NPM1)—one of the most common mutations in AML—with an AUROC of 0.92 using only image data from bone marrow smears. Utilizing occlusion sensitivity maps, we observed so far unreported morphologic cell features such as a pattern of condensed chromatin and perinuclear lightening zones in myeloblasts of NPM1-mutated AML and prominent nucleoli in wild-type NPM1 AML enabling the DL model to provide accurate class predictions.The evaluation of bone marrow morphology by experienced hematopathologists is essential in the diagnosis of acute myeloid leukemia (AML); however, it suffers from a lack of standardization and inter-observer variability. Deep learning (DL) can process medical image data and provides data-driven class predictions. Here, we apply a multi-step DL approach to automatically segment cells from bone marrow images, distinguish between AML samples and healthy controls with an area under the receiver operating characteristic (AUROC) of 0.9699, and predict the mutation status of Nucleophosmin 1 ( Nucleophosmin 1 (NPM1). It plays a critical role in disease initiation and is utilized for molecular risk stratification in the recent European Leukemia Net 2017 (ELN2017) recommendations [NMP1 can be found in a third of all adult AML cases and up to 50–60% in AML with a normal karyotype [NPM1 mutations [A fundamental component in the diagnostic workflow of acute myeloid leukemia (AML) is cytomorphology . The assaryotype . So far,aryotype  have beeutations . Howeverutations , 11. Artutations . Deep leutations , 14. Esputations . These cutations –18.NPM1 mutation status from bone marrow cytomorphology and unveils distinct morphologic features for the prediction of NPM1 mutation status.In our study, we present a CNN-based scalable model that accurately distinguishes between AML cases and healthy subjects from digitalized bone marrow images. Further, our model accurately predicts NPM1 mutations was performed as described previously [We identified 1251 patients who have been newly diagnosed and treated with AML in the previously reported multicentric trials (AML96 ), AML200eviously  and a 5%NPM1 status prediction, transfer learning with a ResNet50 [NPM1 status prediction. DL models were implemented in Python version 3.7.9 with Keras version 2.3.0, TensorFlow version 2.1.2, and PyTorch version 1.5.0. Computations were performed using a high-performance computing system.A multi-step machine learning workflow with individual DL models for different tasks was set up as shown in Fig. ResNet50  pretrainNPM1 VAF the Mann–Whitney U test was used. Computational and statistical analysis was performed in Python (version 3.7.9) and R (version 4.0.3).For performance analysis of the classification models, we used precision–recall curves and receiver operating characteristics (ROC) with the area under the curve (AUC). Precision is the fraction of true positives among all positive predictions of the DL model while recall is the fraction of all positive predictions of the DL model among all relevant events. The final models were evaluated on the validation set that was withheld from model training. To compare NPM1 according to molecular analyses. Detailed information on patient characteristics and controls is provided in Table We retrospectively identified 1251 AML patients, 386 of which harbored mutated NPM1 from bone marrow morphology. BMS image classification at a 50-fold magnification was performed using a ResNet50-architecture using transfer learning. Mirroring and random cropping plus resizing was used to increase sample size and to balance the data. Weight decay of 0.0003 is utilized to prevent overfitting and the data was divided into training and test set with a split of 4:1. The model achieved an accuracy of 0.86 for NPM1 prediction and an AUROC of 0.92 . We further analyzed patient samples from the validation set grouped by NPM1 mutation status and true or false predictions given by the DL model regarding clinical and molecular data. NPM1-mutated AML samples that were correctly identified by the model (true positives) had a significantly higher median VAF than NPM1-mutated AML samples that were misclassified   and false negatives had a significantly lower median white blood cell count (WBC)  and a trend for lower blast counts in peripheral blood . No significant differences for age, sex, ELN2017 risk category, absence or presence of a complex karyotype, bone marrow blast count, Hb, and platelet count were detected. For patients with wild-type NPM1 AML, there was a trend for lower median WBC for true negatives , but age, sex, ELN2017 risk category, AML type , absence or presence of a complex karyotype, bone marrow or peripheral blast count, Hb and platelet count showed no differences. As another internal sanity check, we applied the pretrained classifier to the healthy bone marrow donor image data set and found that 214/236 (91%) of cases were correctly identified as NPM1 wild type while only 22/236 (9%) were labeled as NPM1 mutated . However, we want to point out that the NPM1 classifier has never been trained on healthy controls. Considering its AML-specific training, the very low false-positive rate on newly presented and differently structured image data of healthy controls underlines the distinct morphology picked up by the classifier for correct predictions in NPM1-mutated AML. Interestingly, when we reviewed patient chart data and molecular results in the validation set, we found one sample that was incorrectly labeled as mutated NPM1, but was in fact wild-type NPM1, and the corresponding BMS image was correctly identified as such by the DL model.Further, we investigated whether DL could predict the mutational status of We here present a machine learning approach for cell segmentation and image classification which provides a fast, scalable, and highly accurate method to identify AML samples from bone marrow cytomorphology. Our FRCNN achieved a cell segmentation accuracy of 0.97 from BMS. The binary classification model showed an AUC of 0.97 for both the ROC and the precision–recall curve and a micro-average accuracy of 0.91 distinguishing between AML and healthy bone marrow donor samples. Our model can potentially be applied in initial diagnosis when a case of suspected AML is evaluated upon first contact in a hematologic center. It operates autonomously once BMS images are uploaded and detects AML with high accuracy. The model could operate synchronously with lab technicians to flag cases that are highly suspicious of AML for fast evaluation by experts while results from other diagnostic procedures like flow cytometry, cytogenetics, and molecular genetics are still pending. However, a human-in-the-loop approach is still needed as we manually selected representative regions of the BMS for evaluation by the DL model. Also, it is to be noted that bone marrow donors in our cohort were substantially younger than AML patients. Increased age is associated with observable changes in the bone marrow microenvironment such as cellularity, proliferative activity, and apoptosis  and suchNPM1 from cytomorphology alone. For NPM1 prediction our DL model achieved a high accuracy of 0.86 in predicting mutation status. AML with mutated NPM1 has previously been associated with cup-like blast morphology [NPM1 classification with occlusion sensitivity maps, we found so far unreported features like a pattern of condensed chromatin accompanied by perinuclear lightening zones for NPM1-mutated blasts. We observed prominent nucleoli in myeloblasts as a feature the DL model derived to predict wild-type NPM1 AML while these could only rarely be observed in NPM1-mutated AML samples and then led to misclassification by the model. Further, we found a significantly higher VAF in NPM1 true positives while the group of false negatives was comprised of a significantly higher rate of tAML. Wild-type NPM1 serves as a critical structural protein of the nucleolus, but mutations lead to a delocalization to the cytoplasm [NPM1 AML and the absence thereof in mutated NPM1 AML—both detected as highly predictive features by our DL model.Furthermore, we used DL to predict the mutation status of rphology , 37. Wheytoplasm . This prytoplasm , 40. Weaytoplasm . Arguabl12 deficiency, or hematologic neoplasms such as MDS are associated with subtle changes of cell morphology [Our study shows that DL can derive morphologic features from cytomorphology that predict mutation status. Future work will focus on other clinically important mutations and their morphologic imprint that DL may be able to pick up. In line with our findings, a recent study showed that DL can associate the morphology of myelodysplastic syndromes (MDS) with distinct genetic imprints . Howeverrphology , 44. In rphology  recentlyrphology . NeverthNPM1 mutation status and derived so far unreported morphologic features that indicate absence or presence of NPM1 mutations from myeloblast morphology. This approach can be implemented to aid in clinical decision making, accelerate diagnosis, and may serve as a proof-of-concept for further studies of genetic imprints on disease morphology using DL.In conclusion, we here present a DL approach for the fast and accurate detection of AML from bone marrow cytomorphology. Our DL model accurately predicts


Cells achieve this through segregation of the initiators and modulators to specific plasma membrane (PM) subdomains, such as lipid rafts and caveolae, as well as by dynamic close contacts between the endoplasmic reticulum (ER) membrane and other intracellular organelles, including the PM. Especially, these membrane contact sites (MCSs) are currently receiving a lot of attention as their large influence on cell signaling regulation and cell physiology is increasingly appreciated. Depletion of ER Ca2+ stores activates ER membrane STIM proteins, which activate PM-residing Orai and TRPC Ca2+ channels at ER–PM contact sites. Within the MCS, Ca2+ fluxes relay to cAMP signaling through highly interconnected networks. However, the precise mechanisms of MCS formation and the influence of their dynamic lipid environment on their functional maintenance are not completely understood. The current review aims to provide an overview of our current understanding and to identify open questions of the field.In eukaryotic cells, ultimate specificity in activation and action—for example, by means of second messengers—of the myriad of signaling cascades is primordial. In fact, versatile and ubiquitous second messengers, such as calcium (Ca For example, PKA-mediated phosphorylation of IP3Rs at multiple sites affects its activity in an isoform- and cell type-specific manner. In fact, whereas the output of IP3R1 is enhanced through PKA-mediated phosphorylation on Ser1589 and Ser1755 i clearance. In fact, PKA-mediated phosphorylation of the SERCA inhibitor phospholamban (PLN) at Ser16 dissociates the PLN/SERCA complex and results in SERCA-mediated clearance of [Ca2+]i into the ER i regulates the fraction of E-Syt1 present throughout the ER or concentrated at ER–PM contact sites [2+]i and PM PIP2 levels regulate the formation/maintenance of E-Syt-dependent ER–PM contact sites [2+]i concentrations—low micromolar Ca2+ range—required for E-Syt1 recruitment to the PM are established through activation of extracellular Ca2+ influx, including SOCE, E-Syts-dependent contacts are not required for SOCE itself i concentration stimulates E-Syts-mediated bidirectional transport of glycerolipids—including DAG—between ER and PM driven by the lipid concentration gradient in the membranes [For example, upon association of E-Syts with the PM, micromolar [Caembranes ,217. As embranes . Additioembranes . Additioembranes ,190. Simembranes ,150. Subembranes ,150. Simembranes .Membrane composition is crucial in defining ion channel structure and function, either through specific interactions between lipids and proteins or nonspecific through changes in membrane physicochemical properties—thickness, fluidity, and curvature—that affect ion channel dynamics. Lipids directly interacting with membrane-embedded proteins are defined as annular lipids, those lipids in the first ring surrounding the protein, and non-annular lipids, lipids present in clefts or at the protein subunit interface. For example, molecular dynamics (MD) simulations emphasize the crucial influence of membrane structure and thickness on the function of membrane channels and provide important insights into the influence of the lipid microenvironment on protein function P2-poor regions. Due to the lack of PIP2, SARAF is unable to interact with STIM1, thus allowing maximal Ca2+ influx. Next, the STIM1-Orai1 complex migrates into PIP2-rich domains P2 translocates to the STIM1-Orai1 complex), allowing SARAF to interact with STIM1 followed by SCDI-mediated Ca2+ influx attenuation [2+ influx through Orai1 operates by translocation between PIP2 domains.Anionic lipids, such as PI species, are asymmetrically distributed over the inner and outer leaflets of the PM by active transporters maintaining a higher concentration of these lipid species in the inner leaflet, a process of high physiological importance . Due to reserved . In factreserved , suggestreserved . Interesenuation . Thus, rAdditionally, TRPC channels are regulated by PIP2 ,252,253.2+-regulated adenylyl cyclases—AC1, -5, -6, and -8—are targeted to plasma membrane rafts, whereas Ca2+-independent adenylyl cyclases—AC2 and -7—are not [Ca—are not . Cyclode—are not . The und—are not ,48, the —are not . In fact—are not ,258. Mor—are not . The res—are not . Moreove—are not .+, Ca2+, TRP, ANO1, and ENaC Na+ channels [+/H+ exchangers [+.HCO3− cotransporters [2 through, for example, direct interaction of PIP2 with the transporter [2 levels [Similar to Orai, additional ion transporters and channels are structurally and functionally modulated through alterations in their lipid environment ,262,263.channels ; Na+/H+ changers ; Na+.HCOsporters ,268; andsporters , are regnsporter ,271 or r2 levels . Togethe2+ and cAMP. Therefore, a better understanding of this complex regulation may also reveal unknown aspects of crucial cell functions, such as ion and fluid secretion [It is clear that, through dynamic modulations of its lipid environment and associated proteins, the ER–PM contact site plays a huge regulatory role on the spatiotemporal activity of the versatile second messengers Caecretion ,277,278.


We previously showed that RyR refractoriness plays a key role in the onset of SR Ca2+ alternans in the intact rabbit heart, which contributes to arrhythmogenic action potential duration (APD) alternans. Recent studies have also implicated impaired SERCA function, a key feature of heart failure, in cardiac alternans and arrhythmias. However, the relationship between reduced SERCA function and SR Ca2+ alternans is not well understood. Simultaneous optical mapping of transmembrane potential (Vm) and SR Ca2+ was performed in isolated rabbit hearts (n = 10) using the voltage-sensitive dye RH237 and the low-affinity Ca2+ indicator Fluo-5N-AM. Alternans was induced by rapid ventricular pacing. SERCA was inhibited with cyclopiazonic acid . SERCA inhibition  resulted in dose-dependent slowing of SR Ca2+ reuptake, with the time constant (tau) increasing from 70.8 ± 3.5 ms at baseline to 85.5 ± 6.6, 129.9 ± 20.7, and 271.3 ± 37.6 ms, respectively . At fast pacing frequencies, CPA significantly increased the magnitude of SR Ca2+ and APD alternans, most strongly at 10 μM . SERCA inhibition also promoted the emergence of spatially discordant alternans. Notably, at all CPA doses, alternation of SR Ca2+ release occurred prior to alternation of diastolic SR Ca2+ load as pacing frequency increased. Simultaneous optical mapping of SR Ca2+ and Vm in the intact rabbit heart revealed that SERCA inhibition exacerbates pacing-induced SR Ca2+ and APD alternans magnitude, particularly at fast pacing frequencies. Importantly, SR Ca2+ release alternans always occurred before the onset of SR Ca2+ load alternans. These findings suggest that even in settings of diminished SERCA function, relative refractoriness of RyR Ca2+ release governs the onset of intracellular Ca2+ alternans.Sarcoplasmic reticulum (SR) Ca At rapid rates, beat-to-beat alternation in the amount of Ca2+ released from the SR has been demonstrated to underlie the development of repolarization alternans  duration (APD) alternans  . At stealternans , which clternans . Severallternans . For exalternans . Indeed,lternans . Severallternans , whereaslternans . However2+ alternans, presumably due to insufficient Ca2+ reuptake during diastole and subsequent alternation of SR Ca2+ load   and is ka2+ load . Becausea2+ load , 2014, i release . Indeed, release . Studieslternans , 2012. Olternans , 2016. Iion mice . A recenion mice . These c2+ alternans and subsequent arrhythmogenic APD alternans, we performed optical mapping of free intra-SR Ca2+ concomitantly with transmembrane potential (Vm) in intact rabbit hearts. We assessed the impact of reduced SERCA function using cyclopiazonic acid (CPA), a SERCA inhibitor  were housed on a 12 h light–dark cycle and given access to food and water ad libitum.All procedures involving animals were approved by the Animal Care and Use Committee of the University of California, Davis (Reference No. 20991), and adhered to the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health . Male New Zealand White rabbits . Following cannulation of the aorta, Langendorff perfusion was initiated with oxygenated  Tyrode’s solution of the following composition (in mmol/L): NaCl 128.2, CaCl2 1.3, KCl 4.7, MgCl2 1.05, NaH2PO4 1.19, NaHCO3 20, and glucose 11.1 (pH 7.4 ± 0.05). The perfusate was pumped from a reservoir with 2 L of Tyrode’s solution through an in-line filter and two bubble traps before passing via the cannula to the heart and then recirculated from the perfusion chamber back to the reservoir and re-gassed. Flow rate (∼30 ml/min) was adjusted to maintain a perfusion pressure of 60–70 mmHg. The heart was securely positioned supine in the perfusion chamber. The perfusion apparatus was temperature controlled with heated baths used for the perfusate and a water-jacketed perfusion chamber. Two Ag/AgCl disc electrodes were positioned in the bath to record an electrocardiogram (ECG) analogous to a lead I configuration. A bipolar pacing electrode was positioned on the base of the left ventricular (LV) epicardium.Rabbit hearts were Langendorff-perfused as described previously . Briefly2+] and transmembrane potential (Vm) was performed as previously described in detail . The emitted fluorescence signals were recorded using two CMOS cameras  with a sampling rate of 0.5–1 kHz and 100 × 100 pixels with a field of view of 31 × 31 mm.The anterior epicardial surface was excited using LED light sources centered at 470 nm  and bandpass filtered from 475 to 495 nm . The emitted fluorescence was collected through a THT-macroscope (SciMedia) and split with a dichroic mirror at 545 nm . The longer wavelength moiety, containing the V2+ release was measured as recovery of SR Ca2+ release with a single extra-stimulus (S1–S2 pacing protocol).Baseline electrophysiological parameters were determined during LV epicardial pacing at a pacing cycle length (PCL) of 300 ms using a 2-ms pulse at twice the diastolic threshold. To induce alternans, the PCL was decremented in 20-ms steps. The effects of pharmacological SERCA inhibition were determined by adding CPA (1–10 μM) to the perfusate. Relative refractoriness of RyR CaBV_Analyze, Brainvision, Tokyo, Japan; and Optiq, Cairn, United Kingdom) and a free optical mapping analysis software ElectroMap  and processed with a Gaussian spatial filter (radius 3 pixels). For both APs and SR Ca2+ transients, activation time was determined at 50% of the maximal amplitude (or time at 50% of nadir for SR Ca2+). For APs, repolarization time at 80% return to baseline was used to calculate APD (APD80). SERCA function was quantified using the time constant (τ) of a single exponential fit to the recovery portion of the SR Ca2+ trace (from 5 to 90% recovery).Data analysis was performed using two commercially available analysis programs , where n is the beat number and t is time. A fast Fourier transform (FFT) was used to compute the power spectra across beats for each t, and the spectra were summed. The alternans magnitude was then defined as the resulting amplitude of the summed spectra at 0.5 cycles/beat. This approach allowed us to determine if an area within the mapping field of view was experiencing significant APD or SR Ca2+ alternans (greater than the background noise levels) as well as the spatial extent of significant alternans. A magnitude of ≥ 2 was used as the minimum threshold for significant APD or SR Ca2+ alternans, corresponding to a beat-to-beat change in APD90 ≥ 5 ms or beat-to-beat change in SR Ca2+ release amplitude ≥ 5%, respectively.The spectral method, which has been used clinically for detecting micro-volt T-wave alternans , was useescribed . The spe2+ load alternans and SR Ca2+ release alternans, quantification of the SR Ca2+ transient was also performed, as previously described  release amplitude to the average large beat (L) release amplitude (1 - S/L) during a 1- to 2-s recording. The amplitude of diastolic SR Ca2+ load alternans was calculated as the average difference between diastolic levels (D) of S and L beats divided by the average L amplitude (D/L) during a 1- to 2-s recording. Data are expressed as mean ± standard deviation (SD) and were compared using a one-way ANOVA with Bonferroni post-hoc test for multiple groups or a Student t-test when only two groups were compared. p < 0.05 was considered statistically significant.To more precisely differentiate between the onset of diastolic SR Caescribed . The ampm and SR Ca2+, increasing doses of CPA, a specific SERCA inhibitor (m) and Fluo-5N (SR Ca2+) was performed at 300-ms PCL . CPA did not impact mean conduction velocity . The marked prolongation of APD in the presence of CPA suggests that alterations in SR Ca2+ reuptake feed back onto Vm to influence AP dynamics, potentially due to a reduction in Ca2+-dependent inactivation of L-type Ca2+ current (ICaL) and consequent increased ICaL during the AP plateau.To determine the role of SERCA function on Vnhibitor , were ad0-ms PCL . As expe0-ms PCL  and decrt manner . These ft manner . Furtherime tau, , with ta2+ alternans   increased the incidence and magnitude of both APD and SR Calternans . Example+ traces . Consister PCLs) . In the t manner . However2+ alternans, it does not differentiate SR Ca2+ diastolic load from release alternans. To more precisely investigate the role of SERCA inhibition, detailed quantification of the SR Ca2+ transient was performed as shown in 2+ release alternans always occur at slower PCLs, prior to the onset of SR Ca2+ load alternans, indicating the prominent role of RyR refractoriness in governing the onset of alternans  CPA , indicating that SR Ca2+ content does not appreciably alternate from beat to beat and that at this pacing frequency, availability of RyR for Ca2+ release is a key contributor to alternans both with and without CPA.To determine if SERCA inhibition modifies relative RyR refractoriness and recovery of SR Ca μM) CPA . At S1 =h groups . Notablyrecovery . This pr2+ alternans with increasing doses of CPA and progressively faster PCLs. Spatial discordance occurs when a clear nodal line is observed (5 μM of CPA at 200 ms and 10 μM of CPA at 220 and 200 ms) and was confirmed by assessing alternans phase  even when the heart is spatially out-of-phase . Spatially discordant alternans are known to create very large gradients in repolarization, setting the stage for unidirectional conduction block and the induction of reentry  around 400 μM and therefore exhibits minimal fluorescence in the cytosol compared to the SR lumen, where the Ca2+ content is at or near the millimolar range. Thus, SR Ca2+ dynamics can be assessed independently from transmembrane Ca2+ flux. Both fluorescent indicators are excited with blue light (488 nm), and the emission wavelengths are split and recorded on two separate high-speed detectors, allowing for precise beat-to-beat mapping of Vm and SR Ca2+ dynamics at high spatial and temporal resolutions (2+ reuptake (tau), SERCA inhibition also dose-dependently prolonged APD and decreased the relative amplitude of SR Ca2+ release. Decreased SR Ca2+ transient amplitude is suggestive of decreased SR Ca2+ content, which would be expected with SERCA inhibition; however, Fluo-5N Ca2+ signals are uncalibrated, and therefore, signal amplitudes can only be used to ascertain relative differences.We previously developed methodology using the voltage-sensitive dye, RH237, and low-affinity Cact heart . Fluo-5Nolutions . As show2+ load varies little from beat to beat, indicating a precise balance between RyR Ca2+ release and SERCA reuptake during each ECC cycle. We have previously shown in normal rabbit hearts that as heart rate increases, beat-to-beat alternation of SR Ca2+ release occurs prior to appreciable alternation of SR Ca2+ load and that relative refractoriness of RyR governs the onset of arrhythmogenic alternans  and yet promotes SR Ca2+ alternans at faster pacing . One possible explanation is that greatly impaired SERCA function , CPA tended to increase the magnitude of SR Ca emerged , 3. High emerged , 5. Thes μM, CPA atically , especiancreases , which ireuptake . Thus, tintracellular Ca2+ alternans will also depend on transmembrane Ca2+ flux  and intracellular Ca2+ concentration, which were not assessed here but are also dependent on SERCA and RyR activity. Indeed, a previous study in the isolated rabbit working heart showed that the relationship between SERCA activity and whole heart mechanical function is highly non-linear and also involves significant changes in intracellular Ca2+ flux . The results of this study suggest that simply changing SR Ca2+ regulation can shift this entire continuum of mechanisms, in this case, to occur at slower pacing frequencies. Consistent with previous studies, we posit that dynamical mechanisms  likely still govern the onset of spatial discordance  of ICaL, which would tend to prolong APD. However, a smaller Ca2+ transient also tends to reduce the magnitude of inward Na+–Ca2+ exchange current (INCX), which would tend to shorten APD. Therefore, the net change in APD is a balance between these two mechanisms. The fact that CPA prolongs APD suggests that the net effect of reduced SERCA function  on APD is via reduced CDI of ICaL. This may seem somewhat at odds with the positive SR Ca2+/Vm coupling observed during alternans (large SR Ca2+ transient corresponds to long APD, 2+ transient, intrinsic SERCA function is the same during large and small beats, and in this case, inward INCX depends directly on [Ca]i and predominates over ICa inactivation on APD.SR Ca2+ in the progression from normal rhythms to arrhythmogenic cardiac alternans. While SERCA inhibition caused SR Ca2+ and subsequent APD alternans to appear at longer PCLs, this was not directly due to altered diastolic [Ca2+]SR. However, the reduced SERCA function and consequent lower SR Ca2+ load prolonged RyR refractoriness, which is how alternans is promoted by SERCA inhibition. Indeed, SR Ca2+ release alternans occurred prior to SR Ca2+ load alternans under all conditions, and that can lead secondarily to load alternans, APD alternans, and ultimately spatial discordance. These findings may provide important insight into underlying mechanisms governing alternans onset and severity in failing hearts, where reduced SERCA function is a common phenotype.These findings shed new light on the role of SR Ca2+ and APD alternans but did not specifically address susceptibility to VT/VF nor how SERCA inhibition with CPA may alter VT/VF dynamics. This remains an important area for future study. Although we did not observe appreciable changes in diastolic SR Ca2+ load prior to the onset of SR Ca2+ release alternans, it is possible that very small changes in diastolic SR Ca2+ occur at the luminal side of RyR and govern release. Our imaging approach does not have the spatial resolution nor sensitivity to definitively rule this out.This study assessed mechanisms of SR CaThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The animal study was reviewed and approved by the UC Davis Institutional Animal Care and Use Committee.LW and CR conceived the study, designed and conducted experiments, analyzed data, generated figures, and wrote the manuscript. IL analyzed, reviewed and interpreted data, and edited the manuscript. RM and DB contributed to study conception and design, critically reviewed and interpreted data, and edited the manuscript. All authors have reviewed the data and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The organization and distribution of these two groups of cardiac calcium channel clusters critically underlies the uniform contraction of the myocardium. Ca2+ signaling between these two sets of adjacent clusters produces Ca2+ sparks that in health, cannot escalate into Ca2+ waves because there is sufficient separation of adjacent clusters so that the release of Ca2+ from one RyR2 cluster or supercluster, cannot activate and sustain the release of Ca2+ from neighboring clusters. Instead, thousands of these Ca2+ release units (CRUs) generate near simultaneous Ca2+ sparks across every cardiomyocyte during the action potential when calcium induced calcium release from RyR2 is stimulated by depolarization induced Ca2+ influx through voltage dependent CaV1.2 channel clusters. These sparks summate to generate a global Ca2+ transient that activates the myofilaments and thus the electrical signal of the action potential is transduced into a functional output, myocardial contraction. To generate more, or less contractile force to match the hemodynamic and metabolic demands of the body, the heart responds to β-adrenergic signaling by altering activity of calcium channels to tune excitation-contraction coupling accordingly. Recent accumulating evidence suggests that this tuning process also involves altered expression, and dynamic reorganization of CaV1.2 and RyR2 channels on their respective membranes to control the amplitude of Ca2+ entry, SR Ca2+ release and myocardial function. In heart failure and aging, altered distribution and reorganization of these key Ca2+ signaling proteins occurs alongside architectural remodeling and is thought to contribute to impaired contractile function. In the present review we discuss these latest developments, their implications, and future questions to be addressed.The architectural specializations and targeted delivery pathways of cardiomyocytes ensure that L-type Ca The level of calcium influx through CaV1.2 channels triggers a graded amount of calcium induced calcium release (CICR) from the type 2 ryanodine receptors (RyR2) on the juxtaposed junctional sarcoplasmic reticulum (jSR) at specialized nanodomains between the sarcoplasmic reticulum (SR) and the t-tubule or surface plasma membrane (PM). These SR-PM junctions termed dyads or calcium release units (CRUs), house the fundamental ion channel machinery for EC-coupling, bringing clusters of CaV1.2 channels into nanometer proximity of their intracellular Ca2+ channel partners RyR2  signaling). We also highlight nanoscale reorganization of these two calcium channels that occurs during heart failure (HF) and aging and discuss the implications for contractile function.During action potential (AP)-driven membrane depolarization in ventricular myocytes, Caers RyR2 . These t1C subunit (~250 kDa), associated with CaVβ (~52–72 kDa depending on the isoform), CaVα2δ (~175 kDa), and sometimes CaVγ (~32 kDa) auxiliary subunits in a 1:1:1:1 stoichiometry  has revealed that CaV1.2 channels are not just homogenously distributed along the sarcolemma but rather, preferentially form clusters in both the t-tubules  and sarcÅ 10 nm; , 2016. Aer areas  so a siminotropy .via microtubules anchored by a protein called bridging integrator 1  intracellular, Cast cells . These lst cells . Electrost cells . The 3D st cells  and formst cells .in silico finding that RyRs located within <100 nm of an activated cluster are exposed to sufficiently high levels of Ca2+ (>10 μM) to permit their activation  activity and downstream phosphorylation of multiple molecular targets and effectors as recently reviewed (Po) and gating properties are altered in favor of increased activity and longer mode 2 openings  bridging of neighboring channels via their C-terminal tails  to the dyad, shown with diffraction-limited confocal microscopy  around HF can be defined as the progressive inability of the heart to adequately pump blood to meet the demands of the body. We focus here on changes observed in CaV1.2 and RyR2 calcium channels during HF with reduced ejection fraction (HFrEF) which commonly occurs after cardiac injury eg. ischemia, or with sustained stress eg. hypertension. There remains a limited understanding of the effects of HF with preserved ejection fraction (HFpEF) on CaV1.2 and RyR2 channel function and regulation, hampered by difficulties in generating an accurate animal model . The curThe effects of HF and myocardial infarction (MI) on the t-tubule network, dyad integrity, and SR positioning has been previously  and very2+ leak  motifs bind phospholipids to form the sarcolemmal anchor . Acute k2+ leak . Loss ofach them . This dy channel . Loss ofhythmias . Transgehythmias .Proteomic analyses have further revealed the serine/threonine protein kinase SPEG  as a binding partner of both JPH2 and RyR2, and thus another component of this dyadic protein complex . In that2+ leak, exerting a cardioprotective effect P3, and to a lesser extent PIP2 P2 . Furtherjph2 transcript expression in left ventricular tissue samples from patients with dilated cardiomyopathy  to RyR2  that was transduced into cardiomyocytes ive mice . Triadin to RyR2 . Photoac to RyR2 . Further to RyR2 , adult m to RyR2 , and adu to RyR2 . The mob to RyR2 . Envisio to RyR2 . If inde to RyR2 . It seemV4.2 and KV4.3 potassium channels in mouse ventricular myocytes  was unchanged compared to healthy control myocytes and it was prophetically posited that the ‘increased activity of superficial channels compensates for a reduced channel expression in the t-tubules’ (ICa in failing rat ventricular myocytes post coronary artery ligation (ICa density in intact (tubulated) failing myocytes compared to healthy sham controls, detubulation revealed that the proportion of channels contributing to that current had shifted in their distribution from a predominantly t-tubular locale in health (24:76% crest:t-tubule ratio) to a more even distribution in failure  model) the phosphorylating kinase was identified as PKA and the channel population displaying increased activity was on the t-tubules, not the crest . Interesd in ICM  suggestiICa was found to be maintained in HF myocytes but calcium transient amplitude and the ability to trigger Ca2+ sparks was reduced. Spatial remodeling was postulated as the cause of this reduced EC-coupling gain with three scenarios proposed, (1) mismatch where the t-tubule network remained unaltered by the CaV1.2 and RyR2 channels were misaligned; (2) increased width of the dyadic cleft; and (3) altered t-tubule networks resulting in ‘orphaned’ RyR2 whereby spatial reorganization of the t-tubule network would leave stranded jSR elements behind without juxtaposed CaV1.2 cluster partners. Remodeling of the t-tubule network had already been reported by Tim Kamp’s group .The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The intensification of livestock production in response to the growing demand for food has disrupted the balance between the quantity and quality of animal-based products, contributing to changes in consumer preferences. Modern consumers show an increasing interest in extensive farming and niche products that are regarded as more unique, tastier, and healthier due to the absence of harmful compounds and a higher content of valuable components . However, consumers often formulate their views and opinions based on widely-held beliefs that are not always supported by research findings. Therefore, science-based evidence is needed to draw reliable conclusions about meat products. The fatty acid composition of meat from rabbits raised under intensive and extensive production systems was compared in this study. It was found that intensively farmed rabbits can produce meat of superior quality.2) and were fed a commercial pelleted diet. In group EPS, the rabbits were housed in free-standing cages on straw litter  and were fed a conventional farm-made diet . The FA profile of intramuscular fat (IMF) was analyzed in samples of Longissimus thoracis et lumborum (LTL) muscle. The analyzed production systems had no significant effect on the content of most FAs in IMF. However, the differences between group means contributed to more desirable values of the quality indicators of IMF (in particular in the LTL muscle) in group IPS. The study demonstrated that the claim that meat produced under less intensive farming systems is of superior quality could be an oversimplified generalization that should be validated in research.The aim of this study was to compare the fatty acid (FA) profile of meat from New Zealand White rabbits raised from 30 to 90 days of age under intensive (IPS) and extensive (EPS) production systems. In group IPS, the rabbits were housed in wire mesh cages with a slatted floor (16.7 animals/m In recent decades, the high demand for food on global markets has contributed to the intensification and standardization of food production. In the coming years, this trend is likely to be maintained due to rapid population growth . HoweverRabbits are generally regarded as an animal species that are typically raised in small farms and traditional production systems . MeanwhiModern rabbit breeds differ in their suitability for extensive and intensive production . The folExtensive (and organic) farming methods are highly varied, and they are difficult to compare with intensive production systems in terms of output . The limThe aim of this study was to compare the FA profile of meat from New Zealand White rabbits raised under intensive and extensive production systems.2), indoor . They were fed ad libitum a commercial pelleted diet , supplemented with the coccidiostat robenidine (robenidine hydrochloride—65 mg/kg). The chemical composition and FA profile of the experimental diet are presented in The experiment was approved by the local Ethics Committee for Animal Experimentation  as part of long-term comprehensive studies investigating the factors that affect the quality of rabbit meat. The experiment was conducted in spring and summer, in two rabbit farms (A and B) located in north-eastern Poland, on 40 New Zealand White rabbits born in farm A. The animals were weaned at 30 days of age, and they were divided into a group representing an intensive production system (IPS) and a group representing an extensive production system (EPS). Throughout the experiment, group ISP rabbits were kept on farm A, in wire mesh cages with a slatted floor (0.4 m × 0.6 m × 0.32 m), with 4 animals per cage . They were fed ad libitum a conventional, farm-made diet composed of green fodder (70%), barley grain (10%), stale bread (10%), and hay (10%). Feed was supplied twice daily, and the daily ration was increased as the animals grew older, taking into account the number of leftovers. Group EPS rabbits did not receive any coccidiostat. The chemical composition and FA profile of the experimental diet are presented in Group EPS rabbits were transported to a small-scale farm (B), located at a distance of 25 km from farm A, where they were housed in free-standing cages (1 m × 2 m × 0.6 m) on wheat straw litter, with 5 animals per cage  muscle was removed from the intermediate part, and leg muscles (LM) were removed from the hind part of each carcass. The muscles were transported to the laboratory, where they were passed three times through a 3 mm plate in a meat grinder and thoroughly mixed to obtain samples for analyses.The rabbits  were slaughtered at 91 days of age, after 24 h of fasting. Chilled carcasses  were divided into the following parts: head , fore part (cut between the last thoracic vertebra and the first lumbar vertebra), intermediate part (cut behind the last lumbar vertebra) and hind part (carcass section that remains after the intermediate part had been cut off the fore part). The The FA profile was determined in intramuscular fat (IMF) extracted from LTL muscle and LM samples by the Soxhlet method . Fatty ap ≤ 0.05.The results were analyzed statistically with the use of STATISTICA ver. 13.3 software . The effect of the experimental factor (IPS/EPS) on the FA profile of rabbit meat (in the LTL muscle and LM) was determined by one-way analysis of variance (ANOVA). The significance of differences between group means was estimated by Bonferroni’s test at p = 0.018) in the LTL muscle of rabbits in group EPS.The SFA profile of IMF is presented in p > 0.05) content of lauric acid (C12:0) , myristic acid (C14:0) , and palmitic acid (C16:0) , as well as stearic acid (C18:0) in the LTL muscle (p = 0.131). As a result, the muscles of extensively farmed rabbits had a higher total content of SFAs, but a significant (p = 0.039) difference between group means was noted only in the LTL muscle.The meat of extensively farmed rabbits had higher  in the LTL muscle of rabbits in group ESP. The concentrations of individual MUFAs and, consequently, their total content were comparable in the LTL muscle (p = 0.798) and LM (p = 0.467) of rabbits raised under both production systems.The UFA profile of IMF is presented in p < 0.001) was noted only in the content of arachidonic acid (C20:4) in the LTL muscle. The differences in the concentrations of individual MUFAs in the muscles of rabbits raised under two production systems resulted in their higher total content  in the IMF of intensively farmed rabbits. The differences between group means, determined in the LTL muscle, were significant at p = 0.019 and p = 0.039, respectively.An analysis of the content of PUFAs in rabbit meat revealed that it was higher in group IPS . Howeverp = 0.045) and PUFA/SFA (p = 0.026) ratios, a more desirable ratio of hypocholesterolemic to hypercholesterolemic FAs (DFA/OFA) (p = 0.048), and a higher proportion of essential FAs (EFAs) (p = 0.037). The MUFA/SFA ratio  and the nutritional value of IMF  were similar in rabbits raised under both production systems.The indicators characterizing the nutritional value of IMF were calculated based on its FA profile . The val2) and were fed ad libitum a commercial pelleted diet. In the organic group, rabbits were housed in collective pens on straw litter  and were fed ad libitum a mixture of organic products . The hind leg meat of organically produced rabbits had a lower (p < 0.05) content of SFAs and MUFAs (p < 0.001), a higher (p < 0.001) content of PUFAs, and a more favorable (p < 0.001) PUFA/SFA ratio, relative to the meat of conventionally produced rabbits. These findings differ from the results of this study in terms of SFA and MUFA concentrations in the meat of rabbits in groups ISP and ESP. According to the cited authors, the differences in the proportions of the analyzed FA groups in the meat of conventionally and organically raised rabbits could result from different feeding diets (differences in the SFA and PUFA content of diets) and/or different levels of locomotor activity.Different rabbit production systems are difficult to compare due to the presence of numerous factors and variables specific to each system  and their interactions . Such di2) were located in the experimental rabbitry. The outdoor colony cages (5 rabbits/m2) were placed in an outdoor pen. The rabbits were fed ad libitum an organic  or a conventional (pelleted feed) diet. The study revealed that meat (Longissimus lumborum) from rabbits housed indoors and fed conventional pellets was richer in PUFAs (p < 0.05) and poorer (p > 0.05) in SFAs and MUFAs, as compared with meat from rabbits housed indoors and outdoors and fed an organic diet. These findings are consistent with the results of this study in terms of the SFA and PUFA content of meat from intensively and extensively farmed rabbits.Paci et al.  evaluateThe effect of rabbit diets on the FA profile of IMF, described by Pla  and PaciM. Longissimus thoracis et lumborum and M. Biceps femoris) of rabbits housed on deep-litter straw had lower PUFA content than the meat of rabbits housed on wire- or plastic-mesh floors. Moreover, the use of litter in rabbit farming may be associated with certain health problems (mostly coccidiosis), which affect productive performance. Therefore, Lambertini et al. [When analyzing the influence of rabbit diets on the FA composition of meat , attention should be paid to the fact that rabbits often eat their bedding material. According to Dal Bosco et al. , the coni et al.  concludeM. longissimus dorsi (MLD) and M. vastus lateralis (MVL) muscles, relative to the control group. However, the levels of stearic acid (C18:0) and arachidonic acid (C20:4 n-6) decreased significantly in the MVL muscle after the exercise. Changes in the FA profile resulting from the physically loaded condition showed the same tendency in both muscles, but it was more pronounced in the MVL muscle, probably because this muscle was exposed to more intensive exercise.According to Pla , the FA 2 may affect locomotion, but research results are inconclusive. D’Agata et al. [2 vs. indoor—colony cages at a density of 15 rabbits/m2) on meat quality (hind leg) in local grey-colored rabbits (agouti and wild type) and found that meat from outdoor rabbits contained fewer SFAs (p < 0.05) and more MUFAs (p < 0.01). In the group of PUFAs, differences between the two systems were noted only in the content of C20:4 n-6 and C22:5 n-3, which was higher (p < 0.05) in the meat of indoor rabbits. The differences could be due to the lower (by 0.28 percentage points) amount of IMF in their meat and, consequently, a higher percentage of phospholipids which, as noted by Enser [The potential effect of the locomotor activity of rabbits on the FA composition of their meat can be indirectly inferred from studies investigating production systems with different stocking densities. It appears that the number of animals per ma et al.  analyzedby Enser , are ric2 per rabbit) on a polyphyta natural pasture, and a conventional indoor rearing system, where rabbits were kept in conventional bi-cellular cages (0.07 m2 per rabbit) on the FA composition of meat. The above authors demonstrated that meat (hind leg) from rabbits of the “Leprino of Viterbo” synthetic breed reared outdoors had higher (p < 0.05) SFA content, lower (p < 0.01) MUFA content, and higher (p < 0.05) PUFA (including n-6 PUFA) content. The higher long-chain PUFA content of rabbit meat could be a consequence of the lower muscle fat content in meat from open-air reared rabbits compared with the indoor group. In view of the contradictory results reported by D’Agata et al. [Different results were reported by Cavani et al. , who anaa et al.  and Cavaa et al. , it is dIt appears that factors other than nutrition and locomotion (associated with different production systems) exert only limited effects on the FA profile of rabbit meat. This hypothesis is confirmed by the results of studies investigating the influence of floor type (wire mesh and plastic net) ,32, housThe analyzed production systems had no significant effect on the content of most FAs in the IMF of New Zealand White rabbits. However, the differences between group means revealed certain tendencies, which were reflected in the indicators characterizing the FA profile and nutritional value of meat. Their values were more desirable in IMF extracted from the LTL muscle of intensively farmed rabbits. A tendency towards higher values of these indicators was also observed in IMF extracted from the LM in group IPS, but no significant differences were found relative to group EPS. The results of this study indicate that the claim that meat produced under less intensive farming systems is of superior quality could be an oversimplified generalization. Therefore, it should be validated in research accounting for highly differentiated conditions of extensive production, with particular emphasis on rabbit diets.


Mitochondrial or metabolic stress induced mitophagy through lysosomal Ca2+ release, increased cytosolic Ca2+ and TFEB activation. Lysosomal Ca2+ replenishment by ER- > lysosome Ca2+ refilling was essential for mitophagy. β-cell-specific Tfeb knockout (TfebΔβ-cell) abrogated high-fat diet (HFD)-induced mitophagy, accompanied by increased ROS and reduced mitochondrial cytochrome c oxidase activity or O2 consumption. TfebΔβ-cell mice showed aggravation of HFD-induced glucose intolerance and impaired insulin release. Metabolic or mitochondrial stress induced TFEB-dependent expression of mitophagy receptors including Ndp52 and Optn, contributing to the increased mitophagy. These results suggest crucial roles of lysosomal Ca2+ release coupled with ER- > lysosome Ca2+ refilling and TFEB activation in mitophagy and maintenance of pancreatic β-cell function during metabolic stress.Although autophagy is critical for pancreatic β-cell function, the role and mechanism of mitophagy in β-cells are unclear. We studied the role of lysosomal Ca 2+ release promotes mitophagy via activation of the transcription factor EB (TFEB) and loss of β-cell TFEB aggravates glucose intolerance during high-fat diet.Autophagy is important for pancreatic β-cell function, however, the role of mitophagy and mechanism for mitophagy in β-cells are unclear. Here the authors report that in stressed β-cells, lysosomal Ca Mitochondrial function is maintained by several factors such as mitochondrial biogenesis, fission, fusion, and selective autophagy (mitophagy). The importance of autophagy in the maintenance of β-cell function and mitochondria has been demonstrated in previous studies using β-cell-specific autophagy knockout (KO) (Atg7Δβ-cell) mice which had reduced β-cell mass and mitochondrial abnormalities2. However, the role and mechanism of mitophagy in pancreatic β-cells have not been clearly demonstrated.Mitochondrial function is essential for insulin release and survival of pancreatic β-cells3. TFEB family members also play a role in mitophagy5. In TFEB activation and mitophagy by mitochondrial stressors, lysosomal Ca2+ release plays an important part5. However, the mechanism that perpetuates lysosomal Ca2+ release and enables the full progression of mitophagy despite the small lysosomal volume6 is not clear.Transcription factor EB (TFEB) acts as a master regulator of lysosome biogenesis and autophagy gene expression2+ release through calcineurin. We also obtained data indicating the importance of ER → lysosome Ca2+ refilling in the sustained release of lysosomal Ca2+, TFEB activation and mitophagy by mitochondrial stressors. Finally, we observed that TFEB contributes to β-cell mitophagy through induction of mitophagy receptors such as Calcoco2 (Ndp52) and Optn.We investigated the mechanism and functional role of mitophagy in pancreatic β-cells and found that TFEB is activated by mitochondrial reactive oxygen species (ROS)-induced lysosomal Ca4. When we transfected cells with pMito-Keima encoding a pH-sensitive fluorescent probe with a mitochondria-targeting sequence7, tubular mitochondrial Keima fluorescence excited by 440-nm laser was observed. After rotenone treatment, a mitochondrial complex I inhibitor, many cells showed punctate fluorescence excited by a 590-nm laser (acidic puncta), indicating lysosomal delivery of mitochondrial Keima and mitophagy 8, TFE3 nuclear fluorescence was clearly observed after rotenone or O/A treatment as well , a cathepsin C substrate inducing osmotic lysis of lysosomes12, was significantly reduced by rotenone or O/A pretreatment, suggesting preemptying or release of lysosomal Ca2+ by rotenone or O/A  using Oregon Green 488 BAPTA-1 Dextran. [Ca2+]Lys of INS-1 cells was significantly reduced by rotenone or O/A  of cytoplasmic Ca2+ fluorescence ([Ca2+]i) tracing using Fura-2 after GPN treatment13, also showed significantly reduced [Ca2+]Lys by rotenone or O/A , an antioxidant, markedly reduced ROS accumulation by rotenone or O/A  antagonist, significantly suppressed [Ca2+]Lys recovery after mitochondrial stressor removal ethylene diamine (TPEN) that cannot chelate cytosolic Ca2+ due to low Ca2+ affinity21, both [Ca2+]Lys recovery after mitochondrial stressor removal and [Ca2+]i increase by mitochondrial stressors were reduced  during mitophagy in INS-1 cells transfected with GEM-CEPIA1er23. When [Ca2+]ER was determined after rotenone or O/A treatment in a Ca2+-free Krebs-Ringer bicarbonate (KRB) buffer inhibiting possible store-operated Ca2+ entry (SOCE), a significant decrease of [Ca2+]ER was observed  inhibition due to rotenone- or O/A-induced decrease of ATP in [Ca2+]ER decrease after mitochondrial stressor treatment without extracellular Ca2+ . Confocal microscopy after incubation with specific antibodies (Abs) and rolling circle amplification showed close contact between VAPA on ER and ORP1L on lysosome26 by rotenone or O/A -VAPA and STARD3-BirA(G79-C)-HA were treated with rotenone or O/A, biotin conjugate formation was observed after incubation with FITC-streptavidin, indicating contact between ER (VAPA) and lysosome (STARD3) 28. Indeed, STIM1 puncta were clearly observed after rotenone or O/A treatment 32, mitophagy by rotenone or O/A was again significantly reduced  mice to RIP-Cre mice, and imposed metabolic stress by feeding HFD. In this model, we wanted to confirm that metabolic stress can cause mitochondrial stress leading to mitophagy. We thus treated INS-1 cells with palmitic acid (PA), the most abundant saturated fatty acid in vivo and an effector of insulin resistance or β-cell failure34. PA treatment of INS-1 cells induced mitochondrial stress, as shown by mitochondrial ROS accumulation 39, Triacsin C (an acyl-CoA synthetase inhibitor), Fumonisin B1 (a sphingosine N-acyltransferase inhibitor) or Myriocin 40 did not reduce PA-induced mitochondrial ROS accumulation or TFEB nuclear translocation  familyParkin -fed TfebF/F or HFD-fed TfebΔβ-cell mice  activity in pancreatic islets was increased by HFD probably as an adaptation to metabolic stress, which was significantly reduced by β-cell Tfeb KO  staining that was increased in islets of  KO Fig. . Regardi KO Fig. , suggest KO Fig. , supportTfebΔβ-cell mice showed no abnormality in glucose profile up to 20 weeks of age   and body weight did not differ between TfebΔβ-cell and TfebF/F mice on either NCD or HFD  Fig. , while nTT) Fig. . Aggravadex Fig.  without dex Fig. , suggestTfebΔβ-cell mice to Tfe3-KO mice. While TfebF/F/Tfe3-KO and TfebΔβ-cell/Tfe3-KO mice had slightly impaired glucose tolerance compared to TfebF/F/Tfe3-WT mice and TfebΔβ-cell/Tfe3-WT, respectively, these differences were statistically insignificant in both NCD- and HFD-fed conditions , Clcn7 and Ctsa was upregulated in pancreatic islets of HFD-fed mice together with increased expression of Tfeb and Tfe3 themselves  assay. To this end, we studied promoters of human NDP52 and OPTN that are well characterized48. When 1.1B4 human islet cells generated by electrofusion of human primary pancreatic islet cells to pancreatic adenocarcinoma cells49 were treated with rotenone or O/A, NDP52 or OPTN expression was significantly increased, which was downregulated by siTFEB  can be activated by TFEB or ROS, and Ndp52 and Optn could be Nfe2l2 target genes51, we studied the role of Nfe2l2 in Ndp52 or Optn expression employing pancreatic islets from Nfe2l2-KO mice. Induction of Ndp52 or Optn in primary islet cells by rotenone or O/A was not significantly reduced by Nfe2l2 KO P2, ROS can also activate TRPML1 channel but not TRPML2 or TRPML3 channel5. Since ML-SI3 is not specific for TRPML1, ML-SI3-mediated abrogation of mitochondrial stressor-induced [Ca2+]i increase does not prove the role of TRPML1 in TFEB activation and mitophagy by mitochondrial stressors. However, lysosomal Ca2+ release by mitochondrial ROS that does not activate TRPML2 or TRPML3 channel and abrogation of mitochondrial stressor-induced TFEB activation by siMcoln1 strongly support the role of TRPML1 in mitochondrial stressor-induced lysosomal Ca2+ release. Following lysosomal Ca2+ release through TRPML1, increased [Ca2+]i dephosphorylated TFEB and TFE3 via calcineurin activation. We also observed notably decreased total TFEB and TFE3 following O/A treatment. Although the mechanism of this phenomenon is unknown, it could be due to changes in the phosphorylation of amino acid residues related to the stability of TFEB family members67.When we studied the mechanism of mitophagy in β-cells undergoing mitochondrial stress, we observed significantly increased Lys is comparable to [Ca2+]ER, lysosome is not a major Ca2+ reservoir because its volume is 1/10 of ER volume6. Previous papers demonstrated occurrence of ER → lysosome Ca2+ refilling for completion of events requiring lysosomal Ca2+20. ER Ca2+ channels such as InsP3R or ryanodine receptor channel could act as ER Ca2+ exit channels in ER → lysosome Ca2+ refilling. Indeed, genetic studies have shown the participation of InsP3R in mitophagy68; however, the role of ER Ca2+ or InsP3R in lysosomal Ca2+ refilling and the relationship between ER Ca2+ and lysosomal Ca2+ in mitophagy have not been studied. Our data show that ER → lysosome Ca2+ refilling occurs during mitochondrial stress, which is necessary for replenishment of lysosomal Ca2+ reservoir and completion of TFEB activation or mitophagy.Although the initial lysosomal Ca2+ refilling could be facilitated by ER-lysosome contact25. In addition to well-characterized ER-mitochondrial contact, ER-lysosome contact could have a functional role in cellular responses69. When ER Ca2+ emptying occurs due to ER → lysosome flux6, SOCE is likely to occur through CRAC activation, which is characterized by STIM1 oligomerization and translocation to ER-plasma junction where it interacts with Orai channels28. Thus, STIM1 oligomerization by mitochondrial stressors strongly supports that ER Ca2+ emptying occurs due to ER → lysosome Ca2+ flux, which also can explain the suppression of mitophagy by extracellular Ca2+ depletion70.ER → lysosome Ca2+ release coupled with ER → lysosome Ca2+ refilling during mitochondrial or metabolic stress, and TFEB-dependent induction of mitophagy, facilitated by transactivation of mitophagy receptors such as Ndp52 or Optn, plays an important role in β-cell adaptation to metabolic stress through improved mitochondrial function.Altogether, these results suggest that TFEB is activated by lysosomal CapMito-Keima (MBL), and then treated with rotenone or O/A for 18 h. Keima fluorescence at neutral pH was observed at an excitation wavelength of 430 ± 20 nm and an emission wavelength of 624 ± 20 nm. Keima fluorescence in acidic conditions was observed at an excitation wavelength of 562 ± 20 nm and emission wavelength of 624 ± 20 nm. To visualize mitophagy in another way, cells transfected with the mRFP-LC3 construct were treated with mitochondrial stressors and then incubated with anti-TOM20 Ab . Images were obtained using an LSM780 confocal microscope (Zeiss) from more than five different fields for each experiment. Colocalization of puncta or fluorescence in vitro was evaluated manually in a blinded manner.To visualize mitophagy using a mitophagy-specific probe, INS-1 cells were transfected with a 2+ probe12. After 48 h, lysosomal Ca2+ release was measured in a zero Ca2+ solution  with or without rotenone or O/A pretreatment for 1 h by monitoring fluorescence intensity at 470 nm with a LSM780 confocal microscope (Zeiss). GPN was added at the indicated time.INS-1 cells were transfected with a plasmid encoding a perilysosomal GCaMP3-ML1 Ca2+]i, cells were loaded with 2 μM of the acetoxymethyl ester form of Fura-2 (Invitrogen) in RPMI-1640 at 37 °C for 30 min and then rinsed with fresh solution. Measurements were performed using MetaFluor (Molecular devices) on an Axio Observer A1 (Zeiss) equipped with a 150 W xenon lamp Polychrome V (Till Photonics), a CoolSNAP-Hq2 digital camera (Photometrics), and a Fura-2 filter set. [Ca2+]i was determined in a phenol red-free RPMI-1640 using the 340/380 nm fluorescence ratio and the following equation71.Kd = Fura-2 dissociation constant (224 nM at 37 °C), Fmin(380) = the 380 nm fluorescence in the absence of Ca2+, Fmax(380) = 380 nm fluorescence with saturating Ca2+, R = 340/380 nm fluorescence ratio, Rmax = 340/380 nm ratio with saturating Ca2+, and Rmin = 340/ 380 nm ratio in the absence of Ca2+.For ratiometric measurement of [Ca2+]i after GPN treatment as an estimate of lysosomal Ca2+ content, cells were incubated in a zero Ca2+ solution containing 145 mM NaCl, 5 mM KCl, 3 mM MgCl2, 10 mM glucose, 1 mM EGTA and 20 mM HEPES (pH 7.4)20.To monitor [Ca2+]Lys, cells were loaded with 100 μg/mL of Oregon Green 488 BAPTA-1 Dextran (Invitrogen) at 37 °C for 12 h. Medium was then changed to one without Oregon Green 488 BAPTA-1 Dextran, and cells were chased for an additional 4 h. After treating cells with rotenone or O/A for the last 1 h of chasing, cells were washed in HEPES-buffered saline  for confocal microscopy20.To measure ER23. D1ER fluorescence intensity ratio (F540/F490) was determined using an LSM980 confocal microscope (Zeiss)INS-1 cells were transfected with GEM-CEPIA1er, INS-1 cells were loaded with Oregon Green 488 BAPTA-1 Dextran for 12 h and chased for 4 h. After treating cells with rotenone or O/A for 1 h, cells were monitored for [Ca2+]Lys and [Ca2+]ER in a Ca2+-free KRB buffer in the presence or absence of rotenone or O/A using an LSM780 confocal microscope.Twenty-four h after transfection with 2, 1.5 mM CaCl2, 11.5 mM glucose, 11.5 mM HEPES, pH 7.3) for 10 min in 37 °C incubator, LysoSensor green intensity was acquired using a confocal microscope and converted to pH using a standard curve obtained after treating cells with 10 μM nigericin for 20 min in 105 mM KCl buffer  adjusted to different pH72.After incubating cells with mitochondrial stressors or PA and 1 μM LysoSensor green DND-189 in a pre-warmed HBSS buffer  according to the manufacturer’s protocol. Briefly, cells were incubated with primary Abs against ORP1L  and VAPA  at 4 °C overnight. After washing, cells were incubated with PLA plus and minus probes at 37 °C for 1 h. After ligation reaction to close the circle and rolling circle amplification (RCA) of the ligation product, fluorescence-labeled oligonucleotides hybridized to the RCA products were observed by fluorescence microscopy.Full-length DNA fragments of mouse VAPA and STARD3 were produced from cDNA from J774A.1  cells by PCR. DNA fragments encoding VAPA and STARD3 were inserted into Age1-XhoI site of Flag-BirA(N-G78) and Hind3-BamH1 site of BirA(G79-C)-HA vectors, respectively, using T4 DNA ligase (Takara). Nucleotide sequences of all constructs were confirmed by automatic DNA sequencing. Twenty-four h after transfection of INS-1 cells with Flag-BirA-VAPA and STARD3-BirA-HA using Lipofectamine 2000, 50 µM biotin was added for labeling overnight. After treatment with rotenone or O/A for 1 h, cells were fixed with 4% paraformaldehyde and then permeabilized with 0.5% Triton X-100-PBS. After 10% goat serum blocking, cells were sequentially incubated with anti-FLAG  and -HA Abs  diluted in PBS-1% BSA, secondary Abs diluted in PBS-1% BSA for 30 min, and then with FITC-streptavidin diluted in PBS-1% BSA for 30 min at room temperature. .YFP-STIM1 and 3 x FLAG-mCherry Red-Orai1/P3XFLAG7.1 (provided by Yuan J through Cha S-G) were treated with mitochondrial stressors for 30 min and then subjected to confocal microscopy to visualize STIM1 puncta and their colocalization with Orai1. Colocalization between STIM1 and Orai1 was confirmed by calculating Pearson’s correlation coefficient.INS-1 cells transfected with N-acetylcysteine (Sigma) and 100 μM MitoTEMPO (Sigma), 2 μM Thapsigargin (Sigma), 10 μM DC260126 (Cayman), 10 μM Fumonisin B1 (Sigma), 10 μM Myriocin (Sigma) and 5 μM Triacsin C (Abcam). PA stock solution (50 mM) was prepared by dissolving in 70% ethanol and heating at 55 °C40. The working solution was made by diluting PA stock solution in 2% fatty acid-free BSA-RPMI 1640. To impose mitochondrial stress on primary pancreatic islets, higher doses of mitochondrial stressors  were employed.INS-1 cells (kindly provided by Wollheim C) were cultured in RPMI-1640 containing 11.2 mM glucose and 2 mM glutamine supplemented with 10% FBS, 1 mM pyruvate, 10 mM HEPES, 50 μM 2-mercaptoethanol, 100 U/mL penicillin and 100 μg/mL streptomycin. INS-1 cells were seeded onto wells of Poly-L-ornithine-coated plates. 1.1B4 cells obtained from ECACC  through Abdul Majid FA were cultured in RPMI-1640-10% FBS, 100 U/mL penicillin and 100 μg/mL streptomycin. For drug treatment, the following concentrations were used: 100 nM rotenone (Sigma), 200 nM oligomycin (Millipore), 125 nM antimycin A (Sigma), 400 μM PA (Sigma), 50 μM BAPTA-AM (Invitrogen), 20 μM ML-SI3, 200 μM GPN (Sigma), 3 μM Xesto-spongin C (Abcam), 10 μM Dantrolene (Sigma), 10 μM TPEN (Sigma), 10 μM BTP2 (Sigma), 2 mM EGTA (Sigma), 5 mM MT-Keima, TFEB-GFP, TFE3-GFP, mRFP-LC3, mRFP-GFP-LC3, pcDNA3.1-HA, pcDNA3.1-HA-ΔCnA-H151Q, GCaMP3-ML1, GEM-CEPIA1er, YFP-STIM1, D1ER or mCherry-Orai1 using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol. In the case of TetOn-mCherry-GFP-RAMP4 (Addgene), cells were treated with 4 μg/ml Doxycycline for 48 h after transfection for 24 h before use. When HA-ΔCnA-H151Q was used, the same dose of regulatory calcineurin B subunit was transfected together. To evaluate autophagic and ER-phagic fluxes, the numbers of red puncta of transfected mRFP-GFP-LC3 and TetOn-mCherry-GFP-RAMP437 were counted, respectively.Cells were transiently transfected with the following plasmids: Mcoln1 siRNA (CCCACAUCCAGGAGUGUAA), Ndp52 siRNA (CACACAGCAGAAAAUCCAA), Optn siRNA (CAGCUUACAGAUCUCAGUU) or control siRNA using Lipofectamine RNAiMAX (Invitrogen), according to the manufacturer’s protocol. 1.1B4 cells were transfected with TFEB siRNA (UGUAAUGCAUGACAGCCUG) or control siRNA using Lipofectamine RNAiMAX. Efficacy of Mcoln1 or TFEB siRNA KD was evaluated by RT-PCR. Primer sequences are listed in the Supplementary Tables INS-1 cells were transfected with Cells were solubilized in a lysis buffer containing protease inhibitors. Protein concentration was determined using the Bradford method. Samples (10–30 μg) were separated on 4–12% Bis-Tris gel  and transferred to nitrocellulose membranes for immunoblot analysis using the ECL method (Dongin LS). For immunoblotting, Abs against the following proteins were used: Parkin , TFEB , TFE3 , phospho-S142-TFEB , phospho-(Ser) 14-3-3 binding motif , HSP70 , Tom20 , HSP90  and ACTB .Tfeb- or Tfe3-GFP-transfected INS-1 cells in an ice-cold lysis buffer  containing protease and phosphatase inhibitors, lysates were centrifuged at 12,000 g for 10 min in microfuge tubes, and supernatant was incubated with anti-GFP  in binding buffer  with constant rotation at 4 °C for 1 h. After the addition of 50 μL of 50% of Protein-G bead (Roche Applied Science) slurry to lysates and incubation with rotation at 4 °C overnight, resins were washed with binding buffer. After resuspending pellet in a sample buffer (Life Technology) and heating at 100 °C for 3 min, supernatant was collected by centrifugation at 12,000 g for 30 sec for electrophoretic separation in a NuPAGE® gradient gel (Life Technology). Immunoblotting was conducted by sequential incubation with anti-phospho-(Ser) 14-3-3 binding motif Ab  as the primary Ab and horseradish peroxidase-conjugated anti-rabbit IgG. Bands were visualized using an ECL kit for the detection of chemiluminescence.After lysis of For TFEB and TFE3 immunofluorescence, cells treated with mitochondrial stressors were fixed with 4% paraformaldehyde and permeabilized with 0.5% Triton X-100. Cells were then blocked with 10% bovine serum albumin in PBS. After sequential incubation of cells with anti-TFEB  or anti-TFE3 Ab  at 4 °C overnight and then with anti-rabbit secondary Abs conjugated to Alexa Fluor 488 (Invitrogen) for 1 h, fluorescence was visualized with an LSM780 confocal microscope (Zeiss).9. The number of test points was selected using a nomogram to set the probable error of volume density to less than 10% of the calculated value.Relative β-cell mass was determined by point counting morphometry after insulin immunohistochemistry using anti-insulin Ab 2PO4/Na2HPO4 phosphate buffer (pH 7.2) and phosphate buffer washing, pancreas tissues were post-fixed with 1% OsO4 and embedded in Epon812. Ultrathin sections were mounted and stained with uranyl acetate/lead citrate . Aspect ratio was calculated as the ratio between the length of the major axis and that of the minor axis of mitochondria42. The number of mitochondria surrounded by isolation membrane or autophagosome was counted in 7~8 fields of magnified images  per mouse for a total of 25 fields as an index of mitophagy. The number of double-membrane structure with a luminal density similar to the cytoplasm but without identifiable organelle structure representing bulk autophagy was also counted in the same fields.After fixation in 2.5% glutaraldehyde-0.1 M NaHS18, GAPDH or Rpl32 mRNA level. Sequences of primers used for real-time RT-PCR are listed in Supplementary Tables Total RNA was extracted using TRIzol (Invitrogen), and cDNA was synthesized using M-MLV Reverse Transcriptase (Promega) according to the manufacturer’s protocol. Real-time RT-PCR was performed using SYBR green (Takara) in a QuantStudio3 Real-Time PCR System (Applied Biosystems). Expression values were normalized to the To determine intracellular ROS, cells were treated with rotenone or O/A for 1 h after NAC pretreatment for 1 h. After incubation with 5 μM CM-H2DCFDA (Invitrogen) at 37 °C for 30 min in culture medium without FBS and recovery in complete medium for 10 min, confocal microscopy was conducted. To study mitochondria-specific ROS, cells were stained with 5 μM MitoSOX (Invitrogen) at 37 °C for 30 min. Cells were suspended in PBS-1% FBS, and then were subjected to flow cytometry by FACSVerse (BD Biosciences) using FlowJo software (TreeStar). The gating strategies for flow cytometric data analysis are shown in Supplementary Fig. TFEB-GFP-transfected 1.1B4 cells treated with vehicle, rotenone, or O/A for 8 h by addition of 1% formaldehyde and incubation at room temperature for 10 min. Cell extracts were then sonicated to shear DNA and immunoprecipitated using anti-GFP  or IgG (1:50) with constant rotation at 4 °C overnight. After the addition of 30 μL of Protein-G bead slurry to the lysates and incubation with rotation at 4 °C for 2 h, resins were washed with the ChIP buffer supplied with the kit. After elution of chromatin from the Ab/Protein-G beads with ChIP Elution Buffer and reversing cross-linking by adding 5 M NaCl and 2 μL of 20 mg/mL proteinase K, DNA was purified for PCR using primers designed to cover the putative TFEB CLEAR sequence (CACGTG)50 in the human NDP52  and OPTN promoters (−6885 from the 5’ UTR) . Briefly, the chromatin/DNA protein complexes were prepared by cross-linking chromatin of TfebF/F mice were generated by breeding Tfebtm1a(EUCOMM)Wtsi mice (Mutant Mouse Resource and Research Center) with FLPeR mice (Wellcome Trust Sanger Institute). Tfeb∆β-cell mice were generated by crossing TfebF/F mice with RIP-Cre mice (Jackson Laboratory). Eight-week-old male Tfeb∆β-cell mice were maintained in a 12-h light/12-h dark cycle and fed HFD for 12 weeks. During the observation period, mice were monitored for glucose profile and weighed. Tfe3-KO mice were from the Jackson Laboratory. All animal experiments were conducted in accordance with the Public Health Service Policy in the Humane Care and Use of Laboratory Animals. The mouse breeding temperature was 22 ± 2 °C, and the humidity was 50 ± 10%. Mouse experiments were approved by the IACUC of the Department of Laboratory Animal Resources of Yonsei University College of Medicine, an AAALAC-accredited unit.2. Briefly, after injecting 2.5 ml of collagenase P (0.8 mg/ml) into the common bile duct, the pancreas was procured and incubated in a collagenase solution at 37 °C for 13 min 20 s. After cessation of enzymatic digestion with cold HBSS-5% FBS, tissue was passed through a 400 μm sieve and then centrifuged on 1.10, 1.085, 1.069, and 1.037 g/ml Biocoll gradients (Biochrom). Islets were collected from the interface using micropipettes.Primary murine islets were isolated from fasted mice using the collagenase digestion technique15 min (pM)/Δglucose15 min (mM). ITT was conducted by injecting intraperitoneally 1 U/kg of regular insulin to fasted mice and measuring blood glucose levels at 0, 15, 30, 60, and 120 min. Serum insulin was measured using Mouse insulin ELISA kit (Shibayagi).Intraperitoneal GTT was performed by intraperitoneal injection of 1 g/kg of glucose after overnight fasting. Blood glucose concentrations were determined using a One Touch glucometer (Lifescan) before (0 min) and 15, 30, 60, 120, and 180 min after glucose injection. Insulinogenic index was calculated as follows: Δinsulinc, 5 mg of diaminobenzidine tetrahydrochloride (DAB), 9 ml of sodium phosphate buffer  and 1 ml of catalase (20 μg/ml). COX activity was visualized with a standard DAB reaction. Images were acquired with a light microscope and analyzed using Fiji73. To evaluate mitophagy in vivo, colocalization between LC3 and TOM20 or that between LAMP2 and TOM20 was assessed by calculating Pearson’s correlation coefficient. To evaluate autophagic flux in vivo, colocalization between LC3 and LAMP2 was determined in the same way.Tissue samples were fixed in 10% buffered formalin and embedded in paraffin. Sections of 4 μm thickness were stained for immunohistochemical analyses using anti-LC3 , -LAMP2 , -TOM20 , -insulin  or 4-HNE Abs , and then with anti-rabbit secondary Abs conjugated to Alexa Fluor 647 (Invitrogen), Alexa Fluor 594 (Invitrogen), Alexa Fluor 568 (Invitrogen), Alexa Fluor 405 (Invitrogen) or Alexa Fluor 488 (Invitrogen). COX staining was done by incubating unfixed cryosections of the pancreas in a reaction buffer containing 10 mg of cytochrome 2 consumption of isolated mouse islets was measured using a Seahorse Extracellular Flux (XFe96) Analyzer (Agilent Technologies) equipped with a spheroid microplate-compatible thermal tray according to a modification of the manufacturer’s protocol. Briefly, islets were seeded into the wells of a poly-L-lysine-coated XF96 spheroid microplate (25 islets/well). Islet seeding was done by inserting a pipette tip directly into the wells of the spheroid microplate. Islets then were incubated with pre-warmed XF assay medium  at 37 °C for 1 h in a non-CO2 incubator. Basal respiration was measured in XF assay medium containing 3 mM glucose. Islets were then sequentially exposed to 20 mM glucose, 5 μM oligomycin, 1 μM FCCP and 5 μM rotenone/antimycin A at the indicated time points to measure glucose-stimulated, ATP-coupled, maximal and non-mitochondrial O2 consumption, respectively. O2 consumption was analyzed using the WaveTM software (Agilent Technologies).Ot test was used to compare values between two groups. One-way ANOVA with Tukey’s test was used to compare values between multiple groups. Two-way repeated-measures ANOVA with Bonferroni’s post-hoc test was used to compare multiple repeated measurements between groups. P values < 0.05 were considered to represent statistically significant differences.All values are expressed as the means ± SEM of ≥ 3 independent experiments performed in triplicate. For densitometric analysis of immunoblot bands, the mean from 2 independent experiments was calculated. Two-tailed Student’s Further information on research design is available in the Supplementary InformationPeer Review FileDescription of Additional Supplementary InformationReporting Summary


ORAI1 and STIM1 accomplish store-operated Ca2+ entry (SOCE) and play a key role in platelet activation. Signaling linking phosphate to upregulation of ORAI1 and STIM1 includes transcription factor NFAT5 and serum and glucocorticoid-inducible kinase SGK1. In vascular smooth muscle cells, the effect of hyperphosphatemia on ORAI1/STIM1 expression and SOCE is suppressed by Mg2+ and the calcium-sensing receptor (CaSR) agonist Gd3+. The present study explored whether sustained exposure to Mg2+ or Gd3+ interferes with the phosphate-induced upregulation of NFAT5, SGK1, ORAI1,2,3, STIM1,2 and SOCE in megakaryocytes. To this end, human megakaryocytic Meg-01 cells were treated with 2 mM ß-glycerophosphate for 24 h in the absence and presence of either 1.5 mM MgCl2 or 50 µM GdCl3. Transcript levels were estimated utilizing q-RT-PCR, protein abundance by Western blotting, cytosolic Ca2+ concentration ([Ca2+]i) by Fura-2 fluorescence and SOCE from the increase in [Ca2+]i following re-addition of extracellular Ca2+ after store depletion with thapsigargin (1 µM). As a result, Mg2+ and Gd3+ upregulated CaSR and blunted or virtually abolished the phosphate-induced upregulation of NFAT5, SGK1, ORAI1,2,3, STIM1,2 and SOCE in megakaryocytes. In conclusion, Mg2+ and the CaSR agonist Gd3+ interfere with phosphate-induced dysregulation of [Ca2+]i in megakaryocytes.In chronic kidney disease, hyperphosphatemia upregulates the Ca For determination of SOCE, cells were exposed to thapsigargin (1 µM), a sarco/endoplasmic reticulum Ca2+/ATPase (SERCA) inhibitor, and Ca2+-free solutions to deplete intracellular Ca2+ stores. In the following, extracellular Ca2+ was re-added in the continued presence of thapsigargin to quantify SOCE from the increase in [Ca2+]i. As illustrated in 2. Again, in the absence of ß-glycerophosphate, MgCl2 did not significantly modify SOCE. Neither ß-glycerophosphate nor MgCl2 significantly modified Ca2+ release.Cytosolic Ca2 were mimicked by calcium-sensing receptor agonist Gd3+. As illustrated in CaSR transcription was significantly upregulated by the exposure to 50 µM GdCl3 both in the absence and presence of ß-glycerophosphate. The upregulation of NFAT5, SGK1, ORAI1, ORAI2, ORAI3, STIM1 and STIM2 transcript levels by 2 mM ß-glycerophosphate was significantly blunted by additional exposure to 50 µM GdCl3. In the absence of ß-glycerophosphate, 50 µM GdCl3 did not significantly modify the transcript levels of NFAT5, SGK1, ORAI1, ORAI2, ORAI3, STIM1 and STIM2  for 24 h in the absence and presence of 1.5 mM MgCl2 or 50 µM GdCl3 . In analysis of vascular calcification, phosphate donor ß-glycerophosphate is widely used as a substitute for phosphate and a well-established stimulator of tissue calcification i), Fura-2 fluorescence was utilized [2+ activity, ratiometer (340 nm/380 nm)-based analysis was employed. SOCE was determined following extracellular Ca2+ removal causing store depletion and subsequent Ca2+ re-addition in constant presence of SERCA inhibitor thapsigargin . For quantification of Ca2+ entry, the slope (delta ratio/s) and peak (delta ratio) were determined following re-addition of Ca2+. Experiments were performed with HEPES solution containing (in mM): 125 NaCl, 5 KCl, 1.2 MgSO4, 2 Na2HPO4, 32 HEPES, 5 glucose, 1 CaCl2, pH 7.4. Ca2+-free conditions were achieved by using Ca2+-free HEPES solution containing (in mM): 125 NaCl, 5 KCl, 1.2 MgSO4, 2 Na2HPO4, 32 HEPES, 5 glucose, 0.5 EGTA, pH 7.4.To determine the cytosolic Cautilized ,53,54,55n represents the number of independent experiments . All data were tested for significance using Student’s t test or ANOVA. Results with p < 0.05 were considered statistically significant.Statistical analysis was conducted using SPSS software . Data are provided as means ± SEM, and


Despite advances in understanding of carcinogenesis and of treatment of acute myeloid leukemia, this neoplasm still has a lethality of at least 30%. The search for biomarkers that can predict the response to treatment in the early stages of the disease is still necessary. In recent years, a new form of cellular communication between tumor and non-neoplastic cells has been discovered: the exchange of information through extracellular vesicles. These are small vesicles released by membrane-coated cells that carry proteins, lipids, messenger RNAs, microRNA and DNA, which can be internalized and promote biological changes in target cells. Exosomes are qualified as a type of extracellular vesicle and, in tumors, carry immunoinhibitory signals that promote the escape of immune control. Recent studies have showed their involvement in communication with the cells of the tumor microenvironment and with chemoresistance in several tumors. To date, there is no information about immunoregulatory microRNAs transported by exosomes and their correlation with clinical evolution during chemotherapy for acute myeloid leukemia. Knowledge about immunomodulatory microRNAs obtained by leukemic cells and transported by exosomes can direct us towards the design of new diagnostic and treatment tools in this type of leukemia. The overall survival rates for adult and childhood acute myeloid leukemia (AML), in addition to being suboptimal, have been stagnant for more than 20 years. This demonstrates the efficacy of chemotherapeutic regimens appears to have reached its upper limit. The need for the search for new therapeutic weapons against the disease is urgent. The way in which the tumor cell acts on the immune system and its ability to produce exosomes that influence the tumor niche has drawn attention of the scientific community. Thus, the transfer of non-coding RNAs by exosomes and the modulation of gene expression in target cells present new horizons to be explored.Acute myeloid leukemia is a hematologic malignancy of complex pathogenesis, characterized by abnormal proliferation and differentiation of clonal populations of myeloid stem cells in the bone marrow. In children under 15 years of age, it accounts for only 15% to 20% of acute leukemia cases.The worldwide incidence of AML is 2.5 to three new cases per 100,000 individuals per year, and although it can occur at any age, it is most commonly diagnosed in individuals aged over 65 years.Some genetic and environmental risk factors seem to be associated with a higher frequency of AML. Among the constitutional genetic syndromes that may be correlated with AML, Down syndrome, Fanconi anemia, Blackfan-Diamond anemia, Shwachman-Diamond syndrome, and congenital neutropenia (Kostmann syndrome) stand out. Among the environmental factors, exposure to ionizing radiation, alkylating agents, and topoisomerase-II inhibitors are of epidemiological importance.Despite the high remission rate, 90% of patients relapse if they do not continue the protocol with consolidation and intensification. Consolidation is performed in patients classified as low risk on the 15th day of induction, using prednisone, thioguanine (Lanvis™), vincristine, doxorubicin (Adriamycin), cytarabine, intrathecal cytarabine, cyclophosphamide, and granulocyte colony-stimulating factor (G-CSF).The other patients start consolidation after high-dose cytarabine and etoposide intensification, while low-risk patients perform intensification after consolidation.The treatment protocol of the Berlin-Frankfurt-Münster (BFM) Group is the most widely used for treatment of AML in Brazil. The treatment is based on induction chemotherapy, with the infusion of cytarabine (ARA-C), idarubicin, and etoposide (VP-16), in addition to the administration of intrathecal cytarabine.A considerable part of the increase in survival rates of these patients, however, is due to the improvement in supportive treatment for the complications of chemotherapy. Allogeneic hematopoietic stem cell transplantation in the first remission is also an option, especially for those who are poor responders to induction therapy or who have poor prognostic cytogenetic and molecular characteristics. New therapies are already being used for gene variants of AML, such as FLT3 (midostaurin and gilterinib), BCL2 (venetoclax), and IDH1/IDH2 (ivosidenib and enasidenib) inhibitors. However, such alternative therapies are not yet widely used, either because of their cost or because they require approval for use in Brazil.Despite the advances made in the last 40 years, the survival rates for AML are less than desirable, remaining around 70% at 5 years, even for good responders to induction therapy.,5 As mediators of intercellular communication, the extracellular vesicles can transmit proteins and lipids, besides different types of RNAs and DNA.Extracellular vesicles are secreted by almost all cell types and have the function of performing intercellular communication, which can occur through ligand-receptor interaction, by fusion, and/or internalization.,6 Recently, exosomes were subclassified into Exo-L (large exosome vesicles) and Exo-S , with 90nm-150nm and 60nm-80nm, respectively. In addition, the term “exomere” was created for classification of non-membranous nanoparticles with approximately 35nm.Extracellular vesicles are classified according to their size into exosomes (30nm-150nm), microvesicles (0.1μm-2μm) and apoptotic bodies (0.1μm-5μm). Subsequently, these endosomes are fused to the plasma membrane, releasing the exosomes into the extracellular environment. As a consequence of their endosomal origin, exosomes contain proteins involved in membrane transport and fusion , as well as in the biogenesis of multivesicular bodies , and integrins and tetraspanins .Besides being differentiated by size, extracellular vesicles have different densities and origins. While microvesicles and apoptotic bodies are formed from the direct evagination of the plasma membrane, exosomes are formed from a more complex process, which involves the incorporation of protein or molecular content by invagination into cytoplasmic multivesicular endosomes.,8Various physiological and pathological stimuli can increase the production of extracellular vesicles, which can be detected in body fluids. This feature has opened the horizons for investment in research aimed to use these changes as possible biomarkers in various diseases.Moreover, exosomes are involved in communication with cells in the tumor microenvironment and chemoresistance in several solid neoplasms.,8With respect to exosomes, tumor-derived exosomes are known to carry immunoinhibitory signals that promote escape from immune control. Evidence shows that tumor cell-derived exosomes affect proliferation, apoptosis, and cytokine production, and are able to reprogram cells of innate and adaptive immunity. It has been described, for example, that exosomes containing miRNA-21-3p, miRNA-185d-5p, and miRNA-1246 have the potential to reprogram macrophages into tumor support agents. Furthermore, exosomes expressing transforming growth factor β1 (TGF-β1) and binding to NKG2D may downregulate NKG2D expression, reducing natural killer (NK)-induced cytotoxicity.Caenorhabditis elegans. Later, they were also associated with several types of cancer and with various cellular processes, such as DNA methylation, cell growth, differentiation, and apoptosis. Abnormal levels of these miRNAs may play a role in carcinogenesis, since certain overexpressed miRNAs can lead to silencing of tumor suppressors.The imbalance of these miRNAs has been described in several types of cancers and in hematological neoplasms. Additionally, miRNAs can induce molecular alterations in target cells, affecting the regulation of specific genes, making it pathologically possible to alter processes, such as differentiation, proliferation, and apoptosis.Exosomes can carry inside them microRNAs (miRNAs), small endogenous non-coding RNAs (19 to 25 nucleotides), originally identified as regulators of larval development in  and Li et al,. as shown on Recent studies suggest that extracellular vesicles carry an important amount of non-coding RNA that is relevant to the pathogenesis of leukemias. Some miRNAs have already been described as involved in the pathogenesis of AMLs, as reviewed by Trino et al., In addition to conventional markers for exosomes such as tetrapanins, AML exosomes contain TGF-β1, MICA/MICB, FasL, and markers of myeloid blast cells .Ex vivo, these exosomes decrease NK cell cytotoxicity by decreasing expression of NKG2D. In addition, TGF-β1 is partially responsible for NK cell dysfunction in AML. Thus, it can be said that exosomes derived from leukemic cells in AML have the potential to modulate the immune system and may influence disease progression as well as response to therapy.A summary of functional studies demonstrating the relation between altered exosome production and AML is shown on -19Exosomes originating from leukemic cells in AML patients have a distinct molecular profile when compared to healthy individuals. Exosomes appear to be involved in the deregulation of hematopoiesis during the conversion from a homeostatic microenvironment to a leukemic niche. In the in vivo model,exosomes of AML cell lines have been observed to migrate to the bone marrow, decreasing the colony-forming capacity of hematopoietic progenitor stem cells, and the expression of several transcription factors of genes involved in hematopoiesis. The influence of extracellular vesicles on the tumor niche was also observed by Kumar et al., bone marrow preconditioning with exosomes from AML cells accelerated tumor engraftment and growth, promoted increased expression of DKK1 in bone marrow (a negative regulator of osteoblastic development), and caused reduced trabecular bone volume. In patients with AML, plasma levels of the bone formation indicator gene (OCN) are decreased. Interestingly, the number of circulating exosomes in plasma is significantly increased in patients with AML when compared to healthy individuals. In view of these findings, the authors emphasize the potential need to treat the induced changes in the bone marrow stroma as well, and not only to eradicate leukemic cells. Viola et al., corroborated these observations and showed that monocyte-derived exosomes from patient stroma protect the AML cell line against chemotherapy treatment with cytarabine or FLT3 inhibitor.The first study to show that AML cell lines release exosomes was published in 2013. and released by bone marrow monocytes, and apoptotic proteins found in exosomes derived from bone marrow blasts have been described. In patients with AML refractory to treatment with adoptive cell therapy using NK-92 cells, exosomes isolated from plasma have been shown to reduce the cytolytic activity of NK-92 cells and to decrease their migration capacity. Exosomes have also been found to carry TGF-β and PD-L1 (programmed cell death ligand 1), an important T cell inhibitor.Among the investigations that evaluated the content of extracellular vesicles in AML, miRNA in exosomes circulating in plasma Thus, in clinical practice, flow cytometry assays may incorporate the search for extracellular vesicles as biomarkers of disease activity.In addition, AML blasts can secrete extracellular vesicles that express myeloid cell line-specific markers distinct from those present in normal cells. The expression of CD13, CD34, CD117, and CD33 on extracellular vesicles isolated from plasma of AML patients may indicate the presence of active leukemic cells.Although some studies have already evaluated some of the content of extracellular vesicles at specific times in acute myeloid leukemia, no research has shown whether there is modification of this content over the course of treatment. In addition, some immunoregulatory microRNAs have not yet been evaluated and, perhaps, they may have relevance in the pathogenesis of acute myeloid leukemia. Likewise, very little is known about the dynamics of extracellular vesicles in childhood leukemias. indicate the possibility that in the future, extracellular vesicles will be used as a substrate in laboratory assays for patient follow-up, prognosis determination, and therapeutic response.The importance of circulating exosomes and cellular communication by microRNA in acute leukemias remains poorly elucidated, but recent studies