Methylmercury induces the opening of the permeability transition pore in rat liver mitochondria

Interactions of methylmercury (CH(3)HgCl) with non-energized mitochondria from rat liver (non-respiring mitochondria) have been investigated in this paper. It has been shown that CH(3)HgCl induces swelling in mitochondria suspended in a sucrose medium. Swelling has also been induced by detergent compounds and by phenylarsine, a chemical compound which induces opening of the permeant transition pore (MTP). Opening of the MTP is inhibited by means of cyclosporine A. Results indicate that the swelling induced by CH(3)HgCl, as in the case of phenylarsine, is inhibited by cyclosporine A and Mg(2+), while swelling induced by detergent compounds is not cyclosporine sensitive. This comparison suggests that CH(3)HgCl induces opening of a permeability transition pore (MTP). Since the opening of an MTP induces cell death, this interaction with MTP could be one of the causes of toxicity of CH(3)HgCl.     

Possible Mechanism for Formation and Regulation of the Palmitate-Induced Cyclosporin A-Insensitive Mitochondrial Pore

The mechanism of the palmitate-induced opening of the mitochondrial Ca2+-dependent cyclosporin A (CsA)-insensitive pore was studied, as well as the influence on this process of well-known modulators of the CsA-sensitive Ca2+-dependent pore. Palmitic acid, which can bind Ca2+ with high affinity, induced the cyclosporin A-insensitive swelling of mitochondria, whereas palmitoleic and 2-bromopalmitic acids, which have no such affinity for Ca2+, failed to induce the pore opening. The palmitate-induced Ca2+-dependent swelling of mitochondria was not affected by a well-known inhibitor of the CsA-sensitive pore (ADP) and an activator of this pore (inorganic phosphate, P(i)). However, this swelling was inhibited by physiological concentrations of ATP ([I]50 = 1.3 mM), but 100 microM ATP increased by 30% the rate of mitochondria swelling if Ca2+ had been added earlier. The effects of ATP (inhibition and activation) manifested themselves from different sides of the inner mitochondrial membrane. Mg2+ inhibited the palmitate-induced Ca2+-dependent swelling of mitochondria with [I]50 = 0.8 mM. It is concluded that palmitic acid induces the opening of the CsA-insensitive pore due to its ability for complexing with Ca2+. A possible mechanism of the pore formation and the influence of some modulators on this process are discussed.     

Mitochondrial permeability transition in the crustacean Artemia franciscana: absence of a calcium-regulated pore in the face of profound calcium storage

When mammalian mitochondria are exposed to high calcium and phosphate, a massive swelling, uncoupling of respiration, and release of cytochrome c occur. These changes are mediated by opening of the mitochondrial permeability transition pore (MPTP). Activation of the MPTP in vivo in response to hypoxic and oxidative stress leads to necrotic and apoptotic cell death. Considering that embryos of the brine shrimp Artemia franciscana tolerate anoxia for years, we investigated the MPTP in this crustacean to reveal whether pore opening occurs. Minimum molecular constituents of the regulated MPTP in mammals are believed to be the voltage-dependent anion channel, the adenine nucleotide translocators, and cyclophilin D. Western blot analysis revealed that mitochondria from A. franciscana possess all three required components. When measured with a calcium-sensitive fluorescent probe, rat liver mitochondria are shown to release matrix calcium after addition of >/=100 microM extramitochondrial calcium (MPTP opening), whereas brine shrimp mitochondria continue to take up extramitochondrial calcium and do not release internal stores even up to 1.0 mM exogenously added calcium (no MPTP opening). Furthermore, no swelling of A. franciscana mitochondria in response to added calcium was observed, and no release of cytochrome c could be detected. HgCl(2)-dependent swelling and cytochrome c release were readily confirmed, which is consistent with the presence of an "unregulated pore." Although the absence of a regulated MPTP in A. franciscana mitochondria could contribute to the extreme hypoxia tolerance in this species, we speculate that absence of the regulated MPTP may be a general feature of invertebrates.     

Physiological modulators of cyclosporin A-insensitive nonspecific permeability of the inner membrane of liver mitochondria induced by calcium ions and α,ω-hexadecanedioic acid

Long-chain saturated α,ω-dioic acids can induce nonspecific permeability of the inner membrane (pore opening) of liver mitochondria loaded with Ca²⁺ or Sr²⁺ by the mechanism insensitive to cyclosporin A (CsA). In this work we found that 200 μM Ca²⁺ and 20 μM α,ω-hexadecanedioic acid (HDA) in the presence of 1 μM CsA induced high-amplitude swelling of liver mitochondria (pore opening) only in the presence of succinate as oxidation substrate. Under these conditions protonophore uncoupler of oxidative phosphorylation 2,4-dinitrophenol at the concentration of 75 μM, which is optimal for its uncoupling activity, inhibited mitochondrial swelling induced by Ca²⁺ and HDA, despite the presence of succinate in the incubation medium. Natural uncouplers of oxidative phosphorylation, oleic and linoleic acids, produced a similar effect. These data suggest that energization of organelles, which promotes Ca²⁺ transport into the matrix, is one of the basic requirements of pore opening in liver mitochondria induced by Ca²⁺ and HDA. It is shown that ATP at the physiological concentration of 2 mM inhibits HDA-induced high-amplitude swelling of mitochondria by reducing free Ca²⁺ concentration in the medium. ADP at the same concentration had a similar effect. This modulating effect of nucleotides apparently is attributable to their ability to chelate calcium ions. Polycation spermine, which is known as an inhibitor of the classical CsA-sensitive pore, at the physiological concentration of 1 mM inhibited CsA-insensitive swelling of liver mitochondria induced by sequential addition of Ca²⁺ and HDA. It is assumed that such action of spermine is due to its ability to shield the negative surface charges on the inner membrane of mitochondria. Bovine serum albumin (BSA), which is able to bind free fatty acids and thus prevent the induction of Ca²⁺-dependent pore, inhibited HDA-induced swelling of mitochondria. However, at the same BSA/fatty acid molar ratio inhibitory effect of BSA was much less pronounced if HDA was used as the pore inducer instead of palmitic acid. Apparently, this can be accounted by the fact that BSA binds α,ω-dioic acids weaker than their monocarboxylic analogues.     

Inorganic phosphate stimulates the toxic effects of Tl+ in rat liver mitochondria

We studied action of inorganic phosphate (P(i)) on toxic effects of Tl+ in isolated rat liver mitochondria. This is a convenient model to study the toxicity of heavy metals. P(i) markedly retarded contraction of energized mitochondria swollen in the TlNO3 medium and even stronger stimulated swelling and state 4 of succinate-energized mitochondria in the TlNO3 medium. A valinomycin-induced decrease of K+-diffusion potential was also accelerated by Tl+ in the presence of P(i). The mitochondrial permeability transition pore in the medium containing Ca2+, TlNO3, and nitrates of univalent cations was distinctly stimulated by P(i). However, P(i) did not affect both the Tl+-stimulated swelling of nonenergized mitochondria in the TlNO3 medium and swelling of energized mitochondria in the Tl acetate medium. Respiration stimulated by 2,4-dinitrophenol and monoamine oxidase activity of energized mitochondria were not affected by Tl+ regardless of the presence of P(i). We suggested that stimulation by P(i) of toxic action of Tl+ in mitochondria and cells could be due to even greater enhancement of uncoupling of mitochondria as shown by an additional increase of swelling and state 4, and in the greater probability of opening of MPTP in the presence of P(i) and Ca2+.     

Mitochondrial oxidative stress induced by Ca2+ and monoamines: different behaviour of liver and brain mitochondria in undergoing permeability transition

Mitochondrial permeability transition (MPT) is correlated with the opening of a nonspecific pore, the so-called transition pore, that triggers bidirectional traffic of inorganic solutes and metabolites across the mitochondrial membrane. This phenomenon is caused by supraphysiological Ca(2+) concentrations and by other compounds leading to oxidative stress, while cyclosporin A, ADP, bongkrekic acid, antioxidant agents and naturally occurring polyamines strongly inhibit it. The effects of polyamines, including the diamine agmatine, have been widely studied in several types of mitochondria. The effects of monoamines on MPT have to date, been less well-studied, even if they are involved in a variety of neurological and neuroendocrine processes. This study shows that in rat liver mitochondria (RLM), monoamines such as tyramine, serotonin and dopamine amplify the swelling induced by calcium, and increase the oxidation of thiol groups and the production of hydrogen peroxide, effects that are counteracted by the above-mentioned inhibitors. In rat brain mitochondria (RBM), the monoamines do not amplify calcium-induced swelling, even if they demonstrate increases in the extent of oxidation of thiol groups and hydrogen peroxide production. In these mitochondria, the antioxidants are not at all or scarcely effective in suppressing mitochondrial swelling. In conclusion, we hypothesize that different mechanisms induce the MPT in the two different types of mitochondria evaluated. Calcium and monoamines induce oxidative stress in RLM, which in turn appears to induce and amplify MPT. This process is not apparent in RBM, where MPT seems resistant to oxidative stress.     

Dopamine Oxidation Alters Mitochondrial Respiration and Induces Permeability Transition in Brain Mitochondria

Both reactive dopamine metabolites and mitochondrial dysfunction have been implicated in the neurodegeneration of Parkinson's disease. Dopamine metabolites, dopamine quinone and reactive oxygen species, can directly alter protein function by oxidative modifications, and several mitochondrial proteins may be targets of this oxidative damage. In this study, we examined, using isolated brain mitochondria, whether dopamine oxidation products alter mitochondrial function. We found that exposure to dopamine quinone caused a large increase in mitochondrial resting state 4 respiration. This effect was prevented by GSH but not superoxide dismutase and catalase. In contrast, exposure to dopamine and monoamine oxidase-generated hydrogen peroxide resulted in a decrease in active state 3 respiration. This inhibition was prevented by both pargyline and catalase. We also examined the effects of dopamine oxidation products on the opening of the mitochondrial permeability transition pore, which has been implicated in neuronal cell death. Dopamine oxidation to dopamine quinone caused a significant increase in swelling of brain and liver mitochondria. This was inhibited by both the pore inhibitor cyclosporin A and GSH, suggesting that swelling was due to pore opening and related to dopamine quinone formation. In contrast, dopamine and endogenous monoamine oxidase had no effect on mitochondrial swelling. These findings suggest that mitochondrial dysfunction induced by products of dopamine oxidation may be involved in neurodegenerative conditions such as Parkinson's disease and methamphetamine-induced neurotoxicity.     

Calcium- and phosphate-dependent release and loading of glutathione by liver mitochondria

The status of glutathione (GSH) was studied in isolated rat liver mitochondria under conditions which induce a permeability transition. This transition, which is inhibited by cyclosporin A (CyA), requires the presence of Ca2+ and an inducing agent such as near physiological levels (3 mM) of inorganic phosphate (Pi). The transition is characterized by an increased inner membrane permeability to some low molecular weight solutes and by large amplitude swelling under some experimental conditions. Addition of 70 microM Ca2+ and 3 mM Pi to mitochondria resulted in mitochondrial swelling and extensive release of GSH that was recovered in the extramitochondrial medium as GSH. Both swelling and the efflux of mitochondrial GSH were prevented by CyA. Incubation of mitochondria in the presence of Ca2+, Pi, and GSH followed by addition of CyA provided a mechanism to load mitochondria with exogenous GSH that was greater than the rate of uptake by untreated mitochondria. Thus, GSH efflux from mitochondria may occur under toxicological and pathological conditions in which mitochondria are exposed to elevated Ca2+ in the presence of near physiological concentrations of Pi through a nonspecific pore. Cyclical opening and closing of the pore could also provide a mechanism for uptake of GSH by mitochondria.     

Discrimination between two steps in the mitochondrial permeability transition process

It is well known that a lag phase generally elapses between the addition of inducers of the mitochondrial permeability transition and the opening of the pore. To advance our present understanding as regards the significance of this phenomenon, we used experimental approaches which are sensitive to different aspects of the permeability transition process. The pore conformation was sensed by the fluorescence anisotropy changes of hematoporphyrin-labelled mitochondria. Membrane permeabilization was ascertained by following the matrix swelling consequent to external solute equilibration. We show that the anisotropy changes of mitochondria-bound hematoporphyrin precede both membrane depolarization (proton permeation) and matrix swelling (solute permeation), thus sensing a step of the permeability transition process that involves the pore in its closed state. We suggest that the opening of the pore is preceded by a structural remodelling of mitochondrial domains containing hematoporphyrin-near, pore-regulating histidines. Such a perturbation is strongly inhibited at acidic matrix pH and completely blocked by cyclosporin A. In sucrose-based media the opening of the pore can be strongly delayed, as compared to salt-based media, a fact which probably reflects perturbation of mitochondrial membranes by sugar. We conclude that the mitochondrial permeability transition could be described as an at least two-step process which is mainly regulated by conformational changes of the pore components.     

Taurine chloramine, an oxidant derived from neutrophils, induces apoptosis in human B lymphoma cells through mitochondrial damage

Taurine chloramine (TN-Cl) is one of the most abundant compounds generated by activated neutrophils. In contrast to HOCl, which causes necrosis, TN-Cl is a potent inducer of apoptosis in tumor cells. Here we show that the apoptosis induced by TN-Cl in human B lymphoma cells is dependent upon oxidant-mediated mitochondrial damage, a decrease in mitochondrial membrane potential, and caspase-9 activation. Further, we show that TN-Cl is taken up into the cells and is concentrated in the mitochondria, where it induces opening of the permeability transition pore and mitochondrial swelling. Identical activity is seen upon treatment of isolated mitochondria with TN-Cl and is blocked by the permeability transition pore inhibitors bongkrekic acid and cyclosporin A, as well as by the sulfhydryl-reducing agent tris(2-carboxyethyl)-phosphine. The data suggest that TN-Cl causes apoptosis through direct damage to the mitochondria.     

Commitment to apoptosis by GD3 ganglioside depends on opening of the mitochondrial permeability transition pore.

We have studied the effects of GD3 ganglioside on mitochondrial function in isolated mitochondria and intact cells. In isolated mitochondria, GD3 ganglioside induces complex changes of respiration that depend on the substrate being oxidized. However, these effects are secondary to opening of the cyclosporin A-sensitive permeability transition pore and to the ensuing swelling and cytochrome c depletion rather than to an interaction with the respiratory chain complexes. By using a novel in situ assay based on the fluorescence changes of mitochondrially entrapped calcein (Petronilli, V., Miotto, G., Canton, M., Colonna, R., Bernardi, P., and Di Lisa, F. (1999) Biophys. J. 76, 725-734), we unequivocally show that GD3 ganglioside also induces the mitochondrial permeability transition in intact cells and that this event precedes apoptosis. The mitochondrial effects of GD3 ganglioside are selective, in that they cannot be mimicked by either GD1a or GM3 gangliosides, and they are fully sensitive to cyclosporin A, which inhibits both the mitochondrial permeability transition in situ and the onset of apoptosis induced by GD3 ganglioside. These results provide compelling evidence that opening of the permeability transition pore is causally related to apoptosis.     

Physiological aspects of the mitochondrial cyclosporin A-insensitive palmitate/Ca<Superscript>2+</Superscript>-induced pore: tissue specificity,age profile and dependence on the animal’s adaptation to hypoxia

Earlier we found that being added to rat liver mitochondria, palmitic acid (Pal) plus Ca²⁺ opened a cyclosporin A-insensitive pore, which remained open for a short time. Apparently, this pore is involved in the Pal-induced apoptosis and may also take part in the mitochondrial Ca²⁺ recycling as a Ca²⁺ efflux system (Belosludtsev et al. J Bioenerg Biomembr 38:113–120, 2006; Mironova et al. J. Bioenerg. Biomembr. 39:167–174, 2007). In this paper, we continue studying physiological and regulatory aspects of the pore. The following observations have been made. (1) Cardiolipin has been found to facilitate the Ca²⁺-induced formation of pores in the Pal-containing liposomal membranes. (2) The opening of Pal/Ca²⁺-induced pore is accompanied by the release of apoptosis-induced factor (AIF) from mitochondria. (3) The rate of Pal/Ca²⁺-induced swelling of rat liver mitochondria increases substantially with the age of animals. (4) Although the Pal/Ca²⁺-induced pore opens both in the liver and heart mitochondria, the latter require higher Pal concentrations for the pore to open. (5) The pore opening depends on the resistance of animals to hypoxia: in the highly resistant to hypoxia rats, the mitochondrial Pal/Ca²⁺-induced pore opens easier than in the low resistant animals, this being opposite for the classical, cyclosporin A-sensitive MPT pore. The adaptation of the low resistant rats to oxygen deficiency increases the sensitivity of their mitochondria to PalCaP inductors. The paper also discusses a possible role of the mitochondrial Pal/Ca²⁺-induced pore in the protection of tissues against hypoxia.     

Mitochondrial permeability transition induced by dinuclear gold(I)-carbene complexes: potential new antimitochondrial antitumour agents

Seven dinuclear gold(I) complexes of bidentate N-heterocyclic carbene ligands have been evaluated for their ability to induce mitochondrial membrane permeabilisation (MMP) in isolated rat liver mitochondria. Six of the compounds, at concentrations of 10 microM, induced Ca(2+)-sensitive MMP as evidenced by mitochondrial swelling. In the absence of low concentrations of exogenous Ca(2+), the compounds were either inactive or their activity was significantly decreased. The mitochondrial swelling was completely blocked by the addition of cyclosporin A, a well established inhibitor of the mitochondrial permeability transition pore (MPT) that is believed to be responsible for MMP. The rates and levels of uptake of these compounds into mitochondria were estimated by measuring mitochondrial Au levels using inductively coupled plasma optical emission spectroscopy. Significant differences were found in the levels at which the different compounds accumulated in the mitochondria, but these differences did not correlate with the rate at which they induced mitochondrial swelling. These results suggest that the mechanism by which MMP is induced by these lipophilic cationic Au(I)-carbene complexes is not purely a function of the level of compound accumulation. Instead, a more specific mechanism, possibly involving disruption of the function of a particular enzyme, or interaction with a MPT component, appears to be more likely.     

Mersalyl prevents the Tl+-induced permeability transition pore opening in the inner membrane of Ca2+-loaded rat liver mitochondria

It was earlier shown that the calcium load of rat liver mitochondria in medium containing TlNO₃ and KNO₃ resulted in the Tl⁺-induced mitochondrial permeability transition pore (MPTP) opening in the inner membrane. This opening was accompanied by an increase in swelling and membrane potential dissipation and a decrease in state 3, state 4, and 2,4-dinitrophenol-uncoupled respiration. This respiratory decrease was markedly leveled by mersalyl (MSL), the phosphate symporter (PiC) inhibitor which poorly stimulated the calcium-induced swelling, but further increased the potential dissipation. All of these effects of Ca²⁺ and MSL were visibly reduced in the presence of the MPTP inhibitors (ADP, N-ethylmaleimide, and cyclosporine A). High MSL concentrations attenuated the ability of ADP to inhibit the MPTP. Our data suggest that the PiC can participate in the Tl⁺-induced MPTP opening in the inner membrane of Ca²⁺-loaded rat liver mitochondria.     

Mitochondrial DNA mutations cause resistance to opening of the permeability transition pore

The age-related accumulation of mitochondrial DNA mutations has the potential to impair organ function and contribute to disease. In support of this hypothesis, accelerated mitochondrial mutagenesis is pathogenic in the mouse heart, and there is an increase in myocyte apoptosis. The current study sought to identify functional alterations in cell death signaling via mitochondria. Of particular interest is the mitochondrial permeability transition pore, opening of which can initiate cell death, while pore inhibition is protective. Here, we show that mitochondria from transgenic mice that develop mitochondrial DNA mutations have a marked inhibition of calcium-induced pore opening. Temporally, inhibited pore opening coincides with disease. Pore inhibition also correlates with an increase in Bcl-2 protein integrated into the mitochondrial membrane. We hypothesized that pore inhibition was mediated by mitochondrial Bcl-2. To test this hypothesis, we treated isolated mitochondria with Bcl-2 antagonistic peptides (derived from the BH3 domain of Bax or Bid). These peptides released the inhibition to pore opening. The data are consistent with a Bcl-2-mediated inhibition of pore opening. Thus, mitochondrial DNA mutations induce an adaptive-protective response in the heart that inhibits opening of the mitochondrial permeability pore.     

Age-dependent changes of mitochondrial functions in Ca<Superscript>2+</Superscript>-induced opening of permeability transition pore

Mitochondria are intracellular organelles, which provide cells with energy and participate in multiple processes of cell vital functions. Within one of the numerous theories of aging, dysfunction of mitochondria is considered to lead to tissue degeneration and induce the initial stage in developing of degenerative diseases. Since mitochondria play a clue role in apoptosis/necrosis processes, it was suggested that dysfunction of mitochondria observed under aging is related with disturbance of programmed cell death regulation. In the present study, a comparative examination of parameters of the functional states of mitochondria isolated from young (2–3-months old) and old (20–22-months old) rats under conditions of opening of unselective pore (PTP, permeability transition pore) has been performed. Ca²⁺ accumulation rate in mitochondria isolated from old rats was found to be decreased by 25–30%, threshold calcium concentration was lowered to 50%, and the swelling of mitochondria loaded by calcium was stimulated 3–4-fold. Production of reactive oxygen species (ROS) has been also determined in these mitochondria. In old mitochondria superoxide anion level was increased. In addition, H₂O₂ content was found to be 2 times higher in mitochondria with PTP opened. Using electron microscopy method, a decreased amount of cristae in mitochondria was revealed under aging.     

Palmitic acid opens a novel cyclosporin A-insensitive pore in the inner mitochondrial membrane

An assortment of agents can induce mitochondria to undergo a permeability transition, which results in the inner mitochondrial membrane becoming nonselectively permeable to small (<1500 Da) solutes. This mitochondrial permeability transition (MPT) is characterized by a strict dependence on matrix Ca2+ and sensitivity to cyclosporin A (CsA). However, it is becoming increasingly clear that other experimental conditions can elicit increases in mitochondrial permeability that are distinct from this classic MPT. For example, butylated hydroxytoluene (BHT; Sokolove, P. M., and Haley, L. M. (1996) J. Bioenerg. Biomembr. 28, 199-206) and signal peptides (Sokolove, P. M., and Kinnally, K. W. (1996) Arch. Biochem. Biophys. 336, 69-76) promote increases in mitochondrial permeability that are CsA-insensitive. It has been suggested (Gudz, T., Eriksson, O., Kushnareva, Y., Saris, N.-E., and Novgorodov, S. A. (1997) Arch. Biochem. Biophys. 342, 143-156) that BHT might be opening a CsA-insensitive pore by increasing phospholipase A2 activity and thereby producing an accumulation of free fatty acids and lysophospholipids. We have therefore examined the effect of the saturated free fatty acid, palmitic acid (PA), on the permeability of isolated rat liver mitochondria. The following results were obtained: (1) In the absence of additional triggers, PA (20-60 microM) induced concentration-dependent, CsA-insensitive mitochondrial swelling. (2) Swelling required mitochondrial energization. (3) PA-induced swelling was fast and occurred without a lag. (4) Both Ca2+ and Sr2+ supported PA-induced swelling; the site of cation action was the matrix. (5) EGTA and BSA were potent inhibitors of PA-induced swelling. (6) PA opened a pore rather than disrupting mitochondrial membrane structure. (7) The pore opened by PA closed spontaneously. These results suggest that palmitic acid promotes a nonclassic permeability increase that is clearly distinguishable from the occurrence of the MPT.     

Effects of N-acylethanolamines on mitochondrial energetics and permeability transition

Effects of N-acylethanolamines (NAEs): N-arachidonoylethanolamine (anandamide), N-oleoylethanolamine and N-palmitoylethanolamine, on energy coupling and permeability of rat heart mitochondria were investigated. In nominally Ca2+-free media, these compounds exerted a weak protonophoric effect manifested by dissipation of the transmembrane potential and stimulation of resting state respiration. The strongest action was exhibited by N-arachidonoylethanolamine, followed by N-oleoylethanolamine, whereas N-palmitoylethanolamine was almost inactive. These protonophoric effects were resistant to cyclosporin A (CsA) and were much weaker than those of corresponding nonesterified fatty acids. In uncoupled mitochondria N-arachidonoylethanolamine and N-oleoylethanolamine partly inhibited mitochondrial respiration with glutamate and succinate but not with tetramethyl-p-phenylenediamine (TMPD) plus ascorbate as respiratory substrates. In mitochondria preloaded with small amounts of Ca2+, NAEs produced a much stronger dissipation of the membrane potential and a release of accumulated calcium, both effects being inhibited by CsA, indicative for opening of the mitochondrial permeability transition pore (PTP). Again, the potency of this action was N-arachidonoylethanolamine>N-oleoylethanolamine>N-palmitoylethanolamine. However, in spite of making the matrix space accessible to external [14C]sucrose, N-arachidonoylethanolamine and N-oleoylethanolamine resulted in only a limited swelling of mitochondria and diminished the rate of swelling produced by high Ca2+ load.     

Opening of the mitochondrial permeability transition pore causes depletion of mitochondrial and cytosolic NAD+ and is a causative event in the death of myocytes in postischemic reperfusion of the heart

The opening of the mitochondrial permeability transition pore (PTP) has been suggested to play a key role in various forms of cell death, but direct evidence in intact tissues is still lacking. We found that in the rat heart, 92% of NAD(+) glycohydrolase activity is associated with mitochondria. This activity was not modified by the addition of Triton X-100, although it was abolished by mild treatment with the protease Nagarse, a condition that did not affect the energy-linked properties of mitochondria. The addition of Ca(2+) to isolated rat heart mitochondria resulted in a profound decrease in their NAD(+) content, which followed mitochondrial swelling. Cyclosporin A(CsA), a PTP inhibitor, completely prevented NAD(+) depletion but had no effect on the glycohydrolase activity. Thus, in isolated mitochondria PTP opening makes NAD(+) available for its enzymatic hydrolysis. Perfused rat hearts subjected to global ischemia for 30 min displayed a 30% decrease in tissue NAD(+) content, which was not modified by extending the duration of ischemia. Reperfusion resulted in a more severe reduction of both total and mitochondrial contents of NAD(+), which could be measured in the coronary effluent together with lactate dehydrogenase. The addition of 0.2 microm CsA or of its analogue MeVal-4-Cs (which does not inhibit calcineurin) maintained higher NAD(+) contents, especially in mitochondria, and significantly protected the heart from reperfusion damage, as shown by the reduction in lactate dehydrogenase release. Thus, upon reperfusion after prolonged ischemia, PTP opening in the heart can be documented as a CsA-sensitive release of NAD(+), which is then partly degraded by glycohydrolase and partly released when sarcolemmal integrity is compromised. These results demonstrate that PTP opening is a causative event in reperfusion damage of the heart.     

Silencing of cardiac mitochondrial NHE1 prevents mitochondrial permeability transition pore opening

Inhibition of Na(+)/H(+) exchanger 1 (NHE1) reduces cardiac ischemia-reperfusion (I/R) injury and also cardiac hypertrophy and failure. Although the mechanisms underlying these NHE1-mediated effects suggest delay of mitochondrial permeability transition pore (MPTP) opening, and reduction of mitochondrial-derived superoxide production, the possibility of NHE1 blockade targeting mitochondria has been incompletely explored. A short-hairpin RNA sequence mediating specific knock down of NHE1 expression was incorporated into a lentiviral vector (shRNA-NHE1) and transduced in the rat myocardium. NHE1 expression of mitochondrial lysates revealed that shRNA-NHE1 transductions reduced mitochondrial NHE1 (mNHE1) by ～60%, supporting the expression of NHE1 in mitochondria membranes. Electron microscopy studies corroborate the presence of NHE1 in heart mitochondria. Immunostaining of rat cardiomyocytes also suggests colocalization of NHE1 with the mitochondrial marker cytochrome c oxidase. To examine the functional role of mNHE1, mitochondrial suspensions were exposed to increasing concentrations of CaCl(2) to induce MPTP opening and consequently mitochondrial swelling. shRNA-NHE1 transduction reduced CaCl(2)-induced mitochondrial swelling by 64 ± 4%. Whereas the NHE1 inhibitor HOE-642 (10 μM) decreased mitochondrial Ca(2+)-induced swelling in rats transduced with nonsilencing RNAi (37 ± 6%), no additional HOE-642 effects were detected in mitochondria from rats transduced with shRNA-NHE1. We have characterized the expression and function of NHE1 in rat heart mitochondria. Because mitochondria from rats injected with shRNA-NHE1 present a high threshold for MPTP formation, the beneficial effects of NHE1 inhibition in I/R resulting from mitochondrial targeting should be considered.