochondria from ischemic/reperfused myocardium, ADP-stimulated complex I respiration was similar between CysA (116 ±11 nmol O2/min/mg protein) and IFR (117 ±8), but better preserved with PoCo (160 ±9; p < 0.05). Calcium retention capacity was greater with both PoCo and CysA (1096 ±45 and 1287 ±128 nmol Ca2+/mg protein) than with IFR (756 ±103; p < 0.05).
Cyclosporine A's protection is not associated with improved mitochondrial respiration. Protection is unlikely related to an upstream calcineurin inhibition, but is indeed secondary to mPTP inhibition.
cyclosporine A; infarct size; ischemic postconditioning; mitochondrion; myocardial ischemia/reperfusion
Permeabilization of the mitochondrial membranes is a crucial step in apoptosis and necrosis. This phenomenon allows the release of mitochondrial death factors which trigger or facilitate different signaling cascades ultimately causing the execution of the cell. The mitochondrial permeability transition pore (mPTP) has long been known as one of the main regulators of mitochondria during cell death. mPTP opening can lead to matrix swelling, subsequent rupture of the outer membrane and a nonspecific release of intermembrane space proteins into the cytosol. While mPTP was purportedly associated with early apoptosis, recent observations suggest that mitochondrial permeabilization mediated by mPTP is generally more closely linked to events of late apoptosis and necrosis. Mechanisms of mitochondrial membrane permeabilization during cell death, involving three different mitochondrial channels, have been postulated. These include the mPTP in the inner membrane, and the mitochondrial apoptosis-induced channel (MAC) and voltage dependent anion-selective channel (VDAC) in the outer membrane. New developments on mPTP structure and function, and the involvement of mPTP, MAC, and VDAC in permeabilization of mitochondrial membranes during cell death are explored.
mPTP, mitochondrial permeability transition pore; MAC, mitochondrial apoptosis-induced channel; VDAC, voltage dependent anion-selective channel; Bcl-2 family proteins; patch clamp, pharmacology
Alteration in endoplasmic reticulum (ER) stress in diabetic hearts and its effect on cytoprotective signaling are unclear. Here, we examine the hypothesis that ER stress in diabetic hearts impairs phospho–glycogen synthase kinase (GSK)-3β–mediated suppression of mitochondrial permeability transition pore (mPTP) opening, compromising myocardial response to cytoprotective signaling.
RESEARCH DESIGN AND METHODS
A rat model of type 2 diabetes (OLETF) and its control (LETO) were treated with tauroursodeoxycholic acid (TUDCA) (100 mg · kg−1 · day−1 for 7 days), an ER stress modulator. Infarction was induced by 20-min coronary occlusion and 2-h reperfusion.
Levels of ER chaperones (GRP78 and GRP94) in the myocardium and level of nonphoshopho–GSK-3β in the mitochondria were significantly higher in OLETF than in LETO rats. TUDCA normalized levels of GRP78 and GRP94 and mitochondrial GSK-3β in OLETF rats. Administration of erythropoietin (EPO) induced phosphorylation of Akt and GSK-3β and reduced infarct size (% risk area) from 47.4 ± 5.2% to 23.9 ± 3.5% in LETO hearts. However, neither phosphorylation of Akt and GSK-3β nor infarct size limitation was induced by EPO in OLETF rats. The threshold for mPTP opening was significantly lower in mitochondria from EPO-treated OLETF rats than in those from EPO-treated LETO rats. TUDCA restored responses of GSK-3β, mPTP opening threshold, and infarct size to EPO receptor activation in OLETF rats. There was a significant correlation between mPTP opening threshold and phospho–GSK-3β–to–total GSK-3β ratio in the mitochondrial fraction.
Disruption of protective signals leading to GSK-3β phosphorylation and increase in mitochondrial GSK-3β are dual mechanisms by which increased ER stress inhibits EPO-induced suppression of mPTP opening and cardioprotection in diabetic hearts.
Cardioprotection by volatile anesthetic-induced preconditioning (APC) involves activation of protein kinase C (PKC). The current study investigated the importance of APC-activated PKC in delaying mitochondrial permeability transition pore (mPTP) opening.
Rat ventricular myocytes were exposed to isoflurane in the presence or absence of nonselective PKC inhibitor chelerythrine or isoform-specific inhibitors of PKC-δ (rottlerin) and PKC-ε (myristoylated PKC-ε V1-2 peptide), and the mPTP opening time was measured using confocal microscopy. Ca2+-induced mPTP opening was measured in mitochondria isolated from rats exposed to isoflurane in the presence and absence of chelerythrine, or in mitochondria directly treated with isoflurane after isolation. Translocation of PKC-ε was assessed in APC and control cardiomyocytes by Western blotting.
In cardiomyocytes, APC prolonged time necessary to induce mPTP opening (261±26 s APC vs. 216±27 s control; P<0.05), while chelerythrine abolished this delay to 213±22 s. The effect of isoflurane was also abolished when PKC-ε inhibitor was applied (210±22 s), but not in the presence of PKC-δ inhibitor (269±31 s). Western blotting revealed translocation of PKC-ε toward mitochondria in APC cells. The Ca2+ concentration required for mPTP opening was significantly higher in mitochondria from APC rats (45±8 μM mg-1 control vs. 64±8 μM mg-1 APC), and APC effect was reversed with chelerythrine. In contrast, isoflurane did not protect directly treated mitochondria.
APC induces delay of mPTP opening through PKC-ε-mediated inhibition of mPTP opening, but not through PKC-δ. These results point to the connection between cytosolic and mitochondrial components of cardioprotection by isoflurane.
Bnip3 is a pro-apoptotic BH3-only protein which is associated with mitochondrial dysfunction and cell death. Bnip3 is also a potent inducer of autophagy in many cells. In this study, we have investigated the mechanism by which Bnip3 induces autophagy in adult cardiac myocytes. Overexpression of Bnip3 induced extensive autophagy in adult cardiac myocytes. Fluorescent microscopy studies and ultrastructural analysis revealed selective degradation of mitochondria by autophagy in myocytes overexpressing Bnip3. Oxidative stress and increased levels of intracellular Ca2+ have been reported by others to induce autophagy, but Bnip3-induced autophagy was not abolished by antioxidant treatment or the Ca2+ chelator BAPTA-AM. We also investigated the role of the mitochondrial permeability transition pore (mPTP) in Bnip3-induced autophagy. Although the mPTP has previously been implicated in the induction of autophagy and selective removal of damaged mitochondria by autophagosomes, mitochondria sequestered by autophagosomes in Bnip3-treated cardiac myocytes had not undergone permeability transition and treatment with the mPTP inhibitor cyclosporine A did not inhibit mitochondrial autophagy in cardiac myocytes. Moreover, cyclophilin D (cypD) is an essential component of the mPTP and Bnip3 induced autophagy to the same extent in embryonic fibroblasts isolated from wild-type and cypD-deficient mice. These results support a model where Bnip3 induces selective removal of the mitochondria in cardiac myocytes and that Bnip3 triggers induction of autophagy independent of Ca2+, ROS generation and mPTP opening.
Bnip3; autophagy; cardiac myocytes; mitochondria; permeability transition pore; cyclophilin D
Mitochondria have been shown to play an important role in apoptosis using mammalian cell lines. However, this seems not to be the case in Drosophila, an insect model organism; thus more in-depth studies of insect cell apoptosis are necessary. In the present study, mitochondrial involvement during azadirachtin- and camptothecin-induced apoptosis in Spodoptera frugiperda Sf9 cells (isolated from Spodoptera frugiperda pupal ovarian tissue) was investigated. The results showed that both azadirachtin and camptothecin could induce apoptosis in Sf9 cells. Reactive oxygen species (ROS) generation, activation of mitochondrial permeability transition pores (MPTPs) and loss of mitochondrial membrane potential (MMP) were observed very early during apoptosis and were followed subsequently by the release of cytochrome-c from the mitochondria. Furthermore, the results also revealed that the opening of MPTPs and the loss of MMP induced by azadirachtin could be significantly inhibited by the permeability transition pore (PTP) inhibitor cyclosporin A (CsA), which was used to identify the key role of mitochondria in the apoptosis of Sf9 cells. However, in camptothecin-treated Sf9 cells, CsA could not suppress the opening of MPTPs and the loss of MMP when apoptosis was induced. The data from caspase-3 and caspase-9 activity assays and detection of apoptosis by morphological observation and flow cytometry also uncovered the different effect of CsA on the two botanical apoptosis inducers. Although different mechanisms of apoptosis induction exist, our study revealed that mitochondria play a crucial role in insect cell line apoptosis.