However, the intimate mechanisms underlying the pathology remain obscure. We have reported recently that long-term cultures of rat hippocampal neurons resembling aging neurons are prone to damage induced by Aβ oligomers (Aβo) while short-term cultured cells resembling young neurons are not. In addition, we have also shown that aging neurons display critical changes in intracellular Ca2+ homeostasis including increased Ca2+ store content and Ca2+ transfer from the endoplasmic reticulum (ER) to mitochondria. Aging also promotes the partial loss of store-operated Ca2+ entry (SOCE), a Ca2+ entry pathway involved in memory storage. Here, we have addressed whether Aβo treatment influences differentially intracellular Ca2+ homeostasis in young and aged neurons. We found that Aβo exacerbate the remodeling of intracellular Ca2+ induced by aging. Specifically, Aβo exacerbate the loss of SOCE observed in aged neurons. Aβo also exacerbate the increased resting cytosolic Ca2+ concentration, Ca2+ store content and Ca2+ release as well as increased expression of the mitochondrial Ca2+ uniporter (MCU) observed in aging neurons. In contrast, Aβo elicit none of these effects in young neurons. Surprisingly, we found that Aβo increased the Ca2+ transfer from ER to mitochondria in young neurons without having detrimental effects. Consistently, Aβo increased also colocalization of ER and mitochondria in both young and aged neurons. However, in aged neurons, Aβo suppressed Ca2+ transfer from ER to mitochondria, decreased mitochondrial potential, enhanced reactive oxygen species (ROS) generation and promoted apoptosis. These results suggest that modulation of ER—mitochondria coupling in hippocampal neurons may be a novel physiological role of Aβo. However, excess of Aβo in the face of the remodeling of intracellular Ca2+ homeostasis associated to aging may lead to loss of ER—mitochondrial coupling and AD.Alzheimer’s disease (AD) is the most common neurodegenerative disorder and strongly associated to aging. AD has been related to excess of neurotoxic oligomers of amyloid β peptide (Aβo), loss of intracellular Ca Consequently, a wide amount of studies has shown that the disturbance of Ca2+ homeostasis is associated with the neurotoxicity that occurs in AD  and mitochondria plays an important role in the regulation of Ca2+ during aging and AD  is the most common form of dementia, accounting for 60%–80% of all dementia cases. Despite of all the efforts, the mechanisms underlying the pathology are still unknown. The two main hallmarks of AD are extracellular plaques composed of amyloid β (Aβ) peptide and intracellular neurofibrillary tangles composed of hyper-phosphorylated and misfolded tau. Most AD cases are sporadic (late-onset AD) and affect elderly people, but some cases (around 5%) are inherited in an autosomal dominant fashion, caused by mutations in the genes that encode presenilin1 (PS1), PS2 and amyloid precursor protein . One of the most accepted hypothesis of AD is the “amyloid cascade hypothesis,” which postulates that the deposition of the Aβ is a central event to the pathology of AD  will open, mitochondrial membrane potential will collapse and proapoptotic factors such as cytochrome c will be released, activating the caspase pathway and triggering apoptosis. In addition, an excessive Ca2+ increase will enhance the production of reactive oxygen species (ROS), that will also contribute to the opening of the PTPm . They play a central role in the processes that occur between ER and mitochondria, such as communication between the two organelles, which includes Ca2+ into the cell, causing mitochondrial Ca2+ overload and cell death by apoptosis  in rat hippocampal neurons , are able to alter: (1) resting levels of cytosolic free Ca2+ concentration ([Ca2+]cyt); (2) SOCE; (3) the content and release of Ca2+ from the internal stores, as well as; (4) the Ca2+ transfer from ER-to mitochondria; (5) the interaction ER-mitochondria; and (6) the expression of the mitochondrial Ca2+ uniporter (MCU) and inositol trisphosphate receptors (IP3Rs) in young and aged hippocampal neurons. In order to model aging, we have employed an in vitro culture of hippocampal rat neurons maintained for several days in vitro (DIV).We have previously described an intraorganellar Cain vitro model of aging, such as accumulation of ROS, lipofuscin granules, heterochromatic foci, activation of the Jun N-terminal protein kinase (pJNK) and p53/p21 pathways and changes in NMDA receptor expression  and aged  hippocampal neurons cultured in vitro and exposed to Aβo as a model of AD and in vitro aging.Primary rat hippocampal neurons in long-term culture have been previously used as a model of aging by our group and others . IP3R3 primary antibody is from BD Transduction Laboratories . ER tracker, mitotracker, tetramethylrhodamine, methyl ester (TMRM) and CM-H2DCFDA are from ThermoFisher Scientific. Aβ1–42 peptide is from Bachem AG . Other reagents and chemicals were obtained either from Sigma or Merck.Wistar rat pups (newborn P0–1) were obtained from the Valladolid University animal facility. All animals were handled according to the ethical standard of Valladolid University under protocols approved by the animal housing facility and in accordance with the European Convention 123/Council of Europe and Directive 86/609/EEC. Fura-2/AM and Rhod-5N are from Invitrogen . Fetal bovine serum (FBS) is from Lonza . Horse serum, neurobasal medium, HBSS medium, B27, L-glutamine and gentamycin are from Gibco . Papain solution is from Worthington . The poly-D-lysine and annexin V are from BD . DNase I and antibody against the MCU are from Sigma . IPg for 5 min and pellet was suspended in Neurobasal medium. Hippocampal cells were plated onto poly-D-lysine-coated, 12 mm diameter glass coverslips at 30 × 103 cells/dish , and grown in Neurobasal media supplemented with L-glutamine (2 mM), gentamicin (1 μg/ml), 2% B27 and 10% FBS, maintained in a humidified 37°C incubator with 5% CO2 without further media exchange. Cells were cultured for 4–8 DIV for young cultures, and 15–21 DIV for aged cultures. Other details have been reported in detail elsewhere  were prepared as previously reported by a new procedure , and separated into aliquots in sterile microcentrifuge tubes. The solution was then incubated for 2 h at room temperature (RT) to allow monomerization of the peptide. HFIP was removed under vacuum in a speed vacuum (800 g × 10 min at RT), and the peptide film was stored desiccated at −20°C. For aggregation, the peptide was first suspended in dry DMSO at 5 mM concentration. For complete suspension of the peptide, it was subjected to ultrasounds for 10 min, aliquoted in propylene non-siliconized tubes, and stored at −20°C until use. MEM (Gibco) medium supplemented with 0.5 mg/ml Fe2+, 0.5 mg/ml Cu2+ and 0.5 mg/ml Zn2+ was added to bring the peptide to a concentration of 80 μM. Finally, it was incubated at 37°C for 24 h. For experiments, Aβ1–42 was solved in the culturing media to 1 μM final concentration. Composition of these oligomers has been characterized previously  and apoptosis was evaluated using annexin V  in annexin binding buffer 1× (in mM) NaCl, 140; CaCl2, 2.5; Hepes, 10 (pH 7.4) and assessed by fluorescence microscopy using a Nikon Eclipse TS100 microscope (objective 40×).2 1, MgCl2 1, glucose 10 and Hepes/NaOH 10 (pH 7.4). Then cells were incubated in the same medium containing fura-2 (4 μM) for 60 min at RT in the dark. Then coverslips were placed on the perfusion chamber of a Zeiss Axiovert 100 TV, perfused continuously with the same pre-warmed medium at 37°C and epi-illuminated alternately at 340 and 380 nm using a filter wheel. Light emitted at 520 nm was recorded every 1–5 s with a Hamamatsu ER camera (Hamamatsu Photonics France). Pixel by pixel ratios of consecutive frames were captured and cytosolic Ca2+ concentration ([Ca2+]cyt) values from regions of interest (ROIs) corresponding to individual neurons were averaged and expressed as the ratio of fluorescence emission following excitation at 340 and 380 nm as reported in detail previously  were averaged from responsive neurons . Fraction was calculated by dividing responsive cells for the total cell number in the field, considering responsive cells the ones showing a change in the slope of the Ca2+ recording after application of the stimulus.Hippocampal cells were cultured for 4–8 DIV (short-term) or 15–21 DIV (long-term) and exposed to either vehicle (supplemented MEM) or 1 μM Aβo for 24 h before the experiment. The day of the experiment, hippocampal cells were washed in standard external medium (SEM) containing (in mM) NaCl 145, KCl 5, CaCl2+ ATPase (SERCA) pump blocker thapsigargin (1 μM) for 15 min in the same SEM devoid of extracellular Ca2+ before imaging as reported elsewhere  added in the absence of extracellular Ca2+ was monitored as reported previously  values from ROIs corresponding to individual neurons were averaged and expressed as normalized fluorescence emission (F/F0). For estimation of Ca2+ increase, responses  were averaged from responsive neurons . Fraction was calculated by dividing responsive cells for the total cell number in the field, considering responsive cells the ones showing a change in the slope of the Ca2+ recording after application of the stimulus.Hippocampal cells cultured for 4–8 DIV (short-term) or 15–21 DIV (long-term) were exposed to either vehicle or 1 μM Aβo for 24 h before the experiment. The day of the experiment, hippocampal cells were washed in SEM and incubated in the same medium containing Rhod-5N AM (1 μM) for 30 min at RT in the dark and washed in SEM for additional 30 min. Then coverslips were placed on the perfusion chamber of a Zeiss Axiovert 100 TV, perfused continuously with the same pre-warmed medium at 37°C and epi-illuminated in the red channel (551 nm). Light emitted at 576 nm was recorded every 5 s with a Hamamatsu ER camera (Hamamatsu Photonics France). Pixel by pixel ratios of consecutive frames were captured and mitochondrial Ca3 receptors IP3R1 (1:50), IP3R2 (1:50) and IP3R3 (1:50) at 4°C overnight. Immunopositive cells were revealed using Alexafluor 488-tagged antibodies (1:300). Optical density was measured in selected ROIs corresponding to individual neurons using ImageJ software. Further details have been reported elsewhere , fixed with p-formaldehyde 4% and incubated with antibodies against MCU (1:200) and IPCells in every condition were stained for 10 min with 200 nM MitoTracker green and ERTracker red and imaged directly using a confocal microscope. High resolution images of cells were recorded by using a Leica TCS SP5 confocal microscope . The Mitotracker and ER-tracker were excited with 488 and 543 laser lines, respectively, and emission was acquired with a charged CCD camera. The images were analyzed in LAS AF Lite software . Background was subtracted from all images. Colocalization analysis for the two markers was performed on Z-stacks acquired with steps of 0.8 μm. For colocalization analysis, green and red channel images were acquired independently. Analysis were carried out on single-plane images using ImageJ plug-ins. Manders2 and Pearson’s coefficients were calculated applying the ImageJ Colocalization analysis plug-in.For mitochondrial membrane potential measurements, hippocampal neurons were exposed to either vehicle or Aβo 1 μM for 24 h. Then, cells were washed in SEM and loaded with the mitochondrial potential probe  for 30 min at RT in the dark. Then coverslips containing cells were placed on a Zeiss Axiovert 100 TV inverted microscope and subjected to fluorescence imaging. Fluorescence images were captured with the rhodamine filter set with a Hamamatsu ER-Orca fluorescence camera as reported previously -chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate (CM-H2DCFDA 2 μM) for 40 min at RT in the dark. Then coverslips containing cells were placed on a Zeiss Axiovert 100 TV inverted microscope and subjected to fluorescence imaging. Fluorescence images were captured with the FITC filter set with a Hamamatsu ER-Orca fluorescence camera.t-test was used. For more than two groups, statistical significance of the data was assessed by one-way or multifactorial analysis of variance (ANOVA), depending on the number of factors considered. Differences were considered significant at p < 0.05, where 0.05 is the signification level.Changes in fluorescence ratio are expressed as area under the curve (AUC) and maximum increase in ratio (Δratio). Calculation of AUC and Δratios were performed using Origin Lab 7.0. Data are presented as mean ± SEM. When only two means were compared, Student’s 1–42 oligomers (Aβo) on the rate of apoptosis in rat hippocampal neurons cultured for 4–8 DIV representing young neurons, or in neurons cultured for 15–21 DIV that represent many characteristics of aged neurons as reported previously  when compared to the control neurons (2+]cyt oscillations compared to the control group measured as oscillations index (OI), a parameter representing both the frequency and amplitude of [Ca2+] oscillations  or Aβo , and SOCE was monitored by fluorescence imaging of [Ca2+]cyt. For this end, cultured neurons were loaded with fura-2 and treated with Tg (1 μM) for 15 min in medium devoid of extracellular Ca2+ (0 Ca) in order to fully deplete the intracellular Ca2+ stores. Subsequently, medium containing Ca2+ 5 mM and TTX (500 nM) was added for 5 min to activate SOCE. TTX was added to block the electrical connectivity of cultured neurons. La3+ (100 μM) was added later to block this pathway. Pictures in 2+]cyt before  and after addition of 5 mM Ca2+ (5 Ca). Representative recordings of fluorescence ratios of individual short-term , and compared to the controls . These experiments were always carried out in medium devoid of extracellular Ca2+ (0 Ca), in order to avoid the entry of Ca2+ from the external medium. The stimulation of hippocampal neurons with 400 nM ionomycin in Ca2+ free medium . Exposure of the short-term cultured cells to Aβo did not modify the increase in [Ca2+]cyt induced by ionomycin . In order to cancel the contribution of the entry of Ca2+ and just focus on the release from the stores, all the experiments were carried out in the absence of free Ca2+ in the extracellular medium (0 Ca). Application of ACh to cultured hippocampal neurons in 0 Ca produced an increase in the [Ca2+]cyt, as described previously  of the Ca2+ response  was calculated. For this end we multiplied the AUC of the cells that showed an increase in the [Ca2+]cyt by the fraction of cells that responded to the stimulus. 2+ release from the stores among the four populations. Notice that whereas aging increases the fraction of neurons responsive to ACh, treatment with Aβo enhances instead the release of Ca2+ in each individual cell. Thus, aging increases the fraction of neurons responsive to ACh and Aβo enhances the rise in Ca2+ elicited by ACh in aged neurons. No effect was observed in young neurons.In order to understand the functional consequences of the increase in the levels of Ca neurons . In contol cells . Figuresresponse . The frao showed . It can 2+ release induced by activation of ryanodine receptors (RyRs) in short-term and long-term cultured neurons. Caffeine  was employed as an agonist of the RyRs. Caffeine is an agonist of the RyRs, which are sensitive to Ca2+-induced Ca2+ released (CICR) processes. In this case, nominally free Ca2+ (without adding EGTA) was used as external cellular medium to be able to activate CICR, while minimizing the effect of Ca2+ entry from the extracellular medium into the cytosol. To avoid high osmotic variation following the application of 20 mM Caff, 20 mM glucose was added to the extracellular medium prior to its addition, subsequently replaced by 20 mM Caff . ER Ca2+ mobilized by caffeine was similar in the young neurons (exposed or not exposed to 1 μM Aβo for 24 h) and in the non-exposed cultured-aged neurons. However, in cultured-aged neurons previously exposed to Aβo , the increase in [Ca2+]cyt was significantly higher than in the control group. 2+ increase × fraction) produced by stimulation with caffeine under these conditions. Therefore, Ca2+ release induced by both IP3 receptor and RyR agonists is further enhanced by Aβo treatment in aged neurons but not in young neurons.Next, we assessed the effects of Aβo on Caobserved . The bar2+ transfer from ER to mitochondria. For this end, we measured the increase in mitochondrial [Ca2+] ([Ca2+]mit) induced by Ca2+ release elicited by ACh in medium devoid of Ca2+ (0 Ca), in the same manner as for 2+ probe Rhod-5N, a fluorescent calcium probe that targets mitochondria  to induce the release of Ca2+ from the ER. 2+]mit obtained in single cells before and after stimulation with ACh are also shown. Bars represent the quantification of the rises in [Ca2+]mit induced by ACh in 0 Ca (left), the fraction of responsive cells (middle) and the global response by taking into account both factors (right). As shown previously using mitochondria-targeted aequorin  and the fraction of responsive cells (middle panel) corresponding to a large global ER-mitochondria cross talk in aged neurons relative to young neurons  and this effect was not due to changes in the fraction of responsive cells (middle panel). Therefore, Aβo increase ER-cross talk in young neurons  or Aβo . Then, immunostaining against MCU was evaluated by immunofluorescence. Ca2+ release from intracellular stores takes place after activation of IP3 receptors at the ER. In addition, IP3 receptors are an ER component of the MAMs  changes during in vitro aging. 3 receptor isoforms obtained in short-term (young) and long-term (aged) cultured hippocampal neurons. 3R isoforms in young and aged neurons. Results show that optical density of all three IP3R isoforms is significantly increased in aged neurons relative to young neurons. These results indicate that changes in expression of IP3 receptors may contribute to enhanced response to Aβo in aged neurons and enhanced ER-mitochondria cross talking in aging.CaWe investigated ER-mitochondria colocalization using confocal fluorescence imaging of young and aged rat hippocampal neurons loaded with both MitoTracker green and ER-Tracker red. 2+ transfer from ER to mitochondria in young neurons . Then cells were loaded with the ROS probe CM-H2DCFDA and subjected to fluorescence imaging. Accordingly, the present results show that Aβo enhance ER-mitochondrial coupling in short-term cultured hippocampal neurons corresponding to young neurons but this is not associated to a detrimental effect. In contrast, in long-term cultured hippocampal neurons, corresponding to aged neurons, Aβo also increases ER-mitochondrial colocalization, but this effect is associated to loss of ER-mitochondrial coupling and detrimental effect .2+ plays a fundamental role in learning and memory processes, and participates in survival and neuronal death. Intracellular Ca2+ dyshomeostasis has been extensively related to many neurodegenerative diseases, including AD  with Aβ1–42, where an excess release of Ca2+ from intracellular stores was shown after stimulation with thapsigargin in free calcium media (Pannaccione et al., 2+ stores in AD compared with WT mice in vivo, mainly in hyperactive AD cells. In accordance with this line, the chronic exposure of cultured hippocampal neurons to the Aβo notably increases the peak of [Ca2+]cyt induced by both ACh and Caff. This is again pointing to an increased concentration of Ca2+ in the ER after exposure to Aβo. Consistently, previous studies in fibroblast from AD patients (Gibson et al., 2+ release through IP3Rs. In addition, aberrant Ca2+ increases mediated by IP3 in fibroblasts of asymptomatic members of families with AD have also been described (Etcheberrigaray et al., 2+ in animals carrying the PS1 mutation (Chan et al., 1–42 peptides could be responsible for an increase of the isoform RyR3 in transgenic mouse models of AD (Supnet et al., 2+ that is released from the stores when they are activated. Therefore, Ca2+ store content is increased in aged neurons and this process is further enhanced by Aβo. Again, Aβo do not influence Ca2+ store content in young neurons.PSs are integral proteins located in the ER membrane that may form low conductivity channels in the ER, known as 2+ homeostasis by shaping neuronal Ca2+ signaling. Mitochondria and ER are structurally and functionally connected in contact points enriched in MAMs (Csordás and Hajnóczky, 2+ transfer from ER to mitochondria. It has been previously shown that MAM function is altered in the pathology of AD. Area-Gomez et al. (in vitro aging is associated to enhanced Ca2+ transfer from ER to mitochondria in rat hippocampal neurons (Calvo-Rodríguez et al., 2+] induced by ACh in Ca2+ free medium in aged neurons compared to young neurons. Second, the increased rise in [Ca2+]cyt induced by ACh in the presence of the mitochondrial uncoupler FCCP in aged neurons compared to young neurons (Calvo-Rodríguez et al., 2+, Rhod-5N.Mitochondria play also an important role in intracellular Caz et al.  showed a2+ transfer from mitochondria to ER in young neurons. This view is supported by the finding that Aβo enhance the rise in [Ca2+]mit elicited by Ca2+ release induced by ACh without increasing either the resting levels of [Ca2+]cyt, the Ca2+ store content or the fraction of cells responsive to ACh. This effect is also independent of changes in the expression of MCU, the calcium channel involved in mitochondrial Ca2+ uptake. Thus, the most likely explanation for our results is that Aβo oligomers reinforce the relative position and/or coupling between ER and mitochondria. Consistently with this view, it has been shown that Aβ peptide increases inositol-1,4,5-triphosphate receptor and voltage-dependent anion channel protein expression and elevates the number of ER-mitochondria contact points and mitochondrial calcium concentrations in cultured hippocampal neurons (Hedskog et al., 3 receptor isoforms, including IP3R1, IP3R2, and IP3R3, is increased in aged neurons, which may enhance ER-mitochondrial coupling in aging. In addition, confocal imaging of living neurons and colocalization analysis indicated increased ER-mitochondria colocalization in aged neurons relative to young neurons. Moreover, colocalization was intensified by Aβo in both young and aged neurons. The effects of Aβo on ER—mitochondria coupling in young neurons are not necessarily detrimental since they may favor increased Ca2+ transfer between ER and mitochondria to support increased energy demands and Aβo do not induce apoptosis in young neurons, suggesting a physiological role for Aβo in the modulation of ER—mitochondria coupling.Interestingly, we show now that Aβo enhance Ca2+]mit induced by ACh. These results cannot be explained by lack of ACh receptors as [Ca2+]cyt responses to ACh are indeed enhanced in aged neurons treated with Aβo. They cannot be explained either by depletion of Ca2+ stores since Ca2+ stores are actually enhanced in aged neurons treated with Aβo. Finally, lack of Ca2+ transfer from ER to mitochondria in aged neurons treated with Aβo cannot be attributed either to MCU downregulation as MCU expression is actually enhanced in aged neurons treated with Aβo. These results suggest that, contrary to our expectations, Aβo nearly abolish ER—mitochondria cross talk in aged neurons and this effect, together with exacerbated remodeling of intracellular Ca2+ induced by aging, is highly detrimental as Aβo induces apoptosis to a large fraction of aged neurons.Surprisingly, and in striking contrast with the results in young neurons, exposure of aged neurons to Aβo decreased rather than increased the rise in [Ca2+ transfer from ER to mitochondria is required to maintain the Krebs cycle (Cárdenas et al., 2+ transfer from ER to mitochondria in aged neurons treated with Aβo may compromise OXPHOS activity leading to mitochondrial damage and collapse of the mitochondrial potential. However, excess of Ca2+ transfer associated to enhanced Ca2+ store content may lead to mitochondrial Ca2+ overload.It is well established that an adequate flux of Cain vitro aging is associated to a partial loss of mitochondrial membrane potential and this loss is further decreased by Aβo in both young and aged neurons. The effect is mild in young neurons. However, aged neurons treated with Aβo display a quite large loss of mitochondrial membrane potential, thus reducing rather dramatically the driving force for mitochondrial Ca2+ uptake. In addition, we also found that aged neurons show increased ROS generation compared with young neurons. Moreover, Aβo increase further ROS generation in aged neurons having no effect in young neurons. Accordingly, loss of mitochondrial membrane potential and enhanced ROS generation induced by Aβo in aged neurons may constrain Ca2+ transfer from ER to mitochondria.Several studies by others have proposed that mitochondrial dysfunction is an early event the pathology of AD. Yao et al.  describe2+ transfer from ER to mitochondria despite Ca2+ stores are overloaded and the expression of molecular players involved in Ca2+ transfer increases, thus leading to mitochondrial damage and apoptosis. It has been reported that MAM-associated proteins are up-regulated in AD brain and APP Swe/Lon mouse models (Hedskog et al., 2+ store content associated to age and/or AD-related mutations, the further rise in ER-mitochondria coupling likely induced by Aβo may somehow collapse the own coupling leading to neuron damage. Further research is required to address this critical question. In addition, all the above results have been obtained in identified neurons in an in vitro model of neuronal aging. As stated above, the fraction of glial cells in hippocampal cultures increases with in vitro aging. Whether glial cell may contribute to Ca2+ remodeling in aged neurons treated with Aβo remains to be established.In summary, we show that Aβo modulate differentially ER–mitochondria cross talk in young and aged neurons. In young neurons, Aβo increase ER–mitochondria coupling without having a detrimental effect. In contrast, in aged neurons, although Aβo increase also ER-mitochondria colocalization, the loss of mitochondrial membrane potential and enhanced ROS generation impairs Ca2+ remodeling is a hallmark of the aging that is exacerbated in the presence of Aβo. Aging decreases SOCE required for spine stability and increases Ca2+ store content and MCU and IP3 receptors expression that favors Ca2+ transfer from ER to mitochondria. Most of these effects are exacerbated by Aβo but only in aged neurons. In contrast, in young neurons Aβo increase ER-mitochondrial colocalization and Ca2+ transfer from ER to mitochondria without having effects on ROS generation or apoptosis and only a minor decrease in mitochondrial membrane potential, probably related to increased use of this potential for ATP synthesis. However, the effect of Aβo may become highly detrimental in the aged neuron and the mechanism for this switch may play a fundamental role in AD and warrants further consideration.Taken together, our data show that intraorganellar CaMC-R, LN and CV designed the study and wrote the manuscript. MC-R and EH-P performed research and analyzed data.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 tumor microenvironment (TME) has a strong influence on the progression, therapeutic response, and clinical outcome of acute myeloid leukemia (AML), one of the most common hematopoietic malignancies in adults. In this study, we identified TME-related genes associated with AML prognosis. Gene expression profiles from AML patients were downloaded from TCGA database, and immune and stromal scores were calculated using the ESTIMATE algorithm. Immune scores were correlated with clinical features such as FAB subtypes and patient’s age. After categorizing AML cases into high and low score groups, an association between several differentially expressed genes (DEGs) and overall survival was identified. Functional enrichment analysis of the DEGs showed that they were primarily enriched in the immune response, inflammatory response, and cytokine activity, and were involved in signaling processes related to hematopoietic cell lineage, B cell receptor, and chemokine pathways. Two significant modules, dominated respectively by CCR5 and ITGAM nodes, were identified from the PPI network, and 20 hub genes were extracted. A total of 112 DEGs correlated with poor overall survival of AML patients, and 11 of those genes were validated in a separate TARGET-AML cohort. By identifying TME-associated genes, our findings may lead to improved prognoses and therapies for AML. Acute myeloid leukemia (AML) is one of the most prevalent and aggressive blood cancers in adults, accounting for about 1% of all cancers . In the AML prognosis is currently determined by increasing age, white blood cell counts at diagnosis, cytogenetic abnormalities, and AML-specific molecular genetic lesions , 12. AltMuch attention has been devoted in recent years to the role of the tumor microenvironment (TME) in cancer development . ConsequAlgorithms that evaluate and rank immune and stromal populations within the TME, such as the Estimate of STromal and Immune cells in MAlignant Tumor tissues using Expression data (ESTIMATE) , have beIn the present study, gene expression profiles and clinical information of AML cohorts were downloaded from TCGA, and the ESTIMATE algorithm was then used to calculate immune and stromal scores for these AML cases. Following classification into high- and low-score groups, we identified a subset of TME-associated genes that predict outcome in patients with AML. The correlation between the expression of these genes and AML prognosis was independently validated in a TARGET AML cohort from UCSC Xena database. These findings may contribute to better understand the role of the TME in AML and might lead to improved prognosis and therapies for this disease.Gene expression profiles and associated clinical data of 173 AML patients were retrieved from TCGA database. Among patients, 80 (46.2%) were female and 93 (53.8%) were male. Pathological diagnosis identified 16 cases of FAB M0 (undifferentiated subtype), 42 FAB M1 cases, 39 FAB M2 cases, 16 FAB M3 cases, 35 FAB M4 cases, and 19 cases of FAB M5 . Immune To assess the correlation between immune and stromal scores and overall survival, AML patients were divided into corresponding high- and low-score groups. We found that cases with a low immune score had significantly longer overall survival than those with a high immune score  was achieved for a total of 531 GO terms of biological process, 64 GO terms of molecular function, and 61 GO terms of cellular component. The top 30 GO biological process terms indicated that the DEGs were primarily enriched in ‘regulation of immune response process’, ‘activity of neutrophils and leukocytes’, ‘cytokine secretion’, ‘inflammatory response’, and ‘regulation of tumor necrosis’ . MoleculTo analyze potential connectivity patterns between the transcripts of our DEG set, a protein-protein interaction (PPI) network was constructed using the STRING database. The network revealed two significant modules . We callTo explore the prognostic utility of individual DEGs on overall AML survival, Kaplan-Meier (K-M) survival curves were generated by the survival package in R. In total, 112 genes  is a differentiation marker for cells of the myeloid-monocytic lineage . UpregulCorrelation analysis of individual DEGs identified 112 genes, including 9/20 top hub genes in the PPI network, in association with poor overall AML survival. Functional enrichment analysis confirmed the involvement of these hub genes in immune-related processes. A separate cohort (TARGET-AML) of 187 AML cases from UCSC Xena database was used to validate the survival analysis. Results showed that 11 DEGs from our TCGA AML cohort were also significantly linked to poor overall survival in the TARGET-AML cohort. Most of the validated genes, such as IL15 , ITGAM , PDK4 5, 53, andIL15 and ITGAM, two hub genes from the main PPI modules, are of particular interest. As a proinflammatory cytokine, IL15 has a variety of functions in the immune system and in the generation of multiple lymphocyte subsets , affectiITGAM  was the In summary, our study used the ESTIMATE algorithm to define a set of TME-related DEGs based on immune and stromal scores from TCGA AML data. Correlation analysis of the expression of these genes with patients’ overall survival was performed and results independently validated in the TARGET-AML cohort. Further studies on the DEGs identified here should help clarify the mechanisms by which gene expression within the TME influences the prognosis and progression of AML, and guide the development of more effective therapies.https://portal.gdc.cancer.gov/). Clinical data, including gender, age, French-American-British (FAB) classification [https://xenabrowser.net/datapages/). Clinical follow-up information was also downloaded.Gene expression profiles of 173 AML patients were downloaded from TCGA database  were identified between high and low score groups stratified by the median value of immune scores and stromal scores using limma package . Genes whttps://biit.cs.ut.ee/clustvis/) [Heatmap and clustering were performed using the online tool ClustVis (ustvis/) .Functional enrichment analysis of DEGs was conducted by clusterProfiler  R packagTo explore potential relationships among DEGs, a protein-protein interaction (PPI) network was retrieved from the STRING database  and recoThe survival R package was used to analyze the relationship between the expression of DEGs (including hub genes) and patients’ overall survival using the log-rank test. In addition, Pearson’s correlations between expression data of the validated genes and CXCR4, VLA-4, and E-selectin expression were also obtained. P < 0.05 was considered statistically significant.This article does not contain either human nor animal experiments.Supplementary FiguresSupplementary TablesSupplementary Table 2


Mentoring nurtures a mentee’s personal and professional development. Yet conflation of mentoring approaches and a failure to contend with mentoring’s nature makes it difficult to study mentoring processes and relationships. This study aims to understand of mentee experiences in the Palliative Medicine Initiative (PMI). The PMI uses a consistent mentoring approach amongst a homogeneous mentee population offers a unique opportunity to circumnavigate conflation of practices and the limitations posed by mentoring’s nature. The data will advance understanding of mentoring processes.Sixteen mentees discussed their PMI experiences in individual face-to-face audio-recorded interviews. The two themes identified from thematic analysis of interview transcripts were the stages of mentoring and communication.The 6 stages of mentoring are the ‘pre-mentoring stage’, ‘initial research meetings’, ‘data gathering’, ‘review of initial findings, ‘manuscript preparation” and ‘reflections’. These subthemes sketch the progression of mentees from being dependent on the mentor for support and guidance, to an independent learner with capacity and willingness to mentor others. Each subtheme is described as stages in the mentoring process (mentoring stages) given their association with a specific phase of the research process.Mentoring processes also pivot on effective communication which are influenced by the mentor’s characteristics and the nature of mentoring interactions.Mentoring relationships evolve in stages to ensure particular competencies are met before mentees progress to the next part of their mentoring process. Progress is dependent upon effective communication and support from the mentor and appropriate and timely adaptations to the mentoring approach to meet the mentee’s needs and goals. Adaptations to the mentoring structure are informed by effective and holistic evaluation of the mentoring process and the mentor’s and mentee’s abilities, goals and situations. These findings underline the need to review and redesign the way assessments of the mentoring process are constructed and how mentoring programs are structured. Mentoring in Palliative Medicine has been shown to enhance clinical skills –5, inculdynamic, context dependent, goal sensitive, mutually beneficial relationship between an experienced clinician (mentor) and junior clinicians and/or undergraduates (mentee) that is focused upon advancing the development of the mentee” the PMI’s novice mentoring circumnavigates the limitations posed by mentoring’s nature and conflation of mentoring practices and provides the basis for the PMI’s novice mentoring approach  would readily take the research load off me so that I could settle all these issues without having to worry about the research tasks.” (Mentee 7)Mentee’s situation.“When we first started, Dr X’s instructions seemed to be targeted to a higher level. But when Dr X realized that me and my colleague were doing this for the first time Dr X tailored the guidance to our level.” (Mentee 10)Mentor’s evaluation of the mentee’s abilities.This stage saw mentees take on more responsibilities and ‘ownership of the process’ as they analyzed the data accrued and discussed the findings with the mentor.“The mentor assigned me research roles and tasks that are suited for my level of experience. Over time these tasks required more reflection and analysis.” (Mentee 3)Manuscript and/or poster preparation motivated mentees.“… as we went along, I realized that this was something real. I can get something out of it. I can churn out paper. I can do a poster, I can present at some conference.” (Mentee 13)Mentee reflections following the completion of the PMI projects focused upon appreciation of different aspects of clinical care and new aspirations.Recognizing the skills needed“medicine is ..an art-form.” (Mentee 9)Other mentees described learning the importance of being sensitive to non-verbal communication and the need to listen to the perspectives of patients and their families.Sociocultural aspects“My mentor brought extra perspective in terms of social cultural factors and how to actually balance patient goals with clinical goals” (Mentee 11)Patient-centered care“It has helped me become more sensitive to the reactions of patients for example.” (Mentee 4)Realizing the need to understand the patient as a person was a common outcome which several mentees described.Holistic care“[the mentoring process has] helped me to be more open-minded and understanding in clinical practice, I’ve learnt to see things from various angles.” (Mentee 6)Another mentee described how the process changed her approach to patient care to orientated around patient’s goals and priorities.Greater appreciation of research“Research does seem more meaningful now, like, they actually have significant impact on medical practice and patient care.” (Mentee 7)Appreciation of clinical care‘Pay it forward’“I used to think that I’ll never ever, ever want to teach students. But I think after going through Dr X. I actually wouldn’t mind mentoring a few students ‘cause I think it provides a positive effect on someone’s life too.” (Mentee 5)Mentees expressed the desire to commit to mentoring others in future, realizing how mentoring can shape the future of medicine.Aspiration to be effective educators and mentors in future.“..now I find myself thinking of new ideas, and thinking of new potential projects that I could undertake” (Mentee 16)Another mentee discussed around mentoring styles, and the need for mentors to respect the mentee and be interested in their well-being in order to be effective mentors.Aspire to be good clinicians“[the program] showed me that I could do much more for patients as a physician than I had previously thought” (Mentee 14)Mentees describing learning from mentors perspective taking and the importance of honoring the humanity of patients as one interacts with them.AspirationsThe three subthemes delineated under this theme were the characteristics of mentors, the nature of communication and the obstacles to mentee-mentor interactions.Characteristics of mentor influenced communication. These includeWelcoming“I felt welcomed as a colleague and a fellow investigator” (Mentee 8)Mentees described feeling a sense of belonging to the mentoring team and felt valued as a team member.Concern“My mentor genuinely cared about our well-being” (Mentee 16)Several mentees expressed that mentors’ constant enquires about their well-being helped mentees to feel supported.Motivating“..you can see that he’s very enthusiastic. The enthusiasm you know, it rubs off” (Mentee 13)Mentees described that the mentors’ consistently encouraged and motivated them to persevere on to complete their projects.Individualized“If my psychosocial issues outside of research weren’t settled, and on not too few occasions without my mentor’s help, I would have given up on the projects long ago.” (Mentee 11)Other mentees described how the mentors were understanding when they experienced other life stressors and how help was rendered during these periods to lighten their workload.Encouraging“He encourages you, he doesn’t pressurize you” (Mentee 13)Several mentees expressed appreciating the mentors’ affirmation and positive feedback which validated their efforts. Mentees also shared that the mentors’ provided much needed moral support through the mentoring process.Constructive“..it was very important to have the feedback, so that I can do it better”(Mentee 8)Several mentees emphasized the importance of feedback as they developed through the mentoring process.nature of mentor-mentee interactions revealed a number of unique featuresEvolved from a formal hierarchical to a more casual style“My text[s] became less formal … it becomes a lot easier to ask questions” (Mentee 15)“…slowly it becomes more comfortable. Cause at first, we don’t really know each other. After that we became friends” (Mentee 10)Reciprocal, where feedback from mentees shaped the mentoring approach‘When the mentor realized that, it’s the first time doing this, he tailored things to my level’ …the [mentor’s] feedback allowed me to gain confidence” (Mentee 7)Responsive“I could get a little bit stressed, ‘cause I didn’t know what to do” (Mentee 13).Nearly all mentees were anxious at each new stage of the mentoring process. “My mentor’s guidance made me feel more confident in contributing to the paper and have a greater sense of ownership over the project.” (Mentee 1)These concerns were overcome by specific guidance and personalized support provided. A sense of professional ‘distance’ remained“My mentor managed to maintain a professional enough relationship, without becoming too friendly, which can sometimes be detrimental for the mentoring process” (Mentee 5)Sounding board for the mentee’s new concepts and proposals.“I also liked how he is open to hearing my random ideas… basically I bounce ideas off him, which helps me learn a lot really” (Mentee 3)Other mentees described a non-judgmental platforms where they were free to express their ideas.The Obstacles to mentee-mentor interactions pivot on poor communication and feedback and a lack of organizational support for the mentoring process. Poor communication and feedback saw mentees struggle to comprehend the tasks assigned to them and the expectations upon them leading to poor mentoring relationships.“The mentor’s instructions were targeted at someone at higher level of competency rather than a novice” (Mentee 13)It also saw mentees intimidated by the views and had difficulty expressing their own ideas.“I still felt that I’m not good enough to contribute” (Mentee 8)A lack of protected time and a lack of recognition of a student’s participation in the mentoring process threatened the mentoring process. Mentees cited a lack of time and competing commitments as key obstacles to effective participation in the PMI program.The 2 themes identified in this study provide the first clinical evidence of mentoring’s nature proposed by recent reviews of mentoring practice in Palliative Medicine, Family Medicine, Internal Medicine, nursing, social work, occupational therapy and physiotherapy , 71, 72.Mentoring stages may be viewed as ‘circumscribed sequential projects’ with ‘specific goals and competency requirements’ that build on one another to achieve an overarching goal. Each stage requires the mentee to attain specific skills, insights and abilities, making progress from one mentoring stage to the next, highlighting a competency-based process. This is in stark contrast to time-based progress in mentoring and has a number of ramifications.Competency-based mentoring stages are driven by assessments of a mentee’s progress, ability and motivation. Whilst use of assessments runs contrary to some prevailing mentoring theories a competency-based approach necessitates such evaluations the mentee’s and mentor’s psychosocial, academic, research and clinical situations and modifications in the host organization’s situation given their impact upon progress and attainment of in competencies , 72, 79.A personalized and competency-based approach to the PMI’s novice mentoring approach underscores its need to adapt its mentoring approach to accommodate to diverse influences upon the mentoring process without compromising mentoring experiences and breaching prevailing standards of practice and codes of conduct. Adaptability within the mentoring approach is evident at all mentoring stages, from the determination of specific goals and outcomes within agreed upon timelines and roles and responsibilities at the pre-mentoring stage to personalization’s of the initial research meeting, data gathering, review of findings and manuscript preparations stages. Adaptability within the PMI novice mentoring approach is also apparent in the characteristics of communication at different junctures of the mentoring process. Both examples highlight the influence of assessments in informing adaptations to the mentoring process.Such adaptability however does not threaten the consistency and oversight of the PMI mentoring process courtesy of its structured research program. The presence of clear objectives and outcomes of the project and the agreed upon goals, roles and responsibilities, timelines and a code of conduct based upon professional and academic standards of practice ensures consistency in mentoring experiences, interactions, success and reflections.Whilst an interview approach to appraising mentoring relationships and programs have been determined to be the gold standard, use of retrospective data at a single time point a variable distance from the end of the mentoring process invites recall bias . The preThe small sample size and a uniquely structured approach around a research process may limit the applicability of these findings in other settings.We believe that this study successfully answers the question “what are the experiences of mentees in a novice mentoring program?” providing new insights into mentoring experiences and process in a structured novice mentoring program that employs a consistent mentoring approach. This study shows mentoring to be a dynamic progressive process through which mentors advance through various stages to complete a research project. This study also found that communication is the main vehicle through which growth and maturation occurs.Cartesian reductionism and Newtonian principles of linearity” [Whilst there is more to be understood about the role of the mentor, it is clear that for mentors to be responsive, provide holistic and timely support and address the evolving needs of the mentee and the mentoring relationship, mentors must be trained to evaluate the mentoring relationship and the mentee’s needs and progress. Effective tools that move beyond prevailing reliance upon “nearity”  are requIn addition, the mentoring program should be guided by a balance between flexibility to meet the changing needs of the mentee, mentor, the host organization and their mentoring relationship and structure in the form of mentoring standards, codes of conduct and standards of practice that confines practice to acceptable parameters. Whilst the clearly demarcated research phases around which the PMI is created aids in ensuring that any changes to the mentoring program remains within the structure of the program, there is little reason to assume that similarly structured programs would not be equally successful in other clinical settings beyond the Palliative Medicine setting.S1 Table(XLSX)Click here for additional data file.


In their intracellular environment, RyR channels are regulated by a variety of cytosolic and luminal factors so that their output signal (Ca2+) induces finely-graded cell contraction without igniting cellular processes that may lead to aberrant electrical activity (ventricular arrhythmias) or cellular remodeling. The importance of RyR dysfunction has been recently highlighted with the demonstration that point mutations in RYR2, the gene encoding for the cardiac isoform of the RyR (RyR2), are associated with catecholaminergic polymorphic ventricular tachycardia (CPVT), an arrhythmogenic syndrome characterized by the development of adrenergically-mediated ventricular tachycardia in individuals with an apparently normal heart. Here we summarize the state of the field in regards to the main arrhythmogenic mechanisms triggered by RyR2 channels harboring mutations linked to CPVT. Most CPVT mutations characterized to date endow RyR2 channels with a gain of function, resulting in hyperactive channels that release Ca2+ spontaneously, especially during diastole. The spontaneous Ca2+ release is extruded by the electrogenic Na+/Ca2+ exchanger, which depolarizes the external membrane (delayed afterdepolarization or DAD) and may trigger untimely action potentials. However, a rare set of CPVT mutations yield RyR2 channels that are intrinsically hypo-active and hypo-responsive to stimuli, and it is unclear whether these channels release Ca2+ spontaneously during diastole. We discuss novel cellular mechanisms that appear more suitable to explain ventricular arrhythmias due to RyR2 loss-of-function mutations.Ryanodine receptors (RyRs) are the calcium release channels of sarcoplasmic reticulum (SR) that provide the majority of cal-cium ions (Ca In ventricular cardiomyocytes, depolarization of the sarcolemma and its invaginations, the T-tubules, induces the opening of voltage-dependent L-type Ca2+ channels , which permit the entry of a small amount of Ca2+ (ICa) into the cell. In adult cardiomyocytes, ICa is insufficient to elicit full contractions, but is capable of triggering massive release of Ca2+ from the sarcoplasmic reticulum (SR), which then bathes the contractile filaments, binds to Ca2+-binding proteins, and finally induces contraction  promotes stronger contractions (increased inotropism), a characteristic benefit of the fight-or-flight response. But in the setting of CPVT, this Ca2+ overload constitutes a powerful incentive for a group of mutation-deranged hyperactive RyR2 channels to release Ca2+ on their own, that is, to spew Ca2+ in the absence of ICa stimulation. When spontaneous Ca2+ release occurs during diastole, Ca2+ is rapidly extruded by the Na+/Ca2+ exchanger, as mentioned above, generating an inward current and gradually depolarizing the membrane potential, a phenomenon termed “delayed afterdepolarization”, or DAD . Note also that, although hyperactive, mutant RyR2 channels are not portrayed malfunctioning during the systolic phase of Ca2+ release. Most studies that used mice as models of CPVT remark the excessive diastolic Ca2+ leak [2+]i transient.The vast majority of the RyR2-linked CPVT mutations studied to date have been found to endow RyR2 channels with a gain of function, that is, the mutations generate hyper-active or hyper-reactive RyR2 channels, and this finding restricts the cellular basis of the cardiac arrhythmias to a well-defined cascade of events, succinctly described as follows: during sympathetic stimulation, enhanced a2+ leak 17, but 1.32+ during diastole and cause DADs. A reduction in the threshold for activation of mutant RyR2 channels by luminal Ca2+ is one of the most prominent mechanisms [2+ on their own because the luminal [Ca2+] that is reached during basal conditions and even during sympathetic stimulation is below their threshold for activation. In contrast, a CPVT mutation presumably lowers the RyR2 channel’s threshold for luminal Ca2+ activation, and although mutant channels remain “silent” under basal conditions, they “ignite” during the normal sympathetic Ca2+ overload. This scheme has been demonstrated in a great number of, but not all, RyR2 mutations linked to CPVT i) in isoproterenol (300 nmol L−1)-stimulated RyR2-A4860G+/− ventricular myocytes showed prolonged action potentials and interspaced bursts of altered Ca2+ release consisting of a normal peak followed by a second, prolonged phase of release during the systolic phase. Remarkably, early afterdepolarizations (EADs) were observed only during the prolonged phase of release, and were abolished by either the NCX inhibitor CB-DMB extracellularly applied or the Ca2+ chelator EGTA . The RyR2-A4860G loss-of-function mutation thus reveals novel mechanisms of arrhythmogenesis in CPVT: first, mutant RyR2 channels decrease the peak of Ca2+ release during systole and thus impair L-type Ca2+ channel inactivation (faulty retroactive Ca2+ release→AP feedback), both of which gradually overload the SR. The resultant SR overload then causes bursts of prolonged Ca2+ release, which activate electrogenic Na+/Ca2+ exchange activity during the plateau and descending phases of the AP, triggering EADs  and in heterologous expression systems (HEK293 cells), which are poor substitutes for cardiac myocytes . We gene


Unlike other CaCCs, ANO6 requires a non-physiological intracellular free calcium concentration ([Ca2+]i > 1 μM) and several minutes for full activation under a whole-cell patch clamp. Therefore, its physiological role as an ion channel is uncertain and it is more commonly considered a Ca2+-dependent phospholipid scramblase. Here, we demonstrate that physiological temperature (37 °C) increases ANO6 Ca2+ sensitivity under a whole-cell patch clamp; V1 was activated by 1 μM [Ca2+]i, whereas V2 and V5 were activated by 300 nM [Ca2+]i. Increasing the temperature to 42 °C led to activation of all ANO6 variants by 100 nM [Ca2+]i. The delay time for activation of the three variants was significantly shortened at 37 °C. Notably, the temperature-dependent Ca2+-sensitisation of ANO6 became insignificant under inside-out patch clamp, suggesting critical roles of unknown cytosolic factors. Unlike channel activity, 27 °C but not 37 °C  induced the scramblase activity of ANO6 at submicromolar [Ca2+]i (300 nM), irrespective of variant type. Our results reveal a physiological ion conducting property of ANO6 at 37 °C and suggest that ANO6 channel function acts separately from its scramblase activity.Anoctamin-6 (ANO6) belongs to a family of calcium (Ca The most well-known CaCCs in this group, TMEM16A (ANO1) and TMEM16B (ANO2), play fundamental roles in various physiological processes. ANO1 mediates transepithelial secretions in several gland tissues, possesses pacemaker potential for gastrointestinal motility, and acts as a heat sensor in nociception6, whereas ANO2 is believed to be associated with olfactory sensory transduction and photoreceptor signalling8.The TMEM16 family of proteins, also known as anoctamins (ANOs), comprise several members that function as calcium . The reported half-maximal Ca2+ concentrations (EC50) for ANO6 are quite variable, ranging from several to 106 μM22. Furthermore, even with a high [Ca2+]i, appearance of the ANO6 current is generally delayed and requires several minutes for activation. These properties are largely different from those of the ANO1 current that is activated with a submicromolar EC50 (~400 nM) of [Ca2+]i, showing no delayed activation in response to Ca2+ signals24. Because of the relatively low Ca2+ sensitivity and delayed activation properties of ANO6, unlike ANO1, which is activated directly by ER calcium release during G protein coupled receptor (GPCR) stimulation, ANO6 might be activated by sustained calcium influx through store-operated calcium entry after GPCR stimulation25.In contrast, ANO6 (TMEM16F) has a dual function, acting as a phospholipid scramblase as well as a CaCC. As a scramblase, ANO6 plays essential roles in platelet coagulant activity, induces pro-apoptotic signals in lymphocytes, induces phagocytosis as a downstream target of P2X7 receptors in macrophages, deposits hydroxyapatite in osteoblasts, and restricts T cell responses to maintain the balance of the immune reaction2+ sensitivity and time delay for peak current generation: V2 showed a shorter delayed time than V1 and V5, whereas V2 and V5 showed a higher Ca2+ sensitivity than V1. However, even the relatively more sensitive variants of ANO6 still required several micromolar [Ca2+]i and >100 s delayed time for significant activation to be detected26.Although there are several alternative splicing variants of ANO6, most previous studies have dealt with variant type 1 (V1). According to a recent study, three types  of ANO6 show CaCC activity with differential Ca28. In response to a temperature change, ANO1 showed increased activated current densities, with a Q10 (the 10 °C temperature coefficient) value of approximately 19.4, and an increase in the [Ca2+]i lowered the temperature threshold for ANO1 activation5. Based on this background, we hypothesised that the low Ca2+ sensitivity and the delayed activation of ANO6 might be due to the experimental temperature  applied in previous studies. Therefore, in this study, we investigated the effects of physiological temperature  on the activity of ANO6 variants in terms of their Ca2+ sensitivity and delayed activation. We found that although each variant differed, all variants could be activated at temperatures between 37 and 42 °C under submicromolar Ca2+ concentrations with short delays. In contrast, the [Ca2+]i-dependent scramblase activity of ANO6 was contrary altered by temperature. This work is expected to clarify the role of ANO6 as a chloride channel under physiological conditions beyond its known function as a scramblase.Experimental temperature generally affects the activities of ion channels by increasing the maximum current amplitudes and kinetics of activation/inactivation2+]i, which is known as the maximal Ca2+ concentration for ANO6 variant activation28. Ramp-like depolarisation from −100 mV to +100 mV was applied every 20 s after the membrane break-in to attain the whole-cell configuration followed by a step pulse from −100 mV to +100 mV with 20 mV increments after the activated current reached a steady state. As shown in Fig. ANO6) at 100 μM [Ca2+]i, whereas V3 elicited insignificant membrane currents with the same level as mock. Even in maximal [Ca2+]i for ANO6, ANO6 variants showed characteristic delayed activation, with the different time delays to reach half of the peak amplitude  for variants being 260.9 ± 41.2, 69.4 ± 14.5, and 67.8 ± 21.7 s for V1, V2, and V5, respectively  recordings, as observed by Scudieri et al.24  , whereas V1 did not  on IANO6 under inside-out patch clamp conditions with symmetrical NMDG-Cl solution . After membrane excision, IANO6,i-o could be immediately activated by raising the [Ca2+]i , which differed from the results of whole-cell patch recordings. However, at 1 µM free Ca2+, V2 and V5 were significantly activated upon a temperature increase to 37 °C, whereas V1 activation did not reach significance. At 37 °C, V2 and V5 showed approximately 25-fold increases versus those activated at room temperature, even though the current values were numerically small . In addition, the amplitude of IANO6,i-o with 3 µM of [Ca2+]i was significantly increased by raising the temperature by approximately 7-, 3-, and 4-fold for V1, V2, and V5, respectively . Moreover, the ANO6 variants could not be activated, even at low cytosolic Cl− at 37 °C with resting intracellular Ca2+ (100 nM) , contained in a pipette solution, or 50 μM NEM (PLA2 activator) could not activate the ANO6 variants at 27 °C with 1 μM [Ca2+]i. Conversely, at 37 °C, treating IANO6 with the PLA2 inhibitor (ACA 20 μM and MAFP 5 μM) did not affect the t1/2,peak  Fig. . Further2+ sensitivity of IANO6 at 37 °C raised a question whether the scramblase activities of the ANO6 variants are similarly enhanced under physiological temperature. Thus, we conducted a scramblase assay in the V1-, V2-, and V5-overexpressing HEK293T cells. To determine the extracellular free calcium concentration corresponding to the approximate [Ca2+]i in intact cells, we first measured [Ca2+]i in ANO6-overexpressing HEK293T cells treated with 10 μM ionomycin using a Fura-2 acetoxymethyl ester (Fura-2 AM) probe31. Then, for the scramblase assay, we estimated the required free calcium concentration of normal tyrode (NT) solution that induces [Ca2+]i close to that attained by the whole-cell patch recordings (300 nM and 1 μM). The free calcium concentrations of NT solution for 300 nM and 1 μM [Ca2+]i were 100 μM and 200 µM with ionomycin at 27 °C, respectively, and 30 μM and 100 μM at 37 °C, respectively  resulting from membrane damage, we compiled the statistics using only Annexin V-positive/propidium iodide (PI)-negative cells (Q4-region) , the scramblase activity of ANO6 was induced with both 300 nM and 1 μM [Ca2+]i, the concentrations at which channel activation of ANO6 at 27 °C could not be induced.The notable increase in the Caon) Fig. . The adjon) Fig. . As show32, the physiological roles of ANO6 have been identified in only limited types of cells such as immunocytes, platelets, and osteoblasts15, all of which are thought to be related to its PS scramblase activity35. Conversely, owing to its low calcium channel sensitivity, the potential of ANO6 to function as an ion channel under physiological conditions remains controversial. The present results provide evidence that a prolonged physiological increase in [Ca2+]i indeed activates ionic currents through ANO6 (IANO6) above 37 °C, and there is a differential temperature-dependence of ANO6 variants. Intriguingly, the IANO6 of V2 and V5 was detected without significant induction of the scramblase activities of ANO6 variants with 300 nM [Ca2+]i at 37 °C. Although a recent report by Schreiber et al. showed that ANO1 and ANO6 (V1) could be activated at resting Ca2+ concentrations at 37 °C29, our study shows wider ranges of Ca2+-sensitivity depending on the variants of ANO6.Despite the expression of ANO6 (TMEM16F) in various cell types2+]i required for IANO6 activation, with V2 and V5 being activated even by 300 nM [Ca2+]i at 37 °C. Moreover, a temperature increase to 42 °C further increased the calcium sensitivity of ANO6, such that V1 could be activated by 300 nM [Ca2+]i. At 42 °C, all variants could be slightly activated even with resting Ca2+ concentrations of 100 nM, while there was no activation with Ca2+-free pipette solution even at the higher temperature of 42 °C. The temperature-dependence of IANO6 was reversible . Comparison of IANO6,i-o with the whole-cell current (IANO6) revealed that unknown cytosolic factor(s) might underlie the unique slow activation (see below).Another remarkable finding was the acceleration of I2+]i. Such a property was markedly different from that of ANO1, which shows linearization of the I/V curve at higher [Ca2+]i. Surprisingly, Schreiber et al. showed that IANO6 could produce a linear I/V curve, i.e. large inward current, at 37 °C with resting [Ca2+]i29. The same group  also reported that a sudden increase in intracellular Ca2+ concentration by ionophore leads to a rapid activation of IANO6 and development of a linear I/V curve13. However, in our experience, IANO6 always shows outward rectification even at a very high [Ca2+]i (>50 μM), which has been confirmed by other research groups36. Shimizu et al. also reported that the ionomycin-induced [Ca2+]i increase still showed delayed activation of IANO6 and a persistent outwardly rectifying I-V relationship21. As shown by Stolz et al., hANO6 overexpressed in Xenopus oocytes could not be activated by ionophore (A23187)-induced calcium increase or calcium influx through ATP-activated P2X7 receptors. This may indicate that ANO6 activation requiures long-term exposure (delayed activation) to high Ca2+ levels and that these high Ca2+ levels may not be achieved in Xenopus oocytes through the application of A23187 or the activation of hP2X7R by ATP37.In our study, irrespective of the variant types, the outwardly rectified I/V curves of ANO6 were not altered by temperature even with 100 µM [Ca2+]i for activating IANO6, the peak outward current amplitudes were not significantly increased by raising the temperature in all three variants at 3 μM [Ca2+]i. The delta amplitude of IANO6 detected between 27 °C and 37 °C only increased by approximately 2-fold compared to those detected at room temperature in V2 and V5, whereas V1 showed the same level of activation currents between room temperature (27 °C) and 37 °C. The results differ from the higher temperature sensitivity of ANO1, a type of heat-sensing ion channel, in terms of the current amplitude changes. It has been reported that the Q10 (the 10 °C temperature coefficient) value of ANO1 is approximately 19.45. Nevertheless, the shortened t1/2,peak as well as submicromolar [Ca2+]i threshold in the ANO6 variants at physiological temperatures (37 °C–42 °C) are plausible conditions in specific physiological contexts, such as under GPCR stimulations for numerous Ca2+-sensitive processes38, implying the likely electrical roles of ANO6 under physiological conditions.Despite the marked changes in threshold i combined with the physiological temperature. The candidate intrinsic factors include ATP, phosphorylation states of signalling molecules, cytoskeleton- or calmodulin-dependent signals, and alterations in membrane phospholipids, among others. PLA2 is reported to activate ANO6 only in low cytosolic chloride conditions30. However, in our study, we conclude that the temperature-dependent calcium sensitivity and acceleration of ANO6 variant activation were not due to PLA2 both in symmetrical and low cytosolic chloride conditions . A similar case can be observed in the mammalian Na+/H+ exchanger isoform 1 (NHE1), which has seven consecutive acidic residues in the distal region of the cytosolic tail40. This site is critical for the maintenance of NHE1 activity and calmodulin binding. Thus, V2 and V5 may be weaker or less affected by the specific regulatory factor than V1. In many cases, as with NHE1, consecutive negative acidic amino acids are often used to regulate the activity of ion channels in the cytoplasm. Therefore, it is necessary to identify the role of this domain in future studies through mutation analyses. As a whole, these findings suggest that the cytosolic initial amino acids of the N-terminus play an important role for sensing Ca2+ and determining the delayed time for activation. Nevertheless, the exact mechanism of ANO6 activation and the reason for the observed delayed activation remain unknown.The altered domain structures in the N- and C-termini of the variants have been schematically compared34. A recent elaborate study demonstrated that the ionic flow through ANO6 owes to a leak current associated with lipid transport via scramblase activity. Although the experimental results were obtained with 200 μM [Ca2+]i, this might imply that the ions and phospholipids share the same pathway41. Additionally, the most recent research established that ion conductance and lipid scrambling, which require a high calcium concentration with EC50 of 8 μM for activation, were activated by the same Ca2+ activation mechanisms42. However, the temperature-dependence of lipid scramblase activity appears inconsistent; temperature reductions did not slow lipid transport43, while another report established that ANO6-induced PS exposure was sufficiently rapid at 25 °C (RT), although the rate was faster at 37 °C44. However, in our study, the channel and scramblase activities of ANO6 seemed to be separately induced as the mechanism of the temperature-dependent increase in the calcium sensitivity of the ANO6 channel was not observed for the scramblase activity. When comparing the 27 °C condition and the physiological temperature (37 °C) condition, the calcium sensitivity was higher than the scramblase activity at 27 °C, since the PS exposure occurred at 300 nM [Ca2+]i at 27 °C, whereas at 37 °C, scramblase activity was activated with 1 μM [Ca2+]i but not with submicromolar [Ca2+]i.ANO6 is also well recognized as a membrane phospholipid scramblasein vivo under physiological conditions, further supporting the physiological function of ANO6 as an ion channel  at submicromolar [Ca2+]i might have resulted from the compensatory effects of flippase, which may be stronger at 37 °C than at room temperature (27 °C).Although the results require careful interpretation, the scramblase and channel activities of ANO6 might utilize discriminable molecular mechanisms nel Fig. . However2+]i with a short t1/2,peak. At 42 °C, all ANO6 variants show discernible activity even with 100 nM [Ca2+]i. Thus, we provide an experimental foundation for the functional exploration of ANO6 as an ion channel. The temperature-dependent calcium sensitivity and accelerated activation of ANO6 require additional detailed study to fully understand their precise mechanisms. Interestingly, even at physiological temperatures, the phospholipid scramblase activity of ANO6 seems to require higher calcium concentrations (1 μM) than the ionic current activation. Furthermore, in contrast to the Ca2+-dependence of the ionic currents, the scramblase activities of V1, V2, and V5 showed similar patterns. We cautiously suggest that the channel function of ANO6 acts separately from the scramblase activity. Further quantitative analysis of ANO6 variant expression would be necessary for elucidating their physiological roles in different tissues expressing ANO6.In summary, we provide the first demonstration that the ANO6 isoforms V2 and V5 can be activated within a submicromolar range of cytosolic free calcium at 37 °C, and that V1 can be activated by 1 μM [Ca2/5% CO2, and were subcultured every 2 or 3 days. HEK293T cells were cultured in 35-mm dishes and 75-T flasks for electrophysiology, calcium measurements, and FACS analysis.HEK293T cells  were cultured in high-glucose Dulbecco’s modified Eagle’s medium  supplemented with 10% foetal bovine serum . Cells were maintained in a 37 °C humidified incubator at 20% O22. The cDNAs of hANO6 V2, V3, and V5 transcripts were purchased from GeneCopoeiaTM . The coding regions of hANO6 V1 (GenBank accession no. NM_001025356.2), V2 (NM_001142678.1), V3 (NM_001142679.1), and V5 (NM_001204803.1) were subcloned into the mammalian expression vector pcDNA 3.1(+) (Thermo Fisher Scientific) with a Kozak consensus sequence (GCC ACC) immediately upstream of the initiation codon using polymerase chain reaction amplification. HEK293T cells were maintained in DMEM-HG supplemented with 10% FBS and 1% penicillin and streptomycin (Thermo Fisher Scientific). The plasmids were transiently transfected to HEK293T cells using Turbofect transfection reagent (Thermo Fisher Scientific) according to the manufacturer’s instructions. For electrophysiological experiments, HEK293T cells (cultured in 35-mm dishes) were co-transfected with 0.9 μg of the hANO6 variant plasmids along with 0.1 μg of the GFP expression plasmid to visualise the transfected cells. For western blotting, calcium measurements, and FACS analysis, HEK293T cells (grown in 75-T flasks) were transfected with 4.5 μg of each ANO6 variant plasmid. Experiments were performed within 24–36 h after transfection.The mammalian expression plasmid used for expressing the human ANO6 (hANO6) V1 transcript was described previouslyWhole-cell and inside-out patch clamp techniques were applied for measuring the channel activities of ANO6 variant-transfected HEK293T cells. Cells were transferred to a bath mounted on the stage of an IX-50 inverted microscope  equipped with a light source set to green fluorescence excitation wavelengths. The patch-clamp experiment was performed at 27 °C or 37 °C. Each temperature was maintained by a circulating water bath and was continuously measured in a bath chamber to be maintained at exactly 27 °C or 37 °C . Microglass pipettes  were fabricated using a PP-830 single-stage glass microelectrode puller , with a resistance of 2.5–3.5 MΩ and 5–6 MΩ for whole-cell and inside-out patch recordings, respectively, using an MF-830 microforge . Currents were recorded using an Axopatch 200B amplifier and Digidata 1332 A interface, digitised at 10 kHz and low pass-filtered at 5 kHz by pClamp software 10.3 . The series resistance and junction potential were compensated by an offset circuit in Axopatch 200B. All voltage and current trace data were analysed using Clampfit 10.3 and Origin 8.0 software . The detailed stimulation voltage protocols are described in the relevant figure legends.2, 1 mM MgCl2, 10 mM HEPES, and 5 mM glucose (adjusted to pH 7.4 with NMDG-OH). The basal pipette solution contained 10 mM EGTA, 5 mM HEPES, 0.5 mM MgCl2, and 1 mM Mg-ATP; an appropriate amount of CaCl2 was added to the pipette solution to obtain a Ca2+ concentration of 0.1, 0.3, 1, 3, and 100 µM for the 27 °C, 37 °C, and 42 °C experimental condition (adjusted to pH 7.2 with NMDG-OH). WEBMAX-C software  was used to calculate the accurate amount of CaCl2 for each free Ca2+ concentration at 27 °C, 37 °C, and 42 °C. Because Ca2+ chelation by EGTA is weak when the free Ca2+ concentration is 100 µM, 10 mM HEDTA was used. Given that two Cl− ions bind to one Ca2+ ion, appropriate amounts of NMDG-Cl were added to the basal pipette solution to adjust the Cl− concentration to 150 mM.The whole-cell patch clamp analysis of ANO6 was conducted using a basal extracellular solution containing 146 mM NMDG-Cl, 1 mM CaCl2+, 10 mM EGTA or HEDTA was added to the bath solution, and an appropriate amount of CaCl2 was added to change the free Ca2+ concentration as described above.For the inside-out patch clamp experiment, the basal pipette solution contained 150 mM NMDG-Cl, 1 mM EGTA, and 5 mM HEPES (pH 7.4). The bath solution for the intracellular side of the patch contained 150 mM NMDG-Cl, 1 mM EGTA, and 5 mM HEPES (pH 7.2). To obtain different concentrations of free Ca46. In brief, transfected HEK293T cells were washed three times with ice-cold phosphate-buffered saline (PBS). The cells were then treated with sulpho-NHS-SS-biotin  containing buffer for 30 min at 4 °C to biotinylate the plasma membrane proteins. After biotinylation, the cells were washed with quenching buffer to remove the excess biotin and washed twice again with PBS. The cells were harvested and incubated overnight with avidin solution  at 4 °C. Avidin-bound complexes were washed three times and the biotinylated proteins were eluted in 2 × sample buffer. The protein samples were suspended in a sodium dodecyl sulphate (SDS) buffer and separated by SDS–polyacrylamide gel electrophoresis. The separated proteins were transferred to a nitrocellulose membrane and blotted with appropriate primary and secondary antibodies. Anti-ANO6  antibody was used as the primary antibody, and horseradish peroxidase-conjugated anti-rabbit IgG (Thermo Fisher Scientific) was used as the secondary antibody. Protein bands were detected by enhanced chemiluminescence .Surface biotinylation and immunoblotting were performed using conventional methods as described previously2+ concentration of 0, 30, 100, or 200 μM. The cells were then resuspended with 100 μL Annexin V binding buffer  in 5-mL polystyrene round-bottom tubes. The tubes were then gently vortexed and incubated in the dark for 15 min at 4 °C, and 400 μL of the binding buffer was added to each tube before flow cytometry analysis using the FACSCalibur system . To avoid the influence of ionomycin on the cells, the treated cells were maintained at 4 °C for all processes until the FACS analysis.hANO6 variant-transfected HEK293T cells were incubated with 10 µM of ionomycin for 15 min at 27 °C or 37 °C. The incubation NT solution contained 3.6 mM KCl, 10 mM HEPES, 10 mM glucose, and 10 mM HEDTA (adjusted to pH 7.4 with NaOH), with a fixed CaP < 0.05 was considered statistically significant.The results are expressed as the means ± SEM. Statistical analysis was performed by one-way ANOVA with post-hoc Tukey’s HSD tests and two-way ANOVA with Bonferroni post-tests for multiple comparisons. Supplementary InformationSupplementary Material


HIPK3, SMAC, and BCL‐X, at a lower concentration than amiloride. This splicing regulation involved various splicing factors, and it was accompanied by a change in the phosphorylation state of serine/arginine‐rich proteins (SR proteins). RNA sequencing was performed to reveal that AS of many other apoptotic gene transcripts, such as AATF, ATM, AIFM1, NFKB1, and API5, was also modulated by BS008. In vivo experiments further indicated that treatment of tumor‐bearing mice with BS008 resulted in a marked decrease in tumor size. BS008 also had inhibitory effects in vitro, either alone or in a synergistic combination with the cytotoxic chemotherapeutic agents sorafenib and nilotinib. BS008 enabled sorafenib dose reduction without compromising antitumor activity. These findings suggest that BS008 may possess therapeutic potential for cancer treatment.Alternative splicing (AS) is a process that enables the generation of multiple protein isoforms with different biological properties from a single mRNA. Cancer cells often use the maneuverability conferred by AS to produce proteins that contribute to growth and survival. In our previous studies, we identified that amiloride modulates AS in cancer cells. However, the effective concentration of amiloride required to modulate AS is too high for use in cancer treatment. In this study, we used computational algorithms to screen potential amiloride derivatives for their ability to regulate AS in cancer cells. We found that 3,5‐diamino‐6‐chloro‐N‐carbamimidoyl)pyrazine‐2‐carboxamide (BS008) can regulate AS of apoptotic gene transcripts, including  ASalternative splicingDSBsDNA double‐strand breaksHCChepatocellular carcinomaHIPK3homeodomain‐interacting protein kinase 3hnRNPsheterogeneous RNPsOAokadaic acidPP1protein phosphatase‐1PTMspost‐translational modificationsRBPsRNA‐binding proteinsSiMMapssite‐moiety mapsSMAC/DIABLOsecond mitochondria‐derived activator of caspasesSR proteinsserine/arginine‐rich proteinsSREssplicing regulatory sequence elementsSRPKsSR protein‐specific kinases1et al., et al., et al., In eukaryotes, alternative splicing (AS) is a crucial mechanism for generating transcriptome diversity and can result in single‐gene coding for multiple proteins. Currently, 20 000 coding genes are estimated to be able to produce nearly 80 000 proteins and probably many more through AS in humans (Hahn BCL‐X and homeodomain‐interacting protein kinase 3 (HIPK3), with a concomitant effect in many splicing factors, such as SRSF1, hnRNP A1, hnRNP A2/B1, and hnRNP C1/C2, finally resulting in apoptosis  of size C × R was used to present the profile, where C is the number of docked compounds and R is the number of interacting residues of a protein. An entry (Mij), representing the interacting energy between compound i and residue j, was considered significant if it had a Z‐score of ≥ 1.65. Then, spatially neighboring interacting residues and their interactive moieties with statistical significance were assigned as an anchor , FBS, penicillin, and streptomycin from Gibco‐BRL ; AKT inhibitor from Millipore ; and 3‐‐2,5‐diphenyltetrazolium bromide (MTT), DMSO, Kolliphor EL, nilotinib, OA, propidium iodide (PI), and sorafenib from Sigma‐Aldrich .2.4Antibodies were purchased from the following companies: anti‐H3K27me1, anti‐H3K27me3, anti‐H3K79me1, anti‐H3K79me2, anti‐histone H3, anti‐hnRNP I, anti‐KDM4A, anti‐KDM5A, anti‐KDM5C, anti‐snRNP70, anti‐tubulin, and anti‐U2AF2 from Abcam ; anti‐hnRNP A2/B1 from Acris ; anti‐SR protein‐specific kinases (SRPK)1 and anti‐SRPK2 from BD Biosciences ; anti‐AKT, anti‐H3K27me2, anti‐protein phosphatase‐1 (PP1), anti‐phospho‐PP1 at Thr320, anti‐phospho‐AKT at Ser473, and anti‐cleaved‐CASPASE‐3 from Cell Signaling Technology ; anti‐H3K4me1, anti‐H3K4me2, anti‐H3K4me3, and anti‐H3K36me3 from Millipore; anti‐pro‐CASPASE‐3 and anti‐KDM7A from GeneTex ; anti‐hnRNP C1/C2, anti‐BAX, and anti‐BCL2L1 from Santa Cruz Biotechnology ; anti‐hnRNP A1 from Sigma; and anti‐SRSF1 and anti‐SRSF3 from Zymed .2.5et al., −1 penicillin and 100 μg·mL−1 streptomycin) at 37 °C in a humidified atmosphere of 5% CO2. To perform snRNP70 or hnRNP I knockdown, antisense oligonucleotide sequences of snRNP70, hnRNP I, or a scrambled control  was provided by Michael W. Deininger (O'Hare ol Table  were tra2.6−1) was added to each well. After 2 h, the medium was removed and cells were lysed with 100 μL of DMSO. The absorbance at 565 nm was measured using a microplate reader, and the results are presented as a percentage of the control results.Cells were prepared in a 96‐well plate, and 10 μL of MTT solution  and separated by sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS/PAGE). After proteins were transferred onto polyvinylidene difluoride membranes (Millipore), the membranes were blocked with 5% BSA (Santa Cruz Biotechnology) and then exposed at 4 °C overnight to the indicated primary antibodies, followed by horseradish peroxidase‐conjugated secondary antibody for detection by an ECL chemiluminescence detection system .Proteins were extracted using a cell lysis solution  and then converted into cDNA using High‐Capacity cDNA Reverse Transcription Kits (Applied Biosystems) according to the manufacturer's instructions. PCR was performed using specific pairs of primers Table .2.9−1 RNase A, and 500 μg·mL−1 PI in phosphate‐buffered saline) at 37 °C for 30 min. Data were collected and analyzed using a BD FACSAria™ Fusion flow cytometer (BD Biosciences).Cells were collected and fixed in 70% (v/v) ethanol at 4 °C overnight and then stained with 1 mL of PI staining buffer  according to the manufacturer's instructions. The raw sequencing data were deposited in the National Center for Biotechnology Information's Gene Expression Omnibus (GEO), and they can be obtained through GEO Series with the accession number 2.11nu/CrlNarl mice  were purchased from the National Laboratory Animal and Research Center ; 5 × 106 Huh‐7 cells were subcutaneously injected into the right flank of the mice. Therapy started after 3–4 weeks when the tumors had reached an average volume of 300 mm3. The mice were assigned to groups of six and subcutaneously injected daily with 0.1 mL of solvent  for the control group or 30.9 μg/10 g of BS008 for the treatment group. The longest axis (a), shortest axis (b), and thickness (c) of the tumor were measured with calipers, and the tumor volume (V) was evaluated using the formula V = πabc/6. All animal experiments were performed in accordance with the guidelines established by the Institutional Animal Care and Use Committee (IACUC) of China Medical University (CMU). All animals were housed in the Laboratory Animal Center of CMU under a 12‐h light/dark (08:00/20:00) cycle with free access to food and water. The mice were sacrificed using CO2, and the tumor tissues were subsequently harvested. All breeding and subsequent use of animals in this study, including sacrifice, was approved by the IACUC of CMU .BALB/cAnN.Cg‐Foxn12.12t‐test, with a probability of less than 5% (P < 0.05) considered significant.Differences between the control and treatment were analyzed using Student's 33.1et al., et al., et al., 4PKD), hnRNP I (PDB code 2AD9), and U2AF2 (PDB code 4TU8)  Fig. A. Their et al., et al., et al., et al., et al., et al., After identifying these potential target proteins, we integrated molecular docking  of BS008 being 0.128 mm  in determining the sensitivity of cancer cells to apoptotic death, it was also chosen to investigate whether its splicing variants could be regulated by BS008. Huh‐7 cells treated with BS008 showed a decrease of the antiapoptotic splice variants, BCL‐XL and HIPK3 U(+), while relative maintenance of the proapoptotic splice variants BCL‐XS and HIPK3 U(−). Western blot analysis further confirmed a reduced level of BCL‐XL protein after the treatment with BS008 . Besides BCL‐X and HIPK3, we also found SMAC was regulated by BS008 with an increase in the proapoptotic splice variant SMAC‐3  with a concomitant decrease in the antiapoptotic splice variant HIPK3 U(+). Notably, using highly imatinib‐resistant BaF3/Bcr‐Abl T315I cells, we observed that BS008 slightly increased the proapoptotic splice variants BCL‐XS and SMAC‐3, but not HIPK3 U(−). On the contrary, we detected no alteration in the BCL‐X, HIPK3, and SMAC splicing patterns in normal mononuclear cells from healthy individuals  cDNA  intron containing mRNA resulted in the protection of AS of HIPK3 U(+) regulated by BS008, which might be a cause for the competition between pre‐mRNAs for the splicing machinery  splicing could partially rescue the cell death induced by BS008  variants, but it had no significant effect on the AS of BCL‐X and SMAC transcripts. Furthermore, snRNP70 knockdown increased the proportion of HIPK3 U(+) and SMAC‐3 variants, but it had no significant effect on the AS of BCL‐X transcripts. The combination of hnRNP I and snRNP70 knockdown increased the proportion of SMAC‐3 variants, but it had no significant effect on the AS of BCL‐X and HIPK3 transcripts in the Huh‐7 cells  was validated by western blot   and AS. Specific histone marks can affect the splicing outcome by recruiting splicing factors to the site of transcription  and further validated the changes in AS. As shown in Fig. The ability of BS008 to modulate histone‐tail PTM patterns and the phosphorylation and expression levels of splicing‐related proteins implied that it might have a wide range of effects on the AS of pre‐mRNA. Therefore, we used whole‐RNA sequencing to evaluate global genes in BS008‐treated Huh‐7 cells and found that BS008 could affect the AS of various gene transcripts and the frequency of AS  and BS008 (right) is shown in Fig. 3, respectively  or genotoxic stress, thereby acting as a DNA damage sensor. In response to DNA damage, ATM phosphorylates various downstream proteins, such as CHK2, histone H2AX, and TP53, to start a complex signaling cascade, and it then induces cell cycle arrest, increases DNA repair, and inhibits apoptosis . This suggested that BS008 might potentiate the growth‐inhibitory effect of sorafenib through modulation of these gene transcripts. However, detailed examination is still required to explore sorafenib resistance‐related genes in BS008‐induced AS.Sorafenib is a multikinase inhibitor that represses tumor‐cell proliferation and angiogenesis and promotes tumor‐cell apoptosis by inhibiting the Raf/MEK/ERK signaling pathway and VEGF receptor tyrosine kinase. Although sorafenib is the standard first‐line systemic drug for HCC, primary and acquired resistance to sorafenib results in limited benefits. Primary resistance of HCC to sorafenib is caused by genetic heterogeneity and may be associated with overexpression of EGFR and abnormal activation of its downstream molecules Ras/Raf/MEK/ERK. Acquired resistance is possibly related to activation of the compensatory pathways, such as cross talk involving JAK–STAT pathways  BCL‐X, (B) HIPK3, and (C) SMAC RNAs. Messenger RNA was extracted and detected using RT‐PCR for the AS in K562, T315I and normal mononuclear cells.Click here for additional data file.Fig. S2. Effects of HIPK3 splicing on BS008‐induced cell death. (A) Experimental design and vectors. (B) K562 cells were transfected with the expression plasmids as indicated for 24 h prior to BS008 treatment. RT‐PCR validation of endogenous HIPK3 alternative splicing (upper panel). Cell viability was analyzed using MTT assay followed by 24‐h treatment with BS008 (lower panel). Data are presented as mean ± standard deviation from six independent experiments (*P < 0.05 using unpaired two‐tailed Student's t‐test).Click here for additional data file.Fig. S3. Effects of BS008 on alternative splicing of (A) snRNP70, (B) SRSF1, (C) SRSF3, (D) hnRNP C, and (E) hnRNP I RNAs. Cells were treated with BS008 for 24 h and then harvested for RT‐PCR analysis.Click here for additional data file.Fig. S4. Effects of PP1 phosphatase on BS008‐induced alternative splicing. RT‐PCR results from K562 cells pretreated with (+) or without (‐) okadaic acid and then exposed to BS008 for 24 h.Click here for additional data file.Table S1. Information about the Small interfering RNA used in the study.Table S2. Information about the primers used in reverse transcription polymerase chain reaction.Table S3. Ten known target proteins of amiloride collected from BindingDB.Table S4. Alternative splicing‐related proteins.Click here for additional data file.


We therefore hypothesised that EMPA would directly modify [Na+]c, [Ca2+]c and [Ca2+]m in cardiomyocytes.Empagliflozin (EMPA), an inhibitor of the renal sodium–glucose cotransporter (SGLT) 2, reduces the risk of cardiovascular death in patients with type 2 diabetes. The underlying mechanism of this effect is unknown. Elevated cardiac cytoplasmic Na+]c, [Ca2+]c, [Ca 2+]m and Na+/H+ exchanger (NHE) activity were measured fluorometrically in isolated ventricular myocytes from rabbits and rats.[Na+]c and [Ca2+]c levels. EMPA treatment directly inhibited NHE flux, caused a reduction in [Na+]c and [Ca2+]c and increased [Ca2+]m. After pretreatment with the NHE inhibitor, Cariporide, these effects of EMPA were strongly reduced. EMPA also affected [Na+]c and NHE flux in the absence of extracellular glucose.An increase in extracellular glucose, from 5.5 mmol/l to 11 mmol/l, resulted in increased [Na+]c and [Ca2+]c and enhancing [Ca2+]m, through impairment of myocardial NHE flux, independent of SGLT2 activity.The glucose lowering kidney-targeted agent, EMPA, demonstrates direct cardiac effects by lowering myocardial [NaThe online version of this article (doi:10.1007/s00125-016-4134-x) contains peer-reviewed but unedited supplementary material, which is available to authorised users. SGLT2 i failure , we inveGuide for the Use and Care of Laboratory Animals.Animal handling was in accordance with the Institutional Animal Care and Use Committee of the VU Medical Center and Academic Medical Center Amsterdam, and was conducted according to the 4Cl (NH+) (Sigma-Aldrich Chemie), either alone or in combination. [Na+]c and [Ca2+]c were fluorometrically measured in rabbit cardiomyocytes using SBF1 and indo-1, respectively, at 2 Hz field stimulation. NHE activity was measured in rabbit cardiomyocytes by recording SNARF fluorescence following an NH4+ pulse. Using adenoviral transfection, a ratiometric mitochondrially targeted fluorescence resonance energy transfer (FRET)-based Ca2+ indicator  was expressed in rat cardiomyocytes and free [Ca2+]m was measured using the fluorescence ratio, yellow fluorescent protein intensity/cyan fluorescent protein intensity (YFP/CFP), in cultured adult rat cardiomyocytes.For detailed methods, see + and Ca2+. ANOVA with Dunnett’s post hoc tests was used to compare group means with the control ([EMPA] 0) group. Additionally, unpaired t tests were used to evaluate EMPA effects on [Ca2+]c and EMPA effects on the NHE flux in the absence of glucose, ANOVA with post hoc testing with Bonferroni corrections were used to compare cariporide and EMPA effects on the NHE flux in the presence of glucose and two-way ANOVA for repeated measures followed by post hoc contrast with Bonferroni correction at one time point were performed to detect EMPA effects on [Ca2+]m.Data are reported as mean ± SE. Kolmogorov–Smirnov normality testing was applied to decide the use of non-parametric vs parametric testing. Mann–Whitney tests were used to evaluate the effects of glucose on Na+]c within 10 min  decreased [Namin Fig. . Vehicle+]c Fig. .Fig. 1EM+]c, in the presence of both 11 mmol/l  and systolic [Ca2+]c  (data not shown). EMPA preincubation of cardiomyocytes at 11 mmol/l glucose also significantly lowered diastolic and systolic [Ca2+]c  demonstrated that 1 μmol/l EMPA had no effect on action potential duration (data not shown). The downstream effects of the cytosolic changes of [Na+]c and [Ca2+]c on [Ca2+]m were subsequently tested. Acute EMPA (1 μmol/l) administration significantly increased [Ca2+]m  EMPA incubation at clinically relevant concentrations (0.25–1 μmol/l) also reduced [Nal/l Fig.  and 5.5 l/l Fig.  glucose.+]c Fig. . Patch c+]m Fig. .+]c and [Ca2+]c and increases [Ca2+]m, whereas increases in glucose are associated with elevated [Na+]c and [Ca2+]c.Collectively, these data show that EMPA reduces myocardial [Na+]c and [Ca2+]c [+]c. Following the reduction in [Na+]c with EMPA, additional application of Cariporide had only a minimal effect on [Na+]c . Finally, we evaluated whether SGLTs were involved in the effects of EMPA on NHE by repeating the experiments in the absence of glucose; in glucose-free conditions, EMPA also reduced [Na+]c , respectively c with increased circulating glucose. It was recently reported that SGLT1 within the diseased human heart is mainly localised in capillaries and not in cardiomyocytes [+]c were detected in isolated cardiomyocytes; therefore, it is unlikely that glucose release from the capillaries plays a role in this effect. However, further research is needed to study the role of SGLT1 within the intact heart. We used EMPA concentrations in the range of clinically measured plasma concentrations (≤1 μmol/l) c results in decreased [Ca2+]m through increased mitochondrial efflux via the mitochondrial NCX [2+]m, observed in the present study. Mitochondrial Ca2+ is considered to be an important activator of ATP synthesis and of the antioxidant enzymatic network [2+]m (e.g. increasing [Ca2+]m that is otherwise reduced due to Na+ loading) is predicted to be beneficial in this condition. Indeed, in a recent study, it was demonstrated that increasing mitochondrial Ca2+ during heart failure development was associated with the prevention of sudden death and overt heart failure [2+]m is already elevated, such as during reperfusion injury phenomena, in which further increases in [Ca2+]m can be detrimental.Previous studies have demonstrated that increasing c and [Ca2+]c with EMPA treatment, and the downstream effect of elevated [Ca2+]m contribute to the primary mechanisms underlying the cardiovascular benefits observed in the EMPA-REG OUTCOME trial needs to be examined in future research.Previous studies have demonstrated that chronic inhibition of NHE prevents or mitigates heart failure in animal models , 17. Alt+]c and [Ca2+]c and increases cardiac [Ca2+]m via inhibition of the cardiac NHE.In conclusion, our data demonstrate that the kidney-targeted therapeutic agent EMPA has direct cardiac effects, decreases cardiac [NaESM 1(PDF 511 kb)Below is the link to the electronic supplementary material.