Mitochondrial Calcium Uptake Research Articles

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.

The mitochondrial calcium uniporter complex in trypanosomes.

The presence of a conserved mechanism for mitochondrial calcium uptake in trypanosomatids was crucial for the molecular identification of the mitochondrial calcium uniporter (MCU), a long-sought channel present in most eukaryotic organisms. Since then, research efforts to elucidate the role of MCU and its regulatory elements in different biological models have multiplied. MCU is the pore-forming subunit of a multimeric complex (the MCU complex or MCUC) and its predicted structure in trypanosomes is simpler than in mammalian cells, lacking two of its subunits and probably possessing other unidentified components. MCU protein has been characterized in Trypanosoma brucei and Trypanosoma cruzi, the causative agents of African and American trypanosomiasis, respectively. Contrary to its mammalian homolog, TbMCU was found to be essential for cell growth and survival, while its paralog MCUb is an essential protein in T. cruzi. These findings could be further exploited for chemotherapeutic purposes. The emergence of new molecular tools for the genetic manipulation of trypanosomatids has been determinant for the functional characterization of the MCUC components in these organisms. However, further research has to be done to determine the role of each component in intracellular calcium signaling and cell bioenergetics. In this mini-review we summarize the original results on mitochondrial calcium uptake in trypanosomes, how did they contribute to the molecular identification of the MCU, and the functional characterization of the MCUC subunits that has so far been studied in these peculiar eukaryotes.

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.

Fluorophore-Based Mitochondrial Ca2+ Uptake Assay.

The physiological importance of mitochondrial calcium uptake, observed in processes such as ATP production, intracellular calcium signaling, and apoptosis, makes desirable a simple, straightforward way of investigating this event with unambiguous results. The following protocol uses a calcium-sensitive, membrane-impermeable fluorophore to monitor extra-mitochondrial calcium levels in the presence of permeabilized mammalian cells harboring activated mitochondria.

INF2-mediated actin polymerization at the ER stimulates mitochondrial calcium uptake, inner membrane constriction, and division.

Mitochondrial division requires division of both the inner and outer mitochondrial membranes (IMM and OMM, respectively). Interaction with endoplasmic reticulum (ER) promotes OMM division by recruitment of the dynamin Drp1, but effects on IMM division are not well characterized. We previously showed that actin polymerization through ER-bound inverted formin 2 (INF2) stimulates Drp1 recruitment in mammalian cells. Here, we show that INF2-mediated actin polymerization stimulates a second mitochondrial response independent of Drp1: a rise in mitochondrial matrix calcium through the mitochondrial calcium uniporter. ER stores supply the increased mitochondrial calcium, and the role of actin is to increase ER-mitochondria contact. Myosin IIA is also required for this mitochondrial calcium increase. Elevated mitochondrial calcium in turn activates IMM constriction in a Drp1-independent manner. IMM constriction requires electron transport chain activity. IMM division precedes OMM division. These results demonstrate that actin polymerization independently stimulates the dynamics of both membranes during mitochondrial division: IMM through increased matrix calcium, and OMM through Drp1 recruitment.

Impaired mitochondrial calcium uptake caused by tacrolimus underlies beta-cell failure

BackgroundOne of the most common side effects of the immunosuppressive drug tacrolimus (FK506) is the increased risk of new-onset diabetes mellitus. However, the molecular mechanisms underlying this association have not been fully clarified.MethodsWe studied the effects of the therapeutic dose of tacrolimus on mitochondrial fitness in beta-cells.ResultsWe demonstrate that tacrolimus impairs glucose-stimulated insulin secretion (GSIS) in beta-cells through a previously unidentified mechanism. Indeed, tacrolimus causes a decrease in mitochondrial Ca2+ uptake, accompanied by altered mitochondrial respiration and reduced ATP production, eventually leading to impaired GSIS.ConclusionOur observations individuate a new fundamental mechanism responsible for the augmented incidence of diabetes following tacrolimus treatment. Indeed, this drug alters Ca2+ fluxes in mitochondria, thereby compromising metabolism-secretion coupling in beta-cells.

Synthesis and Evaluation of a Ruthenium-based Mitochondrial Calcium Uptake Inhibitor

We detail the synthesis and purification of a mitochondrial calcium uptake inhibitor, [(OH2)(NH3)4Ru(µ-O)Ru(NH3)4(OH2)]5+. The optimized synthesis of this compound commences from [Ru(NH3)5Cl]Cl2 in 1 M NH4OH in a closed container, yielding a green solution. Purification is accomplished with cation-exchange chromatography. This compound is characterized and verified to be pure by UV-vis and IR spectroscopy. The mitochondrial calcium uptake inhibitory properties are assessed in permeabilized HeLa cells by fluorescence spectroscopy.

Synthesis and Evaluation of a Ruthenium-based Mitochondrial Calcium Uptake Inhibitor

Synthesis and Evaluation of a Ruthenium-based Mitochondrial Calcium Uptake Inhibitor

Mitochondrial Calcium Dysregulation Contributes to Dendrite Degeneration Mediated by PD/LBD-Associated LRRK2 Mutants.

Mutations in leucine-rich repeat kinase 2 (LRRK2) contribute to development of late-onset familial Parkinson's disease (PD), with clinical features of motor and cognitive dysfunction indistinguishable from sporadic PD. Calcium dysregulation plays an important role in PD pathogenesis, but the mechanisms of neurodegeneration remain unclear. Recent reports indicate enhanced excitatory neurotransmission in cortical neurons expressing mutant LRRK2, which occurs before the well-characterized phenotype of dendritic shortening. As mitochondria play a major role in the rapid buffering of cytosolic calcium, we hypothesized that altered mitochondrial calcium handling contributes to dendritic retraction elicited by the LRRK2-G2019S and -R1441C mutations. In primary mouse cortical neurons, we observed increased depolarization-induced mitochondrial calcium uptake. We found that expression of mutant LRRK2 elicited transcriptional upregulation of the mitochondrial calcium uniporter (MCU) and the mitochondrial calcium uptake 1 protein (MICU1) with no change in levels of the mitochondrial calcium antiporter NCLX. Elevated MCU and MICU1 were also observed in LRRK2-mutated patient fibroblasts, along with increased mitochondrial calcium uptake, and in postmortem brains of sporadic PD/PDD patients of both sexes. Transcriptional upregulation of MCU and MICU1 was caused by activation of the ERK1/2 (MAPK3/1) pathway. Inhibiting ERK1/2 conferred protection against mutant LRRK2-induced neurite shortening. Pharmacological inhibitors or RNAi knockdown of MCU attenuated mitochondrial calcium uptake and dendritic/neuritic shortening elicited by mutant LRRK2, whereas expression of a constitutively active mutant of NCLX that enhances calcium export from mitochondria was neuroprotective. These data suggest that an increased susceptibility to mitochondrial calcium dysregulation contributes to dendritic injury in mutant LRRK2 pathogenesis.SIGNIFICANCE STATEMENT Cognitive dysfunction and dementia are common features of Parkinson's disease (PD), causing significant disability. Mutations in LRRK2 represent the most common known genetic cause of PD. We found that PD-linked LRRK2 mutations increased dendritic and mitochondrial calcium uptake in cortical neurons and familial PD patient fibroblasts, accompanied by increased expression of the mitochondrial calcium transporter MCU. Blocking the ERK1/2-dependent upregulation of MCU conferred protection against mutant LRRK2-elicited dendrite shortening, as did inhibiting MCU-mediated calcium import. Conversely, stimulating the export of calcium from mitochondria was also neuroprotective. These results implicate increased susceptibility to mitochondrial calcium overload in LRRK2-driven neurodegeneration, and suggest possible interventions that may slow the progression of cognitive dysfunction in PD.

HINT2 triggers mitochondrial Ca2+ influx by regulating the mitochondrial Ca2+ uniporter (MCU) complex and enhances gemcitabine apoptotic effect in pancreatic cancer

In early studies, it was shown that HINT2, which sensitizes cells to mitochondrial apoptosis, is down-regulated in hepatocellular carcinoma (HCC) cells (Martin et al., 2006). However, the molecular mechanism of this effect is unknown. Immunohistochemistry revealed that HINT2 expression is relatively low in pancreatic cancer tissues, compared to that in adjacent tissues (P < 0.05). Furthermore, its expression was related to pathological grade and lymph node metastasis (P = 0.0161 and 0.0108, respectively); in addition, down-regulation of HINT2 was found to be associated with relatively poor prognosis in pancreatic cancer patients. Up-regulation of HINT2 was shown to trigger pancreatic cancer cell apoptosis, decrease mitochondrial membrane potential (ΔΨm), promote intracellular reactive oxygen species (ROS) production, and elevate mitochondrial Ca2+ levels. However, co-treatment of HINT2 overexpressing BxPC-3 cells with ruthenium red partially inhibited HINT2-induced apoptosis, which was associated with a reduction in ΔΨm and an increase in intracellular ROS and mitochondrial Ca2+. According to our results, mitochondrial calcium uptake1 and 2 (MICU1 and MICU2) were down-regulated and the essential MCU regulator (EMRE) was up-regulated in cells transduced with Adv-HINT2. Therefore, we deduced that HINT2 triggers apoptosis in pancreatic cancer cells by regulating mitochondrial Ca2+ influx through the mitochondrial calcium uniporter (MCU). In addition, we found that HINT2 can sensitize BxPC-3 and L3.6pl cells to gemcitabine-induced apoptosis and that gemcitabine up-regulates HINT2 expression. This indicates that gemcitabine-induced apoptosis is related to HINT2 levels.

Mitochondrial calcium uniporter in Drosophila transfers calcium between the endoplasmic reticulum and mitochondria in oxidative stress-induced cell death

Mitochondrial calcium plays critical roles in diverse cellular processes ranging from energy metabolism to cell death. Previous studies have demonstrated that mitochondrial calcium uptake is mainly mediated by the mitochondrial calcium uniporter (MCU) complex. However, the roles of the MCU complex in calcium transport, signaling, and dysregulation by oxidative stress still remain unclear. Here, we confirmed that Drosophila MCU contains evolutionarily conserved structures and requires essential MCU regulator (EMRE) for its calcium channel activities. We generated Drosophila MCU loss-of-function mutants, which lacked mitochondrial calcium uptake in response to caffeine stimulation. Basal metabolic activities were not significantly affected in these MCU mutants, as observed in examinations of body weight, food intake, body sugar level, and starvation-induced autophagy. However, oxidative stress-induced increases in mitochondrial calcium, mitochondrial membrane potential depolarization, and cell death were prevented in these mutants. We also found that inositol 1,4,5-trisphosphate receptor genetically interacts with Drosophila MCU and effectively modulates mitochondrial calcium uptake upon oxidative stress. Taken together, these results support the idea that Drosophila MCU is responsible for endoplasmic reticulum-to-mitochondrial calcium transfer and for cell death due to mitochondrial dysfunction under oxidative stress.

Increased mitochondrial calcium uniporter in adipocytes underlies mitochondrial alterations associated with insulin resistance.

Intracellular calcium influences an array of pathways and affects cellular processes. With the rapidly progressing research investigating the molecular identity and the physiological roles of the mitochondrial calcium uniporter (MCU) complex, we now have the tools to understand the functions of mitochondrial Ca2+ in the regulation of pathophysiological processes. Herein, we describe the role of key MCU complex components in insulin resistance in mouse and human adipose tissue. Adipose tissue gene expression was analyzed from several models of obese and diabetic rodents and in 72 patients with obesity as well as in vitro insulin-resistant adipocytes. Genetic manipulation of MCU activity in 3T3-L1 adipocytes allowed the investigation of the role of mitochondrial calcium uptake. In insulin-resistant adipocytes, mitochondrial calcium uptake increased and several MCU components were upregulated. Similar results were observed in mouse and human visceral adipose tissue (VAT) during the progression of obesity and diabetes. Intriguingly, subcutaneous adipose tissue (SAT) was spared from overt MCU fluctuations. Furthermore, MCU expression returned to physiological levels in VAT of patients after weight loss by bariatric surgery. Genetic manipulation of mitochondrial calcium uptake in 3T3-L1 adipocytes demonstrated that changes in mitochondrial calcium concentration ([Ca2+]mt) can affect mitochondrial metabolism, including oxidative enzyme activity, mitochondrial respiration, membrane potential, and reactive oxygen species formation. Finally, our data suggest a strong relationship between [Ca2+]mt and the release of IL-6 and TNFα in adipocytes. Altered mitochondrial calcium flux in fat cells may play a role in obesity and diabetes and may be associated with the differential metabolic profiles of VAT and SAT.

Improved Survival in a Long-Term Rat Model of Sepsis Is Associated With Reduced Mitochondrial Calcium Uptake Despite Increased Energetic Demand

To investigate the relationship between prognosis, changes in mitochondrial calcium uptake, and bioenergetic status in the heart during sepsis. In vivo and ex vivo controlled experimental studies. University research laboratory. Male adult Wistar rats. Sepsis was induced by intraperitoneal injection of fecal slurry. Sham-operated animals served as controls. Confocal microscopy was used to study functional and bioenergetic parameters in cardiomyocytes isolated after 24-hour sepsis. Electron microscopy was used to characterize structural changes in mitochondria and sarcoplasmic reticulum. The functional response to dobutamine was assessed in vivo by echocardiography. Peak aortic blood flow velocity measured at 24 hours was a good discriminator for 72-hour survival (area under the receiver operator characteristic, 0.84 ± 0.1; p = 0.03) and was used in ex vivo experiments at 24 hours to identify septic animals with good prognosis. Measurements from animals with good prognostic showed 1) a smaller increase in mitochondrial calcium content and in nicotinamide adenine dinucleotide fluorescence following pacing and 2) increased distance between mitochondria and sarcoplasmic reticulum on electron microscopy, and 3) nicotinamide adenine dinucleotide redox potential and adenosine triphosphate/adenosine diphosphate failed to reach a new steady state following pacing, suggesting impaired matching of energy supply and demand. In vivo, good prognosis animals had a blunted response to dobutamine with respect to stroke volume and kinetic energy. In situations of higher energetic demand decreased mitochondrial calcium uptake may constitute an adaptive cellular response that confers a survival advantage in response to sepsis at a cost of decreased oxidative capacity.

Abstract 140: Role of miR-181c in Mitochondrial Matrix Calcium Accumulation During Ischemia/Reperfusion Injury in the Heart

Background: We have identified a microRNA, miR-181c, which can translocate into the mitochondria of cardiomyocytes, and regulate the mitochondrial gene mt-COX1. Recently, we have also demonstrated that miR-181c/d -/- mice are protected against ischemia/reperfusion (I/R) injury by attenuating oxidative stress in the heart. Previous data also suggest that overexpression of miR-181c in the heart can activate Ca 2+ entry into mitochondria. Here, we investigate the mechanism by which miR-181c regulates Ca 2+ influx into the mitochondrial matrix. Methods and Results: We found both Mitochondrial Calcium Uptake 1 (MICU1) and mitochondrial respiratory complex IV (COX IV) expression are markedly higher in the miR-181c/d -/- mouse heart. Immunoprecipitated with MICU1, and then immunoblot for different sub-units of COX IV confirmed a protein-protein interaction between MICU1 and COX IV. We have also found significantly less Pyruvate Dehydrogenase (PDH) activity in neonatal mouse ventricular myocytes (NMVMs) isolated from miR-181c/d -/- mouse compared to C57BL6 (WT), suggesting significantly lower mitochondrial Ca 2+ -concentrations in the miR-181c/d -/- group. Utilizing a coverslip induced I/R-model, we observe that siRNAs against MICU1 (si-MICU1) during the ischemic phase significantly increase Ca 2+ -entry into the mitochondria of the NMVMs. Lowering MICU1 also significantly increases Ca 2+ -entry into the mitochondria after 30 min of ischemia in miR-181c/d -/- NMVMs. Furthermore, 30 min ischemia followed by 30 min reperfusion in NMVM monolayers led to significantly less oscillatory instability in mitochondrial inner membrane potential (ΔΨ m ) in miR-181c/d -/- NMVMs compared with WT NMVMs. However, using si-MICU1 in the miR-181c/d -/- NMVM group attenuated mitochondrial protection against I/R-injury. Conclusions: MICU1 is directly associated with complex IV. Thus, miR-181c can regulate mitochondrial Ca 2+ -entry by targeting mt-COX1 during I/R injury.

Abstract 205: Overexpression of the Mitochondrial F o F 1 ATP Synthase regulators ATP12 and ATP5J are Protective against Ischemia-Reperfusion Injury in the Heart

Loss of cardiomyocytes by necrotic cell death is a substantial underlying problem in progressive heart failure. One crucial step that is required for regulated necrosis to ensue is the opening of the mitochondrial permeability transition pore (MPTP), which is triggered by elevations in mitochondrial matrix calcium and reactive oxygen species levels. Opening of the MPTP causes permeabilization of the inner mitochondrial membrane and collapse of the electrochemical proton gradient required for ATP production. The latest model of the MPTP consists of the F o F 1 ATP synthase as the pore forming component within the inner mitochondrial membrane, although the mechanistic aspects of how the ATP synthase generates the pore is unknown. Two regulators of the ATP synthase, ATP12 and ATP5J, were identified in an unbiased calcium overload necrotic cell death screen using a lenti-viral gain-of-function library containing over 13,000 human cDNAs. ATP12 is an assembly factor of the F 1 component of the ATP synthase where it directly binds to the α-subunit and prevents homo-oligomerization. ATP5J is a component of the peripheral stalk that links the F o and F 1 portions of the ATP synthase together. To test if overexpression of ATP12 or ATP5J is indeed protective as we identified in the screen, here we generated cardiac-specific transgenic mice expressing each of these proteins. When subjected to ischemia-reperfusion injury, both ATP5J and ATP12 overexpressing mice were significantly protected. Additionally, mitochondria isolated from the overexpressing hearts demonstrated increased mitochondrial calcium uptake, a hallmark of MPTP desensitization. These data provide evidence that ATP12 and ATP5J are novel inhibitors of MPTP opening through their association with the F o F 1 ATP synthase and thus protect against necrotic cell death.

Regulation of calcium release from the endoplasmic reticulum by the serine hydrolase ABHD2

The serine hydrolase inhibitors pyrrophenone and KT195 inhibit cell death induced by A23187 and H2O2 by blocking the release of calcium from the endoplasmic reticulum and mitochondrial calcium uptake. The effect of pyrrophenone and KT195 on these processes is not due to inhibition of their known targets, cytosolic phospholipase A2 and α/β-hydrolase domain-containing (ABHD) 6, respectively, but represent off-target effects. To identify targets of KT195, fibroblasts were treated with KT195-alkyne to covalently label protein targets followed by click chemistry with biotin azide, enrichment on streptavidin beads and tryptic peptide analysis by mass spectrometry. Although several serine hydrolases were identified, α/β-hydrolase domain-containing 2 (ABHD2) was the only target in which both KT195 and pyrrophenone competed for binding to KT195-alkyne. ABHD2 is a serine hydrolase with a predicted transmembrane domain consistent with its pull-down from the membrane proteome. Subcellular fractionation showed localization of ABHD2 to the endoplasmic reticulum but not to mitochondria or mitochondrial-associated membranes. Knockdown of ABHD2 with shRNA attenuated calcium release from the endoplasmic reticulum, mitochondrial calcium uptake and cell death in fibroblasts stimulated with A23187. The results describe a novel mechanism for regulating calcium transfer from the endoplasmic reticulum to mitochondria that involves the serine hydrolase ABHD2.

Enhanced Mitochondrial Transient Receptor Potential Channel, Canonical Type 3-Mediated Calcium Handling in the Vasculature From Hypertensive Rats.

BackgroundMitochondrial Ca2+ homeostasis is fundamental to the regulation of mitochondrial reactive oxygen species (ROS) generation and adenosine triphosphate production. Recently, transient receptor potential channel, canonical type 3 (TRPC3), has been shown to localize to the mitochondria and to play a role in maintaining mitochondrial calcium homeostasis. Inhibition of TRPC3 attenuates vascular calcium influx in spontaneously hypertensive rats (SHRs). However, it remains elusive whether mitochondrial TRPC3 participates in hypertension by increasing mitochondrial calcium handling and ROS production.Methods and ResultsIn this study we demonstrated increased TRPC3 expression in purified mitochondria in the vasculature from SHRs, which facilitates enhanced mitochondrial calcium uptake and ROS generation compared with Wistar‐Kyoto rats. Furthermore, inhibition of TRPC3 by its specific inhibitor, Pyr3, significantly decreased the vascular mitochondrial ROS production and H2O2 synthesis and increased adenosine triphosphate content. Administration of telmisartan can improve these abnormalities. This beneficial effect was associated with improvement of the mitochondrial respiratory function through recovering the activity of pyruvate dehydrogenase in the vasculature of SHRs. In vivo, chronic administration of telmisartan suppressed TRPC3‐mediated excessive mitochondrial ROS generation and vasoconstriction in the vasculature of SHRs. More importantly, TRPC3 knockout mice exhibited significantly ameliorated hypertension through reduction of angiotensin II–induced mitochondrial ROS generation.ConclusionsTogether, we give experimental evidence for a potential mechanism by which enhanced TRPC3 activity at the cytoplasmic and mitochondrial levels contributes to redox signaling and calcium dysregulation in the vasculature from SHRs. Angiotensin II or telmisartan can regulate [Ca2+]mito, ROS production, and mitochondrial energy metabolism through targeting TRPC3.

Expression of MICU1 after experimental focal cerebral ischemia in adult rats

BackgroundMitochondrial Ca2+ uptake is a pivotal pathophysiological process for neuronal survival when subjected to ischemic insult. Mitochondrial calcium uptake 1 (MICU1) has been demonstrated as a key regulator of the mitochondrial calcium uniporter (MCU), identified as a tetrameric highly specific channel that modulates mitochondrial Ca2+ uptake.MethodsAdult male Sprague–Dawley (SD) rats underwent middle cerebral artery occlusion (MCAO) to create the standard focal cerebral ischemia model. The permanent MCAO approach utilized the intraluminal approach. Neurological examination, and subsequent histological characterization of cerebral infarcts using triphenyltetrazolium chloride staining, as well as Western blot, immunohistochemical staining, and real-time quantitative polymerase chain reaction assays were employed to assess the functional effects of MICU1 and its expression in the brain.ResultsAnimals exposed to MCAO displayed the typical neurological deficit accompanied by cortical and subcortical infarction at 72 h post-stroke. The expression of MICU1, with co-localization with neurons, was detected at different time points (6 h and 12 h) after ischemic damage. Altogether, these observations revealed an up-regulation of MICU1 expression in the early stages of cerebral ischemia.ConclusionThe results demonstrated that MICU1 was upregulated in neurons at the acute phase of ischemic stroke. Because MICU1 has been previously shown to participate in mitochondrial Ca2+ uptake mediated by MCU, our study further implicates the involvement of MICU1 in calcium overload-induced cell death which is closely associated with stroke.

MICU1 drives glycolysis and chemoresistance in ovarian cancer

Cancer cells actively promote aerobic glycolysis to sustain their metabolic requirements through mechanisms not always clear. Here, we demonstrate that the gatekeeper of mitochondrial Ca2+ uptake, Mitochondrial Calcium Uptake 1 (MICU1/CBARA1) drives aerobic glycolysis in ovarian cancer. We show that MICU1 is overexpressed in a panel of ovarian cancer cell lines and that MICU1 overexpression correlates with poor overall survival (OS). Silencing MICU1 in vitro increases oxygen consumption, decreases lactate production, inhibits clonal growth, migration and invasion of ovarian cancer cells, whereas silencing in vivo inhibits tumour growth, increases cisplatin efficacy and OS. Mechanistically, silencing MICU1 activates pyruvate dehydrogenase (PDH) by stimulating the PDPhosphatase-phosphoPDH-PDH axis. Forced-expression of MICU1 in normal cells phenocopies the metabolic aberrations of malignant cells. Consistent with the in vitro and in vivo findings we observe a significant correlation between MICU1 and pPDH (inactive form of PDH) expression with poor prognosis. Thus, MICU1 could serve as an important therapeutic target to normalize metabolic aberrations responsible for poor prognosis in ovarian cancer.

A Select Subset of Electron Transport Chain Genes Associated with Optic Atrophy Link Mitochondria to Axon Regeneration in Caenorhabditis elegans

The role of mitochondria within injured neurons is an area of active interest since these organelles are vital for the production of cellular energy in the form of ATP. Using mechanosensory neurons of the nematode Caenorhabditis elegans to test regeneration after neuronal injury in vivo, we surveyed genes related to mitochondrial function for effects on axon regrowth after laser axotomy. Genes involved in mitochondrial transport, calcium uptake, mitophagy, or fission and fusion were largely dispensable for axon regrowth, with the exception of eat-3/Opa1. Surprisingly, many genes encoding components of the electron transport chain were dispensable for regrowth, except for the iron-sulfur proteins gas-1, nduf-2.2, nduf-7, and isp-1, and the putative oxidoreductase rad-8. In these mutants, axonal development was essentially normal and axons responded normally to injury by forming regenerative growth cones, but were impaired in subsequent axon extension. Overexpression of nduf-2.2 or isp-1 was sufficient to enhance regrowth, suggesting that mitochondrial function is rate-limiting in axon regeneration. Moreover, loss of function in isp-1 reduced the enhanced regeneration caused by either a gain-of-function mutation in the calcium channel EGL-19 or overexpression of the MAP kinase DLK-1. While the cellular function of RAD-8 remains unclear, our genetic analyses place rad-8 in the same pathway as other electron transport genes in axon regeneration. Unexpectedly, rad-8 regrowth defects were suppressed by altered function in the ubiquinone biosynthesis gene clk-1. Furthermore, we found that inhibition of the mitochondrial unfolded protein response via deletion of atfs-1 suppressed the defective regrowth in nduf-2.2 mutants. Together, our data indicate that while axon regeneration is not significantly affected by general dysfunction of cellular respiration, it is sensitive to the proper functioning of a select subset of electron transport chain genes, or to the cellular adaptations used by neurons under conditions of injury.