Inhibition Of Mitochondrial Ca2 Research Articles

Ru360 protects against vitrification-induced oocyte meiotic defects by restoring mitochondrial function

Oocyte vitrification has been widely application in female fertility preservation. Recent studies found that vitrification of immature (germinal vesicle stage, GV) oocytes increased the risk of aneuploidy during meiotic maturation; however, the underlying mechanisms and the strategies to prevent this defect remain unexplored. In this study, we found that vitrification of GV oocytes decreased the first polarbody extrusion rate (90.51 ± 1.04% vs. 63.89 ± 1.39%, p < 0.05) and increased the aneuploid rate (2.50% vs. 20.00%, p < 0.05), accompanied with a series of defects during meiotic maturation, including aberrant spindle morphology, chromosome misalignment, incorrect Kinetochore-Microtubule attachments (KT-MTs) and weakened spindle assembly checkpoint protein complex (SAC) function. We also found that vitrification disrupted mitochondrial function by increasing mitochondrial Ca2+ levels. Importantly, inhibition of mitochondrial Ca2+ entry by 1 μM Ru360 significantly restored mitochondrial function and rescued the meiotic defects, indicating that the increase of mitochondrial Ca2+, at least, was a cause of meiotic defects in vitrified oocytes. These results shed light on the molecular mechanisms of oocyte vitrification-induced adverse effects of meiotic maturation and provided a potential strategy to improve oocyte cryopreservation protocols further.

Differential modulation of short-term plasticity at hippocampal mossy fiber and Schaffer collateral synapses by mitochondrial Ca2.

Presynaptic mitochondrial Ca2+ plays a critical role in the regulation of synaptic transmission and plasticity. The presynaptic bouton of the hippocampal mossy fiber (MF) is much larger in size than that of the Schaffer collateral (SC) synapse. Here we compare the structural and physiological characteristics of MF and SC presynaptic boutons to reveal functional and mechanistic differences between these two synapses. Our quantitative ultrastructural analysis using electron microscopy show many more mitochondria in MF presynaptic bouton cross-section profiles compared to SC boutons. Consistent with these results, post-tetanic potentiation (PTP), a form of presynaptic short-term plasticity dependent on mitochondrial Ca2+, is reduced by inhibition of mitochondrial Ca2+ release at MF synapses but not at SC synapses. However, blockade of mitochondrial Ca2+ release results in reduction of PTP at SC synapses by disynaptic MF stimulation. Furthermore, inhibition of mitochondrial Ca2+ release selectively decreases frequency facilitation evoked by short trains of presynaptic stimulation at MF synapses, while having no effect at SC synapses. Moreover, depletion of ER Ca2+ stores leads to reduction of PTP at MF synapses, but PTP is unaffected by ER Ca2+ depletion at SC synapses. These findings show that MF and SC synapses differ in presynaptic mitochondrial content as well as mitochondrial Ca2+ dependent synaptic plasticity, highlighting differential regulatory mechanisms of presynaptic plasticity at MF and SC synapses.

Endoplasmic reticulum Ca2+ release causes Rieske iron-sulfur protein-mediated mitochondrial ROS generation in pulmonary artery smooth muscle cells.

Mitochondrial reactive oxygen species (ROS) cause Ca2+ release from the endoplasmic reticulum (ER) via ryanodine receptors (RyRs) in pulmonary artery smooth muscle cells (PASMCs), playing an essential role in hypoxic pulmonary vasoconstriction (HPV). Here we tested a novel hypothesis that hypoxia-induced RyR-mediated Ca2+ release may, in turn, promote mitochondrial ROS generation contributing to hypoxic cellular responses in PASMCs. Our data reveal that application of caffeine to elevate intracellular Ca2+ concentration ([Ca2+]i) by activating RyRs results in a significant increase in ROS production in cytosol and mitochondria of PASMCs. Norepinephrine to increase [Ca2+]i due to the opening of inositol 1,4,5-triphosphate receptors (IP3Rs) produces similar effects. Exogenous Ca2+ significantly increases mitochondrial-derived ROS generation as well. Ru360 also inhibits the hypoxic ROS production. The RyR antagonist tetracaine or RyR2 gene knockout (KO) suppresses hypoxia-induced responses as well. Inhibition of mitochondrial Ca2+ uptake with Ru360 eliminates N- and Ca2+-induced responses. RISP KD abolishes the hypoxia-induced ROS production in mitochondria of PASMCs. Rieske iron–sulfur protein (RISP) gene knockdown (KD) blocks caffeine- or NE-induced ROS production. Taken together, these findings have further demonstrated that ER Ca2+ release causes mitochondrial Ca2+ uptake and RISP-mediated ROS production; this novel local ER/mitochondrion communication-elicited, Ca2+-mediated, RISP-dependent ROS production may play a significant role in hypoxic cellular responses in PASMCs.

Excessive Accumulation of Ca2 + in Mitochondria of Y522S-RYR1 Knock-in Mice: A Link Between Leak From the Sarcoplasmic Reticulum and Altered Redox State.

Mice (Y522S or YS), carrying a mutation of the sarcoplasmic reticulum (SR) Ca2+ release channel of skeletal muscle fibers (ryanodine receptor type-1, RyR1) which causes Ca2+ leak, are a widely accepted and intensively studied model for human malignant hyperthermia (MH) susceptibility. Since the involvement of reactive oxygen species (ROS) and of mitochondria in MH crisis has been previously debated, here we sought to determine Ca2+ uptake in mitochondria and its possible link with ROS production in single fibers isolated from flexor digitorum brevis (FDB) of YS mice. We found that Ca2+ concentration in the mitochondrial matrix, as detected with the ratiometric FRET-based 4mtD3cpv probe, was higher in YS than in wild-type (WT) fibers at rest and after Ca2+ release from SR during repetitive electrical stimulation or caffeine administration. Also mitochondrial ROS production associated with contractile activity (detected with Mitosox probe) was much higher in YS fibers than in WT. Importantly, the inhibition of mitochondrial Ca2+ uptake achieved by silencing MCU reduced ROS accumulation in the matrix and Ca2+ release from SR. Finally, inhibition of mitochondrial ROS accumulation using Mitotempo reduced SR Ca2+ release in YS fibers exposed to caffeine. The present results support the view that mitochondria take up larger amounts of Ca2+ in YS than in WT fibers and that mitochondrial ROS production substantially contributes to the increased caffeine-sensitivity and to the enhanced Ca2+ release from SR in YS fibers.

Pseudomonas aeruginosa quorum-sensing molecule N-(3-oxo-dodecanoyl)-L-homoserine lactone triggers mitochondrial dysfunction and apoptosis in neutrophils through calcium signaling.

Pseudomonas aeruginosa is an opportunistic pathogen that utilizes the quorum-sensing (QS) process to regulate the production of different virulence factors and biofilm. N-3-oxo-dodecanoyl-L-homoserine lactone (C12) is a key QS molecule of P. aeruginosa which interacts with the mammalian immune cells and modulates their function. Here, we investigated the molecular mechanism of C12-induced apoptosis in neutrophils. Our data show that C12 causes apoptosis in neutrophils through an elevation in cytosolic and mitochondrial Ca2+ levels. Besides, C12 induces phosphatidylserine (PS) exposure,mitochondrial membrane potential (MMP) depolarization, mitochondrial permeability transition pore (MPTP) formation andmitochondrial reactive oxygen species (mROS) generation. C12-induced rise in intracellular Ca2+ level is majorly contributed by endoplasmic reticulum store through the activation of inositol 1, 4, 5-triphosphate receptor. Intracellular calcium chelation inhibited C12-induced mitochondrial dysfunction and apoptosis. Further, inhibition of mitochondrial Ca2+ uniporter by ruthenium red or Ru360 abrogated C12-induced mitochondrial Ca2+ uptake, MMP loss, MPTP opening, mROS production, and PS exposure. These mechanistic insights are expected to provide a better understanding of the role of C12 in P. aeruginosa pathogenesis.

Substrate-dependent and cyclophilin D-independent regulation of mitochondrial flashes in skeletal and cardiac muscle

Mitochondrial flashes (mitoflashes) are stochastic events in the mitochondrial matrix detected by mitochondrial-targeted cpYFP (mt-cpYFP). Mitoflashes are quantal bursts of reactive oxygen species (ROS) production accompanied by modest matrix alkalinization and depolarization of the mitochondrial membrane potential. Mitoflashes are fundamental events present in a wide range of cell types. To date, the precise mechanisms for mitoflash generation and termination remain elusive. Transient opening of the mitochondrial membrane permeability transition pore (mPTP) during a mitoflash is proposed to account for the mitochondrial membrane potential depolarization. Here, we set out to compare the tissue-specific effects of cyclophilin D (CypD)-deficiency and mitochondrial substrates on mitoflash activity in skeletal and cardiac muscle. In contrast to previous reports, we found that CypD knockout did not alter the mitoflash frequency or other mitoflash properties in acutely isolated cardiac myocytes, skeletal muscle fibers, or isolated mitochondria from skeletal muscle and the heart. However, in skeletal muscle fibers, CypD deficiency resulted in a parallel increase in both activity-dependent mitochondrial Ca2+ uptake and activity-dependent mitoflash activity. Increases in both mitochondrial Ca2+ uptake and mitoflash activity following electrical stimulation were abolished by inhibition of mitochondrial Ca2+ uptake. We also found that mitoflash frequency and amplitude differ greatly between intact skeletal muscle fibers and cardiac myocytes, but that this difference is absent in isolated mitochondria. We propose that this difference may be due, in part, to differences in substrate availability in intact skeletal muscle fibers (primarily glycolytic) and cardiac myocytes (largely oxidative). Overall, we find that CypD does not contribute significantly in mitoflash biogenesis under basal conditions in skeletal and cardiac muscle, but does regulate mitoflash events during muscle activity. In addition, tissue-dependent differences in mitoflash frequency are strongly regulated by mitochondrial substrate availability.

AMPK-mediated activation of MCU stimulates mitochondrial Ca2+ entry to promote mitotic progression.

The capacity of cells to alter bioenergetics in response to the demands of various biological processes is essential for normal physiology. The coordination of energy sensing and production with highly energy-demanding cellular processes, such as cell division, is poorly understood. Here, we show that a cell cycle-dependent mitochondrial Ca2+ transient connects energy sensing to mitochondrial activity for mitotic progression. The mitochondrial Ca2+ uniporter (MCU) mediates a rapid mitochondrial Ca2+ transient during mitosis. Inhibition of mitochondrial Ca2+ transients via MCU depletion causes spindle checkpoint-dependent mitotic delay. Cellular ATP levels drop during early mitosis, and the mitochondrial Ca2+ transients boost mitochondrial respiration to restore energy homeostasis. This is achieved through mitosis-specific MCU phosphorylation and activation by the mitochondrial translocation of energy sensor AMP-activated protein kinase (AMPK). Our results establish a critical role for AMPK- and MCU-dependent mitochondrial Ca2+ signalling in mitosis and reveal a mechanism of mitochondrial metabolic adaptation to acute cellular energy stress.

Calcium signals between the ryanodine receptor- and mitochondria critically regulate the effects of arsenite on mitochondrial superoxide formation and on the ensuing survival vs apoptotic signaling

A low concentration of arsenite (6 h), selectively stimulating the intraluminal crosstalk between the inositol-1, 4, 5-triphosphate receptor and the ryanodine receptor (RyR), increased the mitochondrial transport of RyR-derived Ca2+ through the mitochondrial Ca2+ uniporter. This event was characterized in intact and permeabilized cells, and was shown to be critical for mitochondrial superoxide (mitoO2.-) formation. Inhibition of mitochondrial Ca2+ accumulation therefore prevented the effects of arsenite, in both the mitochondrial (e.g., cardiolipin oxidation) and extramitochondrial (e.g., DNA single- strand breakage) compartments, and suppressed the Nrf2/GSH survival signaling. The effects of arsenite on Ca2+ homeostasis and mitoO2.- formation were reversible, as determined after an additional 10 h incubation in fresh culture medium and by measuring long-term viability. A 16 h continuous exposure to arsenite instead produced a sustained increase in the cytosolic and mitochondrial Ca2+ concentrations, a further increased mitoO2.- formation and mitochondrial permeability transition. These events, followed by delayed apoptosis (48 h), were sensitive to treatments/manipulations preventing mitochondrial Ca2+ accumulation. Interestingly, cells remained viable under conditions in which the deregulated Ca2+ homeostasis was not accompanied by mitoO2.-formation.In conclusion, we report that the fraction of Ca2+ taken up by the mitochondria in response to arsenite derives from the RyR. Mitochondrial Ca2+ appears critical for mitoO2.- formation and for the triggering of both the cytoprotective and apoptotic signaling. The effects of arsenite were reversible, whereas its prolonged exposure caused a sustained increase in mitochondrial Ca2+ and mitoO2.- formation, and the prevalence of the apoptotic vs survival signaling.

Is it All Said for NSAIDs in Alzheimer’s Disease? Role of Mitochondrial Calcium Uptake

Epidemiological data suggest that non-steroidal anti-inflammatory drugs (NSAIDs) may protect against Alzheimer's disease (AD). Unfortunately, recent trials have failed in providing compelling evidence of neuroprotection. Discussion as to why NSAIDs effectivity is uncertain is ongoing. Possible explanations include the view that NSAIDs and other possible disease-modifying drugs should be provided before the patients develop symptoms of AD or cognitive decline. In addition, NSAID targets for neuroprotection are unclear. Both COX-dependent and independent mechanisms have been proposed, including γ-secretase that cleaves the amyloid precursor protein (APP) and yields amyloid β peptide (Aβ). We have proposed a neuroprotection mechanism for NSAIDs based on inhibition of mitochondrial Ca2+ overload. Aβ oligomers promote Ca2+ influx and mitochondrial Ca2+ overload leading to neuron cell death. Several non-specific NSAIDs including ibuprofen, sulindac, indomethacin and Rflurbiprofen depolarize mitochondria in the low µM range and prevent mitochondrial Ca2+ overload induced by Aβ oligomers and/or N-methyl-D-aspartate (NMDA). However, at larger concentrations, NSAIDs may collapse mitochondrial potential (ΔΨ) leading to cell death. Accordingly, this mechanism may explain neuroprotection at low concentrations and damage at larger doses, thus providing clues on the failure of promising trials. Perhaps lower NSAID concentrations and/or alternative compounds with larger dynamic ranges should be considered for future trials to provide definitive evidence of neuroprotection against AD.

Mitochondrial Ca2+ Influx Contributes to Arrhythmic Risk in Nonischemic Cardiomyopathy

Introduction: It had been reported that mitochondrial Ca2+ was overloaded in ischemia-reperfusion injury. Inhibition of mitochondrial Ca2+ uniporter (MCU) prevented cell death. In this study, pharmacological block or genetic knockdown MCU inhibited mitochondrial Ca2+ uptake, depressed early afterdepolarizations (EADs) and reduced ventricular fibrillation in nonischemic heart failure (NI-HF) mice. Methods: A model of NI-HF was induced in CD1 mice by hypertension. Echocardiography, patch-clamp (and Rhod-2 fluorescence density recorded synchronously), telemetry monitoring, real-time PCR and Western blotting were employed in this study. Computer simulations were carried out. Results: Echocardiography showed an impairment of systolic function as the average EF value decreased from 63 ± 2% to 51 ± 3% in NI-HF mice (p<0.05). Isolated myopathic myocytes showed decreased cytoplasmic Ca2+ transients, increased mitochondrial Ca2+ transients, and increased action potential duration at 90% (APD90) repolarization. The alteration of APD90 was consistent with in vivo QTc prolongation and could be explained by augmented L-type Ca2+ currents, increased NCX and decreased total K+ currents. Sixty-six percent of myopathic ventricular myocytes showed EADs compared with 17% of sham myocytes (p<0.05). Intracellular application of 1 µM Ru360 could reduce mitochondrial Ca2+ transients, decrease APD90 and ameliorate EADs. Furthermore, genetic knockdown of MCU inhibited mitochondrial Ca2+ uptake, reduced NCX, decreased APD90, depressed EADs, and reduced ventricular fibrillation in NI-HF mice. Inhibiting mitochondrial NCX slowed mitochondrial Ca2+ uptake, decreased APD90, and depressed EADs. Computer simulations showed that EADs were promoted by heart failure remodeling, which were abolished by blocking either the MCU or the L-type Ca2+ current. Simulations revealed that EADs were promoted via a positive feedback loop among APD, SR Ca2+, and mitochondrial Ca2+, resulting in an all-or-none behavior. Conclusions: Mitochondrial Ca2+ handling plays an important role in EADs seen with chronic and moderate nonischemic cardiomyopathy.

Jolkinolide B induces apoptosis of colorectal carcinoma through ROS-ER stress-Ca2+-mitochondria dependent pathway.

Colorectal carcinoma (CRC) remains one of the leading causes of death in cancer-related diseases. In this study, we aimed to investigate the anticancer effect of Jolkinolide B (JB), a bioactive diterpenoid component isolated from the dried roots of Euphorbia fischeriana Steud, on CRC cells and its underlying mechanisms. We found that JB suppressed the cell viability and colony formation of CRC cells, HT29 and SW620. Annexin V/PI assay revealed that JB induced apoptosis in CRC cells, which was further confirmed by the increased expression of cleaved-caspase3 and cleaved-PARP. iTRAQ-based quantitative proteomics was performed to identify JB-regulated proteins in CRC cells. Gene Ontology (GO) analysis revealed that these JB-regulated proteins were mainly involved in ER stress response, which was evidenced by the expression of ER stress marker proteins, HSP90, Bip and PDI. Moreover, we found that JB provoked the generation of reactive oxygen species (ROS), and that inhibition of the ROS generation with N-acetyl L-cysteine could reverse the JB-induced apoptosis. Confocal microscopy and flow cytometry showed that JB treatment enhanced intracellular and mitochondrial Ca2+ level and JC-1 assay revealed a loss of mitochondrial membrane potential in CRC after JB treatment. The mitochondrial Ca2+ uptake and depolarization can be blocked by Ruthenium Red (RuRed), an inhibitor of mitochondrial Ca2+ uniporter. Taken together, we demonstrated that JB exerts its anticancer effect by ER stress-Ca2+-mitochondria signaling, suggesting the promising chemotherapeutic potential of JB for the treatment of CRC.

Abstract 5665: Physical crosstalk between CD8+T and natural killer cells elicits antitumor effector response

Abstract The interaction between the innate and adaptive immune components is fundamental for an effective antitumor immunity. Our studies in murine solid tumor models showed that productive antitumor effector response relies on functional crosstalk between innate immune effectors—natural killers (NK), and adaptive immune effectors—cytolytic CD8+T lymphocytes. We found that this lymphocyte cooperativity between CD8+T and NK cells can prevent the development of antigen-escape tumor variants. In this study, we first investigated the role of physical contacts in during the functional crosstalk between CD8+T and NK cells. Since, studying dynamic lymphocyte interactions present extreme challenges, in this study, we engineered a 3D nanofiber matrix to provide lymphocytes a 3D culture environment for a controlled interaction. Confocal imaging showed that CD3/CD28-activated CD8+T cells (CD69+CD25+) formed multiple intercellular contacts with several naïve NK cells upon coculture, while naïve CD8+T cells made single or no contact with NK cells. In lymphocyte coculture (physical contact possible) we found that activated CD8+T and NK cells cross-regulate each other phenotype wherein NK cells polarize activated CD8+T cells towards “T central memory phenotype” and activated CD8+T lymphocytes induce acquisition of “effector/regulatory phenotype” by naïve NK cells. This cross-regulation of lymphocytes disappeared in trans-well system (no physical contact) indicating the necessity of cell-to-cell physical interaction during CD8+T—NK crosstalk. Notably, intercellular physical interaction led to cross regulation of mitoCa2+ oscillations in both activated CD8+T and NK cells. Inhibition of mitochondrial Ca2+ uptake or Na+/Ca2+ exchanger with Ru360 and CGP37157, respectively, mimicked observed alterations in both lymphocytes. Further, NK cells displayed increased oxidative signaling, Tyk2, Jak 1 and 3, Stat2 and Stat6 phosphorylation while inhibiting TCR- and various cytokine receptors-mediated signaling. In turn, NK cells selectively restrain IL-2 signaling in CD8+T cells by dampening activation-induced up-regulation of CD25, Stat5 phosphorylation, IL-2 synthesis and elevation in IL-2 uptake. These data underscore a novel mitochondrial Ca2+ transport-regulated acquisition of activation/regulatory phenotype by NK and CD8+T cells upon their interaction. Understanding the critical factors involved in this NK—CD8+T cells immunological synapse allowing their functional remodeling with the intact tissues in tumor settings will lead to novel strategies for effective cancer immunotherapies, with a potential of relapse-free survival in cancer patients. Citation Format: Roman V. Uzhachenko, Ashutosh Singhal, Shawn J. Goodwin, William H. Hofmeister, Anil Shanker. Physical crosstalk between CD8+T and natural killer cells elicits antitumor effector response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5665. doi:10.1158/1538-7445.AM2017-5665

Lithocholic acid induces two different calcium-dependent inner membrane permeability systems in liver mitochondria

The effect of the most hydrophobic bile acid–lithocholic–as an inducer of two different Ca2+-dependent inner membrane permeability systems was studied on isolated rat liver mitochondria. It is shown that the addition of lithocholic acid at a concentration of 20 μM to the Ca2+-loaded mitochondria leads to swelling of the organelles, rapid release of Ca2+ from the matrix and almost complete collapse of Δψ. Mitochondrial pore blocker cyclosporin A (CsA) eliminates mitochondrial swelling but has no effect on the process of Ca2+ release and Δψ collapse. In the absence of Ca2+ lithocholic acid causes only a transient decrease of Δψ with subsequent complete recovery. Ruthenium red, inhibitor of mitochondrial Ca2+ uniporter, which blocks Ca2+ influx into the matrix, prevents mitochondrial swelling induced by lithocholic acid. At the same time, ruthenium red, which is added before lithocholic acid to the Ca2+-preloaded mitochondria, does not affect the swelling of the organelles but reduces the CsA-insensitive drop in Δψ. It is concluded that lithocholic acid is able to induce two Ca2+-dependent energy dissipation systems in the inner membrane of liver mitochondria: CsA-sensitive mitochondrial pore and CsA-insensitive permeability, which exhibits sensitivity to ruthenium red. It is found that the effect of this bile acid as an inductor of CsA-sensitive mitochondrial pore is not associated with the modulation of Pi effects. It is assumed that CsA-insensitive action of lithocholic acid is associated with the induction of Ca2+ efflux from the matrix in exchange for protons. In this case, the energy-dependent Ca2+ transport in the opposite direction with the participation of mitochondrial calcium uniporter sensitive to ruthenium red leads to the formation of calcium cycle and thereby to energy dissipation.

Presenilins regulate synaptic plasticity and mitochondrial calcium homeostasis in the hippocampal mossy fiber pathway

BackgroundPresenilins play a major role in the pathogenesis of Alzheimer’s disease, in which the hippocampus is particularly vulnerable. Previous studies of Presenilin function in the synapse, however, focused exclusively on the hippocampal Schaffer collateral (SC) pathway. Whether Presenilins play similar or distinct roles in other hippocampal synapses is unknown.MethodsTo investigate the role of Presenilins at mossy fiber (MF) synapses we performed field and whole-cell electrophysiological recordings and Ca2+ imaging using acute hippocampal slices of postnatal forebrain-restricted Presenilin conditional double knockout (PS cDKO) and control mice at 2 months of age. We also performed quantitative electron microscopy (EM) analysis to determine whether mitochondrial content is affected at presynaptic MF boutons of PS cDKO mice. We further conducted behavioral analysis to assess spatial learning and memory of PS cDKO and control mice at 2 months in the Morris water maze.ResultsWe found that long-term potentiation and short-term plasticity, such as paired-pulse and frequency facilitation, are impaired at MF synapses of PS cDKO mice. Moreover, post-tetanic potentiation (PTP), another form of short-term plasticity, is also impaired at MF synapses of PS cDKO mice. Furthermore, blockade of mitochondrial Ca2+ efflux mimics and occludes the PTP deficits at MF synapses of PS cDKO mice, suggesting that mitochondrial Ca2+ homeostasis is impaired in the absence of PS. Quantitative EM analysis showed normal number and area of mitochondria at presynaptic MF boutons of PS cDKO mice, indicating unchanged mitochondrial content. Ca2+ imaging of dentate gyrus granule neurons further revealed that cytosolic Ca2+ increases induced by tetanic stimulation are reduced in PS cDKO granule neurons in acute hippocampal slices, and that inhibition of mitochondrial Ca2+ release during high frequency stimulation mimics and occludes the Ca2+ defects observed in PS cDKO neurons. Consistent with synaptic plasticity impairment observed at MF and SC synapses in acute PS cDKO hippocampal slices, PS cDKO mice exhibit profound spatial learning and memory deficits in the Morris water maze.ConclusionsOur findings demonstrate the importance of PS in the regulation of synaptic plasticity and mitochondrial Ca2+ homeostasis in the hippocampal MF pathway.

A role for TSPO in mitochondrial Ca2+ homeostasis and redox stress signaling

The 18 kDa translocator protein TSPO localizes on the outer mitochondrial membrane (OMM). Systematically overexpressed at sites of neuroinflammation it is adopted as a biomarker of brain conditions. TSPO inhibits the autophagic removal of mitochondria by limiting PARK2-mediated mitochondrial ubiquitination via a peri-organelle accumulation of reactive oxygen species (ROS). Here we describe that TSPO deregulates mitochondrial Ca2+ signaling leading to a parallel increase in the cytosolic Ca2+ pools that activate the Ca2+-dependent NADPH oxidase (NOX) thereby increasing ROS. The inhibition of mitochondrial Ca2+ uptake by TSPO is a consequence of the phosphorylation of the voltage-dependent anion channel (VDAC1) by the protein kinase A (PKA), which is recruited to the mitochondria, in complex with the Acyl-CoA binding domain containing 3 (ACBD3). Notably, the neurotransmitter glutamate, which contributes neuronal toxicity in age-dependent conditions, triggers this TSPO-dependent mechanism of cell signaling leading to cellular demise. TSPO is therefore proposed as a novel OMM-based pathway to control intracellular Ca2+ dynamics and redox transients in neuronal cytotoxicity.

Absence of physiological Ca2+ transients is an initial trigger for mitochondrial dysfunction in skeletal muscle following denervation

BackgroundMotor neurons control muscle contraction by initiating action potentials in muscle. Denervation of muscle from motor neurons leads to muscle atrophy, which is linked to mitochondrial dysfunction. It is known that denervation promotes mitochondrial reactive oxygen species (ROS) production in muscle, whereas the initial cause of mitochondrial ROS production in denervated muscle remains elusive. Since denervation isolates muscle from motor neurons and deprives it from any electric stimulation, no action potentials are initiated, and therefore, no physiological Ca2+ transients are generated inside denervated muscle fibers. We tested whether loss of physiological Ca2+ transients is an initial cause leading to mitochondrial dysfunction in denervated skeletal muscle.MethodsA transgenic mouse model expressing a mitochondrial targeted biosensor (mt-cpYFP) allowed a real-time measurement of the ROS-related mitochondrial metabolic function following denervation, termed “mitoflash.” Using live cell imaging, electrophysiological, pharmacological, and biochemical studies, we examined a potential molecular mechanism that initiates ROS-related mitochondrial dysfunction following denervation.ResultsWe found that muscle fibers showed a fourfold increase in mitoflash activity 24 h after denervation. The denervation-induced mitoflash activity was likely associated with an increased activity of mitochondrial permeability transition pore (mPTP), as the mitoflash activity was attenuated by application of cyclosporine A. Electrical stimulation rapidly reduced mitoflash activity in both sham and denervated muscle fibers. We further demonstrated that the Ca2+ level inside mitochondria follows the time course of the cytosolic Ca2+ transient and that inhibition of mitochondrial Ca2+ uptake by Ru360 blocks the effect of electric stimulation on mitoflash activity.ConclusionsThe loss of cytosolic Ca2+ transients due to denervation results in the downstream absence of mitochondrial Ca2+ uptake. Our studies suggest that this could be an initial trigger for enhanced mPTP-related mitochondrial ROS generation in skeletal muscle.

Endothelial Mitochondria Regulate Local Calcium Signalling in Mesenteric Arteries From Mice

Local calcium (Ca2+) signals are crucial for endothelial functions, including regulation of underlying smooth muscle cell contractility. Ca2+ pulsar is an endothelial IP3R‐dependent Ca2+ signal restricted to myoendothelial projections (MEP). Targets for this spatially‐discrete Ca2+ signal such as KCa3.1 channels, NOS3 and CaMKII have been previously described. However, determinants for Ca2+ pulsar kinetics and stochasticity remains poorly understood. Mitochondria are an important regulator of intracellular Ca2+ dynamics in several cell types such as pancreatic acinar cells. It has thus been hypothesized that mitochondria are an important modulator of Ca2+ pulsar. Mitochondrial influence on Ca2+ pulsar was explored on cut‐opened mesenteric resistance arteries using a high‐speed confocal microscope. Inhibition of mitochondrial uniporter‐dependent Ca2+ uptake (Ru360) increased the surface covered by a Ca2+ pulsar (+30%). Conversely, stimulation of mitochondrial Ca2+ uptake (kaempferol) reduced Ca2+ pulsar's spreading (−20%), suggesting that mitochondria could limit Ca2+ pulsars propagation. Interestingly, inhibition of mitochondrial Ca2+ efflux (CsA for mPTP; CGP37157 for NCLX) had little if any effect on spatial dispersion of a Ca2+ pulsar. However, de novo Ca2+ pulsar sites were observed following exposure to CsA/CGP. Mitochondria might thus control subcellular Ca2+ levels within MEP to modulate IP3R activity. Electron microscopy showed clustering of mitochondrial distribution near the MEP. These results suggest that mitochondria might play a significant role in regulating local endothelial Ca2+ signalling and then, modulate vascular tone.Support or Funding InformationFRQS, CFI, SQHA and CIHR

Mitochondria Modulates Calcium Pulsars Kinetics in Native Endothelial Cells from Mouse Mesenteric Arteries

Endothelial control of vascular tone is modulated by local calcium (Ca2+) signaling events like Ca2+ pulsars, an IP3R‐dependant Ca2+ release restricted to myoendothelial projections (MEP). Aside from the recent report of Ca2+ pulsars being modulated by CaMKII, mechanisms regulating endothelial spontaneous Ca2+ release remain unidentified. Mitochondria are important modulators of intracellular Ca2+ dynamics in various cell types and, we hypothesized mitochondria may control endothelial Ca2+ pulsars. High‐speed confocal microscopy was used to investigate mitochondria's influence on endothelial Ca2+ pulsar in cut‐open mouse mesenteric arteries (en face configuration). Preventing mitochondrial Ca2+ uptake by uncoupling with FCCP uncovered a new Ca2+ pulsar site population that had a lower frequency (≈+30%) but larger diffusional spread (+57%) than controls. Inhibition of mitochondrial Ca2+ uptake via the uniporter using Ru360 increased the spread of Ca2+ pulsars (≈+30%), though kinetics were not significantly altered. Significantly, inhibition of mitochondrial Ca2+ export (CGP37157, CsA) increased the number of active Ca2+ pulsar sites, each with a decreased bursting frequency. In summary, these results suggest that mitochondrial uptake and release of Ca2+ acts as a critical local modulator of Ca2+ pulsar kinetics and may therefore be crucial to endothelial control of vascular tone.Support or Funding InformationFRQS, HSFC, CFI, SQHA and CIHR

Abstract 12435: Mitochondrial Calcium Flux Modulates Pacemaker Activity in Embryonic Stem Cell-Derived Cardiomyocytes

Introduction: Ca2+ release from the sarcoplasmic reticulum (SR) is known to contribute to spontaneous activity via the cytoplasmic Na+-Ca2+ exchanger (NCX). Mitochondria are known to participate in Ca2+ cycling. We studied the role of mitochondrial Ca2+ flux in spontaneously activity of embryonic stem cells (ESC)-derived CMs. Methods: CMs were derived from wild type (Wt) and ryanodine receptor type 2 knockout (RYR2-/-) mouse ESCs and differentiated for 19-21 days. Action potentials (APs) were recorded by perforated whole cell current-clamp. Cytoplasmic Ca2+ transients were determined by fluorescent imaging. Mitochondrial Ca2+ transients were monitored simultaneously with APs. Results: While If blocker, 10μM ivabradine, had no significant effect on the automaticity, SR Ca2+ handling inhibitors, 10 μM ryanodine and 2 μM 2-APB, reduced the spontaneous beating rate to 56% and 73% respectively in Wt CMs. Inhibition of mitochondrial Ca2+ flux by 10 μM Ru360 showed a similar inhibitory effect on the pacemaker activity.To isolate the contribution of mitochondrial Ca2+, we used RYR2-/- CMs. In RYR2-/- CMs, beating rate was dependent on SR Ca2+ uptake and release from IP3 and the Na+/Ca2+ exchange current but was independent of sarcolemmal Ca2+ influx through L-type Ca2+ channels. In these cells, MCU inhibition by pharmacological or molecular biological means reduced beating rate. Depolarizing mitochondria prevented spontaneous beating. The mitochondrial NCX blocker, 1 or 3μM CGP-37157, terminated AP firings. Conclusions: Mitochondrial Ca2+ cycling plays a role in spontaneous beating activity in embryonic stem (ES) cell-derived cardiomyocytes. Mitochondrial dysfunction may contribute to altered pacemaker activity in cardiomyopathy.

Abstract B45: Mitochondrial Ca2+ transport fine-tunes functional cross-talk between antitumor CD8+T lymphocytes and natural killer cells

Abstract Tumor antigen-specific T cell and natural killer (NK) cell collaboration is indispensable for the elimination of tumor cells, including antigen-deficient tumor escape variants before metastasis. While mechanistic details are available for the innate instruction of T lymphocyte responses, little is known for the adaptive control of NK cell activity. We observed in a mouse model of mastocytoma expressing a self tumor antigen P1A that effector CD8+T cells provided a necessary “help” to dormant NK cells in eliciting their antitumor effector function. Bioluminescence imaging of mastocytoma tumors following adoptive transfer of P1A-specific T cells in RAG-/- and RAG-/-gamma chain-/- mice showed that NK cell anti-tumor activity requires cytolytic T cells, whereas T cells can function independent of NK cells. The goal of present study was to dissect the molecular mechanisms underlying functional cross-talk between activated CD8+T lymphocytes and naïve NK cells. In 2D and 3D co-culture systems, we observed that activated CD8+T cells form multiple intercellular contacts with naïve NK lymphocytes. Moreover, co-culture of activated T cells with naïve NK cells results in the selective down-regulation of CD25 molecule in T cells while elevating CD25 and CD69 expression on naïve NK cells. Further, CD8+T and NK cells cross-regulate their mitochondrial homeostasis including calcium transport. This effect is dependent on both cytokines and intercellular contacts, and partially involves natural killer group 2 member D (NKG2D) receptor activation. Data also indicate that activated CD8+T cells may exchange mitochondria and activation molecules such as CD25 and CD69 with naïve NK cells. Alterations in phosporylation status of multiple signaling proteins during CD8+T-NK interaction suggest a cellular remodeling whereby NK cells shift activated CD8+T cells towards T central-memory (TCM) phenotype and activated CD8+T lymphocytes alter the naïve state of NK cells towards effector/regulatory phenotype. Inhibition of mitochondrial Ca2+ uptake (mCU) or Na+/Ca2+ exchanger (mNCE) with Ru360 and CGP37157, respectively, mimicked observed alterations in CD8+ T and NK cell phenotypes upon their interaction. These results highlight a novel role of mitochondrial Ca2+ homeostasis in the re-modeling of memory differentiation and activation signaling of interacting CD8+T cells and NK cells, respectively. Improved mechanistic understanding of CD8+T-NK interplay will help refine cellular immunotherapy approach against cancer. Citation Format: Roman V. Uzhachenko, J Shawn Goodwin, Lino Costa, Alexander Terekhov, Menaka C. Thounaojam, William H. Hofmeister, Anil Shanker. Mitochondrial Ca2+ transport fine-tunes functional cross-talk between antitumor CD8+T lymphocytes and natural killer cells. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr B45.