These prior studies showed that stimulation of the leading edge Ca2+ signal can strongly activate PI3K, thereby triggering a larger PIP3 signal, but did not elucidate the mechanistic link between Ca2+ and PIP3 signaling. A hypothesis explaining this link emerged, postulating that Ca2+-activated PKC displaces the MARCKS protein from plasma membrane PIP2, thereby releasing sequestered PIP2 to serve as the target and substrate lipid of PI3K in PIP3 production. In vitro single molecule studies of the reconstituted pathway on lipid bilayers demonstrated the feasibility of this PKC-MARCKS-PI3K regulatory module linking Ca2+ and PIP3 signals in the reconstituted system. The present study tests the model predictions in live macrophages by quantifying the effects of: (a) two pathway activators—PDGF and ATP that stimulate chemoreceptors and Ca2+ influx, respectively; and (b) three pathway inhibitors—wortmannin, EGTA, and Go6976 that inhibit PI3K, Ca2+ influx, and PKC, respectively; on (c) four leading edge activity sensors—AKT-PH-mRFP, CKAR, MARCKSp-mRFP, and leading edge area that report on PIP3 density, PKC activity, MARCKS membrane binding, and leading edge expansion/contraction, respectively. The results provide additional evidence that PKC and PI3K are both essential elements of the leading edge positive feedback loop, and strongly support the existence of a PKC-MARCKS-PI3K regulatory module linking the leading edge Ca2+ and PIP3 signals. As predicted, activators stimulate leading edge PKC activity, displacement of MARCKS from the leading edge membrane and increased leading edge PIP3 levels, while inhibitors trigger the opposite effects. Comparison of the findings for the ameboid chemotaxis of leukocytes with recently published findings for the mesenchymal chemotaxis of fibroblasts suggests that some features of the emerging leukocyte leading edge core pathway (PLC-DAG-Ca2+-PKC-MARCKS-PIP2-PI3K-PIP3) may well be shared by all chemotaxing eukaryotic cells, while other elements of the leukocyte pathway may be specialized features of these highly optimized, professional gradient-seeking cells. More broadly, the findings suggest a molecular mechanism for the strong links between phospho-MARCKS and many human cancers.The leukocyte chemosensory pathway detects attractant gradients and directs cell migration to sites of inflammation, infection, tissue damage, and carcinogenesis. Previous studies have revealed that local Ca Leuack loop ,15. Simiing edge –19. In sing edge ,14–19.2+ signal triggers dramatic amplification of leading edge PI3K activity and rapid accumulation of its product signaling lipid PIP3, as well as expansion of the leading edge region [2+ influx through plasma membrane channels blocks the leading edge PIP3 signal and collapses the leading edge [2+ is also a central player in neutrophil chemotaxis, have demonstrated a strong link between Ca2+ signaling, PIP3 signaling, and leading edge growth control in leukocyte ameboid chemotaxis [2+-activated PKCα, together with the abundant signaling protein myristoylated alanine-rich C kinase substrate  molecules of plasma membrane phosphotidylinositol--diphosphate (PIP2), which serves as both the membrane target and substrate lipid of PI3K. Subsequently, PI3K would be activated when the sequestered PIP2 is released by PKCα phosphorylation of 3 specific sites on MARCKS [2 for PI3K membrane binding and lipid kinase activity. Recent in vitro single molecule studies of the reconstituted pathway on supported lipid bilayers mimicking the plasma membrane surface demonstrated that MARCKS binding to PIP2 does indeed inhibit PI3K activity [2+-activated PKCα or CaM displaces MARCKS from PIP2 thereby restoring PI3K activity and PIP3 production [Prior studies of polarized macrophages revealed that stimulation of the Cae region . Moreoveing edge . These femotaxis ,14,15,20 MARCKS ,22) coul coul2+ s signals ,14,20. Tn MARCKS ,23, or bn MARCKS ,25, theractivity . Moreoveoduction .2+- PKCα-MARCKS- PIP2-PI3K-PIP3 signaling module  and three inhibitors  on four leading edge membrane activities . The results define a perturbation matrix of 20 effects on leading edge physiology. Overall, the findings provide strong evidence of the central importance of the Ca2+- PKCα-MARCKS- PIP2-PI3K-PIP3 signaling module in regulating leading edge expansion and contraction in polarized leukocytes. The findings, together with other evidence in the field, yield a more comprehensive model of positive feedback in ameboid cell migration.Further studies in live leukocytes are needed to test the hypothesized leading edge Cag module . Previou2+-PKCα-MARCKS-PIP2-PI3K-PIP3 signaling module hypothesis predicts that activators of the leading edge positive feedback loop will stimulate PI3K and PIP3 production, and will also increase PKCα activity and phosphorylation of MARCKS [2, the phospho-MARCKS population will release sequestered PIP2 and dissociate from membranes. At the other extreme, inhibitors of the positive feedback loop are predicted to downregulate PI3K and PIP3 production, and to decrease PKCα activity and MARCKS phosphorylation, thereby shifting the MARCKS population towards membrane association and PIP2 sequestration.The Caf MARCKS ,14,20. S3, (ii) PKC activity, and (iii) MARCKS binding to the membrane, all at the leading edge of polarized, actively ruffling macrophages. AKT-PH (pleckstrin homology) domain is a standard PI3K activity sensor [3 even in the presence of PIP2. This domain was fused to the fluorescent protein mRFP to yield the PIP3 sensor AKT-PH-mRFP. Protein kinase C activity reporter (CKAR) is a soluble PKC activity sensor that possesses a specific target sequence phosphorylated by PKC and an intramolecular CFP-YFP FRET output that reports the phosphorylation state [2 binding region of MARCKS, and is known to bind to the plasma membrane surface until its target Ser residues are phosphorylated by PKC [Three sensors were employed to test these predictions by monitoring the effects of activators and inhibitors on (i) PI3K production of PIPy sensor ,35,36 thon state . The MARd by PKC ,34,38–41d by PKC . Each sed by PKC ,44.A significant advantage of sensor studies of signaling at the leukocyte leading edge is the straightforward identification of cells that both express the chosen sensor and retain a functional leading edge pathway. For each of the four sensors, confocal fluorescence microscopy revealed the cells that possessed sufficient sensor expression for imaging, and further examination identified adequate numbers of native, polarized cells with active, ruffling leading edges suitable for studies of the effects of modulators on leading edge signaling. The ability to limit the experimental pool to only those native, highly polarized cells with a functional leading edge greatly enhanced the reproducibility of subsequent studies of the effects of modulators on the leading edge signaling pathway.(i)Platelet-derived growth factor (PDGF) is a widely used attractant in live cell studies. PDGF binds to and activates the PDGF receptor (PDGFR), a receptor tyrosine kinase (RTK) that serves as a chemotaxis receptor and directs leukocyte migration to sites of inflammation in animal models and humans [3 production at the leading edge membrane [(ii)ATP is known to activate a global Ca2+ signal by binding multiple receptors, including both P2X receptors  and P2Y receptors (GPCRs that trigger slower downstream Ca2+ release from intracellular stores) [2+ signals, ATP can also serve as a leukocyte attractant [(iii)Wortmannin is a cell permeant PI3K suicide inhibitor known to block PIP3 production and the leading edge feedback loop [(iv)EGTA is an extracellular Ca2+ chelator used to inhibit Ca2+ influx through plasma membrane Ca2+ channels, and is also known to inactivate the leading edge feedback loop since the Ca2+ influx is an essential component [(v)Go6976 is a PKC inhibitor known to block the kinase activity of conventional (Ca2+-dependent) PKCs [Previous live cell studies have demonstrated the usefulness of five modulators—two activators and three inhibitors—to perturb leading edge physiology. Activators stimulate the positive feedback loop and trigger leading edge expansion, while inhibitors downregulate the feedback loop and trigger leading edge contraction. d humans ,46. Actimembrane ,48. (ii) stores) –54. In atractant –52. nt) PKCs , includint) PKCs . EvidencTo directly test the specificity of wortmannin and Go6976 as inhibitors of PI3K and PKC, respectively, both inhibitors were tested in quantitative assays of PI3Kα lipid kinase activity and PKCα protein kinase activity, respectively. Each inhibitor effectively blocked the activity of its target enzyme and had no detectable effect on the activity of the other enzyme see  Figs.The effects of each modulator on leading edge area was determined by imaging the functional leading edge regions of robust, highly polarized macrophages exhibiting actively ruffling, leading edge membranes . As prevNotably, the same effects of modulators on leading edge area are observed, within error, in cells expressing each of the different fluorescent sensors . These o3 lipid at the leading edge membrane 5.5 min after modulator addition. As previously observed for leukocyte attractants and Ca2+ influx activators [3 sensor (AKT-PH-mRFP) to the leading edge membrane, indicating that leading edge PIP3 production and accumulation is significantly increased. This stimulation of the leading edge PIP3 signal is consistent with the known ability of both attractant and Ca2+ to activate the leading edge positive feedback loop and thus stimulate PI3Kα lipid kinase activity and PIP3 production [2+ influx inhibitor (EGTA) trigger decreased recruitment of the PIP3 sensor, indicating that leading edge PI3K activity is inhibited by these drugs as previously shown [3 signal. This inhibitor significantly reduces the leading edge PIP3 signal, providing additional strong evidence supporting the hypothesis [The effects of modulators on leading edge activity sensors were quantified in polarized, actively ruffling cells as illustrated in tivators ,64,65 PDoduction ,51,66–68ly shown . Not prepothesis ,20 that 2+ signals recruit PKC to the leading edge membrane [2+ influx (ATP) activators yield increased PKC-specific phosphorylation of the CKAR FRET sensor at the leading edge, as detected by a characteristic change in intramolecular FRET upon sensor phosphorylation. By contrast, the Ca2+ influx inhibitor (EGTA) and PKC inhibitor (Go6976) trigger decreased PKC activity and CKAR phosphorylation as expected since these inhibitors block the Ca2+ and ATP loading, respectively, needed to activate PKC. Similarly, the PI3K inhibitor (wortmannin) inhibits the leading edge PKC activity, providing strong new support for the linkage between PKC and PI3K in the positive feedback loop [While previous studies have shown that attractant and Camembrane , their eack loop ,14,20.2 at the leading edge of macrophages has not previously been investigated. 2+-activated PKC is known to displace MARCKS from its tightly bound PIP2 by phosphorylating its PIP2 binding region [2+ influx (ATP) activators that enhance PKC activity, as shown above (2+ influx inhibitor (EGTA) and PI3K inhibitor (wortmannin) each yield increased recruitment of MARCKSp-mRFP to the leading edge membrane  controls the PIP2 and membrane binding of MARCKSp-mRFP at the leading edge of stably polarized, immobilized macrophages.In contrast, modulators have no effects on the membrane localization of a control sensor (MARCKSp-SA4-mRFP) in which all four Ser residues serving as specific PKC phosphorylation sites are mutated to Ala , confirmmembrane ,69,70. Tmembrane . (Instea binding ). Thus, 3 as sensed by AKT-PH-mRFP, which approach a new, lower steady state level within ~2 min. The slowest changes are observed for MARCKSp-mRFP membrane association, especially for attractant-triggered dissociation from membrane that may take as long as 5+ min to approach a new steady state.2+-PKCα-MARCKS-PIP2-PI3K-PIP3 signaling module hypothesis [3 production, and stimulate expansion of the leading edge region. These results are predicted by the model, in which activators stimulate the leading edge positive feedback loop, thereby (i) increasing the local Ca2+ signal and PKC activity, which in turn (ii) increases MARCKS phosphorylation and displaces MARCKS from its PIP2 binding sites on the membrane, thereby releasing sequestered PIP2 that (iii) increases net PI3K activity and production of PIP3. Together, (iv) the increased PKC activity, PIP2 availability, and PIP3 production drive expansion of the leading edge region via complex downstream reactions that expand the actin mesh and membrane surface area. At the other extreme, inhibitors of the Ca2+-PKCα-MARCKS-PIP2-PI3K-PIP3 signaling module are observed to decrease PKC activity, increase MARCKS binding to the membrane, and decrease PIP3 production, thereby yielding contraction of the leading edge region as the model predicts [The present findings fully support the Capothesis ,14,20. Apredicts ,14,20.2+ influx and PKCα are strongly localized to the leukocyte leading edge, where they are both essential components of the leading edge positive feedback loop [The findings include new evidence supporting the linkage of PKC and PI3K signaling in the leading edge positive feedback loop ,14,20, sack loop .2+-PKC kinase activity rather than Ca2+-CaM binding dominates MARCKS regulation of free PIP2 density. In vitro studies have shown that both Ca2+-PKC-catalyzed MARCKS phosphorylation and Ca2+-CaM binding to MARCKS can displace MARCKS from PIP2, thereby increasing the density of free PIP2 and net PI3K activity [2+-PKC regulation, but do not modify the CaM recognition sequence and thus should not disrupt Ca2+-CaM regulation [2 binding as expected [2+-PKC kinase activity, rather than Ca2+-CaM binding, dominate MARCKS regulation under these cellular conditions. The in vitro studies have demonstrated that Ca2+-PKC phosphorylation of the MARCKS population is slow, on the timescale of approximately 2–5 min at physiological concentrations, while Ca2+-CaM binds MARCKS in seconds [2+-CaM regulation of MARCKS is important in gradient detection and chemotactic migration on attractant trails, while the slow Ca2+-PKC regulation of MARCKS observed herein dominates in the steady state leading edge pathway operating in the absence of an attractant gradient . Further studies are needed in cells migrating up attractant gradients to ascertain whether Ca2+-CaM plays a role in the rapidly responding compass that guides chemotactic behavior.The new results further suggest that at the leading edge of polarized, actively ruffling macrophages, Caactivity ,26. The gulation . The MARexpected ,41,71, b seconds ,26. It t2+-PKC-MARCKS-PIP2-PI3K-PIP3 signaling module may well be universally important in eukaryotic cell chemotaxis, while other features may be unique to leukocytes due to their advanced, highly specialized gradient sensing and chemotaxis. In mammalian systems, the chemotaxis pathways of leukocytes (ameboid cells) and fibroblasts  are best studied. It now appears likely these ameboid and mesenchymal pathways likely share a PLC-DAG-Ca2+-PKC-MARCKS-PIP2 signaling module that includes phospholipase C (PLC) to generate the diacylglycerol (DAG) needed to activate the kinase activity of leading edge conventional Ca2+-PKCs. In fibroblasts DAG, as well as other elements of the PLC-DAG-Ca2+-PKC-MARCKS-PIP2 signaling module, have been observed to be localized at the leading edge [2+-PKC-MARCKS-PIP2 signaling module likely generates positive feedback since the output PIP2 is a substrate for PLC in DAG production [3 signals are not essential for chemotaxis [3 signals in leukocyte chemotaxis [2+-PKC-MARCKS-PIP2-PI3K-PIP3, while in mesenchymal cells the simpler PLC-DAG-Ca2+-PKC-MARCKS-PIP2 module appears to suffice. Much remains to be learned about key regulatory mechanisms within these leading edge modules, as well as the upstream receptor-triggered reactions that activate the modules, and the downstream reactions controlled by the modules to regulate actin and membrane remodeling in directed cell migration.Certain elements of the observed Caing edge –19. DAG oduction , and is oduction –79. In femotaxis –19, in cemotaxis ,14,80,813 signaling regulates other key cell pathways, including and excessive PIP3 production has long been linked to many human cancers . Recentiewed in ). The prRAW 264.7 murine macrophage cells were obtained from the American Type Culture Collection . These cells (lot 70000171) were analyzed by ATCC and confirmed as RAW 264.7 based on morphology, growth properties and test for contamination. Cells were typically passaged 6–10 times, never more than 12 times. Only cells retaining characteristic, strong leukocyte polarization with a ruffling leading edge were utilized in the studies described herein. Wortmannin, ATP, PDGF-ββ, EGTA and HEPES were from Sigma . Go6976 was from Tocris Bioscience . Neon Transfection System, anhydrous DMSO and CellMask Green and Deep Red plasma membrane stains were from Thermo Fischer Scientific . Cell culture media components included FluoroBrite DMEM media ), fetal bovine serum (FBS) from Sigma , Pen Strep (penicillin streptomycin) from Gibco  and GlutaminePlus  form Atlanta Biologicals . Matriplate plates with 96 culture wells and 0.17mm glass bottom were from Matrical, Inc . C kinase activity reporter (CKAR) mammalian expression plasmid was a kind gift from the lab of Alexandra C. Newton . AKT-PH 2 to ~80% confluency in DMEM media supplemented with 10% heat-inactivated FBS, 20mM HEPES, 2 mM GlutaminePlus and 100 U/mL penicillin, 100 μg/mL streptomycin. Cells were washed in room-temperature Dulbecco's PBS (D-PBS), scraped in a volume of room temperature 10 mL D-PBS and counted using a hemocytometer. After counting, cells were pelleted and resuspended to a concentration of 5x106 cells/mL in prewarmed (37°C) Neon Buffer T and mixed with 30μg of the relevant reporter-XFP plasmid (plasmid addition diluted the cell suspension ≤10%). The cell and plasmid DNA suspension were aspirated using 100 μL Neon tips and electroporated using the Neon system set for a single pulse at 1680 volts for 20 ms. Electroporation mixtures were immediately mixed into 15 mL of prewarmed (37°C) antibiotic-free DMEM supplemented with FBS, 20mM HEPES and L-glutamine dipeptide. 0.5 mL of the resulting cell suspension was added to 0.6 mL capacity Matriplate wells and incubated for 4 hours at 37°C, 5% CO2, whereupon the media was aspirated and replaced with prewarmed (37°C) DMEM supplemented with FBS, HEPES, L-glutamine and antibiotics as described above. Transfected cells were incubated at 37°C, 5% CO2 for at least 24, and up to 48 hours after transfection.RAW264.7 cells were cultured and passaged at 37°C, 5% COμg/mL streptomycin. Cells were then allowed to acclimate at 37°C, 5% CO2 for 3–4 hours. Where indicated, prior to imaging, adherent cell plasma membranes were stained for 10 minutes at 37°C with a 1:1000 dilution of CellMask dye, diluted into Fluorobrite DMEM with supplements described above, followed by three aspiration-rinse cycles using prewarmed (37°C) FluoroBrite DMEM with supplements.At least three hours before imaging experiments, cells were rinsed twice with prewarmed (37°C) FluoroBrite DMEM supplemented with 10% heat-inactivated FBS, 20mM HEPES, 0.292 mg/mL L-glutamine dipeptide and 100 U/mL penicillin, 100 2. Cells selected for imaging and further experimentation were both (i) visibly expressing the transfected fluorescent fusion protein, and (ii) clearly polarized and displaying an actively ruffling leading edge. After an initial image was collected, a selected drug, or control drug vehicle (FluoroBrite DMEM or DMSO), was mixed into the media at zero time and data collection was initiated at t = 30 sec. At 30 second increments, a 0.5 μm Z-stack was collected using Nikon Elements, typically resulting in a Z-stack of 11 images. Exposure time for acquisitions were typically set as follows: YFP 300 ms, RFP 300 ms, GFP 300 ms and CFP 500 ms. Final drug concentrations were: PDGFββ 2.1 μM ; ATP 25 μM (added in DMEM supplemented as indicated above); EGTA 3 mM (added in DMEM supplemented as indicated above); wortmannin 500 nM (added in DMSO); Go6976 1 μM (added in DMSO).Live-cell microscopy experiments were carried out on a Nikon TiE Confocal microscope equipped with a Nipkow Yokogawa CSU-X1 spinning disc unit, a 60x 1.4 N.A, oil immersion objective, seven laser lines and an Andor iXon Ultra 888 EMCCD camera. The excitation laser lines used for imaging were: 488 nm (CFP), 514 nm (YFP), 561 nm (CellMask Green plasma membrane stain), 594 nm (RFP), 640 nm (CellMask Deep Red plasma membrane stain). The quad emission filters employed were: 447/38 nm (CFP), 520/22 nm (YFP and cellmask green), 598/40 nm (RFP), 705/68 nm (cellmask deep red). The microscope was fully enclosed in a humidified Oko Labs environmental chamber at 37°C, 5% COImages were analyzed with Fiji, the open-source processing program distribution of ImageJ . A Z-prodecreases as PKC kinase activity increases the steady state phosphorylation level of the CKAR population. To generate a parameter proportional to PKC kinase activity, a standard procedure was employed [For the simple fluorescence sensors employed  the above procedure yielded the fractional change in the leading edge signal, while for the FRET sensor CKAR additional data analysis was needed. The soluble, cytoplasmic FRET sensor CKAR was imaged near the leading edge membrane (see above) since the plasma membrane-targeted PMCKAR did not yield adequate signal to detect PKC activity changes. The CKAR FRET signal employed . Image semployed . FiguresS1 Fig3 product molecule generated via the binding of a high affinity fluorescent PIP3 sensor (GRP PH domain labeled with AF 555). (A) Representative single particle tracks for GRP1 PH-PIP3 complexes. Such tracks were evaluated and identified by a stringent set of criteria based on size, brightness and diffusion speed to count the number of product PIP3 molecules generated by PI3K as a function of time. (B) Timecourse of PI3K-catalyzed PIP3 production showing the linear accumulation with time of single particle tracks identified as fluorescent GRP PH-PIP3 complexes. The timecourse of PIP3 production is slowed dramatically by the PI3K inhibitor wortmannin, but not by the PKC inhibitor Go6976. (C) The rate of each reaction in (B) determined from the slope of its timecourses. The rates confirm that the PKC-specific inhibitor Go6976 has no significant effect on PI3K kinase activity while the PI3K-specific inhibitor wortmannin efficiently suppresses PIP3 production by the lipid kinase. In all cases, error bars are standard errors of the mean (n ≥ 9), and measurements were 21.5 ± 0.5°C in 100 mM KCl, 20 mM HEPES pH 6.9 , 15 mM NaCl, 5 mM glutathione, 2.0 mM EGTA, 1.9 mM Ca2+, 1.9 mM Mg2+, 1.0 mM ATP, 100 μg ml-1, and 0.05% CHAPS. Under these conditions, the EGTA-ATP-Ca2+ buffering system yields 10 μM free Ca2+ and 0.5 mM free Mg2+.To compare and quantify the inhibitory effects of wortmannin and Go6976 on PI3K lipid kinase activity, our previously described single molecule TIRFM assay , 26, 85 (TIFF)Click here for additional data file.S2 FigA) Raw data of separated phosphorylated and nonphosphorylated PKC substrate after a 30 minute incubation at 30°C. (B) Optical density of the phosphorylated (lower) bands are quantitated using the ImageJ [C) To ensure that the PKC activity remained in the linear, initial rate phase of the reaction, several reaction times between 1–60 min were tested. The reaction was linear for at least 30 min, in accordance with the manufacturer protocol. The rates confirm that the PKC-specific inhibitor Go6976 blocked PKC kinase activity while the PI3K-specific inhibitor wortmannin had little or no effect on PKC. Kinase assays were performed at 30°C per manufacturer protocol, except the PKC lipid activator (phosphatidylserine) was replaced with 200 μg/ml sonicated unilamellar vesicles (SUV) comprised of PC:PS:PIP2:DAG ) at lipid mole percentages of 73:23:2:2, respectively, closely matching the lipid composition employed in the single molecule studies of 2, 26 μM CaCl2, 20 μM EGTA, 1 mM EGTA, 1 mM DTT, 1 mM ATP and 20 mM HEPES pH 7.4).To determine the effect of wortmannin and Go6976 on the activity of PKCα, a modified PepTag assay was employed to quantify bulk PKCα activity . PKe ImageJ  gel anal(TIFF)Click here for additional data file.


Based on the anomalous subdiffusion of Ca2+ in the cytoplasm, a mathematical model was developed to investigate the effects of rogue RyRs on Ca2+ sparks in cardiac myocytes. Ca2+ quarks and sparks from the stochastic opening of rogue and clustered RyRs are numerically reproduced and agree with experimental measurements. It is found that the stochastic opening Ca2+ release units (CRUs) of clustered RyRs are regulated by free Ca2+ concentration in the JSR lumen (i.e. [Ca2+]lumen). The frequency of spontaneous Ca2+ sparks is remarkably increased by the rogue RyRs opening at high [Ca2+]lumen, but not at low [Ca2+]lumen. Hence, the opening of rogue RyRs contributes to the formation of Ca2+ sparks at high [Ca2+]lumen. The interplay of Ca2+ sparks and Ca2+ quarks has been discussed in detail. This work is of significance to provide insight into understanding Ca2+ release mechanisms in cardiac myocytes.Ca On the other hand, mathematical models have been developed to investigate the dynamics of Ca2+ sparks and quarks. Sobie et al. [2+]lumen to support the existence of rogue RyRs. Models of Ca2+ leak from JSRs have been used to characterize differences between Ca2+ sparks and quarks lumen could not recruit Ca2+ sparks from Ca2+ release units (CRUs). However, the effects of rogue RyRs on Ca2+ sparks at high [Ca2+]lumen remain unknown. The anomalous subdiffusion of cytoplasmic Ca2+ and the random distribution of JSR RyRs, unnoticed in these models, could unveil the temporal and spatial properties of Ca2+ sparks and quarks.Zima et al.  demonstre et al.  proposedd quarks . Walker  T represent the total concentrations of indicator and buffers, respectively. [CaF] and [CaBn] are the concentrations of Ca2+-bound complexes; Kpump is the affinity constant, and Jrogue and Jclustered are the Ca2+ fluxes released by rogue and clustered RyRs respectively, which can be written as:δ is the Dirac delta function and S is a stochastic function for the opening of clustered and rogue RyRs; Trogue= 20 ms NSR is 1.0 mM, time constant for Ca2+ transfer between JSR and NSR −11 µl [2+]lumen is less than the beginning level 1.0 mM as a result of Ca2+ release at a certain time. 2+ fluxes due to indicator dye (i.e. Fluo-5N) and buffer  in a JSR lumen, respectively. Their expressions are similar to that in the cytoplasm. Various parameters of dyes  and buf  or clustered (m = 10) RyRs.The firing probability per unit time of RyRs is determined by CaSR lumen –35, whic channel . Here, Φ2.5.2+ release events from a single Ca2+ release site. Moreover, a computational domain of 20 µm × 20 µm was meshed with a size of 0.1 µm to simulate Ca2+ release events from multiple Ca2+ release positions. For the fractional differential term in equation (2.1), the shifted Grünwald formula of centre difference lumen on the firing frequency of Ca2+ sparks and quarks are quantified in a computational domain of 20 × 20 µm2, as shown in 2+]lumen is set from 0.2 to 1.0 mM. The computational results show a higher firing frequency of spontaneous QCR events than that of spontaneous Ca2+ sparks consistent with previous experimental observations [2+ sparks despite a slight change in the firing frequency of spontaneous QCR events with the increase of [Ca2+]lumen [2+]lumen (i.e. less than 0.3 mM) where spontaneous Ca2+ quarks become a major pathway of SR Ca2+ leak. The present study shows the incidence of 12.2 ± 1.1 Ca2+ sparks [100 µm]−1 s−1 under physiological conditions ([Ca2+]lumen = 1.0 mM), which agrees with experimental measurements [figure 2c.The effects of [Carvations . A compu2+]lumen . Moreoveurements . The sta3.4.2+ sparks at different levels of [Ca2+]lumen. A comparison of computational line-scan Ca2+ release events with consideration of rogue RyRs or not is displayed in a square of 20 × 20 µm2  for 2 s (figure 6a versus figure 6b) at different [Ca2+]lumen. The Ca2+ spark frequency has values of 1.3 ± 0.4 and 1.2 ± 0.3 [100 µm]−1 s−1 with consideration of rogue RyRs or not when [Ca2+]lumen = 0.2 mM. Therefore, the stochastic opening of rogue RyRs at low [Ca2+]lumen fails to trigger spontaneous Ca2+ sparks owing to the decreased driving force ([Ca2+]lumen − [Ca2+]cyto) and sensitivity of clustered RyRs as well as the shortened firing possibility of neighbouring RyRs. Conversely, the frequency of spontaneous Ca2+ sparks increases with consideration of rogue RyRs at 1.0 mM [Ca2+]lumen. QCR events are hence responsible for the formation of Ca2+ sparks at high [Ca2+]lumen. Moreover, sensitivity analysis on the firing frequency of Ca2+ sparks was performed with respect to the number of rogue RyRs varying in the range of 2–14 in figure 6c. The opening of clustered RyRs monotonically increases with the increase of the number of rogue RyRs at high [Ca2+]lumen. But it does not change obviously at low [Ca2+]lumen. The firing frequency of Ca2+ sparks has a bigger slope at higher [Ca2+]lumen.We examined how rogue RyRs affect Ca3.5.et al. [2+ spark when [Ca2+]lumen is above a threshold. Walker et al. lumen, but not at low [Ca2+]lumen. This supports the conclusion of Sato et al. lumen. The frequency of spontaneous Ca2+ sparks is remarkably affected by the rogue RyRs opening at high [Ca2+]lumen, but not at low [Ca2+]lumen. This study is of importance to understand basic mechanisms of Ca2+ release events in cardiac myocytes.A mathematical model is developed to investigate Ca


The Deepwater Horizon disaster drew global attention to the toxicity of crude oil and the potential for adverse health effects amongst marine life and spill responders in the northern Gulf of Mexico. The blowout released complex mixtures of polycyclic aromatic hydrocarbons (PAHs) into critical pelagic spawning habitats for tunas, billfishes, and other ecologically important top predators. Crude oil disrupts cardiac function and has been associated with heart malformations in developing fish. However, the precise identity of cardiotoxic PAHs, and the mechanisms underlying contractile dysfunction are not known. Here we show that phenanthrene, a PAH with a benzene 3-ring structure, is the key moiety disrupting the physiology of heart muscle cells. Phenanthrene is a ubiquitous pollutant in water and air, and the cellular targets for this compound are highly conserved across vertebrates. Our findings therefore suggest that phenanthrene may be a major worldwide cause of vertebrate cardiac dysfunction. Polycyclic aromatic hydrocarbons (PAHs), consisting of two or more fused benzene rings, are ubiquitous pollutants in aquatic systems. Crude oils typically produce PAH mixtures with high concentrations of 2-, 3-, and 4-ringed PAHs families and correspondingly low levels of ≥5-ring compounds, while combustion of fossil fuels and other carbon sources generate mixtures that may be dominated by larger 4–6 ring compounds. Although industrial PAH emissions have declined in the developed world, loadings to aquatic habitats have increased with human population growth and increased motor vehicle use11As expected for a large and complex family of structurally related compounds, the biological activities of PAHs are both overlapping and multifaceted. A major challenge has been determining which individual compounds contribute to the overall toxicity of complex mixtures with different chemical compositions. The carcinogenicity of 4–6-ringed PAHs such as benzo(a)pyrene was recognized more than half a century ago9cyp1aStudies in zebrafish embryos have demonstrated cardiotoxic effects of many single PAH compounds and PAH mixtures, but also revealed a minimal binary division of mechanisms. PAHs that are strong carcinogens are potent agonists of the aryl hydrocarbon receptor (AHR), inducing their own metabolism and carcinogenic activation by cytochrome P4501A1113141518192021181920212+) entry into the cell through L-type Ca2+ channels (ICaL). This extracellular Ca2+ influx triggers the release of additional Ca2+ from the internal Ca2+ stores of the sarcoplasmic reticulum (SR) via a process termed ‘Ca2+-induced Ca2+-release’ (CICR). The consequent systolic Ca2+ transient, which activates the contractile machinery within the heart muscle cells, is the spatial and temporal sum of such local Ca2+ releases302+ is returned to resting levels by reuptake into the SR via the SR Ca2+ ATPase (SERCA) and extrusion from the cell via the sodium calcium exchanger (NCX). Critical for AP repolarization are the opening and closing of voltage-gated sodium (Na+), Ca2+ and potassium (K+) channels, which renew the EC coupling process at every heartbeat. Our earlier workWe have recently demonstrated that complex PAH mixtures from crude oil affect excitation-contraction (EC) coupling in fish hearts2628Scomber japonicas), the yellowfin tuna  and the Pacific bluefin tuna . We then reveal the mechanism for this disruption in atrial and ventricular myocytes from these pelagic predators.Despite evidence that the tricyclic PAH fraction causes the crude oil heart failure syndrome in developing fish2+ dynamics were assessed using confocal microscopy. 2+ transients recorded using a calcium-sensitive dye, Fluo-4. Cellular Ca2+ dynamics were unaffected by PAHs with two benzene rings fused to thiophene and pyrrole rings. By contrast, the 3-ringed PAH, phenanthrene, significantly decreased the Ca2+ transient amplitude and slowed the decay  through L-Type Ca2+ channels and/or a smaller Ca2+ release from SR internal Ca2+ stores. To clarify this point, we first used whole-cell voltage-clamp to investigate extracellular Ca2+ influx via ICaL. We found that phenanthrene decreased the amplitude of ICaL  internal SR stores.Similar to Pacific mackerel, phenanthrene (5 μM) impaired Cafin tuna  and B anfin tuna . Thus, i of ICaL –C. ICaL 2+ release  and SR Ca2+ uptake (2 μM thapsigargin to inhibit SERCA) for 30 minutes before exposing to phenanthrene. As anticipated from earlier work2+ transient , which causes the ryanodine receptors to open and empty SR Ca2+ into the cytosol. We found SR Ca2+ content was significantly decreased in myocytes exposed to phenanthrene   and significant prolongation of AP duration (APD) associated with a dose-dependent increase in triangulation (APD0-APD30) . Such pr+ channel current (IKr), is the key ion current responsible for AP repolarization. To determine whether the observed AP prolongation was due to an inhibition of outward K+ conductance, we evaluated the effects of phenanthrene on IKr. As shown in Kr was significantly reduced by ascending concentrations of phenanthrene. In addition to explaining the prolongation of the AP, this mechanism is consistent with the phenanthrene- and crude oil-induced arrhythmogenesis and atrioventricular conduction block previously reported in other fish species1820The delayed rectifier K+ efflux from the cardiomyocyte via IKr. Second, phenanthrene reduces Ca2+ influx into the cell by reducing ICaL. This reduces myofilament activation and thus myocyte contractility. Third, inhibition of ICaL has a knock-on effect as it reduces Ca2+ release from the SR, which further impairs cardiac contractility. Combined, these disruptions to EC coupling in the myocyte can lead to contractile failure and abnormal contractile rhythm. Our findings are in agreement with previous studies showing that SERCA function is altered in phenanthrene-exposed zebrafishatp2a2/SERCA2 and ryr2/ryanodine receptor), these effects must be downstream of AHR activation38Our data demonstrate that impairment of EC coupling in cardiomyocytes by phenanthrene is a key determinant of cardiotoxicity from crude oil. This disruption of excitable cell pathways now establishes a mechanism for crude oil cardiotoxicity that has proven elusive for decades. First, phenanthrene affects membrane excitability by prolonging AP duration by inhibiting Ke.g. refs 17By applying single PAHs directly to isolated cardiomyocytes, we have identified AHR-independent cellular mechanisms that likely underpin the whole-heart cardiotoxicity phenotypes previously observed in oil-exposed fish embryos. Because of the very small size of the embryonic fish heart  is proportionally more toxic to the fish heart. Our results also help explain why certain geologically distinct and phenanthrene-enriched crude oils have more significant cardiotoxic impacts, although we acknowledge that many PAHs and mixtures of PAHs exert their toxicity via multiple different mechanisms. This new insight may simplify the interpretation of water samples collected during oil spills for natural resource injury assessments. Future assessments should give particular weight to measured and modeled levels of phenanthrenes, in addition to complex PAH mixtures that dynamically shift in space and time throughout regions impacted by spilled oil.Kr is generated by the voltage-gated potassium channels hERG1 or hERG24344Lastly, given that phenanthrenes are a near-ubiquitous component of complex environmental PAH mixtures in the oceans and the atmosphere, our findings have implications for humans that extend well beyond the Deepwater Horizon spill. A major area of public health research over the last decade has focused on the acute cardiac impacts of urban air pollution, but the precise etiology of these effects remains elusiveWe have identified a cardio-toxicant compound prominent in crude oil, and shown how it alters cardiac force and cardiac rhythm in pelagic fish. This provides a new framework for evaluating the effects of PAHs on cardiac function that we believe can be extended across a wide range of vertebrates, including humans. Reducing future releases of this pro-arrhythmic chemical should substantively benefit human and ecological health.F/V Shogun in seawater wells, and then transported by truck to the Tuna Research and Conservation Center . Mackerel and tunas were held in a 30-m3 and 109-m3 tank respectively at 20 ± 1 °C and fed a diet of squid, sardines, and enriched gelatin, as previously described48Mackerel , bluefin tuna  and yellowfin tuna  were captured off San Diego, CA, held aboard the All solutions were prepared using ultrapure water supplied by a Milli-Q system . Chemicals were reagent grade and purchased from Sigma  except for TTX , ryanodine , Fluo-4  and E-4031 . Purities for all PAHs were at least 99%. Stock PAH solutions were constituted in dimethyl sulfoxide  at 5 mM.2+ imaging and electrophysiology was based on previous work2, 3.2 CaCl2, 10 glucose and 10 HEPES, with pH set to 7.7 with NaOH. To avoid overlapping ion currents when recording IKr and ICaL, this solution was modified. For ICaL measurement, KCl was replaced by CsCl to inhibit K+ channels. For IKr recording, tetrodotoxin , nifedipine (10 μM), and glibenclamide (10 μM) were included in the Ringer solution to inhibit Na+, Ca2+, and ATP-sensitive K+ channels, respectively.The composition of the extracellular physiological solution (Ringer) used for both Ca2, and 10 HEPES, pH adjusted to 7.2 with KOH. For ICa measurement, the pipette solution contained (in mM) 130 CsCl, 15 TEA-Cl, 5 MgATP, 1 MgCl2, 5 Na2-phosphocreatine, 0.025 EGTA, 10 HEPES, and 0.03 Na2GTP, pH adjusted to 7.2 with CsOH. CsCl and TEA-Cl were included to inhibit K+ currents. The low concentration of EGTA was included to approximate physiological Ca2+ buffering capacityKr, the pipette solution contained (in mM): 10 NaCl, 140 KCl, 5 MgATP, 5 EGTA, 1 MgCl2, and 10 HEPES, pH adjusted to 7.2 with KOH. The high concentration of EGTA was included to block Na+ -Ca2+ exchanger current.Pipette solutions were optimized for each electrophysiological recording. For AP measurement, the pipette solution contained (in mM): 10 NaCl, 140 KCl, 5 MgATP, 0.025 EGTA, 1 MgCl2+ imaging was performed using a laser-scanning unit attached to an Olympus inverted microscope. Control and PAH-exposed myocytes were loaded with 4 μM Fluo-4 AM (Molecular Probes) for 20 min, washed via dilution to de-esterify and then perfused with standard Ringer solution. The dye was excited at 488 nm and fluorescence measured at >500 nm. Transverse line scans were acquired at 5 ms intervals. Cells were electrically stimulated at 0.5 Hz via extracellular electrodes. Batches of myocytes were incubated with 5 μM of PAH for at least 1 hour. Control experiments were performed on time-matched untreated cells. Some cells from both control and PAH-exposure groups were incubated with SR inhibitors (5 μM ryanodine and 2 μM thapsigargin) for at least 30 minutes prior to imaging. In some experiments, a caffeine pulse (20 mM) was applied via a home-built rapid solution system. All line scan images are presented as original raw fluorescence (F). Background fluorescence (F0) was measured in each cell in a region that did not have localized or transient fluorescent elevation.Confocal Ca2+ channel current (ICaL) was elicited by a pulse to 0 mV (the approximate peak of the current-voltage relationship in cardiomyocytes) after a pre-pulse to −40 mV to inactivate Na+ current. ICaL was measured as the difference between peak and the end of pulse current. Trains of depolarizing pulses were applied at 0.2 Hz. Action potentials (APs) were evoked using 10 ms sub-threshold current steps at a frequency of 0.5 Hz. The delayed rectifier K+ current (IKr) was measured using an established protocol adapted from previous studies on Pacific bluefin tunaKr was activated by a pre-pulse to +40 mV (to fully activate K+ channels) and measured as the tail current at −20 mV, the maximum tail current in tuna myocytes+ current (IKr), tail IK amplitude was measured as the current sensitive to a specific IKr inhibitor (2 μM E-4031). Trains of depolarizing pulses were applied 0.2 Hz every 20 seconds. Data was recorded via a Digidata 1322 A A/D converter  controlled by an Axopatch 200B  amplifier running pClamp software . Signals were filtered at 1–10 kHz using an 8-pole Bessel low pass filter before digitization at 10–20 kHz and storage. Patch pipette resistance was typically 1.5–3 MΩ when filled with intracellular solution (below). Cell membrane capacitance was measured using the “membrane test module” in Clampex .Electrophysiological data was recorded as previously described2+ transients from confocal experiments were analyzed using Image J, Clampfit , and Origin . At least three traces at steady state were analyzed and averaged. The decay of the Ca2+ transient was fitted with a single exponential to calculate Tau of decay . The amplitude of the Ca2+ transient is defined as peak increase and basal Ca2+ (F/F0)CaL was measured as the difference between the peak inward current and the current at the end of the depolarizing pulse. IKr amplitude was measured as the current sensitive to E-4031.CaData are presented as mean ± SEM Statistical analysis was performed using SigmaStat software . Electrophysiological data with a confirmed normal distribution and equal variance were analyzed using One Way Repeated Measures Analysis of Variance or Friedman Repeated Measures Analysis of Variance on Ranks followed by a Student-Newman-Keuls test for post hoc analysis. For confocal data, unpaired Student’s t-test were used within the same species of tuna, one-way ANOVAs were used to test for the effect of PAHS in mackerel, and two-way ANOVAs were used to determine interactions between species, SR inhibition and PAHs. P < 0.05 was considered significant.How to cite this article: Brette, F. et al. A Novel Cardiotoxic Mechanism for a Pervasive Global Pollutant. Sci. Rep.7, 41476; doi: 10.1038/srep41476 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Previously, we have found that intracellular calcium homeostasis is altered in platelets from an experimental model of liver cirrhosis, the bile-duct ligated (BDL) rat; these alterations are compatible with the existence of a hypercoagulable state and related to an enhanced intracellular calcium release evoked by thrombin and an increased amount of calcium stored in the intracellular organelles. In the present study we have investigated the role of bile acids in those alterations of the BDL cirrhotic model. Cholic acid (CA) or deoxycholic acid (DCA) did not change P-selectin expression or platelet aggregation in any group but elevated baseline platelet calcium levels. Incubation with both bile acids reduced calcium release after stimulation with thrombin in the absence of extracellular calcium. Pretreatment with CA but not with DCA reduced significantly thrombin-induced calcium entry in all three experimental groups. The capacitative calcium entry was also significantly lower in platelets pretreated with both bile acids. The simultaneous addition of thapsigargin and ionomycin to estimate the total amount of calcium in platelet internal stores was decreased by pretreatment with both CA and DCA, although these changes were significantly different in the control rats only with CA and in the BDL platelets with DCA. These results indicate that CA and DCA reduce calcium movements in platelets of control and BDL animals, thus suggesting that bile acids do not participate in the alterations observed in the BDL cirrotic model. Regarding platelet activation, the necessary increase in cytoplasmic Ca2+ levels, both by release from internal stores and entry of extracellular Ca2+, has been reported to be defective both in experimental models of liver cirrhosis and patients  has been observed to cause profound Ca2+ release from intracellular stores and entry from the extracellular medium of intact or permeabilized fibroblast cells . The animals were kept and treated according to the guidelines established by the European Union for the protection of animals used in experiments (86/609/EEC). All procedures were approved by the Animal Care and Use Committee of the University of Murcia.ad libitum. Experiments were performed 21 days later (BDL non-ascitic group) and at least 28 days after surgery, when ascites was present (BDL ascitic group).Animals weighing ~250 g were subjected to bile-duct ligation (BDL) and excision or sham operation (controls), as described previously  and 5 μl CD62P-PE (for detection of P-selectin). The acquisition was set at 50,000 events in the FAC-sort cytometer (Becton-Dickinson). The software used was CELLQuestTM. These experiments were performed in platelets obtained from six animals per group.2+ measurements were performed. Platelets were placed in fluorescence-free cuvettes (Sigma) in the optical field of a fluorescence spectrometer , and excited alternatively with light at 340 and 380 nm and the light emitted at 510 nm was collected. Changes in cytosolic free Ca2+ concentration ([Ca2+]i) were obtained by using the fura-2 340/380 fluorescence ratio and calibrated as described previously  and presence (1 mmol/l CaCl2) of extracellular Ca2+.The response to thrombin (0.3 U/ml) was studied in the absence (no Ca2+ entry (CCE) was determined by incubating platelets in a Ca2+-free medium with thapsigargin (1 μmol/l). After 180 s, Ca2+ (1 mmol/l) was added and changes in [Ca2+]i were monitored for another 180 s.Capacitative Ca2+ accumulation into intracellular stores was estimated by suspending platelets in a Ca2+-free medium (+100 μmol/l EGTA) and challenging with ionomycin (5 μmol/l) and thapsigargin (1 μmol/l).CaThese methods were carried out as described previously . Then, these three protocols were performed again.All the products used were from Sigma, except where indicated. Fura-2 AM  and Thapsigargin (Invitrogen) were dissolved in DMSO. Appropriate dilutions were prepared freshly every day in measurement buffer  of the individual Ca2+ responses were calculated by summation of all experimental values (180 s) corrected by substraction of the averaged baseline (estimated during 30 s). The resulting values as well as baseline values were compared between groups by one-way analysis of variance and a Student-Newman-Keuls as post-hoc test. A probability level of p < 0.05 was considered to be a significant difference.Data are expressed as the mean ± S.E.M. In order to compare the CaThe BDL rats used in the present study had the typical features of this model, jaundice, enlarged liver and spleen, and mesenteric edema. Ascites was present in variable amounts only in the animals included in this group. Sham-operated animals did not have any of these alterations.2+ levels was significantly elevated by incubation with both CA and DCA .Baseline P-selectin expression was significantly greater in the BDL group as compared to the controls and the BDL-ascitic rats, and CA or DCA did not affect these values in any group Table . Platele2+ release after stimulation with thrombin (0.3 U/ml) in the absence of extracellular Ca2+  and Ca2+ entry from the extracellular space, although only CA, but not DCA, showed a significantly lower response in all three groups, control and BDL-ligated rats. This result agrees with previous data (Bowen et al., 2+ release from internal stores and persistent entry at the plasma membrane (Lau et al., Regarding thrombin-induced intracellular Ca2+ mobilization, secretion, fibrinogen binding, and aggregation. However, bile acids also activate a number of cellular Ca2+ related channel receptors (Dopico et al., 2+ regulation is critical for multiple platelet functions (Sage, 2+ signaling. Clearly, more experiments would be necessary in order to prove this hypothesis.Our results are in keeping with more recent data indicating that bile acids also act as regulatory molecules (Hylemon et al., ns Sage, , we sugg2+ entry, again suggesting an interference with the channels involved in those responses. Interestingly, bile acids did not reduce the intracellular Ca2+ elevation induced by thapsigargin, an inhibitor of SERCA Ca2+ ATP-ase, that although modest compared to the capacitative entry, was significantly elevated with DCA in the platelets of the control animals.A similar effect of bile acids was found when we studied the capacitative Ca2+ in internal stores, as estimated with the use of thapsigargin and ionomycin, was again reduced by the bile acids, suggesting again that bile acids interfere with Ca2+ signaling receptors both at internal and cellular membranes. Clearly, further studies will be necessary to analyze the platelets receptors involved in these alterations.Finally, total amount of Ca2+ release evoked by agonists in activated platelets, secondary to an increased amount of Ca2+ stored in the intracellular organelles. These alterations are not related to bile acids, since they reduce the amount of internal Ca2+ and the entry from the extracellular space, both necessary to induce platelet aggregation.Patients with liver cirrhosis and experimental models show, in the compensated phase prior to the development of ascites, platelet alterations compatible with a hyperaggregatory state. This is related to enhanced CaPR was the main performer of most of the experiments, EN is our lab technician and without her nothing can be done, MO and DI played a key role with the flow cytometry technique, JG was the main writer of the manuscript and NA supervised all the experimental protocols and procedure laboratories and directed the research.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.


Inositol 1,4,5-triphosphate receptors (IP3Rs) and ryanodine receptors (RyRs), which are located on the ER surface, are the main endogenous Ca2+ release channels and play crucial roles in regulating Ca2+ homeostasis. However, the roles of these endogenous Ca2+ release channels in PD and their effects on the function and survival of DA neurons remain unknown. In this study, using a 6-hydroxydopamine (6-OHDA)-induced in vitro PD model (SN4741 Cell line), we found that 6-OHDA significantly increased cytoplasmic Ca2+ levels ([Ca2+]i), which was attenuated by pretreatment with 4-phenyl butyric acid  or ryanodine (a RyRs blocker). In addition, in acute midbrain slices of male Sprague-Dawley rats, we found that 6-OHDA reduced the spike number and rheobase of DA neurons, which were also reversed by pretreatment with 4-PBA and ryanodine. TUNEL staining and MTT assays also showed that 4-PBA and ryanodine obviously alleviated 6-OHDA-induced cell apoptosis and devitalization. Interestingly, a IP3Rs blocker had little effect on the above 6-OHDA-induced neurotoxicity in DA neurons. In conclusion, our findings provide evidence of the different roles of IP3Rs and RyRs in the regulation of endogenous Ca2+ homeostasis, neuronal excitability, and viability in DA neurons, and suggest a potential therapeutic strategy for PD by inhibiting the RyRs Ca2+ channels in the ER.Calcium (Ca The molecular mechanisms of selective DA neuronal loss include genetic mutations, mitochondrial dysfunction, oxidative stress, and ER stress. Reports have indicated the ER as a crucial participant in the pathology of PD . The accth ERAD; .2+, the ER is vital in intracellular Ca2+ regulation. Cytoplasmic Ca2+ levels are mostly affected by ER-localized Ca2+ channels, including IP3Rs, RyRs, and the sarco/endoplasmic reticulum Ca2+-ATPase  and inhibited (>1 μM) by modest increases in Ca2+ concentrations compared with normal cytoplasmic Ca2+ concentrations  in Ca2+ homeostasis and viability in SN4741 cells since it is a DA neuronal cell line of embryonic SN origin which is widely accepted more accessible to molecular analysis and used as an in vitro model system for study SNc DA neurons ] for more than 30 min. Then, the midbrain was blocked in a coronal plane, fixed to the stage of a VT1000P vibratome  and sliced. We maintained slices at room temperature for 1 h before experimentation.All animal work was performed according to the guidelines of the Institutional Animal Care and Use Committee at the Fourth Military Medical University. Carefully prepared slices containing the SNc (300 μm thick) were separated from male Sprague-Dawley rats (12–14 days of age) as described previously . BrieflyThe following chemical agents were used in the experiment: 6-OHDA , 4-PBA , BAPTA-AM , RY , and Xes .2, 10 HEPES, 1 EGTA, 0.3 CaCl2, 0.3 Na-GTP, and 2.1 Mg-ATP, pH 7.3 using NaOH (290–300 mOsm). Experiments were discarded if the access resistance crossed 25 MΩ. The procedures for insulation of DA neurons were performed according to previous reports  puller, was used to patch the neurons. The intracellular solution contained (in mM) 125 K-gluconate, 1.2 MgCl reports . Whole-cData were recorded by filtering at 3 kHz and digitizing at 10 kHz at room temperature (20–24°C) and analyzed in pCLAMP 10.2 software . We used current-clamp recordings to evaluate evoked action potentials in the experiments in which we normalized the resting membrane potential to –60 mV in prepared slices. Neurons were considered to be ‘stable’ and included in the data analysis when their input resistance oscillated by no more than 20%. The current step was set from –200 to 400 pA, with an increment of 25 pA, a duration of 1 s, and a 10 s interpulse interval. Rheobase current amplitude was the minimal injected current capable of evoking an action potential.2 in high-glucose Dulbecco’s minimum essential medium (DMEM) supplemented with 1% D-glucose, 2 mM L-glutamine, and 10% heat-inactivated fetal bovine serum (FBS).SN4741 cells were derived from the embryonic SNc of a transgenic mouse. We cultured the cells at 33°C and 5% CO2+ was measured by fluorescence imaging using the Ca2+ indicator dye fluo-3AM  based on previously detailed methods (5 cells/well) for 24 h, then cells were incubated with 5 μM fluo-3AM at 33°C for 30 min. Cells were then washed twice with fresh buffer . The dye-loaded cells were incubated in fresh buffer or pretreated with 4-PBA, RY or Xes for another 20 min before imaging. Before exposed to 6-OHDA (fresh buffer was applied for control group), the cells were scanned for 3 min to obtain a basal fluorescence intensity level of intracellular Ca2+ (F0) and another 27 min under the treatments to obtain the real-time fluorescence intensity (F), the ratio was F/F0. Cells were imaged with a laser scanning confocal microscope . The image was recorded every 30 s  and the fluorescence intensity was obtained by the Nikon A1 analysis system. Data were presented as the ratio of fluo-3AM fluorescence intensity  which was normalized to control.Free Ca methods . SN4741 5 cells/well) for 24 h. After treatment, the cells were incubated with 10 nM MitoTracker Red CMXRos (Life Technologies) for 20 min at 33°C. Mitochondria were imaged under a laser scanning confocal microscope . Quantification of the mitochondria morphology was performed as described previously  for 24 h. After designed treatment, cells were incubated with 5 μM dye for 30 min at 33°C. Cells were washed three times with prewarmed PBS at 33°C to remove excess dye. ROS-positive cells were visualized via laser scanning confocal microscopy  and counted using Image J software  was used in the measurement of the ROS production. SN4741 cells were cultured on in the 3.5 mm plates  was utilized to measure cell apoptosis. SN4741 cells were pretreated with 4-PBA, ryanodine, or Xes for 20 min before exposure to 6-OHDA for 2 h. The cells were then fixed with 4% paraformaldehyde, and the TUNEL reaction was performed according to the manufacturer’s protocol. TUNEL-positive cells were visualized via laser scanning confocal microscopy  and counted using Image J software (the whole cells were accounted according to DAPI staining) and the positive cells were calculated according to TUNEL staining. The ratio is the specific value of positive cells and whole cells.5 cells/ml) were seeded into a 96-well polystyrene tissue culture plate and incubated for 24 h at 37°C and 5% CO2. After treatment, the culture medium was replaced, and 20 μl of MTT solution (5 mg/ml in PBS) was added to every well. Then, 100 μl of dimethyl sulfoxide (DMSO) was added to each well for 4 h, after which the plate was read at 490 nm. All assays were performed in ten replicates per dose.SN4741 cells (6 × 10t-tests or one-way ANOVA for the current data (rheobase) and two-way ANOVA for spike number vs. injection current(the row factor was injection current) and calcium imaging fluorescence intensity vs. time(the row factor was time). Statistical significance was two-sided, and the level of statistical significance was set at P < 0.05.Patch clamp data obtained from all treatments were analyzed using pClamp 10.2 software  and Origin 8.0 software . The fluorescence intensity of Calcium imaging results from cells with various treatments was obtained using a Nikon A1 analysis system and analyzed with graphpad prism software. Two-way ANOVA was used to compare the differences of changes in calcium level as we select time as row factor. Data were presented as the means ± S.E.M. Statistical analysis of the results was performed using paired 2+ plays an important role in increased cytoplasmic Ca2+ levels (2+ is involved in 6-OHDA-induced cytoplasmic Ca2+ dyshomeostasis is still unclear. Western blot supported that treatment of 6-OHDA (1 μM) can induced ER stress and this effect can be reversed by pretreatment of ER stress inhibitor 4-PBA . Thus, we treated SN4741 cells with fresh buffer solution lacking Ca2+  and then measured the fluorescence in the presence or absence of 6-OHDA (1 μM) for 30 min. Fluorescence staining analysis revealed that 6-OHDA produced a significant [Ca2+]i increase in the absence of extracellular Ca2+ . Next, we tested whether the increase in [Ca2+]i was related to the ER Ca2+ stores. Pretreated cells with the ER stress inhibitor 4-PBA  reversed the 6-OHDA-induced [Ca2+]i increase . Interestingly, the change of [Ca2+]i did not recover, even after a 20 min drug washout .Previous studies have indicated that extracellular Ca+ levels . However2+ release from the ER mainly occurs via two Ca2+ channels: IP3Rs and RyRs  or the IP3Rs inhibitor Xes (800 nM) for 20 min followed by incubation with 6-OHDA (1 μM). Calcium imaging revealed that 6-OHDA significant increased [Ca2+]i levels (P < 0.01) and that treatment with RY alone failed to induce significant changes in [Ca2+]i levels in SN4741 cells . However, pretreatment with RY markedly decreased the 6-OHDA-induced [Ca2+]i increase in SN4741 cells . In contrast, treating with Xes alone did not alter [Ca2+]i levels in SN4741 cells , and pretreatment with Xes also showed little effect on the 6-OHDA-induced [Ca2+]i increase . These data suggest that the RyRs channel rather than the IP3Rs channel primarily mediates the 6-OHDA-induced Ca2+ overload in DA neurons.Caand RyRs . Here, wFigures 4A,B, n = 10, two-way ANOVA of spike number, the row factor was injection current, t-test of rheobase). In addition, pretreatment with the ER stress inhibitor 4-PBA significantly reversed the 6-OHDA-induced decrease in spike number and the rheobase, which indicated that these changes in neuronal excitability might be closely related to ER stress in DA neurons  for 10 min, the electrical activity of DA neurons was recorded. The results showed that the spike number vs. injection current was obviously decreased while the average rheobase current was significantly increased, which represented the excitability level of the neurons was reduced.  and pretreatment of ER stress inhibitor 4-PBA (2 mM) or blockade of [Ca2+]i overload by BAPTA-AM (10 μM) can protect the mitochondrial from 6-OHDA induced dysfunction as well as ROS generation . These data implies that calcium may serves as a critical mediator linking changes in ER homeostasis to the onset of mitochondrial dysfunction.Several studies have confirmed that 6-OHDA can induce oxidative stress R stress . Our datP < 0.01; Figure 7A, n = 10, one-way ANOVA). This effect was significantly reversed by 4-PBA or RY pretreatment  but not by Xes pretreatment . In addition, exclusive use of RY or Xes had no effect on cell viability. TUNEL staining  also showed that 6-OHDA (60 μM) markedly increased cell apoptosis (P < 0.01), which was significantly reversed by 4-PBA or RY pretreatment (P < 0.01) but not by Xes pretreatment.6-Hydroxydopamine is reported as a neurotoxin in cellular models of PD and has been proven to induce cell apoptosis. Here, we treated SN4741 cells with a range of 6-OHDA concentrations for 2 h. MTT assays showed that 60 μM 6-OHDA induced a significant decrease in cell viability compared with the control group  in Ca2+ homeostasis, cellular excitability, and viability in a 6-OHDA-induced PD model. Our data are the first to demonstrate that RyRs rather than IP3Rs play a leading role in the response to 6-OHDA-mediated Ca2+ dyshomeostasis in DA neurons, contributing to a novel mechanism of neurotoxic injury in the 6-OHDA-induced PD model. Our findings also suggested a potential therapeutic strategy for PD by blocking the RyRs Ca2+ channels in the ER.Parkinson’s disease is initially characterized by the loss of DA neurons in the SNc of the midbrain . Studies2+ store, the ER is vital in intracellular Ca2+ regulation. ER stress is reported to disrupt Ca2+ homeostasis in response to multiple extracellular stimuli  and indicate that 6-OHDA can significantly increase cytoplasmic Ca2+ levels in DA neurons  Additionally, we found that the above effect was significantly attenuated by the blockade of ER stress using 4-PBA . Furthermore, we demonstrated that the effect of 6-OHDA on Ca2+ overload was sustained, even with immediate washout following 5 min of treatment in DA neurons . These findings suggest that the sustained and irreversible Ca2+ dyshomeostasis in DA neurons induced by 6-OHDA may contribute to the pathophysiology of PD.As an endogenous Ca stimuli . 6-OHDA, stimuli , which a neurons   significantly alleviated the 6-OHDA-induced cytosolic Ca2+ increases , whereas an IP3Rs blocker (Xes) had no observable effect .ER-mediated Cand IP3Rs . Previoul artery . However neurons , in contle cells . The exi2+ channels  rather than Na+ channels for action potential generation , which then reduced neuronal activity in otic ganglion cells (2+-release channels with high doses of RY (100 μM) suppressed the rhythm in [Ca2+]i and partially reduced the amplitude of the rhythm in the spike frequency of suprachiasmatic nucleus neurons in acute rat slices  and a subsequent increase in basal autophagic flux as a compensatory pro-survival response , which implies that the increase cytoplasmic Ca2+ levels induced by 6-OHDA may related to the dysfunction of mitochondrial  and pretreatment with the ER stress inhibitor 4-PBA protected DA neurons from 6-OHDA-induced apoptosis . Interestingly, IP3Rs inhibition failed to improve the viability of neurons exposed to 6-OHDA, whereas the RyRs inhibitor RY significantly alleviated the neurotoxic effects of 6-OHDA. Thus, these data suggest that RyRs Ca2+ channels in the ER may play a major role in 6-OHDA-induced Ca2+ dyshomeostasis and neurotoxic injury in PD.The Caiability . RyRs aclity  Act and the Institutional Animal Care and Use Committee at the Fourth Military Medical University.GG and QY: Contributed to the conception or design of the work and drafting the work or revising it critically for important intellectual content. LH, YX, and DF: Substantial contributed to the conception or design of the work and acquisition, analysis, as well as interpretation of data for the work. RY and TN: Revised the work critically for important intellectual content. KT and GZ: Final proofreaded of the version to be published.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.


We found that high glucose  altered Ca2+ homeostasis by reducing lysosomal Ca2+ concentration that appeared to be resulted from Ca2+ moving out of lysosomes into cytosol in human monocytic cell lines, U937 and THP-1 cells. Moreover, HG-induced lysosomal Ca2+-dependent mature IL-1β release was strongly correlated with the activation and upregulation of two lysosomal marker proteins, cathepsin D and lysosomal-associated membrane protein-1 (LAMP-1). This involved calcineurin/transcription factor EB (TFEB) pathway and its target genes, cathepsin B, cathepsin D, and LAMP-1, to mediate lysosomal exocytosis. Therefore in this study, we revealed a novel mechanism of HG-induced lysosomal exocytosis which was regulated by lysosomal Ca2+ signals through calcineurin/TFEB pathway, thus contributing to IL-1β secretion in human monocytic cells.Aberrant activation of the innate immune system, including NOD-like receptor pyrin domain containing 3 (NLRP3) inflammasome-dependent interleukin-1β (IL-1β) secretion, has been implicated in the pathogenesis of type 2 diabetes mellitus (T2DM) and its complication. Our previous study demonstrated that hyperglycemia, a hallmark characteristic of T2DM, induced NLRP3 inflammasome-dependent caspase-1 activation and IL-1β maturation in human monocytic cells. In this study, we examined the underlying mechanisms of secreting IL-1β during hyperglycemia, with a focus on the alteration of Ca Interleukin-1β (IL-1β) is one of the pro-inflammatory cytokines that is involved in the pathogenesis of type 1 diabetes, type 2 diabetes mellitus (T2DM), and diabetic vascular complication, such as atherosclerosis . IL-1β mvia non-classical secretory pathway into the extracellular milieu (2+ concentration ([Ca2+]i) rise triggers secretory lysosomes to fuse with the PM and release secretory proteins  in human monocytic cells  could cooperate with endoplasmic reticulum (ER) Ca2+ store and resulted in lysosomal exocytosis and IL-1β secretion in human monocytic cells  is an essential transcriptional regulator for lysosomal function , 21, whiocytosis –24. Furtic cells , 25. Therocesses , it is uin vitro. Treatment with 30 mM glucose for 48 h was regarded as the HG model in U937 and THP-1 monocytic cells. In this study, we demonstrated that HG could induce change in [Ca2+]i and affect lysosomal Ca2+ homeostasis, and mediate lysosomal exocytosis. We also found that this lysosomal Ca2+ signaling by HG could trigger calcineurin/TFEB pathway and its target genes cathepsin D and lysosomal-associated membrane protein-1 (LAMP-1), and then subsequently release IL-1β in human monocytic cells.Here, we used hyperglycemic environment to mimic the diabetic condition 2O2), d-mannitol, and lipopolysaccharides were purchased from Sigma-Aldrich, USA. Bafilomycin A1 and GPN were from Santa Cruz Biotechnology, while BAPTA-AM, cyclosporin A, FK506, ionomycin, nicotinic acid adenine dinucleotide phosphate (NAADP), trans-Ned-19 (Ned-19), and U18666A were from Tocris Biosciences, USA. Thapsigargin (TG) was bought from Almone Labs, USA, while LysoTracker Red DND-99 Dye and Rhod dextran were from Invitrogen, USA. Antibodies used for immunoblotting and immunostaining were as follows: anti-mouse lysosome-associated membrane protein-1 , anti-rabbit cathepsin D , anti-rabbit caspase-1 , anti-rabbit TFEB , anti-rabbit histone H3 (D1H2) , anti-rabbit Integrin β1 , anti-rabbit GAPDH , and anti-rabbit α/β-tubulin .Carbonyl cyanide 3-chlorophenylhydrazone (CCCP), ethylene glycol tetra acetic acid (EGTA), hydrogen peroxide solution .U937  and THP-1  monocytic cell lines were grown in RPMI 1640  supplemented with 10% FBS, 2 mM ® RNAiMAX Transfection reagent . The protocol was synthesized according to the manufacturer’s protocol. GAPDH siRNA was used as a control . Transfection efficiency was >70% assessed by BLOCK-iT™ Alexa Fluor® Red Fluorescent Control  and western blotting. Cells were transfected with siRNA for 24 h before experiments.Cells were transiently transfected with TFEB siRNA , by using Lipofectamine2+ concentration ([Ca2+]i) was measured in single cells as previously described  for 30 min at 37°C. Fluo-4 fluorescence intensity  was sampled at 5 s intervals using a Cell® system . To enable comparisons between cells, the maximal change in fluorescence intensity was measured before and after GPN (400 µM), NAADP (1 µM), Baf A1 (500 nM), or TG (1 µM) was added.The intracellular Caescribed . Cells w2+ concentration was measured as previously described  for 12 h after treatment as indicated in results, while for all cytosolic Ca2+ measurements, the cells were incubated with Fluo-4 (2 µM) for 30 min. The median fluorescence intensity (MFI) was determined using a FACS Canto flow cytometer , and the data were analyzed using FlowJo software .The lysosomal Caescribed . For lysTotal protein was extracted with ice-cold lysis buffer, the nuclear/cytosolic proteins were extracted by using the Nuclear and Cytoplasmic Extraction Kit , and the PM/cytosolic proteins were extracted by using the Mem-PER™ Plus Membrane Protein Extraction Kit . The protein concentrations of the lysates were measured by the bicinchoninic acid kit . 40 µg proteins were used and separated by 10% SDS-PAGE gels and were transferred onto the nitrocellulose membranes. Membranes were incubated with primary antibodies  overnight at 4°C, and secondary antibodies  for 1 h at room temperature, and the immunoblots were developed by enhanced chemiluminescence  with a ChemiDoc™ MP System . GAPDH, β-actin, α/β-Tubulin, Histone H3, and Integrin β1 were used as housekeeping controls.Total RNA was extracted using RNeasy Mini Kit , and cDNA was synthesized using High-Capacity cDNA Reverse Transcription Kit . cDNA was quantified using Taqman assays by ViiA 7 Real-Time PCR System . The Taqman probes  used were as follows: TFEB (Hs00292981_m1), Cathepsin B (Hs00947433_m1), Cathepsin D (Hs00157205_m1), LAMP-1 (Hs00174766_m1), IL-1β (Hs00174097_m1), and β-actin (4326315E). β-Actin was used as an endogenous control. All gene expressions were calculated using the ΔΔCt method and were normalized to control.For cells labeling with lysotracker, the cells were incubated with LysoTracker DND-99 Dye (250 nM) for 45 min at 37°C after treatment as indicated in results. The MFI was determined using a FACS Canto flow cytometer , and the data were analyzed using FlowJo software .The LAMP-1 level on the PM was measured as previously described . After t2+-free buffer for 3 h and then mixed with 200 ml of 1 mM 4-methylumbelliferyl N-acetyl-β-d-glucosaminide  in 0.1 M citrate buffer  for 1 h at 37°C. The reaction was stopped with 400 ml 0.1 M sodium carbonate buffer , and the absorbance was measured at 405 nm. To determine the total cellular content of β-hexosaminidase, the cells were lysed with 1% (v/v) Triton X-100, and 10 µl of the cell extracts were used for the enzyme activity reaction. The percentage of β-hexosaminidase release was calculated from the enzyme activity of the supernatants and lysates.β-Hexosaminidase secretion was measured as previously described . After tCells were extracted with 200 µl of chilled Cell Lysis Buffer following the manufacturer’s instruction. Cathepsin D activity was measured by using a flourimetric assay kit  and was normalized to control.After treatment as indicated in results, the cells were fixed with 4% PFA for 15 min, followed by permeabilization with 0.1% Triton X-100 for 5 min, and were blocked in 20% goat serum  for 30 min. Next, the cells were incubated with TFEB antibody (1/50 dilution) overnight at 4°C, and stained with secondary antibody (1/400 dilution) for 1 h and DAPI for 10 min. For the acquisition of the images, at least six images were taken per well of the 96-well plate by IN Cell Analyzer 2000 , and quantitative analysis was performed with ImageJ software.The cells were seeded onto confocal dishes  and were treated with indicated conditions as described. The cells were fixed with 4% PFA for 15 min, blocked in 20% goat serum  for 30 min, and incubated with primary antibodies (1/50 dilution) overnight at 4°C, and then secondary antibodies (1/400 dilution) for 1 h. Images were captured with a confocal microscope , and quantitative analysis was performed with the ImageJ software.P < 0.05 was considered as significant. Sample size (n) represented the number of independent experiments.All data were expressed as mean ± SEM and were analyzed by GraphPad Prism 5.0 . Significant differences were determined by one-way ANOVA followed by a Dunnett’s test. 2+ homeostasis could lead to lysosomal dysfunction  or 30 mM glucose for 24, 48, or 72 h significantly reduced GPN-evoked Ca2+ release  in U937 cells, this strongly suggested that HG decreased lysosomal Ca2+ concentration and affected cytosolic Ca2+ homeostasis . Therefore, this lysosomal Ca2+ contributed to secreting IL-1β into extracellular milieu in human monocytic cells.Interleukin-1β is one of the major inflammatory cytokines that is critical for chronic inflammatory response during metabolic disorders, including obesity and T2DM. The secretion of IL-1β is primarily from monocytes and macrophages , and HG,olipases , 41. Ourocytosis , 33, 51.2+, particularly lysosomal Ca2+. Interestingly, our results demonstrated that several internal Ca2+ activators, such as ionomycin, GPN, NAADP, and TG, were capable of inducing TFEB nuclear translocation. Therefore, it was likely that HG induced TFEB activation as a consequence of Ca2+ release from the lysosomes. Moreover, in other various cells, such as fibroblasts, neuronal cells, and osteoclasts, it was reported that overexpression of TFEB could mediate lysosomal exocytosis by raising [Ca2+]i  to allow lysosomal Ca2+ release under physiological level.Regarding to the function of lysosomes, prolonged HG treatment was shown to inhibit lysosomal function in different cell types , 56–58. function , 59, we ysosomes , 33. Our studies , 33. Bes2+ influx could contribute to HG-induced ROS overproduction and NLRP3 inflammasome activation, leading to IL-1β maturation and release (2+ signals was critical for IL-1β secretion induced by variety of stimulus, which was not only mediated through NLRP3 inflammasome activation, but also by lysosomal exocytosis (2+ release played a vital role in HG-induced secreting IL-1β into extracellular milieu via lysosomal exocytosis/TFEB pathway. Second, TFEB could promote pro-IL-1β synthesis induced by HG. Taken together, our previous and the present study suggested that TRPM2-mediated Ca2+ influx regulate NLRP3 inflammasome activation, whereas internal Ca2+, particularly lysosomal Ca2+ release, was associated with triggering TFEB activation, which contributed to pro-IL-1β synthesis and secretion. Moreover, lysosomal Ca2+ signals was also responsible for secreting IL-1β into extracellular milieu via lysosomal exocytosis in human monocytic cells. The important role of IL-1β in T2DM has been recognized in the recent years (In our previous work, we identified some novel mechanisms involved in the activation of NLRP3 inflammasome under HG in human monocytic cells. We demonstrated that TRPM2-mediated Ca release . Notablyocytosis , 60, 61.2+ homeostasis, particularly lysosomal Ca2+ homeostasis, to trigger the activation of calcineurin and TFEB, a master gene for lysosomal function, in monocytic cells. Hence, TFEB could modulate lysosomal exocytosis by enhancing [Ca2+]i and contributed to secreting IL-1β into extracellular milieu under HG. Our results also demonstrated that lysosomal Ca2+ release by GPN or NAADP was sufficient for TFEB activation and induction of lysosomal exocytosis, suggesting that lysosomal Ca2+ signals was crucial for lysosomal exocytosis-dependent IL-1β release in monocytic cells. These findings provided an understanding of the underlying mechanisms of secreting IL-1β into extracellular milieu by HG, with a focus on the involvement of lysosomal Ca2+ signals in lysosomal exocytosis in monocytic cells.In conclusion, we demonstrated that HG could alter intracellular CaYK, SL, and MH conceived and designed the study; HT and CV performed the experiments; HT and MH drafted 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.


The mitochondria organelle has a crucial cellular role as a powerhouse generating energy in the currency of adenosine triphosphate (ATP). Mitochondria are known to be implicated in disease, where genetic defects in mitochondrial DNA (mtDNA) result in debilitating mitochondrial disease. Even though mutations in mtDNA are known to occur in cancer cells, the malignant cell still relies on mitochondrial based pathways to generate the vast quantity of ATP required to fuel the rapidly proliferating cell. This is increasingly relevant in acute myeloid leukemia (AML) where, contrary to the Warburg hypothesis, the malignant cell relies on oxidative phosphorylation rather than aerobic glycolysis. This revelation is due to these AML blasts having increased mitochondrial mass compared to their non- malignant counterparts  2]. Rec. Rec2]. in vitro that DPI causes apoptosis in AML blasts cultured with BMSC, with no effect seen on normal CD34+ hematopoietic stem cells. We therefore believe targeting mitochondrial transfer provides a potential novel therapeutic angle for the treatment of AML, aimed at the malignancy specifically. However, the effect of NOX2 inhibitors have yet to be tested in vivo and is an important step in turning the therapeutic idea into reality. However, we do show that in vivo inhibition of NOX2 on AML blasts, through shRNA knockdown (KD), reduces tumour burden and disease progression. The survival of animals engrafted with NOX2 KD AML blasts have significantly increased survival times in addition to reduced mitochondrial levels, highlighting the beneficial effect of mitochondrial transfer to AML in vivo.We showed that NOX2 activity on the AML blast stimulates mitochondrial transfer and also that the mitochondria move via AML-formed tunnelling nanotubes (TNTs) . These tThe bone marrow (BM) microenvironment of AML is known to be crucial in the survival of the malignancy, where ex vivo AML blasts undergo apoptosis which can be prevented by co-culture with BMSC . TargetiThe AML blasts are now capable of increased mitochondrial based energy production, as they have gained the extra metabolic machinery from their microenvironment. However, the malignant still needs to acquire the fuel to feed the imported mitochondria, which generates increased ATP levels. Recently our group has shown that adipocytes, from the bone marrow microenvironment, release free fatty acids (FFA) which are transferred to the AML blasts via the fatty acid binding protein 4 (FABP4) . These FAML blasts orchestrate the bone marrow microenvironment by getting BMSC to transfer their mitochondria and BM adipocytes to transfer their FFA. This allows the AML blast to cycle without control. Further studies to determine the impact of chemotherapy on FFA and mitochondrial transfer are now underway as it may provide an understanding of how AML blasts can survive under high doses of chemotherapy, within the protective microenvironment. The three cell system we describe is highlighted in schematic form in Figure We observe that AML requires both mitochondrial transfer and FFA transfer to generate the high ATP levels needed for its rapid proliferation. This highlights a potential dual therapeutic window, whereby the plug is pulled on both the fuel and machinery source leaving the blasts susceptible to chemotherapy regimens. Now the onus passes onto the pharmaceutical industry to develop compounds that target both mitochondrial transfer and FFA transfer. Mitochondrial transfer is however the most likely starting point as our recent publication in Blood, presents both NOX2 and TNTs, which could be targets for small molecule inhibitors.