Mitochondrial Calcium Uniporter Expression Research Articles

Double Braking Effects of Nanomedicine on Mitochondrial Permeability Transition Pore for Treating Idiopathic Pulmonary Fibrosis.

Mitochondrial permeability transition pore (mPTP) opening is a key hallmark of injured type II alveolar epithelial cells (AECIIs) in idiopathic pulmonary fibrosis (IPF). Inhibiting mPTP opening in AECIIs is considered a potential IPF treatment. Herein, a "double braking" strategy on mPTP by cyclosporin A (CsA) derived ionizable lipid with 3D structure (3D-lipid) binding cyclophilin D (CypD) and siRNA downregulating mitochondrial calcium uniporter (MCU) expression is proposed for treating IPF. 3D-lipid and MCU targeting siRNA (siMCU) are co-assembled to form stable 3D-LNP/siMCU nanoparticles (NPs), along with helper lipids. In vitro results demonstrated that these NPs effectively inhibit mPTP opening by 3D-lipid binding with CypD and siRNA downregulating MCU expression, thereby decreasing damage-associated molecular patterns (DAMPs) release and suppressing epithelial-to-mesenchymal transition (EMT) process in bleomycin-induced A549 cells. In vivo results revealed that 3D-LNP/siMCU NPs effectively ameliorated collagen deposition, pro-fibrotic factors secretion, and fibroblast activation in bleomycin-induced pulmonary fibrosis (PF) mouse models. Moreover, compared to the commercial MC3-based formulation, optimized Opt-MC3/siRNA NPs with incorporating 3D-lipid as the fifth component, showed superior therapeutic efficacy against PF due to their enhanced stability and higher gene silencing efficiency. Overall, the nanomedicine containing 3D-lipid and siMCU will be a promising and potential approach for IPF treatment.

The H222P-Lamin mutation induces heart failure via impaired mitochondrial calcium uptake in human cardiac laminopathy

Abstract Background Mutations in LMNA gene, which encodes lamins A/C, cause a variety of diseases, called laminopathies. Some mutations are particularly associated with the occurrence of dilated cardiomyopathy. The pathological mechanisms are still unclear limiting the development of specific therapies. Purpose Here, we focused on a mutation in the LMNA gene (c.665A>C, p.His222Pro), associated with the Emery-Dreyfuss Muscular dystrophy. We used LMNA H222P mutant human induced pluripotent stem cells (hiPSCs) and CRISPR/Cas9 corrected isogenic control hiPSCs to better characterize the cardiac phenotype associated with the mutation. Methods LMNA H222P mutant and the isogenic control hiPSCs clones were differentiated into cardiomyocytes (CMs). Immunofluorescence staining was performed on hiPSC-CMs to quantify their sarcomere organization (SarcOrgScore) using a Matlab code. Ring-shaped cardiac organoids were generated to compare the contractile properties of the two clones. Calcium transients in mutant and corrected hiPSC-CMs were measured by live confocal imaging. Mitochondrial respiration was measured by Seahorse. Results hiPSC-CMs were generated from the LMNA H222P mutant and the corrected hiPSCs with no difference in the differentiation yield (proportion of troponin-positive cells: 92.74% for LMNA H222P vs. 89.38% for Ctrl-iso1, p=0.1038). The nuclei of LMNA H222P mutant hiPSC-CMs showed morphological abnormalities, typically observed with Lamin A/C mutations. hiPSC-CMs displayed well-formed sarcomeres and their organization was similar between the two cell lines. However, 3D cardiac organoids generated with LMNA H222P hiPSC-CMs showed an impaired contractility compared to control organoids. Calcium transient recordings in LMNA H222P mutant hiPSC-CMs showed a significantly higher calcium transient amplitude with a significantly slower calcium re-uptake. Transcriptomic analyses suggested a global mitochondrial dysfunction and in particular an impaired mitochondrial calcium uptake with a significantly decreased expression of the mitochondrial calcium uniporter (MCU). This decrease in MCU expression was confirmed by Western blot and was accompanied by an increased MICU1 : MCU ratio, as well as an increased PDH Ser232 and Ser300 phosphorylation, indicating a decreased mitochondrial calcium uptake in the LMNA H222P mutant hiPSC-CMs. Finally, measurement of mitochondrial respiration using Seahorse showed lower basal and maximal respiration in LMNA H222P hiPSC-CMs. Conclusions LMNA H222P mutant hiPSC-CMs exhibit contractile dysfunction associated with mitochondrial dysfunction with impaired MCU complex activity, decreased mitochondrial calcium homeostasis, and reduced mitochondrial energy production.

Shikonin ameliorated LPS-induced acute lung injury in mice via modulating MCU-mediated mitochondrial Ca2+ and macrophage polarization

BackgroundMacrophages play a pivotal role in the development and recovery of acute lung injury (ALI), wherein their phenotypic differentiation and metabolic programming are orchestrated by mitochondria. Specifically, the mitochondrial calcium uniporter (MCU) regulates mitochondrial Ca2+ (mCa2+) uptake and may bridge the metabolic reprogramming and functional regulation of immune cells. However, the precise mechanism on macrophages remains elusive. Shikonin, a natural naphthoquinone, has demonstrated efficacy in mitigating ALI and suppressing glycolysis in macrophages, yet which mechanism remains to be fully elucidated. PurposeThis study explored whether Shikonin ameliorated ALI via modulating MCU-mediated mCa2+ and macrophage polarization. MethodsThis study firstly examined the protective effects of Shikonin on LPS-induced ALI mice, and investigated whether it is depends on macrophage by depleting macrophage using clodronate liposomes. The regulatory effect of Shikonin on macrophage polarization and mitochondrial MCU/Ca2+ signal was testified on RAW264.7 cells, and further validated by knocking-down MCU expression or by using RU360, an MCU inhibitor. Additionally, the crucial role of MCU in the therapeutic effect of Shikonin, along with its regulation on macrophage polarization was validated in mice with LPS-induced ALI under the intervention of RU360. ResultsShikonin alleviated LPS-induced mice ALI, down-regulated inflammatory cytokines and inhibited the pro-inflammatory polarization of macrophages. Intravenous injection of clodronate liposomes on mice abolished the protective effects of Shikonin on ALI. On RAW264.7 cells, LPS&IFN decreased the protein expression of MCU, while induced pro-inflammatory polarization and glycolytic metabolism. In contrast, Shikonin increased MCU expression, activated MCU-mediated mCa2+ signal, promoted the polarization of macrophages to anti-inflammatory M2 phenotype, and driven a metabolic shift from glycolysis to oxidative phosphorylation. Either knocking-down MCU expression or pharmacological inhibiting MCU by using RU360 mitigated the effects of Shikonin on Raw 264.7 cells. Furthermore, RU360 counteracted the ameliorative effect of Shikonin on ALI mice. ConclusionThe current data showed that Shikonin alleviated LPS-induced mice ALI by activating mitochondrial MCU/mCa2+ signal and regulating macrophage metabolism.

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.

The mitochondrial calcium uniporter inhibitor Ru265 increases neuronal excitability and reduces neurotransmission via off-target effects.

Excitotoxicity due to mitochondrial calcium (Ca2+) overloading can trigger neuronal cell death in a variety of pathologies. Inhibiting the mitochondrial calcium uniporter (MCU) has been proposed as a therapeutic avenue to prevent calcium overloading. Ru265 (ClRu(NH3)4(μ-N)Ru(NH3)4Cl]Cl3) is a cell-permeable inhibitor of the mitochondrial calcium uniporter (MCU) with nanomolar affinity. Ru265 reduces sensorimotor deficits and neuronal death in models of ischemic stroke. However, the therapeutic use of Ru265 is limited by the induction of seizure-like behaviours. We examined the effect of Ru265 on synaptic and neuronal function in acute brain slices and hippocampal neuron cultures derived from mice, in control and where MCU expression was genetically abrogated. Ru265 decreased evoked responses from calyx terminals and induced spontaneous action potential firing of both the terminal and postsynaptic principal cell. Recordings of presynaptic Ca2+ currents suggested that Ru265 blocks the P/Q type channel, confirmed by the inhibition of currents in cells exogenously expressing the P/Q type channel. Measurements of presynaptic K+ currents further revealed that Ru265 blocked a KCNQ current, leading to increased membrane excitability, underlying spontaneous spiking. Ca2+ imaging of hippocampal neurons showed that Ru265 increased synchronized, high-amplitude events, recapitulating seizure-like activity seen in vivo. Importantly, MCU ablation did not suppress Ru265-induced increases in neuronal activity and seizures. Our findings provide a mechanistic explanation for the pro-convulsant effects of Ru265 and suggest counter screening assays based on the measurement of P/Q and KCNQ channel currents to identify safe MCU inhibitors.

MCU inhibition protects against intestinal ischemia‒reperfusion by inhibiting Drp1-dependent mitochondrial fission

Intestinal ischemia‒reperfusion (IIR) injury is a common complication of surgery, but clear molecular insights and valuable therapeutic targets are lacking. Mitochondrial calcium overload is an early sign of various diseases and is considered a vital factor in ischemia‒reperfusion injury. The mitochondrial calcium uniporter (MCU), which is located on the inner mitochondrial membrane, is the primary mediator of calcium ion entry into the mitochondria. However, the specific mechanism of MCU in IIR injury remains to be clarified. In this study, we generated an IIR model using C57BL/6 mice and Caco-2 cells and found increases in the calcium levels and MCU expression following IIR injury. The specific inhibition of MCU markedly attenuated IIR injury. Moreover, MCU knockdown alleviates mitochondrial dysfunction by reducing oxidative stress and apoptosis. Mechanistically, MCU knockdown substantially reduced the translocation of Drp1 and thus its binding to Fis1 receptors, resulting in decreased mitochondrial fission. Taken together, our findings demonstrated that MCU is a novel upstream regulator of Drp1 in ischemia‒reperfusion and represents a predictive and therapeutic target for IIR.

Nicotine aggravates pancreatic fibrosis in mice with chronic pancreatitis via mitochondrial calcium uniporter.

This study aimed to investigate the effects of nicotine on the activation of pancreatic stellate cells (PSCs) and pancreatic fibrosis in chronic pancreatitis (CP), along with its underlying molecular mechanisms. This was an in vivo and in vitro study. In vitro, PSCs were cultured to study the effects of nicotine on their activation and oxidative stress. Transcriptome sequencing was performed to identify potential signaling pathways involved in nicotine action. And the impact of nicotine on mitochondrial Ca2+ levels and Ca2+ transport-related proteins in PSCs was analyzed. The changes in nicotine effects were observed after the knockdown of the mitochondrial calcium uniporter (MCU) in PSCs. In vivo experiments were conducted using a mouse model of CP to assess the effects of nicotine on pancreatic fibrosis and oxidative stress in mice. The alterations in nicotine effects were observed after treatment with the MCU inhibitor Ru360. In vitro experiments demonstrated that nicotine promoted PSCs activation, characterized by increased cell proliferation, elevated α-SMA and collagen expression. Nicotine also increased the production of reactive oxygen species (ROS) and cellular malondialdehyde (MDA), exacerbating oxidative stress damage. Transcriptome sequencing revealed that nicotine may exert its effects through the calcium signaling pathway, and it was verified that nicotine elevated mitochondrial Ca2+ levels and upregulated MCU expression. Knockdown of MCU reversed the effects of nicotine on mitochondrial calcium homeostasis, improved mitochondrial oxidative stress damage and structural dysfunction, thereby alleviating the activation of PSCs. In vivo validation experiments showed that nicotine significantly aggravated pancreatic fibrosis in CP mice, promoted PSCs activation, exacerbated pancreatic tissue oxidative stress, and increased MCU expression. However, treatment with Ru360 significantly mitigated these effects. This study confirms that nicotine upregulates the expression of MCU, leading to mitochondrial calcium overload and exacerbating oxidative stress in PSCs, and ultimately promoting PSCs activation and exacerbating pancreatic fibrosis in CP.

Spatial and single-cell explorations uncover prognostic significance and immunological functions of mitochondrial calcium uniporter in breast cancer

The mitochondrial calcium uniporter (MCU) is a transmembrane protein facilitating the entry of calcium ions into mitochondria from the cell cytosol. Maintaining calcium balance is crucial for enhancing cellular energy supply and regulating cell death. The interplay of calcium balance through MCU and the sodium-calcium exchanger is known, but its regulation in the breast cancer tumor microenvironment remains elusive. Further investigations are warranted to explore MCU’s potential in BRCA clinical pathology, tumor immune microenvironment, and precision oncology. Our study, employing a multi-omics approach, identifies MCU as an independent diagnostic biomarker for breast cancer (BRCA), correlated with advanced clinical status and poor overall survival. Utilizing public datasets from GEO and TCGA, we discern differentially expressed genes in BRCA and examine their associations with immune gene expression, overall survival, tumor stage, gene mutation status, and infiltrating immune cells. Spatial transcriptomics is employed to investigate MCU gene expression in various regions of BRCA, while spatial transcriptomics and single-cell RNA-sequencing methods explore the correlation between MCUs and immune cells. Our findings are validated through the analysis of 59 BRCA patient samples, utilizing immunohistochemistry and bioinformatics to examine the relationship between MCU expression, clinicopathological features, and prognosis. The study uncovers the expression of key gene regulators in BRCA associated with genetic variations, deletions, and the tumor microenvironment. Mutations in these regulators positively correlate with different immune cells in six immune datasets, playing a pivotal role in immune cell infiltration in BRCA. Notably, high MCU performance is linked to CD8 + T cells infiltration in BRCA. Furthermore, pharmacogenomic analysis of BRCA cell lines indicates that MCU inactivation is associated with increased sensitivity to specific small molecule drugs. Our findings suggest that MCU alterations may be linked to BRCA progression, unveiling new diagnostic and prognostic implications for MCU in BRCA. The study underscores MCU's role in the tumor immune microenvironment and cell cycle progression, positioning it as a potential tool for BRCA precision medicine and drug screening.

Mitochondrial Calcium Uniporter (MCU) that Modulates Mitochondrial Calcium Uptake and Facilitates Endometrial Cancer Progression through Interaction with VDAC1.

Although endometrial cancer represents a frequently diagnosed malignancy of the female reproductive tract, we know very little about the factors that control endometrial cancer. Our study was presented to investigate the function of MCU in endometrial tumorigenesis and the molecular mechanisms involved. A total of 94 endometrial cancer patients were recruited into our cohort. MCU and VDAC1 expression was examined in tumor and normal tissues via immunohistochemistry and immunofluorescence. Associations of MCU and VDAC1 expression with clinicopathological characteristics were evaluated. After transfection with shRNA targeting MCU or full-length MCU plasmids, clone formation, wound healing, transwell and MitoTracker Red staining were separately presented in Ishikawa and RL95-2 cells. Moreover, Western blotting or immunofluorescence was utilized to examine the expression of MCU, VDAC1, Na+/Ca2+/Li+ exchanger (NCLX), and β-catenin under VDAC1 knockdown and/or MCU overexpression or knockdown. MCU and VDAC1 expression were prominently up-regulated in endometrial cancer tissues and were significantly associated with histological grade, depth of myometrial invasion and lymph node status. MCU up-regulation enhanced clone formation, migration, and mitochondrial activity of endometrial cancer cells. The opposite results were investigated when MCU was silenced. MCU or VDAC1 silencing reduced the expression of MCU, VDAC1, NCLX, and β-catenin. Moreover, VDAC1 knockdown alleviated the promoting effect of MCU overexpression on the above proteins. This investigation demonstrated that MCU-induced mitochondrial calcium uptake plays a critical role in endometrial tumorigenesis through interaction with VDAC1.

MCU promotes the migration of glioma cells by activating p38 through TFEB-mediated autophagy.

Changes in calcium signalling are crucial for the development of glioma cells. Whether mitochondrial calcium balance is involved in glial cell development is still unknown. Mitochondrial Calcium Uniporter (MCU) plays an important role in regulating glioma progression. In this work, we found that MCU and p38 expression were positively correlated with glioma grade and the degree tumour progression. MCU increases glioma cell migration by upregulating p38. Furthermore, p38 promotes glioma progression by activating Transcription Factor EB (TFEB)-mediated autophagy. Thus, MCU promotes glioma cell migration by activating autophagy in a p38/TFEB pathway-dependent manner, which provides a theoretical basis for new therapeutic targets for gliomas.

Regulation of mitochondrial calcium uniporter expression and calcium-dependent cell signalling by lncRNA Tug1 in cardiomyocytes.

Cardiomyocyte calcium homeostasis is a tightly regulated process. The mitochondrial calcium uniporter (MCU) complex can buffer elevated cytosolic Ca2+ levels and consists of pore-forming proteins including MCU, and various regulatory proteins such as mitochondrial calcium uptake proteins 1 and 2 (MICU1/2). The stoichiometry of these proteins influences the sensitivity to Ca2+ and activity of the complex. However, the factors that regulate their gene expression remain incompletely understood. Long non-coding RNAs (lncRNAs) regulate gene expression through various mechanisms, and we recently found that the lncRNA Tug1 increased the expression of Mcu and associated genes. To further explore this, we performed antisense LNA knockdown of Tug1 (Tug1 KD) in H9c2 rat cardiomyocytes. Tug1 KD increased MCU protein expression, yet pyruvate dehydrogenase dephosphorylation, which is indicative of mitochondrial Ca2+ uptake, was not enhanced. However, RNA-seq revealed that Tug1 KD increased Mcu along with differential expression of >1000 genes including many related to Ca2+ regulation pathways in the heart. To understand the effect of this on Ca2+ signalling, we measured phosphorylation of Ca2+/calmodulin-dependent protein kinase II (CaMKII) and its downstream target cAMP Response Element-Binding protein (CREB), a transcription factor known to drive Mcu gene expression. In response a Ca2+ stimulus, the increase in CAMKII and CREB phosphorylation was attenuated by Tug1 KD. Inhibition of CaMKII, but not CREB, partially prevented the Tug1 KD-mediated increase in Mcu. Together, these data suggest that Tug1 modulates MCU expression via a mechanism involving CAMKII and regulates cardiomyocyte Ca2+ signalling which could have important implications for cardiac function.

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.

Reduced mitochondrial calcium uptake in macrophages is a major driver of inflammaging

Mitochondrial dysfunction is linked to age-associated inflammation or inflammaging, but underlying mechanisms are not understood. Analyses of 700 human blood transcriptomes revealed clear signs of age-associated low-grade inflammation. Among changes in mitochondrial components, we found that the expression of mitochondrial calcium uniporter (MCU) and its regulatory subunit MICU1, genes central to mitochondrial Ca2+ (mCa2+) signaling, correlated inversely with age. Indeed, mCa2+ uptake capacity of mouse macrophages decreased significantly with age. We show that in both human and mouse macrophages, reduced mCa2+ uptake amplifies cytosolic Ca2+ oscillations and potentiates downstream nuclear factor kappa B activation, which is central to inflammation. Our findings pinpoint the mitochondrial calcium uniporter complex as a keystone molecular apparatus that links age-related changes in mitochondrial physiology to systemic macrophage-mediated age-associated inflammation. The findings raise the exciting possibility that restoring mCa2+ uptake capacity in tissue-resident macrophages may decrease inflammaging of specific organs and alleviate age-associated conditions such as neurodegenerative and cardiometabolic diseases.

Research Note: Heat stress affects immune and oxidative stress indices of the immune organs of broilers by changing the expressions of adenosine triphosphate-binding cassette subfamily G member 2, sodium-dependent vitamin C transporter-2, and mitochondrial calcium uniporter

This study aimed to determine the mechanisms of heat-induced oxidative stress in the thymus and spleen of broilers. After 28 d, 30 broilers were randomly divided into the control (25°C ± 2°C; 24 h/d) and heat-stressed (36°C ± 2°C; 8 h/d) groups; the experiment lasted for 1 wk. The broilers in each group were euthanized, and some samples were collected and analyzed at 35 d. The results showed that the birds subjected to heat stress reduced the weight (P < 0.01) and the indices of thymus (P < 0.01), the activities of T-AOC (P < 0.01) and SOD (P < 0.05) of spleen, and levels of IL-10 (P < 0.05) and the GSH-PX (P < 0.05) in thymus and spleen, and increased the IL-6 content of thymus (P < 0.05), the MDA content (P < 0.01), and the reactive oxygen species (ROS) levels (P < 0.01) in thymus and spleen. Moreover, the expression of the IgG gene in the thymus and spleen of heat-stressed broilers was increased (P < 0.05); however, the expression of the IgM gene in the spleen was increased (P < 0.05), with no difference (P > 0.05) in the thymus of heat-stressed broilers compared with the control. Furthermore, the relative expression of adenosine triphosphate-binding cassette subfamily G member 2 (ABCG2) in the thymus and spleen both increased (P < 0.05). The sodium-dependent vitamin C transporter-2 (SVCT-2) (P < 0.01) and mitochondrial calcium uniporter (MCU) (P < 0.01) mRNA levels in the thymus of heat-stressed broilers increased, and the expression of ABCG2 (P < 0.05), SVCT-2 (P < 0.01), and MCU (P < 0.01) proteins in the thymus and spleen of heat-stressed broilers increased compared with the control group. This study confirmed that heat stress-induced oxidative stress in the immune organs of broilers, further reducing immune function.

The effect of regulating MCU expression on experimental ischemic brain injury

Mitochondrial calcium uniporter (MCU) is a critical channel for Ca2+ influx into mitochondria. The present study aimed to determine if MCU knockdown has beneficial effects on ischemic brain injury and to explore the underlying mechanisms. The present study demonstrated that MCU knockdown but not total knockout (KO) attenuated ischemia infarction volume and primary cortical neuronal cells' ischemic damage. MCU knockdown maintained mitochondrial ultrastructure, alleviated calcium overload, and reduced mitochondrial apoptosis. Moreover, MCU knockdown regulated the changes of MICU1 and MICU2 after cerebral infarction, while no changes were observed in other mitochondrial calcium handling proteins. Based on metabolomics, MCU knockdown reversed middle cerebral artery occlusion (MCAO)-induced up-regulated phosphoenolpyruvate and down-regulated GDP to protect energy metabolism after cerebral infarction. Furthermore, a total of 87 and 245 differentially expressed genes (DEGs) were detected by transcriptome sequencing among WT mice, MCU KO mice and MCU knockdown mice in the MCAO model, respectively. Then, NR4A1 was identified as one of the DEGs in different MCU expressions in vivo ischemia stroke model via transcriptomic screening and genetic validation. Furthermore, MCU knockdown downregulated the ischemia-induced upregulation of NR4A1 expression. Together, this is the further evidence that the MCU knockdown exerts a protective role after cerebral infarction by promoting calcium homeostasis, inhibiting mitochondrial apoptosis and protecting energy metabolism.

MCU Upregulation Overactivates Mitophagy by Promoting VDAC1 Dimerization and Ubiquitination in the Hepatotoxicity of Cadmium.

Cadmium (Cd) is a high-risk pathogenic toxin for hepatic diseases. Excessive mitophagy is a hallmark in Cd-induced hepatotoxicity. However, the underlying mechanism remains obscure. Mitochondrial calcium uniporter (MCU) is a key regulator for mitochondrial and cellular homeostasis. Here, Cd exposure upregulated MCU expression and increased mitochondrial Ca2+ uptake are found. MCU inhibition through siRNA or by Ru360 significantly attenuates Cd-induced excessive mitophagy, thereby rescues mitochondrial dysfunction and increases hepatocyte viability. Heterozygous MCU knockout mice exhibit improved liver function, ameliorated pathological damage, less mitochondrial fragmentation, and mitophagy after Cd exposure. Mechanistically, Cd upregulates MCU expression through phosphorylation activation of cAMP-response element binding protein at Ser133(CREBS133 ) and subsequent binding of MCU promoter at the TGAGGTCT, ACGTCA, and CTCCGTGATGTA regions, leading to increased MCU gene transcription. The upregulated MCU intensively interacts with voltage-dependent anion-selective channel protein 1 (VDAC1), enhances its dimerization and ubiquitination, resulting in excessive mitophagy. This study reveals a novel mechanism, through which Cd upregulates MCU to enhance mitophagy and hepatotoxicity.

Mitochondrial calcium uniporter activates TFEB-driven autophagy to promote migration of breast cancer cells.

Tumor metastasis is the leading cause of death in breast cancer (BC) patients and is a complicated process. Mitochondrial calcium uniporter (MCU), a selective channel responsible for mitochondrial Ca2+ uptake, has been reported to be associated with tumorigenesis and metastasis. The molecular mechanisms of MCU contributing to the migration of BC cells are partially understood. This study investigated the role of MCU in BC cell metastasis and explored the underlying mechanism of MCU-mediated autophagy in BC cell migration. The Kaplan-Meier plotter database was used to analyze the prognostic value of MCU mRNA expression. Western blotting was used to examine the expression level of MCU in 4 paired BC and adjacent normal tissues. The cellular migration capability of BC was measured by transwell migration assay and wound healing assay. Western blotting and reverse transcription-quantitative polymerase chain reaction were performed to detect the expression levels of autophagy-related markers. The effects of MCU activation or inhibition on TFEB nuclear translocation in BC cells were detected by laser scanning confocal microscopy. Expression of MCU was found to be negatively correlated with BC patient prognosis in the Kaplan-Meier plotter database. Compared with the adjacent normal tissues, MCU was markedly up-regulated in the BC tissues. MCU overexpression promoted cellular migration, activated autophagy, and increased TFEB nuclear translocation in BC cells, whereas its knockdown produced the opposite effects. MCU activates TFEB-driven autophagy to promote BC cell metastasis and provides a potential novel therapeutic target for BC clinical intervention.

Ruthenium red attenuates acute pancreatitis by inhibiting MCU and improving mitochondrial function

Acute pancreatitis (AP) is a common inflammatory disease of the digestive system. Mitochondrial calcium uniporter (MCU) mediates mitochondrial uptake of Ca2+ and plays an important role in calcium homeostasis. However, it is undefined whether AP can be relieved by inhibiting MCU. This study aimed to study the therapeutic potential of Ruthenium red (RuR), a MCU inhibitor, in AP mice model and primary acinar cells. Cell injury and AP mice model was induced by caerulein. RuR alleviated CER-AP evidenced by reduced serum lipase, TNF-α, and pancreatic MPO levels, less severe pancreatic pathology damage, and decreased inflammatory cell infiltration. In freshly isolated pancreatic acinar cells, RuR diminished cell necrosis with effect on suppressing the expression of MCU. RuR also decreased levels of cytosolic calcium and ROS, preventing mitochondrial membrane potential loss, ATP depletion and MPTP opening. The present findings indicate that inhibit MCU by RuR has a beneficial effect in AP by preventing calcium overload, mitochondrial dysfunction, and cell necrosis.

Abstract P3038: Upregulation Of The Mitochondrial Calcium Uniporter Abolishes Mitochondrial Dysfunction Associated With Obesity In Vascular Smooth Muscle Cells

Obesity is a growing national epidemic that is associated with an increased prevalence of arterial disease. We found that aortic medial hypertrophy during obesity was accompanied by upregulation of serum and glucocorticoid-inducible kinase 1 (SGK1) and reduced mitochondrial oxidative capacity of vascular smooth muscle cells (VSMC). Interestingly, VSMC-specific deletion of SGK1 attenuated obesity-related aortic remodeling and restored mitochondrial oxidative capacity in VSMC implicating a mediator role for SGK1 in mitochondrial dysfunction during obesity. The mechanistic basis for impaired mitochondrial respiration during obesity in VSMC is unknown. Thus, due to its fundamental role in stimulation of mitochondrial oxidative phosphorylation, the current study will investigate the mitochondrial calcium uniporter (MCU) in obesity-related mitochondrial dysfunction in VSMC. As SGK1 is known to regulate the expression and/or activity of ion channels, we hypothesize that upregulation of SGK1 during obesity negatively regulates MCU expression, mitochondrial oxidative activity, and ATP production. MCU protein levels were assessed by western blot analysis. Mitochondrial oxidative activity and ATP production rate were measured using the Agilent Seahorse XF Cell Mito Stress Test and the ATP Rate Assay, respectively in cultured wildtype and SGK1 knockout aortic VSMC from low-fat (LF) control and high-fat (HF) obese mice. To assess the importance of MCU, both assays were performed in the presence or absence of the MCU inhibitor, Ru360. Obesity caused a significant decrease in MCU abundance, but SGK1 deletion resulted in enrichment of MCU in VSMC. Ru360 treatment reversed the increase in basal mitochondrial respiration. We found that obesity reduced overall ATP production by diminishing ATP production from glycolysis and mitochondrial respiration. However, SGK1 deletion enhanced ATP production from glycolysis and mitochondrial respiration during obesity and this effect was attenuated by Ru360. These findings demonstrate that SGK1 negatively regulates MCU expression and activity and imply that augmentation of MCU-driven mitochondrial oxidative activity may underlie the protective effects of SGK1 inhibition on the vascular during obesity.

Mitochondrial Calcium Transporters Regulate Autophagy

Autophagy is an essential cellular process regulated by intracellular calcium signals, which play an important role in autophagic activation during metabolic changes. Calcium transporters are present in mitochondria, an organelle able to uptake and release calcium ions, thus participating in cellular calcium signaling. Uptake by mitochondria is mediated by the mitochondrial calcium uniporter (MCU), while extrusion occurs through the mitochondrial sodium/lithium/calcium exchanger (NCLX). The aim of this work was to investigate how MCU and NCLX affect autophagy. To do so, we evaluated makers of autophagic activity in murine Aml‐12 hepatic cells transfected with siRNAs targeting either MCU or NCLX expression, leading to genetic knockdown (KD). Additionally, we investigated the autophagic response to serum/amino acid starvation and rapamycin (mTORC1 inhibitor) treatment in Aml‐12 cells with NCLX KD or pharmacological inhibition by CGP37157 (CGP). Using a cell line stably expressing the autophagic probe LC3‐GFP‐mCherry, we observed that NCLX KD leads to impaired autophagosome formation under basal conditions. Curiously, this effect was associated with a significant decrease in mRNA expression of LC3A and LC3B genes, while the expression of other autophagy‐related genes, such as TFEB, ATG5, ATG12, and ATG7, was upregulated or unchanged. Conversely, MCU KD led to an apparent increase in autophagosome and autolysosome numbers, indicating enhanced autophagic activity. Interestingly, MCU KD also led to decreased expression of LC3A, but not LC3B. The expression of TFEB, ATG12, ATG5, and ATG7 were decreased or unchanged by MCU KD. After autophagic stimulation by serum/amino acid starvation, the levels of LC3 II were lower in NCLX KD cells compared to negative control in the presence or absence of bafilomycin A1, which indicates a reduction of autophagic flux. The levels of LC3 I were significantly lower in NCLX KD cells under basal and stimulated conditions, corroborating the decreased LC3 mRNA levels observed. Importantly, these effects were also observed using CGP to inhibit NCLX. The reduction of autophagic flux by NCLX KD or CGP was not observed in cells treated with rapamycin. We also measured the levels of phosphorylated 4E‐BP1 as an indication of mTORC1 activity. As expected, serum/amino acid starvation and rapamycin decreased the levels of p‐4E‐BP1; however, this decrease was modulated by CGP only in starved cells, indicating that NCLX may affect mTORC1 activity in an upstream pathway. In conclusion, we show that mitochondrial calcium transporters are novel autophagy‐regulating pathways: MCU modulates autophagic activation under basal conditions, while NCLX maintains autophagic activity under basal and stimulated conditions.