Inhibitor Of Mitochondrial Permeability Transition Pore Research Articles

Mitochondrial dysfunction is involved in P2X7 receptor‐mediated neuronal cell death

P2X7 receptor (P2X7R) is known to be a 'death receptor' in immune cells, but its functional expression in non-immune cells such as neurons is controversial. Here, we examined the involvement of P2X7R activation and mitochondrial dysfunction in ATP-induced neuronal death in cultured cortical neurons. In P2X7R- and pannexin-1-expressing neuron cultures, 5 or more mM ATP or 0.1 or more mM BzATP induced neuronal death including apoptosis, and cell death was prevented by oxATP, P2X7R-selective antagonists. ATP-treated neurons exhibited Ca(2+) entry and YO-PRO-1 uptake, the former being inhibited by oxATP and A438079, and the latter by oxATP and carbenoxolone, while P2X7R antagonism with oxATP, but not pannexin-1 blocking with carbenoxolone, prevented the ATP-induced neuronal death. The ATP treatment induced reactive oxygen species generation through activation of NADPH oxidase and activated poly(ADP-ribose) polymerase, but both of them made no or negligible contribution to the neuronal death. Rhodamine123 efflux from neuronal mitochondria was increased by the ATP-treatment and was inhibited by oxATP, and a mitochondrial permeability transition pore inhibitor, cyclosporine A, significantly decreased the ATP-induced neuronal death. In ATP-treated neurons, the cleavage of pro-caspase-3 was increased, and caspase inhibitors, Q-VD-OPh and Z-DEVD-FMK, inhibited the neuronal death. The cleavage of apoptosis-inducing factor was increased, and calpain inhibitors, MDL28170 and PD151746, inhibited the neuronal death. These findings suggested that P2X7R was functionally expressed by cortical neuron cultures, and its activation-triggered Ca(2+) entry and mitochondrial dysfunction played important roles in the ATP-induced neuronal death.

Andrographolide induces autophagic cell death in human liver cancer cells through cyclophilin D-mediated mitochondrial permeability transition pore

Liver cancer is the third leading cause of cancer death worldwide and about half of the patients with liver cancer require adjuvant therapy after surgical resection. Therefore, development of novel agents to eradicate cancer cells may constitute a viable approach to treat patients with liver cancer. Andrographolide, a diterpenoid lactone isolated from Andrographis paniculata, is known to possess potent antioxidant, anti-inflammatory, antineoplastic and antiviral properties. In this study, we investigated the cytotoxic effect of andrographolide on human liver cancer cells and explored the cell death mechanism. Andrographolide induced a cell death distinct from apoptosis in multiple human liver cancer cells. The death was characterized by autophagy as evidenced by the accumulation of LC3 II and autophagosomes, and the formation of puncta GFP-LC3. This autophagy as well as cytotoxicity caused by andrographolide could be effectively prevented by 3-methyladenine (a chemical inhibitor of autophagy). Mechanistic study indicated that andrographolide induced autophagic cell death by disruption of mitochondrial transmembrane potential and elevation of reactive oxygen species, which were correlated with mitochondrial permeability transition pore Inhibition of cyclophilin D (a component of MPTP) by cyclosporin A or abrogation of its expression by small interfering RNA significantly suppressed the cytotoxicity of andrographolide, suggesting that cyclophilin D may play an important role in mediating andrographolide-induced cytotoxicity. Taken together, our findings unveil a novel mechanism of drug action by andrographolide in liver cancer cells and suggest that andrographolide may represent a promising novel agent in the treatment of liver cancer.

Inhibition of Mitochondria- and Endoplasmic Reticulum Stress-Mediated Autophagy Augments Temozolomide-Induced Apoptosis in Glioma Cells

Autophagy is a crucial process for cells to maintain homeostasis and survival through degradation of cellular proteins and organelles, including mitochondria and endoplasmic reticula (ER). We previously demonstrated that temozolomide (TMZ), an alkylating agent for brain tumor chemotherapy, induced reactive oxygen species (ROS)/extracellular signal-regulated kinase (ERK)-mediated autophagy to protect glioma cells from apoptosis. In this study, we investigated the role of mitochondrial damage and ER stress in TMZ-induced cytotoxicity. Mitochondrial depolarization and mitochondrial permeability transition pore (MPTP) opening were observed as a prelude to TMZ-induced autophagy, and these were followed by the loss of mitochondrial mass. Electron transport chain (ETC) inhibitors, such as rotenone (a complex I inhibitor), sodium azide (a complex IV inhibitor), and oligomycin (a complex V inhibitor), or the MPTP inhibitor, cyclosporine A, decreased mitochondrial damage-mediated autophagy, and therefore increased TMZ-induced apoptosis. TMZ treatment triggered ER stress with increased expression of GADD153 and GRP78 proteins, and deceased pro-caspase 12 protein. ER stress consequently induced autophagy through c-Jun N-terminal kinases (JNK) and Ca2+ signaling pathways. Combination of TMZ with 4-phenylbutyrate (4-PBA), an ER stress inhibitor, augmented TMZ-induced cytotoxicity by inhibiting autophagy. Taken together, our data indicate that TMZ induced autophagy through mitochondrial damage- and ER stress-dependent mechanisms to protect glioma cells. This study provides evidence that agents targeting mitochondria or ER may be potential anticancer strategies.

116 Matrix metalloproteinase inhibition attenuates reperfusion injury, independently of and additive to mitochondrial permeability transition pore inhibition

While matrix-metalloproteinase (MMP) inhibitors appear to protect against myocardial ischaemia/reperfusion injury, the mechanisms are poorly understood. We hypothesised that cardioprotection resultant from MMP inhibition is independent of mitochondrial permeability transition...

DNase activation by hypoxia–acidosis parallels but is independent of programmed cell death

AimsHypoxia, acidosis and programmed cell death are each hallmarks of acute myocardial infarction (AMI). We previously described a death pathway of cardiac myocytes mediated by hypoxia–acidosis that was characterized by activation of the Bcl2-family protein Bnip3 and programmed necrosis. The pathway included extensive DNA fragmentation that was sensitive to inhibition of the mitochondrial permeability transition pore (mPTP) and calpain inhibitors, but not caspase inhibitors. We did not identify the DNases responsible for DNA cleavage. Main methodsNeonatal rat cardiomyocytes were subjected to hypoxia with and without concurrent acidosis, and the cellular localization of apoptosis-inducing factor (AIF), DNase II and caspase-dependent DNase (CAD) were determined. Key findingsHere we report the occurrence of biphasic pH-dependent translocations of AIF and DNase II but no change in CAD or its inhibitor ICAD. AIF co-localized with the mitochondria under aerobic and hypoxia-neutral conditions but translocated to the nucleus at pH ~6.7 coincident with a decrease of the mitochondrial membrane potential. DNase II co-localized with lysosomes under normoxia and hypoxia-neutral conditions, and translocated to the nucleus at pH ~6.1 coincident with the appearance of single strand DNA cuts. Inhibition of the mPTP pore with BH4-TAT peptide, calpain inhibition with PD150606, or knockdown (KD) of Bnip3 failed to prevent nuclear translocation of these DNase although Bnip3 KD blocked mitochondrial fission. SignificanceThese results suggest that caspase-independent DNA fragmentation is precisely regulated and occurs in parallel but independently from programmed necrosis mediated by hypoxia–acidosis.

Proteasome inhibitor MG132 induces selective apoptosis in glioblastoma cells through inhibition of PI3K/Akt and NFkappaB pathways, mitochondrial dysfunction, and activation of p38-JNK1/2 signaling

Proteasome inhibitors are emerging as a new class of anticancer agents. In this work, we examined the mechanisms underlying cytotoxicity, selectivity and adjuvant potential of the proteasome inhibitor MG132 in a panel of glioblastoma (GBM) cells (U138MG, C6, U87 and U373) and in normal astrocytes. MG132 markedly inhibited GBM cells growth irrespective of the p53 or PTEN mutational status of the cells whereas astrocytic viability was not affected, suggesting a selective toxicity of MG132 to cancerous glial cells. Mechanistically, MG132 arrested cells in G2/M phase of the cell cycle and increased p21(WAF1) protein immunocontent. Following cell arrest, cells become apoptotic as shown by annexin-V binding, caspase-3 activation, chromatin condensation and formation of sub-G1 apoptotic cells. MG132 promoted mitochondrial depolarization and decreased the mitochondrial antiapoptotic protein bcl-xL; it also induced activation of JNK and p38, and inhibition of NFkappaB and PI3K/Akt survival pathways. Pre-treatment of GBMs with the mitochondrial permeability transition pore inhibitor, bongkrekic acid, or pharmacological inhibitors of JNK1/2 and p38, SP600125 and SB203580, attenuated MG132-induced cell death. Besides its apoptotic effect alone, MG132 also enhanced the antiglioma effect of the chemotherapeutics cisplatin, taxol and doxorubicin in C6 and U138MG cells, indicating an adjuvant/chemosensitizer potential. In summary, MG132 exerted profound and selective toxicity in GBMs, being a potential agent for further testing in animal models of the disease.

The calcium uniporter regulates the permeability transition pore in isolated cortical mitochondria.

To investigate the influence of the mitochondrial calcium uniporter on the mitochondrial permeability transition pore, the present study observed mitochondrial morphology in cortical neurons isolated from adult rats using transmission electron microscopy, and confirmed the morphology and activity of isolated mitochondria by detecting succinic dehydrogenase and monoamine oxidase, two mitochondrial enzymes. Isolated mitochondria were treated with either ruthenium red, an inhibitor of the uniporter, spermine, an activator of the uniporter, or in combination with cyclosporin A, an inhibitor of the mitochondrial permeability transition pore. Results showed that ruthenium red inhibited CaCl2-induced mitochondrial permeability transition pore opening, spermine enhanced opening, and cyclosporin A attenuated the effects of spermine. Results demonstrated that the mitochondrial calcium uniporter plays a role in regulating the mitochondrial permeability transition pore in mitochondria isolated from the rat brain cortex.

Mitochondrial Electron Transport Chain in Heavy Metal-Induced Neurotoxicity: Effects of Cadmium, Mercury, and Copper

To clarify the role of mitochondrial electron transport chain (mtETC) in heavy-metal-induced neurotoxicity, we studied action of Cd2+, Hg2+, and Cu2+ on cell viability, intracellular reactive oxygen species formation, respiratory function, and mitochondrial membrane potential of rat cell line PC12. As found, the metals produced, although in a different way, dose- and time-dependent changes of all these parameters. Importantly, Cd2+ beginning from 10 [mu]M and already at short incubation time (3 h) significantly inhibited the FCCP-uncoupled cell respiration; besides, practically the complete inhibition of the respiration was reached after 3 h incubation with 50 [mu]M Hg2+ or 500 [mu]M Cd2+, whereas even after 48 h exposure with 500 [mu]M Cu2+, only a 50% inhibition of the respiration occurred. Against the Cd2+-induced cell injury, not only different antioxidants and mitochondrial permeability transition pore inhibitors were protective but also such mtETC effectors as FCCP and stigmatellin (complex III inhibitor). However, all mtETC effectors used did not protect against the Hg2+- or Cu2+-induced cell damage. Notably, stigmatellin was shown to be one of the strongest protectors against the Cd2+-induced cell damage, producing a 15–20% increase in the cell viability. The mechanisms of the mtETC involvement in the heavy-metal-induced mitochondrial membrane permeabilization and cell death are discussed.

Abstract P169: A Novel Mitochondrial Permeability Transition Pore Inhibitor, Bendavia, Reduces Microvascular Obstruction (No-Reflow) due to Myocardial Ischemia/Reperfusion Injury in the Rabbit

Background: The no-reflow phenomenon is an important detrimental component of reperfusion injury with clinical implications, as it is an independent predictor of outcome after myocardial infarction. Whether the mitochondrial permeability transition pore (MPTP) is involved in vascular integrity and no-reflow post reperfusion is unknown. Therefore, we tested Bendavia, a compound that is known to inhibit the MPTP, for its effects on no-reflow. Methods: Anesthetized rabbits were subjected to 30 min coronary artery occlusion and 3 hrs reperfusion. Treated animals (n=49) received Bendavia (0.05 mg/kg/hr) after the onset of ischemia, 1, 10 or 20 min before reperfusion. Control animals (n=15) received saline. Risk zone was measured by Unisperse blue staining, anatomic no-reflow area using thioflavin S and necrosis by triphenyltetrazolium chloride. Results: The ischemic insult was similar in both groups (31% of left ventricle (LV) in Bendavia and 30% in control). Bendavia treatment resulted in a non-significant 11% reduction in necrosis, expressed as a percentage of the risk zone. However for any given risk zone size, no-reflow was significantly reduced by Bendavia, as there was a significant group effect on the relationship between the extent of the no-reflow zone and that of the risk zone (ANCOVA, p = 0.0085). Overall, the no-reflow zone, expressed as a % of the risk zone was 22 ± 2% in treated hearts vs. 28 ± 3% in controls (p=0.07). In hearts with less severe risk zones (below the mean value of 31% of LV), Bendavia treatment significantly reduced the no-reflow zone by 35% (17±2 vs. 26±4% of risk zone, p <0.05); in hearts with large risk zone, Bendavia non-significantly reduced no-reflow by 17%. Bendavia treatment had no effect on hemodynamic parameters or regional myocardial blood flow during the ischemic period. Conclusion: These data suggest that the MPTP may play a role in the pathophysiology of no-reflow after myocardial ischemia/reperfusion. Bendavia reduced the extent of the no-reflow zone for any given risk zone size, suggesting that it helped prevent microvascular obstruction.

Inhibition of mitochondrial permeability transition pore opening contributes to the neuroprotective effects of ischemic postconditioning in rats

Ischemic postconditioning (IPost) has been shown to attenuate cerebral ischemia–reperfusion injury. However, the mechanism remains elusive. Because opening of the mitochondrial permeability transition pore (MPTP) is a crucial determinant of cell death after ischemia–reperfusion, we hypothesized that the neuroprotective effect of IPost may be associated with inhibition of MPTP opening. In part 1 of this study, pentobarbital-anesthetized rats subjected to middle cerebral artery occlusion for 90min, followed by reperfusion for 72h, were assigned to receive one of the following treatments: three cycles of IPost (15s each), intracerebroventricular injection of saline (control), administration of the MPTP inhibitor cyclosporin A (CsA) (2μmol/L, 15μL) or its vehicle alcohol, administration of the MPTP opener atractyloside (Atr) (2mmol/L, 15μL), or IPost plus CsA/Atr treatment. Neurological deficit scores (NDS) and infarct volumes were assessed. Mitochondrial ultrastructure and swelling were also examined after reperfusion. In part 2, control and IPost groups underwent ischemia (90min) and reperfusion (15min). CsA and Atr groups were treated as described in part 1. Brain mitochondria were isolated after reperfusion and MPTP activity was evaluated. IPost or CsA treatment significantly improved NDS and reduced infarction volume, while Atr reversed the neuroprotective effects of IPost, and attenuated the decrease in mitochondrial swelling induced by IPost or CsA. Thus, inhibiting MPTP opening may play a crucial role in the neuroprotective effects of IPost, which may have potential clinical value against cerebral ischemia–reperfusion injury.

16 Matrix metalloproteinase inhibition is a parallel pathway to protection against reperfusion injury, both independent and additive to mitochondrial permeability transition pore inhibition

While matrix-metalloproteinase (MMP) inhibition protects against myocardial ischaemia/reperfusion injury, the mechanisms are poorly understood. We hypothesised that this cardioprotection is independent of mitochondrial permeability transition pore (mPTP) inhibition, the end-effector...

State of the Science of Cardioprotection: Challenges and Opportunities— Proceedings of the 2010 NHLBI Workshop on Cardioprotection

The National Heart, Lung, and Blood Institute convened a Workshop on September 20-21, 2010, "New Horizons in Cardioprotection," to identify future research directions for cardioprotection against ischemia and reperfusion injury. Since the early 1970s, there has been evidence that the size of a myocardial infarction could be altered by various interventions. Early coronary artery reperfusion has been an intervention that consistently reduces myocardial infarct size in animal models as well as humans. Most cardiologists agree that the best way to treat acute ST-segment elevation myocardial infarction is to reperfuse the infarct artery as soon as possible and to keep the infarct artery patent. In general, stenting is superior to angioplasty, which is superior to thrombolysis. There is no accepted adjunctive therapy to acutely limit myocardial infarct size along with reperfusion that is routinely used in clinical practice. In the Kloner experimental laboratory, some adjunctive therapies have reproducibly limited infarct size (regional hypothermia, preconditioning, cariporide, combinations of the above, remote preconditioning, certain adenosine agonists, and late sodium current blockade). In clinical trials, a host of pharmacologic adjunctive therapies have failed to either reduce infarct size or improve clinical outcome. Potential reasons for the failure of these trials are discussed. However, some adjunctive therapies have shown promise in data subanalyses or subpopulations of clinical trials (adenosine, therapeutic hypothermia, and hyperoxemic reperfusion) or in small clinical trials (atrial natriuretic peptide, ischemic postconditioning, and cyclosporine, the mitochondrial permeability transition pore inhibitor). A recent clinical trial with remote conditioning induced by repetitive inflation of a brachial artery cuff begun prior to hospitalization showed promise in improving myocardial salvage and there are several reports in the cardiothoracic literature, suggesting that remote preconditioning protects hearts during surgery. Thus, in 2011, there is hope that applying some of the body's own conditioning mechanisms may provide protection against ischemic damage.

Post-Injury Administration of the Mitochondrial Permeability Transition Pore Inhibitor, NIM811, Is Neuroprotective and Improves Cognition after Traumatic Brain Injury in Rats

Mitochondrial dysfunction is known to play a pivotal role in cell death mechanisms following traumatic brain injury (TBI). N-methyl-4-isoleucine-cyclosporin (NIM811), a non-immunosuppressive cyclosporin A (CsA) analog, inhibits the mitochondrial permeability transition pore (mPTP) and has been shown to be neuroprotective following TBI in mice. However, the translation of the neuroprotective effects of mPTP inhibitors, including CsA and NIM811, into improved cognitive end points has yet to be fully investigated. Therefore, to build upon these results, a severe unilateral controlled cortical impact model of TBI was used in the present study to establish a dose-response curve for NIM811 in rats. The findings demonstrate that the neuroprotection afforded by NIM811 is dose dependent, with the 10 mg/kg dose being the most effective dose. Once the dose response was established, we evaluated the effect of the optimal dose of NIM811 on behavior, mitochondrial bioenergetics, and mitochondrial oxidative damage following TBI. For behavioral studies, rats were administered NIM811 at 15 min and 24 h post-injury, with cognitive testing beginning 10 days post-injury. Mitochondrial studies involved a single injection of NIM811 at 15 min post-injury followed by mitochondrial isolation at 6 h post-injury. The results revealed that the optimal dose of NIM811 improves cognition, improves mitochondrial functioning, and reduces oxidative damage following TBI.

Effects of FK506 and cyclosporin a on calcium ionophore‐induced mitochondrial depolarization and cytosolic calcium in astrocytes and neurons

This study tested the hypothesis that sensitivity to the Ca(2+) -induced loss of mitochondrial membrane potential (ΔΨ(m)) and the sensitivity of the loss of ΔΨ to mitochondrial permeability transition pore (PTP) inhibitors are different for neurons and astrocytes. Primary cultures of rat cortical neurons and astrocytes were exposed to the Ca(2+) ionophore 4-Br-A23187, and ΔΨ(m) was monitored with the fluorescent probe tetramethylrhodamine methyl ester (TMRM). Ca(2+) ionophore caused a decline in ΔΨ(m) in both cell types that was partially inhibited by cyclosporin A (CsA) in astrocytes but not in neurons. Another PTP inhibitor, 2-aminoethoxy-diphenylborate, was ineffective at protecting against mitochondrial depolarization, but depolarization was inhibited by FK506, an immunosuppressant drug similar to CsA that does not inhibit the PTP. CsA and FK506 both significantly reduced the ionophore-induced rise in [Ca(2+) ](i) in both neurons and astrocytes. We conclude that the protective effects of CsA and FK506 against Ca(2+) ionophore-induced mitochondrial membrane depolarization in intact astrocytes is not due to PTP inhibition but possibly is a consequence of inhibiting the rise in [Ca(2+) ](i).

Mitochondrial Pathway Mediates the Antileukemic Effects of Hemidesmus Indicus, a Promising Botanical Drug

BackgroundAlthough cancers are characterized by the deregulation of multiple signalling pathways, most current anticancer therapies involve the modulation of a single target. Because of the enormous biological diversity of cancer, strategic combination of agents targeted against the most critical of those alterations is needed. Due to their complex nature, plant products interact with numerous targets and influence several biochemical and molecular cascades. The interest in further development of botanical drugs has been increasing steadily and the FDA recently approved the first new botanical prescription drug. The present study is designed to explore the potential antileukemic properties of Hemidesmus indicus with a view to contributing to further development of botanical drugs. Hemidesmus was submitted to an extensive in vitro preclinical evaluation.Methodology/Principal FindingsA variety of cellular assays and flow cytometry, as well as a phytochemical screening, were performed on different leukemic cell lines. We have demonstrated that Hemidesmus modulated many components of intracellular signaling pathways involved in cell viability and proliferation and altered the protein expression, eventually leading to tumor cell death, mediated by a loss of mitochondrial transmembrane potential and increased Bax/Bcl-2 ratio. ADP, adenine nucleotide translocator and mitochondrial permeability transition pore inhibitors did not reverse Hemidesmus-induced mitochondrial depolarization. Hemidesmus induced a significant [Ca2+]i raise through the mobilization of intracellular Ca2+ stores. Moreover, Hemidesmus significantly enhanced the antitumor activity of three commonly used chemotherapeutic drugs (methotrexate, 6-thioguanine, cytarabine). A clinically relevant observation is that its cytotoxic activity was also recorded in primary cells from acute myeloid leukemic patients.Conclusions/SignificanceThese results indicate the molecular basis of the antileukemic effects of Hemidesmus and identify the mitochondrial pathways and [Ca2+]i as crucial actors in its anticancer activity. On these bases, we conclude that Hemidesmus can represent a valuable tool in the anticancer pharmacology, and should be considered for further investigations.

Інгібітор мітохондріального Na+-Ca2+-обмінника CGP37157 деполяризує ендотеліальні клітини та викликає осциляції мембранного потенціалу

We explored the effect of inhibitor of mitochondrial Na(+)-Ca(2+)-exchanger CGP37157 and the effect of mitochondria permeability transition pore inhibitor, cyclosporine A, on the membrane potential and electrical responses to endothelium-dependent dilators in intact endothelial cells from excised rat aorta and EA.hy 926 endothelial cells. Cyclosporin A did not affect the resting membrane potential and hyperpolarization to acetylcholine and histamine in intact and cultured cells. In contrast, CGP37157 (20 mcM) evoked membrane depolarization in unstimulated cells and suppressed the sustained component of hyperpolarization to acetylcholine and histamine both in intact and cultured endothelial cells, respectively. This was accompanied by a pronounced depolarization with membrane potential oscillations, which were not observed in the absence of Ca2+. We conclude that CGP37157 modulates endothelial membrane potential at rest and electrical responses to endothelium-dependent dilators. Possible mechanisms accompanying the CGP37157 action are discussed.

Protective Effects of Cyclo(L-Leu-L-Tyr) against Postischemic Myocardial Dysfunction in Guinea-Pig Hearts

The protective effects of cyclic dipeptides in alcoholic beverages were investigated in the perfused guinea-pig hearts subjected to ischemia and reperfusion. Subsequently, in order to determine the importance of cyclic dipeptide structure, the effects of cyclo(L-Leu-L-Tyr) (cLY) were compared with those of the newly synthesized non-cyclic dipeptides, L-Leu-L-Tyr (LY) and L-Tyr-L-Leu (YL). After reperfusion, pressure recovery (%) in the left ventricle reached a peak of over 90% in the presence of cLY (10(-6) M and 10(-5) M) (control: 22.9%). The recovery by LY and YL was significantly lower than that by cLY, and ATP levels simultaneously monitored using (31)P-NMR were already lower during the ischemic end period than those observed with cLY treatment. In perfused mitochondrial preparations, cLY significantly inhibited mitochondrial Ca(2+) ([Ca(2+)](m)) elevation in a similar way to that of the mitochondrial permeability transition pore (MPTP) inhibitor cyclosporin A. In vitro electron paramagnetic resonance (EPR) revealed that the active oxygen radicals quenching activity of cLY was greater than those of non-cyclic dipeptides. cLY inhibited caspase-3-induced apoptosis. The cyclic dipeptide structure inhibits opening of the MPTP by preventing [Ca(2+)](m) overload-induced apoptosis related to mitochondrial active oxygen radical accumulation in ischemia-reperfusion hearts.

The MPTP Status During Early Reoxygenation is Critical for Cardioprotection

Previous studies have revealed that the mitochondrial permeability transition pore (MPTP) plays a critical role in necrotic and apoptotic cell death. Given the opposed roles of the MPTP in cardioprotection (transient versus sustained opening) the primary aim of this study was to determine how two structurally different MPTP inhibitors (cyclosporine A and bongkrekic acid) administered for varying time regimes influenced ischemia/reperfusion (I/R)-induced injury in myocardial slices from rat left ventricle. A second objective was to explore how pharmacologic MPTP opening (using atractyloside) at different time points during I/R modulated myocardial injury. Myocardial slices from rat left ventricle were subjected to 90 min ischemia/120min reoxygenation at 37°C. MPTP inhibitors and openers were added at various time points during the experimental regime. Tissue injury was assessed by creatine kinase (CK) released and determination of cell necrosis and apoptosis. Myocardial caspase 3 activity was also determined. The results show that the status of MPTP can dramatically influence ischemic/reoxygenation induced injury and protection of the rat left ventricular myocardium. Importantly, the status of the MPTP during first 10 min of reoxygenation is of critical importance with both opening and closing of the pore being as protective as ischemic preconditioning. The present study has shown that both formation and inhibition of the MPTP can be exploited for therapeutic purposes and that there is a defined therapeutic window, with the first few minutes of reoxygenation being a crucial period to achieve cardioprotection.

Inhibition of permeability transition pore opening by mitochondrial STAT3 and its role in myocardial ischemia/reperfusion

The signal transducer and activator of transcription 3 (STAT3) contributes to cardioprotection by ischemic pre- and postconditioning. Mitochondria are central elements of cardioprotective signaling, most likely by delaying mitochondrial permeability transition pore (MPTP) opening, and STAT3 has recently been identified in mitochondria. We now characterized the mitochondrial localization of STAT3 and its impact on respiration and MPTP opening. STAT3 was mainly present in the matrix of subsarcolemmal and interfibrillar cardiomyocyte mitochondria. STAT1, but not STAT5 was also detected in mitochondria under physiological conditions. ADP-stimulated respiration was reduced in mitochondria from mice with a cardiomyocyte-specific deletion of STAT3 (STAT3-KO) versus wildtypes and in rat mitochondria treated with the STAT3 inhibitor Stattic (STAT3 inhibitory compound, 6-Nitrobenzo[b]thiophene 1,1-dioxide). Mitochondria from STAT3-KO mice and Stattic-treated rat mitochondria tolerated less calcium until MPTP opening occurred. STAT3 co-immunoprecipitated with cyclophilin D, the target of the cardioprotective agent and MPTP inhibitor cyclosporine A (CsA). However, CsA reduced infarct size to a similar extent in wildtype and STAT3-KO mice in vivo. Thus, STAT3 possibly contributes to cardioprotection by stimulation of respiration and inhibition of MPTP opening.

Mechanistic study of mitochondria-dependent programmed cell death induced by aluminium phytotoxicity using fluorescence techniques

Recent studies have suggested that aluminium (Al) induces programmed cell death (PCD) in plants. To investigate possible mechanisms, fluorescence techniques were used to monitor the behaviour of mitochondria in vivo, as well as the activation of caspase-3-like activity during protoplast PCD induced by Al. A quick burst of mitochondrial reactive oxygen species (ROS) was detected in Al-treated protoplasts. The mitochondrial swelling and mitochondrial transmembrane potential (MTP) loss occurred prior to cell death. Pre-incubation with ascorbic acid (AsA, antioxidant molecule) retarded mitochondrial swelling and MTP loss. The real-time detection of caspase-3-like activation was achieved by measuring the degree of fluorescence resonance energy transfer (FRET). At 30 min after exposure to Al, caspase-3-like protease activation, indicated by the decrease in the FRET ratio, occurred, taking about 1 h to reach completion in single living protoplasts. The mitochondrial permeability transition pore (MPTP) inhibitor, cyclosporine (CsA) gave significant protection against MTP loss and subsequent caspase-3-like activation. Our data also showed that Al-induced mitochondrial ROS possibly originated from complex I and III damage in the respiratory chain through the interaction between Al and iron-sulphur (Fe-S) protein. Alternative oxidase (AOX), the unique respiratory terminal oxidase in plants, was demonstrated to play protective roles in Al-induced protoplast death. Our results showed that mitochondrial swelling and MTP loss, as well as the generation of mitochondrial ROS play important roles in Al-induced caspase-3-like activation and PCD, which provided new insight into the signalling cascades that modulate Al phytotoxicity mechanism.