Overexpressed Mdm2 and its 7homolog MdmX impair p53 activity in many cancers. Small molecules mimicking a p53 peptide can effectively inhibit Mdm2 but not MdmX. Here, we show a strategy for improving lead compounds for Mdm2 and MdmX inhibition based on the multivalency of the p53 peptide. Crystal structures of MdmX complexed with nutlin-3a, a strong Mdm2 inhibitor but a weak one for MdmX, reveal that nutlin-3a fits into the ligand binding pocket of MdmX mimicking the p53 peptide. However, due to distinct flexibility around the MdmX ligand binding pocket, the structures are missing many important intermolecular interactions that exist in the MdmX/p53 peptide and Mdm2/nultin-3a complexes. By targeting these flexible regions, we identify allosteric and additive fragments that enhance the binding affinity of nutlin-3a for MdmX, leading to potent Mdm2/MdmX inhibitors with anticancer activity. Our work provides a practical approach to drug design for signal transduction therapy. Peptide fragments derived from the interfaces of protein-protein interactions (PPIs) provide useful templates for designing small molecule PPI inhibitors. Here, the authors utilize the multivalency of an MdmX-binding p53 peptide to develop a weak inhibitor of MdmX into potent Mdm2/MdmX inhibitors. Many PPI modulators including easily accessible peptides and small molecules developed with more effort, have entered clinical studies, and some have been approved for marketing4, but the transformation of peptide modulators to small molecules is not yet sufficiently well-understood8.Protein–protein interactions (PPIs) are fundamental mechanisms of cellular signaling pathways but present a challenge to drug design11. Mdm2/MdmX are overexpressed in many cancers to impair p53 activity via binding of their N-terminal domains (N-Mdm2 and N-MdmX) to the p53 transactivation domain (p53p)12. The discovery of effective small-molecule inhibitors of Mdm2 targeting N-Mdm2 is one of the most successful cases of the transformation of peptide modulators into small molecules14. N-Mdm2 and N-MdmX are homologous and exhibit submicromolar affinity for p53p16, but these two structurally homologous proteins exhibit different selectivity for Mdm2 small-molecule inhibitors19. How the conformation of N-MdmX differentially recognized Mdm2 small-molecule inhibitors remains elusive. For example, nutlin-3a is a first-in-class Mdm2 inhibitor with nanomolar binding affinity but a weak MdmX inhibitor20, providing an excellent model system for exploring mechanistic questions regarding how homologous proteins differentially recognize ligands and how the flexibility of ligand-binding pockets determine their interaction specificity20.The p53-Mdm2/MdmX signaling axis plays an important role in maintaining genome integrity to prevent cancersIn this study, we determined crystal structures of MdmX in complex with nutlin-3a. The configuration of nutlin-3a molecule docked in the ligand-binding pocket of MdmX and its intramolecular and intermolecular interactions into MdmX were in detail examined in comparison with those of the MdmX/p53 peptide and Mdm2/nultin-3a complexes. We also determined a crystal structure of MdmX in complex with p53 analog to analyze the p53p multivalency. Furthermore, we identified allosteric and additive fragments that enhanced the binding affinity of nutlin-3a for MdmX, leading to potent Mdm2/MdmX inhibitors with anticancer activity. Thus, our work provides a practical strategy for drug design targeting aberrant protein–protein interactions, via transforming peptide templates and lead compounds into potent small-molecule inhibitors.21 reveal that the ligand-binding pockets on N-MdmX and N-Mdm2 are surrounded by four intrinsic flexible regions  and two helixes24  and timescale of motion (τe) for each observable backbone amide N-H bond vector. S2 can range from 0 (completely unrestricted) to 1 (completely rigid), indicating the degree of motional restriction. The S2 values for residues in N-MdmX in complex with nutlin-3a ranged from 0.20 to 0.82 with an average of 0.60 , and R-4 region (residues 86–97). Notably, many 15N-1H amide signals in the R-3 and R-4 regions were still invisible , F90, and L98 (I98)  Fig. . We obseY99 Fig. , formingY99 Fig. , which wps~ns timescale , as previously reported by Chen et al.27, so that its F19p53p subsite remained empty, as shown in Fig. p53p from p53ΔF19 fragment is only one tryptophan residue, functioning as not only a key residue for binding of p53p fragment to N-MdmX but also an intrinsic fluorescence signal to probe the association/dissociation of p53p fragment with N-MdmX. As depicted in Supplementary Fig. p53p subsite, the W23p53p fluorescence signal of p53ΔF19 fragment will be enhanced at 324 nm. If a fragment displaces the p53ΔF19 fragment, the W23p53p fluorescence signal of will be attenuated and its maximum emission wavelength will be shifted to 350 nm. Therefore, in this fusion protein model, the intrinsic fluorescence signal of the W23p53p side chain was sensitive to protein conformational change caused by ligand binding. By screening a previously constructed small compound library of Mdm2 inhibitors27, we identified four compounds 28. In this study, for a proof of concept, the overexpressed Mdm2 and MdmX in H1299 cells were achieved by the transient transfection of the RFP-tagged MdmX and Mdm2 expression plasmids (see “Methods“). The overexpressed Mdm2 and/or MdmX in the H1299p53+ were strong inhibitors to cellular p53 protein. The cells were exposed to H203, nutlin-3a (the positive control) or nutlin-3b (the negative control) for 48 h, and cell growth/viability was measured. The results showed that H203 significantly reduced cell viability in the cells overexpressing both Mdm2 and MdmX . Lipofectamine 2000 was purchased from Invitrogen . The peptides including p53p, fluorescein-labeled p53p (Flu-p53p), and a mutant p53 peptide (p53pAAA) where three key residues were substituted by alanine were synthesized by TOPE Biotech . Peptide analogs were prepared by Huazheng Biotech . 1-chloro-4-ethynylbenzene (CAS#: 873-73-4), 1,3-dichloro-5-iodobenzene (CAS#3032-81-3), 2-amino-4-methylbenzoic acid (CAS#: 2305-36-4), 1,2-Bis (4-chlorophenyl) ethane-1,2-diamine (CAS#: 86212-34-2), 2,5-dioxo-2,3,4,5-tetrahydro-1H-benzodiazepine-8-carboxylic acid (CAS#: 195985-12-7), 1-(tert-Butoxycarbonyl)-5-oxopiperazine-2-carboxylic acid (CAS#: 1246553-28-5) were purchase from Bidepharm . 1-(4-chlorophenyl)-2- ethane-1,2-diamine and 2,5-dioxo-1-phenyl-2,3,4,5-tetrahydro-1H-benzo-diazepine-8-carboxylic acid were synthesized by Huazheng Biotech .HCT116, RKO, H164a, and H1299 cells were purchased from ATCC and were cultured in a 37 °C incubator with 5% CO24. The p53pΔF19 gene was synthesized by GenScript  and sub-cloned to the pET28-MdmX plasmid.A modified pET28b plasmid containing the p53-binding domain of human MdmX  and human Mdm2  were constructed previously in our laboratoryE. coli BL21 (DE3) cells. Cells were grown in LB medium containing kanamycin (34 μg/mL) and induced with 0.4 mM IPTG at 18 °C for 12 h. For heteronuclear NMR experiments, protein samples were uniformly labeled with 15N and 13C in MOPS medium with BME vitamins containing 1 g/L (15NH4)2SO4 and 2 g/L [13C6] glucose as the sole sources of nitrogen and carbon, respectively. Cells were harvested by centrifugation at 5000 × g for 30 min, resuspended in a buffer containing 10 mM Tris-HCl, 40 mM NaCl, 2 mM β-mercaptoethanol, 2 mM imidazole, pH 8.0 (Buffer A), and lysed by sonication and homogenization, followed by spinning at 18,000 × g for 30 min. The supernatant was loaded onto a 5 mL Ni-NTA agarose column  and His-tagged protein was competitively eluted using a gradient of Buffer A mixed with Buffer B containing 10 mM Tris-HCl, 40 mM NaCl, 2 mM β-mercaptoethanol, 300 mM imidazole, pH 8.0. The eluate was diluted in 20 times with a buffer containing 20 mM sodium citrate (pH 6.5), 10% glycerol, 2 mM β-mercaptoethanol. The supernatant was loaded onto a MonoS10/100 cation exchange column  which was pre-equilibrated with 10 column volumes (CV) of Buffer MA . The protein was eluted with a linear gradient of NaCl from Buffer MA to Buffer MB . Peak fractions were separately collected, and mixed with 2 mM DTT and 2% 18-crown-ether.Recombinant N-MdmX and N-Mdm2 proteins were prepared in ΔF19-MdmX fusion protein essentially followed the same procedure as above, except that the pH value of samples was adjusted to 7.16 by adding a buffer containing 50 mM Tris-HCl (pH 8.0), 200 mM NaCl, 10 mM imidazole, 2 mM β-mercaptoethanol.The purification of the p53pΔF19-MdmX protein were respectively concentrated to 0.4–0.8 mg/mL and 10–25 mg/mL, and freshly frozen in liquid nitrogen, and kept at −80 °C.The purified N-MdmX, N-Mdm2, and the p53pTM, PEGRx, and Crystal ScreenTM  and the AmSO4 Suite, the JCSG+ Suite, and the PEGs Suite  using GRYPHON station . Diamond crystals appeared within two days in the condition.The conditions of protein crystallization were screened against different kits including IndexProtein crystallization was further optimized using a sitting drop method. Briefly, 0.6 μL protein drop was composed of 0.3 μL protein sample (10 mg/mL), 0.3 μL reservoir solution. The drop was incubated at 18 °C. Further, large amounts of protein crystal were reproduced by seeding procedure. New drops of mother liquid were inoculated with crushed needle crystal suspended in a slurry mother solution. Diamond crystals grew after 2 days. The crystals were transferred to a cryoprotection solution containing 50% (v/v) reservoir solution and 25% (v/v) glycerin and quickly cooled in liquid nitrogen.25. The datasets were integrated, scaled, reduced, and phased using AutoPROC, XDS, DIALS, Porpoise, and Xia2 programs. Model was built with Basic Molecular Replacement - PHASER in Phenix package using Mdm2 structure from 4J3E as a template for N-Mdm2, and MdmX structures from 6Q9W and 6V4F as a template for N-MdmX. Refinement was done with REFMAC5 in the CCP4i2 package46.The X-ray diffraction data were collected by HKL 2000 on BL17U1 at Shanghai Synchrotron Radiation Facility (SSRF) using the EigerX16M detector15N−1H HSQC NMR spectra were recorded in the States-TPPI mode for quadrature detection. All the NMR samples were prepared in a buffer containing 20 mM sodium phosphate, 200 mM NaCl, 2 mM DTT, and 95% H2O/5% D2O at pH 6.8. The final datasets were acquired with 2048 complex points in t2 and 128 complex points in t1. All datasets were processed using Topspin or NMRPipe. Spectral display, assignments, and analysis were performed using the NMRViewJ software package. 15N T1 and T2 relaxation times and the heteronuclear NOE values were measured using 15N-labeled protein complexed with nutlin-3a pr p53p using the pulse sequences described by Farrow et al.47. Relaxation delays of 2.8 s were used for the T1 and T2 datasets, and a 5 s recycle delay was used for the heteronuclear NOE experiment. Longitudinal relaxation delays of 10, 70 (2*), 150, 250, 320, 520 (2*), 760, 1100 (2*), 1500, 2000, and 3600 ms (where 2* represents a duplicate measurement) and T2 delays of 14.4 (2*), 28.8, 43.2, 57.6, 72 (2*), 86.4, 100.8, 115.2 (2*), 144, and 172.8 ms were sampled for all complexes. Steady-state heteronuclear NOE data were obtained in an interleaved manner with and without proton pre-saturation (3 s). To minimize heating between the T2 time points, the total number of CPMG pulses was kept constant for each time point by introducing the appropriate number of dummy CPMG pulses49. Errors in the heteronuclear NOE values were estimated from the root-mean-square variation of noise in empty regions of the two spectra as previously described50.Protein samples were prepared through buffer exchange with the NMR buffer. The concentration of the protein complex was 0.3–0.4 mM. All NMR spectra were collected at 25 °C on a Bruker Avance 600 MHz or 800 MHz spectrometer equipped with a triple-resonance pulsed-field gradient probe. R1 and R2 measurements were estimated from duplicate datasets. Values for R1 and R2 and the uncertainties were determined by nonlinear least-squares fitting of experimental data to mono-exponential functions using GUARDD with MATLAB 2018a. Errors for the heteronuclear NOE values were estimated from the root-mean-square variation of noise in empty regions of the two spectra as described previously51.Uncertainties in peak heights for 15N-1H HSQC titrations were performed by stepwise addition of compounds (at a high concentration) into the 15N-labeled component  to a final 1-5-fold excess. Minimal changes in volume and pH were ensured throughout the NMR sample preparations.AAA) was used as the negative control. All FP data were fitted with Origin 2017 for obtaining Kd values.All fluorescence polarization assays were done using black, low-protein-binding 96-well plates  in a total volume of 100 μL per well of 20 mM phosphate (pH 6.8), 200 mM NaCl, and 1 mM DTT. After a preincubating of 75 nM fluorescein-p53p  with 1.5 μM N-MdmX for 30 min, a compound was then added and the mixture incubated for an additional 30 min. FP readings were taken with a 555 nm excitation filter and a 632 nm static and polarized filter on a BioTek H1 multiplate reader with Gen5 software. The unlabeled p53p peptide and nutlin-3a were used as positive controls. A mutant p53 peptide (p53pH) plotted as a function of the molar ratio of the ligand. When necessary, prior to the integration procedures, the baseline was manually adjusted to minimize the background noise. The Origin 7.0-based software was used for data analysis with the one-set-of-binding-sites model. The disassociation constant of Kd was determined from the slope of the central linear part of the fractional saturation curve. The Gibbs free energy change (ΔG) and the entropy change (ΔS) were calculated on the basis of the following equations: ΔG =−RT ln Kd = ΔH − TΔS, where ΔH was derived from the original ΔH/molar ratio plots.The protein−ligand interactions were characterized using a model ITC-200 isothermal titration microcalorimeter  at 25 °C. A typical experiment included the injection of 19 aliquots (2.0 μL each) containing an ~0.2 mM ligand solution into an ~10–20 μM protein solution in the ITC cell (volume of ∼200 μL). Titrations were performed with a stirring speed of 750 rpm and a spacing time of 120 s. A control experiment was run by injecting a ligand solution into buffer instead of a protein solution in the cell. Before data analysis, the control values were subtracted from the experimental data. The binding isotherms were integrated to give the enthalpy change  simulations were performed to investigate the conformational preference of H72. The initial structures of N-MdmX and N-Mdm2 were isolated from the N-MdmX/p53p (3DAB) and N-Mdm2/p53p complex (1YCR), respectively.53 and TIP3P54 water model using GROMACS56. One N-MdmX or N-Mdm2 molecule was placed in cubic box with periodic boundary conditions. Counter ions were added to neutralize the net charges. All bonds were constrained using the LINCS algorism57. A time step of 2 fs was used. Long-range electrostatic interactions were calculated by particle-mesh Ewald method58 with a fourth-order interpolation and a grid spacing of 0.16 nm. The cutoff distances were set to 10 Å for short-range electrostatic and van der Waals interactions. The solute and solvent were coupled separately to a temperature bath of 298 K using a velocity-rescaling thermostat with a relaxation time of 0.1 ps59. The pressure was maintained at 1 bar using the Parrinello–Rahman algorithm60 with a relaxation time of 2 ps and isothermal compressibility of 4.5 ×  10−5 bar−1. The systems were relaxed by 1000 steps of the steepest-descent energy minimization followed by 1 ns equilibration at NVT and NPT ensembles. Production simulations with 1 μs duration were carried out in the NVT ensemble. Coordinates were saved every 5 ps and the side chain dihedral angles were calculated.We performed standard MD simulations with the AMBER99SB-ILDN force field61. The residues H72 and K93 around the ligand pocket on N-MdmX and N-Mdm2 were set up as flexible residues. The receptor grid box was set directly on the MdmX crystal structure obtained in this study after extraction of the included nutlin-3a molecule.Potent inhibitors were generated with ChemBioDraw14 and further energetically minimized with ChemBio3D . The minimized structures were exported in pdb format and manually evaluated using the ADT tool program harboring the AutoDock 4 program® Mini kit from Qiagen . The concentration of RNAs was measured with Nanodrop-2000c. Real-time qPCR was performed with a BaldStar TaqMan One-Step RT-qPCR Kit from Biorab  on a BioRad CFX96 qPCR instrument . The primers and probes for qPCR were designed using Primer Express 3.0 software . The sequences of primers for p21 were RT-p21-L1 (5′-CTTTGTCACCGAGACACCAC-3′) and p21-R1 (5′-CAGGTCCACATGGTCTTCCT-3′). The sequence of the probe for p21 was p21-P1 (5′-ACTCATCCCGGCCTCGCCGG-3′). The sequences of primers for p53 were RT-p53-L1 (5′-GTCCAGATGAAGCTCCCAGA-3′) and RT-p53-R1 (5′-CAAGAAGCCCAGACGGAAAC-3′). The sequence of the probe for p53 was p53-P1 (5′-AGCTCCTACACCGGCGGCCC-3′). TaqMan probes were labeled with 5′-FAM and 3′-TAMRA. All primers and probes were synthesized by GenScript . Samples were analyzed in triplicate and normalized to the control.Cancer cells with wild-type p53  were treated with H203 for 8 h, and the change in the level of transcription was measured by quantitative PCR and expressed as fold induction compared with the untreated control. Total RNAs were extracted using a Trizol protocol with RNeasy2 in Dulbecco’s modified eagle medium supplemented with 10% serum, penicillin and streptomycin for 3 days, refreshed the medium with RPMI 1640 supplemented with 3% FBS. Cells were plated at a density of 1 × 104/cm2. After 2 h, added H203 and nutlin-3a for 24 h. After adding 10 µl/well MTT (5 mg/ml) solution, the cells were incubated for another 4 h at 37 °C in a CO2 incubator. After the supernatant was discarded, the cells were washed with PBS, and 100 µl DMSO was added to each well. The plates were agitated on a plate shaker for 10 min and then were read the OD at 520 nm. Data were processed with MicroCal Origin software .MTT assay was performed on a Synergy H1 multiplate reader  using H1299 cells containing engineered-inducible wild-type p53 gene, encoding p53 protein tagged with GFP protein. Overexpression of Mdm2 and MdmX was achieved by transfecting pCMV plasmids harboring a gene encoding the full-length Mdm2 and MdmX tagged with RFP protein. Cells were grown at 37 °C with 5% CO280nm and the cellular β-actin contents were then calibrated with mouse anti-β-actin monoclonal antibody from Proteintech  with a dilution ratio of 1:10000. Calibrated samples were separated by SDS-PAGE, while a normal molecular marker was used. Each SDS-PAGE gel was electrically transferred to a polyvinylidene difluoride (PVDF) membrane . In order to minimize the usage of antibodies, each PVDF membrane was cut into different sections, based on the molecular weights of cellular p53, Mdm2, MdmX, p21, PUMA, and β-actin and the features of their corresponding antibodies provided by their manufacturers. Each section of PVDF membranes was blocked with a 5% skimmed milk powder dissolved in TBST buffer for 1 h at room temperature, followed by incubation individually with their corresponding primary antibodies for 2 h in a sealed plastic bag. After being washed three times with TBST, the membranes were incubated with HRP-conjugated secondary antibody for 1 hr in a sealed plastic bag. Multiple sections of PVDF membranes were collected and the protein bands were visualized using enhanced chemiluminescence (ECL) reagents from BioSharp  on a Tanon 5200 Chemiliminescent Imager . Each blotting was repeated until a good quality image was achieved. Cellular p53 protein was blotted with a rabbit anti-TP53 polyclonal antibody IgG from CUSABIO  with a dilution ratio of 1:4000; Cellular Mdm2 and MdmX proteins tagged with RFP were assayed with a mouse anti-RFP monoclonal antibody from Solarbio  with a dilution ratio of 1:10,000. Cellular p21 protein was detected with a rabbit anti-p21 polyclonal antibody from Elabscience  with a dilution ratio of 1:500. Cellular PUMA protein was detected with a rabbit anti-human PUMA monoclonal antibody from Beyotime  with a dilution ratio of 1:1000; Secondary antibodies were detected with either HRP-conjugated goat anti-rabbit IgG(H + L) which was from Proteintech  with a dilution ratio of 1:10000 or HRP-conjugated goat anti-mouse IgG(H + L) which was form Biosharp  with a dilution ratio of 1:10,000, respectively.Total cellular proteins were extracted with a RPMI lysis buffer containing 50 mM Tris (pH 7.4), 150 mM NaCl, 1% Triton-X100, 0.1% sodium deoxycholate, 0.1% SDS and 0.1% PMSF. The concentration of the protein extracts was firstly measured with Nanodrop-2000c at ODFurther information on research design is available in the Supplementary informationReporting Summary


TP53 mutations), overlooking to some extent, acquired mechanisms of resistance through extrinsic processes. In the bone marrow microenvironment (BMME), leukaemic cells interact with the surrounding bone resident cells, driving acquired therapy resistance in AML. Growing evidence suggests that macrophages, highly plastic immune cells present in the BMME, play a role in the pathophysiology of AML. Leukaemia-supporting macrophage subsets (CD163+CD206+) are elevated in preclinical in vivo models of AML and AML patients. However, the relationship between macrophages and therapy resistance in AML warrants further investigation. In this review, we correlate the potential links between macrophages, the development of therapy resistance, and patient outcomes in AML. We specifically focus on macrophage reprogramming by AML cells, macrophage-driven activation of anti-cell death pathways in AML cells, and the association between macrophage phenotypes and clinical outcomes in AML, including their potential prognostic value. Lastly, we discuss therapeutic targeting of macrophages, as a strategy to circumvent therapy resistance in AML, and discuss how emerging genomic and proteomic-based approaches can be utilised to address existing challenges in this research field.Acute Myeloid Leukaemia (AML) is a commonly occurring severe haematological malignancy, with most patients exhibiting sub-optimal clinical outcomes. Therapy resistance significantly contributes towards failure of traditional and targeted treatments, disease relapse and mortality in AML patients. The mechanisms driving therapy resistance in AML are not fully understood, and approaches to overcome therapy resistance are important for curative therapies. To date, most studies have focused on therapy resistant mechanisms inherent to leukaemic cells (e.g.,  NPM1), fms like tyrosine kinase 3 (FLT3), and CCAAT/enhancer-binding protein alpha (CEBPA), are currently being used in routine practice, and have significantly improved individual prognosis and clinical management through more targeted therapeutic approaches  and treatment sensitivity in AML  inhibitor, selectively blocking the function of CDK2 and CDK9 (Acute Myeloid Leukaemia (AML) is a genetically diverse haematological malignancy common in older adults, with 5-year survival rates less than 20% for the majority of AML patients, aged between 60 and 74 . Importaproaches . Despiteproaches . To a cey in AML . The driy in AML . Two of and CDK9 . Inhibitand CDK9 . Importaand CDK9 , with eland CDK9 , and alsand CDK9 .in vitro, via the rapid loss of MCL-1 protein expression, and reduces leukaemia burden in xenograft models of MLL (EOL-1) and non-MLL (HL-60) AML, exhibiting superiority over the conventional AML therapeutic cytarabine/ara-C, in this setting .The environment that arises is suboptimal to support the haematopoietic functions of HSPCs. Overall, LSPCs co-opt the HSPC niches to ensure their own survival and proliferation . Leukaemvia the phosphoinositide-3-kinase (PI3K)/Akt pathway. In line with these findings, via their release of growth factors, pro-inflammatory cytokines and chemokines. These factors promote growth/expansion , recruitment , differentiation  and polarisation  of monocytes and Mφs, towards an M2-like phenotype  compared to BM-MSCs isolated from healthy counterparts . CXCL8 chenotype . Evidenc206+ Mφs  occurrin206+ Mφs . It is w206+ Mφs . Howevervia the actions of interferon gamma (IFN-γ), granulocyte-macrophage colony-stimulating factor (GM-CSF) and lipopolysaccharide (LPS), which signal via signal transducer and activator of transcription (STAT) 1 and 2 (STAT1/STAT2)  and chitinase-3-like protein 1 (CHI3L1/YKL-40), and the anti-inflammatory cytokines IL-10 and TGF-β displaying leukaemia supporting and immunosuppressive properties  . Further1/STAT2) . At the operties , as welloperties . To add operties . These Moperties .via cell-to-cell contact, AML blasts have the capacity to re-educate monocytes and Mφs towards an M2-like leukaemia-supporting phenotype. This occurs through secreted factors and signal transduction modulation of transcription factors. Using in vitro and in vivo models, + BM monocytes. Importantly, via immunohistochemical analysis of BM samples from AML patients, they also showed high expression of CD206+ cells and arginase II. Providing further evidence that AML blasts have the ability to re-educate Mφs to a leukaemia supporting phenotype, +Ly6G–MHCII–Ly6C–  expression, essential for myeloid differentiation, and shown to be involved in the ability of AML cells to preferentially polarise Mφs to an M2-like phenotype, at the expense of M1-like Mφs. The authors showed that BMDMs derived from Gfi1-KO mice were enriched for M1-like vs. M2-like Mφs, when exposed to M1 and M2-polarising regimes, as determined by an increase in Ly6C–CD206– cells , isolated from the BM of secondary recipient mice, originally from MSCV-MLL-AF9-IRES-GFP AML mice, are enriched for M1 gene signatures, including iNOS, TNF, IL-6, and IL-1 (–F4/80+CD169+VCAM1+ and –GR1lowMCSFRintF4/80hiSSClow Mφs (–F4/80+CD169+VCAM1+) persists within these AML mice. Discrepancies in the Mφ subsets identified could be due to key factors, such as disease severity/level of disease and time to analysis. Mechanistic insights into the leukaemia-driven AAM phenotype in the leukaemic mice, was provided through transcription factor growth factor independence 1 , and the general myeloid regulator microRNA miR-223 were inferred to be involved  displayed higher MOZ and lower pri-miR-223 expression following exposure to LPS with the opposite observed after IL-4 treatment. Importantly, low levels of MOZ were demonstrated to be present in the M5 monocytic AML FAB subtype, correlating with inferior overall survival (OS) in retrospective data collected from the cancer genome atlas (TCGA) cohort  cohort . Findingvia direct Mφ-to-AML cell contact and/or Mφ secreted factors, thereby reducing drug sensitivity in AML cells. This is driven by the ability of Mφs to upregulate pro-survival/anti-apoptotic pathways in AML cells  pathway and upregulate MCL-1 protein expression  f206+ Mφs . Chemorepression . It is tin situ from circulating CCR2+CD14++CD16– classical monocytes  region of MCL-1  in the BM of AML patients compared to normal BM counterparts. Furthermore, M2-like Mφs represented a major component of the BMME, compared to other haematological malignancies, including chronic myeloid leukaemia. M2-like Mφ enrichment in AML patients, was confirmed through the algorithm, xCell, via data from two additional independent AML patient cohorts. Cognizant that in solid cancers the frequencies/levels of M2-like Mφs can predict clinical outcomes, the authors then conducted CIBERSORT analysis of two independent AML patient cohorts with available survival data. In one dataset , increased levels of M2-like Mφs correlated with inferior OS and event-free survival in AML patients, which they validated by xCell analysis.Findings from recent studies show that the number of M2 and M1-like Mφs in the BM correlates with important patient outcomes, providing prognostic value in AML patients. Utilising the algorithmic program CIBERSORT, via an Affymetrix GeneChipTM Human Genome U133 Plus 2.0 Array), arose as the sole and significant prognosticator for prolonged survival in AML patients. These findings indicate the ratio of M2-like to M1-like Mφs (M2/M1) could be of clinical importance to AML patients, as This is in line with NPM1 and IDH1. A potential caveat to their study is that significant correlations were shown between inactive/resting dendritic cells (DCs) and CD206 expression. Therefore, the authors could not completely rule out the possible contribution of CD206+ DCs in their study. Complementing this work, is the study by + DCs, associated with recruitment of regulatory T-cell populations, and T-cell suppressive CX3CR1+ DCs. This study highlights the power of single-cell sequencing in resolving and better understanding the complex immunological composition of the BMME of individual AML patients, and importantly how this information could potentially be utilised, to select patient-specific treatments targeting resident immune cell populations. Furthermore, this study highlights the importance of distinguishing CD206+ macrophages from CD206+ DCs, as these different immune cell subtypes may have different impacts on clinical outcomes in AML patients. Another consideration not fully recognised by + and CD206+ macrophage populations were found to be infrequently enriched in AML BM samples, analysis at the single cell level will aid in identifying rare cell types that, when found alone or in association with other immune cell types, may exacerbate disease pathogenesis and therefore negatively impact patient prognosis , via CCL2-CCR2 blockade; specific deletion/depletion via anti-CD115/M-CSFR antibodies; and phenotypic/functional reprogramming to M1-like anti-leukaemia Mφs, through DICER inhibition-mediated upregulation of IFN-γ/STAT1-inducible genes and modulation of microRNAs. An overview of TAM targeting strategies are eloquently discussed in recent review articles (Numerous therapeutic strategies have been devised to target M2-like Mφs/TAMs. These include: inhibiting the recruitment of CCR2articles . Deciphearticles . PI3K isFLT3-ITD)  from amassing in the tumours  Mφ infiltration, while suppressing M2-like (CD206+) Mφ infiltration into tumours. Reduced TAM infiltration likely resulted from the inhibition of PI3Kγ-dependant migration signals, that have been documented to occur through G-protein coupled receptor induction in Mφs and their precursors  , with coLT3-ITD) , 2020. T0, TGF-β . However tumours . In agreted mice . In contecursors . The repStudies to date have reported that AML blasts always express p110δ, whereas the incidence of p110γ expression and other isoforms, are highly variable, owing to the heterogenic molecular landscape observed in AML .via the selective inhibitor IPI-145/duvelisib, has been demonstrated to decrease survival and induce cellular apoptosis in AML cell lines and primary AML blasts (n = 6) was included in this initial trial, and AML patients were treated only with the 75 mg dose, and not at the higher 100 mg dose. Moreover, the cytogenetic and mutational status of the patients are not provided, and it is uncertain whether treatment naïve (TN) or relapsed/refractory (R/R) AML patients were included in this study. This has now been extended to a Phase II trial , is necessary to fully ascertain the therapeutic potential of duvelisib and other PI3K isoform-selective inhibitors of AML.Dual targeting of PI3Kγ and PI3Kδ L blasts . This obL blasts . It is ivia phagocytosis. These observations, along with previous reports that CD47 binding to phagocyte/macrophage expressed signal regulatory protein alpha (SIRPα) negatively regulates phagocytosis . However, these myeloid markers are now known to be expressed and shared by both non-malignant monocytes and Mφs and AML-Mφs. Encouragingly, the recent applications of single-cell RNA-seq and genetic profiling, have been able to detect differences in transcript expression and mutations within malignant AML cells, which are now allowing researchers to specifically distinguish and characterise non-malignant and malignant-Mφs within the AML BMME. Nevertheless, in comparison to the progress made in the understanding of M2-like Mφs/TAMs in solid malignancies, our basic understanding of Mφ biology in AML is still in its infancy. Many fundamental questions surrounding the frequency and composition of Mφ phenotypes within the BMME and their contributions to disease processes, such as therapy resistance and disease relapse/progression, have still to be addressed. With the advent of new enabling technologies, including multiplex imagining modalities  platform) (+ protein or FLT3), will enable researchers to distinguish and study the interaction of bona fide non-malignant M2-like NPM1c–/FLT3–CD163+CD206+ Mφs with NPM1c+/FLT3+CD163+/–CD206+/– AML blasts in the BMME of NPM1-mutant and FLT3 AML patients, with the potential to apply this technology to other molecular subtypes of AML. Furthermore, we are currently in the profiling era of advanced technology, in which we can undertake profiling of the secretome using Luminex, SWATH proteomics, as well as scRNA sequencing and metabolomics, enabling us to understand AML pathogenesis at the single cell level.Studies investigating the role of Mφs in AML have been hindered to some extent, due to challenges surrounding the ability to accurately distinguish non-malignant latform) , researcWith the advent of a recently developed macrophage annotation platform, MacSpectrum, which draws upon single-cell RNA-seq data, and has demonstrated the ability to resolve distinct and heterogeneous BM-derived and adipose tissue-derived macrophage populations , it is cvia the upregulation of pro-survival/anti-apoptotic pathways, such as ERK1/2 and MCL-1 in AML cells, treatment combinations comprising selumetinib and/or AZD5991 or CYC065, could represent novel therapeutic strategies to overcome Mφ, and more specifically, MCL-1-driven therapy resistance in AML.It is crucial that LAMs and tissue-resident Mφs in the BMME of AML patients, are fully characterised at the phenotypic and functional level, in order to determine molecular (transcript/protein) differences, that could be exploited therapeutically, for the specific targeting of LAMs. This will be paramount in the ability to generate future Mφ targeting strategies, exhibiting desirable pharmacological properties, such as enhanced efficacy/on-target effects, and reduced toxicity owing to off-target effects. Given that Mφ-mediated therapy resistance likely occurs Finally, with the advent of new immune-oncology based companies established to develop first-in-class compounds to target Mφ-mediated immunomodulation, the future holds much promise for the generation of novel therapeutic strategies, capable of safely and effectively modulating/targeting Mφ-driven disease processes in AML and other malignancies.KM and MW wrote the manuscript. KM prepared the figures. KM, MW, MG, and HW contributed to the discussion of the draft. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Phospholamban p.Arg14del mutation is found in patients with dilated or arrhythmogenic cardiomyopathy. This mutation triggers cardiac contractile dysfunction and arrhythmogenesis by affecting intracellular Ca2+ dynamics. Little is known about the physiological processes preceding induced cardiomyopathy, which is characterized by sub-epicardial accumulation of fibrofatty tissue, and a specific drug treatment is currently lacking. Here, we address these issues using a knock-in Phospholamban p.Arg14del zebrafish model. Hearts from adult zebrafish with this mutation display age-related remodeling with sub-epicardial inflammation and fibrosis. Echocardiography reveals contractile variations before overt structural changes occur, which correlates at the cellular level with action potential duration alternans. These functional alterations are preceded by diminished Ca2+ transient amplitudes in embryonic hearts as well as an increase in diastolic Ca2+ level, slower Ca2+ transient decay and longer Ca2+ transients in cells of adult hearts. We find that istaroxime treatment ameliorates the in vivo Ca2+ dysregulation, rescues the cellular action potential duration alternans, while it improves cardiac relaxation. Thus, we present insight into the pathophysiology of Phospholamban p.Arg14del cardiomyopathy.The heterozygous  Arrhythmogenic cardiomyopathy is a genetically inherited disease caused by deletion of an arginine in Phospholamban (PLN), a small protein that regulates intracellular calcium levels. Here the authors show that zebrafish with the PLN p.Arg14del mutation develop severe cardiomyopathy which is preceded by contractile dysfunction and altered calcium dynamics and istaroxime is identified as a small molecule that can rescues some of these phenotypes. Sudden cardiac death most commonly occurs in young patients, during the concealed stage of the disease, when no overt cardiomyopathy is detectable yet. Little is known about the underlying cause of cardiac remodeling, eventually culminating in contractile dysfunction, heart failure, and sudden cardiac death in ACM patients2. Myocardial biopsies of late-stage ACM patients also display inflammatory infiltrates, which are associated with the fibrofatty replacement of myocardial tissue2. Most cases of familial ACM are caused by mutations in desmosomal genes . However, non-desmosomal gene mutations such as the Phospholamban (PLN) p.Arg-14 deletion (R14del) have recently been identified as a cause for ACM as well3.Arrhythmogenic cardiomyopathy (ACM) is a genetically inherited disease that occurs in 1:1000–1:5000 of the general population. ACM is characterized by the accumulation of fibrous tissue and fat at the sub-epicardial region of the ventricles, where it infiltrates the myocardial wall. In addition, malignant ventricular arrhythmias and a high propensity for sudden cardiac death have been observedPLN R14del is a Dutch founder mutation and the most prevalent cardiomyopathy-related mutation in the Netherlands. It has been identified in 10–15% of all Dutch patients with dilated cardiomyopathy and/or ACM and it is estimated that there are more than 1000 PLN-R14del Dutch carriers4. PLN mutations, including PLN R14del, have also been detected in several other countries, including Canada, the USA, Spain, Germany, and Greece8. Patient phenotypes are very heterogeneous not only between families but also within families10. Up to today, no homozygous carriers have been identified11. Severely affected PLN R14del mutation carriers display profound fibrofatty infiltration in the sub-epicardial region of both ventricular walls, along with malignant ventricular arrhythmias and end-stage heart failure12. Currently, specific drugs to treat this disease are lacking.13. During excitation–contraction coupling, free cytosolic Ca2+ levels increase, causing more Ca2+ to bind to the cardiac myofilaments, which generates contractile force. After contraction, free cytosolic Ca2+ levels must be diminished to resting levels in order to induce cardiomyocyte relaxation. Ca2+ is pumped back into the SR via the sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA2a) and extruded out of the cell via the Na+–Ca2+ exchanger (NCX). Activity of SERCA2a, the cardiac SERCA isoform, is tightly regulated by its scaffolding protein PLN. Under physiological conditions, PLN inhibits SERCA2a activity and thereby tempers the rate at which Ca2+ flows back into the SR. Upon the inhibition of PLN activity, such as by phosphorylation on Ser16, SR Ca2+ cycling is enhanced, leading to an increase in SR Ca2+ reuptake and improved cardiac function16. The PLN R14del mutation results in a protein that has a stronger affinity for SERCA2a6, which has been correlated with the disruption of the Ser16 phosphorylation motif. It was suggested that disruption of this motif could lead to an increased inhibition of the SERCA2a, cytoplasmic Ca2+ overload, and increased risk for malignant ventricular arrhythmias18. Whether changes in intracellular Ca2+ dynamics also occur in vivo and affect cardiac structure and function remains unaddressed. Studying these processes in depth requires accurate in vivo models. A recent knock-in mouse model demonstrated severe cardiac remodeling and heart failure when homozygous for PLN R14del. Heterozygous PLN R41del mice only showed mild fibrosis, whereas an inflammatory response or heart failure was never observed19.PLN is a small 52 amino-acid transmembrane sarcoplasmic reticulum (SR) protein and a crucial regulator of SR function within cardiomyocytesDanio rerio) has emerged as a powerful, cost-efficient, and easy-to-use vertebrate model to study human disease20. Its conserved genome , physiology, and pharmacology has made the zebrafish a highly valuable model for the resolution of human disease-related mechanisms and for novel drug (target) discovery23. In addition, CRISPR/Cas genome editing in the zebrafish has led to the opportunity of introducing of patient-specific gene mutations in the zebrafish genome26.During the past decades, the zebrafish  duration (APD)  was identified in isolated plna R14del cardiomyocytes. Since these irregular APs can be caused by altered Ca2+ dynamics, we analyzed intracellular Ca2+ dynamics in vivo. Corroborating this, we found evidence for delayed SR Ca2+ reuptake in plna R14del cardiomyocytes. Importantly, istaroxime ameliorated the in vivo Ca2+ dysregulation and contractile impairment in plna R14del zebrafish, and rescued the observed irregular APDs in plna R14del cardiomyocytes.In this study, we used zebrafish with a R14del variant in the endogenous PLN has been annotate on chromosome 20 (ENSDARG00000069404), which we refer to as plna , which we refer to as plnb  would be the best genotype to represent the heterozygous PLN R14del mutation in patients. We will refer to this genotype as homozygous plna R14del fish or simply as plna R14del mutants. Adult fish with either a heterozygous or homozygous plna R14del mutation had a normal appearance and showed normal behavior. To investigate cardiac morphological changes, we isolated hearts from 2-year-old wild-type control, plna R14del heterozygous, and homozygous zebrafish. Importantly, we observed that 23% of homozygous plna R14del fish displayed severe cardiac morphological changes, such as an increased size of the heart and a white flocculent layer of tissue that lined the outside of the heart. We did not observe any of these changes in wild-type siblings nor in heterozygous plna R14del fish  and observed one case with a similar altered tissue organization, which was accompanied by cell death, in the sub-epicardial region  expression. Corroborating this finding, an accumulation of immune cells in the sub-epicardial region was observed as well . Consistent with the increased fibrosis, we observed periostin expression in cells located in the sub-epicardial region . T-box18 (Tbx18) is a transcription factor that is weakly expressed in the epicardium of the adult heart, but its expression is induced upon cardiac injuryion Fig. . Strikinyl7 Fig. . In addipes Fig. ; boxes. ell Fig. . As Picrion Fig. . Togethe28. To examine cardiac contractility and pump function in plna R14del mutant zebrafish, and relate this to heart morphology, we performed echocardiographic measurements combined with histological analysis of the cardiac tissue. For echocardiography fish were positioned ventral side up- and a long-axis view, which included the two chambers of the heart, was imaged accordingly  between beats. These variations were already observed in 6-month-old plna R14del mutant fish, and became more apparent in 10-month-old fish, resulting in a significantly higher extent of VOT PV variation in plna R14del mutant fish, compared to their wild-type siblings . To examine cardiac electrophysiology in plna R14del mutant zebrafish we first recorded electrocardiograms (ECGs) from 10-month-old fish. The ECGs revealed normal PR- and PQ interval times and normal amplitudes of the P- and R-waves  and Phase-3 repolarization velocities between the two groups  gating properties. Current density was not affected by the plna R14del mutation , and slower decay of the Ca2+ transient, which is consistent with a slower SR Ca2+ reuptake . Analysis of GCaMP6f (cpEGFP) signal intensity over time allowed examination of Ca2+ transient amplitudes, diastolic Ca2+ levels, and the speed of intracellular Ca2+ release and reuptake/clearance, as described earlier   and significantly slower decay of the Ca2+ transient, as indicated by an increased decay time constant  between wild-type siblings and plna R14del mutants  Fig. . A similgin Fig. , suggestnts Fig. . Diastolyos Fig. . TogethePLN R14Del cardiomyopathy, we looked for a small molecule with the potential to rescue the above-described electrophysiological phenotypes observed in plna R14del mutants. Istaroxime is proposed to stimulate the activity of SERCA2a, thereby enhancing SR Ca2+ sequestration34. In addition, it inhibits Na+/K+ ATPase (NKA) activity36. Since our results indicate that SR Ca2+ sequestration is impaired in plna R14del mutant zebrafish, we tested whether the treatment of plna R14del mutant embryos with istaroxime could rescue the Ca2+ abnormalities. Wild type and plna R14del mutant embryos with GCaMP6f were first imaged to record baseline Ca2+ dynamics. After baseline recording, 100 µM istaroxime was added to the E3-MS222 medium and after 30 min of incubation another recording was performed  plna R14del mutant fish we observed cardiac remodeling, which was characterized by the accumulation of immune cells, fibroblast and fat, features reminiscent of those seen in PLN R14del patients. Importantly, our results demonstrate that drug treatment with istaroxime improves cardiac Ca2+ dynamics, enhances cardiac relaxation and reverses APD alternans in plna R14del cardiomyocytes.In this study, we utilized the zebrafish to investigate the in vivo consequence of a plna R14del in 2-year-old zebrafish, as some fish appeared completely healthy while others displayed end-stage heart disease characterized by the accumulation of fibroblasts, immune cells and fat in the sub-epicardial region. A similar heterogenous effect of the PLN R14del mutation is seen in patients, with fibrofatty infiltrates that are usually located in the posterolateral region of the left ventricular myocardium, right underneath the epicardium. Cardiac fibrofatty replacement in PLN R14del patients has a likely (sub)epicardial origin, which implicates that the underlying mechanisms of disease might be comparable between zebrafish and man37. The epicardium is a mesothelial cell layer that acts as an important source of trophic signals to maintain continued growth and differentiation of the developing heart. The importance of the epicardium as a driver of morphological changes in the heart is increasingly recognized38. In the adult zebrafish the epicardium responds to injury by inducing embryonic epicardial gene expression (e.g. tbx18) and proliferation39. After cardiac injury, the epicardial cells undergo a process called epithelial-to-mesenchymal transformation (EMT). They migrate into the cardiac tissue, where they can give rise to cardiac smooth muscle cells and fibroblasts40. Epicardial cells can also give rise to fat cells via mesenchymal transformation and peroxisome proliferator-activated receptor γ (PPARγ) activation41. As the remodeled tissue within plna R14del hearts showed a strong expression of tbx18, it suggests that it may have an epicardial origin. This should be studied further by lineage tracing experiments.We observed a heterogeneous effect of plna R14del zebrafish presented with a remarkable higher variation in ventricular outflow peak velocity, visible as alternating strong and weak beads in the echocardiography data. Pulsus alternans result primarily from an alternating contractile state of the ventricle42. Importantly, a recent clinical study revealed that pre-symptomatic PLN R14del carriers display mechanical alterations in the left ventricle and that this change in mechanical properties precedes structural remodeling43. This is consistent with our observation that mechanical variations are observed in non-remodeled plna R14del zebrafish hearts. In accordance with our echocardiography measurements, we noticed a clear alternans between consecutive APs in plna R14del cardiomyocytes during patch clamp experiments at the high pacing frequency of 4 Hz. Alternans may be a membrane voltage or AP-driven phenomenon or due to Ca2+I44. In the present study, we found no differences in average APD in a wide range of frequencies between WT and plna R14del myocytes, which is supported by the virtually overlapping of major ion currents underlying in the cardiac AP. This strongly argues against a membrane voltage or AP-driven mechanisms of our observed APD alternans31. A plausible mechanism underlying the alternans is a functional change in SR Ca2+ pumps, resulting in alternating strong and weak beats45. Also, concomitant slow transportation of Ca2+ from the uptake compartment to the release compartment in the SR has been suggested as a cause of alternans44. Corroborating such a model, we observed a slower Ca2+ reuptake in plna R14del cardiomyocytes and by application of istaroxime we were able to restore reduced Ca2+ amplitudes and decay phase, and prevent electrical alternans. Istaroxime is a drug that acts by releasing PLN from SERCA and thereby stimulates SR Ca2+ loading and release34. We did not find Ca2+ transient alternans, possibly because we were not able to reach the required 4 Hz stimulation with the use of field stimulation during the measurement of cytoplasmic [Ca2+] after Indo-1 loading in adult myocytes.Interestingly, 2+ transient amplitude decreased in embryonic plna-R14del mutant fish and the Ca2+ reuptake duration was clearly prolonged, potentially caused by a hampered SR Ca2+ sequestration6. While the diastolic Ca2+ concentration was unaffected in embryonic hearts of plna R14del mutants, it was increased in adult plna R14del cardiomyocytes. Importantly, in embryonic zebrafish hearts ncx1 is strongly expressed and loss of Ncx1 activity results in Ca2+ overload46. Differences in embryonic and adult ncx1 expression may therefore explain observed differences in diastolic Ca2+ levels in embryonic and adult plna R14del hearts. In line with literature, we demonstrated that istaroxime improves intracellular Ca2+ transient amplitude and has a positive inotropic effect on zebrafish heart function. More efficient reuptake of Ca2+ into the SR, by mitigated PLN-SERCA inhibition, will improve SR Ca2+ load, Ca2+ release, and as a consequence elevates the Ca2+ transient amplitude. The enhanced Ca2+ transient amplitudes did not directly translate into improved contractility of the zebrafish cardiomyocyte in our study. This may be explained by high myofilament affinity for Ca2+, which may lead to complete myofilament saturation in both the absence and presence of istaroxime. Zebrafish have the ability to adapt their myofilament Ca2+ sensitivity extensively in order to maintain cardiac function under a large range of temperatures (6–38 °C), demanding a high Ca2+ (ref. 47). In this study, we show that istaroxime predominantly improves the relaxation, relaxation time, and filling of the zebrafish heart, rather than affecting the contractile state of the heart. The positive effect of istaroxime on cycle length relaxation time was primarily present in plna R14del mutants. These data in combination with the clearly reduced Ca2+ transient tau of decay confirm that istaroxime can improve SR Ca2+ re-uptake34. Ouabain is a widely used inotropic agent in heart failure therapy. Unlike istaroxime, ouabain did not affect the Ca2+ transient amplitudes. This suggests a predominant lusitropic effect of istaroxime via enhanced SR Ca2+ sequestration. Clinical trials with 6–24 h istaroxime infusion in heart failure patients, however, did not demonstrate improved left ventricular diastolic volumes49, which is expected from improved SR Ca2+ reuptake. This may be explained by the Na/K-ATPase inhibiting activity of istaroxime, which leads to increased diastolic Ca2+.In line with our presumptions, the in vivo Capln R14del cardiomyocytes, a phenomenon previously reported in vitro and in vivo in the chronic atrioventricular block dog model50. Under physiological conditions, the removal of Ca2+ from the cytosol is achieved via SR Ca2+ reuptake and via the forward mode of the NCX. Upon istaroxime exposure, the enhanced SR sequestration of Ca2+ through SERCA2A during diastole likely reduces the amount of Ca2+ extruded by the NCX and reduces its depolarizing pump current. A reduced amplitude of NCX current, in combination with enhanced Ca2+-induced ICa,L inactivation, therefore results in shortening of repolarization. Impaired NKA Na+ extrusion by istaroxime elevates intracellular Na+ levels and favors the reverse mode of NCX, further shortening AP repolarization. More important, in this study we show that APD alternans caused by the plna R14del mutation can be reversed. This highlights Ca2+ handling defects as underlying cause for the APD alternans in isolated ventricular plna R14del cardiomyocytes. APD alternans are likely not related to a prolonged repolarization, as APD90 did not differ between wild type and pln R14del cardiomyocytes. We appreciate that the Ca2+ handling defects in plna R14del embryonic hearts and adult cardiomyocytes cannot yet be linked directly towards the structural remodeling at later stages. In PLN R14del patients, the disease symptoms predominantly manifest at later ages (50–60 years of age), implying that APD alternans and impaired Ca2+ transient dynamics are an early phenotypical characteristic of the disease, which over time predisposes to a pathogenic Ca2+ handling related cardiomyopathy.This study has also demonstrated that istaroxime shortens AP repolarization in the zebrafish heart and isolated zebrafish pln gene has been duplicated in the zebrafish due to the teleost-specific genome51. Duplicated genes can act redundant or may diverge in their function, which may complicate interpolations to mammalian models. As both plna and plnb are highly expressed in the ventricle and our studies show their genetic interaction, we favor the hypothesis that plna and plnb have redundant roles in the zebrafish ventricle. A second limitation is that istaroxime used in this study has a dual function as it stimulates SERCA2 activity and inhibits Na+/K+ ATPase (NKA) activity36. The plna R14del zebrafish model may help to screen for more selective compounds that only improve SERCA2a activity without affecting Na/K-ATPase activity to benefit PLN R14del carriers.Some limitations of this study should be considered. First, the plna gene we generated a relevant zebrafish cardiomyopathy model which shows in vivo cardiac Ca2+ dysregulation and morphological features reminiscent to those observed in cardiac specimen from patients. Our data present possible early disease mechanisms in PLN R14del cardiomyopathy and provide usefulness of this model to explore patient-specific drug treatment. Naturally, extrapolation of our findings to the human situation should be done with caution.In conclusion, by introducing the R14del mutation in the endogenous zebrafish 52. All experiments were conducted in accordance with the ethical guidelines and approved by the local ethics committee of the Royal Dutch Academy of Sciences (KNAW).Fish used in this study were housed under standard conditionsPlna R14del fish were previously generated using CRISPR/Cas9 technology with homologous recombination25. In short, one-cell-stage zebrafish embryos were microinjected with an injection mixture consisting of  150 ng/μl nuclear Cas9 (nCas9) mRNA, 20–40 ng/μl sgRNA, 10% (v/v) Phenol Red, and 25 ng/μl template oligo. Each putative founder adult fish was crossed with a wild-type adult fish (F1). Homozygous fish (F2) were generated by inbreeding heterozygous mutant carriers.The R14del mutation was generated in the wild-type Tupfel Longfin (TL) strain zebrafish using CRISPR/Cas9 technology. For paraffin sections, adult zebrafish hearts were dissected and fixed in 4% paraformaldehyde (dissolved in phosphate buffer containing 4% sucrose) at 4 °C overnight, washed twice in PBS, dehydrated in EtOH, and embedded in paraffin. Serial sections were made at 10 µm using a microtome . For cryosections, zebrafish hearts were extracted and fixed in 4% paraformaldehyde (in phosphate buffer) for 4 h at room temperature (RT). Three washes of 30 min were performed using 4% sucrose (in phosphate buffer) followed by overnight incubation at 4 °C in 30% sucrose (in phosphate buffer). Hearts were embedded in tissue freezing medium , frozen on dry ice and kept at −80 °C. Cryo-sectioning using Cryostar NK70 (Thermo Scientific) was performed to obtain 10 µm thin sections. Images of extracted whole hearts were acquired using a Leica M165 FC stereo microscope.53, with the exception that the hybridization buffer did not contain heparin and yeast total RNA. Briefly, slides were dehydrated, tissue was digested with proteinase K and then (pre-) hybridized with the probe of interest overnight. After a series of washes with SSC, SCC with formamide, and 0.1% PBS-Tween 20, slides were incubated in α-DIG-AP  overnight for antibody detection. Staining was developed using NBT-BCIP . Primer sequences for the grn1, postnb probes are shown in Table myl7 and tbx18 ISH probes were previously generated55. Hematoxylin and eosin (H&E) staining was performed on cryosections and paraffin sections according to the standard laboratory protocol, with the exception that cryosections were fixed in 4% paraformaldehyde for 1 h at RT beforehand. Picro-sirius Red staining on cryosections and paraffin sections was performed in accordance with the standard laboratory protocol. Briefly, dewaxed and hydrated the paraffin sections, stained nuclei in hematoxylin for 8 min, washed slides in running water for 10 min, and stained with picro-sirius red  for 1 h. Then, washed 2× in acidified water  in 1 L of water), removed all water by vigorous shaking of slides, dehydrated 3× in 100% EtOH, cleared in xylene before mounting. Oil Red O (ORO) staining was performed on cryosections according to the standard protocol57. Briefly, cryosections slides were first air dried and rinsed in 70% ethanol. Slides were stained with Oil Red O solution  for 2–3 h, rinsed in 70% ethanol and then distilled water. Counter staining was performed with hematoxylin for 5 min, and washed the well in distilled water. Imaging of stained sections was performed using a Leica DM4000 B LED upright automated microscope. Terminal dUTP nick-end labeling (TUNEL) apoptosis staining was performed on cryosections and detected using In Situ Cell Death Detection Kit, fluorescein from Roche  according to the manufacturer’s instructions. Nuclei were stained with DAPI  from Molecular Probes. Confocal images were acquired using a Leica Sp8 confocal microscope and processed using Imaris image analysis software (version 9.3.1).ISH was performed on paraffin sections according to standard protcol58 and Visual Sonics Inc. Every parameter was determined as the average measurement of at least five cardiac cycles. Ventricular outflow surface was calculated by the equation 2; VOT diameter was measured in three consecutive beats per fish. Stroke volume was calculated by multiplying the VOT VTI with the ventricular outflow surface. Variation in outflow peak velocity was determined by the discrepancy in VOT PV between two consecutive heart beats and averaged over at least six beats per fish. VOT PV variation was corrected for the average total OT PV per fish. Hearts of these fish were then individually isolated and processed as frozen samples for generation of cryosections.Adult zebrafish were anesthetised with 16 mg/ml of MS-222 in aquarium water. When the zebrafish were unresponsive to a slight tail pinch with a forceps, it was determined that the appropriate anesthetised condition for echocardiography was reached. Anesthetized zebrafish were placed in a custom-made mold and placed ventral side up in a large petridish filled with 4% MS-222 in aquarium water. Breathing was monitored by visual tracking of opercular movement to ensure fish health throughout the protocol. Transthoracic echocardiography was performed using a Vevo2100 Imaging System  with a 50 MHz ultrasound transducer fixed above the ventral side of the zebrafish and parallel to the longitudinal axis plane. Recordings were performed within 3 min after induction of anesthesia to preserve optimal cardiac performance. Quantitative measurements were assessed offline using the Vevo2100 analytical software. Color Doppler images were used for measuring heart rate, ventricular outflow diameter , ventricular outflow tract velocity time integral (VOT VTI) and ventricular outflow peak velocity (VOT PV), as validated by Wang et al.59. Here, ventricles from three to four adult fishes (6–7 months) were pooled and stored at RT in a modified Tyrode’s solution containing (in mmol/l): NaCl 140, KCl 5.4, CaCl2 1.8, MgCl2 1.0, glucose 5.5, HEPES 5.0; pH 7.4 (set with NaOH). Subsequently, the ventricles were cut in small pieces, which were transferred to Tyrode’s solution with 10 μmol/l CaCl2 (30 °C). The solution was refreshed one time before the addition of Liberase TM research grade ) and Elastase from porcine pancreas ) for 12–15 min. During the incubation period, the tissue was triturated through a pipette (tip diameter: 2.0 mm). The dissociation was stopped by transferring the ventricular pieces into a modified Kraft–Brühe solution (30 °C) containing (in mmol/l): KCl 85, K2HPO4 30, MgSO4 5.0, glucose 5.5, pyruvic acid 5.0, creatine 5.0, taurine 30, β-hydroxybutyric acid 5.0, succinic acid 5.0, BSA 1%, Na2ATP 2.0; pH 6.9 (set with KOH). The tissue pieces were triturated (pipette tip diameter: 0.8 mm) in Kraft–Brühe solution (30 °C) for 4 min to obtain single cells. Finally, the cells were stored for at least 45 min in modified Kraft–Brühe solution before they were transferred into a recording chamber on the stage of an inverted microscope (Nikon Diaphot), and superfused with Tyrode’s solution (28 °C). Quiescent single cells with smooth surfaces were selected for electrophysiological measurements. APs and net membrane currents were recorded using the amphotericin-B perforated patch-clamp technique and an Axopatch 200B amplifier . Voltage control and data acquisition were realized with custom-made software Scope  and analysis was performed with the custom-made software, MacDaq . Pipettes (resistance 3–4 MΩ) were pulled from borosilicate glass capillaries  using a custom-made microelectrode puller, and filled with solution containing (in mmol/l): K-gluconate 125, KCl 20, NaCl 10, amphotericin-B 0.44, HEPES 10; pH 7.2 (set with KOH). Potentials were corrected for the calculated liquid junction potential60. Signals were low-pass-filtered with a cut-off of 5 kHz and digitized at 40 and 5 kHz for APs and membrane currents, respectively. Cell membrane capacitance (Cm) was estimated by dividing the time constant of the decay of the capacitive transient in response to 5 mV hyperpolarizing voltage clamp steps from –40 mV by the series resistance. APs were elicited at 0.2–4 Hz by 3 ms, ~1.2× threshold current pulses through the patch pipette. Susceptibility to delayed afterdepolarization (DAD) generation was tested using fast burst pacing (20 APs at 3 Hz) which was followed by an 8-sec pause. After the 8 s pause, a single AP was evoked to test the susceptibility of early afterdepolarizations generation. APs were characterized by RMP, maximum AP amplitude (APAmax), AP duration at 20, 50 and 90% of repolarization , maximal velocity (dV/dt) of the AP upstroke (Phase-0) and phase-3 repolarization (Phase-3), and plateau amplitude . Averages were taken from 10 consecutive APs. DADs were defined as spontaneous depolarization of >1 mV. The AP measurements were alternated by a general voltage clamp protocol to elucidate the ionic mechanism underlying the AP changes. For K+ current measurements, 500 ms depolarizing and hyperpolarizing voltage clamp steps were applied from a holding potential of −50 mV with a cycle length of 2 s  and rapid delayed rectifier K+ current IKr were defined as the quasi-steady-state current at the end of the voltage-clamp steps at potentials negative or positive to −30 mV, respectively. The L-type Ca2+ current  was measured with a two-pulse voltage clamp protocol  was used to analyze the inactivation properties of ICa,L. ICa,L was defined as the difference between peak current and steady-state current. Current densities were obtained by normalizing to Cm. After baseline recordings, isolated ventricular cardiomyocytes were treated for 5 min with 5 μM istaroxime . The effect on APD was measured at 1 and 4 Hz. [Ca2+]i were measured at 25 °C in HEPES solution ((mmol/l): [Na+] 156, [K+] 4.7, [Ca2+] 1.3, [Mg2+] 2.0, [Cl−] 150.6, [HCO3] 4.3, [HPO2−] 1.4, [HEPES] 17, [Glucose] 11, and 1% fatty acid free albumin, pH 7.3) using the fluorescent probe Indo-1 as described previously61. In brief, isolated myocytes were exposed to 5 µmol/l of the acetoxymethyl esters of indo-1 during 30 min at 25 °C. Myocytes were attached to a poly-d-lysine (0.1 g/l)-treated coverslip placed on a temperature-controlled microscope stage of an inverted fluorescence microscope (Nikon Diaphot) with quartz optics. A temperature-controlled perfusion chamber , with two needles at opposite sides for perfusion purposes, was tightly positioned over the coverslip. The contents of the chamber could be replaced within 100 ms. Bipolar square pulses for field stimulation (40 V/cm) were applied through two thin parallel platinum electrodes at a distance of 8 mm. One quiescent single myocyte was selected (myocytes with more than one spontaneous oscillation per 10 s were excluded) and the measuring area was adjusted to the cell surface with a rectangular diaphragm. The wavelength of excitation of Indo-1 was 340 nm, applied with a stabilized xenon-arc lamp (100 W). Fluorescence was measured in dual emission mode at 410 and 516 nm. Emitted light passed a barrier filter of 400 nm, a dichroic mirror (450 nm) and respective narrow band interference filters in front of two photomultipliers (Hamamatsu R-2949). Signals were digitized at 1 kHz and corrected for background signals recorded from Indo-1-free myocytes. Ten subsequent Ca2+ transients were averaged from which apparent [Ca2+]i was calculated according to the ratio equation62.Single ventricular cardiomyocytes were isolated by an enzymatic dissociation procedure as used previously for zebrafish sinoatrial node and atrial cells2 s Fig. . To ensucol Fig.  from a hπ. End-diastolic and end-systolic volume (EDV/ESV) were calculated by: (1/6) × (π) × (major axis) × (minor axis2). Stroke volume (SV) by EDV−ESV. Ejection fraction (EF) by SV/EDV. Cardiac output (CO) by SV × heart rate.Embryos were placed in 1-phenyl-2-thiourea (PTU) 20–24 h post fertilization (hpf) to prevent pigmentation. Three days postfertilization (dpf) embryos were embedded in 0.3% agarose prepared in E3 medium containing 16 mg/ml MS-222. Recordings were performed at 150 frames per seconds (fps) using a high-speed inverted light microscope at 28 °C. Basal parameters were recorded first. Subsequently 3 ml of 100 µM istaroxime  or ouabain  in E3-MS-222 solution was added, incubated for 30 min, and parameters were measured for a second time. Heart rate measurements and contractility parameters were analyzed using ImageJ . Contraction time, relaxation time, and contraction cycle time were analyzed by scrolling through the recorded movies frame by frame, and by identifying (1) the moment the ventricular wall moved inward and the ventricle started expelling blood (start of contraction); (2) the moment the ventricular wall moved back outward (start of relaxation); and (3) the moment that the ventricular wall reached its most dilated position (maximum relaxation). This process was repeated three times for each heart and values were averaged. The hemodynamic parameters such as surface area and volumes were analyzed using ImageJ by drawing an ellipse on top of the ventricle at end-diastole and end-systole. Per heart six ellipses were analyzed: three at diastole and three at systole. Values were averaged. ImageJ provided the values for the minor and major axis of each ellipse. Surface area was calculated using the following formula: (0.5 × major axis) × (0.5 × minor axis) × PLN R14del fish were crossed to tg fish33 to obtain PLN R14del fish with a genetically encoded cardiac Ca2+ sensor. Wild-type fish expressing GCaMP6f or a genetically encoded voltage sensor (VSFP Butterfly CY) were used as controls33. A morpholino (MO) oligomer targeted against tnnt2a (5′-CATGTTTGCTCTGATCTGACACGCA-3′) from Genetools was injected at the one-cell stage to uncouple contraction from excitation in embryos, thereby preventing motion artifacts in our recordings of intracellular cardiac Ca2+ handling63. Embryos were placed in PTU after 20–24 h to keep them transparent. In all, 3 dpf embryos were embedded in 0.3% agarose prepared in E3 medium containing 16 mg/ml MS-222 and placed in a heated (28 °C) recording chamber. Recordings were performed with Micro-manager 1.4.17 (Image software) using a custom-built upright widefield microscope (Cairn Research) equipped with a ×20 1.0 NA objective (Olympus XLUMPLFLN20X W). Blue LED excitation light (470 nm) was filtered using a 470/40 nm filter (Chroma ET470/×40) and reflected towards the objective using a 515 nm dichroic mirror . Emitted fluorescence was filtered by a 514 long-pass filter  and images were projected on a high-speed camera (Andor Zyla 4.2 plus sCMOS). Recordings were performed at 100 fps for 1000 frames. Basal parameters  were recorded first. Subsequently, drug stocks were diluted in 28 °C E3-MS-222 medium  and the medium was mixed vigorously to assure a homogeneous concentration of the drug. Embryos were incubated for 30 min in the E3-MS222-istaroxime mixture and parameters were measured again. Recordings were analyzed using ImageJ and Matlab .n = 3) and atrial (n = 3) samples. ef1α was used as a reference gene for the qPCR. Samples were loaded on a 96-well PCR plate and run in a CFX real-time PCR System (Bio rad) using KiCStart SYBR Green qPCR ReadyMix with ROX as recommended by the manufacturer (Sigma-Aldrich: KCQS02). Three technical replicates were performed for each sample together with no-template control (NTC) for each gene. The qPCR was performed with a 2-min hold at 50 °C and a 10 min hot start at 95 °C, followed by the amplification step for 43 cycles of 15 s denaturing at 95 °C and 1 min annealing/extension at 58 °C, and the melting curve was obtained by increments of 1 °C/ 10 s from 63 to 95 °C. Primers for qPCR are described in detail in Table −(∆∆Ct), which is based on calculating the averaged changed between the pln values in relation to ef1α, the reference gene64.RNA was isolated from hearts of adult TL wild-type fish. Adult hearts were separated into ventricular  was inserted in the submerged sponge, the reference electrode (2) placed on the abdominal tissue and recording electrode, and silver wire placed in a glass capillary filled with 1 mM NaCl (3) was placed on the heart region. Data were amplified using a DAGAN EX-1 amplifier, digitized with a National Instruments USB-6210 and recorded using LabscribeNI. Signals were amplified 100× and a 3-100 Hz bandpass filter was applied. Recordings lasted 3 min in total and heart rate was calculated using the LabscribeNI PRS detector function. Single Labscribe files were exported and Matlab was used to calculate average ECG complexes to generate a single wild type and mutant trace on which interval and amplitude analysis could be performed.Adult zebrafish were anesthetized for 5 min in 16 mg/ml MS-222 in aquarium water and placed ventrally on a submerged sponge. ECG recordings were performed using a local field potential recording chamber as described previouslyt-test, comparisons between experimental groups were analyzed by one-way ANOVA for non-parametric variables with Tukey’s post-test for intergroup comparisons. Comparisons between experimental groups in combination with an intervention were analyzed by two-way ANOVA for non-parametric variables with Tukey’s post-test for intergroup comparisons. All data are presented as mean ± SEM, and p < 0.05 was considered significant. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001, n.s. p > 0.05. All comparisons with no statistical indications on the figures are non-significant. N denotes the number of fish used per dataset.Statistical analysis and drawing of graphs and plots were carried out in GraphPad Prism  and SigmaStat 3.5 software. Normality and equal variance assumptions were tested with the Kolmogorov–Smirnov and the Levene median test, respectively. Differences between two groups were analyzed using the paired Student’s Further information on research design is available in the Supplementary InformationPeer Review FileReporting Summary


N-methyl-D-aspartate receptors (NMDARs) are an essential target for the analgetic action of tricyclic antidepressants (TCAs). Their therapeutic blood concentrations achieve 0.5–1.5 μM, which, however, are insufficient to cause in vitro the open-channel block known as the only effect of TCAs on NMDARs. Whereas structures of amitriptyline (ATL), desipramine (DES), and clomipramine (CLO) are rather similar these compounds manifest different therapeutic profiles and side effects. To study structure-activity relationships of DES and CLO on NMDARs, we measured IC50s as a function of extracellular calcium ([Ca2+]) and membrane voltage (Vm) of NMDAR currents recorded in cortical neurons. Here two components of TCA action on NMDARs are described, which could be characterized as the Ca2+-dependent inhibition and the open-channel block. DES demonstrated a profound Ca2+-dependent inhibition of NMDARs, while the CLO effect was weak. DES IC50 exhibited an e-fold change with a [Ca2+] shift of 0.59 mM, which is consistent with ATL. The Ca2+ dependence of NMDAR inhibition by DES disappeared in BAPTA loaded neurons, suggesting that Ca2+ acts from the inside. Since CLO differs from DES and ATL by the presence of Cl-atom in the structure, most likely, this is the atom which is responsible for the loss of pronounced [Ca2+] dependence. As for the NMDAR open-channel block, both DES and CLO were about 5-folds more potent than ATL due to their slow rates of dissociation either from open and closed states. DES demonstrated stronger Vm-dependence than CLO, suggesting a deeper location of the DES binding site within the ion pore. Because DES and CLO differ from ATL by the nitrogen-containing tricycle, presumably this moiety of the molecules determines their high-affinity binding with the NMDAR channel, while the aliphatic chain mono-methyl amino-group of DES allows a deep permeation in the channel. Thus, different structure-activity relationships of the Ca2+-dependent inhibition and Vm-dependent open-channel block of NMDARs by DES and CLO suggest that these processes are independent and most likely may represent an action on different molecular targets. The proposed model of TCA action on NMDARs predicts well the experimental values of IC50s at physiological [Ca2+] and within a wide range of Vms. These neurons in primary culture express NMDARs of GluN1/GluN2A and GluN1/GluN2B subunit compositions  and were approved by the Animal Care and Use Committees of Sechenov Institute. Briefly, 17 days pregnant Wistar rats  were sacrificed by 1 min CO earlier . Neurons earlier .2; 10 HEPES, at pH 7.2–7.4, osmolarity 310 mOsm. The intrapipette solution contained (in mM): 120 CsF, 10 CsCl, 10 EGTA, and 10 HEPES, osmolarity 300 mOsm, with pH adjusted to 7.4 with CsOH. Patch pipettes of 4–6 MΩ were pulled from Sutter BF150-89-10 borosilicate capillaries. Experiments were performed at 22–25°C. Under control conditions, neurons were voltage clamped at −70 mV. Data are reported without corrections for liquid junction potential, which was measured as −11 mV. To activate NMDARs, 100 µM NMDA was always co-applied with 10 µM glycine as co-agonist. Some experiments were performed on neurons loaded with BAPTA. To load BAPTA, cortical neuron cultures were incubated in a bathing solution containing 5 µM BAPTA-AM for 1 h.Whole-cell currents were recorded from cultured rat cortical neurons using a MultiClamp 700B patch-clamp amplifier. Recordings were low-pass filtered at 400 Hz and digitized at the acquisition rate of 20,000 samples per second by Digidata 1440A controlled by pClamp v10.6 software (Molecular Devices). Solution exchange was performed by means of a fast solution application system as described earlier . Unless Compounds were acquired from Sigma-Aldrich, St. Louis, MO, United States.b) were normalized to maximal current response in control (Ic). The IC50 is the concentration of TCA that cause 50% of the maximal inhibition of currents, and the Hill coefficient (h) were estimated by fitting concentration-inhibition curves with the Hill equation:To determine the blocking potency of TCAs, NMDA elicited currents were measured in the absence and presence of different TCA concentrations ([B]). Amplitudes of currents measured in the presence of blocker  or offset . From these measurements, the rate constants of block (k+) and unblock (k−) was estimated. k+ was obtained from τon measured at 20 and 90 μM DES (C1 and C2 respectively) using the equation:NMDAR current relaxations during block or unblock by TCA were fit with the single exponential equation:− was the reciprocal to measured τoff:The kd) for DES was calculated as follows:The equilibrium dissociation constant  and δ were set as free parameters. IC50 (0 mV) is the value of IC50 at Vm = 0 mV, δ is the fraction of the membrane voltage field exerting force on TCA at its binding site. z = 1 is a molecule electric charge for both DES and CLO. For Faraday constant, gas constant, and room temperature of 28°C, the RT/F ≈ 26 mV. RT/(zδF) is equal to Vm, producing an e-fold change of IC50, and X is equal to [Ca2+], producing an e-fold change of IC50.To obtain the voltage-dependence of NMDAR block by TCAs, the concentration-ihibition curves were generated at three membrane holding potentials Vn [Ca2+]  Woodhull1 and I2 are amplitudes of the exponential and τ1 and τ2 are time constants of the exponential and C equals I1 + I2 + Ib.The rise time of currents activated by the testing NMDA + Gly application in trapping channel block experiments was fitted in ClampFit  using the double exponential function:n) refers to the number of recorded cells. Groups were compared using ANOVA with Bonferroni correction and Student’s two-tailed t-test. Statistical significance is reported in the figures according to the following symbols *** and ****, which indicate p values below (<) 0.001 and 0.0001, respectively. Curve fitting was performed using OriginPro software (OriginLab Corp.). IC50s obtained from individual experiments performed in the same experimental conditions were averaged to get mean ± SEM values.Data are presented as representative measurements as well as mean values ± standard error of the mean (SEM). Sample number  of both substances was measured for extracellular [Ca2+] of 0, 1 and 4 mM, at Vm = −70 mV. Increasing DES concentrations were applied on the top of the steady-state currents induced by 100 μM NMDA  or low (20 μM) concentrations (on) and offset (τoff) were well fit by a single exponential function  (f and Is) for the fast and slow components. The time constants did not depend on the washout duration. The fast component rate describes the opening of unblocked channels in response to agonist action and has an If amplitude (off) is 25 s and corresponds to the kinetics of DES release by blocked channels. An increase of If in the total current amplitude (If + Is) with the channel closed state time may indicate a gradual release of blocker molecules from the channels in the closed state  . Then, tb) . The bipmplitude . The timed state . Single 50 of DES at three Vms of −100, −70, and −30 mV. Since increasing extracellular [Ca2+] from 0 to 1 mM caused a 5-fold drop of DES IC50, the voltage-dependence was studied at both [Ca2+]. Examples of currents recorded at outermost Vms in Ca2+-free solution and in [Ca2+] = 1 mM are shown in ms. The concentration-inhibition curves  = 60 ± 5.7 µM (n = 10) and an e-fold change of 36.9 ± 3.4 mV for the IC50 voltage-dependence and an e-fold change of 0.59 ± 0.07 mM for the IC50 Ca2+ dependence.Interpretation of the voltage-dependence of the DES open-channel block within the framework on the Woodhull model  yields ams of −30 and −100 mV in [Ca2+] = 1 mM are illustrated in 2+]  = 91 ± 8.8 µM (n = 5) and an e-fold change of 50 ± 5.5 mV regardless of the presence of extracellular Ca2+.We studied the voltage-dependence of the NMDAR block by CLO using similar approaches as for DES. Examples of currents obtained at Vd in 2+] , which id in 2+] . The Vm-2+-dependent inhibition within the range of physiologically relevant [Ca2+].These unmask a great difference in the pharmacological action on NMDARs between DES and CLO. First, the voltage-dependence of the DES open-channel block of NMDARs is much stronger than those for CLO and, second, unlike DES, CLO does not demonstrate the Ca2+-dependent inhibition of NMDAR currents described here is much more pronounced for DES than for CLO (2+-dependence of these two compounds may differ.The Ca for CLO . This ob2+-dependent desensitization (CDD) of NMDARs which earlier was assumed to explain the Ca2+-dependence of ATL effects on NMDARs  obtained in a calcium-free solution in the absence of CDD. In agreement, 7.41 μM DES displaces MK-801 from the NMDAR channels due to the competition of these compounds for the ion pore , the DES IC50 at Vm = −70 mV was found to be 2.7 µM , we first studied this mechanism at [Ca2+] = 0 and Vm = −70 mV, using a similar approach as for ATL (m and [Ca2+] was tested since the Vm-dependence represents an inherent property of the block (To come out with the “proper” parameters of NMDAR open-channel block by DES and CLO and to avoid a concomitant Ca for ATL . Then thhe block .50 (0 mV) values, derived from measurements of the Vm-dependence. Clearly, while DES  and CLO (of 90 µM) are rather similar, these compounds are almost 5-folds more powerful than ATL. This difference could be determined by a low rate of DES dissociation (τoff is about 25 s) from channels which is of about 5-folds lesser than the rate for ATL, suggesting that the molecule of ATL occupies the channel at the least one-fifth of the occupancy time by DES and presumably CLO during the open-channels block. DES, as well as ATL 2), reveal weaker voltage-dependence than those for DES containing a smaller amino group (-NH2CH3) 2 of ATL and CLO, probably facilitates deeper DES penetration to the channel.The traditional interpretation of open-channel block within the framework of the Woodhull model implies that the voltage dependence is determined by the charge of blocking molecules and a deepness of the binding site location in the channel on which the electric field drop occurs. For example, Mgf NMDARs . The Mg2 channel . As for  channel  exhibit  channel . In acco-NH2CH3) . This ob-NH2CH3)  and alloL or CLO . The δ vL or CLO , and CLOL or CLO , the sma2+-dependent inhibition and the Vm-dependent open-channel block of NMDARs suggest that these processes are independent and most likely may represent an action on different molecular targets. The observation that the absence or presence of Ca2+-dependent inhibition did not alter the voltage-dependence for DES, CLO as well as ATL  = IC50 (0 mV) • exp (Vm/(e-fold change Vm)) • exp (- [Ca2+]/(e-fold change [Ca2+])). The dependences of TCA IC50s on [Ca2+] (m (2+], it allows an estimation of IC50 values that are consistent to the experimental data for DES (2+] of 4 mM and Vm = −70 mV, the model predicts IC50 = 0.01 μM which deviates from experimental data of 1.8 μM. Therefore, we cannot exclude that the direct extracellular Ca2+ action on NMDARs (2+]. This contribution perhaps could be evaluated considering the [Ca2+] effect on CLO IC50.Clearly, the parameters of NMDAR inhibition obtained in experiments result from a combination of two or even more processes. Different structure-activity relationships of TCAs with respect to the Cal as ATL  further l as ATL  for a den [Ca2+]  and Vm (Ca2+] (m  derived  for DES , CLO (Fi for DES , and ATL for DES . Neverthn NMDARs  may cont2+] of 2.5 mM and Vm = −70 mV, the model described by 50 = 0.13 μM for DES and IC50 = 1.6 μM for ATL. These IC50s correspond well to the TCAs therapeutic concentrations of 0.5–1.5 μM (2+-dependence of NMDAR inhibition. At [Ca2+] = 2.5 mM and Vm = −70 mV the model provides CLO IC50 = 14 μM, which strongly exceeds CLO therapeutic blood concentrations of 0.3–1 μM (At physiological [Ca5–1.5 μM , which c0.3–1 μM . Therefo2+-dependent inhibition (2+-dependent inhibition of NMDARs by ATL and DES occurs at therapeutic concentrations and may arise from a disruption of Ca2+ export from neurons (+/Ca2+-exchanger inhibitors like lithium ions or KB-R7943 demonstrate Ca2+-dependent inhibition of NMDARs (+/Ca2+-exchanger and the Ca2+-dependent inhibition of NMDARs represent promising targets to treat neuropathic pain pharmacologically.Thus, we demonstrated here two components of TCA action on NMDARs, which are represented by the Cahibition  and the hibition . We obsehibition . The conhibition , and MK-hibition . Howeverhibition . Convers neurons  (Lavoie  neurons , which i neurons . Therefof NMDARs  and exhif NMDARs . It is l2+-dependent inhibition augments with glutamate elicited Ca2+ entry into the cytoplasm of depolarized neurons. These two modes of TCAs action on NMDARs most likely govern the different therapeutic profiles and side effects of these drugs.The contribution of one or other mechanism of NMDAR inhibition is activity-dependent because the voltage-dependent block weakens with glutamate-induced membrane depolarization, but the Ca


There is an urgent medical need for improving treatment of AML.Emerging targeted therapies for patients with AML are successfully entering the clinic.Many immunotherapeutic approaches in the treatment of AML are currently explored.Acute myeloid leukemia (AML) is a clonal neoplasm derived from myeloid progenitor cells with a varying outcome depending on risk profile mainly defined by cytogenetic and mutational abnormalities. The initial treatment goal is the achievement of complete remission (CR) defined already for over 40 years by morphology. Preferably this should be CR without measurable residual disease (MRD) as it is now defined in the ELN 2017 recommendation.For over 50 years the standard of care (SOC) consisted of a combination of an anthracycline and cytosine-arabinoside (7 + 3) for those patients that can be treated intensively.Many new drugs are currently in development, quite a number have been approved by the EMA and/or FDA in the past 2 years (see Table –Vyxeos (CPX-351) is a liposomal formulation of cytosine-arabinoside and daunorubicin at a 5:1 molar ratio with a preferential uptake in leukemic blasts. It has been shown to significantly improve median overall survival vs 7 + 3  through day 30 and day 60, .–FLT3 mutations and IDH mutations are examples that have led to the development of specific inhibitors. Midostaurin is the first FLT3 inhibitor that in addition to intensive chemotherapy has shown of benefit in FLT3mutated AML.–IDH is a critical metabolic enzyme in the citric acid cycle. IDH1 is located in cytoplasm and IDH2 in mitochondria. IDH1/2 mutation produces 2-hydroxyglutarate (2-HG) that acts as an oncogene and blocks normal cellular differentiation important in the pathophysiology of AML. Enasidenib and Ivosidenib are IDH2 and IDH1 mutation inhibitors, respectively, that as monotherapy have proven to be highly effective in refractory and relapsed AML. These drugs are now also under investigation in the upfront setting in fit and unfit elderly either as monotherapy or in combination with low intensity therapy.–BCL-2 protein is another promising therapeutic target for AML. Overexpression enhances survival of AML cells and is associated with chemotherapy resistance in AML and poor survival.–Aberrant Hedgehog signaling is critical for leukemia stem-cell survival and expansion. Overexpression of Hedgehog pathway components has been shown in chemotherapy-resistant myeloid leukemia cells. Inhibition of this pathway enhanced the sensitivity to chemotherapy. The addition of glasdegib, a Hedgehog pathway inhibitor, to low dose ARA-C (LDAC) resulted in an improvement in OS compared with the standard therapy of LDAC. Glasdegib is approved in November 2018 in the USA for use in combination with low-dose cytarabine for the treatment of newly-diagnosed acute myeloid leukemia (AML) in patients aged ≥75 years or those who have comorbidities that preclude use of intensive induction chemotherapy.The list of new drugs currently investigated in AML is extended and although many are in early development the future looks bright for the patient with AML.Besides transplant-based immunotherapy other forms of immunotherapy are emerging.Leukemic cells express antigens that can be recognized by monoclonal antibodies: naked as well as conjugated antibodies (ADC) are used for targeted tumor eradication. Until now only gemtuzumab ozogamycin (mylotarg) has been proven to be successful especially in good risk AML.Bispecific antibodies targeting a leukemia specific antigen on the one end and a T cell recognizing antigen on the other end bring together T cells and tumor cells in order to eradicate the leukemia cells.Immune checkpoint inhibitors are in early stage of development in AML as is also the case for CART cell therapy and the application of AML dendritic cell vaccines.Results of ongoing studies have to be awaited.In summery many new drugs are emerging in AML. Many targeted agents are successfully entering the clinic. For immunotherapy apart from the alloSCT it is still early days and we have to await the outcomes of the multitude of studies that are ongoing.