Coenzyme Q10 reduces ethanol-induced apoptosis in corneal fibroblasts

Dilute ethanol (EtOH) is a widely used agent to remove the corneal epithelium during the modern refractive surgery. The application of EtOH may cause the underlying corneal fibroblasts to undergo apoptosis. This study was designed to investigate the protective effect and potential mechanism of the respiratory chain coenzyme Q(10) (CoQ(10)), an electron transporter of the mitochondrial respiratory chain and a ubiquitous free radical scavenger, against EtOH-induced apoptosis of corneal fibroblasts. Corneal fibroblasts were pretreated with CoQ(10) (10 μM) for 2 h, followed by exposure to different concentrations of EtOH (0.4, 2, 4, and 20%) for 20 s. After indicated incubation period (2-12 h), MTT assay was used to examine cell viability. Treated cells were further assessed by flow cytometry to identify apoptosis. Reactive oxygen species (ROS) and the change in mitochondrial membrane potential were assessed using dichlorodihydrofluorescein diacetate/2',7'-dichlorofluorescein (DCFH-DA/DCF) assays and flow-cytometric analysis of JC-1 staining, respectively. The activity and expression of caspases 2, 3, 8, and 9 were evaluated with a colorimetric assay and western blot analysis. We found that EtOH treatment significantly decreased the viability of corneal fibroblasts characterized by a higher percentage of apoptotic cells. CoQ(10) could antagonize the apoptosis inducing effect of EtOH. The inhibition of cell apoptosis by CoQ(10) was significant at 8 and 12 h after EtOH exposure. In EtOH-exposed corneal fibroblasts, CoQ(10) pretreatment significantly reduced mitochondrial depolarization and ROS production at 30, 60, 90, and 120 min and inhibited the activation and expression of caspases 2 and 3 at 2 h after EtOH exposure. In summary, pretreatment with CoQ(10) can inhibit mitochondrial depolarization, caspase activation, and cell apoptosis. These findings support the proposition that CoQ(10) plays an antiapoptotic role in corneal fibroblasts after ethanol exposure.     

Concentration-dependent effects of nitric oxide on mitochondrial permeability transition and cytochrome c release

The mitochondrial permeability transition pore (PTP) and associated release of cytochrome c are thought to be important in the apoptotic process. Nitric oxide (NO( small middle dot)) has been reported to inhibit apoptosis by acting on a variety of extra-mitochondrial targets. The relationship between cytochrome c release and PTP opening, and the effects of NO( small middle dot) are not clearly established. Nitric oxide, S-nitrosothiols and peroxynitrite are reported to variously inhibit or promote PTP opening. In this study the effects of NO( small middle dot) on the PTP were characterized by exposing isolated rat liver mitochondria to physiological and pathological rates of NO( small middle dot) released from NONOate NO( small middle dot) donors. Nitric oxide reversibly inhibited PTP opening with an IC(50) of 11 nm NO( small middle dot)/s, which can be readily achieved in vivo by NO( small middle dot) synthases. The mechanism involved mitochondrial membrane depolarization and inhibition of Ca(2+) accumulation. At supraphysiological release rates (>2 micrometer/s) NO( small middle dot) accelerated PTP opening. Substantial cytochrome c release occurred with only a 20% change in mitochondrial swelling, was an early event in the PTP, and was also inhibited by NO( small middle dot). Furthermore, NO( small middle dot) exposure resulted in significantly lower cytochrome c release for the same degree of PTP opening. It is proposed that this pathway represents an additional mechanism underlying the antiapoptotic effects of NO( small middle dot).     

Bcl-x(L) blocks a mitochondrial inner membrane channel and prevents Ca2+ overload-mediated cell death

Apoptosis is an active process that plays a key role in many physiological and pathological conditions. One of the most important organelles involved in apoptosis regulation is the mitochondrion. An increase in intracellular Ca(2+) is a general mechanism of toxicity in neurons which occurs in response to different noxious stimuli like excitotoxicity and ischemia producing apoptotic and necrotic cell death through mitochondria-dependent mechanisms. The Bcl-2 family of proteins modulate the release of pro-apoptotic factors from the mitochondrial intermembrane space during cell death induction by different stimuli. In this work, we have studied, using single-cell imaging and patch-clamp single channel recording, the mitochondrial mechanisms involved in the neuroprotective effect of Bcl-x(L) on Ca(2+) overload-mediated cell death in human neuroblastoma SH-SY5Y cells. We have found that Bcl-x(L) neuroprotective actions take place at mitochondria where this antiapoptotic protein delays both mitochondrial potential collapse and opening of the permeability transition pore by preventing Ca(2+)-mediated mitochondrial multiple conductance channel opening. Bcl-x(L) neuroprotective actions were antagonized by the Bcl-x(L) inhibitor ABT-737 and potentiated by the Ca(2+) chelator BAPTA-AM. As a consequence, this would prevent free radical production, mitochondrial membrane permeabilization, release from mitochondria of pro-apoptotic molecules, caspase activation and cellular death.     

Exercise induces a cardiac mitochondrial phenotype that resists apoptotic stimuli

Ischemia-reperfusion-induced calcium overload and production of reactive oxygen species can trigger apoptosis by promoting the release of proapoptotic factors via the mitochondrial permeability transition pore. While it is clear that endurance exercise provides cardioprotection against ischemia-reperfusion-induced injury, it is unknown if exercise training directly alters mitochondria phenotype and confers protection against apoptotic stimuli in both subsarcolemmal (SS) and intermyofibrillar (IMF) mitochondria. We hypothesized that exercise training increases expression of endogenous antioxidant enzymes and other antiapoptotic proteins, resulting in a SS and IMF mitochondrial phenotype that resists apoptotic stimuli. Mitochondria isolated from hearts of sedentary (n = 8) and exercised-trained (n = 8) adult male rats were studied. Endurance exercise increased the protein levels of primary antioxidant enzymes in both SS and IMF mitochondria. Furthermore, exercise increased the levels of antiapoptotic proteins in the heart, including the apoptosis repressor with a caspase recruitment domain and inducible heat shock protein 70. Importantly, our findings reveal that endurance exercise training attenuates reactive oxygen species-induced cytochrome c release from heart mitochondria. These changes are accompanied by a lower maximal rate of mitochondrial permeability transition pore opening (V(max)) and prolonged time to V(max) in both SS and IMF cardiac mitochondria. These novel findings reveal that endurance exercise promotes biochemical alterations in cardiac SS and IMF mitochondria, resulting in a phenotype that resists apoptotic stimuli. Furthermore, these results are consistent with the concept that exercise-induced mitochondrial adaptations contribute to exercise-induced cardioprotection.     

Antioxidant MCI-186 inhibits mitochondrial permeability transition pore and upregulates Bcl-2 expression

Reperfusion after a period of ischemia is associated with the formation of reactive oxygen species (ROS) and Ca2+ overload resulting in the opening of a nonspecific pore in the inner membrane of the mitochondria, called the mitochondrial permeability transition pore (PTP), leading to cell damage. Although endogenous antioxidants are activated because of oxidative stress following ischemia, their levels are not high enough to prevent reperfusion injury. Hence there is always a need for exogenous supplement of antioxidants, especially after acute ischemia. Here we demonstrated the effects of the antioxidant 3-methyl-1-phenyl-2-pyrazolin-5-one (MCI-186) in preventing reperfusion injury of the heart by inhibition of PTP opening. Ischemia (30 min) by left coronary artery (LCA) occlusion and reperfusion (120 min) in Wistar rats after pretreatment with MCI-186 (10 mg/kg iv) infusion starting from 30 min before LCA occlusion resulted in 1) less area of myocardial infarction (19.2% vs. 61.6%), 2) well-maintained myocardial ATP content (P < 0.03 vs. control), 3) decreased mitochondrial swelling and reduced cytochrome c release, 4) increased expression of BCl-2, 5) lower prevalence of apoptotic cells (14.3% vs. 2.9%), and 6) reduced DNA fragmentation in the MCI-186-treated group. These cytoprotective effects of MCI-186 were inhibited on opening PTP before MCI-186 treatment with the PTP activators lonidamine (10 mg/kg iv) or atractyloside (5 mg/kg iv) but failed to inhibit the protective effects exerted by another antioxidant, allopurinol, suggesting that the PTP inhibiting property is specific for MCI-186. These results demonstrate that the radical scavenger MCI-186, by inhibiting the opening of the PTP, prevents necrosis and cytochrome c release and hence pathological apoptosis.     

Vaccinia Virus Infection Disarms the Mitochondrion-Mediated Pathway of the Apoptotic Cascade by Modulating the Permeability Transition Pore

Many viruses have evolved strategies that target crucial components within the apoptotic cascade. One of the best studied is the caspase 8 inhibitor, crmA/Spi-2, encoded by members of the poxvirus family. Since many proapoptotic stimuli induce apoptosis through a mitochondrion-dependent, caspase 8-independent pathway, we hypothesized that vaccinia virus would encode a mechanism to directly modulate the mitochondrial apoptotic pathway. In support of this, we observed that Jurkat cells, which undergo Fas-mediated apoptosis exclusively through the mitochondrial route, were resistant to Fas-induced death following infection with a crmA/Spi-2-deficient strain of vaccinia virus. In addition, vaccinia virus-infected cells subjected to the proapoptotic stimulus staurosporine exhibited decreased levels of both cytochrome c released from the mitochondria and caspase 3 activation. In all cases we found that the loss of the mitochondrial membrane potential, which occurs as a result of opening the multimeric permeability transition pore complex, was prevented in vaccinia virus-infected cells. Moreover, vaccinia virus infection specifically inhibited opening of the permeability transition pore following treatment with the permeability transition pore ligand atractyloside and t-butylhydroperoxide. These studies indicate that vaccinia virus infection directly impacts the mitochondrial apoptotic cascade by influencing the permeability transition pore.     

Apoptosis driven by IP(3)-linked mitochondrial calcium signals

Increases of mitochondrial matrix [Ca(2+)] ([Ca(2+)](m)) evoked by calcium mobilizing agonists play a fundamental role in the physiological control of cellular energy metabolism. Here, we report that apoptotic stimuli induce a switch in mitochondrial calcium signalling at the beginning of the apoptotic process by facilitating Ca(2+)-induced opening of the mitochondrial permeability transition pore (PTP). Thus [Ca(2+)](m) signals evoked by addition of large Ca(2+) pulses or, unexpectedly, by IP(3)-mediated cytosolic [Ca(2+)] spikes trigger mitochondrial permeability transition and, in turn, cytochrome c release. IP(3)-induced opening of PTP is dependent on a privileged Ca(2+) signal transmission from IP(3) receptors to mitochondria. After the decay of Ca(2+) spikes, resealing of PTP occurs allowing mitochondrial metabolism to recover, whereas activation of caspases is triggered by cytochrome c released to the cytosol. This organization provides an efficient mechanism to establish caspase activation while mitochondrial metabolism is maintained to meet ATP requirements of apoptotic cell death.     

Glutamate Interacts with VDAC and Modulates Opening of the Mitochondrial Permeability Transition Pore

The amino acid glutamate, synthesized in the mitochondria, serves multiple functions, including acting as a neurotransmitter and participating in degradative and synthetic pathways. We have previously shown that glutamate modulates the channel activity of bilayer-reconstituted voltage-dependent anion channel (VDAC). In this study, we demonstrate that glutamate also modulates the opening of the mitochondrial permeability transition pore (PTP), of which VDAC is an essential component. Glutamate inhibited PTP opening, as monitored by transient Ca2+ accumulation, mitochondrial swelling and accompanying release of cytochrome c. Exposure to L-glutamate delayed the onset of PTP opening up to 3-times longer, with an IC50 of 0.5 mM. Inhibition of PTP opening by L-glutamate is highly specific, not being mimicked by D-glutamate, L-glutamine, L-aspartate, or L-asparagine. The interaction of L-glutamate with VDAC and its inhibition of VDAC's channel activity and PTP opening suggest that glutamate may also act as an intracellular messenger in the mitochondria-mediated apoptotic pathway.     

Mitochondrial calcium, oxidative stress and apoptosis in a neurodegenerative disease model induced by 3-nitropropionic acid

Intracellular calcium homeostasis is important for cell survival. However, increase in mitochondrial calcium (Ca2+m) induces opening of permeability transition pore (PTP), mitochondrial dysfunction and apoptosis. Since alterations of intracellular Ca2+ and reactive oxygen species (ROS) generation are involved in cell death, they might be involved in neurodegenerative processes such as Huntington's disease (HD). HD is characterized by the inhibition of complex II of respiratory chain and increase in ROS production. In this report, we studied the correlation between the inhibitor of the complex II, 3-nitropropionic acid (3NP), Ca2+ metabolism, apoptosis and behavioural alterations. We showed that 3NP (1 mm) is able to release Ca2+m, as neither Thapsigargin (TAP, 2 microm) nor free-calcium medium affected its effect. PTP inhibitors and antioxidants inhibited this process, suggesting an increase in ROS generation and PTP opening. In addition, 3NP (0.1 mm) also induces apoptotic cell death. Behavioural changes in animals treated with 3NP (20 mg/kg/day for 4 days) were also attenuated by pre- and co-treatment with vitamin E (VE, 20 mg/kg/day). Taken together, our results show that complex II inhibition could involve Ca2+m release, oxidative stress and cell death that may precede motor alterations in neurodegenerative processes such as HD.     

Cyclosporin A inhibits caspase-independent death of NGF-deprived sympathetic neurons: a potential role for mitochondrial permeability transition

Opening of the permeability transition pore (PTP) has been implicated as an important mitochondrial event that occurs during apoptosis. We examined the role of the PTP in the well-characterized cell death of rat sympathetic neurons deprived of nerve growth factor (NGF) in vitro. Removal of NGF causes these neurons to undergo either a classic apoptotic cell death or, when treated with a broad-spectrum caspase inhibitor such as boc-aspartyl(OMe)-fluoromethylketone (BAF), a delayed, nonapoptotic cell death. The PTP inhibitor, cyclosporin A (CsA), blocked commitment-to-die in the presence of BAF, as defined by the ability of NGF readdition to rescue cells, but had little effect on commitment-to-die in the absence of BAF. CsA did not have trophic effects on BAF-saved cells, but did block the decrease in mitochondrial membrane potential. These data suggest that PTP opening is a critical event in caspase-independent, nonapoptotic (but not caspase-dependent, apoptotic) death of NGF-deprived rat sympathetic neurons.     

Akt is activated in response to an apoptotic signal

Akt is a serine-threonine kinase known to exert antiapoptotic effects through several downstream targets. Akt is cleaved during mitochondrial-mediated apoptosis in a caspase-dependent manner. The reason for this is not clear, however, because Akt has not been demonstrated to be activated in response to mitochondrial apoptotic stimuli. Accordingly, we explored whether the well described mitochondrial apoptotic stimuli staurosporine (STS) and etoposide activate Akt and whether such activation impacts apoptosis. Both STS and etoposide activated Akt in NIH 3T3 cells, maximally at 8 and 2 h, respectively, preceding the onset of apoptosis and poly(ADP-ribose) polymerase cleavage. The overexpression of Akt delayed STS-induced apoptosis with an even more pronounced delay observed with overexpression of constitutively active Akt. Akt activation by proapoptotic stimuli lay upstream of mitochondria, because neither caspase inhibitors nor overexpression of Bcl-2 or Bcl-x(L) could prevent it. Activation depended on phosphatidylinositol 3-kinase activity, however. Conversely, inhibition of phosphatidylinositol 3-kinase with wortmannin sensitized cells to apoptosis initiated by STS. These data demonstrate that mitochondrial apoptotic stimuli also activate Akt and such activation modulates apoptosis in this setting.     

Mitochondrial membrane permeabilization produced by PTP, Bax and apoptosis: a 1H-NMR relaxation study

To analyze the involvement of structured water (bound to macromolecules) in apoptosis-induced mitochondrial outer-membrane permeability, we compared the dynamics of water protons from nuclear magnetic resonance (NMR) data in apoptotic liver mitochondria with that of control mitochondria incubated in vitro with free Ca(2+) (opening of the permeability transition pore, PTP) or with Bax alpha. Our results demonstrate that water molecules in apoptotic mitochondria exhibit an accelerated translational motion of structured water common with that induced by the opening of the PTP, but limited in amplitude. On the other hand, no significant quantitative change in structured water was observed in apoptotic mitochondria, a phenomenon also observed with Bax alpha-induced permeability. We conclude that the changes observed in the different water phases differ both quantitatively and qualitatively during the opening of the PTP and the Bax alpha-induced permeability, and that the apoptotic mitochondria exhibit mixed properties between these model situations.     

Mitochondrial permeability transition pore opening as an endpoint to initiate cell death and as a putative target for cardioprotection.

In recent years, mitochondria have been recognized as regulators of cell death via both apoptosis and necrosis in addition to their essential role for cell survival. Cellular dysfunctions induced by intra- or extracellular insults converge on mitochondria and induce a sudden increase in permeability of the inner mitochondrial membrane, the so-called mitochondrial permeability transition. The mitochondrial permeability transition is caused by the opening of permeability transition pores (PTP) in the inner mitochondrial membrane with subsequent loss of ionic homeostasis, matrix swelling and outer membrane rupture. The detailed molecular mechanisms underlying the PTP-induced cellular dysfunction during cardiac pathology such as ischemia/reperfusion or post-infarction remodeling remain to be elucidated. However, a growing body of evidence supports the concept that pharmacological inhibition of the PTP is an effective and promising strategy for the protection of the heart against ischemia/reperfusion injury and for attenuation of the remodeling process which contributes to heart failure. This review summarizes and discusses current data on i) the structure and function of the PTP, ii) possible mechanisms and consequences of PTP opening and iii) the inhibition of PTP opening as a therapeutic approach for treatment of heart disease.     

A tale of two mitochondrial channels,MAC and PTP,in apoptosis

The crucial step in the intrinsic, or mitochondrial, apoptotic pathway is permeabilization of the mitochondrial outer membrane. Permeabilization triggers release of apoptogenic factors, such as cytochrome c, from the mitochondrial intermembrane space into the cytosol where these factors ensure propagation of the apoptotic cascade and execution of cell death. However, the mechanism(s) underlying permeabilization of the outer membrane remain controversial. Two mechanisms, involving opening of two different mitochondrial channels, have been proposed to be responsible for the permeabilization; the permeability transition pore (PTP) in the inner membrane and the mitochondrial apoptosis-induced channel (MAC) in the outer membrane. Opening of PTP would lead to matrix swelling, subsequent rupture of the outer membrane, and an unspecific release of intermembrane proteins into the cytosol. However, many believe PTP opening is a consequence of apoptosis and this channel is thought to principally play a role in necrosis, not apoptosis. Activation of MAC is exquisitely regulated by Bcl-2 family proteins, which are the sentinels of apoptosis. MAC provides specific pores in the outer membrane for the passage of intermembrane proteins, in particular cytochrome c, to the cytosol. The electrophysiological characteristics of MAC are very similar to Bax channels and depletion of Bax significantly diminishes MAC activity, suggesting that Bax is an essential constituent of MAC in some systems. The characteristics of various mitochondrial channels and Bax are compared. The involvement of MAC and PTP activities in apoptosis of disease and their pharmacology are discussed.     

The mitochondrial effects of small organic ligands of BCL-2: sensitization of BCL-2-overexpressing cells to apoptosis by a pyrimidine-2,4,6-trione derivative

We have investigated the mitochondrial effects of BH3I-2', Chelerythrine, and HA14-1, small organic molecules that share the ability to bind the BH3 domain of BCL-2. All compounds displayed a biphasic effect on mitochondrial respiration with uncoupling at low concentrations and respiratory inhibition at higher concentrations, the relative uncoupling potency being BH3I-2' (half-maximal uncoupling at about 80 nm) > Chelerythrine (half-maximal uncoupling at about 2 microm) > HA14-1 (half-maximal uncoupling at about 20 microm). At concentrations lower than required for uncoupling all compounds sensitized the permeability transition pore (PTP) to opening both in isolated mitochondria and intact cells. To assess whether the effects on BCL-2 binding, PTP induction and respiration could be due to different structural determinants we have tested a set of HA14-1 analogs from the Hoffmann-La Roche chemical library. We have identified 5-(6-chloro-2,4-dioxo-1,3,4,10-tetrahydro-2H-9-oxa-1,3-diaza-anthracen-10-yl)-pyrimidine-2,4,6-trione (EM20-25) as a molecule devoid of effects on respiration that is able to induce PTP opening, to disrupt the BCL-2/BAX interactions in situ and to activate caspase-9 in BCL-2-overexpressing cells. EM20-25 neutralized the antiapoptotic activity of overexpressed BCL-2 toward staurosporine and sensitized BCL-2-expressing cells from leukemic patients to the killing effects of staurosporine, chlorambucil, and fludarabine. These results provide a proof of principle that the potentially toxic effects of BCL-2 ligands on mitochondrial respiration are not essential for their antiapoptotic activity and represent an important step forward in the development of tumor-selective drugs acting on BCL-2.     

Mitochondrial permeability transition in Ca(2+)-dependent apoptosis and necrosis

A variety of stimuli utilize an increase of cytosolic free Ca²⁺ concentration as a second messenger to transmit signals, through Ca²⁺ release from the endoplasmic reticulum or opening of plasma membrane Ca²⁺ channels. Mitochondria contribute to the tight spatiotemporal control of this process by accumulating Ca²⁺, thus shaping the return of cytosolic Ca²⁺ to resting levels. The rise of mitochondrial matrix free Ca²⁺ concentration stimulates oxidative metabolism; yet, in the presence of a variety of sensitizing factors of pathophysiological relevance, the matrix Ca²⁺ increase can also lead to opening of the permeability transition pore (PTP), a high conductance inner membrane channel. While transient openings may serve the purpose of providing a fast Ca²⁺ release mechanism, persistent PTP opening is followed by deregulated release of matrix Ca²⁺, termination of oxidative phosphorylation, matrix swelling with inner membrane unfolding and eventually outer membrane rupture with release of apoptogenic proteins and cell death. Thus, a rise in mitochondrial Ca²⁺ can convey both apoptotic and necrotic death signals by inducing opening of the PTP. Understanding the signalling networks that govern changes in mitochondrial free Ca²⁺ concentration, their interplay with Ca²⁺ signalling in other subcellular compartments, and regulation of PTP has important implications in the fine comprehension of the main biological routines of the cell and in disease pathogenesis.     

Necroptosis Interfaces with MOMP and the MPTP in Mediating Cell Death

During apoptosis the pro-death Bcl-2 family members Bax and Bak induce mitochondrial outer membrane permeabilization (MOMP) to mediate cell death. Recently, it was shown that Bax and Bak are also required for mitochondrial permeability transition pore (MPTP)-dependent necrosis, where, in their non-oligomeric state, they enhance permeability characteristics of the outer mitochondrial membrane. Necroptosis is another form of regulated necrosis involving the death receptors and receptor interacting protein kinases (RIP proteins, by Ripk genes). Here, we show cells or mice deficient for Bax/Bak or cyclophilin D, a protein that regulates MPTP opening, are resistant to cell death induced by necroptotic mediators. We show that Bax/Bak oligomerization is required for necroptotic cell death and that this oligomerization reinforces MPTP opening. Mechanistically, we observe mixed lineage kinase domain-like (MLKL) protein and cofilin-1 translocation to the mitochondria following necroptosis induction, while expression of the mitochondrial matrix isoform of the antiapoptotic Bcl-2 family member, myeloid cell leukemia 1 (Mcl-1), is significantly reduced. Some of these effects are lost with necroptosis inhibition in Bax/Bak1 double null, Ppif^-/-, or Ripk3^-/- fibroblasts. Hence, downstream mechanisms of cell death induced by necroptotic stimuli utilize both Bax/Bak to generate apoptotic pores in the outer mitochondrial membrane as well as MPTP opening in association with known mitochondrial death modifying proteins.     

Effect of chronic contractile activity on SS and IMF mitochondrial apoptotic susceptibility in skeletal muscle

Chronic contractile activity of skeletal muscle induces an increase in mitochondria located in proximity to the sarcolemma [subsarcolemmal (SS)] and in mitochondria interspersed between the myofibrils [intermyofibrillar (IMF)]. These are energetically favorable metabolic adaptations, but because mitochondria are also involved in apoptosis, we investigated the effect of chronic contractile activity on mitochondrially mediated apoptotic signaling in muscle. We hypothesized that chronic contractile activity would provide protection against mitochondrially mediated apoptosis despite an elevation in the expression of proapoptotic proteins. To induce mitochondrial biogenesis, we chronically stimulated (10 Hz; 3 h/day) rat muscle for 7 days. Chronic contractile activity did not alter the Bax/Bcl-2 ratio, an index of apoptotic susceptibility, and did not affect manganese superoxide dismutase levels. However, contractile activity increased antiapoptotic 70-kDa heat shock protein and apoptosis repressor with a caspase recruitment domain by 1.3- and 1.4-fold (P<0.05), respectively. Contractile activity elevated SS mitochondrial reactive oxygen species (ROS) production 1.4- and 1.9-fold (P<0.05) during states IV and III respiration, respectively, whereas IMF mitochondrial state IV ROS production was suppressed by 28% (P<0.05) and was unaffected during state III respiration. Following stimulation, exogenous ROS treatment produced less cytochrome c release (25-40%) from SS and IMF mitochondria, and also reduced apoptosis-inducing factor release (approximately 30%) from IMF mitochondria, despite higher inherent cytochrome c and apoptosis-inducing factor expression. Chronic contractile activity did not alter mitochondrial permeability transition pore (mtPTP) components in either subfraction. However, SS mitochondria exhibited a significant increase in the time to Vmax of mtPTP opening. Thus, chronic contractile activity induces predominantly antiapoptotic adaptations in both mitochondrial subfractions. Our data suggest the possibility that chronic contractile activity can exert a protective effect on mitochondrially mediated apoptosis in muscle.     

Propagation of the apoptotic signal by mitochondrial waves

Generation of mitochondrial signals is believed to be important in the commitment to apoptosis, but the mechanisms coordinating the output of individual mitochondria remain elusive. We show that in cardiac myotubes exposed to apoptotic agents, Ca2+ spikes initiate depolarization of mitochondria in discrete subcellular regions, and these mitochondria initiate slow waves of depolarization and Ca2+ release propagating through the cell. Traveling mitochondrial waves are prevented by Bcl-x(L), involve permeability transition pore (PTP) opening, and yield cytochrome c release, caspase activation and nuclear apoptosis. Mitochondrial Ca2+ uptake is critical for wave propagation, and mitochondria at the origin of waves take up Ca2+ particularly effectively, providing a mechanism that may underlie selection of the initiation sites. Thus, apoptotic agents transform the mitochondria into an excitable state by sensitizing PTP to Ca2+. Expansion of the local excitation by mitochondrial waves propagating through the whole cell can be especially important in activation of the apoptotic machinery in large cells.     

Effects of curcumin and curcumin derivatives on mitochondrial permeability transition pore

Curcumin (1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione) is a natural compound with antiproliferative properties. Recent studies suggest that these properties might be due to the ability of curcumin to induce apoptosis in tumor cells by increasing the permeability of the mitochondrial membrane. In the present study, we confirm these observations and provide a molecular mechanism for the action of curcumin in rat liver mitochondria. Curcumin induced mitochondrial swelling, the collapse of Deltapsi, and the release of cytochrome C, events associated with the opening of the permeability transition pore (PTP). Experiments were performed with chemically substituted curcumin derivatives. Some derivatives were obtained by modification of groups on the terminal aromatic rings, and others were obtained by substitution of the diketone function with the cyclohexanone function. They demonstrated that phenol and methoxy groups were essential to promote PTP opening. Curcumin and curcumin derivatives that open the PTP were able to oxidize thiol groups. In addition, PTP opening was abolished in medium devoid of O2 and decreased in the presence of catalase, ferrozine, o-phenanthroline, mannitol, or N-ethylmaleimide. These data suggest that the mechanism by which curcumin promotes PTP opening involves the reduction of Fe3+ to Fe2+, inducing hydroxyl radical (HO*) production and oxidation of thiol groups in the membrane, leading to pore opening.