Mitochondrial Ca2+ Uniporter Complex Research Articles

The structural remodeling of the mitochondrial Ca2+ uniporter complex underlies its functional impairment during metabolic cardiomyopathy

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): ANR Introduction Calcium (Ca2+) uptake by the mitochondrial Ca2+ uniporter complex (MCUC) controls key steps of oxidative metabolism to adapt energy production to cardiac demand. To ensure this critical function, MCUC assembly and maturation is a tightly regulated process involving the AAA proteases. We recently demonstrated that mitochondrial Ca2+ uptake is impaired during the early stages of metabolic cardiomyopathy in the atria. Purpose Our aim was to investigate mitochondrial Ca2+ handling in the left ventricle during the early stages of metabolic cardiomyopathy and to characterize the mechanisms underlying the functional remodeling of the MCUC. Methods Male C57Bl6/J mice were fed a high-fat sucrose diet (HFS) for two weeks to induce metabolic cardiomyopathy. Mitochondrial Ca2+ uptake was then studied (1) in isolated ventricular cardiomyocytes using whole-cell patch-clamp coupled with confocal microscopy and (2) in isolated mitochondria using Ca2+ Green-5N. The biophysical properties of MCUC were recorded in a planar lipid bilayer. MCUC structure and AAA-proteases expression were assessed by Western blot and co-immunoprecipitation. Control mice were treated with AAV9 harboring shRNA to downregulate the expression of MAIP1. Results Metabolic cardiomyopathy was associated with reduced beat-to-beat mitochondrial Ca2+ uptake in ventricular cardiomyocytes, which was further confirmed in isolated mitochondria. Single-channel recordings of the native cardiac MCUC embedded in a planar lipid bilayer revealed a reduction in ionic conductance and open probability, demonstrating functional impairment of the MCUC. Biochemical analysis of the MCUC revealed an accumulation of the unprocessed form of the EMRE subunit and a decreased interaction of the EMRE-MICU1 subcomplex with the pore-forming MCU subunits, indicating a structural remodeling of the MCUC. The expression of the AAA proteases YME1L and AFG3L2/SPG7, which are responsible for EMRE maturation and insertion into the MCUC, was unchanged. However, the expression of m-AAA protease-interacting protein 1 (MAIP1), which orchestrates EMRE processing, was reduced. In control mice, shRNA-mediated downregulation of MAIP1 recapitulated the structural and functional changes of the MCUC. Conclusions The structural remodeling of the MCUC integrity by downregulation of its assembly affects mitochondrial Ca2+ homeostasis in the early stages of metabolic cardiomyopathy. MAIP1 is a key regulator of MCUC assembly and its downregulation underlies the functional remodeling of the MCUC. These findings provide a novel post-translational paradigm of MCUC regulation in pathophysiology and suggest novel therapeutic targets for the treatment of the metabolic cardiomyopathy.

Gene Expression Profile of the Mitochondrial Calcium Uniporter and Antioxidant Enzymes throughout the Brain During Alzheimer’s Disease

In 2023, there were an estimated ~6.7 million Americans ≥65 years old living with Alzheimer’s disease (AD), a prominent form of dementia. Development of AD has been associated with impaired blood flow to and throughout the brain. In turn, there is diminished delivery of oxygen and nutrients in tandem with reduced clearance of neurotoxic cellular byproducts, notably reactive oxygen species (ROS; e.g., superoxide, peroxide/hydroxyl radicals). As a primary source of cerebrovascular ROS, endothelial mitochondria become progressively dysfunctional in producing ATP relative to ROS in response to aging and cell senescence. A primary mechanism is described by enhanced Ca2+ sequestration into the mitochondrial matrix through the mitochondrial Ca2+ uniporter (MCU) complex ( Mcu, Mcub, Mcur1, Micu1, Micu2, Micu3) and a resulting overproduction of ROS up to conditions of cell death. The primary antioxidants that eliminate ROS are superoxide dismutase ( Sod2), catalase ( Cat), and glutathione peroxidase ( Gpx1). We tested the hypothesis that upregulation of the MCU complex occurs in tandem with decreasing expression of antioxidant enzymes throughout the brain during progression of AD pathology. To investigate this hypothesis, we employed a triple mutation mouse model of AD ( 3xTg-AD) comprising young control (YC; 1-2 mo), mild cognitive impairment (MCI; 4-5 mo), presence of extracellular amyloid-β plaques (Aβ; 6-8 mo), and extracellular amyloid-β plaques with neurofibrillary tangles composed of tau (AβT; ≥12 mo) [n ≥3 per group, males & females]. Quantitative polymerase chain reaction (qPCR), Nanostring, and/or RNA-Seq have been performed on cortex, hippocampus, cerebral vessels, cerebrovascular endothelial cells (ECs), and peripheral blood. Primary highlights of our findings for each of the AD study groups are described below. With detectable presence of all target genes throughout tissue compartments, the YC group is particularly characterized by a higher MCU complex and antioxidant expressions in cortex relative to hippocampus. During the MCI phase, the MCU complex and Cat are upregulated in ECs relative to the YC group. Upon onset of AD (Aβ stage), there is a downregulation and upregulation of MCU complex in the brain and peripheral blood respectively. Further, Gpx1 expression is upregulated in ECs in the Aβ group. Relative to YC, the MCU complex and antioxidants are downregulated in cortex but generally stable in hippocampus during AβT conditions with exception of upregulation in peroxide scavenger mRNAs Cat and Gpx1. In contrast for ECs, the MCU complex increases, while Cat and Gpx1 decrease, in the AβT group relative to YC. Altogether, these findings support an intricate expression profile of the MCU complex and key antioxidant enzymes across brain tissue and cell compartments preceding and accompanying AD pathology. A remarkable observation is that expression profiles of cerebrovascular vessels and blood indicate MCI and early onset of AD, whereas those of the cortex and hippocampus best accompany late-stage AD pathology. This research was supported by the National Institutes of Health (R01AG073230). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Neither too much nor too little: mitochondrial calcium concentration as a balance between physiological and pathological conditions.

Ca2+ ions serve as pleiotropic second messengers in the cell, regulating several cellular processes. Mitochondria play a fundamental role in Ca2+ homeostasis since mitochondrial Ca2+ (mitCa2+) is a key regulator of oxidative metabolism and cell death. MitCa2+ uptake is mediated by the mitochondrial Ca2+ uniporter complex (MCUc) localized in the inner mitochondrial membrane (IMM). MitCa2+ uptake stimulates the activity of three key enzymes of the Krebs cycle, thereby modulating ATP production and promoting oxidative metabolism. As Paracelsus stated, "Dosis sola facit venenum,"in pathological conditions, mitCa2+ overload triggers the opening of the mitochondrial permeability transition pore (mPTP), enabling the release of apoptotic factors and ultimately leading to cell death. Excessive mitCa2+ accumulation is also associated with a pathological increase of reactive oxygen species (ROS). In this article, we review the precise regulation and the effectors of mitCa2+ in physiopathological processes.

Preserving and enhancing mitochondrial function after stroke to protect and repair the neurovascular unit: novel opportunities for nanoparticle-based drug delivery.

The neurovascular unit (NVU) is composed of vascular cells, glia, and neurons that form the basic component of the blood brain barrier. This intricate structure rapidly adjusts cerebral blood flow to match the metabolic needs of brain activity. However, the NVU is exquisitely sensitive to damage and displays limited repair after a stroke. To effectively treat stroke, it is therefore considered crucial to both protect and repair the NVU. Mitochondrial calcium (Ca2+) uptake supports NVU function by buffering Ca2+ and stimulating energy production. However, excessive mitochondrial Ca2+ uptake causes toxic mitochondrial Ca2+ overloading that triggers numerous cell death pathways which destroy the NVU. Mitochondrial damage is one of the earliest pathological events in stroke. Drugs that preserve mitochondrial integrity and function should therefore confer profound NVU protection by blocking the initiation of numerous injury events. We have shown that mitochondrial Ca2+ uptake and efflux in the brain are mediated by the mitochondrial Ca2+ uniporter complex (MCUcx) and sodium/Ca2+/lithium exchanger (NCLX), respectively. Moreover, our recent pharmacological studies have demonstrated that MCUcx inhibition and NCLX activation suppress ischemic and excitotoxic neuronal cell death by blocking mitochondrial Ca2+ overloading. These findings suggest that combining MCUcx inhibition with NCLX activation should markedly protect the NVU. In terms of promoting NVU repair, nuclear hormone receptor activation is a promising approach. Retinoid X receptor (RXR) and thyroid hormone receptor (TR) agonists activate complementary transcriptional programs that stimulate mitochondrial biogenesis, suppress inflammation, and enhance the production of new vascular cells, glia, and neurons. RXR and TR agonism should thus further improve the clinical benefits of MCUcx inhibition and NCLX activation by increasing NVU repair. However, drugs that either inhibit the MCUcx, or stimulate the NCLX, or activate the RXR or TR, suffer from adverse effects caused by undesired actions on healthy tissues. To overcome this problem, we describe the use of nanoparticle drug formulations that preferentially target metabolically compromised and damaged NVUs after an ischemic or hemorrhagic stroke. These nanoparticle-based approaches have the potential to improve clinical safety and efficacy by maximizing drug delivery to diseased NVUs and minimizing drug exposure in healthy brain and peripheral tissues.

Mitochondrial Ca2+ dynamics differ between the left and right ventricles in murine and porcine hearts

Despite their differences in metabolism, mechanics, and developmental origin, the right ventricle (RV) is often assumed to function similarly to the left ventricle (LV). Specifically, though Ca 2+ transport into mitochondria is well established to play a crucial role in matching cardiac energy production to demand, little is known regarding whether mitochondrial Ca 2+ (mtCa 2+ ) handling is distinct in the RV and LV. Our objective was to test the hypothesis that the regulation of mtCa 2+ differs in the RV and LV. By isolating mitochondria from RV and LV free wall tissues from wild-type mice and healthy pigs, we compared several parameters indicative of Ca 2+ -regulated mitochondrial function. Basal mtCa 2+ levels were higher in RV mitochondria than in LV mitochondria. When successive boluses of Ca 2+ were administered to isolated mitochondria, RV mitochondria took up fewer Ca 2+ boluses, showing lower Ca 2+ retention capacity. RV mitochondria displayed relatively more protein carbonylation, suggesting oxidative stress. Interestingly, ATP production rate was higher in RV mitochondria relative to LV mitochondria; however, only LV mitochondria exhibited an increase in ATP production rate in the presence of Ca 2+ . We also compared the protein expression of the subunits of the mitochondrial Ca 2+ uniporter complex (MCUC) and the mitochondrial Na+/Ca 2+ exchanger (NCLX) and found that levels of the MCUC subunit Essential MCU Regulator (EMRE) and NCLX were higher in the LV than the RV, potentially facilitating more dynamic Ca 2+ transport in and out of LV mitochondria. Collectively, these data suggest that mtCa 2+ is calibrated to higher but more static levels in the RV, whereas in the LV basal mtCa 2+ is lower to ensure dynamic range for physiological stimuli to increase mitochondrial bioenergetics, corresponding to the larger changes in workload experienced by the LV. This study was supported by the National Institutes of Health (1K22HL137901 to Julia C. Liu) and by the Department of Integrative Biology and Physiology (to Jae Hwi Sung). This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Beyond the matrix: structural and physiological advancements in mitochondrial calcium signaling.

Mitochondrial calcium (Ca2+) signaling has long been known to regulate diverse cellular functions, ranging from ATP production via oxidative phosphorylation, to cytoplasmic Ca2+ signaling to apoptosis. Central to mitochondrial Ca2+ signaling is the mitochondrial Ca2+ uniporter complex (MCUC) which enables Ca2+ flux from the cytosol into the mitochondrial matrix. Several pivotal discoveries over the past 15 years have clarified the identity of the proteins comprising MCUC. Here, we provide an overview of the literature on mitochondrial Ca2+ biology and highlight recent findings on the high-resolution structure, dynamic regulation, and new functions of MCUC, with an emphasis on publications from the last five years. We discuss the importance of these findings for human health and the therapeutic potential of targeting mitochondrial Ca2+ signaling.

The mitochondrial calcium uniporter complex–A play in five acts

Mitochondrial Ca2+ (mitCa2+) uptake controls both intraorganellar and cytosolic functions. Within the organelle, [Ca2+] increases regulate the activity of tricarboxylic acid (TCA) cycle enzymes, thus sustaining oxidative metabolism and ATP production. Reactive oxygen species (ROS) are also generated as side products of oxygen consumption. At the same time, mitochondria act as buffers of cytosolic Ca2+ (cytCa2+) increases, thus regulating Ca2+-dependent cellular processes. In pathological conditions, mitCa2+ overload triggers the opening of the mitochondrial permeability transition pore (mPTP) and the release of apoptotic cofactors. MitCa2+ uptake occurs in response of local [Ca2+] increases in sites of proximity between the endoplasmic reticulum (ER) and the mitochondria and is mediated by the mitochondrial Ca2+ uniporter (MCU), a highly selective channel of the inner mitochondrial membrane (IMM). Both channel and regulatory subunits form the MCU complex (MCUC). Cryogenic electron microscopy (Cryo-EM) and crystal structures revealed the correct assembly of MCUC and the function of critical residues for the regulation of Ca2+ conductance.

Targeting the mitochondrial Ca2+ uniporter complex in cardiovascular disease.

Cardiovascular diseases (CVDs), the leading cause of death worldwide, share in common mitochondrial dysfunction, in specific a dysregulation of Ca2+ uptake dynamics through the mitochondrial Ca2+ uniporter (MCU) complex. In particular, Ca2+ uptake regulates the mitochondrial ATP production, mitochondrial dynamics, oxidative stress, and cell death. Therefore, modulating the activity of the MCU complex to regulate Ca2+ uptake, has been suggested as a potential therapeutic approach for the treatment of CVDs. Here, the role and implications of the MCU complex in CVDs are presented, followed by a review of the evidence for MCU complex modulation, genetically and pharmacologically. While most approaches have aimed within the MCU complex for the modulation of the Ca2+ pore channel, the MCU subunit, its intra- and extra- mitochondrial implications, including Ca2+ dynamics, oxidative stress, post-translational modifications, and its repercussions in the cardiac function, highlight that targeting the MCU complex has the translational potential for novel CVDs therapeutics.

Modulation of the mitochondrial Ca2+ uniporter complex subunit expression by different shear stress patterns in vascular endothelial cells

Mitochondrial calcium (m Ca2+ ) uptake occurs via the Mitochondrial Ca2+ Uniporter (MCU) complex and plays a critical role in mitochondrial dynamics, mitophagy, and apoptosis. MCU complex activity is in part modulated by the expression of its regulatory subunits. Cardiovascular disease models demonstrated altered gene/protein expression of one or multiple subunits in different cells, including vascular endothelial cells (ECs). MCU complex activity was found necessary for stable flow (s-flow)-induced mitophagy and promotion of an atheroprotective EC phenotype. Disturbed flow (d-flow) is known to lead to an atheroprone phenotype. Despite the role of MCU in flow-regulated EC function, flow-induced alterations in MCU complex subunit expression are currently unknown. We exposed cultured human ECs to atheroprotective (steady shear stress, SS) or atheroprone flow (oscillatory shear stress, OS) and measured mRNA and protein levels of the MCU complex members. SS and OS differentially modulated subunit expression at gene/protein levels. Protein expression changes of the core MCU, m Ca2+ uptake 1 (MICU1) and MCU regulator 1 (MCUR1) subunits in SS- and OS-exposed, compared to static, ECs suggested an enhanced m Ca2+ influx under each flow and a potential contribution to EC dysfunction under OS. In silico analysis of a single-cell RNA-sequencing dataset was employed to extract transcript values of MCU subunits in mouse carotid ECs from regions exposed to s-flow or d-flow. Mcu and Mcur1 genes showed significant differences in expression after prolonged exposure to each flow. The differential expression of MCU complex subunits indicated a tight regulation of the complex activity under physiological and pathological hemodynamic conditions.

Enhanced Mitochondrial Calcium Uptake Suppresses Atrial Fibrillation Associated With Metabolic Syndrome

BackgroundPatients with metabolic syndrome (MetS) have an increased risk of atrial fibrillation (AF). Impaired Ca2+ homeostasis and mitochondrial dysfunction have emerged as an arrhythmogenic substrate in both patients and animal models of MetS. Whether impaired mitochondrial Ca2+ handling underlies AF associated with MetS remains poorly explored. ObjectivesThe aim of this study was to determine the initial mechanisms related to AF susceptibility and mitochondrial dysfunction encountered in metabolic cardiomyopathy. MethodsA total of 161 mice and 34 patients were studied. Mitochondrial Ca2+ and mitochondrial Ca2+ uniporter complex (MCUC) were investigated in right atrial tissue of patients with (n = 18) or without (n = 16) MetS and of C57Bl/6J mice fed with a high-fat sucrose diet (HFS) for 2 (n = 42) or 12 (n = 39) weeks. Susceptibility to AF was evaluated in isolated sinoatrial tissue and in vivo in mice. ResultsIncreased expression of the MICUs subunits of the MCUC (1.00 ± 0.33 AU vs 1.29 ± 0.23 AU; P = 0.034) was associated with impaired mitochondrial Ca2+ uptake in patients (168.7 ± 31.3 nmol/min/mg vs 127.3 ± 18.4 nmol/min/mg; P = 0.026) and HFS mice (0.10 ± 0.04 ΔF/F0 × ms-1 vs 0.06 ± 0.03 ΔF/F0 × ms-1; P = 0.0086, and 0.15 ± 0.07 ΔF/F0 × ms-1 vs 0.046 ± 0.03 ΔF/F0 × ms-1; P = 0.0076 in 2- and 12-week HFS mice, respectively). HFS mice elicited a 70% increased susceptibility to AF. The MCUC agonist kaempferol restored MCUC activity in vitro and abolished the occurrence of AF in HFS mice. ConclusionsImpaired MCUC activity and mitochondrial Ca2+ homeostasis from the early stage of metabolic cardiomyopathy in mice lead to AF. Given that similar defects in cardiac mitochondrial Ca2+ handling are present in MetS patients, the modulation of the MCUC activity represents an attractive antiarrhythmic strategy.

Abstract 9399: Elevated MCU Expression by CaMKIIδB Limits Pathological Cardiac Remodeling

Introduction: Calcium (Ca 2+ ) is a key regulator of energy metabolism. Impaired Ca 2+ homeostasis damages mitochondria, causing cardiomyocyte death, pathological hypertrophy, and heart failure. This study investigates the regulation and the role of the mitochondrial Ca 2+ uniporter (MCU) in chronic stress-induced pathological cardiac remodeling. Methods: MCU knockout or transgenic mice were infused with isoproterenol (ISO; 10 mg/kg per day, 4 weeks). Cardiac hypertrophy and remodeling were evaluated by echocardiography and histology. Primary cultured rodent adult cardiomyocytes were treated with ISO (100 nmol/L, 48 hours). Intracellular Ca 2+ handling and cell death pathways were monitored. Adenovirus-mediated gene manipulations were used in vitro. Results: Chronic administration of the β-adrenergic receptor agonist ISO increased the levels of the MCU and the MCU complex in cardiac mitochondria, raising mitochondrial Ca 2+ concentrations, in vivo and in vitro. ISO also upregulated MCU without affecting its regulatory proteins in adult cardiomyocytes. It is interesting that ISO-induced cardiac hypertrophy, fibrosis, contractile dysfunction, and cardiomyocyte death were exacerbated in global MCU knockout mice. Cardiomyocytes from knockout mice or overexpressing a dominant negative MCU exhibited defective intracellular Ca 2+ handling and activation of multiple cell death pathways. Conversely, cardiac-specific overexpression of MCU maintained intracellular Ca 2+ homeostasis and contractility, suppressed cell death, and prevented ISO-induced heart hypertrophy. ISO upregulated MCU expression through activation of Ca 2+ /calmodulin kinase II δB (CaMKIIδB) and promotion of its nuclear translocation via calcineurin-mediated dephosphorylation at serine 332. Nuclear CaMKIIδB phosphorylated CREB (cAMP-response element binding protein), which bound the Mcu promoter to enhance Mcu gene transcription. Conclusions: The β-adrenergic receptor/CaMKIIδB/CREB pathway upregulates Mcu gene expression in the heart. MCU upregulation is a compensatory mechanism that counteracts stress-induced pathological cardiac remodeling by preserving Ca2+ homeostasis and cardiomyocyte viability.

Sex-Specific Differences in Endothelial Function Are Driven by Divergent Mitochondrial Ca2+ Handling.

BackgroundSex‐specific differences in vasodilation are mediated in part by differences in cytosolic Ca2+ handling, but how variations in mitochondrial Ca2+ contributes to this effect remains unknown. Here, we investigated the extent to which mitochondrial Ca2+ entry via the MCU (mitochondrial Ca2+ uniporter) drives sex differences in vasoreactivity in resistance arteries.Methods and ResultsEnhanced vasodilation of mesenteric resistance arteries to acetylcholine (ACh) was reduced to larger extent in female compared with male mice in 2 genetic models of endothelial MCU ablation. Ex vivo Ca2+ imaging of mesenteric arteries with Fura‐2AM confirmed higher cytosolic Ca2+ transients triggered by ACh in arteries from female mice versus male mice. MCU inhibition both strongly reduced cytosolic Ca2+ transients and blocked mitochondrial Ca2+ entry. In cultured human aortic endothelial cells, treatment with physiological concentrations of estradiol enhanced cytosolic Ca2+ transients, Ca2+ buffering capacity, and mitochondrial Ca2+ entry in response to ATP or repeat Ca2+ boluses. Further experiments to establish the mechanisms underlying these effects did not reveal significant differences in the expression of MCU subunits, at either the mRNA or protein level. However, estradiol treatment was associated with an increase in mitochondrial mass, mitochondrial fusion, and the mitochondrial membrane potential and reduced mitochondrial superoxide production.ConclusionsOur data confirm that mitochondrial function in endothelial cells differs by sex, with female mice having enhanced Ca2+ uptake capacity, and that these differences are attributable to the presence of more mitochondria and a higher mitochondrial membrane potential in female mice rather than differences in composition of the MCU complex.

Elevated MCU Expression by CaMKIIδB Limits Pathological Cardiac Remodeling.

Calcium (Ca2+) is a key regulator of energy metabolism. Impaired Ca2+ homeostasis damages mitochondria, causing cardiomyocyte death, pathological hypertrophy, and heart failure. This study investigates the regulation and the role of the mitochondrial Ca2+ uniporter (MCU) in chronic stress-induced pathological cardiac remodeling. MCU knockout or transgenic mice were infused with isoproterenol (ISO; 10 mg/kg per day, 4 weeks). Cardiac hypertrophy and remodeling were evaluated by echocardiography and histology. Primary cultured rodent adult cardiomyocytes were treated with ISO (1 nmol/L, 48 hours). Intracellular Ca2+ handling and cell death pathways were monitored. Adenovirus-mediated gene manipulations were used in vitro. Chronic administration of the β-adrenergic receptor agonist ISO increased the levels of the MCU and the MCU complex in cardiac mitochondria, raising mitochondrial Ca2+ concentrations, in vivo and in vitro. ISO also upregulated MCU without affecting its regulatory proteins in adult cardiomyocytes. It is interesting that ISO-induced cardiac hypertrophy, fibrosis, contractile dysfunction, and cardiomyocyte death were exacerbated in global MCU knockout mice. Cardiomyocytes from knockout mice or overexpressing a dominant negative MCU exhibited defective intracellular Ca2+ handling and activation of multiple cell death pathways. Conversely, cardiac-specific overexpression of MCU maintained intracellular Ca2+ homeostasis and contractility, suppressed cell death, and prevented ISO-induced heart hypertrophy. ISO upregulated MCU expression through activation of Ca2+/calmodulin kinase II δB (CaMKIIδB) and promotion of its nuclear translocation via calcineurin-mediated dephosphorylation at serine 332. Nuclear CaMKIIδB phosphorylated CREB (cAMP-response element binding protein), which bound the Mcu promoter to enhance Mcu gene transcription. The β-adrenergic receptor/CaMKIIδB/CREB pathway upregulates Mcu gene expression in the heart. MCU upregulation is a compensatory mechanism that counteracts stress-induced pathological cardiac remodeling by preserving Ca2+ homeostasis and cardiomyocyte viability.

An unexpected effect of risperidone reveals a nonlinear relationship between cytosolic Ca2+ and mitochondrial Ca2+ uptake.

Mitochondria actively contribute to cellular Ca2+ homeostasis. The molecular mechanisms of mitochondrial Ca2+ uptake and release are well characterized and are attributed to the multi-protein assembly of the mitochondrial Ca2+ uniporter complex (MCUC) and the mitochondrial sodium-calcium exchanger (NCLX), respectively. Hence, Ca2+ transfer from the endoplasmic reticulum (ER) and store-operated Ca2+ entry (SOCE) into the mitochondrial matrix has been quantitatively visualized on the subcellular level using targeted fluorescent biosensors. However, a correlation between the amplitude of cytosolic Ca2+ elevation with that in the mitochondrial matrix has not been investigated in detail so far. In the present study, we combined the Ca2+-mobilizing agonist histamine with the H1-receptor antagonist risperidone to establish a well-tunable experimental approach allowing the correlation between low, slow, high, and fast cytosolic and mitochondrial Ca2+ signals in response to inositol 1,4,5-trisphosphate (IP3)-triggered ER Ca2+ release. Our present data confirm a defined threshold in cytosolic Ca2+, which is necessary for the activation of mitochondrial Ca2+ uptake. Moreover, our data support the hypothesis of different modes of mitochondrial Ca2+ uptake depending on the source of the ion (i.e., ER vs SOCE).

The effect of DS16570511, a new inhibitor of mitochondrial calcium uniporter, on calcium homeostasis, metabolism, and functional state of cultured cortical neurons and isolated brain mitochondria

BackgroundDisorders of mitochondrial Ca2+ homeostasis play a key role in the glutamate excitotoxicity of brain neurons. DS16570511 (DS) is a new penetrating inhibitor of mitochondrial Ca2+ uniporter complex (MCUC). The paper examines the effects of DS on the cultivated cortical neurons and isolated mitochondria of the rat brain. MethodsThe functions of neurons and mitochondria were examined using fluorescence microscopy, XF24 microplate-based сell respirometry, ion-selective microelectrodes, spectrophotometry, and polarographic technique. ResultsAt the doses of 30 and 45 μM, DS reliably slowed down the onset of glutamate-induced delayed calcium deregulation of neurons and suppressed their death. 30 μM DS caused hyperpolarization of mitochondria of resting neurons, and 45 μM DS temporarily depolarized neuronal mitochondria. It was also demonstrated that 30–60 μM DS stimulated cellular respiration. DS was shown to suppress Ca2+ uptake by isolated brain mitochondria. In addition, DS inhibited ADP-stimulated mitochondrial respiration and ADP-induced decrease in the mitochondrial membrane potential. It was found that DS inhibited the activity of complex II of the respiratory chain. In the presence of Ca2+, high DS concentrations caused a collapse of the mitochondrial membrane potential. ConclusionsThe data obtained indicate that, in addition to the inhibition of MCUC, DS affects the main energy-transducing functions of mitochondria. General significanceThe using DS as a tool for studying MCUC and its functional role in neuronal cells should be done with care, bearing in mind multiple effects of DS, a proper evaluation of which would require multivariate analysis.

Circulating mitochondrial genes detect acute cardiac allograft rejection: Role of the mitochondrial calcium uniporter complex.

Acute rejection after heart transplantation increases the risk of chronic dysfunction. Disturbances in mitochondrial function may play a contributory role, however, the relationship between histological signs of rejection in the human transplanted heart and expression levels of circulating mitochondrial genes, such as the mitochondrial Ca2+ uniporter (MCU) complex, remains unexplored. We conducted an RNA‐sequencing analysis to identify altered mitochondrial genes in serum and to evaluate their diagnostic accuracy for rejection episodes. We included 40 consecutive samples from transplant recipients undergoing routine endomyocardial biopsies. In total, 112 mitochondrial genes were identified in the serum of posttransplant patients, of which 28 were differentially expressed in patients with acute rejection (p < .05). Considering the receiver operating characteristic analysis with an area under the curve (AUC) >0.900 to discriminate patients with moderate or severe degrees of rejection, we found that the MCU system showed a strong capability for detection: MCU (AUC = 0.944, p < .0001), MCU/MCUR1 ratio (AUC = 0.972, p < .0001), MCU/MCUB ratio (AUC = 0.970, p < .0001), and MCU/MICU1 ratio (AUC = 0.970, p < .0001). Mitochondrial alterations are reflected in peripheral blood and are capable of discriminating between patients with allograft rejection and those not experiencing rejection with excellent accuracy. The dysregulation of the MCU complex was found to be the most relevant finding.

Molecular nature and physiological role of the mitochondrial calcium uniporter channel.

Calcium (Ca2+) signaling is critical for cell function and cell survival. Mitochondria play a major role in regulating the intracellular Ca2+ concentration ([Ca2+]i). Mitochondrial Ca2+ uptake is an important determinant of cell fate and governs respiration, mitophagy/autophagy, and the mitochondrial pathway of apoptosis. Mitochondrial Ca2+ uptake occurs via the mitochondrial Ca2+ uniporter (MCU) complex. This review summarizes the present knowledge on the function of MCU complex, regulation of MCU channel, and the role of MCU in Ca2+ homeostasis and human disease pathogenesis. The channel core consists of four MCU subunits and essential MCU regulators (EMRE). Regulatory proteins that interact with them include mitochondrial Ca2+ uptake 1/2 (MICU1/2), MCU dominant-negative β-subunit (MCUb), MCU regulator 1 (MCUR1), and solute carrier 25A23 (SLC25A23). In addition to these proteins, cardiolipin, a mitochondrial membrane-specific phospholipid, has been shown to interact with the channel core. The dynamic interplay between the core and regulatory proteins modulates MCU channel activity after sensing local changes in [Ca2+]i, reactive oxygen species, and other environmental factors. Here, we highlight the structural details of the human MCU heteromeric assemblies and their known roles in regulating mitochondrial Ca2+ homeostasis. MCU dysfunction has been shown to alter mitochondrial Ca2+ dynamics, in turn eliciting cell apoptosis. Changes in mitochondrial Ca2+ uptake have been implicated in pathological conditions affecting multiple organs, including the heart, skeletal muscle, and brain. However, our structural and functional knowledge of this vital protein complex remains incomplete, and understanding the precise role for MCU-mediated mitochondrial Ca2+ signaling in disease requires further research efforts.

Different Sensitivity of Control and MICU1- and MICU2-Ablated Trypanosoma cruzi Mitochondrial Calcium Uniporter Complex to Ruthenium-Based Inhibitors.

The mitochondrial Ca2+ uptake in trypanosomatids shares biochemical characteristics with that of animals. However, the composition of the mitochondrial Ca2+ uniporter complex (MCUC) in these parasites is quite peculiar, suggesting lineage-specific adaptations. In this work, we compared the inhibitory activity of ruthenium red (RuRed) and Ru360, the most commonly used MCUC inhibitors, with that of the recently described inhibitor Ru265, on Trypanosoma cruzi, the agent of Chagas disease. Ru265 was more potent than Ru360 and RuRed in inhibiting mitochondrial Ca2+ transport in permeabilized cells. When dose-response effects were investigated, an increase in sensitivity for Ru360 and Ru265 was observed in TcMICU1-KO and TcMICU2-KO cells as compared with control cells. In the presence of RuRed, a significant increase in sensitivity was observed only in TcMICU2-KO cells. However, application of Ru265 to intact cells did not affect growth and respiration of epimastigotes, mitochondrial Ca2+ uptake in Rhod-2-labeled intact cells, or attachment to host cells and infection by trypomastigotes, suggesting a low permeability for this compound in trypanosomes.

Structural and Functional Features of Ca2+ Transport Systems in Liver Mitochondria of Rats with Experimental Hyperthyroidism.

We analyzed structural and functional features of the main mitochondrial Ca2+-transporting systems, mitochondrial Ca2+ uniporter complex (MCUC) and Ca2+-dependent cyclosporin A-sensitive mitochondrial permeability transition pore (MPT pore), in rats with hyperthyroid state. It was found that, the rate of Ca2+ accumulation by rat liver mitochondria in this pathology increases by 1.3 times, which can be associated with higher level of the channel-forming subunit of the uniporter MCU and lower content of dominant-negative subunit of this complex MCUb. At the same time, the level of the regulatory subunit MICU1 remained unchanged. It was shown that calcium retention capacity of liver mitochondria in rats with experimental hyperthyroidism decreased by 2 times in comparison with the control, which attested to reduced resistance of liver mitochondria of hyperthyroid rats to induction of the MPT pore. The observed changes are consistent with the data on increased amount of cyclophilin D, a mitochondrial matrix peptidyl-prolyl isomerase that is known to modulate the MPT pore opening and expression of the Ppif gene that encodes mitochondrial cyclophilin D in rats with experimental hyperthyroidism.

Identification of Critical MCUR1 Domains in the Mitochondrial Calcium Uniporter Complex that Regulates Cellular Metabolism

Mitochondria are multivariate signal processors that decode diverse cellular inputs into various bioenergetic outputs. One such signal is the calcium (Ca2+) flux between the cytosol and mitochondria which modulates the organelle’s function. Under physiologic cytosolic [Ca2+], energy output (ATP) can be adjusted to meet cellular demands. To maintain the Ca2+ homeostatic circuit, a selective mitochondrial Ca2+ Uniporter (MCU) complex promotes Ca2+ uptake. MCUR1 is an integral mitochondrial inner membrane protein which serves as a scaffold factor for the MCU complex and regulates its functional activity. The resolved NMR structure of the human MCUR1 protein revealed a high degree of conservation for 3 residues within the “head domain” of the protein, A170, L174, V203, and Y204.ObjectiveWe hypothesized that alterations of these critical residues would affect MCUR1 function, and thereby, MCU complex activity.MethodsTo test this hypothesis, MCU‐mediated mitochondrial calcium (mCa2+) uptake, resting matrix mCa2+ levels, and mitochondrial bioenergetics such as NAD(P)H production and oxygen consumption rate (OCR) were evaluated.ResultsRemarkably, cells expressing the MCUR1A170R/L174R mutant demonstrated near complete ablation of MCU‐mediated mCa2+ uptake, NAD(P)H production and OCR. Conversely, cells expressing MCURWT, or the MCURV203R/Y204R mutant demonstrated similar effects on MCU activity and mitochondrial bioenergetics suggesting a nominal change at the structural level.ConclusionCollectively, our results indicate that the MCUR1 head domain is necessary for proper MCU complex activity, and that MCU‐mediated mCa2+ uptake modulates mitochondrial bioenergetics and cell function.Support or Funding InformationThis research was funded by the National Institutes of Health R25R25DK078381 program. (NIHR01GM109882, R01HL086699, R01HL142673, DOD/CDMRP PR181598P and NIH1S10RR027327 to M. Madesh and R25DK078381 to W.B.R. B.T.E. is supported by the National Center For Advancing Translational Sciences of the National Institutes of Health under Award Number TL1TR002647. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.