We used mice with cardiac‐specific Sirt1 knockout (Sirt1−/−). Sirt1flox/flox mice were served as control. Sirt1−/− mice showed impaired cardiac ejection fraction with increased ventricular spontaneous activity and burst firing compared with those in control mice. The arrhythmic events were suppressed by KN93 and ranolazine. Reduction in Ca2+ transient amplitudes and sarcoplasmic reticulum (SR) Ca2+ stores, and increased SR Ca2+ leak were shown in the Sirt1−/− mice. Electrophysiological measurements were performed using patch‐clamp method. While L‐type Ca2+ current  was smaller in Sirt1−/− myocytes, reverse‐mode Na+/Ca2+ exchanger (NCX) current was larger compared with those in control myocytes. Late Na+ current  was enhanced in the Sirt1−/− mice, alongside with elevated cytosolic Na+ level. Increased cytosolic and mitochondrial reactive oxygen species (ROS) were shown in Sirt1−/− mice. Sirt1−/− cardiomyocytes showed down‐regulation of L‐type Ca2+ channel α1c subunit (Cav1.2) and sarcoplasmic/endoplasmic reticulum Ca2+ ATPase 2a (SERCA2a), but up‐regulation of Ca2+/calmodulin‐dependent protein kinase II and NCX. In conclusions, these findings suggest that deficiency of Sirt1 impairs the regulation of intracellular Ca2+ and Na+ in cardiomyocytes, thereby provoking cardiac dysfunction and arrhythmogenesis.This study addressed the hypothesis that cardiac Sirtuin 1 (Sirt1) deficiency alters cardiomyocyte Ca It has recently been reported that Sirt1‐deficient mice exhibit abnormalities in cardiac conduction and arrhythmia‐induced premature death that leads to the hyperacetylation of Na2+ and Na+ regulation, resulting in cardiac dysfunction and predisposition to arrhythmia. Here, we used cardiac‐specific Sirt1 knockout mice to identify the Ca2+ and Na+ regulatory mechanisms affected. We assessed a variety of indices that mandate cardiac function and ionic regulation to establish a clear picture of pathological cellular Ca2+ and Na+ homeostasis in the heart with Sirt1 deficiency. Our results suggest deficiency of cardiac Sirt1 promote dysregulation of Ca2+ and Na+, leading to contractile dysfunction and providing proarrhythmic substrates.The aim of the present study was to assess whether Sirt1 deficiency in the heart of mice alters intracellular Ca22.1Animal experiments were all conducted with the approval of the Institutional Animal Care and Use Committee (IACUC 18‐056) of the National Defense Medical Center, Taipei, Taiwan and in accordance with the National Institutes of Health guidelines, ‘Guide for the Care and Use of Laboratory Animals’, on the operation of experimental animals.Sirt1 exon 4 knockout (Sirt1−/−) were created by crossing Sirt1flox/flox mice (Sirt1flox/flox was the control mice that were purchased from Jackson Laboratory) with α‐MHC (myosin heavy chain) promoter‐driven Cre mice with C57BL/6J background  and are currently in use in the laboratory.flox/flox (control) and Sirt1−/− 40‐week‐old mice were killed, and the hearts were procured for subsequent experiments. Animals were kept at temperature of 21 ± 1°C under controlled 12:12 h light‐dark lighting cycle with ad libitum access to standard chow  and deionized drinking water before use.Mice with Cardiac‐specific 2.23 (D = linear LV diameter). LV ejection fraction (EF) was calculated as following equation: EF = (LV end‐diastolic volume ‐ LV end‐systolic volume)/LV end‐diastolic volume and expressed in %. Internal diameter of LVs at systolic (LVIDs) and diastolic (LVIDd) phase were recorded for calculating fractional shortening (FS) as following equation: FS = (LVIDd ‐ LVIDs)/LVIDd. The average was calculated from measurements taken from three consecutive cardiac cycles.A Mindray M9 ultrasound machine  equipped with a 12MHz probe was used to measure the cardiac functional changes in the experimental mice. Mice were subjected to echocardiography under anaesthesia with ketamine  and xylazine  during echocardiography. In short‐axis view, M‐mode traces were obtained to measure left ventricle (LV) wall thickness and chamber dimensions at diastole and systole and echocardiography‐calculated LV mass. The Teichholz formula was used to calculate LV volumes: 7/ (2.4 + D) × D2.33, 0.5 NaH2PO4, 0.5 MgCl2, 2.7 CaCl2 and 11 dextrose at a constant rate (3 ml/min), saturated with a 97% O2 ‐ 3% CO2 gas mixture. The bath temperature was maintained at 37°C. Before the electrophysiological assessments, the preparations were allowed to equilibrate in the bath for 1 h.Mice were anesthetized by intraperitoneal injections of Zoletil 50 (5 mg/kg) and xylazine (5 mg/kg) with isoflurane inhalation (5% in oxygen) in a vaporizer. The hearts were harvested from the mice by performing a midline thoracotomy as described previously.20, APD50 and APD90. Spontaneous electrical activity and arrhythmia, including burst firing, delayed after depolarizations (DADs), and ventricular tachycardia were recorded and analysed. Ventricle preparations were perfused with KN93, a calmodulin‐dependent protein kinase II (CaMKII) inhibitor, (1 μmol/L) or ranolazine,+ current  inhibitor, (10 μmol/L) at a constant rate to determine pharmacological responses.Transmembrane action potentials (APs) were recorded using 3M KCl‐filled glass microelectrodes connected to a WPI Duo 773 electrometer as described previously.2.42+‐free solution containing 1 mg/mL collagenase  and 0.06 mg/mL proteinase . After perfusion, the heart was taken down from the cannula, cut into small pieces, gently triturated with a plastic transfer pipette and filtered through a nylon mesh. The dissociated cells were stored in NT at 20‐22°C. Rod‐shaped cells with clear striations and no granulation were used within 6‐8 hours for all the experiments.Ventricular myocytes were enzymatically dissociated as previously described with modifications.2.52.5.12, 0.5 mmol/L MgCl2, 5.4 mmol/L KCl, 10 mmol/L glucose and 10 mmol/L 4‐(2‐Hydroxyethyl)piperazine‐1‐ethanesulfonic acid (HEPES) (pH adjusted to 7.4 with NaOH).Tyrode's solution contained 137 mmol/L NaCl, 1.8 mmol/L CaCl2.5.22+‐free solution comprised 120 mmol/L NaCl, 5.4 mmol/L KCl, 1.2 mmol/L MgSO4, 1.2 mmol/L KH2PO4, 6 mmol/L HEPES, 10 mmol/L glucose and 10 mmol/L taurine (pH adjusted to 7.4 using NaOH).Ca2.5.3ICa,L was composed of 130 mmol/L CsCl, 1 mmol/L MgCl2, 5 mmol/L Mg ATP, 10 mmol/L HEPES, 0.1 mmol/L NaGTP and 5 mmol/L Na2‐phosphocreatine (pH adjusted to 7.2 with CsOH). For nickel‐sensitive Na+/Ca2+ exchanger (NCX) current, the solution comprised 20 mmol/L NaCl, 110 mmol/L CsCl, 0.4 mmol/L MgCl2, 20 mmol/L TEACl, 1.75 mmol/L CaCl2, 5 mmol/L 1,2‐Bis(2‐aminophenoxy)ethane‐N,N,N′,N′‐tetraacetic acid (BAPTA), 5 mmol/L Mg ATP, 5 mmol/L glucose and 10 mmol/L HEPES (pH adjusted to 7.25 using CsOH). For the INa, the solution was composed of 133 mmol/L CsCl, 5 mmol/L NaCl, 10 mmol/L ethylene glycol tetraacetic acid (EGTA), 5 mmol/L Mg2 ATP, 20 mmol/L TEACl and 5 mmol/L HEPES (pH adjusted to 7.3 with CsOH). For INa,L, the solution was composed of 130 mmol/L CsCl, 4 mmol/L Na2 ATP, 10 mmol/L EGTA, 1 mmol/L MgCl2 and 5 mmol/L HEPES (pH adjusted to 7.3 with NaOH).Micropipettes solution for 2.5.42, 1 mmol/L MgCl2, 5 mmol/L HEPES and 10 mmol/L glucose with 10 μmol/L strophanthidin to block the Na+/K+ pump, and 10 μmol/L nitrendipine and 100 μmol/L niflumic acid to block Ca2+‐activated Cl− currents (pH adjusted to 7.4 with NaOH). For INa, the solution contained 5 mmol/L NaCl, 133 mmol/L CsCl, 2 mmol/L MgCl2, 1.8 mmol/L CaCl2, 0.002 mmol/L nifedipine, 5 mmol/L glucose and 5 mmol/L HEPES (pH adjusted to 7.3 with NaOH). For INa,L, the solution contained 130 mmol/L NaCl, 5 mmol/L CsCl, 1 mmol/L MgCl2, 1 mmol/L CaCl2, 10 mmol/L glucose and 10 mmol/L HEPES (pH adjusted to 7.4 with NaOH). For sarcoplasmic reticulum (SR) Ca2+ leak, the 0 Na+/0 Ca2+ solution had the same composition with NT but no added Ca2+, 10 mmol/L EGTA and 140 mmol/L LiCl substituted for NaCl (pH adjusted to 7.4 with LiOH).The external solution for NCX experiment contained 140 mmol/L NaCl, 2 mmol/L CaCl2.6−/− mice were loaded with Ca2+ dye (10 μmol/L Fluo‐3 AM) at room temperature for 30 minutes and imaged as previously described method.F) were normalized against the baseline fluorescence (F0) to obtain reliable information about transient intracellular Ca2+ changes (Ca2+ transient = [F − F0]/F0) and to correct the variations in the fluorescence intensity due to different amount of dye uptake into cells. The Ca2+ transient was measured with 1‐Hz field stimulation. After achieving a steady‐state Ca2+ transients with the repeated pulses (1 Hz for 15 seconds), the superfusate was rapidly switched to 0 Na+/0 Ca2+ solution with 1 mmol/L tetracaine for a minimum of 20 seconds. The SR Ca2+ leak was measured as the tetracaine (1 mmol/L)‐reduced intracellular Ca2+ as previously described.2+ stores were assessed by rapid application of 20 mmol/L caffeine after a pule stimulation train at 1 Hz for 30 seconds. The SR Ca2+ stores were estimated from the peak amplitudes of the caffeine‐provoked Ca2+ transient. The integral of the inward NCX current induced by fast application of 20 mmol/L caffeine to cells voltage‐clamped at −40 mV was used to calculate SR Ca2+ content as previously described,2+ content (μmol/L/L cytosol) = [(1 + 0.12) × Ccaff/F × 1000)]/ (Cm × 8.31 × 8.44), where Ccaff is the integral of the inward NCX current induced by caffeine, F is Faraday's number, Cm is the membrane capacitance, and cell surface‐to‐volume ratio was 8.44 pF/pL.Cardiomyocytes from control and Sirt1escence F0 to obtaescence F0 to obta2.72.7.1ICa,L was determined as an inward current during voltage‐clamp steps from a holding potential of −50 mV to potentials from −40 to +60 mV in 10‐mV steps for 300 ms at a frequency of 0.1 Hz. ICa,L was assessed between 5‐15 minutes after membrane patch rupture in each cardiomyocyte to avoid ‘run‐down’ effects. The current of NCX was measured using voltage‐clamp potentials between −100 and +100 mV from a holding potential of −40 mV in 20‐mV steps for 300 ms at a frequency of 0.1 Hz. NCX current amplitudes were determined as Nickel (10 mmol/L NiCl2)‐sensitive currents as previously described.INa was elicited during potential steps from a holding potential of −120 mV to testing potentials from −80 to 0 mV in 10‐mV steps for 40 ms at a frequency of 3 Hz. INa,L was measured using a step/ramp protocol as described below: start with a potential of −100 mV stepping to +20 mV for 100 ms afterwards ramp back to −100 mV for 100 ms The INa,L was determined as tetrodotoxin (30 µmol/L TTX)‐sensitive current obtained when the potential was ramped back to −100 mV.Electrophysiological properties of ventricular myocytes were obtained by whole‐cell configuration patch‐clamp techniques with Axopatch 1D amplifier  as described previously.2.8+ level. Experiments were conducted using an inverted laser‐scanning confocal microscope  with a 63x1.25 objective as previously described.Ventricular myocytes were incubated in NT solution with 10 μmol/L CellROX green and 2 μmol/L MitoSOX Red (Life Technologies) to assess cytosolic and mitochondria reactive oxygen species (ROS) production, respectively. Myocytes incubated with 5 μmol/L Asante NaTRIUM Green‐2 AM (Teflabs) was used to measure the cytosolic Na2.9The protein extraction buffer contained 100 mmol/L Tris‐HCl (pH 8.0), 0.1% sodium dodecyl sulphate, 1% Triton X‐100, 150 mmol/L NaCl and protease inhibitor cocktail (Roche). The cardiac protein extracts were separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis and then transferred to polyvinylidene difluoride membranes (Millipore) that were incubated with the listed antibodies: Cav1.2 , CaMKII , NCX , SERCA2a  and α‐tubulin . Subsequently, the membranes were incubated with anti‐mouse  secondary IgG antibodies at a dilution of 1:10 000. Immunoreactive proteins were detected by enhanced chemiluminescence  and quantified using the ImageJ software.2.10t test, or Pearson's chi‐square test were used to compare the differences. The SigmaPlot version 12  was used for statistical comparisons. The ‘n’ stands for the total cells from the total number of hearts (n = cells/hearts) and the ‘N’ is the animal numbers. Statistical significance was represented as *, **, and *** for P < 0.05, P < 0.01 and P < 0.005, respectively.Continuous values have been expressed as mean ± SEM. Student's 33.1−/− mice possessed larger LVIDs than those in the control group  in the Sirt1−/− myocytes was greater than that in the control myocytes  in the Sirt1−/− cardiomyocytes was higher compared with that in the control cardiomyocytes  to cause more Ca2+ leak from the SR.−/− mice. The Sirt1−/− ventricular myocytes showed increased Ca2+ leakage from SR, which would cause diastolic calcium overload. Hyperphosphorylation of RyR2 by CaMKII increases diastolic SR Ca2+ leak2+ stores and may increase intracellular Ca2+ level in Sirt1−/− myocytes. Moreover, the increased reverse‐mode NCX current in Sirt1−/− myocytes may also contribute to the loading of cytosolic [Ca2+]. Overloading intracellular Ca2+ in cardiomyocytes may trigger ectopic activity, DADs, and, consequently life‐threatening ventricular tachyarrhythmia.−/− mice. Calmodulin‐dependent protein kinase II were up‐regulated in the Sirt1−/− ventricles. Using KN93, ventricular arrhythmic events were ameliorated. KN93, one of the CaMKII inhibitors, has been shown to have effect against arrhythmias via reduced SR Ca2+ leak, Ca2+ waves and probabilities of EADs/DADs in various settings of cardiovascular diseases.Sirt1ICa,L was measured to determine its contribution in the decrease of SR Ca2+ content. The current density of ICa,L was lower in Sirt1−/− myocytes compared with that of the control myocytes. This can be attributed to the change in the relative protein levels of L‐type Ca2+ channel subunits α1c. The decrease in Ca2+ influx via ICa,L implies that the reduced SR Ca2+ content levels result from decreased ICa,L in the Sirt1−/− mice. Increased protein levels of NCX may contribute to the increased reverse‐mode NCX current in Sirt1−/− myocytes. While the amount of Ca2+ influx increased through reverse‐mode NCX in Sirt1−/− myocytes, which could help to improve the SR Ca2+ content, larger Ca2+ transient amplitudes, and therefore enhance contractility,2+ loading effect may be compromised resulting in accumulation of cytosolic Ca2+ in the face of reduced SERCA2a function, and leaky RYR2.INa, L are relatively low but contribute substantially to [Na+]i levels when enhanced due to its slow inactivation characteristics.INa,L increases in various pathological cardiac conditions, including ischaemia/reperfusion, myocardial infarction and heart failure+ level. Moreover, intracellular Na+ homeostasis is tightly connected with Ca2+ handling since Na+ modulates the operational direction of NCX and increases the diastolic intracellular Ca2+ concentration.2+ impairs contractility and is arrhythmogenic.−/− mice is abrogated by ranolazine . Ranolazine improves the regulation of Ca2+ levels and decreases pro‐arrhythmic events by indirectly reducing diastolic Ca2+ overload and [Ca2+]i accumulation.INa,L results from enhanced activity of CaMKII.v1.5 to regulate its magnitude and other properties, including INa,L inactivation and recovery from inactivation.+ and Ca2+ by phosphorylating various target proteins and channels.−/− cardiomyocytes increase INa, L and production of ROS that quickly enhances late INa, L, thereby stimulating arrhythmogenesis.The amplitudes for −/− mice. Sirt1 controls intracellular ROS production by multiple pathways, such as NF‐κB signalling2+ leak by CaMKII may increase cytosolic levels of Ca2+, thereby enabling an overload of mitochondrial Ca2+ and facilitating ROS production. Moreover, mitochondria‐derived ROS induces local release of ER Ca2+ in cardiomyocytes. ROS‐activated CaMKII enhances INa,L that results in an overload of cellular Na+ and enhances Ca2+ influx via reversed mode of NCX, thereby enabling arrhythmia.2+ handling in cardiomyocytes. Sirt1−/− mice hearts exhibited increased arrhythmia that was inhibited by KN93 and ranolazine. The Sirt1‐deficient mice showed lower ICa,L and Ca+ transient together with poor cardiac function. Enhanced INa,L and reversed mode of NCX were observed in Sirt1‐deficient cardiomyocytes with increased production of ROS and CaMKII expression. These findings provide insights into novel mechanisms underlying arrhythmia associated with Sirt1 deficiency.Cytosolic and mitochondrial synthesis of ROS increased in Sirt12+ and Na+ regulation in mice cardiomyocytes. Dysregulated Ca2+ handling and Na+ regulation leads to a higher frequency of ventricular arrhythmia and cardiac dysfunction. INa,L was enhanced alongside with increased cytosolic Na+ level. ROS production and expression of CaMKII were higher in the Sirt1−/− mice. The CaMKII inhibitor KN93 and ranolazine prevented arrhythmia. These findings suggest that the deficiency of Sirt1 in the cardiomyocytes leads to dysregulation of intracellular Ca2+ and Na+ that provide proarrhythmic substrates.In conclusion, Sirt1 deficiency in the cardiac tissues resulted in detrimental effects on CaThe authors declare no conflict of interest.H.‐Y. Yang, F.‐Z. Lin and H.‐W. Yang performed experiments; H.‐Y. Yang, F.‐Z. Lin, P.‐L. Yu and S.‐M. Huang analysed data. H.‐Y. Yang, Y.‐C. Chen, C.‐S. Tsai and C.‐Y. Lin interpreted results of experiments; H.‐Y. Yang, F.‐Z. Lin and Y.‐C. Chen prepared figures; H.‐Y. Yang drafted manuscript; H.‐Y. Yang and Y.‐C. Chen edited and revised manuscript; Y.‐C. Chen, C.‐S. Tsai and C.‐Y. Lin conceived and designed research. All authors approved final version of manuscript.


An encoded mitochondrial matrix Ca2+ probe (Mt-GCaMP6s) was introduced to observe mitochondrial Ca2+ ([Ca2+]m) dynamic changes during oocyte maturation and activation. We found that active mitochondria surrounding the nucleus showed a higher [Ca2+]m than those distributed in the cortex during oocyte maturation. During oocyte partheno-activation, the patterns of Ca2+ dynamic changes were synchronous in the cytoplasm and mitochondria. Such higher concentration of mitochondrial matrix Ca2+ was closely related to the distribution of mitochondrial calcium uptake (MICU) protein. We further showed that higher [Ca2+]m mitochondria around the chromosomes in oocytes might have a potential role in stimulating mitochondrial energy for calmodulin-responsive oocyte spindle formation, while synchronizing Ca2+ functions in the cytoplasm and nuclear area are important for oocyte activation.Mitochondrial energy insufficiency is strongly associated with oocyte activation disorders. Ca According to the WHO 2016 report, at least one of 10 couples in developed countries cannot have children within 5 years of marriage, half of which are due to female infertility . Oocyte 2+ is one of the major signaling molecules regulating several cell functions, such as celldivision, arrest, and apoptosis. At fertilization, oocyte activation begins with a series of crucial steps triggered by periodical repetitive increases and decreases in intracellular Ca2+ ([Ca2+]i) concentrations known as [Ca2+]i oscillations  in Ca2+-free CZB for 4 h. After partheno-activation, eggs were transferred into KSOM .Female mice were injected with 10 IU pregnant mare serum gonadotropin . After 44–48 h, GV oocytes were collected by ovarian mincing. Female mice were injected with 10 IU PMSG followed 44–48 h later by injection of 10 IU human chorionic gonadotropin . After 15 h of hCG, ovulated MII oocytes were collected and denuded in 1 mg/mL hyaluronidase. Matured oocyte i oscillations regulation on mitochondrial activity during oocyte activation. [Ca2+]i oscillations were observed by staining with 2μM Fluo4 AM  in the partheno-activation system. Rhod2 is a classical dye for determining mitochondrial Ca2+ concentration ([Ca2+]m) with a higher Ca2+ affinity  in a partheno-activation system. Mt-GCaMP6s was introduced to obtain the patterns of mitochondrial matrix Ca2+ dynamic changes. Real-time images were obtained using a time-lapse confocal laser microscope  and recorded at 2 frames per minute. [Ca2+]i and [Ca2+]m intensities were detected using an argon laser.We set out to confirm i) and mitochondria ([Ca2+]m) by co-staining with Fluo4 AM    to deter GCaMP6s . In comp2+]m changes in mitochondria during GV oocyte in vitro maturation . 1st polar body non-extruded oocytes did not show any Ca2+ spike. The second spike occurred 4–5 h after the first polar body emission, i.e., at about 14 h of culture in 33.2 ± 6.2% of oocytes which exhibited the first spike  to observe the changes of [Ca2+]m and activated mitochondria distribution during oocyte activation   has multiple subunits, and its Cas (MICU) . In vitr oocytes . In PA oIn vitro transcription of VDAC was constructed to observe the distribution of VDAC in mouse oocytes and to confirm the relationship between [Ca2+]m and VDAC. The distribution of [Ca2+]m and VDAC were obviously different. VDAC distributed in most parts of oocytes (VDACs (voltage-dependent anion channels) are a widespread on the outer mitochondrial membranes of most organisms . The OMM oocytes .2+ concentration. CaMs are expected to distribute in oocytes around the most sensitive organelles for Ca2+. Firstly, we cloned the CaM1 and CaM2 cDNA sequences and linked them with mCherry as pGEMHE-CaM1-mCherry and pGEMHE-CaM2-mCherry plasmids , including two members termed CaM1 and CaM2, are thought to be the initial responding proteins in the cell to sense dynamic changes in Caplasmids ; then weplasmids , which iplasmids .2+]m and CaM2. Higher [Ca2+]m mitochondria surrounded the spindle, while CaM2 was localized on the spindle  dynamics plays vital roles in regulating fundamental cellular and organelle functions including bioenergetics  to detect the patterns of [Ca2+]m (2+]m still exhibited oscillation patterns synchronized with Rhod2. Because Rhod2 displayed exactly the same pattern as [Ca2+]i oscillations (2+]m exhibited a same pattern in synchrony with [Ca2+]i. We also found that there are some differences in the position of Rhod2 and Mt-GCaMP6s; Rhod2-stained mitochondria are increased in quantity compared to Mt-GCaMP6s positive mitochondria. Indeed, most mitochondria can be stained by Rhod2, while only some of them displaying much higher [Ca2+]m mitochondria will be stained by Mt-GCaMP6s (2+ dyes, Mt-GCaMP6s may be more suitable to distinguish the changes of Ca2+ concentration in mitochondria. Further studies are needed to better understand the difference between Rhod2 and Mt-GCaMP6s positive mitochondria.Although Rhod2 is used as a classic Cachondria , it is n [Ca2+]m . After illations , [Ca2+]m-GCaMP6s . Compara2+]m during GV oocyte maturation. We found that higher [Ca2+]m mitochondria distributed around the germinal vesicle (2+]m was higher in the area surrounding the spindle. During emission of the first polar body, [Ca2+]m in the equatorial plate area increased significantly. Mitochondrial metabolism is much stronger in the energy-demanding part of the oocyte. Interestingly, there were two spikes of [Ca2+]m around the time of first polar body extrusion (2+]m dynamic changes pattern as that matured MII oocytes. The irradiation of the laser caused failure of the first polar body extrusion in some oocytes (2+]m was found in MII oocytes in preparation for activation. These two time points are likely the most energy requiring critical periods for oocytes.Next, we wanted to detect the dynamic changes of [Ca vesicle . After Gxtrusion . Right c oocytes . The 2nd2+]m related to activated mitochondria? To address this question the relationship between [Ca2+]m and the distribution of active mitochondria was studied. MitoRed is a dye localized to activated mitochondria (in vitro maturation, we observed [Ca2+]m by Mt-GCaMP6s and activated mitochondria by MitoRed (2+]m and activated mitochondria, we performed a short 4-h observation with double concentration of MitoRed (2+]m and activated mitochondria showed a much clearer co-localization than during long term observation. If higher [Ca2+]m was co-localized with activated mitochondria, Ca2+ dynamic entry into mitochondria may participate in mitochondrial metabolism transfer from the resting to the activated state. There was a clear co-localization between higher [Ca2+]m and MitoRed-positive mitochondria surrounding the chromosomes (2+]m and MitoRed-positive mitochondria indicated that higher activated mitochondria required a higher [Ca2+]m. The localization relationship between [Ca2+]m and active mitochondria during oocyte partheno-activation was also observed next (2+]m and MitoRed still had clear co-localizations in the resting state. With [Ca2+]i oscillations, all of the mitochondria in the entire oocyte have a higher [Ca2+]m, increasing in the flashing state, which was consistent with the result of 2+ flashing state, the [Ca2+]m increases, and the MitoRed positive mitochondria remain in a stable distribution pattern. One part of the mitochondria maintained a higher [Ca2+]m around the nucleus, and the other mitochondrial [Ca2+]m will increase instantaneously carried by [Ca2+]i, resulting in a pulse-like function enhancement. The role of [Ca2+]m oscillations on mitochondrial metabolism needs to be further studied.Is this dynamic change in mitochondrial m2+ channel (2+ across the inner mitochondrial membrane, playing critical roles in various mitochondrial functions (2+ influx activity is controlled by regulatory subunits (2+ binding (2+]m mitochondria in the GV oocytes (2+]i oscillations. This may indicate that MICU is related to mitochondrial Ca2+ and mitochondrial activity. We also observed that VDAC (2+]m mitochondria, indicating that the process of regulating Ca2+ entry and exit from the mitochondrial matrix into and out of the mitochondria is not related to VDAC.The MCU, which is identified as a highly selective Ca channel , uptakesunctions . MCU consubunits , includisubunits  and MICUsubunits . MICU1is binding . In our  oocytes , howeverhat VDAC , which i2+ responds to its corresponding functions, and this change is determined by a protein initiation signal pathway with [Ca2+]i sensing. Most studied Ca2+-responsive proteins are members of the calmodulin family. The calmodulin family contains CaM1 and CaM2. We examined the relationship between CaMs and higher [Ca2+]m of mitochondria. We cloned CaM1 and CaM2 linked with mCherry (2+]m were examined to show the relationship between cytoplasmic Ca2+sensor CaM2 and active mitochondria. It can be seen in 2+]m mitochondria are distributed surrounding the spindle, however, CaM2 is associated with the spindle (2+]m and activated mitochondria around the spindle also indicates that the change of calcium-regulated spindle dynamic changes requires a continuous supply of energy by higher [Ca2+]m stimulated mitochondria. Cortical region Mito-Red signals are independent of [Ca2+]m dynamic changes, which was not our expected result. Cortical [Ca2+]m oscillations may have some other function of mitochondria regulations. There may be another possibility that another probe but not Mito-Red is suitable for detecting the mitochondrial state during partheno-activation. An accurate distribution of higher [Ca2+]m mitochondria in oocytes is important for oocyte spindle reformation.Dynamic Ca mCherry . In GV o mCherry . CaM2 an spindle . When ac spindle . The mai spindle . The dis2+]m and mitochondrial metabolism in oocytes will provide more possibilities for improving the efficiency of assisted reproduction.Deepening our understanding and basic knowledge about oocyte maturation/activation and energy metabolism is of great value for solving clinical problems such as insufficient oocyte cytoplasmic maturation and activation as well as insufficient energy supply caused by mitochondrial damage. Studying the regulatory mechanisms between [CaThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.All mice were handled in accordance with the institutional animal care policies of the Institute of Zoology, Chinese Academy of Sciences. The Laboratory Animal Care and Use Committee of the Institute of Zoology approved this study.FW, Q-YS, and X-HO conceived and designed the experiments. FW and others conducted experiments. FW and Q-YS analyzed the data. FW, HS, X-HO, and Q-YS wrote the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Objectives: The COVID-19 pandemic has led to major adjustments in health care systems and significantly affected medical education. Accordingly, our mentoring program MeCuM-Mentor had to expand its virtual elements, in order to continue to meet the needs for mentoring at the medical faculty of the Ludwig-Maximilians-University Munich.Methods: Here we report on our recently implemented online formats to facilitate training for currently coached peer mentors, as well as the introduction of an online consultation hour and a new social mentoring event called PubQuiz.Results: First results demonstrated feasibility of the above-mentioned virtual formats, which were positively rated by the participants in small voluntary evaluation questionnaires. Utilization rates indicate existing need for mentoring during the pandemic. In addition, the new event PubQuiz promotes social interaction among peers during isolation due to COVID-19.Conclusion: With the transition to online formats, mentoring at the Medical Faculty could be continued during COVID-19. The newly introduced mentoring event PubQuiz will be repeated. However, it remains unclear to what extent online formats can replace in-person one-to-one mentoring conversations or peer mentoring meetings. MeCuM-Mentor , [9, [9MeCuMy Munich , all menFirst important step was to convert the pre-planned training for the currently coached peer mentors into a monthly virtual course via Zoom. Subsequently, they could accompany digital implementation of mentoring events. An online evaluation questionnaire was sent by e-mail to all currently trained peer mentors at the end of the semester.Due to an above-average number of inquiries from students via e-mail to the coordinators of the mentoring program, a weekly consultation hour via Zoom was initiated in a second step. There, students could regularly and easily address their questions in individual one-on-one talks with a coordinator of the mentoring program.PubQuiz with regard to the psychosocial consequences of COVID-19 was initiated. As already pointed out by Gotian 2020: “Most importantly, remember the human and empathetic part of the mentoring relationship” [Furthermore, a new event called Utilization rate of all online implemented elements showed existing demand for mentoring among students. In addition, feedback was positive:In the virtual peer mentoring course, topics were: adaptation by COVID-19, evaluation of mentoring relationships, planning of the upcoming semester as well as discussions on new projects. In a small voluntary evaluation questionnaire among the currently trained peer mentors , the majority pleaded for future continuation of the meetings with the peer mentor group not only in person (75%) but also via Zoom (66.7%). In the context of their one-to-one peer mentoring, student mentors stated that they continued meetings virtually , video calls e.g. via Zoom (50%), text message (25%)).The weekly consultation hour via Zoom was regularly used by students. Topics included advice on mentoring and matching, information on how to apply as a peer mentor, and questions about different projects.PubQuiz was evaluated with a small questionnaire , in which 85.7% rated the event as excellent. The vast majority also pleaded for repetition – both in virtual form (85.7%) and face-to-face (60%). Nine participants (42.9%) also indicated that this encouraged them to find their own mentor.The PubQuiz showed that physician mentors and students enjoy socializing even in a virtual casual atmosphere. In future, such event could be both offered virtually and in-person, in order to accommodate all preferences and foster a more informal exchange between mentors and mentees.The reorganization of our mentoring program by means of online platforms made it possible to keep mentoring running and to stay connected with all our stakeholders despite COVID-19. Moreover, training of peer mentors was warranted, which helped maintaining established projects and organizing new ones. Online formats offer the option to plan mentoring conversations more flexible and thus exchange experiences more often. However, in such virtual meetings the impact of an in-person contact is missing. Evaluation results of PubQuiz, a new event was created that allows mentoring conversations in a novel context, focusing on the dimension of social interaction. Thus, we provide students with a valuable reference point even during COVID-19. Due to the continuing interest in the PubQuiz, a repetition is already planned. To what extent virtual meetings can achieve similar outcomes and therefore replace face-to-face events in the mentoring context has to be investigated in future prospective randomized trials.A transition into online mentoring was successfully demonstrated and positively evaluated by participants. Both new mentoring consultation and training of the peer mentors could be smoothly implemented online. With The authors declare that they have no competing interests.


The aim of this study was to evaluate the pathogenic role of newly identified long non-coding (lnc)-RNA LINCO1268 in acute myeloid leukemia (AML), and investigate its therapeutic potential. The expression level of LINC01268 in AML was measured by quantitative PCR (qPCR). The viability, cell cycle progression, and apoptosis of AML cells were measured by CCK-8 assay and flow cytometry, respectively. The interaction between LINC01268 and miR-217 were predicted by the miRDB website, and then verified by luciferase reporter assay and RNA immunoprecipitation (RIP) assay. The relationship between miR-217 and SOS1 was predicted by TargetScan website, and verified by luciferase reporter assay. LINC01268 was significantly upregulated by 1.6 fold in bone marrow samples of AML patients, which was associated with poor prognosis. LINC01268 was also significantly upregulated in AML cells. LINC01268 knockdown inhibited viability and cell cycle progression but promoted apoptosis of AML cells. Furthermore, LINC01268 functioned as a ceRNA via competitively binding to miR-217, and SOS1 was identified as a target of miR-217. Moreover, LINC01268 positively regulated SOS1 expression to promote AML cell viability and cell cycle progression but inhibited apoptosis via sponging miR-217. LINC01268 promoted cell growth and inhibited cell apoptosis through modulating miR-217/SOS1 axis in AML. This study offers a novel molecular mechanism for a better understanding of the pathology of AML. LINC01268 could be considered as a potential biomarker for the therapy and diagnosis of AML. Acute myeloid leukemia (AML), as a highly heterogeneous disorder, is the most common acute leukemia in adults . The genLong non-coding RNAs (lncRNAs) are defined as non-protein coding transcripts larger than 200 nucleotides . AccumulLong intergenic non-protein coding RNA 1268 (LINC01268), also known as LOC285758, is a newly identified lncRNA . LINC012The diagnosis of AML was confirmed based on morphological, cytochemical, immunophenotyping, and cytogenetic analysis of bone marrow samples, and AML was classified according to the FAB (French-American-British) criteria . Before 2.Human bone marrow stromal cell line HS-5 and human AML cell lines  were obtained from American Type Culture Collection . Cells were incubated in RPMI 1640 medium  containing 100 U/mL penicillin, 0.1 mg/mL streptomycin, and 10% fetal bovine serum  at 37°C in a humidified incubator with 5% COTwo different kinds of siRNAs for LINC01268 (si-LINC01268 #1 and si-LINC01268 #2) and control siRNA (si-NC) were purchased from GenePharma (China). miR-217 mimic, miRNA scrambled control (control mimic), miR-217 inhibitor, and inhibitor control (NC inhibitor) were synthesized by RiboBio (China). After growing to 80% confluence, cells were transfected with the above compounds using Lipofectamine 3000 reagent  following the manufacturer's instructions.−ΔΔCt method and was normalized to the internal control (β-actin). The primers are shown in Total RNA from tissues and cells were isolated using Trizol reagent (Invitrogen), and reversely transcribed to cDNA with M-MLV reverse transcriptase (Invitrogen). Next, qRT-PCR was carried out using SYBR Green Mixture RT-PCR kits  on the ABI 7500 Fast Real-Time PCR system. Relative mRNA expression was calculated by the 24 cells per well and incubated in RPMI 1640 medium (Gibco) with 10% FBS for 0, 24, 48, and 72 h. Next, CCK8 solution (10 μL) was added into each well and incubated for 4 h at 37°C. Subsequently, the sample's absorbance was detected at 450 nm by a microplate reader .Cell viability was determined by CCK-8 assay. Cells were separately seeded onto 96-well plates at a density of 1×10For cell cycle analysis, the transfected cells were harvested, re-suspended in the binding buffer, stained with propidium iodide  solution, and analyzed by a FACSCalibur flow cytometer .For cell apoptosis analysis, the harvested cells were re-suspended in the binding buffer and stained with FITC-Annexin V (Sigma-Aldrich) and PI solution for 30 min in the dark. Then, the sample was evaluated by the flow cytometer. A quadrant diagram of flow cytometry indicated the discrimination between living cells (Q4), early apoptotic cells (Q3), late apoptotic cells (Q2), and necrotic cells (Q1). The corresponding histograms of flow-cytometry showed the cellular apoptotic rates including early apoptotic cells (Q3) and late apoptotic cells (Q2).Total protein was extracted from transfected cells using RIPA buffer supplemented with phosphatase inhibitors and protease inhibitors on ice for 30 min. The BCA method was utilized to determine the concentration of protein. Next, the protein was equally separated on 10% SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and then transferred to PVDF (polyvinylidene difluoride) membranes. After blocking with 5% skimmed milk for 2 h, the membranes were incubated with primary antibodies against CDK2, p21, Bax, Bcl-2, cleaved caspase-3, GAPDH, and SOS1 overnight at 4°C. Then, the membranes were washed with TBST  buffer, incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 h at 25°C, and visualized using the ECL detection system . The semi-quantitative analysis of the protein expression was performed with Quantity One software . GAPDH was used as an internal control.The interaction between miR-217 and LINC01268 or SOS1 was detected by luciferase reporter assay. LINC01268 or SOS1 with putative binding sequences of wild-type (WT) miR-217 was amplified by qPCR and then cloned into the pmirGLO vector  to obtain LINC01268-WT or SOS1-WT, respectively. The corresponding mutated sequences (MUT) of putative binding sequences were also cloned into the pmirGLO vector (Promega) to obtain LINC01268-MUT and SOS1-MUT. Cells were incubated for 24 h, and then co-transfected with LINC01268-MUT, SOS1-MUT, LINC01268-WT, or SOS1-WT and miR-217 mimic or control mimic using Lipofectamine 3000 reagent (Invitrogen). After 48 h of transfection, the dual-luciferase reporter assay system (Promega) was utilized to examine the luciferase activity, which was normalized to Renilla luciferase activity.g for 10 min at 4°C, and the supernatant was collected for immunoprecipitation. Then, the supernatant was mixed with RIP buffer containing a magnetic bead conjugated with human anti-Ago2 antibody or mouse immunoglobulin G . Next, beads were pelleted by centrifuging briefly at 10,000 g at 4°C, washed with RIPA buffer and PBS, and re-suspended with proteinase k. The mixture was centrifuged at 20,000 g for 10 min at room temperature, and the supernatant was divided into a 3:1 ratio for RNA and protein extraction.The binding relationship between miR-217 and LINC01268 was further validated by the RIP assay. Cells were lysed using RIP lysis buffer with a protease inhibitor cocktail and RNase inhibitor in a Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit . The lysate was centrifuged at 20,000 t-test identified the significance of the difference between two groups, and one-way ANOVA analyzed the difference among multiple groups. P<0.05 was considered to be statistically significant. Survival curves were estimated by the Kaplan-Meier method followed by the log-rank test for the comparison of differences in survival distributions. A chi-squared test was conducted to evaluate the relationship between LINC01268 expression and clinical-pathological characteristics.Data are reported as means±SD and were evaluated by SPSS 13.0. Each experiment was repeated at least three times. Student's The expression of LINC01268 was significantly upregulated by 1.6-fold in bone marrow samples from AML patients compared to that from healthy donors . The expThe expression level of LINC01268 was positively correlated with the number of white blood cells (P=0.001), hemoglobin level (P=0.007), and platelet count (P=0.001). There was no significant correlation between LINC01268 level and age, gender, FAB subtype, or complete remission .Based on the median expression value of LINC01268, AML patients were classified into two groups: LINC01268 high expression group (n=23) and LINC01268 low expression group (n=27). AML patients with high expression of LINC01268 had a worse overall survival compared to that with low expression of LINC01268 .To explore the role of LINC01268 in AML cells, the cell viability, cell cycle stage, and cell apoptosis were determined in HL-60 and Kasumi-1 cells. The siRNAs for LINC01268  were transfected into HL-60 and Kasumi-1 cells, and si-LINC01268 #2 possessed a lower expression level than si-LINC01268 #1, which was used in the following experiments .LINC01268 knockdown significantly inhibited the viability of AML cells . For cycLINC01268 knockdown significantly increased the apoptotic rates of AML cells compared to the control group . Additiohttp://www.mirdb.org/), miR-217 was predicted as a target of LINC01268  predicted that SOS1 was a target of miR-217  inhibited the viability of AML cells, but enhanced the cell viability after co-transfection with si-LINC01268 and miR-217 inhibitor .For cycle progression analysis, compared with the control group, LINC01268 knockdown increased the percentage of AML cells in the G1 stage but decreased it in the G2 and S stages. This result was reversed after co-transfection with si-LINC01268 and miR-217 inhibitor .The apoptosis analysis suggested that the increased apoptotic rate of AML cells caused by LINC01268 knockdown was reversed after transfection of miR-217 inhibitor .Finally, Given the limited therapeutic methods and poor prognosis of AML patients, it is urgent to find new therapies for AML ,6. lncRNlncRNAs regulate cellular processes through diverse molecular mechanisms in various diseases ; specifiSOS1, a guanine nucleotide exchange factor, catalyzes the exchange of GDP for GTP and activates Ras . SOS1 coIn conclusion, these results suggested that LINC01268 promoted AML cell viability and cell cycle progression but inhibited cell apoptosis by regulating miR-217/SOS1 axis. LINC01268 could be considered a potential biomarker for the therapy and diagnosis of AML. This study may offer a novel molecular mechanism for a better understanding on the pathology of AML. Certainly, this is a preliminary study considering the roles of LINC01268 in AML, and the promotion of the LINC01268/miR-217/SOS1 axis in the progression of AML still needs to be further validated by animal experiments and clinical trials.


NPM1- and FLT3-mutated acute myeloid leukemia (AML) is still a matter of debate. Here, we combined in vitro clonogenic assays with targeted sequencing to gain further insights into the cell-of-origin of NPM1 and FLT3-ITD-mutated AML in diagnostic bone marrow (BM) from nine NPM1+/FLT3-ITD (+/−) AMLs. We reasoned that individually plucked colony forming units (CFUs) are clonal and reflect the progeny of a single stem/progenitor cell. NPM1 and FLT3-ITD mutations seen in the diagnostic blasts were found in only 2/95 and 1/57 individually plucked CFUs, suggesting that BM clonogenic myeloid progenitors in NPM1-mutated and NPM1/FLT3-ITD-mutated AML patients do not harbor such molecular lesions. This supports previous studies on NPM1 mutations as secondary mutations in AML, likely acquired in an expanded pool of committed myeloid progenitors, perhaps CD34−, in line with the CD34−/low phenotype of NPM1-mutated AMLs. This study has important implications on the cell-of-origin of NPM1+ AML, and reinforces that therapeutic targeting of either NPM1 or FLT3-ITD mutations might only have a transient clinical benefit in debulking the leukemia, but is unlikely to be curative since will not target the AML-initiating/preleukemic cells. The absence of NPM1 and FLT3-ITD mutations in normal clonogenic myeloid progenitors is in line with their absence in clonal hematopoiesis of indeterminate potential.The cell-of-origin of  Acute myeloid leukemia (AML) represents a heterogeneous group of malignant hematological disorders characterized by the rapid expansion of immature myeloid cells (blasts) in the bone marrow (BM). There is a wide disease heterogeneity in AML and patient risk-stratification principally relies on cytogenetic-molecular data . AML freVirtually all cancers are clonal and reflect the progeny of a single cell , but theNPM1-mutated AML has long been a matter of debate. NPM1-mutated AMLs commonly lack expression of both CD34 [neg phenotype of NPM1-mutated AML raises questions as to whether the NPM1 mutation occurs in a CD34− committed myeloid progenitor or whether a rare pool of CD34+CD38− NPM1-mutated early progenitor/HSC exists. Falini’s group showed that MACS-sorted bulk CD34+ cells, and immature CD34+CD38− cells were NPM1-mutated by PCR and Western blot [NPM1 mutations are secondary to other AML driver mutations, and that the CD34+ cell fraction contains preleukemic subclones lacking NPM1 mutations [The cell-of-origin of oth CD34 . The CD3ern blot . Later oern blot . Additioutations .NPM1-mutated and NPM1/FLT3-ITD-mutated AML patients do not harbor either the NPM1 or the FLT3-ITD mutation present in the AML blasts (Here, we combined in vitro clonogenic assays with targeted sequencing of both NPM1 and FLT3-ITD to gain further insights into the cell-of-origin of NPM1-mutated and FLT3-ITD-mutated AML in diagnostic BM from nine AMLs, five NPM1+/FLT3-ITD+, and four NPM1+/FLT3-ITD . We reasL blasts D. This s−/low phenotype of NPM1-mutated AML patients. Third, despite leukemia-induced hematopoietic displacement, clonogenic/CFU assays from diagnostic BM from AML patients read out normal myeloid progenitors with multilineage representation rather than leukemic CFUs. Of clinical relevance, therapeutic targeting of either NPM1 or FLT3-ITD mutations might thus have a transient benefit in restraining disease progression and debulking the leukemia but is unlikely to be curative since will not target the AML-initiating/preleukemic cells. Finally, the absence of NPM1 and FLT3-ITD mutations in normal clonogenic myeloid progenitors is in line with the fact that these particular mutations are rarely found in clonal hematopoiesis of indeterminate potential.This study has implications on the cell-of-origin of NPM1+ AML. First, our data provide relevant information about the mutational status of NPM1 and FLT3-ITD in a clonal progeny derived from single HSC/myeloid progenitors. Second, our data supports previous studies suggesting that NPM1 mutations are secondary to other AML driver mutations acquired in an expanded pool of committed myeloid progenitors, perhaps CD34−, further supporting the CD34


By direct physical interaction with ORAI1, a plasma membrane Ca2+ channel, STIM1 activates the CRACI current, whereas the binding with the voltage-operated Ca2+ channel CaV1.2 inhibits the current through this latter channel. In this way, STIM1 is a key regulator of Ca2+ signaling in excitable and non-excitable cells, and altered STIM1 levels have been reported to underlie several pathologies, including immunodeficiency, neurodegenerative diseases, and cancer. In both sporadic and familial Alzheimer’s disease, a decrease of STIM1 protein levels accounts for the alteration of Ca2+ handling that compromises neuronal cell viability. Using SH-SY5Y cells edited by CRISPR/Cas9 to knockout STIM1 gene expression, this work evaluated the molecular mechanisms underlying the cell death triggered by the deficiency of STIM1, demonstrating that STIM1 is a positive regulator of ITPR3 gene expression. ITPR3 (or IP3R3) is a Ca2+ channel enriched at ER-mitochondria contact sites where it provides Ca2+ for transport into the mitochondria. Thus, STIM1 deficiency leads to a strong reduction of ITPR3 transcript and ITPR3 protein levels, a consequent decrease of the mitochondria free Ca2+ concentration ([Ca2+]mit), reduction of mitochondrial oxygen consumption rate, and decrease in ATP synthesis rate. All these values were normalized by ectopic expression of ITPR3 in STIM1-KO cells, providing strong evidence for a new mode of regulation of [Ca2+]mit mediated by the STIM1-ITPR3 axis.STIM1 is an endoplasmic reticulum (ER) protein that modulates the activity of a number of Ca This transport is tightly regulated to maintain a cytosolic free Ca2+ concentration ([Ca2+]i) close to 100 nM in resting conditions, and the transient increase over this basal level acts as an effector for many stimuli. There are multiple signaling pathways that become deregulated during cytosolic Ca2+ dyshomeostasis, which is the basis for the proposal of the Ca2+ hypothesis of neurodegenerative diseases that suggests a direct causal link between altered Ca2+ signaling and the onset of Alzheimer’s mit). While it is well known that this [Ca2+]mit is essential to control mitochondria functionality [2+]mit observed in wild-type cells.These results suggest that aberrant STIM1 levels could underlie the pathogenesis in FAD. In this regard, we recently reported that there is a significant reduction of STIM1 levels in human brain tissues  from patients diagnosed with sporadic  Alzheimer’s disease (SAD) . The reaionality , it rema2+ [2+]mit by analyzing the gene expression of some of the most important Ca2+ transport systems. Our data revealed that the expression of IP3 receptor type 3 (IP3R3 or ITPR3) is significantly lowered in STIM1-deficient cells. This alteration in ITPR3 expression was found to be responsible for the low [Ca2+]mit since the ectopic overexpression of ITPR3 in STIM1-KO cells normalized [Ca2+]mit, as well as basal oxygen consumption rate, mitochondrial coupling efficiency, ATP production rate, and cell viability.Interestingly, mitochondrial dysfunction is a key feature in Alzheimer’s disease, as mitochondria from Alzheimer’s disease (AD) patients show decreased uptake of Ca2+ . In this2+]mit is critical to support mitochondrial function. We recently reported that human brain samples from SAD patients present significant decreases in the levels of STIM1 [2+]mit and the inhibition of mitochondrial complex I (NADH-coenzyme Q oxidoreductase) activity. However, the molecular mechanism underlying these alterations is not clear.As stated above, mit found in STIM1-KO cells was caused by reductions in the expression levels of ITPR3. To confirm the TaqMan results, we analyzed mRNA levels for ITPR1, ITPR2, and ITPR3 genes by quantitative RT-PCR in non-differentiated SH-SY5Y cells. In agreement with the results shown above, we found ~90% reduction in ITPR3 gene transcripts, whereas the products of ITPR1 and ITPR2 did not change significantly  ,28,29. Wficantly b. Remark ITPR1-2 c. A simi ITPR1-2 . On the  ITPR1-2 d,e, as inscripts b.2+ signaling in the cell. However, the comparison of ITPR3 expression between SH-SY5Y and U2OS cells, where ITPR3 is the most abundant isoform, suggests that this specific variant is weakly expressed in SH-SY5Y cells ) in different cell lines ,28,29, whis work .2+ from the ER in fura-2-loaded cells stimulated with 100 μM carbachol (CCh) in Ca2+-free medium. This stimulation, which activates the IP3 pathway, revealed a partial decrease in the amplitude of the Ca2+ release from the ER in response to a 30 s pulse of CCh  in resting conditions by transfecting the genetically-encoded Ca2+ sensor ER-GCaMP6-210 [2+]ER in STIM1-deficient cells (from 246 ± 10.8 μM in wild-type cells to 202 ± 11.7 μM in STIM1-KO cells) (2+ concentration ([Ca2+]i) in wild-type cells was greater than that found in STIM1-KO cells  a. Howeve2+]ER in response to CCh. This ER emptying follows a first-order exponential decay  of the kinetics of mit was markedly decreased in STIM1-KO . Our data confirmed that stable overexpression of ITPR3 rescued the defects in [Ca2+]mit triggered by the deletion of STIM1, which strongly suggests that ITPR3 deficiency was the cause of [Ca2+]mit dysregulation in STIM1-KO cells.We were able to recapitulate this phenotype in STIM1-KO cell lines stably overexpressing ITPR3-Myc i,j, wher2 consumption was non-mitochondrial  sensitive to oligomycin, carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP), and rotenone + antimycin A, as a readout of the mitochondrial functionality and metabolic status . The methondrial a. Indeed2+]mit in STIM1-KO cells, normalized basal oxygen consumption, coupling efficiency, and ATP production . Converd cancer , again s2+ regulators and transporters, finding a strong decrease in ITPR3 transcripts and ITPR3 protein expression in STIM1-deficient cells, as is reported here. ITPRs are most abundantly expressed in the ER, but they are also found in the nuclear envelope, Golgi apparatus, secretory vesicles, and plasma membrane . Theferences ,29,40, achondria ,42.2+ transfer is essential for mitochondrial function because Ca2+ positively regulates three enzymatic reactions of the Krebs cycle  mit. This conclusion is supported by the normalization of [Ca2+]mit, as well as the recovery of the basal OCR, ATP production, and cell viability, observed in STIM1-KO cells overexpressing ectopic ITPR3. For this reason, our results indicate that STIM1 has a key role in the normalization of [Ca2+]mit by controlling ITPR3 protein levels. Interestingly, postmortem cerebella from patients with the PSEN1-E280A mutation, typical of FAD, showed much lower levels of ITPR3 compared to control patients, and the expression of PSEN1-E280A in a neuronal cell line altered ER-mitochondria tethering compared with that in cells expressing wild-type PSEN1 [2+]mit must be considered to explain the modulation of neurodegeneration and survival.The present results in SH-SY5Y demonstrate that the reduced ITPR3 levels observed in STIM1-deficient cells are responsible for the reduced Cape PSEN1 . Becausepe PSEN1 , the axiall-trans-retinoic acid (RA), collagen type I solution (Ref. #C3867), Dulbecco’s modified Eagle’s medium (DMEM), DMEM:F-12 Ham’s medium, and carbachol (carbamoylcholine chloride) were purchased from Sigma-Aldrich. Fura-2-acetoxymethyl ester (fura-2-AM) was from Merck Millipore ; Clarity Max™ Western ECL substrate was from Bio-Rad .SH-SY5Y cells were purchased from ECACC (European Collection of Authenticated Cell Cultures) and distributed by Sigma-Aldrich ; The rabbit polyclonal anti-STIM1 antibody (#4119) was from ProSci Inc. ; the mouse monoclonal anti-red fluorescent protein  was from Chromotek ; the mouse anti-beta tubulin antibody (clone TUB2.1) was from Sigma-Aldrich; the mouse anti-ITPR3 antibody (#610312), was from BD Biosciences ; the mouse anti-ITPR2 antibody , the mouse anti-ITPR1/2/3 antibody , the mouse anti-ASCL4 , mouse anti-VDAC1 , and the mouse monoclonal anti-GAPDH antibodies were from Santa Cruz Biotechnology ; the rabbit anti-ITPR1 antibody (RBT-03) was a kind gift from Dr. Jan B. Parys (KU Leuven) who generated and characterized the antibody elsewhere . The antITPR3 cDNA (NM_002224.4) into the pmCherry-N1 vector using the cDNA from the Harvard plasmid clone HsCD00399229 as a template. The following primers were used to insert SmaI flanking sites (underlined): ITPR3-fwd: 5′-TCCCCCGGG-ATGAGTGAAATGTCCAGCTTTC, and ITPR3-rev: 5′-TCCCCCGGG-GGCGGCTAATGCAGTTCTGGAC. The construct for the expression of mouse Itpr3-Myc was purchased from OriGene  (clone #MR225699).The construct for the expression of ITPR3-mCherry was made by inserting the human Transfection of cells with DNA constructs was performed with 1–2 μg plasmid DNA per 10-cm dish and polyethylenimine  in serum-containing medium.v/v) heat-inactivated fetal bovine serum (FBS) (Ref. 10500-064 from ThermoFisher Scientific), 2 mM L-glutamine, 100 U/mL penicillin, and 0.1 mg/mL streptomycin in a humidified atmosphere of 95% air/5% CO2 at 37 °C, as described elsewhere i) was measured in fura-2-AM-loaded cells as described elsewhere mit was assessed in cells transfected with the genetically-encoded Ca2+ sensor 4mtD3cpv. CFP, YFP, and FRET efficiency between the two channels was measured with the dual CFP/YFP-2×2M-B filter set (Semrock). All measurements were performed in Ca2+-containing HBSS for 4–5 min. Spectral unmixing  was performed by determining the bleed-through coefficients as described in References [ROI − FRETbackground)/(CFPROI − CFPbackground) was converted to [Ca2+]mit as described in Reference ER) was measured using the genetically-encoded Ca2+ sensor ER-GCaMP6-210 [2+.Endoplasmic reticulum free calcium concentration  attached to a Nikon Eclipse Ti-E inverted microscope . Illumination was performed with a xenon arc lamp. All measurements were performed in a DH-35iL culture dish incubator, and the temperature was set at 36 °C . In all cases, excitation and emission conditions were controlled by the NIS-Elements AR software.Cells were plated on 8-well μslide plates from Ibidi  at 40,000 cells/well density. Forty-eight hours after plating, cells were stained with the Live/Dead Cell Imaging kit (ThermoFisher Scientific), according to the manufacturer protocol. Ten images from randomly chosen fields were recorded for every experimental condition. Imaging was performed with a 20× objective on a Nikon Eclipse Ti-E inverted microscope.t-test). Analyses were performed with the GraphPad software. Differences between groups of data were taken statistically significant for p < 0.05. The p-values are represented as follows: (*) p < 0.05, (**) p < 0.01, and (***) p < 0.001.Statistical analyses between pairs of data groups were done using the Mann–Whitney test of data (non-parametric unpaired


DNA methyltransferase 3 alpha (DNMT3A) mutation was one of the most frequent genetic alterations in acute myeloid leukemia (AML), which was associated with poor prognosis and appeared to be a potential biomarker. Herein, we aimed to identify the key genes and pathways involved in adult AML with DNMT3A mutations and to find possible therapeutic targets for improving treatment.  The RNA sequencing datasets of 170 adult AML patients were obtained from The Cancer Genome Atlas (TCGA) database. EdgeR of the R platform was used to identify the differentially expressed genes (DEGs). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed by Metascape and DAVID. And protein-protein interaction (PPI) network and clustering modules were analyzed with the STRING database and Cytoscape software.  Mutated DNMT3A resulted in a shorter overall survival (OS) in AML patients and obviously associated with age, blast percentage in peripheral blood, and FLT3 mutation. A total of 283 DEGs were detected, of which 95 were upregulated and 188 were downregulated. GO term analysis showed that DEGs were significantly enriched in neutrophil degranulation, myeloid cell differentiation, stem cell proliferation, positive regulation of neurological system process, leukocyte migration, and tissue morphogenesis. KEGG pathway enrichment analysis indicated that the pathway of cancer, PI3K-Akt signaling pathway, and transcriptional misregulation in cancer may play a crucial role in DNMT3A mutation AML. Seven hub genes  had a significant prognostic value.  Bioinformatics analysis in the present study provided novel targets for early diagnosis and new strategies for treatment for AML with DNMT3A mutation. Acute myeloid leukemia (AML) is a common type of hematopoietic neoplasm characterized with molecular characteristics . MoleculAs an epigenetic modification, DNA methylation is important for mammalian development. Three genes  have been found to encode proteins with DNA methyltransferase activity , 8. Howehttps://gdc-portal.nci.nih.gov/) [http://xena.ucsc.edu/).Information of 170 adult AML patients, including RNA-seq dataset, corresponding survival profiles, and gene mutation information, was obtained from The Cancer Genome Atlas (TCGA) database (2  | fold change (FC) | ≥1 and P value < 0.05. A heat map and volcano plot of DEGs were drawn by the ggplots package in the R platform.EdgeR was used to screen DEGs between DNMT3A mutation and wild-type AML patients according to the user's guide , 20. DEGhttp://metascape.org), including biological process (BP), molecular function (MF), and cellular component (CC) [https://david.ncifcrf.gov/tools.jsp) [P value < 0.05 was considered statistically significant.Gene Ontology (GO) term enrichment was analyzed by Metascape (ent (CC) , while tols.jsp) . P valuehttp://string.embl.de/) [An online tool, the Search Tool for the Retrieval of Interacting Genes (STRING) database (mbl.de/)  was usedmbl.de/) . The cytmbl.de/) , 26. GO t-test was used to evaluate the gene expression level between DNMT3A mutation and wild-type AML. Chi-square analysis was used to evaluate the relationship between DNMT3 mutation and clinicopathological parameters. The Kaplan-Meier method with the log-rank test was used to calculate the overall survival (OS) of patients. Hazard ratio (HR) and 95% confidence intervals (CIs) were analyzed by the Cox proportional hazards regression model. P value < 0.05 was indicated as statistically significant.All the statistical analyses were conducted with SPSS version 20.0 and GraphPad Prism version 8.0. The A total of 170 AML patients were enrolled in the article from TCGA database. The inclusion criteria were as follows: (1) age ≥ 18 years old, (2) providing information of DNMT3A mutation, and (3) covering RNA-seq dataset and corresponding survival profiles and clinicopathological parameters. There were 43 AML patients with DNMT3A mutation (25%).P < 0.001, P = 0.004). And univariate Cox proportional hazards regression analysis observed that DNMT3A mutation was correlated with poor prognosis and an increased risk of death , blast percentage in peripheral blood (P = 0.006), and FLT3 mutation (P = 0.034). However, there was no statistical difference between DNMT3A mutation and sex (P = 0.862), FAB type (P = 0.326), blast percentage in bone marrow (P = 0.28), and white blood cell (P = 0.475).To further understand the role of DNMT3 mutation in AML, chi-square analysis was used to evaluate the relationship between DNMT3 mutation and clinicopathological parameters . The res2  | fold change (FC) | ≥1 and P value < 0.05. Altogether, 283 DEGs were detected, which 95 were upregulated and 188 were downregulated . We selected the top 3 modules to further analyze , MPO (P = 0.002), THBS1 (P = 0.014), APP (P = 0.034), ELANE (P = 0.042), HOXA7 (P = 0.027), and VWF (P = 0.015) had a significant prognostic value in AML  in DNMT3A mutation and wild-type AML patients is shown in e in AML .Multiple genetic alterations with a prognostic value can be suggested as biomarkers for AML to improve diagnosis and treatments. DNMT3A high-frequency mutation has been reported as a dangerous element in AML. DMNT3A might be a novel prognostic factor and therapeutic target of AML. Our study showed that DNMT3A expression was lower in wild-type AML compared with DNMT3A mutation AML. Survival analysis indicated that mutated leukemia patients had a shorter overall survival (OS) and increased risk of poor clinical outcome. This result was consistent with previous studies , 18, 27.However, the changes in biological processes and signal pathway DNMT3A mutation cause had not been reported. Herein, we used an RNA-seq dataset of adult AML from TCGA database to identify the key genes and pathways associated with DNMT3A mutation via bioinformatics analysis. Altogether, 283 DEGs were detected, which 95 were upregulated and 188 were downregulated. GO analyses showed that upregulated and downregulated DEGs were notably abundant in neutrophil degranulation, cell morphogenesis involved in differentiation, stem cell proliferation, myeloid cell differentiation, leukocyte migration, tissue morphogenesis, definitive hemopoiesis, and positive regulation of myeloid cell differentiation. It was suggested that DNMT3A mutations may contribute to disease progression and affect prognosis by influencing cell proliferation, differentiation, morphogenesis, and hemopoiesis in AML patients. The KEGG enrichment analysis revealed that DEGs were enriched in pathways of cancer, PI3K-Akt signaling pathway, and transcriptional misregulation in cancer. Consistent with previous studies, the above pathways have been reported that affect the pathogenesis and prognosis of AML –33.What is more, we built the protein-protein interactome networks and selected some hub genes with high connectivity involved in DNMT3A mutation AML. The results about GO enrichment analysis of modules were similar to previous analysis of DEGs. The genes of the top 3 modules were enriched in hemopoiesis, myeloid cell differentiation, myeloid leukocyte activation, and migration which were closely associated with the pathogenesis of AML. The top hub genes included ELANE, APP, MMP9, BMP4, MPO, THBS1, VWF, OLFM4, LCN2, and HOXA7, which significantly enriched in regulation of leukocyte migration, myeloid cell differentiation, extrinsic apoptotic signaling pathway, and monocyte differentiation, associated with cancer. Corresponding survival analyses suggested that seven genes  had a significant prognostic value in AML. Vandenberghe and Beel reported that ELANE mutation associated with severe congenital neutropenia increased the risk of AML . And ELAOur study indicated that mutated DNMT3A resulted in a shorter OS which was in line with previous reports. Bioinformatics analyses showed DNMT3A mutation may contribute to disease progression and affect prognosis by influencing cell proliferation, differentiation, morphogenesis, and hemopoiesis in AML patients. Cancer pathway, PI3K-Akt signaling pathway, and transcriptional misregulation in cancer may play a crucial role in DNMT3A mutation AML. Seven hub genes  had a significant prognostic value in AML. These findings provided novel targets for early diagnosis and new strategies for treatment for AML associated with DNMT3A mutation. But further experiments are still needed to support our results.


The cells were loaded with Fura-2 to detect changes in the [Ca2+]i and with Mag-fluo-4 to measure variations in the [Ca2+]ER or with TMRE to follow modifications in the mitochondrial membrane potential in response to five different inducers of CGN cell death. These inducers were staurosporine, thapsigargin, tunicamycin, nifedipine and plasma membrane repolarization by switching culture medium from 25 mM KCl (K25) to 5 mM KCl (K5). Additionally, different markers of ER stress were determined and all these parameters were correlated with the activation of caspase-3. The different inducers of cell death in CGN resulted in three different levels of activation of caspase-3. The highest caspase-3 activity occurred in response to K5. At the same time, staurosporine, nifedipine, and tunicamycin elicited an intermediate activation of caspase-3. Importantly, thapsigargin did not activate caspase-3 at any time. Both K5 and nifedipine rapidly decreased the [Ca2+]i, but only K5 immediately reduced the [Ca2+]ER and the mitochondrial membrane potential. Staurosporine and tunicamycin increased the [Ca2+]i and they decreased both the [Ca2+]ER and mitochondrial membrane potential, but at a much lower rate than K5. Thapsigargin strongly increased the [Ca2+]i, but it took 10 min to observe any decrease in the mitochondrial membrane potential. Three cell death inducers -K5, staurosporine, and thapsigargin- elicited ER stress, but they took 30 min to have any effect. Thapsigargin, as expected, displayed the highest efficacy activating PERK. Moreover, a specific PERK inhibitor did not have any impact on cell death triggered by these cell death inducers. Our data suggest that voltage-gated Ca2+ channels, that are not dihydropyridine-sensitive, load the ER with Ca2+ and this Ca2+ flux plays a critical role in keeping the mitochondrial membrane potential polarized. A rapid decrease in the [Ca2+]ER resulted in rapid mitochondrial membrane depolarization and strong activation of caspase-3 without the intervention of the ER stress in CGN.In this study we evaluated the effect of the reduction in the endoplasmic reticulum calcium concentration ([Ca It is well documented that a sustained reduction in the luminal [Ca2+] of the ER can result in a condition known as ER stress that eventually can lead to cell death  is one of the most important intracellular Call death . This coll death . We havell death . The pos2+ from the ER to the mitochondria could be the reason behind CGN apoptotic death-induced by the decreased [Ca2+]i. It has been calculated that about half of the Ca2+ released from the ER is taken up by the mitochondria  are involved in the apoptotic death of CGN. These cells need a sustained Ca2+ influx for survival, which can be achieved by culturing cells in high K+ , which in turn activates voltage-gated Ca2+ channels (VGCC) resulting in a continuous Ca2+ influx and increased intracellular calcium concentration ([Ca2+]i)  .In support of the role played by sustained calcium influx in avoiding apoptosis in CGN, it has been shown that apoptotic cell death of CGN chronically cultured in K25 can be induced by blocking their VGCC . Thus, C+ leads to the deactivation of VGCC. This, in turn, results in the inhibition of Ca2+ influx and subsequent cell death. A result that can be prevented by increasing the [Ca2+]i with the addition of a Ca2+ ionophore . All efforts were made to minimize animal suffering and to reduce the number of animals used.6 or 2 × 106 cells/ml in 12 or 6 well culture plates with or without coverslips coated with poly-L-lysine (5 μg/ml). They were maintained for 6–8 days in vitro. Culture medium contained basal Eagle’s medium supplemented with 10% (v/v) heat-inactivated fetal calf serum, 2 mM glutamine, 25 mM KCl, 50 U/ml penicillin, and 50 mg/ml streptomycin. This medium is denoted in the text as K25 and is the control condition. The culture dishes were incubated at 37 °C in a humidified 5% CO2/95% air atmosphere. Cytosine arabinoside (10 mM) was added 24 h after seeding to avoid the presence of non-neuronal cells. To trigger cell death, CGN were treated with staurosporine (0.5 μM), thapsigargin (2 μM), tunicamycin (20 μg/ml), and nifedipine (10 μM) or they were transferred to a medium identical to K25 except that KCl was 5 mM (it is denoted as K5).CGN cultures were prepared as previously described . Briefly6 or 2 × 106 cells/ml) were incubated with MagFluo4-AM (0.25 μM) in the dark and at room temperature for 1 h. Subsequently, Fura2-AM (1.5 μM) was added for 1 h, without removing the first dye. Cells were washed with K25 for 10–30 min and the coverslips were mounted in a device into the cuvette. The dyes were excited at 340, 360, and 380 nm wavelength for Fura-2 and 495 nm wavelength for MagFluo-4 (2+]i (fura-2) and [Ca2+]ER (Magfluo-4) in response to the addition of staurosporine, thapsigargin, nifedipine, or tunicamycin were carried out in CGN maintained in K25. Ca2+ release from the ER for CGN in K25 was stimulated with either histamine or glutamate. Changes in the [Ca2+]i and the [Ca2+]ER were induced by exchanging K25 with K5 or vice versa.CGN (6–7 days) seeded on coverslips  to get Rmin. The background fluorescence was determined at the end by adding Mn2+, and this value was subtracted before doing the ratio 340/380. To correct for coverslip movements and the autofluorescence of staurosporine, the 360 nm fluorescence signal was subtracted from the 340 and 380 nm signals, as previously indicated (To convert Fura-2 fluorescence ratios into [Candicated . Brieflyexc 340 - λexc 360) + C = λexc 340 correctedEc (1): (λexc 380 - λexc 360) + C = λexc 380 correctedEc (2):   :2+ = Kd β [(R - Rmin)/(Rmax – R)]Ec (3): Ca2+]ER in CGN, then changes in the [Ca2+]ER are indicated as ΔF/Fo.MagFluo-4 fluorescence was normalized using the initial signal as Fo. Since we do not know the resting level of the [Ca2+] and for this reason, CGN were incubated with 1 μM rhod-2/AM for 1 h in the dark and at room temperature. However, this dye, in agreement with previous reports  in the dark and at room temperature for 30 min. The cells were washed with K25 for 5–10 min and the coverslip was placed in a cuvette using a homemade holder. The dye was excited at 549 nm and emission was recorded at 575 nm using a QM-8 spectrofluorometer (PTI). Initial fluorescence (Fo) was recorded, and then changes in mitochondrial membrane potential were induced by switching solutions (K25 to K5) and by adding different inducers of apoptosis in K25. At the end of the experiment, FCCP (20 μM) was added as a depolarization control. Negative values of ΔF/Fo reflect depolarization of mitochondrial membrane potential, as shown by the application of FCCP. Simultaneous changes in the ) and incubated overnight at 4 °C with the following specific primary antibodies: 1:1000 rabbit anti-eIF2α, 1:1000 mouse anti-GRP78, and 1:2000 mouse anti-β-Tubulin. β-Tubulin was used as a loading control.Bands were visualized using chemiluminescence according to the manufacturer’s recommendations and exposed to Kodak BioMax-LightFilm. The optical density (O.D.) of each band was divided by the corresponding O.D. for β-tubulin and this ratio was compared to the one obtained from CGN extracts maintained in K25 .CGN grown for 6–7 days on coverslips were incubated with the combination of TMRE  and MagFluo-4  or with Rhod-2  in the dark and at room temperature. Cells were washed with K25 for 5–10 min. The coverslip was mounted to a recording chamber placed on the stage of the microscope. The fluorescence images were collected using a confocal microscope Carl Zeiss LSM 700 with a 63x oil immersion objective. The excitation wavelengths were 488 nm for Mag-fluo-4 and 555 nm for Rhod-2 and TMRE.post hoc test or by the non-parametric analysis Kruskal–Wallis followed by the Dunnett post hoc test; p-values less than 0.05 were considered statistically significant.Statistical analysis was done by using SigmaPlot 12.1 software. Data are expressed as means ± SEM. Pairwise comparison within multiples groups was made by analysis of variance (ANOVA) followed by the Holm Sidak 2+] that did not colocalize with the mitochondrial membrane potential indicator . This elevation was followed by a slow rise in the [Ca2+]ER (green trace) without any significant change in the mitochondrial membrane potential (red trace) for the initial 10 min after membrane depolarization. The application of thapsigargin, to inhibit the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) pump, resulted in a slow reduction in the [Ca2+]ER. In this case, the plasma membrane depolarization with K25 derived in a much larger increase in the [Ca2+]i than the one observed in those CGN with active SERCA pump and as expected, no increase in the [Ca2+]ER was attained  or glutamate (200 μM) to verify the ER Ca2+ store. Both agonists produced a similar initial increase in the [Ca2+]i. Nevertheless, the ER Ca2+ reduction was smaller and slower with histamine  to inhibit this Ca2+ entry. This dihydropyridine reduced the [Ca2+]i similarly, as seen with K5  -a potent and specific inhibitor of SERCA pump - to test2+]i that might result from the partial inhibition of the SERCA pump activity . This is an inhibitor of protein glycosylation, which also produces ER stress . Tunicam2+]i , a nonspecific inhibitor of kinases, which also has a well-established role as an inducer of apoptosis . This kiBoth K5 and staurosporine induce apoptosis in these cells. We have studied the increase in caspase-3 activity caused by K5 in comparison with other inducers of cell death. CGN cultured in K25 were either switched to K5 for 6 h  red bar,Mitochondria is a critical element in the activation of caspase-3, thus, we decided to compare the effect of these chemicals on the mitochondrial membrane potential that had been previously recorded. The same pattern emerged as for activation of caspase-3 when compared with the initial (1 min) mitochondrial membrane depolarization induced by K5 , red bar2+]i , staurosporine, thapsigargin, nifedipine, and tunicamycin, have different mechanisms to induce cell death. Additionally, the ER stress does not seem to be associated with the activation of caspase-3 in CGN. Our data support the scenario where cell depolarization (25K), which is necessary for CGN survival, associates with sustained activation of VGCCs, increased [Ca2+]i, a higher level of the luminal [Ca2+]ER, and a larger mitochondrial membrane potential.We have studied the role played by Ca2+]i in depolarizing conditions (K25). This is the case since both conditions, i.e., the addition of nifedipine in depolarizing medium (K25) or the repolarization of CGN by switching from K25 to K5, produced precisely the same reduction in the [Ca2+]i. These data argue that only L-type VGCCs are involved in the increase in the [Ca2+]i induced by K25. Indeed, it has been shown that L-type VGCCs have a voltage activation curve more negative than the dihydropyridine-insensitive channels .EB-R carried out the experiments, contributed to analyze and interpret the data, participated in the design of the study, and to the draft of the manuscript. MO-A and GD-M carried out some of the experiments. ML-M contributed to obtaining and processing of images. AG-H and JM conceived the study, participated in its design, and contributed to draft the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.