However, its role in the spindle organisation in mitotic cells remains controversial. Here, we investigated the role of Ca2+ on kinetochore fibres in living cells. We found that depletion of Ca2+ during mitosis reduced kinetochore fibre stability. Reduction of kinetochore fibre stability was not due to direct inhibition of microtubule polymerisation by Ca2+-depletion but due to elimination of one dynamic component of kinetochore, CENP-F from the kinetochore. This compromised the attachment of kinetochore fibres to the kinetochore which possibly causes mitotic defects induced by the depletion of Ca2+.The attachment of spindle fibres to the kinetochore is an important process that ensures successful completion of the cell division. The Ca The attachment of microtubules to kinetochores of each sister chromatid is crucial for successful cell division. Efficient kinetochore-microtubule attachment is determined by the assembly of intact kinetochore and microtubule dynamics, including de-/polymerisation. Defects in these components impair attachment and chromosome alignment at the spindle equator, which possibly leads to mitotic arrest.During the mitosis of animal cells and some fungi, spindle microtubules grow from the microtubule-organising centre to capture and align chromosomes at the cell equator. Microtubules attach to the kinetochore located at the chromosome centromere and these microtubules are called kinetochore microtubules (or K-fibres)2+ is a universal secondary messenger involved in several processes inside cells. Concentration of Ca2+ in cells is critical for the cell cycle progression. During mitosis, intracellular Ca2+ increases and induces chromosome condensation, spindle fibre formation and chromosome segregation, which are processes required for successful cell division4. The precise role of Ca2+ on spindle fibre formation during mitosis remains unresolved, although several previous studies imply their relationship. According to Xu et al., the mitotic spindle structure is affected by Ca2+-depletion in the concentration dependent manner, in which the mitotic spindle continues to shrink under the treatment, suggesting microtubule disassembly5. An increase in the Ca2+ concentration during the mitosis is thought to induce microtubule depolymerisation leading to anaphase onset4, however the injection of Ca2+ saturated calmodulin during prometaphase prolongs the duration to anaphase onset3 similar to the injection of EGTA-CaCl2 solution to lower intracellular Ca2+ concentration beneath 100 nM2, and the shortened spindle microtubules was observed in both treatments. Factors required for microtubule polymerisation have been characterised using in vitro systems and it was found that self-assembly of microtubules from brain tubulin extracts requires the presence of Mg2+ and GTP8, and Ca2+ should be removed using chelating reagents9. However this might not fully explain the effect of Ca2+ on microtubule polymerisation in a living cell which is more complicated. According to Ochieng et al., human breast epithelial cells displayed extensively polymerised cytoplasmic microtubule complexes when cultured in high Ca2+ medium. While in low Ca2+ medium, tubulins in majority of the cells were in depolymerised state10. This suggests that high level of Ca2+ favours either microtubule polymerization or stabilises the polymerised state of tubulin.Ca2+ during mitosis also causes global chromatin decondensation11. Because the assembly of kinetochore occurs in stepwise manner from the innermost to outermost layer, decondensed chromatin might interfere the formation of heterochromatin which makes up centromeric chromatin that supports kinetochore structure13, or interrupting the organisation of the constitutive centromere associated network (CCAN) by affecting centromeric chromatin folding similar to CENP-depleted chromosome14.Depletion of intracellular Ca2+ during mitosis, particularly the effects of Ca2+-depletion on spindle fibre formation and stability in the cells. We found that the depletion of Ca2+ during mitosis reduced the stability of kinetochore microtubules. The effects of Ca2+-depletion on spindle fibre formation were confirmed for the first time, with live cell imaging showing that the formation of spindle microtubules was not inhibited by the depletion of Ca2+. The loss of spindle fibre stability under Ca2+-depletion was the most likely due to the loss of CENP-F (mitosin), a dynamic component of the fibrous corona, from the kinetochores.In this study, we present an additional role of Ca2+ on the stability of kinetochore fibre, microtubules with acetylated tubulin (Ac-tubulin) which have slower turnover rate than non-acetylated microtubules, indicating higher stability15, was detected. HeLaWT cells were arrested at metaphase using MG132, a proteasome inhibitor, without disruption to spindle fibres, and immunofluorescence assay against Ac-tubulin was performed. In control metaphase cells, intense signal of Ac-tubulin was observed through the length of kinetochore fibres, showing stable microtubules  within 10 min after the release from nocodazole treatment and remained above 2-fold increase until the end of the observation. A similar phenomenon was observed in the treatment with BAPTA and ionomycin, in which 3-fold increase in total mCherry-α-tubulin intensity was also observed within 10 min after the release of nocodazole in BAPTA-treated cells. In BAPTA-AM treatment, although the total mCherry-α-tubulin intensity increased after the release of nocodazole, the increment was obviously small compared with cells in control and BAPTA-ionomycin treatments. These results clearly indicate that Ca2+-depletion alone does not prevent microtubule polymerisation and the inhibition of microtubule repolymerisation by BAPTA-AM might be independent from its Ca2+-chelation activity (see Discussion).Total mCherry-α-tubulin intensity at each time point after the release of nocodazole was measured and compared with the intensity at the first time point after release  localises at the centromere by recognising and binding to trimethylated histone H3 lysine 9 (H3K9me3), and provides itself as a mark to anchor other inner kinetochore proteins19. Mislocalisation of H3K9me3 due to chromosome decondensation may prevent the localisation of other centromeric proteins, including HP1α, to the centromere20. H3K9me3 signals were detected at pericentromeric regions  of chromosomes, consistent with previous studies  direct effects of Ca2+-depletion on the de-/polymerisation of microtubules and (ii) defects in kinetochore structure. For the first possibility, Ca2+-depletion by treatment of the cells with BAPTA-AM, BAPTA-AM derivatives or EGTA may inhibit microtubule polymerisation because these treatments decrease the total amount of ATP inside cells23, and in the presence of GTP, ATP increased the rate and extent of microtubule assembly24. However, as demonstrated by live cell imaging, Ca2+-depletion did not interfere with the microtubule polymerisation process  responsible for H3K9me3 but not H3K9me1 and H3K9me2, increases in mitotic cells26. H3K9me3 is lost from the centromeric regions of the cells with chromosome instability. Reduction of H3K9me3 and HP1 from chromatin centromeric regions was detected after TSA treatment, which caused global chromatin decompaction in HTC116 nuclei20. Pericentromeric localisation of H3K9me3 could be regulated by altering SUV39H1 activity. Disruption of the SUV39H1 gene led to the loss of H3K9 methylation from pericentric heterochromatin. SUV39H1 activity is inhibited by the binding of deleted in breast cancer 1 (DBC1) to its catalytic domain, preventing SUV39H1 methylating histone H3. Factors, for example, tumour suppresser gene, KLLN, can also regulate H3K9me3 by altering SUV39H1 activity through interaction with DBC127. Interestingly, the C-terminus of DBC1 contains an EF hand domain that binds Ca2+. However, this EF hands may not be functional because it is unlikely to bind to Ca2+, as observed for the Nudix domain28. In addition, Ca2+ contributes to chromosome condensation after NEB, unlike other chromosome condensation factors, such as Topoisomerase IIα and histone post translational modifications. H3K9 may already be methylated by SUV39H1 and localise to the centromere before Ca2+ gains access to chromosomes to promote condensation. Therefore, the localisation of H3K9me3 in less compact chromosomes-induced by Ca2+-depletion remained unchanged, and the localisation of HP1, which recognises and binds to H3K9me3, was not affected by Ca2+-depletion.For the second possibility, kinetochore structures might be impaired owing to Ca2+-depletion. Some conserved kinetochore components, such as Ndc80, KNL1 and Dsn1, which are crucial for kinetochore assembly, require Aurora B for their localisation30. Since Aurora B remained at the centromere under the condition of Ca2+-depletion, the localisation of those kinetochore proteins should not be impaired.Similar to H3K9me3 and HP1α, the localisation of Aurora B kinase to the centromere was not affected by Ca2+ also did not prevent the localisation of static components of kinetochore, Mis12 and Hec1, but affected kinetochore localisation of CENP-F. Mis12 directly interacts with HP1α31 and the localisation of Mis12 to the outer kinetochore requires HP1α, because a reduction of HP1α by RNAi abolishes kinetochore localisation of Mis1232. Because of the observation that HP1α could localise normally to the centromere when Ca2+ was depleted, the recruitment and localisation of Mis12 to the kinetochore was not disrupted. Hec1 is another static outer kinetochore component that directly interacts with spindle fibres at the kinetochore-microtubule interface. Hec1 requires Aurora B phosphorylation at its N-terminus to regulate microtubule attachment and plus-end microtubule polymerisation33. Loss of Hec1 from the kinetochore results in complete loss of kinetochore-microtubule attachment34. However, when Ca2+ was depleted, Aurora B localised normally to the centromere and some microtubules remained attached to the kinetochore. Thus, the localisation and function of Hec1 should not be affected by the depletion of Ca2+.Depletion of Ca2+ was depleted. CENP-F is essential for the firm assembly of the kinetochore-microtubule interface because CENP-F depleted cells exhibited mitotic delay and a decrease in the stability of kinetochore microtubules38. Although the interaction between CENP-F and microtubules, and its importance for microtubule–kinetochore attachment are well studied, the recruitment of this protein to the kinetochore is still inconclusive. A spindle checkpoint protein Bub1 and a kinetochore scaffold KNL1 were reported to be crucial for localisation of CENP-F to the kinetochore40. The recruitment of CENP-F to kinetochore could also be mediated by a small GTPase Rab541. Rab5-silenced cells exhibited mitotic delay and chromosome misalignment, similar to CENP-F-depleted cells41. Rab5 formed a complex with CENP-F, and promoted efficient localisation of CENP-F to kinetochore41. Besides Rab5, the contribution of Rab11, the other Rab family protein, to the organisation of mitotic spindle was also reported42. Depletion of Ca2+ might inhibit small GTPase activity either through guanine nucleotide exchange factors possessing EF hands, or the binding of Ca2+/calmodulin to the small GTPases43. Severe reduction of the tubulin acetylation and cold-stable microtubule in BAPTA-AM treatment  supplemented with 10% fetal bovine serum (Biosera) at 37 °C with 5% CO2. The following drugs were used for cell treatments: thymidine , nocodazole , MG132 , BAPTA-AM , BAPTA , ionomycin  and Fura-2 am .HeLaThe following primary antibodies were used for indirect immunostaining: mouse monoclonal anti-α-tubulin , mouse monoclonal anti-acetylated tubulin , rabbit polyclonal anti-CENP-B , rabbit polyclonal anti-histone H3K9me3 , mouse monoclonal anti-HP1α , rabbit polyclonal anti-Aurora B , mouse monoclonal anti-HEC1 , rabbit polyclonal anti-Mis12 , mouse monoclonal anti-CENP-F , and human anti-centromere autoantibody .2+ was measured using Fura-2 am as previously described in Phengchat et al.11. In brief, before the treatment with BAPTA-AM or BAPTA and ionomycin, cells were incubated with Fura-2 am for 45 min at room temperature and further 20 min at 37 °C to allow completely hydrolysis of AM ester. Medium was replaced with fresh phenol red-free medium containing BAPTA-AM or BAPTA and ionomycin. After 2 h incubation, the cells were placed on of the inverted microscope equipped with a temperature-controlled chamber set at 37 °C with CO2 supply (Olympus IX71) and a 40× 1.35 UApo/340 Iris oil immersion objective lens. Fluorescence images of Fura-2 were sequentially acquired by 340 and 380 nm excitation wavelength and measured emission at 510 nm. Quantification of fluorescence at 510 nm after excitation at 340 and 380 nm (F340/F380 ratio) was used as an index for intracellular Ca2+ levels using ImageJ .Levels of intracellular CaHeLa cells were cultured on glass coverslips and synchronised with double thymidine block. Cells were arrested at metaphase using MG132 for 1 h then treated with either BAPTA-AM or BAPTA and ionomycin for 2 h. For microtubule stability (cold-treatment), all coverslips were transferred to ice-cold medium and incubated for 20 min on ice before fixation. For choromosome spreads, HeLa cells were synchronised with double thymidine blocks and arrested in prometaphase with nocodazole for 12 h then treated with BAPTA-AM for 2 h. Cells were collected for the metaphase-chromosome spread using the following method. Cells were treated with hypotonic solution (75 mM KCl) for 15 min at 37 °C and were cytospun onto coverslips. Cells were fixed with either 4% para-formaldehyde for 10 min and permeabilised using 0.5% Triton X-100/PBS for 10 min, or ice-cold methanol at −20 °C for 10 min. Cells were blocked with 3% BSA/PBS for 1 h. All primary antibody reactions were performed at 4 °C overnight, except for HP1α, which was performed at 37 °C for 20 min. Secondary antibody reactions were performed at room temperature for 1 h. DNA was counterstained with Hoechst 33342. Samples were mounted in Vectorshield mounting medium (Vector Laboratories).Images were taken with a DeltaVision deconvolution microscope  using a 1.4 NA PlanApo 100× oil immersion objective (Olympus). The z-stack distance was 0.5–1 µm and the total z-stack thickness was 15–18 µm. Raw 3D images were deconvoluted using constrained iterative deconvolution and converted to 2D images with maximum projection using softWoRx (AppliedPrecision). Images were processed by background subtraction and fluorescence intensities were individually scaled to 0–255. Image processing and intensity measurements were performed using ImageJ.2-chamber set at 37 °C with a 100× 1.4 NA PlanApo 100× oil immersion objective (Olympus). For the microtubule depolymerisation/repolymerisation assay, metaphase cells with intense mCherry-α-tubulin and CENP-A-GFP signals were chosen. The cells were treated with 0.5 or 1 µg/ml nocodazole for 10–15 min, washed with PBS three times then released into pre-warmed fresh medium (containing indicated drugs) to allow microtubule repolymerisation. The z-stack images were taken every 2 min during release. Images were processed by background subtraction and fluorescence intensities were individually scaled to 0–255. Spindle microtubule regions were specified by segmented the grey scale images of mCherry-α-tubulin with auto-threshold function. Relative mCherry-α-tubulin intensity in spindle microtubule regions was calculated by normalising total fluorescence intensity of mCherry-α-tubulin at each time point after the release of nocodazole with that of the first time point (t = 0 min).U2OS cells cultured in 35 mm poly-L-lysine coated glass bottom dishes were synchronised with double thymidine blocking and DNA was stained with Hoechst 33342. Before performing time-lapse observations, the medium was discarded and replaced with phenol red-free DMEM containing 10% fetal bovine serum, 4 mM L-glutamine and 10 mM HEPES for microscopic observations. The cells were monitored under the temperature-controlled stage of the DeltaVision deconvolution microscope equipped with a COp < 0.05, 0.01 and 0.001, respectively.Statistical analysis was carried out in R using the Mann-Whitney-Wilcoxon test to compare the mean values. ‘*’, ‘**’ and ‘***’ indicate


However, current modalities for drug testing, such as electrophysiology and fluorescence imaging have inherent drawbacks. To circumvent these problems, we report the development of a bioluminescent Ca2+) gives a 140% signal change with Ca2+, and can image drug-induced changes of Ca2+ dynamics in cultured cells. Since bioluminescence requires application of a chemical substrate, which is consumed over ~ 30 min we made a dedicated microscope with automated drug dispensing inside a light-tight box, to control drug addition. To overcome thermal instability of the luminescent substrate, or small molecule, dual climate control enables distinct temperature settings in the drug reservoir and the biological sample. By combining GmNL(Ca2+) with this adaptation, we could image spontaneous Ca2+ transients in cultured cardiomyocytes and phenotype their response to well-known drugs without accessing the sample directly. In addition, the bioluminescent strategy demonstrates minimal perturbation of contractile parameters and long-term observation attributable to lack of phototoxicity and photobleaching. Overall, bioluminescence may enable more accurate drug screening in a high-throughput manner.GmNL(CaThe online version of this article (10.1186/s13104-018-3421-7) contains supplementary material, which is available to authorized users. For decades, a major bottleneck in drug development has been limited availability of patient-derived tissues or cells. This changed with the establishment of human-induced pluripotent stem cells (hiPSC) , 2, whicDrug-induced changes in hiPSC-CMs can be detected by many methods including classical and automated electrophysiology or established fluorescence imaging modalities , 8. HoweBy contrast biocompatibility of bioluminescence emission is robustly demonstrated in many eukaryotic phyla, and as such may provide a better short or long term imaging solution. Bioluminescent proteins (BPs) generate detectable emissions by catalyzing a chemical reaction which consumes a bioluminescent substrate while releasing a photon in the process. This makes them totally independent of external light, reducing phototoxicity. Recent developments of bright BPs such as Nano-lantern, a chimera of Renilla luciferase (Rluc) variant and a fluorescent protein (FP), enable bioluminescence imaging with high signal-to-noise ratio comparable to fluorescence imaging , 10. How2+ indicator GmNL(Ca2+) for hiPSC-CM imaging together with a customized light-tight box which contains a liquid dispenser and regional temperature control and can easily be installed on existing fluorescence microscopy. Combining GmNL(Ca2+) with the environmental modifications we demonstrate prolonged visualization of Ca2+ transients is improved by bioluminescence compared to fluorescence imaging. Consequently this strategy in hiPSC-CMs may bring value to drug development, particularly in chronic toxicology studies where there is an unmet need.Here we develop a new bioluminescent CaBamHI and KpnI. The digested PCR fragments were cloned in-frame into the KpnI/EcoRI sites of Nano-lantern(Ca2+)_600/pRSETB (Addgene#51976) for bacterial expression. To express the GmNL(Ca2+) in mammalian cells, PCR-amplified GmNL(Ca2+) genes were inserted into a pcDNA3 mammalian expression vector using BamHI and EcoRI sites.General molecular biology experiments were conducted as described . The seq2+)_variant proteins with N-terminal polyhistidine tags were expressed in E. coli. (JM109(DE3)), purified, and spectroscopically characterized as described  signals during spontaneous hiPSC-CM contraction in the customized microscope. GmNL(Ca2+) was expressed in cardiomyocyte spheroids by Adeno associated virus infection  was imaged at 20 Hz for 15 min, detecting periodic changes in bioluminescence signal during synchronized contractions . The amplitude of GmNL(Ca2+) did not change significantly during observation , in contrast to that of Fluo4 which reduced significantly under all illumination conditions tested (− 50 ± 8.5% at 62 mW cm−2 to − 99 ± 0.51% at 500 mW cm−2) indicative of dye loss by export or photobleaching   and Fluo4 at the lowest power density (62 mW cm−2) . At higher power density reduction in peak-to-peak interval  was seen suggesting extrinsic light illumination can cause significant phototoxicity in this system  improves long-term signal stability without physiological perturbations arising from extrinsic illumination. Interestingly the variation between hiPSC-CMs from GmNL(Ca2+) was greater than that from cells labelled with Fluo4.In addition, stability of beating parameters between the start and the end of the observation window was only seen with GmNL(Ca2+) can identify expected Ca2+ transient changes in hiPSC-CMs induced by well characterized drugs. After addition of isoproterenol, a non-selective β-adrenergic agonist used clinically to increase the heart rate, the mean peak-to-peak interval reduced   could correctly elicit the rate related Ca2+ transient alterations induced by Dopamine, and Doxazosin  is able to report bidirectional drug effects in hiPSC-CMs using a drug dispensing system based inside the on-stage environmental control conditions needed for bioluminescence imaging.Next we tested whether GmNL(Ca2+) enables imaging free from the problems of phototoxicity and photobleaching, which plague fluorescence imaging. In contrast to our expectations, GmNL(Ca2+) was slightly dimmer than Nano-lantern(Ca2+) at saturating Ca2+ concentrations. Saturation mutagenesis at the junction between the light donor and the FRET acceptor might improve the performance of GmNL(Ca2+). Although the GmNL(Ca2+) measurements in hiPSC-CM show stable Ca2+ transients between the beginning and the end of the observation window, the variation between hiPSC-CMs appears high in comparison with that from Fluo4. The observed variability might be attributable to heterogeneous infection of AAV or reduced penetration of the luminescent substrate into the spheroid culture model as either may lower bioluminescence signal.GmNL is intensiometric indicator, the oscillation from GmNL(Ca2+) in hiPSC-CMs should include the fraction of motion artefact in addition to Ca2+-dependent signal as previously shown [2+-insensitive probe will give the insightful information about motion artefact.As GmNL A series of pseudo-coloured ratio images of HeLa cells expressing GmNL(Ca2+), following 10 μM histamine stimulation (arrow). Scale bar, 10 μm. (b) Time course of the B/B0 ratio change at an ROI (white box in (a)). Number indicates the time point of each image in (a).Additional file 4. Evaluation of mean beat interval before and after treatment with either 10 μM Dopamine or 10 μM Doxazosin. Two-tailed Student’s t-test was performed. **p < 0.01; Data are presented as mean ± S.D.; n = 3.


Based on Ca2+ regulation from the junctional sarcoplasmic reticulum (JSR), a mathematical model was developed to investigate the interplay of clustered and rogue RyRs on Ca2+ waves. The model successfully reproduces Ca2+ waves in cardiac myocytes, which are in agreement with experimental results. A new wave propagation mode of “spark-diffusion-quark-spark” is put forward. It is found that rogue RyRs greatly increase the initiation of Ca2+ sparks, further contribute to the formation and propagation of Ca2+ waves when the free Ca2+ concentration in JSR lumen ([Ca2+]lumen) is higher than a threshold value of 0.7 mM. Computational results show an exponential increase in the velocity of Ca2+ waves with [Ca2+]lumen. In addition, more CRUs of rogue RyRs and Ca2+ release from rogue RyRs result in higher velocity and amplitude of Ca2+ waves. Distance between CRUs significantly affects the velocity of Ca2+ waves, but not the amplitude. This work could improve understanding the mechanism of Ca2+ waves in cardiac myocytes.Ca Based on the Fickian diffusion of cytoplasmic Ca2+, a computational model was developed to show the effects of rogue RyRs on Ca2+ waves under heart failure   model of Ca2+ waves in the cytoplasm was proposed with considering the distribution of clustered and rogue RyRs on the JSR membrane. The anomalous subdiffusion of Ca2+ in the cytoplasm and JSR Ca2+ regulation were also included. The stochastic opening Ca2+ release units (CRUs) of rogue and clustered RyRs was regulated by free Ca2+ concentrations in both cytoplasm and JSR lumen. With these features, we showed the importance of rogue RyRs on the initiation and propagation of Ca2+ waves.The objective of the study is to quantify the interplay of rogue and clustered RyRs on regulating Ca2+ in the cytoplasm  and Dy (= 150 μm2s−1) denote the Ca2+ diffusion coefficients for anisotropic diffusion. The anomalous subdiffusion order β is 2.25. Jdye is the flux due to the Ca2+ fluorescent indicator dye, Fluo-4-AM, in the cytoplasm. Jbuffer−cyto is the flux due to the endogenous stationary buffers. Jpump is the pumping rate of SR Ca2+-ATPase. SR pumps will be started when 2+ concentration level (0.1 μM). The detailed description is in the Appendix 2+ blinks in each JSR is written as:where [Ca2+]lumen is the free Ca2+ concentration in the lumen of a JSR. Jrelease−lumen denotes the Ca2+ release flux caused by opening of clustered RyRs (Jclustered) and rogue RyRs (Jrogue). Jbuffer−lumen is the Ca2+ flux due to the buffer, calsequestrin, in the JSR. Jrefill is the refilled Ca2+ flux to the JSR. The detailed description is in the Appendix where cyto.We have recently studied the effects of rogue RyRs on single Ca2+ waves could be triggered in a domain where [Ca2+]cyto is higher than the resting Ca2+ concentration level , , , and  with considering rogue RyRs. A comparison of Figures 2+ waves and higher amplitude when the effects of rogue RyRs are included in the computational model. Moreover, we found the “spark-diffusion-quark-spark” mode. Ca2+ released from clustered RyRs diffuses to a neighbor JSR, rogue RyRs are firstly activated in a stochastic manner to form Ca2+ quarks, and subsequently they make activation of clustered RyRs to produce a Ca2+ spark. The CRUs on the next z-line repeat the process to release Ca2+ in the cytoplasm.Ca2+ concentration with and without considering rogue RyRs at the line of y = 16.8. The results reveal that rogue RyRs accelerate the propagation of waves by triggering more Ca2+ sparks. The longitudinal propagating velocity has mean ± SD value of 95.9 ± 8.0 μm/s, which agrees with the experimental records   exists for generation of steady Ca2+ waves. Figure 2+ waves linearly increase with [Ca2+]lumen because of the large driving force ([Ca2+]lumen-[Ca2+]cyto) of Ca2+ sparks and quarks.In previous mathematical models of Ca2+ waves was performed with respect to the changes in CRU number of rogue RyRs in a JSR (Nrogue). Figure 2+]cyto at the line of y = 16.8 when CRU number of rogue RyRs in a JSR are 2, 8, and 14. As shown in Figure 2+ quarks and Ca2+ sparks.There are a large number of randomly-distributed rogue RyRs near clustered RyRs. Sensitivity analysis on Ca2+ release per CRU of rogue RyRs is mainly determined by the current through rogue RyRs (Irogue) and the duration of current flow (Trogue). The current and duration of rogue RyRs are 0.15 pA and 20 ms in Figures 2+ waves for different values of (Irogue × Trogue). There is a strong correlation between Ca2+ release through rogue RyRs and wave properties. When the release time of rogue RyRs decreases by half to 10 ms with the current of 0.15 pA, the longitudinal velocity and amplitude of waves decrease. When the current increases from 0.15 to 0.3 pA with the duration of 20 ms, Ca2+ waves have higher values of amplitude and longitudinal velocity. Hence, the release amount of rogue RyRs characterized by (Irogue × Trogue) is a significant parameter to affect wave properties.The Ca2+ sparks  restitution on alternans and spiral breakup. On the other hand, the present results suggest that Ca2+ release from JSRs is prone to be initiated to cause Ca2+ waves with the help of rogue RyRs when [Ca2+]lumen is large enough. Moreover, when the CRU number of rogue RyRs in a JSR or Ca2+ release per CRU of rogue RyRs is large enough, or the distance between CRUs is small, the membrane potential would be elevated significantly to trigger action potential in single myocytes.When a myocyte is under paced, several spontaneous Ca2+ waves in cardiac myocytes. According to our simulations, JSR Ca2+ overload could increase RyR opening probability and generate Ca2+ waves in heart (Williams et al., 2+ waves are significantly affected by the parameters of rogue RyRs, which may contribute to the formation of fibrillation (Macquaide et al., 2+ waves or dyssynchronous Ca2+ transients in myocytes of congestive heart failure (Louch et al., 2+ quarks through rogue RyRs may be a promising therapeutic target to prevent fibrillation in congestive heart failure.This study shows some implications to heart diseases relevant to Ca2+ waves relevant to the interplay of rogue and clustered RyRs. It addressed the importance of rogue RyRs to increase the initiation of Ca2+ sparks, the incidence and propagation of Ca2+ waves when [Ca2+]lumen is large. The amplitude and velocity of Ca2+ waves are in agreement with the experimental measurements. However, the model should be improved such that more parameters are added to investigate the mechanisms of Ca2+ waves. This study used the fixed release time of rogue and clustered RyRs. It should be a random variable depending on JSR regulation. The detailed structure of clustered RyRs should be taken into consideration because it can influence the frequency of Ca2+ sparks (Walker et al., 2+ waves in future studies.The present study showed Ca2+ waves in the cytoplasm. The computational results on Ca2+ waves agree with experimental measurements in cardiac myocytes. It shows that four neighbor Ca2+ sparks at the corner of a cardiac myocyte could induce Ca2+ waves. Ca2+ quarks increase the probabilities of triggering Ca2+ sparks and speed up the propagation of Ca2+ waves at high [Ca2+]lumen. A new wave propagation mode of “spark-diffusion-quark-spark” is put forward. Particularly, Ca2+ waves could occur only when [Ca2+]lumen is higher than a threshold value of 0.7 mM. More rogue RyRs in a JSR result in more opening CRUs of rogue and clustered RyRs. Besides, Ca2+ release from CRUs of rogue RyRs is a strong factor of wave properties. The velocity of Ca2+ waves is affected significantly by the distance between CRUs. This study helps to understand basic mechanisms of Ca2+ waves in cardiac myocytes.We developed a mathematical model to investigate the interplay of rogue and clustered RyRs on regulating CaXC, YH, and WT designed and performed the numerical calculation; YF analyzed and interpreted data; XC, YH, and WT wrote the manuscript.The 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.


Cold urticaria consists of an allergic immune response to cold temperatures with symptoms ranging from pruritic wheals to life-threatening angioedema, bronchospasm, or anaphylactic shock. Adequate planning to maintain normothermia perioperatively is vital due to impaired hypothalamic thermoregulation and overall depression of sympathetic outflow during deep sedation and general anesthesia. This case report describes the successful perioperative management of a 45-year-old female with a history of cold urticaria undergoing a laparoscopic Nissen fundoplication for refractory gastroesophageal reflux disease and discusses how to appropriately optimize the care of these patients. Cold urticaria, a subset of chronic urticarias, was first described in the 1860s and is characterized by the presence of chronically recurring wheals for a period longer than 6 weeks after exposure to cold stimuli . ChronicUrticaria represents a type I hypersensitivity reaction. Through an unclear mechanism, cold stimuli lead to mast cell or basophil degranulation, followed by the release of histamine and other inflammatory mediators , 7. The Cold urticarias are rarely associated with underlying disease. However, some infectious diseases, medications, and other pathologies [notably cryoglobulinemia and cryopyrin-associated periodic syndrome (CAPS)] have been associated with cold urticaria . In cryoA 45-year-old white female with a long-standing history of uncontrolled gastroesophageal reflux disease presented for laparoscopic Nissen fundoplication. The patient had a 14-year history of cold urticaria in addition to osteoarthritis, diverticulitis, and restless leg syndrome. Prior triggers for the patient’s cold urticaria included consuming ice chips, being in cold environments and colonoscopy bowel preparation. She was also allergic to naproxen, guaifenesin, and menthol. Her previous cold exposures had led to complications as varied as extremity numbness to airway compromise, anaphylaxis, and cardiac arrest.2 100% on room air.Her home medications included escitalopram, pramipexole, cetirizine, and ranitidine. Her surgical history consisted of a cesarean delivery and cholecystectomy, both prior to the diagnosis of cold urticaria. Neither she nor her family had a history of adverse reactions to anesthesia. At presentation for surgery, the patient’s physical exam was unremarkable, including a Mallampati class II oropharynx. Vital signs before induction were normal: heart rate 82 beats/min, blood pressure 122/80 mmHg, respiratory rate 16/min, SpOThirty minutes prior to induction, diphenhydramine 25 mg IV, famotidine 20 mg IV, hydrocortisone 100 mg IV, and midazolam 2 mg IV were administered. Intubation was uneventful after induction of general anesthesia with lidocaine, propofol, and fentanyl. Rocuronium was used for neuromuscular blockade, and anesthesia was maintained with desflurane. Normothermia was maintained with two warm blankets pre-intubation upon arriving to the OR and 2 separate forced air warming blankets over the lower and upper body after intubation. The OR temperature was maintained above 70°F. A fluid warmer was used on all intravenous fluids flowing into an 18-G peripheral venous catheter. Body temperature was monitored with both an oropharyngeal probe and a bladder probe. Epinephrine was prepared in advance and kept immediately on hand. Just before incision, the patient developed hypotension with a blood pressure of 69/30 mmHg, which was successfully treated with epinephrine. There was no evidence of urticarial rash or bronchospasm during this period. Hypertension and tachycardia resulting from epinephrine administration were treated with esmolol and by increasing the depth of anesthesia. There were no more episodes of hemodynamic instability for the remainder of the case.The surgical procedure progressed without complication. Paralysis was reversed with neostigmine, and extubation was uneventful. Hydromorphone was used for pain control during and after the case. In the postoperative care unit, the patient initially showed delirious disorientation to time and place, but this resolved after haloperidol 2 mg IV. Her vital signs and temperature remained stable during the recovery period with no evidence of an urticarial rash. Warm blankets were utilized to maintain normothermia while recovering from general anesthesia.Patients with cold urticaria undergoing general anesthesia risk developing anaphylaxis unless strict perioperative normothermia is maintained. Redistribution of core body temperature peripherally occurs within the first 30 min to an hour following induction of general or regional anesthesia as a result of sympathoplegia and vasodilation . This ca1 and H2 receptor blockers) and a single dose of corticosteroids. When providing general anesthesia for a patient with cold urticaria, prevention of the initial as well as final mechanism of anaphylaxis is a primary goal. To that end, famotidine, diphenhydramine, and a single dose of hydrocortisone administered intravenously 30 min prior to induction may deter immune histamine release. In addition, medications known to induce histamine release should be avoided as much as possible. While histamine release from drugs is not known to trigger cold urticaria, an urticarial reaction from any source can confuse the clinical picture of a patient with cold urticaria. Neuromuscular blocking agents such as rocuronium have been implicated in up to 58.2% of anesthetic-associated anaphylaxis , immunosuppressant agents  and biological agents such as intravenous immunoglobulin and antibodies to IgE  have all yielded promising results in the treatment of patients with antihistamine-resistant chronic urticaria –16. For Cold urticaria is a rare and potentially deadly disease that poses an easily underestimated challenge for anesthesiologists. Proper preoperative management is critical to prevent catastrophic events, such as angioedema or anaphylaxis. The risk of severe reaction can be mitigated through careful planning for seamless perioperative normothermia, and preoperative prophylactic use of histamine 1 and histamine 2 receptor blockers and corticosteroids. Normothermia is best achieved with a single or multiple forced air warming blankets and careful monitoring of core and ambient temperature in the operating room. Avoidance of potentially allergenic medications may simplify recognition of the true cause of any reaction. These aggressive steps will minimize the perioperative morbidity and mortality in patients with cold urticaria.We have written informed consent to use the patient’s information for publication of this paper.PA, first author: drafting, acquisition, and interpretation of data. NS, AR, and LS: revision of content. KM, corresponding author: drafting, acquisition, analyzing, and interpretation of data.The 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.


Ca2+ uptake into mitochondria modulates a number of Ca2+-dependent proteins and enzymes participating in fatty acids metabolism, tricarboxylic acid cycle, oxidative phosphorylation and apoptosis in response to physiological and pathophysiological conditions. Mitochondrial calcium uniporter (MCU) complex has been identified as a major channel located on the inner membrane to regulate Ca2+ transport into mitochondria. Recent studies of MCU complex have increased our understanding of the modulation of mitochondrial function and retrograde signaling to the nucleus via regulation of the mitochondrial Ca2+ level. Mitochondria couple cellular metabolic state by regulating not only their own Ca2+ levels, but also influence the entire network of cellular Ca2+ signaling. The mitochondria-associated ER membranes (MAMs), which are specialized structures between ER and mitochondria, are responsible for efficient communication between these organelles. Defects in the function or structure of MAMs have been observed in affected tissue cells in metabolic disease or neurodegenerative disorders. We demonstrated that dysregulation of intracellular Ca2+ homeostasis due to mitochondrial dysfunction or defects in the function of MAMs are involved in the pathogenesis of insulin insensitivity and T2D. These observations suggest that mitochondrial dysfunction and disturbance of Ca2+ homeostasis warrant further studies to assist the development of therapeutics for prevention and medication of insulin resistance and T2D.Metabolic diseases such as obesity, type 2 diabetes (T2D) and insulin resistance have attracted great attention from biomedical researchers and clinicians because of the astonishing increase in its prevalence. Decrease in the capacity of oxidative metabolism and mitochondrial dysfunction are a major contributor to the development of these metabolic disorders. Recent studies indicate that alteration of intracellular Ca Intracellular Ca2+ levels should be tightly controlled in response to the timely demands of target cells. This regulation relies on an array of Ca2+ channels, transporters and exchangers located on the plasma membrane, the ER and mitochondrial membranes [Caembranes .2+ homeostasis is related to metabolic diseases such as obesity, insulin resistance and type 2 diabetes (T2D) in the human and animals. Higher intracellular Ca2+ level has been found in primary adipocytes isolated from obese human subjects with insulin resistance [2+ level is positively correlated with the fasting blood glucose and insulin resistance index in the human [2+ ATPase (SERCA) [2+ homeostasis, are associated with the susceptibility to higher body mass index (BMI) and diabetes. Moreover, chelation of Ca2+ ions could improve insulin sensitivity of rats fed on the high-fat diet [It has been proven that dysregulation of Casistance . Besideshe human . Genome- (SERCA)  and inos (SERCA) , which rfat diet .2+ homeostasis is a key factor in the dysregulation of metabolism. Intracellular Ca2+ fluctuation has been substantiated to play a role in the downstream signaling of insulin stimulation. The cytosolic Ca2+ level of adipocytes was found to increase upon insulin stimulation [2+ signaling either by treatment of calmodulin (CaM) antagonists [2+ influx by 2-aminoethoxydiphenyl borate (2-APB), an inhibitor of IP3R and TRP channels, ameliorated insulin-stimulated glucose uptake in skeletal muscle while there was no change in the phosphorylation of Akt [2+ level and the activation of Ca2+ sensing proteins may directly or indirectly modulate Glut4 exocytosis, which is the most important step for glucose utilization of muscle cells in response to insulin.Many studies have shown that disturbance of Camulation . Inhibitagonists  in adipoagonists , respectn of Akt . Thus, a2+-mediated insulin action. Recent studies revealed that in adipocytes, synaptotagmin VII (Syt VII) can modulate the translocation of Glut4 and glucose utilization in response to insulin [2+ signaling to regulate the insulin signaling pathway. Secondly, an actin-binding protein, Myo1c, has been shown to participate in the insulin-stimulated Glut4 translocation, which is regulated by Ca2+/CaM signaling because the effect was diminished by treatment with trifluoperazine, a CaM inhibitor [2+/CaM kinase II (CaMKII) contributes to insulin-triggered regulation of Glut4 translocation in 3 T3-L1 pre-adipocytes [2+/CaM signaling is required for the FAM3A-mediated Akt activation [2+-dependent effectors or Ca2+/CaM signaling cascades in the regulation of insulin action under different conditions.The change in the distribution of some proteins has been demonstrated to play a role in Ca insulin . This finhibitor , 13. Thiipocytes . Moreoveipocytes . Moreovetivation . In ligh2+ ions are also involved in adiponectin-mediated regulation of metabolism. Adiponectin has received increasing attention than other adipokines due to the observation that its level is negatively associated with metabolic syndrome and its beneficial effect on cellular bioenergetic metabolism in diabetic mouse models [2+ flux into cytoplasm and activation of Ca2+/CaM-dependent protein kinase kinase β (CaMKKβ). In turn, CaMKKβ could further stimulate AMPK activation to induce glucose uptake and β-oxidation of fatty acids. On the other hand, CaMK could also be activated by CaMKKβ, which transcriptionally regulates the expression of PGC-1α to increase the biogenesis and function of mitochondria in muscle cells [2+-dependent signaling cascade is involved in the action of adiponectin to improve not only glucose homeostasis but also lipid metabolism of muscle and other peripheral tissues.In addition to their role in the action of insulin, Cae models , 17. Brile cells . These f2+ can cause defects in lipid metabolism in mammalian cells. Functional genetic screens in Drosophila demonstrated the importance of dSERCA and the ryanodine receptor (dRyR) [STIM1 or ORAI1 gene revealed defects in the mobilization of fatty acids from lipid droplets, lipolysis, and β-oxidation of fatty acids [Abundant evidence has substantiated that dysregulation of intracellular Car (dRyR) , dIP3R [r (dRyR) , and dStr (dRyR)  in lipidr (dRyR) . Fibroblty acids .2+ ions to alter both the amplitude and the spatio-temporal distribution pattern of the intracellular Ca2+ levels. The mitochondrial membrane potential produces a large electrochemical gradient  of the inner membrane of mitochondria so that Ca2+ ions can freely cross the outer membrane of mitochondria (OMM). However, there are distinct systems to import or efflux Ca2+ through the inner membrane of mitochondria (IMM). Mitochondrial Ca2+ uniporter machinery facilitates the entry of Ca2+ ions to the matrix. H+/Ca2+ and Na+/Ca2+ exchangers (NCX) efflux Ca2+ ions from matrix to the cytosol. Tight regulation of these proteins is important to increase the Ca2+ level to activate mitochondrial enzymes and to prevent accumulation of Ca2+ ions and Ca2+ overload within the mitochondria [Mitochondria are able to modulate influx and efflux of Cachondria .2+ between mitochondria must be balanced. Disruption of this balance may result in the opening of the mitochondrial permeability transition pore (mPTP) and the induction of cell death [2+ ions taken up into the mitochondrial matrix can increase ATP production via Ca2+-dependent activation of three important metabolic enzymes in the matrix, which include the pyruvate dehydrogenase (PDH), α-ketoglutarate dehydrogenase (αKGDH) and isocitrate dehydrogenase (IDH) [2+ uptake will affect Ca2+ signaling at local and the global levels. The Ca2+ ions released through the activation of IP3 receptor of ER in response to external stimuli can activate a series of signal transductions, but these activations need to be shut down at the right moment by sequestration of Ca2+ ions into mitochondria. This regulation highly depends on the efficiency of the functional coupling between mitochondria and ER and on the subcellular distribution of mitochondria [2+ ions by mitochondria plays a crucial role in the modulation of the Ca2+-dependent signaling and in the pathophysiology of a wide spectrum of diseases [The influx and efflux rates of Call death . Ca2+ iose (IDH) . The mitchondria . Thus, tdiseases , 28.2+ uptake of mitochondria, consists of both pore-forming and regulatory subunits  in the intermembrane space of mitochondria have been identified as regulatory proteins to control the Ca binding . MICU1 a binding . MICU2 f the MCU .a2+-dependent dehydrogenases in the mitochondrial matrix, modulation of the duration of cytosolic Ca2+ signals by buffering cytosolic Ca2+ ions. The identification of the molecular components of the uniporter provides an unprecedented opportunity to unravel the role of mitochondrial Ca2+ ions in the regulation of cellular metabolism in more detail using genetic tools  activated by Ca2+ ions in the matrix can dephosphorylate PDH and increase its activity. The MCU complex activity is positively correlated with the PDH activity and oxidative phosphorylation in mitochondria. A deficiency of MCU in skeletal muscle resulted in an increase of phosphorylation of PDH and concomitant decrease of PDH activity [2+ ions in the mitochondrial matrix, decreased the phosphorylation and increased the activity of PDH in HeLa cells [It is accepted that MCU plays a role in excitation-energetic coupling through the activation of mitochondrial matrix dehydrogenases. Manipulation of components in the MCU complex could alter the activity of the PDH complex and intracellular ATP levels of human cells. Caactivity . KnockdoLa cells . FurtherLa cells . This reLa cells . Thus, t2+ ions to modulate the cellular metabolism. It was demonstrated that Ca2+ signaling downstream of the leukotriene receptor is influenced by MCU [2+ uptake but also in the suppression of Ca2+-dependent gene expression following stimulation of the leukotriene receptor with leukotriene C4 (LTC4). The MCU seems to involve in two processes that are relevant to the immune signaling: store-operated calcium entry (SOCE) and activation of the NLR family pyrin domain containing 3 (NLRP3) inflammasome. Lack of MCU has been shown to reduce the SOCE response after inositol trisphosphate-mediated Ca2+ ions release from ER [Pseudomonas aeruginosa in airway epithelial cells from patients with cystic fibrosis [2+ signaling and substantiate its importance in the immune response.It has been shown that alteration of the MCU complex is involved in regulating transient fluxes of cytosolic Cad by MCU . In rat  from ER , which ifibrosis . Taken t2+ signaling events associated with muscle contraction [2+ uniporter activity and cellular energetics [2+ uniporter, including its role in skeletal muscle contraction and in the response of cardiac muscle to adrenergic stimulation.MCU deficiency can be tolerated in mice with a mixed genetic background. MCU knockout was found to be lethal in C57BL/6 mice, whereas the knockout mice with an outbred CD1 background were viable. MCU-knockout CD1 mice displayed no obvious phenotype but exhibited impaired tolerance to exercise. This is consistent with a cellular role of MCU in stimulating the activity of the TCA cycle during Catraction . In addiergetics . Althougergetics . Notablyergetics . Taken t2+ overload in mitochondria and increase of ROS production. Interestingly, the impairment of Ca2+ regulation could be restored by age-dependent decline of the EMRE expression. This indicates that the remodeling of MCU complex may help to maintain the Ca2+ homeostasis [Loss of MICU1 expression by truncating mutations in the human could lead to skeletal muscle myopathy, learning disability and movement disorder . The pateostasis . Hepatoceostasis . Future 2+ signaling from mitochondria in the regulation of cell metabolism. Dysregulation of intracellular Ca2+ homeostasis due to ATP depletion and release of Ca2+ ions from the mitochondria have been proposed as a principal cause for insulin resistance, but no detailed studies have been performed yet on the mitochondrial and cellular Ca2+ transport processes to clarify this issue. Although it is established that transcriptional control of metabolism by Ca2+ is exerted indirectly via Ca2+-dependent kinases and phosphatases, such as CaMK and calcineurin, which regulate the expression of PGC-1α [2+ signaling networks and what are the components involved in these processes.The retrograde signals from mitochondria can trigger gene transcription in the nucleus to induce adaptive responses or modulate cellular metabolism . Althougf PGC-1α , the und2+ level can inhibit the differentiation and maturation of human mesenchymal stem cells and 3 T3-L1 preadipocytes. For example, increases of the intracellular Ca2+ level by incubation of adipocytes in the culture media containing a high concentration of Ca2+ ions [2+ ion channels or receptors [2+-dependent enzymes including calcineurin (CaN), a Ca2+-dependent phosphatase, CaMKII, a Ca2+/CaM kinase 2 as well as calreticulin , a Ca2+-buffering chaperone in the ER, have been demonstrated, respectively, to play important roles in adipogenesis [2+ ions and activated Ca2+-calcineurin-dependent signaling, which inhibited the transcriptional cascades at the late stage of adipogenesis in mice [2+ homeostasis and normal mitochondrial function by Cisd2 are essential for adipogenic differentiation and function of adipocytes, which in turn regulates systemic glucose homeostasis in mice. Dysregulation of Ca2+ homeostasis and insulin insensitivity could be similarly induced in mouse progenitor cells-derived adipocytes with genetic manipulation of TFAM [2+ homeostasis caused by mitochondrial dysfunction plays an important role in T2D and insulin resistance in mice. Regulation of mitochondrial Ca2+ ions also modulates the morphology of the skeletal muscle. MCU overexpression by adeno-associated viral vectors induced muscle hypertrophy and MCU silencing triggered muscle hypotrophy in mice [2+-dependent mitochondria-to-nucleus route that links mitochondrial function to the control of muscle mass [Many studies have revealed that an increase in the intracellular Caa2+ ions , activateceptors , 55, andeceptors  have beeogenesis –59. In a in mice . These f of TFAM  or down- of TFAM . These g in mice . The con in mice . In addicle mass .2+ ions between the two organelles, which are the two largest Ca2+ storage sites in human cells. MAMs are responsible for dynamic and efficient transmission of physiological and pathological Ca2+ signals between the ER and the mitochondria. Due to the enrichment of Ca2+ handling proteins present in the MAMs, the functional coupling at the ER-mitochondria interface is very important for the regulation of intracellular Ca2+ homeostasis during metabolic reprogramming and cellular adaptation to various physiological and environmental stimuli [Mitochondria-associated ER membranes (MAMs) are the contact sites between the mitochondrial outer membrane and ER membrane, which are defined as structural membranes between the two organelles . This sp stimuli . In addi stimuli , 66.2+ ions to mitochondria and increase of cytosolic level of Ca2+ ions, which could further activate PP2A. Thus, inhibition of PP2A by okadaic acid could prevent high glucose-induced disruption of MAMs and restored the morphology and bioenergetic function of mitochondria [2+ homeostasis have also been observed in the liver of mice with deficiency of cyclophilin D (CypD), which is a mitochondrial protein that regulates mPTP and was recently found in MAMs fractions. Conversely, restoration of MAMs integrity by overexpression of CypD could improve insulin sensitivity and insulin signaling cascade [2+ ions overloading, which could impair the mitochondrial bioenergetic function and increase the ROS production in the liver of obese mice [2+ homeostasis, the common conclusion is that MAMs structure should be flexible and dynamic for an efficient control of the Ca2+ level in response to stimuli or the change of nutrients.Defects in MAMs have been suggested to play a role in the pathogenesis of diseases such as Alzheimer’s disease, insulin resistance and T2D , 67, 68.chondria . Impairm cascade . In contese mice . Althoug2+ ions level in the ER [2+ ions, which in turn regulate the intracellular Ca2+ homeostasis. This novel role of Cisd2 in MAMs is crucial for adipogenic differentiation and function of adipocytes, and even in the glucose tolerance and insulin sensitivity of the mouse [2+ level and mitochondrial function, which may participate in the modulation of glucose homeostasis and insulin sensitivity. It is worth mentioning that MAMs formation is a dynamic process to support efficient transmission of Ca2+ ions and lipid biosynthesis, which culminates in an increase of mitochondrial function to meet the cellular energy demand under stress conditions. The fluctuating feature of MAMs in cooperation between ER and mitochondria provides an inter-organelle communication for tissue cells to adapt to specific physiological and environmental conditions.It has been reported that Cisd2 is localized on both ER and mitochondrial membranes , 74. Cishe mouse . Taken t2+ homeostasis in the pathogenesis of metabolic diseases such as insulin resistance and T2D . The insight gained from studies of the inter-organelle communications can help us better understand the pathogenesis of the complicated and multifactorial disorders such as T2D. This line of research will also provide us novel information for the development of therapeutic agents to improve the function and/or structural integrity of MAMs. We have demonstrated that dysregulation of Ca2+ homeostasis is a novel mechanism underlying the mitochondrial dysfunction-related insulin insensitivity of adipocytes and possibly an etiology factor of T2D. We believe that simultaneous improvement of the structure and function of mitochondria and ER may be a useful strategy to restore and maintain glucose homeostasis in the human and animals.Most importantly, we discuss in this review the importance of mitochondria-ER cross-talk in the maintenance of Ca


Salvianolic acid B  is cardioprotective against ATO and enhances ATO’s anticancer activities. The present study assessed whether the Sal B protective effect was related to maintenance of Ca2+ homeostasis and inhibition of ER stress. Male BALB/c mice were injected with ATO or ATO+Sal B once a day via the tail vein for 2 weeks. We then detected the effects of Sal B in real time using adult rat ventricular cardiomyocytes in vitro using an IonOptix MyoCam system. Sal B treatment alleviated ATO-induced abnormal cardiac contractions and Ca2+ homeostasis imbalance. Sal B increased sarcoplasmic reticulum Ca2+-ATPase (SERCA) activity, regulated Ca2+ handling protein expression, and decreased expression of ERS proteins. Our results demonstrate that the cardioprotective effect of Sal B correlates with SERCA modulation, maintenance of Ca2+ homeostasis, and inhibition of ER stress. These findings suggest Sal B may ameliorate ATO cardiotoxicity during clinical application.Arsenic trioxide (ATO) is a potent anticancer agent used to treat acute promyelocytic leukemia. However, its cardiotoxicity limits ATO’s widespread clinical use. Previous studies demonstrated that ATO may aggravate Ca Increased [Ca2+]I is also a critical sign of cell apoptosis, and has been associated with a number of abnormalities in cardiac tissues, such as ventricular arrhythmias and contractile dysfunction [Ca, L density, triggering intracellular calcium overload and disorder, and ultimately inducing arrhythmia and cell apoptosis [Arsenic trioxide (ATO) was recently found to induce complete remission in relapsed or refractory acute promyelocytic leukemia (APL) patients, and the US Food and Drug Administration subsequently approved ATO as the frontline agent for APL treatment -3. Howevfunction . ATO canpoptosis -11.2+ reservoir. Ca2+-binding chaperones mediate proper folding of proteins in the ER lumen [The endoplasmic reticulum (ER) is the major intracellular CaER lumen . PreviouER lumen -15. We rER lumen . TherefoSalvia miltiorrhiza Bunge  is a popular traditional Chinese medicine, and has been used extensively in both Asian and Western countries to treat various diseases, including cerebrovascular diseases, coronary artery diseases, and myocardial infarction [The dried root of farction . Salvianfarction . Our prefarction . Sal B afarction . These d2+ channels and Ca2+ influx in rat coronary artery vascular smooth muscle cells [2+ transients in ARVMs in real time using an IonOptix MyoCam system. We repeated previous BALB/c mouse model experiments to elucidate the Sal B cardioprotective mechanism. We then assessed sarcoplasmic reticulum Ca2+-ATPase (SERCA) activity, and Ca2+-handling- and ER stress signaling-related proteins in heart tissue and ARVMs. Thus, we assessed whether Sal B protects against ATO-induced cardiotoxicity via calcium homeostasis maintenance and ER stress inhibition.Previous studies confirmed that Sal B inhibits Cale cells . Sal B rle cells . We hypo90  for 7 min after ATO treatment (100 μM) for 20 min. Sal B treatment alone did not affect resting cardiomyocyte contractile function compared with controls Figure , -dL/dt 0 Figure , and TPS0 Figure , while A2+ transients. As a calcium channel blocker [2+ ratio and the amplitude of Ca2+ transients  vesicles extracted from heart tissue and ARVMs. ATO attenuated SERCA activity, whereas Sal B treatment effectively reversed SERCA activity compared with controls , ER stress sensors (protein kinase RNA-like ER kinase (PERK), activating transcription factor 6 (ATF6), inositol-requiring enzyme-1α (IRE1), and eukaryotic initiation factor 2 α (eIf2α), and ER stress-initiated proapoptotic factors (C/EBP homologous protein (CHOP) and Caspase12) in heart tissue and ARVMs via western blotting. GRP78, p-PERK, p-eIf2α, IRE1, and ATF6 were upregulated in the ATO group compared with controls , which has two phosphorylation sites, Ser16 and Thr17 [2+ removal, respectively [2+ transport and modulates myocardial cell contractile and electrical activities [2+ overload in cells and trigger malignant ventricular arrhythmia [2+ overload in ARVMs by regulating SERCA expression and activity.Camyocytes . The [Caytoplasm . SERCA ind Thr17 . Dephospectively . Calciumtivities . A recenrhythmia , 31. We ATO treatment can induce ATP depletion, oxidative stress, and calcium imbalance, leading to accumulation and aggregation of unfolded proteins in the ER lumen, and ultimately the unfolded protein response (UPR) and ER stress , 33. GluAlthough the significance of ER stress in the ATO anticancer effect has been studied, few studies focused on alleviating ATO cardiotoxicity by suppressing ER stress, and whether ATO-induces ER stress in the heart requires further study . Our dat2+-handling protein regulation and ER stress-associated apoptosis inhibition are important in Sal B cardioprotection in vivo and in vitro. Sal B treatment blocked Ca2+ overload and upregulated SERCA activity and expression, ameliorating ATO-induced calcium homeostasis imbalance. Finally, Sal B inhibited ATO-induced ER stress by downregulating GRP78, ERS, PERK, ATF6, and IRE1. Our results suggest that Sal B may prevent ATO cardiotoxicity during clinical application. However, our data do not fully explain the direct relationship between calcium homeostasis imbalance and ER stress. Therefore, the exact mechanisms of Sal B cardioprotective activity require further study.In conclusion, Sal B treatment protected against ATO-induced cardiotoxicity by improving contractile recovery and maintaining calcium homeostasis. We found that Ca2+-ATPase (SERCA) activity detection kit was acquired from Nanjing Jiancheng Bioengineering Institute . All antibodies were obtained from Santa Cruz Biotechnology except for SERCA and NCX, which were purchased from Abcam . All chemicals were obtained from Sigma Chemical Co., Ltd. .Sal B (≥98%) was purchased from Shanghai Winherb Medical S&T Development Co., Ltd. . ATO was acquired from Harbin YI-DA Pharmaceutical Ltd. . Collagenase Type II and Fura-2/AM were purchased from Life Technologies Corporation . The sarcoplasmic reticulum CaSixty male BALB/c mice weighing 18–20 g each  were used in the experiment. All procedures were approved by the local animal committee. All animal care procedures and interventions were performed in accordance with the Guidelines and Policies for Animal Surgery provided by the Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China, and were approved by the Institutional Animal Use and Care Committee (registration number: #IMPLAD2015090216). Experimental protocols were previously described [2+-containing Tyrode’s solution equilibrated with O2  for 2 min at a rate of 6 mL/min. Hearts were then perfused with Ca2+-free Tyrode’s solution for 5 min along with the components mentioned above, except for CaCl2. Hearts were then perfused with Ca2+-free Tyrode’s solution with collagenase Type II (210.00 units/mg) and 0.7 g/l bovine serum albumin until they became flaccid. The digested tissues were separated and filtered through a nylon mesh (300 mm) after perfusion. The filtered cell suspension was rinsed several times with Ca2+-containing Tyrode’s solution to progressively increase extracellular Ca2+ to 1.2 mM. Only viabilities over 85% and rod-shaped ARVMs with clear edges were used in this study.ARVMs were isolated using a modified enzymatic method as previously described . Briefly2+ transients were assessed simultaneously upon field stimulation  using a video-based sarcomere contractility and calcium-recording module in a SoftEdge MyoCam system . To measure intracellular Ca2+, freshly isolated myocytes were incubated with Fura-2/AM (2 μM) for 15 min at 37°C and then washed twice with Ca2+-containing Tyrode’s solution to remove residual Fura-2/AM. ARVMs were then placed in a Warner chamber mounted on the stage of an inverted microscope  and superfused with the following: (I) Ca2+-containing Tyrode’s solution for 27 min (control group), (II) Ca2+-containing Tyrode’s solution with Sal B (1 μM) for 7 min , (III) Ca2+-containing Tyrode’s solution with ATO (100 μM) for 20 min (ATO group), and (IV) Ca2+-containing Tyrode’s solution with ATO (100 μM) for 20 min before Ca2+-containing Tyrode’s solution with Sal B (1 μM) for 7min  at a rate of 1.5 mL/min. Myocytes (n=30–40 cells from 5 rats per group) were allowed to equilibrate for about 10 min before measurement. Data were recorded and analyzed using IonWizard software (version 6.2.0.59) [Sarcomere shortening and Ca.2.0.59) .As previously described , SERCA aAfter exposure to different treatment solutions, ARVM and heart tissue homogenates were lysed on ice with tissue or cell protein extraction reagent containing 1% phenylmethylsulfonyl fluoride . Supernatants were collected after centrifuging at 12,000 rpm at 4°C for 20 min. Protein concentrations were determined using a BCA kit . Equal amounts of protein were separated via 8–12% SDS-PAGE and then transferred to nitrocellulose membranes  in a Tris-glycine buffer at 100 V for 1 h in an icebox. Membranes were blocked with 5% (w/v) non-fat milk powder in Tris-buffered saline containing 0.1% (v/v) Tween-20 (TBST) for 3 h at room temperature, and then incubated overnight with appropriate primary antibodies (1:200) at 4°C. Membranes were washed with TBST three times, incubated with corresponding secondary HRP-conjugated antibodies (1:1000) for 2 h on a shaking table, and then washed again three times with TBST. Membranes were developed using an enhanced chemiluminescence solution. Protein levels were visualized with Image Lab Software . Western blot analysis was performed as previously described .Data were expressed as means ± SD from at least three independent experiments. Statistical comparisons between different groups were determined by one-way ANOVA or the Student-Newman-Keuls method with GraphPad Prism 5.0 software . P<0.05 was considered statistically significant.


We examined the involvement of mitochondrial calcium uniporter, which mediates mitochondrial Ca2+ uptake, in endothelial cell dysfunction resulting from high-glucose treatment.Mitochondrial Ca2+ concentrations were analysed by laser confocal microscopy, and cytoplasmic and mitochondrial oxidative stress was detected using 2′,7′-dichlorofluorescein diacetate and MitoSOX Red, respectively. Apoptosis was assessed by annexin V-fluorescein isothiocyanate/propidium iodide staining, and a wound-healing assay was performed using an in vitro model.Human umbilical vein endothelial cells were exposed to various glucose concentrations and to high glucose (30 mM) following mitochondrial calcium uniporter inhibition or activation with ruthenium red and spermine, respectively. Subsequently, mitochondrial calcium uniporter and mitochondrial calcium uniporter regulator 1 messenger RNA and protein expression was measured by real-time polymerase chain reaction and western blotting. Ca2+ and reactive oxygen species levels, increased apoptosis and compromised wound healing . These effects were enhanced by spermine and completely negated by ruthenium red, which are known to activate and inhibit mitochondrial calcium uniporter, respectively.High glucose markedly upregulated mitochondrial calcium uniporter and mitochondrial calcium uniporter regulator 1 messenger RNA expression, as well as protein production, in a dose- and time-dependent manner with a maximum effect demonstrated at 72 h and 30 mM glucose concentration. Moreover, high-glucose treatment significantly raised both mitochondrial and cytoplasmic CaMitochondrial calcium uniporter plays an important role in hyperglycaemia-induced endothelial cell dysfunction and may constitute a therapeutic target to reduce vascular complications in diabetes. Therefore, understanding the molecular mechanism by which vascular EC dysfunction arises under HG conditions may be useful for both the prevention and treatment of diabetic vascular complications.Endothelial cells (ECs) play a crucial role in maintaining vascular homeostasis,68 and mitochondria constitute one of the largest intracellular calcium pools. However, the key signalling events linking hyperglycaemia with mitochondrial Ca2+ concentration in ECs are not known. Mitochondrial calcium uniporter (MCU) is the pore-forming subunit of the Ca2+ uniporter ion channel located on the mitochondrial inner membrane that mediates Ca2+ uptake into the matrix, regulating cytoplasmic Ca2+ signalling.911 MCU is essential for glucose-induced increases in adenosine triphosphate (ATP) concentration in pancreatic β-cells,12 and its knockdown reduces agonist- and depolarization-induced mitochondrial Ca2+ sequestration, ATP production, and d-glucose-stimulated insulin secretion.13 However, the role of MCU in hyperglycaemia-induced EC dysfunction has not been explored.Many reports have shown that calcium is an important regulator of endothelial function in diabetes,2+ overload in EC dysfunction caused by hyperglycaemia. Mitochondrial calcium uniporter regulator 1 (MCUR1) is also essential for the formation of MCU complex and may regulate its function, enabling mitochondrial Ca2+ uptake and the maintenance of normal cellular bioenergetics.14 Reactive oxygen species (ROS) overproduction, increased apoptosis and compromised wound-healing ability contribute to the development of pathological conditions associated with endothelial dysfunction.16 Thus, in this study, we used human umbilical vein endothelial cells (HUVECs) to investigate the involvement of MCU and MCUR1 in hyperglycaemia-induced mitochondrial Ca2+ overload, ROS overproduction, apoptosis and decreased wound-healing ability.We propose that MCU is the critical protein responsible for mitochondrial Ca2. Cells of passages 3–7 were used for experiments. As described in previous studies, the cells were treated with different concentrations of glucose  for 72 h and exposed to HG (30 mM) for different lengths of time .18 Treatment with 5.5 mM glucose was considered the ‘normal glucose’ (NG) condition. Once the optimal glucose exposure time and concentration had been established, experiments involving 30-min pre-treatment with the MCU inhibitor ruthenium red   or MCU activator spermine  (Sigma) were carried out. As an osmotic control, treatment with 24.5 mM mannitol (Mnt) and 5.5 mM glucose was used.HUVECs were purchased from the American Type Culture Collection  and were cultured in low-glucose Dulbecco’s Modified Eagle’s medium (DMEM) containing 10% foetal bovine serum, 100 U/L penicillin and 100 U/L streptomycin at 37°C in 5% COMCU , MCUR1  and β-actin . Values for each unknown were determined by the 2−ΔΔCt method, using β-actin as a reference sequence and standard curves derived from samples of known RNA quantities.Total RNA was isolated from HUVECs using Total RNA Kit I  and reverse transcribed into complementary DNA (cDNA) with a ReverTra Ace qPCR RT Master Mix with gDNA Remover kit . In all, 50-ng samples of this cDNA were then subjected to real-time polymerase chain reaction (PCR) on a Mastercycler nexus X2 PCR System  using SYBR Green I and TaqMan probes and primers targeting Total protein extracts (30 µg) were electrophoresed on a 10% sodium dodecyl sulphate–polyacrylamide gel. The separated proteins were transferred electrophoretically to a polyvinylidene difluoride (PVDF) membrane, which was blocked with a 5% skimmed milk solution for 2 h at 25°C and incubated with primary antibodies against MCU and MCUR1  overnight at 4°C. After washing, the blots were incubated with a goat anti-rabbit IgG horseradish peroxidase (HRP)-conjugated secondary antibody  for 2 h at 25°C. Antigen–antibody complexes were visualized using ECL Western Blotting Detection Reagents and an ImageQuant 350 instrument . Once stripped, the membranes were further probed with an antibody against β-actin as a loading control. Greyscale values were calculated from blots using ImageJ software.2+]mito and [Ca2+]cyt were detected by incubating HUVECs for 30 min at 37°C in the dark with the Ca2+ indicators Rhod-2 AM  and Fluo-3 AM  at final concentrations of 2 and 5 µmol/L, respectively. After incubation, the cells were washed twice with phosphate-buffered saline (PBS) and analysed by capturing fluorescence images using a confocal microscope equipped with an argon–krypton laser. Fluorescence deriving from Rhod-2 AM and Fluo-3 AM was assessed with 559/578 and 488/515 nm excitation/emission filters, respectively.Changes in mito and [Ca2+]cyt were significantly elevated in cells of the HG group (p < 0.05) (p < 0.05)  (mito was significantly reduced in the HG + RR group and elevated in the HG + sper group (p < 0.05)  ; howeverAs shown in 2+ overload has been implicated as a critical mediator of cellular dysfunction under hyperglycaemic conditions,7 but the key signalling events linking hyperglycaemia with increased mitochondrial Ca2+ have so far remained elusive. Here, we demonstrate for the first time that hyperglycaemia-induced mitochondrial Ca2+ overload depends on MCU activation, leading to increased mitochondrial ROS levels and HUVEC dysfunction.Mitochondrial Ca2+ and ROS levels in these cells, increased their rate of apoptosis and compromised their wound-healing ability. Hyperglycaemia was thus found to induce EC dysfunction, as reported in previous studies.19 Furthermore, our data suggest that MCU and MCUR1 are upregulated in ECs after HG exposure, providing evidence for the role of the MCU complex in diabetic vascular diseases.First, our results showed that HG increased HUVEC MCU and MCUR1 expression at transcriptional and translational levels in a dose- and time-dependent manner. Moreover, HG resulted in elevated Ca2+ and controls its concentration within this organelle.20 Previous studies have shown that excessive mitochondrial Ca2+ uptake has deleterious effects, including ROS overproduction,21 sensitization to apoptosis,22 and activation of the permeability transition pore and subsequent cell death pathways.24 Our results demonstrate that hyperglycaemia results in increased MCU expression and impaired cell function. However, little is known of the significance of MCU in HG-induced EC dysfunction. In order to address this gap in our knowledge, we exposed HUVECs to an inhibitor or an activator of MCU prior to HG treatment. The Ca2+ overload, heightened oxidative stress, increased apoptosis rate and decreased wound-healing ability induced by hyperglycaemia, and these effects were negated by MCU inhibition. Conversely, the MCU activator spermine was found to exacerbate HG-induced EC dysfunction. These results support the conclusion that MCU is clearly an important mediator of the detrimental effects of HG on EC function.MCU is a mitochondrial inner membrane uniporter that mediates mitochondrial uptake of Ca26 we found MCU to be associated with oxidative stress-induced apoptosis. Knocking down endogenous MCU has been shown to decrease mitochondrial Ca2+ uptake and attenuate the death of HeLa cells and primary cerebellar granule neurons resulting from oxidative stress.25 In addition, Sripetchwandee et al.26 established that iron overload could result in cardiac mitochondrial dysfunction, including increased ROS production, membrane depolarization and swelling, and showed that these effects could be prevented by MCU blockers. MCU is also necessary for store-operated Ca2+ entry-dependent breast cancer cell migration.27 Our results confirm an essential role for MCU in the Ca2+ overload that leads to deleterious consequences for ECs. However, further investigation is required to identify the signalling pathways downstream of MCU involved in EC dysfunction.Consistent with previous results,2+ overload. Subsequent excessive generation of ROS due to heightened mitochondrial activity may then eventually cause endothelial dysfunction. We propose that MCU plays an important role in diabetic vascular diseases. Therefore, targeting MCU or up- or down-stream factors may constitute a novel therapeutic approach for patients with diabetes and vascular complications.To conclude, our study suggests that hyperglycaemia results in enhanced expression of MCU and MCUR1, leading to mitochondrial CaHigh-glucose conditions result in endothelial cell (EC) dysfunction.2+ overload may be responsible for the effects of hyperglycaemia on ECs.CaMitochondrial calcium uniporter (MCU) and mitochondrial calcium uniporter regulator 1 (MCUR1) are upregulated under high-glucose conditions in ECs.Inhibiting and activating MCU, respectively, negates and exacerbates high glucose–induced EC dysfunction.MCU may be a key factor and drug target in diabetic vascular injury.


In most non-excitable cells, store-operated Ca2+ entry (SOCE) is an important mechanism by which intracellular Ca2+ signaling is regulated. However, few drugs can selectively modulate SOCE. 2-Aminoethoxydiphenyl borate (2APB) and its analogs (DPB162 and DPB163) have been reported to inhibit SOCE. Here, we examined the effects of another 2-APB analog, DPB161 on SOCE in acutely-isolated rat submandibular cells. Both patch-clamp recordings and Ca2+ imaging showed that upon removal of extracellular Ca2+ ([Ca2+]o=0), rat submandibular cells were unable to maintain ACh-induced Ca2+ oscillations, but restoration of [Ca2+]o to refill Ca2+ stores enable recovery of these Ca2+ oscillations. However, addition of 50 μM DPB161 with [Ca2+]o to extracellular solution prevented the refilling of Ca2+ store. Fura-2 Ca2+ imaging showed that DPB161 inhibited SOCE in a concentration-dependent manner. After depleting Ca2+ stores by thapsigargin treatment, bath perfusion of 1 mM Ca2+ induced [Ca2+]i elevation in a manner that was prevented by DPB161. Collectively, these results show that the 2-APB analog DPB161 blocks SOCE in rat submandibular cells, suggesting that this compound can be developed as a pharmacological tool for the study of SOCE function and as a new therapeutic agent for treating SOCE-associated disorders.Cellular Ca In many non-excitable cells, intracellular Ca2+ signals respond to extracellular agonist stimulation in an oscillatory, rather than sustained, manner o=0) conditions, continuous bath-application of ACh (20 nM) induced persistent Ca2+ oscillatory responses as measured by the Ca2+-dependent Cl- current using patch-clamp whole-cell recordings in mouse pancreatic acinar cells  using Fura-2 imaging in isolated rat submandibular cells. Application of high concentration (1 μM) of ACh resulted in a spike in [Ca2+]i that declined much faster under [Ca2+]o=0 conditions , 1 μM ACh induced [Ca2+]i spike, with the same rise and decline, regardless of whether the bath solutions contained Ca2+ or not  and 3542.4 ± 231.4 for the [Ca2+]o=0 conditions . The difference was significant . In the presence of 50 μM DPB161, the ACh-induced current areas under 1 mM [Ca2+]o  and [Ca2+]o=0 conditions  were 1575.4 ± 80.2 and 1377.8 ± 190.8 . These results support the patch-clamp finding that DPB161 blocks the refilling of the Ca2+ store through SOCE channels.To further evaluate the role of DPB161 in inhibiting SOCE, we measured intracellular Cas Figure  than unds Figure , suggestt Figure . Statist2+]i twice with 5-10 min intervals with or without external Ca2+. As shown in Figure 2+]o=0 without external Ca2+ refilling, the second ACh-induced elevation of [Ca2+]i was eliminated, suggesting that the first ACh exposure (1 μM for ∼4 min) has already depleted the Ca2+ store. However, with [Ca2+]o perfusion (1 mM for ∼ 4 min), the Ca2+ store was able to refill as the second ACh exposure induced [Ca2+]i elevation to a similar extent  and first [Ca2+]i response with different concentrations of DPB161 under conditions of 1 mM [Ca2+]o perfusion. One-way ANOVA showed a statistical difference in S2/S1 ratio after perfusion of DPB161 (F(4)=56.1, p<0.001). Tukey analysis showed little effect of 5 μM DPB161 in prevention of [Ca2+]o entry compared to 0 μM DPB161 , and 50 μM DPB161 reduced S2/S1 ratio to 23.0±4.8% -induced elevation of [Cat Figure . Thereaf2+]i elevation under conditions of DPB161 treatment. To measure SOC channel-mediated [Ca2+]i elevation, we depleted intracellular Ca2+ store by pre-treating cells with thapsigargin (TG) under [Ca2+]o=0 conditions. Depletion of Ca2+ stores was demonstrated by the inability of ACh application to induce [Ca2+]i elevation -induced Ca2+ oscillations world terminate in the absence of external Ca2+ due to an inability to refill the emptied Ca2+ stores resulting from persistent ACh exposure. However, our data shows that even under [Ca2+]o=0 conditions, ACh-induced Ca2+ oscillations continued for more than 30 min, suggesting that ACh-induced Ca2+ oscillations are not strongly dependent on SOCE to refill Ca2+ stores in pancreatic acinar cells. The precise reason is still unclear, but could be due to the highly expressed Ca2+ pump on the membrane of Ca2+ stores, which may be strong enough to reuptake released Ca2+ back to Ca2+ stores to maintain the persistent Ca2+ oscillations during exposure of ACh. Then, we selected rat submandibular cells based on previous work showing that ACh-induced Ca2+ oscillations in rat submandibular cells are dependent on external Ca2+ entry since ACh only induces a short-lasting Ca2+ oscillations if the external Ca2+ is removed o=0 conditions, suggesting that rat submandibular cells are an excellent cell model to evaluate the pharmacological effects of agents that modulate SOCE.Since previous experiments showed that pharmacological manipulation of SOCE using 2APB analogs in different cell lines , 12, in  removed , 19. In 2+]o=0 conditions, we exposed cells to either ACh or thapsigargin to deplete Ca2+ stores and open SOC channels, and tested the effects of DPB161 on SOCE. Patch-clamp experiments showed that persistent exposure of ACh (20 nM) induces a short-lasting oscillatory response (measured Ca2+-dependent Cl- currents) under [Ca2+]o=0 conditions. This is due to the ACh has released all releasable Ca2+ from Ca2+ stores without extracellular Ca2+ refilling. After emptying Ca2+ stores, exposure to 1 mM external Ca2+ enables the refilling of Ca2+ stores and recovery of the 20 nM ACh-induced oscillatory responses  indicate that the duration of the [Ca2+]i response is remarkably short in the absence of extracellular Ca2+, suggesting that during ACh depletion of Ca2+ stores, the refilling of Ca2+ stores requires the presence of external Ca2+  induces [Ca2+]i elevations of similar size  in InsP3 receptor-deficient DT40 cells i with Fura-2, the excitation wavelengths were 340 nm and 380 nm, a dichroic mirror of 440 nm and an emission filter of 510 nm were used. [Ca2+]i was calculated from the ratio (R) of the two fluorescence intensities, F340/F380 [2+]i was calculated by subtracting [Ca2+]i at the peak response level from that at the pre-stimulated level.Isolated cells were preloaded with a standard external solution containing 2 μM Fura-2/AM  for 40 min at 37°C. Ca340/F380 . The cha2+-free external solution ([Ca2+]o = 0 mM), CaCl2 was removed from and 1 mM EGTA was routinely added to the standard extracellular solution. The standard intracellular (pipette) solution contained (in mM) KC1 140, MgCl2 1.13, HEPES 10, glucose 10, ATP 1.0, and EGTA 0.25, titrated with KOH to pH 7.2.The standard extracellular (bath) solution contained (in mM) NaCl 130, KCl 14.7, CaC12 1.0, MgC12 1.13, HEPES 10, and glucose 10, titrated with NaOH to pH 7.2. To make a CaCells were perfused continuously with a stream of the extracellular solution (∼0.5 ml/min). A computer-controlled U-tube system was used for drug application . The reagents used for the present study were acetylcholine and thapsigargin , collagenase , and Fura-2/AM . The 2-APB analog DPB161 , then the drug-induced changes were normalized to the current response (induced by ACh) before testing drug  exposure (Baseline). If data were obtained from the same recorded cell and the changes of ACh response were compared prior to, during and after testing drug exposure, a paired t-test was used. To analyze multiple effects , one-way ANOVA with Tukey’s post hoc tests were used.For patch-clamp experiments, the Ca


Treatment of acute myeloid leukemia (AML), an aggressive and heterogeneous hematological malignancy, remains a challenge. Despite advances in our understanding of the complex genetics and biology of AML pathophysiology, these findings have been translated to the clinic with only limited success, and poor outcomes persist for the majority of patients. Thus, novel treatment strategies are clearly needed for achieving deeper and prolonged remissions and for avoiding the development of resistance. Due to its profound role in (cancer) stem cell biology and differentiation, the Hedgehog (HH)/Glioma-associated Oncogene Homolog (GLI) signaling pathway may be an attractive novel therapeutic target in AML. In this review, we aim to provide a critical and concise overview of the currently known potential and challenges of HH/GLI targeting. We describe the biological role of the HH/GLI pathway in AML pathophysiology. We specifically focus on ways of targeting non-canonical HH/GLI signaling in AML, particularly in combination with standard treatment regimens, which may overcome some hurdles observed with approved HH pathway inhibitors in solid tumors. Acute myeloid leukemia (AML) is an aggressive hematological neoplasm with a highly diverse clinical course. Known prognostic factors include age, complex karyotype, mutations, presence of elevated white blood cell counts, antecedent hematologic disease, and prior chemo/radiotherapy for another malignancy . With thIn order to be successful, AML treatment mainly requires management of the BM and systemic disease. Therefore, AML therapy is based on systemic combination chemotherapy and usually includes two treatment phases: firstly, the achievement of remission (induction) and secondly, the consolidation of remission. Current treatment for previously untreated AML in fit/younger patients is composed of two therapeutics, cytarabine (Ara-C) and an anthracycline such as daunorubicin (“7 + 3 induction therapy”), with a complete response/remission (CR) rate of about 65% . This caA randomized study by the Eastern Cooperative Oncology Group (ECOG) showed that after only a short term CR all patients without consolidation therapy relapsed  and onlyOlder or unfit patients, however, often do not endure a high dose remission initiating treatment. Thus, those patients benefit more from receiving low-dose Ara-C  or hypomDespite achievement of CR after initiation/consolidation therapy the majority of AML patients eventually relapse, either due to a lack of response or the development of drug resistance. Thus, relapsed/refractory AML (rrAML) is rather common and unfortunately very difficult to manage due to limited availability of effective therapies . CurrentMoreover, since there is increasing evidence that levels of minimal residual disease (MRD) after induction therapy are a relevant risk factor, the monitoring of MRD during remissions has already entered the clinical trial stage in AML . In the AML is based on a differentiation defect of hematopoietic stem and progenitor cells (HSPCs) in the BM, resulting in accumulation of immature blast cells that displace the normal hematopoietic system. Within the BM microenvironment, AML blasts interact and communicate with stromal and immune cells, thereby impacting on the pathogenesis of the disease. In particular, leukemic blasts create their own protective niche by reprogramming mesenchymal stromal cells to selectively support leukemic cells, while simultaneously suppressing the normal hematopoiesis . A commoStem cell pathways such as Wnt, Notch or Hedgehog (HH)/GLI signaling have been implicated in cellular self-renewal and resistance to chemotherapy of various cancer stem cell types .a priori resistance to SMO inhibitors [The recent approval of small molecule inhibitors of HH/GLI signaling for the treatment of advanced and metastatic non-melanoma skin cancer has sparked high expectations that HH/GLI targeting may prove an efficient and even curative therapeutic approach for a range of solid and hematological malignancies –38. Howehibitors –44.In the following chapters, we aim to provide a concise overview of recent studies addressing the role of HH/GLI signaling in AML pathogenesis and its possible therapeutic implications. We summarize selected key mechanisms of non-canonical HH/GLI signal transduction, concentrating on novel insights into SMO-independent regulation of GLI activity by multiple oncogenic signal cues. Based on these cross-talk signaling events, we discuss possible therapeutic approaches tackling AML by targeting oncogenic GLI proteins with novel compounds and rational combination treatments.With regard to AML biology and pathogenesis, the HH pathway has recently received much attention for its implication in leukemic stem cell regulation and in the orchestration of acquired drug resistance of poor prognostic AML  . The autIn addition, HH/GLI targeting also bears potential for those patients that do not tolerate aggressive therapeutic regimes. In particular, a combination of these antagonists with 5-Aza can be envisaged. Tibes and colleagues conducted an RNA interference sensitizer screen to identify gene targets of distinct regions presumably enhancing 5-Aza therapy . Severalhttp://clinicaltrials.org, NCT01842646, NCT01841333, NCT01546038, NCT02367456). It is noteworthy that in a phase 2 trial with untreated AML and high-risk MDS patients, low dose Ara-C chemotherapy in combination with glasdegib improved overall survival when compared to chemotherapy only [In fact, several clinical trials using SMO inhibitors alone or in combination with compounds blocking driver mechanisms in AML have already been initiated. For instance, the potency of the SMO inhibitor glasdegib (PF-04449913) alone or in combination with e.g. 5-Aza or chemotherapy is being investigated in several clinical trials for hematologic malignancies including MDS and AML , a glycoprotein binding to and thus inhibiting HH ligand function, is produced by healthy BM stromal cells and has been shown to have the potential of suppressing proliferation of leukemic cells. In contrast, HHIP expression in BM stromal cells derived from AML and MDS patients was reduced , which wMore recently, GLI1 expression has been shown to correlate with increased expression of DNA methyltransferase 1 (DNMT1) and high-risk MDS. Of note, knock-down of GLI1 in MDS cells not only decreased survival, proliferation and DNMT1 expression but also enhanced the demethylating efficacy of 5-Aza, resulting in lower methylation of the tumor suppressor gene p15 promoter and enhanced p15 expression, respectively . CombineIn line with an oncogenic role of HH/GLI in MDS and AML, a positive correlation between GLI1 expression and percentage of AML blasts, as defined by CD34 expression, has recently been observed in BM . Also inWhile canonical activation of the GLI transcription factors depends on activation of the essential pathway effector SMO either by ligand-dependent or genetic inactivation of the HH receptor Patched (PTCH) Fig.  reviewe.Fig. 2CaIn the following section, we will provide an overview of mechanistic models that can account for SMO inhibitor resistance and will summarize several representative and seminal studies that have revealed SMO-independent positive or negative regulation of GLI activity by oncogenic signaling pathways and epigenetic factors . GiThe PI3K/AKT/mTOR and RAS/RAF/MEK/ERK signaling cascades control multiple cellular functions such as transcription, translation, proliferation, growth and survival. The aberrant activation of these oncogenic signals plays major roles in numerous malignant entities and therapeutic targeting of PI3K/AKT/mTOR and MEK/ERK signaling is a promising approach, intensely tested in clinical trials using selective small-molecule inhibitors (reviewed in –72). CroAnother study dealing with human pancreatic cancer revealed an inhibitory effect on tumorigenic cancer stem cells through the combined blockade of HH and mTOR signaling using SMO and mTOR inhibitors together with standard chemotherapy . These fMoreover, the signal integration of non-canonical GLI1/2 activation by PI3K/AKT was also deciphered as a novel potential therapeutic target because combination of the GLI inhibitor GANT61 and the AKT inhibitor perifosine resulted in synergistically suppressed tumor growth and induced apoptosis in renal cell carcinoma models . RecentlGLI transcriptional activity is also positively regulated by RAS/RAF/MEK/ERK signaling, for instance in melanoma and pancreatic cancer , 85, 86.Additional druggable kinases modulating oncogenic GLI activity include atypical Protein Kinase C (aPKC or PKC iota/gamma) and members of the dual-specificity tyrosine phosphorylation regulated kinase (DYRK) family. aPKC has been shown to directly phosphorylate GLI1 at amino acid residues located in the zinc finger DNA binding domain, thereby enhancing DNA binding and maximum transcriptional activity of GLI. Notably, SMO inhibitor resistance can be mediated by hyperactivation of aPKC, suggesting that aPKC targeting in patients unresponsive to SMO inhibitors may overcome SMO inhibitor resistance, as shown in in vitro models , 88. DYRCasein kinase 1-alpha (CK1A) and protein kinase A (PKA) can be considered additional potential therapeutic targets. Activation of CK1A by pyrvinium can promote GLI repressor formation and GLI degradation . HoweverIn addition to phosphorylation, other post-translational modifications of GLI proteins control the oncogenic activity of GLI. Canettieri et al. have shown that acetylation of GLI1 and GLI2 represses, while histone deacetylase (HDAC)-mediated deacetylation increases their transcriptional activity. This interplay is further regulated by a positive feed-forward loop involving HH-induced upregulation of HDAC1 . The rolAs another epigenetic regulator of HH/GLI, the BET family member bromodomain 4 (BRD4) protein has been shown to modulate HH signaling. BRD4 can bind to acetylated lysines in histones, enhance target gene expression via stimulation of RNA polymerase II activity and can be efficiently inhibited by the BRD antagonists JQ1 and I-BET , 101. BRIn summary, the still very high occurrence of AML relapses upon therapy reflects the need for novel treatment strategies. In this regard, targeting the HH/GLI pathway in AML can be a promising therapeutic approach, since this signaling cascade is crucially involved not only in the regulation of cancer stem/leukemia initiating cells, but also in the development of drug resistance. The possibility of inhibiting multiple key players in this pathway  as well as the combination with other agents targeting important mechanisms involved in AML pathology  provide a multitude of new treatment options. Targeting the non-canonical HH/GLI signaling pathway by directly interfering with the activity of the GLI transcription factors as well as their cross-talk with other signaling pathways (e.g. kinases) may be particularly promising, since this alternate approach might prevent the development of resistance and severe side effects as seen for SMO inhibitors.