NPC1 (95% of cases) or NPC2. Reduced late endosome/lysosome calcium (Ca2+) levels and the accumulation of unesterified cholesterol and sphingolipids within the late endocytic system characterize this disease. We previously reported impaired lysosome‐related organelle (LRO) function in Npc1−/− Natural Killer cells; however, the potential contribution of impaired acid compartment Ca2+ flux and LRO function in other cell types has not been determined. Here, we investigated LRO function in NPC1 disease platelets. We found elevated numbers of circulating platelets, impaired platelet aggregation and prolonged bleeding times in a murine model of NPC1 disease. Electron microscopy revealed abnormal ultrastructure in murine platelets, consistent with that seen in a U18666A  treated megakaryocyte cell line (MEG‐01) exhibiting lipid storage and acidic compartment Ca2+ flux defects. Furthermore, platelets from NPC1 patients across different ages were found to cluster at the lower end of the normal range when platelet numbers were measured and had platelet volumes that were clustered at the top of the normal range. Taken together, these findings highlight the role of acid compartment Ca2+ flux in the function of platelet LROs.Niemann‐Pick disease type C (NPC) is a neurodegenerative lysosomal storage disorder caused by mutations in either  Defects in lysosome‐related organelles, specifically in platelets, are observed in the mouse model and patients with Niemann‐Pick disease type C1.11.1NPC1 patients were enrolled in a longitudinal observational study (NCT00344331) at the National Institutes of Health . Participants data was excluded if on an anticoagulant or post splenectomy. The NICHD Institutional Review Board approved this study; normal ranges are from the NIH Clinical Center Department of Laboratory Medicine reference ranges. Informed consent was obtained for all subjects as well as assent, when appropriate. The diagnosis was established by biochemical testing/mutation analysis.1.2Npc1m1N/J, Npc1−/− mice) are null for Npc1 and were from an established colony. All mice were bred under sterile conditions, with food and water available ad lib. Pups were weaned 3 weeks post‐partum. All animal studies were conducted using protocols approved by the UK Home Office for the conduct of regulated procedures under license . Npc1−/− mice have a lifespan of 10 to 12 weeks (average 10.5 weeks) with neurological symptoms presenting from 5 to 6 weeks of age.Niemann‐Pick disease type C1 mice .1.4Npc1−/− and age‐matched control littermates. For preparation of washed platelets, mice were anaesthetized and blood was collected by cardiac puncture. A total of 500 μL of blood per mouse was collected in a tube containing 100 μL acid citrate‐dextrose solution  and mixed with 3 mL 100 mM EGTA (pH 6.8) in a modified Tyrode's Ca2+ free buffer  supplemented with NaHCO3 and BSA. Platelet‐rich plasma (PRP) was obtained by centrifugation at 180g for 10 minutes at room temperature. For preparation of washed platelets, PRP was washed at 1000 g for 8 minutes at room temperature, and the pellet was resuspended in the modified Tyrodes buffer containing prostaglandin E1 (0.25 μmol/L). Isolated mouse platelets were washed twice and either fixed for EM analysis or incubated at 37°C for 30 minutes before use.Platelets were harvested at indicated time points of 1.5Mice were anaesthetized with isoflurane and placed onto a heated mat (37°C). The tail bleeding time was determined by removing 3 mm of the distal tail tip and immersing the tail into sterile DPBS solution (37°C). The time to cessation of bleeding was measured.1.6g for 10 minutes at room temperature. The platelet count was adjusted with autologous plasma. Aggregation from PRP platelets was monitored by assessing light transmission in a microplate reader  with continuous stirring at 1100 rpm at 37°C.Platelet‐rich plasma (PRP) was prepared by centrifugation at 2501.72.The human megakaryoblastic leukemia cell line MEG‐01 was obtained from the ATCC and cultured in RPMI‐1640 supplemented with 10% (vol/vol) fetal bovine serum, 1% penicillin/ streptomycin, and 0.3 μg/mL glutamine in a humidified atmosphere at 37°C and 5% CO1.8DMSO or U18666A treated MEG‐01 cells were prepared for electron microscopy as previously described.1.97 platelets/mL in modified Tyrode's buffer and DMSO or U18666A treated MEG‐01 cells were resuspended in HBSS solution prior to incubation with LysoTracker Green DND‐26 for 15 minutes at room temperature.Washed murine platelets were diluted to 2.5 × 101.10l‐lysine‐coated glass coverslips and loaded with 5 μM fura‐2/AM in the presence of 0.03% Pluronic F127 in extracellular midum  for 45 minutes at room temperature, followed by a 15 minutes de‐esterification. Experiments were conducted in Ca2+‐free medium: cells were washed once with Ca2+‐free ECM supplemented with 1 mM EGTA, followed by two washes in Ca2+‐free ECM containing 100 μM EGTA. Cells were imaged using an Olympus IX71 microscope equipped with a 40× UApo/340 objective and excited alternately by 350 nm and 380 nm light using a Cairn monochromator; emission was collected at 480 to 540 nm. Experiments were conducted at room temperature with an image collected every 3 seconds. Autoflourescence was determined at the end of each experiment by addition of 1 μM ionomycin with 4 mM MnCl2 to quench fura‐2. Images were analyzed using custom‐written Magipix software  on a single‐cell basis and the data expressed as the mean ± SEM of the maximum peak fluorescence changes (∆350/380).MEG‐01 cells were treated either with DMSO or 2 μM U18666A for 72 hours and then allowed to adhere to poly‐1.11Glycosphingolipid (GSL) extraction, ceramide glycanase digestion, and 2‐aminobenzoic acid (2‐AA) labeling were performed as described.1.12Normal‐phase HPLC analysis for glycosphingolipids profiles was performed as described.1.13t test or multiple t test using GraphPad Prism 5 for Mac OS X (version 5.0d). P ≤ .05 was considered significant.Data were expressed as the mean ± SEM. For group comparisons, the statistical significance of differences in mean values was determined by a two‐tailed single‐factor ANOVA, Student's 22+ store and releases Ca2+ in response to the potent second‐messenger nicotinic acid adenine dinucleotide phosphate (NAADP).2+ store was made in specialized lysosome‐related organelles (LROs) found in sea urchin oocytes (yolk platelets/reserve granules).2+ stores was subsequently replicated in conventional lysosomes, giving rise to the field of acidic store Ca2+ signaling.2+ remain incompletely understood, but there is growing evidence that they are important in the homeostasis of the late endocytic system and in signaling cross‐talk with the endoplasmic reticulum (ER) at IP3 receptors.The lysosome is a regulated Ca2+ is to study inborn errors of metabolism in which this store is compromised. It has previously been identified that there is a lysosomal Ca2+ defect in the rare neurodegenerative lysosomal storage disease, Niemann‐Pick disease type C (NPC).NPC1 or NPC2.2+ in LE/Lys of NPC disease cells, likely the result of a Ca2+ store‐filling defect or enhanced Ca2+ leak.2+ release from late endosomes/lysosomes in response to the second messenger NAADP, blocking fusion, and leading to the secondary storage of multiple lipids in late endosomes (LE)/Lys, including cholesterol, sphingosine, sphingomyelin and multiple glycosphingolipids.2+ in LRO function and in NPC remains incompletely understood.One approach to better understand the physiological functions of acidic store Ca2+ defect found in NPC disease also affects LRO function more generally focusing on platelets, as they have both LROs and conventional lysosomes. Defects in platelets, including reduced aggregation responses to collagen, absent secondary response to epinephrine, and mean platelet volumes at the lower normal range limits, were previously observed in three NPC patients.2+ defect.We previously reported that LRO function in Natural Killer (NK) cells was compromised in a mouse model of NPC disease, reducing the ability of these cells to kill target cells.33.1Npc1−/− mice. There was a significant increase in the circulating platelet count (PLT) , as was the plateletcrit compared to wild type controls . However, there was no significant change in mean platelet volume (MPV) in Npc1−/− platelets compared with control (Npc1+/+) platelets at any age (data not shown). There was a significant decrease in Npc1−/− platelet aggregation by 40.7% in response to thrombin stimulation (1 U/mL) compared with control (Npc1+/+) mouse platelets . However, there were no significant differences in Npc1−/− platelet aggregation in response to the Ca2+ ionophore A23187 compared with controls .To measure hematological parameters, whole blood was collected from presymptomatic (5 weeks), early symptomatic (7 week), and late stage (9 week) ) Figure  and plate Figure . The num3.2Npc1−/− mice have an abnormal circulating platelet count and impaired thrombin induced‐platelet aggregation, we next investigated whether this would lead to bleeding abnormalities. A tail‐bleeding assay was performed on anaesthetized 10.5‐week‐old Npc1−/− mice (late‐stage disease) and we found a marked hemostatic defect in Npc1−/− mice  mice, tail‐bleeding time was markedly increased in 10.5‐week‐old Npc1−/− mice .As e Figure . Whereas3.3Npc1−/− platelets we analyzed them by transmission electron microscopy. Compared to control wild type (Npc1+/+) platelets  to induce NPC1 disease‐associated cellular phenotypes in the human megakaryocyte line MEG‐01, as these cells are responsible for the production of platelets. U18666A is an inhibitor of NPC1 and has recently been shown to directly bind to the NPC1 protein.P < .0001) .To further investigate the underlying mechanisms of ) Figure . Transmi) Figure  suggesti3.52+, we monitored Ca2+ content in MEG‐01 cells indirectly by releasing Ca2+ from the lysosome lumen to the cytosol with the lysomotropic agent glycyl‐l‐phenylalamine 2‐napthylamide (GPN).2+‐free medium to eliminate Ca2+ influx. We have previously shown that GPN responses faithfully reflect lysosomal Ca2+ levels in NPC1 patients‐derived human fibroblasts and U18666A‐treated RAW cells.2+ release compared to vehicle (DMSO)‐treated cells , consistent with less Ca2+ within the lysosomes of U18666A‐treated MEG‐01 cells. The major effect of U18666A on the GPN response is on the maximum amplitude of the Ca2+ response (48% reduction), whilst there is only a small (13%) difference in the kinetics (time from GPN addition to the maximum amplitude) .To assess the effect of U18666A treatment on lysosomal Ca2+ release from both the lysosome and the endoplasmic reticulum (ER),2+ content. To discount this explanation, the ER content was assessed with the Ca2+ ionophore, ionomycin. Ionomycin was used rather than the SERCA inhibitor, thapsigargin, because it is a more reliable assessment of the ER content: thapsigargin releases ER Ca2+ so slowly that other Ca2+ removal processes dampen both its kinetics and amplitude,2+ with such rapid kinetics that it is less susceptible to Ca2+ buffering processes. In contrast to GPN‐dependent Ca2+ release, ionomycin‐dependent Ca2+ release from the ER was unaffected by U18666A treatment . Together, these data are consistent with U18666A‐treatment reducing the lysosome (but not ER) Ca2+ content in MEG‐01 cells.Because GPN responses can be a summation of Ca3.6P = .0345). Platelet counts in the NPC1 patients also significantly correlate with severity score,r = −.2361, P = .0111, Figure Platelet counts were measured in NPC1 patients. As illustrated in Figure 4Npc1−/− disease mice. Electron microscopy revealed abnormal ultrastructure consistent with lipid storage in Npc1−/− platelets and in the U18666A‐treated MEG‐01 cell line. Furthermore, U18666A‐treated MEG‐01 cells had reduced levels of acidic store Ca2+ and glycosphingolipid storage. Taken together, these data suggested that megakaryocytes/platelets have functional defects in NPC disease.We have found an elevated number of circulating platelets, defective platelet aggregation, abnormal platelet morphology and prolonged bleeding times in Npc1−/− platelets exhibit functional defects specifically when simulated with thrombin. It has been suggested that platelet lysosome secretion is only activated by potent agonists, such as thrombin.Npc1−/− platelets were stimulated with A23187, a Ca2+ ionophore selectively transporting Ca2+ across cell membranes into the cytoplasm or releasing the divalent cation from intracellular storage sites, the Npc1−/− platelets did not exhibit aggregation defects.3‐signaling, resulting in an increased intracellular Ca2+ flux.Npc1−/− platelets due to lysosomal calcium defects.We found that Npc1−/− mice. The reason for this difference is unknown and could be a species‐specific difference or reflect the fact that the mouse model is completely null for Npc1 whereas the patients typically have some residual NPC1 expression leading to less extreme cellular phenotypes. Another possible explanation is that there could be a platelet clearance defect and altered physiological distribution of platelets in NPC1 disease, since enlargement and fibrosis of the spleen occurs in NPC1 patients and Npc1−/− mice.As anticipated,1 and S1P4 receptors, have also been suggested to be involved in the regulation of megakaryopoiesis, pro‐platelet formation and shedding in vivo and in vitro models.4 receptor‐null megakaryocytes exhibited abnormal cellular morphology, which was characterized by cytoplasmic vacuolation and nuclear‐ploidy.Npc1−/− platelet phenotypes could also be associated with abnormal intracellular lipid trafficking and storage phenotypes during megakaryopoiesis which could impair down‐stream lipid‐mediated intracellular signaling.However, we still cannot exclude a potential platelet production defect due to impaired megakaryocyte function in NPC1, and in fact it has been suggested that NPC1 plays a role in the regulation of thrombocyte formation in zebrafish.Npc1−/− platelets functional defects could also primarily be associated with acidic compartment Ca2+ signaling defects. Although the physiological functions and activation mechanisms of platelet LROs remain incompletely understood, our current studies highlight the importance of acidic store Ca2+ signaling in the regulation of platelet function. NAADP is a novel Ca2+‐mobilizing second messenger, which selectively targets acidic organelles.2+ signaling, which suggest a crucial role of NAADP and acidic store Ca2+ release during platelet activation.Npc1−/− platelet functional defects are primarily associated with impaired NAADP‐mediated acidic store Ca2+ signaling.2+ storage. Additionally, one patient with Tangier disease treated with miglustat showed a clinical response to this NPC therapeutic suggesting pathogenic convergence.Furthermore, similar observations of abnormal platelet granule morphology and platelet functional defects have been reported in other lysosomes/LROs dysfunctional diseases, such as Tangier disease, Chediak‐Higashi syndrome and Hermansky‐Pudlak syndrome.2+ homeostasis defects, either in the ER or LE/Lys Ca2+ stores.2+‐mediated signaling is crucial for intracellular vesicle fusion and fission events, acidic compartment‐mediated Ca2+ homeostasis defects could lead to abnormal intracellular vesicle trafficking and storage in these diseases. Therefore, it would be reasonable to hypothesize that acidic stores Ca2+ homeostasis defects in NPC1 disease could impair subcellular trafficking, affect granules content secretion and lead to the activation/aggregation defects observed in Npc1−/− megakaryocytes/platelets. Better understanding the role of calcium signaling in platelets is important, as both NPC1 and Tangier patients have decreased plasma HDL‐C cholesterol levels, which are associated with an increased risk for all forms of atherosclerotic diseases, including myocardial infarction and stroke.One of the common features of these lysosomes/LROs dysfunction disorders is that they all have intracellular CaNpc1−/− platelets have functional defects in a murine model of NPC1 disease. Furthermore, our studies suggest that Npc1−/− platelet functional defects could be associated with impaired acidic stores Ca2+ signaling in NPC1 disease model and implicates acidic store Ca2+ to be further examined in better understanding the homeostasis of LROs.Taken together, our current studies have demonstrated that The authors have no conflict of interest.All procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation  and with the Helsinki Declaration of 1975, as revised in 2000.All institutional and national guidelines for the care and use of laboratory animals were followed.O.C.‐W.C., A.C., L.C.D., F.N.K., and A.O.S. developed methods, devised, and performed experiments; O.C.‐W.C., A.C., L.C.D., and F.N.K., analyzed the data and performed statistical analysis; D.A.S. and L.M. helped with animal studies; P.T. and E.E. performed the transmission electron microscopy. F.D.P. and N.Y.F. obtained ethical permission, collected clinical samples, and provided data; O.C.‐W.C., A.O.S., G.C.C., A.G., F.D.P., and F.M.P. devised and designed the research; O.C.‐W.C., A.C., and F.M.P. wrote the article.NPC1 patients were enrolled in a longitudinal observational study (NCT00344331) at the National Institutes of Health . The NICHD Institutional Review Board approved this study. All procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation  and with the Helsinki Declaration of 1975, as revised in 2000. All animal studies were conducted to comply with the ARRIVE guidelines using protocols approved by the UK Home Office for the conduct of regulated procedures under license .No identifiable personal data is included in this article.Data sharing is not applicable to this article as no datasets were generated or analyzed during the current study.


However, the mechanism for this action of resveratrol remains to be clarified. The purpose of this study was to elucidate the Ca2+ entry and subsequent aggregation of human platelets induced by different stimulants including thrombin, thapsigargin, and 1-oleoyl-2-acetylglycerol (OAG) were measured by the fluorescence method and light transmittance method, respectively. Each stimulant was added to a nominally Ca2+-free medium containing platelets, and then CaCl2 was added to the medium to induce Ca2+ influx into platelets.Ca2+ entry into platelets and subsequent platelet aggregation were significantly inhibited in the presence of resveratrol at 6.25 μM or higher concentrations, while OAG-induced Ca2+ entry and subsequent platelet aggregation were not affected by resveratrol at concentrations up to 50 μM. In the nominally Ca2+-free medium, thrombin induced a small transient increase in intracellular Ca2+ concentrations, which was attenuated in the presence of resveratrol at 12.5 μM or higher concentrations. Thrombin-induced Ca2+ entry into platelets and subsequent platelet aggregation were significantly inhibited in the presence of resveratrol at 12.5 μM or higher concentrations.Thapsigargin-induced Ca2+ release from its stores and inhibiting store-operated Ca2+ influx into platelets.The results suggest that resveratrol inhibits thrombin-induced platelet aggregation through decreasing Ca The French paradox is based on epidemiological evidence that the incidence of ischemic heart disease in France is relatively low among western countries despite the saturated fat-rich diet of French people . The Fre2+ concentration ([Ca2+]i) is a crucial signal for the regulation of cellular functions in various cells including platelets [2+]i. However, there have been few studies in which the effects of resveratrol on the Ca2+ signal and its associated functions of platelets were investigated. To the best of our knowledge, there has been only one research group that investigated the effects of resveratrol on Ca2+ signal in platelets: It was shown that resveratrol inhibited Ca2+ influx in platelets stimulated with thrombin and thapsigargin i. Thrombin activates its receptors that are linked with the breakdown of phosphoinositides and induces Ca2+ influx mediated by two second messengers, inositol trisphosphate and diacylglycerol i was determined by using the fluorescent Ca2+ indicator fura-2. Washed platelets were loaded with fura-2/AM  at 37 °C for 30 min. After loading, the platelets were washed once with Ca2+- and Mg2+-free Tyrode solution buffered by Hepes  (pH 7.4) and containing 1 mM EGTA, and they were resuspended in 2 ml of a Ca2+-free Tyrode solution buffered by Hepes  (pH 7.4) .[Ca2+]i were determined by using a ratio (R) of fluorescence intensity (F) of F340/F380. The fluorescence after sequential addition of 0.25% Triton X-100 and EGTA (5 mM) to the platelet suspension provided the maximum fluorescence ratio (Rmax) and minimum fluorescence ratio (Rmin), respectively. [Ca2+]i was calculated using the following formula i calculated by subtraction of the basal [Ca2+]i level from the maximum [Ca2+]i level after stimulation.where 2+-free Tyrode solution buffered by Hepes  (pH 7.4) and evaluated using an aggregometer  that measures increases in light transmission through a cuvette (0.2 ml) containing a stirred platelet suspension. The light transmission through a washed-platelet suspension without any treatment and that through a suspended buffer not containing platelets were considered as 0 percent and 100 percent, respectively. The percentage of aggregation during the course of each experiment was calculated. The experimental conditions were the same as those for [Ca2+]i measurement except for the volume of the cuvettes.Platelet aggregation was measured using platelets suspended in Ca2+-free medium in the cuvette for 3 min and then pretreated with various concentrations of resveratrol or a vehicle for 3 min. Platelets were then stimulated with each stimulant . At 1 min after the addition of each stimulant, Ca2+ entry and aggregation were induced by adding CaCl2 (0.5 mM) to the cuvette.Platelets were stabilized in nominally CaResveratrol  was dissolved in dimethylsulfoxide to make a stock solution of 50 mM and diluted with distilled water at the time of use. Thapsigargin (Sigma), OAG (Sigma), and fura-2/AM  were dissolved in dimethylsulfoxide to make stock solutions of 1 mM, 100 mM, and 5 mM, respectively, and stored at −80 °C. Bovine thrombin  was dissolved in distilled water to make a stock solution of 1 U/μl and was stored at − 80 °C.F test. P values less than 0.05 were regarded as significant.Data are presented as means ± standard deviations. Statistical analysis was performed using analysis of variance followed by Scheffé 2+-free medium, OAG caused a slight aggregation, while no aggregation was induced by thrombin and thapsigargin. The addition of CaCl2 induced a large aggregation by each of the agonists. Pretreatment of platelets with resveratrol (12.5 μM) attenuated the aggregatory responses to CaCl2 in the presence of thrombin  than in its absence   to the medium induced an increase in [Ca2+]i (Ca2+ entry), which was much greater in thapsigargin-stimulated platelets  of thrombin was examined. The thrombin-induced transient increase in [Ca2+]i in nominally Ca2+-free medium was significantly inhibited by resveratrol in a concentration-dependent manner at concentrations from 12.5 to 50 μM .Since the thrombin 0.025 U/ml)-induced transient increase in i in a nominally Ca2+-free solution, which corresponds to inositol trisphosphate-induced release of Ca2+ from its stores, agrees with the finding in a previous study that phospholipase C activity in platelets was inhibited by resveratrol [2+]i independently of inositol trisphosphate. Therefore, the findings of inhibitory effects of resveratrol on increase in [Ca2+]i and aggregation of platelets induced by thapsigargin suggest that resveratrol inhibits SOCI channels. Thus, resveratrol is thought to hamper two sites contributing to Ca2+-related signal transduction, phospholipase C and SOCI channels of platelets, resulting in a decrease in thrombin-induced elevation of [Ca2+]i and attenuation of subsequent aggregation. Diacylglycerol, the other product of hydrolysis of phosphoinositides, also induces transmembraneous Ca2+ influx through Ca2+ channels that are independent of Ca2+ stores (non-SOCI channels) [2+]i and aggregation induced by OAG, a mimic of diacylglycerol, were not affected by resveratrol. When platelets are stimulated by thrombin, two different channels are activated through inositol trisphosphate and diacylglycerol. Thus, resveratrol is thought to selectively inhibit Ca2+ entry, namely, to inhibit only SOCI, in platelets. Figure 2+]i and thereby attenuation of the Ca2+-dependent aggregatory response of platelets.Thrombin activates phospholipase C, resulting in hydrolysis of phosphoinositides and production of two intracellular messengers, inositol trisphosphate and diacylglycerol. Inositol trisphosphate causes release of Casmalemma . Our finveratrol . Thapsighannels) . HoweverResveratrol has been demonstrated to show inhibitory action on platelet aggregation both in vitro and in vivo . However2+-related signals in platelets remains to be clarified. Since resveratrol has a potent antioxidant action, it would be interesting to elucidate the relation of antioxidant action of resveratrol to Ca2+ signals in platelets. In fact, resveratrol has been shown to stimulate nitric oxide production in platelets [2+ entry through SOCI channels, which are also called capacitative Ca2+ channels, was inhibited by nitric oxide in vascular smooth muscle cells [Further studies including experiments to test the effects of administration of resveratrol on platelet aggregability in vivo are needed to clarify the reason for the discrepancy regarding the effective concentrations of resveratrol and to determine whether the inhibitory action of resveratrol on platelet aggregation is clinically significant. Moreover, the detailed mechanism for inhibition of Calatelets  and to ilatelets . In addile cells  and endole cells . Therefo2+ release from its stores and inhibiting SOCI into platelets. Diacylglycerol-induced Ca2+ entry through non-SOCI channels is not affected by resveratrol.Resveratrol inhibits thrombin-induced platelet aggregation through decreasing Ca


Na,late) is the small sustained component of the sodium current active during the plateau phase of the action potential. Several studies demonstrated that augmentation of the current can lead to cardiac arrhythmias; therefore, INa,late is considered as a promising antiarrhythmic target. Fundamentally, enlarged INa,late increases Na+ influx into the cell, which, in turn, is converted to elevated intracellular Ca2+ concentration through the Na+/Ca2+ exchanger. The excessive Ca2+ load is known to be proarrhythmic. This review describes the behavior of the voltage-gated Na+ channels generating INa,late in health and disease and aims to discuss the physiology and pathophysiology of Na+ and Ca2+ homeostasis in context with the enhanced INa,late demonstrating also the currently accessible antiarrhythmic choices.The cardiac late sodium current (I This is followed by a tightly regulated process, in which L-type Ca2+ channels open to let the Ca2+ ions flow into the cell , so that the contraction can occur in the process called Ca2+-induced Ca2+ release (CICR) by opening the ryanodine receptors (RyR). Besides the depolarizing inward currents, a number of outwardly driven K+ currents repolarize the membrane. During repolarization, Ca2+ is removed from the cytoplasm; therefore, complete relaxation of the cell occurs while the membrane potential returns to its resting value so that the next AP can be elicited . As . As + cuxtrusion ,7. INa,logenesis . In the + channels are known to be regulated by changes in the actual membrane potential Na,late iv1.5 have been described in ventricular cells , a fraction of Na+ channels can recover from inactivation and might reopen. However, considering the voltage range of this window current (approximately – 70 mV), it is far below the physiological plateau potential of the AP, so it is unlikely to play a major role in the INa,late i, observed at high frequency, is mainly determined by the early component as INa,late recovers slowly from inactivation at rapid pacing i. The concomitant abnormal conduction can cause sudden death in HF patients. Conduction velocity is determined also by the Na+ channel function i is going to increase with a concomitant rise in the [Ca2+]i, which is considered to be arrhythmogenic i i . Na+ chaproteins . In rabb+ levels . In addiates RyR  i, slows inactivation of INa,early and recovery from inactivation, while shifting steady-state inactivation to more negative membrane potential in a Ca2+-dependent manner [+ current density was required to achieve a significant increase in [Na+]i [et al. demonstrated that phosphorylation of CaMKII and the expression of Nav1.5 channel protein has been significantly elevated in the left ventricle upon treatment with the Ca2+ channel activator Bay K 8644 and the Na+ channel activator sea anemone toxin II (ATX-II). These effects were readily reversible by the application of TTX [2+]i and high [Na+]i potentiates their arrhythmogenic activities.In HF  expression and activity of CaMKII are increased, which may be proarrhythmogenic . Furthert manner . All then [Na+]i ,123. Wein of TTX . Bay K 82+ buffering stores i sensitive transporter i, when exceeding the functional reserve of the NKP, increases [Ca2+]i by switching NCX to a reverse mode operation with a consequent loading of the SR Ca2+ stores (+]i slows Ca2+ extrusion by NCX i and [Ca2+]i which was associated with a reduction in the forward mode and an increase in the reverse mode activity of NCX i shifts the steady-state inactivation curve of the Na+ current to more positive voltages and increases the availability of the channels at more positive potentials [2+]i should decrease INa,late. Our experiments, however, showed that INa,late is rather influenced by the shape and voltage profile of the AP than by Ca2+ itself i, and [Ca2+]i is arrhythmogenic; therefore, an effective antiarrhythmic treatment is necessary under these conditions. An obvious objective is the inhibition of voltage-gated Na+ channels. However, the disappointing results of the CAST and SWORD studies clarified that blocking a single specific ion channel alone can lead to unexpected adverse effects. Recently, a need for selective inhibitors that are able to distinguish between INa,early and INa,late is rather emerging. The selectivity here is critically important since blocking the early component of the current can lead to a decrease in conduction velocity and might lead to conduction block and reentrant arrhythmias . T. T153].  greater .Table 1+ channel blockers including ranolazine and mexiletine, reduces APD and suppresses EAD or DAD formation and generation of Torsade de Pointes (TdP) type ventricular tachyarrhythmias i and [Ca2+]i. A second feature of the INa,late blockade is the normalization of repolarization, allowing restoring the repolarization reserve [It has been demonstrated that suppression of Ioverload ,177–179. reserve .Na,late  may also bring a better therapeutic protocol closer, since each gating mechanism has its own characteristic drug affinity and sensitivity profile [Characterization of the true gating mechanism of I profile ,180,181.+ current and the concomitant elevation of intracellular Na+ and Ca2+ concentrations. In spite of the tremendous work that had been done on understanding the background of the Ca2+ related arrhythmias, there is still a need for more research to better design antiarrhythmic treatments and drugs. It became clear that more attention has to be paid to INa,late in patients with Ca2+-dependent arrhythmias, especially in the case of bradycardia.In summary, we have reviewed the arrhythmogenic behavior of the augmented late Na2+ overload and the therapeutic options are rather complex if we take into account the augmented INa,late. Hence, a selective INa,late inhibitor may only treat a part of the issue. To date, the most selective drug to INa,late in the market is ranolazine; however, it exerts electrophysiological effects on other ion currents as well. Therefore, more work is necessary to gain a better understanding of the role of INa,late and Ca2+ handling in cardiac arrhythmias and to develop novel antiarrhythmic therapies with a focus on translational aspects.Various processes can lead to Ca


NPM1 mutations. A deep knowledge of the distribution of selected antigens on the surface of NPM1-mutated AML cells may help optimizing new therapies for this frequent AML subtype. CD123 is known to be expressed on leukemic cells but also on healthy hematopoietic and endothelial cells, although at lower levels. Differences in antigen densities between AML and healthy cells may enlighten therapeutic windows, where targeting CD123 could be effective without triggering “on-target off-tumor” toxicities. Here, we perform a thorough analysis of CD123 expression demonstrating high expression of this antigen on both NPM1-mutated bulk leukemic cells and CD34+CD38− cells.One-third of adult acute myeloid leukemia (AML) harbors NPM1-mutated (NPM1mut) acute myeloid leukemia (AML) comprises about 30% of newly diagnosed AML in adults. Despite notable advances in the treatment of this frequent AML subtype, about 50% of NPM1mut AML patients treated with conventional treatment die due to disease progression. CD123 has been identified as potential target for immunotherapy in AML, and several anti-CD123 therapeutic approaches have been developed for AML resistant to conventional therapies. As this antigen has been previously reported to be expressed by NPM1mut cells, we performed a deep flow cytometry analysis of CD123 expression in a large cohort of NPM1mut and wild-type samples, examining the whole blastic population, as well as CD34+CD38− leukemic cells. We demonstrate that CD123 is highly expressed on NPM1mut cells, with particularly high expression levels showed by CD34+CD38− leukemic cells. Additionally, CD123 expression was further enhanced by FLT3 mutations, which frequently co-occur with NPM1 mutations. Our results identify NPM1-mutated and particularly NPM1/FLT3 double-mutated AML as disease subsets that may benefit from anti-CD123 targeted therapies. Acute myeloid leukemia (AML) is an aggressive cancer of hematopoietic stem and progenitor cells (HSPCs) , affectiA significant fraction of AML relapses is thought to be secondary to the persistence of residual leukemic cells, able to re-establish the full tumor bulk . In thisNovel approaches aimed to eradicate residual disease in AML are under development ,9. Parti+CD38− putative LSCs [CD123, the alfa-subunit of the interleukin-3 (IL-3) receptor, is an attractive target, which has been reported to be expressed by the majority of AML patients, both on bulk leukemic cells and CD34ive LSCs ,15. Howeive LSCs ,17. We b+ cells [+CD38− cells have been analyzed only in a relatively small number of patients [+CD38− than bulk AML cells and whether it correlates with specific risk categories is still controversial [In the past years, several groups have investigated the landscape of CD123 expression in AML. However, the majority of studies have evaluated CD123 expression only on bulk and CD34+ cells ,19, whilpatients ,21,22. Moversial ,20.NPM1 mutations [NPM1-mutated AML the designation as a distinct entity of the World Health Organization classification of hematopoietic tumors [NPM1-mutated AML has a relatively favorable prognosis, about 50% of patients eventually die due to relapse and disease progression [FLT3 internal tandem duplications (FLT3-ITD) coexist [NPM1mut putative LSC express CD123 [NPM1mut patients, no study had been designed to specifically analyze this AML subgroup or to compare CD123 fluorescence intensities between bulk AML cells and the CD34+CD38− population [About one-third of adult AML patients harbor utations . Specific tumors . Althouggression ,26. The  coexist . Interesss CD123 . Althougpulation ,15,19.NPM1 mutational status, with the aim to explore whether NPM1mut AML could represent an entity that could particularly benefit from anti-CD123 therapies.Here, we investigate CD123 expression in a large number of newly diagnosed AML, focusing on the correlation between CD123 expression and Between October 2010 and October 2020, 151 samples  from 80 female and 71 male adult patients with newly diagnosed AML  were studied.+CD38− putative LCS in 115 samples containing at least 50 events in this rare subpopulation. CD123 expression levels were reported as the percent of positive cells (PPC) and as the median fluorescence intensity (MFI). PPC was available for all 151 samples, while MFI for 123 samples (see Methods). An arbitrary cut-off of 20% PPC was set to assign CD123 positivity. A summary of all results is presented in CD123 expression was studied by multiparameter flow cytometry on bulk leukemic cells in all samples. Additionally, we also analyzed CD123 levels on CD34+CD38− cells, finding frequencies of CD123 positivity similar to those observed in bulk cells (mean PPCs 68 ± 30 and mean MFI 48 ± 60)   C,D. Alth 0.0072) E, indicap = 0.00181 and PPC p = 0.0938) in female patients as compared to males when analyzing CD34+CD38− LSC , and 97% (66/68) NPM1mut samples were CD123-positive. When compared with NPM1wt samples, NPM1mut cases displayed significantly higher CD123 expression levels on bulk AML cells (PPC and MFI p < 0.0001)  A,B.NPM1mut CD34+CD38− AML putative LSCs. CD123 was positive in 91% of NPM1mut samples (49/54). Both PPCs and MFIs were significantly higher in NPM1mut putative LSC compared to NPM1wt samples (PPCs p = 0.0066 and MFI p < 0.0001) (+CD38− putative LSC than in bulk NPM1mut AML cells (p = 0.0029), confirming that CD123 is highly expressed on the surface of NPM1mut AML LSCs (We then looked at CD123 expression in  0.0001) C,D. AverAML LSCs E.NPM1mut patients at diagnosis and relapse. In all patients, CD123 expression increased at relapse, compared to diagnosis (PPC p = 0.0068 and MFI p = 0.0195) (+CD38− cells at diagnosis (<50 events), no comparison between diagnosis and relapse was possible in this subpopulation. Nonetheless, all six samples showed very high CD123 expression levels on CD34+CD38− cell at relapse (average PPC 96 and average MFI 139). Altogether, these data confirm that CD123 is consistently expressed on NPM1mut AML putative LSCs.As several studies suggest that relapse frequently derives from residual LSCs after treatment ,5, we an 0.0195) A–C. As tNPM1 frequently harbors FLT3 mutations, which have been previously reported to be associated with higher CD123 expression [FLT3 mutational status in 122 samples. FLT3 was mutated in 46% (24/52) NPM1mut and in 19% (13/70) of NPM1wt cases. Two patients were positive for the FLT3-D835 mutation, while all others harbored FLT3-ITD. We first compared CD123 in FLT3-mutated (FLT3mut) and FLT3 wild-type (FLT3wt) samples. FLT3mut samples showed significantly higher CD123 levels than FLT3wt cases. This was true for bulk cells (PPC p < 0.0001 and MFI p < 0.0001) and, though with a lesser degree of certainty, for CD34+CD38− cells (PPC p = 0.013 and MFI p = 0.0483)  A–D.NPM1/FLT3-ITD genotypes. The NPM1mut/FLT3-ITD, NPM1mut/FLT3wt, NPM1wt/FLT3-ITD, and NPM1wt/FLT3wt genotypes accounted, respectively, for 21/120 (18%), 29/120 (24%), 13/120 (11%), and 57/120 (47%). The highest CD123 expression was detected in double-mutated patients, while NPM1wt/FLT3wt cases displayed the lowest expression levels   E, furtheImmunotherapy targeting antigens expressed on AML cells represents a promising approach with the potential to reproduce the outstanding results achieved in B-ALL . DespiteCD123 shows several features that prioritize this antigen among all promising potential target in AML. Previous studies have shown that the majority of AML patients express CD123 ,19,20,22However, CD123 expression on HSPCs and endothelium still represents a major obstacle . Althouglow endothelial cell lines while holding high activity against CD123high AML cells lines [CD123 expression density is higher on AML cells than normal tissues. An important challenge is to find an optimal strategy where anti-CD123 CAR-T cells can still efficiently recognize leukemic cells while avoiding capillary leak syndrome and prolonged myelosuppression. Stevens et al. recently showed, by epitope density analysis, that anti-CD123 CAR-T cells could be able to eliminate tumor cells with higher CD123 antigen density, while sparing normal hematopoietic stem cells with lower antigen densities . Arcangels lines . In thisNPM1mut AML cells. CD123 expression on NPM1mut cells was already reported by our group while characterizing NPM1mut CD34+CD38− leukemia-initiating cells in xenograft experiments [NPM1mut CD34+CD38− cells in patients, suggesting that anti-CD123 immunotherapies could be particularly effective in NPM1mut AML. The molecular mechanisms behind the higher CD123 expression and NPM1 mutations in AML are unclear. However, NPM1 seems to play an important role in the differentiation of myeloid progenitors into mature myeloid cells through the IL-3/CD123/JAK2/STAT5 pathway [NPM1 mutation and high CD123 expression warrants further investigations.In this study, we demonstrate that CD123 is highly expressed on eriments . Here, w pathway ,45,46. TNPM1mut/FLT3-ITD samples. This was observed both on bulk cells, as well as in CD34+CD38− cells. As NPM1mut/FLT3-ITD AML patients have relatively poor outcomes due to high relapse rates [CD123 expression was found to be particularly high in se rates , we enviNPM1mut AML at diagnosis and relapse. We therefore hypothesize that CD123 plays a major role in the maintenance of this AML entity, given the importance of the IL-3 receptor for stem cell biology. This hypothesis is in line with several studies demonstrating the engraftment failure of CD123-negative AML-LSCs [NPM1mut AML.We also found consistently higher CD123 levels at relapse when comparing CD123 expression in AML-LSCs ,47,48. TAML-LSCs . Future NPM1mut AML cells both at diagnosis and relapse, with the highest levels detected in NPM1mut/FLT3-ITD samples. This study establishes NPM1mut AML as an attractive candidate for CD123-targeted therapeutic strategies laying the groundwork for the design of clinical trials in this specific AML subset.In conclusion, this work demonstrates that CD123 is highly expressed on the surface of BMs and PBs were collected in seven different Italian centers and subsequently analyzed in our laboratory within 24 h from sampling. In 6 cases, paired BM or PB samples were collected at both diagnosis and relapse. To compare CD123 expression in normal and pathologic samples, we also studied 4 bone marrows from healthy donors. All patients and donors gave their written informed consent before either BM aspiration or PB collection.NPM1 and FLT3 mutational status were performed as previously described [NPM1 mutations were also confirmed by Western blot analysis [NPM1 and FLT3 mutational status was available, respectively, for 151/151 and 122/151 samples. In 111 cases, a successful karyotype analysis was obtained through standard G-banding [Routine morphologic, immunohistochemistry, cytogenetic, and molecular studies to assess escribed . NPM1 muanalysis . NPM1 an-banding . Cytogendim-side scatter (SSC)low cells in samples with no- or early myeloid differentiation . Blasts were gated as CD45dim-SSClow and CD123 analysis was performed on both whole immature cell population and CD34posCD38neg precursors. Data were reported as both CD123 percentage of positive cells (PPC) and median fluorescence intensity (MFI).To set the cut-off point to distinguish between CD123 negative and positive cells, we used the “Fluorescence Minus One” technique, as described by Perfetto et al. . A singlIn all specimens, cell doublets and debris were excluded from analysis by forward scatter (FSC) vs. SSC dot-plot examination. Analysis was performed on either a FACSCalibur or FACSCanto II flow cytometers using the CellQuest Pro 6.0 or FACS Diva 7.0 analysis software . PPC was determined on all samples . MFI was also studied in all samples; however, only those samples acquired on FACSCalibur were reported , as MFI values are not comparable between these two machines (four decades vs. five decades).t-test, depending on the experiment performed . Differences among three or more groups were compared with an unpaired ANOVA test performing post-hoc multiple comparison tests. In all tests, p-values were considered statistically significant if <0.05.Statistical analysis was carried out using GraphPad Prism 7 . Differences between two groups were determined using paired or unpaired two-tailed NPM1mut AML could be particularly sensitive to anti-CD123 target therapies, for the differences in CD123 antigen densities between NPM1mut AML and healthy cells expressing low levels of CD123  could reveal a therapeutic window where immunotherapeutic strategies could effectively eliminate NPM1mut LSCs (NPM1mut AML.Our results suggest that mut LSCs  while av


Oppositely, induced cell proliferation and decreased cell apoptosis were observed in pcDNA-3.1-TRIM31 transfected cells. Furthermore, knockdown of TRIM31 suppressed the activation of Wnt/β-catenin pathway in AML cells. Activation of Wnt/β-catenin pathway by LiCl abolished the effects of si-TRIM31 on cell proliferation, apoptosis and sensitivity to daunorubicin in AML cells. In conclusion, the results indicated that TRIM31 promoted leukemogenesis and chemoresistance to daunorubicin in AML. The oncogenic role of TRIM31 in AML was mediated by the Wnt/β-catenin pathway. Thus, TRIM31 might serve as a therapeutic target for the AML treatment.Tripartite motif (TRIM) 31 is a member of TRIM family and exerts oncogenic role in the progression and drug resistance of several cancers. However, little is known about the relevance of TRIM31 in acute myeloid leukemia (AML). Herein, we investigated the role of TRIM31 in AML. We examined the expression levels of TRIM31 in the blood samples from 34 patients with AML and 34 healthy volunteers using qRT-PCR. The mRNA levels of TRIM31 in human bone marrow stromal cells (HS-5) and five AML cell lines were also detected. Loss/gain-of-function assays were performed to assess the role of TRIM31 in AML cells proliferation, apoptosis and sensitivity to daunorubicin. The expression levels of pro-caspase 3, cleaved caspase 3, Wnt3a, β-catenin, cyclin D1 and c-Myc were measured using Western blot. TRIM31 expression levels were significantly up-regulated in AML patients and cell lines. Knockdown of TRIM31 suppressed cell proliferation and promoted apoptosis in AML-5 and U937 cells. The IC Acute myeloid leukemia (AML) is a kind of leukemia that starts in the myeloid cells, which have the capacities of self-renewing, sustaining malignant populations and producing subclones . AML is Currently, the management of AML is divided into induction phase and consolidation phase ,5. ChemoTripartite motif (TRIM) 31, a member of the TRIM protein family, was found to exert oncogenic role in the development and progression of several tumors. Overexpression of TRIM31 is observed in high-grade glioma tissues and associated with short survival time. Overexpressing TRIM31 promotes the proliferation, invasion and migration of glioma cells . TRIM31 Considering the oncogenic roles of TRIM31 in these types of cancers, we speculated that TRIM31 might exhibit similar biological functions in AML. Thus, the aim of the present study was to uncover the role of TRIM31in AML.The study was approved by the Ethics Committee of Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology . A total of 34 patients diagnosed with AML and 34 healthy volunteers were enrolled. Prior informed consents were obtained from all participants. Up to 5 ml whole blood sample was obtained from each participant and used for the detection of TRIM31.Human bone marrow stromal cells (HS-5) and four AML cell lines including AML-5, U937, K-562 and THP-1 cells were obtained from the American Type Culture Collection . The cells were cultured in RPMI-1640 or DMEM . The medium was supplemented with 10% FBS (Life Technologies).The TRIM31 siRNA (si-TRIM31), non-targeting scrambled siRNA (si-NC) and TRIM31-overexpressing plasmid (pcDNA-3.1-TRIM31) were procured from GenePharma . Cell transfections were carried out using Lipofectamine 2000  based on the manufacturer’s instructions.Ct−ΔΔ method.Total RNA was isolated from blood samples using RNAprep Pure Blood Kit . Total RNA was extracted from the cell lines using a Total RNA Purification kit (Invitrogen) in accordance with the manufacturer’s protocols. After the assessment of quantity and concentration of RNA, total RNA was used for the reverse transcription reaction with the Prime-Script RT reagent kit . Quantitative RT-PCR was performed using an miScript SYBR green PCR kit . The relative mRNA level of TRIM31 was calculated using the comparative 2Total proteins were extracted from AML-5 and U937 cells using radioimmunoprecipitation assay (RIPA) lysis buffer . The protein samples were loaded for electrophoresis and then subjected to Western blot analysis for the measurement of pro-caspase 3, cleaved caspase 3, Wnt3a, β-catenin, cyclin D1 and c-Myc expression levels. The specific primary and corresponding secondary antibodies were obtained from Santa Cruz Biotechnology . The protein bands were detected by enhanced chemiluminescence (ECL) reagent  and analyzed by ImageJ Software .4 cells/well) were plated in a 96-well plate and cultured for adhesion. After incubating for 0, 24, 48, 72, 96 h, each well was added with 20 µl MTT  and incubated for additional 4 h. Then, 150 μl DMSO was added the cells to dissolve formazan. Finally, the optical density (OD) at 490 nm was measured using a Microplate Reader .MTT assay was performed to assess cell proliferation and sensitivity to daunorubicin. For the cell proliferation assay, AML-5 and U937 cells (1 × 104 cells/well) were incubated with a series of concentrations of daunorubicin  for 24 h. Then the cell viability was measured using MTT assay as described above.For the chemosensitivity assay, AML-5 and U937 cells (1 × 10Cell apoptosis was detected by flow cytometry using AnnexinV-FITC/Propidium Iodide (PI) staining kit (Invitrogen). In brief, after transfection, cells were harvested and resuspended in binding buffer. Subsequently, AnnexinV-FITC and PI were added and stained for 15 min at room temperature in dark. Finally, cells were subjected to flow cytometry .Caspase-3 activity of AML-5 and U937 cells was measured using a caspase-3 activity assay kit (Nanjing Jiancheng Bioengineering Institute). According to the instructions, cell lysates were incubated with 50 μl Ac-DEVD-pNA for 4 h at 37°C. The absorbance at 450 nm was measured using a microplate Reader (Bio-Rad).t test or one-way ANOVA was respectively used to assess the significant differences between two groups or among multiple groups. Differences were considered statistically significant when P-value <0.05.All statistical analyses were performed using Student GraphPad Prism 5 . The Student’s In the present study, we first validated the relative expression of TRIM31 in AML patients. Results in Loss-of-function assays were then performed in AML-5 and U937 cells through transfection with si-TRIM31 or si-NC A,B. MTT Subsequently, gain-of-function assays were performed through transfection with TRIM31-overexpressing plasmid (pcDNA-3.1-TRIM31) or pcDNA-3.1 empty plasmid A,B. As iAs shown in To further investigate the mechanism of TRIM31, the expression levels of Wnt3a and β-catenin in U937 cells were measured using Western blot. As shown in To further confirm the role of Wnt/β-catenin signaling pathway, U937 cells were treated with LiCl, which is an activator of Wnt/β-catenin signaling pathway. We found that LiCl increased the protein expression levels of Wnt3a and β-catenin in si-TRIM31-transfected U937 cells A. TreatmAs described in the present study, TRIM31 protein could promote cell proliferation and induce cell apoptosis, as well regulate chemoresistance of AML cells. In addition, the roles of TRIM31 were mediated by the Wnt/β-catenin signaling pathway.TRIM family is a group of proteins characterized by three novel functional domains: a ‘really interesting new gene’ (RING) domain in the N-terminus, 1 or 2 zinc-finger domains called B boxes (B1 box and B2 box) and a coiled-coil motif ,12. Due Overexpression of TRIM32 in the human promyelogenous leukemic cell line HL60 suppresses cellular proliferation and induces granulocytic differentiation, implying that TRIM32 may be a potentially therapeutic target for APL . TRIM62 Transduced Wnt signals are highly conserved and regulate various developmental processes ,24. β-caIn conclusion, our findings demonstrated that TRIM31 was involved in leukemogenesis and chemoresistance. Wnt/β-catenin signaling pathway played a critical role in the oncogenic potential of TRIM31 in AML. Collectively, TRIM31 might serve as a therapeutic target for the AML treatment.Click here for additional data file.


Introduction: Mentorship is a key component to success in academic medicine. Women are under-represented in leadership positions within medicine. Women are less likely to identify mentors than men. Speed mentoring is an innovative strategy to facilitate mentorship in academic medicine.Materials and Methods: A speed mentoring event for women faculty members in an academic anesthesiology department was held, followed by a second event for trainees. Attendees completed surveys about mentorship experiences at baseline and in follow-up. Questions were rated on a 7-point Likert scale with 1 = strongly disagree and 7 = strongly agree with values reported as median .Results: Baseline surveys indicated poor satisfaction with mentoring in the prior 6 months as 4.5 . Twelve months later, mentees reported increased satisfaction with mentoring 6 . Mentors and mentees felt their time was well spent during both events. There was an increase in the number of mentors identified after the events by both groups.Conclusions: Our results suggest speed mentoring is well received and impactful with minimal time and monetary investment. The attendees of the events identified an increased number of mentors after speed mentoring events, and this effect was maintained at 6–12 months. Speed mentoring may be one path to providing support for women to advance their careers in academic medicine. More research is warranted to better evaluate effectiveness of formats such as speed mentoring to facilitate improved mentorship for women in academic anesthesiology. Mentorship has been identified as a key component of success in business as well as in academic medicine.5 Initiating a mentoring relationship may be difficult, particularly for junior faculty and trainees. A previous research survey completed among faculty within our department found that only 30% of respondents identified a mentor. Thus, speed mentoring events were scheduled to encourage multiple mentor sessions that would hopefully lead to further mentor–mentee relationships to enhance work satisfaction and retain and advance women faculty in their careers.Efforts to address these discrepancies should include improvement in recruitment, retention, and mentorship to ensure career satisfaction.6 and is an event that creates multiple pairings of mentees with mentors in a structured environment.This study was deemed exempt by the Mayo Clinic Institutional Review Board. The gap in mentorship in our department led a group of women faculty to create a formal event to foster mentor–mentee relationships among women. Speed mentoring was selected as the format for our department's event since it allows multiple high-yield interactions in a short period of time. This approach was originally described by Cook et al.The aim of the intervention was to engage in discussion of mentee-identified goals, including clinical practice, research, education, and work–life integration. We also sought to increase interdepartmental satisfaction with mentorship and to facilitate sponsorship.via e-mail 6 weeks before the event. Six women faculty, with leadership and research experience were invited to serve as mentors and nine junior faculty attended as mentees.The pilot intervention was open to all women who were junior faculty (academic rank of instructor or assistant professor) in the Department of Anesthesiology in Rochester, Minnesota, and was held on a weekday evening in May 2018. Participants were invited The event lasted 2 hours and 30 minutes, during which every mentee met with three mentors for 30 minutes each. Each mentee was given a schedule with color-coded time slots to speak with mentors. The mentor–mentee encounters were not structured, but mentees were encouraged to prepare questions before the event. The invitation e-mail stated “in preparation to the event, we ask you to reflect on 2–3 questions/goals for the mentors at the event. These can be broad or very specific, and will help to create the pairing grid for the event.”Each participant completed baseline surveys on paper before and after the speed mentoring event. A seven-question follow-up survey was input into REDCap and a link was e-mailed to all participants at 1, 3, 6, and 12 months .via e-mail to attend. Six senior faculty mentors and 12 (mentees) residents, fellows, and junior faculty attended the event. Each mentor and mentee was given a schedule with four time slots to meet for 30 minutes with a different senior faculty member.The trainee event was organized in a similar manner and was held in December 2018. All residents, fellows, and junior faculty in our department were invited via e-mail links to REDCap. Twelve months follow-up was not conducted due to several trainees completing training and relocating. Questions on surveys were rated on a 7-point Likert scale with 1 = strongly disagree, 4 = neutral, and 7 = strongly agree. All values are reported as median . Data for the faculty and trainee events were combined. Themes for the free responses were categorized by author consensus and not analyzed further.All attendees completed baseline surveys before and after the event in person on paper. Follow-up surveys were collected at 1, 3, and 6 months Twenty-one mentees and 12 mentors participated in the events. Three mentors participated in both events. The mentees comprised 11 junior faculty (52%), 6 residents (29%), and 4 fellows (19%). Eighteen mentees were women and three mentees were men. Baseline surveys revealed participants desired mentorship in the following areas: clinical practice = 6 , research = 7 , academic advancement = 7 , and work–life integration = 7 . The mentees were asked to rate their satisfaction with mentoring at baseline and on follow-up surveys and the results are reported in The number of mentors for each mentee increased after the speed mentoring event, and remained stable for the follow-up period . At baseAll participants felt their time was well spent during the event . In addition, all participants indicated they would slightly agree, agree, or strongly agree with recommending a similar event to a colleague.Surveys included space for free text comments to garner opinions about the utility of the events, interactions with mentors, and suggestions for improvement. Mentees found the speed mentoring event to be useful and shared that it provided an opportunity to establish connections and relationships with senior leaders in the department. In addition, participants thought encouraging formal follow-up would be beneficial. A representative sample of comments has been summarized in 7 Various mentoring modalities have reported improvements in academic and career development in addition to personal satisfaction.7 These have predominately involved dyadic, peer, and group mentorship models.Reviews of mentoring studies have found that mentorship programs for women can lead to increased success in academic medicine, although there is minimal literature on speed mentoring outcomes.6 They reported high event satisfaction from both mentors and mentees, yet satisfaction with mentoring did not change 12 months after the event, from a mean score of 4.7 at baseline on a 7-point scale to a mean of 4.6 at 12 months.6 In comparison, mentees in our study indicated increased satisfaction with mentorship after 12 months, from a baseline median of 4.5  to a median of 6.The first speed mentoring event in academic medicine was published by Cook et al.Speed mentoring is a low-resource modality that may provide increased opportunities for development of mentoring relationships. As there are less women who advance in academic careers, this may allow for the ability to provide mentorship for multiple women with limited need for longer time and resource needs. Our events were viewed as an efficient use of time and all participants indicated they would recommend a similar event to a colleague.2 In addition, mentorship has been associated with career satisfaction, and may help increase retention of women faculty in academic departments.8 Implementation of a minimal resource event such as speed mentoring may allow for early identification of possible mentors for women to address bias and improve retention and academic progress.Lack of mentorship has been cited as a reason for the gap in academic achievement between women and men.9Mentorship can be beneficial for both the mentor and mentee, with the primary focus on the development of the mentee. Recently, it has been suggested that mentorship may not be enough to level the playing field for women.10 can help junior talent advance while also expanding the impact of senior leadership, but only when both parties stand to benefit.11 Women in business “are likely to be over-mentored and under-sponsored,” and the literature thus far suggests the same phenomenon exists in medicine.12 One possible hypothesis is that women mentees may have less powerful mentors who are, therefore, unable to act as sponsors.13Sponsorship (active support by someone in the organization with significant influence)11Developing mentorship relationships may be the first step along the continuum of professional engagement that can lead to sponsorship and career advancement. Hopefully, working on systemic leveling of the mentorship and sponsorship gap will also help alleviate some of the well-documented gender disparities in leadership positions and pay.The findings from our department's initial speed mentoring interventions have several limitations. The sample sizes are small and potentially biased. There were many mentees who did not complete follow-up during the study and 12-month follow-up was not offered to attendees of the trainee event. Mentors and mentees who participated all responded by e-mail, implying they are motivated to invest their time and efforts into mentorship at baseline. We had no control group; therefore, the junior faculty and trainees may have found more mentors over time on their own without the intervention of a speed mentoring event. Furthermore, detailed information about mentoring relationships or outcomes such as collaborations or projects were not collected.The strengths of our interventions include high levels of reported satisfaction with the events and the low cost and small time commitment required of all participants for the event. The successes of our initial speed mentoring events have resulted in further events within our department.Future research is needed to determine optimal interventions to improve mentorship and sponsorship for women anesthesiologists in all career stages. Future interventions should include a larger number of mentees as well as a control group to better evaluate the intervention. Once more data are known regarding when and how to best intervene, training programs and academic departments should endorse successful interventions to improve access of mentorship to all individuals seeking such relationships.In conclusion, speed mentoring is a low-cost intervention requiring minimal time commitment for both mentees and mentors. After two speed mentoring events, our survey data suggest high event satisfaction and increased satisfaction with mentoring. Many academic medicine departments and academic training programs promote mentorship through assigned mentors. The positive results of our intervention suggest this could be an additional tool to facilitate compatible mentor–mentee relationships. In addition, speed mentoring provides an opportunity for junior faculty and trainees to seek out mentorship from senior faculty members.


The pitching motion is an asymmetric action by which coordination of scapular rotation in the dominant arm might be affected in time and in comparison with the non-dominant arm. The study aimed to compare asymmetry and the evolution of scapular upward rotation over a one-year period. Data were collected twice, before and after a one-year period, from 92 participants . Scapular motion was tracked at different glenohumeral angles of elevation in the scapular plane: anatomical position (0°), 45°, 90° and 135°. Scapular upward rotation was calculated as the angle between the spinae scapula and the spine. Scapular upward rotation of the dominant arm was 5.1° (95% CI: 2.1°−8.1°) more compared to the non-dominant arm. Age group or glenohumeral angles of elevation did not affect this difference. Scapular upward rotation of the dominant arm decreased 1.9° (95% CI: −0.5° to 4.3°) after a one-year period, however, neither this observation, nor the interaction with age group or elevation angle was significant. These findings may indicate that pitchers could be at risk to develop shoulder injuries especially those that have been associated with scapular asymmetry. Overhead throwing athletes solve a complex full body problem in order to generate high throwing velocities. Baseball (170 km/h), handball (130 km/h) and javelin (113 km/h) are all examples of sports that involve very high throwing velocities. Such high speeds can only be reached with a perfected technique that involves the entire body Putnam . On of tIt is often considered problematic when differences in the kinematics between the left and right scapula are found in athletes performing overhead throws, even though in many asymptomatic athletes such asymmetry exists  arm and the non-dominant arm, whether scapular upward rotation of the dominant arm changes over a one-year period, and whether these potential effects are different for players of different ages. It was hypothesized that pitchers will demonstrate a greater upward rotation in the dominant arm than in the non-dominant arm and that the asymmetry decreases with age.n = 56, age = 13.8 SD 0.99 years) and an older  group. The research design and protocol were approved by the local ethical committee of the Department of Human Movement Sciences before its conductance. Informed consent was obtained from the parents of the participants before participating in the study.Data were collected from 92 male baseball pitchers , who were playing in the Dutch baseball academies and the national youth (U18) baseball team. Participants were tested twice. The pre-test was conducted in April 2014 and the post-test in March 2015. A division in two age groups was made, based on the team division in the local baseball competition, a younger  and at thoracic vertebrae 8 (T8) and normed to length one (The trigonum spinae (SM), angulus acromialis (SL) and the angulus inferior (AI)  of the sngth one .(1)Yaxiy-axis (The x-axis was defined perpendicular to the y-axis .(2)XaxispinaeV) from SM to SL (The scapula was Ↄ defined as a vector (SM to SL .(3)Vspiβ was calculated as the angle between the vector of the y-axis and the vector spinaeV (The angle  Vspinae .(4)β=coβ. In this way, when the spinae scapula was horizontal, the angle for upward rotation was 0°.The upward rotation was calculated as 90° – Z-values of skewness and kurtosis, and a Shapiro-Wilks test were also performed on the data. Homogeneity of variance was checked using Levene’s test. There were no violations of these assumptions. All statistical analyses were performed in SPSS v23.0.0.2  and a p-value below 0.05 was considered significant.To determine whether the amount of scapular upward rotation  was different between the dominant and non-dominant arm and whether these differences were affected by the level of arm elevation  and age group (young and old), data from the pre-test were analyzed statistically using a three-way mixed design ANOVA. A three-way mixed design ANOVA was also used to examine whether the scapular upward rotation of the dominant arm was different between the pre-test and post-test and whether these differences were affected by the level of arm elevation and age group. One-way within- and between-subjects ANOVAs with Bonferroni correction were used to examine if the interaction effects were significant. The assumption of normality was checked by means of visual inspection of the histogram, q-q plot and the box plot of the data within the groups. F = 11.36, p < 0.001), with the dominant arm showing more upward rotation than the non-dominant arm. There was no significant interaction effect of age group  = 1.13, p = 0.293) or angle of arm elevation  = 1.43, p = 0.235) on the asymmetry. Dominant and non-dominant scapular upward rotation for all studied angles of arm elevation is displayed in Asymmetry between the dominant and non-dominant arm was 5.1° on average  = 2.53, p = 0.120). The scapular upward rotation of the dominant arm is shown for both age groups and the four angles of GH-elevation in F = 1.53, p = 0.224). Also, the interaction between time (pre-test/post-test) and angle of arm elevation for scapular upward rotation was not significant  = 1.58, p = 0.213)  .10.1080As hypothesized, an asymmetry was observed in scapular upward rotation between the dominant and non-dominant shoulder in elite youth baseball pitchers. However, no significant differences were observed between the group of 12–15 years of age and the group of 16–19 years or for different angles of GH-elevation. Also no significant evolution of the scapular upward rotation was observed over the one-year study period.The magnitude of asymmetry in upward rotation observed in the present study (5.10° on average) is similar to that in a couple of other studies . A decrease in scapular upward rotation in the 16–19 years old age group was expected because an increase of the posterior capsule thickness of the dominant arm has a positive relationship with upward scapular rotation, leading to fatigue-related inhibition of the scapular muscles on the long term (Thomas et al. This study showed that the dominant arms of elite youth baseball pitchers exhibit more scapular upward rotation compared to their non-dominant arm. However, scapular upward rotation was not greater for the older pitchers as compared to the younger pitcher and it also did not increase over the one-year study period in either age group. As a result of the asymmetry in upward scapular rotation, these athletes could be at risk to develop shoulder injuries, and thus in the training program of these athletes specific exercises could be incorporated to minimize scapular upward rotation of the throwing arm.


Leukemia stem cells (LSCs) in play an important role in the initiation, relapse, and progression of acute myeloid leukemia (AML), and in the development of chemotherapeutic drug resistance in AML. Studies regarding the detection of LSCs and the development of novel therapies for targeting them are extensive. The identification of LSCs and targeting therapies for them has been continuously under investigation.dimCD34+CD38−CD133+ cells in bone marrow samples from patients with hematological malignancies and healthy controls, using four-color flow cytometry.We examined the levels of CD45dimCD34+CD38−CD133+ cells were highly expressed in the bone marrow of patients with AML compared to that in healthy controls (HC). Moreover, the proportions of CD45dimCD34+CD38−CD133+ cells were also examined in diverse hematological malignancies, including AML, CML, DLBCL, MM, MDS, HL, ALL, and CLL. LSCs were prominently detected in the BMCs isolated from patients with AML and CML, but rarely in BMCs isolated from patients with DLBCL, MM, MDS, ALL, CLL, and HL. Additionally, the high CD45dimCD34+CD38−CD133+ cell counts in AML patients served as a significantly poor risk factor for overall and event free survival.Interestingly, the CD45dimCD34+CD38−CD133+ cells in AML might potentially serve as LSCs. In addition, this cell population might represent a novel therapeutic target in AML.Therefore, our results suggest that CD45 Acute myeloid leukemia (AML) is generally regarded as a stem cell disease. It originates from a class of leukemic stem cells that are capable of self-renewal , 2. AML +CD38− stem cells at diagnosis significantly correlated with a high minimal residual disease frequency and subsequently to relapse in AML patients. These cell populations directly correlated with poor survival , mouse anti-human CD38-PE-Cy™5 , and appropriated isotype control antibodies were purchased from BD Biosciences . Mouse anti-human CD133-APC  was obtained from Miltenyi Biotec .n = 40), chronic myeloid leukemia , diffuse large B-cell lymphoma , multiple myeloma , myelodysplastic syndrome , Hodgkin lymphoma , acute lymphocytic leukemia , or chronic lymphocytic leukemia . Control bone marrows were obtained to rule out hematologic disorders but proven to be normal marrows from 27 healthy donors at Ulsan University Hospital, Ulsan South Korea. Baseline clinical characteristics of 40 patients with AML are summarized in Supplementary Table We analyzed bone marrow samples collected from 87 patients who were newly diagnosed with AML . They were washed with phosphate-buffered saline (PBS).The bone marrow cells (BMCs) were isolated by the density gradient method, as previously described . In brie3). The total bone marrow cell number used in the experiment was 4 × 106 cells. Cells were incubated with four antibodies against each cell surface antigen, including CD45, CD34, CD38, and CD133 on ice for 30 min. First, live BMCs were collected, and SSClow and CD45dim cells were gated, as shown in Fig. 1–102; R3 Gate: FL2-H, 102–104, FL3-H, 100–101. The BMCs were incubated with three combinations of monoclonal antibodies (mAbs) on ice for 30 min; these included isotype control 1 , isotype control 2 , and sample , as shown in Fig. dimCD34+CD38−CD133+ cells, CD133 positive cells among the R1, R2, R3-gated cells were measured, and the results were expressed as percentage change from the basal conditions including the isotype control 2. The 40,000 cells were used for flow cytometric acquisition in each sample tube.The BMCs were collected and washed twice with FACS buffer (PBS containing 0.3% BSA and 0.1% NaNCell-free plasma from bone marrow samples of patients with AML was collected and frozen at − 80 °C. Plasma interleukin (IL)-1β, IL-6, IL-17, and IL-23 levels were measured using ELISA kits according to the manufacturer’s introductions (R&D Systems).P < 0.05. For patients with AML, continuous variables were compared using the Student’s t-test, whereas categorical variables were analyzed using the Pearson chi-square test or Fisher’s exact test. Overall survival (OS) was calculated from the date of HCT to the date of death or last follow-up. Event-free survival (EFS) was defined from the date of HCT to the date of relapse or death from any cause. Survival probabilities were estimated by the Kaplan–Meier method. Univariate and multivariate analyses for OS, EFS, and relapse probability were performed using the log rank test and Cox proportional hazards model, respectively. The following variables were included in univariate analyses: CD45dimCD34+CD38−CD133+ cell proportion, age, white blood cell (WBC) count, platelet count, bone marrow blast percentage, cytogenetic risk groups, chemotherapeutic regimens, and immunophenotyping including CD7, CD33, CD34, and HLA-DR. Variables with a P-value < 0.1 in the univariate analyses were included in the multivariate analyses. The statistical analyses were performed with SPSS version 21.0 software . For all analyses, the P-values were two-sided; a P-value of < 0.05 was considered statistically significant.The data presented here represent the mean ± standard error of mean (SEM) of at least three independent experiments. All values were evaluated by one-way analysis of variance followed by Turkey range tests implemented in GraphPad Prism 7.0. Differences were considered significant at low/CD45dim cells were obtained. The BMCs were stained with various combinations of monoclonal antibodies for 30 min such as isotype 1, isotype 2, and sample  is shown in Fig. Recently, Th17 related cytokines such as IL-1β, IL-6, IL-17, IL-21, IL-22, and IL-23 play crucial roles in the pathogenesis of many diseases, including inflammatory diseases, autoimmune diseases, and cancers . They hadimCD34+CD38−CD133+ cells were examined by four-color flow cytometry in diverse hematological malignancies including AML (n = 40), CML (n = 6), DLBCL (n = 19), MM (n = 10), MDS, (n = 5), HL (n = 4), ALL (n = 3), and CLL (n = 2). These cells are significantly detected in the bone marrow of patients with AML and CML, but not in those with DLBCL, MM, MDS, ALL, CLL, and HL. These results indicated that CD45dimCD34+CD38−CD133+ cells in bone marrow are potential of AML stem cells. In addition, these cells might be used for the detection of AML stem cells.As shown in Fig. + AML and CD34− AML among 36 AML patients evaluable for CD34 expression was noted in 30 patients and 6 patients, respectively. The proportion of CD45dimCD34+CD38−CD133+ cells in CD34- AML were significantly lower than CD34+ AML . And CD34− AML showed tendency to have lower proportion of CD45dimCD34+CD38−CD133+ cells. FLT3-ITD mutation was rarely found in AML patients with higher counts of CD45dimCD34+CD38−CD133+ cells (≥10%) than in patients with fewer CD45dimCD34+CD38−CD133+ cells (< 10%) . In addition, higher counts of CD45dimCD34+CD38−CD133+ cells (≥ 20%) were significantly associated with lower levels of IL-17, as compared to lower CD45dimCD34+CD38−CD133+ cells (< 20%) . However, there was no significant difference in IL-1β, L-6, and IL-23 levels based on the population of CD45dimCD34+CD38−CD133+ cells.CD34dimCD34+CD38−CD133+ cells , univariate analysis revealed that the 2-year OS rate was 64.3, 57.9, and 0%, respectively (P < 0.001) and the 2-year EFS was 62.3, 37.2, and 0% (P = 0.002), respectively , no significant differences were observed in baseline clinical factors including age (P = 0.085), white blood cell count (P = 0.397), platelet count (P = 0.737), and chemotherapy intensity (P = 0.158). Univariate analyses for OS and EFS in patients with AML revealed that older age (> 60 years) was significantly associated with worse OS than younger age  (Supplementary Table P = 0.038) (Supplementary Table P = 0.012). When we took into consideration other clinical parameters in univariate and multivariate analyses, higher percentage of CD45dimCD34+CD38−CD133+ cells (≥ 40%) was found to be an independent prognostic factor for OS  and EFS   and chemotherapy intensity (hypomethylating agents vs. intensive chemotherapy)  were significant prognostic factors for OS in multivariate analysis.When we divided AML patients into three groups based on the percentage of CD45The hypothesis that cancer stem cells including LSCs are responsible for the initiation, relapse, and drug resistance of cancers has caused a great deal of excitement in this area of research. The importance of cancer stem cells has been demonstrated in a variety of tumors , 25–29. AML is a hematopoietic disease that is characterized by clonal growth and the accumulation of myelopoietic progenitor cells . It is a+CD38− immunophenotype [+CD38− compartment was shown to contain both CD34+CD38− LSCs and normal hematopoietic stem cells (HSCs) [+CD38−CD123+ cells are AML LSCs [dimCD34+CD38−CD133+ cells were examined by four-color flow cytometry to define a more specific and prognostically significant LSC population  . Mawali AML LSCs . In the + AML was found in 75% of patients with AML and 6 patients had CD34− AML. The clinical implications of CD45dimCD34+CD38−CD133+ cells might be different in CD34− AML. The CD34−AML had lower proportion of CD45dimCD34+CD38−CD133+ cells. Even though we could not evaluate prognostic impact of CD45dimCD34+CD38−CD133+ cells in CD34− AML due to small number of patients, some portion of CD45dimCD34+CD38−CD133+ cells in CD34− AML might contain normal hematopoietic stem cells as well as LSCs.In the present study, CD34dimCD34+CD38−CD133+ cells are present in more numbers in the bone marrow of patients with AML, but not in healthy controls (Fig. +CD38− might be a significant marker for discriminating LSC and a prognostic biomarker. Moreover, the asynchronism of CD133+ expression should be also evaluated in CD34− AML in the future. In other lymphoid hematologic malignancies such as lymphoma, MM, ALL, and CLL than AML, there could be some differences according to the percentages of both malignant cells and CD34+CD38− compartments within bone marrows because of niche competition between two cell populations.CD133 has been reported to be a cancer stem cell marker in solid tumors , 32–34. dimCD34+CD38−CD133+ LSCs which was shown to be a significant negative prognostic marker, considering together clinical factors. There is little data regarding IL-23 levels in AML, although IL-23 levels have been reported to be associated with AML leukemogenesis and disease susceptibility in a previous study [We also found increased production of IL-1β, IL-6, IL-17 and IL-23 in the bone marrow microenvironment of AML patients at the time of diagnosis (Fig. us study . Based odim population using the same criteria. Also, our results showed that the individual differences were large for CD45dim population (Supplementary Fig. dimCD34+CD38−CD133+ cells were prominently detected in the bone marrow of patients with AML and CML, but not in those with DLBCL, MM, MDS, ALL, CLL, and HL (Fig. +CD45−LAP+ cell proportions were related to poor survival [dimCD34+CD38−CD133+ cells predict poor OS and EFS in AML (Fig. dimCD34+CD38−CD133+ cell compartment in the bone marrow could help discriminate between LSCs and normal hematopoietic stem cells, and can serve as a strong prognostic marker. Therefore, targeting CD45dimCD34+CD38−CD133+ cells could serve as a novel therapeutic strategy in AML. Future studies will focus on the elimination of the CD45dimCD34+CD38−CD133+ cells in patients with AML. Also, it needs to make sure that CD45dimCD34+CD38−CD133+ cells actually work as LSCs in the future. And it is necessary to assess whether the CD45dimCD34+CD38−CD133+ cells have the same characteristics as the stem cells. Therefore, our results indicate that CD45dimCD34+CD38−CD133+ cells have the potential of leukemic stem cells in acute myeloid leukemia.We applied the gate of CD45dimCD34+CD38−CD133+ cells in AML might potentially serve as LSCs. Moreover, the high CD45dimCD34+CD38−CD133+ cell counts in AML patients served as a significantly poor risk factor for overall and event free survival. In addition, this cell population might represent a novel therapeutic target in AML.CD45Additional file 1: Table S1. Baseline characteristics of AML patients.Additional file 2: Table S2. Patient characteristics.Additional file 3: Table S3. Univariate analysis for AML patients.Additional file 4: Figure S1. The expression of CD45dim population on bone marrow cells in the study.


In the recent years, expression and function of Orai1 have gained recognition in excitable cardiomyocytes, albeit controversial. Even if its cardiac physiological role in adult is still elusive and needs to be clarified, Orai1 contribution in cardiac diseases such as cardiac hypertrophy and heart failure (HF) is increasingly recognized. The present review surveys our current arising knowledge on the new role of Orai1 channels in the heart and debates on its participation to cardiac hypertrophy and HF.The archetypal store-operated Ca This is used to determine which both short- and long-term cellular functions are activated and when. This fine control of Ca2+ handling is notably setup by various ion channels at the plasma membrane, including the store-operated Ca2+ channels (SOCs) and their corresponding store-operated Ca2+ entry (SOCE).Spatiotemporal modulation of intracellular Ca2+ entry” (2+ influx to the depletion of endoplasmic reticulum (ER) Ca2+ stores. Ca2+ influx into the cell via SOCs can be pumped back into the ER to replenish depleted stores and restore ER Ca2+ homeostasis.The notion of SOCE was first proposed in salivary gland cells as a “capacitative Ca+ entry” , 1990, w2+ release-activated Ca2+ current (ICRAC) activated by Ca2+ store depletion in mast cells was provided by patch-clamp experiments  forming the classical SOCs, which is highly selective for Ca2+. The participation of the transient receptor potential canonical (TRPCs), which are non-Ca2+ selective channels, as SOCs remains a highly contentious issue. The mechanism for SOCE activation was then elucidated by a series of elegant studies conducted primarily in non-excitable cells. Upon ER Ca2+ depletion, Ca2+ dissociates from the STIM1 EF-hand domain resulting in dimerization or oligomerization of STIM1 proteins and their translocation to the ER-plasma membrane (PM) junctions where they interact and activate Orai1 channels  and cytosolic NH2- and COOH-tails . M1 linetermined . This sttermined .ORAI1, which are associated with severe combined immunodeficiency (SCID)-like disease, tubular aggregate myopathy (TAM), and Stormorken syndrome   and thenExpression of Orai1 protein was first detected in 1-day-old neonate mice heart . Later oOf note, one research group could not detect the Orai1 expression in isolated adult rat cardiomyocytes . This miOrai1 expression is more abundant in human myocardial tissue , in humaLimited numbers of studies have investigated the cellular distribution of Orai1. The Orai1 location seems somewhat inconsistent depending on the cell types studied, in particular between neonatal and adult cardiomyocytes. This is probably due to the different techniques for cell fixation and permeabilization and/or the antibody specificities.2+ SR depletion, Orai1 was redistributed as puncta in the SR-PM junctions, with STIM1 promoting their interaction 4 channels at the surface membrane  . Furthermembrane .2+ entry into the heart remains somewhat unclear and enigmatic. As for other excitable cells, it has long been considered that SOCE contribution to cardiac Ca2+ homeostasis is rather limited or non-existent, taking into account that Ca2+ influx during each beat through voltage-dependent Ca2+ channels is large enough to maintain cardiac excitation-contraction coupling (ECC). However, even a modest change in Ca2+ signaling can progressively alter heart function if sustained. For a prolonged period, a source of Ca2+ through SOCs can therefore potentially affect the heart.Overall, most of the reports have detected Orai1 expression in cardiomyocytes. However, the physiological role of the Orai1-mediated Ca2+ level or the SR Ca2+ load , calmodulin-dependant kinase II (CaMKII), or extracellular signal-regulated kinases (ERK1/2), signaling pathways involved in cardiac hypertrophy, and HF. Conversely, the reduction of SOCE in the HL-1 cell line with Orai1 KD decreased the diastolic Ca2+ level and SR Ca2+ content, suggesting a role of Orai1 in the SR Ca2+ load maintenance (2+ level via the serum and glucocorticoid-regulated kinase 1 (SGK1) in NRVMs  in NRVMs did not affect the diastolic Caa2+ load . Howeverntenance . More rein NRVMs . In hESCnization . In human of Akt .2+ levels during development therefore raises the question of its potential involvement in the adult heart.The role of Orai1 in maintaining cytosolic and intrareticular Ca2+ transients nor cell contraction  transcript expression, those results suggested that Orai1 is not instrumental for the ECC and cardiac function at rest. In addition, it has been shown that dn-Orai1R93W mice display abnormal amounts of lipid droplets in the heart, providing a potential role in lipid metabolism  to the initiation of atrial and ventricular arrhythmias . Indeed,2+ content, the diastolic Ca2+, as well as cell growth during cardiac development. However, in adulthood, additional studies are clearly necessary to clarify the controversy over the role of SOCE machinery in the heart.Taken together, it is generally accepted that SOCE carried by Orai1 channels regulates the SR CaWhether Orai1 role in heart physiology is still elusive, its involvement in pathophysiological situation has been more documented.2+ homeostasis is an important and proximal player underlying the pathogenesis of heart diseases. As the main regulator of the cardiac ECC, mishandling of Ca2+ is directly related to the mechanical dysfunction and certain arrhythmias associated with hypertrophy and HF. Moreover, the Ca2+-dependent intracellular signaling pathways activation, notably CaN/NFAT, CaMKII, and ERK promotes pro-hypertrophic gene expression, leading to pathological growth, cardiac remodeling, and dysfunction  or phenylephrine (PE)-induced hypertrophy of NRVMs or hESC-CMs . Increasfarction . In righfarction . Surprisfarction . Interesfarction . By contfarction .In NRVMs, non-selective inhibitors of SOCs, such as glucosamine or SKF-96365, prevented the increase of NFAT nuclear translocation and cellular hypertrophy after a 48-h treatment with hypertrophic stressors (AngII or PE) . FurtherIn a mouse model and cultured cardiomyocyte model treated with AngII or PE, gastrodine, a polyphenol with anti-inflammatory properties used in traditional Chinese medicine, is protective against the development of cellular and cardiac hypertrophy by attenuating the SOCE and reducing the expression of STIM1 and Orai1 .G98A or by nitric oxide (NO) via activation of PKG prevented PE-induced cellular hypertrophy , or in vivo JPIII-treated (a new selective Orai1 inhibitor) mice were protected from left ventricular systolic dysfunction and from interstitial fibrosis deposition, even if increased cardiac hypertrophy was observed. This was correlated with a protection from pressure overload-induced cellular Ca2+ signaling alterations , SERCA2a downregulation, and Pyk2/MEK/ERK overactivation , and digoxin are indicated for acute systolic HF patients with decreased cardiac contractility and evidence of end-organ hypoperfusion -3 inhibitors , β-adrenergic receptor agonists , Caerfusion . Howevererfusion . Moreoveerfusion . There iThis uncomfortable dilemma has led to expand new and far more powerful methods of preventing and treating HF. Currently, HF drug treatment research focuses on interrupting intracellular signaling pathways that are injurious to cardiovascular tissue and on stimulating signaling pathways that protect cardiovascular tissues.There is an arsenal of pharmacological selective inhibitors that modulate Orai1 function, and several have now advanced into human clinical trials for psoriasis, pancreatitis, asthma, or Hodgkins lymphoma . ConsequOver the past few years, as described herein, STIM1/Orai1-mediated SOCE has emerged as a promising target to treat HF. Here arises a question of which STIM1 and/or Orai1 should be targeted since both molecules operate in the same pathway. From a safety or toxicological perspective, Orai1 may be a more attractive target than STIM1. Firstly, compared with STIM1, Orai1 appears to be more restricted in its function of mediating SOCE. STIM1 appears to be involved in the activity of other proteins than Orai1, such as Cav1.2  and TRPCin vivo pharmacological Orai1 small-molecule inhibition by JPIII have little impact on Ca2+ homeostasis related to ECC and left ventricular systolic performance. After chronic pressure overload, JPIII markedly improves the left ventricular systolic function and Ca2+ handling by preventing the Ca2+ cycling mishandling, SERCA2a downregulation and fibrosis, without causing adverse effect. Our findings suggest that Orai1 inhibition has a potential favorable hemodynamic value to protect the heart from maladaptive hypertrophy and might represent a new inotropic support to help to relieve systolic dysfunction. This plausible therapeutic intervention need to be extended to other pathological models (According to the study conducted in our lab , under pl models . In addil models . Currentl models . Nonethe2+-responsive signaling pathways. This pathological importance of Orai1 echoed its role during the embryonic and neonatal phase of cardiac development since hypertrophic cardiac remodeling is characterized by reactivation of the fetal gene program. Hence, Orai1-mediated SOCE may be a novel therapeutic target to consider in heart disease.In conclusion, although the Orai1 function in adult cardiac physiology remains elusive, converged experimental results pointed out a detrimental effect of Orai1 upregulation in hypertrophy and HF, including fibrosis, ventricular contractility, and pro-hypertrophic CaRL drafted the manuscript under the supervision of J-PB and JS. J-PB, JS, and A-MG edited the manuscript. All authors contributed to the article and approved the submitted version.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.