Therapy for acute myeloid leukemia (AML) involves intense cytotoxic treatment and yet approximately 70% of AML are refractory to initial therapy or eventually relapse. This is at least partially driven by the chemo-resistant nature of the leukemic stem cells (LSCs) that sustain the disease, and therefore novel anti-LSC therapies could decrease relapses and improve survival. We performed in silico analysis of highly prognostic human AML LSC gene expression signatures using existing datasets of drug–gene interactions to identify compounds predicted to target LSC gene programs. Filtering against compounds that would inhibit a hematopoietic stem cell (HSC) gene signature resulted in a list of 151 anti-LSC candidates. Using a novel in vitro LSC assay, we screened 84 candidate compounds at multiple doses and confirmed 14 drugs that effectively eliminate human AML LSCs. Three drug families presenting with multiple hits, namely antihistamines (astemizole and terfenadine), cardiac glycosides  and glucocorticoids , were validated for their activity against human primary AML samples. Our study demonstrates the efficacy of combining computational analysis of stem cell gene expression signatures with in vitro screening to identify novel compounds that target the therapy-resistant LSC at the root of relapse in AML. Despite the achievement of remission for a majority of patients, up to 70% of adults and 30% of children will not survive beyond 5 years after initial clinical response due to relapsing disease5. This highlights the urgent and unmet need for novel drugs to enable a sustainable recovery in patients with AML. Repurposing of drugs, which consists of using a known drug to treat a new indication, is an approach with high potential for the rapid introduction of new therapeutics into the clinic7. This strategy exploits the known toxicology and pharmacological properties of approved drugs to accelerate regulatory approval. Repurposing is possible because many drugs target multiple pathways, in addition to those that have been described for their current clinical use. For example, thalidomide, once used to treat morning sickness and withdrawn for triggering phocomelia, has been repurposed as a highly effective treatment for myeloma8.For more than four decades, the standard of care for acute myeloid leukemia (AML) has not changed significantly and consists of an intensive combined chemotherapy with cytarabine plus an anthracycline as the cornerstone drugs9. However, LSCs are relatively resistant to standard therapies12. For example, these cells are less sensitive to killing by daunorubicin and cytarabine, partially due to increased expression of multidrug resistance genes  and their quiescent state, which reduces the effects of cytotoxic agents that target rapidly replicating cells14. There have been some examples of success in developing anti-LSC compounds18. However, in general the current methodologies to identify chemotherapeutic agents effective in AML are based upon readouts of general toxicity in the bulk of leukemic cells, and thereby paradoxically ignore LSCs and their unique features. In addition, LSCs are very similar to their normal hematopoietic stem cell (HSC) counterparts. Like HSCs, they utilize similar molecular mechanisms to self-renew, maintain an undifferentiated state and produce progeny that differentiate into more mature progenitor cells9. Thus, the biological parallels between AML and normal hematopoiesis highlight the difficulty of developing compounds that target LSCs for elimination without also eradicating HSCs.In AML, leukemia stem cells (LSCs) produce all the leukemic cells in the patient and therefore a lasting cure for this disease is dependent on eradication of these cells20. In silico analysis of gene expression can be used to identify drug candidates22. The molecular pathways represented in our gene signatures are therefore targets for the development of novel therapeutics against AML through the elimination of LSCs. Despite the substantial similarity between leukemic and healthy stem cells, we generated an HSC-specific gene expression signature as well19. Utilizing both LSC and HSC signatures allows for a strategy for the identification of drugs that target LSCs without harming normal HSCs.We have identified two gene expression signatures for LSCs from analysis of primary human AML samples that were sorted into LSC and non-LSC fractions and validated by xenotransplantation. The high expression of each of these signatures is tightly linked to poor survival in AML and failure of standard therapy across all AML subtypesTo identify anti-LSC compounds, we probed our LSC and HSC gene expression signatures against an existing database of drug–gene interactions to identify compounds predicted to negatively affect the gene expression program of leukemia while sparing normal hematopoietic function. Candidates identified in the in silico analysis were screened in vitro to assess their anti-leukemic and anti-LSC effects. Using this approach, we identified several compounds that display anti-LSC activity in vitro.19.All AML specimens used in this study were from adult patients at the University Health Network . Human cord blood cells from full-term deliveries were provided from consenting healthy donors at the Cord Blood Bank of Centre hospitalier universitaire Sainte-Justine . Briefly, mononuclear cells were isolated using Ficoll  and were enriched 60–90% for CD34+ hematopoietic and progenitor cells (HSPCs) (STEM CELL Technologies). Informed consent was obtained according to the procedures approved by the Research Ethics Board of the respective institutions. Collection of AML samples and mononuclear cell isolation have been previously described19) and an additional signature (LSC-Ng) derived from 78 additional AML  as well as HSC-R . The LSC-R up and down probes sets are the 100 probes most significantly correlated with LSCs and 100 probes most correlated with non-LSCs from Eppert et al.19. The LSC-Ng probes sets are derived from an early build of the LSC expression data from Ng et al.20. The top and bottom 250 genes associated with LSCs (determined by t-statistic) were converted to Affymetrix probe IDs  with a p value of ≤0.1 for the LSC signatures and that were not associated with a negative ES in HSC-R were considered for in vitro screening.The list of drug candidates was obtained by querying the Connectivity Map database (CMap build 02) with both LSC-R  with growth factors (Life Technologies) (AMLs: 10 ng/mL interleukin (IL)-3, IL-6 and granulocyte colony-stimulating factor (G-CSF), 25 ng/mL thrombopoietin (TPO), 50 ng/mL stem cell factor (SCF) and FLT3 ligand (FLT3L); cord blood: 10 ng/mL IL-6 and G-CSF, 100 ng/mL SCF, FLT3L and 15 ng/mL TPO), and penicillin–streptomycin (Life Technologies). Then, 500 nM of SR1 was included in the culture media for AMLs 9706 and 9642. The MOLM-13 cell line was obtained and cultured per the specification of Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ). AML 8227 was cultured for up to 16 weeks under the same conditions as other primary AMLs described above23. All cells were incubated at 37 °C with 5% CO2.Primary AML and cord blood samples were cultured using StemSpanCompounds were purchased from Tocris Bioscience, Cedarlane or Sigma-Aldrich. Primary AML cells or CD34+ enriched human cord blood cells were plated as described above. Candidate molecules or dimethyl sulfoxide  were added to the cells at specified concentrations and incubated for 6 days for 8227 AML cells and 4 days for primary AML and cord blood samples. Cells were analyzed by flow cytometry. Briefly, for AML cells, phenotype and viability were assessed using CD34-APC or APC-Cy7 (581), CD38-PE (HB-7), CD15-FITC (HI98), SYTOX Blue (Life Technologies) and when necessary CD33-APC (WM53) and CD14-AlexaFluor 700 (HCD14). HSC phenotype and viability were assessed using CD34-APC-Cy7, CD33-APC, CD38-PE, CD19-PerCP-Cy5.5 (HIB19), CD15-FITC and SYTOX Blue (Life Technologies). All antibodies were purchased from Biolegend. Flow cytometry was performed using a LSRFortessa fitted with a high-throughput sampler (BD Biosciences).Cells were treated with drugs or DMSO as control for 4 days. The same volume of cell suspension was used to perform the assay for each condition as determined by the cell count of DMSO control. Cells were diluted with Iscove's modified Dulbecco's medium (Life Technologies), 2% fetal bovine serum , seeded in MethoCult media  in duplicate. The assay duration was 12 days prior to counting colonies.TM Kit (BD Biosciences). Cells were fixed in 1% (w/v) paraformaldehyde  in phosphate-buffered saline (Life Technologies). Washed cells were suspended in 70% (v/v) ethanol. DNA labeling and staining (FITC-labeled anti-BRDU and propidium iodine/RNase staining buffer) were performed as described by the manufacturer (BD Biosciences). DNA breaks and cell cycle phase distribution were evaluated by flow cytometry. To discriminate between G0/G1, cells were fixed and permeabilized using the BD Cytofix/Cytoperm kit (BD Biosciences). Cells were stained with Ki-67 AlexaFluor 700 (Ki-67) and Hoechst 33342 (ThermoFisher Scientific) and analyzed by flow cytometry.MOLM-13 cells were grown in serum-free RPMI 1640 medium (Life Technologies) for 24 h followed by 12 h of incubation in medium containing 20% FBS (Wisent) and were then treated with 10 μM astemizole or DMSO. The effect of a 24 h treatment on the cell cycle distribution and late apoptosis was evaluated using the APO-BRDU24. The enrichment map was generated from the GSEA above using Cytoscape 3.6.0 and the Enrichment Map and AutoAnnotate apps26. GSEA analysis of 8227 fractions and the LSC signatures was performed using the control sample data from GSE55814. GEO2R was used to generate a ranked list of LSC-related genes (6 LSC CD34+ CD38− samples vs 12 non-LSC CD34− samples) used in GSEA analysis.Functional enrichment analysis was performed by integrating the astemizole transcriptomic data from CMap. Data rank matrix was exported from CMap and instances of cells treated with astemizole  were extracted and probes converted to gene symbols. The ranked expression of probes was summed by genes and then ordered highest to lowest to perform a gene-set enrichment analysis  using Molecular Signatures Database (MSigDB) Collections (c2.cp.reactome.v6.0.symbols.gmt). The number of permutations was fixed at 1000, maximum size at 1000 and minimum size at 850) were performed using Prism version 6.00 for Mac, GraphPad Software . Data are represented as mean ± s.d. and significant differences (p ≤ 0.05) between groups were determined by a two-way unpaired Student’s t-test. Flow cytometry data analysis was performed using FlowJo (v X10.1).The concentration effect curve graph and the calculation of half lethal concentration  to identify compounds predicted to inhibit the LSC gene expression programs. We then assayed the CMap data with the HSC-R signature to identify compounds that may inhibit HSC gene programs and filtered the LSC results to remove the former compounds. This resulted in a total of 151 compounds predicted to target LSC-specific gene profiles, including 133 molecules expected to have no effect on HSC-specific gene expression and 18 compounds predicted to enhance the HSC-specific gene profile . The cultured LSCs maintain the same LSC gene expression profile as the primary patient LSCs. The LSC signatures are preferentially expressed in 8227 LSCs and it is therefore an excellent model to examine CMap predicted compounds , (2) those that target the LSC-containing population (n = 15) with lower effectiveness against non-LSC cells and (3) those that had other targeting patterns (n = 3)  Fig. . General 3) Fig. . We rete 3) Fig. . We then 3) Fig. . Assessi50 in CD34+ CD38− of 10.21 μM and 4.06 μM for astemizole and terfenadine, respectively, with equal efficacy against the LSC population (CD34+ CD38−) and the total leukemic cell population , diphenhydramine and cetirizine do not significantly alter the AML 8227 cell count compared to DMSO Fig. . While ap ≤ 0.01); this inhibition was associated with a higher level of apoptosis compared to other cycle phases    Fig. . To dete02) Fig. . FurtherµM) Fig. , indicat50: strophanthidin = 23.25 nM, digoxin = 23.19 nM, ouabain = 11.61 nM) , while AML 116, AML 9642 and AML 9706 were killed at higher doses (LC50: 33.73–77.04 nM)  Fig. . A colonnM) Fig. . To assenM) Fig. . Sensiti50 for AML 8227 and cord blood were respectively: strophanthidin = 29.59 nM and 68.41 nM; digoxin = 29.57 nM and 43.40 nM and ouabain = 15.17 nM and 31.45 nM)  Fig. .50 budesonide = 1.41 nM, mometasone = 0.57 nM, halcinonide = 1.20 nM)  Fig. . To confnM) Fig. . FurthernM) Fig. .Fig. 6Th50 for AML 8227 and cord blood: Budesonide = 4.04 nM, and 69.95 nM; Mometasone = 0.86 nM and 50.76 nM and Halcinonide = 3.81 nM and 68.28 nM, Fig. To investigate whether the glucocorticoids might differentiate HSPCs, we counter-screened all three steroids against CD34+ enriched normal human cord blood samples at doses shown to be effective against AML cells. All three drugs displayed lower toxicities against CD34+ HSPCs, shown by a minimal loss of CD34+ cells and expansion of differentiated CD15+ myeloid cells, than against AML cells   Fig. . We furtnM) Fig. .Fig. 7Ca28. This is advantageous because the translation of these drugs to clinical use is substantially less difficult compared to a novel compound that has never been clinically tested7. A critical feature of our strategy is to use a cell system that enables the analysis of LSC populations in vitro. This allows for the dissection of the effect of the drugs on bulk cells, CD15+ blasts, CD34+ leukemic progenitors and the LSC-containing population in a screen. As we hypothesized, we identified drugs that act against LSC-enriched fractions and, in some cases, all cell populations tested.We demonstrate that querying CMap with functionally validated LSC and HSC signatures can identify relevant candidates to be repositioned for use in AML in a timely and economic manner. Indeed, from more than 1000 small bioactive molecules listed in CMap, the cross-comparison of drug lists identified using both LSC and HSC signatures reduced the potential candidates by nearly 10-fold. Over half of the drugs identified from this approach successfully demonstrated activity against AML in vitro. CMap includes well-established Food and Drug Administration (FDA)-approved drugs and 9/14 of our primary candidates have been approved in a jurisdiction for human use35. The evaluation of three additional molecules in this class, namely diphenhydramine and cetirizine  and fexofenadine (not included in CMap), did not alter leukemia cell viability even at high micromolar concentrations. This suggests that engagement of HRH1 is not the causal mechanism explaining the action of terfenadine and astemizole on LSCs36. This conclusion was further strengthened by the fact that co-treatment of AML cells with histamine together with either astemizole or terfenadine did not reverse the cell death phenotype. Rather, this combination led to potentiation of cell mortality. Similarly, Jakhar et al.37 reported that histamine synergizes with the antiproliferative effect of astemizole on MCF7 breast cancer cells. They demonstrated that combined use of these molecules triggered endoplasmic reticulum (ER) stress-induced apoptotic and autophagic cell death by disruption of calcium (Ca2+) homeostasis37. Furthermore, terfenadine has been shown to induce apoptosis and autophagy by the depletion of ER Ca2+ stores and Ca2+ influx from extracellular medium in melanoma cells38. Cell proliferation is another process regulated by Ca2+-dependent signaling pathways, including expression of cell cycle regulator controlling of the G1/S cell cycle transition39. The H1-antihistamines terfenadine and cyproheptadine were shown to cause G0/G1 cell cycle arrest and apoptosis in leukemia40. Our gene-set enrichment analysis of transcriptomic fingerprints of astemizole in cancer cells was consistent with a possible alteration of G1/S cell cycle progression. This was further supported by our in vitro results showing an apparent cell cycle arrest in G0/G1 associated with increased apoptosis. The ability of astemizole to cause cell death mainly in G0/G1 phase suggests that this molecule can eradicate quiescent cells, such as LSCs. However, this remains to be further investigated. From a clinical point of view, it could be argued that the repositioning of astemizole and terfenadine would be limited by their potential severe adverse effects41. Both drugs were removed from the market following commercialization due to causing prolonged QT time and ventricular tachycardia. Thus, the concentration required to suppress cancer in a patient will need to be clarified with regard to the proarrhythmic risk deemed acceptable to treat a potential life-threatening disease. The combination of low doses of terfenadine or astemizole synergizes with chemotherapy, and this could be a feasible option for their repurposing in clinic since risk of cardiac events is decreased at lower doses34. Furthermore, use of cationic amphiphilic drug antihistamines, comprising astemizole and terfenadine, was linked to lower mortality in a large pharmacoepidemiological study of Danish cancer patients, supporting the use of these compounds32. Development of novel astemizole or terfenadine-related compounds with higher potency and more favorable toxicity profile is another possible strategy.We first focused on a group of positive hits comprising drugs sharing an H1-antihistaminine activity, which could potentially be the common mechanism targeting the leukemic cells. In particular, we observed that astemizole and terfenadine, both commercialized for their action against HRH1, were effective against AML LSC and bulk cell populations. There is growing evidence supporting the therapeutic potential of these two drugs as well as other H1-antihistamines for a variety of malignancies, including leukemia, myeloma, breast, prostate, colon, lung and liver cancers44. These drugs are inhibitors of the Na+/K+-ATPase pump, which, in addition to transporting ions across the cell membrane, contributes to intracellular signaling involved in cellular functions such as cell proliferation and apoptosis47. In particular, the binding of cardiac glycosides to the Na+/K+-ATPase protein was shown to affect intracellular calcium oscillation and the RAS signaling cascade48. Recently, it was elegantly demonstrated that cardiac glycosides can reactivate cancer suppressor genes at least partially through calcium-linked epigenetic regulation, which may participate in the anticancer activity mediated by these drugs43. We observed that sensitivity of AML cells toward the cardiac glycosides was 1.5 to 2 times higher compared to normal HSCs under these in vitro conditions, and thus there is the potential that they would preserve, in part, the normal hematopoietic system if used in vivo to treat AML. In support of our findings for cardiac glycosides, ouabain has shown activity against myeloid leukemia cell lines50, and in vitro effectiveness against primary patient CD34+ AML cells, although the elimination of primary LSCs was not established51. Furthermore, in support of the use of cardiac glycosides in leukemia treatment, Haux et al.52 observed a link between higher digitoxinemia and protection against leukemia/lymphoma in digitoxin users in Norway. Overall, the cardiac glycosides strophanthidin, digoxin and ouabain are likely anti-LSC candidates for further study as treatment options for AML.Cardiac glycosides of the cardenolide functional class are another class of drugs for which we demonstrated the ability to alter LSC viability in vitro. This finding is in line with the anti-tumor capability reported for cardiac glycosides in multiple tumor types including breast, lung and melanoma, among otherstrans retinoic acid (ATRA), the highly successful treatment of acute promyelocytic leukemia (APL) that overrides the block in differentiation of the leukemic blasts5. The discovery of ATRA has significantly increased cure rates for APL to over 80%53. Furthermore, glucocorticoids, such as prednisone and dexamethasone, are part of the standard of care for acute lymphoblastic leukemia (ALL). They are largely responsible for the survival rates of approximately 90% in pediatric patients as glucocorticoid resistance has been shown to be an adverse prognostic factor in ALL55. Rather than inducing differentiation, glucocorticoids have been described to trigger apoptosis in ALL56. In AML, glucocorticoids induce cell death and differentiation, particularly in chemorefractory disease and RUNX1-mutated AML60. Dexamethasone was included in older AML treatment regimens, but these had high toxicity, most likely due to the other standard drugs in the combination and the aggressive scheduling63. Furthermore, dexamethasone and prednisone are included in CMap but were predicted to not be as effective against the LSC signature as our candidate glucocorticoids. Therefore, the three candidates we have identified might offer an improvement over traditionally used glucocorticoids.The third subset of drugs that we evaluated further were the glucocorticoids that triggered the differentiation of the leukemic progenitor and LSC-enriched CD34+ populations into terminally differentiated CD15+ cells. Differentiation of blasts follows the archetype of all-Our approach of combining in silico analysis with an in vitro LSC assay can efficiently identify candidate drugs with anti-leukemia properties among therapeutics commonly used for non-malignant diseases. By probing LSC and HSC profiles in CMap we identified positive hits in a variety of therapeutic classes, including H1-antihistamines, cardiac glycosides and glucocorticoids. The ability of a subset of these candidate molecules to differentially decrease cell populations of the leukemic hierarchy was demonstrated using 8227 AML cells. We confirmed drug efficacy in primary, cytogenetically normal AML. Next, further validation in pre-clinical in vitro and in vivo assays using multiple AML subtypes will be needed to determine the efficacy of these drugs across the broad spectrum of AML. The elucidation of the pathways involved in their effects has a great potential for discovery of novel drugable targets. In addition, medicinal chemistry approaches could be utilized to develop analogs with improved anticancer properties and more favorable safety profiles through the modification of their chemical structures. Our approach is not limited to CMap, and could be adapted to query other large-scale datasets of transcriptional profiles of compounds from multiple cell lines to identify additional drugs to be tested. Overall, promising anti-leukemia molecules were identified and further investigations in pre-clinical models are warranted.Supplemental Table 1Supplemental Table 2Supplemental Table 3Supplemental Figure 1Supplemental Figure 2Supplementary Legends


Many NCS proteins ) form functional dimers under physiological conditions. The dimeric NCS proteins have similar amino acid sequences (50% homology) but each bind to and regulate very different physiological targets. Retinal recoverin binds to rhodopsin kinase and promotes Ca2+-dependent desensitization of light-excited rhodopsin during visual phototransduction. The guanylyl cyclase activating proteins (GCAP1–5) each bind and activate retinal guanylyl cyclases (RetGCs) in light-adapted photoreceptors. VILIP1 binds to membrane targets that modulate neuronal secretion. Here, I review atomic-level structures of dimeric forms of recoverin, GCAPs and VILIP1. The distinct dimeric structures in each case suggest that NCS dimerization may play a role in modulating specific target recognition. The dimerization of recoverin and VILIP1 is Ca2+-dependent and enhances their membrane-targeting Ca2+-myristoyl switch function. The dimerization of GCAP1 and GCAP2 facilitate their binding to dimeric RetGCs and may allosterically control the Ca2+-dependent activation of RetGCs.Neuronal calcium sensor (NCS) proteins are EF-hand containing Ca The Ca2+-induced dimerization of recoverin enhances membrane binding by creating a dual pronged myristoyl anchor  compared to the affinity of a monomeric Ca2+-myristoyl switch ) is equal to the square of the dissociation constant of the monomer: Kd(dimer) = Kd(monomer)2 = (10–4 M)2 = 10–8 M. Thus, the membrane binding affinity of Ca2+-myristoyl switch proteins is predicted here to be dramatically enhanced by the combined effect of both Ca2+-binding and protein dimerization. Dimerization of Ca2+-myristoyl switch proteins may also entropically enhance its binding to dimeric protein targets, as was suggested for the binding of dimeric Ca2+-bound recoverin to dimeric rhodopsin  that each weaken dimerization also abolish activation of the cyclase , which may help explain the steep CaJA wrote and conceived the entire manuscript.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.


Forkhead box (FOX) proteins are a group of transcriptional factors implicated in different cellular functions such as differentiation, proliferation and senescence. A growing number of studies have focused on the relationship between FOX proteins and cancers, particularly hematological neoplasms such as acute myeloid leukemia (AML). FOX proteins are widely involved in AML biology, including leukemogenesis, relapse and drug sensitivity. Here we explore the role of FOX transcription factors in the major AML entities, according to “The 2016 revision to the World Health Organization classification of myeloid neoplasms and acute leukemia”, and in the context of the most recurrent gene mutations identified in this heterogeneous disease. Moreover, we report the new evidences about the role of FOX proteins in drug sensitivity, mechanisms of chemoresistance, and possible targeting for personalized therapies. Drosophila Melanogaster  by Weigel et al. [Forkhead box (FOX) proteins are an extended group of transcriptional factors characterized by the presence of an evolutionary conserved DNA-binding domain (DBD) named “winged-helix” or “fork-head”. The family name “fork-head” derives by the first gene discovered in l et al.  in 1989 l et al. .FOX family members are now categorized, on the basis of sequence homology, into nineteen subgroups, from FOXA to FOXS, and reach at today the number of at least 50 genes distributed almost on all human chromosomes, forkhead domain, FOX proteins may exert different roles in the fine regulation of downstream genes, acting as repressors or activators of gene expression [Transcription factors are sequence-specific DNA-binding proteins (DBP) that control the rate of transcription of genetic information from DNA to messenger RNA, by binding to a specific DNA sequences (promoters and/or enhancers). As transcription factors, FOX proteins are responsible for the fine-tuning of gene expression during all stages of embryonic development and are guardians of the homeostasis in adult tissues. FOX proteins have been reported as active regulators of several networks, the main of which are: development, differentiation, maintenance of multipotency, proliferation, metabolism, DNA repair, cell cycle progression, migration, senescence, survival and apoptosis ,11,12,13pression .The mechanisms of gene expression regulation controlled by FOX proteins are, in some cases, so intricate that some FOX proteins are themselves the target of other members of the same gene family, as shown by Karadedou et al. that described the mechanisms by which FOXO3A and FOXM1 antagonize the activity of one another by regulating the transcription of downstream target genes . The finThe ability of FOX proteins to contribute to the control of several fundamental signaling pathways and of all the aspects of development and cell fate allows this superfamily of transcription factors to be heavily implicated in cancer initiation and progression. Indeed, FOX factors have been shown to play a role as either oncogenes or tumour suppressors, as well as active regulators of cellular resistance to chemotherapy and actionable targets in cancer therapy.Myeloid neoplasms are a complex and heterogeneous group of hematopoietic diseases characterized by uncontrolled proliferation and/or blockage of differentiation of abnormal myeloid progenitor cells, and variable prognosis. “The 2016 revision to the World Health Organization classification of myeloid neoplasms and acute leukemia” categorizes myeloid malignancies into five primary types: myeloproliferative neoplasms (MPN), myeloid/lymphoid neoplasms with eosinophilia and rearrangement of PDGFRA , PDGFRB (platelet derived growth factor receptor beta), or FGFR1 (fibroblast growth factor receptor 1), or with PCM1-JAK2 , myelodysplastic/myeloproliferative neoplasms (MDS/MPN), myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) and related neoplasms .Accumulating evidence suggests that FOX proteins are profoundly involved in the maintenance of multipotency of hematopoietic stem cells (HSC) and in critical mechanism driving aberrant self-renewal in preleukemic cells .In this review, we try to highlight the crucial role that FOX transcription factors play in acute myeloid leukemia development and progression, their role as potential direct and/or indirect therapeutic targets and as biomarkers of drug response and/or resistance.Acute myeloid leukemia (AML) is a heterogeneous group of clonal disorder of the hematopoietic compartment characterized by abnormal proliferation of undifferentiated myeloid progenitors, impaired hematopoiesis, bone marrow failure and variable response to therapy.Although AML arises in bone marrow hematopoietic stem cells, it may involve other extramedullary sites as lymph nodes, brain, spinal cord, liver, spleen, testicles and other parts of the body.AML is classified according to “The World Health Organization (WHO) Classification of Tumours of Haematopoietic and Lymphoid Tissues”, which was last updated in 2008 . The curFOX proteins deregulation has been found closely related to several aspects of leukemia development, progression and therapy resistance.One of the most frequent initiating alterations in AML is the AML1-ETO translocation t, accounting for about 10% of total AML . AlthougAcute promyelocytic leukemia (APL) is another subgroup of acute myeloid leukemia characterized by a unique t translocation generating the PML/RARA fusion gene.The PML/RARA oncoprotein synthesis is the key pathogenetic event of APL specifically targeted by all-trans retinoic acid (ATRA) and arsenic trioxide (ATO), two non-chemotherapeutic agents that synergistically act inducing oncoprotein degradation. Although it is well known that this fusion protein blocks granulocytic differentiation by direct transcriptional inhibition of retinoic acid target genes , the detArsenic trioxide in combination with ATRA is currently considered the standard of care for adults with low-to-intermediate-risk APL, with a complete remission rate near to 100% . InteresFOXO subfamily members are not the solely forkhead transcription factors involved in APL pathogenesis. In 2015, Somerville et al. reported the overexpression of FOXC1 in nearly 20% of primary non-APL AML samples, showing its involvement in the monocyte/macrophage differentiation block and in the increased clonogenic potential of AML cells . FOXC1 iAcute myeloid leukemia and related neoplasms are not only classified according to cytogenetic alterations, but also according to specific gene mutations, such as nucleophosmin 1 (NPM1), biallelic mutation of CCAAT/enhancer-binding protein alpha (CEBPA) and tunt-related transcription factor 1 (RUNX1) as provisional entity. Other recurrent mutations, such as FLT3-ITD , TP53 (tumor protein p53), JAK2 (Janus kinase 2), K-RAS , N-RAS , ASXL1 , TET2 (tet methylcytosine dioxygenase 2), DNMT3A , IDH1 (isocitrate dehydrogenase type 1), IDH2 (isocitrate dehydrogenase type 2), SRSF2 (serine and arginine rich splicing factor 2), SF3B1 (splicing factor 3b subunit 1), U2AF1 , etc,  do not dNew insights show that many FOX proteins are downstream targets of some recurrent somatic mutations in myeloid neoplasm. In this line, future studies may be helpful to identify FOX proteins as predictive marker of survival, early biomarkers of response and actionable targets for AML therapy. Here we report about the interaction between FOX proteins and FLT3-ITD, NPM1 and IDHs somatic mutations in AML.Leukemias with mutated NPM1 have been recognized as a specific subgroup of AML . NPM1 isp < 0.001), whereas they do not find statistically significant differences in FOXO3 and FOXO4 expression [Fms-related tyrosine kinase 3 internal tandem duplication (FLT3-ITD) gene mutations were first reported in 1996 by Nakao et al.  and werepression . The Autpression . Thus, fpression ,75. In apression ,77. HoweBeyond FOXC1 and FOXO subfamily members, FOXM1 expression levels were found deregulated in FLT3-ITD cell lines and patients. In particular, Liu et al. showed that expression of FOXM1 in AML patients was correlated with the presence of FLT3-ITD and overall survival . They alIsocitrate dehydrogenases (IDHs) are enzymes mainly involved in epigenetic regulation. The term epigenetic refers to a set of heritable phenotype changes that do not involve alterations in the DNA sequence, but can switch genes on or off and consequently determine which proteins will be transcribed and which others will be silenced, respectively. The connection between epigenetic changes and the occurrence and development of tumors has been extensively studied in the last decades ,79. In pIn particular, isocitrate dehydrogenases 1 (IDH1) and isocitrate dehydrogenases 2 (IDH2), the cytoplasmic and the mitochondrial isoform, respectively are enzymes of Krebs cycle, converting isocitrate, generated in the tricarboxylic acid cycle (TCA), to alpha-ketoglutarate (α-KG) leading to restoration of cellular NADPH (Nicotinamide adenine dinucleotide phosphate-H) . MutatioDiscovered approximately 10 years ago by Marcucci et al. , hot spoIDH2-R140 mutations occur more frequently than IDH2-R172 mutations together with other epigenetic somatic mutations, identifying two distinct subgroups of patients with different prognosis (the former with better prognosis) and mutational status. These mutations are targeted by enasidenib, which, like ivosidenib, may cause a differentiation syndrome with a clinical picture similar to that caused by ATRA in APL ,95.IDH1 is directly regulated by FOXO transcription factors and, in case of mutations, this regulation leads to the upregulation of 2-hydroxyglutarate (2-HG). Charitou et al. firstly discovered the dual function of FOXOs in mediating both tumor suppression and promotion in the case of IDH1 mutation . In wildRecently, the two novel aforementioned agents selective for these mutations have been approved by the FDA (Food and Drug Administration), ivosidenib for IDH1 and enasidenib for IDH2 mutations . Other IOne of the first genes belonging to the FOX superfamily that was associated with the pathogenesis of AML is FOXM1. Already mentioned in the case of NPM1 and FLT3 mutated AML, it stimulates expression of cyclins as CCNA2 and CCNB1 and regulates different pathways involved in cell proliferation and division . The impFOXOs are one of the largest subgroups of forkhead family members, including FOXO1, FOXO3, FOXO4 and FOXO6, bona fide tumor suppressors. Their aberrant expression plays an important role in leukemogenesis. Moreover, FOXO3 is implicated in BCR/ABL fusion gene leukemogenesis, a translocation occurring in chronic myeloid leukemia, acute lymphoblastic leukemia and more rarely AML . FOXO3A FOXP1 is one of the FOXP subfamily members which, as the other forkhead box transcription factors, regulates cell differentiation and proliferation . FOXP1 sFinally, FOXP3, together with CD4 and CD25, is the marker of regulatory T cells (Tregs), a subset of T lymphocytes known to have a critical role in cancer development. In the setting of AML patients, it has been shown that FOXP3 polymorphisms in the donor may have a prognostic impact on clinical outcome of pediatric patients undergoing hematopoietic stem cell transplant (HSCT) in terms of higher incidence of veno-occlusive disease (VOD), Cytomegalovirus (CMV) infection and OS . MoreoveAs summarized in this review, in the last decades, accumulating evidences have suggested wide implications of forkhead box proteins in AML pathogenesis, prognosis and treatment response, beginning to dissect the complex panoplia of their multifunctional and subcellular interactions. Nevertheless several points remain to be addressed.First of all, as previously described, FOX family members have been identified as both tumor suppressors and oncogenes, depending on cell type and context. This ambiguous function urges to be better-unraveled trough future studies on well-characterized subgroups of AMLs.Nowadays, the study of bone marrow microenvironment is emerging as an important actor in AML biology ,112. In Furthermore, single nucleotide polymorphisms (SNP) were widely implicated in AML pathogenesis. SNPs have been identified as risk factors for AML development ,116,117 Beyond the aforementioned function of DNA methylation in AML biology, miRNAs are emerging as important players in epigenetic modulation of gene expression. However, only few studies have tried to elucidate the interaction between miRNAs and FOX family members in AML pathophysiology.Already mentioned in the case of APL, TRAIL and its receptors are often deregulated in AML resulting in drug-resistance, probably due to a deficiency of NF-κβ pathway via PU1 and upregulation of anti-apoptotic genes . The disMoreover, understanding more about FOX protein-mediated mechanisms of chemosensitivity and resistance will address the unmet clinical need of patients unfit for standard treatment or resistant to conventional chemotherapy. The novel evidences about FOX proteins-related chemoresistance may lead to combine FOX protein regulators with lower doses of chemotherapeutic drugs extending the possibility of being treated also to unfit and elderly patients.Thus, targeting these pathways may represent a new way of treating acute myeloid leukemia, moving towards a more specific and personalized medicine approach. The study of FOX proteins at different disease specific time points, such as diagnosis and relapse, will ameliorate the prognosis of patients with AML together with other well recognized prognostic markers like molecular biology and cytogenetics in order to better characterize the individual risk and have more information about the drug sensitivity profile.


Such stiffness-dependent calcium response is associated with the induced selectin related to β2-integrin activation through the Syk/Src signaling pathway, and F-actin/myosin II are also involved in this. Moreover, tension-activated calcium ion channels displayed critical roles in initiating stiffness-dependent calcium spike. These results provide an insight into understanding how the stiffening of vascular walls could regulate the calcium flux of adhered neutrophils, and thus the immune responses in atherosclerosis.Atherosclerosis is caused by chronic inflammation associated with the adhesion of neutrophils and endothelial cells (ECs) that is mediated by their respective cellular adhesive molecules to stiffened blood vessel walls. However, the stiffness dependence of calcium flux on neutrophils remains unclear yet. Here, the effect of substrate stiffness by ECs on neutrophils' calcium spike was quantified when the individual neutrophils that adhered to the human umbilical vascular endothelial cell (HUVEC) monolayer were pre-placed onto a stiffness-varied polyacrylamide substrate (5 or 34.88 kPa) or glass surface. Our data indicated that E-/P-selectins and intercellular adhesion molecule 1 (ICAM-1) on HUVECs and β As chronic vascular inflammation, atherosclerosis presents increased arterial stiffening, which is a hallmark of atherosclerosis and also the consequence of many diseases such as diabetes and chronic renal compromise. In atherosclerosis, sub-endothelial matrices of arteries are much stiffer [∼28 kPa Ref. ] than he2-integrins  and CD44 in neutrophils are key adhesive receptors in these processes. For example, neutrophil rolling on E-selectin forms catch bonds with L-selectin that transduces high affinity β2-integrin bond formation with ICAM-1, inducing the neutrophil arrest on the inflamed endothelium under shear flow.2-integrins to high affinity via binding to their ligands in a differential way.Neutrophils are major constituents of the cellular immune system and serve as the first-line defense to invading pathogens. In atherosclerotic vessels, neutrophils are exposed to widely varied physiological and mechanical microenvironments including shear force, chemo-attractants, and mechanical properties , driving neutrophils to adapt and respond to the varied environmental challenges for functioning properly.Substrate stiffness is known to have a significant impact on cellular behaviors for both endothelial cells and neutrophils. In the past decades, attention has been focused on the effects of substrate stiffness on cell adhesion, migration and morphology. For example, ECs on a stiff substrate exerting larger traction forces have a more stretched and reorganized actin cytoskeleton than those on a soft substrate,2+ is an intracellular second messenger and has long been known as the key events of cellular immunity, i.e., chemotaxis, phagocytosis and activation.2+ concentration contributes to neutrophil activation and essentially regulates important cellular events in neutrophils including degranulation, chemotaxis, and migration. Changes in the spatial and frequency distribution of calcium spike affect multiple aspects of neutrophil activity.2+ response helps define the direction of cell movement.Cavia pre-monolayered human umbilical vascular endothelial cells (HUVECs). We hypothesized that the matrix stiffening during atherosclerosis stiffens the ECs, which in turn alters the interactions between ECs and neutrophils via the cellular adhesive molecules involved. High stiffness allows further activation of β2-integrins through physical linkage of these molecules, and thus enhances neutrophil calcium response. To test this, neutrophil calcium flux was quantitatively compared among stiffness-varied polyacrylamide (PA) hydrogels or on glass, and the potential effects of selectins and ICAM-1s on ECs or PSGL-1, CD44 and β2-integrins on neutrophils were elucidated separately. The underlying signaling pathways of this stiffness-dependent regulation in calcium response were also discussed.Here, we focused on understanding how substrate stiffness affects neutrophil calcium response in vitro cellular inflammation model by pre-culturing HUVECs on a PA gel with a thickness of 100 μm to from a monolayer. To examine the potential involvement of adhesive molecules in the Ca2+ spike of neutrophils, the HUVECs were treated with lipopolysaccharide (LPS) for 4 h to induce the expression of selectins and ICAM-1s before tests, as previously reported.supplementary material, Fig. S1(a)]. Fluorescence intensity analysis confirmed the above observations, yielding a 16.3-, 2.0- or 13.7-fold increase in E- and P-selectins, or ICAM-1 expression . Moreover, this LPS-induced enhancement of cellular adhesive molecules was not positively correlated with substrate stiffness  protocol. Here, a quantitative nanomechanics (QNM) mode of AFM allowed us to examine the height, the topography and the elastic modulus of the live HUVEC monolayer simultaneously . The obtsupplementary material, Fig. S2(a)], and the Ca2+ spikes in neutrophils were monitored by time-lapsed imaging . By defining a specific Ca2+ spike event with Ca2+ intensity >50 and the spike frequency as the Ca2+ peak number within a 25-min duration , it was found that LPS treatment on HUVECs upregulated the Ca2+ intensity and the spike number in spreading neutrophils onto the HUVEC monolayer , and the total numbers of P-selectin and ICAM-1 molecules on a single HUVEC cell were determined via flow cytometry after being digested with trypsin [Figs. S3(a)–S3(d)], respectively. The molecular densities of P-selectins and ICAM-1s on HUVECs [black bar in Figs. S3(k) and S3(l)] on each substrate were then obtained by dividing the total number of those molecules of a single HUVEC cell [Figs. S3(b) and S3(d)] by the average spreading area of a single HUVEC cell [Fig. S3(i)]. Similarly, the numbers of PSGL-1s, CD44s and CD18s on a single neutrophil [Figs. S3(e)–S3(h)], the spreading area of a single neutrophil on different substrates via the HUVEC monolayer [Fig. S3(j)] and the molecular densities of CD18, PSGL-1, and CD44s on neutrophils [white and dotted bars in Figs. S3(k) and S3(l)] were also obtained separately. The results [Fig. S3(k)] indicated that, for ICAM-1s on HUVECs, although the expression level of ICAM-1s on the 5-kPa substrate is lower than that on the 34.88-kPa substrate, the corresponding β2 integrin-ICAM-1 interaction is sufficient enough to mediate calcium response in the same contact area, which is mainly governed by the receptors on neutrophils. For P-selectin [black bar in Fig. S3(l)], as its expression is very low , its role in mediating calcium response in neutrophils on different substrates is approximately at the same level. Thus, the expression levels of P-selectins and ICAM-1s would not be a primary factor that differentially regulates the neutrophil Ca2+ on 5-kPa and 34.88-kPa gels, and such stiffness-dependent manner of calcium response is most likely mechanically specific.As shown in kPa gels , insteadAdditionally, to exclude the potential impacts of the PA gel itself or the thickness of the gel on the measured fluorescence intensity of the calcium spike, fluorescein isothiocyanate (FITC)-labeled polystyrene beads were placed on the three substrates separately and their fluorescence intensities were compared. The results indicated that neither the gel itself nor the thickness of the gel would affect the fluorescent signals [Fig. S4], convincing the data collected above.2-integrins is known to mediate neutrophil rolling and adhesion on endothelium.2-integrins in neutrophils,2-integrins are responsible for stiffness-dependent Ca2+ spike, we tested Ca2+ flux in neutrophils by blocking or activating β2-integrins. Cooperative blockage of β2-integrins using anti-CD11a (MEM25) and anti-CD11b (ICRF44) mAbs dramatically reduced Ca2+ intensity to the basal level and completely abolished the spike at all stiffnesses  were the same as those by inhibiting Syk. Taken together, these results suggested that selectin-induced β2-integrin activation is responsible for stiffness-dependent Ca2+ spike in neutrophils on a 4-h LPS-treated HUVEC monolayer.It is known that the above βffnesses , while tffnesses , thus di2+ signals in sensing the substrate stiffness. To investigate the roles of the cytoskeletal network, cytochalasin D (Cyto D) was applied to depolymerize actin filaments. As shown in 2+ spike in neutrophils in all stiffnesses and both Ca2+ intensity and peak number were significantly inhibited by Cyto D treatment. These results suggested that intracellular cytoskeletal support through actin filaments is necessary for the induction of stiffness-dependent Ca2+ spikes. Surprisingly, inhibiting myosin II using blebbistatin [2+ intensity and the peak number on the glass substrate and had no effects on Ca2+ spike on PA gels at 5 kPa and 34.88 kPa substrates. Collectively, these results implied that cytoskeletal support is essential for Ca2+ spike, and active actomyosin contractility may be more sensitive to substrate stiffness that is responsible for tension-induced Ca2+ spike in neutrophils on glass.As substrate stiffness is known to affect the cell morphology, the cytoskeletal structure and the cell adhesion,bistatin  only att2+ spike is usually governed by Ca2+ trafficking between the plasma membrane and the endoplasmic reticulum (ER) membrane. Using 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA) to chelate intracellular Ca2+ seemingly had no effects on Ca2+ intensity and spike number of stiffness-dependent Ca2+ spike, especially for neutrophils on glass [2+ influx from the extracellular environment dominates the stiffness-dependent Ca2+ spike in neutrophils.Cytoplasmic Caon glass . This ob2+ spike, we first used 2-amino-ethoxydiphenylborate (2-APB) to inhibit IP3R proteins on the membrane and found that inhibiting IP3R lowered Ca2+ intensity and peak at all stiffnesses [2+ (SOC) channel plays an important role in Ca2+ spike. We further applied a potent inhibitor of tension-activated calcium permeable channels, gadolinium chloride (GdCl3), or an inhibitor of L-type Ca2+ channel, Nifedipine. As shown in 3 specifically inhibited Ca2+ intensity and spike number in neutrophils on glass, whereas inhibiting the L-type Ca2+ channel almost had no effect on Ca2+ spike in neutrophils. Taken together, our results suggested that Ca2+ entry via tension-activated calcium permeable channels from the extracellular environment upon substrate stiffness is essential for the stiffness-dependent Ca2+ spike in neutrophils.To examine whether calcium influx from the extracellular environment is involved in the stiffness-dependent Caffnesses , indicatin vitro cellular inflammation model to elucidate the impact of substrate stiffness on Ca2+ spike in neutrophils adhering to HUVECs that are pre-layered on varied substrates. A complicated, coordinated feature of Ca2+ response regulation in neutrophils was observed, in which selectin-induced β2-integrin activation via the Syk/Src signaling pathway is dominant in initiating the neutrophil Ca2+ flux, and a rise in cytoplasmic Ca2+ concentration may activate Ca2+-sensitive secondary kinases (such as Rho GTPases) to direct actin polymerization and actomyosin contraction.2-integrin-ICAM-1 bonds provide a physical linkage between the ECM and the actin cytoskeleton, and thus play a critical role in cellular mechano-transduction.via β2-integrin bonds to directly activate those tension-sensitive Ca2+ channels on the plasma membrane. A working model was proposed to illustrate how Ca2+ spike in neutrophils is regulated by substrate stiffness via adhering to the HUVEC monolayer  and of endothelium (as a leukocyte substrate) collectively contribute to the process of leukocyte transmigration. HUVEC morphology and neutrophil migration are regulated by substrate elasticity.2+ spike plays an important role in functional responses of neutrophils. The cytosol Ca2+ level is balanced by the extracellular calcium influx and the ER-derived calcium release, which is associated with the Orail-3/STIM1–2 signaling pathway to promote neutrophil or lymphocyte recruitment in inflammation.in vivo wound healing or drug-induced chemotaxis.2+ spike triggers the disassembly of focal adhesion with similar kinetics of stress fiber end movement and disrupts integrin-ECM interactions, supporting a key role for Ca2+-sensitive inside-out signaling in cell migration.2+ spike in neutrophils is mediated by selectin-induced high-affinity β2-integrins via Syk/Src signaling and actin/myosin II remodeling, which is governed by tension-related gating of multiple calcium channels from extracellular Ca2+ influx  kinase. And, cytoskeletal dynamics and MLCK-driven actomyosin contractility have a significant impact on cellular tension.2+ spike in neutrophils. Surprisingly, although depolymerizing the actin fibers via Cyto D completely abolished Ca2+ spike in all stiffness, inhibiting the myosin-II proteins via blebbistatin only decreased Ca2+ spike on glass  can enhance the expressions of E- or P-selectin expression on HUVECs, which could also manipulate the intensities of calcium responses.2+ spike manner in neutrophils, which is mediated by mechanical regulation of substrate stiffness via layered HUVECs. Binding of E-/P-selectin-CD44/PSGL-1 activates β2-integrins to a high-affinity state and β2-integrin-ICAM-1 binding dominates the calcium responses in neutrophils via Syk/Src signaling. Multiple calcium ion channels are dispensable in this process, mainly attributed to the extracellular Ca2+ influx. This work provides an insight into elucidating Ca2+ spike in atherosclerosis from the viewpoint of mechanotransduction.Collectively, we presented a stiffness-dependent Ca3), nifedipine and 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA) were from Sigma-Aldrich . Anti-CD11b (ICRF44) and anti-PSGL-1 (PL-1) blocking mAbs were from Santa Cruz Biotechnology . Alexa Fluor 594-conjugated goat anti-mouse secondary Abs for immunofluorescence staining were from Abcam . FITC-labeled anti-P-selectin blocking mAbs (AK-6) were from Thermo Fisher Scientific . Medium 199, Dulbecco’s Phosphate buffered Saline (DPBS), trypsin, and 4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) were from GE Healthcare Life Sciences . Amphotericin B was from Amresco , the basic fibroblast growth factor (bFGF) was from R&D Systems  and fatal bovine serum (FBS) was from Gibco . Fluo-4-AM dye was from Invitrogen .Allophycocyanin (APC)-labeled anti-human CD44 (BJ18), Alexa Fluor® 647-labeled anti-human CD54 (HCD54), and phycoerythrin (PE)-labeled anti-human CD162 (KPL-1) monoclonal antibodies (mAbs), as well as isotype-matched irrelevant antibodies (Abs) LEAF™ purified mouse IgG1 (MOPC-21), purified rat IgG2a (RTK2758), and LEAF purified mouse IgM (MM-30) were obtained from BioLegend . Mouse anti-human E-selectin (S9555) and anti-CD11a blocking mAbs (MEM25), as well as thymidine, heparin sodium, LPS, fMLP, cytochalasin D, PP1, piceatannol, blebbistatin, 2-amino-ethoxydiphenylborate (2-APB), gadolinium chloride  and cultured in Medium 199 supplemented with 20% FBS, 100 U/ml penicillin, 20 mM HEPES, 3 2+ spike.Whole human blood was obtained from healthy human donors after the informed consent was obtained, as approved by the Animal and Medicine Ethics Committee of the Institute of Mechanics with the ethics approval ID number 2018–03, Chinese Academy of Sciences, in accordance with the Declaration of Helsinki. All the donors were unmedicated and selected randomly. Neutrophils were then isolated by a density gradient centrifugation protocol using Histopaque-1077 and Histopaque-1119 (Sigma-Aldrich), as previously described.μg/ml FITC-anti-human CD18, APC-anti-human CD44, PE-anti-human CD162 mAbs, PE-anti-human E-selectin, PE-anti-human P-selectin, Alexa Fluor 647-labeled anti-human CD54 or isotype-matched irrelevant mouse IgG1s and IgG2a, respectively, on ice for 45 min. Washed neutrophils were resuspended in DPBS and analyzed by a FACS Calibur cytometer .Isolated neutrophils were incubated with 5 To determine the site densities of these adhesive molecules on HUVECs or neutrophils, the fluorescence intensities of the stained cells were quantified using standard fluorescence calibration beads  to determine the mean number of molecules of equivalent soluble fluorochrome (MESF) per cell. The MESF value was divided by the F/P value of FITC-labeled mAbs and the surface area of the cell, and then converted into the site density of the target molecules.via a soft-contact lithography technique described in our previous work.N,N,N′,N′-tetramethylethylenediamine  into the mixture, 20 μl of the mixture was quickly sucked with a pipette and transferred to a chloro-silanated, 35-mm glass-bottom dish . The glass bottom was covered with one 18-mm diameter circular glass to allow the gel to be polymerized for 10 min and rinsed with distilled H2O for at least 4 h. After adding 0.2 mg/ml sulfo-SANPAH solution (cross-linking agent) in an adequate volume, the mixture was irradiated with ultra-violet light for 25 min to form a planar PA hydrogel. Two sets of concentrations were used for 25% acrylamide + 15% bis-acrylamide (34.88 kPa) and 25% acrylamide + 1.5% bis-acrylamide (5 kPa), where the Young’s modulus of the gel was determined using a self-weighing assay.μm in thickness was coated with collagen I (2 ml of 20 μg/ml) prior to seeding the cells. A glass coverslip with the same diameter and thickness was used as control.Biopolymer substrates were constructed using PA hydrogel μm2 with a resolution of 512 × 512 pixels, and a 0.15 Hz scanning rate was used. An AFM force curve of the target cell was obtained by indenting the cell surface using AFM probes equipped with a Nanoscope V controller and Nanoscope software version 8.15. The QNM-Live Cell (PFQNM-LC) probes  with a spring constant of 0.07 N/m, a tip length of 17 μm, and a tip radius of 65 nm were used. For all the measurements, an indentation depth of 400–500 nm was applied to determine the local cell elasticity. The probe’s spring constant was pre-calibrated before each measurement using the thermal fluctuation protocol with Bruker’s polystyrene test samples, as described previously.μm was also used to convince the robustness of HUVEC moduli measured, and the cell modulus was estimated from fitting the collected force-displacement curves with the Hertz model. All measurements were carried out in HUVEC culture medium at room temperature. On each PA gel or glass, at least six scanning fields (100 × 100 μm2 with 512 × 512 pixels) were examined in three independent repeats. To reduce the labor consumption, a region of 25 μm2 was randomly selected from each scanned image to calculate the average Young’s modulus in this region for further data analysis. All data were analyzed with Nanoscope Analysis version 1.8.Topographical and modulus images were scanned with a Dimension Icon atomic force microscope  using the PeakForce Quantitative Nanomechanics (QNM) mode. The scanning size was 100 × 100 μl of HUVEC suspension with ∼5 × 104 cells was placed onto PA gel or glass coverslip pre-coated with collagen I overnight to form a monolayer. After 4-h of LPS-treatment, the HUVEC monolayer was washed twice with PBS and replaced with culture media. Human neutrophils were loaded with 1.25 μg/ml of Fluo-4-AM for 15 minutes at a concentration of 5 × 105/ml in calcium-free PBS at 4 °C as described previously.5) were added onto the HUVEC monolayer and allowed to adhere for 5 min before imaging. Cells were imaged on an automatic inverted microscope  equipped with an electron-multiplying charge-coupled device (EMCCD) camera , that was controlled by MetaMorph software . To image the calcium response in those adhered neutrophils on HUVECs, the Fluo-4-AM loaded neutrophils were excited by a 100-W xenon lamp through a 480AF30 filter  and a 60×/NA1.35 oil immersion objective lens was used to observe the changes in intracellular calcium in individual neutrophils on HUVECs at a 500-ms interval for 25 min. Here, the dish containing the cells was placed in a custom-made heating device that provides temperature control (37 ± 0.5 °C) and a 5% CO2 supply during live cell imaging.100 μm diameter polystyrene beads  were placed on the three substrates, excited by a 100-W xenon lamp via a 480AF30 filter , and imaged through a 60×/NA1.35 oil immersion objective lens . Three independent repeats are performed and at least three FOVs are acquired in each repeat. Collected images are analyzed using the same protocol described above.In some cases, FITC-labeled 7.1-For immunostaining experiments, HUVECs cultured on a PA hydrogel or glass coverslip were rinsed in PBS and fixed in 4% paraformaldehyde at room temperature (RT) for 10 min. After blocking non-specific epitopes using 1% bovine serum albumin (BSA)  at 37 °C for 1 h, the collected cells were stained with primary mAbs at 37 °C for 1 h, incubated at 4 °C overnight, and rinsed five to seven times with PBS. Then, the adequate amount of labeled secondary Abs in 1% BSA/PBS was added and incubated at 37 °C for 1 h. After being washed five to seven times with PBS, the collected samples were incubated with Hoechst 33342 for 10–15 min at RT, washed three to five times with PBS, and then stored at 4 °C for examination by confocal laser scanning microscopy  with a 40×/0.95 NA objective. All images were analyzed using ImageJ .2+ signals of a single cell.black arrows in Fig. S2). To protect the potential overestimation of calcium signals, a threshold value of 50 was set in the current work to explicit those remarkable calcium spikes in neutrophils. For simplicity, a spike with the peak value over 50 was counted as a specific event . The number of spiking Ca2+ peaks was counted as the corresponding peak number in each neutrophil and then averaged for all the cells counted. Also, it was noticed that no photobleaching of calcium signals was found within the observation window in the current experimental settings.A custom-developed automated image analysis method was used to track the movement of a single neutrophil and quantify the Cat test between paired data or one-way ANOVA analysis followed by Tukey’s post-hoc test for grouped data. Prism statistical software  was used by setting statistical difference as *p < 0.05, **p < 0.01, ***p < 0.001, and **** p < 0.0001.Data are presented as mean ± SE under different conditions. Statistical tests were performed using unpaired two-tailed Student’s supplementary material for Fig. S1–S5.Fig. S1 indicates that selectin or ICAM-1 expression is increased on the 4-h LPS-treated HUVEC monolayer compared to that for untreated cells. Figure S2 illustrates the stiffness-dependent Ca2+ spike in neutrophils on 4-h LPS-treated HUVECs with the definition of Ca2+ spike in a neutrophil. Figure S3 compares the expressions of cellular adhesive molecules on HUVECs and neutrophils on three distinct substrates. Figure S4 presents the mean fluorescence intensity of FITC-labeled polystyrene beads on three substrates. Figure S5 demonstrates that the Src signaling pathway is responsible for stiffness-dependent Ca2+ spike in neutrophils on 4-h LPS-treated HUVECs.See


As reactivation of the fetal gene program has been implicated in pathological remodelling during heart failure (HF), we examined whether cardiomyocyte subcellular structure and function revert to an immature phenotype during this disease.2+ channels and ryanodine receptors during development, and ‘unpacked’ during HF.Surface and internal membrane structures appeared gradually during development, and returned to a juvenile state during HF. Similarly, dyadic junctions between the cell membrane and sarcoplasmic reticulum were progressively ‘packed’ with L‐type Ca2+ in failing cardiomyocytes.Despite similarities in subcellular structure, dyads were observed to be functional from early developmental stages, but exhibited an impaired ability to release Ca2+ homeostasis observed in HF.Thus, while immature and failing cardiomyocytes share similarities in subcellular structure, these do not fully account for the marked impairment of Ca2+ channels and RyRs into dyadic junctions during development, and orderly unpacking during HF. These findings support a ‘last in, first out’ paradigm, as the latest stages of dyadic structural development are reversed during disease. Paired imaging of t‐tubules and Ca2+ showed that the disorganized arrangement of dyads in immature and failing cells promoted desynchronized and slowed Ca2+ release in these two states. However, while developing cells exhibited efficient triggering of Ca2+ release at newly formed dyads, dyadic function was impaired in failing cells despite similar organization of Ca2+ handling proteins. Thus, pathologically deficient Ca2+ homeostasis during HF is only partly linked to the re‐emergence of immature subcellular structure, and additionally reflects lost dyadic functionality.Reactivation of the fetal gene programme has been implicated as a driver of pathological cardiac remodelling. Here we examined whether pathological remodelling of cardiomyocyte substructure and function during heart failure (HF) reflects a reversion to an immature phenotype. Using scanning electron microscopy, we observed that Z‐grooves and t‐tubule openings at the cell surface appeared gradually during cardiac development, and disappeared during HF. Confocal and super‐resolution imaging within the cell interior revealed similar structural parallels; disorganization of t‐tubules in failing cells was strikingly reminiscent of the late stages of postnatal development, with fewer transverse elements and a high proportion of longitudinal tubules. Ryanodine receptors (RyRs) were observed to be laid down in advance of developing t‐tubules and similarly ‘orphaned’ in HF, although RyR distribution along Z‐lines was relatively sparse. Indeed, nanoscale imaging revealed coordinated packing of L‐type Ca As reactivation of the fetal gene program has been implicated in pathological remodelling during heart failure (HF), we examined whether cardiomyocyte subcellular structure and function revert to an immature phenotype during this disease.2+ channels and ryanodine receptors during development, and ‘unpacked’ during HF.Surface and internal membrane structures appeared gradually during development, and returned to a juvenile state during HF. Similarly, dyadic junctions between the cell membrane and sarcoplasmic reticulum were progressively ‘packed’ with L‐type Ca2+ in failing cardiomyocytes.Despite similarities in subcellular structure, dyads were observed to be functional from early developmental stages, but exhibited an impaired ability to release Ca2+ homeostasis observed in HF.Thus, while immature and failing cardiomyocytes share similarities in subcellular structure, these do not fully account for the marked impairment of Ca During the action potential, opening of L‐type Ca2+ channels (LTCCs) in the surface sarcolemma triggers release of Ca2+ from ryanodine receptors (RyRs) in the sarcoplasmic reticulum  and performed in accordance with the National Institute of Health guidelines  and European Directive 2010/63/EU.ad libitum. Following birth, pups were collected at 15, 17.5, 20 and 30 days after birth and heart tissue was extracted while under anaesthesia  for various experimental purposes. Eight‐ to ten‐week‐old rats were considered adults.Animals were purchased from Janvier Labs  and housed in temperature‐regulated rooms with a 12 h light–dark cycle. Pregnant female Wistar rats were housed (one per cage) with access to food and water et al. 2O, 32% O2, and 2–3% isoflurane. Myocardial infarction was induced via ligation of the left anterior descending coronary artery and HF progression was assessed via echocardiography and magnetic resonance imaging after 6 weeks. SHAM‐operated controls underwent the same procedure minus coronary ligation. Buprenorphine was used for analgesia during postoperative care.Myocardial infarction was induced in male Wistar rats  as described previously : 130 NaCl, 25 Hepes, 0.5 MgCl2, 5.4 KCl, 0.4 NaH2PO4, 22 glucose. Hearts were quickly cannulated via the aorta and retrogradely perfused with 37°C isolation buffer at a rate of 3 ml/min. Once clear of blood, the myocardium was digested for a period of 5–12 min (depending on age and size of heart) by perfusion of 0.3–1.8 mg/ml of collagenase, type 2  in oxygenated isolation buffer supplemented with 10 μmol/l Ca2+. Following digestion, the ventricles were trimmed away from the remaining tissue and submerged in collagenase buffer. Right ventricular tissue was removed and the remaining left ventricle was dissected into approximately 3 mm3 chunks. Tissue was gently teased apart with forceps then transferred to a conical tube containing 0.2 mg DNase (Worthington) and 20 mg/ml BSA  in dH2O. A plastic Pasteur pipette was used to agitate tissue chunks for further digestion. The cell solution was filtered through open mesh fabric with a pore size of 255 μm into a 50 ml conical tube. After the isolated cells had settled, the supernatant was removed and the cell pellet was washed twice with a room‐temperature solution of 1 mg/ml BSA in isolation buffer. [Ca2+] was gradually increased to the desired amount, starting from 0.1 mmol/l Ca2+.Individual cardiomyocytes were isolated according to a previously described protocol (Hodne l‐lysine (Sigma‐Aldrich) for 15 min at room temperature (RT), followed by 10 μg/ml natural mouse laminin  in Medium 199 (Sigma‐Aldrich) overnight at 4°C. Isolated cardiomyocytes were seeded on coated coverslips for 30–45 min then fixed in solution containing 2.5% glutaraldehyde/4% paraformaldehyde in isolation buffer (see ‘Cell isolation’) containing 100 mmol/l Hepes for 10 min at RT. After initial incubation, fixative was replenished and samples were maintained in the same fixative solution at 4°C for various amounts of time. Fixed samples were washed with dH2O three times then dehydrated with a graded series of ethanol washes . All remaining ethanol was removed by critical point drying in a Bal‐Tec CPD 030 Critical Point Dryer . Dried samples were mounted on metal stubs with conductive carbon adhesive pads and sputter coated with approximately 6 nm of platinum in a Cressington 308UHR Sputter Coater . Images were acquired on a Hitachi S‐4800 field emission scanning electron microscope .Eight millimetre round glass coverslips  were autoclaved then coated with 0.01% poly‐The proportion of Z‐groove per Z‐spine was calculated by measuring the length of elevated membrane crests, which define each groove, and then dividing this value by the total length of the Z‐spine extending from the base of the groove. The proportion of Z‐spines with t‐tubules was obtained by dividing the number of Z‐spines with a visible t‐tubule lumen by the total number of Z‐spines analysed per cell.Isolated cardiomyocytes were incubated with 25 μmol/l di‐8‐ANEPPS (Thermo Fisher Scientific) for 25 min at RT. After 15 min of labelling, cardiomyocytes were transferred to a perfusion chamber containing a 30 mm coverslip coated in 10 μg/ml natural mouse laminin (Thermo Fisher Scientific) in Medium 199 (Sigma‐Aldrich). Following dye wash‐out, the t‐tubule network was visualized on a Zeiss LSM710  scanning confocal microscope equipped with a C‐Apochromat ×40/1.2 water immersion lens. Pixel sizes ranged from 0.148 to 0.259 μm with a dwell time of 12.6 μs per pixel. Optical sections had a Z‐width of 0.4 μm.T‐tubule density and organization was measured with a custom image processing macro. In short, excess background was removed  prior to rotating the image so that the long axis of the cell was horizontal. Contrast was enhanced to 0.8% saturated pixels before application of Mexican hat (radius = 3) and median (radius = 1) filters. Following segmentation via the Otsu algorithm Otsu, , t‐tubul2+ release and t‐tubule localization. Dye was washed out and subsequent experiments were performed with superfusion of Hepes‐Tyrode buffer containing (in mmol/l): 140 NaCl, 5 Hepes, 0.5 MgCl2, 5.4 KCl, 0.4 NaH2PO4, 5.5 glucose, 1.0 CaCl2. Cardiomyocytes were field stimulated at 1 Hz and all recordings were taken on an LSM 7Live scanning confocal microscope (Zeiss) equipped with a C‐Apochromat ×40/1.20 water‐immersion lens. Fluo‐4 and FM 1‐43FX were excited at 488 nm and resulting signals were passed through a long‐pass 505 nm filter. Small intracellular areas of the Fluo‐4/FM 1‐43FX loaded cells were scanned every 1.5 ms, with pixel size and dwell time set at 160 nm and 17.1 μs, respectively. Each optical slice had a thickness of 1.7 μm. Local Ca2+ transients were analysed within small, 1 × 1 μm sections of the recordings, centred on transverse or longitudinal t‐tubules. Comparison was made with Ca2+ release at ‘orphaned’ RyR sites in developing and failing cells, positioned at irregular gaps between transversely oriented t‐tubules. Transient timing was calculated by measuring the period from the start of Ca2+ rise within the 1 × 1 μm section to the intensity at half‐maximal fluorescence (F50) measured for the overall transient.Cells were seeded on laminin pre‐coated 30 mm coverslips, as described above. Attached cells were loaded with 20 μmol/l fluo‐4 AM (Thermo Fisher Scientific) and 1.8 μmol/l FM 1‐43FX (Thermo Fisher Scientific) for 10 min at RT for simultaneous assessment of Ca2+ imaging, but with a broader scan area encompassing the entire cell scanned every 10 ms. The percentage of depolarized t‐tubules was calculated by first segmenting the t‐tubule network as described in ‘Confocal imaging of t‐tubules’. Pixels corresponding to the location of t‐tubules were then isolated by subtracting an inverted image of the t‐tubule skeleton from the three frames prior to cell contraction. The resulting image stack was then divided by the first frame in the stack, and FluoVolt signals above background were segmented using the Otsu algorithm  for 20 min at RT, before wash‐out and superfusion with Hepes‐Tyrode buffer. Microscope settings were as described above for Cahm Otsu, . The skel‐lysine (Sigma‐Aldrich) and laminin (Thermo Fisher Scientific) as described in ‘Scanning electron microscopy’. Isolated cardiomyocytes were seeded on a coated coverglass for 30–45 min prior to fixation with 4% paraformaldehyde in 100 mmol/l Hepes isolation buffer (see ‘Cell isolation’) for 5 min at RT. Cells were subsequently washed with phosphate‐buffered saline (PBS) and fixation was quenched with 100 mmol/l glycine in PBS. Membranes were permeabilized with 0.5% Triton X‐100 (Sigma) in PBS and non‐specific antigens were blocked by incubating in a solution containing 15.1 mmol/l NaCl, 1.75 mmol/l Na3C6H5O7, 5% normal goat serum, 3% BSA and 0.02% NaN3 for 2 h at RT. Primary and secondary antibodies were diluted in a solution containing 15.1 mmol/l NaCl, 1.75 mmol/l Na3C6H5O7, 2% normal goat serum, 1% BSA and 0.02% NaN3 and applied to cells over‐night at 4°C and for 2 h at RT, respectively. Cells were washed with PBS between most steps following fixation, but not prior to incubation with primary antibody. Finally, samples were mounted in ProLong® Diamond Antifade with 4′,6‐diamidino‐2‐phenylindole  and allowed to cure for 24 h at RT. The following antibodies were used: α‐actinin , cacna1c , caveolin‐3 , RyR1 , F(ab′)2‐goat anti‐mouse IgG (H+L) secondary antibody, Alexa Fluor 488 , F(ab′)2‐goat anti‐rabbit IgG (H+L) secondary antibody, Alexa Fluor 546 .Eight‐well LAB‐TEK® chambered coverglasses (Thermo Fisher Scientific) were coated with poly‐2/3.66 μs and 71 nm2/3.54 μs, respectively. Airyscan post‐processing in Zen 2.1  was applied to each image prior to export and subsequent analysis.All images were captured on an LSM‐800 scanning confocal microscope (Zeiss) equipped with a Plan‐Apochromat ×63/1.4 oil immersion lens and Airyscan (Zeiss) detector. Excitation and emission wavelengths of 493/517 and 557/572 were used for AF488 and AF546 fluorophores, respectively. Pixel size and pixel dwell time for 15‐day‐old and adult cardiomyocytes were 34 nmFor analysis, LTCC and RyR channels were separated and background was removed . A region of interest was traced encompassing the cell's interior and all extracellular signal was cleared from the image. Values for fractional overlap  were calculated in JACoP, and the resulting automatically generated thresholds were used when calculating overall protein density within the cell in Fiji. For orientation‐specific analysis, the RyR channel was processed, binarized then skeletonized prior to being separated into transverse and longitudinal elements via a custom macro. The transverse skeleton was then subjected to both a vertical and horizontal convolution filter  and binarized. The resultant transverse mask was then used to isolate the fluorescence signal in either the transverse or the longitudinal direction of processed Airyscan images, and MCCs were calculated as described above. The density of intact dyads in each cell was calculated as follows:et al. To measure RyR and LTCC density, Airyscan micrographs were processed as described above then automatically segmented using the default algorithm in Fiji . Blots were blocked in non‐fat dairy milk (Sigma‐Aldrich) or casein  in tris‐buffered saline‐tween buffer (TBS‐T) and incubated overnight with primary antibody at 4°C, followed by species‐specific horseradish peroxidase‐conjugated secondary antibody for 1 h at room temperature. Blots were developed with ECL prime  and visualized on an LAS‐4000 Luminescent Image Analyzer . Membranes were stripped for re‐probing using Restore Western Blot stripping buffer (Thermo Fisher Scientific). Image processing and protein signal quantification were performed in ImageQuant TL  and ImageJ , caveolin‐3 , junctophilin‐2 , vinculin .et al. t test. Multiple groups were compared using one‐way ANOVA with the Tukey post hoc test. Differences with a P‐value < 0.05 were deemed significant. With the exception of Figs E and n’ used for statistical tests relates to the number of analysed cells. In Fig. E the ‘n’ relates to number of hearts, while in Fig. All image processing and analysis was conducted in Fiji , formed by elevated membrane crests on both sides of an underlying Z‐disc , features that previous work has suggested may be attributed to sarcomeric structures .We employed the unparalleled resolving power of scanning electron microscopy to investigate changes in cardiomyocyte surface topography during postnatal development, adulthood and heart failure. Figure A and B). Interestingly, this surface topography of failing cells was very similar to that of immature cardiomyocytes described above, as the measured proportion of Z‐grooves was comparable to that of cells isolated 17.5 days after birth . T‐tubule openings within Z‐grooves were also less apparent in failing cells, in resemblance of the surface of developing cells . These findings support that external cardiomyocyte topography develops gradually in the postnatal period and is dismantled as HF progresses. This paradigm is illustrated schematically in Fig. Next, we investigated whether programmed remodelling of cardiomyocyte surface topography occurs during heart failure (HF). Myocytes isolated from HF rats 6 weeks following myocardial infarction were compared with sham‐operated controls (SHAM). We observed marked loss of Z‐grooves at the surface of failing cardiomyocytes, resulting in a smoother external membrane with easily visible Z‐spines Fig. A and B.B), and initial longitudinal elements were replaced by transverse t‐tubules . Indeed, we found that longitudinal t‐tubules were largely transient in nature, reaching peak levels between 17.5 to 30 days after birth before decreasing in density as the cell reached adulthood.We next investigated whether changing surface morphology during development and HF progression was mirrored at internal sites. T‐tubule density and organization were assessed by confocal microscopy in cardiomyocytes stained with di‐8‐ANEPPS Fig. . T‐tubulles Fig. C and D.et al. et al. et al. D). Thus, as with external membrane structures, t‐tubules are restructured during HF, resulting in a remodelled network reminiscent of that present in immature cells .While pathological remodelling of t‐tubules during the progression to HF is a well‐documented phenomenon  containing LTCCs, which were frequently colocalized with RyR clusters. Thus, distances measured from each RyR location to the nearest LTCC were generally short, and only a minority of RyRs could be clearly identified as being non‐junctional or ‘orphaned’ . Quantification of the signal densities for these two proteins and colocalization analysis are illustrated in Fig. A–D. Of note, we calculated that more than 80% of LTCCs located along transversely oriented tubules were colocalized with RyRs in healthy adult cells . Although there are relatively few longitudinal tubules in healthy adult cells, these tubules were also observed to contain LTCCs and form dyadic junctions with RyRs. However, association between the two proteins was markedly lower along longitudinal elements than at their transversely oriented counterparts, with only a minority of longitudinal LTCCs observed to colocalize with RyRs . This finding suggests that there is distinct organization of dyadic proteins along the two types of t‐tubules.To understand the functional implications of changes in t‐tubule structure in development, adulthood and HF, we examined the organization of t‐tubules and LTCCs relative to RyRs using super‐resolution AiryScan imaging. Representative images in Fig. ed’ Fig. A and B.lls Fig. D. AlthoyRs Fig. D. This A and B, respectively). Thus, measured distances from RyR locations to the nearest LTCCs were right‐shifted in comparison with mature cells, consistent with a greater proportion of orphaned RyRs . However, RyR clusters showed a more sparse distribution along Z‐lines at this stage than present in adult cardiomyocytes. Indeed, quantification revealed increasing RyR density with further maturation . LTCC density in transverse t‐tubules similarly increased towards adulthood , and colocalization analyses revealed an increasing density of transversely oriented dyads progressively packed with LTCCs and RyRs . Of note, where present in developing cells, LTCCs already exhibited a high degree of colocalization with transversely oriented RyRs , suggesting that these dyads may be functional even at early stages.To address how these dyads are first assembled, we chose to examine 17.5‐day‐old cardiomyocytes as a developmental time point since they contain a nearly equal proportion of longitudinal and transverse t‐tubules. We observed that transversely aligned RyRs preceded the arrival of t‐tubules (Cav3 staining) and LTCCs . While there was also a significant fraction of longitudinally oriented RyRs in these cells , colocalization analysis showed that, as in adult cells, only a minority of LTCCs were associated with RyRs at these sites . Thus, longitudinal tubules and dyads may be viewed as rather immature structures, which are prominent in developing cells but still found at lower density in healthy adult cells. The two dyadic arrangements are schematically illustrated in Fig. E.Mirroring t‐tubule morphology in developing cells, LTCC imaging revealed greater allocation to longitudinally oriented t‐tubules than is observed in mature cardiomyocytes . Indeed, measurements of RyR distances to nearest LTCC tended to be right‐shifted in failing cells , and significantly fewer dyadic junctions were observed . However, the transversely oriented RyR network remained only partially in place in failing cells, as quantification revealed an overall loss of RyR staining which paralleled changes in LTCCs . Importantly, where t‐tubules and LTCCs remained present, proportional colocalization with RyRs remained high , indicating that loss of LTCCs and RyRs occurred predominantly at the same sites. Thus, the SR appears to reorganize in tandem with t‐tubule structure during HF, resulting in an unpacking of both RyRs and LTCCs from transversely oriented dyads. In further similarity to developing cells, an increased allotment of LTCCs and RyRs to newly formed longitudinal dyads was also observed in failing cells . As in the other cell groups examined, LTCCs and RyRs exhibited only modest colocalization along longitudinal tubules in these cells . These findings are summarized in Fig. E.During HF progression, dyadic organization showed a reversion to an immature phenotype. In agreement with previous work, we observed that disorganization of t‐tubular structure appeared to result in the formation of orphaned RyRs at gaps between transverse elements Fig. A and B.lls Fig. A and B,ved Fig. C. HowevCCs Fig. A and B.igh Fig. D, indiclls Fig. A–C. As lls Fig. D. These2+ handling components during development and HF, we next investigated how these changes affect Ca2+ release in immature and failing cardiomyocytes. Ca2+ release was assessed by means of a 2D confocal time series of isolated cardiomyocytes loaded with Fluo‐4 AM. As expected based on their disorganized t‐tubule networks, both developing and HF cardiomyocytes were observed to produce slower, more dyssynchronous Ca2+ transients than their respective controls (Supporting information C) and Ca2+ release was more heterogeneous, as evidenced by dyssynchrony maps  and increased dyssynchrony indices .Having characterized changes to both sarcolemmal organization and key Caces Fig. D.2+ release in 2D confocal scans. This was done in experiments with simultaneous labelling of t‐tubules (FM 1‐43FX) and Ca2+ (Fluo‐4), to enable assessment of local Ca2+ release within small (1 × 1 μm) regions of interest corresponding to t‐tubules or orphaned RyRs . In healthy adult cells, Ca2+ release was initiated rapidly from RyRs located along both transverse and longitudinal t‐tubules, as indicated by representative videos  and local Ca2+ transients . Thus, although LTCC and RyR colocalization is lower at longitudinal compared to transverse elements , both types of dyads can efficiently trigger Ca2+ release. At t‐tubule sites in developing cells, Ca2+ release was also efficiently triggered . This finding indicates that the overall desynchronized and slowed Ca2+ transient observed in these cells results rather from the disorganized and low density of t‐tubules, which creates abnormal gaps between them. Indeed, local transients measured at these gaps showed delayed Ca2+ release , in agreement with previous work showing that orphaned RyRs are triggered only after Ca2+ propagates from intact dyads . This deficit was manifested as a markedly slower local Ca2+ transient, particularly at transverse elements where time to F50 values were significantly prolonged in comparison with equivalent regions in Sham . Thus, slow and dyssynchronous Ca2+ transients observed in failing cardiomyocytes are attributed to both immature organization of t‐tubules and dyadic dysfunction.We further characterized the function of t‐tubules themselves by examining local CayRs Fig. A. In hents Fig. B. Thus,nts Fig. D, both red Fig. B and C.ase Fig. B and C,tes Fig. B and C.ham Fig. C. Thus,et al. A). Unlike previous reports, we did not observe any differences in the extent of action potential propagation in failing cardiomyocytes. As illustrated in Fig. B and C, a similar fraction of the t‐tubule network was depolarized in failing, immature and control t‐tubule networks. Rather, the non‐functionality of t‐tubules appears to be consistent with reduced t‐tubular Ca2+ current, a phenomenon recently reported by others in failing cells . These structures have been described by Gorelik et al.  to the membrane crest between adjacent Z‐grooves  and are also readily lost during HF Fig. B. Thesek et al. , using set al. et al. et al. et al. et al. The mechanisms behind Z‐groove formation and destruction remain elusive; however, others have argued that they form in part due to translocation of mitochondria during cardiac development, as illustrated in Fig. et al. et al. et al. et al. et al. et al. et al. et al. et al. et al. et al. et al. et al. T‐tubules are also extremely versatile structures capable of adapting to different developmental and pathological stimuli. Although species like cow . High colocalization of LTCCs with RyRs from early stages suggests that dyadic packing is a carefully controlled process in the developing heart, which allows t‐tubules to become functional shortly after they are grown .Beyond t‐tubule structure, our data provide additional insight into the composition of dyads during development, adulthood and disease. In agreement with previous work . While this differential dyadic arrangement does not appear to dramatically alter the ability of longitudinal dyads to trigger Ca2+ release . Furthermore, we did not observe marked changes in Jph2 or Bin1 expression in failing hearts, suggesting that loss of these proteins is not a critical initiator of dyadic remodelling. Nevertheless, we believe that with even greater severity of HF in some animal models and patients  under grant agreement No. 647714. Additional support was provided by The South‐Eastern Norway Regional Health Authority, Anders Jahre's Fund for the Promotion of Science, The Norwegian Institute of Public Health, Oslo University Hospital Ullevål and the University of Oslo.+ transientsVideo S1. 2D Confocal time‐lapse recordings of Ca2+ transients and t‐tubulesVideo S2. 2D Confocal time‐lapse recordings of Ca2Click here for additional data file.


When these cotyledons are placed in culture, their adaxial epidermal cells trans-differentiate to a TC phenotype regulated by auxin, ethylene, extracellular hydrogen peroxide (apoH2O2), and cytosolic Ca2+ ([Ca2+]cyt) arranged in series. Staining cultured cotyledons with the sterol-specific dye, Filipin III, detected a polarized sterol-enriched domain in the plasma membrane of their trans-differentiating epidermal transfer cells (ETCs). Ethylene activated sterol biosynthesis while extracellular apoH2O2 directed sterol-enriched vesicles to fuse with the outer periclinal region of the ETC plasma membrane. The sterol-enriched domain was essential for generating the [Ca2+]cyt signal and orchestrating construction of both the uniform wall layer and wall ingrowth papillae. A model is presented outlining how the sterol-enriched plasma membrane domain forms and functions to regulate wall labyrinth assembly.Transfer cells (TCs) facilitate high rates of nutrient transport into, and within, the plant body. Their transport function is conferred by polarized wall ingrowth papillae, deposited upon a specialized uniform wall layer, that form a scaffold supporting an amplified area of plasma membrane enriched in nutrient transporters. We explored the question of whether lipid-enriched domains of the TC plasma membrane could serve as organizational platforms for proteins regulating the construction of the intricate TC wall labyrinth using developing  Transfer cells (TCs) exhibit an enhanced capacity for nutrient transport conferred by an amplified plasma membrane (PM) surface area, enriched in membrane nutrient transporters, supported on an intricate wall labyrinth  trans-differentiation to a TC morphology and function comparable with their in planta abaxial epidermal TC (ETC) counterparts  production that switches on, and provides the positional cue for, the localized deposition of the UWL to the outer periclinal wall (apoH2O2 induce the formation of a polarized cytosolic Ca2+ ([Ca2+]cyt) signal  that localize en tubes .V. faba cotyledons contribute to assembling their reticulate wall labyrinths. To this end, we show that the PM lining the outer periclinal region of developing ETCs was selectively and rapidly enriched in sterols during the early phases of trans-differentiation to a TC morphology. Ethylene switched on sterol biosynthesis and apoH2O2 directed trafficking of sterol-enriched membrane vesicles to fuse with the PM lining the outer periclinal ETC region. In turn, the sterol-enriched domain (SED) within the PM directly regulated assembly of the UWL. Influence over formation of WI papillae was mediated through the SED functioning as an organizing platform for Ca2+-permeable channels to generate the polarized plumes of elevated [Ca2+]cyt that direct construction of WI papillae.Given these precedents, we explored the proposition that lipid-enriched domains in the PM of ETCs of Vicia faba L. cv. Fiord plants were raised under controlled environmental conditions according to Vicia faba cotyledons were aseptically cultured on a modified Murashige and Skoog (MS) liquid medium (http://rsbweb.nih.gov/ij/), and corrected for background fluorescence measured in unstained cells.Sterol-enriched membrane domains in ETCs were visualized by staining freshly prepared tissue sections of cotyledons with the UV fluorescent probe, Filipin III (hereafter referred to as Filipin), that binds to the 3'-β-OH group of sterols, following a protocol for live cell imaging described by To determine the subcellular localization of Filipin fluorescence, whole cotyledons were stained with 20 µM RH-414, a PM marker (2 nm−1=nm) using ImageJ software (http://rsbweb.nih.gov/ij/). A Zeiss VP field emission scanning electron microscope  was used to visualize WI papillae on the cytoplasmic faces of the outer periclinal walls of fractured ETCs, prepared as described by Ultrathin transverse sections of ETCs were visualized with a JEOL 1200 EX II transmission electron microscope  as previously described  of apoH2O2 generated in the outer periclinal wall of ETCs was measured using Amplex Red reagent  as described by Flux .The histochemical reagent, 3,3'-diaminobenzidine , was used to localize 2+]cyt were obtained by loading cultured cotyledons with a membrane-permeable Ca2+-sensitive dye, Oregon Green BAPTA-1 acetoxymethyl ester (OGB-1), while the cellular distribution of Ca2+-permeable channels relied on staining cultured cotyledons with DM-BODIPY(–)-dihydropyridine  Calcofluor White to outline the ETC walls. An Olympus FV1000 CLSM set with a 405 nm laser and a 440–490 nm emission filter set was used to visualize Calcofluor white fluorescence, while a 473 nm laser and a 510–550 nm emission filter set was used to visualize OGB-1 and fl-DHP fluorescence. Fluorescence densities (pixels per unit area) of OGB-1 in the outer periclinal cytosol of ETCs were measured using ImageJ and converted to estimates of cyt  operating in the pathway generating the largest group of plant sphingolipids, glycosyl inositol phosphoceramides  data set was interrogated for ETC-specific differentially expressed genes (DEGs) encoding enzymes known to participate in the biosynthetic pathways for phytosterols . EthylenVfEBF1 and VfEBF2, were found to be ethylene sensitive between 3 h and 12 h of cotyledon culture /Golgi to the outer periclinal portion of the ETC PM was directed by an 2 signal ; Fig. 3 2 signal , a featu2 signal . The cytapoH2O2 dependent  proteins, VfSYT1, VfSYT2, and VfSYT3  to provide a platform for docking and exocytosis of vesicles carrying cargoes of matrix polysaccharides (apoH2O2 signal continues to define the outer periclinal domain for SED incorporation during WI papillae assembly (apoH2O2 remains to be determined. The SED clusters define sites for insertion of the Ca2+-permeable channels (2+]cyt that remodel the actin network to deliver vesicles, carrying cargoes of cell wall materials, to the Ca2+-defined loci for WI papillae assembly (The SED-dependent assembly of the polarized ETC wall labyrinth , D joinscharides . In contcharides . While tassembly , whetherchannels . These cassembly  could imJXB online.Supplementary data are available at Fig. S1. Filipin staining co-localizes with the PM of ETCs.Fig. S2. Temporal pattern of SED formation.apoH2O2 distribution.Fig. S3. Effect of the SED on 2+ signalling and Ca2+ channel distributionFig. S4. Effect of the SED on CaTable S1. Lengths of specified PM domains in ETCs and SPCs.Table S2. Effect of blocking vesicle trafficking on SED distribution.Table S3. Effect of sphingolipid, sterol, and brassinosteroid inhibitors on SED formation.Table S4. Levels of biological control exercised by ethylene on SED formation.Table S5. Impact of AVG on transcript abundance of sphingolipid- and phytosterol-related genes.apoH2O2 signalling.Table S6. Effect of vesicle trafficking on the regulation of sterol distribution by Table S7. Impact of ascorbic ascid on transcript abundance of vesicle trafficking genes.Supplementary DataClick here for additional data file.