Mitochondrial Calcium Uniporter Regulator Research Articles

MiR‑124 inhibits cardiomyocyte apoptosis in myocardial ischaemia‑reperfusion injury by activating mitochondrial calcium uniporter regulator 1.

Mitochondria‑mediated apoptosis is the primary cause of cardiomyocyte death. Therefore, mitochondria are a key target for treating myocardial injury. Mitochondrial calcium uniporter regulator 1 (MCUR1)‑mediated mitochondrial calcium homeostasis markedly promotes cell proliferation and resistance to apoptosis. However, whether MCUR1 is involved in regulation of cardiomyocyte apoptosis during myocardial ischaemia‑reperfusion remains unknown. microRNA‑124 (miR‑124) is upregulated in cardiovascular disease, suggesting a key role for miR‑124 in the cardiovascular system. Whether miR‑124 affects cardiomyocyte apoptosis and myocardial infarction is not well understood. Western blot showed that miR‑124 and MCUR1 were upregulated in cardiomyocyte apoptosis induced by hydrogen peroxide (H2O2). Flow cytometry assay of cell apoptosis showed that miR‑124 inhibited cardiomyocyte apoptosis by activating MCUR1 following H2O2 treatment. The dual‑luciferase reporter assay confirmed binding of miR‑124 to MCUR1 3'‑UTR and subsequent activation of MCUR1. FISH assay revealed the entry of miR‑124 into the cell nucleus. Therefore, MCUR1 was identified as a novel target of miR‑124, and it was shown that the miR‑124‑MCUR1 axis modulated cardiomyocyte apoptosis induced by H2O2 invitro. The results indicated induced expression of miR‑124 during acute myocardial infarction and its transport to the nucleus. In the nucleus, miR‑124 transcriptionally activated MCUR1 by binding to its enhancers. These findings reveal a role of miR‑124 as a biomarker for myocardial injury and infarction.

Mitochondrial Calcium Uniporter Regulator 1 (MCUR1) Relieves Mitochondrial Damage Induced by Lipopolysaccharide by Mediating Mitochondrial Ca2+ Homeostasis in Bovine Mammary Epithelial Cells

The metabolic stress triggered by negative energy balance after calving induces mitochondrial damage of bovine mammary epithelial cells. Mitochondrial calcium uniporter regulator 1 (MCUR1) is a key protein-coding gene that mediates mitochondrial calcium ion (Ca2+) uptake and plays an important role in mediating homeostasis of mitochondria. The aim of the present study was to elucidate the effects of MCUR1-mediated Ca2+ homeostasis on mitochondria of bovine mammary epithelial cells in response to an inflammatory challenge with lipopolysaccharide (LPS). Exogenous LPS resulted in upregulation of the MCUR1 mRNA and protein abundance, mitochondrial Ca2+ content, and mitochondrial reactive oxygen species (Mito-ROS) content while decreasing mitochondrial membrane potential, causing mitochondrial damage, and increasing the rate of apoptosis. Ryanodine pretreatment attenuated the upregulation of the mitochondrial Ca2+ content and Mito-ROS content induced by LPS. Overexpression of MCUR1 increased the mitochondrial Ca2+ content and Mito-ROS content, while it decreased mitochondrial membrane potential, damaged mitochondria, and induced cell apoptosis. In addition, knockdown of MCUR1 by small interfering RNA attenuated LPS-induced mitochondrial dysfunction by inhibiting mitochondrial Ca2+ uptake. Our results revealed that exogenous LPS induces MCUR1-mediated mitochondrial Ca2+ overload in bovine mammary epithelial cells, which leads to mitochondrial injury. Thus, MCUR1-mediated Ca2+ homeostasis may be a potential therapeutic target against mitochondrial damage induced by metabolic challenges in bovine mammary epithelial cells.

MiR‑4732‑5p promotes ovarian cancer mobility by targeting MCUR1.

MicroRNAs (miRNAs/miRs) play critical roles in tumor progression. However, the role of miR-4732 and its underlying molecular mechanism in ovarian cancer (OC) remain unclear. In the present study, the high expression of miR-4732 was confirmed to be associated with the mortality of patients with OC following surgery, according to The Cancer Genome Atlas Ovarian Cancer database (TCGA-OV). Additionally, the expression of miR-4732 was positively associated with an increased tendency to exhibit an early TNM stage (IIA, IIB and IIC) of OC, indicating its promotional role in the early stages of tumorigenesis. By performing in vitro gain-of-function experiments, the transient transfection of IGROV1 cells with miR-4732-5p mimics enhanced cell viability according to Cell Counting Kit-8 assay, and cell migration and invasion in Transwell assays. However, though the application of loss-of-function experiments, the transient transfection of IGROV1 cells with miR-4732-5p inhibitors hindered cell viability, cell migration and invasion in vitro. Mitochondrial calcium uniporter regulator 1 (MCUR1) was validated as a downstream direct target of miR-4732-5p through bioinformatics analysis, western blotting and luciferase assays. Therefore, the results of the present study provide evidence that miR-4732-5p may promote OC cell mobility through the direct targeting of the tumor suppressor, MCUR1.

Netrin-1 attenuates cerebral ischemia/reperfusion injury by limiting mitochondrial ROS and Ca2+ levels via activation of AKT phosphorylation and mitochondrial m-AAA protease AFG3L2.

Cerebral ischemia-reperfusion (I/R) injury as the consequence of revascularization after ischemic stroke is associated with mitochondrial dysfunction, oxidative stress, and neuron loss. In this study, we used a deprivation/reoxygenation (OGD/R) model to determine whether interactions between Netrin-1, AKT, and the mitochondrial AAA protease AFG3L2 could influence mitochondrial function in neurons after I/R. We found that Netrin-1 protects primary cortical neurons from OGD/R-induced cell death and regulates mitochondrial reactive oxygen species (ROS) and Ca2+ levels. The accumulation of mitochondrial calcium uniporter (MCU) subunits was monitored in cells by immunoblot analysis. Although the regulatory subunits MICU1 and MICU2 were relatively unaffected, the accumulation of the essential MCU regulator (EMRE) subunit was impaired. In OGD/R-induced cells, the 7kDa form of EMRE was significantly reduced. Netrin-1 inhibited the accumulation of EMRE and mitochondrial Ca2+ levels by upregulating AFG3L2 and AKT activation. Loss of AFG3L2 or inhibition of AKT increased levels of 7kDa EMRE. Moreover, overexpression of AKT increased the expression of AFG3L2 in Netrin-1-knockdown neurons after OGD/R. Our results demonstrate that Netrin-1 enhanced AFG3L2 protein expression via activation of AKT. We also observed that overexpression of Netrin-1 significantly reduced infarction size in an I/R-induced brain injury model in rats but not when AKT was inhibited. Our data suggest that AFG3L2 is a protein substrate of AKT and indicate that Netrin-1 attenuates cerebral I/R injury by limiting mitochondrial ROS and Ca2+ levels through activating AKT phosphorylation and AFG3L2.

A brown fat-selective mechanism of mitochondrial calcium import?

In this issue of Cell Metabolism, Xue etal. propose that the mitochondrial calcium uniporter (MCU) binds uncoupling protein 1 (UCP1) via the MCU regulator (EMRE) to form a protein complex that the authors term the "thermoporter." Through gain- and loss-of-function experiments, the authors infer that the thermoporter promotes calcium influx into the mitochondrial matrix to enhance NADH production, which supports thermogenesis in brown adipose tissue (BAT).

The mitochondrial calcium uniporter engages UCP1 to form a thermoporter that promotes thermogenesis

Uncoupling protein 1 (UCP1)-mediated adaptive thermogenesis protects mammals against hypothermia and metabolic dysregulation. Whether and how mitochondrial calcium regulates this process remains unclear. Here, we show that mitochondrial calcium uniporter (MCU) recruits UCP1 through essential MCU regulator (EMRE) to form an MCU-EMRE-UCP1 complex upon adrenergic stimulation. This complex formation increases mitochondrial calcium uptake to accelerate the tricarboxylic acid cycle and supply more protons that promote uncoupled respiration, functioning as a thermogenic uniporter. Mitochondrial calcium uptake 1 (MICU1) negatively regulates thermogenesis probably through inhibiting thermogenic uniporter formation. Accordingly, the deletion of Mcu or Emre in brown adipocytes markedly impairs thermogenesis and exacerbates obesity and metabolic dysfunction. Remarkably, the enhanced assembly of the thermogenic uniporter via Micu1 knockout or expressing linked EMRE-UCP1 results in opposite phenotypes. Thus, we have uncovered a "thermoporter" that provides a driving force for the UCP1 operation in thermogenesis, which could be leveraged to combat obesity and associated metabolic disorders.

Quantitative analysis of mitochondrial calcium uniporter (MCU) and essential MCU regulator (EMRE) in mitochondria from mouse tissues and HeLa cells

Mitochondrial calcium homeostasis plays critical roles in cell survival and aerobic metabolism in eukaryotes. The calcium uniporter is a highly selective calcium ion channel consisting of several subunits. Mitochondrial calcium uniporter (MCU) and essential MCU regulator (EMRE) are core subunits of the calcium uniporter required for calcium uptake activity in the mitochondria. Recent 3D structure analysis of the MCU‐EMRE complex reconstituted in nanodiscs revealed that the human MCU exists as a tetramer forming a channel pore, with EMRE bound to each MCU at a 1 : 1 ratio. However, the stoichiometry of MCU and EMRE in the mitochondria has not yet been investigated. We here quantitatively examined the protein levels of MCU and EMRE in the mitochondria from mouse tissues by using characterized antibodies and standard proteins. Unexpectedly, the number of EMRE molecules was lower than that of MCU; moreover, the ratios between MCU and EMRE were significantly different among tissues. Statistical calculations based on our findings suggest that a MCU tetramer binding to 4 EMREs may exist, but at low levels in the mitochondrial inner membrane. In brain mitochondria, the majority of MCU tetramers bind to 2 EMREs; in mitochondria in liver, kidney, and heart, MCU tetramers bind to 1 EMRE; and in kidney and heart, almost half of MCU tetramers bound to no EMRE. We propose here a novel stoichiometric model of the MCU‐EMRE complex in mitochondria.

Key Role of MCUR1 in Malignant Progression of Breast Cancer.

BackgroundMitochondrial calcium uniporter regulator 1 (MCUR1, also known as CCDC90A) is a protein-coding gene that plays a key role in mitochondrial calcium uptake. However, knowledge about its clinical significance in breast cancer is still limited.MethodsThe expression profile of MCUR1 in various cancers was analyzed via the ONCOMINE and Tumor Immune Estimation Resource databases. The correlation between MCUR1 expression and the clinical features of breast cancer was investigated using UALCAN and MEXPRESS. Immunohistochemical analysis was applied to verify the expression of MCUR1 in breast cancer. The prognostic significance of MCUR1 in breast cancer was evaluated using Kaplan–Meier plotter and the PrognoScan database. Gene Set Enrichment Analysis (GSEA) was performed to explore the possible biological functions of MCUR1. In addition, the function of MCUR1 was examined by gene silencing in vitro. Western blotting was applied to detect the expression of proteins.ResultsMCUR1 was overexpressed in breast cancer and significantly related to the clinical characteristics of breast cancer. Results from the public databases and IHC analysis indicated that MCUR1 expression was the highest in triple-negative breast cancer (TNBC). The high expression of MCUR1 was associated with poor overall survival, distant metastasis-free survival, and recurrence-free survival. GSEA showed that the hypoxia pathway, cellular reactive oxygen species (ROS) pathway, epithelial–mesenchymal transition pathway, and notch signaling pathway were differentially enriched in the high MCUR1 expression phenotype. In vitro experiments showed that MCUR1 knockdown in TNBC cell lines led to a decrease in cellular ROS and weakened cell migration and invasion abilities. Moreover, Western blotting showed that MCUR1 knockdown inhibited the epithelial–mesenchymal transition of TNBC cells via the ROS/Nrf2/Notch pathways.ConclusionOur study suggests that MCUR1 plays a pivotal role in the malignant progression of breast cancer.

The Molecular Mechanisms of Mitochondrial Calcium Uptake by Calcium Uniporter

Mitochondria play a role as intracellular calcium stores as well as energy conversion functions. Excessive calcium accumulation in mitochondria induces cell death and induces diseases such as ischemia-reperfusion injury. Mitochondrial calcium uptake is considered to be mediated by calcium uniporters, which have attracted much attention as potential drug targets. Although calcium uniporter was shown to function as an ion channel, the molecular mechanisms have long been unclear. In this decade, the molecular composition of the calcium uniporter complex was discovered; the calcium uniporter consists of the 7 subunits. Each subunit has no structural similarity to other Ca ion channels; thus, the novel molecular mechanism of the Ca2+ uptake by calcium uniporter is of interest. Although calcium uniporter is conserved in human to warm, yeast lack mitochondrial calcium uptake activity. In the previous study, various subunits of mammalian calcium uniporter were expressed in the yeast mitochondria. As a result, although the expression of each subunit alone did not affect on the mitochondrial calcium uptake activity, the co-expression of mitochondrial calcium uniporter (MCU) and essential MCU regulator (EMRE) enabled to reconstitute calcium uptake activity in yeast mitochondria. This indicated that MCU and EMRE are key factors of the calcium uptake activity in mitochondria. This yeast reconstitution technique has also enabled us to perform detailed structure-function analysis of the MCU and EMRE. In this paper, we will discuss the molecular mechanism of Ca2+ uptake and the prospects for drug discovery.

Evolutionary divergence reveals the molecular basis of EMRE dependence of the human MCU.

The mitochondrial calcium uniporter (MCU) is a calcium-activated calcium channel critical for signaling and bioenergetics. MCU, the pore-forming subunit of the uniporter, contains two transmembrane domains and is found in all major eukaryotic taxa. In amoeba and fungi, MCU homologs are sufficient to form a functional calcium channel, whereas human MCU exhibits a strict requirement for the metazoan protein essential MCU regulator (EMRE) for conductance. Here, we exploit this evolutionary divergence to decipher the molecular basis of human MCU's dependence on EMRE. By systematically generating chimeric proteins that consist of EMRE-independent Dictyostelium discoideum MCU and Homo sapiens MCU (HsMCU), we converged on a stretch of 10 amino acids in D. discoideum MCU that can be transplanted to HsMCU to render it EMRE independent. We call this region in human MCU the EMRE dependence domain (EDD). Crosslinking experiments show that EMRE directly interacts with HsMCU at its transmembrane domains as well as the EDD. Our results suggest that EMRE stabilizes the EDD of MCU, permitting both channel opening and calcium conductance, consistent with recently published structures of MCU-EMRE.

Abstract 381: Mcur1 is a Potential Target to Modulate Cardiac Contractility and Alleviate Cardiac Function During Heart Failure

Cardiac contractility is regulated by the intracellular Ca 2+ concentration fluxes which are actively regulated by multiple channels and transporters. Ca 2+ uptake into the mitochondrial matrix is precisely controlled by the highly Ca 2+ selective channel, Mitochondrial Calcium Uniporter (MCU). Earlier studies on the cardiac-specific acute MCU knockout and a transgenic dominant-negative MCU mice have demonstrated that mitochondrial Ca 2+ ( m Ca 2+ ) signaling is necessary for cardiac ‘‘fight-or-flight’’ contractile response, however, the role of m Ca 2+ buffering to shape global cytosolic Ca 2+ levels and affect E-C coupling, particularly the Ca 2+ transient, on a beat-to-beat basis still remains to be solved. Our earlier studies have demonstrated that loss of MCU Regulator 1 (MCUR1) in cardiomyocytes results in the impaired m Ca 2+ uptake. We have now employed the cardiac-specific MCUR1 knockout mouse to dissect the precise role of MCU in regulating cytosolic Ca 2+ transients associated with excitation-contraction (E-C) coupling and cardiac function. Results from our studies including the in vivo analyses of cardiac physiology during normal and pressure-overloaded mouse models and in vitro experiments including single-cell cardiac contractility, calcium transients, and electrophysiology measurements demonstrate that MCUR1/MCU regulated m Ca 2+ buffering in cardiomyocytes, although insignificant under basal condition, becomes critical in stress induced conditions and actively participates in regulating the c Ca 2+ transients. Also, the ablation of MCUR1 in cardiomyocytes during stress conditions prevents m Ca 2+ overload and subsequent mROS overproduction. Our data indicate that MCUR1 ablation offers protection against pressure-overload cardiac hypertrophy. In summary, our results provide critical insights into the mechanisms by which the MCU channel contributes in regulating the contractile function of the cardiomyocytes and the role of m Ca 2+ in the development and progression of heart failure.

Structural Characterization of the N-Terminal Domain of the Dictyostelium discoideum Mitochondrial Calcium Uniporter.

The mitochondrial calcium uniporter (MCU) plays a critical role in mitochondrial calcium uptake into the matrix. In metazoans, the uniporter is a tightly regulated multicomponent system, including the pore-forming subunit MCU and several regulators (MICU1, MICU2, and Essential MCU REgulator, EMRE). The calcium-conducting activity of metazoan MCU requires the single-transmembrane protein EMRE. Dictyostelium discoideum (Dd), however, developed a simplified uniporter for which the pore-forming MCU (DdMCU) alone is necessary and sufficient for calcium influx. Here, we report a crystal structure of the N-terminal domain (NTD) of DdMCU at 1.7 Å resolution. The DdMCU-NTD contains four helices and two strands arranged in a fold that is completely different from the known structures of other MCU-NTD homologues. Biochemical and biophysical analyses of DdMCU-NTD in solution indicated that the domain exists as high-order oligomers. Mutagenesis showed that the acidic residues Asp60, Glu72, and Glu74, which appeared to mediate the interface II, as observed in the crystal structure, participated in the self-assembly of DdMCU-NTD. Intriguingly, the oligomeric complex was disrupted in the presence of calcium. We propose that the calcium-triggered dissociation of NTD regulates the channel activity of DdMCU by a yet unknown mechanism.

Functional analysis of coiled-coil domains of MCU in mitochondrial calcium uptake

The mitochondrial calcium uniporter (MCU) complex is a highly-selective calcium channel. This complex consists of MCU, mitochondrial calcium uptake proteins (MICUs), MCU regulator 1 (MCUR1), essential MCU regulator element (EMRE), etc. MCU, which is the pore-forming subunit, has 2 highly conserved coiled-coil domains (CC1 and CC2); however, their functional roles are unknown. The yeast expression system of mammalian MCU and EMRE enables precise reconstitution of the properties of the mammalian MCU complex in yeast mitochondria. Using the yeast expression system, we here showed that, when MCU mutant lacking CC1 or CC2 was expressed together with EMRE in yeast, their mitochondrial Ca2+-uptake function was lost. Additionally, point mutations in CC1 or CC2, which were expected to prevent the formation of the coiled coil, also disrupted the Ca2+-uptake function. Thus, it is essential for the Ca2+ uptake function of MCU that the coiled-coil structure be formed in CC1 and CC2. The loss of function of those mutated MCUs was also observed in the mitochondria of a yeast strain lacking the yeast MCUR1 homolog. Also, in the D. discoideum MCU, which has EMRE-independent Ca2+-uptake function, the deletion of either CC1 or CC2 caused the loss of function. These results indicated that the critical functions of CC1 and CC2 were independent of other regulatory subunits such as MCUR1 and EMRE, suggesting that CC1 and CC2 might be essential for pore formation by MCUs themselves. Based on the tetrameric structure of MCU, we discussed the functional roles of the coiled-coil domains of MCU.

Abstract 570: Mechanism Of Mitochondrial Calcium Uniporter Regulation

Calcium uptake by mitochondria plays an important role in sequestering cytosolic calcium, regulating ATP production and maintaining the redox homeostasis. Mitochondrial calcium uniporter (MCU), a macromolecular channel complex that resides in the inner mitochondrial membrane, is the primary electrogenic pathway for calcium influx into the matrix. Because calcium uptake by mitochondria is dictated by the kinetic equilibrium between the influx and efflux machinery, the patch clamp is the best method to measure electrogenic MCU activity in isolation from the efflux mechanisms. Here we report the development of a system for heterologous expression of MCU and its associated subunits, essential MCU regulator (EMRE), MCUb and MICU1–3. Using this system, we investigated the biophysical properties of the MCU pore and its regulation. The system is based on a recombinant cell line (Drp1 -/- mouse embryonic fibroblasts) in which knockouts for all subunits of the MCU complex have been generated. This system allowed both optical and direct electrophysiological studies of the structure-function relationships in the MCU complex with a relative ease in comparison to other cell lines. We confirmed the essential role of pore-forming MCU and EMRE subunits in mediating the MCU current. Importantly, fast-step solution exchange and single-channel recordings allowed us to gain novel insights into the biophysical properties of MCU e.g. inward rectification, kinetics of activation and selectivity to monovalent vs divalent ions.

Progress in understanding mitochondrial calcium uniporter complex-mediated calcium signalling: A potential target for cancer treatment.

Due to its Ca2+ buffering capacity, the mitochondrion is one of the most important intracellular organelles in regulating Ca2+ dynamic oscillation. Mitochondrial calcium uniporter (MCU) is the primary mediator of Ca2+ influx into mitochondria, manipulating cell energy metabolism, ROS production, and programmed cell death, all of which are critical for carcinogenesis. The understanding of the uniporter complex was significantly boosted by recent groundbreaking discoveries that identified the uniporter pore-forming subunit MCU and its regulatory molecules, including MCU-dominant negative β subunit (MCUb), essential MCU regulator (EMRE), MCU regulator 1 (MCUR1), mitochondrial calcium uptake (MICU) 1, MICU2, and MICU3. These provide the means and molecular platform to investigate MCU complex (uniplex)-mediated impaired Ca2+ signalling in physiology and pathology. This review aims to summarize the progress of the understanding regulatory mechanisms of uniplex, roles of uniplex-mediated Ca2+ signalling in cancer, and potential pharmacological inhibitors of MCU.

MCUR1 facilitates epithelial-mesenchymal transition and metastasis via the mitochondrial calcium dependent ROS/Nrf2/Notch pathway in hepatocellular carcinoma

BackgroundMitochondrial Ca2+ plays a critical role in tumorigenesis, including cell proliferation and metastasis. Mitochondrial calcium uniporter regulator 1 (MCUR1) has been shown to be frequently upregulated in HCC and promote cancer cell survival. However, whether MCUR1 is involved in the metastasis of HCC and its underlying mechanisms remain unknown.MethodsThe effect of MCUR1 expression on epithelial-mesenchymal transition (EMT) in HCC cells was first evaluated by immunofluorescent staining and Western blot. Then, in vitro invasion and in vivo metastasis assays were used to evaluate the function of MCUR1 in HCC metastasis. The underlying mechanism has also been explored by investigating the effect of MCUR1 on ROS/Nrf2/Notch1 pathway.ResultsMCUR1 expression was significantly higher in HCC with metastasis and associated with tumor progression. MCUR1 promoted in vitro invasion and in vivo metastasis of HCC cells by promoting EMT via Snail. Mechanistically, MCUR1-mediated mitochondrial Ca2+ signaling promoted the EMT of HCC cells by activating ROS/Nrf2/Notch1 pathway. Inhibition of ROS production, mitochondrial Ca2+ uptake, Nrf2 expression or Notch1 activity significantly suppressed MCUR1-induced EMT of HCC cells. In addition, treatment with the mitochondrial Ca2+-buffering protein parvalbumin significantly inhibited ROS/Nrf2/Notch pathway and MCUR1-induced EMT and HCC metastasis.ConclusionsOur study provides evidence supporting a metastasis-promoting role for MCUR1-dependent mitochondrial Ca2+ uptake in HCC. Our findings suggest that MCUR1 may be a potential therapeutic target for HCC treatment.

Characterization of MCU-Binding Proteins MCUR1 and CCDC90B — Representatives of a Protein Family Conserved in Prokaryotes and Eukaryotic Organelles

Membrane-bound coiled-coil proteins are important mediators of signaling, fusion, and scaffolding. Here, we delineate a heterogeneous group of trimeric membrane-anchored proteins in prokaryotes and eukaryotic organelles with a characteristic head-neck-stalk-anchor architecture, in which a membrane-anchored coiled-coil stalk projects an N-terminal head domain via a β-layer neck. Based on sequence analysis, we identify different types of head domains and determine crystal structures of two representatives, the archaeal protein Kcr-0859 and the human CCDC90B, which possesses the most widespread head type. Using mitochondrial calcium uniporter regulator 1 (MCUR1), the functionally characterized paralog of CCDC90B, we study the role of individual domains, and find that the head interacts directly with the mitochondrial calcium uniporter (MCU) and is destabilized upon Ca2+ binding. Our data provide structural details of a class of membrane-bound coiled-coil proteins and identify the conserved head domain of the most widespread type as a mediator of their function.

Abstract 366: The Role of Mitochondrial Calcium Uniporter Complex in Cardiac Contractility and Function

Cardiac contractility is regulated by the intracellular Ca 2+ concentration fluxes which are maintained by multiple channels and transporters. Mitochondrial Calcium Uniporter (MCU) is the highly selective Ca 2+ ion channel responsible for the electrophoretic Ca 2+ uptake into the mitochondrial matrix. Earlier studies on the cardiac-specific acute MCU knockout and transgenic dominant-negative MCU mice have demonstrated that mitochondrial Ca 2+ ( m Ca 2+ ) signaling is necessary for cardiac ‘‘fight-or-flight’’ contractile response, however, the role of m Ca 2+ in the beat-to-beat regulation of cardiac contractility is highly debated. The MCU channel function is regulated by varied molecular regulatory components as well as ROS mediated modification at the Cys-96 residue of the core MCU protein. Our earlier studies have demonstrated that loss of MCU Regulator 1 (MCUR1) in cardiomyocytes results in the impaired m Ca 2+ uptake. To dissect the precise role of MCU in regulating cytosolic Ca 2+ transients associated with excitation-contraction (E-C) coupling and cardiac functions, we have employed the loss-of-function, cardiac-specific MCUR1 knockout mouse and a gain-of-function MCU C96A knockin mouse generated by CRISPR/Cas9 approach. Using these novel mouse models, both by in vivo analyses of cardiac physiology and in vitro experiments including single-cell cardiac contractility, calcium transients, and electrophysiology measurements, our study demonstrates that m Ca 2+ buffering actively participates in E-C coupling. MCUR1/MCU regulated m Ca 2+ buffering in cardiomyocytes, although insignificant under basal condition, becomes critical in stress-induced conditions and actively participates in regulating the c Ca 2+ transients during adrenergic stimulation. Our data indicate that MCUR1 ablation offers protection against pressure-overload cardiac hypertrophy and MCU C96A KI mice exhibit significantly decreased LV ejection fraction at baseline. In summary, our results provide critical insights into the mechanisms by which the MCU channel contributes in regulating the contractile function of the cardiomyocytes and the role of m Ca 2+ in the development and progression of heart failure.

Fine‐tuning of hepatocyte calcium signaling and liver regeneration by the mitochondrial calcium uniporter

Calcium (Ca2+) signaling regulates a variety of cellular processes such as metabolism, cell proliferation, division and differentiation, gene transcription, stimulus‐secretion coupling and cell death. Ca2+ is able to regulate these diverse cellular processes due to the versatility of the Ca2+ signaling mechanism in terms of speed, amplitude and spatiotemporal patterning. Mitochondria form an integral part of this machinery, both as regulators and targets of Ca2+ signaling. In recent years, leading‐edge research in the field of mCa2+ (mitochondrial Ca2+) handling and its implications for cellular Ca2+ homeostasis opened up the possibility to investigate the functional implications of mCa2+ uptake in shaping the spatiotemporal dynamics of Ca2+ signaling, and regulating cellular metabolism (through activation of Ca2+ sensitive TCA cycle enzymes) and mitochondria‐mediated cell death (through mitochondrial permeability transition). mCa2+ is mediated by the mitochondrial uniporter complex, consisting of the pore forming MCU (Mitochondrial Calcium Uniporter) and regulators such as MICU1 (mitochondrial calcium uptake 1), MICU2 and EMRE (essential MCU regulator). MCU deletion abolishes mCa2+ uptake in mouse hepatocytes driven by vasopressin‐evoked cCa2+ (cytosolic Ca2+) increases. This lack of mCa2+ uptake resulted in alterations in cCa2+ dynamics, including an elevated resting [cCa2+] and a reduced cCa2+ peak and the total Ca2+ mobilization in response to vasopressin stimulation. These changes in cCa2+ dynamics could lead to alterations in downstream Ca2+ responsive signaling events. Indeed, we see suppression of AMPK activation through CAMKK2, in response to hormone‐evoked Ca2+ signals in MCU KD hepatocytes. Activation of Creb, CAMKK2, AMPKα and ERK1/2 are also more transient in the MCU KD hepatocytes compared to controls. Our previous studies have highlighted the importance of regulating MCU (through MICU1) under stress conditions involving enhanced mCa2+ entry, especially during liver regeneration following partial hepatectomy (PHx). Enhanced mCa2+ entry during liver regeneration maybe vital to cellular signaling events and increased mitochondrial ATP production. The lack of mCa2+ uptake in hepatocyte‐specific MCU KD mice resulted in decelerated kinetics of cell cycle progression after PHx reflected by the inhibition in BrdU incorporation and Ki67 upregulation at 30h post‐PHx, which recovers by 36h. Interestingly, apart from an inhibition in Cyclin D1 expression, many cell cycle regulatory proteins remained unchanged in the KD compared to WT controls. Cell signaling events such as AMPK, CAMKK2 and mTOR activation post‐PHx are altered. Gene expression analysis at 30h post‐PHx revealed massive changes in gene expression profiles between KD and control livers, especially in genes involving metabolic processes and protein synthesis in response to PHx. Overall, the lack of mCa2+ uptake affects multiple signaling and metabolic pathways in hepatocytes and impacts the kinetics of the liver regeneration response.Support or Funding InformationAA018873 and U01EB023224 (JBH)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Single channel recording of a mitochondrial calcium uniporter

Mitochondrial calcium uniporter (MCU) is the pore-forming subunit of the entire uniporter complex and plays an important role in mitochondrial calcium uptake. However, the single channel recording of MCU remains controversial. Here, we expressed and purified different MCU proteins and then reconstituted them into planar lipid bilayers for single channel recording. We showed that MCU alone from Pyronema omphalodes (pMCU) is active with prominent single channel Ca2+ currents. In sharp contrast, MCU alone from Homo sapiens (hMCU) is inactive. The essential MCU regulator (EMRE) activates hMCU, and therefore, the complex (hMCU-hEMRE) shows prominent single channel Ca2+ currents. These single channel currents are sensitive to the specific MCU inhibitor Ruthenium Red. Our results clearly demonstrate that active MCU can conduct large amounts of calcium into the mitochondria.