Ca Efflux Research Articles

Rasagiline and selegiline suppress calcium efflux from mitochondria by PK11195-induced opening of mitochondrial permeability transition pore: a novel anti-apoptotic function for neuroprotection

Rasagiline and selegiline, inhibitors of type B monoamine oxidase (MAO-B), protect neurons from cell death in cellular and animal models. Suppression of mitochondrial membrane permeabilization and subsequent activation of apoptosis cascade, and induction of anti-apoptotic, pro-survival genes are proposed to contribute the anti-apoptotic function. Rasagiline suppresses neurotoxin- and oxidative stress-induced membrane permeabilization in isolated mitochondria, but the mechanism has been not fully clarified. In this paper, regulation of the mitochondrial permeability transition pore by rasagiline and selegiline was examined in apoptosis induced by PK11195, a ligand of the outer membrane translocator protein 18 kDa (TSPO) in SH-SY5Y cells. The pore opening was quantitatively measured using a simultaneous monitoring system for calcium (Ca(2+)) and superoxide (O2(-)) (Ishibashi et al. in Biochem Biophys Res Commun 344:571-580, 2006). The association of the pore opening with Ca(2+) efflux and ROS increase was proved by the inhibition of Bcl-2 overexpression and cyclosporine A treatment. Potency to release Ca(2+) was correlated with the cytotoxicity of TSPO antagonists, PK11195, FGIN-1-27 and protoporphyrin IX, whereas a TSPO agonist, 4-chloro-diazepamine, did not significantly increase Ca(2+) or cause cell death. Rasagiline and selegiline inhibited mitochondrial Ca(2+) efflux through the mitochondrial permeability transition pore dose dependently. Ca(2+) efflux was confirmed as the initial signal in mitochondrial apoptotic cascade, and the suppression of Ca(2+) efflux may account for the neuroprotective function of rasagiline and selegiline. The quantitative measurement of Ca(2+) efflux can be applied to determine anti-apoptotic activity of neuroprotective compounds. The role of mitochondrial Ca(2+) release in neuronal death and also in neuroprotection by MAO-B inhibitors is discussed.

Experimental Colitis Is Associated with Transcriptional Inhibition of Na+/Ca2+ Exchanger Isoform 1 (NCX1) Expression by Interferon γ in the Renal Distal Convoluted Tubules

NCX1 is a Na(+)/Ca(2+) exchanger, which is believed to provide a key route for basolateral Ca(2+) efflux in the renal epithelia, thus contributing to renal Ca(2+) reabsorption. Altered mineral homeostasis, including intestinal and renal Ca(2+) transport may represent a significant component of the pathophysiology of the bone mineral density loss associated with Inflammatory Bowel Diseases (IBD). The objective of our research was to investigate the effects of TNBS and DSS colitis and related inflammatory mediators on renal Ncx1 expression. Colitis was associated with decreased renal Ncx1 expression, as examined by real-time RT-PCR, Western blotting, and immunofluorescence. In mIMCD3 cells, IFNγ significantly reduced Ncx1 mRNA and protein expression. Similar effects were observed in cells transiently transfected with a reporter construct bearing the promoter region of the kidney-specific Ncx1 gene. This inhibitory effect of IFNγ is mediated by STAT1 recruitment to the proximal promoter region of Ncx1. Further in vivo study with Stat1(-/-) mice confirmed that STAT1 is indeed required for the IFNγ mediated Ncx1 gene regulation. These results strongly support the hypothesis that impaired renal Ca(2+) handling occurs in experimental colitis. Negative regulation of NCX1- mediated renal Ca(2+) absorption by IFNγ may significantly contribute to the altered Ca(2+) homeostasis in IBD patients and to IBD-associated loss of bone mineral density.

Life after the birth of the mitochondrial Na+/Ca2+ exchanger, NCLX.

Powered by the mitochondrial membrane potential, Ca(2+) permeates the mitochondria via a Ca(2+) channel termed Ca(2+) uniporter and is pumped out by a Na(+)/Ca(2+) exchanger, both of which are located on the inner mitochondrial membrane. Mitochondrial Ca(2+) transients are critical for metabolic activity and regulating global Ca(2+) responses. On the other hand, failure to control mitochondrial Ca(2+) is a hallmark of ischemic and neurodegenerative diseases. Despite their importance, identifying the uniporter and exchanger remains elusive and their inhibitors are non-specific. This review will focus on the mitochondrial exchanger, initially describing how it was molecularly identified and linked to a novel member of the Na(+)/Ca(2+) exchanger superfamily termed NCLX. Molecular control of NCLX expression provides a selective tool to determine its physiological role in a variety of cell types. In lymphocytes, NCLX is essential for refilling the endoplasmic reticulum Ca(2+) stores required for antigen-dependent signaling. Communication of NCLX with the store-operated channel in astroglia controls Ca(2+) influx and thereby neuro-transmitter release and cell proliferation. The refilling of the Ca(2+) stores in the sarcoplasmic reticulum, which is controlled by NCLX, determines the frequency of action potential and Ca(2+) transients in cardiomyocytes. NCLX is emerging as a hub for integrating glucose-dependent Na(+) and Ca(2+) signaling in pancreatic β cells, and the specific molecular control of NCLX expression resolved the controversy regarding its role in neurons and β cells. Future studies on an NCLX knockdown mouse model and identification of human NCLX mutations are expected to determine the role of mitochondrial Ca(2+) efflux in organ activity and whether NCLX inactivation is linked to ischemic and/or neurodegenerative syndromes. Structure-function analysis and protein analysis will identify the NCLX mode of regulation and its partners in the inner membrane of the mitochondria.

Involvement of palmitate/Ca2 +(Sr2 +)-induced pore in the cycling of ions across the mitochondrial membrane

The palmitate/Ca2+-induced (Pal/Ca2+) pore, which is formed due to the unique feature of long-chain saturated fatty acids to bind Ca2+ with high affinity, has been shown to play an important role in the physiology of mitochondria. The present study demonstrates that the efflux of Ca2+ from rat liver mitochondria induced by ruthenium red, an inhibitor of the energy-dependent Ca2+ influx, seems to be partly due to the opening of Pal/Ca2+ pores. Exogenous Pal stimulates the efflux. Measurements of pH showed that the Ca2+-induced alkalization of the mitochondrial matrix increased in the presence of Pal. The influx of Ca2+ (Sr2+) also induced an outflow of K+ followed by the reuptake of the ion by mitochondria. The outflow was not affected by a K+/H+ exchange blocker, and the reuptake was prevented by an ATP-dependent K+ channel inhibitor. It was also shown that the addition of Sr2+ to mitochondria under hypotonic conditions was accompanied by reversible cyclic changes in the membrane potential, the concentrations of Sr2+ and K+ and the respiratory rate. The cyclic changes were effectively suppressed by the inhibitors of Ca2+-dependent phospholipase A2, and a new Sr2+ cycle could only be initiated after the previous cycle was finished, indicating a refractory period in the mitochondrial sensitivity to Sr2+. All of the Ca2+- and Sr2+-induced effects were observed in the presence of cyclosporin A. This paper discusses a possible role of Pal/Ca2+ pores in the maintenance of cell ion homeostasis.

NCLX Protein, but Not LETM1, Mediates Mitochondrial Ca2+ Extrusion, Thereby Limiting Ca2+-induced NAD(P)H Production and Modulating Matrix Redox State

Mitochondria capture and subsequently release Ca(2+) ions, thereby sensing and shaping cellular Ca(2+) signals. The Ca(2+) uniporter MCU mediates Ca(2+) uptake, whereas NCLX (mitochondrial Na/Ca exchanger) and LETM1 (leucine zipper-EF-hand-containing transmembrane protein 1) were proposed to exchange Ca(2+) against Na(+) or H(+), respectively. Here we study the role of these ion exchangers in mitochondrial Ca(2+) extrusion and in Ca(2+)-metabolic coupling. Both NCLX and LETM1 proteins were expressed in HeLa cells mitochondria. The rate of mitochondrial Ca(2+) efflux, measured with a genetically encoded indicator during agonist stimulations, increased with the amplitude of mitochondrial Ca(2+) ([Ca(2+)]mt) elevations. NCLX overexpression enhanced the rates of Ca(2+) efflux, whereas increasing LETM1 levels had no impact on Ca(2+) extrusion. The fluorescence of the redox-sensitive probe roGFP increased during [Ca(2+)]mt elevations, indicating a net reduction of the matrix. This redox response was abolished by NCLX overexpression and restored by the Na(+)/Ca(2+) exchanger inhibitor CGP37157. The [Ca(2+)]mt elevations were associated with increases in the autofluorescence of NAD(P)H, whose amplitude was strongly reduced by NCLX overexpression, an effect reverted by Na(+)/Ca(2+) exchange inhibition. We conclude that NCLX, but not LETM1, mediates Ca(2+) extrusion from mitochondria. By controlling the duration of matrix Ca(2+) elevations, NCLX contributes to the regulation of NAD(P)H production and to the conversion of Ca(2+) signals into redox changes.

A role for transcription factor glial cell missing 2 in Ca2+ homeostasis in zebrafish, Danio rerio.

The present study investigated the role of the transcription factor, glial cell missing 2 (gcm2), in Ca(2+) regulation in zebrafish larvae. Translational gene knockdown of gcm2 decreased Ca(2+) uptake and the density of ionocytes expressing the epithelial Ca(2+) channel (ecac), and disrupted the overall Ca(2+) balance. Ca(2+) uptake and the expression of gcm2 messenger RNA (mRNA) were significantly elevated in larvae acclimated to low Ca(2+) water (25 μM); the stimulation of Ca(2+) uptake was not observed in fish experiencing gcm2 knockdown. Acclimation to acidic water (pH 4) significantly reduced whole-body Ca(2+) content owing to reduced Ca(2+) uptake and increased Ca(2+) efflux. However, ecac mRNA levels and the density of ecac-expressing ionocytes were increased in fish acclimated to acidic water, and maximal Ca(2+) uptake capacity (J MAX) was significantly increased when measured in control water (pH ~7.4). Acclimation of larvae to acidic water significantly increased gcm2 mRNA expression, and in gcm2 morphants, no such stimulation in Ca(2+) uptake was observed after their return to control water. Overexpression of gcm2 mRNA resulted in a significant increase in the numbers of ecac-expressing ionocytes and Ca(2+) uptake. These observations reveal a critical role for gcm2 in Ca(2+) homeostasis in zebrafish larvae.

Regulation of calcium ion and its effect on growth and developmental behavior in wild type and ntcA mutant of Anabaena sp. PCC 7120 under varied levels of CaCl2

A study of calcium ion regulation in Anabaena 7120 and its derivative mutant (CSE2) strain impaired in ntcA gene were investigated in terms of altered morphological and physiological responses against various levels of calcium stress (0–100 mM). Calcium concentration of 10 mM was found to be inhibitory while 100 mM proved lethal for both wild type and mutant strain. The involvement of Ca2+ in the regulation of cellular processes has been described in terms of an influx or efflux of Ca2+ from the cytosol. A biphasic calcium uptake with difference in calcium influx and efflux rate was responsible for differential amount of remaining calcium which followed a decreasing trend both for wild type and mutant. Low K s 0.5 and high V max in mutant suggest heavy and less restricted influx of calcium ion. Further, the interactive effect of calcium influx/efflux rate, remaining Ca2+ and intracellular levels of Na+ and K+ may be attributed for the degree of membrane damage and growth sustenance during exogenous supply of calcium salt. Widening in heterocyst spacing pattern, decreased heterocyst frequency and formation of abnormal cell structures at higher concentration (100 mM CaCl2) suggest that calcium mediated regulatory process modulate heterocyst frequency and maintenance of cell structure. Further, poor regulation of calcium ion homeostasis in ntcA suggests that the calcium level and ntcA gene expression are inter-related.

Polyamines cause plasma membrane depolarization, activate Ca2+-, and modulate H+-ATPase pump activity in pea roots.

Polyamines regulate a variety of cation and K(+) channels, but their potential effects on cation-transporting ATPases are underexplored. In this work, noninvasive microelectrode ion flux estimation and conventional microelectrode techniques were applied to study the effects of polyamines on Ca(2+) and H(+) transport and membrane potential in pea roots. Externally applied spermine or putrescine (1mM) equally activated eosin yellow (EY)-sensitive Ca(2+) pumping across the root epidermis and caused net H(+) influx or efflux. Proton influx induced by spermine was suppressed by EY, supporting the mechanism in which Ca(2+) pump imports 2 H(+) per each exported Ca(2+). Suppression of the Ca(2+) pump by EY diminished putrescine-induced net H(+) efflux instead of increasing it. Thus, activities of Ca(2+) and H(+) pumps were coupled, likely due to the H(+)-pump inhibition by intracellular Ca(2+). Additionally, spermine but not putrescine caused a direct inhibition of H(+) pumping in isolated plasma membrane vesicles. Spermine, spermidine, and putrescine (1mM) induced membrane depolarization by 70, 50, and 35 mV, respectively. Spermine-induced depolarization was abolished by cation transport blocker Gd(3+), was insensitive to anion channels' blocker niflumate, and was dependent on external Ca(2+). Further analysis showed that uptake of polyamines but not polyamine-induced cationic (K(+)+Ca(2+)+H(+)) fluxes were a main cause of membrane depolarization. Polyamine increase is a common component of plant stress responses. Activation of Ca(2+) efflux by polyamines and contrasting effects of polyamines on net H(+) fluxes and membrane potential can contribute to Ca(2+) signalling and modulate a variety of transport processes across the plasma membrane under stress.

Reduced IRE1α mediates apoptotic cell death by disrupting calcium homeostasis via the InsP3 receptor.

The endoplasmic reticulum (ER) is not only a home for folding and posttranslational modifications of secretory proteins but also a reservoir for intracellular Ca2+. Perturbation of ER homeostasis contributes to the pathogenesis of various neurodegenerative diseases, such as Alzheimer's and Parkinson diseases. One key regulator that underlies cell survival and Ca2+ homeostasis during ER stress responses is inositol-requiring enzyme 1α (IRE1α). Despite extensive studies on this ER membrane-associated protein, little is known about the molecular mechanisms by which excessive ER stress triggers cell death and Ca2+ dysregulation via the IRE1α-dependent signaling pathway. In this study, we show that inactivation of IRE1α by RNA interference increases cytosolic Ca2+ concentration in SH-SY5Y cells, leading to cell death. This dysregulation is caused by an accelerated ER-to-cytosolic efflux of Ca2+ through the InsP3 receptor (InsP3R). The Ca2+ efflux in IRE1α-deficient cells correlates with dissociation of the Ca2+-binding InsP3R inhibitor CIB1 and increased complex formation of CIB1 with the pro-apoptotic kinase ASK1, which otherwise remains inactivated in the IRE1α–TRAF2–ASK1 complex. The increased cytosolic concentration of Ca2+ induces mitochondrial production of reactive oxygen species (ROS), in particular superoxide, resulting in severe mitochondrial abnormalities, such as fragmentation and depolarization of membrane potential. These Ca2+ dysregulation-induced mitochondrial abnormalities and cell death in IRE1α-deficient cells can be blocked by depleting ROS or inhibiting Ca2+ influx into the mitochondria. These results demonstrate the importance of IRE1α in Ca2+ homeostasis and cell survival during ER stress and reveal a previously unknown Ca2+-mediated cell death signaling between the IRE1α–InsP3R pathway in the ER and the redox-dependent apoptotic pathway in the mitochondrion.

CGP37157, an inhibitor of the mitochondrial Na+/Ca2+ exchanger, protects neurons from excitotoxicity by blocking voltage-gated Ca2+ channels.

Inhibition of the mitochondrial Na+/Ca2+ exchanger (NCLX) by CGP37157 is protective in models of neuronal injury that involve disruption of intracellular Ca2+ homeostasis. However, the Ca2+ signaling pathways and stores underlying neuroprotection by that inhibitor are not well defined. In the present study, we analyzed how intracellular Ca2+ levels are modulated by CGP37157 (10 μM) during NMDA insults in primary cultures of rat cortical neurons. We initially assessed the presence of NCLX in mitochondria of cultured neurons by immunolabeling, and subsequently, we analyzed the effects of CGP37157 on neuronal Ca2+ homeostasis using cameleon-based mitochondrial Ca2+ and cytosolic Ca2+ ([Ca2+]i) live imaging. We observed that NCLX-driven mitochondrial Ca2+ exchange occurs in cortical neurons under basal conditions as CGP37157 induced a decrease in [Ca2]i concomitant with a Ca2+ accumulation inside the mitochondria. In turn, CGP37157 also inhibited mitochondrial Ca2+ efflux after the stimulation of acetylcholine receptors. In contrast, CGP37157 strongly prevented depolarization-induced [Ca2+]i increase by blocking voltage-gated Ca2+ channels (VGCCs), whereas it did not induce depletion of ER Ca2+ stores. Moreover, mitochondrial Ca2+ overload was reduced as a consequence of diminished Ca2+ entry through VGCCs. The decrease in cytosolic and mitochondrial Ca2+ overload by CGP37157 resulted in a reduction of excitotoxic mitochondrial damage, characterized here by a reduction in mitochondrial membrane depolarization, oxidative stress and calpain activation. In summary, our results provide evidence that during excitotoxicity CGP37157 modulates cytosolic and mitochondrial Ca2+ dynamics that leads to attenuation of NMDA-induced mitochondrial dysfunction and neuronal cell death by blocking VGCCs.

Calcium Efflux Activity of Plasma Membrane Ca2+ ATPase-4 (PMCA4) Mediates Cell Cycle Progression in Vascular Smooth Muscle Cells

We explored the role played by plasma membrane calcium ATPase-4 (PMCA4) and its alternative splice variants in the cell cycle of vascular smooth muscle cells (VSMC). A novel variant (PMCA4e) was discovered. Quantitative real-time-PCR-quantified PMCA4 splice variant proportions differed in specific organs. The PMCA4a:4b ratio in uninjured carotid arteries (∼1:1) was significantly reduced by wire denudation injury (to ∼1:3) by modulation of alternative splicing, as confirmed by novel antibodies against PMCA4a/e and PMCA4b. Laser capture microdissection localized this shift to the media and adventitia. Primary carotid VSMC from PMCA4 knock-out (P4KO) mice showed impaired [(3)H]thymidine incorporation and G1 phase arrest as compared with wild type (P4WT). Electroporation of expression constructs encoding PMCA4a, PMCA4b, and a PMCA4b mutant lacking PDZ binding rescued this phenotype of P4KO cells, whereas a mutant with only 10% of normal Ca(2+) efflux activity could not. Microarray of early G1-synchronized VSMC showed 39-fold higher Rgs16 (NFAT (nuclear factor of activated T-cells) target; MAPK inhibitor) and 69-fold higher Decorin (G1 arrest marker) expression in P4KO versus P4WT. Validation by Western blot also revealed decreased levels of Cyclin D1 and NFATc3 in P4KO. Microarrays of P4KO VSMC rescued by PMCA4a or PMCA4b expression showed reversal of perturbed Rgs16, Decorin, and NFATc3 expression levels. However, PMCA4a rescue caused a 44-fold reduction in AP-2β, a known anti-proliferative transcription factor, whereas PMCA4b rescue resulted in a 50-fold reduction in p15 (Cyclin D1/Cdk4 inhibitor). We conclude that Ca(2+) efflux activity of PMCA4 underlies G1 progression in VSMC and that PMCA4a and PMCA4b differentially regulate specific downstream mediators.

Measurement of Intracellular Ca2+ Release in Permeabilized Cells Using 45Ca2+

This protocol describes a technique to measure Ca(2+) release from the nonmitochondrial intracellular Ca(2+) stores in monolayers of saponin-permeabilized cells cultured in 12-well 4-cm(2) clusters. The (45)Ca(2+)-flux technique described here can only be applied to cell types that still adhere to the plastic after exposing them to saponin. We describe the permeabilization procedure, the loading of the nonmitochondrial Ca(2+) stores with (45)Ca(2+), and the subsequent (45)Ca(2+) efflux.

The plasma membrane-localised Ca2+-ATPase ACA8 plays a role in sucrose signalling involved in early seedling development in Arabidopsis

Arabidopsis Ca (2+) -ATPase ACA8 plays a role in sucrose signalling during early seedling development by integrating developmental signals with carbon source availability. Calcium (Ca(2+)) is an essential signal transduction element in eukaryotic organisms. Changes in the levels of intracellular Ca(2+) affect multiple developmental processes in plants, including cell division, polar growth, and organogenesis. Here, we report that the plasma-membrane-localised Arabidopsis Ca(2+)-ATPase ACA8 plays a role in sucrose signalling during early seedling development. Disruption of the ACA8 gene elevated the expression of genes that encode transporters for Ca(2+) efflux. The seedlings that carried a T-DNA insertion mutation in ACA8 experienced water stress during early development. This response was unrelated to inadequate osmoregulatory responses and was most likely caused by disruption of cell membrane integrity and severe ion leakage. In addition, aca8-1 seedlings displayed a significant decline in photosynthetic performance and arrested root growth after removal of sucrose from the growth medium. The two phenomena resulted from impaired photosynthesis, reduced cell proliferation in the root meristem and the sucrose control of cell-cycle events. All of the stress-response phenotypes were rescued when expression of ACA8 was restored in aca8-1 mutant. Taken together, our results indicate that ACA8-mediated Ca(2+) signalling contributes to modulate early seedling development and coordinates root development with nutrient availability.

Measurement of Intracellular Ca2+ Release in Intact Cells Using 45Ca2+

This protocol describes a technique using (45)Ca(2+) to measure the release of Ca(2+) from the intracellular stores in monolayers of intact cells cultured in 12-well 4-cm(2) clusters. The (45)Ca(2+)-flux technique described here can only be applied to cell types that adhere to plastic. We describe the loading of the stores with (45)Ca(2+), and the subsequent (45)Ca(2+) efflux.

Clusianone, a naturally occurring nemorosone regioisomer, uncouples rat liver mitochondria and induces HepG2 cell death

Clusianone is a member of the polycyclic polyprenylated acylphloroglucinol family of natural products; its cytotoxic mechanism is unknown. Clusianone is a structural isomer of nemorosone, which is a mitochondrial uncoupler and a well-known cytotoxic anti-cancer agent; thus, we addressed clusianone action at the mitochondria and its potential cytotoxic effects on cancer cells. In the HepG2 hepatocarcinoma cell line, clusianone induced mitochondrial membrane potential dissipation, ATP depletion and phosphatidyl serine externalization; this later event is indicative of apoptosis induction. In isolated mitochondria from rat liver, clusianone promoted protonophoric mitochondrial uncoupling. This was evidenced by the dissipation of mitochondrial membrane potential, an increase in resting respiration, an inhibition of Ca2+ influx, stimulation of Ca2+ efflux in Ca2+-loaded mitochondria, a decrease in ATP and NAD(P)H levels, generation of ROS, and swelling of valinomycin-treated organelles in hyposmotic potassium acetate media. The cytotoxic and uncoupling actions of clusianone were appreciably less than those of nemorosone, likely due to the presence of an intra-molecular hydrogen bond with the juxtaposed carbonyl group at the C15 position. Therefore, clusianone is capable of pharmacologically increasing the leakage of protons from the mitochondria and with favorable cytotoxicity in relation to nemorosone.

Lipid Modulation of Calcium Flux through CaV2.3 Regulates Acrosome Exocytosis and Fertilization

Membrane lipid regulation of cell function is poorly understood. In early development, sterol efflux and the ganglioside GM1 regulate sperm acrosome exocytosis (AE) and fertilization competence through unknown mechanisms. Here, we show that sterol efflux and focal enrichment of GM1 trigger Ca(2+) influx necessary for AE through CaV2.3, whose activity has been highly controversial in sperm. Sperm lacking CaV2.3's pore-forming α1E subunit showed altered Ca(2+) responses, reduced AE, and a strong subfertility phenotype. Surprisingly, AE depended on spatiotemporal information encoded by flux through CaV2.3, not merely the presence/amplitude of Ca(2+) waves. Using studies in both sperm and voltage clamp of Xenopus oocytes, we define a molecular mechanism for GM1/CaV2.3 regulatory interaction, requiring GM1's lipid and sugar components and CaV2.3's α1E and α2δ subunits. Our results provide a mechanistic understanding of membrane lipid regulation of Ca(2+) flux and therefore Ca(2+)-dependent cellular and developmental processes such as exocytosis and fertilization.

Association of the RYR3 gene polymorphisms with atherosclerosis in elderly Japanese population

BackgroundThe Ryanodine receptor 3 gene (RYR3) encodes an intracellular calcium channel that mediates the efflux of Ca2+ from intracellular stores. Two single-nucleotide polymorphisms (SNPs) in the RYR3 gene have been shown to associate with stroke (rs877087) and carotid intima-media thickness (rs2229116) in two independent genome-wide association studies (GWAS) in Caucasian. We investigated the effect of these two SNPs as well as the 31.1 kilobases spanning region on atherosclerosis in Japanese population.MethodsAtherosclerotic severity was assessed by carotid artery (n = 1374) and pathological atherosclerosis index (PAI) (n = 1262), which is a macroscopic examination of the luminal surfaces of 8 systemic arteries in consecutive autopsy samples. 4 tag SNPs in the 31.1 Kb region, rs877087, rs2132207, rs658750 and rs2229116, were genotyped and haplotypes were inferred to study the association with atherosclerotic indices.Resultsrs877087 and rs2229116 were associated with PAI (OR = 2.07 [1.04-4.12] (95% CI), p = 0.038; and OR = 1.38 [1.02-1.86], p = 0.035, respectively). rs2229116 was also associated with common carotid atherosclerosis (OR = 1.45 [1.13-1.86], p = 0.003). The risk allele of rs2229116 was opposite from the original report. The haplotype block of this 31.1 Kb region was different between Caucasian and Japanese. Haplotype analysis revealed that only TAGG haplotype was associated with PAI (OR = 0.67 [0.48-0.94], p = 0.020) and atherosclerosis of common carotid artery (OR = 0.75 [0.58-0.98], p = 0.034).Conclusionrs877087 and rs2229116 of RYR3 gene are associated with atherosclerosis severity in Japanese. The functional difference caused by rs2229116 needs to be investigated.

Redox controls UPR to control redox.

In many physiological contexts, intracellular reduction-oxidation (redox) conditions and the unfolded protein response (UPR) are important for the control of cell life and death decisions. UPR is triggered by the disruption of endoplasmic reticulum (ER) homeostasis, also known as ER stress. Depending on the duration and severity of the disruption, this leads to cell adaptation or demise. In this Commentary, we review reductive and oxidative activation mechanisms of the UPR, which include direct interactions of dedicated protein disulfide isomerases with ER stress sensors, protein S-nitrosylation and ER Ca(2+) efflux that is promoted by reactive oxygen species. Furthermore, we discuss how cellular oxidant and antioxidant capacities are extensively remodeled downstream of UPR signals. Aside from activation of NADPH oxidases, mitogen-activated protein kinases and transcriptional antioxidant responses, such remodeling prominently relies on ER-mitochondrial crosstalk. Specific redox cues therefore operate both as triggers and effectors of ER stress, thus enabling amplification loops. We propose that redox-based amplification loops critically contribute to the switch from adaptive to fatal UPR.

Transient Mitochondrial Permeability Transition Pore Opening in Cardiac Myocytes During SR Ca Release

The opening of a high-conductance and long-lasting mitochondrial permeability transition pore (mPTP) induces uncoupling of respiration from ADP phosphorylation, and causes mitochondrial injury and cell death. However, low-conductance and transient openings of mPTP may limit mitochondrial calcium load and mediate mitochondrial reactive oxygen species (ROS) signaling. Transient openings of mPTP have been proposed, and could be cardioprotective, but evidence for this in cells is indirect and not thoroughly studied. To address the cellular mechanism, we measured mitochondrial [Ca] ([Ca]Mito) with Rhod-2 AM and membrane potential (Δψm) in isolated single permeablized myocytes during cyclical sarcoplasmic reticulum (SR) Ca release using 2-D confocal imaging where individual mitochondria can be seen. Rapid and transient decreases in both mitochondrial [Ca] and Δψm were observed during SR Ca release. The frequency of these candidate transient mPTP openings increased at higher [Ca] and with H2O2 (1μM) exposure, but were typically observed in << 1% of individual mitochondria being imaged. This suggests that both Ca and ROS modulate transient pore openings. These [Ca]mito and Δψm oscillations and H2O2 effects were sensitive to mPTP inhibitor cyclosporine A (CsA, 10μM) and we conclude that they are mediated by transient mPTP openings and closings. The duration of these openings was 47 ± 15 s. The size of the pore did not allow Rhod-2 or calcien (M.W. 600 Da) permeation, indicating that only small solutes can freely move across. Our data are consistent with the idea that rare transient mPTP openings allow individual mitochondrial Ca efflux, but with minimal perturbation of global average [Ca]mito or Δψm. These events may be an important physiological protective mechanism to limit [Ca]Mito overload.

Burst Pacemaker Activity in NCX1 Knockout Mice: Is it Funny Current?

The cardiac Sodium-Calcium exchanger (NCX1) is the dominant Ca efflux protein in SinoAtrial Node (SAN) cells. NCX1 has also been implicated in the generation of pacemaker activity, through the “Ca clock”. To study SAN pacing in the absence of NCX1, we created an atrial-specific NCX1 KO mouse using a Cre/loxP system under the control of the endogenous sarcolipin promoter. NCX1 KO mice have complete AV block and no P waves. The latter is caused by conduction interference between SAN and atria. To test the hypothesis that SAN pacemaking can be maintained by funny current (If) in the absence of NCX1, we recorded Ca transients (using Cal 520 and high speed 2D confocal imaging) in an ex vivo tissuepreparation that included the SAN, right atrium and left atrium. At ∼36°C, the SAN pacemaker rate was 5.3±0.3Hz in WT (n=14) but only 2±0.2Hz (n=17) in KO. The pattern of pacing in KO was characterized by frequent bursts of Ca transients alternating with long pauses. During bursts in KO, the rate was comparable to WT (4.1±0.3Hz). WT SANs (n=5) showed a 70.5±6.1% increase in rate after exposure to the β-adrenergic agonist ISO (10μM), while KO showed no increase in average rate. However, when considering only rate during burst activity in KO, 6 out of 10 KO SANs responded significantly to ISO 10μM (52±16% increase). The specific If inhibitor ivabradine (IVA, 9μM) reduced the spontaneous pacemaker rate in both WT (n=6) and KO (n=8) SANs (44±9% and 58.9±6.8% decrease, respectively), and even during the bursts in the KO (36±17.9% decrease). Thus, NCX1 KO SANs can generate bursts of pacemaker activity similar to WT. These bursts are responsive to both ISO and IVA, consistent with If-mediated pacemaker activity despite the absence of NCX.