Structural Mechanism of EMRE-Dependent Gating of the Human Mitochondrial Calcium Uniporter.

Mitochondrial calcium uptake is crucial to the regulation of eukaryotic Ca2+ homeostasis and is mediated by the mitochondrial calcium uniporter (MCU). While MCU alone can transport Ca2+ in primitive eukaryotes, metazoans require an essential single membrane-spanning auxiliary component called EMRE to form functional channels; however, the molecular mechanism of EMRE regulation remains elusive. Here, we present the cryo-EM structure of the human MCU-EMRE complex, which defines the interactions between MCU and EMRE as well as pinpoints the juxtamembrane loop of MCU and extended linker of EMRE as the crucial elements in the EMRE-dependent gating mechanism among metazoan MCUs. The structure also features the dimerization of two MCU-EMRE complexes along an interface at the N-terminal domain (NTD) of human MCU that is a hotspot for post-translational modifications. Thus, the human MCU-EMRE complex, which constitutes the minimal channel components among metazoans, provides a framework for future mechanistic studies on MCU.

Systematic mapping of mitochondrial calcium uniporter channel (MCUC)-mediated calcium signaling networks.

The mitochondrial calcium uniporter channel (MCUC) mediates mitochondrial calcium entry, regulating energy metabolism and cell death. Although several MCUC components have been identified, the molecular basis of mitochondrial calcium signaling networks and their remodeling upon changes in uniporter activity have not been assessed. Here, we map the MCUC interactome under resting conditions and upon chronic loss or gain of mitochondrial calcium uptake. We identify 89 high-confidence interactors that link MCUC to several mitochondrial complexes and pathways, half of which are associated with human disease. As a proof-of-concept, we validate the mitochondrial intermembrane space protein EFHD1 as a binding partner of the MCUC subunits MCU, EMRE, and MCUB. We further show a MICU1-dependent inhibitory effect of EFHD1 on calcium uptake. Next, we systematically survey compensatory mechanisms and functional consequences of mitochondrial calcium dyshomeostasis by analyzing the MCU interactome upon EMRE, MCUB, MICU1, or MICU2 knockdown. While silencing EMRE reduces MCU interconnectivity, MCUB loss-of-function leads to a wider interaction network. Our study provides a comprehensive and high-confidence resource to gain insights into players and mechanisms regulating mitochondrial calcium signaling and their relevance in human diseases.

MICU1 and MICU2 control mitochondrial calcium signaling in the mammalian heart.

Activating Ca2+-sensitive enzymes of oxidative metabolism while preventing calcium overload that leads to mitochondrial and cellular injury requires dynamic control of mitochondrial Ca2+ uptake. This is ensured by the mitochondrial calcium uptake (MICU)1/2 proteins that gate the pore of the mitochondrial calcium uniporter (mtCU). MICU1 is relatively sparse in the heart, and recent studies claimed the mammalian heart lacks MICU1 gating of mtCU. However, genetic models have not been tested. We find that MICU1 is present in a complex with MCU in nonfailing human hearts. Furthermore, using murine genetic models and pharmacology, we show that MICU1 and MICU2 control cardiac mitochondrial Ca2+ influx, and that MICU1 deletion alters cardiomyocyte mitochondrial calcium signaling and energy metabolism. MICU1 loss causes substantial compensatory changes in the mtCU composition and abundance, increased turnover of essential MCU regulator (EMRE) early on and, later, of MCU, that limit mitochondrial Ca2+ uptake and allow cell survival. Thus, both the primary consequences of MICU1 loss and the ensuing robust compensation highlight MICU1's relevance in the beating heart.

MICU1 occludes the mitochondrial calcium uniporter in divalent-free conditions.

Mitochondrial Ca2+ uptake is mediated by the mitochondrial uniporter complex (mtCU) that includes a tetramer of the pore-forming subunit, MCU, a scaffold protein, EMRE, and the EF-hand regulatory subunit, MICU1 either homodimerized or heterodimerized with MICU2/3. MICU1 has been proposed to regulate Ca2+ uptake via the mtCU by physically occluding the pore and preventing Ca2+ flux at resting cytoplasmic [Ca2+] (free calcium concentration) and to increase Ca2+ flux at high [Ca2+] due to cooperative activation of MICUs EF-hands. However, mtCU and MICU1 functioning when its EF-hands are unoccupied by Ca2+ is poorly studied due to technical limitations. To overcome this barrier, we have studied the mtCU in divalent-free conditions by assessing the Ru265-sensitive Na+ influx using fluorescence-based measurement of mitochondrial matrix [Na+] (free sodium concentration) rise and the ensuing depolarization and swelling. We show an increase in all these measures of Na+ uptake in MICU1KO cells as compared to wild-type (WT) and rescued MICU1KO HEK cells. However, mitochondria in WT cells and MICU1 stable-rescued cells still allowed some Ru265-sensitive Na+ influx that was prevented by MICU1 in excess upon acute overexpression. Thus, MICU1 restricts the cation flux across the mtCU in the absence of Ca2+, but even in cells with high endogenous MICU1 expression such as HEK, some mtCU seem to lack MICU1-dependent gating. We also show rearrangement of the mtCU and altered number of functional channels in MICU1KO and different rescues, and loss of MICU1 during mitoplast preparation, that together might have obscured the pore-blocking function of MICU1 in divalent-free conditions in previous studies.

Systematic Identification of MCU Modulators by Orthogonal Interspecies Chemical Screening.

The mitochondrial calcium uniporter complex is essential for calcium (Ca2+) uptake into mitochondria of all mammalian tissues, where it regulates bioenergetics, cell death, and Ca2+ signal transduction. Despite its involvement in several human diseases, we currently lack pharmacological agents for targeting uniporter activity. Here we introduce a high-throughput assay that selects for human MCU-specific small-molecule modulators in primary drug screens. Using isolated yeast mitochondria, reconstituted with human MCU, its essential regulator EMRE, and aequorin, and exploiting a D-lactate- and mannitol/sucrose-based bioenergetic shunt that greatly minimizes false-positive hits, we identify mitoxantrone out of more than 600 clinically approved drugs as a direct selective inhibitor of human MCU. We validate mitoxantrone in orthogonal mammalian cell-based assays, demonstrating that our screening approach is an effective and robust tool for MCU-specific drug discovery and, more generally, for the identification of compounds that target mitochondrial functions.

The mitochondrial calcium uniporter: Balancing tumourigenic and anti-tumourigenic responses.

Increased malignancy and poor treatability associated with solid tumour cancers have commonly been attributed to mitochondrial calcium (Ca2+) dysregulation. The mitochondrial Ca2+ uniporter complex (mtCU) is the predominant mode of Ca2+ uptake into the mitochondrial matrix. The main components of mtCU are the pore-forming mitochondrial Ca2+ uniporter (MCU) subunit, MCU dominant-negative beta (MCUb) subunit, essential MCU regulator (EMRE) and the gatekeeping mitochondrial Ca2+ uptake 1 and 2 (MICU1 and MICU2) proteins. In this review, we describe mtCU-mediated mitochondrial Ca2+ dysregulation in solid tumour cancer types, finding enhanced mtCU activity observed in colorectal cancer, breast cancer, oral squamous cell carcinoma, pancreatic cancer, hepatocellular carcinoma and embryonal rhabdomyosarcoma. By contrast, decreased mtCU activity is associated with melanoma, whereas the nature of mtCU dysregulation remains unclear in glioblastoma. Furthermore, we show that numerous polymorphisms associated with cancer may alter phosphorylation sites on the pore forming MCU and MCUb subunits, which cluster at interfaces with EMRE. We highlight downstream/upstream biomolecular modulators of MCU and MCUb that alter mtCU-mediated mitochondrial Ca2+ uptake and may be used as biomarkers or to aid in the development of novel cancer therapeutics. Additionally, we provide an overview of the current small molecule inhibitors of mtCU that interact with the Asp residue of the critical Asp-Ile-Met-Glu motif or through other allosteric regulatory mechanisms to block Ca2+ permeation. Finally, we describe the relationship between MCU- and MCUb-mediating microRNAs and mitochondrial Ca2+ uptake that should be considered in the discovery of new treatment approaches for cancer.

Mitochondrial Calcium Uniporter (MCU) is Involved in an Ischemic Postconditioning Effect Against Ischemic Reperfusion Brain Injury in Mice.

The phenomenon of ischemic postconditioning (PostC) is known to be neuroprotective against ischemic reperfusion (I/R) injury. One of the key processes in PostC is the opening of the mitochondrial ATP-dependent potassium (mito-KATP) channel and depolarization of the mitochondrial membrane, triggering the release of calcium ions from mitochondria through low-conductance opening of the mitochondrial permeability transition pore. Mitochondrial calcium uniporter (MCU) is known as a highly sensitive transporter for the uptake of Ca2+ present on the inner mitochondrial membrane. The MCU has attracted attention as a new target for treatment in diseases, such as neurodegenerative diseases, cancer, and ischemic stroke. We considered that the MCU may be involved in PostC and trigger its mechanisms. This research used the whole-cell patch-clamp technique on hippocampal CA1 pyramidal cells from C57BL mice and measured changes in spontaneous excitatory post-synaptic currents (sEPSCs), intracellular Ca2+ concentration, mitochondrial membrane potential, and N-methyl-D-aspartate receptor (NMDAR) currents under inhibition of MCU by ruthenium red 265 (Ru265) in PostC. Inhibition of MCU increased the occurrence of sEPSCs (p = 0.014), NMDAR currents (p < 0.001), intracellular Ca2+ concentration (p < 0.001), and dead cells (p < 0.001) significantly after reperfusion, reflecting removal of the neuroprotective effects in PostC. Moreover, mitochondrial depolarization in PostC with Ru265 was weakened, compared to PostC (p = 0.004). These results suggest that MCU affects mitochondrial depolarization in PostC to suppress NMDAR over-activation and prevent elevation of intracellular Ca2+ concentrations against I/R injury.

From passage to inhibition: Uncovering the structural and physiological inhibitory mechanisms of MCUb in mitochondrial calcium regulation.

Mitochondrial calcium (Ca2+ ) regulation is critically implicated in the regulation of bioenergetics and cell fate. Ca2+ , a universal signaling ion, passively diffuses into the mitochondrial intermembrane space (IMS) through voltage-dependent anion channels (VDAC), where uptake into the matrix is tightly regulated across the inner mitochondrial membrane (IMM) by the mitochondrial Ca2+ uniporter complex (mtCU). In recent years, immense progress has been made in identifying and characterizing distinct structural and physiological mechanisms of mtCU component function. One of the main regulatory components of the Ca2+ selective mtCU channel is the mitochondrial Ca2+ uniporter dominant-negative beta subunit (MCUb). The structural mechanisms underlying the inhibitory effect(s) exerted by MCUb are poorly understood, despite high homology to the main mitochondrial Ca2+ uniporter (MCU) channel-forming subunits. In this review, we provide an overview of the structural differences between MCUb and MCU, believed to contribute to the inhibition of mitochondrial Ca2+ uptake. We highlight the possible structural rationale for the absent interaction between MCUb and the mitochondrial Ca2+ uptake 1 (MICU1) gatekeeping subunit and a potential widening of the pore upon integration of MCUb into the channel. We discuss physiological and pathophysiological information known about MCUb, underscoring implications in cardiac function and arrhythmia as a basis for future therapeutic discovery. Finally, we discuss potential post-translational modifications on MCUb as another layer of important regulation.

Inhibition of mitochondrial calcium transporters alters adp-induced platelet responses.

INTRODUCTION: ADP-stimulated elevation of cytosolic Ca2+ is an important effector mechanism for platelet activation. The rapidly elevating cytosolic Ca2+ is also transported to mitochondrial matrix via Mitochondrial Ca2+ Uniporter (MCU) and extruded via Na+/Ca2+/Li+ Exchanger (NCLX). However, the exact contribution of MCU and NCLX in ADP-mediated platelet responses remains incompletely understood. METHODS AND RESULTS: The present study aimed to elucidate the role of mitochondrial Ca2+ transport in ADP-stimulated platelet responses by inhibition of MCU and NCLX with mitoxantrone (MTX) and CGP37157 (CGP), respectively. As these inhibitory strategies are reported to cause distinct effects on matrix Ca2+ concentration, we hypothesized to observe opposite impact of MTX and CGP on ADP-induced platelet responses. Platelet aggregation profiling was performed by microplate-based spectrophotometery while p-selectin externalization and integrin αIIbß3 activation were analyzed by fluorescent immunolabeling using flow cytometery. Our results confirmed the expression of both MCU and NCLX mRNAs with relatively low abundance of NCLX in human platelets. In line with our hypothesis, MTX caused a dose-dependent inhibition of ADP-induced platelet aggregation without displaying any cytotoxicity. Likewise, ADP-induced p-selectin externalization and integrin αIIbß3 activation was also significantly attenuated in MTX-treated platelets. Concordantly, inhibition of NCLX with CGP yielded an accelerated ADP-stimulated platelet aggregation which was associated with an elevation of p-selectin surface expression and αIIbß3 activation. CONCLUSION: Together, these findings uncover a vital and hitherto poorly characterized role of mitochondrial Ca2+ transporters in ADP-induced platelet activation.

Evidence for crosstalk between the aryl hydrocarbon receptor and the translocator protein in mouse lung epithelial cells.

Cellular homeostasis requires the use of multiple environmental sensors that can respond to a variety of endogenous and exogenous compounds. The aryl hydrocarbon receptor (AHR) is classically known as a transcription factor that induces genes that encode drug metabolizing enzymes when bound to toxicants such as 2,3,7,8-tetrachlorodibenzo-ρ-dioxin (TCDD). The receptor has a growing number of putative endogenous ligands, such as tryptophan, cholesterol, and heme metabolites. Many of these compounds are also linked to the translocator protein (TSPO), an outer mitochondrial membrane protein. Given a portion of the cellular pool of the AHR has also been localized to mitochondria and the overlap in putative ligands, we tested the hypothesis that crosstalk exists between the two proteins. CRISPR/Cas9 was used to create knockouts for AHR and TSPO in a mouse lung epithelial cell line (MLE-12). WT, AHR-/-, and TSPO-/- cells were then exposed to AHR ligand (TCDD), TSPO ligand (PK11195), or both and RNA-seq was performed. More mitochondrial-related genes were altered by loss of both AHR and TSPO than would have been expected just by chance. Some of the genes altered included those that encode for components of the electron transport system and the mitochondrial calcium uniporter. Both proteins altered the activity of the other as AHR loss caused the increase of TSPO at both the mRNA and protein level and loss of TSPO significantly increased the expression of classic AHR battery genes after TCDD treatment. This research provides evidence that AHR and TSPO participate in similar pathways that contribute to mitochondrial homeostasis.

Mitochondrial calcium uniporter promotes kidney aging in mice through inducing mitochondrial calcium-mediated renal tubular cell senescence.

Renal tubular epithelial cell senescence plays a critical role in promoting and accelerating kidney aging and age-related renal fibrosis. Senescent cells not only lose their self-repair ability, but also can transform into senescence-associated secretory phenotype (SASP) to trigger inflammation and fibrogenesis. Recent studies show that mitochondrial dysfunction is critical for renal tubular cell senescence and kidney aging, and calcium overload and abnormal calcium-dependent kinase activities are involved in mitochondrial dysfunction-associated senescence. In this study we investigated the role of mitochondrial calcium overload and mitochondrial calcium uniporter (MCU) in kidney aging. By comparing the kidney of 2- and 24-month-old mice, we found calcium overload in renal tubular cells of aged kidney, accompanied by significantly elevated expression of MCU. In human proximal renal tubular cell line HK-2, pretreatment with MCU agonist spermine (10 µM) significantly increased mitochondrial calcium accumulation, and induced the production of reactive oxygen species (ROS), leading to renal tubular cell senescence and age-related kidney fibrosis. On the contrary, pretreatment with MCU antagonist RU360 (10 µM) or calcium chelator BAPTA-AM (10 µM) diminished D-gal-induced ROS generation, restored mitochondrial homeostasis, retarded cell senescence, and protected against kidney aging in HK-2 cells. In a D-gal-induced accelerated aging mice model, administration of BAPTA (100 µg/kg. i.p.) every other day for 8 weeks significantly alleviated renal tubuarl cell senescence and fibrosis. We conclude that MCU plays a key role in promoting renal tubular cell senescence and kidney aging. Targeting inhibition on MCU provides a new insight into the therapeutic strategy against kidney aging.

A MICU1 Splice Variant Confers High Sensitivity to the Mitochondrial Ca2+ Uptake Machinery of Skeletal Muscle.

Skeletal muscle is a dynamic organ, characterized by an incredible ability to rapidly increase its rate of energy consumption to sustain activity. Muscle mitochondria provide most of the ATP required for contraction via oxidative phosphorylation. Here we found that skeletal muscle mitochondria express a unique MCU complex containing an alternative splice isoform of MICU1, MICU1.1, characterized by the addition of a micro-exon that is sufficient to greatly modify the properties of the MCU. Indeed, MICU1.1 binds Ca2+ one order of magnitude more efficiently than MICU1 and, when heterodimerized with MICU2, activates MCU current at lower Ca2+ concentrations than MICU1-MICU2 heterodimers. In skeletal muscle in vivo, MICU1.1 is required for sustained mitochondrial Ca2+ uptake and ATP production. These results highlight a novel mechanism of the molecular plasticity of the MCU Ca2+ uptake machinery that allows skeletal muscle mitochondria to be highly responsive to sarcoplasmic [Ca2+] responses.

Perfluorooctane sulfonate induces ferroptosis-dependent non-alcoholic steatohepatitis via autophagy-MCU-caused mitochondrial calcium overload and MCU-ACSL4 interaction.

The incidence of nonalcoholic steatohepatitis (NASH) is related with perfluorooctane sulfonate (PFOS), yet the mechanism remains ill-defined. Mounting evidence suggests that ferroptosis plays a crucial role in the initiation of NASH. In this study, we used mice and human hepatocytes L-02 to investigate the role of ferroptosis in PFOS-induced NASH and the effect and molecular mechanism of PFOS on liver ferroptosis. We found here that PFOS caused NASH in mice, and lipid accumulation and inflammatory response in the L-02 cells. PFOS induced hepatic ferroptosis in vivo and in vitro, as evidenced by the decrease in glutathione peroxidase 4 (GPX4), and the increases in cytosolic iron, acyl-CoA synthetase long-chain family member 4 (ACSL4) and lipid peroxidation. In the PFOS-treated cells, the increases in the inflammatory factors and lipid contents were reversed by ferroptosis inhibitor. PFOS-induced ferroptosis was relieved by autophagy inhibitor. The expression of mitochondrial calcium uniporter (MCU) was accelerated by PFOS, leading to subsequent mitochondrial calcium accumulation, and inhibiting autophagy reversed the increase in MCU. Inhibiting mitochondrial calcium reversed the variations in GPX4 and cytosolic iron, without influencing the change in ACSL4, induced by PFOS. MCU interacted with ACSL4 and the siRNA against MCU reversed the changes in ACSL4，GPX4 and cytosolic iron systemically. This study put forward the involvement of hepatic ferroptosis in PFOS-induced NASH and identified MCU as the mediator of the autophagy-dependent ferroptosis.

A Ca2+-Dependent Mechanism Boosting Glycolysis and OXPHOS by Activating Aralar-Malate-Aspartate Shuttle, upon Neuronal Stimulation.

Calcium is an important second messenger regulating a bioenergetic response to the workloads triggered by neuronal activation. In embryonic mouse cortical neurons using glucose as only fuel, activation by NMDA elicits a strong workload (ATP demand)-dependent on Na+ and Ca2+ entry, and stimulates glucose uptake, glycolysis, pyruvate and lactate production, and oxidative phosphorylation (OXPHOS) in a Ca2+-dependent way. We find that Ca2+ upregulation of glycolysis, pyruvate levels, and respiration, but not glucose uptake, all depend on Aralar/AGC1/Slc25a12, the mitochondrial aspartate-glutamate carrier, component of the malate-aspartate shuttle (MAS). MAS activation increases glycolysis, pyruvate production, and respiration, a process inhibited in the presence of BAPTA-AM, suggesting that the Ca2+ binding motifs in Aralar may be involved in the activation. Mitochondrial calcium uniporter (MCU) silencing had no effect, indicating that none of these processes required MCU-dependent mitochondrial Ca2+ uptake. The neuronal respiratory response to carbachol was also dependent on Aralar, but not on MCU. We find that mouse cortical neurons are endowed with a constitutive ER-to-mitochondria Ca2+ flow maintaining basal cell bioenergetics in which ryanodine receptors, RyR2, rather than InsP3R, are responsible for Ca2+ release, and in which MCU does not participate. The results reveal that, in neurons using glucose, MCU does not participate in OXPHOS regulation under basal or stimulated conditions, while Aralar-MAS appears as the major Ca2+-dependent pathway tuning simultaneously glycolysis and OXPHOS to neuronal activation.SIGNIFICANCE STATEMENT Neuronal activation increases cell workload to restore ion gradients altered by activation. Ca2+ is involved in matching increased workload with ATP production, but the mechanisms are still unknown. We find that glycolysis, pyruvate production, and neuronal respiration are stimulated on neuronal activation in a Ca2+-dependent way, independently of effects of Ca2+ as workload inducer. Mitochondrial calcium uniporter (MCU) does not play a relevant role in Ca2+ stimulated pyruvate production and oxygen consumption as both are unchanged in MCU silenced neurons. However, Ca2+ stimulation is blunt in the absence of Aralar, a Ca2+-binding mitochondrial carrier component of Malate-Aspartate Shuttle (MAS). The results suggest that Ca2+-regulated Aralar-MAS activation upregulates glycolysis and pyruvate production, which fuels mitochondrial respiration, through regulation of cytosolic NAD+/NADH ratio.

Distinct effects of cardiac mitochondrial calcium uniporter inactivation via EMRE deletion in the short and long term.

Transport of Ca2+ into mitochondria is thought to stimulate the production of ATP, a critical process in the heart's fight or flight response, but excess Ca2+ can trigger cell death. The mitochondrial Ca2+ uniporter complex is the primary route of Ca2+ transport into mitochondria, in which the channel-forming protein MCU and the regulatory protein EMRE are essential for activity. In previous studies, chronic Mcu or Emre deletion differed from acute cardiac Mcu deletion in response to adrenergic stimulation and ischemia/reperfusion (I/R) injury, despite equivalent inactivation of rapid mitochondrial Ca2+ uptake. To explore this discrepancy between chronic and acute loss of uniporter activity, we compared short-term and long-term Emre deletion using a novel conditional cardiac-specific, tamoxifen-inducible mouse model. After short-term Emre deletion (3 weeks post-tamoxifen) in adult mice, cardiac mitochondria were unable to take up Ca2+, had lower basal mitochondrial Ca2+ levels, and displayed attenuated Ca2+-induced ATP production and mPTP opening. Moreover, short-term EMRE loss blunted cardiac response to adrenergic stimulation and improved maintenance of cardiac function in an ex vivo I/R model. We then tested whether the long-term absence of EMRE (3 months post-tamoxifen) in adulthood would lead to distinct outcomes. After long-term Emre deletion, mitochondrial Ca2+ handling and function, as well as cardiac response to adrenergic stimulation, were similarly impaired as in short-term deletion. Interestingly, however, protection from I/R injury was lost in the long-term. These data suggest that several months without uniporter function are insufficient to restore bioenergetic response but are sufficient to restore susceptibility to I/R.

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.

Nicorandil protects podocytes via modulation of antioxidative capacity in acute puromycin aminonucleoside-induced nephrosis in rats.

Nephrotic syndrome, characterized by proteinuria and hypoalbuminemia, results from the dysregulation of glomerular podocytes and is a significant cause of end-stage kidney disease. Patients with idiopathic nephrotic syndrome are generally treated with immunosuppressive agents; however, these agents produce various adverse effects. Previously, we reported the renoprotective effects of a stimulator of the mitochondrial ATP-dependent K+ channel (MitKATP), nicorandil, in a remnant kidney model. Nonetheless, the cellular targets of these effects remain unknown. Here, we examined the effect of nicorandil on puromycin aminonucleoside-induced nephrosis (PAN) rats, a well-established model of podocyte injury and human nephrotic syndrome. PAN was induced using a single intraperitoneal injection. Nicorandil was administered orally at 30 mg/kg/day. We found that proteinuria and hypoalbuminemia in PAN rats were significantly ameliorated following nicorandil treatment. Immunostaining and ultrastructural analysis under electron microscopy demonstrated that podocyte injury in PAN rats showed a significant partial attenuation following nicorandil treatment. Nicorandil ameliorated the increase in the oxidative stress markers nitrotyrosine and 8-hydroxy-2-deoxyguanosine in glomeruli. Conversely, nicorandil prevented the decrease in levels of the antioxidant enzyme manganese superoxide dismutase in PAN rats. We found that mitochondrial Ca2+ uniporter levels in glomeruli were higher in PAN rats than in control rats, and this increase was significantly attenuated by nicorandil. We conclude that stimulation of MitKATP by nicorandil reduces proteinuria by attenuating podocyte injury in PAN nephrosis, which restores mitochondrial antioxidative capacity, possibly through mitochondrial Ca2+ uniporter modulation. These data indicate that MitKATP may represent a novel target for podocyte injury and nephrotic syndrome.NEW & NOTEWORTHY Our findings suggest that the mitochondrial Ca2+ uniporter may be an upstream regulator of manganese superoxide dismutase and indicate a biochemical basis for the interaction between the ATP-sensitive K+ channel and Ca2+ signaling. We believe that our study makes a significant contribution to the literature because our results indicate that the ATP-sensitive K+ channel may be a potential therapeutic target for podocyte injury and nephrotic syndrome.

Mitochondrial calcium uniporter deficiency in dentate granule cells remodels neuronal metabolism and impairs reversal learning.

The mitochondrial calcium uniporter (MCU) is the main route of calcium (Ca2+ ) entry into neuronal mitochondria. This channel has been linked to mitochondrial Ca2+ overload and cell death under neurotoxic conditions, but its physiologic roles for normal brain function remain poorly understood. Despite high expression of MCU in excitatory hippocampal neurons, it is unknown whether this channel is required for learning and memory. Here, we genetically down-regulated the Mcu gene in dentate granule cells (DGCs) of the hippocampus and found that this manipulation increases the overall respiratory activity of mitochondrial complexes I and II, augmenting the generation of reactive oxygen species in the context of impaired electron transport chain. The metabolic remodeling of MCU-deficient neurons also involved changes in the expression of enzymes that participate in glycolysis and the regulation of the tricarboxylic acid cycle, as well as the cellular antioxidant defenses. We found that MCU deficiency in DGCs does not change circadian rhythms, spontaneous exploratory behavior, or cognitive function in middle-aged mice (11-13 months old), when assessed with a food-motivated working memory test with three choices. DGC-targeted down-regulation of MCU significantly impairs reversal learning assessed with an 8-arm radial arm water maze but does not affect their ability to learn the task for the first time. Our results indicate that neuronal MCU plays an important physiologic role in memory formation and may be a potential therapeutic target to develop interventions aimed at improving cognitive function in aging, neurodegenerative diseases, and brain injury.

Layer-specific mitochondrial diversity across hippocampal CA2 dendrites.

CA2 is an understudied subregion of the hippocampus that is critical for social memory. Previous studies identified multiple components of the mitochondrial calcium uniporter (MCU) complex as selectively enriched in CA2. The MCU complex regulates calcium entry into mitochondria, which in turn regulates mitochondrial transport and localization to active synapses. We found that MCU is strikingly enriched in CA2 distal apical dendrites, precisely where CA2 neurons receive entorhinal cortical input carrying social information. Furthermore, MCU-enriched mitochondria in CA2 distal dendrites are larger compared to mitochondria in CA2 proximal apical dendrites and neighboring CA1 apical dendrites, which was confirmed in CA2 with genetically labeled mitochondria and electron microscopy. MCU overexpression in neighboring CA1 led to a preferential localization of MCU in the proximal dendrites of CA1 compared to the distal dendrites, an effect not seen in CA2. Our findings demonstrate that mitochondria are molecularly and structurally diverse across hippocampal cell types and circuits, and suggest that MCU can be differentially localized within dendrites, possibly to meet local energy demands.

Deficiency of mitochondrial calcium uniporter abrogates iron overload-induced cardiac dysfunction by reducing ferroptosis.

Iron overload associated cardiac dysfunction remains a significant clinical challenge whose underlying mechanism(s) have yet to be defined. We aim to evaluate the involvement of the mitochondrial Ca2+ uniporter (MCU) in cardiac dysfunction and determine its role in the occurrence of ferroptosis. Iron overload was established in control (MCUfl/fl) and conditional MCU knockout (MCUfl/fl-MCM) mice. LV function was reduced by chronic iron loading in MCUfl/fl mice, but not in MCUfl/fl-MCM mice. The level of mitochondrial iron and reactive oxygen species were increased and mitochondrial membrane potential and spare respiratory capacity (SRC) were reduced in MCUfl/fl cardiomyocytes, but not in MCUfl/fl-MCM cardiomyocytes. After iron loading, lipid oxidation levels were increased in MCUfl/fl, but not in MCUfl/fl-MCM hearts. Ferrostatin-1, a selective ferroptosis inhibitor, reduced lipid peroxidation and maintained LV function in vivo after chronic iron treatment in MCUfl/fl hearts. Isolated cardiomyocytes from MCUfl/fl mice demonstrated ferroptosis after acute iron treatment. Moreover, Ca2+ transient amplitude and cell contractility were both significantly reduced in isolated cardiomyocytes from chronically Fe treated MCUfl/fl hearts. However, ferroptosis was not induced in cardiomyocytes from MCUfl/fl-MCM hearts nor was there a reduction in Ca2+ transient amplitude or cardiomyocyte contractility. We conclude that mitochondrial iron uptake is dependent on MCU, which plays an essential role in causing mitochondrial dysfunction and ferroptosis under iron overload conditions in the heart. Cardiac-specific deficiency of MCU prevents the development of ferroptosis and iron overload-induced cardiac dysfunction.