Role of mitochondrial calcium uniporter‐mediated Ca2+ and iron accumulation in traumatic brain injury

Previous studies have suggested that the cellular Ca²⁺ and iron homeostasis, which can be regulated by mitochondrial calcium uniporter (MCU), is associated with oxidative stress, apoptosis and many neurological diseases. However, little is known about the role of MCU‐mediated Ca²⁺ and iron accumulation in traumatic brain injury (TBI). Under physiological conditions, MCU can be inhibited by ruthenium red (RR) and activated by spermine (Sper). In the present study, we used RR and Sper to reveal the role of MCU in mouse and neuron TBI models. Our results suggested that the Ca²⁺ and iron concentrations were obviously increased after TBI. In addition, TBI models showed a significant generation of reactive oxygen species (ROS), decrease in adenosine triphosphate (ATP), deformation of mitochondria, up‐regulation of deoxyribonucleic acid (DNA) damage and increase in apoptosis. Blockage of MCU by RR prevented Ca²⁺ and iron accumulation, abated the level of oxidative stress, improved the energy supply, stabilized mitochondria, reduced DNA damage and decreased apoptosis both in vivo and in vitro. Interestingly, Sper did not increase cellular Ca²⁺ and iron concentrations, but suppressed the Ca²⁺ and iron accumulation to benefit the mice in vivo. However, Sper had no significant impact on TBI in vitro. Taken together, our data demonstrated for the first time that blockage of MCU‐mediated Ca²⁺ and iron accumulation was essential for TBI. These findings indicated that MCU could be a novel therapeutic target for treating TBI.     

A critical role of the transient receptor potential melastatin 2 channel in a positive feedback mechanism for reactive oxygen species-induced delayed cell death

Transient receptor potential melastatin 2 (TRPM2) channel activation by reactive oxygen species (ROS) plays a critical role in delayed neuronal cell death, responsible for postischemia brain damage via altering intracellular Zn²⁺ homeostasis, but a mechanistic understanding is still lacking. Here, we showed that H₂O₂ induced neuroblastoma SH-SY5Y cell death with a significant delay, dependently of the TRPM2 channel and increased [Zn²⁺]_i, and therefore used this cell model to investigate the mechanisms underlying ROS-induced TRPM2-mediated delayed cell death. H₂O₂ increased concentration-dependently the [Zn²⁺]_i and caused lysosomal dysfunction and Zn²⁺ loss and, furthermore, mitochondrial Zn²⁺ accumulation, fragmentation, and ROS generation. Such effects were suppressed by preventing poly(adenosine diphosphate ribose, ADPR) polymerase-1-dependent TRPM2 channel activation with PJ34 and 3,3′,5,5′-tetra-tert-butyldiphenoquinone, inhibiting the TRPM2 channel with 2-aminoethoxydiphenyl borate (2-APB) and N-(p-amylcinnamoyl)anthranilic acid, or chelating Zn²⁺ with N,N,N,N-tetrakis(2-pyridylmethyl)-ethylenediamine (TPEN). Bafilomycin-induced lysosomal dysfunction also resulted in mitochondrial Zn²⁺ accumulation, fragmentation, and ROS generation that were inhibited by PJ34 or 2-APB, suggesting that these mitochondrial events are TRPM2 dependent and sequela of lysosomal dysfunction. Mitochondrial TRPM2 expression was detected and exposure to ADPR-induced Zn²⁺ uptake in isolated mitochondria, which was prevented by TPEN. H₂O₂-induced delayed cell death was inhibited by apocynin and diphenyleneiodonium, nicotinamide adenine dinucleotide phosphate hydrogen (NADPH) oxidase (NOX) inhibitors, GKT137831, an NOX1/4-specific inhibitor, or Gö6983, a protein kinase C (PKC) inhibitor. Moreover, inhibition of PKC/NOX prevented H₂O₂-induced ROS generation, lysosomal dysfunction and Zn²⁺ release, and mitochondrial Zn²⁺ accumulation, fragmentation and ROS generation. Collectively, these results support a critical role for the TRPM2 channel in coupling PKC/NOX-mediated ROS generation, lysosomal Zn²⁺ release, and mitochondrial Zn²⁺ accumulation, and ROS generation to form a vicious positive feedback signaling mechanism for ROS-induced delayed cell death.     

Regulation of mitochondrial fission by intracellular Ca2+ in rat ventricular myocytes

Mitochondria are dynamic organelles that constantly undergo fission, fusion, and movement. Increasing evidence indicates that these dynamic changes are intricately related to mitochondrial function, suggesting that mitochondrial form and function are linked. Calcium (Ca²⁺) is one signal that has been shown to both regulate mitochondrial fission in various cell types and stimulate mitochondrial enzymes involved in ATP generation. However, although Ca²⁺ plays an important role in adult cardiac muscle cells for excitation–metabolism coupling, little is known about whether Ca²⁺ can regulate their mitochondrial morphology. Therefore, we tested the role of Ca²⁺ in regulating cardiac mitochondrial fission. We found that neonatal and adult cardiomyocyte mitochondria undergo rapid and transient fragmentation upon a thapsigargin (TG)- or KCl-induced cytosolic Ca²⁺ increase. The mitochondrial fission protein, DLP1, participates in this mitochondrial fragmentation, suggesting that cardiac mitochondrial fission machinery may be regulated by intracellular Ca²⁺ signaling. Moreover, the TG-induced fragmentation was also associated with an increase in reactive oxygen species (ROS) formation, suggesting that activation of mitochondrial fission machinery is an early event for Ca²⁺-mediated ROS generation in cardiac myocytes. These results suggest that Ca²⁺, an important regulator of muscle contraction and energy generation, also dynamically regulates mitochondrial morphology and ROS generation in cardiac myocytes.     

Mitochondrial Calcium Uniporter MCU Supports Cytoplasmic Ca2+ Oscillations,Store-Operated Ca2+ Entry and Ca2+-Dependent Gene Expression in Response to Receptor Stimulation

Ca²⁺ flux into mitochondria is an important regulator of cytoplasmic Ca²⁺ signals, energy production and cell death pathways. Ca²⁺ uptake can occur through the recently discovered mitochondrial uniporter channel (MCU) but whether the MCU is involved in shaping Ca²⁺ signals and downstream responses to physiological levels of receptor stimulation is unknown. Here, we show that modest stimulation of leukotriene receptors with the pro-inflammatory signal LTC₄ evokes a series of cytoplasmic Ca²⁺ oscillations that are rapidly and faithfully propagated into mitochondrial matrix. Knockdown of MCU or mitochondrial depolarisation, to reduce the driving force for Ca²⁺ entry into the matrix, prevents the mitochondrial Ca²⁺ rise and accelerates run down of the oscillations. The loss of cytoplasmic Ca²⁺ oscillations appeared to be a consequence of enhanced Ca²⁺-dependent inactivation of InsP₃ receptors, which arose from the loss of mitochondrial Ca²⁺ buffering. Ca²⁺ dependent gene expression in response to leukotriene receptor activation was suppressed following knockdown of the MCU. In addition to buffering Ca²⁺ release, mitochondria also sequestrated Ca²⁺ entry through store-operated Ca²⁺ channels and this too was prevented following loss of MCU. MCU is therefore an important regulator of physiological pulses of cytoplasmic Ca²⁺.     

Cytosolic and mitochondrial Ca2+ concentrations in primary hepatocytes change with ageing and in consequence of an mtDNA mutation

Mitochondrial Ca²⁺ flux is crucial for the regulation of cell metabolism. Ca²⁺ entry to the mitochondrial matrix is mediated by VDAC1 and MCU with its regulatory molecules. We investigated hepatocytes isolated from conplastic C57BL/6NTac-mt^NODLtJ mice (mtNOD) that differ from C57BL/6NTac mice (controls) by a point mutation in mitochondrial-encoded subunit 3 of cytochrome c oxidase, resulting in functional and morphological mitochondrial adaptations. Mice of both strains up to 12 months old were compared using mitochondrial GEM-GECO1 and cytosolic CAR-GECO1 expression to gain knowledge of age-dependent alterations of Ca²⁺ concentrations. In controls we observed a significant increase in glucose-induced cytosolic Ca²⁺ concentration with ageing, but only a minor elevation in mitochondrial Ca²⁺ concentration. Conversely, glucose-induced mitochondrial Ca²⁺ concentration significantly declined with ageing in mtNOD mice, paralleled by a slight decrease in cytosolic Ca²⁺ concentration. This was consistent with a significant reduction of the MICU1 to MCU expression ratio and a decline in MCUR1. Our results can best be explained in terms of the adaptation of Ca²⁺ concentrations to the mitochondrial network structure. In the fragmented mitochondrial network of ageing controls there is a need for high cytosolic Ca²⁺ influx, because only some of the isolated mitochondria are in direct contact with the endoplasmic reticulum. This is not important in the hyper-fused elongated mitochondrial network found in ageing mtNOD mice which facilitates rapid Ca²⁺ distribution over a large mitochondrial area.     

A comparison of Zn2+- and Ca2+-triggered depolarization of liver mitochondria reveals no evidence of Zn2+-induced permeability transition

Intracellular Zn²⁺ toxicity is associated with mitochondrial dysfunction. Zn²⁺ depolarizes mitochondria in assays using isolated organelles as well as cultured cells. Some reports suggest that Zn²⁺-induced depolarization results from the opening of the mitochondrial permeability transition pore (mPTP). For a more detailed analysis of this relationship, we compared Zn²⁺-induced depolarization with the effects of Ca²⁺ in single isolated rat liver mitochondria monitored with the potentiometric probe rhodamine 123. Consistent with previous work, we found that relatively low levels of Ca²⁺ caused rapid, complete and irreversible loss of mitochondrial membrane potential, an effect that was diminished by classic inhibitors of mPT, including high Mg²⁺, ADP and cyclosporine A. Zn²⁺ also depolarized mitochondria, but only at relatively high concentrations. Furthermore Zn²⁺-induced depolarization was slower, partial and sometimes reversible, and was not affected by inhibitors of mPT. We also compared the effects of Ca²⁺ and Zn²⁺ in a calcein-retention assay. Consistent with the well-documented ability of Ca²⁺ to induce mPT, we found that it caused rapid and substantial loss of matrix calcein. In contrast, calcein remained in Zn²⁺-treated mitochondria. Considered together, our results suggest that Ca²⁺ and Zn²⁺ depolarize mitochondria by considerably different mechanisms, that opening of the mPTP is not a direct consequence of Zn²⁺-induced depolarization, and that Zn²⁺ is not a particularly potent mitochondrial inhibitor.     

Oxidative Stress Underlies the Mechanism for Ca2+-induced Permeability Transition of Mitochondria

Recent studies demonstrated that the generation of intracellular reactive oxygen species (ROS) was enhanced prior to the onset of mitochondrial membrane permeability transition (MPT), a critical step for the induction of DNA fragmentation and apoptosis. Although Ca²⁺ induces typical MPT that involves depolarization and swelling of mitochondria and finally releases cytochrome c into cytosol, the mechanism by which ROS induce MPT remains unclear. In the presence of inorganic phosphate, Ca²⁺ increased the oxygen consumption and ROS production by isolated mitochondria as determined by a chemiluminescence (CHL) method using L-012. Ca²⁺ increased the generation of H²O² by some mechanism that was inhibited by cyclosporin A but not by superoxide dismutase (SOD) and trifluoperazine. Ca²⁺ decreased the content of free thiols in adenine nucleotide translocase (ANT) in mitochondrial membranes with concomitant increase in ROS generation. The presence of cyclosporin A, trifluoperazine, or SOD inhibited the Ca²⁺-induced increase of L-012 CHL and decrease in the free thiols of ANT. These results indicate that Ca²⁺ increases the generation of ROS which oxidize the free thiol groups in mitochondrial ANT, thereby inducing MPT to release cytochrome c.     

Subsarcolemmal mitochondrial flashes induced by hypochlorite stimulation in cardiac myocytes

Abstract

Mitochondrial superoxide flash (mitoflash) reflects quantal and bursting superoxide production and concurrent membrane depolarization triggered by transient mitochondrial permeability transition in many types of cells, at the level of single mitochondria. Here we investigate reactive oxygen species (ROS)-mediated modulation of mitoflash activity in cardiac myocytes and report a surprising finding that hypochlorite ions potently and preferentially triggered mitoflashes in the subsarcolemmal mitochondria (SSM), whereas hydrogen peroxide (H₂O₂) elicited mitoflash activity uniformly among SSM and interfibrillar mitochondria (IFM). The striking SSM mitoflash response to hypochlorite stimulation remained intact in cardiac myocytes from NOX2-deficient mice, excluding local NOX2-mediated ROS as the major player. Furthermore, it occurred concomitantly with SSM Ca²⁺ accumulation and local Ca²⁺ and CaMKII signaling played an important modulatory role by altering frequency and unitary properties of SSM mitoflashes. These findings underscore the functional heterogeneity of SSM and IFM and the oxidant-specific responsiveness of mitochondria to ROS, and may bear important ramifications in devising therapeutic strategies for the treatment of oxidative stress-related heart diseases.     

Hormonal stimulation, mitochondrial Ca2+ accumulation, and the control of the mitochondrial permeability transition in intact hepatocytes

Ca²⁺ functions as an intracellular signal to transfer hormonal messages to different cellular compartments, including mitochondria, where it activates intramitochondrial Ca²⁺-dependent enzymes. However, excessive mitochondrial Ca²⁺ uptake can promote the mitochondrial permeability transition (MPT), a process known to be associated with cell injury. The factors controlling mitochondrial Ca²⁺ uptake and release in intact cells are poorly understood. In this paper, we investigate mitochondrial Ca²⁺ accumulation in intact hepatocytes in response to the elevation of cytosolic Ca²⁺ levels ([Ca²⁺]_c) induced either by a hormonal stimulus (vasopressin), or by thapsigargin, an inhibitor of the endoplasmic reticulum Ca²⁺ pump. After stimulation, cells were rapidly permeabilized for the determination of the mitochondrial Ca²⁺ content (Ca^2+_m) and to analyze the susceptibility of the mitochondria to undergo the MPT. Despite very similar levels of [Ca²⁺]_c elevation, vasopressin and thapsigargin had markedly different effects on mitochondrial Ca²⁺ accumulation. Vasopressin caused a rapid (< 90 sec), but modest (< 2 fold) increase in Ca²⁺m that was not further increased during prolonged incubations, despite a sustained [Ca²⁺]_c elevation. By contrast, thapsigargin induced a net Ca²⁺ accumulation in mitochondria that continued for up to 30 min and reached Ca^2+_m levels 10–20 fold over basal. Accumulation of mitochondrial Ca²⁺ was accompanied by a markedly increased susceptibility to undergo the MPT. Both mitochondrial Ca²⁺ accumulation and MPT activation were modulated by treatment of the cells with inhibitors of protein kineses and phosphatases. The results indicate that net mitochondrial Ca²⁺ uptake in response to hormonal stimulation is regulated by processes that depend on protein kinase activation. These controls are inoperative when the cytosol is flooded by Ca²⁺ through artificial means, enabling mitochondria to function as a Ca²⁺ sink under these conditions. (Mol Cell Biochem 174: 173–179, 1997)     

Transport of Ca2+ and Ca2+-dependent permeability transition in heart mitochondria in the early stages of Duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD) is a progressive skeletal muscle disease that is associated with severe cardiac complications in the late stages. Significant mitochondrial dysfunction is reportedly responsible for the development of cardiomyopathy with age. At the same time, adaptive changes in mitochondrial metabolism in cardiomyocytes were identified in the early stages of DMD. In this work, we evaluate the functioning of calcium transport systems (MCU and NCLX), and MPT pore in the heart mitochondria of young dystrophin-deficient mice. As compared to wild-type animals, heart mitochondria of mdx mice have been found to be more efficient both in respect to Ca²⁺ uniport and Na⁺-dependent Ca²⁺ efflux. The data obtained indicate that the increased rate of Ca²⁺ uptake by heart mitochondria of mdx mice may be due to an increase in the ratio of MCU and MCUb subunits. In turn, an increase in the rate of Ca²⁺ efflux from organelles in DMD may be the result of a significant increase in the level of NCLX. Moreover, the heart mitochondria of mdx mice were more resistant to MPT pore opening, which may be due to an increase in the microviscosity of mitochondrial membranes of DMD mice. At the same time, the level of putative MPT pore proteins did not change. The paper discusses the effect of rearrangements of the mitochondrial proteome involved in the transport and accumulation of calcium on the adaptation of this organ to DMD.     

Mitochondrial Ca2+ Uptake 1 (MICU1) and Mitochondrial Ca2+ Uniporter (MCU) Contribute to Metabolism-Secretion Coupling in Clonal Pancreatic ��-Cells

In pancreatic β-cells, uptake of Ca²⁺ into mitochondria facilitates metabolism-secretion coupling by activation of various matrix enzymes, thus facilitating ATP generation by oxidative phosphorylation and, in turn, augmenting insulin release. We employed an siRNA-based approach to evaluate the individual contribution of four proteins that were recently described to be engaged in mitochondrial Ca²⁺ sequestration in clonal INS-1 832/13 pancreatic β-cells: the mitochondrial Ca²⁺ uptake 1 (MICU1), mitochondrial Ca²⁺ uniporter (MCU), uncoupling protein 2 (UCP2), and leucine zipper EF-hand-containing transmembrane protein 1 (LETM1). Using a FRET-based genetically encoded Ca²⁺ sensor targeted to mitochondria, we show that a transient knockdown of MICU1 or MCU diminished mitochondrial Ca²⁺ uptake upon both intracellular Ca²⁺ release and Ca²⁺ entry via L-type channels. In contrast, knockdown of UCP2 and LETM1 exclusively reduced mitochondrial Ca²⁺ uptake in response to either intracellular Ca²⁺ release or Ca²⁺ entry, respectively. Therefore, we further investigated the role of MICU1 and MCU in metabolism-secretion coupling. Diminution of MICU1 or MCU reduced mitochondrial Ca²⁺ uptake in response to d-glucose, whereas d-glucose-triggered cytosolic Ca²⁺ oscillations remained unaffected. Moreover, d-glucose-evoked increases in cytosolic ATP and d-glucose-stimulated insulin secretion were diminished in MICU1- or MCU-silenced cells. Our data highlight the crucial role of MICU1 and MCU in mitochondrial Ca²⁺ uptake in pancreatic β-cells and their involvement in the positive feedback required for sustained insulin secretion.     

Regulation of the Na+/Ca2+ Exchanger by Pyridine Nucleotide Redox Potential in Ventricular Myocytes

The cardiac Na⁺/Ca²⁺ exchanger (NCX) is the major Ca²⁺ efflux pathway on the sarcolemma, counterbalancing Ca²⁺ influx via L-type Ca²⁺ current during excitation-contraction coupling. Altered NCX activity modulates the sarcoplastic reticulum Ca²⁺ load and can contribute to abnormal Ca²⁺ handling and arrhythmias. NADH/NAD⁺ is the main redox couple controlling mitochondrial energy production, glycolysis, and other redox reactions. Here, we tested whether cytosolic NADH/NAD⁺ redox potential regulates NCX activity in adult cardiomyocytes. NCX current (I_NCX), measured with whole cell patch clamp, was inhibited in response to cytosolic NADH loaded directly via pipette or increased by extracellular lactate perfusion, whereas an increase of mitochondrial NADH had no effect. Reactive oxygen species (ROS) accumulation was enhanced by increasing cytosolic NADH, and NADH-induced I_NCX inhibition was abolished by the H₂O₂ scavenger catalase. NADH-induced ROS accumulation was independent of mitochondrial respiration (rotenone-insensitive) but was inhibited by the flavoenzyme blocker diphenylene iodonium. NADPH oxidase was ruled out as the effector because I_NCX was insensitive to cytosolic NADPH, and NADH-induced ROS and I_NCX inhibition were not abrogated by the specific NADPH oxidase inhibitor gp91ds-tat. This study reveals a novel mechanism of NCX regulation by cytosolic NADH/NAD⁺ redox potential through a ROS-generating NADH-driven flavoprotein oxidase. The mechanism is likely to play a key role in Ca²⁺ homeostasis and the response to alterations in the cytosolic pyridine nucleotide redox state during ischemia-reperfusion or other cardiovascular diseases.     

Keeping zombies alive: The ER-mitochondria Ca2+ transfer in cellular senescence

Cellular senescence generates a permanent cell cycle arrest, characterized by apoptosis resistance and a pro-inflammatory senescence-associated secretory phenotype (SASP). Physiologically, senescent cells promote tissue remodeling during development and after injury. However, when accumulated over a certain threshold as happens during aging or after cellular stress, senescent cells contribute to the functional decline of tissues, participating in the generation of several diseases. Cellular senescence is accompanied by increased mitochondrial metabolism. How mitochondrial function is regulated and what role it plays in senescent cell homeostasis is poorly understood. Mitochondria are functionally and physically coupled to the endoplasmic reticulum (ER), the major calcium (Ca²⁺) storage organelle in mammalian cells, through special domains known as mitochondria-ER contacts (MERCs). In this domain, the release of Ca²⁺ from the ER is mainly regulated by inositol 1,4,5-trisphosphate receptors (IP3Rs), a family of three Ca²⁺ release channels activated by a ligand (IP3). IP3R-mediated Ca²⁺ release is transferred to mitochondria through the mitochondrial Ca²⁺ uniporter (MCU), where it modulates the activity of several enzymes and transporters impacting its bioenergetic and biosynthetic function. Here, we review the possible connection between ER to mitochondria Ca²⁺ transfer and senescence.Understanding the pathways that contribute to senescence is essential to reveal new therapeutic targets that allow either delaying senescent cell accumulation or reduce senescent cell burden to alleviate multiple diseases.     

Mitochondrial Ca<Superscript>2+</Superscript> uptake pathways

Calcium (Ca²⁺) plays diverse roles in all living organisms ranging from bacteria to humans. It is a structural element for bones, an essential mediator of excitation-contraction coupling, and a universal second messenger in the regulation of ion channel, enzyme and gene expression activities. In mitochondria, Ca²⁺ is crucial for the control of energy production and cellular responses to metabolic stress. Ca²⁺ uptake by the mitochondria occurs by the uniporter mechanism. The Mitochondrial Ca2+ Uniporter (MCU) protein has recently been identified as a core component responsible for mitochondrial Ca²⁺ uptake. MCU knockout (MCU KO) studies have identified a number of important roles played by this high capacity uptake pathway. Interestingly, this work has also shown that MCU-mediated Ca²⁺ uptake is not essential for vital cell functions such as muscle contraction, energy metabolism and neurotransmission. Although mitochondrial Ca²⁺ uptake was markedly reduced, MCU KO mitochondria still contained low but detectable levels of Ca²⁺. In view of the fundamental importance of Ca²⁺ for basic cell signalling, this finding suggests the existence of other currently unrecognized pathways for Ca²⁺ entry. We review the experimental evidence for the existence of alternative Ca²⁺ influx mechanisms and propose how these mechanisms may play an integral role in mitochondrial Ca²⁺ signalling.     

Molecularly Distinct Routes of Mitochondrial Ca2+ Uptake Are Activated Depending on the Activity of the Sarco/Endoplasmic Reticulum Ca2+ ATPase (SERCA)

The transfer of Ca²⁺ across the inner mitochondrial membrane is an important physiological process linked to the regulation of metabolism, signal transduction, and cell death. While the definite molecular composition of mitochondrial Ca²⁺ uptake sites remains unknown, several proteins of the inner mitochondrial membrane, that are likely to accomplish mitochondrial Ca²⁺ fluxes, have been described: the novel uncoupling proteins 2 and 3, the leucine zipper-EF-hand containing transmembrane protein 1 and the mitochondrial calcium uniporter. It is unclear whether these proteins contribute to one unique mitochondrial Ca²⁺ uptake pathway or establish distinct routes for mitochondrial Ca²⁺ sequestration. In this study, we show that a modulation of Ca²⁺ release from the endoplasmic reticulum by inhibition of the sarco/endoplasmatic reticulum ATPase modifies cytosolic Ca²⁺ signals and consequently switches mitochondrial Ca²⁺ uptake from an uncoupling protein 3- and mitochondrial calcium uniporter-dependent, but leucine zipper-EF-hand containing transmembrane protein 1-independent to a leucine zipper-EF-hand containing transmembrane protein 1- and mitochondrial calcium uniporter-mediated, but uncoupling protein 3-independent pathway. Thus, the activity of sarco/endoplasmatic reticulum ATPase is significant for the mode of mitochondrial Ca²⁺ sequestration and determines which mitochondrial proteins might actually accomplish the transfer of Ca²⁺ across the inner mitochondrial membrane. Moreover, our findings herein support the existence of distinct mitochondrial Ca²⁺ uptake routes that might be essential to ensure an efficient ion transfer into mitochondria despite heterogeneous cytosolic Ca²⁺ rises.     

Intracellular targeting of the photoprotein aequorin: A new approach for measuring,in living cells,Ca<Superscript>2+</Superscript> concentrations in defined cellular compartments

We here present a novel method, based on the targeting of the photoprotein aequorin, for measuring the concentration of Ca²⁺ ions in defined cellular compartments of intact cells. In this contribution we will discuss the application to mitochondria. A chimaeric cDNA was constructed by fusing in frame the aequorin cDNA with that for a mitochondrial protein. The cDNA encoded a “mitochondrially-targeted” aequorin, composed of a typical mitochondrial targeting signal at the N-terminus and the photoprotein at the C-terminus. The cDNA, inserted in the expression vector pMT2, was co-transfected into bovine endothelial and HeLa cells together with the selectable plasmid pSV2-neo and stable transfectants, selected for high aequorin production, were analyzed. In subcellular fractionations, aequorin was shown to be localized in mitochondria; in intact cells, the first direct measurement of mitochondrial free Ca²⁺, [Ca²⁺]_m, were obtained, which showed that [Ca²⁺]_m is low at rest (<0.5 μM), but rapidly increases to the micromolar range upon cell stimulation [1]. These data indicate that mitochondria “sense” very accurately the cytosolic Ca²⁺ concentration ([Ca²⁺]_i), and after cell stimulation [Ca²⁺]_m rises to values capable of activating the Ca²⁺-sensitive mitochondrial dehydrogenases.     

ADP requirement for prevention by a cytosolic factor of Mg2+ and Ca2+ release from rat liver mitochondria

One hundred micromolar Ca²⁺ added to rat liver mitochondria induces a transient uptake of Ca²⁺ plus a rapid efflux of the mitochondrial Mg²⁺. Addition of a cytosolic molecule, cytosolic metabolic factor, to mitochondria prevents the efflux of the two divalent cations. ADP is required for this cytosolic metabolic factor action. This requirement for ADP is specific as it is shown by experiments with traps for nucleotides and inhibitors of the translocase. The implication of cytosolic metabolic factor in the mitochondrial regulation process is discussed.     

Mitochondrial dynamics in human NADH:ubiquinone oxidoreductase deficiency

Mitochondrial NADH:ubiquinone oxidoreductase or complex I (CI) is a frequently affected enzyme in cases of mitochondrial disorders. However, the cytopathological mechanism of the associated pediatric syndromes is poorly understood. Evidence in the literature suggests a connection between mitochondrial metabolism and morphology. Previous quantitative analysis of mitochondrial structure in cultured fibroblasts of 14 patients revealed that mitochondria were fragmented and/or less branched in patients with severe CI deficiency. These patient cells also displayed greatly increased levels of reactive oxygen species (ROS) and marked aberrations in mitochondrial and cellular Ca²⁺/ATP handling upon hormone stimulation. Here, we discuss the interrelationship between these parameters and demonstrate that the hormone-induced increase in mitochondrial Ca²⁺ and ATP concentration, as well as the rate of cytosolic Ca²⁺ removal, are not related to mitochondrial length and/or degree of branching, but decrease as a function of the number of mitochondria per cell. This suggests that the amount of mitochondria, and not their shape, is important for Ca²⁺-induced stimulation of mitochondrial ATP generation to feed cytosolic ATP-demanding processes.     

Translocation of iron from lysosomes to mitochondria during ischemia predisposes to injury after reperfusion in rat hepatocytes

The mitochondrial permeability transition (MPT) initiated by reactive oxygen species (ROS) plays an essential role in ischemia–reperfusion (IR) injury. Iron is a critical catalyst for ROS formation, and intracellular chelatable iron promotes oxidative injury-induced and MPT-dependent cell death in hepatocytes. Accordingly, our aim was to investigate the role of chelatable iron in IR-induced ROS generation, MPT formation, and cell death in primary rat hepatocytes. To simulate IR, overnight-cultured hepatocytes were incubated anoxically at pH 6.2 for 4 h and reoxygenated at pH 7.4. Chelatable Fe²⁺, ROS, and mitochondrial membrane potential were monitored by confocal fluorescence microscopy of calcein, chloromethyldichlorofluorescein, and tetramethylrhodamine methyl ester, respectively. Cell killing was assessed by propidium iodide fluorimetry. Ischemia caused progressive quenching of cytosolic calcein by more than 90%, signifying increased chelatable Fe²⁺. Desferal and starch–desferal 1 h before ischemia suppressed calcein quenching. Ischemia also induced quenching and dequenching of calcein loaded into mitochondria and lysosomes, respectively. Desferal, starch–desferal, and the inhibitor of the mitochondrial Ca²⁺ uniporter (MCU), Ru360, suppressed mitochondrial calcein quenching during ischemia. Desferal, starch–desferal, and Ru360 before ischemia also decreased mitochondrial ROS formation, MPT opening, and cell killing after reperfusion. These results indicate that lysosomes release chelatable Fe²⁺ during ischemia, which is taken up into mitochondria by MCU. Increased mitochondrial iron then predisposes to ROS-dependent MPT opening and cell killing after reperfusion.