The mitochondrial permeability transition pore and ischemia-reperfusion injury

Mitochondrial dysfunction is an underlying cause of ischemia-reperfusion injury. In particular, ischemic injury induces dramatic increases in mitochondrial permeability, thereby instigating a chain of events that leads to both apoptotic and necrotic cardiomyocyte death. The mitochondrial permeability transition (MPT) pore, a large, non-specific channel that spans the inner mitochondrial membrane, is known to mediate the lethal permeability changes that initiate mitochondrial-driven cardiomyocyte death. The purpose of this review is to focus on the role of the MPT pore in ischemia-reperfusion injury, the mechanisms involved, and, in particular, what we do and do not know regarding the pore’s molecular composition.     

Adenine nucleotide translocase-1 induces cardiomyocyte death through upregulation of the pro-apoptotic protein Bax

Overexpression of the adenine nucleotide translocase (ANT) has been shown to be cytotoxic in several cell types. Although ANT was originally proposed to be a critical component of the mitochondrial permeability transition (MPT) pore, recent data have suggested that this may not be the case. We therefore hypothesized that the cytotoxic actions of ANT are through an alternative mechanism, independent of the MPT pore. Infection of cultured neonatal cardiomyocytes with an ANT1-encoding adenovirus induced a gene dosage-dependent increase in cell death. However, ANT1 overexpression failed to induce MPT, and neither pharmacological nor genetic inhibition of the MPT pore was able to prevent ANT1-induced cell death. These data suggested that ANT1-induced death progressed through an MPT pore-independent pathway. Somewhat surprisingly, we observed that protein levels of Bax, a pro-apoptotic Bcl protein, were consistently elevated in ANT1-infected cardiomyocytes. Membranes isolated from ANT1-infected myocytes exhibited significantly increased amounts of membrane-inserted Bax, and immunocytochemistry revealed increased Bax activation in ANT1-infected myocytes. Co-expression with the Bax antagonist Bcl2 was able to greatly reduce the degree of ANT1-induced cell death. Furthermore, Bax/Bak-deficient fibroblasts were resistant to the cytotoxic effects of ANT1 overexpression. Interestingly, ANT1 overexpression was also associated with enhanced production of reactive oxygen species (ROS), and the antioxidant MnTBAP was able to significantly attenuate both the ANT1-induced upregulation of Bax and cell death. Taken together, these data indicate that ANT mediates cell death, not through the MPT pore, but rather via a ROS-dependent upregulation and activation of Bax.     

Cardiac mitochondrial alterations observed in hyperglycaemic rats--what can we learn from cell biology?

Diabetes mellitus is one of the most common metabolic diseases in the world. The complications associated with this disease are often responsible for a decreased quality of life in many patients. For example, the diabetic population has a greater probability to suffer from cardiovascular problems and heart failure than the general population. Due to the importance heart mitochondria have in the context of the bioenergetics of the myocardium, it appears logical to explore mitochondrial dysfunction as an important link between hyperglycaemia and heart alterations observed during diabetes. One important factor that can lead to mitochondrial dysfunction is the mitochondrial permeability transition (MPT), caused by the formation of poly-protein pores (MPT pores), occurring with mitochondrial calcium overload and increased oxidative stress, conditions already described to exist in myocytes exposed to hyperglycaemia. The MPT has been involved as determinant in the survival of myocytes after anoxia and reoxigenation, as well as in triggering cell death. The present review deals with cardiac mitochondrial alterations observed in drug-induced hyperglycaemic animals or in the GK rat, a hereditary model of hyperglycaemia. Respiration rates, susceptibility to oxidative stress, protein expression and MPT induction are altered in hyperglycaemic animals, which in extreme conditions can alter the bioenergetics of the diabetic myocardium and even cause myocardial cell death. The study of the cardiac mitochondrial function of hyperglycaemic animals offer an important insight, not only to explain cardiac alterations found in diabetic patients, but also in the design of new therapeutic approaches to reduce mitochondrial dysfunction and cell death typically associated with diabetes.     

The Mitochondrial Permeability Transition: Role in Ischemia/Reperfusion Injury

The mitochondrial permeability transition (MPT) occurs when a non-specific pore opens in the inner mitochondrial membrane and converts the mitochondrion from an organelle whose ATP production sustains the normal function of the cell to an instrument of death. Conditions favouring the MPT including high [Ca2+], oxidative stress and adenine nucleotide depletion, all of which occur when a tissue is reperfused following a period of ischemia. Cyclosporin A (CsA) and low pH (<7.0) are potent inhibitors of the MPT. Methods have been devised to demonstrate directly that the MPT pores open upon reperfusion but not during ischemia. The mechanism of the MPT appears to involve binding of mitochondrial cyclophilin (CyP) to the adenine nucleotide translocase (ANT) followed by a calcium-mediated conformational change that converts the ANT into a non-specific pore. Understanding the molecular mechanism has assisted in devising strategies that can be used to protect tissues from damage caused by reperfusion injury. These might also be of benefit in the prevention of multiple organ failure for which reperfusion injury of the gut is thought to be the initial trigger. Protective regimes include the pretreatment of tissues prior to ischemia/reperfusion with CsA (binds to CyP), free radical scavengers that reduce oxidative stress (e.g., pyruvate and propofol) and agents that decrease pHi (e.g., pyruvate or amelioride derivatives). Reperfusion injury can produce both immediate cell death by necrosis or delayed apoptotic cell death and it appears that the mitochondria determine which route is taken. Prolonged opening leads to rapid cell death by necrosis, whilst transient opening leads to cytochrome c release and subsequent apoptosis hours or days later.     

Carvedilol protects ischemic cardiac mitochondria by preventing oxidative stress.

Ischemia negatively affects mitochondrial function by inducing the mitochondrial permeability transition (MPT). The MPT is triggered by oxidative stress, which occurs in mitochondria during ischemia as a result of diminished antioxidant defenses and increased reactive oxygen species production. It causes mitochondrial dysfunction and can ultimately lead to cell death. Therefore, drugs able to minimize mitochondrial damage induced by ischemia may prove to be clinically effective. We analyzed the effect of carvedilol, a beta-blocker with antioxidant properties, on mitochondrial dysfunction. Carvedilol decreased levels of TBARS (thiobarbituric acid reactive substances), an indicator of oxidative stress, which is consistent with its antioxidant properties. Regarding cell death by apoptosis, although ischemia did increase caspase-8-like activity, there were no changes in caspase-3-like activity, which is activated downstream of caspase-8; this may indicate that the apoptotic cascade is not activated by 60 minutes of ischemia. We conclude that carvedilol protects ischemic mitochondria by preventing oxidative mitochondrial damage, and, by so doing, it may also inhibit the formation of the MPT pore.     

NHE-1 inhibition improves impaired mitochondrial permeability transition and respiratory function during postinfarction remodelling in the rat

The mechanism by which Na+-H+ exchange (NHE) inhibition results in attenuation and reversal of postinfarction remodelling and heart failure remains controversial. In this study, we investigated the possible contribution of mitochondrial involvement by determining the effect of the NHE-1-specific inhibitor EMD-87580 (EMD) on mitochondrial permeability transition (MPT) and respiratory function during the postinfarction remodelling process. Male Sprague-Dawley rats were subjected to either 12 or 18 weeks of coronary artery ligation (CAL) or sham procedure. EMD was provided in the diet immediately after ligation. MPT pore opening was determined by perfusing hearts with 2-deoxy-[3H]-glucose ([3H]-DOG) and measurement of mitochondrial [3H]-DOG entrapment. The respiratory function of isolated mitochondria was measured using Clark-type oxygen electrode. Remodelling was associated with significant hypertrophy and there was an increase in MPT pore opening in hearts both 12 and 18 weeks following CAL. Mitochondrial respiratory function, especially state 2 and state 3 rates were significantly decreased in hearts subjected to CAL. EMD suppressed MPT pore opening by 40% (P <0.01) and 35% (P <0.01) 12 and 18 weeks after ligation, respectively. Mitochondria isolated from EMD treated hearts exhibited increased respiratory chain activity for oxidation of substrates at complex I and II. These beneficial effects of EMD were associated with decreased mitochondrial vulnerability to exogenous Ca2+. We conclude that NHE-1 inhibition has a protective effect on mitochondrial function, attenuating MPT pore opening and improving the respiratory function, which may contribute to the salutary effect of NHE-1 inhibitors in heart failure.     

Carvedilol: relation between antioxidant activity and inhibition of the mitochondrial permeability transition.

OBJECTIVES: The mitochondrial permeability transition (MPT) is an event related to severe oxidative stress (for example, during myocardial ischemia and reperfusion) and excessive mitochondrial calcium accumulation, also being implicated in cell death. In this study, we compared the effect of carvedilol on the cardiac MPT induced by calcium and phosphate (Ca/Pi) and calcium/carboxyatractyloside (Ca/Catr). Oxidative stress plays a major role in MPT induction by Ca/Pi, leading to the oxidation of protein thiol groups, in contrast with Ca/Catr, where such oxidation is secondary to MPT induction and is not caused by oxidative stress. MATERIALS AND METHODS: Mitochondria were isolated from rat hearts and parameters related to MPT induction were evaluated (n = 5 for each inducer): mitochondrial swelling and oxidation of protein thiol groups (both measured by spectrophotometry). RESULTS: Using Ca/Pi, carvedilol protected mitochondria from MPT induction, particularly in its high conductance form. Its effect was demonstrated by analyzing the decrease in mitochondrial swelling amplitude. Simultaneously, we observed inhibition of protein thiol group oxidation (p < 0.001). By contrast, carvedilol did not show any protective effect with Ca/Catr. CONCLUSIONS: Carvedilol was only effective against the MPT when the oxidation of protein thiol groups was the cause and not the consequence of the MPT phenomenon. The results clearly show that during myocardial aggressions (ischemia and reperfusion, for example), the protective effect of carvedilol is primarily due to an antioxidant mechanism, inhibiting the production and effects of reactive oxygen species.     

Specific inhibition of the mitochondrial permeability transition prevents lethal reperfusion injury

The aim of the present study was to determine whether specific inhibition of mitochondrial permeability transition (MPT) by NIM811 at the time of reperfusion following acute myocardial infarction may protect the heart. MPT pore opening appears to be a pivotal event in cell death following acute myocardial infarction. Recently, MPT pore opening has been involved in ischemic preconditioning. In protocol 1, NZW rabbits underwent either no intervention (sham) or 10 min of ischemia followed by 5 min of reperfusion, preceded (preconditioned, PC) or not (control, C) by 5 min of ischemia and 5 min of reperfusion. Additional rabbits were treated by cyclosporin A (CsA) or its non-immunosuppressive and more specific derivative (NIM811) (10 mg kg(-1), IV bolus), either 10 min before ischemia or 1 min before reperfusion. Hearts were excised and mitochondria isolated for further assessment of Ca(2+)-induced MPT. In protocol 2, animals were randomly assigned into similar experimental groups and underwent 30 min of ischemia and 4 h of reperfusion. Infarct size was assessed by TTC staining, and apoptosis by TUNEL assay. The Ca2+ overload required to induce MPT pore opening was significantly higher in NIM811, CsA and PC groups than in controls. Both necrotic and apoptotic cardiomyocyte death were significantly reduced by NIM811, CsA and PC. In both protocols, administration of NIM811 at reperfusion provided full protection. These data indicate that specific inhibition of MPT pore opening at reperfusion following acute myocardial infarction provides a powerful antinecrotic and antiapoptotic protection.     

Advantages in the use of carvedilol versus propranolol for the protection of cardiac mitochondrial function.

BACKGROUND: Carvedilol is a neurohormonal antagonist of multiple action which is used in clinical practice for the treatment of congestive heart failure, mild to moderate hypertension and myocardial infarction. Previous results from our group have demonstrated that one of the main targets for the protective effect of carvedilol is the cardiac mitochondrial network. In-this work, we compare the effect of carvedilol with propranolol in different models of mitochondrial dysfunction and in the generation of transmembrane electric potential (EP). We further tested if carvedilol was able to inhibit the mitochondrial permeability transition (MPT) induced by doxorubicin and calcium-dependent cytochrome c release, a phenomenon frequently associated with apoptotic cell death. METHODS: Cardiac mitochondria were isolated by differential centrifugation. Oxygen consumption and mitochondrial EP were determined using an oxygen electrode and a tetraphenylphosphonium-sensitive electrode, respectively. Changes in mitochondrial volume and the release of cytochrome c were measured with spectrophotometric techniques. RESULTS: Propranolol, compared with carvedilol, had only a marginal effect, not only in protection against MPT induction, but also against oxygen consumption linked to the oxidation of external NADH, a process that is considered by several authors as key in the cardiotoxicity of doxorubicin. Regarding EP generation, propranolol had no effect, in contrast to carvedilol, which was confirmed to act as a protonophore. For the first time we also show that carvedilol inhibits the MPT induced by doxorubicin and calcium-dependent cytochrome c release. CONCLUSIONS: With this work, we further support the notion that carvedilol is effective in several models of mitochondrial dysfunction, particularly those involving oxidative stress. The results demonstrate that for some pathological conditions, carvedilol and propranolol have different mechanisms of action at the sub-cellular level, as propranolol seems to lack effectiveness in the protection of cardiac mitochondria.     

Ethanol potentiates tumor necrosis factor-alpha cytotoxicity in hepatoma cells and primary rat hepatocytes by promoting induction of the mitochondrial permeability transition

In the present study, tumor necrosis factor-alpha (TNF-alpha) cytotoxicity is shown to be potentiated by ethanol exposure in vitro in the human hepatoma cell line, HepG2, and in rat primary hepatocytes. Exposure of HepG2 cells and primary hepatocytes for 48 hours to concentrations of ethanol ranging between 50 and 100 mmol/L significantly increased TNF-alpha cytotoxicity compared with cells treated with TNF-alpha alone. The cell killing was associated with, and dependent on, the development of the mitochondrial permeability transition (MPT). Two inhibitors of MPT pore opening, cyclosporin A and bongkrekic acid, prevented TNF-alpha cytotoxicity in the presence of ethanol. In addition to inhibiting cell death caused by TNF-alpha, blockade of MPT pore opening prevented mitochondrial depolarization, cytochrome c redistribution from the mitochondria to the cytosol, caspase 3 activation, and oligonucleosomal DNA fragmentation. Unlike the potentiation of TNF-alpha cytotoxicity by the translational inhibitor cycloheximide, ethanol promoted TNF-alpha-induced cell killing by a mechanism that was independent of caspase-8 activity. HepG2 cells overexpressing cytochrome-P4502E1 were even more sensitized by ethanol to induction of the MPT by TNF-alpha and the resultant cytotoxicity than wild-type HepG2 cells. In addition, primary hepatocytes isolated from chronically ethanol-fed rats showed enhanced susceptibility to TNF-alpha cytotoxicity compared with their isocalorically matched controls. Again as with the HepG2 cells, inhibiting MPT pore opening prevented the cytotoxicity of TNF-alpha in the primary hepatocytes isolated from ethanol-fed animals.     

Critical role of reactive oxygen species and mitochondrial permeability transition in microcystin-induced rapid apoptosis in rat hepatocytes

Microcystin-LR (M-LR) is a specific hepatotoxin. At present, the exact toxic mechanism of its action remains unclear though apoptosis is believed to be involved. This study was designed to investigate the role of reactive oxygen species (ROS) and mitochondrial permeability transition (MPT) in the M-LR-induced apoptotic process. Morphologic changes such as cell shrinkage, externalization of cell membrane phosphatidylserine, DNA fragmentation, and nuclear condensation suggest that M-LR causes rapid apoptosis in hepatocytes. Confocal microscopy revealed that M-LR exposure led to the onset of MPT and mitochondrial depolarization, evidenced by (1) redistribution of calcein fluorescence from cytosol to mitochondria, and (2) loss of mitochondrial tetramethyrhodamine methyl ester (TMRM) fluorescence; both occurred before apoptosis. Moreover, there was a significant and rapid increase of ROS level before the onset of MPT and loss of MMP, indicating a critical role of ROS in M-LR-induced apoptosis. Deferoxamine (DFO), an iron chelator, prevented the increase of ROS production, delayed the onset of MPT, and, subsequently, cell death. In addition, a specific MPT inhibitor, cyclosporin A (CsA), blocked the M-LR-induced ROS formation, onset of MPT, and mitochondrial depolarization as well as cell death. Thus, we conclude that the M-LR-induced ROS formation leads to the onset of MPT and apoptosis.     

Melatonin preserves the transient mitochondrial permeability transition for protection during mitochondrial Ca(2+) stress in astrocyte

Cells have two modes of mitochondrial permeability transition (MPT) which produce virtually opposite pathophysiological outcomes of survival or death when responding to apoptotic insults. The transient-MPT (t-MPT) protects mitochondria, whereas the prolonged-MPT (p-MPT), once activated, triggers the 'point of no return' for apoptosis or necrosis. Our previous studies show that in addition to scavenging mitochondrial reactive oxygen species, melatonin targets mitochondrial Ca(2+) (mCa(2+))-mediated MPT for protection during mCa(2+)-mediated apoptosis in astrocytes. The precise mechanism for how melatonin modulates the MPT during mCa(2+) stress, however, remains unelucidated. With the application of fluorescence laser scanning imaging microscopy, this study demonstrated for the first time that melatonin does not inhibit the MPT pore, rather it crucially preserves the pore in its protective mode of t-MPT during mCa(2+) stress. Melatonin-preserved t-MPT importantly maintained mitochondrial membrane potential (ΔΨ(m)) which not only prevented depolarized ΔΨ(m)-induced p-MPT but also retained ΔΨ(m)-dependent ATP formation during disturbed Ca(2+) homeostasis. Additionally, the melatonin-preserved t-MPT allowed mitochondria to release the toxic overload of mCa(2+) to sublethal levels, which prevented mCa(2+)-mediated fission and mCa(2+)-dependent p-MPT and possibly also improved mCa(2+)-dependent ATP synthesis. Melatonin's effect in reducing the Ca(2+) load greatly diminished when the MPT was inhibited by cyclosporine A, suggesting its pore dependency as well as that a preserved t-MPT may be superior to a MPT inhibition in protecting mCa(2+)-mediated apoptosis. The unique modulation on the MPT provided by melatonin may have extraordinary therapeutic potential in the treatment of mCa(2+)-mediated astrocyte-associated neurodegenerative pathologies and diseases.     

Apoptosis of ventricular myocytes: a means to an end

One of the most compelling issues to impact on contemporary cardiology is arguably the phenomenon of programmed cell death or apoptosis. Studies in the nematode Caenorhabditis elegans provided the first indication that determinants of cell fate crucial for normal worm development were under genetic influences of the ced-3 and ced-9 genes, which promote or prevent cell death, respectively. Extrapolation of these seminal findings led to the discovery of the mammalian ced-3 and ced-9 homologs, which broadly encompass a family of cellular cysteine proteases known collectively as caspases and the Bcl-2 proteins. In quiescent cells, caspases exist as inactive zymogens that are readily activated by autocatalytic processes or by other caspases following a death signal. The caspase-dependent cleavage of intracellular substrates results in the biochemical dismantling of the cell and morphological features characteristic of apoptosis. Recently, a mitochondrial death pathway for apoptosis has been proposed. Perturbations to mitochondria resulting in the loss of mitochondrial membrane potential, DeltaPsim, permeability transition pore (PTP) opening and the release of pro-apoptotic factors by mitochondria including cytochrome c, second mitochondrial activator of caspases/direct IAP binding protein with low pI (Smac/DIABLO), AIF, and others are considered terminal events in the apoptotic pathway. Bcl-2 and related family members are characterized by their ability to promote or prevent cell death. These proteins exert their pro- or anti-apoptosis function by impinging on components of the cell death pathway that underlie caspase activation, mitochondrial dysfunction or both. The limited regenerative potential of the adult cardiac muscle itself, together with the heightened and exciting possibility of regenerating cardiac muscle with cardiac progenitor cells, acknowledges the need for new strategies to suppress and/or prevent inappropriate cardiac cell death in patients with ischemic heart disease or heart failure patients as a therapeutic means of preserving cardiac pump function after injury.     

Mitochondrial complex I and NAD(P)H oxidase are major sources of exacerbated oxidative stress in pressure-overloaded ischemic-reperfused hearts

We tested the hypothesis that pressure overload exacerbates oxidative stress associated with augmented mitochondrial permeability transition (MPT) pore opening and cell death in ischemic-reperfused hearts. Pressure overload decreased the level of reduced glutathione but increased nitrotyrosine and 8-hydroxydeoxyguanosine levels in ischemic-reperfused hearts. The activity of catalase, but not superoxide dismutase (SOD), was lower in ischemic-reperfused hearts perfused at higher pressure. Mitochondria from ischemic-reperfused hearts subjected to higher perfusion pressure displayed significantly greater [³H]-2-deoxyglucose-6-P entrapment suggestive of greater MPT pore opening and consistent with greater necrosis and apoptosis. Tempol (SOD mimetic) reduced infarct size in both groups but it remained greater in the higher pressure group. By contrast, uric acid (peroxynitrite scavenger) markedly reduced infarct size at higher pressure, effectively eliminating the differential between the two groups. Inhibition of xanthine oxidase, with allopurinol, reduced infarct size but did not eliminate the differential between the two groups. However, amobarbital (inhibitor of mitochondrial complex I) or apocynin [inhibitor of NAD(P)H oxidase] reduced infarct size at both pressures and also abrogated the differential between the two groups. Consistent with the effect of apocynin, pressure-overloaded hearts displayed significantly higher NAD(P)H oxidase activity. Furthermore, pressure-overloaded hearts displayed increased nitric oxide synthase activity which, along with increased propensity to superoxide generation, may underlie uric acid-induced cardioprotection. In conclusion, increased oxidative and nitrosative stress, coupled with lack of augmented SOD and catalase activities, contributes importantly to the exacerbating impact of pressure overload on MPT pore opening and cell death in ischemic-reperfused hearts.     

Contribution of increased mitochondrial free Ca2+ to the mitochondrial permeability transition induced by tert-butylhydroperoxide in rat hepatocytes

Previously, we showed that the oxidant chemical, tert-butylhydroperoxide (t-BuOOH), induces a mitochondrial permeability transition (MPT) in intact hepatocytes, causing lethal cell injury. Here, we investigated the role of mitochondrial free Ca2+ in t-BuOOH cytotoxicity to 1-day-cultured rat hepatocytes using confocal microscopy of autofluorescence and parameter-indicating fluorophores. t-BuOOH (100 micromol/L) caused an early increase of mitochondrial free Ca2+, as assessed by confocal microscopy of Rhod-2 fluorescence. Increased mitochondrial Ca2+ was followed by onset of the MPT, as evidenced by permeation of cytosolic calcein into mitochondria and loss of the mitochondrial membrane potential-indicating dye, tetramethylrhodamine methylester. Preincubation with an intracellular Ca2+ chelator (BAPTA-AM and its derivatives) partially blocked the late phase of mitochondrial NAD(P)H oxidation after t-BuOOH, but failed to prevent the early oxidation of mitochondrial NAD(P)H. Ca2+ chelation also prevented the increase of mitochondrial Ca2+, generation of mitochondrial reactive oxygen species (ROS), onset of the MPT, and subsequent cell death. Confocal images showed that protection occurred when loading of the Ca2+ chelator was predominantly mitochondrial. The antioxidant, desferal, also diminished increased mitochondrial Ca2+ after t-BuOOH and prevented cell death. We conclude that oxidative stress induced by t-BuOOH enhances mitochondrial Ca2+ uptake, leading to increased matrix Ca2+, increased ROS formation, onset of the MPT, and cell death.     

Marked differences between atrial and ventricular gene-expression remodeling in dogs with experimental heart failure

Under conditions of mitochondrial calcium overload, especially when accompanied by oxidative stress, elevated phosphate concentrations and adenine nucleotide depletion, a non-specific pore, the mitochondrial permeability transition pore (MPTP), opens in the inner mitochondrial membrane. MPTP opening enables free passage into the mitochondria of molecules of < 1.5 kDa including protons. The resulting uncoupling of oxidative phosphorylation leads to ATP depletion and necrotic cell death and it is now widely recognised that MPTP opening is a major cause of reperfusion injury and an effective target for cardioprotection. The properties of the MPTP are well defined, but despite extensive research in many laboratories, its exact molecular identity remains uncertain. Knockout studies have confirmed a role for cyclophilin-D (CyP-D), probably mediated by its peptidyl-prolyl cis–trans isomerase activity facilitating a conformational change of an inner membrane protein. However, the identity of the membrane component(s) remains controversial. Knockout studies have eliminated an essential role for either the voltage dependent anion channel (VDAC) or the adenine nucleotide translocase (ANT), although a regulatory role for the ANT was confirmed. Our own studies implicate the mitochondrial phosphate carrier (PiC) in MPTP formation and are consistent with a calcium-triggered conformational change of the PiC, facilitated by CyP-D, inducing pore opening. We propose that this is enhanced by an association of the PiC with the “c” conformation of the ANT. Agents that modulate pore opening may act on either or both the PiC and the ANT. However, knockdown and reconstitution studies are awaited to confirm or refute this model.     

Purified photoproducts of merocyanine 540 trigger cytochrome C release and caspase 8-dependent apoptosis in human leukemia and melanoma cells.

If the interplay between caspase proteases and mitochondria decide the fate of the cell during apoptosis, they may constitute useful molecular targets for novel drug design. We have shown that photoactivated merocyanine 540 (pMC540) triggers caspase-mediated apoptosis in HL60 leukemia and M14 melanoma cells. Because pMC540 is a mixture of photoproducts, we set out to purify the biologically active component(s) from this mixture and to investigate their ability to directly activate intracellular caspases and/or trigger mitochondrial events associated with apoptosis. Two photoproducts, namely C1 and C2, purified and characterized by mass spectroscopy and nuclear magnetic resonance (NMR) analysis, effectively induced apoptosis in HL60 and M14 cells. Interestingly, both C1 and C2 induced non-receptor-dependent activation of caspase 8, which was responsible for the downstream activation of caspase 3 and cell death. Both compounds induced the release of cytochrome C from mitochondria of tumor cells and from purified rat liver mitochondria; however, different mechanisms were operative in cytochrome C translocation in response to C1 or C2. C1-induced cytochrome C release was mediated by the mitochondrial permeability transition (MPT) pore and accompanied by a decrease in mitochondrial transmembrane potential (triangle uppsim), whereas cytochrome C release in response to C2 was independent of MPT pore opening. These findings do not exclude the possibility that changes in mitochondrial triangle uppsim are critical for apoptosis in some instances, but support the notion that this may not be a universal step in the apoptotic process. Thus, identification of two novel anticancer agents that directly activate effector components of the apoptotic pathway could have potential implications for the development of newer chemotherapeutic drugs.     

Mitochondrial Involvement in Cardiac Apoptosis During Ischemia and Reperfusion: Can We Close the Box?

Myocardial ischemia is the main cause of death in the Western societies. Therapeutic strategies aimed to protect the ischemic myocardium have been extensively studied. Reperfusion is the definitive treatment for acute coronary syndromes, especially acute myocardial infarction; however, reperfusion has the potential to exacerbate tissue injury, a process termed reperfusion injury. Ischemia/reperfusion (I/R) injury may lead to cardiac arrhythmias and contractile dysfunction that involve apoptosis and necrosis in the heart. The present review describes the mitochondrial role on cardiomyocyte death and some potential pharmacological strategies aimed at preventing the opening of the box, i.e., mitochondrial dysfunction and membrane permeabilization that result into cell death. Data in the literature suggest that mitochondrial disruption during I/R can be avoided, although uncertainties still exist, including the fact that the optimal windows of treatment are still fairly unknown. Despite this, the protection of cardiac mitochondrial function should be critical for the patient survival, and new strategies to avoid mitochondrial alterations should be designed to avoid cardiomyocyte loss.     

Mitochondrial calcium uniporter regulator 1 (MCUR1) regulates the calcium threshold for the mitochondrial permeability transition

During the mitochondrial permeability transition, a large channel in the inner mitochondrial membrane opens, leading to the loss of multiple mitochondrial solutes and cell death. Key triggers include excessive reactive oxygen species and mitochondrial calcium overload, factors implicated in neuronal and cardiac pathophysiology. Examining the differential behavior of mitochondrial Ca²⁺ overload in Drosophila versus human cells allowed us to identify a gene, MCUR1, which, when expressed in Drosophila cells, conferred permeability transition sensitive to electrophoretic Ca²⁺ uptake. Conversely, inhibiting MCUR1 in mammalian cells increased the Ca²⁺ threshold for inducing permeability transition. The effect was specific to the permeability transition induced by Ca²⁺, and such resistance to overload translated into improved cell survival. Thus, MCUR1 expression regulates the Ca²⁺ threshold required for permeability transition.The mitochondrial permeability transition (MPT) pore is large, and its opening collapses the mitochondrial membrane potential (ΔΨ), depleting the matrix of solutes <1.5 kDa. The osmotic imbalance swells and disrupts mitochondria, leading to cell death. The molecular structure of the MPT pore is unknown, although cyclophilin D [peptidyl-prolyl isomerase F (PPIF)], the ADP/ATP translocase, the F1-FO-ATP synthase, and spastic paraplegia 7 are key for its function (1–5).Key triggers for the MPT include oxidative damage and Ca²⁺ overload. Reactive oxygen species attack a cysteine residue in mammalian PPIF (6, 7), but how Ca²⁺ overload activates the pore is unknown. Elimination of the known regulators typically inhibits the sensitivity of the MPT globally, not favoring any particular trigger (8–10). Because Ca²⁺ overload promotes cell death in excitable cells, targeting this pathway selectively may prove beneficial.To discover novel regulators specific to mitochondrial Ca²⁺ overload, we studied MPT in Drosophila S2R+ cells, a system where screens have identified molecules involved in Ca²⁺ transport (11–13). We found that mitochondria within these cells were resistant to Ca²⁺ overload (14) but did possess an MPT. Moreover, we identified a mammalian gene, mitochondrial calcium uniporter regulator 1 (MCUR1), with no known Drosophila homolog, which is able to alter the MPT Ca²⁺ threshold. Inhibiting this gene confers resistance from cell death mediated by mitochondrial Ca²⁺ overload.