Mitochondrial Calcium Uniporter Research Articles

Liver knockout of MCU leads to greater dysregulation of lipid metabolism in MAFLD.

Metabolic-associated fatty liver disease (MAFLD) is a common chronic condition that poses a significant threat to human health. Mitochondrial dysfunction, particularly involving the mitochondrial Ca2+ uniporter (MCU), plays a key role in its pathogenesis. This study aimed to investigate the impact of the MCU gene on hepatic lipid metabolism in mice fed a high-fat diet. Using MCU knockout and wild-type mice, subjected to either a high-fat or normal diet for 14weeks, we observed notable Steatosis and liver weight gain in MCU-deficient mice. Liver function markers, serum triglycerides, very low-density lipoprotein (VLDL) levels, and ApoB protein expression were all significantly elevated. Mechanistic studies revealed that MCU deletion led to mitochondrial dysfunction, increased oxidative stress. These findings highlight the critical role of the MCU gene in maintaining hepatic lipid balance and suggest its potential as a therapeutic target for managing nonalcoholic fatty liver disease.

Genome-Wide Identification and Expression Profiling Analysis of the Mitochondrial Calcium Uniporter Gene Family Under Abiotic Stresses in Medicago sativa

The mitochondrial calcium uniporters (MCUs) are a family of calcium unidirectional transporters important for cytoplasmic Ca2+ signals. Though the MCU proteins in several plant species have been investigated, genome-wide analysis of MCUs in alfalfa is lacking. Here, via genome-wide analysis, a total of 5, 20, and 6 MCU genes were identified in three different alfalfa cultivars, namely Medicago truncatula Jemalong A17, Medicago sativa XinJiangDaYe, and M. sativa Zhongmu No. 1, respectively. They were further phylogenetically classified into three subfamilies. Most MCU genes have only one intron, and gene duplication events of MCU genes were observed within each alfalfa accession and between different accessions. All alfalfa MCU proteins contained a highly conserved MCU domain and 10 conserved motifs, featuring two transmembrane domains and a DI/VME motif. According to the tissue expression data of M. sativa Zhongmu No. 1, MsMCU6.2 was the most abundant transcript with the highest expression in the leaf, and MsMCU5 and MsMCU1.2 showed higher expression levels in the stem than other tissues. We analyzed the expression profiles of five MCU genes (MsMCU1.1/1.2/5/6.1/6.2) under salt, drought, and cold stresses via qRT-PCR assays. All five MCU genes were induced by drought stress, except MsMCU5, whose expression was up-regulated by salt stress, while cold stress slightly altered MsMCU expression. Nine potential interacting proteins and three miRNAs targeting MtMCUs were predicted. These results provide detailed knowledge of alfalfa MCU genes and suggest their potential functions in abiotic stress response.

Mitochondrial calcium uptake orchestrates vertebrate pigmentation via transcriptional regulation of keratin filaments.

Mitochondria regulate several physiological functions through mitochondrial Ca2+ dynamics. However, role of mitochondrial Ca2+ signaling in melanosome biology remains unknown. Here, we show that pigmentation requires mitochondrial Ca2+ uptake. In vitro gain and loss of function studies demonstrate that mitochondrial Ca2+ uniporter (MCU) is crucial for melanogenesis while MCU rheostat, MCUb negatively control melanogenesis. Zebrafish, MCU+/- and MCUb-/- mice models show that MCU complex drives pigmentation in vivo. Mechanistically, MCU silencing activates transcription factor NFAT2 to induce expression of keratin (5, 7, and 8) filaments. Interestingly, keratin5 in turn augments mitochondrial Ca2+ uptake and potentiates melanogenesis by regulating melanosome biogenesis and maturation. Hence this signaling module acts as a negative feedback loop that fine-tunes both mitochondrial Ca2+ signaling and pigmentation. Notably, mitoxantrone, an FDA approved drug that inhibits MCU, reduces pigmentation thereby highlighting therapeutic potential of targeting mitochondrial Ca2+ uptake for clinical management of pigmentary disorders. Taken together, we reveal an MCU-NFAT2-Keratin5 driven signaling axis that acts as a critical determinant of mitochondrial Ca2+ uptake and pigmentation. Given the vital role of mitochondrial Ca2+ signaling and keratin filaments in cellular physiology, this feedback loop could be operational in a variety of other patho-physiological processes.

Knockdown of hepatic mitochondrial calcium uniporter mitigates MASH and fibrosis in mice

BackgroundMitochondrial calcium uniporter (MCU) plays pleiotropic roles in cellular physiology and pathology that contributes to a variety of diseases, but the role and potential mechanism of MCU in the pathogenesis of metabolic dysfunction-associated steatohepatitis (MASH) remain poorly understood.Methods and resultsHere, hepatic knockdown of MCU in C57BL/6J mice was achieved by tail vein injection of AAV8-mediated the CRISPR/Cas9. Mice were fed a Choline-deficient, L-amino acid-defined high-fat diet (CDAHFD) for 8 weeks to induce MASH and fibrosis. We find that expression of MCU enhanced in MASH livers of humans and mice. MCU knockdown robustly limits lipid droplet accumulation, steatosis, inflammation, and hepatocyte apoptotic death during MASH development both in vivo in mice and in vitro in cellular models. MCU-deficient mice strikingly mitigate MASH-related fibrosis. Moreover, the protective effects of MCU knockdown against MASH progression are accompanied by a reduced level of mitochondrial calcium, limiting hepatic oxidative stress, and attenuating mitochondrial dysfunction. Mechanically, RNA sequencing analysis and protein immunoblotting indicate that knockdown MCU inhibited the Hippo/YAP pathway activation and restored the AMP-activated protein kinase (AMPK) activity during MASH development both in vitro and in vivo.ConclusionsMCU is up-regulated in MASH livers in humans and mice; and hepatic MCU knockdown protects against diet-induced MASH and fibrosis in mice. Thus, targeting MCU may represent a novel therapeutic strategy for MASH and fibrosis.Graphical

PINK1-deficiency facilitates mitochondrial iron accumulation and colon tumorigenesis

ABSTRACT Mitophagy, the process by which cells eliminate damaged mitochondria, is mediated by PINK1 (PTEN induced kinase 1). Our recent research indicates that PINK1 functions as a tumor suppressor in colorectal cancer by regulating cellular metabolism. Interestingly, PINK1 ablation activated the NLRP3 (NLR family pyrin domain containing 3) inflammasome, releasing IL1B (interleukin 1 beta). However, inhibiting the NLRP3-IL1B signaling pathway with an IL1R (interleukin 1 receptor) antagonist or NLRP3 inhibitor did not hinder colon tumor growth after PINK1 loss. To identify druggable targets in PINK1-deficient tumors, ribonucleic acid sequencing analysis was performed on colon tumors from pink1 knockout and wild-type mice. Gene Set Enrichment Analysis highlighted the enrichment of iron ion transmembrane transporter activity. Subsequent qualitative polymerase chain reaction and western blot analysis revealed an increase in mitochondrial iron transporters, including mitochondrial calcium uniporter, in PINK1-deficient colon tumor cells and tissues. Live-cell iron staining demonstrated elevated cellular and mitochondrial iron levels in PINK1-deficient cells. Clinically used drugs deferiprone and minocycline reduced mitochondrial iron and superoxide levels, resulting in decreased colon tumor cell growth in vitro and in vivo. Manipulating the mitochondrial iron uptake protein MCU (mitochondrial calcium uniporter) also affected cell and xenograft tumor growth. This study suggests that therapies aimed at reducing mitochondrial iron levels may effectively inhibit colon tumor growth, particularly in patients with low PINK1 expression.

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.

MICU2 up-regulation enhances tumor aggressiveness and metabolic reprogramming during colorectal cancer development.

The mitochondrial Ca2+ uniporter (MCU) plays crucial role in intramitochondrial Ca2+ uptake, allowing Ca2+-dependent activation of oxidative metabolism. In recent decades, the role of MCU pore-forming proteins has been highlighted in cancer. However, the contribution of MCU-associated regulatory proteins mitochondrial calcium uptake 1 and 2 (MICU1 and MICU2) to pathophysiological conditions has been poorly investigated. Here, we describe the role of MICU2 in cell proliferation and invasion using in vitro and in vivo models of human colorectal cancer (CRC). Transcriptomic analysis demonstrated an increase in MICU2 expression and the MICU2/MICU1 ratio in advanced CRC and CRC-derived metastases. We report that expression of MICU2 is necessary for mitochondrial Ca2+ uptake and quality of the mitochondrial network. Our data reveal the interplay between MICU2 and MICU1 in the metabolic flexibility between anaerobic glycolysis and OXPHOS. Overall, our study sheds light on the potential role of the MICUs in diseases associated with metabolic reprogramming.

RNA-seq analysis of mitochondria-related genes regulated by AMPK in the human trophoblast cell line BeWo.

How AMP activated protein kinase (AMPK) signaling regulates mitochondrial functions and mitophagy in human trophoblast cells remains unclear. This study was designed to investigate potential players mediating the regulation of AMPK on mitochondrial functions and mitophagy by next generation RNA-seq. We compared ATP production in protein kinase AMP-activated catalytic subunit alpha 1/2 (PRKAA1/2) knockdown (AKD) and control BeWo cells using the Seahorse real-time ATP rate test, then analyzed gene expression profiling by RNA-seq. Differentially expressed genes (DEG) were examined by Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment. Then protein-protein interactions (PPI) among mitochondria related genes were further analyzed using Metascape and Ingenuity Pathway Analysis (IPA) software. Both mitochondrial and glycolytic ATP production in AKD cells were lower than in the control BeWo cells (CT), with a greater reduction of mitochondrial ATP production. A total of 1092 DEGs were identified, with 405 upregulated and 687 downregulated. GO analysis identified 60 genes associated with the term 'mitochondrion' in the cellular component domain. PPI analysis identified three clusters of mitochondria related genes, including aldo-keto reductase family 1 member B10 and B15 (AKR1B10, AKR1B15), alanyl-tRNA synthetase 1 (AARS1), mitochondrial ribosomal protein S6 (MRPS6), mitochondrial calcium uniporter dominant negative subunit beta (MCUB) and dihydrolipoamide branched chain transacylase E2 (DBT). In summary, this study identified multiple mitochondria related genes regulated by AMPK in BeWo cells, and among them, three clusters of genes may potentially contribute to altered mitochondrial functions in response to reduced AMPK signaling.

Effects of Salicylic and Acetylsalicylic Acids in Mitochondrial and Erythrocyte Membranes

For further clarification of the mechanisms of pharmacological effects of salicylic and acetylsalicylic acids, the interactions of these acids with mitochondrial and erythrocyte membranes were studied and the role of calcium ions in the effects of salicylic and acetylsalicylic acids was examined. Salicylic acid and to a lesser extent acetylsalicylic acid at 0.5−2.0 mM concentration effectively inhibited the respiratory activity of isolated rat liver mitochondria, by uncoupling respiration and phosphorylation processes, induced depolarization of the mitochondrial membrane and potentiated Ca2+-stimulated formation of mitochondrial permeability transition pores in EGTA-free media. Cyclosporine A and ruthenium red partially inhibited the mitochondrial pore opening process induced by salicylic and acetylsalicylic acids both in the absence and presence of Ca2+ ions. Salicylic acid (180–360 µM) markedly accelerated proton-induced lysis of human erythrocytes (at pH 3.2) and caused hyperpolarization of erythrocyte membranes (at pH 5.5, but not at pH 7.4), probably as a result of proton transfer to the cytoplasm of the cell. Thus, salicylic and acetylsalicylic acids interact with mitochondrial and plasma membranes, act as effective proton/Ca2+ ionophores and stimulate the mitochondrial calcium uniporter.

Compound heterozygous mutation of AFG3L2 causes autosomal recessive spinocerebellar ataxia through mitochondrial impairment and MICU1 mediated Ca2+ overload.

Autosomal recessive spinocerebellar ataxias (SCARs) are one of the most common neurodegenerative diseases characterized by progressive ataxia. Although SCARs are known to be caused by mutations in multiple genes, there are still many cases that go undiagnosed or are misdiagnosed. In this study, we presented a SCAR patient, and identified a probable novel pathogenic mutation (c.1A>G, p.M1V) in the AFG3L2 start codon. The proband's genotype included heterozygous mutations of the compound AFG3L2 (p.[M1V]; [R632X] (c.[1A>G]; [1894.C>T])), which were inherited from the father (c.1A>G, p.M1V) and mother (c.1894C>T, p.R632X). Functional studies performed on hiPSCs (human induced pluripotent stem cells) generated from the patients and HEK293T cells showed that the mutations impair mitochondrial function and the unbalanced expression of AFG3L2 mRNA and protein levels. Furthermore, this novel mutation resulted in the degradation of the protein and the reduction of the stability of the AFG3L2 protein, and MCU (mitochondrial calcium uniporter) complex mediated Ca2+ overload.

Loss of mitochondrial Ca2+ response and CaMKII/ERK activation by LRRK2R1441G mutation correlate with impaired depolarization-induced mitophagy

BackgroundStress-induced activation of ERK/Drp1 serves as a checkpoint in the segregation of damaged mitochondria for autophagic clearance (mitophagy). Elevated cytosolic calcium (Ca2+) activates ERK, which is pivotal to mitophagy initiation. This process is altered in Parkinson’s disease (PD) with mutations in leucine-rich repeat kinase 2 (LRRK2), potentially contributing to mitochondrial dysfunction. Pathogenic LRRK2 mutation is linked to dysregulated cellular Ca2+ signaling but the mechanism involved remains unclear.MethodsMitochondrial damages lead to membrane depolarization. To investigate how LRRK2 mutation impairs cellular response to mitochondrial damages, mitochondrial depolarization was induced by artificial uncoupler (FCCP) in wild-type (WT) and LRRK2R1441G mutant knockin (KI) mouse embryonic fibroblasts (MEFs). The resultant cytosolic Ca2+ flux was assessed using live-cell Ca2+ imaging. The role of mitochondria in FCCP-induced cytosolic Ca2+ surge was confirmed by co-treatment with the mitochondrial sodium-calcium exchanger (NCLX) inhibitor. Cellular mitochondrial quality and function were evaluated by Seahorse™ real-time cell metabolic analysis, flow cytometry, and confocal imaging. Mitochondrial morphology was visualized using transmission electron microscopy (TEM). Activation (phosphorylation) of stress response pathways were assessed by immunoblotting.ResultsAcute mitochondrial depolarization induced by FCCP resulted in an immediate cytosolic Ca2+ surge in WT MEFs, mediated predominantly via mitochondrial NCLX. However, such cytosolic Ca2+ response was abolished in LRRK2 KI MEFs. This loss of response in KI was associated with impaired activation of Ca2+/calmodulin-dependent kinase II (CaMKII) and MEK, the two upstream kinases of ERK. Treatment of LRRK2 inhibitor did not rescue this phenotype indicating that it was not caused by mutant LRRK2 kinase hyperactivity. KI MEFs exhibited swollen mitochondria with distorted cristae, depolarized mitochondrial membrane potential, and reduced mitochondrial Ca2+ store and mitochondrial calcium uniporter (MCU) expression. These mutant cells also exhibited lower cellular ATP: ADP ratio albeit higher basal respiration than WT, indicating compensation for mitochondrial dysfunction. These defects may hinder cellular stress response and signals to Drp1-mediated mitophagy, as evident by impaired mitochondrial clearance in the mutant.ConclusionsPathogenic LRRK2R1441G mutation abolished mitochondrial depolarization-induced Ca2+ response and impaired the basal mitochondrial clearance. Inherent defects from LRRK2 mutation have weakened the cellular ability to scavenge damaged mitochondria, which may further aggravate mitochondrial dysfunction and neurodegeneration in PD.

The intricacies of mitochondrial calcium and enzyme regulation in liver metabolism

Mitochondrial Ca2+ plays a positive role in regulating pyruvate dehydrogenase, as well as the TCA cycle enzymes isocitrate dehydrogenase and α-ketoglutarate dehydrogenase. This regulation boosts the production of reducing equivalents that fuel the electron transport chain, ultimately driving ATP production. The Mitochondrial Calcium Uniporter (MCU) is the highly selective channel responsible for mitochondrial Ca2+ uptake when local Ca2+ levels reach the threshold for channel activation. In a recent study, LaMoia et al. used an innovative [13C5]glutamine-based metabolic flux analysis method (Q-flux) to measure in vivo hepatic metabolic fluxes in liver-specific MCU-/- mice. Surprisingly, they observed increased flux through isocitrate dehydrogenase and α-ketoglutarate dehydrogenase. Metabolic pathways are continuously reorganized in response to intrinsic cellular signals, as well as hormonal and nutritional inputs. Integrating metabolic flux analysis into complex systems can provide deeper insights into how metabolic adaptations occur under different conditions.

CD38 and the mitochondrial calcium uniporter contribute to age-related hematopoietic stem cell dysfunction.

Hematopoietic stem cells (HSCs) are the multipotent progenitors of all immune cells. During aging, their regenerative capacity decreases for reasons that are not well understood. Recently, Song et al investigated the roles of two metabolic proteins in age-related HSC dysfunction: CD38 (a membrane-bound NADase) and the mitochondrial calcium uniporter that transports calcium into the mitochondrial matrix. They found that the interplay between these proteins is deranged in aged HSCs, contributing to their diminished renewal capacity. These findings implicate compromised nicotinamide adenine dinucleotide metabolism as underlying HSC dysfunction in aging.

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.

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.

Conditional ablation of MCU exacerbated cardiac pathology in a genetic arrhythmic model of CPVT

BackgroundCatecholaminergic polymorphic ventricular tachycardia (CPVT) is a genetic arrhythmic syndrome caused by mutations in the calcium (Ca2+) release channel ryanodine receptor (RyR2) and its accessory proteins. These mutations make the channel leaky, resulting in Ca2+-dependent arrhythmias. Besides arrhythmias, CPVT hearts typically lack structural cardiac remodeling, a characteristic often observed in other cardiac conditions (heart failure, prediabetes) also marked by RyR2 leak. Recent studies suggest that mitochondria are able to accommodate more Ca2+ influx to inhibit arrhythmias in CPVT. Thus, we hypothesize that CPVT mitochondria can absorb diastolic Ca2+ to protect the heart from cardiac remodeling. Methods and resultsThe Mitochondrial Ca2+ uniporter (MCU), the main mitochondrial Ca2+ uptake protein, was conditionally knocked out in a CPVT model of calsequestrin 2 (CASQ2) KO. In vivo cardiac function was impaired in the CASQ2−/−-MCUCKO model as assessed by echocardiography. Cardiac dilation and cellular hypertrophy were also observed in the CASQ2−/−-MCUCKO hearts. Live-cell imaging identified altered Ca2+ handling and increased oxidative stress in CASQ2−/−-MCUCKO myocytes. The activation status of Ca2+-dependent remodeling pathways (CaMKII, Calcineurin) was not altered in the CASQ2−/−-MCUCKO model. RNAseq identified changes in the transcriptome of the CASQ2−/−-MCUCKO hearts, distinct from the classic cardiac remodeling program of fetal gene re-expression. ConclusionsWe present genetic evidence that mitochondria play a protective role in CPVT. MCU-dependent Ca2+ uptake is crucial for preventing pathological cardiac remodeling in CPVT.

C-Src Is Responsible for Mitochondria-Mediated Arrhythmic Risk in Ischemic Cardiomyopathy.

Increased mitochondrial Ca2+ uptake has been implicated in the QT prolongation and lethal arrhythmias associated with nonischemic cardiomyopathy. We attempted to define the role of mitochondria in ischemic arrhythmic risk and to identify upstream regulators. Myocardial infarction (MI) was induced in wild-type FVB/NJ mice by ligation of the left anterior descending coronary artery. Western blot, immunoprecipitation, ECG telemetry, and patch-clamp techniques were used. After MI, c-Src (proto-oncogene tyrosine-protein kinase Src) and its active form (p-Src Y416) were increased. The activation of c-Src was associated with increased diastolic Ca2+ sparks, action potential duration prolongation, and arrhythmia in MI mice. c-Src upregulation and arrhythmia could be reversed by treatment of mice with the Src inhibitor PP1 but not with the inactive analogue PP3. Tyrosine phosphorylated mitochondrial Ca2+ uniporter (MCU) was upregulated in the heart tissues of MI mice and patients with ischemic cardiomyopathy. In a heterologous expression system, c-Src could bind MCU and phosphorylate MCU tyrosines. Overexpression of wild-type c-Src significantly increased the mitochondrial Ca2+ transient while overexpression of dominant-negative c-Src significantly decreased the mitochondrial Ca2+ transient. c-Src inhibition by PP1, MCU inhibition by Ru360, or MCU knockdown could reduce the action potential duration, Ca2+ sparks, and arrhythmia after MI. The human heart tissue showed that patients with ischemic cardiomyopathy had significantly increased c-Src active form associated with increased MCU tyrosine phosphorylation and ventricular arrhythmia. MI leads to increased c-Src active form that results in MCU tyrosine phosphorylation, increased mitochondrial Ca2+ uptake, QT prolongation, and arrhythmia, suggesting c-Src or MCU may represent novel antiarrhythmic targets.

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.

Cytosolic calcium regulates hepatic mitochondrial oxidation, intrahepatic lipolysis, and gluconeogenesis via CAMKII activation

To examine the roles of mitochondrial calcium Ca2+ ([Ca2+]mt) and cytosolic Ca2+ ([Ca2+]cyt) in the regulation of hepatic mitochondrial fat oxidation, we studied a liver-specific mitochondrial calcium uniporter knockout (MCU KO) mouse model with reduced [Ca2+]mt and increased [Ca2+]cyt content. Despite decreased [Ca2+]mt, deletion of hepatic MCU increased rates of isocitrate dehydrogenase flux, α-ketoglutarate dehydrogenase flux, and succinate dehydrogenase flux invivo. Rates of [14C16]palmitate oxidation and intrahepatic lipolysis were increased in MCU KO liver slices, which led to decreased hepatic triacylglycerol content. These effects were recapitulated with activation of CAMKII and abrogated with CAMKII knockdown, demonstrating that [Ca2+]cyt activation of CAMKII may be the primary mechanism by which MCU deletion promotes increased hepatic mitochondrial oxidation. Together, these data demonstrate that hepatic mitochondrial oxidation can be dissociated from [Ca2+]mt and reveal a key role for [Ca2+]cyt in the regulation of hepatic fat mitochondrial oxidation, intrahepatic lipolysis, gluconeogenesis, and lipid accumulation.