To enable study of local Ca2+ signaling that regulates plasticity, we developed a GCaMP Ca2+ biosensor fused to the electrical synapse protein Connexin 36 (Cx36). Cx36-GCaMP transfected into mammalian cell cultures formed gap junctions at cell-cell boundaries and supported Neurobiotin tracer coupling that was regulated by protein kinase A signaling in the same way as Cx36. Cx36-GCaMP gap junctions robustly reported local Ca2+ increases in response to addition of a Ca2+ ionophore with increases in fluorescence that recovered during washout. Recovery was strongly dependent on Na+-Ca2+ exchange activity. In cells transfected with NMDA receptor subunits, Cx36-GCaMP revealed transient and concentration-dependent increases in local Ca2+ on brief application of glutamate. In HeLa cells, glutamate application increased Cx36-GCaMP tracer coupling through a mechanism that depended in part on Ca2+, calmodulin-dependent protein kinase II (CaMKII) activity. This potentiation of coupling did not require exogenous expression of glutamate receptors, but could be accomplished by endogenously expressed glutamate receptors with pharmacological characteristics reminiscent of NMDA and kainate receptors. Analysis of RNA Sequencing data from HeLa cells confirmed expression of NMDA receptor subunits NR1, NR2C, and NR3B. In summary, Cx36-GCaMP is an effective tool to measure changes in the Ca2+ microenvironment around Cx36 gap junctions. Furthermore, HeLa cells can serve as a model system to study glutamate receptor-driven potentiation of electrical synapses.A variety of electrical synapses are capable of activity-dependent plasticity, including both activity-dependent potentiation and activity-dependent depression. In several types of neurons, activity-dependent electrical synapse plasticity depends on changes in the local Ca This tool will be valuable to investigate the dynamic changes in Ca2+ that are responsible for some forms of electrical synapse plasticity. Furthermore, we have discovered that a widely used model system for in vitro studies, the HeLa cell, endogenously expresses glutamate receptors that effectively drive intracellular Ca2+, calmodulin-dependent protein kinase II (CaMKII) signaling. This signaling can be exploited in many types of studies.We have developed a Connexin 36 (Cx36)-GCaMP3 fusion construct that effectively reports the CaElectrical synapses provide a means to organize neurons into networks from which high-order activity emerges. Plasticity is a fundamental property of electrical synapses, altering the strength of electrical communication between coupled neurons and potentially playing a large role in regulation of network states . Electri2+ and activation of Ca2+, calmodulin-dependent protein kinase II (CaMKII) activity  with primers T7 and JOB 284 . The vector was modified to accept a second user-derived sequence by deleting the GFP open reading frame driven by the EF1a promoter by digestion with Bsu36I and BgmBI and inserting a linker containing PacI and SbfI restriction sites followed by an IRES sequence derived from the vector pIRES DsRedT3-KR24 . The modThe full open reading frame of Cx36-GCaMP, including the SV40 poly A signal, was amplified by PCR using primers KBM 7 and KBM 8 and inserted into the SwaI site following the CMV promoter of the modified PB513 vector using Gibson Assembly. The full coding region of NMDA receptor NR1 was amplified by PCR using primers JOB 405 and JOB 406 and cloned into the PacI and SbfI sites following the EF1a promoter of the same construct to generate a dual-expression construct expressing both NR1 and Cx36-GCaMP. The NR2 subunits were each cloned into the SwaI site of the modified PB513 vector using Gibson Assembly as described above to generate separate single-expression clones. Resulting clones were fully sequenced.M APV  to prevent toxicity from activation of transfected NMDA receptors. Cells were cultured for 24 h following transfection, rinsed twice with MEM without APV, and used for experiments.Human Embryonic Kidney 293T/17 cells  used for calcium imaging and immunolabeling experiments were grown in DMEM with sodium pyruvate + 10% fetal bovine serum (FBS) with penicillin/streptomycin/fungizone. Cells for all experiments were used between passages 4 and 10 relative to the original cell line obtained from ATCC. Cell culture reagents were obtained from Gibco/ThermoFisher. Cells were plated onto 35-mm culture dishes containing coverslips coated with poly-L-lysine and Laminin (Sigma) and grown to 70–80% confluency. Cells were transiently transfected with Cx36-GCaMP/NR1 and various combinations of NR2 clones in Piggybac vectors, up to a maximum of 4 μg of DNA per dish, using GenePorter H (Genlantis) following the manufacturer’s protocol. For all transfections including clones in the modified PB513 vector, Piggybac transposase  was included in the transfection mix. Following transfection, cells were grown in MEM  + sodium pyruvate, 10% FBS, penicillin/streptomycin/fungizone and 100 μHeLa cells  used for tracer coupling experiments were grown in MEM with essential amino acids + sodium pyruvate, 10% FBS and penicillin/streptomycin/fungizone. Cells for all experiments were used between passages 7 and 20 relative to the original cell line obtained from ATCC. Cells were plated onto tissue culture coated coverslips (ThermoFisher) and grown to 70–80% confluency. Cells were transiently transfected with clones in pcDNA vectors or Piggybac vectors as were HEK293 cells and cultured for 24 h. Before use in experiments, cells were rinsed twice with MEM.HEK293 cells transfected with Cx36-GCaMP and NMDA receptor clones were fixed in 4% formaldehyde in 0.1 M phosphate buffer (pH 7.5) for 30 min, washed with 0.1 M phosphate buffer, and blocked 1 h in 5% normal donkey serum in 0.1 M phosphate buffer, 0.3% Triton X-100. Cells were then immunolabeled using polyclonal antibodies for rabbit anti-NR2A , rabbit anti-NR2B , rabbit anti-NR2C , and rat anti-NMDA NR1 . Cx36-GCaMP labeling was enhanced using anti-GFP . Cy3 and Cy5-labeled secondary antibodies were purchased from Jackson ImmunoResearch. Imaging was performed on a Zeiss LSM510 Meta confocal microscope using a 40×/1.3 NA objective or a Zeiss LSM780 confocal microscope using a 40×/1.4 NA objective.M NaCl, 6.2 mM KCl, 1. 2 mM NaH2PO4, 1.2 mM MgSO4, 2. 5 mM CaCl2, 10 mM glucose, 10 mM HEPES (pH 7.4), and 1 mM glycine (CMS + glycine) at 37°C using a VC3 8-channel gravity perfusion system (ALA Scientific Instruments). Cells were imaged using a Zeiss Axiovert 200 microscope with 40×/0.5 NA objective and an ORCA 100 digital camera (Hamamatsu Photonics) using 470BP40 excitation and 535BP40 emission filters. Live images were captured using HCImage software (Hamamatsu) set to record with 4 × 4 binning and 0.4- to 0.8-s time intervals. Two to five individual gap junctions on each coverslip were marked as regions of interest for recording. Approximately 20-s baseline measurement was obtained under perfusion with CMS + glycine before switching into solutions containing glutamate  for 40 s, followed by return to CMS + glycine. The same perfusion timing was used for applications of 5 μM ionomycin  in CMS + glycine. Ionomycin experiments also included 5 min pre-incubation with 200 nM Na+-Ca2+ exchanger antagonist SEA 0400 methoxy]phenoxy]−5-ethoxybenzenamine; R&D Systems) or 100 nM SR-ER Ca2+-ATPase antagonist thapsigargin (R&D Systems) in CMS + glycine, followed by a perfusion experiment with drug in both perfusion solution and ionomycin solution.Both HeLa and HEK293 cells were tested in perfusion and imaging experiments. HEK293 cells were superior for this application since most of the transfected Cx36-GCaMP was present at gap junctions and these were well organized at vertical cell-cell interfaces A. HeLa c0 to calculate ΔF/F0 without further correction for background fluorescence, since background regions of interest often showed some changes in fluorescence intensity due to scattered signals from nearby fluorescent Cx36-GCaMP structures. For each response, we calculated the peak value of ΔF/F0 and the integrated area under the response curve, consisting of the sum of ΔF/F0 values for each time point contained within the response normalized to 1-s time intervals.Raw fluorescence intensity data from regions of interest were exported from HCImage as tabular data and analyzed in Excel (Microsoft) and Prism (GraphPad). Declining fluorescence intensity of the baseline due to photobleaching was fit with a first-order exponential function or a linear function for each region of interest. This baseline function was used as FM protein kinase A antagonist Rp-8-cpt-cAMPs (Axxora LLC), 10 μM protein kinase A agonist Sp-8-cpt-cAMPs (Axxora), 30 μM glutamate + 1 mM glycine, or 100 μM glutamate + 1 mM glycine for 5 min. Additional experiments investigating glutamate receptor contribution to tracer coupling employed the 100 μM glutamate + 1 mM glycine incubation for 5 min with or without 100 nM selective kainate receptor antagonist ACET , 20 μM AMPA/kainate receptor antagonist CNQX , 40 μM AMPA and kainate receptor antagonist GYKI 53 655 (1-(4-aminophenyl)−3-methylcarbamyl-4-methyl-3,4-dihydro-7,8-methylenedioxy-5H-2,3-benzodiazepine hydrochloride; R&D Systems), or 20 μM selective NMDA receptor antagonist (R)-CPP . Fresh solutions of appropriate drug were added to each dish with Neurobiotin at a 0.1% concentration. Cells were scraped with a 26-gauge needle, allowed to incubate 10 min, rinsed three times in CMS, and fixed for 1 h with 4% formaldehyde in 0.1 M phosphate buffer. Fixed cells were rinsed briefly in 0.1 M phosphate buffer, permeabilized with PBS, 0.1% Triton X-100, 0.1% Na azide (PBSTA) for 1 h, and labeled with Cy3-strepavidin  for 1.5 h. Coverslips were washed with PBSTA for 1 h and mounted on slides using Vectashield mounting medium (Vector Laboratories) and fluorescently imaged at 40× magnification.Gap junction coupling was analyzed by measuring tracer diffusion of Neurobiotin (Vector Laboratories) loaded into cells by scrape loading . HEK293 k. Independent measurements of k were made for each of the five to eight loaded regions examined within a single experiment. Three to six experiments were performed and all values of k used for comparisons.In each experiment, a minimum of five loaded regions along the scraped edge of the cells were identified for complete single cell loading of Neurobiotin with identifiable transfer of tracer away from the loaded cell as an indicator of a coupled region for measurement. Images were collected using HCImage software and analyzed with SimplePCI (Hamamatsu) and MATLAB (MathWorks) software. Intensity of Neurobiotin/Cy3-streptavidin signal was measured in 2-μm circles, with the brightest regions of individual cells selected. Cell-to-cell distance was measured from center to center of adjacent cells. Tracer diffusion was estimated by fitting data from regions of cells extending out from a loaded cell along the scraped edge of the coverslip B using ahttps://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSM759888) deposited in GEO  0.00074–0.00112), similar to the increase in coupling in Cx36-EGFP-transfected cells , indicating that Cx36-GCaMP forms functional gap junction channels similar to those made by Cx36-EGFP. Stimulation of endogenous protein kinase A activity with 10 μM Sp-8-cpt-cAMPS did not significantly change tracer coupling in cells transfected with any of the constructs. None of the treatments significantly affected tracer coupling in EGFP-N1-transfected cells indicating that the endogenous gap junction channels in HeLa cells were not regulated by PKA activity. The pattern of regulation in response to altered PKA activity observed with Cx36-GCaMP is similar to that observed with other Cx36 constructs . Ca2+ remained elevated while ionomycin perfusion continued . Application of 5 μM ionomycin after 5-min pretreatment with thapsigargin (n = 15 gap junctions in three experiments) resulted in a delayed and reduced relative rise in intracellular Ca2+ with delayed recovery . Note that the intracellular Ca2+ used as the baseline F0 is elevated, so the peak Ca2+ may not be substantially lower than in control conditions. Treatment with the Na+/Ca2+ exchange inhibitor SEA 0400 (200 nM) also delayed the rise in intracellular Ca2+ and prevented recovery (n = 14 gap junctions in three experiments). Thus, both Ca2+ stores and plasma membrane Ca2+ handling significantly influence the kinetics of Ca2+ rise and recovery.When Cx36-GCaMP-transfected HEK293 cells were superfused with CMS containing 2.5 montinued A, but re2+ responses, despite quite robust responses observed in cells diffusely expressing Cx36-GCaMP.To investigate the coupling of NMDA receptor activation to calcium signals in the vicinity of Cx36 we transfected HEK293 cells with Cx36-GCaMP, NMDA receptor NR1 subunit and one of the NMDA receptor NR2 subunits NR2A, NR2B or NR2C. M, 100 μM, or 1 mM glutamate. CMS in all conditions contained 1 mM glycine to act as a co-agonist for NMDA receptor activation. M glutamate stimulation. NMDA receptors containing NR2A, NR2B or NR2C all drove transient increases in GCaMP fluorescence, indicative of Ca2+ increases in the microenvironment surrounding the gap junction. Baseline-subtracted average responses from 4 to 11 gap junctions are shown in 2+ within the first few seconds of glutamate application followed by gradual declines. There were no consistent differences in the kinetics of Ca2+ signals recorded at Cx36-GCaMP gap junctions in response to glutamate stimulation of NMDA receptors containing NR2A, NR2B or NR2C. M, 100 μM, and 1 mM glutamate. Both NMDA receptor types drove concentration-dependent increases in peak response that were largely similar to each other, with both appearing to saturate between 100 μM and 1 mM glutamate. Because changes in signaling to the gap junction are likely to depend on the total Ca2+ encountered during NMDA receptor responses, we also compared integrated areas under the response curve . There was no change in coupling in EGFP-transfected controls . Surprisingly, 100 μM glutamate significantly increased coupling when only the NR1 subunit, which does not by itself form a functional NMDA receptor, was transfected with Cx36-GCaMP . Very much to our surprise, HeLa cells transfected only with Cx36-GCaMP also showed a significant increase in coupling with the 100 μM glutamate incubation . Furthermore, there were no significant differences in the 100 μM glutamate condition between cells transfected with Cx36-GCaMP + NR1 and cells transfected with Cx36-GCaMP alone or with Cx36-GCaMP + NR1 + NR2A (two-way ANOVA), and all were significantly greater than the EGFP control . These results suggest that HeLa cells express an endogenous glutamate receptor that contributes to signaling that increases coupling in Cx36-GCaMP.NMDA receptor activation in retinal AII amacrine cells  and infeM glutamate on tracer coupling of Cx36-GCaMP-transfected cells as an assay. M glutamate (one-way ANOVA with Tukey’s multiple comparison tests). Each of the following significantly reduced coupling below the 100 μM glutamate condition: 100 nm of the selective kainate receptor antagonist ACET , 10 μM of the poorly selective AMPA/kainate receptor antagonist CNQX , 40 μM of the poorly selective AMPA/kainate receptor antagonist GYKI 53 655 , or 10 μM of the selective NMDA receptor antagonist CPP . Of these treatments, only CNQX did not fully block the increase caused by 100 μM glutamate, yielding a very slight increase in coupling . This indicates that glutamate receptors in HeLa cells that influence Cx36 coupling have pharmacological characteristics of kainate and NMDA receptors, and may as well have some characteristics of AMPA receptors.We further characterized the glutamate receptors endogenously expressed in HeLa cells through pharmacological experiments using the effect of 5-min incubation with 100 μTo further investigate the glutamate receptor expression in HeLa cells, we analyzed HeLa RNA Sequence dataset GSM759888 deposited in GEO , searchiWe also examined the expression of connexin genes to gain insight into the connexin background that contributes to coupling in HeLa cells . SubstanM CaMKII inhibitor KN-93 significantly reduced the increase in coupling caused by 100 μM glutamate incubation , although coupling was still slightly enhanced compared with control . In contrast, while coupling was significantly increased by inhibition of PKA with 10 μM Rp-8-cpt-cAMPS , 10 μM KN-93 did not block this increase . Thus, the enhancement of coupling caused by activation of endogenous glutamate receptors depended in part on CaMKII, while the coupling revealed by PKA inhibition did not.Because glutamate receptor signaling that increases coupling in Cx36 gap junctions depends, at least in part, on CaMKII activity , we examPlasticity is a fundamental property of electrical synapses  and a gr2+ and reports dynamic changes in its concentration in the immediate vicinity of the gap junction. The dynamic nature of the Ca2+ microenvironment is particularly apparent in the response to administration of ionomycin to HEK cells expressing Cx36-GCaMP (2+ rises to a peak shortly after onset of ionomycin administration, its level plateaus and is rapidly reduced on washout by cellular processes, which include both extrusion via Na+-Ca2+ exchangers and sequestration in intracellular compartments such as endoplasmic reticulum and mitochondria. It is likely that the plasma membrane Ca2+-ATPase, which we did not examine, also plays a significant role in extrusion of Ca2+ from the cell. Intracellular stores in the ER play a complex role. Inhibiting the SERCA pump with thapsigargin raised intracellular Ca2+ only a small amount, but potently damped the rate of rise of Ca2+ on ionomycin addition. This suggests that the rise in control conditions includes elements of Ca2+-induced Ca2+ release from the ER stores, or that the elevated cytoplasmic Ca2+ has activated the plasma membrane Ca2+-ATPase or Na+-Ca2+ exchanger. It is likely that all of these processes are involved in the dynamics of Ca2+ in the vicinity of the Cx36 gap junctions.Cx36-GCaMP responds robustly to influx of extracellular Ca36-GCaMP . While C2+ responses at Cx36-GCaMP gap junctions. Responses peaked in the first few seconds of glutamate administration and often declined in the continued presence of glutamate. The decline no doubt reflects both the desensitization kinetics of the NMDA receptors and the Ca2+ removal processes described above. Despite these processes, Ca2+ remained elevated above the baseline level throughout the duration of the glutamate application for glutamate concentrations of 100 μM or higher. This suggests that signaling driven by elevated Ca2+ may be activated continuously when glutamate is present, at least at higher concentrations.Pertinent to understanding activity-dependent plasticity are the responses of Cx36-GCaMP to glutamate administration in the presence of NMDA receptors. All three types of NMDA receptors tested, those containing NR2A, NR2B, and NR2C, drove qualitatively similar Ca2+-mediated response, suggesting the presence of an endogenous glutamate receptor that supports Ca2+ influx in HeLa cells. Furthermore, the CaMKII activity in HeLa cells was not constitutive. The intervention routinely performed in our group of inhibiting PKA activity with Rp-8-cpt-cAMPS, which appears to increase coupling by reducing PKA-stimulated protein phosphatase 2A activity (activity , increas2+ on break-in was proposed. Thus, in both expression systems (In Neuro2A cells,  systems , CaMKII  systems . Its rol systems . In addi systems . Thus, C2+-calmodulin binding (CaMKII is comprised of four isoforms, each with several splice variants, that have distinct cell type distributions, activity and protein binding characteristics, and effects on various aspects of learning and memory . The dis binding . In this2+ and CaMKII activity, although it is not clear how the electrical synaptic depression would be achieved. More in keeping with current understanding is the depression of thalamic reticular neuron electrical synapses by burst activity (2+ entry through T-type voltage-gated Ca2+ channels activates calcineurin, with the predicted reduction in Cx36-mediated coupling. The bidirectional modulation of electrical synapse strength by different Ca2+-dependent processes is highly analogous to the differences in synaptic activity that drive long-term potentiation and long-term depression in central glutamatergic synapses. The Cx36-GCaMP biosensor we have designed will be useful to investigate these differences.Potentiation is not the only outcome of neuronal activity surrounding electrical synapses. In contrast to the NMDA receptor and CaMKII-dependent potentiation of coupling in inferior olive neurons resulting from NMDA application or selective paired-pulse presynaptic stimulation , low freactivity . In thes


However, anion exchange resins employed for the removal of negatively charged target molecules including bilirubin may also deplete these anticoagulants due to their negative charge. The aim of this study was to evaluate the adsorption of citrate by anion exchange resins and the impact on extracorporeal Ca2+ concentrations were measured pre and post albumin filter as well as pre and post adsorbents. In addition, batch experiments were performed to quantify citrate adsorption.Liver support treatments were simulated in vitro. Citrate and Ca2+ concentrations were reached well with only minor deviations. Citrate was adsorbed by anion exchange resins, resulting in a higher Ca2+ concentration downstream of the adsorbent cartridges during the first hour of treatment.Pre albumin filter target Ca2+ concentration in the extracorporeal circuit, which may cause an increased risk of clotting during the first hour of treatment. An increase of citrate infusion during the first hour of treatment should therefore be considered to compensate for the adsorption of citrate.The anion exchange resin depletes citrate, leading to an increased Ca In regional citrate anticoagulation (RCA), the inhibition of the coagulation cascade as well as platelet activation are limited to the extracorporeal circuit, where the Ca2+ concentration is reduced by chelation with citrate. Before the blood is re-infused into the patient, the Ca2+ level is restored by an infusion of Ca2+. Furthermore, metabolization of citrate by the patient during and after the treatment leads to release of Ca2+ and contributes to the restoration of physiological Ca2+ level. Beyond anticoagulation, citrate exerts anti-inflammatory properties in extracorporeal blood purification25 and is associated with longer filter lifetimes.7In patients suffering from liver disease with impaired coagulation, heparin can further contribute to hemorrhagic complications during extracorporeal liver support therapy,810 the KDIGO Working Group even for patients with low risk of bleeding. Meanwhile, RCA is widely applied also in extracorporeal liver support systems.1115 Several authors have reported clotting events during adsorbent-based liver support therapy.16 In a recent study, Prometheus treatments of patients suffering from alcoholic liver disease had to be occasionally terminated prematurely due to filter clotting.15 Filter clotting was also reported during MARS treatment at a citrate concentration in blood of 3.1 mmol/L, where Ca2+ post filter was maintained at 0.22–0.44 mmol/L.12 Besides the disturbed coagulation in liver failure, interactions between adsorbents and anticoagulants in the extracoporeal circuit should be considered to contribute to these complications.Therefore, the European Renal Association, the British Renal Association, as well as the KDIGO  Working Group have recommended RCA for patients with increased bleeding risk,17 The adsorption of these negatively charged molecules to the anion exchange resins may induce further imbalances of the clotting system. Since citrate is negatively charged, anion exchangers should be considered to deplete this anticoagulant. Therefore, we aimed to assess the depletion of citrate in extracorporeal circuits of liver support.Both, in the Prometheus and the MARS liver support systems, anion exchange resins are used to eliminate negatively charged unconjugated bilirubin. However, in addition to the adsorption of their target molecules, clotting factors II and X as well as protein C were shown to be depleted by anion exchange resins in vivo and in vitro.All experiments were performed in accordance with the Declaration of Helsinki 2013. The study was approved by the Institutional Review Board of Danube University Krems.n = 3) with 1.2 L heparinized (9 IU/mL) human whole blood , withdrawn 1 day prior to the experiments.To test the influence of adsorbents on the adsorption of citrate in extracorporeal circuits, in vitro experiments were performed ; n = 3 wFor the experiments, the Prometheus Fresenius 4008H with an albumin permeable high cut-off filter  and a high-flux dialysis filter  with calcium-free dialysis solution  were used. In the filtrate circuit, two columns containing a neutral resin  and an anion exchange resin  were incorporated. The blood flow rate was 200 mL/min, dialysis fluid flow 500 mL/min, and filtrate flow 300 mL/min.2+ concentration in the blood pool, an additional high-flux dialysis filter  was integrated in the venous line after calcium substitution with a dialysis fluid flow rate of 800 mL/min to allow the maximum possible exchange rate. Dialysis fluid calcium was adjusted by mixing calcium-free  with calcium containing  dialysis fluid to reach concentrations of 0.9, 1.2, or 1.5 mmol/L in order to simulate different Ca2+ plasma levels.To avoid citrate accumulation and to maintain a defined Ca2 solution  were infused using an algorithm to adjust Ca2+ pre albumin filter to 0.2 or 0.3 mmol/L and to substitute calcium loss via the dialysis filter.18For RCA, 0.5 M tri-sodium citrate solution and 0.5 M CaCl2+ (n = 3).Samples were drawn from the blood circuit pre citrate infusion as well as pre and post albumin filter, and from the filtrate circuit pre and post adsorbent cartridges at the beginning as well as after 0.5, 1, 2, 3, 4, 5, 6, 7, and 8 turn-overs of the pool volume. All samples were analyzed for citrate and Can = 6).To quantify and differentiate adsorption characteristics, adsorption isotherms of each adsorbent were determined in batch experiments with different quantities (0–10 mL) of adsorbents in 10 mL fresh frozen plasma with a citrate concentration of about 20 mmol/L . Citrate concentration at steady state was measured in supernatant after incubation for 6 h . Citrate was quantified by a colorimetric test  on an automated analyzer .Ca2+ was > 0.1 mmol/L. Statistical analysis was conducted using SPSS 19 .For the blood purification experiments, only data with a valid citrate dosage were included. Data were excluded if the difference between the intended and the measured patient CaFor adsorption isotherm determination, the following formulas were used:The distribution volume of citrate consists of the plasma volume, the volume of pores plus interadsorbent volume and differs depending on adsorbent volume used.. . . . . . . . . citrate distribution volume [mL]. . . . . . . . . plasma volume [mL]. . . . . . . . . pore and interadsorbent volume [mL]Pore and interadsorbent volume was estimated by mass difference of dry versus wet adsorbent of each sample. Initial citrate concentration was calculated from plasma citrate concentration measured without adsorbent by considering the different distribution volumes due to different adsorbent volumes.. . . . . . . initial citrate concentration [mmol/L]. . . . . . initial citrate concentration without adsorbent [mmol/L]Adsorbed citrate was calculated by the difference of citrate concentrations in supernatant at steady state and initial citrate concentration proportional to distribution volume.. . . . . adsorbed citrate [µmol]citrate concentration of supernatant at steady state [mmol/L]Adsorption capacity was calculated as adsorbed citrate concentration at equilibrium per g of dry adsorbent.. . . . . . . citrate adsorbed at equilibrium per g of adsorbent [µmol/g]. . . . . . dry mass of adsorbent [g]f(x) at x = 0.To be able to extrapolate to infinite, the reciprocal of adsorbed citrate per g of adsorbent and citrate concentration at steady state were used in adsorption isotherm. The reciprocal of adsorption capacity was determined by the resulting linear function 2+ concentrations pre and post albumin filter and pre and post adsorbents are summarized in Overall 148 out of 166 samples of the blood circuit and 82 out of 88 samples of the filtrate circuit were included in data analysis. Ca2+ pre albumin filter could be reached well, whereas post albumin filter Ca2+ were above the target concentrations.As shown in 2+ was reduced over time , 22.7% in the anticoagulation group 0.2 mmol/L , and 45.2% (0.49 ± 0.07 mmol/L) in 0.3 mmol/L anticoagulation group. After the first 15 min the ratio of Ca2+ >0.4 mmol/L is reduced to 17.2% (Ca2+: 0.45 ± 0.05 mmol/L), to 1.8% (n = 1) in anticoagulation group 0.2 mmol/L (Ca2+: 0.61 mmol/L) but still to 32.2% in anticoagulation group 0.3 mmol/L (Ca2+: 0.44 ± 0.03 mmol/L).As shown in For neutral resin, citrate concentrations in the supernatant corrected by the distribution volume remained high with increasing adsorbent quantities whereas a steep decline of citrate concentrations was observed for the anion exchange resin .The citrate adsorption capacity for anion exchange resin calculated by the adsorption isotherm  was 1.9717 Although RCA is increasingly applied in extracorporeal liver support, the extent of citrate adsorption to anion exchange resins has not been evaluated systematically to date.Clotting problems during adsorbent-based extracorporeal liver support can be due to disturbances of the clotting system in patients suffering from liver diseases. Moreover, adsorption of negatively charged clotting factors and its regulators by anion exchange resins can aggravate the imbalance of the coagulation system, favoring its activation.2+ concentrations were achieved well with only minor deviations, while citrate adsorption by the anion exchange cartridges resulted in a higher Ca2+ post albumin filter. Post albumin filter, a dialysis filter is incorporated into the system to eliminate water-soluble toxins. Insufficient anticoagulation at the dialyzer entrance may be detrimental. At the filter entrance, blood cells are exposed to increased shear stress and flow disturbances, which force activation of platelets and raise the risk of coagulation activation. To inhibit the activation of coagulation, a reduction of Ca2+ to ⩽0.4 mmol/L is widely applied in RCA.19 In our study, for more than one-third of all analyzed samples, the post albumin filter anticoagulation status was not satisfactory. Especially when targeting a Ca2+ concentration of 0.3 mmol/L in the extracorporeal circuit, a clinically relevant target concentration,20 almost one-half of the measured samples exceeded a Ca2+ concentration of 0.4 mmol/L. For the 0.2 mmol/L anticoagulation group – a concentration only used by few dialysis centers21 – still about 23% of all samples showed an increased risk of clotting.In this study, in vitro blood purification set ups were used to assess the anticoagulation in the extracorporeal circuit. Pre albumin filter target Ca2+ concentrations pre and post albumin filter was negligible. The risk of clotting based on elevated Ca2+ concentrations thus mainly exists during the first hour of treatment.Adsorption of citrate almost reached steady state after 1 h of treatment, since after 1 h the difference between Ca2+ concentration ≤0.4 mmol/L should be targeted to assure an adequate anticoagulation status in the whole extracorporeal system. Another option would be to equilibrate the adsorbent with citrate solution before the treatment. However, this would require further studies as well as a risk assessment. Unpublished data from our group show that pretreatment of the adsorbent with citrate does not significantly affect its adsorption capacity for toxins such as bilirubin.To quantify the adsorption of citrate, we calculated the adsorption capacity based on adsorption isotherms. We observed a strong reduction of citrate by the anion exchange resin, whereas the slight decrease of citrate, which was detected for the neutral resin, was reflecting dilution by different quantities of adsorbent. Theoretically, one column of anion exchange resin can bind up to 240 mmol citrate. While this calculated adsorption capacity is a theoretical value, it clearly shows a strong adsorption of citrate by the anion exchange resin. To reduce the risk of clotting during the first hour of treatment, an increase of the citrate infusion rate in order to compensate for citrate adsorption could be considered. Therefore, a pre dialyzer CaIn contrast to the anion exchange resin, the neutral resin adsorbent did not adsorb citrate. Therefore, adsorption of citrate by neutral resins, which are applied for cytokine adsorption in whole blood in sepsis and other severe inflammatory diseases, such as the current COVID-19, seems also unlikely.2+ concentration in the extracorporeal circuit and an increased risk of clotting during the first hour of treatment.In conclusion, we found that the anion exchange resin adsorbed citrate, resulting in an increased Ca


Therefore, the present work develops a new human ventricular model (BPS2020), based on ORd, able to simulate the inverse dependence of AP duration (APD) on extracellular Ca2+ concentration ([Ca2+]o), and APD rate dependence at 4 mM extracellular K+. The main changes needed with respect to ORd are: (i) an increased sensitivity of L-type Ca2+ current inactivation to [Ca2+]o; (ii) a single compartment description of the sarcoplasmic reticulum; iii) the replacement of Ca2+ release. BPS2020 is able to simulate the physiological APD-[Ca2+]o relationship, while also retaining the well-reproduced properties of ORd . We also used BPS2020 to generate an experimentally-calibrated population of models to investigate: (i) the occurrence of repolarization abnormalities in response to hERG current block; (ii) the rate adaptation variability; (iii) the occurrence of alternans and delayed after-depolarizations at fast pacing. Our results indicate that we successfully developed an improved version of ORd, which can be used to investigate electrophysiological changes and pro-arrhythmic abnormalities induced by electrolyte variations and current block at multiple rates and at the population level.The importance of electrolyte concentrations for cardiac function is well established. Electrolyte variations can lead to arrhythmias onset, due to their important role in the action potential (AP) genesis and in maintaining cell homeostasis. However, most of the human AP computer models available in literature were developed with constant electrolyte concentrations, and fail to simulate physiological changes induced by electrolyte variations. This is especially true for Ca One of the most productive fields of application is cardiac physiology: modeling has provided physiological insights through the prediction of phenomena and mechanisms, later confirmed or disproved experimentally  model . In order to understand these mechanisms, and taking advantage of the increasing availability of specific data on ionic currents gathered from human cardiomyocytes, several mathematical models were developed to describe the biophysical mechanisms underlying the human ventricular AP. Currently, the “gold standard” for d) model . In the es e.g.,  and selees e.g., . However2+ concentration. This is especially meaningful, e.g., when considering new in vitro experiments with cardiomyocytes and ion concentrations set to values different than those considered for developing and tuning the model. At the same time, we sought to reproduce all the experimental protocols reported in the original ORd paper (+ current (IKr) that can capture drug-channel dynamic interactions  affects the cardiac AP: in fact, an increase of [Ca2+]o shortens the AP while a decrease lengthens it. This has been observed in different species  and different cell types   . Since b therapy , patholo therapy . Multipl the ORd  and the  the ORd , models.in silico results and in vitro data. On the contrary, it would be incorrect to use the same concentrations when the analysis of in vivo pathophysiological mechanisms is the ultimate aim of simulation  is one clear shortcoming of the model and of its usage for replicating specific in vitro and in vivo conditions. For this reason, we have modified the ORd model so that it can be used to simulate conditions where [Ca2+]o changes and consequently also APD.When simulating the electrical activity of cardiac cells, it is also fundamental to set the same extracellular ionic concentrations used in experimental protocols, in order to properly compare mulation , since t2+]o dependence of the ORd model, while still reproducing the large amount of experimental human data that was used for its development and validation. We paid particular attention in setting in silico the same extracellular concentrations used for in vitro experiments. Moreover, we constructed a population of models  which corrects the APD-[Caf models  to show f models . The pop2+]o dependence, we first targeted the components of the model most likely contributing to this relationship. In particular, our earlier studies, based on the Ten Tusscher-Noble-Noble-Panfilov model (2+ current (ICaL) was primarily responsible for APD-[Ca2+]o dependence. Two contrasting mechanisms are involved. When [Ca2+]o is higher, it increases the ICaL driving force which, by itself, would enhance the current density, and prolong the APD. On the other hand, a larger ICaL also increases its Ca2+-dependent inactivation (CDI), which would reduce the current, and shorten the APD. Since the physiologically-observed outcome is APD shortening, we hypothesized that the increase in CDI must play the predominant role. Based on this hypothesis, the first change to the ORd model was a new ICaL formulation (see section L-type Ca2+ Current), which strengthens the sensitivity of CDI to [Ca2+]o compared to the ORd model. With this formulation, the increase in CDI induced by variations in [Ca2+]o overcomes the effect of the increase in driving force, thus achieving the inverse APD-[Ca2+]o relation.The BPS2020 model was developed starting from the ORd model. Since one of the motivations of this study was to reproduce the physiological APD-o) to match the one used in the experiments . More details on the optimization procedure and the cost function are available in After changing the Int model . The cos2+ Current to Other Changes), while for others we only tuned the maximal conductances.We reformulated a few currents/fluxes compared to the ORd model, as described below  states. Each loop includes four transitions: activation (from C to O), fast inactivation (from O to I1), slow inactivation (from I1 to I2) and recovery (from I2 to C). Activation and recovery rates  are the same in the two loops; they were mainly derived from the ORd time constant and steady state values of the corresponding gating variables. In particular, the activation rates (α/β) were derived from the formulation of the d activation gate of ORd  as well, but their range was limited to 0.2 to take into account the incomplete fast inactivation typical for ICaL and the time constant was adjusted to reproduce voltage-clamp data. Finally, the rates η/θ modulate the slow inactivation from I1 to I2, and the equations were obtained by imposing the microscopic reversibility to the Markov model (see equations in CDI times faster than the ones in the VDI loop. This closely reflects the observation by 2+. The CDI and VDI loops are connected by up/down rates (rup/rdown), modulated by intracellular Ca2+ concentration, and controlled by the n gate. The KCDI factor was initially set to 10, that is CDI was supposed to be 10 times faster than VDI. In fact, no published data are available which directly quantify this factor in the undiseased human ventricle. CDI was then automatically optimized, getting the final value of 9.The Iov model . This neamp data . The γ/δCaL state variable directly dependent on Ca2+ concentration, and its formulation is based on the interaction between Ca2+ and the Calmodulin (CaM) bound to ICaL channels (2+ ions bind to CaM (k1/k–1 rates), the Ca2+-CaM complex may activate CDI (k2/k–2 rates). In the BPS2020 model, the n gate controls rup/rdown, thus modulating the fraction of channels in the VDI and CDI loop. Kinetics rates Kmn and k–2n  and the background Ca2+ currents were increased by factors of 3.13 and 4, respectively. The Ca2+ diffusion from subspace to bulk myoplasm was speeded up, by reducing the corresponding time constant. The sarcoplasmic reticulum (SR) was reduced to a single compartment, and its total volume was reduced by 5%, in agreement with other human computational models (2+ Variations and Implications on Intracellular Ca2+ Handling). Consequently, Calsequestrin concentration ([CSQN]) was re-distributed in the whole SR: this reduced its concentration from 10 to 1 mM.In addition to the Il models   via the ryanodine (RyR)-sensitive channels was replaced by the phenomenological formulation used by 2+ concentrations of BPS2020  at the electrode tip. In fact, in the IK1 experiments from +]o = 5.4 mM despite the actual experimental concentration of 4 mM. The Nernst potential difference between the cases of 4 and 5.4 mM extracellular K+ is approximately +8 mV. Therefore, we introduced a positive shift equal to +8 mV (approximately corresponding to LJP) in the current equations built using the experimental data with [K+]o = 4 mM. The affected equations are: the steady-state activation/inactivation and time constant of the transient outward K+ current (Ito), IK1 formulation, and the steady state activation/deactivation and time constant of IKs. In addition, we set [K+]o = 4 mM in all the simulation protocols which are compared to these experimental data . The extracellular Na+ concentration ([Na+]o) was also changed from 140 to 144 mM.All the s online , the in +/Ca2+ exchanger (INaCa) formulation is the same as in the ORd model. We increased the maximum conductance by a factor of 2.4 upon automatic optimization.The NaKr  maximum conductance was decreased by 29% and the steady state rectification slope was increased by 9%, following parameter optimization.The inward rectifier K+ current (IKs) conductance was doubled, as in Kr channel block.The slow delayed rectifier K+ current (INaL) conductance was increased by a factor of 2.8, as in The late NaNaF (hss and jss) were modified as in NaF conductance was reduced by 73% compared to its original value to preserve the peak current magnitude.The steady state inactivation and recovery from inactivation gates for INaK maximum current was increased (twofold) to improve APD rate dependence (see +/K+ ATPase current (INaK) in non-failing or healthy human ventricular cells are available, as also reported by Ience see . We consThe current stimulus duration was set to 1 ms, with −53 μA/μF amplitude (twice the diastolic threshold), as in X was measured once membrane voltage reached X% of the resting value  on a Win 10 PC with an Intel Core i7. Numerical integration was performed with the Matlab function ode15s, a variable-step, variable-order solver, based on numerical differentiation formulas . Pacing ue as in . The codAs in NaL, INaF, ICaL, Ito, IKr, IKs, IK1, INaCa, INaK, Jrel, and Jup. The parameters were sampled in the range [20–200%] of their original values, using the Latin Hypercube Sampling , AP triangulation , maximum upstroke velocity (dV/dtmax), AP peak voltage (Vpeak) and resting potential (RMP). Each biomarker calibration range was built considering its minimum and maximum in vitro values. As an additional condition for calibration, we required that the models simulated correctly the inverse APD-[Ca2+]o dependence, as detailed in section APD – [Ca2+]o Dependence. Only models showing all the AP biomarkers within their in vitro ranges and an inverse APD-[Ca2+]o dependence were accepted in the final calibrated population. The other models were discarded. As in +]i in the range  mM and the sarcoplasmic Ca2+ concentration ([Ca2+]SR) in  mM (in steady state) were added to the calibrated population.The random population was then calibrated to select those models whose APs were in agreement with the sed data  (summarised data . The AP To assess the occurrence of delayed afterdepolarizations (DADs) we used the same protocol as in +]o = 5 mM, [Ca2+]o = 2 mM, [Na+]o = 137 mM) were used. The drug effect was simulated using the IKr drug binding values reported by We also used the population approach to study the occurrence of repolarization abnormalities, such as EADs and repolarization failures (RFs). The occurrence of repolarization abnormalities in the whole population was assessed after the administration of 0.1 μM dofetilide at a cycle length (CL) of 4,000 ms. The same extracellular concentration experimentally used by in silico population with high pacing rate to assess variability in rate dependence and potential alternans occurrence: each model underwent increasing pacing rates, as in the 1998 work by +]o = 5.4 mM, [Ca2+]o = 1.2 mM and [Na+]o = 140 mM. For this protocol, we performed an additional calibration step to filter the models displaying unphysiological concentrations as consequence of fast pacing. The upper bounds of [Na+]i and [Ca2+]SR were expanded by 50% with respect to the values used to calibrate the population in basal conditions. Therefore, models showing [Na+]i and [Ca2+]SR over 22.5 and 15 mM, respectively, were not considered for further analysis. Models were grouped in three classes: (i) adapting to the pacing rate increment without developing alternans (ADAPT), (ii) failing to adapt (ADAPT FAIL), and (iii) developing alternans (ALT).Finally, we challenged our To generate the population of models and running the DADs, dofetilide and alternans simulations, we used the Taito supercluster of CSC – IT Center for Science (Finland). We used Matlab R2017b on 50 CPU cores, using 3 GB/core memory. Generating the population took 21 h, while running DADs, dofetilide and alternans took 3, 15, and 8 h, respectively.2+]o dependence in computational cardiac models was first described by 2+]o, i.e., opposite of what observed in vitro and in vivo , simulations with BPS2020 and ORd produce very similar APs: APD90 at 1 Hz is 274 ms and 285 ms, for BPS2020 and ORd, respectively  and CDI , before CDI starts playing a role. The same mechanisms are involved when considering a decrease in [Ca2+]o, which is causing AP shortening in ORd and prolongation in BPS2020 (2+]o dependence (+]o and [Na+]o) was performed  of APD is caused by increasing [Na+]o over a very large range. Finally, the bottom panels show that the inverse APD dependence on [Ca2+]o is even larger for APD70 and APD50 than for APD90, which is consistent with the progressive increase in CDI with [Ca2+]o and therefore the progressive reduction in ICaL, which exerts its predominant role in the phase 2 of the AP. On the other hand, the slight increase in AP peak is consistent with the progressive increase in ICaL peak shown in ectively . Howeverpendence , not welrmed see . The upp2+]o dependence is due to the new ICaL formulation, or rather dependent on the new Jrel formulation, we ran two additional simulations with the BPS2020 model, separately restoring ICaL and Jrel to the ORd formulation , followed by a single S2 extra-systolic stimulus, delivered at various diastolic intervals (DIs). APDs at 30, 50, 70, and 90% of repolarization were compared against the same human experimental data used for ORd validation  and peak [Ca2+]i (middle and right panels). We compared simulation results against the human experimental data by +]i and 2+]i, normalized to 0.25 Hz and 0.5 Hz, respectively. To facilitate a quantitative comparison between BPS2020 and ORd, simulated [Ca2+]i data are also shown as non-normalized  than in vitro . Simulations with the ORd model are different from those shown in 2+ cycling, its effects are reported in 2+ concentration in the SR of the BPS2020 model compared to the Ca2+ concentration in the SR junctional and network compartments in the ORd model.In addition, since CaMK is important for controlling rate dependence of Cain vivo experimental recordings , by scaling specific ionic current conductances in the endocardial version (ENDO) of the model described until now. Changes were based on the same mRNA and protein expression data used for the ORd model , and ful90 data , to faci2+ transients are shown in The experimental calibration selected a population of 342 models out of the initial 5,000; their APs and Cap < 0.05) differences in the sampled parameters among the REP, EAD, and RF classes are reported in Ks, denoting smaller repolarization reserve, compared to the REP models. The RF models showed smaller IK1 than the REP ones, in line with the IK1 role of stabilizing the resting potential. Models developing EADs showed significantly higher ICaL than the other classes; in fact, it is well-known that one of the mechanisms leading to EADs is actually ICaL reactivation during phase three of the AP , it provides less contribution to AP repolarization. However, in 2+ release from SR activates an inward current  that carries the depolarizing charge for EADs (Administration of 0.1 μM dofetilide induced early afterdepolarizations (EADs) in 9 models, repolarization failure (RF) in 40 models and simple AP prolongation with no pro-arrhythmic events (REP) in 293 models. Illustrative AP traces for the three groups are reported in f the AP . Anotherfor EADs . These mfor EADs , 2017.2+ accumulation in the SR , causing a slow Ca2+ efflux from SR before the DAD/anticipated AP by means of the Ca2+ leakage flux from SR (Jleak), that slowly increased [Ca2+]i and the Ca2+ concentration in the subspace ([Ca2+]SS). Consequently, the RyR-sensitive channels sensed an increased [Ca2+]SS and opened spontaneously (transition 0 to 1 of the RyRo gating variable), allowing a Ca2+ efflux from SR via Jrel. This Ca2+ release triggered a remarkable inward current by INaCa that depolarized the membrane potential, thus resulting in the DAD/anticipated AP.We also identified models that, in spite their regular AP profiles when paced at CL = 1,000 ms, developed DADs with fast pacing at CL = 300 ms and one long beat at CL = 10,000 ms . Figures95 in response to the pacing rate increment. rel failed to recover from inactivation .The fast pacing rate protocol we used to trigger alternans resulted in 287 ADAPT models, 28 ADAPT FAIL models and 14 ALT models. 13 models were excluded from this analysis since their intracellular ion concentrations were out of the boundaries defined in the Materials and Methods section. tivation , or that2+]o dependence, observed both in vitro and in vivo . The major changes in BPS2020 compared to ORd are: (i) a novel Markov formulation for ICaL; (ii) a single compartment description of SR; (iii) a modified Jrel formulation, as in Kr formulation from in vitro data from In this study we present the BPS2020 model, an updated version of the ORd human ventricular AP model : our pri in vivo , and to 2+]o variations should be considered in all clinical contexts where electrolyte modifications occur, since APD changes are very important triggers for arrhythmia onsets. Unfortunately, most of the published human AP models do not take this dependence into account, and therefore respond in a non-physiological way: [Ca2+]o increases lengthen APD, and vice versa.The APD dependence on [Ca2+]o dependence has been corrected: the original L-type Ca2+ current has been replaced by a new Markov model, where the CDI sensitivity to intracellular Ca2+ has been strengthened. Indeed, we found that the APD-[Ca2+]o inverse relation is mainly mediated by the sensitivity of CDI to [Ca2+]i, in turn modulated by [Ca2+]o variations. Upon changes of [Ca2+]o two counteracting effects are elicited, both affecting ICaL: an increase in the driving force, which would promote APD prolongation, and an increase in CDI, which would promote a faster ICaL inactivation and APD shortening. The latter prevails, thus leading to the physiological inverse relation. By quantitatively analyzing CDI using the approach by 2+ range. These results are shown in 2+]o = 1.8 mM, fail to reproduce the physiological APD-[Ca2+]o dependence.In our BPS2020 model, the APD-[Ca2+]o dependence, following the same approach used by 2+ dependence from the IKs formulation, and varying the INaCa amplitude . In both cases, the correct APD-[Ca2+]o dependence was maintained . When considering short diastolic intervals in the restitution protocol, the junctional SR doesn’t have time to refill, thus leading to a very small Ca2+ release. This has not a big effect in ORd, while in BPS2020 – where the sensitivity of CDI to changes in intracellular Ca2+ is increased – a smaller Ca2+ release leads to a significant decrease of CDI, with a consequent, unphysiological, AP prolongation at short diastolic intervals. A very fast diffusion (as described by a single SR compartment) was sufficient to restore the physiological behavior at all the pacing rates, and this is why in BPS2020 we decided to use a single SR compartment. To enforce this observation we tested the role of Ca2+ diffusion within the SR by restoring a SR two-compartments (JNS+NSR) in BPS2020, with the original diffusion time constant used in ORd (100 ms), and with faster diffusion (10 and 1 ms). As shown in By itself, the increased CDI sensitivity in BPS2020 had a negative impact on the restitution curve (S1S2 protocol). This was caused by the relative slow Ca2+ diffusion within the SR , although the in vitro data were mainly recorded with [KCl] = 4 mM in the bath solution.It is worth noting that many Although several human ventricle AP models were developed and published in the past two decades, the ORd model is currently considered as the state-of-the-art in the field, based on its extensive validation with human data. Therefore, we have performed a stringent comparison between the two models, while we considered a systematic benchmarking with all the available models beyond the scope of this paper.Ks is increased by 5.75 in Mann et al. model  as done by Mann et al. (2.95 and 9.12)  . In addi2+]o relationship for ORd, BPS2020, ORd CiPA . As a matter of fact, both models have been used to investigate and integrate into the equations additional knowledge of basic cardiac cell electrophysiology (some of them in the form of model-generated hypotheses), e.g., sensitivity of CDI to intracellular [Ca2+], extraction of the activation curve from I–V data, to mention only a couple. However, a rigorous comparison of the two models, as well as the possibility of combining them into a comprehensive AP model, is far beyond the scope of the present work.Another modification of the ORd model has been very recently published by Kr block. The first goal was achieved by first varying the maximum conductances of the main ionic currents around their nominal values , and that are not easy to reproduce with the average undiseased cell model. In vivo, EADs, DADs, and alternans are elicited by specific ‘stressfull’ protocols . But even in these challenging conditions only few cells show them , providing further validation of the BPS2020 model.The population of models approach  has beenl values , under tl values . Then, ml values . Of notel values . This suCaL I–V curve does not perfectly fit the positive potentials and this remains the case for the BPS2020: this shortcoming could lead to ICaL overestimation at positive potentials. Although the new Jrel formulation, with no direct dependence on ICaL, was still able to reproduce graded Ca2+ release in good agreement for negative voltages, for positive voltages the model strays from experimental data: a detailed investigation of this phenomenon, including a comparison with local control models of Ca2+ release, would be necessary but is beyond the scope of the present work. Another limitation is that in our formulation, the RyR opening dependence on Ca2+ is very steep  could be taken into account, once more human data will be available.2+]o dependence, generally not considered by the human ventricular models available in literature, including ORd. BPS2020 accurately simulates a variety of in vitro experiments, and it also takes into consideration the actual electrolyte concentrations used in the experimental setup. These are the experiments used to develop and validate the ORd model, of which BPS2020 represents a modified version. Furthermore, the new Ca2+ release formulation enables BPS2020 to produce DADs. In conclusion, the BPS2020 model expands the domain of applicability of the current ORd model, by adding the possibility to explore changes in ventricular electrophysiology induced by electrolyte changes, e.g., effects of hemodialysis or pathological changes in Ca2+ concentrations. Therefore, the BPS2020 model can be used to simulate and investigate a variety of conditions, and constitutes what we propose to be deemed an advanced general-purpose model of human ventricular cardiac electrophysiology.The new BPS2020 model correctly simulates the inverse APD-[CaAll datasets generated for this study are included in the article/in silico model. MP and JH enabled the access to parallel computing facilities, developed the population of models, and run the in silico experiments on the population. CB, EP, MP, and SS analyzed the in silico data, prepared the figures, and drafted the manuscript. All the authors interpreted the results and revised the manuscript.CB, EP, and SS conceived and designed the study. CB and EP developed and validated the 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.


Execution of the intrinsic apoptotic pathway is controlled by the BCL-2 proteins at the level of the mitochondrial outer membrane (MOM). This family of proteins consists of prosurvival  and proapoptotic  members, the functional balance of which dictates the activation of BAX and BAK. Once activated, BAX/BAK form pores in the MOM, resulting in cytochrome c release from the mitochondrial intermembrane space, leading to apoptosome formation, caspase activation, and cleavage of intracellular targets. This pathway is induced by cellular stress including DNA damage, cytokine and growth factor withdrawal, and chemotherapy/drug treatment. A well-documented defense of leukemia cells is to shift the balance of the BCL-2 family in favor of the prosurvival proteins to protect against such intra- and extracellular stimuli. Small molecule inhibitors targeting the prosurvival proteins, named ‘BH3 mimetics’, have come to the fore in recent years to treat hematological malignancies, both as single agents and in combination with standard-of-care therapies. The most significant example of these is the BCL-2-specific inhibitor venetoclax, given in combination with standard-of-care therapies with great success in AML in clinical trials. As the number and variety of available BH3 mimetics increases, and investigations into applying these novel inhibitors to treat myeloid leukemias continue apace the need to evaluate where we currently stand in this rapidly expanding field is clear. Dysregulation of prosurvival BCL-2 proteins is highly implicated in the oncogenesis, progression, and therapy-resistance of myeloid leukemias.BH3 mimetics inhibit prosurvival BCL-2 proteins and re-balance the apoptotic pathway.The BCL-2-specific BH3 mimetic venetoclax has had significant clinical success in acute myeloid leukemia in combination with standard therapy.Various BH3 mimetics in combination with standard-of-care therapies are currently under investigation in myeloid leukemias.Could BH3 mimetics be useful in the treatment of chronic myeloid leukemia, either alone or in combination with tyrosine kinase inhibitors?Will the recent clinical success of the BCL-2 inhibitor venetoclax in combination with cytarabine or hypomethylating agents in acute myeloid leukemia encourage further combinatorial studies of venetoclax with other standard-of-care therapies in this disease?Is there scope for using MCL-1 and BCL-xL inhibitors in myeloid leukemias in a clinical setting?2. An apoptotic cell displays morphological changes including nucleus shrinkage and membrane blebbing. Apoptotic cells undergo DNA degradation, cleavage of intracellular structures, and loss of mitochondrial function1. The term ‘apoptosis’ refers to two pathways distinct in initiation. The extrinsic apoptotic pathway is triggered via death receptor binding at the cellular membrane3, while the intrinsic, or ‘mitochondrial’, the pathway is regulated by B-cell leukemia/lymphoma-2 (BCL-2) family of proteins5. The two pathways crosstalk at the level of truncated BH3-interacting domain death agonist (tBID) activation, which occurs concurrently with the instigation of a caspase cascade in the context of the extrinsic pathway, and prior to activation of the multidomain proapoptotic effector proteins in the case of the intrinsic pathway.Apoptosis is the best-described form of programmed cell death, discrete from other forms of cell death such as autophagy, necroptosis, ferroptosis, and pyroptosis6. One extensively studied example is an over-reliance on the BCL-2 family7. BCL-2 was first described in relation to survival from cell death due to its role as a driver of follicular lymphoma (FL)8. Other prosurvival members of the BCL-2 family have been implicated to varying degrees in the pathogenesis of other hematological malignancies including acute myeloid leukemia (AML)10, chronic myeloid leukemia (CML)15, multiple myeloma16, diffuse large B-cell lymphoma17, and acute lymphoblastic leukemia (ALL)18.During neoplastic transformation cells face numerous signals, including DNA damage, which would initiate apoptosis in healthy cells; however malignant cells hijack the apoptotic machinery to evade cell deathIn recent years, the implications of BCL-2 family dependence in hematological malignancies has resulted in widespread and sustained effort to investigate whether this can be exploited to selectively eliminate cancerous cells.19. The family can be divided primarily by function , and latterly by structure  and is dictated by a balance between opposing factions within the BCL-2 family20. The result is MOM permeabilisation (MOMP), the efflux of proteins from the mitochondrial intermembrane space and subsequent loss of membrane potential. Among the proteins released from the mitochondria through the BAX/BAK pores is cytochrome c, which combines with apoptotic protease-activating factor 1 (APAF1) and caspase-9 to form the apoptosome, a multi-protein complex that activates the effector caspases-3, -6 and -721.When activated, BAX and BAK oligomerize to form toroidal structures within the membrane22. The direct activation model suggests that BAX and BAK exist in an inactive conformation until activated by a subset of BH3-only proteins termed ‘direct activators’, including BIM, PUMA, and BID. These direct activators are sequestered by the prosurvival proteins and released upon inhibition of the latter by further ‘sensitiser’ BH3-only proteins . Conversely, the indirect activation model postulates that BAX/BAK are constitutively active and are inhibited by the prosurvival BCL-2 proteins; in response to a death stimulus, BH3-only proteins in turn inhibit the prosurvival proteins, thereby lifting the inhibition of BAX/BAK. In both models, the inhibition of the prosurvival proteins and subsequent release of either direct activator BH3-only proteins or BAX/BAK is the initiating step in triggering apoptosis23.The mechanism of BAX and BAK activation may occur via the direct activation and/or the indirect activation model Fig. 22. The d24. Along with the evidence that malignant cells can evade apoptosis through over-reliance on the prosurvival BCL-2 proteins, this has led to the development of highly specific small molecule inhibitors of the prosurvival proteins. The inhibitors are rationally designed to mimic the BH3-only protein known to inhibit the prosurvival protein of interest and, as such, are termed ‘BH3 mimetics’25.BH3-only proteins display differing binding affinities to the prosurvival proteins In this review, we focus on the use of BH3 mimetics within the myeloid leukemias, specifically CML and AML. We highlight the dependencies on these proteins, the compounds developed to take advantage of these discoveries and investigations conducted which combine BH3 mimetics with standard-of-care therapies, concluding with future directions for the field.27. This constitutively active tyrosine kinase sits at the epicenter of a complex signaling network that contributes to the malignant transformation of HSC into leukemic stem cells (LSC) which overpopulate the hematopoietic system with a myeloid bias28.CML is typified by the Philadelphia chromosome, a ) chromosomal translocation arising in a hematopoietic stem cell (HSC), leading to the expression of the fusion oncoprotein BCR-ABL29. For patients whose cancer becomes TKI-refractory, the disease may progress to blast phase (BP), with a 6 to 11-month survival rate and few treatment options available30. The BCL-2 family of proteins has been implicated in both CML development and progression; therefore, targeting the intrinsic apoptotic pathway may be a viable therapeutic option  CML is BCR-ABL-specific tyrosine kinase inhibitors (TKIs) such as imatinib (Gleevec®)31. BCR-ABL downregulates BIM transcription and labels the BIM protein for degradation through mitogen-activated protein kinase (MAPK) phosphorylation, with BIM levels restored by inhibiting BCR-ABL with TKIs, such as imatinib. Silencing of BIM effectively rescues CML cells from apoptosis caused by imatinib32. BCR-ABL therefore supports CML cell survival, at least in part, through the downregulation of BIM.Evasion of apoptosis in response to cytokine withdrawal is one of the most consistently observed effects of BCR-ABL; this withdrawal results in BIM upregulation in normal hematopoietic progenitors, but the effect is abolished in BCR-ABL-transformed cellsMCL-1 mRNA and MCL-1 protein are expressed constitutively in a BCR-ABL-dependent manner in CML regardless of disease stage12. Upregulation of BCL-xL has been observed in BCR-ABL-transformed HL-60 and BaF3 cells, while inhibition of the Akt/Protein kinase B pathway was found to reverse the upregulation of BCL-xL in the latter34. Investigations using apoptosis-resistant BCR-ABL+ mice suggest BCL-2 mutations in myeloid progenitors may be critical in the transition of BCR-ABL+ leukemias to advanced stage disease13. Further, inhibiting both BCL-2 and BCR-ABL is sufficient to induce apoptosis in CML stem cells in a murine CML model and TKI-resistant BP-CML patient samples35.37. In CML cells expressing high levels of BCR-ABL, this movement of BAX is prevented38.The serine/threonine-specific protein kinase Akt/Protein kinase B, a downstream target of BCR-ABL, is constitutively active in CP-CML and BP-CML cells; Akt inhibits a conformational change in BAX required for translocation to the mitochondrial membrane, thus hindering MOMP in response to cellular stress40. Overexpression of miR-29b in the CML cell line K562 has been shown to halt proliferation and induce apoptosis, indicating an important role for this miR in regulating cell death39.The microRNA miR-29b, able to increase the expression of BAX, is inhibited by BCR-ABL and is downregulated in BP-CMLThus, CML cells can hijack BAX both at the translational and conformational levels, thereby decreasing sensitivity to cytotoxic stimuli and a further balance shift of the BCL-2 family proteins in favor of cell survival.41.AML is the most common myeloid malignancy in adults, with an incidence rate of 3–5 cases per 100,000 per year and a median age of 68 years at diagnosis. AML covers a genetically heterogenous group of disorders of myelopoiesis with immature myeloid blasts in the bone marrow, blood, and extramedullary tissues. These blast cells out-compete normal hematopoiesis leading to the disease phenotype of fever, infection, anemia, bruising, and bleeding42. Conventional karyotyping is the mainstay of risk stratification in AML and is complemented by fluorescence in situ hybridization analysis and RT-PCR for the targeted detection of specific recurrent genetic abnormalities43.Classification of AML is based on the World Health Organization and European Leukemia Network criteria, which rely on morphology, immunophenotyping, and the detection of underlying genetic lesions including both recurrent cytogenetic and molecular abnormalities44. The cytogenetic and molecular abnormalities present at diagnosis influence prognosis and clinical management and are used to subtype patients appropriately into favorable, intermediate, and adverse prognostic categories42.Next-generation sequencing can further stratify AML based on the presence or absence of cooperating mutations involved in driving the disease, encompassing epigenetic regulators, cell signaling and proliferation pathways, master hematopoietic transcription factors, and tumor suppressors41. LDAC and HMA may achieve remissions in a minority of patients, but are not curative and almost all patients will relapse. Small molecule inhibitors may also be included for patients with specific genetic lesions, e.g. midostaurin for patients with a fms-like tyrosine kinase 3 (FLT3) mutation45.This complex genomic landscape, combined with other co-morbidities and age at onset, makes treatment and management of AML patients challenging and, increasingly, an individualized approach is required. The therapeutic pathway taken will depend not only on the underlying genomic lesions, but also the age and fitness of the patient. Younger and fit older patients will receive high-intensity induction chemotherapy, followed by either consolidation chemotherapy or a stem cell transplant, dependent on response to therapy and genetic lesions present at diagnosis. Until recently, patients deemed unfit to tolerate intensive chemotherapy would receive either low dose cytarabine (LDAC), hypomethylating agents (HMA), or palliative treatment such as hydroxyurea in addition to supportive care+ progenitor cells and promyelocytes while this expression was absent in their heathy counterparts, and evidence was presented that induction chemotherapy resulted in selection for leukemic CD34+ cells expressing high levels of BCL-246. Later, it was shown that BCL-2 is essential for the maintenance of cancer cells in a murine model of leukemia, in the first example of the functional removal of a BCL-2 family prosurvival protein resulting in cancer regression47.The journey from the identification of BCL-2 dependence in AML to the successful development and clinical application of the BCL-2-specific inhibitor venetoclax is a triumph of modern cancer therapy. BCL-2 was found to be expressed in AML CD3450. Patients with elevated BCL-2 tend to present with higher percentage of peripheral blasts48, with over-expression also correlating with CD34 and CD117 positivity and poorer response to chemotherapy50, suggesting a more primitive phenotype.BCL-2 expression has also been shown to be significantly upregulated in newly diagnosed AML patients (range of 34–87%) and relapsed AML patients51. This was followed swiftly by the description of HA14-1, a small molecule compound able to bind to the BCL-2 surface pocket and capable of inducing caspase-dependent apoptosis in HL-60 cells52.An early investigation saw the application of a cell-permeable BCL-2 binding peptide, based on the structure of BAD, in HL-60 cells in vitro and human myeloid leukemia cells in a murine model, resulting in leukemic cell death55. BCL-xL and BAD, along with BCL-2, are upregulated in the majority of AML stem/progenitor cell populations, compared to normal hematopoietic stem/progenitor cells (HSPCs), with induction chemotherapy resulting in a further upregulation of BCL-2 and BCL-xL54. MCL-1 is consistently high in the majority of newly diagnosed AML patients and has been associated with relapse57. MCL-1 is also linked to stem cell survival, especially in FLT3-internal tandem duplication (FLT3-ITD) AML stem cells58.Other BCL-2 family proteins, including BCL-xL and MCL-1 have been implicated in the pathogenesis of AML57.A critical role of MCL-1 in cell survival was demonstrated in an elegant study using bone marrow HSCs/HSPCs transformed with the oncogenes mixed-lineage leukemia (MLL)-eleven nineteen leukemia (MLL-ENL) and MLL-ALL1-fused gene from chromosome 9 (MLL-AF9), and corresponding AML mouse models. Depletion of Mcl-1 led to the death of cells in vitro and reduced disease burden in AML-afflicted mice, with cell death being rescued by overexpressing Bcl-2 or Mcl-159. If BH3 mimetics are to be used successfully clinically in the management of AML, patient-specific prediction of BCL-2 family dependency, potentially by BH3 profiling may well be essential61.Due to the heterogeneity of AML, studies indicate that cells may be ‘addicted’ to BCL-2, MCL-1, or both depending on the genomic landscape of the patient at diagnosis62 and Leverson et al.63. One of the first BH3 mimetics developed, following HA14-1, was ABT-737, a small molecule with high binding affinity to BCL-2, BCL-xL, and BCL-w64. ABT-737 represents the first example of an anti-cancer drug designed specifically to target a protein-protein interaction, and was identified through the structure-activity relationships (SAR) by nuclear magnetic resonance (NMR) method and site-directed parallel synthesis, a triumph of modern, rational cancer therapy design64.The developmental journey of BH3 mimetics to clinical use has been extensively covered, including the excellent reviews by Lessene et al.65. Navitoclax is a dual inhibitor of BCL-2/BCL-xL, and its application as a monotherapy in relapsed/refractory (R/R) CLL was promising, with a 35% partial response rate, though 28% of patients experienced grade 3/4 thrombocytopenia due to the requirement of BCL-xL in the development of platelets67. The serious adverse effects associated with BCL-xL inhibition in vivo was addressed with the development of ABT-199 , a highly specific BCL-2 inhibitor that induced less thrombocytopenia68.The major limitation of ABT-737 was the lack of oral bioavailability, prompting the development of ABT-263 (navitoclax)68, and since has been approved in the US for the treatment of CLL with 17p deletion (2016), in combination with rituximab (Rituxan®) for previously untreated CLL (2018), newly diagnosed AML in combination with HMA or LDAC where intensive induction chemotherapy is not possible  and in combination with non-chemotherapeutics for previously untreated CLL (2019). The path to these approvals in AML will be addressed further in this review.Venetoclax was first described in 2013Of interest in the context of the current COVID-19 pandemic, NHS England granted temporary emergency approval of venetoclax in specific AML patient groups. Venetoclax treatment can be delivered on an outpatient basis, allowing for reduced attendance at the clinic for the duration of the pandemic until regular treatment can be resumed.71. To this end, and especially when targeting cancers in which BCL-2 is not the primary prosurvival BCL-2 protein, other BH3 mimetics may come to the fore of clinical studies in the future 80, reducing leukemic burden and long-term engraftment potential and increasing overall survival in CML murine models 35, and increasing apoptosis in BP-CML progenitors 35, 81 and CP-CML progenitors 82.More recently, BH3 mimetics in combination with TKIs have been used to target the CML progenitor compartment, with notable success in a number of CML disease modelsIn summary, for TKI-resistance mediated by the proapoptotic or prosurvival proteins, BH3 mimetics appear effective in redressing the balance, and re-sensitizing CML cells to TKIs.83. To date, no results are available for this trial. A second phase 2 study combining decitabine, ponatinib and venetoclax in blast phase CML is also underway, with a primary endpoint of overall response rate (NCT04188405)84. To date, there are no clinical trials in CML of MCL-1 or BCL-xL inhibitors.However, due to the effectiveness of TKIs alone, there is very little clinical trial activity investigating combinations of BH3 mimetics with TKIs. One phase 2 clinical trial is currently recruiting, combining venetoclax with the TKI dasatinib in early CP-CML, with the primary endpoint of assessing the proportion of patients achieving major molecular response after 12 months of therapy (NCT02689440)68 to full approval by the FDA for venetoclax plus HMA or LDAC in older, unfit AML patients in 2020. This speaks to the substantial and convincing work into the BCL-2 family in AML, through pre-clinical studies of venetoclax alone and in combination with other therapies, to the large international trials that resulted in directly improving patient care. Figure The success story of venetoclax in AML is one that cannot be understated, especially for the exceptionally short timeframe from the first description of venetoclax in 20139, with particular sensitivity to venetoclax seen in AML cells harboring the MLL fusion genes and in acute promyelocytic leukemia (APL) cells85. Interestingly, venetoclax is especially potent in AML cells with isocitrate dehydrogenase 1 and 2 (IDH1/2) gene mutations; these proteins have been implicated in increasing BCL-2 dependence86. In a phase 2 clinical trial in relapsed/refractory AML, single-agent venetoclax had an overall response rate of 19%, while 33% (4 out of 12) of patients with IDH1/2 mutations demonstrated CR88.Along with mounting evidence for the role of BCL-2 in AML cell survival, early preclinical studies into venetoclax as a monotherapy in AML cell lines, patient samples, and a murine xenograft model demonstrated on-target cell killing89.There is also increasing use and success with the MCL-1 inhibitors AMG-176, AMG-397, and S64315 in pre-clinical models of AML, regardless of the presence of specific genetic lesions+ compartment46 and high de novo expression of BCL-2 is correlated with poor response to treatment91, indicating scope for venetoclax combinations in AML.Despite the limited clinical success of venetoclax as a monotherapy in AML, evidence supporting the combination of venetoclax with standard-of-care therapies in AML is encouraging. For example, treatment of AML patients with cytarabine and idarubicin has been shown to increase BCL-2 expression in the CD3494. RNA-interference screening identified the proapoptotic BCL-2 proteins as potential targets for enhancing the effects of azacitidine92, and later it was shown that ABT-737 was a more potent agent than venetoclax when used in combination with azacitidine, due to the variable expression of the prosurvival BCL-2 proteins between patients93.Pre-clinical synergistic cytotoxic effects were shown by several groups when combining ABT-737 or venetoclax with the HMA azacitidine in AML cell lines and primary patient samples in vitro97. These results led, in 2016, to the FDA granting venetoclax Breakthrough Therapy Designation in combination with HMA in older patients with treatment-naive AML.These promising findings led to the development of clinical trials investigating a BCL-2-targeting BH3 mimetic in combination with azacitidine in myeloid leukemias. A phase 1b trial comparing the combination of venetoclax with azacitidine or decitabine in AML patients over 65 years of age with treatment-naïve AML, and who were ineligible for intensive chemotherapy, demonstrated an extremely promising 73% CR or CR with incomplete count recovery (CRi) for the cohort receiving HMA and 400 mg venetoclax98. At an interim analysis, overall survival (OS) and CR were increased from the control  to the azacitidine plus venetoclax group 99. By 2018, the FDA had granted accelerated approval for venetoclax in combination with HMA in patients who cannot receive induction chemotherapy, and full approval granted for this indication in 2020.The large VIALE-A phase 3 trial that followed combined azacitidine with 400 mg venetoclax and compared against an azacitidine plus placebo control group, enrolling 443 untreated AML patients who were either over the age of 75 or could not tolerate standard chemotherapy, or both100. Pre-clinical inhibition of BCL-2 by antisense oligonucleotides, obatoclax or venetoclax in combination with cytarabine has been shown to significantly enhance cell death in AML cell lines and patient samples102. Cytarabine-mediated reduction of MCL-1 expression may also contribute to the synergistic action of BCL-2 and/or BCL-xL inhibition in these cells71.Incorporation of cytarabine into the DNA of rapidly dividing cells induces cell cycle arrest in the S phase through inhibition of DNA synthesis104.In phase 1b/2 clinical trials with patients over the age of 65, the combination of venetoclax with LDAC had a CR rate of 54% and OS of 10.1 months, though these rates were increased to 62% and 13.5 months respectively for patients with no prior HMA treatment105. Enrolled participants were ineligible for intensive chemotherapy and were treated either with venetoclax or placebo with LDAC. As of summer 2020, OS was 4.1 and 7.2 months and CR rates were 13% and 48% in the control and venetoclax arms, respectively106. This trial continues in follow-up, but in 2020 these promising initial results led to FDA full approval of LDAC with venetoclax in treatment-naive AML patients.As with the azacitidine plus venetoclax success, a large, international phase 3 clinical trial, VIALE-C, was quick to followThe success of venetoclax in AML in combination with the standard-of-care therapies HMA and LDAC has led to investigations into combining this BH3 mimetic with other clinically available therapies. Here we will briefly describe some of these pre-clinical investigations.107. FLT3-ITD upregulates MCL-1 through STAT5 activation and the Akt pathway; therefore, inhibition of FLT3-ITD and treatment with venetoclax concomitantly removes the protection of both MCL-1 and BCL-2, rendering the cell sensitive to apoptosis108.The apoptotic response to the FLT3 kinase inhibitor midostaurin in FLT3-ITD-positive primary AML samples and cell lines is enhanced in the presence of venetoclax110. Further sensitizing cells to BIM with BH3 mimetics potentiates the apoptotic effect of sorafenib, as seen in combination with obatoclax and venetoclax111.Sorafenib is a multi-kinase inhibitor targeting RAF, PDGFRB, VEGFR2, FLT3, and KIT, and induces apoptosis in AML cells via BIM and downregulation of MCL-1112. However, the combination of JY-1-106 with ATRA was shown in one study to have little effect on reducing cell proliferation in HL-60, an APL cell line113.MCL-1 overexpression impedes the ability of ATRA to induce growth arrest and differentiation in APL and combining ATRA with an MCL-1-interfering BH3 mimetic has been postulated to induce a greater cytotoxic response than ATRA alone114. Overexpression of BCL-2 has been shown to prevent daunorubicin-induced apoptosis in AML cell lines through inhibition of X-linked inhibitor of apoptosis protein (XIAP) and degradation of Akt116. Removal of this BCL-2-mediated protection against daunorubicin has been shown to be effective at synergistically inducing apoptosis and growth inhibition in cell lines and in patient samples, using either ABT-737 or venetoclax117.Daunorubicin is a DNA-intercalating chemotherapeutic able to induce sphingomyelin hydrolysis and ceramide generation119. Combining BH3 mimetics has the advantage of disabling the cell’s ability to ‘switch’ between prosurvival proteins, a commonly reported resistance mechanism to BCL-2 inhibition, and thus overcoming the redundancy in the BCL-2 family system122.The possibility of combining BH3 mimetics with different target specificities is also under scrutiny in both CML and AML studies, although toxicity concerns have potentially held back investigations of this nature123. Recently, however, 4 agents  with activity against MCL-1 entered phase 1 clinical trials as single agents in AML  or azacitidine (NCT02675452129), once dose-finding studies are completed. Importantly, CDK9 inhibitors  indirectly inhibit MCL-1. These agents have preclinical activity in AML, and a number of early phase clinical trials are ongoing. It will be important to determine if they have efficacy with a favorable safety profile.In contrast to the clinical success of venetoclax, clinical trials of MCL-1 inhibitors have been more problematic. Initially, there were difficulties in developing MCL-1 inhibitors as the binding site is shallower and less flexible than that of BCL-2 or BCL-xL67.The BCL-2/BCL-xL inhibitor navitoclax has undergone extensive clinical trial evaluation in solid tumor, lymphoid malignancies and myeloproliferative neoplasms, but not AML. Further development has been limited by the predicted and on-target side effect of thrombocytopenia88.Heterogenous responses to BH3 mimetics occurs in patients, indicating a need for personalizing treatment approaches when considering these drugs. BH3 profiling, a technique developed to predict relative dependency on BCL-2, MCL-1, and BCL-xL, has been shown to be useful in predicting responses of patients with AML after treatment with venetoclax, as well as highlighting potential resistance mechanisms141, but also in trials focusing on CLL142 and ALL143. Further, combining BH3 profiling results with basal expression data for the prosurvival BCL-2 proteins  has also been shown to be effective in indicating BCL-2 dependence144. In the case of the clinical trial NCT02520011145, demonstrable MCL-1 dependence in AML, as determined by mitochondrial profiling, was used to identify eligible patients, although this trial was later terminated due to slow enrollment.The success of BH3 profiling in this regard has led to the incorporation of this technique in several clinical trials as a prognostic marker and determinant of response, most notably for myelodysplastic syndrome and AML147 and expression of BH3-only proteins such as BIM148 have been shown to correlate with response to BH3 mimetics.In addition, protein and gene expression profiles of the target BCL-2 family membersBH3 and mitochondrial profiling, along with gene and protein expression data, represent high-throughput methods with a fast turnaround time, often requiring few cells and with limited ex vivo exposure of patient samples. As these techniques are refined, there is a trend in the literature towards assays with high specificity and sensitivity for identifying patients who may benefit from BH3 mimetic treatment, warranting investigations into the point-of-care applications of these assays.With substantial progress being made in the field of BCL-2-targeted therapies and our increasing understanding of dysregulation of this family in the myeloid leukemias, great strides have been made in bringing these areas together, as highlighted in this review.A number of unanswered questions and areas for further investigation remain to be addressed. With the difficulties in targeting MCL-1 and BCL-xL, the identification of a therapeutic window is required, which could be addressed through sequential or alternating treatment strategies to allow time for healthy tissue recovery, or through potent combinations that would allow for a substantial reduction in the concentrations of BH3 mimetic required. Further, measuring BCL-2 family dependency in a point-of-care setting to refine treatment deserves additional scrutiny to determine clinical utility.As clinical trials advance and standard treatment regimens incorporate BH3 mimetics to a greater degree, these novel therapeutic combinations may represent a significant step in the direction of targeted, personalized therapy for patients with myeloid leukemias.