MCU Expression Research Articles

Effects of a proinflammatory response on metabolic function of cultured, primary ruminal epithelial cells

Inflammation of ruminal epithelium may occur during ruminal acidosis as a result of translocation and interaction of ruminal epithelial cells (REC) with molecules such as lipopolysaccharide (LPS). Such inflammation has been reported to alter cellular processes such as nutrient absorption, metabolic regulation, and energy substrate utilization in other cell types but has not been investigated for REC. The objectives of this study were to investigate the effects of LPS on metabolism of short-chain fatty acids by primary REC, as well as investigating the effects of media containing short-chain fatty acids on the proinflammatory response. Ruminal papillae from 9 yearling Speckle Park beef heifers were used to isolate and culture primary REC. Cells were grown in minimum essential medium (MEM) for 12 d before use and then reseeded in 24-well culture plates. The study was conducted as a 2 × 2 factorial, where cells were grown in unaltered MEM (REG) or medium containing 2 mM butyrate and 5 mM propionate (SCFA) with (50,000 EU/mL; +LPS) or without LPS (-LPS) for 24 h. Supernatant samples were collected for analysis of glucose and SCFA consumption. Cells were collected to determine the expression of mRNA for genes associated with inflammation (TNF, IL1B, CXCL2, CXCL8, PTGS2, and TLR4), purinergic signaling (P2RX7, ADORAB2, and CD73), nutrient transport [SLC16A1 (MCT1), SLC16A3 (MCT4), SLC5A8, and MCU], and cell metabolism [ACAT1, SLC2A1 (GLUT1), IGFBP3, and IGFBP5]. Protein expression of TLR4 and ketogenic enzymes (BDH1 and HMGCS1) were also analyzed using flow cytometry. Statistical analysis was conducted with the MIXED model of SAS version 9.4 (SAS Institute Inc., Cary, NC) with medium, LPS exposure, and medium × LPS interaction as fixed effects and animal within plate as a random effect. Cells tended to consume more glucose when exposed to LPS as opposed to no LPS exposure (31.8 vs. 28.7 ± 2.7), but consumption of propionate and butyrate was not influenced by LPS. Expression of TNF and IL1B was upregulated when exposed to LPS, and expression of CXCL2 and CXCL8 increased following LPS exposure with SCFA (medium × LPS). For cells exposed to LPS, we found a downregulation of ACAT1 and IGFBP5 and an upregulation of SLC2A1, SLC16A3, MCU, and IGFBP3. Medium with SCFA led to greater expression of MCU. SLC16A1 was upregulated in cells incubated with SCFA and without LPS compared with the other groups. Protein expression of ketogenic enzymes was not affected; however, BDH1 mean fluorescence intensity (MFI) expression tended to be less in cells exposed to LPS. These data are interpreted to indicate that when REC are exposed to LPS, they may increase glucose metabolism. Moreover, transport of solutes was affected by SCFA in the medium and by exposure to LPS. Overall, the results suggest that metabolic function of REC in vitro is altered by a proinflammatory response, which may lead to a greater glucose requirement.

Variable Assembly of EMRE and MCU Creates Functional Channels with Distinct Gatekeeping Profiles.

SummaryMCU is a Ca2+-selective channel that mediates mitochondrial Ca2+ influx. The human channel contains tetrameric pore-forming MCU, regulatory subunits MICU1/2, and EMRE that is required both for channel function and MICU1/2-mediated Ca2+ regulation. A structure of MCU with EMRE revealed a 4:4 stoichiometry, but the stoichiometry in vivo is unknown. Expression of tagged EMRE and MCU at a 1:10 ratio in cells lacking EMRE and MCU restored channel activity but not full channel gatekeeping. Increasing EMRE expression enhanced gatekeeping, raising the cytoplasmic Ca2+ concentration ([Ca2+]c) threshold for channel activation. MCU-EMRE concatemers creating channels with 1EMRE:4MCU restored Ca2+ uptake in cells, whereas cells expressing concatemers that enforced a 4EMRE:4MCU stoichiometry demonstrated enhanced channel gatekeeping. Concatemers enforcing 2EMRE/4MCU recapitulated the activity, gatekeeping, and size of endogenous channels. Thus, MCU does not require four EMRE, with most endogenous channels containing two, but complexes with 1–4 EMRE have activity with full or partial gatekeeping.

The Role of MCU Expression Regulation in Pathogenesis

The Role of MCU Expression Regulation in Pathogenesis

Transport of Ca2+ and Ca2+-Dependent Permeability Transition in Rat Liver Mitochondria under the Streptozotocin-Induced Type I Diabetes

Although diabetes mellitus is known to be a disease associated with mitochondrial dysfunction, not everything is clear about mitochondrial Ca2+ transport and Ca2+-induced permeability transition in diabetic cells. The objective of this work was to study the operation of MCU and Ca2+-dependent mitochondrial permeabilization in the liver cells of Sprague-Dawley rats under the streptozotocin-induced type I diabetes. It was shown that two weeks after the induction of diabetes, the rate of Ca2+ uptake by the mitochondria of diabetic animals increased ~1.4-fold. The expression of MCU and MICU1 subunits did not change, yet the quantity of dominant-negative MCUb channel subunits was almost twice as lower. The organelles also became more resistant to the induction of CsA-sensitive MPT pore and less resistant to the induction of CsA-insensitive palmitate/Ca2+-induced pore. The mitochondria of diabetic liver cells also showed changes in the lipid matrix of their membranes. The content of fatty acids in the membranes grew, and microviscosity of the lipid bilayer (assessed with laurdan) increased. At the same time, lipid peroxidation (assessed by the production of malonic dialdehyde) was stimulated. The paper discusses the consequences of the diabetes-related changes in mitochondria in the context of cell physiology.

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

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

A1964 TRPP3 knockout promotes cardiac hypertrophy through modulation of mitochondrial calcium homeostasis in mice

Objectives: High intake of salt is an independent risk factor for cardiac hypertrophy. TRPP3 acts as a “sour” sensor in taste cells, and its potential role in the cardiovascular system is not known. We investigated the role of TRPP3 in high salt-induced cardiac hypertrophy. Methods: Two-month male TRPP3 knockout (TRPP3−/−) mice and their wild-type (WT) littermates were fed a normal diet (0.4% NaCl) or high-salt diet (HSD; 8% NaCl) diet for 24 weeks. Cardiac hypertrophy was examined and cardiomyocyte hypertrophy assayed by wheat germ agglutinin (WGA) staining and detection of protein levels of hypertrophic markers. H9c2 cardiomyocytes were stained with Fura-2AM or Rhod-2AM fluorescence indicators for detection of cytosolic and cardiac mitochondrial calcium ions (Ca2+), respectively. Expression of the mitochondrial Ca2+channelsNCX1, NHE1, MCU and LETM1wasdetected by western blotting. Results: High salt intake increased the HW/BW ratio and HW/TL ratio of WT mice significantly, and this was exacerbated by TRPP3 knockout. Consistently, WGA staining revealed that the cardiomyocyte hypertrophy induced by a HSD was exacerbated markedly in TRPP3−/− mice, and this was accompanied by higher expression of the hypertrophic markers β-MHC, ANP and BNP, compared with WT mice. Cytosolic and mitochondrial Ca2+uptake in H9c2 cardiomyocytes was reduced significantly by TRPP3 knockdown. TRPP3 deficiency resulted in increased expression of NCX1 and NHE1 without affecting expression of MCU and LETM1 in cardiomyocytes. Conclusion: We identified TRPP3 as a novel regulator of pathologic cardiac hypertrophy. The reduced Ca2+influx in cardiomyocytes and upregulation of expression of NCX1 and NHE1 contribute to the pro-hypertrophic effect of TRPP3 knockout.

Privileged crosstalk between TRPV1 channels and mitochondrial calcium shuttling machinery controls nociception

The nociceptive noxious heat-activated receptor - TRPV1, conducts calcium and sodium, thus producing a depolarizing receptor potential, leading to activation of nociceptive neurons. TRPV1-mediated calcium and sodium influx is negatively modulated by calcium, via calcium-dependent desensitization of TRPV1 channels. A mitochondrial Ca2+ uniporter - MCU, controls mitochondrial Ca2+ entry while a sodium/calcium transporter - NCLX shapes calcium and sodium transients by mediating sodium entry into and removing calcium from the mitochondria. The functional interplay between TRPV1, MCU and NCLX, in controlling the cytosolic and mitochondrial calcium and sodium transients and subsequently the nociceptive excitability, is poorly understood. Here, we used cytosolic and mitochondrial fluorescent calcium and sodium imaging together with electrophysiological recordings of TRPV1-induced currents in HEK293T cells and nociceptor-like dissociated rat dorsal root ganglion neurons, while modulating NCLX or MCU expression using specific small interfering RNA (siNCLX). We show that the propagation of the TRPV1-induced cytosolic calcium and sodium fluxes into mitochondria is dependent on coordinated activity of NCLX and MCU. Thus, knocking-down of NCLX triggers down regulation of MCU dependent mitochondrial Ca2+ uptake. This in turn decreases rate and amplitude of TRPV1-mediated cytosolic calcium, which inhibits capsaicin-induced inward current and neuronal firing. TRPV1-mediated currents were fully rescued by intracellular inclusion of the fast calcium chelator BAPTA. Finally, NCLX controls capsaicin-induced cell death, by supporting massive mitochondrial Ca2+ shuttling. Altogether, our results suggest that NCLX, by regulating cytosolic and mitochondrial ionic transients, modulates calcium-dependent desensitization of TRPV1 channels, thereby, controlling nociceptive signaling.

The mitochondrial calcium uniporter is involved in mitochondrial calcium cycle dysfunction: Underlying mechanism of hypertension associated with mitochondrial tRNAIle A4263G mutation

Recent studies have shown that the mitochondrial DNA mutations are involved in the pathogenesis of hypertension. Our previous study identified mitochondrial tRNAIle A4263G mutation in a large Chinese Han family with maternally-inherited hypertension. This mutation may contribute to mitochondrial Ca2+ cycling dysfuntion, but the mechanism is unclear. Lymphoblastoid cell lines were derived from hypertensive and normotensive individuals, either with or without tRNAIle A4263G mutation. The mitochondrial calcium ([Ca2+]m) in cells from hypertensive subjects with the tRNAIle A4263G mutation, was lower than in cells from normotension or hypertension without mutation, or normotension with mutation (P<0.05). Meanwhile, cytosolic calcium ([Ca2+]c) in hypertensive with mutation cells was higher than another three groups. After exposure to caffeine, which could increase the [Ca2+]c by activating ryanodine receptor on endoplasmic reticulum, [Ca2+]c/[Ca2+]m increased higher than in hypertensive with mutation cells from another three groups. Moreover, MCU expression was decreased in hypertensive with mutation cells compared with in another three groups (P<0.05). [Ca2+]c increased and [Ca2+]m decreased after treatment with Ru360 (an inhibitor of MCU) or an siRNA against MCU. In this study we found decreased MCU expression in hypertensive with mutation cells contributed to dysregulated Ca2+ uptake into the mitochondria, and cytoplasmic Ca2+ overload. This abnormality might be involved in the underlying mechanisms of maternally inherited hypertension in subjects carrying the mitochondrial tRNAIle A4263G mutation.

Expression of mRNA Encoding Mcu and Other Mitochondrial Calcium Regulatory Genes Depends on Cell Type, Neuronal Subtype, and Ca2+ Signaling.

Uptake of Ca2+ into the mitochondrial matrix controls cellular metabolism and survival-death pathways. Several genes are implicated in controlling mitochondrial Ca2+ uptake (mitochondrial calcium regulatory genes, MCRGs), however, less is known about the factors which influence their expression level. Here we have compared MCRG mRNA expression, in neural cells of differing type (cortical neurons vs. astrocytes), differing neuronal subtype (CA3 vs. CA1 hippocampus) and in response to Ca2+ influx, using a combination of qPCR and RNA-seq analysis. Of note, we find that the Mcu-regulating Micu gene family profile differs substantially between neurons and astrocytes, while expression of Mcu itself is markedly different between CA3 and CA1 regions in the adult hippocampus. Moreover, dynamic control of MCRG mRNA expression in response to membrane depolarization-induced Ca2+ influx is also apparent, resulting in repression of Letm1, as well as Mcu. Thus, the mRNA expression profile of MCRGs is not fixed, which may cause differences in the coupling between cytoplasmic and mitochondrial Ca2+, as well as diversity of mitochondrial Ca2+ uptake mechanisms.

In vivo Reconstitution of the Mitochondrial Uniporter

The mitochondrial uniporter is a highly selective calcium channel present broadly in eukaryotes, but absent in Saccharomyces cerevisiae. Therefore, we used yeast as a reconstitution system to identify the minimal components sufficient for in vivo uniporter activity. First, we considered Dictyostelium discoideum and showed that it has a highly simplified uniporter machinery: the expression of DdMCU, a single transmembrane component alone is sufficient to reconstitute mitochondrial calcium uniporter activity. Second, to establish human uniporter activity, the coexpression of MCU and - the animal specific protein - EMRE is necessary, whereas expression of MCU alone is insufficient. Our work established yeast as a powerful in vivo reconstitution system for the uniporter to study the evolution and function of this channel.

Unresolved questions from the analysis of mice lacking MCU expression

Entry of mitochondrial calcium is believed to play an essential role in regulating bioenergetics and initiating cell death pathways. We have recently described a mouse model lacking MCU expression. Surprisingly, these mice are viable and the cells and tissues from these animals do not exhibit any marked protection from cell death. Here, we discuss our findings as well as potential explanations for some of the more unexpected results.

Mitochondrial calcium uniporter Mcu controls excitotoxicity and is transcriptionally repressed by neuroprotective nuclear calcium signals

The recent identification of the mitochondrial Ca2+ uniporter gene (Mcu/Ccdc109a) has enabled us to address its role, and that of mitochondrial Ca2+ uptake, in neuronal excitotoxicity. Here we show that exogenously expressed Mcu is mitochondrially localized and increases mitochondrial Ca2+ levels following NMDA receptor activation, leading to increased mitochondrial membrane depolarization and excitotoxic cell death. Knockdown of endogenous Mcu expression reduces NMDA-induced increases in mitochondrial Ca2+, resulting in lower levels of mitochondrial depolarization and resistance to excitotoxicity. Mcu is subject to dynamic regulation as part of an activity-dependent adaptive mechanism that limits mitochondrial Ca2+ overload when cytoplasmic Ca2+ levels are high. Specifically, synaptic activity transcriptionally represses Mcu, via a mechanism involving the nuclear Ca2+ and CaM kinase-mediated induction of Npas4, resulting in the inhibition of NMDA receptor-induced mitochondrial Ca2+ uptake and preventing excitotoxic death. This establishes Mcu and the pathways regulating its expression as important determinants of excitotoxicity, which may represent therapeutic targets for excitotoxic disorders.