We find that prolonged exposure of A7r5 cells to hyperglycemia (weeks) is associated with changes to Ca2+ signaling, including most prominently an inhibition of the passive ER Ca2+ leak and the sarcoplasmic reticulum Ca2+-ATPase (SERCA). To translate these findings to the in vivo condition, we used primary VSM cells from normal and diabetic subjects and find that only the inhibition of the ER Ca2+ leaks replicates in cells from diabetic donors. These results show that prolonged hyperglycemia in isolation alters the Ca2+ signaling machinery in VSM cells. However, these alterations are not readily translatable to the whole organism situation where alterations to the Ca2+ signaling machinery are different. Diabetes is a complex disease that is characterized with hyperglycemia, dyslipidemia, and insulin resistance. These pathologies are associated with significant cardiovascular implications that affect both the macro- and microvasculature. It is therefore important to understand the effects of various pathologies associated with diabetes on the vasculature. Here we directly test the effects of hyperglycemia on vascular smooth muscle (VSM) Ca Using this assay, the ability of the cells to cleave by mitochondrial dehydrogenases the WST-1 tetrazolium salt to the red colored formazan allows assessing the metabolic activity of the cells under different glucose concentration. The absorbance was measured at 440 nm using Envision 2104 Multilabel Reader .μM Fura-2AM  for 30 min at 37°C. After incubation, cells were washed with PBS and incubated with Ca2+ containing Ringer buffer for 10 min at room temperature (RT) before analysis. The Ca2+ containing Ringer buffer contained (in mM) 120 NaCl, 5.0 KCl, 1.0 MgCl2, 2.0 CaCl2, 5.5 glucose, and 20 HEPES (pH 7.4). In Ca2+ free Ringer buffer, CaCl2 was replaced with equimolar MgCl2. For experiments that required low Na+, Ringer buffer NaCl was replaced with equimolar amount of N-methyl-D-glucamine (NMDG+). The imaging system included inverted epifluorescence microscope  connected to a CoolSNAP HQ2 charged coupled device (CCD) camera. Image acquisition was performed using EasyRatioPro calcium imaging system .A7r5 cells were cultured on 35 mm poly-d-lysine coated glass coverslips  and incubated in their respective media at 37°C. When 60–70% confluent, A7r5 cells were loaded with 2 2+ level were determined from the ratio of Fura-2AM fluorescence emission intensities following excitation at 340 and 380 nm.Changes in cytosolic Ca2+/Mg2+-free PBS and incubated in HBS for 10 min prior to live cell imaging. Cell contraction was visualized using an inverted microscope  equipped with a LucPlan FLN 40x/0.60 objective. Images were acquired with a CCD camera  and processed using DP2-BSW software . Cell contraction was quantified by morphometric analysis using NIH software (ImageJ). Briefly, addition of 20 mM KCl induces the formation of contractile fibers that appears as protruded edges on the surface of contracted cells. To quantify contractile responses to KCl, contractile fibers were quantified using the edge detection function in ImageJ on thresholded time-lapse images.Assessment of cell contraction was performed on A7r5 cells cultured on glass coverslips. Cells were washed with prewarmed CaμM DCFH-DA prepared in PBS at 37°C. 30 min after incubation, cells were shifted to HG, NG, or OC for an additional 30 min. Hydrogen peroxide (H2O2)  was used at 250 μM as a positive control. DCF fluorescence was then determined, after cells lysis in 90% DMSO/10% PBS for 10 min in the dark, using a fluorescent plate reader  with 485 nm excitation and 520 nm emission wavelengths.To test whether cells cultured under HG have higher reactive oxygen species (ROS) level than those cultured in NG and OC media, the level of ROS in cells cultured in media with different glucose concentration was examined using 2′,7′dichlorodihydrofluorescein diacetate (DCFH-DA) . In this assay, the DCFH-DA molecule passively diffuses into the cells and is cleaved and oxidized in the intracellular environment by ROS to the green fluorescence emitting compound, 2′,7′-dichlorofluorescein (DCF). Briefly, cells from HG, NG, and OC streams were plated, at equal density, in 96-well plates and after overnight starvation in 1% FBS, cells were incubated with 100 β-Actin, α-tubulin, and the secondary antibodies were from Sigma Aldrich . Following incubation with secondary antibodies, membranes were reacted with enhanced chemiluminescence western blot detection reagent . The luminescent reactivity was then measured using an image acquisition system, Gene SNAP, Geliance 600 Imaging system . All membranes were stripped with Restore Plus Western Blot Stripping Buffer  and equal loading was then verified through reprobing the membranes with ß-actin/α-tubulin. Further densitometry analysis was performed using Gene Tools, Geliance 600 Imaging system .Total protein from HG, NG, and OC streams was extracted using RIPA buffer  supplemented with phosphatase and protease inhibitors. Protein extracts were quantified using Bradford method using Bio-Rad Protein Assay Dye Reagent  according to the manufacturer's protocol. Protein samples were mixed, respectively, with 10% and 25% of reducing agent and LDS buffer containing bromophenol blue for gel electrophoresis . An equal amount of protein lysate was subjected to gel electrophoresis on NuPAGE Bis-Tris or Tris-Acetate Gels  for 50 min at 200 V. Proteins were then transferred to a PVDF transfer membrane  at 30 V for 30 min. After transfer, membranes were immunoblotted with the following primary antibodies: PMCA1, PMCA 4, and PMCA total , SERCA 2 , NCX1 , STIM1 , Orai1 , and IP3R1 . μL of template cDNA. α-Actin was used as housekeeping gene. Quantitect primers were obtained from Qiagen . qRT-PCR was performed with the Fast SYBR Green Master Mix (2x) according to the manufacturer's instructions. Each PCR generated only the expected amplicon as shown by the negative first-deviation plots of the melting curve. Results were normalized to noninduced housekeeping gene-levels. Samples were analyzed in duplicate from three independent experiments.RNA was extracted from A7r5 cells cultured in HG, NG, and OC using Qiagen RNAeasy extraction kit  and reversed transcribed using High-Capacity cDNA reverse transcription kit (Applied Biosystems (AB)), all following the manufacturer's instructions. Quantitative real-time reverse transcriptase (qRT-PCR) was used to analyze the expression of the following genes  using 2 Results are presented as mean ± standard error of the mean (SE) from at least three independent experiments done each in triplicate. Comparison between the different groups was done using Nonparametric/Kruskal-Wallis test using IBM SPSS Statistics 23 software. The level of significance was set at 0.05.2+ signaling in VSM cells, we switched A7r5 cells initially cultured in HG to NG for up to 72 h and tested various Ca2+ signaling modalities, including basal Ca2+ levels, store-operated Ca2+ entry (SOCE), Ca2+ decay, and Ca2+ release but could not detect any alterations in Ca2+ signaling that correlate with the medium glucose concentrations . This data is in accordance with the study by Searls et al., who failed to show significant effect on Ca2+ when glucose levels were switched for a short time between 5 and 25 mM, prompting them to switch to 75 mM for short-term studies to replicate effect observed in diabetic mice [A7r5 rat aortic smooth muscle cells are typically cultured in high glucose concentration of 25 mM. This concentration is hyperglycemic (HG) as the level of glucose in the blood of diabetic rats is around 17 mM, in contrast to the normoglycemic (NG) levels of 5.5 mM . To begi2+ signaling. We therefore incubated A7r5 cells for a minimum of 4 weeks in physiological glucose (5.5 mM) to mirror normoglycemia (NG) in the animal. Once cells were switched to the NG stream they were maintained under these culture conditions for the duration of the study. Alternatively another stream of A7r5 cells was cultured in 25 mM glucose representing the hyperglycemic group (HG). In addition, an osmotic control for the HG treatment was included (OC), where NG was supplemented with 19.5 mM mannitol, to separate effects due to hyperglycemia from those resulting from changes due to osmolarity at the high glucose concentrations.Because complications associated with diabetes manifest themselves over prolonged periods of time (years in humans), we were interested in determining whether long-term exposure of VSM cells to HG concentrations affects Cap < 0.05) as compared to their respective controls. Similarly, when A7r5 cells were transferred from the HG stream to NG, they exhibited lower metabolic activity (p < 0.05). Furthermore, long-term incubation of A7r5 cells under the different streams NG, HG, or OC does not alter their basal resting cytoplasmic Ca2+ levels measured using Fura-2AM in Ca2+-free or Ca2+-containing extracellular solution . Incubating A7r5 cells in different glucose concentrations for extended time periods does not alter intracellular Ca2+ store content  and generate inositol 1,4,5-triphosphate receptor (IP3). IP3 in turn binds to IP3 receptors (IP3Rs) on the ER membrane to gate them open and release intracellular Ca2+. The maximum levels of PE-induced Ca2+ release in cells cultured under the different glucose conditions were not statistically different arguing that the signal transduction cascade downstream of PE is not altered significantly when cells are cultured under hyperglycemia conditions  (3Rs expression levels.Phenylephrine (PE) is an  Figures . Therefo and OC) , showing2+ results in store depletion and the activation of a ubiquitous Ca2+ influx pathway known as store-operated Ca2+ entry (SOCE) [2+ channel at the plasma membrane, and STIM1, a single pass ER membrane protein [2+ store content [2+ release in response to agonist stimulation lowers Ca2+ store content, which results in STIM1 oligomerization and its translocation to subplasma membrane cortical ER domains; where it recruits Orai1 and gates it open allowing Ca2+ influx [2+ homeostasis by ensuring the replenishment of the ER Ca2+ stores following store depletion.Agonist-mediated release of intracellular Cay (SOCE) . This pa protein , 37. The content , 39. Ca2+ influx –44. Cons2+ readdition protocol after store depletion. For this assay Ca2+ stores were depleted with thapsigargin (TG), a specific nonreversible inhibitor of the sarcoplasmic reticulum Ca2+ ATPase (SERCA) that blocks store refilling and leads to store depletion due to an ill-defined continuous Ca2+ leak from the ER. Store depletion was performed in Ca2+-free conditions after which cells were switched to Ca2+-containing medium, which results in Ca2+ influx mediated by SOCE (p < 0.034) , which mimic the osmotic conditions resulting from increased glucose levels in HG medium  in cells grown under HG as compared to NG and OC  inhibition of the SERCA pump on the ER membrane demonstrates the presence of a passive leak pathway from the ER. The molecular mechanisms controlling this ER leak pathway remain controversial with roles proposed for the IPpathways –53. In o similar , which i similar . These dG and OC . Because2+-ATPase (PMCA) and the sodium/calcium exchanger (NCX) are the primary mechanisms for Ca2+ extrusion out of the cell to maintain cytosolic Ca2+ homeostasis. To test whether Ca2+ extrusion is altered when cells are cultured under hyperglycemia condition, we used TG-dependent Ca2+ release in Ca2+-free conditions and calculated the rate of decay of the Ca2+ transient as a measure of Ca2+ extrusion out of the cell  and for the osmotic control (OC), arguing that the primary Ca2+ extrusion pathway in A7r5 cells is PMCA (p = 0.027), revealed as a faster decay to half-max (p = 0.033) (p = 0.04) but not PMCA1 increased protein expression levels  . Consist= 0.033) , support Figures .2+ store refilling after a Ca2+ transient, we released Ca2+ from intracellular store using phenylephrine and then at the peak of the Ca2+ transient a high concentration of lanthanum  was added to block both Ca2+ influx from the extracellular space and Ca2+ extrusion pathways [2+ extrusion is blocked the decay kinetics of the Ca2+ signal back to baseline reflect SERCA activity   restores HG-induced SERCA compromised activity while no effect was seen on cells grown in NG or OC. These data confirm that oxidative stress is responsible for the decrease in SERCA activity of cells grown under HG. To further confirm the involvement of HG in ROS generation, intracellular ROS levels were determined using the ROS-sensitive fluorescence-generating probe DCF-DA assay. This assay shows that 30 min exposure to HG of cells already grown in NG or OC is sufficient to significantly (∗p < 0.05) increase ROS generation , a universal radical scavenger, for 2 weeks. neration .2+ signaling machinery due to persistent irreversible damage as a result of diabetes and not to the culturing conditions. We used this approach based on the findings from the A7r5 model where alterations in Ca2+ signaling were observed only after prolonged exposure to hyperglycemia, strongly arguing that they are due to long lasting modifications.To test whether the changes observed in the A7r5 model of cells cultured under hyperglycemic conditions apply to human VSM cells under diabetic conditions, we obtained primary aortic VSM cells from normal (NHVSMC) and diabetic (Type 2) individuals (DHVSMC) . Cells from normal and diabetic individuals were cultured in media containing NG levels (5.5 mM) supplemented with FBS and all necessary cytokines and growth factor as recommended by the supplier. Each batch of cells  was used within two weeks of culture. Cells were cultured in normal glucose levels to focus specifically on changes to the Ca2+ signaling machinery in VSM cells from normal and diabetic individuals. No changes in proliferation rate (2+ levels (p = 0.009) in Ca2+ store content, based on the ionomycin releasable pool method, was detected in cells from diabetic individuals as compared to the normoglycemic control (p = 0.044) in SOCE in diabetic VSM cells .To assess the expression levels of the different Ca2+ signaling is an integral signaling module involved in many aspects of cellular physiology. Specificity of Ca2+ signaling stems from the integrated contribution of different channels and transporters that move Ca2+ across disparate subcellular compartments leading to complex spatial and temporal dynamics to encode specific downstream cellular functions [2+ signals are able to encode various cellular responses based on the specific dynamics of Ca2+ transients. As such, alterations in the function or regulation of Ca2+ transporting proteins could have significant consequences on cellular and organismal physiology [Caunctions . As suchysiology –58.2+ signaling, which in turn would contribute to vascular dysfunction (VD) by altering VSM or endothelial cell physiology. Interestingly, a recurring pathway that appears to be involved in the aforementioned mechanisms is the generation of reactive oxygen species (ROS) [Several reports show that vascular dysfunction in the context of hyperglycemia is associated with alterations to multiple signaling pathways, including advanced glycation end-products (AGEs), PKC-DAG, and the hexosamine pathways , 24–28. es (ROS) , 26, 59.2+ signaling. However, long-term culture of A7r5 cells in HG versus NG conditions leads to significant changes to Ca2+ signaling. Most significantly long-term exposure to hyperglycemic conditions is associated with an inhibition of both Ca2+ leak from the ER (2+ reuptake into the ER (Our data show that short-term (up to 72 hrs) exposure of A7r5 vascular smooth muscle cells to hyperglycemic conditions does not result in any detectable changes in Cam the ER  and SERCo the ER . The facFurthermore, the contribution of HG-induced ROS to the observed SERCA inhibition has been previously reported . Tong et2+ signaling observed in A7r5 cells exposed for prolonged periods of time to hyperglycemia are replicated in primary VSM cells from human subjects with diabetes.Culturing VSM cells under hyperglycemic conditions is an easy experimental approach to discern the effects of hyperglycemia on VSM function and has been used by several groups. The conclusion from our experiments and those of others are that there are long-term alterations that are associated with changes to VSM physiology exposed to hyperglycemia. We were as such interested in testing whether changes to Ca2+ store content (2+ lead from the ER (p = 0.02)  . SurprisSM cells . This leSOCE is inhibited in both the A7r5 model and human diabetic VSM cells; however, the fact that a similar inhibition was observed in the osmotic control in the A7r5 model in our hands casts doubt as to whether this inhibition is due to hyperglycemia per se.2+ signaling, we show here that the passive Ca2+ leak from the ER is consistently inhibited in VSM cells exposed to prolonged hyperglycemia both in humans and in culture model. In human diabetic VSM cells, consistently, this is associated with higher Ca2+ store content. However, these changes in Ca2+ dynamics were not associated with alteration to VSM Ca2+ signals in response to PE or to basal cytoplasmic Ca2+ levels. Therefore, their physiological significance remains unclear. Nonetheless, one could envision agonist stimulation in vivo leading to a more pronounced Ca2+ transient given the increased Ca2+ store content, which could result in a more sustained Ca2+ signal, thus affecting VSM physiology and contractility.There is compelling evidence from animal and clinical studies that some of the adverse effects of hyperglycemia on VD associated with diabetes are everlasting and cannot be reversed when glucose levels are improved or controlled . In the Hyperglycemia has also been shown to affect the microvasculature by impacting the activation state of the calcineurin-NFAT signaling cascade in smooth muscle cells of resistance arteries , a proceFinally, an important take-home message from our experiments is that changes observed in a cell culture model of hyperglycemia need to be interpreted with caution as they are not readily translatable to VSM cells from diabetic subjects. This is not surprising given that the type and extent of exposure of VSM to hyperglycemia in the whole organism tend to be intermittent and it occurs in the context of a multitude of additional factors acting on VSM cells.2+ signaling in VSM cells.Supplemental Figure 1: The effects of short term switch in hyperglycemia on CaSupplemental Figure 2: Effect of glucose on protein expression of PMCA isoforms (1 and 4) in A7r5 cells cultured under HG, NG, and OC.2+ channels and transporters in normal and diabetic VSM cells.Supplemental Figure 3: Expression of Ca


It is widely accepted that these Ca2+ oscillations are initiated by a sperm derived phospholipase C isoform, PLCζ that hydrolyses its substrate PIP2 to produce the Ca2+ releasing messenger InsP3. However, it is not clear whether PLCζ induced InsP3 formation is periodic or monotonic, and whether the PIP2 source for generating InsP3 from PLCζ is in the plasma membrane or the cytoplasm. In this study we have uncaged InsP3 at different points of the Ca2+ oscillation cycle to show that PLCζ causes Ca2+ oscillations by a mechanism which requires Ca2+ induced InsP3 formation. In contrast, incubation in Sr2+ media, which also induces Ca2+ oscillations in mouse eggs, sensitizes InsP3-induced Ca2+ release. We also show that the cytosolic level Ca2+ is a key factor in setting the frequency of Ca2+ oscillations since low concentrations of the Ca2+ pump inhibitor, thapsigargin, accelerates the frequency of PLCζ induced Ca2+ oscillations in eggs, even in Ca2+ free media. Given that Ca2+ induced InsP3 formation causes a rapid wave during each Ca2+ rise, we use a mathematical model to show that InsP3 generation, and hence PLCζ's substate PIP2, has to be finely distributed throughout the egg cytoplasm. Evidence for PIP2 distribution in vesicles throughout the egg cytoplasm is provided with a rhodamine-peptide probe, PBP10. The apparent level of PIP2 in such vesicles could be reduced by incubating eggs in the drug propranolol which also reversibly inhibited PLCζ induced, but not Sr2+ induced, Ca2+ oscillations. These data suggest that the cytosolic Ca2+ level, rather than Ca2+ store content, is a key variable in setting the pace of PLCζ induced Ca2+ oscillations in eggs, and they imply that InsP3 oscillates in synchrony with Ca2+ oscillations. Furthermore, they support the hypothesis that PLCζ and sperm induced Ca2+ oscillations in eggs requires the hydrolysis of PIP2 from finely spaced cytoplasmic vesicles.Egg activation at fertilization in mammalian eggs is caused by a series of transient increases in the cytosolic free Ca Such Ca2+ oscillations play the key role in egg activation and the subsequent development of the embryo  that crosses the egg from apparently random points in the egg cortex  which are exclusively of type 1 IP3R in mammalian eggs  production, and all the reproducible studies suggest that this is principally due to the introduction of a sperm specific phospholipase Czeta (PLCζ) into the egg after gamete fusion , can be detected in the plasma membrane of mouse eggs using the PH domain of PLCδ1  and left to capacitate for 2–3 h before adding to eggs  followed by human chorionic gonadotrophin  ~50 h later  (Life Technologies). OGBD was diluted in a KCl HEPES buffer  so that the injection solution contained 0.33 or 0.5 mM OGBD. The OGBD mix was microinjected into eggs using high pressure pulses. In those eggs that were stimulated by adenophostin this was microinjected into eggs along with the OGBD. In this case instead of mixing the OGBD with KCL HEPES it was mixed with KCL HEPES containing 5 μM adenophostin in the same quantities. Where PLCζ cRNA was used this was microinjected alongside OGBD in the same way at a concentration of 0.02 μg/μl. For imaging, eggs were then transferred to a glass-bottomed dish, containing HKSOM media, on an epifluorescence imaging system (Nikon TiU) attached to a cooled CCD camera as described previously  where an image was captured every 10 s. Where IVF was performed, or drugs were later added to the eggs, the zona pellucidas were removed from the eggs using acid Tyrodes treatment prior to imaging. For those experiments that required InsP3 stimulation, NPE-caged-InsP3 (1 mM in the pipette) from ThermoFisher Scienific was microinjected prior to imaging at the same time as the injection of fluorescent dye (OGBD). In order to photo-release InsP3 the eggs were exposed to an electronically gated UV LED light source  that was positioned just above the dish containing the eggs. The duration of the UV pulse was controlled by a time gated TTL pulse that was delivered in between two successive fluorescence acquisitions. All data measuring Ca2+ dynamics were recorded as .tif files using the Micromanger software on the epifluorescence system.In all experiments cytosolic Cay Swann, . Ca2+ dy3, 20 mM HEPES, 0.01 mM EDTA, 0.2 mM Na Pyruvate, 1 mM L-glutamine, 0.2 mM glucose, 10 mM Na Lactate 1.7 mM CaCl2, 0.063 g/l Benzylpenicllin, and 10 mg/l phenol red. Ca2+ free media was made in the same way as HKSOM however CaCl2 was not added and the media was supplemented with 100 μM EGTA. Sr2+ containing media was made in the same way as HKSOM however, instead of adding 1.7 mM CaCl2, 10 mM SrCl2 was added instead.M2 media was purchased from Sigma Aldrich as a working solution. HKSOM was made up to pH 7.4, in cell culture grade water as follows: 95 mM NaCl, 0.35 mM KH2PO4, 0.2 MgSO4, 2.5 mM KCl, 4 mM NaHCOAll drugs and chemicals used, unless otherwise mentioned, were purchased from Sigma Aldrich. Propranolol was used at a working concentration of 300 μM in HKSOM media. A stock of 300 mM propranolol was made up in DMSO which was then diluted 1:100 in HKSOM media. Then 100 μl of this solution was pipetted into the imaging dish containing 900 μl of standard HKSOM. Propranolol was removed by washing out this media and replacing it with fresh HKSOM media using a perfusion system that passed 10 ml of clean HKSOM through the dish containing the eggs to ensure sufficient wash out. In a similar way a stock of 5 mM thapsigarin in DMSO was diluted 1:1,000 to a concentration of 5 μM in HKSOM and then 100 μl of this thapsigargin solution was added to the imaging dish containing 900 μl of HKSOM to give a working concentration of 500 nM of thapsigargin.https://imagej.nih.gov/ij/).In those eggs that were microinjected with PBP10, a solution of 1 mM PBP10  was made up in KCl HEPES and ~4–10 pl of this solution was microinjected into each egg. Following PBP10 microinjection, eggs were imaged on a Leica SP2 Confocal  microscope using a Helium-Neon laser (543 nm) at 30% intensity. Eggs were imaged in M2 media using a x63 oil objective and a pinhole aperture of 91 nm. Images were acquired with a line averaging of 8 and a resolution of 1,058 × 1,058 pixels. For each egg a single z-stack image of (1 μm depth) was captured of an equatorial slice through the egg. All images were exported as .tif files and analyzed using Image J (2+ dynamics of the eggs on the widefield imaging system was extracted from .tif stacks using Image J (https://imagej.nih.gov/ij/). Background fluorescence was first subtracted from the egg fluorescence value. These fluorescence values were then normalized by dividing each fluorescence value in the egg by the baseline fluorescence value at the start of the imaging run to provide a relative change in fluorescence (F/F0) that could be plotted against time. These traces were produced and analyzed using SigmaPlot 12. The Confocal images were also analyzed using Image J software. PIP2 positive vesicle size and distribution was calculated using the particle analysis function on Image J and a nearest neighbor distance (Nnd) plugin in Image J. A bandpass filter function was applied to the images . The threshold was altered to between 2 and 5% so only the fluorescence of the vesicles inside the image of the egg were included in the analysis. The particle analysis function was applied and configured so it recorded area, integrated intensity and coordinates for each fluorescent vesicle in the egg. These areas were used to work out the radius and diameter of the vesicles. The coordinates were fed into a nearest distance neighbor plugin  to give the mean distance between the vesicles. The total fluorescence of the vesicles was calculated by adding all the integrated intensity readings for a single egg which was carried out using the measure tool in ImageJ and background fluorescence values were subtracted. Statistical analysis was carried out using SigmaPlot 12. If not stated otherwise the data is presented as the mean and standard errors of the mean. Shapiro–Wilk tests for normality and tests for equal variances were conducted prior to carrying out group comparison tests. If the data passed both these tests a Student's T-test was conducted. If the data failed either or both of these tests a Mann-Whitney U-test was conducted instead.Quantitative data measuring the Cau; stored calcium, v; and IP3, p,The model and associated parameter values are based on the work of . Essentially, the function S creates a regular grid of squares of size Lon × Lon in which the specified kinetics are active. We are then able to alter the wavelength, or separation distance, L, between these active regions.The actions of the stored Ca2+ spikes can be generated in mouse eggs where no membrane Ca2+ fluxes occur , in the free Ca2+ population.The equations were simulated using a finite element Runge-Kutta method on a two-dimensional disk of diameter 70 μm, which was discretised into 6,550 elements. The 2D assumption is considered valid because any dilution effects of going to three dimensions are off set equally by an increase in the third dimension production. The two-dimensional simulations can be thought of a single slice through a cell and it offers speed, clarity and insight. Finally, the boundary was specified to have a zero-flux condition, meaning that no substances were able to leak out of the domain. This is a simplification considered valid since it is known that PLCζ induced Ca2+ oscillations by using photo-release of caged InsP3 that was microinjected into mouse eggs. In initial experiments we uncaged InsP3 in unfertilized (control) mouse eggs that were not undergoing any Ca2+ oscillations. Figure 2+ increases with the amplitudes that were larger with longer duration pulses. With the protocol we used there was adequate amounts of caged InsP3 for multiple releases of InsP3 even with longer duration pulses of UV light as illustrated by Figure 2+ in control eggs. We then tested the effects of triggering such pulses during Ca2+ oscillations induced by either Sr2+ media or by PLCζ injection. Figure 3 caused no Ca2+ increase. In contrast, Figure 2+ oscillations occurring in response to Sr2+ media and in such eggs there was a rapid and large Ca2+ transient every time a pulse of just 100 ms was used to uncage InsP3. Since the response to 100 ms pulses of UV were minimal in control eggs  caused by uncaging of InsP3 against the time delay (dt) of the InsP3 pulse from the subsequent Ca2+ spike -triggered Ca2+ oscillations “run down” and can cease entirely in Ca2+ free media  of thapsigargin to completely block Ca2+ oscillations in eggs . We noted that this was associated with a decline in the fluorescence of OGBD, suggesting that resting Ca2+ levels were also undergoing a decline  Ca2+ transients in mouse eggs which is ~1 s after fertilization or after PLCζ protein injection  are spread across the egg. However, this may not be the case with PIP2 that is the precursor to InsP3. In most cells PIP2 is in the plasma membrane, and if this is used in Ca2+ waves in eggs then InsP3 diffusion range might constrain the ability to generate fast Ca2+ waves. Recently, the diffusion coefficient of InsP3 in intact cells has been shown to be <10 μm2/s which means that InsP3 may only diffuse <5μm in 1 s  it is not possible to generate a Ca2+ wave through the egg cytoplasm, and only a concentric pattern of Ca2+ release occurs. We previously presented evidence for PIP2 being present in intracellular vesicles spread throughout the cytoplasm in mouse eggs . Our simulations show that when the PIP2 vesicles are within 2 or 3 μm of each other a rapid Ca2+ can be generated, but that once the PIP2 is more than 3 μm the Ca2+ increase fails to occur. These results suggests that PIP2 needs to be present on vesicles spaced <3 μm apart in the cytoplasm in order to propagate a rapid Ca2+ wave of the type seen in fertilizing and PLCζ injected eggs.Previous studies of fertilizing mouse and hamster eggs show that most Ca2 within the cytoplasm of eggs came from studies using antibodies to PIP2  in control eggs to a mean of 2.93 × 104 RFU (n = 7) in those eggs treated with propranolol. This difference was significant following a Student's T-test (p = 0.006). This implies that propranolol is affecting PIP2 levels in cytoplasmic vesicles.We have previously sought to modify the level of PIPl Figure . Furtherl Figure . The ove2 inside eggs, we investigated the effect of propranolol on Ca2+ oscillations. Figure 2+ oscillations in response to fertilization by IVF were rapidly blocked. Figure 2+ levels and the inhibition was reversed upon removal of propranolol . In somatic cells, non-plasma membrane PIP2 has been found in the Golgi apparatus (De Matteis et al., 2+ oscillations triggered by PLCζ and fertilization. This effect was specific in that the same concentration of propranolol did not effect oscillations when added to other Ca2+ releasing agents such as Sr2+ media which causes a pattern of oscillations most similar to fertilization. The small effect on adenosphostin induced Ca2+ oscillations is unlikely to be sufficient to explain the effects of propranolol because it was only a 2-fold decrease in oscillations compared the cessation of oscillations after propranolol in most eggs that were fertilized or injected with PLCζ. It is also noteworthy that the Ca2+ levels remained low in propranolol treated eggs, and that its effects were reversible. In mouse eggs we found that propranolol also decreased the number of the PIP2 containing vesicles and the mean distance between vesicles, therefore presumably, the availability of the vesicular PIP2 to propagate a Ca2+ wave. This effect could be because propranolol disrupts the structure of the vesicles or because trafficking between the Golgi and the ER is inhibited. Whatever the actual mechanism, the loss of PIP2 after treatment with propranolol supports our hypothesis that these vesicles are required for generating Ca2+ oscillations in eggs in response to sperm or PLCζ. Since there is evidence for intracellular PIP2 on organelles in frog and sea urchin eggs, which also show Ca2+ waves at fertilization, it is attractive to speculate that intracellular PIP2 is an important feature that allows eggs to generate the Ca2+ signal needed for egg activation.The precise nature of the PIP2+ measurements and the PIP2 imaging experiments, analyzed data, and co-wrote the manuscript; BA and AM: Performed Ca2+ measurement experiments on eggs and analyzed data; TW: Produced and analyzed the mathematical simulation; KS: Conceived the study, directed experiments and co-wrote the manuscript. All authors approved the final manuscript.JS: Performed some of the CaThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


When considering the results of experiments reported over the last 15 years, one feature has been frequently noted, but rarely discussed: the magnitude of changes in myofilament Ca2+-sensitivity due to myopathy-causing mutations in skeletal or heart muscle seems to be always in the range 1.5–3x EC50. Such consistency suggests it may be related to a fundamental property of muscle regulation; in this article we will investigate whether this observation is true and consider why this should be so. A literature search found 71 independent measurements of HCM mutation-induced change of EC50 ranging from 1.15 to 3.8-fold with a mean of 1.87 ± 0.07 (sem). We also found 11 independent measurements of increased Ca2+-sensitivity due to mutations in skeletal muscle proteins ranging from 1.19 to 2.7-fold with a mean of 2.00 ± 0.16. Investigation of dilated cardiomyopathy-related mutations found 42 independent determinations with a range of EC50 wt/mutant from 0.3 to 2.3. In addition we found 14 measurements of Ca2+-sensitivity changes due skeletal muscle myopathy mutations ranging from 0.39 to 0.63. Thus, our extensive literature search, although not necessarily complete, found that, indeed, the changes in myofilament Ca2+-sensitivity due to disease-causing mutations have a bimodal distribution and that the overall changes in Ca2+-sensitivity are quite small and do not extend beyond a three-fold increase or decrease in Ca2+-sensitivity. We discuss two mechanism that are not necessarily mutually exclusive. Firstly, it could be that the limit is set by the capabilities of the excitation-contraction machinery that supplies activating Ca2+ and that striated muscle cannot work in a way compatible with life outside these limits; or it may be due to a fundamental property of the troponin system and the permitted conformational transitions compatible with efficient regulation.Mutations in striated muscle contractile proteins have been found to be the cause of a number of inherited muscle diseases; in most cases the mechanism proposed for causing the disease is derangement of the thin filament-based Ca Hypertrophic cardiomyopathy and hypercontractile diseases of skeletal muscle, such as distal arthrogryposis and “stiff child syndrome,” have been linked to a higher myofilament Ca2+-sensitivity  and restrictive cardiomyopathy (RCM)-causing mutations. Our literature search found 71 independent measurements of the mutation-induced change of EC50 ranging from 1.15 to 3.8-fold with a mean of 1.87 ± 0.07 (sem)  of 1.86-fold (corresponding to ΔpCa50 = 0.255 ± 0.015), whilst the decreases in Ca2+ sensitivity have a mean EC50 wt/mutant of 0.54-fold  and by loaded assays . The mean magnitude of the Ca2+-sensitivity change is about 20% less when measured in loaded assays.Thus, our extensive literature search, although not necessarily complete, found that, indeed, the changes in myofilament Ca50? We will consider two possible mechanisms that are not necessarily mutually exclusive. Firstly, it could be that the limit is set by the capacity of the EC coupling system that supplies activating Ca2+ and that striated muscle cannot work in a way compatible with life outside these limits; alternatively it may be due to a fundamental property of the troponin system and the permitted conformational transitions compatible with efficient regulation.What could be the underlying reason for this consistent and small effect of mutations on EC2+-sensitivity shifts. Perhaps the most puzzling observation is that there appears to be no correlation between the Ca2+-sensitivity shift and disease severity. Skeletal myopathy mutations that cause life-threating muscle weakness from birth and often require mechanical assistance in breathing ; for the 6 assays in the table the mean is 1.84, close to the mean of all 71 HCM measurements (1.87). The same variability can be seen with other mutations where multiple values are available: ACTC E99K, n = 5, 1.24–2.45 mean 1.85; TPM1 E180G, n = 4, 1.30–2.75, mean 1.78. The second relevant observation is that the physiological modulation of cardiac muscle myofilament Ca2+-sensitivity due to phosphorylation of troponin I by protein kinase A has been known to be a 2–3-fold shift for many years  and so is solely responsible for Ca2+-regulation  or sites that bind Ca2+ 200x tighter (sites III and IV) and are permanently occupied by Ca2+ or Mg2+  and caused a still smaller shift in the Ca2+-sensitivity of force production  could not properly regulate force in skinned fibers since they only produced about 13% of the maximal force of wild-type muscle at saturating Ca2+ concentrations. On the other hand, two less extreme mutations, M81Q and F78Q decreased Ca2+-sensitivity whilst retaining the same maximum force production as wild type. In these cases, again, the increased Ca2+ binding affinity for TnC was substantially greater than the increased Ca2+-sensitivity of skinned fibers (5.9x vs. 1.8x for M81Q and 8.4x vs. 4.2x for F78Q). Thus, thin filament structure seems to limit the possible effects of changes in Ca2+-binding affinity.A slightly different situation arises when Ca2+ sensitivity will affect contractile output in muscle. It is well-established that EC50 for skinned muscle fibers is about 1 μM and that Ca2+-activation of contraction is highly cooperative. Most measurements suggest a five-fold range in free Ca2+ concentration during a cardiac muscle contraction. Peak Ca2+ concentration is about 600 nM at rest and can be substantially higher during adrenergic stimulation, thus normally muscle is only partially activated (Negretti et al., It is self-evident that changing myofilament Ca2+-activation curve for myofibrils and the contractility of intact papillary muscle as well as the Ca2+-transient (Song et al., 2+ transient was the same in Wild-type and ACTC E99K muscle, Ca2+ sensitivity was 0.8 μM for wild-type and 0.34 μM for ACTC E99K with a Hill coefficient of about 4. The increase in Ca2+-sensitivity due to the ACTC E99K HCM mutation corresponds to an approximately four-fold increase in twitch force in the absence of a change in the Ca2+-transient that was actually observed.Figure 2+-sensitivity changed beyond the normal range. If myofilament Ca2+-sensitivity was 4 times normal, maximum force would reach close to 100%, leaving no range for it to be modulated by adrenergic agents. Moreover, it is likely that the muscle would not fully relax, since, based on the five-fold range of the Ca2+ transient even at the lowest Ca2+ level force would be 5–10%, a substantial fraction of the peak force of wild-type muscle, thus the hypercontractile phenotype would impose a major defect in relaxation, much more severe than the diastolic dysfunction associated with HCM mutations with only a 1.8-fold average Ca2+ sensitivity increase.We can use this model to consider what would happen if Ca2+-sensitivity were decreased to half the normal, contractility would be very low indeed. The fact that mutations that decrease Ca2+-sensitivity are not lethal and indeed in transgenic mice, may exhibit little phenotype, is probably due to a compensatory increase in the Ca2+-transient (Du et al., 2+-sensitivity.If myofilament Ca2+-sensitivity of contractility only varies within an narrow range of three-fold above and below the normal EC50 at rest and to investigate why this should be. The high cooperativity of muscle activation by Ca2+ means there is a narrow [Ca2+] range between relaxed and active muscle. It would appear that the excitation-contraction coupling machinery of the cell has limited ability to change the amplitude of the Ca2+-transient or baseline [Ca2+] to compensate for changes in EC50; thus increased Ca2+-sensitivity would be limited by inability to relax and reduced Ca2+-sensitivity would be limited by inability to contract. It is intriguing that the Ca2+-sensitivity range of the thin filament itself is independently limited. Mutations that change Ca2+-binding affinity to TnC by a large amount nevertheless only produce a small change in EC50 for activation of loaded or unloaded contractility in vitro. Whether this property is an evolutionary adaptation that limits the deleterious effects of mutations in thin filaments or simply fortuitous in unknown.The objective of this article was to confirm that CaThe author confirms being the sole contributor of this work and approved it for publication.SM's research is funded by British Heart Foundation programme grant RG/11/20/29266.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Previous studies have demonstrated an association between high body mass index (BMI) and acute myeloid leukemias (AML), particularly acute promyelocytic leukemia (APL). However, the effect of obesity and overweight on the incidence of AML is not supported by all studies, and the relationship between obesity and prognosis of AML and APL has not been established. Thus, we conducted a meta-analysis to determine the role of BMI on the risk and clinical outcome of AML, including APL. Twenty-six eligible studies enrolling 12,971 AML (including 866 APL) patients were retrieved and analyzed. Overweight and obesity was associated with an increased incidence of AML . High BMI did not significantly affect overall survival (OS)  or disease-free survival  in patients with non-APL AML. By contrast, APL patients with high BMI had shorter OS  and a higher risk of differentiation syndrome . Overall, our findings suggest that patients with overweight or obesity have a higher incidence of AML, and high BMI is a predictor of adverse clinical outcomes in APL. Currently, the proportion of overweight or obesity in adults has increased to approximately 40% worldwide1. It has been reported that overweight or obesity is associated with over 20 types of cancer, such as cancers of the breast, colon, uterus, gallbladder, and cervix, as well as leukemia3.The prevalence of obesity has been increasing worldwide over the past decades. According to World Health Organization (WHO), overweight and obesity are defined by a body mass index (BMI) of 25–29.9 kg/m4. Although much effort has been devoted to studying this disease, the etiology and pathophysiology of AML are still poorly understood and the outcome remains unsatisfactory. Epidemiologic observations have suggested that obesity is a risk factor for AML7. However, obesity did not affect risk for AML in several studies10, and Wong et al. even reported that obesity is protective against AML11. Overweight may have a similar effect on the incidence of AML, although this relationship is less certain12. The most recent meta-analysis on this topic was performed in 2012 and suggested that obesity, but not overweight, increases incidence of AML7. However, the number of patients with AML was limited because this meta-analysis included all types of leukemia and only included five studies on AML. Since 2012, five new studies, including two large multinational survey programs, have been published13. Thus, including these new studies in an updated analysis is needed to comprehensively evaluate the association between BMI and risk for AML.Leukemia is a clonal hematopoietic disorder characterized by the aggressive proliferation of immature hematopoietic progenitor cells arrested at an early stage of differentiation. Acute myeloid leukemia (AML) is one of the most common myeloid malignancies in adults16. Reports on the prognostic value of obesity for AML are somewhat inconsistent. Several studies have found that obesity adversely affects the clinical outcome of AML17 whereas others have failed to confirm such an association19.In addition to the incidence of AML, the association between high BMI and the prognosis of AML is unclear due to the lack of a meta-analysis, although several studies have investigated this issue21. APL generally carries the PML/RARα fusion protein linking the retinoic acid receptor alpha gene (RARα) on chromosome 17 with the PML gene on chromosome 15. Historically, APL was one of the most fatal forms of acute leukemia but currently has a cure rate of approximately 80–85% with the introduction of all-trans retinoic acid (ATRA) and arsenic trioxide (ATO)22. Emerging studies have demonstrated that obesity has adverse effects on the treatment outcome of APL23. However, to our knowledge, a quantitative analysis that evaluates this association with APL is not available.Notably, several studies have shown that the percentage of obese patients with acute promyelocytic leukemia (APL), a unique subtype of AML, is higher than that observed in patients with non-APL AMLWith the aim of evaluating the prognostic value of obesity in patients with AML (including APL), we conducted the first comprehensive meta-analysis to clarify the potential association between AML, particularly APL, with overweight and obesity as defined by BMI. Another objective of our study was to investigate the association between obesity and the incidence of AML.2]). According to the criteria from the World Health Organization, BMI was categorized as underweight (<18.5 kg/m2), normal (18.5–25 kg/m2), overweight (25–29.9 kg/m2), and obese (≥30 kg/m2). Most of the studies used 25 and 30 kg/m2 as the thresholds for overweight and obesity, respectively. Studies that did not meet these criteria24 were eligible for analysis of the association based on high BMI but were excluded from the overweight and obese subgroup analysis. Lin et al. used body weight (BW) ≥130% ideal body weight (IBW) as the criteria to define obesity25.BMI was calculated by dividing weight in kilograms by the square of height in meters  was conducted in PubMed, EMBASE, Web of Science and the Cochrane Library for articles assessing the effect of overweight and obesity on the incidence and clinical outcome of AML using the keywords as follows: (obesity OR overweight OR body mass OR BMI OR body weight OR anthropometric) AND leukemia. References lists from the retrieved studies were also examined. The results were limited to peer-reviewed English language studies.et al.26. When the patient populations overlapped between studies, only the most recent or most complete publication was included to avoid duplications.The studies were considered eligible if they reported the effect of overweight and obesity on the incidence or clinical outcome of patients with AML and provided sufficient data to determine an estimate of relative risk (RR) or hazard ratio (HR) and a 95% confidence interval (CI). Odd ratios (OR) were converted to RRs using methods reported by Zhang 7. The NOS score includes an assessment of subject selection (four points), comparability of groups (two points), and exposure or clinical outcome (three points). The maximum score is 9 and the scores of eligible papers included in our meta-analysis is ranged from 5 to 9 et al.27. The potential heterogeneity across studies was evaluated using the Cochran’s Q-test and expressed using the I2 index. The pooled results for RR and HR were calculated by the fixed-effects model (I2 ≤ 50%) or the random-effects model (I2 > 50%). Publication bias was evaluated by the funnel plot and Egger’s and Begg’s tests. The effect of publication bias on the pooled findings was evaluated by trim-and-fill analyses. The stability of the pooled findings was confirmed by one-way sensitivity and subgroup analyses. All statistical analyses were conducted using STATA version 11.0.RR or HR from each article was extracted directly from the original reports or calculated using the method reported by Tierney 31, 11 papers regarding AML survival with 5,505 non-APL AML patients and 538 APL patients34, and three papers on the subject of differentiation syndrome in APL (after excluding duplicate publications36) with 243 patients38.A total of 7,475 studies were retrieved from the preliminary literature search , and an additional two studies were identified from a review of citations. 173 potential relevant publications were retrieved for detailed evaluation. Of these publications, 111 articles were excluded after reading the title and abstract because they were irrelevant to our meta-analysis, or because they were mechanistic studies, case reports, conference abstracts, or reviews. An additional 38 publications were then excluded because they were irrelevant to AML, provided no data for further analysis, or were duplicate publications or reviews. Finally, 26 studies were included in our meta-analysis , the funnel plot suggested the possibility of missing studies . One-way sensitivity analysis further confirmed the stability of our results . Although the funnel plot showed asymmetry . Specifically with respect to obesity, there was still no association between obesity and DFS  were available in three studies about non-APL AML Table . The res2 = 0%; fixed effects; Fig. 32. Moreover, high BMI in patients with APL was associated with an increased risk of developing differentiation syndrome, a severe complication of chemotherapy, 15.APL is a unique subtype of AML. Surprisingly, we found that among patients with APL, those with high BMI had a shorter duration of OS than those with low BMI after pooling three relevant studies  of AML using a meta-analytical methodology. We found that obesity had no effect on the OS or DFS of non-APL AML, whereas overweight or obese patients with APL had a poorer OS and a higher risk of developing differentiation syndrome. Overall, obesity was closely related to AML, especially APL.Increasing evidence has demonstrated that obesity is correlated to the incidence and clinical outcome of AML41. In fact, fatty acid binding protein 4 (FABP4)/IL-6/STAT3/DNA methyltransferase 1 (DNMT1) have been shown to link obesity to the growth of AML cells42. Li et al. demonstrated that the obesity-associated gene FTO is highly expressed in AML with the MLL-fusion gene, thereby promoting leukemogenesis and inhibiting ATRA-induced cell differentiation43. Moreover, studies have shown that leukemia stem cells with a high expression of CD36 (fatty acid transporter) are prone to be chemoresistance44, and CD36+ patients with AML have a poorer leukemia-free survival45.Exploring the association between obesity and AML is important for drug development and the battle against AMLs. However, mechanistic studies investigating the adverse association between obesity and AML are still in the early stages. Recently, epigenome-wide association studies identified that BMI is associated with widespread changes in DNA methylation47, suggesting they may also contribute to the leukemogenesis of AML. Furthermore, as adipocytes are one of the most abundant cell types in blood-producing bone marrow48, direct cell-to-cell interaction cannot be disregarded, as Tabe et al. have demonstrated that the direct cell-to-cell interaction between adipocytes and APL cells is required for adipocyte-induced anti-apoptotic effect49. Also, it should be noted that obese individuals have unique pharmacokinetics compared to normal-weight controls. Reduced efficacy of chemotherapy drugs in overweight and obese patients, probably as a result of altered liver enzyme function, is a likely and often underestimated event related to cancer mortality46. It is also unclear whether the increased incidence and adverse outcome of obese patients with leukemia are associated to complications from obesity-related co-morbidities and a chronic inflammatory state or to obesity itself. In addition, microbiota can modulate the response to cancer therapy and susceptibility to toxic side effects, and obesity-induced gut microbial metabolites alterations may influence the clinical outcomes of AML patients47.Apart from the mechanisms illustrated above, the function of obesity in contributing to leukemogenesis requires more research. Adipose tissue is a complex and active endocrine organ and can secrete various bioactive adipokines, hormones, inflammatory factors, angiogenesis factors, and free fatty acids. These secretory factors have been shown to participate in production and progression of tumorset al. summarized several retrospective analyses and concluded that dosing according to body surface area based on ideal body weight (cap) or actual body weight (not cap) have similar complete remission rates, toxicity, and overall survival in obese patients with AML50.When evaluating the prognosis of patients with AML, the treatment strategies and patient’s characteristics should also be factors to consider, including gender, age, chromosome karyotype, genetic changes, and ECOG. In our meta-analysis, multivariate analysis was conducted to avoid the interference of confounding factors in most studies Table . Another51. This discrepancy may be due to the physiological and metabolic differences between children and adults. However, we did find that obesity was associated with poorer OS in adult patients with APL. Actually, Tabe et al. showed that the leptin receptor is highly expressed on APL cells, and co-culturing of mesenchymal stem cell-derived adipocytes with APL cells significantly reduced ATRA and doxorubicin-induced apoptosis of APL cells49. More mechanistic studies are required to investigate the association between obesity and APL.Our finding that obesity has no effect on the OS of adult patients with AML was somewhat surprising, particularly because a high BMI is associated with poorer OS in pediatric patients with AMLOur meta-analysis may carry inherent limitations within the published literature. First, there is risk for publication, ecological, and racial bias. Second, although the survival data included was primarily obtained through multivariable analyses considering the most influential confounder, some confounders may have still been ignored. Third, BMI is inappropriate for evaluating adiposity in individuals at extreme heights or older ages. Moreover, self-reported height and weight in some studies may be inaccurate and thus result in classification errors. Obese individuals vary in their distribution of visceral and subcutaneous fat. Since the function and metabolism between the two types are different, individuals who have the same BMI may respond differently to risk factors and cancer drugs due to variance in fat distributions. Finally, the research on APL is relatively limited because its incidence is lower than in other subtypes of AML. Further studies should be conducted to sufficiently address these issues.In conclusion, both overweight and obesity were associated with increased risk of AML, with obesity producing more palpable effects. However, obesity does not significantly affect the OS and DFS of non-APL AML. APL patients are, more obese, on average, than those with other forms of AML, and obesity is an adverse prognostic factor for clinical outcome in APL (demonstrated by reduced OS and the increased incidence of differentiation syndrome). Thus, APL is more specifically associated with obesity.Evaluating the underlying mechanisms responsible for their relationship is necessary to develop the novel therapeutic approaches for AML. Also, given that high BMI increases the incidence of AML, controlling body weight may be considered for obese individuals. In addition, for obese APL patients, more attention should be given to possible ATRA-associated complications during induction therapy.Supplementary Information


METTL20-mediated methylation of ETFB influences the oxygen consumption rate in permeabilised mitochondria, suggesting that METTL20-mediated ETFB methylation may also play a regulatory role in mitochondrial metabolism. In this study, we generated Mettl20 knockout (KO) mice to uncover the in vivo functions of METTL20. The KO mice were viable, and a loss of ETFB methylation was confirmed. In vitro enzymatic assays revealed that mitochondrial ETF activity was higher in the KO mice than in wild-type mice, suggesting that the KO mice had higher β-oxidation capacity. Calorimetric analysis showed that the KO mice fed a ketogenic diet had higher oxygen consumption and heat production. A subsequent cold tolerance test conducted after 24 h of fasting indicated that the KO mice had a better ability to maintain their body temperature in cold environments. Thus, METTL20 regulates ETF activity and heat production through lysine methylation when β-oxidation is highly activated.METTL20 is a seven-β-strand methyltransferase that is localised to the mitochondria and tri-methylates the electron transfer flavoprotein (ETF) β subunit (ETFB) at lysines 200 and 203. It has been shown that METTL20 decreases the ability of ETF to extract electrons from medium-chain acyl-coenzyme A (CoA) dehydrogenase (MCAD) and glutaryl-CoA dehydrogenase  Although hundreds of non-histone lysine methylation sites have been reported, the enzymes responsible for methylation of these lysines and the physiological functions of these PTMs remain largely unknown.Protein post-translational modifications (PTMs) are involved in diverse cellular processes such as gene expression, signal transduction, and intracellular interactions. Lysine methylation in histones has been extensively studied for more than a decade; it is known to play important roles in gene expression, replication, and genome stability4. Nonetheless, only a small number of these genes has been characterised so far. METTL20 was the first mitochondrial lysine MTase (KMT) to be discovered (by two independent groups)6. METTL20 specifically methylates the β subunit (ETFB) of electron transfer flavoprotein (ETF), which acts as a mobile carrier of electrons from several flavin adenine dinucleotide–containing dehydrogenases to ETF: quinone oxidoreductase. The two METTL20 methylation sites in ETFB, i.e. K200 and K203, are located in the proximity of the recognition loop responsible for the interaction between ETF and medium-chain acyl-coenzyme A (CoA) dehydrogenase (MCAD)7; it is believed to be involved in interactions with other ETF-dependent dehydrogenases. Accordingly, it has been shown that METTL20 decreases the ability of ETF to extract electrons from MCAD and glutaryl-CoA dehydrogenase in vitro6. Moreover, METTL20-mediated methylation of ETFB influences the oxygen consumption rate in permeabilised mitochondria that are supplied with palmitoyl-L-carnitine5, suggesting that METTL20-mediated ETFB methylation may play a regulatory role in mitochondrial metabolism.There are approximately 40 MTase genes that may participate in mitochondrial functioning because the encoded proteins contain mitochondria-targeting signal sequences9. The second approach takes advantage of methylated lysine-binding domains. It has been reported that an engineered version of the triple malignant-brain-tumour domain region, i.e. L3MBTL1, can serve as a probe for the detection and enrichment of proteins containing mono- and di-methylated lysine residue(s)11. Another approach is to use cofactor analogues for labelling, detection, and pull-down via copper-catalysed azide-alkyne cycloaddition, also known as click chemistry, to enrich PTM targets. Synthetic co-factors for lysine methylation of several SET domain KMTs have been reported, for example, SUV39H212, G9a/GLP13, ESET14, Set7/913, and a seven-β-strand MTase, METTL1015.Several approaches have been developed to probe lysine-methylated proteins specifically. One such approach involves antibodies specific to methylated lysine6. To explore the biological function of METTL20-mediated ETFB methylation, we created Mettl20 knockout (KO) mice and characterised phenotypes related to their metabolism.In the present study, we performed a proteomic substrate screening for METTL20 in mitochondria of cultured cells. We found that ETFB is a major substrate; this result is consistent with other findings6. We also attempted proteomic identification of METTL20 substrates in mitochondria. A proteomic analysis using a synthetic S-adenosylmethionine (AdoMet) analogue, ProSeAM15, has been applied to identify the substrate  of the protein was subjected to western blotting with streptavidin conjugated with horseradish peroxidase  in an experiment and demonstrated that METTL20 can methylate both ETFB and H3 in vitro. It should be noted that because histones are nuclear proteins, they are possibly not a physiological target of METTL20, which is localised to mitochondria. Nevertheless, it is still possible that METTL20 has one or more additional mitochondrial substrates aside from ETFB. We also confirmed that the methylation site in ETFB is exactly the same as in other reports, i.e., K200 and K203  as described previously17. The methylation status of ETFB after the methylation reaction was confirmed . As shown in Fig. Mettl20 KO mice.To understand the physiological function of METTL20 L20 Fig. . The guiain Fig. . Expressain Fig. . Tri-metMettl20 KO mice as reported elsewhere17, with a slight modification. Briefly, the mitochondrial fractions from the WT or KO murine liver were sonicated, and the supernatant was mixed with recombinant MCAD, octanoyl-CoA, and DCPIP. As presented in Fig. Mettl20 KO mouse mitochondria was ~1.5-fold greater than that in the WT. This result is consistent with that of in vitro experiments, suggesting that methylation of ETFB inhibited ETF activity  or KD. Additionally, the differences in the body weight and several parameters such as results of liver function testing , renal function testing , lipid metabolism analyses , glucose, IPGTT, and serum adipocytokine tests were statistically insignificantly different between WT and KO mice (data not shown).The growth of Mettl20 KO mice were better at generating energy from fat, a calorimetric analysis was performed on the mice fed the ND or KD, because the KO mice showed increased ETF activity , whereas these parameters were unchanged when these mice were fed the ND . Moreover, levels of free fatty acids (FFAs) and acyl carnitine were similar between the two strains of mice . In addition to the frameshift mutation that was reported in the Icelandic population21, individuals exist that are homozygous for certain missense mutations in METTL20 , which is also present as a heterozygous mutation in the ExAC database, were expressed in bacteria; an MTase activity assay was conducted in vitro  implies that this gene is not essential. To test whether this is true for humans, the ExAC database was searched for genomic variations in Mettl20 exons. The analysis revealed that METTL20 is classified as a gene tolerant to LoF  containing 5% fat and 25% protein  or a KD containing 91% fat and 9% protein  from the age of 4 weeks to 26 weeks.All the experiments involving mice were carried out according to protocols approved by the Animal Experiment Committee of the RIKEN Brain Science Institute. The animals were maintained in a 12 h light/dark cycle with Mettl20 (NCBI ID: AK132096), Etfb (AK150293), Mcad (AK146444), METTL20 (NM_001135863), ETFA (NM_000126), and ETFB (NM_001985) were obtained either by PCR from a template plasmid, as a clone from the HeLa cDNA library, or as a clone from the Fantom3 clone collection24. The cDNAs of METTL20 and MCAD were cloned into the pET19b vector to generate N-terminally His-tagged constructs . For tandem expression of the ETFA–ETFB complex, mouse Etfb cDNA was cloned at BamHI/NotI sites into MCS1, and human ETFA cDNA was cloned at NdeI/XhoI sites into MCS2 of the pETDuet-1 vector (resulting in pETDuet-1-His-ETFB/ETFA). In addition, the cDNAs for METTL20 and ETFB were cloned into the pcDNA3 vector with a C-terminal FLAG tag (pcDNA3-cFLAG) to generate mammalian expression constructs (pcDNA3-METTL20-cFLAG and pcDNA3-ETFB-cFLAG). The QuikChange Site-Directed Mutagenesis Kit  was used to generate mutant plasmids.Full-length cDNA corresponding to 26 by means of a chemically lysine-di-methylated baculovirus as an antigen. Their specificity for lysine-di-methylation was confirmed by an ELISA as described elsewhere27 , anti-α-tubulin antibody , anti-FLAG M2 antibody (Sigma-Aldrich), anti-COXIV antibody , and anti-6xHis antibody (Wako). Monoclonal anti-di-methylated lysine antibodies (diMeLys) c4930 and c5123 were generated as described previouslyg for 10 min, and the supernatants were incubated with Ni-NTA Agarose  for 1 h at 4 °C with gentle agitation. The agarose beads were washed five times with wash buffer  and then the bound proteins were eluted with elution buffer . The purified proteins were dialyzed against 1× PBS containing 10% of glycerol, and their concentration was measured with the Bradford Protein Assay Kit .BL21 (pLysS) strains were transformed with pET19b plasmids, and the bacteria were cultured in the 2xYT medium with ampicillin (100 µg/ml) and 0.2 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) for 18 h at 16 °C. The cells were pelleted and lysed with 1× PBS containing 0.5% of NP-40 by sonication with a Branson Sonifier  for 5 min on ice. The lysates were centrifuged at 15,000× 12C6) or stable heavy (13C6) isotope in lysine after processing with the SILAC labelling kit . The mitochondrial fractions of each cell type were purified as described previously28. Briefly, cell pellets were resuspended in SEM buffer , and homogenised by means of the Dounce tissue grinder . After that, the supernatants were centrifuged at 700× g for 10 min to separate nuclear pellets from the supernatants containing cytosolic materials and mitochondria. The supernatants were again centrifuged at 700× g to remove residual pellets, and then at 7,000× g for 10 min to separate the mitochondrial pellet from the cytosol-enriched supernatant. The mitochondrial pellets were next washed twice with SEM buffer, and the resulting mitochondrial fractions were stored at −80 °C.For METTL20 target screening, HEK293T cells were cultured for at least six cell doublings in DMEM supplemented with 10% of dialysed FCS containing either a light  containing 150 μg of either light-isotope–labelled mitochondria (12C6-Mito) or heavy-isotope–labelled mitochondria (13C6-Mito) was incubated with 0.4 mM ProSeAM, with (13C-Mito) or without (12C-Mito) His-METTL20 (10 μg) (protein:enzyme at 15:1) in 50 ul of MTase reaction buffer (50 mM Tris-HCl pH 8.0) at 20 °C for 2 h. The reaction was stopped by adding four volumes of ice-cold acetone. The reaction tube was centrifuged at 15,000× g for 5 min, and the precipitate was washed once with ice-cold acetone. The pellet was dissolved in 58.5 µl of 1× PBS containing 0.2% of SDS, after which 15 µl of 5× click reaction buffer and 1.5 µl of 10 mM Azide-PEG4-Biotin (Click Chemistry Tools) were added; the reaction mixture was incubated for 60 min at room temperature. The click reaction was stopped with four volumes of ice-cold acetone. The pellet was dissolved in 75 µl of binding buffer  and sonicated for 10 s. 12C-Mito and 13C-Mito samples were mixed in a tube; 450 µl of IP buffer  with 0.1% Tween 20) containing 3 mg of Dynabeads M-280 Streptavidin  was added into the tube, and it was incubated for 30 min at room temperature . The protein-bound beads were washed thrice with wash buffer  and twice with 100 mM ammonium bicarbonate buffer, and were analysed by western blotting or MS.Labelling of mitochondrial proteins with ProSeAM was carried out by a previously described procedureAcetonitrile (1/10 volume) and dithiothreitol (20 mM) were added to protein-bound Dynabeads in 100 mM ammonium bicarbonate buffer, and the mixture was incubated for 30 min at 56 °C. Then, iodoacetamide was added, and the mixture was incubated for 30 min at 37 °C in the dark. Next, the protein samples were digested with 0.5 µg of Lys-C (Promega). The protein fragments were applied to a liquid chromatograph  coupled to a Q Exactive Hybrid Quadrupole-Orbitrap Mass Spectrometer , with a nanospray ion source in positive mode. The peptides derived from the protein fragments were separated on a NANO-HPLC C18 capillary column . Mobile phase A was composed of water with 0.1% of formic acid, and mobile phase B consisted of acetonitrile with 0.1% of formic acid. Two different slopes were used for gradient elution for 120 min at a flow rate of 300 nl/min: 0–30% of phase B during 100 min and 30–65% of phase B for 20 min. The mass spectrometer was operated in top-10 data-dependent scan mode. The operating parameters of the mass spectrometer were as follows: spray voltage, 2.3 kV; capillary temperature, 275 °C; mass-to-charge (m/z) ratio, 350–1800; normalised collision energy, 28%. Raw data were acquired via the Xcalibur software (Thermo Fisher Scientific). The MS and MS/MS data were subjected to searches in the Swiss-Prot database by means of Proteome Discoverer 1.4 (Thermo Fisher Scientific) in the MASCOT search engine software version No. 2.4.1 . The search parameters were as follows: enzyme, Lys-C; quantitation, SILAC K (+6); static modifications, carbamidomethyl (Cys); dynamic modifications, oxidation (Met); precursor mass tolerance, ± 6 ppm; fragment mass tolerance, ± 20 mDa; and maximum missed cleavages, 1. The proteins were considered identified when their false discovery rate was less than 1%. For substrate identification, proteins manifesting at least a 2-fold increase in levels in two independent experiments were defined as positive hit proteins.14C-labelled AdoMet  from 2 h to overnight at 30 °C. The reaction was stopped by adding Laemmli SDS sample buffer. Proteins were resolved by SDS-PAGE in a 12.5% acrylamide gel. An imaging plate (Fuji-Film) was exposed to the dry gel for 48 h, and the autoradiograph was obtained on a BAS-5000 Image analyser (Fuji-Film).Recombinant histone H3.1 was purchased from NEB. One microgram of the substrate was incubated in 10 ul of 1× Reaction buffer (50 mM Tris-HCl pH 8.0) with His-METTL20 (1 µg) and 17. The recombinant WT and mutant ETF complex were prepared with the pETDuet-1-His-ETFB/ETFA. Four micrograms of the ETF complex was mixed with 4 µg of METTL20 with or without AdoMet and incubated at 30 °C for 3 h in 20 ul of MTase reaction buffer. After that, 4 µg of the ETF complex was mixed on ice with 8 µg of His-MCAD, 50 µM octanoyl-CoA, and 70 µM DCPIP in potassium phosphate buffer (pH 7.6) containing 0.2 mM N-Ethylmaleimide (NEM). For the mitochondrial ETF activity assay, 300 µg of the mitochondrial fraction from the murine liver was sonicated, and the supernatant was mixed with 3 µg of recombinant MCAD, 80 µM octanoyl-CoA, and 70 µM DCPIP in potassium phosphate buffer (pH 7.6) containing 0.2 mM NEM. The initial rate of reduction of DCPIP was calculated from optical density at 600 nm measured at 30 °C every 30 s for 10 min on a microplate reader . Data were obtained in triplicate for each biological sample.ETF enzymatic activity was determined by a previously described DCPIP dye assayMettl20 were screened with CRISPR design tools (http://crispr.mit.edu/). The T7 promoter sequence 5′-TTAATACGACTCACTATAGG-3′ and the 20-mer Mettl20 guide RNA sequence 5′-GAGTGGAGCATCAAAGATCT-3′ were cloned into the AflII site of the gRNA_Cloning Vector, a gift from George Church (Addgene #41824)19. Oligos Insert-F (5′-TTTCTTGGCTTTATATATCTTAATACGACTCACTATAGGAGTGGAGCATCAAAGATCT-3′) and Insert-R (5′-GACTAGCCTTATTTTAACTTGCTATTTCTAGCTCTAAAACAGATCTTTGATGCTCCACTC-3′) were annealed and extended to construct a 100-bp double-stranded DNA fragment using Phusion polymerase . The gRNA_Cloning Vector was linearised with AflII and the inserts were incorporated via Gibson assembly . To prepare hCas9 mRNA and Mettl20 guide RNAs, an in vitro transcription reaction kit with T7 RNA polymerase  was used; the transcribed RNAs were purified with the MEGAclear Kit (Life Technologies). The quality of the guide RNA was checked by an in vitro Cas9 cleavage assay with recombinant hCas9 nuclease (NEB) and a 1 kb PCR product around the guide RNA target site. The Cas9 mRNA and guide RNA were injected into fertilised eggs (mouse strain C57BL/6J), and the mutated Mettl20 gene versions were screened by PCR after MscI digestion, because the MscI site TGGCCA is located immediately after the guide RNA target site. One in 11 heterozygous (+/−) males carried the expected 17 bp deletion. The heterozygous mouse was crossed with a WT C57BL/6J mouse, and the male (+/−) and female (+/−) mice were crossed to obtain Mettl20 KO (−/−) mice and WT (+/+) mice.Guide RNAs that target exon 4 of mouse http://www.mousephenotype.org/impress/protocol/86) of the International Mouse Phenotyping Consortium30. Briefly, mice were acclimated to the test room  and the calorimetry cage 24 h before testing. Into the calorimetry cage, a single mouse was placed; food and water were made available ad libitum during acclimation and calorimetric analysis. The measurements were started 5 h before lights were switched off for 21 h. Input and output levels of gases oxygen (O2) and carbon dioxide (CO2) were measured by an Oxymax indirect calorimetry system . The changes in the CO2 and O2 levels between the input and output were used to compute O2 consumption  and CO2 production .We conducted this analysis according to the standard operating procedure for the indirect calorimetry test ; heat production  = 4.184 × CV × VO2. Water intake was measured by the Food and Water Intake Monitor . Locomotor activity of the mice was quantified by the Infrared XY beam activity system for mice .The respiratory exchange ratio (RER), calorific value (CV), and heat production  were computed from VCO31, with a slight modification. WT and Mettl20 KO mice were either fed the ND or fasted for 24 h before the experiment. Mice in the same cage in terms of conditions and genotype were transferred to the cold room (4 °C), and their core body temperature was measured with a BAT-7001H Thermometer  after 1, 3, and 5 h.This test was performed as described previouslyMettl20 KO male mice (n = 10 for each condition) were fed the ND containing 5% of fat and 25% of protein  or the KD containing 91% fat and 9% protein  from the age of 4 weeks to 26 weeks. Twenty-six-week-old mice were killed, and the serum samples of mice from each group were pooled and kept at −80 °C until measurements. All LC-TOF MS analyses were conducted at Human Metabolome Technologies Inc., Tsuruoka, Japan. Briefly, 500 μl of pooled serum was mixed with 6 μM internal standards in 1.5 ml of the mixture of 1% formic acid and 99% acetonitrile. Samples were centrifuged at 2,300× g and 4 °C for 5 min, and the supernatants were collected and filtered through a solid-phase extraction column. Flow-through fractions were dried and dissolved in 100 μl of 50% 2-propanol. The samples were applied to a liquid chromatograph  coupled to an Agilent LC/MSD TOF Mass Spectrometer , with electrospray ionisation in both positive and negative modes. The samples were separated on an ODS column . Mobile phase A comprised water with 0.1% of formic acid, and mobile phase B comprised 65% of isopropanol, 30% of acetonitrile, 5% of H2O with 0.1% of formic acid, and 2 mM HCOONH4. Three slopes were used for gradient elution for 20 min at a flow rate of 300 μl/min: 0–1% of phase B for 0.5 min, 1–100% of phase B during 13.5 min, and then 100% of phase B for 20 min. The settings of the mass spectrometer were as follows: nebuliser pressure, 40 psi; dry gas flow, 10 l/min; dry gas temperature, 350 °C; capillary voltage, 3500 V; scan range, m/z 100–1700. The obtained data were subjected to searches in the HMT database by means of MasterHands ver.2.17.1.11 (ref.32) (Keio University). The overall data processing flow consisted of the following steps: noise filtering, baseline removal, migration time correction, peak detection, and integration of the peak area from a 0.02 m/z–wide slice of the electropherograms.WT and Mettl20 KO male mice (n = 10 for each condition) were fed the ND or KD from the age of 4 weeks to 23 weeks. The blood samples were collected from the orbital sinus during ad libitum feeding. Concentrations of serum FFAs were analysed with a kit . The obtained data were presented as mean ± SEM, n = 10 per condition.WT and METTL20 gene mutation22 . Positive Z scores indicated increased constraint (intolerance to variation), and therefore the gene had fewer variants than expected. Negative Z scores were given to genes that had more variants than expected.The ExAC Browser was searched for the Regarding LoF, the closer pLI is to 1.0, the more LoF-intolerant the gene is expected to be. If pLI ≥ 0.9, then the set of genes is extremely LoF-intolerant. Among the 106 loss-of-function and missense variants in ExAC , a D188N mutant (RSID: rs143179970) was found to be a homozygous mutation (Supplementary Table Supplementary Figures


DEP-EOM of increasing polarity was obtained by pressurized sequential extraction of DEPs with n-hexane (n-Hex-EOM), dichloromethane (DCM-EOM), methanol, and water. Chemical analysis revealed that the majority of organic matter was extracted by the n-Hex- and DCM-EOM, with polycyclic aromatic hydrocarbons primarily occurring in n-Hex-EOM. The concentration of calcium was measured in human microvascular endothelial cells (HMEC-1) using micro-spectrofluorometry. The lipophilic n-Hex-EOM and DCM-EOM, but not the more polar methanol- and water-soluble extracts, induced rapid [Ca2+]i increases in HMEC-1. n-Hex-EOM triggered [Ca2+]i increase from intracellular stores, followed by extracellular calcium influx consistent with store operated calcium entry (SOCE). By contrast, the less lipophilic DCM-EOM triggered [Ca2+]i increase via extracellular influx alone, resembling receptor operated calcium entry (ROCE). Both extracts increased [Ca2+]i via aryl hydrocarbon receptor (AhR) non-genomic signalling, verified by pharmacological inhibition and RNA-interference. Moreover, DCM-EOM appeared to induce an AhR-dependent reduction in the global plasma membrane order, as visualized by confocal fluorescence microscopy. DCM-EOM-triggered [Ca2+]i increase and membrane alterations were attenuated by the membrane stabilizing lipid cholesterol. In conclusion, lipophilic constituents of DEPs extracted by n-hexane and DCM seem to induce rapid AhR-dependent [Ca2+]i increase in HMEC-1 endothelial cells, possibly involving both ROCE and SOCE-mediated mechanisms. The semi-lipophilic fraction extracted by DCM also caused an AhR-dependent reduction in global membrane order, which appeared to be connected to the [Ca2+]i increase.Exposure to diesel exhaust particles (DEPs) affects endothelial function and may contribute to the development of atherosclerosis and vasomotor dysfunction. As intracellular calcium concentration [Ca Long-term exposure to air pollution, especially particulate matter (PM), is associated with progression and development of cardiovascular disease (CVD) . This haa]pyrene (B[a]P), often used as an indicator of PAHs i) in endothelial cells is an important second messenger regulating blood pressure and flow, angiogenesis, as well as other functions i may also be increased through activation of receptor-operated calcium entry (ROCE) without an initial depletion of internal calcium stores. In this case, receptor activation of TRP channels and other Ca2+ channels result in a direct influx of Ca2+ through the plasma membrane. The cytosolic concentration of calcium  ,32,33. V pathway . Stromaly (SOCE) . SOCE isy (SOCE) ,37. The 2+]i in respiratory epithelial cells by activating TRP vanilloid 4 (TRPV4) channels i regulation i in human microvascular endothelial cells (HMEC-1), and if so, through which mechanisms. The two extracts containing the most lipophilic organic chemicals  and dichloromethane-EOM (DCM-EOM)) triggered rapid and substantial [Ca2+]i increases via AhR signalling. The initial increase in [Ca2+]i triggered by n-Hex-EOM had an intracellular origin followed by extracellular Ca2+ influx, whereas the [Ca2+]i response triggered by DCM-EOM was completely of extracellular origin and seemed related to altered plasma membrane structure. Recently, we reported that lipophilic extractable organic matter of DEPs (DEP-EOM) could trigger pro-inflammatory responses in human endothelial cells . In the n-hexane, followed by DCM, methanol and ending with polar water. The amount of organic carbon (OC) extracted amounted to 58 ± 6% of the total OC-content of the DEPs. Specifically, n-hexane recovered 27 ± 5% of OC, DCM extracted 20 ± 4%, and the methanol extract contained 11 ± 3% of OC. The OC-content of the last water extract was below detection limit. The extracted OC was dominated by aliphatic compounds, of which the majority were unresolved in the gas chromatography—mass spectrometry (GC–MS) analysis  with greater amounts of C20–C26 alkanes. Lower amounts of PAHs and aliphatic hydrocarbons were found in the DCM extract, and none were recovered from the methanol extract. Since DCM and methanol extracts contained high amounts of higher molecular weight OC .To increase recoveries of a wide-polarity range of organic compounds from native DEPs, a sequential pressurized extraction was employed starting with non-polar analysis A. The qu extract . The mos extract B. Besideeight OC , their i2+]i increase [2+]i in endothelial cells. Human micro-vascular endothelial cells were exposed to four fractions of DEP-EOM ranging from non-polar to polar . At concentrations corresponding to extracts from 5 μg/mL (0.6 μg/cm2) of the original DEP, n-Hex- and DCM-EOM triggered substantial and rapid increases of [Ca2+]i in HMEC-1 (2) of the original DEPs, n-Hex-EOM still triggered substantial calcium response, while DCM-EOM had almost no effect  i increase due to n-Hex-EOM was unaffected, while the latter part was abrogated by removing extracellular Ca2+  and by siRNA knock-down. The efficiency of the siRNA-transfection has previously been verified, resulting in an approximately 85% reduction in AhR mRNA levels, loss of AhR protein expression. And a nearly complete abrogation of CYP1B1 inducibility in the HMEC-1 cells i increase was seemingly less affected by the treatment  attenuated the DCM-EOM induced membrane-remodelling  triggered a rapid increase in [Ca2+]i in human microvascular endothelial cells (HMEC-1) via AhR. By contrast, the more polar methanol-EOM and water-EOM did not affect [Ca2+]i at the tested concentrations. The relatively rapid kinetic of this response, occurring within minutes of exposure, and thus preceding gene expression, is consistent with AhR non-genomic Ca2+ signalling [n-Hex-EOM triggered [Ca2+]i increase from intracellular stores followed by extracellular influx, possibly via TRP channels. In contrast, DCM-EOM-triggered [Ca2+]i increase appeared to be totally dependent on extracellular influx, primarily through TRP channels. The DCM-EOM-induced Ca2+ signalling seemed to involve an AhR-dependent reduction of ordered membrane microdomains globally. To the best of our knowledge, the present study is the first to report a potential role of AhR non-genomic signalling in the biological effects of DEPs. It is interesting to note that previous studies have found that PM-induced inflammatory and vascular effects, as well as atherosclerosis, are linked to the more volatile, lipophilic OC of the particles [2+]i in endothelial HMEC-1 cells. The apparently central role of AhR in regulating these [Ca2+]i increases, induced by both n-Hex- and DCM-EOM, further suggest that aromatic compounds may be the prime drivers of the Ca2+ responses by the two fractions. As seen from the chemical analysis, n-Hex-EOM contained approximately 10-fold higher levels of PAHs than the slightly less lipophilic DCM-EOM. Accordingly, only n-Hex-EOM induced a [Ca2+]i-increase in HMEC-1 cells when the exposure was reduced from an extract concentration corresponding to 5 μg/mL of original DEPs to 1 μg/mL.In the current study, we found that relatively low concentrations of lipophilic DEP-EOM   dependent pathway. Interestingly, based on their central role in vascular function, as well as in vascular pathologies, TRP channels have been suggested as potential therapeutic targets in CVD [Based on the results obtained with buffered solution containing Cahibitor)  32], an, an2+ orhibitor) . Since B of SOCE ,57. Howe of SOCE , and has of SOCE . Thus th of SOCE  and to p of SOCE . It is ts in CVD .2+ signalling by two different DEP-EOMs remains to be clarified. However, it should be noted that SOCE and ROCE share many of the same molecular mechanisms and both are activated by phospholipase C (PLC), which has been postulated as a potential downstream mediator of AhR i increase. A possible explanation is that DCM-EOM triggered an AhR-dependent disassembly of structured lipid domains, resulting in localization of TRPC-channels in non-raft regions. According to Liao et al. i, underscores that AhR non-genomic effects may not be restricted to a single signalling pathway. An AhR non-genomic pathway has been proposed to mediate effects via both phospholipase A2/Ca2+ ,64 and F2+ . Accumul2+  cholesteinvolved . Rey-Barinvolved  have fouinvolved  have suglesterol . Thus, to et al. , such a 2+]i increase for long-term effects of PM-exposure on endothelial function should be interpreted with caution. However, it is well known that endothelial [Ca2+]i plays a central role in vasomotor regulation P-coated soot particles showed that 30% of the deposited particle-bound PAH was cleared into the blood within minutes after exposure ethyl]-1H-imidazole hydrochloride (SKF 96365) and N-phenyl]-4-methyl-1,2,3-thiadiazole-5-carboxamide (BtP2) purchased from TOCRIS . 2-Aminoethoxydiphenylborate (2-APB), 2-methyl-2H-pyrazole-3-carboxylic acid (2-methyl-4-o-tolylazo-phenyl)-amide (CH223191), cholesterol, and ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA) were purchased from Sigma-Aldrich . Di-4ANEPPDHQ was purchased from Molecular Probes, Life Technologies .Dimethyl sulfoxide (DMSO) and hydrocortisone were purchased from Sigma-Aldrich . 2HPO4, 1 mM MgCl2, 1 mM CaCl2, 10 mM glucose, 10 mM HEPES, pH 7.4.Calcium cell suspension buffer contained: 134.8 mM NaCl, 4.7 mM KCl, 1.2 mM K2.5 had an OC level of approximately 25%, while ultrafine PM, known to be major contributors to vascular effects, have OC levels around 50% i were analyzed in HMEC-1 cells exposed to all four DEP-OE, by micro-spectrofluorimetry using the Ca2+ sensitive probe Fura-2AM, as previously reported [2+]i imaging involved data acquisition every 10 s (emission at 510 nm) at 340- and 380-nm excitation wavelengths using an LEICA DMIRB microscope with an inverse 40× oil objective.Variations in intracellular Careported . Briefly2+]i were monitored using a DMIRB  inverted microscope-based imaging system equipped with a 40×/1.35 UApo N340 high UV light transmittance oil immersion objective , a CoolSnapHQ fast-cooled monochromatic digital camera (Princeton instrument), a DG-4 Ultra High Speed Wavelength Switcher  for fluorophore excitation, and METAFLUOR software  for image acquisition and analysis. Data were acquired every 10 s (emission at 510 nm) at 340- and 380-nm excitation wavelengths. All images were background-subtracted. Results are presented as normalized calcium level compared to basal [Ca2+]i measured 3 min prior to exposure. Area under the curve (AUC) was calculated from baseline (1.0).Changes in [Ca500–580 − I620–750)/(I500–580 + I620–750). On each GP image generated , a GP value was measured and normalized by subtraction of the mean of all GP values found for DMSO-treated cells . Then, ΔGP values of images from at least three experiments were used to generate a dot blot. Finally, the global mean of ΔGP values for each condition was calculated from a minimum of 10 images.We visualized the plasma membrane order with confocal fluorescence microscopy using di-4 ANEPPDHQ dye  a membrane-order sensitive probe. Di-4 ANEPPDHQ displays a fluorescent spectral blue-shift from 620 nm when incorporated into membranes with a low lipid order , and to 560 nm when inserted into membranes with a high lipid order . Fluorescence images were acquired using confocal fluorescence microscopy. We acquired both disordered and ordered-phase fluorescence images, and a new image indicative of membrane order was obtained by calculating a generalized polarization (GP) value—a ratio-metric measurement of fluorescence intensities for each pixel which is correlated to membrane lipid order . Brieflyt-test based on analysis of independent experiments . Due to the inherent variability in di-4-ANEPPDHQ-staining among individual cells, a very high counting-number is needed to obtain statistical significance in the membrane order measurements by confocal microscopy  and performing statistical comparison with unpaired croscopy . The sta2+ via AhR non-genomic signalling. Considering the variety of effects of Ca2+ signalling on endothelial cell physiology and pathophysiology, we believe these findings warrant further study. This study demonstrated, for the first time in human microvascular endothelial cells, that exposure to lipophilic organic chemicals from diesel exhaust particles increased intracellular Ca