The role of mitochondria in protection of the heart by preconditioning

A prolonged period of ischaemia followed by reperfusion irreversibly damages the heart. Such reperfusion injury (RI) involves opening of the mitochondrial permeability transition pore (MPTP) under the conditions of calcium overload and oxidative stress that accompany reperfusion. Protection from MPTP opening and hence RI can be mediated by ischaemic preconditioning (IP) where the prolonged ischaemic period is preceded by one or more brief (2-5 min) cycles of ischaemia and reperfusion. Following a brief overview of the molecular characterisation and regulation of the MPTP, the proposed mechanisms by which IP reduces pore opening are reviewed including the potential roles for reactive oxygen species (ROS), protein kinase cascades, and mitochondrial potassium channels. It is proposed that IP-mediated inhibition of MPTP opening at reperfusion does not involve direct phosphorylation of mitochondrial proteins, but rather reflects diminished oxidative stress during prolonged ischaemia and reperfusion. This causes less oxidation of critical thiol groups on the MPTP that are known to sensitise pore opening to calcium. The mechanisms by which ROS levels are decreased in the IP hearts during prolonged ischaemia and reperfusion are not known, but appear to require activation of protein kinase Cepsilon, either by receptor-mediated events or through transient increases in ROS during the IP protocol. Other signalling pathways may show cross-talk with this primary mechanism, but we suggest that a role for mitochondrial potassium channels is unlikely. The evidence for their activity in isolated mitochondria and cardiac myocytes is reviewed and the lack of specificity of the pharmacological agents used to implicate them in IP is noted. Some K(+) channel openers uncouple mitochondria and others inhibit respiratory chain complexes, and their ability to produce ROS and precondition hearts is mimicked by bona fide uncouplers and respiratory chain inhibitors. IP may also provide continuing protection during reperfusion by preventing a cascade of MPTP-induced ROS production followed by further MPTP opening. This phase of protection may involve survival kinase pathways such as Akt and glycogen synthase kinase 3 (GSK3) either increasing ROS removal or reducing mitochondrial ROS production.     

MitoK ATP -dependent changes in mitochondrial volume and in complex II activity during ischemic and pharmacological preconditioning of langendorff-perfused rat heart

It has been proposed that activation of the mitochondrial ATP-sensitive potassium channel (mitoK_ATP) is part of signaling pathways triggering the cardioprotection afforded by ischemic preconditioning of the heart. This work was to analyze the mitochondrial function profile of Langendorff-perfused rat hearts during the different phases of various ischemia-reperfusion protocols. Specifically, skinned fibers of ischemic preconditioned hearts exhibit a decline in the succinate-supported respiration and complex II activity during ischemia, followed by a recovery during reperfusion. Meanwhile, the apparent affinity of respiration for ADP (which reflects the matrix volume expansion) is increased during preconditioning stimulus and, to a larger extent, during prolonged ischemia. This evolution pattern is mimicked by diazoxide and abolished by 5-hydroxydecanoate. It is concluded that opening the mitoK_ATP channel mediates the preservation of mitochondrial structure-function via a mitochondrial matrix shrinkage and a reversible inactivation of complex II during prolonged ischemic insult.     

The role of the mitochondrial permeability transition pore in heart disease

Like Dr. Jeckyll and Mr. Hyde, mitochondria possess two distinct persona. Under normal physiological conditions they synthesise ATP to meet the energy needs of the beating heart. Here calcium acts as a signal to balance the rate of ATP production with ATP demand. However, when the heart is overloaded with calcium, especially when this is accompanied by oxidative stress, mitochondria embrace their darker side, and induce necrotic cell death of the myocytes. This happens acutely in reperfusion injury and chronically in congestive heart failure. Here calcium overload, adenine nucleotide depletion and oxidative stress combine forces to induce the opening of a non-specific pore in the mitochondrial membrane, known as the mitochondrial permeability transition pore (mPTP). The molecular nature of the mPTP remains controversial but current evidence implicates a matrix protein, cyclophilin-D (CyP-D) and two inner membrane proteins, the adenine nucleotide translocase (ANT) and the phosphate carrier (PiC). Inhibition of mPTP opening can be achieved with inhibitors of each component, but targeting CyP-D with cyclosporin A (CsA) and its non-immunosuppressive analogues is the best described. In animal models, inhibition of mPTP opening by either CsA or genetic ablation of CyP-D provides strong protection from both reperfusion injury and congestive heart failure. This confirms the mPTP as a promising drug target in human cardiovascular disease. Indeed, the first clinical trials have shown CsA treatment improves recovery after treatment of a coronary thrombosis with angioplasty.     

Cyclosporin A binding in mitochondrial cyclophilin inhibits the permeability transition pore and protects hearts from ischaemia/reperfusion injury

When loaded with high (pathological) levels of Ca²⁺, mitochondria become swollen and uncoupled as the result of a large non-specific increase in membrane permeability. This process, known as the mitochondrial permeability transition (MPT), is exacerbated by oxidative stress and adenine nucleotide depletion. These conditions match those that a heart experiences during reperfusion following a period of ischaemia. The MPT is caused by the opening of a non-specific pore that can be prevented by sub-micromolar concentrations of cyclosporin A (CsA). A variety of conditions that increase the sensitivity of pore opening to [Ca²⁺], such as thiol modification, oxidative stress, increased matrix volume and chaotropic agents, all enhance the binding of matrix cyclophilin (CyP) to the inner mitochondrial membrane in a CsA-sensitive manner. In contrast, ADP, membrane potential and low pH decrease the sensitivity of pore opening to [Ca²⁺] without affecting CyP binding. We present a model of pore opening involving CyP binding to a membrane target protein followed by Ca²⁺-dependent triggering of a conformational change to induce channel opening. Using the ischaemic/reperfused rat heart we have shown that the mitochondrial pore does not open during ischaemia, but does do so during reperfusion. Recovery of heart during reperfusion is improved in the presence of 0.2 µM CsA, suggesting that the MPT may be critical in the transition from reversible to irreversible reperfusion injury. (Mol Cell Biochem 174: 167–172, 1997)     

Mitochondria and cell death

Mitochondria play a central role in both apoptosis and necrosis through the opening of the mitochondrial permeability transition pore (MPTP). This is thought to be formed through a Ca(2+)-triggered conformational change of the adenine nucleotide translocase (ANT) bound to matrix cyclophilin-D and we have now demonstrated this directly by reconstitution of the pure components. Opening of the MPTP causes swelling and uncoupling of mitochondria which, unrestrained, leads to necrosis. In ischaemia/reperfusion injury of the heart we have shown MPTP opening directly. Recovery of hearts correlates with subsequent closure, and agents that prevent opening or enhance closure protect from injury. Transient MPTP opening may also be involved in apoptosis by initially causing swelling and rupture of the outer membrane to release cytochrome c (cyt c), which then activates the caspase cascade and sets apoptosis in motion. Subsequent MPTP closure allows ATP levels to be maintained, ensuring that cell death remains apoptotic rather than necrotic. Apoptosis in the hippocampus that occurs after a hypoglycaemic or ischaemic insult is triggered by this means. Other apoptotic stimuli such as cytokines or removal of growth factors also involve mitochondrial cyt c release, but here there is controversy over whether the MPTP is involved. In many cases cyt c release is seen without any mitochondrial depolarization, suggesting that the MPTP does not open. Recent data of our own and others have revealed a specific outer-membrane cyt c-release pathway involving porin that does not release other intermembrane proteins such as adenylate kinase. This is opened by pro-apoptotic members of the Bcl-2 family such as BAX and prevented by anti-apoptotic members such as Bcl-X(L). Our own data suggest that this pathway may interact directly with the ANT in the inner membrane at contact sites.     

二氮嗪对大鼠供心心肌线粒体结构及通透性的影响

目的:探讨线粒体ATP敏感性钾离子通道(mitoKATPC)开放剂二氮嗪(DE)对离体大鼠供心长时程低温保存时线粒体超微结构及线粒体渗透性转换孔(MPTP)开放的影响。方法:利用Langendorff离体鼠心灌注法,观察供心在4℃含不同浓度DE(15、30、45μmol/L)的Celsior保存液中保存9h后,复灌期心脏作功量(RPP)变化情况。比色法测定MPTP开放情况;透射电子显微镜观察心肌细胞线粒体超微结构的变化。结果:①Celsior保存液中加入30μmol/L的DE对促进长时程低温保存后供心收缩功能的恢复、减轻心肌细胞线粒体超微结构损伤和抑制MPTP开放的作用最显著。②DE的上述作用可分别被mitoKATP特异性阻断剂5-羟基葵酸盐(5-HD)及MPTP开放剂苍术苷(Atr)所取消。结论:DE可通过抑制MPTP开放而减轻由长时程低温保存导致的大鼠供心心肌线粒体超微结构的损伤。     

乙酰胆碱对离体大鼠缺血/复灌心脏的保护作用及其机制

目的:探讨乙酰胆碱(ACh)预处理抗心肌缺血复灌(I/R)损伤作用及其与线粒体渗透性转换孔和/或线粒体ATP敏感性钾通道的关系。方法:采用离体大鼠心脏Langendorff灌流方法进行全心停灌30min,复灌120min复制I/R模型。测定心室力学指标和复灌各时间点冠脉流出液中乳酸脱氢酶(LDH)含量。实验结束测定心肌组织formazan含量的变化。结果:与单纯I/R组相比,ACh(0.1μmol/L,5min)预处理明显提高心肌细胞的formazan含量,降低复灌期间冠脉流出液中LDH含量,明显改善I/R所致的左室发展压、左心室内压最大上升和下降速率、心率与发展压乘积和左室舒张末压力的下降,缓解冠脉流量的减少。线粒体渗透性转换孔开放剂苍术苷(20μmol/L,复灌前给药20min)和线粒体ATP敏感性钾通道抑制剂5-羟基癸酸(100μmol/L,缺血前给药20min)能明显减弱ACh的保护作用。结论:在大鼠离体心脏灌流模型上,ACh预处理具有抗心脏缺血/复灌损伤的作用,这种保护作用可能与其抑制线粒体渗透性转换孔的开放和促进线粒体ATP敏感性钾通道的开放有关。     

Negative regulation of vascular smooth muscle cell migration by blood shear 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.     

Vagal nerve stimulation improves mitochondrial dynamics via an M3 receptor/CaMKKβ/AMPK pathway in isoproterenol‐induced myocardial ischaemia

Mitochondrial dynamics—fission and fusion—are associated with ischaemic heart disease (IHD). This study explored the protective effect of vagal nerve stimulation (VNS) against isoproterenol (ISO)‐induced myocardial ischaemia in a rat model and tested whether VNS plays a role in preventing disorders of mitochondrial dynamics and function. Isoproterenol not only caused cardiac injury but also increased the expression of mitochondrial fission proteins [dynamin‐related peptide1 (Drp1) and mitochondrial fission protein1 (Fis‐1)) and decreased the expression of fusion proteins (optic atrophy‐1 (OPA1) and mitofusins1/2 (Mfn1/2)], thereby disrupting mitochondrial dynamics and leading to increase in mitochondrial fragments. Interestingly, VNS restored mitochondrial dynamics through regulation of Drp1, Fis‐1, OPA1 and Mfn1/2; enhanced ATP content and mitochondrial membrane potential; reduced mitochondrial permeability transition pore (MPTP) opening; and improved mitochondrial ultrastructure and size. Furthermore, VNS reduced the size of the myocardial infarction and ameliorated cardiomyocyte apoptosis and cardiac dysfunction induced by ISO. Moreover, VNS activated AMP‐activated protein kinase (AMPK), which was accompanied by phosphorylation of Ca²⁺/calmodulin‐dependent protein kinase kinase β (CaMKKβ) during myocardial ischaemia. Treatment with subtype‐3 of muscarinic acetylcholine receptor (M₃R) antagonist 4‐diphenylacetoxy‐N‐methylpiperidine methiodide or AMPK inhibitor Compound C abolished the protective effects of VNS on mitochondrial dynamics and function, suggesting that M₃R/CaMKKβ/AMPK signalling are involved in mediating beneficial effects of VNS. This study demonstrates that VNS modulates mitochondrial dynamics and improves mitochondrial function, possibly through the M₃R/CaMKKβ/AMPK pathway, to attenuate ISO‐induced cardiac damage in rats. Targeting mitochondrial dynamics may provide a novel therapeutic strategy in IHD.     

线粒体渗透性转变孔结构和功能的研究进展

非正常生理浓度的Ca^2 和氧化应激等刺激线粒体渗透性转变孔(mitochondria permeability transition pore,MPTP)开放,使线粒体形态功能发生改变,被释放的细胞色素c和凋亡诱导因子(apoptosisinducing factor,AIF)等参与到caspase信号通路中,诱导细胞发生凋亡。本文在MPTP的主要组成成分、两种不同的结构功能模型、抑制剂对MPTP的抑制机制和缺血,再灌注及缺血预适应对MPTP开放的影响等方面的研究进展作一综述。     

Relationship between oxidative stress and mitochondrial function in the post-conditioned heart

The pathways activated by post-conditioning may converge on the mitochondria, in particular on the mitochondrial permeability transition pore. We sought to characterize the inhibition status of the mitochondrial permeability transition early after the post-conditioning maneuver and before long reperfusion was established. We observed that post-conditioning maneuvers applied to isolated rat hearts, after a prolonged ischemia and before reperfusion, promoted cardiac mechanical function recovery and maintained mitochondrial integrity. These effects were evaluated by mitochondrial swelling, calcium transport, and NAD⁺ content measurements; the improvements were established before restoring a long lasting reperfusion period. Mitochondrial integrity was associated with a diminution in oxidative stress, since carbonylation of proteins was prevented and aconitase activity was preserved in the post-conditioned hearts, implying that ROS might mediate mitochondrial dysfunction and mPTP opening. In addition, we found that cytochrome release was significantly abolished in the post-conditioned heart, in contrast with conventionally reperfused hearts.     

Ischemic preconditioning preserves proton leakage from mitochondrial membranes but not oxidative phosphorylation during heart reperfusion

The aim of this study was to evaluate the role of mitochondria in the recovery of cardiac energetics induced by ischaemic preconditioning at reperfusion. Isolated rat hearts were aerobically perfused (control), subjected to global ischaemia and reperfusion (reperfusion), or subjected to 3 brief cycles of ischaemia/reperfusion and then to the protocol of reperfusion (preconditioning). At the end of the perfusion, antimycin A was delivered to the heart for 25 min, to inhibit mitochondrial respiration and stimulate glycolysis. The increased amount of lactate released in the coronary effluent was correlated with the number of viable cells producing this end-product of glycolysis. Preconditioned hearts released 18% more lactate than reperfused hearts (p < 0.05). This result indicates that preconditioning partially preserved cell viability, as was also evidenced by the MTT assay performed on cardiac biopsies. The difference between antimycin A-stimulated and basal lactate concentration, representing the contribution of mitochondria to the overall energetics of cardiac tissue, was also 18% more elevated in the preconditioned hearts than in the reperfused hearts (p < 0.01). The study of the respiratory function of mitochondria isolated at the end of perfusion, showed that preconditioning did not improve the oxygen-dependent production of ATP (state 3 respiration, ADP/O). On the contrary, state 4 respiration, which is related to proton leakage, was 35.0% lower in the preconditioned group than reperfusion group (p < 0.05). Thus, preconditioning ameliorates cardiac energetics by preserving cell death, but without affecting mitochondrial oxidative phosphorylation. Mitochondria can contribute to cell survival by the attenuation of proton leak from inner membrane.     

线粒体钾通道参与葛根素抗心肌细胞缺氧/复氧损伤的作用

目的:探讨线粒体ATP敏感性钾通道和线粒体钙激活钾通道在葛根素预处理抗心肌细胞缺氧/复氧损伤中的作用。方法:采用酶解分离大鼠心肌细胞复制心肌细胞缺氧/复氧模型,台盼蓝拒染法测定心肌细胞存活率;四甲基罗丹明乙酯(TMRE)孵育测定线粒体膜电位值;分离线粒体测定线粒体渗透性转换孔开放程度。结果:与缺氧/复氧组相比,葛根素(0.24mmol/L)预处理5min可明显增加心肌细胞的存活率,线粒体ATP敏感性钾通道抑制剂5-羟基癸酸(100μmol/L,预处理20min)或线粒体钙激活钾通道阻断剂paxilline(1μmol/L,预处理5min)均可拮抗葛根素的作用。葛根素预处理可明显减弱缺氧引起的线粒体膜电位的耗损,5-羟基癸酸和paxilline都能明显拮抗其作用。在分离心肌线粒体模型上,葛根素显著减弱CaCl2诱导的线粒体在A520处吸光度降低,其作用与单独应用线粒体渗透性转换孔抑制剂环孢菌素A相似;5-羟基癸酸和paxilline可拮抗葛根素的保护作用。结论:在大鼠分离心肌细胞模型或分离线粒体模型上,葛根素预处理具有抗缺氧/复氧损伤的作用,这种保护作用可能与其促进线粒体ATP敏感性钾通道和线粒体钙激活钾通道的开放,进而稳定线粒体膜电位,抑制线粒体渗透性转换孔开放有关。     

Preconditioning modulates pulmonary endothelial dysfunction following ischemia-reperfusion injury in the rat lung: role of potassium channels

Ischemic preconditioning (IP) may protect the lung from ischemia-reperfusion (I/R) injury following cardiopulmonary by-pass and lung or heart transplantation. The present study was undertaken to investigate the role of ATP-dependent potassium channels (K(ATP)) in IP in the isolated buffer-perfused rat lung (IBPR) under conditions of elevated pulmonary vasoconstrictor tone (PVT). Since pulmonary arterial perfusion flow and left atrial pressure were constant, changes in pulmonary arterial pressure (PAP) directly reflect changes in pulmonary vascular resistance (PVR). When compared to control value, the pulmonary vasodilator responses to histamine and acetylcholine (ACh) following 2 h of hypothermic ischemia were significantly attenuated, whereas the pulmonary vasodilator response to sodium nitroprusside (SNP) was not altered. IP in the form of two cycles of 5 min of ischemia and reperfusion applied prior to the two-hour interval of ischemia, prevented the decrease in the pulmonary vasodilator responses to histamine and ACh. Pretreatment with glybenclamide (GLB) or HMR-1098, but not 5-hydroxydecanoic acid (5-HD), prior to IP abolished the protective effect of IP. In contrast, GLB or 5-HD did not significantly alter the pulmonary vasodilator response to histamine without IP pretreatment. The present data demonstrate that IP prevents impairment of endothelium-dependent vasodilator responses in the rat pulmonary vascular bed. The present data further suggest that IP may alter the mediation of the pulmonary vasodilator response to histamine and thereby trigger a mechanism dependent on activation of sarcolemmal, and not mitochondrial, K(ATP) channels to preserve endothelial-dependent vasodilator responses and protect against I/R injury in the lung.     

ROS-Ca is associated with mitochondria permeability transition pore involved in surfactin-induced MCF-7 cells apoptosis

The surfactin can inhibit proliferation and induce apoptosis in cancer cells. Moreover, surfactin can induce cell death in human breast cancer MCF-7 cells through mitochondrial pathway. However, the molecular mechanism involved in this pathway remains to be elucidated. Here, the reactive oxygen species (ROS) and Ca²⁺ on mitochondria permeability transition pore (MPTP) activity, and MCF-7 cell apoptosis which induced by surfactin were investigated. It is found that surfactin evoked mitochondrial ROS generation, and the surfactin-induced cell death was prevented by N-acetylcysteine (NAC, an inhibitor of ROS). An increasing cytoplasmic Ca²⁺ concentration was detected in surfactin-induced MCF-7 apoptosis, which was inhibited by 1,2-bis (2-aminophenoxy) ethane-N,N,N′,N′-tetraacetic acid (BAPTA-AM, a chelator of calcium). In addition, the relationship between ROS generation and the increase of cytoplasm Ca²⁺ was determined. The results showed that surfactin initially induced the ROS formation, leading to the MPTP opening accompanied with the collapse of mitochondrial membrane potential (ΔΨ_m). Then the cytoplasmic Ca²⁺ concentration increased in virtue of the changes of mitochondrial permeability, which was prevented by BAPTA-AM. Besides, cytochrome c (cyt c) was released from mitochondria to cytoplasm through the MPTP and activated caspase-9, eventually induced apoptosis. In summary, surfactin has notable anti-tumor effect on MCF-7 cells, however, there was no obvious cytotoxicity on normal cells.     

In vivo hyperoxic preconditioning protects against rat-heart ischemia/reperfusion injury by inhibiting mitochondrial permeability transition pore opening and cytochrome c release

In vivo hyperoxic preconditioning (PC) has been shown to protect against ischemia/reperfusion (I/R) myocardial damage. Mitochondrial permeability transition pore (MPTP) opening is an important event in cardiomyocyte cell death occurring during I/R and therefore a possible target for cardioprotection. We tested the hypothesis that in vivo hyperoxic PC, obtained by mechanical ventilation of animals, could protect heart against I/R injury by inhibiting MPTP opening and cytochrome c release from mitochondria. Mechanically ventilated rats were first exposed to a short period of hyperoxia and isolated hearts were subsequently subjected to I/R in a Langendorff apparatus. Hyperoxic PC significantly improved the functional recovery of hearts on reperfusion, reduced the infarct size, and decreased necrotic damage as shown by the reduced release of lactate dehydrogenase. Mitochondria from hyperoxic PC hearts were less sensitive than mitochondria from reperfused heart to MPTP opening. In addition, hyperoxic PC prevented mitochondrial NAD(+) depletion, an indicator of MPTP opening, and cytochrome c release as well as cardiolipin oxidation/depletion associated with I/R. Together, these results demonstrate that hyperoxic PC protects against heart I/R injury by inhibiting MPTP opening and cytochrome c release. Thus, in vivo hyperoxic PC may represent a useful strategy for the treatment of cardiac I/R injury and could have potential applications in clinical practice.     

Effect of IGF-1 on the balance between autophagy of dysfunctional mitochondria and apoptosis

Mutations in mitochondrial DNA (mtDNA) cause excessive production of mitochondrial reactive oxygen species (ROS) and shorten animal life span. We examined the mechanisms responsible for removal of mitochondria with deleterious mtDNA mutations by autophagy. Incubation of primary cells and cell lines in the absence of serum promotes autophagy of mitochondria with deleterious mtDNA mutations but spares their normal counterparts. The effect of serum withdrawal on the autophagy of dysfunctional mitochondria is prevented by the addition of IGF-1. As a result of the elimination of mitochondria with deleterious mutations, excessive ROS production, characteristic of dysfunctional mitochondria, is greatly reduced. Mitochondrial autophagy shares a common mechanism with mitochondrial-induced cell apoptosis, including mitochondrial transition pore formation and increased ROS production.     

Controlled reperfusion after hypothermic heart preservation inhibits mitochondrial permeability transition-pore opening and enhances functional recovery

We investigated whether low-pressure reperfusion may attenuate postischemic contractile dysfunction, limits necrosis and apoptosis after a prolonged hypothermic ischemia, and inhibits mitochondrial permeability transition-pore (MPTP) opening. Isolated rats hearts (n = 72) were exposed to 8 h of cold ischemia and assigned to the following groups: 1) reperfusion with low pressure (LP = 70 cmH(2)O) and 2) reperfusion with normal pressure (NP = 100 cmH(2)O). Cardiac function was assessed during reperfusion using the Langendorff model. Mitochondria were isolated, and the Ca(2+) resistance capacity (CRC) of the MPTP was determined. Malondialdehyde (MDA) production, caspase-3 activity, and cytochrome c were also assessed. We found that functional recovery was significantly improved in LP hearts with rate-pressure product averaging 30,380 +/- 1,757 vs. 18,000 +/- 1,599 mmHg/min in NP hearts (P < 0.01). Necrosis, measured by triphenyltetrazolium chloride staining and creatine kinase leakage, was significantly reduced in LP hearts (P < 0.01). The CRC was increased in LP heart mitochondria (P < 0.01). Caspase-3 activity, cytochrome c release, and MDA production were reduced in LP hearts (P < 0.001 and P < 0.01). This study demonstrated that low-pressure reperfusion after hypothermic heart ischemia improves postischemic contractile dysfunction and attenuates necrosis and apoptosis. This protection could be related to an inhibition of mitochondrial permeability transition.     

KB-R7943, a plasma membrane Na/Ca exchanger inhibitor,blocks opening of the mitochondrial permeability transition pore

The isothiourea derivative, KB-R7943, inhibits the reverse-mode of the plasma membrane sodium/calcium exchanger and protects against ischemia/reperfusion injury. The mechanism through which KB-R7943 confers protection, however, remains controversial. Recently, KB-R7943 has been shown to inhibit mitochondrial calcium uptake and matrix overload, which may contribute to its protective effects. While using KB-R7943 for this purpose, we find here no evidence that KB-R7943 directly blocks mitochondrial calcium uptake. Rather, we find that KB-R7943 inhibits opening of the mitochondrial permeability transition pore in permeabilized cells and isolated liver mitochondria. Furthermore, we find that this observation correlates with protection against calcium ionophore-induced mitochondrial membrane potential depolarization and cell death, without detrimental effects to basal mitochondrial membrane potential or complex I-dependent mitochondrial respiration. Our data reveal another mechanism through which KB-R7943 may protect against calcium-induced injury, as well as a novel means to inhibit the mitochondrial permeability transition pore.     

Inhibition of Bcl-2 Sensitizes Mitochondrial Permeability Transition Pore (MPTP) Opening in Ischemia-Damaged Mitochondria

Background

Mitochondria are critical to cardiac injury during reperfusion as a result of damage sustained during ischemia, including the loss of bcl-2. We asked if bcl-2 depletion not only leads to selective permeation of the outer mitochondrial membrane (MOMP) favoring cytochrome c release and programmed cell death, but also favors opening of the mitochondrial permeability transition pore (MPTP). An increase in MPTP susceptibility would support a role for bcl-2 depletion mediated cell death in the calcium overload setting of early reperfusion via MPTP as well as later in reperfusion via MOMP as myocardial calcium content normalizes.

Methods

Calcium retention capacity (CRC) was used to reflect the sensitivity of the MPTP opening in isolated cardiac mitochondria. To study the relationship between bcl-2 inhibition and MPTP opening, mitochondria were incubated with a bcl-2 inhibitor (HA14-1) and CRC measured. The contribution of preserved bcl-2 content to MPTP opening following ischemia-reperfusion was explored using transgenic bcl-2 overexpressed mice.

Results

CRC was decreased in mitochondria following reperfusion compared to ischemia alone, indicating that reperfusion further sensitizes to MPTP opening. Incubation of ischemia-damaged mitochondria with increasing HA14-1concentrations increased calcium-stimulated MPTP opening, supporting that functional inhibition of bcl-2 during simulated reperfusion favors MPTP opening. Moreover, HA14-1 sensitivity was increased by ischemia compared to non-ischemic controls. Overexpression of bcl-2 attenuated MPTP opening in following ischemia-reperfusion. HA14-1 inhibition also increased the permeability of the outer membrane in the absence of exogenous calcium, indicating that bcl-2 inhibition favors MOMP when calcium is low.

Conclusions

The depletion and functional inhibition of bcl-2 contributes to cardiac injury by increasing susceptibility to MPTP opening in high calcium environments and MOMP in the absence of calcium overload. Thus, ischemia-damaged mitochondria with decreased bcl-2 content are susceptible to MPTP opening in early reperfusion and MOMP later in reperfusion when cytosolic calcium has normalized.