Tbx5-mutant mice demonstrate spontaneous AF. We report that TBX5 is critical for cellular Ca2+ homeostasis, providing a molecular mechanism underlying the genetic implication of TBX5 in AF. We show that cardiomyocyte action potential (AP) abnormalities in Tbx5-deficient atrial cardiomyocytes are caused by a decreased sarcoplasmic reticulum (SR) Ca2+ ATPase (SERCA2)-mediated SR calcium uptake which was balanced by enhanced trans-sarcolemmal calcium fluxes , providing mechanisms for triggered activity. The AP defects, cardiomyocyte ectopy, and AF caused by TBX5 deficiency were rescued by phospholamban removal, which normalized SERCA function. These results directly link transcriptional control of SERCA2 activity, depressed SR Ca2+ sequestration, enhanced trans-sarcolemmal calcium fluxes, and AF, establishing a mechanism underlying the genetic basis for a Ca2+-dependent pathway for AF risk.Risk for Atrial Fibrillation (AF), the most common human arrhythmia, has a major genetic component. The T-box transcription factor TBX5 influences human AF risk, and adult-specific  The human heart contains four distinct chambers that work together to pump blood around the body. In individuals with a condition called atrial fibrillation, two of the chambers (known as the atria) beat irregularly and are unable to push all the blood they hold into the other two chambers of the heart. This can cause heart failure and increases the likelihood of blood clots, which may lead to stroke and heart attacks.Small molecules called calcium ions play a crucial role in regulating how and when the atria contract by driving electrical activity in heart cells. To contract the atria, a storage compartment within heart cells known as the sarcoplasmic reticulum releases calcium ions into the main compartment of the cells. Calcium ions also enter the cell from the surrounding tissue. As the atria relax, calcium ions are pumped back into the sarcoplasmic reticulum or out of the cell by specific transport proteins.Tbx5 are more likely to develop atrial fibrillation than other people, but it was not clear how such gene mutations contribute to the disease. Here, Dai, Laforest et al. used mice with a mutation in the Tbx5 gene to study how defects in Tbx5 affect electrical activity in heart cells.Individuals with mutations in a gene called Tbx5 gene was critical for calcium ions to drive normal electrical activity in mouse heart cells. Compared with heart cells from normal mice, the heart cells from the mutant mice had decreased flow of calcium ions into the sarcoplasmic reticulum and increased flow of calcium ions out of the cell.The experiments found that the These findings provide a direct link between atrial fibrillation and the flow of calcium ions in heart cells. Together with previous work, these findings indicate that multiple different mechanisms could lead to atrial fibrillation, but that many of these involve changes in the flow of calcium ions. Therefore, personalized medicine, where clinicians uncover the specific mechanisms responsible for atrial fibrillation in individual patients, may play an important role in treating this condition in the future. Atrial fibrillation (AF) is the most common arrhythmia in humans, characterized by irregularly irregular atrial electrical activity, resulting in asynchronous atrial contraction. AF is a global problem, affecting more than 33 million people and approximately 25% of Americans over the age of forty . AF is aTbx5 knockout mice demonstrate primary spontaneous and sustained AF, providing evidence supporting the genetic implication at this locus. GWAS have also implicated multiple genes involved in cardiomyocyte calcium handling, including Atp2a2, encoding the sarcolemmal calcium ATPase SERCA2, and Sln and Pln, encoding direct binding SERCA2 inhibitors sarcolipin and phospholamban, respectively. We have previously demonstrated that these cardiomyocyte calcium control genes are direct TBX5 targets  calcium uptake due to depressed SERCA2 expression, depressed SERCA function, and increased phospholamban expression. Decreased SR calcium uptake is compensated by increased Ca2+ extrusion from cardiomyocytes via sodium-calcium exchanger (NCX) current (INCX), which provides a mechanism for TBX5-dependent action potential (AP) prolongation and the propensity for triggered cellular ectopy. In the setting of enhanced NCX mediated Ca2+ efflux and depressed SR uptake, compensatory increases in L-type calcium current (ICaL) balance calcium extrusion to maintain steady state calcium homeostasis. Together these calcium handling alterations contribute to AP prolongation and triggered activity.We sought to define the Tbx5-dependent cellular mechanisms responsible for abnormal calcium-dependent electrical activity. We found that Tbx5-dependent AF. These results establish a direct link between depressed SR Ca2+ sequestration, enhanced NCX activity, and AF. This model suggests that targeting calcium handling pathways may be a treatment approach for a subpopulation of AF patients.We further demonstrated that calcium handling abnormalities, AP alterations, and triggered activity are all normalized by knockout of phospholamban, which prevents Tbx5-deficient atrial cardiomyocytes demonstrated AP prolongation and myocardial ectopy. We hypothesized that these defects were caused by cellular calcium handling abnormalities. We therefore surveyed the expression of known calcium handling genes in the adult-specific Tbx5 knockout model. We assessed gene expression in fl/fl;R26CreERT2Tbx5 and control CreERT2R26 mice at 10 weeks of age following tamoxifen (TM) treatment at 8 weeks of age. Consistent with previous observations, the adult fl/fl;R26CreERT2Tbx5 but not control mice developed spontaneous AF, showing an irregularly irregular heartbeat, by telemetric electrocardiogram (ECG) recordings  and SERCA2 (Atp2a2), two of the main determinants of sarcoplasmic reticulum (SR) calcium flux, were decreased by 61% and 71% respectively in fl/fl;R26CreERT2Tbx5 mice compared to CreERT2R26 controls (p=0.026 and p=0.001 for Ryr2 and Atp2a2 respectively) consistent with previous studies (Pln) mRNA expression was increased by 69% in fl/fl;R26CreERT2Tbx5 compared to CreERT2R26 (p=0.023), which would be expected to further depress SERCA2 activity. There was no significant difference in mRNA expression of the alpha 1C subunit of the L-type calcium channel (Cacna1c), the cardiac sodium calcium exchanger (Ncx1), or any of the calmodulins 1–3   . These dTbx5-mutant atria. In steady state, with each cardiomyocyte contraction cycle, calcium entering the cardiomyocyte  is extruded from the cell (predominantly via inward INCX). Similarly, calcium leaving the SR via RyR2 release or SR leak pathways is taken back up into the SR via SERCA2. We examined the effect of altered TBX5-dependent gene expression on these aspects of cardiomyocyte calcium flux. Given the observed changes in Ryr2 and Atp2a2 mRNA abundance, we hypothesized that AP prolongation in Tbx5 deficient cardiomyocytes was due to calcium handling defects downstream of initial Ca2+ entry through ICaL. To test this, we recorded APs in the presence and absence of the L-type Ca2+ channel blocker nifedipine. This approach blocks Ca2+ entry into the cell and indirectly removes the effect of Ca2+ entry on downstream Ca2+ handling pathways, including SR Ca2+ release/reuptake as well as the electrogenic effect of calcium transport out of the cell via inward INCX. 30 μM nifedipine completely inhibited L-type calcium current, preventing Ca2+ entry or release of SR calcium in control CreERT2R26 and fl/fl;R26CreERT2Tbx5  was small, with 19 ± 4% shortening of APD at 90% repolarization (APD90) (p=0.008)  was shortened by 16 ± 6% and APD90 by 61 ± 6% (p=0.02 and 0.007 respectively) (V1.2 showed no significant difference in protein expression (CaL current was increased 92 ± 34% (p=0.027) in fl/fl;R26CreERT2Tbx5 atrial cardiomyocytes compared to control CreERT2R26 . Steady-state ICaL inactivation was unchanged  we hypothesized that Tbx5-deficiency disrupts Ca2+ handling pathways downstream of ICaL.We examined the relationship between myocardial electrophysiology deficits and calcium flux in 6CreERT2 . In contp=0.008) . Howeverctively) . Westernpression , in linepression , consist6CreERT2 . The inanchanged . These dRyr2 mRNA was downregulated in Tbx5-mutant atria, we investigated the Tbx5 dependent regulation of RyR2 protein expression and function. RyR2 protein expression was significantly decreased in left atria of fl/fl;R26CreERT2Tbx5 mice compared to CreERT2R26 mice by western blot  using confocal linescans  in fl/fl;R26CreERT2Tbx5 cardiomyocytes compared to CreERT2R26, which suggests that AP prolongation is essential to maintaining peak twitch [Ca2+]i. The latter experiment is also consistent with depressed SR loads in fl/fl;R26CreERT2Tbx5 compared to CreERT2R26 atrial myocytes.We next focused on the balance of diastolic calcium efflux pathways as potential mediators of CaT2 atria . To defimeasured . The i transients are presented as total fluorescence intensity normalized to resting fluorescence (F/F0) obtained from steady-state resting conditions before field stimulation. [Ca2+]i transients and sparks were acquired in line-scan mode  using a Zeiss confocal microscope.Langendorff perfusion with 2 mg/mL of Collagenase Type 2  at 5 ml/min was used to isolate atrial cardiomyocytes. Cardiomyocytes were then plated on laminin coated glass bottom dishes for 30 min prior to incubation with 10 µM Fluo-4/AM (Molecular Probes/Invitrogen) in normal Tyrode’s solution containing (in mM): 140 NaCl, 4 KCl, 10 glucose, 10 HEPES, and 1 MgCl2+]i transients with fixed duration depolarizations. Cardiomyocytes are kept at 37°C and perfused with Tyrode solution . Internal pipette solution composition was (in mM): 20 KCl, 100 K-glutamate, 10 HEPES, 5 MgCl2, 10 NaCl, 5 Mg-ATP, 0.3 Na-GTP. Patch pipettes (World Precision Instruments) were pulled to have a mean resistance of 3.5–5 MΩ. An Ag–AgCl pellet and 3M KCl agar bridge was used to ground the bath. Liquid junction potentials, were always corrected after cell rupture.APs and voltage clamp recordings were recorded using standard ruptured patch protocol . We usedCaL contained (in mM): 120 Tetraethylammonium-chloride, 10 CsCl, 10 Glucose, 10 HEPES, 1.5 MgCl2, 1 CaCl2, pH 7.4 with CsOH. Internal pipette solution contained (in mM): 100 Cs-methanesulfonate, 30 CsCl, 10 HEPES 5 EGTA, 2 MgCl2, 5 Mg-ATP, pH 7.2 with CsOH. ICaL was recorded during 200 ms voltage clamp pulses from a holding potential of −40 mV to test potentials ranging from −40 to 60 mV, with pulses applied every 2 s in 5 mV increments. Peak current amplitudes were normalized to the cell capacitance (Cm) and presented as current density (A/F). Steady-state inactivation of ICaL was investigated using two-pulse protocol. Holding potential was −80 mV. The first pulse depolarized membrane from −60 to 20 mV with 10 mV increments during 500 ms, the second pulse depolarized the membrane to 10 mV for 50 ms. The inactivation curves were fit to a Boltzmann distribution.External solution for IAcquisition was performed using an Axopatch-200B amplifier connected to a Digidata1550A acquisition system . In recording filtering at 2 kHz was performed using the amplifier Bessel and sampled at 10 kHz. Analysis was performed using pCLAMP10 (Axon Instruments) and a home written analysis code.Atrial tissue was collected and homogenized as described previously , in a bu3H]ryanodine (PerkinElmer), 1 mM EGTA and enough CaCl2 to set free [Ca2+] between 10 nM (pCa2+ 8) and 100 µM (pCa2+ 4). The ratio between Ca2+ and EGTA was determined using MaxChelator (WEBMAXCLITE v1.15 http://maxchelator.stanford.edu/webmaxc/webmaxclite115.htm). Following a 2 hr incubation at 36°C, reactions were filtered through Whatman GF/B Filters using a Brandel M24-R Harvester. [3H]ryanodine binding was determined using a Beckman LS6500 scintillation counter and BioSafe II scintillation cocktail (RPI Corp). Non-specific binding was quantified in the presence of 2 µM unlabeled ryanodine  and subtracted.Binding assays were carried out following a protocol previously described . Bindingfl/fl;R26CreERT2Tbx5 and CreERT2R26 mice was removed two weeks after receiving tamoxifen and RNA was isolated using a Trizol (Invitrogen) based method. Reverse transcription reaction was carried out using the qScript cDNA synthesis kit (Quanta) according to the manufacturer’s protocol. Quantitative RT-PCR was performed using the Power SYBR Green PCR Master Mix (Applied Biosystems) and run on an Applied Biosystems AB7500 machine. Relative fold changes were calculated using the comparative threshold cycle method (2-ΔΔCt), using glyceraldehyde-3-phosphate dehydrogenase (Gapdh) gene expression level as internal control.Left atrial tissue of CaL, spark frequency, and [Ca2+]i transients were determined using hierarchical statistical methods . Statistical significance for quantitative metrics of APs, SERCA, NCX, SR load, I methods . Statist In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Richard Aldrich as the Senior Editor. The following individuals involved in review of your submission have agreed to reveal their identity: Robert S. Kass (Reviewer #1); David Eisner (Reviewer #3).Thank you for submitting your article "A calcium transport mechanism for atrial fibrillation in Tbx5 mutant mice" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission. Our goal is to provide the essential revision requirements as a single set of instructions, so that you have a clear view of the revisions that are necessary for us to publish your work.Summary:Tbx5 in adult mice leads to (1) decreased expression of the SR Ca2+ pump SERCA2 and increased expression of phospholamban, leading to a decreased level of SR Ca2+ loading; and (2) increased Na-Ca exchanger (NCX1) expression and activity, providing a mechanism for prolonged action potentials and triggered activity. Additional knockout of phospholamban restored normal SR loading and action potential duration and prevented triggered activity and AF in Tbx5-knockout mice. These results provide a mechanistic link from genetic (GWAS) studies to the cellular mechanisms of AF.This is an interesting study of the effects of the T-box transcription factor TBX5 on the susceptibility to atrial fibrillation (AF), the most common and difficult to treat human cardiac arrhythmia. The authors provide convincing evidence that knockout of Essential revisions:The reviewers all found the paper to be interesting and important, but raised a number of concerns that must be adequately addressed before it can be accepted. Some of the required revisions will require further experimentation within the framework of the presented studies and techniques.Tbx5 KO cells, and the effect of nifedipine to shorten the AP is greatly increased. The authors interpret this as an effect of prolonged and increased NCX current, but a change in Cav1.2 activity may also contribute. Cav1.2 channels could be more active in the KO cells if there is less Ca2+-dependent inactivation due to less CICR (from the reduced SR Ca content). This could contribute to the prolonged APD and to a more pronounced effect of nifedipine in blocking these channels. To address this point, the authors should conduct voltage clamp recordings of the Cav1.2 current to monitor the degree of inactivation in WT vs. KO, with and without nifedipine. Simultaneous measurement of [Ca2+]i would help establish a basis for these effects.1) In Figure 2, the AP is lengthened in the Tbx5 KO cells because of an increased NCX and decreased SERCA, but this needs some qualification. In the steady state, the amount of Ca pumped out of the cell must balance influx through L-type channels (assuming negligible PMCA activity). Thus, changes in SERCA and NCX cannot affect the integral of the NCX current. If the long plateau of the action potential in Figure 2B reflects an increase of delayed NCX current (due to prolongation of the Ca2+ transient by decreased SERCA), then this must be associated with a decrease of NCX in the earlier stage of the action potential. Is there any evidence for this? As suggested above (#1), simultaneous recording of the action potential and [Ca2+]i in control and Tbx5 KO cells would help to clarify this point.2) The authors suggest that a larger NCX current occurs in the Tbx5 KO on the amplitude of the twitch Ca2+ transient is interesting given that SR Ca2+ content is decreased. Two potential compensatory mechanisms should be addressed. 1) It may be a consequence of the prolongation of the AP. Stimulation of the cells with constant-duration square voltage clamp pulses while measuring [Ca2+]i (see #1 above) would show whether the Ca transient is now less in Tbx5 than control. 2) It looks as though, as a consequence of the slowing of decay of the Ca2+ transient , the diastolic [Ca2+]i is elevated. This may reduce cytoplasmic Ca2+ buffering such that a smaller total release of Ca2+ from the SR could result in an unchanged amplitude of the Ca2+ transient. Please comment on this possibility.3) In Figure 4E, the lack of effect of 4) The statistical analysis may need to be modified. In Figure 1, please state the actual numbers of animals used in each group and for the t test. In Figure 2, 3C, 4E-I, and 5C-F, unless each myocyte comes from a different heart, it is invalid to use the number of cells as the "n" values for the t test . Either use hierarchical or nested statistical approaches  or simply average cells from the same heart and reduce n to be the number of hearts.Tbx5 KO mice, the functional contribution of PLB needs to be addressed by examining the phosphorylation status of PLB16 and PLB17.5) Although PLB mRNA is increased in Tbx5-KO mice needs to be tested directly. Can an NCX inhibitor eliminate EADs and DADs in Tbx5-KO myocytes?6) The role of increased NCX in causing AF in Tbx5 KO mouse  are typical heart failure phenotypes. This raises the question of whether the observed phenotype indicates the underlying basis of AF, or is secondary to ventricular changes leading to heart failure. This question may be addressed using the mouse model before it has progressed to the heart failure stage.7) The atrial phenotype of the  Essential revisions:The reviewers all found the paper to be interesting and important, but raised a number of concerns that must be adequately addressed before it can be accepted. Some of the required revisions will require further experimentation within the framework of the presented studies and techniques.v1.2 activity may also contribute. Cav1.2 channels could be more active in the KO cells if there is less Ca2+-dependent inactivation due to less CICR (from the reduced SR Ca content). This could contribute to the prolonged APD and to a more pronounced effect of nifedipine in blocking these channels. To address this point, the authors should conduct voltage clamp recordings of the Cav1.2 current to monitor the degree of inactivation in WT vs. KO, with and without nifedipine. Simultaneous measurement of [Ca2+]i would help establish a basis for these effects.1) In Figure 2, the AP is lengthened in the Tbx5 KO cells, and the effect of nifedipine to shorten the AP is greatly increased. The authors interpret this as an effect of prolonged and increased NCX current, but a change in CaCaL could contribute to APD prolongation, and that calcium influx and efflux must be balanced. To address this, we have measured ICaL. We find peak ICaL is increased 92 ± 34% in fl/fl;R26CreERT2Tbx5 cardiomyocytes in comparison to the CreERT2R26 control. . Additionally, 30 μM nifedipine completely blocked ICa,L and eliminated any [Ca2+]i transient in both TBX5 KO and control cardiomyocytes . These data are consistent with a role of ICaL in AP prolongation. Increased ICaL may augment peak [Ca]i in the setting of depressed SR load  and also approximately balances the increase in NCX mediated calcium efflux . We have added these observations to the Results and Discussion sections of the text.We agree with the reviewers that I2+ transient by decreased SERCA), then this must be associated with a decrease of NCX in the earlier stage of the action potential. Is there any evidence for this? As suggested above (#1), simultaneous recording of the action potential and [Ca2+]i in control and Tbx5 KO cells would help to clarify this point.2) The authors suggest that a larger NCX current occurs in the Tbx5 KO cells because of an increased NCX and decreased SERCA, but this needs some qualification. In the steady state, the amount of Ca pumped out of the cell must balance influx through L-type channels (assuming negligible PMCA activity). Thus, changes in SERCA and NCX cannot affect the integral of the NCX current. If the long plateau of the action potential in Figure 2B reflects an increase of delayed NCX current  and include corresponding discussions. Nevertheless, the reviewers also point out that a decrease in NCX in the earlier stage of the action potential could also contribute to steady state [Ca]i balance. Indeed, in the setting of AP prolongation, a decreased electrochemical drive for inward INCX could exist in the early part of the AP. Therefore, as suggested by the reviewers, simultaneous AP and [Ca2+]i measurements were performed . Peak [Ca]i was not depressed during the AP, consistent with our field stimulation experiments in Figure 4E. Thus, assuming that submembrane calcium in the vicinity of the NCX is also similar, then the electrochemical drive for inward INCX may be decreased in the early part of the AP in fl/fl;R26CreERT2Tbx5 cardiomyocytes relative to CreERT2R26 control. However, in the context of increased ICaL and INCX activity, it is challenging to dissect the individual contributions of calcium-dependent currents at depolarized potentials. In the future we hope to better quantify the influx and efflux pathways for calcium during the AP with Tbx5 deficiency using a combined experimental and computer modeling approach. A discussion of this point has been added to the manuscript.The reviewers correctly point out that calcium influx must match calcium efflux in steady state. We have now considered this point in the context of new experiments to determine I2+ transient is interesting given that SR Ca2+ content is decreased. Two potential compensatory mechanisms should be addressed. 1) It may be a consequence of the prolongation of the AP. Stimulation of the cells with constant-duration square voltage clamp pulses while measuring [Ca2+]i (see #1 above) would show whether the Ca transient is now less in Tbx5 than control. 2) It looks as though, as a consequence of the slowing of decay of the Ca2+ transient , the diastolic [Ca2+]i is elevated. This may reduce cytoplasmic Ca2+ buffering such that a smaller total release of Ca2+ from the SR could result in an unchanged amplitude of the Ca2+ transient. Please comment on this possibility.3) In Figure 4E, the lack of effect of Tbx5 KO on the amplitude of the twitch Cai without the effect of AP prolongation. The square wave experiments are now shown in Figure 4—figure supplement 1. In these experiments fl/fl;R26CreERT2Tbx5 cardiomyocytes have a 23 ± 4% decrease in the calcium transient amplitude compared to controls. Our results suggest that AP duration (possibly coupled with increased ICaL density) can explain the similarities in peak twitch calcium between fl/fl;R26CreERT2Tbx5 and CreERT2R26 cardiomyocytes. It is also interesting to consider that altered buffering capacity could play a role. However, we did not detect any changes in rate of rise of the first 10 ms of the calcium transient as would be expected if a greater portion of calcium buffer was bound or if total calcium buffering capacity was altered. . Furthermore, we want to emphasize that Fo was always recorded at rest without pacing, so measurements of SR load would be unaffected by this possibility. This new data has been added to the manuscript.The new square wave experiment proposed by the reviewers is a very good idea because it allows us to assess the peak [Ca]4) The statistical analysis may need to be modified. In Figure 1, please state the actual numbers of animals used in each group and for the t test. In Figure 2, 3C, 4E-I, and 5C-F, unless each myocyte comes from a different heart, it is invalid to use the number of cells as the "n" values for the t test . Either use hierarchical or nested statistical approaches  or simply average cells from the same heart and reduce n to be the number of hearts.As suggested, we adjusted the statistical analysis using the hierarchical approach described in Sikkel et al [1]. Animal numbers are stated in the text where applicable and all of the figures are corrected to show significance indicators based on the revised analysis.5) Although PLB mRNA is increased in Tbx5 KO mice, the functional contribution of PLB needs to be addressed by examining the phosphorylation status of PLB16 and PLB17.fl/fl;R26CreERT2Tbx5and CreERT2R26 control atria. Phosphorylation of PLN-S16 was significantly increased while that of PLN-T17 was unchanged in fl/fl;R26CreERT2Tbx5compared to CreERT2R26 control . Increased S16 phosphorylation may be a compensatory mechanism for the decrease in SERCA expression. However, based on measurements of SERCA expression and activity , functional effects of PLN phosphorylation are not sufficient to normalize SERCA activity, but knockout of Pln is sufficient . This data and related discussion have been added to the manuscript.We agree that the phosphorylation status of PLN is important. Thus, we performed western blot for PLN-S16 and PLN-T17 in 6) The role of increased NCX in causing AF in Tbx5-KO mice needs to be tested directly. Can an NCX inhibitor eliminate EADs and DADs in Tbx5-KO myocytes?NCX is still detectable in the presence of 5µM ORM-10103 using a square wave voltage clamp pulse to trigger SR release, as described in point 3 . Higher dosages of ORM-10103 or other NCX inhibitors are known to have off-target effects on Na+, Ca2+ and K+ channels, which would complicate interpretation , particularly in the setting of only partial NCX inhibition. Thus, it is unfortunately challenging to reach any conclusions from the NCX inhibitor data. As newer agents become available it would be reasonable to test their ability to inhibit triggered activity and AF in this model, and we address this in the Discussion.As suggested by the reviewers, NCX inhibitors could theoretically reduce the frequency of DADs in our model. In our recordings using a new generation NCX inhibitor, ORM-10103, at 5μM, we observed a tendency towards action potential shortening (not significant) but no effect on frequency of triggered activity (EADs and DADs). A representative trace is shown in  point 3 . INCX in2+ load) are typical heart failure phenotypes. This raises the question of whether the observed phenotype indicates the underlying basis of AF, or is secondary to ventricular changes leading to heart failure. This question may be addressed using the mouse model before it has progressed to the heart failure stage.7) The atrial phenotype of the Tbx5 KO mouse  Sikkel MB, Francis DP, Howard J, et al. Hierarchical statistical techniques are necessary to draw reliable conclusions from analysis of isolated cardiomyocyte studies. Cardiovascular research. Dec 1 2017;113(14):1743-1752.2+ exchanger, decreases early and delayed afterdepolarizations in the canine heart. British journal of pharmacology. Oct 2013;170(4):768-778.2) Jost N, Nagy N, Corici C, et al. ORM-10103, a novel specific inhibitor of the Na+/Ca2+ exchange inhibitor. Cardiovascular drug reviews. Winter 2003;21(4):255-276.3) Amran MS, Homma N, Hashimoto K. Pharmacology of KB-R7943: a Na+-Ca4) Nadadur RD, Broman MT, Boukens B, et al. Pitx2 modulates a Tbx5-dependent gene regulatory network to maintain atrial rhythm. Science translational medicine. Aug 31 2016;8(354):354ra115.


The prognosis of adult T-cell leukemia-lymphoma (ATL) remains very poor, and there is an urgent clinical need to investigate novel therapies for ATL. The expression of phosphatidylinositol 3-kinase-δ (PI3k-δ) is normally restricted to hematopoietic cells and is known as a key determinant of cell survival in certain cancers. The inhibitor of PI3k-δ, idelalisib, has been shown to be effective in the treatment of chronic lymphocytic leukemia. Here, we report the expression of PI3k-δ and the ability of idelalisib to promote apoptosis in ex vivo ATL samples. The activity of PI3K was quantified by a PI3-Kinase Activity ELISA kit. Although there was no significant difference in mean PI3K activity between healthy donors and patients with ATL, certain cases of ATL showed extremely high PI3K activities. The expression of PI3k-δ protein was detectable in most ATL cases. The freshly isolated cells from ATL patients were cultured with or without idelalisib for 0–10 days, and cell survival was then quantified. Idelalisib induced apoptosis in ATL cells in a time-dependent manner, and significantly reduced the frequency of viable ATL cells at 10 days. No time-dependent effects of idelalisib were observed in non-malignant T cells from the same patients. CCL22 has been reported to promote survival of ATL cells in part through the PI3K-AKT pathway. Idelalisib blocked this CCL22-induced phosphorylation of AKT and significantly inhibited the proliferation of ATL cells. These results validate the PI3K-AKT pathway as a potential therapeutic target in ATL.The online version of this article (10.1186/s40364-018-0138-7) contains supplementary material, which is available to authorized users. Adult T-cell leukemia-lymphoma (ATL) is a malignancy of peripheral T lymphocytes caused by infection with human T-lymphotropic virus type-I HTLV-1). Approximately 5% of HTLV-1 carriers develop ATL in their lifetime . Approxi. The chrThe phosphatidylinositol 3-kinase (PI3K) pathway, a key signaling system that links multiple receptors and oncogenes, is commonly activated in human cancers. PI3K-δ regulates the function of cells in the immune system: its expression is mostly restricted to hematopoietic cells, including myeloid cells, B cells, and T cells. In mouse models, inactivated PI3K-δ impaired antigen receptor signaling in B and T cells, and attenuated the immune response .The PI3K-δ inhibitor, idelalisib, significantly improved survival in patients with relapsed chronic lymphocytic leukemia (CLL) with a tolerable safety profile . PI3K-δ The PI3K activity assay was performed on whole-cell lysates from ATL patients and HTLV-1 uninfected donors. Although there was no significant difference in mean PI3K activity between healthy donors and patients with ATL, certain cases of ATL showed extremely high PI3K activities Fig. . The levWe investigated the effect of idelalisib on the viability of ATL cells in vitro. First, frozen and thawed PBMCs of four ATL patients were incubated with or without idelalisib 0.1-100 μM) for 72 h. This concentration range was selected on the basis of prior reports of preclinical activity of idelalisib in CLL [0 μM for CCL22 binding to CCR4 has been reported to promote survival of ATL cells, in part through the PI3K-AKT pathway . ExposurThe findings reported here closely resemble the impact of idelalisib on ex vivo cells from patients with CLL, in which idelalisib has a potent therapeutic benefit , 10. DesIn conclusion, idelalisib significantly reduced the survival of primary ATL cells ex vivo and blocked CCL22-induced phosphorylated AKT. As in CLL, idelalisib monotherapy is unlikely to be sufficient to treat ATL, but these data support the investigation of idelalisib as part of combination therapy or as maintenance therapy for ATL in clinical trials.Additional file 1:Figure S1. Effect of idelalisib on the frozen and thawed samples of ATL patients. Figure S2. The apoptotic ATL cells by treatment with idelalisib. Figure S3. The viability of non-ATL cells by treatment with idelalisib. Figure S4. The apoptotic non-ATL cells by treatment with idelalisib. (DOCX 38 kb)


Over the past decades, our molecular understanding of acute myeloid leukemia (AML) pathogenesis dramatically increased, thanks also to the advent of next-generation sequencing (NGS) technologies. Many of these findings, however, have not yet translated into new prognostic markers or rationales for treatments. We now know that AML is a highly heterogeneous disease characterized by a very low mutational burden. Interestingly, the few mutations identified mainly reside in epigenetic regulators, which shape and define leukemic cell identity. In the light of these discoveries and given the increasing number of drugs targeting epigenetic regulators in clinical development and testing, great interest is emerging for the use of small molecules targeting leukemia epigenome. Together with their effects on leukemia cell-intrinsic properties, such as proliferation and survival, epigenetic drugs may affect the way leukemic cells communicate with the surrounding components of the tumor and immune microenvironment. Here, we review current knowledge on alterations in the AML epigenetic landscape and discuss the promises of epigenetic therapies for AML treatment. Finally, we summarize emerging molecular studies elucidating how epigenetic rewiring in cancer cells may as well exert immune-modulatory functions, boost the immune system, and potentially contribute to better patient outcomes. Acute myeloid leukemia (AML) is an aggressive blood cancer, characterized by the uncontrolled proliferation of poorly differentiated hematopoietic stem and progenitor cells. Even if disease prognosis has improved over the last decades, mainly thanks to decreased treatment-related mortality and wider use of allogeneic hematopoietic cell transplantation  as consolidation therapy, prognostic classification of AML patients remains inaccurate and therapeutic options for high-risk patient are largely unsatisfactory . MoreoveIn spite of being one of the tumors with the lowest mutational load, AML is a highly heterogeneous disease in terms of genetic background and clinical manifestation. Interestingly, although very few, mutations frequently hit genes encoding for proteins that regulate DNA methylation , affect post-translational modifications on histone tails , and drive three-dimensional chromatin conformation of cancer cells (such as CTCF and cohesin complex). In addition to mutations in epigenetic regulators, some of the fusion proteins that are known to drive abnormal transcriptional programs in AML act through aberrant expression or redirected specificity of epigenetic regulators. More broadly, global alterations in DNA methylation patterns and enhancer deregulation were recently linked to AML clonal expansion, further supporting the notion that epigenetic heterogeneity better explains leukemia identity compared to the genetic background . The AMLThe enzymes able to catalyze modifications on DNA or histone proteins are named “epigenetic writers” and can be classified into enzymes impacting on DNA methylation, histone methyltransferases (HMTs), and histone acetyltransferases (HATs). The first category includes all the proteins that directly contribute to the regulation of DNA methylation . The HMT category comprises lysine methyltransferases (KMTs) and arginine methyltransferases (PRMTs). Methylation of histone tails promotes both gene activation and repression depending on the modified histone residue. In detail, methylation of H3K4, H3K36, H3K79, and H4R3 is generally associated to transcriptional activation, while methylation of H3K9, H3K27, and H4K20 induces transcriptional repression. Finally, HATs are epigenetic writers responsible for acetylation of lysine residues on nucleosomes, which is associated to open chromatin and activation of gene expression.de novo methyltransferase, is mutated in 20–25% of AML patients  or the removal (by the indirect action of TET and IDH1/2 proteins) of methyl groups to cytosine or adenine residues. Besides the essential physiological functions, alterations in DNA methylation pattern have been extensively described in different cancer types including AML. Importantly, it has been reported that genetic alleles and epialleles (as assessed by genome-wide DNA methylation profile) can evolve independently from each other during AML progression and that the DNA methylation patterns can be used to stratify patients and predict clinical outcome . Interespatients  and linkpatients . Notablypatients , b. Delepatients .Given the pivotal role of DNA methylation in remodeling AML epigenome at both promoters and distal regulatory elements, DNMTs emerged as attractive therapeutic targets to restore normal DNA methylation patterns in leukemic blasts. Two nucleosidic epigenetic compounds inhibiting DNMT activity, azacytidine (5-azacytidine) and decitabine (5-aza-2′-deoxycytidine) (DNMTi), are currently in clinical use for myeloid malignancies. Azacytidine, upon conversion to decitabine, incorporates into newly synthetized DNA, thwarting the binding of DNMTs. Of notice, azacytidine is predominantly incorporated into RNA with a more evident effect on gene translation . Chemicade novo AML (Another layer of epigenetic regulation of DNA is the oxidation of 5mC (5hmC), which indirectly prevents the addition of methyl groups on cytosine by DNMTs. This modification is catalyzed by the Ten-Eleven-Translocation (TET) enzymes and depends on the action of isocitrate dehydrogenase 1/2 (IDH1/2) proteins, which in turn produce α-ketoglutarate (α-KG) to stimulate TET activity. Somatic mutations in both these classes of enzymes cause aberrant DNA hypermethylation mainly occurring at gene promoters. Specifically, TET2 mutations affect 8–10% of patients with novo AML  and are novo AML . As prevnovo AML . Also, Tnovo AML .Present in around 6 to 13% of AML patients , IDH1/2 in vitro  showed high selectivity for the mutant form of IDH2 sparing the wild-type isoform and have been shown to induce differentiation in AML cells both s of AML . The effs of AML , 2019.in vitro and in vivo  is involved in the methylation of H3K4 residue (H3K4me1/2/3), a transcriptional activation mark. MLL translocations with multiple factors as well as MLL partial tandem duplications (MLL-PTD) are observed with frequencies ranging from 5 to 10% in AML patients, and are associated with poor prognosis . To date in vivo . In the  in vivo . Upon DO in vivo . Based o in vivo . Moreoveo models  and wereo models .SETD2 is a lysine methyltransferase responsible for tri-methylation of the transcriptional elongation mark H3K36 that has been found to be mutated in different tumor types, including hematological malignancies . MutatioFirst discovered in Drosophila, Polycomb group (PcG) proteins are constituents of two chromatin-remodeling complexes (PRC1 and PRC2) that act as transcriptional repressors, regulating key biological processes including cell proliferation, differentiation, and stem cell plasticity . PRC1 coPRC2 is a multi-subunit complex consisting in four core constituents: drosophila enhancer of zeste homolog 2 (EZH2), embryonic ectoderm development (EED), suppressor of zeste 12 homolog (SUZ12), and human retinoblastoma binding protein 4 (RBBP4). Furthermore, accessory molecules favoring PRC2 recruitment and stabilization include JARID2 and YY1. EZH2 is the catalytic subunit of the PRC2 complex and is involved in mono, di, and tri-methylation of H3 lysine 27 (H3K27me1/2/3), leading to chromatin compaction and transcriptional repression of target genes. Acting both as oncogene and tumor suppressor, EZH2 plays contrasting roles in AML pathogenesis . Frequenin vitro and in vivo experiments in MLL-rearranged AML models reverting the repressed PRC2 target gene signatures to an active status  are currently in early stage clinical trials for a variety of hematological malignancies including lymphoma. Recent studies show that PRC2 acts in parallel with MLL rearrangements to sustain leukemia growth . Based oe status . In addie status . While ae status . Other we status .G9a is a lysine methyltransferase belonging to the Su(var)3-9 family, which catalyzes the reaction of mono/di-methylation at H3K9 (H3K9me1/2) triggering gene repression . Even ifArginine residues within histone tails (H3 and H4) can be regulated through methylation by protein arginine methyltransferases (PRMTs). Protein arginine methylation is an abundant post-translational modification, which regulates a plethora of pathways including signal transduction, gene transcription, DNA repair, and mRNA splicing. Crosstalk occurring between PRMT and KMT is necessary to establish appropriate patterns of histone methylation . Among Pin vitro and in vivo   and t . These rearrangements generate the MOZ-CBP and the MOZ-P300 fusion proteins respectively and both have been shown to contribute to leukemogenesis . InhibitEnzymes able to remove epigenetic marks are identified as “epigenetic erasers.” There are two main groups of proteins belonging to this class: histone demethylases (HDMs) and histone deacetylases (HDACs).in vitro  and JMJC domain containing HDMs. Mutations of genes belonging to the latter family are rare in AML and mainly limited to the ones occurring in lysine demethylase 6A (KDMT6A) . The lysin vitro , leadingGiven the importance of histone acetylation in regulating the expression of many genes, it is not surprising that enzymes able to regulate this modification are frequently hit by alterations in different tumor types including hematological malignancies. Thus, HDAC alterations can be linked to both silencing of tumor suppressor genes and activation of oncogenic processes altering the cell cycle progression, activation of the DNA damage response (DDR) pathway, apoptosis, and many others. Although HDAC somatic mutations have been identified so far in several solid tumors with a relatively high frequency , they arIn the last years, several clinical trials have been conducted with HDACi in patients with MDS and AML. The HDACi vorinostat and panobinostat are among the earliest approved by FDA for treatment of cutaneous T cell lymphoma and multiple myeloma, given their capacity to induce cancer cell differentiation. Second-generation HDACi are currently in use in several clinical trials for different tumors, including relapsed AML . In the in vitro studies showed that combinations of panobinostat with other treatments or epigenetic drugs could have synergistically lethal effects on AML cells  is a short-chain fatty acid acting as a powerful HDACi that causes hyperacetylation of the N-terminal tails of H3 and H4 histones by inhibiting the catalytic activity of class I HDACs . VPA hasML cells . HoweverML cells . OverallThe group of proteins able to recognize and bind post-translational modifications are called “epigenetic readers.” These proteins have specialized domains able to recognize a variety of nucleosome modifications acting directly on the transcription or indirectly by serving as scaffold for the recruitment of other epigenetic regulators. The cooperation between these proteins and the chromatin-modifying enzymes is therefore fundamental for gene expression patterns and deregulation of chromatin readers has been frequently reported in cancer.in vitro and in vivo. These inhibitors act by displacing BRD4 from regulatory elements and blocking the RNA Pol II mediated transcriptional elongation at the level of specific oncogenes including c-MYC, BCL2, and CDK6  proteins are a class of chromatin readers that act by binding histone and non-histone acetyl groups at lysine residues . Among tand CDK6 . Similarand CDK6 . Severaland CDK6 . Even ifand CDK6 . Furtherand CDK6 . In addiand CDK6  providinand CDK6  or the Pand CDK6 .The systematic characterization of regulatory regions in normal and AML blasts led to the identification of clusters of transcriptionally active chromatin domains with putative or proven enhancer activity for expression of leukemic genes (defined as “active enhancers” or “super-enhancers”). Enhancer elements present in normal blood cells may accumulate mutations that generate new binding sites for transcription factors and establish new enhancers driving leukemia oncogene expression . SimilarTo date, most of the studies on epigenetic drugs focused on understanding the direct effects of epigenetic therapy on tumor cells. However, interest is now extending to decipher whether epigenome rewiring may as well contribute to cancer cell eradication by triggering changes in the tumor microenvironment and in particular in the interplay between cancer cells and the immune system. In fact, the accumulation of epigenetic alterations during tumorigenesis contributes to profound changes in a plethora of transcriptional signatures including genes regulating antitumor immunity . As descIn addition to CTAs and ERVs, genes involved in antigen presentation, both via HLA class I and class II, have been shown to be under the control of DNA methylation . MoreoveWhereas it might be difficult to leverage on these immune-related effects in patients that are treatment-naive or exposed to a tight schedule of cycles of intensive chemotherapy, boosting an anti-leukemic immune response might be highly desirable in the maintenance setting, especially after allo-HCT. It is in fact well established that most of the therapeutic effect of allo-HCT relies on the transfer from the donor to the patient of a healthy immune system, capable of recognizing and eliminating residual cancer cells. Boosting this “graft-versus-leukemia” effect, possibly without unleashing severe graft-versus-host disease, has indeed over the last decades represented the “holy grail” of transplanters. For instance, the ability of DNMT inhibitors to concomitantly prime T cell-mediated anti-leukemic responses and induce T regulatory cells (Tregs) has attracted considerable interest in the allo-HCT setting . SupportInterestingly, azacytidine, either alone or in combination with donor lymphocyte infusions (DLIs), has shown interesting results also in the more challenging setting of the treatment of AML patients with frank hematological relapse. To date, data on more that 600 patients have been collected, reporting very variable results in terms of clinical outcomes . StartinLike DNMT inhibitors, small molecules targeting HDAC activity are able to induce positive and negative immunomodulatory effects; in fact, they can increase cancer immunogenicity through the expression of HLA class I genes and tumor antigens and/or block T cell response by the upregulation of ligand for checkpoint inhibitor receptors as well as by reducing the number of Tregs .As a result of the evidence that both DNMT and HDACi can contribute to T cell exhaustion, a number of clinical studies are currently testing the combination of these epigenetic drugs with immune checkpoint inhibitors .With these immune-related effects in mind, HDACi alone or in combination with azacytidine and DLI have been tested in a post-transplantation setting. Results on phase I/II trials testing the combinatorial treatment as maintenance therapy after transplantation reported a 2-year rate overall survival and a relapse free survival rate of 81 and 75%, respectively, .Besides their reported effect in suppressing tumor cell proliferation, recently published preclinical studies highlighted the role of EZH2 inhibitors in enhancing antitumor immunity by altering the ratio between effector and regulatory T cells in favor of the first population . Anotherex vivo cultured primary AML (ex vivo and in vivo models will be instrumental to also test epigenetic drugs in combinations with immunotherapy to further enhance their antitumor efficacy. Of note, most epigenetic regulators will have an impact on transcriptional programs fundamental also for normal cell fitness, posing the risk for increased toxicity. Accurate selection of patients that will benefit from a given epigenetic therapy is thus needed to guide a proper choice of epigenetic therapies in AML patients. Additionally, the concomitant presence of distinct leukemia subclones may further challenge the success of epigenetic therapies for AML treatment. The advent of NGS technologies mapping all the different layers of epigenetic regulation at a single-cell level together with transcriptomic and genomic analysis will help to better address these questions in the setting of AML. Altogether, we believe that the application of innovative technologies and more suitable preclinical models that take into consideration the interplay between cancer cells and the immune system will, in the near future, better elucidate biological processes on the basis of tumor response to epigenetic therapy and contribute to move one step forward toward personalized medicine for cancer treatment.AML is a highly heterogeneous cancer type often associated with bad prognosis, with the minority of patients being cured without allo-HCT and for which new therapeutic approaches are urgently needed. Here, we provide a comprehensive overview of studies documenting the effects of epigenetic aberrations in AML pathogenesis and summarized the most recent clinical efforts reporting safety and efficacy of epigenetic compounds for AML treatment. By targeting multiple pathways simultaneously, epigenetic drugs hold great therapeutic promise, although several challenges need to be faced to see in the clinics the full success of such a therapy. One of the most clinically relevant issue still limiting the full implementation of epigenetic therapies in the treatment algorithms for AML patient relates to our incomplete understanding of their interactions with chemo- and immunotherapies. Understanding synergistic and antagonistic effects of epigenetic drugs with current therapeutic paradigms will be fundamental to design new trials incorporating epigenetic therapies based on a specific biological rationale, possibly associated to ancillary studies aimed at determining their effects on leukemic cells and the immune system. For this purpose, it will be necessary to develop new models to test epigenetic therapies, possibly employing actual primary patient samples instead of cell lines and mouse models, that poorly recapitulate the complexity of such a heterogeneous disease. Very recently, the group of Melnick developed a platform to systematically study long-term effects of epigenetic drugs as monotherapy and in combination on a large number of mary AML . Other smary AML . AlthougAll authors contributed to writing the review and discussed its contents.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer IG declared a past co-authorship with one of the authors LV to the handling Editor.


Immediate delivery of a second pulse of the opposite polarity in which the duration and amplitude were the same as the first pulse  or greater than the first pulse  had a stimulatory effect, evoking larger Ca2+ responses than the corresponding unipolar pulse. Progressively decreasing the amplitude of the opposite polarity pulse while also increasing its duration converted stimulation to attenuation, which reached a maximum of 43% when the positive phase was 150 ns at 3.1 kV/cm, and the negative phase was 800 ns at 0.2 kV/cm. When VGCCs were blocked, Ca2+ responses evoked by asymmetrical and even symmetrical bipolar pulses were significantly reduced relative to those evoked by the corresponding unipolar pulse under the same conditions, indicating that attenuation involved mainly the portion of Ca2+ influx attributable to membrane electropermeabilization. Thus, by tuning the shape of the bipolar pulse, Ca2+ entry into chromaffin cells through electropores could be attenuated while preserving Ca2+ influx through VGCCs.Exposing adrenal chromaffin cells to single 150 to 400 ns electric pulses triggers a rise in intracellular Ca In vivo, catecholamine secretion from chromaffin cells occurs when acetylcholine released from splanchnic nerve terminals stimulates nicotinic cholinergic receptors (nAChRs) that are ligand-gated Na+ channels. Na+ influx via nAChRs causes membrane depolarization leading to an elevation in intracellular Ca2+ concentration ([Ca2+]i) orchestrated by Ca2+ entry through several classes of voltage-gated Ca2+ channels 1. This sequence of events culminates in Ca2+-dependent exocytosis3.Neuroendocrine adrenal chromaffin cells are neural crest-derived excitable cells involved in the sympathetic discharge of the catecholamines epinephrine and norepinephrine into the vascular system during the “fight or flight” response. 4. Such short duration electric stimuli, particularly pulse durations that extend into the subnanosecond range, have the potential for remote, targeted delivery to tissues via antennas6, which could eliminate the need for invasive, contact electrodes. Moreover, the biophysical changes caused by nsPEF, one being the reversible formation of nanometer-diameter ion-conducting electropores  in the plasma membrane lipid bilayer11, may reveal novel mechanisms by which cell excitability can be altered. In adrenal chromaffin cells, as an example, we found that a single 5 ns, 5 MV/m pulse can stimulate these cells, producing an increase in [Ca2+]i followed by Ca2+-dependent catecholamine release that is triggered by a mechanism that bypassed nicotinic receptors but still involved Na+ influx, membrane depolarization and activation of VGCCs14. Because tetrotoxin failed to block the response4 ruled out voltage-sensitive Na+ channels as the pathway of Na+ influx and suggested instead that Na+ was most likely entering chromaffin cells via plasma membrane nanopores, causing membrane depolarization4. A similar mechanism of electroporation-induced depolarization may also be involved in the stimulation of rat hippocampal neurons by 200 ns pulses15. Convincing evidence in support of this mechanism of cell excitability in chromaffin cells has been provided by whole-cell patch clamp recordings of nsPEF-evoked inward Na+ currents16, leading us to conclude that nanopores could be subserving a function typically attributed to voltage-gated Na+ channels by causing membrane depolarization to a level that was sufficient to activate VGCCs4. Notably, Ca2+ does not also enter chromaffin cells via this plasma membrane pathway13, indicating that like stimulus-secretion coupling mediated by acetylcholine, activation of VGCCs is solely responsible for Ca2+ influx. Of note also is that nsPEF-evoked cell activation occurs without evidence of the plasma membrane perturbations that promote uptake of non-permeant dyes such as YO-PRO-114, release of Ca2+ from intracellular stores due to electroporation of membranes of Ca2+-storing organelles such the endoplasmic reticulum (ER)13, or adverse effects such as cell blebbing or cell swelling14. In addition to these findings from our group showing the potential for short duration nsPEF to be used for neurostimulation, Casciola et al. reported that 12 ns pulses applied to nerve fibers can elicit action potentials without causing nerve damage. Interestingly, the underlying mechanism of nsPEF-evoked excitability appears to be different in nerve than in chromaffin cells17.As modified post-ganglionic sympathetic neurons that are easy to isolate and maintain in culture, isolated chromaffin cells have played a major role in characterizing mechanisms underlying stimulus-secretion coupling. Accordingly, our group has been using bovine adrenal chromaffin cells as a model system to explore the potential for high intensity, nanosecond pulsed electric fields (nsPEF) to serve as a new modality for neurostimulation2+ responses also without evidence of cell swelling or cell blebbing, or uptake of YO-PRO-118. However, the more pronounced rise in [Ca2+]i evoked by these longer nsPEFs was found to be the result of Ca2+ entering the cells now by two pathways: (1) via VGCCs, which accounted for the bulk of Ca2+ influx (60–70%), and (2) Ca2+ influx through plasma membrane electropores18. Thus, the plasma membrane was no longer impermeable to Ca2+, coinciding with a similar finding of rapid influx of Ca2+ via nanopores in isolated rat hippocampal neurons exposed to 600 ns pulses19.While exploring other nsPEF parameters for stimulating chromaffin cells, a more recent study from our group revealed that exposing chromaffin cells to single longer duration nsPEF (≥150 ns) produced more robust and longer lasting Ca2+ influx through plasma membrane nanopores. In this new nsPEF paradigm, the application of a second pulse of opposite polarity and equivalent duration delivered immediately after the first pulse  could attenuate or even abolish the response produced by the first pulse, a process termed bipolar cancellation. This phenomenon was first described by Pakhomov et al.20 in non-excitable Chinese Hamster Ovary (CHO) cells exposed to single or multiple unipolar or bipolar nsPEF20. In these cells, a 60 ns unipolar pulse triggered a rise in [Ca2+]i that included nsPEF-induced Ca2+ influx from the outside the cell and Ca2+ efflux from the ER, attributed to a short-lived nanoelectroporation of the plasma membrane and ER membrane, respectively. At high stimulus intensities, Ca2+-induced calcium release (CICR) also contributed to the rise in [Ca2+]i20. When exposed to a symmetrical bipolar pulse (60 + 60 ns), CHO cells displayed much smaller Ca2+ transients than cells exposed to a 60 ns unipolar pulse, with each component that contributed to the Ca2+ response attenuated by the bipolar pulse. Bipolar cancellation was also demonstrated for longer duration pulses where a bipolar pulse (300 + 300 ns) was found to be less efficient for evoking increases in [Ca2+]i compared with the corresponding unipolar pulse (300 ns).Recently, a new modality of nsPEF stimulation has been reported that enables fine-tuning of cellular responses that include nsPEF-evoked Caet al.21 evaluated the effects of a single or multiple nanosecond electric field unipolar and bipolar oscillation (referred to as NEFO) on membrane electropermeabilization and changes in membrane conductance in CHO and GH3 cells. Relative to unipolar NEFO, bipolar NEFO yielded lower rates of uptake of YO-PRO-1 and, as determined by whole-cell patch clamp electrophysiology, a reduced membrane conductance21. These results and those of others reporting a similar reduction of electropermeabilization by bipolar pulses were attributed to the ability of these pulses to cause less plasma membrane perturbations24. In light of these results, we considered the possibility that in chromaffin cells exposed to nsPEF ≥ 150 ns, Ca2+ influx due to plasma membrane electroporation might be attenuated/cancelled by applying a bipolar pulse. This could potentially mean eliminating an undesirable effect of longer duration nsPEF while preserving the ability of the cells to be activated in a manner similar to that evoked by a short duration nsPEF. The primary objective of the present study was to evaluate whether bipolar cancellation could be detected in chromaffin cells for nsPEF ≥ 150 ns by exploring the effect of symmetrical bipolar pulses (same duration and peak amplitude for each phase) and asymmetrical bipolar pulses (duration and/or peak amplitude of the negative phase different than the positive phase) on [Ca2+]i. Our results show that depending on the pulse waveform, bipolar pulses could either enhance or attenuate Ca2+ influx evoked by a 150 ns unipolar pulse. Moreover, attenuation of Ca2+ entry by bipolar nsPEFs appeared to involve the portion of Ca2+ influx attributable to membrane electropermeabilization and not Ca2+ influx via VGCCs. Thus, tuning the shape of the bipolar pulse attenuated undesirable Ca2+ entry into chromaffin cells through electropores while maintaining Ca2+ influx through VGCCs. These results have implications for the future development of nsPEF stimulation approaches for modulating cell excitability non-deleteriously since the reduced electroporation efficiency of bipolar compared with unipolar pulses expands the range of nsPEF parameters that can be used for this purpose.In a subsequent report, Gianulis 2+ influx evoked by unipolar nsPEF could be “cancelled” by applying a second pulse of equivalent strength but opposite polarity and identical duration11. To determine whether bipolar cancellation of Ca2+ influx could be similarly achieved in excitable chromaffin cells, a systematic investigation of the impact of unipolar nsPEF on [Ca2+]i was first undertaken followed by an analysis of the effects of the corresponding bipolar nsPEF on [Ca2+]i. Cells were exposed to a single unipolar pulse of 150, 200, 300, 400 or 800 ns duration  and was not associated with adverse effects on cell morphology, such as cell swelling , they unequivocally demonstrate an absolute requirement for a Ca2+ entry pathway to trigger the response.Since VGCC activation is an all-or-none cellular process, symmetrical bipolar pulses were most likely enhancing Ca ns Fig. , ↑200 +  ns Fig.  or ↑400  ns Fig.  bipolar 2+ responses evoked by bipolar pulses, like single unipolar pulses18, increased in amplitude with pulse duration. For example, Ca2+ responses evoked by ↑200 + ↓200 ns or ↑400 + ↓400 ns bipolar pulses were 55% or 33% larger, respectively, than those produced by ↑150 + ↓150 ns bipolar pulses . Thus, the negative pulse phase acted as a 2nd stimulus regardless of pulse duration.The results of the experiments shown in Fig. ses Fig.  with the2+ influx induced by a bipolar pulse of ↑150 + ↓150 ns to a single unipolar pulse of 300 ns, which represents an equivalent amount of energy, the response elicited by the bipolar pulse was significantly smaller than that evoked by the unipolar pulse . Thus, even though the negative phase of the ↑150 + ↓150 bipolar pulse acted as a 2nd stimulus, Ca2+ influx was not comparable to that elicited by a single polarity 300 ns pulse. A possible explanation is that the positive phase of the bipolar pulse limited the extent to which the plasma membrane could be electropermeabilized by the negative phase of the pulse25. We also compared Ca2+ influx induced by a single bipolar pulse of ↑200 + ↓200 or ↑400 + ↓400 ns to a single unipolar pulse of 400 or 800 ns, which represents an equivalent amount of energy for each bipolar pulse, respectively. We found that the response triggered by a ↑200 + ↓200 ns vs a 400 ns pulse, or a ↑400 + ↓400 ns vs a 800 ns pulse was similar. Given the longer duration of these pulses relative to a ↑150 + ↓150 bipolar pulse or a unipolar 300 ns pulse, the Ca2+ indicator may have been saturated in each case, not allowing differences in [Ca2+]i to be detected among the groups.When we compared Ca2+ influx was not attenuated by symmetrical bipolar pulses. Instead, Ca2+ influx was enhanced. The next series of experiments investigated the effect of altering the shape of the bipolar pulse on Ca2+ influx by manipulating the parameters of the negative pulse phase.Taken as a whole, these results demonstrate that Ca2+ influx stimulated by single unipolar pulses increased in magnitude with pulse duration. Thus, increasing the duration of the negative phase of a bipolar pulse would presumably result in more electropermeabilization and hence more Ca2+ influx, resulting in even greater increases in [Ca2+]i when compared to that evoked by a symmetrical ↑150 + ↓150 bipolar pulse. To examine this possibility, the positive pulse phase was kept constant at 150 ns and the duration of the negative pulse phase was varied from 200 up to 800 ns, both phases set at an E-field of 3.1 kV/cm. The specific asymmetrical bipolar pulses used in the analysis were ↑150 + ↓200, ↑150 + ↓400 or ↑150 + ↓800 ns . These results are consistent with the demonstration in CHO cells that a ↑300 + ↓900 bipolar pulse caused more YO-PRO-1 uptake than a 300 ns unipolar pulse22. Table 2+ responses evoked by each asymmetrical bipolar pulse were also greater than those evoked by a symmetrical ↑150 + ↓150 bipolar pulse. Post hoc analysis revealed that Ca2+ responses elicited by ↑150 + ↓200 bipolar pulses were not significantly different from those produced by 150 ns unipolar pulses (p > 0.05) whereas responses to bipolar pulses of ↑150 + ↓400 and ↑150 + ↓800 were significantly different from those evoked by 150 ns unipolar pulses . Although the magnitude of the rise in [Ca2+]i tended to increase in a linear fashion with the duration of the negative phase  and ↑150 + ↓1000 ns at an E-field ≤ 0.4 kV/cm . Figure 2+ response elicited by the 150 ns unipolar pulse, not only was the magnitude of the rise in [Ca2+]i decreased but also [Ca2+]i returned to pre-stimulus levels during the 50-s post-pulse monitoring period. Indeed, the Ca2+ response triggered by these bipolar pulse waveforms now more closely resembled that evoked by the physiologic stimulus, activation of nicotinic cholinergic receptors26. Due to the technical limitations of our bipolar pulse exposure system, we could not determine if maximal cancellation of Ca2+ influx was achieved since this would require negative pulse phases of even lower E-field amplitude and/or longer duration, which are not possible with our setup.Consistent with the experiments described in the previous section, increasing the duration of the negative pulse phase led to enhanced stimulation, especially at higher E-fields  unipolar pulses is passing through VGCCs18, it is possible that bipolar pulses are unable or are less efficient at cancelling the Ca2+ influx component involving VGCCs, or that the Ca2+ that enters the cells via VGCCs affects how the cell responds to a bipolar pulse.As indicated previously, strong bipolar cancellation of Ca2+ influx by symmetrical bipolar pulses, as shown for cells that do not express VGCCs. For this determination, chromaffin cells were pre-treated with a combination (cocktail) of Ca2+ channel blockers and exposed to a ↑150 + ↓150 ns bipolar pulse. The cocktail of Ca2+ channel blockers comprised 100 nM ω-agatoxin GVIA (P/Q-type) + 20 nM ω-conotoxin (N-type) + 20 µM nitrendipine (L-type), which we previously determined was effective for blocking Ca2+ influx mediated by VGCCs in cells exposed to the pulse durations used in this study18. In other experiments, cells were pre-treated with dihydropyridines alone at high concentrations , which we previously also determined caused substantial inhibition of Ca2+ influx triggered by ≥150 ns pulses18. High concentrations of dihydropyridines ensured that both CaV1.2 and CaV1.3 isoforms of L-type channels, which are both expressed in bovine chromaffin cells27, were effectively inhibited since CaV1.3 channels are less sensitive to dihydropyridines than CaV1.228. That this treatment significantly inhibited Ca2+ influx10 most likely meant that the other types of VGCCs present in these cells were partially blocked29.To explore these possibilities, we determined whether conducting experiments under conditions that would eliminate the contribution of VGCCs to nsPEF-evoked responses would show cancellation of Ca2+]i that was larger by around 18% than that elicited by the corresponding 150 ns unipolar pulse. When cells were treated with the cocktail of VGCC blockers, Ca2+ influx elicited by a 150 ns pulse was inhibited by around 66%  reached statistical significance . These results are evidence that bipolar pulses were cancelling the portion of Ca2+ influx attributable to Ca2+ permeation mediated by electronanoporation and not Ca2+ influx via VGCCs. They also beg the question of why Ca2+ influx due to electronanoporation was now achievable when Ca2+ influx via VGCCs was suppressed. The answer to this question awaits future investigation30.Similar to the results shown in Fig. 66% Fig. , which i2+ influx was not achievable with the range of asymmetrical bipolar pulse waveforms available with our current exposure setup. It could also be because any depolarizing stimulus that causes Ca2+ entry via VGCCs in chromaffin cells, such as nicotinic receptor stimulation as well as nsPEF, may also trigger some CICR due to activation of ryanodine receptors33. Thus, blocking Ca2+ influx via VGCCs would also prevent CICR, causing an apparent enhanced cancellation effect relative to cells exposed to asymmetric bipolar pulses with VGCC responses intact. We did not test for the presence of CICR since it is a response that is secondary to Ca2+ influx via VGCCs and thus outside the scope of the present study, which was mainly focused on determining pulse parameters and conditions that would allow us to detect bipolar cancellation of Ca2+ influx.The levels of cancellation achieved by suppressing VGCCs were slightly greater than those achieved by asymmetrical bipolar pulses producing significant and optimal cancellation Table . This di2+ influx by symmetrical bipolar pulses was observed only when VGCCs were blocked, we explored how Ca2+ influx via VGCCs impacted the level of cancellation produced by an asymmetrical bipolar pulse having a long duration (1000 ns), low E-field amplitude (0.4 kV/cm) negative pulse phase. For these experiments, cells were exposed to a 150 ns unipolar pulse or to a ↑150 + ↓1000 ns bipolar pulse in the presence and absence of the VGCC blocker cocktail. As shown in Fig. 2+ influx by the asymmetrical bipolar pulse was significantly greater when VGCCs were blocked . These results again demonstrate that in chromaffin cells, bipolar pulses appear to be cancelling the portion of Ca2+ influx attributable to Ca2+ permeation mediated by electronanoporation. They also provide additional evidence that Ca2+ influx via VGCCs may impact cancellation efficiency.Since cancellation of Caduration 00 ns, loduration 000 ns, l2+ responses in excitable cells. Using neuroendocrine chromaffin cells isolated from the adrenal medulla as a cell model, we had previously reported10 and show here also that exposing these cells to unipolar pulses that are ≥150 ns in duration elicit an increase in [Ca2+]i that is mediated by Ca2+ influx via both VGCCs and putative plasma membrane nanopores, i.e. electropores. We now show that depending on the shape of the pulse waveform, exposing chromaffin cells instead to a bipolar pulse can partially cancel the portion of Ca2+ influx attributed to electropores, thereby attenuating an undesirable pathway of Ca2+ entry into the cells while leaving all-or-none VGCC activation intact. This finding, together with reports from other groups showing that bipolar pulses can cause less cell death of CHO, U937 and Jurkat cells than unipolar pulses24, provides incentive for continuing studies that explore the range of nsPEF parameters that can be used to drive neuromodulation safely and potentially non-invasively in the future.This study provides new insight into the potential application of bipolar pulses for modulating nsPEF-evoked Ca2+ influx, where the second phase of the bipolar pulse acts like a 2nd stimulus, most likely reflects an increase in the number of electropores. However, mechanisms underlying bipolar cancellation are not yet well defined and several have been proposed to explain the phenomenon. They include: (1) assisted membrane discharge20; (2) electropermeabilization of membranes as a two-step chemical process20; (3) reverse electrophoretic ion transfer34; (4) localized membrane charging and discharging22 and (5) nanopore occlusion35. Adding to the complexity of the bipolar cancellation phenomenon in chromaffin cells is the demonstration that for symmetrical bipolar pulses, Ca2+ influx via VGCCs can determine whether there is cancellation or stimulation. Future experiments will be carried out to test these various hypotheses.Regarding mechanisms, bipolar stimulation of Cain vivo.In conclusion, the demonstration in this study that the shape of bipolar nsPEFs could be altered in manner to allow for titrating the response of chromaffin cells may pave the way in the future for remote and non-invasive control of these and other types of excitable cells 2 as previously described36. Cells were used up until 2 weeks in culture. For nsPEF exposure, the large aggregates of cells that form in suspension culture were dissociated into single isolated cells with the protease dispase37 and plated onto fibronectin-coated 35 mm glass bottom dishes. Once attached, cells retained their spherical morphology and were used for a period not exceeding two days after attachment.Fresh bovine adrenal glands were obtained from a local slaughterhouse . After the outer adrenal cortex was removed by dissection, chromaffin cells were isolated by collagenase digestion of the adrenal medulla and maintained in suspension culture in Ham’s F-12 medium supplemented with 10% bovine calf serum, 100 U/ml penicillin, 100 µg/ml streptomycin, 0.25 µg/ml fungizone, and 6 µg/ml cytosine arabinoside at 36.5 °C under a humidified atmosphere of 5% CO2+]i were monitored in cells labeled with the Ca2+-sensitive fluorescent indicator, Calcium Green-1(Ex480 nm and Em535 nm) as previously described18. For dye loading, the cells were incubated with 1 µM Calcium Green-1 for 45 min at 37 °C in a balanced salt solution (BSS) containing 0.1% bovine serum albumin (BSA) and (in mM): 145 NaCl, 5 KCl, 1.2 NaH2PO4, 2 CaCl2, 1.3 MgCl2, 10 glucose, and 15 HEPES, pH 7.4. For fluorescence monitoring, cells were washed twice with dye-free BSS lacking BSA and placed on the stage of a Nikon TE2000 epifluorescence microscope equipped with a 100X objective. For Ca2+ free experiments cells were bathed in Ca2+-free BSS Ca2+ containing 1 mM EGTA. Fluorescence images of the cells were captured before, during and after application of a stimulus by an iXonEM + DU-897 EMCCD camera  using the open source microscopy software Micro-Manager . The exposure time of the camera was set to 100 ms and images were captured at a rate of 7.5 Hz. Bright field images were obtained at the start and end of an experiment, and all experiments were carried out at ambient room temperature.Changes in [Ca18 and complete fabrication details are given in Ryan et al.38. The unit has two independent circuits that deliver positive and negative pulses ranging from 150 to 1000 ns, with the ability to adjust the pulse widths and amplitudes of the positive and negative pulses separately. For cell exposure, single unipolar and bipolar pulses were delivered to an attached cell via a pair of cylindrical tungsten rod electrodes (127 µm diameter) with their tips spaced 100 µm apart. The cell to be investigated was positioned at the center of the gap between the electrode tips, which were positioned 40 µm above the bottom of the dish using a motorized micromanipulator . A LabVIEW program (version 8.01) was used to control the delivery of pulses. Each cell was exposed to a single unipolar or bipolar pulse only once. As reported in Bagalkot et al.18, the E-field distribution in the vicinity and at the location of the target cell was computed using the commercially available Finite-Difference Time-Domain (FDTD) software package SEMCAD X . The simulations showed that the E-field at the location of the cell was essentially uniform and that minor differences in electrode placement among cells would have negligible impact on the E-field amplitude to which a cell was exposed18.The custom-designed high-voltage biphasic nanosecond pulse generator used to expose chromaffin cells to nsPEF has been previously describedhttps://imagej.nih.gov/ij/). Changes in cell fluorescence intensity (ΔF) were calculated by subtracting background fluorescence from the fluorescence of the cell (ΔF = Fcell − Fbackground). The values were then normalized to the fluorescence intensity value at the time when the pulse was applied (F0). All experiments were replicated using cells from different cell preparations and different days in culture. Results were expressed as the mean ± standard error of the mean (s.e.m.). Statistical analyses, which were performed using SigmaPlot 13 software , consisted of an unpaired two-tailed t-test when the means of two groups were compared, or a one-way ANOVA test followed by Tukey post hoc multiple range tests for multiple group comparisons. p < 0.05 was considered statistically significant.Fluorescence images for each cell were continuously recorded during a period spanning 10 s prior to nsPEF exposure to obtain baseline, and then for a period spanning 50 s after nsPEF exposure. Images were analyzed using the public domain image processing software ImageJ (


The receptor tyrosine kinase family of fibroblast growth factor receptors (FGFRs) play crucial roles in embryonic development, metabolism, tissue homeostasis and wound repair via stimulation of intracellular signalling cascades. As a consequence of FGFRs’ influence on cell growth, proliferation and differentiation, FGFR signalling is frequently dysregulated in a host of human cancers, variously by means of overexpression, somatic point mutations and gene fusion events. Dysregulation of FGFRs is also the underlying cause of many developmental dysplasias such as hypochondroplasia and achondroplasia. Accordingly, FGFRs are attractive pharmaceutical targets, and multiple clinical trials are in progress for the treatment of various FGFR aberrations. To effectively target dysregulated receptors, a structural and mechanistic understanding of FGFR activation and regulation is required. Here, we review some of the key research findings from the last couple of decades and summarise the strategies being explored for therapeutic intervention. Ty. Ty50 vain vivo ATP concentrations [To address the toxicity issues of multi-kinase inhibitors, efforts have been made to develop FGFR-selective kinase inhibitors, yielding numerous reversible type I inhibitor compounds with FGFR1–3 and pan-FGFR activities . Of thestrations . Furthertrations –115. Contrations . IntriguFGFR-targeted therapies are not limited to the tyrosine kinase domain only; there have been multiple efforts to target the extracellular domains of FGFRs also, offering further opportunities for isoform-selective inhibition . This isWhile improved selectivity of therapies may be beneficial to target specific FGFR aberrations, it is important to recognise that clinical efficacy and selectivity are not necessarily related. Highly selective FGFR-targeted therapies may also be more prone to resistance development if not used in combination strategies. For example, in addition to the gain of gatekeeper mutations detailed above, resistance to FGFR inhibitors has also been acquired in cell lines harbouring FGFR3 amplification by switching to ErbB family signalling . MoreoveSince their first description, it has been established that FGFRs play crucial roles in a host of physiological processes which when dysregulated result in a plethora of pathologies. From an extensive range of studies covering FGFR expression, structure and function, among others, mechanisms of FGFR regulation and activation have come to light, and good progress has been made in the development of anti-FGFR therapies. Despite this, due to the complexity of FGFR signalling inputs, outputs and FGFR interactomes, and difficulties faced with the biochemical and biophysical characterisation of full-length receptors, we are still far from an integrated understanding of FGFR biology. Crucially, mechanisms of activation in the context of the full-length receptor are unclear and will remain unresolved until structures of full-length FGFRs in autoinhibited, ligand-activated and pathogenically activated modes are solved. In fact, to date there are no high-resolution full-length structures of any receptor tyrosine kinase, severely limiting our understanding of this highly important class of kinases. Equally, while it is recognised that activation of FGFRs can lead to differential activation of intracellular signalling cascades, the underlying molecular basis of how this occurs and of how cellular context influences phenotypic outcome remain poorly understood. We anticipate that advances will be made in addressing these and other remaining questions in the coming decades, and with this, new and improved strategies for treatment of disorders arising due to aberrant FGFR signalling will develop.


There is great discussion about non-confluent pulmonary artery (PA) reconstruction, and several materials have been used. Autologous pericardium is considered feasible for infectious resistance, autoimmune response, extensibility, and growth potential.The patient was born at 39 weeks (body mass = 2550 g). He was diagnosed with tetralogy of Fallot, pulmonary atresia, non-confluent PA, and bilateral patent ductus arteriosus. Right and left Blalock-Taussig shunts with patent ductus arteriosus ligations were placed on day 27 and 3 months, respectively. At 19 months (8.8 kg), definitive repair was performed with tricuspid valved conduit concurrent with PA reconstruction using an autologous pericardium roll conduit. The autologous pericardium was treated with glutaraldehyde . Following an incision on the visceral side of the PAs before the 1st branch, the autologous pericardial roll conduit was anastomosed. Follow-up angiographies on postoperative months 9 and 57 demonstrated that the PA, including the autologous pericardium roll conduit, had spontaneously enlarged.Particularly for non-confluent PA, the patients require increased pulmonary beds at an early age because of hypoplastic PA. While size mismatch between the graft and native PA develops as the child grows, size-adjustable extensibility of the PA graft should be noted.The online version of this article (10.1186/s13019-019-0913-z) contains supplementary material, which is available to authorized users. There is extensive discussion regarding non-confluent pulmonary artery (PA) reconstruction, and several materials have been used , 2. AutoThe patient  was diagnosed with tetralogy of Fallot, pulmonary atresia, non-confluent PA, and bilateral patent ductus arteriosus. Despite intubation on day 19, desaturation progressed even with prostaglandin administration. Right and left Blalock-Taussig shunts with patent ductus arteriosus were ligated on day 27 and 3 months, respectively. The interrupted pulmonary arterial portion was so long (20 mm) that unifocalization of bilateral PAs was impossible.At age 19 months (8.8 kg), definitive repair was performed with tricuspid valved conduit concurrent with PA reconstruction using an autologous pericardium roll conduit. Cardiopulmonary bypass was achieved, and the previous bilateral Blalock-Taussig shunts were removed. After inducing cardiac arrest, ventricular septal defect was closed with a polytetrafluoroethylene patch. Thereafter, the autologous pericardium was treated with glutaraldehyde . Following an incision on the visceral side of the PAs before the 1st branch, the autologous pericardial roll conduit was anastomosed. Finally, the tricuspid valved conduit was anastomosed between the right ventricular outlet and pericardial conduit Autologous pericardium is frequently used in pediatric cardiac surgery. Compared to other materials such as artificial graft  and xenoAutologous pericardium is often treated with glutaraldehyde. Glutaraldehyde treatment is accessible to technical handling and reinforces the pericardium strength while maintaining its configuration. Non-ringed artificial grafts or xenografts may be compressed by other structures. Lee et al. demonstrated that the most feasible glutaraldehyde treatment method was that with 0.5–0.6% glutaraldehyde for 20 min , and indParticularly for non-confluent PA, the patient requires increased pulmonary beds at an early age because of hypoplastic PA. While size mismatch between the graft and native PA develops as the child grows, size-adjustable extensibility of the PA graft should be noted. Long-term outcomes are unclear; therefore, careful observation is needed in the future.


This study reports the intracellular Ca2+ increase that participates in the activation of ERK1/2 and axogenesis induced by E2. Hypothalamic neuron cultures were established from 16-day-old male rat embryos and fed with astroglia-conditioned media for 48 h. E2-induced ERK phosphorylation was completely abolished by a ryanodine receptor (RyR) inhibitor (ryanodine) and partially attenuated by an L-type voltage-gated Ca2+ channel (L-VGCC) blocker (nifedipine), an inositol-1,4,5-trisphosphate receptor (IP3R) inhibitor (2-APB), and a phospholipase C (PLC) inhibitor (U-73122). We also conducted Ca2+ imaging recording using primary cultured neurons. The results show that E2 rapidly induces an increase in cytosolic Ca2+, which often occurs in repetitive Ca2+ oscillations. This response was not observed in the absence of extracellular Ca2+ or with inhibitory ryanodine and was markedly reduced by nifedipine. E2-induced axonal growth was completely inhibited by ryanodine. In summary, the results suggest that Ca2+ mobilization from extracellular space as well as from the endoplasmic reticulum is necessary for E2-induced ERK1/2 activation and axogenesis. Understanding the mechanisms of brain estrogenic actions might contribute to develop novel estrogen-based therapies for neurodegenerative diseases.17β-estradiol (E2) induces axonal growth through extracellular signal-regulated kinase 1 and 2 (ERK1/2)-MAPK cascade in hypothalamic neurons of male rat embryos  For many years, estrogens have been recognized as one of the main orchestrators of the sexual differentiation of the brain, acting during critical periods of development to organize neural circuits in a way that determines the modulatory/activational effects of gonadal hormones in adulthood. Testosterone secreted by male rodent testes during development is aromatized in neurons to 17β-estradiol (E2), which displays multiple cellular processes that finally set the masculine phenotype  that function as ligand-activated transcription factors to regulate the expression of estrogen-responsive genes. Additionally, estrogens generate a wide diversity of rapid “non-classical” effects, which occur in a range from some seconds to a few minutes via membrane-initiated mechanisms  and diacylglycerol  and promoted Ca2+ release through ryanodine receptors (RyRs). This early Ca2+ response underlies E2-induced ERK1/2 activation and axogenesis in hypothalamic neurons. Altogether, these results bring new insights about the mechanism of brain estrogenic actions and might contribute to developing novel estrogen-based therapies for neurodegenerative diseases.In this study, we found that E2 evoked activation of Ca2 anesthesia, and the fetuses were dissected from the uterus. The male fetuses used for cultures were identified by visualization of the spermatic artery on the developing testes. Ventromedial hypothalamic and mesencephalic regions were dissected out and stripped off the meninges for primary neuronal and glial cultures, respectively. At E16, the axogenic effect of E2 is contingent on the presence of astroglia  for morphological studies.Embryos were obtained from pregnant Wistar rats at embryonic day 16 (E16). The day of vaginal plug was set as E0. Experimental procedures for handling and sacrificing animals were approved by the Animal Care and Use Committee at our institution  and followed the NIH guidelines for care and use of laboratory animals. The minimum number of animals required was used for these experiments and suffering was minimized. Primary neuronal and astroglial cultures were prepared as previously described in Cambiasso et al. . Pregnanin vitro (DIV). After a 2 h washout period using BM, neuronal cultures were treated for 1 h with nifedipine , inhibitory ryanodine , 2-APB  or U-73122 , and then pulsed with 100 nM E2  for 15 min. Hormone concentration used was determined by dose dependence (1–100 nM) experiments previously performed by our group . Protein samples (20 μg/lane) were separated by 10% SDS-PAGE and transferred onto polyvinylidene fluoride membrane . ERK1/2 phosphorylation was detected by using a rabbit monoclonal anti-phospho-p44/42 MAPK , which specifically detects both Thr202 and Tyr204 ERK phosphorylation forms of ERK . Total ERK1/2 was detected by using a mouse anti-p44/42 MAPK . Secondary antibodies conjugated to horseradish peroxidase  were used for the detection by enhanced chemiluminescence on X-ray film. After incubation with the antibody against phospho-ERK1/2, blots were stripped and then re-probed with anti-total ERK1/2 to ensure equal protein loading. The resulting film samples were scanned and analyzed with an image analysis program . Data are presented as a ratio of phospho-ERK1/2/total-ERK1/2 of 3–4 different experiments (independent cultures) performed in duplicate.After treatment, hypothalamic neurons were washed and harvested at 4°C in RIPA buffer  with protease and phosphatase inhibitors  for 30 min at 37°C in a Ca2+-containing HEPES buffered salt solution (Ca2+-HBSS) composed of (mM): 135 NaCl, 5.4 KCl, 2 CaCl2, 1 MgCl2, 10 HEPES, and 10 glucose; pH = 7.4 set with NaOH at RT. Following loading, neurons were washed twice with warm Ca2+-HBSS and were imaged in the same buffer. To analyze the participation of extracellular Ca2+, the cells were resuspended in an EGTA-containing buffer composed of (mM): 135 NaCl, 5 KCl, 0.5 CaCl2, 1.2 MgCl2, 5 HEPES, 14 NaHCO3, and 1 EGTA; pH = 7.4 set with NaOH at RT.After 2 DIV, hypothalamic neurons were treated (or not) with 50 μM ryanodine or 2 μM nifedipine for 1 h and incubated with 3 μM acetoxymethyl (AM) ester form of the organic Ca2+ signals was performed using a 60× oil immersion objective of an Olympus IX81 inverted microscope , an ORCA AG (Hamamatsu) CCD camera and OSIS software. Frames were collected at a continuous rate of 2.5 per second during 5 min (790 frames). Cal-520 was excited at a wavelength of 492 nm, and emitted fluorescence was collected at 514 nm. E2 (100 nM) was added 30 s after starting the recording. 10 μM thapsigargin (tg) was added at 3 min of recording as a positive control of normal endoplasmic reticulum Ca2+ content. The fluorescence intensity of the Ca2+ indicator was analyzed using ImageJ  software and plotted as the change in fluorescence (ΔF) of 2 × 2 pixels divided by mean resting fluorescence  over time. We measured both the peak fluorescence value and the integrated area under the ΔF/Fo curve with OriginPro 8 SR0 software . The integrated area roughly corresponds to the total amount of Ca2+ released over the recording period.Imaging of cytosolic CaTo analyze the effect of ryanodine in E2-stimulated axon growth without affecting the normal polarization of neurons, we performed the experiment after 1 DIV (stage III of development). After 2 h in absence of E2, the cultures were treated for 1 h with 50 μM ryanodine before the addition of 10 nM E2 for an additional 24 h. The hormone concentration used to study the neuritogenic effect of E2 was chosen based on previous studies of our laboratory . The details of the immunocytochemical procedure were as specified by Díaz et al. . BrieflyThe morphometric analysis of stained neuronal cultures was performed on digitized video images using JAVA as an image processor  controlled by a host computer. Images were acquired through an optic microscope . Microscope slides were coded, and the person conducting the analysis was blind to the experimental group. All labeled cells that could be identified as one individual neuron were measured in random fields at 40× magnification. Neural processes were classified as minor processes or axons according to accepted morphological criteria  where p < 0.05 was considered statistically significant.Data were statistically evaluated by one-way ANOVA, followed by Fisher’s Least Significant Difference (LSD) 2+-dependent. Here, we further investigated the Ca2+ response involved in ERK activation mediated by E2. Hypothalamic cultures grown with E2 for 48 h were washed in BM for 2 h and pre-treated with specific compounds for 1 h before a pulse of E2 for 15 min. In agreement with previous reports, E2 induced a strong phosphorylation of ERK at 15 min . These Ca2+ events often occur in repetitive oscillations, which display interspike intervals of 21.58 ± 8.83 s (9 out of 17 neurons). The amplitude in terms of ΔF/Fo was 0.154 ± 0.01. The total amount of Ca2+ mobilized, measured as the integrated area under the curve (AUC) for all E2-generated peaks, was 4.69 ± 1.03, which was 90.6% with respect to the control .As E2-activation of the ERK1/2 signaling cascade depends on extracellular as well as intracellular Carocesses . Sevente2+ signal was not observed in the absence of extracellular Ca2+. Moreover, we found that pre-incubation of neuronal cultures with inhibitory ryanodine suppressed E2-evoked Ca2+ release  and 35.05 ± 11.54 (ryanodine pre-incubation), five and nine times greater, respectively, than the amounts of Ca2+ mobilized by tg in E2-induced control neurons , which strongly suggests the participation of L-VGCCs in this signal  or without E2 (control). In agreement with previous results, the morphometric analysis indicated that neurons grown under hormonal treatment show longer axons than neurons in control conditions without E2  mechanism to evoke the larger Ca2+ signaling needed to maintain changes during long-term memory storage  mediated by the hormone (Dolmetsch et al., Our results show that E2-induced cytosolic Ca2+-dependent manner. RyRs inhibition abolished this activation as well as axonal growth. The oscillatory Ca2+ signal generated by E2 required functional RyRs and L-VGCCs. This early Ca2+ response that underlies E2-induced RyRs and MAPK/ERK activation may transmit a finely tuned message into a neuronal development program, reflecting the need for tight control of a critical event during sexual differentiation of the male brain. The conjunction of XY genotype with adequate estrogen exposure levels at the time of hypothalamic neuronal differentiation may induce the growth of axons towards their appropriate targets. A complete and detailed understanding of the intracellular signaling mechanisms and neuronal processes mediated by estrogens will allow to improve current estrogen-based therapies, such as hormone replacement therapy in postmenopausal women, as well as to develop novel treatments to prevent and/or alleviate neurological pathologies based on its widely proven neuritogenic and neuroprotective effects.In conclusion, we have provided new insights into the non-classical mechanisms triggered by estrogens and its axogenic effect in male rat hypothalamic neurons. The hormone induces ERK1/2 activation in a CaLCZ, MB and MC made the conception and design of research, interpreted results of experiments, wrote the manuscript. LCZ performed all experiments and analyzed the data. LCZ and MB prepared the figures. MB and MC edited and revised the manuscript. MC drafted the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.