Ramune Morkuniene

Inhibition of mitochondrial permeability transition prevents mitochondrial dysfunction, cytochrome c release and apoptosis induced by heart ischemia

Ischemia/reperfusion of heart causes contractile dysfunction, necrosis and/or apoptosis and is a major cause of human death, but the molecular mechanisms are unclear. We show that ischemia alone (without reperfusion) is sufficient to induce apoptosis and mitochondrial dysfunction, and we have investigated the mechanism responsible; 30 and 60 min stop-flow ischemia in Langendorff-perfused rat hearts induced progressive (a). release of cytochrome c from mitochondria to cytosol, (b). inhibition of the mitochondrial respiratory functions, (c). activation of caspase-3-like protease activity and (d). DNA strand breaks (however, only 2% of myocyte nuclei were TUNEL positive at 60 min). Fifteen minutes pre-perfusion of hearts with cyclosporin A, an inhibitor of mitochondrial-permeability transition (MPT), largely prevented all these ischemic changes. Pre-perfusion of hearts with FK506, an inhibitor of calcineurin, caused no protection. Pre-perfusion with DEVD-CHO, an inhibitor of caspase-3-like proteases, completely prevented ischemia-induced DNA strand breaks, but only partially blocked cytochrome c release and mitochondrial respiratory inhibition. Reperfusion of hearts after 30 min ischemia further stimulated caspase activity and nuclear apoptosis. We conclude that ischemia-induced MPT causes release of cytochrome c, which then activates the caspases that execute apoptosis and feedback to cause further cytochrome c release. The MPT-induced cytochrome c release is also largely responsible for the ischemic respiratory inhibition, which might contribute to contractile dysfunction or necrosis at reperfusion.

Release of cytochrome c from heart mitochondria is induced by high Ca2+ and peroxynitrite and is responsible for Ca2+-induced inhibition of substrate oxidation

Prolonged heart ischaemia causes an inhibition of oxidative phosphorylation and an increase of Ca2+ in mitochondria. We investigated whether elevated Ca2+ induces changes in the oxidative phosphorylation system relevant to ischaemic damage, and whether Ca2+ and other inducers of mitochondrial permeability transition cause the release of cytochrome c from isolated heart mitochondria. We found that 5 microM free Ca2+ induced changes in oxidative phosphorylation system similar to ischaemic damage: increase in the proton leak and inhibition of the substrate oxidation system related to the release of cytochrome c from mitochondria. The phosphorylating system was not directly affected by high Ca2+ and ischaemia. The release of cytochrome c from mitochondria was caused by Ca2+ and 0.175-0.9 mM peroxynitrite but not by NO, and was prevented by cyclosporin A. Adenylate kinase and creatine kinase were also released after incubation of mitochondria with Ca2+, however, the activity of citrate synthase in the incubation medium with high and low Ca2+ did not change. The data suggest that release of cytochrome c and other proteins of intermembrane space may be due to the opening of the mitochondrial permeability transition pore, and may be partially responsible for inhibition of mitochondrial respiration induced by ischaemia, high calcium, and oxidants.

Size-dependent neurotoxicity of β-amyloid oligomers

The link between the size of soluble amyloid beta (Abeta) oligomers and their toxicity to rat cerebellar granule cells (CGC) was investigated. Variation in conditions during in vitro oligomerization of Abeta(1-42) resulted in peptide assemblies with different particle size as measured by atomic force microscopy and confirmed by dynamic light scattering and fluorescence correlation spectroscopy. Small oligomers of Abeta(1-42) with a mean particle z-height of 1-2 nm exhibited propensity to bind to phospholipid vesicles and they were the most toxic species that induced rapid neuronal necrosis at submicromolar concentrations whereas the bigger aggregates (z-height above 4-5 nm) did not bind vesicles and did not cause detectable neuronal death. A similar neurotoxic pattern was also observed in primary cultures of cortex neurons whereas Abeta(1-42) oligomers, monomers and fibrils were non-toxic to glial cells in CGC cultures or macrophage J774 cells. However, both oligomeric forms of Abeta(1-42) induced reduction of neuronal cell densities in the CGC cultures.

Release of mitochondrial cytochrome c and activation of cytosolic caspases induced by myocardial ischaemia.

It has previously been shown that apoptosis is increased in ischaemic/reperfused heart. However, little is known about the mechanism of induction of apoptosis in myocardium during ischaemia. We investigated whether prolonged myocardial ischaemia causes activation of caspases and whether this activation is related to cytochrome c release from mitochondria to cytosol during ischaemia. Using an in vitro model of heart ischaemia, we show that 60 min ischaemia leads to a significant accumulation of cytochrome c in the cytosol and a decrease in mitochondrial content of cytochrome c but not cytochrome a. The release of cytochrome c from mitochondria was accompanied by activation of caspase-3-like proteases (measured by cleavage of fluorogenic peptide substrate DEVD-amc) and a large increase in number of cells with DNA strand breaks (measured by TUNEL staining). Caspase-1-like proteases (measured by YVAD-amc cleavage) were not activated during ischaemia. Addition of 14 microM cytochrome c to cytosolic extracts prepared from control hearts induced ATP-dependent activation of caspase-3-like protease activity. Our data suggest that extended heart ischaemia can cause apoptosis mediated by release of cytochrome c from mitochondria and subsequent activation of caspase-3.

Nitric oxide donors, nitrosothiols and mitochondrial respiration inhibitors induce caspase activation by different mechanisms.

We investigated to what extent different types of NO donors induce caspase activation by opening of the mitochondrial permeability transition pore (PTP) or inhibition of mitochondrial respiration. We found that nitrosothiols can directly open the PTP in isolated mitochondria and cause cytochrome c release, whereas NONOate donors can not. In macrophages nitrosothiols cause caspase activation that is blocked by cyclosporin A or calcium chelation, both of which prevent PTP opening, whereas caspase activation caused by NONOates is much less sensitive to these agents. Inhibitors of mitochondrial respiration did not promote PTP opening in isolated mitochondria, and although they cause caspase activation in macrophages, this activation was slower than that caused by NO donors, and was relatively insensitive to cyclosporin and calcium chelators suggesting that PTP opening was not involved.

Estrogens prevent calcium-induced release of cytochrome c from heart mitochondria

We investigated the effect of estrogens on heart mitochondrial functions and whether estrogens can prevent calcium‐induced release of cytochrome c from mitochondria. 10 nM–10 μM 17β‐estradiol or 4‐hydroxytamoxifen did not affect mitochondrial respiration rate and membrane potential in state 3 and state 4. Higher concentrations of both agents decreased state 3 respiration rate and membrane potential. 100 nM 17β‐estradiol and 4‐hydroxytamoxifen blocked high calcium‐induced cytochrome c release from mitochondria but not mitochondrial swelling. Thus, at physiological concentrations estrogens do not affect mitochondrial respiratory functions but protect heart mitochondria from high calcium‐induced release of cytochrome c.

Nitric oxide protects the heart from ischemia-induced apoptosis and mitochondrial damage via protein kinase G mediated blockage of permeability transition and cytochrome c release

BackgroundHeart ischemia can rapidly induce apoptosis and mitochondrial dysfunction via mitochondrial permeability transition-induced cytochrome c release. We tested whether nitric oxide (NO) can block this damage in isolated rat heart, and, if so, by what mechanisms.MethodsHearts were perfused with 50 μM DETA/NO (NO donor), then subjected to 30 min stop-flow ischemia or ischemia/reperfusion. Isolated heart mitochondria were used to measure the rate of mitochondrial oxygen consumption and membrane potential using oxygen and tetraphenylphosphonium-selective electrodes. Mitochondrial and cytosolic cytochrome c levels were measured spectrophotometrically and by ELISA. The calcium retention capacity of isolated mitochondria was measured using the fluorescent dye Calcium Green-5N. Apoptosis and necrosis were evaluated by measuring the activity of caspase-3 in cytosolic extracts and the activity of lactate dehydrogenase in perfusate, respectively.Results30 min ischemia caused release of mitochondrial cytochrome c to the cytoplasm, inhibition of the mitochondrial respiratory chain, and stimulation of mitochondrial proton permeability. 3 min perfusion with 50 μM DETA/NO of hearts prior to ischemia decreased this mitochondrial damage. The DETA/NO-induced blockage of mitochondrial cytochrome c release was reversed by a protein kinase G (PKG) inhibitor KT5823, or soluble guanylate cyclase inhibitor ODQ or protein kinase C inhibitors (Ro 32-0432 and Ro 31-8220). Ischemia also stimulated caspase-3-like activity, and this was substantially reduced by pre-perfusion with DETA/NO. Reperfusion after 30 min of ischemia caused no further caspase activation, but was accompanied by necrosis, which was completely prevented by DETA/NO, and this protection was blocked by the PKG inhibitor. Incubation of isolated heart mitochondria with activated PKG blocked calcium-induced mitochondrial permeability transition and cytochrome c release. Perfusion of non-ischemic heart with DETA/NO also made the subsequently isolated mitochondria resistant to calcium-induced permeabilisation, and this protection was blocked by the PKG inhibitor.ConclusionThe results indicate that NO rapidly protects the ischemic heart from apoptosis and mitochondrial dysfunction via PKG-mediated blockage of mitochondrial permeability transition and cytochrome c release.

Estradiol prevents release of cytochrome c from mitochondria and inhibits ischemia-induced apoptosis in perfused heart.

The study investigated whether estradiol can prevent release of cytochrome c from mitochondria and induction of apoptosis after 30 and 60 min stop-flow heart ischemia in Langendorff-perfused female rat hearts. Pre-perfusion of hearts with 100 nM 17beta-estradiol prevented the loss of cytochrome c from mitochondria, its accumulation in cytosol, and inhibition of respiration during ischemia. Estradiol strongly reduced activation of caspase-3-like activity and decreased DNA strand breaks in the nuclei of cardiomyocytes (measured by TUNEL staining). The results show that 17beta-estradiol prevents the ischemia-induced release of cytochrome c from mitochondria, subsequent inhibition of mitochondrial respiration, and inhibits caspase activation and apoptosis. Therefore, inhibition of the intrinsic, mitochondria-mediated apoptotic pathway may be one of the mechanisms by which estrogens protect the heart against ischemic damage.

Estradiol-induced protection against ischemia-induced heart mitochondrial damage and caspase activation is mediated by protein kinase G.

We have previously reported that estradiol can protect heart mitochondria from the ischemia-induced mitochondrial permeability transition pore-related release of cytochrome c and subsequent apoptosis. In this study we investigated whether the effect of 17-beta-estradiol on ischemia-induced mitochondrial dysfunctions and apoptosis is mediated by activation of signaling protein kinases in a Langendorff-perfused rat heart model of stop-flow ischemia. We found that pre-perfusion of non-ischemic hearts with 100nM estradiol increased the resistance of subsequently isolated mitochondria to the calcium-induced opening of mitochondrial permeability transition pore and this was mediated by protein kinase G. Loading of the hearts with estradiol prevented ischemia-induced loss of cytochrome c from mitochondria and respiratory inhibition and these effects were reversed in the presence of the inhibitor of Akt kinase, NO synthase inhibitor L-NAME, guanylyl cyclase inhibitor ODQ and protein kinase G inhibitor KT5823. Estradiol prevented ischemia-induced activation of caspases and this was also reversed by KT5823. These findings suggest that estradiol may protect the heart against ischemia-induced injury activating the signaling cascade which involves Akt kinase, NO synthase, guanylyl cyclase and protein kinase G, and results in blockage of mitochondrial permeability transition pore-induced release of cytochrome c from mitochondria, respiratory inhibition and activation of caspases.

Small Aβ1–42 oligomer-induced membrane depolarization of neuronal and microglial cells: Role of N-methyl-D-aspartate receptors

Although it is well documented that soluble beta amyloid (Aβ) oligomers are critical factors in the pathogenesis of Alzheimers disease (AD) by causing synaptic dysfunction and neuronal death, the primary mechanisms by which Aβ oligomers trigger neurodegeneration are not entirely understood. We sought to investigate whether toxic small Aβ1–42 oligomers induce changes in plasma membrane potential of cultured neurons and glial cells in rat cerebellar granule cell cultures leading to neuronal death and whether these effects are sensitive to the N‐methyl‐D‐aspartate receptor (NMDA‐R) antagonist MK801. We found that small Aβ1–42 oligomers induced rapid, protracted membrane depolarization of both neurons and microglia, whereas there was no change in membrane potential of astrocytes. MK801 did not modulate Aβ‐induced neuronal depolarization. In contrast, Aβ1−42 oligomer‐induced decrease in plasma membrane potential of microglia was prevented by MK801. Small Aβ1–42 oligomers significantly elevated extracellular glutamate and caused neuronal necrosis, and both were prevented by MK801. Also, small Aβ1–42 oligomers decreased resistance of isolated brain mitochondria to calcium‐induced opening of mitochondrial permeability transition pore. In conclusion, the results suggest that the primary effect of toxic small Aβ oligomers on neurons is rapid, NMDA‐R‐independent plasma membrane depolarization, which leads to neuronal death. Aβ oligomers‐induced depolarization of microglial cells is NMDA‐R dependent.