Release Of Mitochondrial Ca2 Research Articles

Mitochondrial Ca2+ Homeostasis: Emerging Roles and Clinical Significance in Cardiac Remodeling.

Mitochondria are the sites of oxidative metabolism in eukaryotes where the metabolites of sugars, fats, and amino acids are oxidized to harvest energy. Notably, mitochondria store Ca2+ and work in synergy with organelles such as the endoplasmic reticulum and extracellular matrix to control the dynamic balance of Ca2+ concentration in cells. Mitochondria are the vital organelles in heart tissue. Mitochondrial Ca2+ homeostasis is particularly important for maintaining the physiological and pathological mechanisms of the heart. Mitochondrial Ca2+ homeostasis plays a key role in the regulation of cardiac energy metabolism, mechanisms of death, oxygen free radical production, and autophagy. The imbalance of mitochondrial Ca2+ balance is closely associated with cardiac remodeling. The mitochondrial Ca2+ uniporter (mtCU) protein complex is responsible for the uptake and release of mitochondrial Ca2+ and regulation of Ca2+ homeostasis in mitochondria and consequently, in cells. This review summarizes the mechanisms of mitochondrial Ca2+ homeostasis in physiological and pathological cardiac remodeling and the regulatory effects of the mitochondrial calcium regulatory complex on cardiac energy metabolism, cell death, and autophagy, and also provides the theoretical basis for mitochondrial Ca2+ as a novel target for the treatment of cardiovascular diseases.

Is the C-Subunit Ring of the F1Fo ATP Synthase the Elusive Mitochondrial Permeability Transition Pore?

Certain metabolic conditions induce an increase in the permeability of the mitochondrial inner membrane, which, if prolonged, ultimately leads to cell death. It is believed that at the onset of this so-called permeability transition, the opening of an ion channel causes the inner membrane to become depolarized, which halts ATP production and has other deleterious effects such as the release of mitochondrial Ca2+. The molecular identity of this channel, refered to as Mitochodrial Permeability Transition Pore (MPTP) remains to be conclusively established and has been the subject of much controversy. A recent electrophysiological study purports to have demonstrated that the elusive MPTP is, surprisingly, the lumen of the c-subunit ring of the F1Fo ATP synthase, under conditions in which the catalytic domain, F1, becomes detached from the ring. Here, we examine this claim through theoretical calculations of the ion conductance of two representative c-rings, which differ in their c-subunit stoichiometry and thus in the width of their lumen. These calculations, which are based on fully-atomistic molecular dynamics simulations, clearly demonstrate that the lumen of the c-ring cannot possibly sustain the conductance levels associated with the MPTP, even under the assumption that the lumen is fully hydrated, rather than occupied by lipid molecules.

Abstract 322: Metformin Inhibits Mitochondrial Permeability Transition Pore Opening in Human Cardiac Mitochondria

Background: Type II diabetes mellitus is a major health problem contributing to increased morbidity and mortality with associated cardiovascular diseases. Metformin, an oral antidiabetic agent, has cardioprotective properties independent of their glucose lowering effect; however, mechanisms underlying cardioprotection remain poorly defined. We hypothesized that the cardioprotective effect of metformin appears to be associated with inhibition of mitochondrial permability transition pore (mPTP) that could be beneficial in diabetic hearts with attenuated endogenous cardioprotective responses. Purpose: The aim of the study was to determine the protective effect of metformin on mPTP opening in mitochondria from human myocardium.. Methods: Mitochondria freshly isolated from the left atrial appendage of nondiabetic patients undergoing cardiac surgery were loaded with calcium- (fluo-3) and membrane potential-sensitive (Safranin-O) fluorescent dyes and challenged with sequential pulses of Ca2+ (10 μM every 3 m) in the absence and presence of different concentrations of metformin (2.5, 5, 10 mM). The sensitivity of mitochondria toward mPTP opening was assessed by abrupt release of mitochondrial Ca2+ (fluo-5N fluorescence), with simultaneous dissipation of mitochondrial membrane potential (safranin O fluorescence) and mitochondrial swelling (decrease in light scattering). Results: Metformin caused a dose-dependent inhibition of Ca2+-induced mPTP opening with delayed mitochondrial Ca2+ release, depolarization and swelling. The tolerance of metformin-treated mitochondria to Ca2+-induced mPTP opening when compared to controls was increased from 457±71 to 600±74 nmol/mg protein at 2.5mM, 674±10 nmol/mg protein at 5 mM and 750±77 nmol/mg protein at 10 mM. Cyclosporin A (1 μM), a known mPTP inhibitor, has marginal incremental effect on metformin-induced inhibition of mPTP opening. Conclusion: In human cardiac mitochondria, metformin inhibited calcium-overload-mediated mPTP opening that otherwise leads to mitochondrial energetic failure and cell death. Thus, metformin may help restore attenuated cardioprotection in diabetic hearts by increasing tolerance to stress-induced mPTP opening and, thus, preventing cell death.

Anoxia-mediated calcium release through the mitochondrial permeability transition pore silences NMDA receptor currents in turtle neurons

Mammalian neurons are anoxia sensitive and rapidly undergo excitotoxic cell death when deprived of oxygen, mediated largely by Ca(2+) entry through over-activation of N-methyl-d-aspartate receptors (NMDARs). This does not occur in neurons of the anoxia-tolerant western painted turtle, where a decrease in NMDAR currents is observed with anoxia. This decrease is dependent on a modest rise in cytosolic [Ca(2+)] ([Ca(2+)]c) that is mediated by release from the mitochondria. The aim of this study was to determine whether the mitochondrial permeability transition pore (mPTP) is involved in NMDAR silencing through release of mitochondrial Ca(2+). Opening the mPTP during normoxia with atractyloside decreased NMDAR currents by releasing mitochondrial Ca(2+), indicated by an increase in Oregon Green fluorescence. Conversely, the mPTP blocker cyclosporin A prevented the anoxia-mediated increase in [Ca(2+)]c and reduction in NMDAR currents. Mitochondrial membrane potential (Ψm) was determined using rhodamine-123 fluorescence and decreased with the onset of anoxia in a time frame that coincided with the increase in [Ca(2+)]c. Activation of mitochondrial ATP-sensitive potassium (mK(+)ATP) channels also releases mitochondrial Ca(2+) and we show that activation of mK(+)ATP channels during normoxia with diazoxide leads to Ψm depolarization and inhibition with 5-hydroxydecanoic acid blocked anoxia-mediated Ψm depolarization. Ψm does not collapse during anoxia but rather reaches a new steady-state level that is maintained via ATP hydrolysis by the F1-F0 ATPase, as inhibition with oligomycin depolarizes Ψm further than the anoxic level. We conclude that anoxia activates mK(+)ATP channels, which leads to matrix depolarization, Ca(2+) release via the mPTP, and ultimately silencing of NMDARs.

The antiestrogen endoxifen protects rat liver mitochondria from permeability transition pore opening and oxidative stress at concentrations that do not affect the phosphorylation efficiency

Endoxifen (EDX) is a key active metabolite of tamoxifen (TAM) with higher affinity and specificity to estrogen receptors that also inhibits aromatase activity. It is safe and well tolerated by healthy humans, but its use requires toxicological characterization. In this study, the effects of EDX on mitochondria, the primary targets for xenobiotic-induced toxicity, were monitored to clarify its potential side effects. EDX up to 30nmol/mg protein did not affect the mitochondrial oxidative phosphorylation. At 50nmol EDX/mg protein, EDX decreased the ADP phosphorylation rate and a partial collapse of mitochondrial membrane potential (Δψ), that parallels a state 4 stimulation, was observed. As the stimulation of state 4 was not inhibited by oligomycin and 50nmol EDX/mg protein caused a slight decrease in the light scattering of mitochondria, these data suggest that EDX promotes membrane permeabilization to protons, whereas TAM at the same concentration induced mitochondrial membrane disruption. Moreover, EDX at 10nmol/mg protein prevented and reversed the Ca2+-induced depolarization of ΔΨ and the release of mitochondrial Ca2+, similarly to cyclosporine A, indicating that EDX did not affect Ca2+ uptake, but directly interfered with the proteins of the mitochondrial permeability transition (MPT) megacomplex, inhibiting MPT induction. At this concentration, EDX exhibited antioxidant activity that may account for the protective effect against MPT pore opening. In conclusion, EDX within the range of concentrations reached in tissues did not significantly damage the bioenergetic functions of mitochondria, contrarily to the prodrug TAM, and prevented the MPT pore opening and the oxidative stress in mitochondria, supporting that EDX may be a less toxic drug for women with breast carcinoma.

On the relationship between calcium and phosphate transport, transmembrane potential and acetoacetate-induced oxidation of pyridine nucleotides in rat-liver mitochondria.

Acetoacetate addition to rat liver mitochondria induces a complete oxidation of pyridine nucleotides, a collapse of membrane potential, a release of mitochondrial Ca2+ and a loss of respiratory control only in the presence of external phosphate. Acetoacetate also enhances the efflux of mitochondrial Mg2+ promoted by phosphate. All these effects are not only prevented but also reversed, except the oxidation of pyridine nucleotides, by the combined addition of Mg2+, ADP and dithioerythritol to damaged mitochondria. It is concluded that acetoacetate, through the oxidation of mitochondrial pyridine nucleotides, potentiates the action of phosphate in altering the mitochondrial permeability barrier, which is closely dependent on the maintenance of membrane thiol groups in a reduced form.

Mechanism of estrogen-mediated neuroprotection: regulation of mitochondrial calcium and Bcl-2 expression.

Estrogens are neuroprotective against glutamate excitotoxicity caused by an excessive rise in intracellular calcium ([Ca(2+)](i)). In this study, we demonstrate that 17 beta-estradiol (E(2)) treatment of hippocampal neurons attenuated the excitotoxic glutamate-induced rise in bulk-free [Ca(2+)](i) despite potentiating the influx of Ca(2+) induced by glutamate. E(2)-induced attenuation of bulk-free [Ca(2+)](i) depends on mitochondrial sequestration of Ca(2+), which is blocked in the presence of the combination of rotenone and oligomycin or in the presence of antimycin, which collapse the mitochondrial membrane potential, thereby preventing mitochondrial Ca(2+) transport. Release of mitochondrial Ca(2+) by carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) after excitotoxic glutamate treatment resulted in a greater [Ca(2+)](i) in E(2)-treated cells, indicating an E(2)-induced increase in the mitochondrial calcium ([Ca(2+)](m)) load. The increased [Ca(2+)](m) load was accompanied by increased expression of Bcl-2, which can promote mitochondrial Ca(2+) load tolerance. These findings provide a mechanism of E(2)-induced neuronal survival by attenuation of excitotoxic glutamate [Ca(2+)](i) rise via increased mitochondrial sequestration of cytosolic Ca(2+) coupled with an increase in Bcl-2 expression to sustain mitochondrial Ca(2+) load tolerance and function.

Acute Exposure to Methylmercury Opens the Mitochondrial Permeability Transition Pore in Rat Cerebellar Granule Cells

Cerebellar granule cells are preferentially targeted during methylmercury (MeHg) poisoning. Following acute MeHg exposure, granule cells in culture undergo an increase in intracellular Ca2+ concentration ([Ca2+]i) that apparently contributes to cell death. This effect consists of several temporally and kinetically distinct phases. The initial elevation arises from release of Ca2+i stores; the second phase results from entry of Ca2+e. In these experiments, we tested the hypothesis that release of mitochondrial Ca2+ through the mitochondrial permeability transition pore (MTP) contributes to the initial release of Ca2+i. Neonatal rat cerebellar granule cells in culture and single cell microfluorimetry were used to examine MeHg-induced changes in [Ca2+]i and mitochondrial membrane potential (Ψm). Pretreatment with the MTP inhibitor cyclosporin A (CsA, 5 μM) delayed the initial phase of increased [Ca2+]i induced by 0.2 and 0.5 μM MeHg, but not 1.0 μM MeHg. CsA (5 μM) also delayed the irreversible loss of Ψm induced by 0.5 μM MeHg. Ca2+e was not required for either effect, because the effect of CsA on the first phase increase in [Ca2+]i and loss of Ψm was not altered in nominally Ca2+-free buffer. Increasing concentrations of MeHg (0.2–2.0 μM) caused increasing incidence of cell death at 24 h postexposure. Treatment with CsA provided protection against cytotoxicity at lower MeHg concentrations (0.2–0.5 μM), but not at higher MeHg concentrations (1.0–2.0 μM). Thus, the MTP appears to play a significant role in the cellular effects following acute exposure of cerebellar granule neurons to MeHg.

α-Latrotoxin Releases Calcium in Frog Motor Nerve Terminals

alpha-Latrotoxin (alpha-LTX) is a neurotoxin that accelerates spontaneous exocytosis independently of extracellular Ca(2+). Although alpha-LTX increases spontaneous transmitter release at synapses, the mechanism is unknown. We tested the hypothesis that alpha-LTX causes transmitter release by mobilizing intracellular Ca(2+) in frog motor nerve terminals. Transmitter release was measured electrophysiologically and with the vesicle marker FM1-43; presynaptic ion concentration dynamics were measured with fluorescent ion-imaging techniques. We report that alpha-LTX increases transmitter release after release of a physiologically relevant concentration of intracellular Ca(2+). Neither the blockade of Ca(2+) release nor the depletion of Ca(2+) from endoplasmic reticulum affected Ca(2+) signals produced by alpha-LTX. The Ca(2+) source is likely to be mitochondria, because the effects on Ca(2+) mobilization of CCCP (which depletes mitochondrial Ca(2+)) and of alpha-LTX are mutually occlusive. The release of mitochondrial Ca(2+) is partially attributable to an increase in intracellular Na(+), suggesting that the mitochondrial Na(+)/Ca(2+) exchanger is activated. Effects of alpha-LTX were not blocked when Ca(2+) increases were reduced greatly in saline lacking both Na(+) and Ca(2+) and by application of intracellular Ca(2+) chelators. Therefore, although increases in intracellular Ca(2+) may facilitate the effects of alpha-LTX on transmitter release, these increases do not appear to be necessary. The results show that investigations of Ca(2+)-independent alpha-LTX mechanisms or uses of alpha-LTX to probe exocytosis mechanisms would be complicated by the release of intracellular Ca(2+), which itself can trigger exocytosis.

Arachidonic acid inhibits thyrotropin-releasing hormone-induced elevation of cytoplasmic free calcium in GH3 pituitary cells.

We have shown that arachidonic acid stimulates 45Ca2+ efflux from prelabeled rat pituitary mammotropic (GH3) cells resuspended in "Ca2+-free" medium (Kolesnick, R. N., Mussachio, I., Thaw, C., and Gershengorn, M. C. (1984) Am. J. Physiol. 246, E458-E462). In this study, we further characterize the effects of arachidonic acid on Ca2+ homeostasis in GH3 cells and demonstrate its antagonism of changes induced by thyrotropin-releasing hormone (TRH). At below 5 microM, arachidonic acid stimulated intracellular for extracellular Ca2+ exchange without affecting cell Ca2+ content. Above 5 microM, arachidonic acid decreased membrane-bound Ca2+, as monitored by chlortetracycline, and decreased total cell 45Ca2+ content by depleting nonmitochondrial and mitochondrial pools. However, arachidonic acid did not elevate cytoplasmic free Ca2+ concentration ([Ca2+]i). Arachidonic acid inhibited TRH-induced 45Ca2+ efflux, loss of membrane-bound Ca2+, mobilization of nonmitochondrial Ca2+, and elevation of [Ca2+]i. Arachidonic acid also lowered elevated [Ca2+]i caused by release of mitochondrial Ca2+ with an uncoupler or by influx of extracellular Ca2+ stimulated with K+ depolarization. Hence, arachidonic acid stimulates Ca2+ extrusion from and depletes Ca2+ stores within GH3 cells. We suggest that arachidonic acid may be an important regulator of cellular Ca2+ homeostasis which may inhibit TRH-induced elevation of [Ca2+]i.