The present study was designed to investigate the role of intracellular Ca2+ on cAMP mediated-decidualization and gland maturation events, which can be identified by the up-regulation of prolactin and IGF-binding protein (IGFBP)1 in human endometrial stromal cells (ESCs), and cyclooxygenase 2 (COX2) and prostaglandin E2 (PGE2) and glandular epithelial EM-1 cells. Increases in decidual prolactin and IGFBP-1 transcript levels, induced by cAMP-elevating agents forskolin or dibutyryl cyclic AMP, were inhibited by Ca2+ influx into ESCs with Ca2+ ionophores  in a dose-dependent manner. Conversely, inhibitors of Ca2+ influx through L-type voltage-dependent Ca2+ channel (VDCC), nifedipine and verapamil, enhanced the decidual gene expression. Furthermore, dantrolene, an inhibitor of Ca2+ release from the intracellular Ca2+ store, up-regulated prolactin and IGFBP-1 expression. Ca2+ ionophores decreased intracellular cAMP concentrations, whereas nifedipine, verapamil or dantrolene increased cAMP concentrations in ESCs. In glandular epithelial cells, similar responses in COX2 expression and PGE2 production were found when intracellular cAMP levels were up-regulated by decreases in Ca2+ concentrations. Thus, a marked decrease in cytosolic Ca2+ levels caused the elevation of cAMP concentrations, resulting in enhanced expression of implantation-related factors including decidual markers. These findings suggest that fluctuation in cytosolic Ca2+ concentrations alters intracellular cAMP levels, which then regulate differentiation of endometrial stromal and glandular epithelial cells.Decidualization of human endometrial stroma and gland development is mediated through cyclic AMP (cAMP), but the role of intracellular calcium ion (Ca Receptive endometrium for implantation is constituted with the luminal epithelium, decidual cells, and glandular epithelial cells which secrete substances that support blastocyst development. Uterine endometrial stromal cells (ESCs) differentiate into decidual cells, called as decidualization during the secretory phase of the menstrual cycle. Decidualization of ESCs occurs spontaneously during the menstrual cycles. This differentiation is indispensable for successful embryo implantation and subsequent placenta formation . One of 2+) have been shown to play an essential role as a second messenger in various physiological and pharmacological systems. Calcium-mobilizing mechanism exists in the cells, including Ca2+ influx from the extracellular region and Ca2+ release into cytoplasm from internal stores such as endoplasmic reticulum (ER) [2+ homeostasis in endometrial differentiation and implantation have been reported in human ESCs [2+ channel (non-VDCC) superfamily, induces IGFBP1 expression via Ca2+ influx [2+-binding protein, is involved in the process of embryo implantation [+ channel triggers Ca2+ influx, and leads to the up-regulation of COX2 expression and PGE2 release via the activation of PKA in mouse uterine epithelial cells [2+ signal could be closely associated with the preparation of endometrium for embryo implantation. Despite the importance of Ca2+ and cAMP on endometrial differentiation, the relationship between Ca2+ and cAMP in the endometrium has not been studied. This study investigated whether Ca2+ plays a role on endometrial differentiation mediated by cAMP signaling in human stromal and glandular epithelial cells.Similar to the cAMP signaling, intracellular calcium ions  . Vital rman ESCs , 20. The+ influx . In uteral cells . These f6, 2′-O-dibutyryladenosine 3′, 5′-cyclic monophosphate (db-cAMP) and various Ca2+ modulators nifedipine, verapamil, dantrolene, alamethicin, and ionomycin were purchased from Sigma-Aldrich . Forskolin, an activator of AC, was obtained from Applichem . O,O'-Bis (2-aminophenyl)ethyleneglycol-N,N,N',N'-tetraacetic acid, tetraacetoxymethyl ester; BAPTA)was commercially provided by Dojindo A cAMP analog N2+ on the production of cAMP signaling-induced factor in human endometrium, ESCs and immortalized human endometrial glandular epithelial cell line  [4 cells/cm2 were grown at 37°C in Dulbecco’s modified Eagle medium and Ham’s F-12 supplemented with 10% (w/v) charcoal-stripped fetal bovine serum, 50 μg/ml penicillin, 50 μg/ml streptomycin, 100 μg/ml neomycin, and 0.5 μg/ml amphotericin B [IGFBP1 and prolactin expression peaked within 5 days (120 h) with progression of decidualization in ESC [COX2 expression and PGE2 accumulation into the media increased at 6 h after PKA activator and reached to the maximum levels between 48 and 72 h in a preliminary experiment. ESCs or EM1 cells were, therefore, treated with the above Ca2+ modulators for 1 h and then stimulated with forskolin or db-cAMP for 48 h.Samples of eutopic endometrial tissue in the proliferative phase (n = 6) were obtained from women undergoing endometriosis surgery. The patient signed an informed consent and accepted to participate to this research project, which was approved by the clinical research ethics committee of the Tokyo Medical University Hospital and the Tokyo University of Pharmacy and Life Sciences. Primary ESCs were obtained as described previously . To elucls; EM1)  were subericin B . In preln in ESC , 24. EpiProlactin; 5’-AAAGGATCGCCATGGAAAG-3’ (S) and 5’-GGTCTCGAAGGGTCACCTG-3’ (AS), IGFBP1; 5’-AATGGATTTTATCACAGCAGACAG-3’ (S) and 5’-GGTAGACGCACCAGCAGAGT-3’ (AS), COX2; 5’-CTTCACGCATCAGTTTTTCAAG-3’ (S) and 5’-TCACCGTAAATATGATTTAAGTCCAC-3’ (AS), and GAPDH; 5’-AGCCACATCGCTCAGACA-3’ (S) and 5’-GCCCAATACGACCAAATCC-3’ (AS). The PCR consisted 40 cycles at 95 C for 10 s, annealing and extension at 60 C at 30s. iQ5 Optical system software was used to collect the data and calculate the threshold cycle (Ct). The expression of each mRNA was normalized with GAPDH and analyzed by the comparative Ct method [Total RNA was extracted from endometrial cells using Isogen reagent  and quantified by A260/A280 measurement using a NanoVue . Real time PCR was performed with the iQ5 Real time PCR Detection System (Bio-Rad) in triplicate in 20 μl volumes containing 100 ng RNA, 10 μl 2 X SYBR Green Master Mix  and 50 nM of primers. Human glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA was used as an internal standard for RNA loading. The following specific sense (S) and antisense (AS) primers were used: t method .EM1 cells were treated with alamethicin, ionomycin, nifedipine, verapamil, or dantrolene for 1 h and then with forskolin or db-cAMP for 48 h. The culture medium (500 μl) was centrifuged at 10,000 x g at 4°C for 10 min, and the concentration of PGE2 in the supernatant was determined using a ELISA kit . One hundred μl of the supernatant was diluted 2-fold with EIA buffer for each sample measurement. Three independent sets of experiments were performed in triplicate. For IGFBP measurement at protein levels, the culture medium was centrifuged as above and IGFBP-1 in the supernatant was determined using a commercially available sandwich ELISA kit , as described before . The con2+ modulators for 48 h, were determined using a competitive EIA kit  according to the manufacturer’s recommendations. Briefly, cells were lysed for 10 min in 80 μl of 0.1 M HCl and were centrifuged at 1,000 x g at 4°C. Sixty μl of supernatant was used for the measurement. The values for inter- and intra-assay coefficients for EIA were below 10%.Total cAMP levels in ESCs and EM1 cells, treated with forskolin in the presence of various Ca3 cells/cm2 in 96 well dish were treated with vehicle or various inhibitors for 48 h in the presence or absence of forskolin (15 μM). After treatment, cell viability was assessed using the WST-8 (the tetrazolium reagent 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5--2H-tetrazolium; Cell counting kit, Dojindo, Kumamoto, Tokyo). Culture medium was removed and 100 μl of WST-8 (1:10 dilution) in phosphate buffered saline were added to each well following each inhibitor treatment. After incubation in CO2 incubator for 1 h, 50 μl from each well were then transferred to a 96-well microplate and read at 450 nm.Cells plated at a density of 6 x 10P-value < 0.05 was considered statistically significant.Data are expressed as the mean ± SEM. Significance was assessed using the Tukey-Kramer multiple comparisons test. A 2+ influx into the cytoplasm affected decidual marker expression, ESCs were pretreated with alamethicin or ionomycin that transports Ca2+ across the lipid bilayer of cell membrane, and were then stimulated with a general activator of AC, forskolin or db-cAMP. Forskolin enhanced prolactin (161 ± 18 fold) and IGFBP1 (1927 ± 443 fold) mRNA levels, compared with control levels. Pretreatment with alamethicin reduced forskolin-induced prolactin , but not IGFBP1 expression induced by forskolin (0.92 ± 0.001) (prolactin expression (0.02 ± 0.005) (IGFBP1 was observed in the ionomycin-treated group (To investigate whether Carolactin  or IGFBP (IGFBP1  expressictively. . Alamethpression . Similar± 0.001) . Pretrea± 0.005) . No effeed group .2+ influx into the cytoplasm from extracellular region, the effect of VDCC blockers of dihydropyridines (DHP) type such as nifedipine or non-DHP of verapamil on the AC/cAMP pathway-mediated decidual marker expression was examined. Nifedipine or verapamil pretreatment promoted forskolin-induced expression of prolactin in ESCs  and IGFBP1 (2.13 ± 0.42) induced by forskolin (prolactin (1.85 ± 0.50) and IGFBP1 (1.95 ± 0.49) expression in the presence of db-cAMP  as well as the ionophore, alamethicin (1 μM) and ionomycin (3 μM) did not affect cell viability, although ionomycin at 10 μM was found to be cytotoxic to ESCs  that inhibits the release of Carolactin  and IGFB . The forskolin-stimulated cAMP levels were significantly enhanced by nifedipine (511.9 ± 55.1 pmol/μg), verapamil (516.6 ± 34.4 pmol/μg), or dantrolene (416.6 ± 20.2 pmol/μg), compared with those in the forskolin-treated group.We hypothesized that increases in cytosolic Ca in ESCs . AlamethCOX2 and then PGE2 production in endometrial glandular epithelial cells [2+ concentration affected cAMP signaling-induced COX2 expression and PGE2 secretion in EM1 cells, the effect of Ca2+ modulators on COX2 expression and PGE2 secretion was examined. Forskolin stimulated COX2 expression  (vs. forskolin)  and PGE2 secretion . Furthermore, as shown in We and others have shown that the activation of cAMP signaling stimulates the expression of al cells , 17. To ctively)  and PGE2rskolin) . However2+ in endometrial differentiation, we examined whether Ca2+ chelator, BAPTA influences the efficiency of alamethicin, nifedipine, or dantrolene on forskolin-induced prolactin and COX2 expression in ESCs and EM1 cell. BAPTA counteracted the down-regulation of forskolin-induced prolactin and COX2 by alamethicin (prolactin (1.49 ± 0.17) and COX2 (1.71 ± 0.11) expression, whereas the expression of prolactin and COX2 induced by nifedipine or dantrolene was suppressed.To explore the role of intracellular Camethicin . Interes2+ levels in the cytoplasm, but up-regulated by decreases in Ca2+ levels. There are two calcium-mobilizing mechanisms that control Ca2+ concentrations in the cells, namely, Ca2+ influx from the extracellular regions and Ca2+ release from internal stores such as ER. Two Ca2+ ionophores, alamethicin and ionomycin, which force external Ca2+ influx into the cell, reduced the expression of decidual markers prolactin and IGFBP1, whereas nifedipine, verapamil or dantrolene, which decrease intracellular Ca2+ levels, stimulated decidual marker expression. Ca2+ chelater up-regulated Ca2+ ionophore-induced inhibition of decidual marker. In addition, continuous increases in Ca2+ influx decreased the cAMP concentrations in endometrial cells. These results indicated that the elevation in intracellular Ca2+ concentrations could have repressed prolactin and IGFBP1 expression through the suppression of cAMP signaling in ESCs. Similarly, expression of implantation-related factor COX2 and the production of PGE2 were inhibited by Ca2+ influx stimulators, but promoted by VDCC and RyR inhibitors in glandular epithelial EM-1 cells. In addition, Ca2+ chelater abolished Ca2+ ionophore-triggered inhibition of prolactin and COX2 expression, and also enhanced forskolin-stimulated prolactin and COX2 expression, suggesting that elevation of Ca2+ in the cytoplasm may affect negatively forskolin-stimulated prolactin and COX2 expression. 2+ channel blocker-triggered reduction in intracellular Ca2+ levels potentiated the cAMP signaling, and suggest that changes in intracellular Ca2+ concentrations play a role on cAMP signaling and its down-stream gene expression in decidualization and glandular maturation in the endometrium.In the present study, we demonstrated that forskolin or db-cAMP-stimulated decidual gene expression in human endometrial stromal cells were down-regulated by increases in Ca2+ ionophore A23187 attenuates prolactin release in decidual cells prepared from patients in early pregnancy [2+ channel blockers inhibit prolactin secretion in human decidual cells [et al. [2+ influx increases the phosphorylation level of CREB and facilitates COX2-dependent up-regulation of PGE2. However, there were no significant changes in the levels of phosphorylated CREB protein among Ca2+ modulator-treated cells in this study. This probably indicates that up-regulation of decidual marker and COX-2 induced by continuous reduction in intracellular Ca2+ levels is not dependent on CREB activation (data not shown). Although the biochemical mechanisms associated with these differences are not known, it is possible that a progressive increase in Ca2+ levels through the use of pharmacological agents may possibly accelerate the degradation of cAMP, or that Ca2+ reduction may have led to the elevation of cAMP in undifferentiated or immature endometrial cells in this study. It has been shown that angiotensin II results in the activation of calcineurin/nuclear factor of activated T-cells (NFAT) and the elevation of COX2 expression via the elevation of intracellular Ca2+ in ESCs [2+/calcineurin/NFAT pathway in decidual cells [2+ may be mediated partially through the pathway including calcineurin/NFAT. However, further investigation is required as to whether the reduction in cAMP levels induced by intracellular Ca2+ influx would influence the calcineurin/NFAT pathway. Interestingly, Ca2+ chelater suppressed Ca2+ influx inhibitor-induced up-regulation of prolactin and COX2 expression in endometrial cells. These results indicate that optimal and pulsatile Ca2+ concentration in the cytoplasm is essential for induction of these expression. Because the spatial and temporal fluctuation of Ca2+ levels in the cytoplasm is necessary for diverse cellular responses including proliferation, differentiation, and gene expression, pulsatile Ca2+ changes in endometrial cells is probably crucial for implantation. Importantly, the conditioned media from developmentally competent embryo, but not impaired embryo induces actively intracellular Ca2+ oscillation [2+ which may link to cAMP levels modulates differentiation of ESCs and EM-1, our study showed only pharmacological effects of Ca2+ modulators on decidualization and glandular maturation. It should be noted that in addition to pharmacological changes in Ca2+/cAMP signaling, this study was not designed to determine a role of endogenous Ca2+ on cAMP- or implantation-related gene expression. The mechanism that modulates intracellular Ca2+ oscillation would be needed to clarify.It was previously shown that the treatment with Caregnancy , and thaal cells . Ruan et [et al.  showed t in ESCs , 28. In al cells –31. Accoillation . Althoug2+ and the inhibitory effect was abolished by a selective PDE1 inhibitor [2+ concentrations has been shown to activate PDE1A or PDE1C and to degrade cAMP in human embryonic kidney cells [2+/CaM possibly activated PDE and then resulted in degradation of cAMP in the ESCs and EM1 cells.cAMP is hydrolyzed into AMP by 3’, 5’- cyclic nucleotide phosphodiesterases (PDE) . The supnhibitor . The eleey cells . These f2+; it activates the function of AC1, AC3 and AC8 but inhibits AC5 and AC6 [2+-mediated inhibition of AC5 and AC6 reduces the cAMP concentrations in vascular smooth muscle cells [2+ influx through VDCC attenuates skeletal muscle contraction via the inhibition of AC5 and AC6 activity [prolactin and IGFBP1 was enhanced only in the presence of forskolin or db-cAMP when cells were treated with nifedipine, verapamil or dantrolene; however, treatment with each reagent without forskolin or db-cAMP had no effect on prolactin and IGFBP1 expression (data not shown). The stimulatory effect of nifedipine, verapamil or dantrolene alone in EM1 cells was also similar to that in ESCs. Thus, significant effect was seen even in the condition with elevated cAMP levels. These results suggest that Ca2+ influx-triggered cAMP reduction in the cytoplasm could be mediated by activation of PDE1, but not by down-regulation of AC5 or AC6 in human endometrial cells. Intracellular Ca2+ might activate PDE1 in human endometrial stromal or glandular epithelial cells.In turn, five of nine AC families are directly regulated by Ca and AC6 . Ca2+-mele cells . AC5 andle cells  and Ca2+activity . However2+ signaling pathway could be a crucial mediator in endometrial development [2+ signaling with Wnt/β-catenin signaling could be required for the induction of proper decidualization and restricted invasion of trophoblast into the pregnant endometrium.It has been proposed that Wnt/β-catenin signaling plays an important role in blastocyst implantation, endometrial decidualization and placental formation , 41. Estelopment . It is p2+ in ER storage induces a Ca2+ influx, called as Ca2+-release activated Ca2+ entry (SOCE) through Ca2+-release activated Ca2+ channel (CRAC) in plasma membrane. The key molecule for the operation of SOCE has been identified as stromal interaction molecules 1 (STIM1), an ER calcium sensor. STIM2 has also been reported to be an inhibitor of STIM2-mediated SOCE [2+ levels are complicated and Ca2+ signaling cascade must be tightly controlled. Although any diseases with increased cytoplasmic calcium and the relationship between abnormal intracellular calcium and implantation failure are not known, the functional loss of intracellular molecules which regulate Ca2+ homeostasis such as STIM as well as IP3-sensitive Ca2+ channel, VDCC would affect normal Ca2+ oscillation in endometrial cells, as described above [2+ homeostasis and proper signaling. More research into the identification of physiological regulators for the Ca2+/cAMP signaling in endometrial cells will be needed to clarify the role of the Ca2+/cAMP pathway in uterine physiology towards pregnancy. We assume that more detail understanding of the exact regulation and function of Ca2+-related molecular machinery can define new clinical targets and effective therapeutic strategies.The exhaustion of Cated SOCE . Thus, red above . It is p2+ levels lowers intracellular cAMP concentrations and the expression of implantation-related factors, and conversely that a decrease in intracellular Ca2+ levels promotes the expression of implantation-related factors in human endometrial cells.In conclusion, we found that continuous elevation of CaS1 Fig3 cells/cm2 in 96 well dish were treated for 1 h with nifedipine (20 μM), verapamil (20 μM), dantrolene (20 μM), alamethicin (1 μM), or ionomycin  and then stimulated for 48 h with forskolin (15 μM). After treatment, cell viability was assessed using the WST-8 cell survival assay. After incubation with WST-8 reagent in CO2 incubator for 1 h, 50 μl from each well were then transferred to a 96-well microplate and read at 450 nm. The data from three independent experiments are presented. **p<0.01 vs. Control- no forskolin, ##p<0.01 vs. forskolin alone. Values represent the mean ± SEM.ESCs plated at a density of 6 x 10(TIF)Click here for additional data file.S2 Figvs. forskolin alone. Values represent the mean ± SEM.ESCs cells were treated for 1 h with nifedipine (20 μM), verapamil (20 μM), dantrolene (20 μM), or alamethicin (1 μM) and then stimulated for 48 h with forskolin (15 μM). The IGFBP1 levels released into media were determined by ELISA. *p<0.05, **p<0.01 (TIF)Click here for additional data file.


Upon action potential firing, the majority of spines are subject to global back-propagating action potential (bAP) Ca2+ transients. These transients translate neuronal suprathreshold activation into intracellular biochemical events. Using a combination of electrophysiology, two-photon Ca2+ imaging, and modelling, we demonstrate that bAPs are electrochemically coupled to Ca2+ release from intracellular stores via ryanodine receptors (RyRs). We describe a new function mediated by spine RyRs: the activity-dependent long-term enhancement of the bAP-Ca2+ transient. Spines regulate bAP Ca2+ influx independent of each other, as bAP-Ca2+ transient enhancement is compartmentalized and independent of the dendritic Ca2+ transient. Furthermore, this functional state change depends exclusively on bAPs travelling antidromically into dendrites and spines. Induction, but not expression, of bAP-Ca2+ transient enhancement is a spine-specific function of the RyR. We demonstrate that RyRs can form specific Ca2+ signalling nanodomains within single spines. Functionally, RyR mediated Ca2+ release in these nanodomains induces a new form of Ca2+ transient plasticity that constitutes a spine specific storage mechanism of neuronal suprathreshold activity patterns.A key feature of signalling in dendritic spines is the synapse-specific transduction of short electrical signals into biochemical responses. Ca A combination of two-photon calcium imaging, electrophysiology, and modelling shows how ryanodine receptors  generate a signalling nanodomain within individual dendritic spines, enabling compartmentalized plasticity of calcium dynamics. 2+ ion. Ca2+ serves both as an electric charge carrier mediating fast voltage changes at the membrane and as a second messenger activating intracellular signalling cascades. Even within the spatial confines of dendritic spines, the specialized domains of dendrites that receive synaptic connections, Ca2+ encodes a versatile array of specific functions. In this study, we first demonstrate that voltage-gated Ca2+ channels and ryanodine receptors, intracellular channels located on the membrane of the endoplasmic reticulum through which Ca2+ can be released into the cytosol, are electrochemically coupled in single dendritic spines. We identify how ryanodine receptors induce enhancement of the Ca2+ influx, mediated by the opening of voltage-gated Ca2+ channels, induced by action potentials in a compartmentalized, spine-specific manner. Within the femtoliter volume of a single spine, specificity of this route of Ca2+-signalling is achieved by a signalling nanodomain centred on the ryanodine receptor. Our work stresses the role of the ryanodine receptor not only as an ion channel releasing Ca2+ from the endoplasmic reticulum but also as a macromolecular complex generating specificity of Ca2+-signalling within the spatial constraints of a single spine.Experiences change neuronal circuits, and these circuit changes outlast the initial experiences. This means that, in neurons, the fast electrical activity encoding experiences needs to be transduced into longer-lived biochemical and structural changes. A key mediator between these two timescales of neuronal activity is the Ca These include changes in synaptic strength, membrane excitability, cell morphology, and gene expression ) in spines , spines are resistant to shrinkage and LTD , Ca2+ binding to the effector is most likely cooperative. We observe enhancement of the bAP-Ca2+ transient predominantly in spines with small baseline amplitudes. This suggests that there is a fixed dynamic range for bAP mediated spine [Ca2+], which limits further enhancement in spines where the downstream effector has already been activated. In this context, it is an interesting observation that we can induce enhancement in the small baseline amplitude spines over a wide range of small amplitude doublets, but fail to do so with single bAPs. One explanation is that doublets will prolong the time window of RyR activation .A contribution of nanodomain signalling of RyRs has to be considered for a number of physiological and pathophysiological processes during synaptic plasticity and memory: RyRs are up-regulated during intense training tasks  and medi2+ release in neurons: RyRs can induce a plastic functional state change in spines that scales with neuronal output levels. This compartmentalized storage of global activity levels provides a new mechanism of how endoplasmic reticulum mediated Ca2+ release contributes to neuronal information processing.In addition to demonstrating that RyRs can form specific intraspine signalling nanodomains, we also propose a new function for intracellular CaAnimal experiments were performed in accordance with institutional and national guidelines. Acute cortical slices were prepared from Wistar rats . Animals were anesthetized with isoflurane and decapitated. See  for detaAR antagonist Gabazine (1 μM) was added to the ACSF. Slices were preincubated in the respective drug solution for at least 30 min before starting the experiment. Control experiments were interleaved with experiments where drugs were applied. Wash-in experiments were performed with 30 μM CPA or a mixture of 30 μM CPA and 10 μM ryanodine to facilitate store depletion.For pre-incubation experiments, 30 μM CPA or 100 μM ryanodine or a synaptic receptor inhibitor cocktail consisting of the NMDAR antagonist 2R-amino-5-phosphonovaleric acid  the AMPA and Kainate receptor antagonist 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo[f]quinoxaline-2,3-dione  and the group I metabotropic glutamate receptor (mGluR) antagonist (S)-α-Methyl-4-carboxyphenylglycine ([S]-MCPG), 500 μM—substituted by 2-Methyl-6-(phenylethynyl)pyridine, (MPEP), 25μM and (S)-(+)-α-Amino-4-carboxy-2-methylbenzeneacetic acid (LY 367385), 100 μM, in some experiments—as well as the GABAWhole-cell current clamp experiments were performed at near physiological temperature (32–34°C) using an Axon Multiclamp 700B amplifier . Signals were low pass filtered at 2 kHz and digitized at a sampling rate of 5 kHz . Pipettes (3–6 MΩ) were filled with an intracellular solution containing 130 K-gluconate, 20 KCl, 10 HEPES, 4 MgATP, 0.3 NaGTP and 10 phosphocreatine  and 30 μM Alexa 594 and 200 μM fluo-4FF or 200 μM fluo-5F or 500 μM fluo-5F. Initial series resistances were between 6 and 20 MΩ. Action potentials were induced with 2 ms square current pulses ranging from 1 to 3 nA. Doublets and quintuplets were delivered at 100 Hz. Experiments were aborted if the holding current exceeded -200 pA at -60 mV.A Femto2D two-photon laser scanning system  equipped with a femtosecond pulsed Ti:Sapphire laser tuned to λ = 805 nm  controlled by the Matlab-based MES software package  was used. Fluorescence was detected in epifluorescence mode with a water immersion objective . Transfluorescence and transmitted infra-red were detected using an oil immersion condenser (Olympus).2+ transients of the doublet test stimulus were measured every 60 s. After six baseline sweeps, we delivered bursts of 5 APs at 100 Hz every 30 s for 5 min . For morphological reconstructions, we performed post-hoc high-resolution z-stacks of the recorded spines with a ∆z of 0.2 μm.Calibration of Gmax/R values was performed at the tip of a sealed pipette in the imaging plane of the slice using a calibration solution consisting of 50 μl recording solution and 50 μl 1M Ca2+] are reported as the ratio of green fluorescence change over red (∆G/R) as described by Yasuda et al.  sink where λ determines the time scale of the extrusion. The above equations for free [Ca2+], dye and endogenous buffers are solved in a rectangular box representing the spine head. Calcium transport through the ER membrane ∂ΩER (left side in PM (right side) is modelled by inhomogeneous Neumann boundary conditions:PRyR, PSERCA, and Pleak control the maximum effect size of those three calcium fluxes. t and whether Rc around the channel location. KSERCA denotes the dissociation coefficient of SERCA pumps. To obtain a resting state with given free Ca2+ concentrations c0 (cytosol) and E0 (endoplasmic reticulum), Pleak has to obey:Here, N identical Gaussian pulses (width σ) separated by a delay τ. A0 controls the number of Ca2+ ions that enter per bAP. In simulations that include RyR efflux, we assumed that a single RyR will open for 2 ms after the second bAP, i.e., t ∈ . For the deduction of the parameters introduced above, please see The openings of VGCCs triggered by a given sequence of bAPs are represented as train of The set of reaction-diffusion equations was numerically solved using the DUNE open source software library for finite element calculations –62. The 2+]. The open and close rates ko and kc of a channel are given in the following way:c and E denote cytosolic and luminal free [Ca2+], respectively. The stationary open probability of a single RyR channel is given byThe RyR gating model is based on the model from Sobie et al. ,64. Hereα and Km parameters have been changed from the values provided in  for one AP (grey) and a doublet (black) at the Ca2+ activation site of the RyR pore in the presence of 500 μM fluo-5F. (d2) Time course of the cumulative open probability of a single RyR tetrameric channel activated by the VGCC mediated bAP-[Ca2+] changes plotted in d1. (e) Time course of the steady state open probability of a single RyR tetrameric channel activated by VGCC mediated bAP-[Ca2+] changes under conditions of reduced buffering in the presence of 200 μM fluo-4FF.(a(EPS)Click here for additional data file.S2 Fign = 650/160 spines/cells; CA1: n = 189/42 spines/cells). Amplitudes in MEC were significantly larger than in CA1 . Using a Shapiro Wilk normality test, both datasets differed significantly from a normal distribution . (b) Baseline amplitudes of all spine measurements in drug-free control conditions with recovered spine morphology for MEC  and CA1 . Amplitudes are plotted against the apparent size of the respective spine . (c) Baseline amplitudes of all spine measurements in drug-free control conditions with recovered spine length for MEC  and CA1 . Amplitudes are plotted against the length of the respective spine .(a) Distribution histogram of baseline amplitudes for CA1 pyramidal cells (green) and layer 2 cells in the MEC (black). All baseline measurements in drug-free control conditions were included (MEC: (EPS)Click here for additional data file.S3 Fig2+ transient enhancement . (b) Apparent spine size is plotted against normalized bAP-Ca2+ transient enhancement . (c) Spine length is plotted against normalized bAP-Ca2+ transient enhancement . (d1) Averaged baseline bAP-Ca2+ transients 0 to 5 min after bAP stimulation onset (grey traces) and averaged bAP-Ca2+ transients 15 to 20 minutes after bAP stimulation onset of a spine (black) and the adjacent dendritic microdomain (red).(d2) Z-projection of the imaged dendritic segment . Asterisks mark imaged spine and dendritic segment. (e) Normalized bAP-Ca2+ transient in spines 15 to 20 min after bAP stimulation onset is plotted against the corresponding dendritic microdomain (±1 μm). Values are not significantly correlated . Dashed lines correspond to enhancement >20%. Averaged values in spines are significantly larger than in dendrites . (f1) For CA1 pyramidal cells in the hippocampus, the averaged baseline amplitude is plotted against bAP-Ca2+ transient enhancement 15 to 20 min after bAP stimulation onset . Spines with baseline amplitudes <0.026 ∆G/R are depicted in green (n = 17/8 spines/cells), spines with baseline amplitudes >0.026 are depicted in orange (n = 42/16 spines/cells). (f2) Time plot of normalized bAP-Ca2+ transients demonstrates selective enhancement of spines with baseline amplitudes <0.026 ∆G/R (green) when compared to spines with baseline amplitudes >0.026 ∆G/R (orange). Dendrites are plotted in red. One doublet was applied every 60 s, the data is plotted in 3 min bins. The interval 15 to 20 min after bAP stimulation onset used for quantification of normalized enhancement is shaded in grey. (f3) Cumulative distribution plot of normalized enhancement 15 to 20 min after bAP stimulation onset. Enhancement for spines with baseline ∆G/R < 0.026  is significantly larger than in spines with baseline ∆G/R > 0.026 . The trend towards smaller enhancement of adjacent dendrites was statistically not significant. . Data are expressed as mean ± SEM, * p < 0.05(a) Spine distance from soma (μm) is plotted against normalized bAP-Ca(EPS)Click here for additional data file.S4 Fig2+ transient enhancement 15 to 20 min after bAP stimulation onset for spines with synaptic transmission blocked  and control spines . (b) The apparent spine size of spines with fluo-5F  and control spines  is plotted against the bAP-Ca2+ transient 15 to 20 min after bAP stimulation onset.(a) The averaged baseline amplitude is plotted against bAP-Ca(EPS)Click here for additional data file.S5 Fig2+ transient enhancement 15 to 20 min after bAP stimulation onset for spines preincubated with 30 μM CPA  and control spines . (b) Averaged baseline amplitude plotted against bAP-Ca2+ transient enhancement 15 to 20 min after bAP stimulation onset for spines preincubated with 100 μM ryanodine  and control spines . (c) Averaged baseline amplitude plotted against bAP-Ca2+ transient enhancement 15 to 20 min after bAP stimulation onset for spines preincubated with 10 μM XestosponginC  and control spines .(a) Averaged baseline amplitude plotted against bAP-Ca(EPS)Click here for additional data file.S6 Fig2+ transient enhancement measured in a 20 ms time interval starting with the second AP in a doublet plotted against measurement in a 70 ms time interval starting with the first AP .bAP-Ca(EPS)Click here for additional data file.S7 Fig2+] reaches about 0.5 μM. Then, the RyR opening adds another 0.25 μM. This test was used to tune parameters for the RyR and VGCCs to comply with constraint (b) given in (a) Considering only the first two bAPs, the free [Ca(EPS)Click here for additional data file.S1 Table2+ ions per doublet, we applied the calibration method described by Yasuda et al. [Ni ≈ 2000. We choose Ni ≈ 500 to comply with the calibrated measurements (b) and constraints (e) and (d). This does not contradict with [A0 is given with Ni as Na is the Avogadro constant and w and d denote the width and depth of the simulation box, respectively. (e) To satisfy constraints (d) we had to reduce the Ryanodine receptor efflux, which could be achieved by shorter open time or reduced efflux. We did this by tuning the current to a slightly smaller value than given in literature, to achieve a mean open time of 2 ms. Note that higher efflux and shorter open time would not contradict with our conclusions of the nanodomain downstream receptor activation. (g) In [ Estimated from a representative subset of own measurements. For estimating the number of Caa et al.  to a suba et al.  to be Nict with  since th. (g) In  the mean(DOCX)Click here for additional data file.


The astrocyte Ca2+ signaling is coordinated by gap junction channels and hemichannels formed by connexins (Cx43 and Cx30) and channels formed by pannexins (Panx-1). The neuronal activity-initiated Ca2+ waves are propagated among neighboring astrocytes directly via gap junctions or through ATP release via connexin hemichannels or pannexin channels. In addition, Ca2+ entry via connexin hemichannels or pannexin channels may participate in the regulation of the astrocyte signaling-mediated neurovascular coupling. Interestingly, nitric oxide (NO) can activate connexin hemichannel by S-nitrosylation and the Ca2+-dependent NO-synthesizing enzymes endothelial NO synthase (eNOS) and neuronal NOS (nNOS) are expressed in astrocytes. Therefore, the astrocytic Ca2+ signaling triggered in neurovascular coupling may activate NO production, which, in turn, may lead to Ca2+ influx through hemichannel activation. Furthermore, NO release from the hemichannels located at astrocytic endfeet may contribute to the vasodilation of parenchymal arterioles. In this review, we discuss the mechanisms involved in the regulation of the astrocytic Ca2+ signaling that mediates neurovascular coupling, with a special emphasis in the possible participation of NO in this process.Neuronal activity must be tightly coordinated with blood flow to keep proper brain function, which is achieved by a mechanism known as neurovascular coupling. Then, an increase in synaptic activity leads to a dilation of local parenchymal arterioles that matches the enhanced metabolic demand. Neurovascular coupling is orchestrated by astrocytes. These glial cells are located between neurons and the microvasculature, with the astrocytic endfeet ensheathing the vessels, which allows fine intercellular communication. The neurotransmitters released during neuronal activity reach astrocytic receptors and trigger a Ca Information processing by the neuronal network in the central nervous system (CNS) is a very complex task that relies on dynamic interactions between neurons and glial cells, but also on functional association among brain cells and cerebral microcirculation, which is intended to be reflected by the concept “neurovascular unit”  precedes vasodilation of cerebral arterioles  and prostaglandin E2 (PGE2), which are synthesized by the cytochrome P450 epoxygenase and by a cyclooxygenase enzyme-dependent pathway, respectively  has been addressed in further studies. Girouard et al. . Astrocytic endfeet express Ca2+-activated K+ channels of large conductance (BKCa) and vascular smooth muscle cells of the parenchymal arterioles express inward rectifier K+ channels (Kir)  eliminates the electrochemical gradient of K+ and produces smooth muscle cell depolarization and vasoconstriction -dependent pathway, with the consequent generation of inositol 1, 4, 5-triphosphate (IP3) from membrane phospholipids, and then, the stimulation of Ca2+ release from the endoplasmic reticulum (ER) via IP3 receptors , a blocker of the sarcoplasmic/ER Ca2+ ATPase  family -mediated intercellular communication   cells or Madin-Darby canine kidney (MDCK) cells were sensitive to short-time application (<30 min) of the connexin blocking peptides 37,43Gap27 (a mimetic peptide of the second extracellular loop of Cx37 and Cx43) or 43Gap26 (a mimetic peptide of the first extracellular loop of Cx43), respectively , a member of a protein family  that forms channels with similar characteristics of connexin hemichannels , deletion of nNOS and specific nNOS inhibition with 7-nitroindazole have been reported to attenuate the increase in sensory cortex cerebral blood flow observed in response to vibratory hindpaw stimulation in mouse  is a widely distributed, pleiotropic signaling molecule synthesized by the enzyme NO synthase (NOS) from the amino acid L-arginine . ATP may be released through hemichannels formed by Cx30 or Cx43 and/or channels formed by Panx-1 and, in addition, activation of these channels provides a direct pathway for Ca2+ influx that may be involved in the regulation of the IP3R-initiated astrocytic Ca2+ signal. However, although connexins and Panx-1 are likely to play a central role in the astrocyte-mediated neurovascular coupling, NO seems to control and orchestrate the development of the Ca2+ response, since NO production is activated by the initial IP3R-mediated Ca2+ release and NO is involved in the generation, propagation and regulation of the Ca2+ signaling. This is because the increase in NO concentration leads to ATP release and activates a Ca2+ influx pathway that contributes to the astrocytic Ca2+ signal observed in response to both ATP or metabotropic glutamate receptor stimulation. The NO-evoked Ca2+ influx seems to be also involved in the regulation of the Ca2+ signaling by contributing to refill the IP3R-associated intracellular Ca2+ store. Although the activation of Cx43 hemichannels by S-nitrosylation may provide the pathway for the NO-dependent ATP release and Ca2+ influx, the participation of connexin- or Panx-1 formed channels in the NO-dependent Ca2+ signals must be confirmed in future investigations.Neurovascular coupling is a complex signaling mechanism that depends on functional and coordinated interactions of astrocyte with neurons and vascular cells. Changes in neuronal activity are transduced into vasomotor responses through astrocytic Ca2+ wave into the astrocyte endfeet is supported and regulated by specialized signaling mechanisms of these subcellular domains. Astrocyte endfeet express Cx43 hemichannels and TRPV4 channels and although the generation of the Ca2+ signal in the endfeet is governed by IP3Rs, Ca2+-dependent activation of Cx43 hemichannels and TRPV4 channels may contribute to enhance the Ca2+ signal at specialized microdomains associated with the activation of vasodilator mechanisms. Interestingly, diffusion or production of NO in the endfeet may be involved in the control of the Ca2+ signal by inducing the opening of Cx43 hemichannels and the inhibition of TRPV4 channels. Furthermore, the NO-mediated Cx43 hemichannel activation may also play an important role in the astrocyte endfoot-elicited vasodilation by providing the pathway for the release of NO and PGE2 into the perivascular space. In addition of Cx43 hemichannels, NO may also induce the activation of BK channels at the astrocytic enfeet, which highlights the relevance of the interaction between NO and Ca2+ in the regulation of the astrocyte-dependent vasodilator signals activated during neurovascular coupling. The specific contribution of eNOS and nNOS to the astrocyte-conducted Ca2+-mediated vasodilator signaling may be determined by the subcellular location and spatial organization of these NOS isoforms in relation to other signaling proteins involved in the regulation of neurovascular coupling. Then, the study of the subcellular distribution of eNOS and nNOS in astrocytes and the possible association of these NO-synthesizing enzymes with connexins, Panx-1, TRPV4 channels and BK channels may be an interesting and fruitful area of investigation that may help to understand the complex and dynamic regulation of neurovascular coupling.The propagation of the neuronal-activated CaThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Biochemical assays reveal how three proteins fit together to form the channel that controls the flow of calcium ions into mitochondria. Related research article Tsai MF, Phillips CB, Ranaghan M, Tsai CW, Wu Y, Williams C, Miller C. 2016. Dual functions of a small regulatory subunit in the mitochondrial calcium uniporter complex. eLife5:e15545. doi: 10.7554/eLife.15545Image Schematic showing calcium ions (gray) flowing through an open ion channel2+). However, the importance of this phenomenon was not immediately clear. Later, in the 1990s, it was revealed that mitochondria inside eukaryotic cells also take up Ca2+ ions  can pass freely through the outer membrane, but not the inner one. The transport of Ca2+ through the inner membrane depends on an ion channel called the “mitochondrial Ca2+ uniporter” (or MCU channel for short). This channel is the most selective Ca2+ channel currently known  form what can be referred to as the “core functional unit of the MCU” . This stmembrane .Figure 12+concentration in the cytosol is at its resting level. This stops Ca2+ ions from flowing into the mitochondria. However, when the Ca2+ concentration in the cytosol increases, Ca2+ ions bind to MICU1and cause it to dissociate from the pore to allow other Ca2+ ions to pass through (2+-sensitive “lid” on the MCU channel complex, which closes and opens the channel in response to changes in the Ca2+ concentration in the cytosol. Notably, the pore-forming subunit cannot work without EMRE (Combined with relevant data from other groups , the res through . Thus MIout EMRE . Thus it2+ uptake by mitochondria and how it could be affected via drugs to control energy production in cells and cell death.Now that we know how the MCU core functional unit is assembled, the stage is set to explore how the structure of the MCU channel relates to its function. This will bring us closer to understanding the phenomenon of Ca


Background: In heart failure, the release of calcium becomes erratic leading to the generation of arrhythmias. Dysregulated Zn2+ homeostasis occurs in chronic heart failure.Results: Zn2+ can directly activate RyR2, removing the dependence of Ca2+ for channel activation.Conclusion: Zn2+ shapes Ca2+ dynamics by directly interacting with and modulating RyR2 function.Significance: This highlights a new role for Zn2+ in cardiac excitation-contraction coupling. 2+ homeostasis is a hallmark of certain cardiomyopathies associated with altered contractile force. In this study, we addressed whether Zn2+ modulates cardiac ryanodine receptor gating and Ca2+ dynamics in isolated cardiomyocytes. We reveal that Zn2+ is a high affinity regulator of RyR2 displaying three modes of operation. Picomolar free Zn2+ concentrations potentiate RyR2 responses, but channel activation is still dependent on the presence of cytosolic Ca2+. At concentrations of free Zn2+ >1 nm, Zn2+ is the main activating ligand, and the dependence on Ca2+ is removed. Zn2+ is therefore a higher affinity activator of RyR2 than Ca2+. Millimolar levels of free Zn2+ were found to inhibit channel openings. In cardiomyocytes, consistent with our single channel results, we show that Zn2+ modulates both the frequency and amplitude of Ca2+ waves in a concentration-dependent manner and that physiological levels of Zn2+ elicit Ca2+ release in the absence of activating levels of cytosolic Ca2+. This highlights a new role for intracellular Zn2+ in shaping Ca2+ dynamics in cardiomyocytes through modulation of RyR2 gating.Aberrant Zn Damaging changes in Ca2+ homeostasis are associated with heart failure, conduction abnormalities, and contractile dysfunction. Abnormal RyR2 function is recognized as an important component in the etiology of such disease states   , 12. Durfor Ca2+ . Other g current , 15. It ide Ca2+ , 13.2+ homeostasis has been shown to be associated with cardiomyopathy including chronic heart failure, and myocardial damage as a result of dysregulated intracellular Ca2+ release, reduced cardiac contractility, and significantly prolonged rises of systolic Ca2+   and N,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) were from Sigma-Aldrich.Chemicals were AnalaR or the best equivalent grade from BDH Chemicals  or Sigma-Aldrich. All solutions were made in deionized water, and those for use in bilayer experiments were filtered through a Millipore membrane filter (0.45-μm pore). 2,2′-(Ethylenedioxy)dianiline-g followed by ultracentrifugation of the supernatant at 100,000 × g. The heavy SR membrane fraction was obtained from loading mixed membranes onto a discontinuous sucrose density gradient. Heavy SR was collected, snap frozen in liquid N2, and stored at −80 °C. As reported previously, SR vesicles fused in a fixed orientation such that the cis-chamber corresponded to the cytosolic face of the channel and the trans-chamber to the SR lumen . The trans-chamber was perfused with 250 mm glutamic acid and 10 mm HEPES (pH to 7.2) with Ca(OH)2 (concentration of free Ca2+ ∼50 mm). The identity of RyR2 was confirmed by the single-channel conductance, and the number of channels gating in the bilayer was assessed by the application of caffeine at the end of the experiment. Zn2+ was added as ZnCl2 to the cis-chamber at the required concentration from an appropriate stock solution. 100 mm stock solution of ZnCl2 was prepared in 0.029 m HCl, and serial ZnCl2 dilutions were made in a Tris/HEPES buffer which contained, 250 mm HEPES, 80 mm Tris, and 10 μm free Ca2+ (pH 7.2) to make appropriate stock solutions. The concentration of each Zn2+ stock was further assessed using a pZn meter  as previously described . Special care was taken when preparing Zn2+ stock solutions. All glassware was prewashed with 100 nm TPEN and then rinsed carefully with MilliQ water passed through a Chelex Resin (Bio-Rad). The use of colored pipette tips was also avoided.Sheep hearts were obtained from a local abattoir, and heavy SR membrane vesicles were prepared and fused with planar phosphatidylethanolamine lipid bilayers, as described previously . BrieflySR lumen , 22. Theescribed  to confi2+ and pH of all of our solutions were determined using a Ca2+ electrode and a pH electrode  as previously described (2+ ≤ 100 μm had no significant effect on the concentration of free Ca2+ ([Ca2+] was 10 ± 3.8 μm in the absence of Zn2+ and 10 ± 3.2 μm in the presence of 100 μm Zn2+ (n = 3). The addition of 1 mm BAPTA reduced the free Ca2+ concentration from 10 ± 2 μm to 10 ± 3.8 nm (n = 3). Subsequent addition of ZnCl2 (≤100 μm) had no significant effect on the measured free Ca2+ concentration  under these conditions. The total concentration of Zn2+ in all of our recording solutions was assessed using inductively coupled plasma optical emission spectrometry (ICP-OES)  and shown to be below the detectable limit (1 ppb). Free Zn2+ was also assessed using a pZn meter and calculated to be ∼9 pm. The addition of concentrations of Ca2+ ≤ 100 μm had no significant effect on the concentration of free Zn2+ . The calculated free Zn2+ concentration following addition of 1 mm cytosolic BAPTA was first estimated using the MaxChelator program and then assessed by actual measurements of free Zn2+ using a pZn meter. When the starting free Ca2+ concentration was 10 μm, the addition of 1 mm BAPTA had no significant effect on free Zn2+ levels.Experiments were carried out at room temperature (22 ± 2 °C). The concentration of free Caescribed . Our meaPo) and lifetime distributions were calculated from 3 min of continuous recording using TACfit 4.2.0 software (Bruxton Corporation). Lifetime analysis was carried out only when a single channel was incorporated into the bilayer. Individual lifetimes were fitted to a probability density function using the method of maximum likelihood . Channel recordings were low pass filtered at 10 kHz with a 4-pole Bessel filter, digitized at 100 kHz using a National Instruments acquisition interface , and recorded on a computer hard drive using WinEDR 3.05 software . The recordings were subsequently filtered at 800 Hz (−3 dB) using a low pass digital filter implemented in WinEDR 3.05. Channel events were detected by the 50% threshold method  using TAkelihood . LifetimAdult male Wistar rats (300–400 g) were killed by concussion followed by cervical dislocation. The care and sacrifice of the animals conformed to the requirements of Directive 2010/63/EU of the European Parliament.m NaCl, 5 mm KCl, 330 μm NaH2PO4, 5 mm glucose, 5 mm sodium pyruvate, 10 mm HEPES, 1 mm MgCl2, and 2 mm CaCl2 (pH 7.4). Nominally Ca2+-free Tyrode's solution used during cardiomyocyte isolation was as outlined above with no added Ca2+.All extracellular solutions were based on a modified Tyrode's solution. The basic solution contained, 135 m2+-free Tyrode's solution (NT). The heart was then cannulated via the aorta on a Langendorff type apparatus and warmed NT buffer at 37 °C was perfused through the heart in a retrograde fashion for 6 min to clear residual blood. The solution was then exchanged for a Ca2+-free NT buffer with enzyme mix (containing 15 mg of collagenase type II (Worthington), with 50 mg of bovine serum albumin prepared from Cohn fraction V albumin and 18 mg of protease (type XIV 15% Ca2+) in 30 ml) for 8–15 min. Identification of rod-shaped cardiomyocytes in the perfusate was used as an indication of digestion being complete. The solution was then exchanged for a 2 mm Ca2+ NT solution, the heart was cut down, and cardiomyocytes were mechanically dispersed from the tissue in a shaking water bath. Typically this method yielded 70–90% rod-shaped cardiomyocytes, which were stored in NT solution at room temperature and used within 18 h of isolating. In intact myocytes, cells were exposed to ZnCl2 in the presence of the zinc ionophore, zinc pyrithione (ZnPy) to enable the entry of Zn2+ into the cell because ZnCl2 is cell-impermeable.The protocol for isolation of cardiomyocytes was as described previously . Brieflym Ca2+ Tyrode's solution for 2 min prior to perfusion with a solution containing 100 μm EGTA, 5 mm ATP, 10 mm HEPES, 150 mm potassium gluconate, 25 μm MgCl2 at 20 °C. The sarcolemmal membrane was permeabilized with saponin (50 μg/ml) for 30 s. After permeabilization, cardiomyocytes were maintained in recording solution containing 1 mm EGTA, 10 mm HEPES, 120 mm potassium gluconate, 5 mm ATP, 1 mm pyruvate, 1 mm free MgCl2, and 100 nm free Ca2+ , and 30 μm Fluo-4, pH 7.2. 1 mm BAPTA was added as indicated in the text, and additional Zn2+ was added to maintain as required.Isolated cardiomyocytes were permeabilized using saponin. Briefly, cardiomyocytes were perfused with 0.5 mm Ca2+, there was no significant change in Fluo-4 fluorescence with increasing concentrations of Zn2+ (ΔFluo-4 F/F0 0.026 ± 0.01 units in 10 nm Zn2+ compared with control Tyrode's solution with no ZnCl2 added). For the cardiomyocyte experiments, ZnCl2 stocks were made directly in Tyrode's solution because the maximum concentration of Zn2+ used was 100 nm. For measurements of Ca2+ or Zn2+ in intact cardiomyocytes using Fluo-4-AM or Zinpyr-1, cells were illuminated with 488 nm using a monochromator  with fluorescence emissions captured above 520 nm using a Cascade 512B CCD camera  and continuously perfused at 32 ± 2 °C. The data were acquired using EasyRatioPro software  and analyzed using Winfluor v3.6.8 software .Images from permeabilized cardiomyoyctes were recorded using an Olympus 1X81 confocal microscope system using Olympus Fluoview software (version 4.0). Line scans were recorded using a 60× oil immersion lens using 2 × 2 pixel binning where each pixel was 0.43 μm in diameter. Fluo-4 indicator was excited using a 488-nm laser, and line scans were acquired at a rate of 333 Hz. The data were analyzed offline using ImageJ software. In our Tyrode's solution alone, in the presence of 100 nSpontaneous contractions were measured from cardiomyocytes perfused with Tyrode's solution at 32 ± 1 °C where videos were recorded to DVD. Analysis of contractions was carried out using edge detection measurements in Winfluor version 3.6.8 software.t test was used to assess the difference between mean values. A p value of 0.05 was taken as significant. Where multiple treatments were compared, ANOVA followed by a Bonferroni post hoc test was used to assess the difference between treatments. A p value of 0.05 was taken as significant.The data were expressed as means ± S.E. Where appropriate, a Student's 2+ can alter RyR2 gating, single RyR2 channels were incorporated into phospholipid bilayers enabling the membrane environment to be carefully controlled, and the direct action of Zn2+ at the cytosolic face of the channel was studied. Using Ca2+ as a permeant ion and holding at a command potential of 0 mV, which is considered to be the resting membrane potential of the sarcoplasmic reticulum  had no further significant effect on channel Po (B). Physiological concentrations of Zn2+ will therefore play a key role in regulating graded Ca2+ release events via RyR2 channels, enabling flexibility in the force of cardiac contraction, permitting the heart to adjust to altered diastolic filling , possibly a consequence of Zn2+ binding to the low affinity divalent inhibitory site of RyR2 (2+ had no significant effect on RyR2 gating (A). To confirm that the level of free Zn2+ in our recording solutions, which was measured at ∼9 pm, was insufficient to influence channel gating, we exposed RyR2 channels to the potent Zn2+ chelator TPEN. In the absence of exogenously added Zn2+, the addition of 100 nm TPEN to the cis-chamber caused no significant change in channel Po (B).To determine whether Zneticulum , the add5 ± 0.04 , indicatannel Po B. Physio filling . The addopenings , A and B of RyR2 . Control2 gating A. To conannel Po B.2+, we performed lifetime analysis on experiments where only a single RyR2 channel was gating in the bilayer. Dwell time histograms of the apparent open and apparent closed states uncovered three modes of Zn2+ operation at the cytosolic face of RyR2 (C). Control channel-gating was best characterized by short openings and long closing as described previously    and then channel . In the openings . These d2+ and Ca2+ at the cytoplasmic face of RyR2  by the addition of 1 mm BAPTA, as expected, the Po value for RyR2 reduced to zero , however, caused a marked increase in both the incidence of spontaneous Ca2+ waves and the amplitude of these events in a Zn2+ concentration-dependent manner (C). Increasing the Zn2+ concentration to >100 nm caused a marked decrease in the amplitude and frequency of waves, and the cardiomyocytes entered an irreversible series of Ca2+ waves often culminating in hypercontracture resulting in a loss of membrane integrity. This concentration of Zn2+ is close to the pathophysiological concentration of Zn2+ previously reported (∼30 nm) (–We then measured the effect of Znironment . In thest manner C. Increa(∼30 nm) –36.2+ to subactivating levels while maintaining the concentration of Zn2+ did not diminish the Zn2+-induced frequency of the Ca2+ waves seen in the permeabilized cells. In the presence of 100 nm Zn2+, removal of Ca2+ by chelation with 1 mm BAPTA markedly reduced the wave amplitude , and a concomitant increase in intracellular Zn2+ measured using the fluorescent indicator Zinpyr-1 (C and D). As shown by edge detection measurements, this resulted in spontaneous contractions compared with control cells (A and B). The observed spontaneous contractions were reversed by chelating intracellular Zn2+ by the addition of 10 μm TPEN (C and D), confirming that these cellular effects are attributed to elevated intracellular Zn2+ levels.Further evidence to suggest that changes in intracellular Zn by Zn2+ , A and BZinpyr-1 , C and Dol cells , A and B μm TPEN , C and D2+ is a high affinity activator of RyR2, effective at low picomolar concentrations. Lifetime analysis of RyR2 reveals unique and distinctive gating characteristics dependent on the concentration of cytosolic Zn2+. When Zn2+ levels are in the range 100 pm to 1 nm, RyR2 activation occurs by sensitization of the channel to cytosolic Ca2+. When the concentration of Zn2+ is elevated above 1 nm, the effect of Zn2+ overrides the influence of cytosolic Ca2+, and removal of activating levels of cytosolic Ca2+ no longer influences channel Po. When Zn2+ is elevated to toxic levels (in the millimolar range), it inhibits RyR2 channel function. Further evidence for the modulation of RyR2 function by Zn2+ in isolated permeabilized cardiomyocytes was acquired through measurement of Ca2+ waves. In these recordings, spontaneous Ca2+ waves were markedly increased in both frequency and amplitude in a Zn2+ concentration-dependent manner (between 100 pm and 10 nm Zn2+). Removal of Ca2+ by chelation with 1 mm BAPTA did not abolish Ca2+ waves in the presence of Zn2+, showing that Zn2+ can directly influence Ca2+ dynamics. These findings suggest an important pathophysiological consequence of dysregulated Zn2+ homeostasis, whereby even a small increase in RyR2 open probability during diastole can have major consequences for normal cardiac function and the generation of arrhythmias   –36, we s2+ is a potent activator of RyR2. Previous studies in which cardiac muscle [3H]ryanodine binding assays were used to assess the effects of Zn2+ on RyR2 function failed to see any effect of Zn2+ between 10 nm and 1 μm in the presence of 50 μm activating Ca2+  was altered and that this type of gating was consistent with Ca2+-independent openings. Our single channel data also provide the first evidence that when the concentration of Zn2+ is elevated above 1 nm, RyR2 becomes directly activated by Zn2+, and the dependence of Ca2+ on channel activation is abolished.Our mechanistic approach has for the first time revealed that Zning Ca2+ . This is2+ following prolonged exposure of cardiomyocytes to micromolar levels of extracellular Zn2+ and that this results in a reduced SR Ca2+ load (2+ removes the Ca2+ dependence of RyR2 causing channels to gate in exceptionally long-lived open states. Further evidence for this is demonstrated in intact cardiomyocytes where, in a 100 pm Zn2+-Tyrode's solution, the caffeine-induced elevation in intracellular Ca2+ was potentiated only after application of 10 μm ZnPy. Potentially this will lead to inappropriate Ca2+ release from the SR and likely result in reduced SR Ca2+ load. Such a mechanism would be particularly problematic under conditions where levels of intracellular Zn2+ are chronically elevated, including diabetes (2+ on RyR2 gating when cytosolic Zn2+ is raised to nonphysiological and toxic millimolar concentrations. It is possible that the inhibitory action of Zn2+ on RyR2 may be a consequence of Zn2+ binding to the low affinity unspecific divalent inhibitory site of RyR2 (It is notable that previous studies have shown a slow accumulation of intracellular Zna2+ load . One pladiabetes  and certdiabetes . In linediabetes , 44, we  of RyR2 , 45.2+ homeostasis has been shown to be associated with cardiomyopathy (–2+ contributes to cardiac pathology is unknown. Chronic oxidative stress is a major contributor in the pathophysiology of heart failure and under these conditions intracellular levels of Zn2+ increase by as much as 30-fold (2+ is primarily the result of altered metallothionein function, which perturbs its binding of Zn2+, resulting in raised levels (2+ release, which consequently reduces cardiac contractility and significantly prolongs the rise of systolic Ca2+ leading to chronic heart failure (2+ from the SR during cardiac excitation-contraction coupling is known to govern contractility of the heart. RyR2 plays a fundamental role as the main pathway for the release of Ca2+ and drives cellular contraction. Consequently dysfunction in the release of Ca2+ and modulatory influences that control RyR2 function are identified as contributory to the pathophysiology of heart failure and fatal cardiac arrhythmias (2+ may contribute to the progression of contractile dysfunction and heart failure through the alteration of RyR2 gating, which will result in perturbed Ca2+ release.Aberrant Znmyopathy –48, but  30-fold . This ind levels , 50. Alsd levels . Diabeti failure . The conhythmias . The pre2+ modulates RyR2 gating is shown in 2+ is able to fine-tune the release of Ca2+ from the SR while in parallel enabling flexibility and control of SR Ca2+ dynamics mediated through RyR2. Under pathophysiological conditions, where Zn2+ homeostasis is disturbed and levels of Zn2+ may reach >1 nm, RyR2 starts to gate in exceptionally long open states, which leads to very high Po potentially leading to arrhythmia. In this conformation, RyR2 is uncoupled from the usual regulatory effects of cytosolic Ca2+ and RyR2 channels are now activated by solely by Zn2+.A simple model to explain how cytosolic Zn2+ homeostasis is intimately related to intracellular Zn2+ levels and provides a mechanistic explanation as to how altered Zn2+ homeostasis can modulate cardiac RyR2 function. We reveal that Zn2+ is a high affinity activator of RyR2 which challenges our understanding of cardiac excitation-contraction coupling. We suggest that under normal physiological conditions, intracellular Zn2+ is essential in fine-tuning the release of Ca2+ from the SR. Pathological perturbations in Zn2+ homoeostasis will lead to inappropriate release of Ca2+ as observed in certain cardiac abnormalities including heart failure and fatal arrhythmias.Our study reveals that RyR2-mediated CaJ. W. and R. D. R. performed experiments and analyzed data; A. J. S. designed the experiments and contributed toward writing the manuscript; and S. J. P. designed the experiments, performed experiments, analyzed data, wrote the manuscript, and supervised the project. All authors discussed the results and implications and commented on the manuscript at all stages.


Interestingly, the CaSR activation-induced increase in [Ca2+]i results not only from intracellular Ca2+ release from internal stores but also from canonical transient receptor potential (TRPC)-dependent Ca2+ influx. This increase in Ca2+ was attenuated by treatment with a nonselective TRPC channel blocker but not by treatment with a voltage-gated calcium blocker or Na+/Ca2+ exchanger inhibitor. Furthermore, stimulation of CaSR by high [Ca2+]o enhanced the expression of TRPC3 and TRPC6 but not TRPC1 and TRPC4, and siRNA targeting TRPC3 and TRPC6 attenuated the CaSR activation-induced [Ca2+]i increase. Further experiments indicate that 1-oleoyl-2-acetyl-sn-glycerol (OAG), a known activator of receptor-operated calcium channels, significantly enhances the CaSR activation-induced [Ca2+]i increase. Moreover, under conditions in which intracellular stores were already depleted with thapsigargin (TG), CaSR agonists also induced an increase in [Ca2+]i, suggesting that calcium influx stimulated by CaSR agonists does not require the release of calcium stores. Finally, our data indicate that pharmacological inhibition and knock down of TRPC3 and TRPC6 attenuates the CaSR activation-induced cell proliferation in human MCs. With these data, we conclude that CaSR activation mediates Ca2+ influx and cell proliferation via TRPC3 and TRPC6 in human MCs.Calcium-sensing receptor (CaSR) has been demonstrated to be present in several tissues and cells unrelated to systemic calcium homeostasis, where it regulates a series of diverse cellular functions. A previous study indicated that CaSR is expressed in mouse glomerular mesangial cells (MCs), and stimulation of CaSR induces cell proliferation. However, the signaling cascades initiated by CaSR activation in MCs are currently unknown. In this study, our data demonstrate that CaSR mRNA and protein are expressed in a human mesangial cell line. Activating CaSR with high extracellular Ca Calcium-sensing receptor (CaSR), a cell-surface protein, is highly expressed in tissues and cells involved in systemic calcium homeostasis, including the parathyroid gland, kidney, and bone, where it contributes to the maintenance of systemic calcium within a narrow physiological window 2+ concentration ([Ca2+]i) 2+ concentration ([Ca2+]o) or a polyamine (such as spermine) activates phospholipase C (PLC), which converts phosphatidylinositol 4,5-bisphosphate into inositol-1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). IP3 triggers Ca2+ release from internal stores, resulting in an increase in [Ca2+]i. However, the concomitant store depletion might mediate store-operated calcium entry (SOCE) through store-operated channels (SOCs) in the plasma membrane. Moreover, DAG can cause receptor-operated calcium entry (ROCE) by activating receptor-operated channels (ROCs). IP3-mediated Ca2+ release, SOCE and ROCE all likely contribute to the increase in [Ca2+]i upon activation of CaSR. IP3-mediated Ca2+ release in response to CaSR stimulation has been widely investigated in many cell types; however, relatively little is known about calcium entry mechanism upon CaSR activation. SOCs and, in many cases ROCs, have been identified as canonical transient receptor potential (TRPC) channels. Furthermore, several studies indicated that TRPC channels are involved in the CaSR stimulation-induced calcium influx in some cell types, such as salivary ductal cells Although downstream effects can be highly varied, the first reactions following CaSR activation are common; stimulation of CaSR evokes an increase in intracellular CaStudies from our laboratory and other laboratories have demonstrated that human MCs express TRPC channel proteins, including isoforms of TRPC1, 3, 4, and 6 2+ supplemented with 10% fetal bovine serum  in 5% CO2 at 37°C. Human MCs between passages 3 and 15 were used. A human breast cancer cell line MCF-7 was obtained from the Cell Bank of the Chinese Academy of Science , maintained in 5% CO2 at 37°C in DMEM medium  containing 10% fetal bovine serum . Human MCs were transiently transfected with human TRPC3 siRNA, TRPC6 siRNA or scrambled siRNA  using the Xtreme GENE siRNA transfection reagent  according to the manufacturer's instructions. The transfected cells were assayed 24 to 48 h post-transfection.An stable human mesangial cell line  was established by transfection and immortalization by the viral oncogene large T-SV40 of human mesangial cells isolated from normal human glomeruli Reverse transcription was performed using an RT system  and a 10 µl reaction mixture. A High Capacity cDNA RT Kit  was used for the initiation of cDNA synthesis. The primer sequences used to amplify CaSR, TRPC1, TRPC3, TRPC4 and TRPC6 were as follows (5′-3′):CGGGGTACCTTAAGCACCTACGGCATCTAA,CaSR sense GCTCTAGAGTTAACGCGATCCCAAAGGGCTC;and antisense CGCCGAACGAGGTGAT,TRPC1 sense GCACGCCAGCAAGAAA;and antisense CGGCAACATCCCAGTG,TRPC3 sense CGTAGAAGTCGTCGTCCTG;and antisense CTCTGGTTGTTCTACTCAACATG,TRPC4 sense CCTGTTGACGAGCAACTTCTTCT;and antisense GCCAATGAGCATCTGGAAAT,TRPC6 sense TGGAGTCACATCATGGGAGA.and antisense −ΔΔCt.PCR cycling conditions for CaSR included one cycle of 10 min at 95°C, 35 cycles of 30 s at 95°C, 30 s at 55°C and 60 s at 72°C, and one cycle of 10 min at 72°C. The PCR products of CaSR were then separated on a 1% agarose gel and stained with ethidium bromide. Reverse transcriptase was omitted as a negative control for the RT-PCR to eliminate amplification from contaminating genomic DNA. All real-time PCR experiments were performed with SYBR Green PCR MasterMix  using an ABI PRISM 7500 . GAPDH was used as the internal control, and ΔΔCt was calculated for each sample with the expression levels indicated by values of 22+]o. At the end of the 24 h incubation, the cells were harvested for western blot analysis. Anti-CaSR antibody , anti-TRPC1, -TRPC3, -TRPC4, or -TRPC6 antibodies  or an anti-actin antibody  were used as primary antibodies. Fluorescence-conjugated goat anti-rabbit or goat anti-mouse IgG antibodies  were used as secondary antibodies. Western blot bands were quantified using the Odyssey infrared imaging system .Western blot was performed using a standard protocol. Human MCs were starved for 24 h in a serum-free medium prior to stimulation with high i was expressed as a pseudo-ratio value of the actual fluorescence intensity divided by the average baseline fluorescence intensity. Calibrations were performed immediately following each experiment. Data from 20 to 40 cells were summarized in a single run, and at least three independent experiments were conducted.MCs were grown on coverslips and loaded in 1% physiological saline solution containing Pluronic F-127  and Fluo-3/AM  at 37°C for 45 min. After washing, the coverslips with cells were placed in a chamber containing HEPES-buffered Na2+]o in the presence or absence of various inhibitors for 24 h. Eight hours before the end of incubation, BrdU was added to the medium, and cells then were continually incubated for 8 hours. The absorbance at 450 nm (reference wavelength 630 nm) was measured with a scanning multi-well spectrophotometer (Amersham Pharmacia Biotech). The absorbance values correlate directly to the amount of DNA synthesis and therefore to the number of proliferating cells in culture. Stimulation is expressed as fold proliferation over basal growth of the control set as unity.Cell proliferation was measured by a Cell Proliferation ELISA BrdU kit  according to the manufacturer's protocol. Cells were seeded in a 96-well plate (5000 cells/well) and cultured for 24 h. After starvation for another 24 h in serum-free medium, cells were then incubated in the same medium supplemented with different o from 1 to 5 mM evoked a rapid peak of [Ca2+]i and a subsequent sustained increase in [Ca2+]i. The increase in the [Ca2+]i due to the [Ca2+]o occurred in a concentration-dependent manner, as shown in 2+]o that was necessary to achieve the [Ca2+]i response in the human MCs was approximately 4.93 mM. A similar effect was observed with the use of 3 mM spermine, another CaSR agonist , an endoplasmic reticulum Ca2+-ATPase inhibitor, further indicating that CaSR agonists stimulate Ca2+ release from intracellular stores  or SN-6  had no apparent effect . Taken together, these results strongly suggest the requirement of TRPC3 and TRPC6 in the CaSR agonist-induced [Ca2+]i increase.To investigate whether TRPC3 and TRPC6 are involved in [Cachannels . Compareincrease . Transfe2+ influx was significantly reduced by transfection with TRPC3 siRNA and TRPC6 siRNA compared with transfection with scrambled siRNA  and other intracellular vesicular store compartments. As shown in 2+]i, which was due to SOCE, and 3 mM spermine evoked an additional substantial increase in [Ca2+]i under conditions where Ca2+ stores were already depleted. However, the additional substantial increase in [Ca2+]i was blocked by NSP2390, a CaSR antagonist. Similar results were obtained with a 5 mM [Ca2+]o-induced calcium influx . The cell viability was not affected under our experimental conditions (data not shown). Consistent with previous reports, the promotion of cell proliferation by [Ca2+]o appeared to act through CaSR stimulation because pretreatment of cells with 10 µM NPS2390 significantly inhibited [Ca2+]o-mediated cell proliferation  2+ binding to CaSR allows CaSR to function as a sensitive detector of Ca2+, thereby quite easily distinguishing small (∼200 µM) fluctuations in the [Ca2+]o2+]o is approximately 1.3 mM CaSR can be activated by two types of agonists. Type I agonists are divalent cations, such as Ca2+]i increase through interactions between CaSR and PLC, which are mediated by the Gq/11 subunits of heterotrimeric G proteins 2+ sources in different cells. Upon CaSR simulation, intracellular Ca2+ is released from internal stores, as has been demonstrated in a variety of cell types; however, relatively little is known about the contribution of Ca2+ entry to the [Ca2+]i increase. Here, we show that CaSR activation by agonists, both high [Ca2+]o and spermine, produces a PLC-dependent [Ca2+]i increase in human MCs. The CaSR activation-induced [Ca2+]i increase is composed of an initial rapid increase and followed by a sustained increase, consistent with a previous study in mouse MCs 2+ mobilization, and the sustained elevation may represent activation of Ca2+ influx. Indeed, our data indicate that CaSR stimulation induces TRPC-dependent calcium entry as well as calcium release from intracellular stores. Further, our results suggest that TRPC3 and TRPC6 may be responsible for the CaSR activation-induced calcium influx because of the following: (i) stimulation of CaSR by high [Ca2+]o enhanced the expression of TRPC3 and TRPC6, rather than of TRPC1 and TRPC4, and (ii) siRNA targeting of TRPC3 and TRPC6 attenuated the CaSR activation-induced calcium influx. Previous patch clamp experiments revealed that stimulation of CaSR induces TRPC-like nonselective cation currents in HEK293 cells stably transfected with CaSR 2+ entry, such as TRPC3 in salivary ductal cells 2+ entry.Generally, exposure of cells to CaSR agonists commonly elicits a [Ca2+]i increase. Moreover, further experiments with TG to deplete intracellular stores before CaSR stimulation revealed that the effects of CaSR agonists on [Ca2+]i still occurred, suggesting that calcium influx stimulated by CaSR agonists do not require depletion of intracellular stores. This idea is supported by the results obtained in osteoclasts and aortic smooth muscle cells, where CaSR activation mediates ROCE 2+ entry in prostate smooth muscle cells 2+ entry in a cell-specific manner. In this study, we cannot exclude the possibility that SOCE occurs simultaneously with ROCE upon CaSR activation because CaSR stimulation induces Ca2+ release from intracellular stores and can thereby directly or indirectly affect the TRPC3 and TRPC6 activities In MCs, as in other cell types, TRPC3 channels and TRPC6 channels are considered to be ROCs 2+]o-induced mesangial cell proliferation. A similar role was described in MCF-7 breast cancer cells. However, whether the role of TRPC3 and TRPC6 in the CaSR stimulation-induced cell proliferation is mediated by changing intracellular calcium or results from multiple postreceptor responses has yet to be determined. Regardless, our study demonstrates that CaSR modulates extracellular calcium entry and proliferation via TRPC3/6 channels in human MCs.Finally, we determined the role of TRPC3 channels and TRPC6 channels in CaSR stimulation-induced cell proliferation, showing that both pharmacological blockage and siRNA knock down of TRPC3 and TRPC6 inhibited high [Ca


KIT mutations in core-binding factor acute myeloid leukemia (CBF-AML), including inv(16) and t AML, is uncertain. We performed a systematic review and meta-analysis of the effect of KIT mutations on the complete remission (CR) and relapse rates and overall survival (OS) of CBF-AML. PubMed, Embase, Web of Science, and the Cochrane Library were searched and relevant studies were included. Negative effect was indicated on relapse risk of CBF-AML  and t AML , not on OS of CBF-AML , CR , relapse risk  or OS  of inv(16) AML. Subgroup analysis of t AML showed negative effect of KIT mutations on CR , relapse risk  and OS  of non-Caucasians, not on CR  or OS  of Caucasians. This study indicates KIT mutations in CBF-AML to be included in the initial routine diagnostic workup and stratification system of t AML. Prospective large-scale clinical trials are warranted to evaluate these findings.The prognostic significance of  KIT mutations, as potential molecular markers, are found in 12–46% of t patients and 9–53% of inv(16) patients  and inv(16)p13q22)/t [abbreviated as inv(16)] genetic abnormalities are termed as core-binding factor (CBF)-AML. To date, patients with CBF-AML are generally recognized as a favorable cytogenetic AML sub-group [q22/t OR free text search terms “KIT” (“kit” OR “CD117” OR “Stem Cell Factor Receptor” OR “SCF Receptor” OR “SCFR”)] AND . Similar search terms were used for the Embase, Web of Science, and Cochrane Library databases. The reference lists of identified articles were manually searched for additional studies of interest.The first direct evidence of  in 1998 ; therefoKIT mutational status. The following exclusion criteria were used: published as an editorial, letter, review, expert opinion, or case report; had no available prognostic data; was a subset of articles by the same author .The following inclusion criteria were used: published as an original article in English; focused on patients with CBF-AML, included inv(16), t(8.21), or both; and evaluated any prognostic outcome, including OS, relapse rate, or both, according to Two reviewers (W.L.C and H.X) independently evaluated the titles and abstracts of the identified publications. Potentially relevant articles were retrieved in full. The final inclusion of articles into our systematic review was based on agreement between both reviewers.et al. [KIT mutations, study design, outcome data, including hematological complete remission (CR) rate, relapse rate, and OS using the KIT mutational status from each study. To minimize publication bias towards articles that only described significant or relevant data, we contacted all authors for additional data on all evaluated CBF-AML prognostic factors.Data on the characteristics of the selected studies were extracted, according to the guidelines presented in “Systematic Reviews of Genetic Association Studies” by Sagoo et al. . BetweenTwo reviewers (W.L.C and F.C.K) independently assessed the study quality. Disagreements were resolved through a joint reevaluation of the original article. The Newcastle-Ottawa Scale (NOS)  was usedhttp://www.cochrane.org/) and with STATA version 12.0 . We calculated the CR, the relapse rates and 5-year OS for each sub-category within the study. The CR, relapse and OS were defined as described previously [KIT mutational status. The risk ratio (RR) was used to determine the probability of relapse rate and 5-year OS based on the KIT mutational status. For relapse risk, we calculated and analyzed the relapse rate prognostic data, including the relapse rate, relapse incidence, cumulative incidence of relapse of the selected studies. An OR or RR > 1 indicated that the KIT mutations were significantly associated with worse outcomes. Given the small number of studies, inter-study heterogeneity, or insufficient reporting, we did not pool the results for other outcomes.Meta-analysis was performed with RevMan version 5.2, according to the Cochrane Collaboration recommendations  , 12, 29 KIT mutations and CBF-AML ; the average NOS score was 7.3 .KIT mutations on CBF-AML . For OS, we evaluated five studies [P = 0.47, I2 = 0%). For relapse risk, nine studies [P = 0.28, I2 = 18%) and 1.43 , respectively. These results showed that the KIT mutations did not impact the CR or OS in CBF-AML cases treated together as an group. However, an analysis of the 2- and 5-year data showed that patients with KIT mutations had a significantly higher relapse risk. The P-values and I2 statistics for the above data indicated low heterogeneity, and the Begg’s and Egger’s tests indicated no publication bias. CBF-AML patient outcomes, according to the KIT mutations, are shown in We analyzed three primary end points  to investigate the prognostic impact of  CBF-AML . For CR, studies , 17, 42  studies , 39, 42  studies , 29, oneThough CBF-AML is often considered together in one prognostic group, it has been reported that t8,21) and inv(16) AML appear to be distinct clinical entities 1 and inv. AccordiP = 0.48, I2 = 0%) and 0.95 l. These data indicate that the KIT mutations did not affect CR in inv(16)AML, and they had a mild but statistically non-significant effect on CR in t AML. These results are consistent with those of previously published cohort studies [For the subgroup analysis of CR, we included nine t8,21) AML studies ,21 AML s, 39, 42 R in inv1AML, and  studies , 39. TheP = 0.52, I2 = 0%) and 1.70 , respectively. These results suggest a significant KIT mutation-related increase in the relapse risk of t AML. There was no evidence of heterogeneity and no publication bias across studies  and 1.18 . Moderate heterogeneity was observed across these studies; publication bias was detected with the Begg’s and Egger’s tests, and the P-values were P = 1.000 and P = 0.324, respectively, suggesting a low probability of publication bias. Trim-and-fill analysis detected one imputed study, which would not have affected our results. In the “leave-one-out” sensitivity analysis of this subgroup, omitting any single study did not lower the I2 further than 47%, with no significant changes in the estimated RRs. These results suggest that, unlike t AML, there was no significant KIT mutation-related increase in relapse risk of inv(16) AML. In addition, when we focused on the 5-year relapse risk, analysis of the subgroup differences between inv(16) and t AML showed that P = 0.02 and I2 = 82% in the fixed-effects model, and P = 0.1 and I2 = 63% in the random-effects model. Based on the subgroup difference, it appears that when KIT mutations are considered, the CBF-AML relapse rate should not be evaluated together but in subgroups. For the subgroup analysis of relapse risk, we used eight t8,21) AML studies ,21 AML s, 39, 42  studies . All sixP = 0.04, I2 = 58%) in the fixed-effects model and 1.42  in the random-effects model. Moderate heterogeneity was found in these studies; we conducted publication bias analysis, and the Begg’s and Egger’s tests P-values were P = 0.452 and P = 0.023, respectively; the Egger’s test P-value indicated publication bias. We used the trim-and-fill method to calculate an adjusted pooled random-effects RR to evaluate the influence of potential publication bias. This method added three estimates to balance the funnel plot  AML. For the inv(16) AML subgroup, the pooled RRs of OS were 1.03  and 1.01  in the fixed- and random-effects models, respectively, suggesting no significant KIT mutation-related decrease in the OS of inv(16) AML. No significant heterogeneity and publication bias were found across the inv(16) AML studies, and the Egger’s and Begg’s tests P-values were P = 0.902 and P = 0.904, respectively. In addition, a subgroup difference between inv(16) and t AML for OS was observed as P = 0.12 and I2 = 58% in the random-effects model and P = 0.03 and I2 = 78% in the fixed-effects model, which indicated that, as with the relapse rate, OS of CBF-AML should be evaluated in subgroups with in terms of the KIT mutations. For the subgroup analysis of OS, six t AML studies (498 patients), 39, 42,nel plot , and theet al. [P = 0.006) and with shorter OS than Caucasians when certain secondary cytogenetic abnormalities were present, indicating that ethnicity is an important predictor in t AML. In addition, the pooled RRs for OS of t AML in the present study were not stable; therefore, we performed a subgroup analysis of t AML according to race. We subdivided patients with t AML into Caucasian and non-Caucasian subgroups, and the impact of KIT mutations on CR, relapse risk, and OS of the subgroups was analyzed separately based on the presence or absence of KIT mutations  and 2.03 , suggesting that the KIT mutation has an adverse impact on the CR of non-Caucasian patients but not on that of Caucasian patients. No heterogeneous data were observed. For relapse risk in non-Caucasians, two studies provided 2-year relapse data [P = 0.26, I2 = 24%), 2.33 , and 2.03 , respectively. However, in Caucasians, three studies only provided a 5-year relapse data [P = 0.70, I2 = 0%). These data indicated a significant KIT mutation-related increase in relapse risk in both subgroups. As for OS, three studies [P = 0.26, I2 = 27%) and 2.26 , respectively. No heterogeneous data were observed. These OS-related results showed that, similar to the CR rate but unlike the relapse risk, the KIT mutations adversely affected the OS of the non-Caucasian patients but not that of the Caucasian patients.Marcucci et al.  reportedutations . For CR,ctor in t,21 AML. ctor in t,21 AML. pse data , and onepse data , and thes P = 0.7, I2 = 0% studies , 39, 42 KIT mutations did not affect inv(16) AML, and ethnicity did not affect the clinical outcomes in this subgroup, nevertheless, we also performed the relevant analysis for the inv(16) AML subgroup. No significant effects of race were observed  and inv(16) AML subgroups, which was insufficient for the meta-analysis but still systematically reviewed  AML. Manara et al. [KIT mutations demonstrated no significant impact on the prognosis of inv(16) AML; however, Shamda et al. [KIT mutation-related risks in the prognosis of t AML also exists in pediatric patients; the systematic review also implied that pediatric CBF-AML should not be treated as a group but rather as t and inv(16) AML subgroups. Thus, these results are consistent with the results of our meta-analysis.In this systematic review, six included studies , 29, 38 d et al. , which i. [et al. and Manal.[et al.  com comKIT m 19–44% , 16), an, anKIT m  and t AML subtypes [KIT mutations did not affect the CR of inv(16) or t AML, and the KIT mutation-related relapse risk of t AML was significantly increased, but it was not increased in inv(16) AML, indicating that the increased relapse risk of CBF-AML may be due to the risk of t AML but not inv(16) AML. Furthermore, in inv(16) AML, the OS was not significantly affected by the KIT mutations; however, the OS tended to be shorter in t AML patients with KIT mutations. To date, inv(16) and t AML have been considered to have relatively favorable prognoses and to have been treated similarly. However, with the distinct effects of KIT mutations in our subgroup analyses, CBF-AML with KIT mutations should be regarded as distinct and heterogeneous entities with different outcomes.This study reports the first meta-analysis (to our knowledge) evaluating the impact of reported , 39, 43, the inv1 and t and t8,1 AML subKIT mutations were found to adversely impact the CR of non-Caucasian patients but not that of Caucasian patients, a finding that has never been reported and is consistent with the findings of a study involving non-Caucasian patients [KIT mutations increased the relapse risk in both non-Caucasian and Caucasian patients, and they adversely affected the OS of non-Caucasian patients but not that of Caucasian patients. A study by Marcucci et al. also reported that the nonwhite patients with t have higher odds of failing induction therapy compared with the corresponding white patient population [KIT mutations should be evaluated according to ethnicity, which is consistent with other studies [KIT mutations in OS of t AML as an entity in the present analysis. A similar analysis for inv(16) AML revealed that ethnicity is not a risk factor for this CBF-AML subtype with KIT mutations; the potential risk factors require further investigation.Because the meta-analysis result of the OS in t8,21) AML was not definitive, using race as a risk factor in t AML [1 AML waspatients . Moreove studies , 11, 39 KIT mutations were of prognostic relevance [KIT mutations in pediatric CBF-AML, which requires future confirmation.In contrast to adult CBF-AML patients, most pediatric studies did not show that the elevance , 13, 38.elevance , 7 from KIT mutations occur mainly on exon 8 or 17, and it is unclear whether the prognostic significance differs according to the subtypes of CBF abnormalities [KIT mutations should be considered as an entity, not in genotypes, for the prognostic effect in CBF-AML, which must be determined in further high-quality studies. Note that the detection method potentially influences the evaluated result in this study, as reported by Wakita et al. [In CBF-AML, malities , 39, whimalities , 7, 15 oa et al. , and morKIT mutations could not be technically analyzed. Allo-SCT, as in the included studies for the meta-analysis, was administrated in pediatric patients if a suitable donor was available [KIT mutations in the reported patients and to the limited patients (with or without KIT mutations) received SCT. In adult patients, most studies excluded the patients who received SCT from the study or analysis [KIT. Another study [KIT was not considered, which was also reported by Zhu et al [KIT mutations has not yet been determined. As for FLT3 mutations, the potentially risky mutations in CBF-AML, they have been evidenced to negatively affect clinical outcome of CBF-AML [FLT3 and KIT mutations have a even worse prognosis [FLT3 mutations in the outcomes of CBF-AML with KIT mutations could not be analyzed or determined. More substantial studies are necessary. For other mutations, such as RAS, JAK2, IDH and WT1, no prognostic significance has been found in CBF-AML [et al.[KIT mutations were associated with higher WBC counts, which subsequent studies did not support [KIT and FLT3 mutations had significantly higher WBC counts compared to patients without the mutations [KIT mutations were significantly associated with higher WBC counts in inv(16) AML but not in t AML (data not shown). In the present analysis, however, the KIT mutations demonstrated obvious effects on the clinical outcomes of t AML patients. These findings suggest that higher WBC counts are not a confounding risk factor for our KIT mutation-related analyses of the clinical outcomes of t AML patients.There might be other associated predictors, such as differences in clinical characteristics, chemotherapy-related AML, transformed AML, WBC count, age and individualized treatment. Increasing age has been reported to be the best predictor for survival of CBF-AML patients aged less than or equal to 60 years at univariate and multivariate analysis ,6,17. Invailable , 4, 7, banalysis , 29, 42.analysis , the pathu et al . Thus, t CBF-AML , 41, and CBF-AML , 41, 49. CBF-AML , 42. EarL [et al. reported support , 17. TheKIT transcript expression levels in CBF-AML were not considered, although Allen et al. reported that the levels of KIT mutations are clinically meaningful for prognoses of CBF-AML [KIT mutations, which may have also resulted in data heterogeneity. Finally, publication bias is inevitable, although we did not detect it directly, and it has been reported that the occurrence of tAML and inv(16) AML differ according to geographical location [One limitation of this meta-analysis is that we used data that were abstracted from published reports, and we performed study-level analysis; the substantial effect of heterogeneity must be considered. There are other potential limitations. First, observational prospective studies for a rare disease are logistically difficult to conduct, and the observational nature of the available studies renders such studies unavoidably open to the influence of residual confounders. Second, a relatively small number of studies could be included. Third,  CBF-AML . Fourth,location . In addiKIT mutations to CBF-AML, performing subgroup analysis according to CBF abnormalities and patient ethnicity, thereby rendering this meta-analysis more powerful than any individual study. Based on the current evidence, these data present the most comprehensive view, to date, of the prognostic significance of CBF-AML. Additionally, we systematically reviewed the summarized association between KIT mutation genotypes and CBF-AML, and KIT mutations and pediatric CBF-AML. We did not exclude any article during the identification and selection process, and only the P-value of the Egger’s test indicated publication bias in terms of OS for t AML as an entity. Similarly, the heterogeneity tests indicated little variability between studies that cannot be explained by chance. Moreover, we have performed several sensitivity analyses to examine the potential sources of heterogeneity and to evaluate robustness in the subgroups. In conclusion, this study indicates that KIT mutations are the key risk factors for the prognosis of t AML but not inv(16) AML, thereby supporting the inclusion of testing for KIT mutational status in the initial routine diagnostic workup and stratification system of t AML. We also demonstrated the negative role of ethnicity  in the CR and OS of t AML with KIT mutations. These results support the early prediction of the worse prognoses, as well as effective minimal residual disease monitoring of patients with t AML with KIT mutations. It would be valuable to include KIT mutations, even the genotypes, as prognostic factors for risk prediction in prospective large-scale clinical trials. Until data from these future studies become available, the present analysis helps to define the prognostic significance of KIT mutations in CBF-AML, particularly the t AML subgroup.Despite these limitations, we specifically evaluated the relevance of S1 Fig(PDF)Click here for additional data file.S2 Fig(PDF)Click here for additional data file.S3 Fig(PDF)Click here for additional data file.S4 Fig(PDF)Click here for additional data file.S5 Fig(PDF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 Table(PDF)Click here for additional data file.S4 Table(PDF)Click here for additional data file.S5 Table(DOCX)Click here for additional data file.