Ca Uniporter Research Articles

Intralipid blocks the entry of the SARS-Cov2/COVID/19 virus into cells by maintaining receptor a leucine-rich repeat containing 15 (LRRC15), an angiotensin-converting enzyme 2 receptor competitor.

The effectiveness of Intralipid against SARS-Cov2/COVID/19 with Multiple Organ Dysfunction Syndrome (MODS) prevention or regression is described in the original scientific paper [1]. Intralipid at Oxidative and Nitro-Galogenic stress in patients with SARS-Cov2 / COVID /19, favors the predominance of the membrane-cytoprotective action of Reactive Oxygen Species (ROS) / Reactive Nitrogen Species (ROS/RNS) over the membrane-to-destructive action, restoring the balance between [ROS /AS] / [RNS / ANOS]. Membrane-cytoprotective mechanism Intralipid is due to a decrease in ROS and RNS and an increase in the activity of the Antioxidant System (AS) and Anti Nitro Oxidant System (ANOS), stopping lipid peroxidation (LPO), reducing Electro-Ion Membrane Distress Syndrome (Maria&Irina Vasilieva syndrome) [2], accelerates the regeneration of endothelial and epithelial cells of the alveolar acinus, restoring gas-respiratory metabolism and the predominance of physiological cell apoptosis over necrosis. Intralipid at SARS-Cov 2 / COVID / 19 opposes Microcirculatory Mitochondrial Distress syndrome (MMDS) by Microcirculatory - Mitochondrial Recruitment; as a result of which pCO2 (AVgap) <6 mm Hg, since LPO decreases and at the level of mitochondrial membranes, improving the function of Mitochondrial permeability transition pore-dependent Ca uniporter, mPT pore, support energy metabolism, eliminating energy deficits, restoring Extreme / Abnormal myelopoiesis and impaired autophagy (mitophagy). Thus, Intralipid has been shown in the strategy of targeted treatment of LPO in Oxidative and Nitro-Galogenic stress in patients with SARS-Cov2 /COVID / 19.

Intralipid blocks the entry of the SARS-Cov2/COVID/19 virus into cells by maintaining receptor a leucine-rich repeat containing 15 (LRRC15), an angiotensin-converting enzyme 2 receptor competitor.

The effectiveness of Intralipid against SARS-Cov2/COVID/19 with Multiple Organ Dysfunction Syndrome (MODS) prevention or regression is described in the original scientific paper [1]. Intralipid at Oxidative and Nitro-Galogenic stress in patients with SARS-Cov2 / COVID /19, favors the predominance of the membrane-cytoprotective action of Reactive Oxygen Species (ROS) / Reactive Nitrogen Species (ROS/RNS) over the membrane-to-destructive action, restoring the balance between [ROS /AS] / [RNS / ANOS]. Membrane-cytoprotective mechanism Intralipid is due to a decrease in ROS and RNS and an increase in the activity of the Antioxidant System (AS) and Anti Nitro Oxidant System (ANOS), stopping lipid peroxidation (LPO), reducing Electro-Ion Membrane Distress Syndrome (Maria&Irina Vasilieva syndrome) [2], accelerates the regeneration of endothelial and epithelial cells of the alveolar acinus, restoring gas-respiratory metabolism and the predominance of physiological cell apoptosis over necrosis. Intralipid at SARS-Cov 2 / COVID / 19 opposes Microcirculatory Mitochondrial Distress syndrome (MMDS) by Microcirculatory - Mitochondrial Recruitment; as a result of which pCO2 (AVgap) <6 mm Hg, since LPO decreases and at the level of mitochondrial membranes, improving the function of Mitochondrial permeability transition pore-dependent Ca uniporter, mPT pore, support energy metabolism, eliminating energy deficits, restoring Extreme / Abnormal myelopoiesis and impaired autophagy (mitophagy). Thus, Intralipid has been shown in the strategy of targeted treatment of LPO in Oxidative and Nitro-Galogenic stress in patients with SARS-Cov2 /COVID / 19.

The mitochondrial Ca uniporter is involved in hypomagnesemia-induced mitochondrial dysfunction and diastolic heart failure

The mitochondrial Ca uniporter is involved in hypomagnesemia-induced mitochondrial dysfunction and diastolic heart failure

Abstract P1101: Mitochondrial Ca Uniporter Complex, ROS And CaT Alternans

Background: Atrial calcium transient (CaT) alternans is defined as beat-to-beat alternation in CaT amplitude and is causally linked to atrial fibrillation (AF). Inhibition of mitochondrial Ca uptake via the mitochondrial Ca uniporter complex (MCUC) facilitates CaT alternans and can lead to an increase in mitochondrial reactive oxygen species(ROS) production that compromises the cellular antioxidative capacity. The objective was to determine the mechanisms through which mitochondrial Ca uptake and ROS production determine the susceptibility of atrial cells to develop CaT alternans. Methods: CaT alternans was measured in single rabbit left atrial myocytes with fluorescence microscopy using the fluorescent Ca indicator Cal-520/AM, and for simultaneous measurements of ROS and alternans, cells were loaded with CM-DCFDA and Cal-590/AM. CaT alternans was induced by increasing the pacing frequency until stable CaT alternans was observed. The degree of alternans was quantified as the alternans ratio (AR = 1 – S/L, where S/L is the ratio of the small to the large amplitude of a pair of alternating CaTs). Results: Exposure of LA myocytes to the specific MCUC inhibitor Ru360 increased the AR by 86%. However, the subsequent stimulation of mitochondrial Ca uptake with the MCUC activator spermine (Spm) decreased AR by 79% (p=0.028). Quercetin (a O2 .- scavenger) and Tempol (a superoxide dismutase mimetic) lowered CaT AR by 96% (p=0.0007) and 79% (p=0.0029), respectively. An oxidative challenge with tert-butylhydroperoxide (tBHP) increased AR by 140% (p=<0.0001), and the reducing agent dithiothreitol (DTT) decreased AR by 39% (p=0.0194). Moreover, DTT treatment rescued CaT alternans previously enhanced by tBHP. During CaT alternans the rate of ROS production increased by 23% (p=0.026), however Spm treatment attenuated the increased ROS production. Conclusion: An oxidizing environment facilitates the development of CaT alternans. During CaT alternans mitochondrial ROS formation is increased and involves MCUC activity.

Metabolic adaptation to the chronic loss of Ca2+ signaling induced by KO of IP3 receptors or the mitochondrial Ca2+ uniporter

Calcium signaling is essential for regulating many biological processes. Endoplasmic reticulum inositol trisphosphate receptors (IP3Rs) and the mitochondrial Ca2+ uniporter (MCU) are key proteins that regulate intracellular Ca2+ concentration. Mitochondrial Ca2+ accumulation activates Ca2+-sensitive dehydrogenases of the tricarboxylic acid (TCA) cycle that maintain the biosynthetic and bioenergetic needs of both normal and cancer cells. However, the interplay between calcium signaling and metabolism is not well understood. In this study, we used human cancer cell lines (HEK293 and HeLa) with stable KOs of all three IP3R isoforms (triple KO [TKO]) or MCU to examine metabolic and bioenergetic responses to the chronic loss of cytosolic and/or mitochondrial Ca2+ signaling. Our results show that TKO cells (exhibiting total loss of Ca2+ signaling) are viable, displaying a lower proliferation and oxygen consumption rate, with no significant changes in ATP levels, even when made to rely solely on the TCA cycle for energy production. MCU KO cells also maintained normal ATP levels but showed increased proliferation, oxygen consumption, and metabolism of both glucose and glutamine. However, MCU KO cells were unable to maintain ATP levels and died when relying solely on the TCA cycle for energy. We conclude that constitutive Ca2+ signaling is dispensable for the bioenergetic needs of both IP3R TKO and MCU KO human cancer cells, likely because of adequate basal glycolytic and TCA cycle flux. However, in MCU KO cells, the higher energy expenditure associated with increased proliferation and oxygen consumption makes these cells more prone to bioenergetic failure under conditions of metabolic stress.

SR-Mitochondria Crosstalk Shapes Ca Signalling to Impact Pathophenotype in Disease Models Marked by Dysregulated Intracellular Ca Release.

Diastolic Ca release (DCR) from sarcoplasmic reticulum (SR) Ca release channel ryanodine receptor (RyR2) has been linked to multiple cardiac pathologies, but its exact role in shaping divergent cardiac pathologies remains unclear. We hypothesize that the SR-mitochondria interplay contributes to disease phenotypes by shaping Ca signalling. A genetic model of catecholaminergic polymorphic ventricular tachycardia (CPVT2 model of CASQ2 knockout) and a pre-diabetic cardiomyopathy model of fructose-fed mice (FFD), both marked by DCR, are employed in this study. Mitochondria Ca (mCa) is modulated by pharmacologically targeting mitochondria Ca uniporter (MCU) or permeability transition pore (mPTP), mCa uptake, and extrusion mechanisms, respectively. An MCU activator abolished Ca waves in CPVT2 but exacerbated waves in FFD cells. Mechanistically this is ascribed to mitochondria's function as a Ca buffer or source of reactive oxygen species (mtROS) to exacerbate RyR2 functionality, respectively. Enhancing mCa uptake reduced and elevated mtROS production in CPVT2 and FFD, respectively. In CPVT2, mitochondria took up more Ca in permeabilized cells, and had higher level of mCa content in intact cells vs. FFD. Conditional ablation of MCU in the CPVT2 model caused lethality and cardiac remodelling, but reduced arrhythmias in the FFD model. In parallel, CPVT2 mitochondria also employ up-regulated mPTP-mediated Ca efflux to avoid mCa overload, as seen by elevated incidence of MitoWinks (an indicator of mPTP-mediated Ca efflux) vs. FFD. Both pharmacological and genetic inhibition of mPTP promoted mtROS production and exacerbation of myocyte Ca handling in CPVT2. Further, genetic inhibition of mPTP exacerbated arrhythmias in CPVT2. In contrast to FFD, which is more susceptible to mtROS-dependent RyR2 leak, in CPVT2 mitochondria buffer SR-derived DCR to mitigate Ca-dependent pathological remodelling and rely on mPTP-mediated Ca efflux to avoid mCa overload. SR-mitochondria interplay contributes to the divergent pathologies by disparately shaping intracellular Ca signalling.

Abstract 15737: Deficiency of Mitochondrial Calcium Uniporter Protects Mouse Hearts From Iron Overload by Attenuating Ferroptosis

Iron (Fe) plays essential roles in many physiological processes. Hereditary hemochromatosis and frequent blood transfusions result in iron overload (IO) and dysfunction of iron-deposited organs including the heart. Although IO-induced cardiomyopathy remains a significant clinical challenge, the underlying mechanism is not well defined. In the present study, we aim to assess the involvement of the mitochondrial Ca uniporter (mCU) in IO-induced cardiac contractile dysfunction and ferroptosis. In a chronic IO model, after receiving Fe (dextran, i.p. 0.6 mg/g, 3 days/wk) for 6 weeks, systolic function (LVEF and LVFS) was reduced in mCU +/+ (WT) compared to mCU -/- (mCU KO). This observation was confirmed in isolated ventricular myocytes where we similarly detected a significant decrease in cell shortening in WT, but not mCU KO myocytes. We found lower Fe levels in mitochondria from mCU KO myocytes compared to WT, while observing the same level of Fe deposition in heart tissue from both groups. The mitochondrial ROS level was lower in mCU KO myocytes vs. WT. Long term cardiac dysfunction may result in myocyte cell death, however, we did not detect apoptosis (TUNEL) in either mCU KO or in WT hearts with cardiac dysfunction after chronic IO. The lipid oxidation level was increased with Fe load suggesting ferroptosis may be involved in IO-induced cardiomyopathy, which was supported by the observation that administration of the selective ferroptosis inhibitor ferrostatin-1 reduced lipid oxidation (4-HNE) and maintained heart function. To further determine the role of mCU in IO-mediated ferroptosis in the heart, we used isolated myocytes from WT and mCU KO and conducted Live/Dead cell viability assays. While Fe (FAC, 0.1-5 mM) induced ferroptosis in a dose-dependent manner, it was prevented by ferrostatin-1 (10 μM), the Fe chelator 2,2'-bipyridyl (2 mM), and MitoTEMPO (5 μM), respectively. Fe-induced ferroptosis in mCU KO myocytes did not occur while the lipid oxidation level (Liperfluo) remained low. In conclusion, mitochondrial Fe uptake, presumably mediated by mCU, accounts for the molecular/cellular mechanisms for Fe-induced myocyte ferroptosis and cardiomyopathy. Cardiac-specific deficiency of mCU prevents the development of Fe induced cardiomyopathy.

Abstract 254: Mitochondrial Permeability Transition Pore, Calcium Uniporter, and Iron Overload in the Heart

Although iron (Fe) overload-induced cardiomyopathy/arrhythmias remain a significant clinical challenge, the underlying mechanism is not well defined. In the present study, we assessed the involvement of mitochondrial permeability transition pore (mPTP) and Ca uniporter (mCU) in Fe overload-induced mitochondrial Ca mishandling and cardiac contractile dysfunction. Firstly, we tested the hypothesis that Fe overload promotes reactive oxygen species (ROS) production and mitochondrial membrane potential (Δ Ψ m ) depolarization that may open the mPTP and promote intracellular Ca dysfunction. In isolated mouse ventricular myocytes, both cytosolic and mitochondrial Fe levels (monitored by using specific Fe dyes) were elevated following acute perfusion with Fe 3+ /8-Hydroxyquinoline (8-HQ) (1-100 μM). Mitochondrial superoxide generation (by MitoSOX Red) and cytosolic ROS levels (by DCF) were significantly enhanced by Fe overload. Furthermore, Fe overload induced Δ Ψ m depolarization as measured by TMRM. The mitochondrial maximal respiration rate and reserve capacity (using the Seahorse XF e 96 analyzer) were significantly reduced by Fe overload. Fe overload significantly increased the rate of Ca waves as imaged with Fluo-4. Fe overload-promoted Ca waves were significantly reduced by the mPTP inhibitor cyclosporine A (CsA). In Langendorff-perfused hearts subjected to programmed S 1 -S 2 stimulation, Fe significantly increased arrhythmia scores compared to the hearts pretreated with CsA, indicating mPTP inhibition can ameliorate the proarrhythmic effects of Fe-overload. These observations were confirmed in cyclophilin D knockout mice, in which mPTP opening is impaired. Furthermore, the involvement of the mCU was evaluated in a chronic Fe overload model (Fe dextran injection, i.p. ) by using conditional mCU knockout (mCU KO) mice. We observed that mCU KO prevented mitochondrial Fe uptake and conferred a significantly higher resistance to Fe-induced mitochondrial ROS generation, Ca mishandling and cardiac contractile dysfunction. In conclusion, mitochondrial Fe uptake and mitochondrial Ca dysregulation, presumably mediated by both mCU and mPTP, account for the molecular/cellular mechanism for Fe-induced cardiomyopathy and arrhythmias.

Structural Insights into Mitochondrial Calcium Uniporter Regulation by Divalent Cations

Calcium (Ca(2+)) flux into the matrix is tightly controlled by the mitochondrial Ca(2+) uniporter (MCU) due to vital roles in cell death and bioenergetics. However, the precise atomic mechanisms of MCU regulation remain unclear. Here, we solved the crystal structure of the N-terminal matrix domain of human MCU, revealing a β-grasp-like fold with a cluster of negatively charged residues that interacts with divalent cations. Binding of Ca(2+) or Mg(2+) destabilizes and shifts the self-association equilibrium of the domain toward monomer. Mutational disruption of the acidic face weakens oligomerization of the isolated matrix domain and full-length human protein similar to cation binding and markedly decreases MCU activity. Moreover, mitochondrial Mg(2+) loading or blockade of mitochondrial Ca(2+) extrusion suppresses MCU Ca(2+)-uptakerates. Collectively, our data reveal that the β-grasp-like matrix region harbors an MCU-regulating acidic patch that inhibits human MCU activity in response to Mg(2+) and Ca(2+) binding.

UCP3 Regulates Single-Channel Activity of the Cardiac mCa1.

Mitochondrial Ca2+ uptake (mCa2+ uptake) is thought to be mediated by the mitochondrial Ca2+ uniporter (MCU). UCP2 and UCP3 belong to a superfamily of mitochondrial ion transporters. Both proteins are expressed in the inner mitochondrial membrane of the heart. Recently, UCP2 was reported to modulate the function of the cardiac MCU related channel mCa1. However, the possible role of UCP3 in modulating cardiac mCa2+ uptake via the MCU remains inconclusive. To understand the role of UCP3, we analyzed cardiac mCa1 single-channel activity in mitoplast-attached single-channel recordings from isolated murine cardiac mitoplasts, from adult wild-type controls (WT), and from UCP3 knockout mice (UCP3–/–). Single-channel registrations in UCP3−/− confirmed a murine voltage-gated Ca2+ channel, i.e., mCa1, which was inhibited by Ru360. Compared to WT, mCa1 in UCP3−/− revealed similar single-channel characteristics. However, in UCP3−/− the channel exhibited decreased single-channel activity, which was insensitive to adenosine triphosphate (ATP) inhibition. Our results suggest that beyond UCP2, UCP3 also exhibits regulatory effects on cardiac mCa1/MCU function. Furthermore, we speculate that UCP3 might modulate previously described inhibitory effects of ATP on mCa1/MCU activity as well.

A new answer to an old problem: The energization of brain mitochondria with pyruvate is regulated by cytosolic calcium via the mitochondrial gas pedal and does not require the mitochondrial Ca uptake via the Ca uniporter- New evidences from experiments with MCU-,- mice

A new answer to an old problem: The energization of brain mitochondria with pyruvate is regulated by cytosolic calcium via the mitochondrial gas pedal and does not require the mitochondrial Ca uptake via the Ca uniporter- New evidences from experiments with MCU-,- mice

MCUR1 Is a Scaffold Factor for the MCU Complex Function and Promotes Mitochondrial Bioenergetics.

Mitochondrial Ca(2+) Uniporter (MCU)-dependent mitochondrial Ca(2+) uptake is the primary mechanism for increasing matrix Ca(2+) in most cell types. However, a limited understanding of the MCU complex assembly impedes the comprehension of the precise mechanisms underlying MCU activity. Here, we report that mouse cardiomyocytes and endothelial cells lacking MCU regulator 1 (MCUR1) have severely impaired [Ca(2+)]m uptake and IMCU current. MCUR1 binds to MCU and EMRE and function as a scaffold factor. Our protein binding analyses identified the minimal, highly conserved regions of coiled-coil domain of both MCU and MCUR1 that are necessary for heterooligomeric complex formation. Loss of MCUR1 perturbed MCU heterooligomeric complex and functions as a scaffold factor for the assembly of MCU complex. Vascular endothelial deletion of MCU and MCUR1 impaired mitochondrial bioenergetics, cell proliferation, and migration but elicited autophagy. These studies establish the existence of a MCU complex that assembles at the mitochondrial integral membrane and regulates Ca(2+)-dependent mitochondrial metabolism.

The mitochondrial Ca2+ uniporter: regulation by auxiliary subunits and signal transduction pathways.

Mitochondrial Ca(2+) homeostasis, the Ca(2+) influx-efflux balance, is responsible for the control of numerous cellular functions, including energy metabolism, generation of reactive oxygen species, spatiotemporal dynamics of Ca(2+) signaling, and cell growth and death. Recent discovery of the molecular identity of the mitochondrial Ca(2+) uniporter (MCU) provides new possibilities for application of genetic approaches to study the mitochondrial Ca(2+) influx mechanism in various cell types and tissues. In addition, the subsequent discovery of various auxiliary subunits associated with MCU suggests that mitochondrial Ca(2+) uptake is not solely regulated by a single protein (MCU), but likely by a macromolecular protein complex, referred to as the MCU-protein complex (mtCUC). Moreover, recent reports have shown the potential role of MCU posttranslational modifications in the regulation of mitochondrial Ca(2+) uptake through mtCUC. These observations indicate that mtCUCs form a local signaling complex at the inner mitochondrial membrane that could significantly regulate mitochondrial Ca(2+) handling, as well as numerous mitochondrial and cellular functions. In this review we discuss the current literature on mitochondrial Ca(2+) uptake mechanisms, with a particular focus on the structure and function of mtCUC, as well as its regulation by signal transduction pathways, highlighting current controversies and discrepancies.

Regulation of mitochondrial calcium in plants versus animals.

Ca(2+) acts as an important cellular second messenger in eukaryotes. In both plants and animals, a wide variety of environmental and developmental stimuli trigger Ca(2+) transients of a specific signature that can modulate gene expression and metabolism. In animals, mitochondrial energy metabolism has long been considered a hotspot of Ca(2+) regulation, with a range of pathophysiology linked to altered Ca(2+) control. Recently, several molecular players involved in mitochondrial Ca(2+) signalling have been identified, including those of the mitochondrial Ca(2+) uniporter. Despite strong evidence for sophisticated Ca(2+) regulation in plant mitochondria, the picture has remained much less clear. This is currently changing aided by live imaging and genetic approaches which allow dissection of subcellular Ca(2+) dynamics and identification of the proteins involved. We provide an update on our current understanding in the regulation of mitochondrial Ca(2+) and signalling by comparing work in plants and animals. The significance of mitochondrial Ca(2+) control is discussed in the light of the specific metabolic and energetic needs of plant and animal cells.

Mitochondrial Ca(2+) Processing by a Unit of Mitochondrial Ca(2+) Uniporter and Na(+)/Ca(2+) Exchanger Supports the Neuronal Ca(2+) Influx via Activated Glutamate Receptors.

The current study demonstrates that in hippocampal neurons mitochondrial Ca(2+) processing supports Ca(2+) influx via ionotropic glutamate (Glu) receptors. We define mitochondrial Ca(2+) processing as Ca(2+) uptake via mitochondrial Ca(2+) uniporter (MCU) combined with subsequent Ca(2+) release via mitochondrial Na(+)/Ca(2+) exchanger (NCX). Our tool is to measure the Ca(2+) influx rate in primary hippocampal co-cultures, i.e. neurons and astrocytes, by fluorescent digital microscopy, using a Fura-2-quenching method where we add small amounts of Mn(2+) in the superfusion medium. Thus, Ca(2+) influx is measured with Mn(2+) in the bath. Ru360 as inhibitor of mitochondrial Ca(2+) uptake through MCU strongly reduces the rate of Ca(2+) influx in Glu-stimulated primary hippocampal neurons. Similarly, the Ca(2+) influx rate in Glu-stimulated neurons declines after suppression of potential-dependent MCU, when we depolarize mitochondria with rotenone. With inhibition of Ca(2+) release from mitochondria via NCX using CGP-37157 the Ca(2+) influx via N-methyl-D-aspartate (NMDA)- and kainate-sensitive receptors is slowed down. Working jointly as mitochondrial Ca(2+) processing unit, MCU and NCX, apparently sustain the Ca(2+) throughput of activated Glu-sensitive receptors. Our results revise the role frequently attributed to mitochondria in neuronal Ca(2+) homeostasis, where mitochondria function mainly as Ca(2+) buffer, and prevent excessively high cytosolic Ca(2+) concentration increase during neuronal activity. The mechanism to control Ca(2+) influx in neurons, as discovered in this study, highlights mitochondrial Ca(2+) processing as a promising pharmacological target. We discuss this pathway in relation to the endoplasmic reticulum-related mechanisms of Ca(2+) processing.

Mitochondrial calcium uniporter inhibition attenuates mouse bone marrow-derived mast cell degranulation induced by beta-1,3-glucan

Mast cells are primary mediators of allergic inflammation. Beta-1,3-glucan (BG) protects against infection and shock by activating immune cells. Activation of the BG receptor induces an increase in intracellular Ca2+, which may induce exocytosis. However, little is known about the precise mechanisms underlying BG activation of immune cells and the possible role of mitochondria in this process. The present study examined whether BG induced mast cell degranulation, and evaluated the role of calcium transients during mast cell activation. Our investigation focused on the role of the mitochondrial calcium uniporter (MCU) in BG-induced degranulation. Black mouse (C57) bone marrow-derived mast cells were stimulated with 0.5 µg/ml BG, 100 µg/ml peptidoglycan (PGN), or 10 µM A23187 (calcium ionophore), and dynamic changes in cytosolic and mitochondrial calcium and membrane potential were monitored. BG-induced mast cell degranulation occurred in a time-dependent manner, and was significantly reduced under calcium-free conditions. Ruthenium red, a mitochondrial Ca2+ uniporter blocker, significantly reduced mast cell degranulation induced by BG, PGN, and A23187. These results suggest that the mitochondrial Ca2+ uniporter has an important regulatory role in BG-induced mast cell degranulation.

Modulation of Calcium Entry by Mitochondria.

The role of mitochondria in intracellular Ca(2+) signaling relies mainly in its capacity to take up Ca(2+) from the cytosol and thus modulate the cytosolic [Ca(2+)]. Because of the low Ca(2+)-affinity of the mitochondrial Ca(2+)-uptake system, this organelle appears specially adapted to take up Ca(2+) from local high-Ca(2+) microdomains and not from the bulk cytosol. Mitochondria would then act as local Ca(2+) buffers in cellular regions where high-Ca(2+) microdomains form, that is, mainly close to the cytosolic mouth of Ca(2+) channels, both in the plasma membrane and in the endoplasmic reticulum (ER). One of the first targets proposed already in the 1990s to be regulated in this way by mitochondria were the store-operated Ca(2+) channels (SOCE). Mitochondria, by taking up Ca(2+) from the region around the cytosolic mouth of the SOCE channels, would prevent its slow Ca(2+)-dependent inactivation, thus keeping them active for longer. Since then, evidence for this mechanism has accumulated mainly in immunitary cells, where mitochondria actually move towards the immune synapse during T cell activation. However, in many other cell types the available data indicate that the close apposition between plasma and ER membranes occurring during SOCE activation precludes mitochondria from getting close to the Ca(2+)-entry sites. Alternative pathways for mitochondrial modulation of SOCE, both Ca(2+)-dependent and Ca(2+)-independent, have also been proposed, but further work will be required to elucidate the actual mechanisms at work. Hopefully, the recent knowledge of the molecular nature of the mitochondrial Ca(2+) uniporter will allow soon more precise studies on this matter.

Mitochondrial Dysfunction and Ca2+ Overload Contributes to Hesperidin Induced Paraptosis in Hepatoblastoma Cells, HepG2

Paraptosis is a programmed cell death which is morphologically and biochemically different from apoptosis. In this study, we have investigated the role of Ca(2+) in hesperidin-induced paraptotic cell death in HepG2 cells. Increase in mitochondrial Ca(2+) level was observed in hesperidin treated HepG2 cells but not in normal liver cancer cells. Inhibition of inositol-1,4,5-triphosphate receptor (IP3 R) and ryanodine receptor also block the mitochondrial Ca(2+) accumulation suggesting that the release of Ca(2+) from the endoplasmic reticulum (ER) may probably lead to the increase in mitochondrial Ca(2+) level. Pretreatment with ruthenium red (RuRed), a Ca(2+) uniporter inhibitor inhibited the hesperidin-induced mitochondrial Ca(2+) overload, swelling of mitochondria, and cell death in HepG2 cells. It has also been demonstrated that mitochondrial Ca(2+) influxes act upstream of ROS and mitochondrial superoxide production. The increased ROS production further leads to mitochondrial membrane loss in hesperidin treated HepG2 cells. Taken together our results show that IP3 R and ryanodine receptor mediated release of Ca(2+) from the ER and its subsequent influx through the uniporter into mitochondria contributes to hesperidin-induced paraptosis in HepG2 cells.

The EF-Hand Ca2+ Binding Protein MICU Choreographs Mitochondrial Ca2+ Dynamics in Arabidopsis.

Plant organelle function must constantly adjust to environmental conditions, which requires dynamic coordination. Ca(2+) signaling may play a central role in this process. Free Ca(2+) dynamics are tightly regulated and differ markedly between the cytosol, plastid stroma, and mitochondrial matrix. The mechanistic basis of compartment-specific Ca(2+) dynamics is poorly understood. Here, we studied the function of At-MICU, an EF-hand protein of Arabidopsis thaliana with homology to constituents of the mitochondrial Ca(2+) uniporter machinery in mammals. MICU binds Ca(2+) and localizes to the mitochondria in Arabidopsis. In vivo imaging of roots expressing a genetically encoded Ca(2+) sensor in the mitochondrial matrix revealed that lack of MICU increased resting concentrations of free Ca(2+) in the matrix. Furthermore, Ca(2+) elevations triggered by auxin and extracellular ATP occurred more rapidly and reached higher maximal concentrations in the mitochondria of micu mutants, whereas cytosolic Ca(2+) signatures remained unchanged. These findings support the idea that a conserved uniporter system, with composition and regulation distinct from the mammalian machinery, mediates mitochondrial Ca(2+) uptake in plants under in vivo conditions. They further suggest that MICU acts as a throttle that controls Ca(2+) uptake by moderating influx, thereby shaping Ca(2+) signatures in the matrix and preserving mitochondrial homeostasis. Our results open the door to genetic dissection of mitochondrial Ca(2+) signaling in plants.

The Ca2+-Dependent Release of the Mia40-Induced MICU1-MICU2 Dimer from MCU Regulates Mitochondrial Ca2+ Uptake

The essential oxidoreductase Mia40/CHCHD4 mediates disulfide bond formation and protein folding in the mitochondrial intermembrane space. Here, we investigated the interactome of Mia40 thereby revealing links between thiol-oxidation and apoptosis, energy metabolism, and Ca(2+) signaling. Among the interaction partners of Mia40 is MICU1-the regulator of the mitochondrial Ca(2+) uniporter (MCU), which transfers Ca(2+) across the inner membrane. We examined the biogenesis of MICU1 and find that Mia40 introduces an intermolecular disulfide bond that links MICU1 and its inhibitory paralog MICU2 in a heterodimer. Absence of this disulfide bond results in increased receptor-induced mitochondrial Ca(2+) uptake. In the presence of the disulfide bond, MICU1-MICU2 heterodimer binding to MCU is controlled by Ca(2+) levels: the dimer associates with MCU at low levels of Ca(2+) and dissociates upon high Ca(2+) concentrations. Our findings support a model in which mitochondrial Ca(2+) uptake is regulated by a Ca(2+)-dependent remodeling of the uniporter complex.