INa activation was examined using 10-ms depolarising, V1, steps to varying voltages 0–80 mV positive to resting membrane potential. The dependence of the subsequent, INa inactivation on V1 was examined by superimposed, V2, steps to a fixed depolarising voltage. Current-voltage activation and inactivation curves indicated that adding 0.5 and 2 mM caffeine prior to establishing the patch seal respectively produced decreased (within 1 min) and increased (after ~2 min) peak INa followed by its recovery to pretreatment levels (after ~40 and ~30 min respectively). These changes accompanied negative shifts in the voltage dependence of INa inactivation (within 10 min) and subsequent superimposed positive activation and inactivation shifts, following 0.5 mM caffeine challenge. In contrast, 2 mM caffeine elicited delayed negative shifts in both activation and inactivation. These effects were abrogated if caffeine was added after establishing the patch seal or with RyR block by 10 μM dantrolene. These effects precisely paralleled previous reports of persistently (~10 min) increased cytosolic [Ca2+] with 0.5 mM, and an early peak rapidly succeeded by persistently reduced [Ca2+] likely reflecting gradual RyR inactivation with ≥1.0 mM caffeine. The latter findings suggested inhibitory effects of even resting cytosolic [Ca2+] on INa. They suggest potentially physiologically significant negative feedback regulation of RyR activity on Nav1.4 properties through increased or decreased local cytosolic [Ca2+], Ca2+-calmodulin and FKBP12.We investigated effects of the ryanodine receptor (RyR) modulator caffeine on Na This is detected by voltage-dependent transverse tubular Cav1.1 that undergoes transitions resulting in an allosterically-activated ryanodine receptor (RyR) mediated release of sarcoplasmic reticular (SR) Ca2+ thereby increasing cytosolic free Ca2+ concentration, [Ca2+]i, Recent findings have been suggestive of feedback mechanisms through which downstream events in this feedforward sequence might conversely affect Nav1.4 function. However, many of these reports provided structural rather than functional evidence, or involved experiments in cell lines or isolated cultured cells. They thus provide necessary rather than sufficient evidence for the actual operation of Ca2+ mediated feedback mechanisms in working muscle in vivo. Few available reports examined the existence and properties of RyR-mediated feedback actions on Nav1.4 function in intact in situ skeletal myocytes particularly those directly targeting the RyR mediated Ca2+ release process.Skeletal muscle excitation-contraction coupling involves triggering of a Nav1.4 possesses several potential Ca2+ and calmodulin (CaM) binding or modulation sites. These include: (a) one or more Ca2+-binding C-terminal EF-like hand motifs1; (b) a C-terminal isoleucine-glutamine (IQ) domain that could bind CaM following combination of Ca2+ with CaM’s own EF-hand motifs2; (c) a III-IV loop Ca2+/CaM-binding site3; (d) sites phosphorylatable by CaM kinase II (CaMKII) themselves regulated by Ca2+/CaM4 and (e) a phosphorylation site for phosphokinase C (PKC)5.First, Na2+, raising [Ca2+]i to ~2 µM, and Ca2+ overspill from neighbouring Ca2+ channels, reduced peak Na+ currents, INa, in Nav1.4-transfected HEK293 cells and skeletal muscle cell lines. These effects were abrogated by intracellular BAPTA6. Additionally, CaM expression in the transgenic cells negatively shifted the voltage-dependences of Nav1.4 inactivation. Both the reduction of peak INa, and this negative shift in voltage dependence, were abolished by mutations in either the Ca2+-binding-EF hands on CaM or the Nav1.4 C-terminal IQ domain8. These results implicated CaM binding to the IQ domain in Nav1.4 modulation by [Ca2+]i.Secondly, single-cell patch-clamp studies reported that both rapid photo-release of caged Cai resulting from reductions in its affinity for RyR subunits associated with RyR opening10, positively shifted activation and inactivation voltage dependences in cardiomyocyte Nav1.511, thought closely homologous to Nav1.44. This likely involves an indirect effect; co-immunoprecipitation assays appear to exclude direct FKBP12–Nav1.5 interactions11.Thirdly, increases in free intracellular concentrations of FK506 binding protein, [FKBP12]2+]i-dependent Nav1.4 modulation in intact in situ skeletal myocytes. Loose-patch clamp studies reported that carbonyl cyanide-3-chlorophenylhydrazone12 and 8-(4-chlorophenylthio)adenosine-3′, 5′-cyclic monophosphate challenge13 reduced INa. These effects were reversed by the Ca2+ buffer BAPTA and RyR blocker dantrolene respectively. However, these agents affected [Ca2+]i indirectly through modifying mitochondrial Ca2+ release or altering RyR activity through CAMKII-dependent pathways via Exchange protein directly activated by cAMP (Epac).However, fewer reports are available on such [Cadirect RyR-SR Ca2+ channel modulator caffeine14 modifies INa in intact mammalian skeletal muscle fibres. We utilised experimental protocols previously shown to accomplish contrasting effects of 0.5 and >1.0 mM caffeine challenge on spectrofluometrically measured resting skeletal myocyte [Ca2+]i17. Thus, in isolated rabbit skeletal SR or murine skeletal muscle fibre preparations, caffeine (0.5 mM) caused increased [Ca2+]i (to ~300 nM) from resting levels . This was persistent over the subsequent 3–10 min intervals investigated16. In contrast, ≥1.0 mM caffeine induced early [Ca2+]i peaks followed by reductions to persistently below-resting [Ca2+]i within 80–90 sec17. The latter was attributed to a slow RyR inactivation reducing channel open probabilities taking place over seconds. This property was previously demonstrated in sheep and rabbit skeletal muscle RyRs reconstituted in lipid bilayers under levels of steady [Ca2+]i that would initially produce RyR activation (~10–100 µM)19.We here test the hypothesis that the INa in intact murine skeletal muscle using the loose-patch clamp approach. This employed relatively low-resistance seals (<2 MΩ) with an undisrupted surface membrane thus leaving intracellular, particularly [Ca2+]i homeostasis unperturbed. In contrast, conventional tight-patch seals often include the Ca2+-sequestrating ethylene glycol-bis(βaminoethyl ether)-N, N, N’, N’-tetraacetic acid (EGTA) in intracellular pipette solutions. In addition, the loose-patch pipettes could be re-used between patches, permitting sequential and multiple recordings from the same cell before and after pharmacological challenge. This permitted standardized comparisons between successive results with the same pipette20. Test results were compared with effects of control challenges using the known specific RyR blocker dantrolene21 to identify caffeine effects specifically attributable to its direct RyR action.The present experiments correspondingly investigated effects of 0.5 mM and 2.0 mM caffeine on + current, INa, including its activation and inactivation, were examined using a double test pulse protocol  mV, first removed any residual INa inactivation at the RMP. This standardized the proportion of activatable Na+ channels prior to application of the subsequent depolarising V1 pulse, itself of duration 10 ms, at time 55–65 ms.The experiments employed a loose-patch clamp preparation and recording layout fully described in the Materials and Methods section Fig. . Voltagecol Fig. . In eachINa in individual patches. This averaged results from 5 sweeps of a pulse protocol imposing a single voltage step to a single voltage V1 at (RMP + 80) mV between 55 and 80 ms. INa features at later time intervals following solution change were studied using the full procedure. This examined effects of a family of V1 amplitudes varied between sweeps in +5 mV increments through test voltages between RMP and (RMP + 80) mV. This elicited currents reflecting the voltage dependence of INa activation. A further V2 pulse was then made to a constant, depolarised voltage of (RMP + 80) mV. This would activate all the Na+ channels within the patch not inactivated by the preceding V1 pulse. This made it possible to explore the voltage dependence of Na+ current inactivation. For each value of V1, currents from 10 sweeps were averaged before examining the next value of V1. Each complete protocol applied to a single patch therefore involved collection of currents from 170 sweeps.At early intervals immediately following solution change, a simplified protocol examined V1 = (RMP + 35) mV following P/4 correction under control conditions. Downward, negative deflections denote inward while upward, positive deflections represent outward membrane currents. The V0 pulse typically yielded no currents. The V1 and associated V2 steps each elicited transient inward currents representing the opening and inactivation of Na+ channels. The magnitudes of their peaks, i1 and i2, were measured relative to the currents measured just before the respective V1 and V2 steps.Figure 22. Furthermore, following every set of sweeps a further bracketing protocol was then imposed using a single V1 step to RMP. The resulting average i2 value from the succeeding V2 step was then compared with the i2 value obtained by the first 10 sweeps of the preceding test protocol, for which V1 was also RMP. Patch stability was confirmed, permitting further analysis, if the bracketing i2 value was within 10% of the test i2 value. Thus, only patches with a stable seal, INa generation, and activation and inactivation characteristics were selected for further analysis.Each patch was only subjected to a single application of the complete set of pulse protocols. This made differences between results arising from prolonged changes in the patch such as membrane blebbing unlikelyINa at the earliest times (1–4 min) following introduction of caffeine. The simplified protocol employing a single V1 test voltage of (RMP + 80) mV permitted prompt assessments of INa at times immediately following solution change. This made it additionally possible to explore the effects of using differing patch-clamping protocols either excluding or permitting caffeine access to membrane beneath the patch seal. All these manoeuvres first obtained control currents in the absence of caffeine  mV under control conditions (A) and at different times following introduction of 0.5 mM caffeine (B). Initial rates of rise of current increased non-linearly with degree of depolarization elicited by the V1 steps. So did the peak INa which then reached a maximum at V1 ≈ (RMP + 75) mV before decreasing with further depolarisation. The subsequent V2 steps yielded currents whose peak INa decreased with increasingly positive V1 consistent with prior voltage-dependent channel inactivation reducing the proportion of remaining activatable Na+ channels. The pulse protocols were repeated on different patches over successive time intervals following the introduction of caffeine. Figure INa initially showed marked, ~80%, reductions within the 10-minute interval following challenge  and inactivation curves plotting dependences of peak INa on V1 in response to the respective V1 and V2 voltage steps. These are plotted for families of records obtained at different intervals following onset of caffeine challenge  time interval  mV and using a V2 step to (RMP + 100) mV. This encompassed the V1 at which maximum peak INa was observed, both under the control conditions  mV at 11–25 min and at (RMP + 95) mV at 26–40 min respectively.Findings of this kind yielded nge Fig. . These cnge Fig. , INa wasval Fig. . The maxons Fig.  and at tons Fig. . TogetheImax. In addition, there was a negative shift in the V* value describing INa inactivation. In contrast, V* values for INa activation and k values for both activation and inactivation then remained unchanged. There then followed marked positive shifts in the V* of both INa activation and INa inactivation. The final INa recovery to pretreatment values, was then accompanied by more gradual negative shifts in the voltage dependence of both INa activation and INa inactivation, with control Imax, and V* values regained at 26–40 and 41–55 min respectively. These changes in Imax and V* contrasted with the constant k values through these manoeuvres. Furthermore, they were abolished, as described fully below, by dantrolene-mediated RyR block.Figure INa changes. Following the initially decreased peak INa within 2 min described above  Their demonstration of an increased INa in the presence of dantrolene alone complements the demonstrations of increased INa produced by 2 mM caffeine in suggesting some background inhibition of INa at even background resting [Ca2+]i.These control experiments thus demonstrate that (a) the RyR inhibitor dantrolene abrogated the contrasting actions of either 0.5 mM or 2.0 mM caffeine in transiently decreasing or increasing v1.4 channel function that would thereby modify surface membrane excitability in skeletal muscle in vivo. This mechanism would complement feedforward, excitation-contraction-coupling, processes by which Nav1.4-mediated surface membrane depolarisation triggers RyR-mediated release of intracellularly stored sarcoplasmic reticular (SR) Ca2+. Feedback alterations in Nav1.4 availability and/or voltage sensitivity are potentially physiologically important particularly under conditions of intense and prolonged muscle activation including tetanic stimulation and metabolic exhaustion. The latter changes could compromise SR Ca2+-ATPase activity through ATP depletion, thereby elevating [Ca2+]i. Any consequent effects of Ca2+ on Nav1.4 function would complement its other actions promoting slow RyR inactivation19 and increasing sarcolemmal Ca2+-activated large conductance K+ channel (KCa1.1) conductance in downregulating excitation-contraction coupling23. Such feedback mechanisms would also bear on clinical conditions affecting skeletal muscle excitability. K+-aggravated myotonia is associated with slowed INa kinetics and impaired INa inactivation that accompany Nav1.4 mutations in its EF-hand-like domain24. Other genetic, K+ and cold-aggravated myotonias are associated with reduced Ca2+-dependent Na+ channel inhibition similarly attributed to Nav1.4 mutations6. Finally, the weakness observed in dystrophic muscle is associated with elevated [Ca2+]i25.The present experiments explored for the existence of physiologically effective feedback loops between ryanodine receptor (RyR) activation by tubular depolarisation and Na2+ release by the RyR agonist caffeine14 on both Nav1.4 channel availability and its voltage-dependent activation and inactivation properties in intact skeletal myocytes. They utilised previously established contrasting effects of different applied caffeine concentrations on spectrofluometrically measured resting [Ca2+]i16. Low concentrations of caffeine (0.5 mM) then increased [Ca2+]i persistently over the subsequent 3–10 min recording intervals in isolated rabbit skeletal SR or murine skeletal muscle fibre preparations15. In contrast, ≥1.0 mM caffeine induced early [Ca2+]i peaks rapidly followed by [Ca2+]idecreases below even resting-levels within 80–90 sec17.The experiments here examined effects of direct pharmacological manipulation of RyR-mediated SR Ca+ currents, INa were measured in intact mammalian gastrocnemius skeletal muscle using the loose patch clamp technique. This avoided membrane disruption and perturbations of [Ca2+]i homeostasis inherent in other experimental measurement procedures involving conventional cell-attached patch clamp studies, which additionally often include the Ca2+-sequestrating EGTA in the pipette solutions. Furthermore, the latter can require study of isolated or cultured cells with enzymatically treated membranes as opposed to the intact in situ skeletal myocytes examined here. The loose-patch configuration also permitted sequential and multiple recordings with the same standardized pipette in successive patches before and after pharmacological challenge as required in the present experiments26. The features of the resulting INa concurred with those obtained on earlier occasions26 and with other voltage clamp methods applied to mammalian skeletal muscle27. The magnitude, voltage-dependence and quantified activation  and inactivation properties of INa were compared at different times following introducing caffeine. Further experiments blocking these effects with further applications of the specific RyR inhibitor dantrolene attributed the observed changes specifically to altered RyR-mediated SR Ca2+ release.Here, NaINa closely paralleled the previously reported [Ca2+]i changes following caffeine challenge. Thus, INa was reduced early (1–2 min) following either 0.5 or 2 mM caffeine challenge. This effect was abrogated by reducing access of caffeine to tubular and therefore tubular-sarcoplasmic reticular triad regions in which RyRs are concentrated, in regions within the patch from which the recordings were obtained. The latter was accomplished by establishing patch seals before rather than after introduction of caffeine to the extracellular space. This would alter direct access of caffeine over a surface membrane area close to the 30 μm2 cross sectional area of the electrode tip in contact with the membrane within which INa was recorded.The observed contrasting alterations in INa followed by their recovery to pretreatment levels. These findings corroborate and extend previous independent reports that INa was reduced by increased RyR activity following 8-CPT induced Epac pathway activation13. The INa changes were quantified by systematically exploring a consistent range of V1 test voltages thereby deriving families of INa records. These in turn yielded activation, I-V, and inactivation curves giving Imax, V* and k values. These respectively quantified total available INa and the position and steepness of their voltage dependences. Sustained reductions in Imax induced by 0.5 mM caffeine paralleled the sustained [Ca2+]i increases previously reported following caffeine mediated activation of RyR-mediated SR Ca2+ release15. The increased INa observed in the presence of 2 mM caffeine similarly paralleled reported contrastingly decreased [Ca2+]i attributed to slow RyR inactivation17. The latter findings further suggested significant inhibition of INa by even normal background [Ca2+]i.Subsequent recordings following the 0.5 and 2 mM caffeine challenge obtained by identical pulse protocols respectively demonstrated contrasting increases and decreases in peak INa were accompanied by time-dependent changes in properties of the I-V and inactivation curves. With 0.5 mM caffeine challenge, the initial decreases in INa were accompanied by negative shifts in inactivation V* over 1–10 min. This was followed, over 11–25 min, by positive shifts in both INa activation and INa inactivation V*. The final INa recovery to pretreatment values between 26–55 min was accompanied by opposite, negative, shifts in activation and inactivation V*. With 2 mM caffeine challenge, the contrasting, declining phases of the transient INa increases accompanied delayed transient negative shifts in both activation and inactivation V*. In contrast, values of k of either activation or inactivation little changed throughout these protocols.These alterations in total available 2+ affinities of RyR activation sites21. Previous reports had demonstrated that it inhibited caffeine-induced increments in mammalian SR Ca2+ permeability28 and caffeine-induced contractures in murine diaphragm muscle29.Finally, the effects of the specific RyR inhibitor dantrolene were investigated both by itself and in combination with identical caffeine concentrations and pulse protocols. Dantrolene specifically stabilises RyR closed states by selectively decreasing CaImax, or activation or inactivation INa or V*. In contrast, dantrolene by itself produced only small increases in Imax without affecting activation and inactivation, V* and k values. Together with the increased INa produced by 2 mM caffeine this latter finding suggests that [Ca2+]i exerts significantly inhibits INa even at background RyR activity. These explorations using dantrolene thus trace all the present actions of caffeine on INa specifically to its effects on RyR-mediated SR Ca2+ release.In the present experiments dantrolene abrogated all the effects of either 0.5 or 2 mM caffeine, whether on Imax and V* combined with previously reported [Ca2+]i changes under similar experimental conditions17 were incompatible with straightforward combined negative shifts in Na+ channel activation and inactivation V* resulting from simple screening effects arising from an increased [Ca2+]i31. However, they directly correlate with previous in vitro biochemical and biophysical reports on skeletal muscle Nav1.4 and RyR. They establish for these a physiological context, possibly occurring within a common microdomain, in working muscle, as opposed to in vitro systems for the first time.These findings together demonstrate and characterise RyR-Nav1.4 feedback phenomena complementing their established feedforward interactions underlying excitation contraction coupling Fig. . The obsINa reductions with 0.5 and 2 mM caffeine, persistent with 0.5 mM caffeine, were consistent with the reported effects of either direct Ca2+-binding to a Nav1.4 EF hand site or indirect Ca2+ binding to Nav1.4 via its binding to CaM in reducing INa  positive shifts in INa activation and inactivation V* directly paralleled reported reductions in FKPB12 binding affinity for RyR following RyR activation. An increased free [FKBP12]i would then accompany the resulting SR Ca2+ release elevating [Ca2+]i. in intact fibres10; FKBP12 is known to positively shift V* activation and inactivation in Nav1.511, thought closely homologous to Nav1.44. This action is probably indirect and delayed: co-immunoprecipitation assays appeared to exclude direct FKBP12–Nav1.5 interactions11. Fourthly, negative shifts in both INa activation and inactivation V* values eventually accompanied the reductions in INa and recoveries from INa elevation, 26–55 or 16–40 min following 0.5 and 2.0 mM caffeine challenge respectively. These were compatible with a subsequent, previously reported, slow RyR inactivation19. This would reduce RyR open channel probabilities and Ca2+ release whilst permitting continued SR Ca2+ re-uptake and possible surface membrane Ca2+ expulsion by Na+/Ca2+ exchange33. These would reverse the increases in [Ca2+]i and [FKBP12]i19  was used in all experiments. Chemical agents were purchased from Sigma-Aldrich  unless otherwise stated. The following variants of the basic KH solution containing caffeine and/or dantrolene including 0.1% dimethyl sulfoxide vehicle were prepared and used in experiments: (1) KH + 0.5 mM caffeine, (2) KH + 2.0 mM caffeine, (3) KH + 10 µM dantrolene, (4) KH + 10 µM dantrolene + 0.5 mM caffeine, and (5) KH + 10 µM dantrolene + 2.0 mM caffeine. Solutions were filtered to remove particles greater than 10 μm in diameter with standard filtration paper .Experiments were performed under the Animals (Scientific Procedures) Act 1986 Amendment Regulations 2012 following ethical review by the University of Cambridge Animal Welfare and Ethical Review Body (AWERB). C57BL6 WT mice aged 3–6 months were used in all experiments. Mice were housed at a stable temperature of 21 °C in a licensed facility, fed sterile rodent chow  and subjected to 12-hour light/dark cycles with free access to water, bedding and environmental stimuli. Krebs–Henseleit (KH) solution  Act 1986. The gastrocnemius and soleus muscles were then immediately isolated, with the distal (Achilles) tendon secured by a knot to a ligature and the proximal tendon still attached to its bony origin. Overlying connective tissue was excised to allow access to the myocyte surface. The isolated muscles were then immediately placed in a Sylgard-bottomed experimental bath containing KH solution with the soleus muscle facing down maximizing electrode access to gastrocnemius fibres. Experiments were carried out at stable temperatures around 20 °C. The muscle was secured in a taut and fixed position by pinning down the bony origin and the ligature with A1 insect pins Fig. .Loose patch clamp pipettes were pulled from borosilicate glass capillaries  using a Brown–Flaming microelectrode puller . They were mounted under a microscope with ×250 magnification with a calibrated eyepiece graticule, and a small indentation made on one side of the pipette using a diamond knife. A transverse force was then applied distal to the indentation, giving a fracture perpendicular to the long axis of the pipette. Squarely-broken tips were then fire‐polished under visual guidance at ×400 magnification using an electrically-heated nichrome filament to smooth the edges of the tip. The internal diameters of the pipettes were measured under ×1000 magnification. Pipettes of internal tip diameters between 28–38 µm were used in the experiments. Such loose patch recordings thus provide a complete representation of the properties of both surface and tubular membrane directly beneath these seals. Finally, the pipettes were bent at about 1 mm from the tip to make a 45° angle with the pipette’s long axis. This allowed the tips to contact the muscle surface at 90° when mounted at an angle of 45° below the horizontal on the recording amplifier head stage Fig. . The pip26. The Ag/AgCl electrode within the pipette both measured the intra-pipette potential relative to the ground reference potential of the bath and passed the clamp current.The pipette was lowered onto the gastrocnemius muscle membrane surface and gentle suction applied to allow seal formation around the resulting membrane patch. The central regions of the muscle , measured current (in nA) and pipette diameter (in µm):Experiments were initially performed with the muscle preparation immersed in control KH solution. Around 2–3 different patches were investigated using the complete set of pulses within 20 minutes after transferring the muscle preparation into the bath. Only muscles that gave stable control results were further studied in the presence of pharmacological challenge. For each pharmacological condition, data from patches collected from at least 3 muscle preparations were used. To achieve conditions comparable to previous studies on ). The inactivation I-V curves were fitted to a similar Boltzmann function relating the peak current density I2 for the V2 step following any given V1 excursion to its maximum value (Imax), the voltage at half maximal current (V*), the excursion of the V1 step (V) and a steepness factor (k): I2 = Imax(1 − 1/[1 + exp{(V* − V)/k}]).The resulting peak Imax, V* and k as obtained from each patch averaged to give means ± SEM for currents obtained within successive time intervals following introduction of pharmacological agents. Curve fitting procedures of the I-V curves used fitting algorithms in the open source programme SciDAVis (Version 1.25-mac). Derived parameters obtained under different conditions were tested for significant differences to a P < 0.05 significance level by ANOVA with a post-hoc Dunnett’s test.Successive families of ionic currents revealed alterations in current characteristics with time following caffeine challenge. These are represented by displaying currents, current-voltage curves and computed values of This research has been regulated under the Animals (Scientific Procedures) Act 1986 Amendment Regulations 2012 following ethical review and approval by the University of Cambridge Animal Welfare and Ethical Review Body (AWERB). All procedures were completed by Home Office-licensed personnel and fell within the scope of Schedule 1 of the UK Animals (Scientific Procedures) Act (1986).


Up to 18% of patients with acute myeloid leukemia (AML) present with a white blood cell (WBC) count of greater than 100,000/µL, a condition that is frequently referred to as hyperleukocytosis. Hyperleukocytosis has been associated with an adverse prognosis and a higher incidence of life-threatening complications such as leukostasis, disseminated intravascular coagulation (DIC), and tumor lysis syndrome (TLS). The molecular processes underlying hyperleukocytosis have not been fully elucidated yet. However, the interactions between leukemic blasts and endothelial cells leading to leukostasis and DIC as well as the processes in the bone marrow microenvironment leading to the massive entry of leukemic blasts into the peripheral blood are becoming increasingly understood. Leukemic blasts interact with endothelial cells via cell adhesion molecules such as various members of the selectin family which are upregulated via inflammatory cytokines released by leukemic blasts. Besides their role in the development of leukostasis, cell adhesion molecules have also been implicated in leukemic stem cell survival and chemotherapy resistance and can be therapeutically targeted with specific inhibitors such as plerixafor or GMI-1271 (uproleselan). However, in the absence of approved targeted therapies supportive treatment with the uric acid lowering agents allopurinol and rasburicase as well as aggressive intravenous fluid hydration for the treatment and prophylaxis of TLS, transfusion of blood products for the management of DIC, and cytoreduction with intensive chemotherapy, leukapheresis, or hydroxyurea remain the mainstay of therapy for AML patients with hyperleukocytosis. Acute myeloid leukemia (AML) is characterized by the clonal proliferation of hematopoietic stem cells (HSC) leading to bone marrow failure as well as complications related to the excess production of undifferentiated myeloid blasts such as leukostasis, tumor lysis syndrome (TLS), and disseminated intravascular coagulation (DIC). ,2 The raFLT3-ITD mutations, and abnormalities involving the MLL gene on 11q23 have been identified as risk factors for hyperleukocytosis [Hyperleukocytosis in AML has been previously associated with various cytogenetic and molecular characteristics. Monocytic and myelomonocytic AML subtypes as defined by the French-American-British (FAB) classification (AML M4 and M5) and patients with ocytosis ,16,17. Hocytosis . Especiaocytosis ,19,20,21The HSC niche in the bone marrow consists of stromal cells, HSCs, and various stages of lineage-specific hematopoietic progenitor cells that interact via complex signaling mechanisms ,23,24. IA major limitation to studying the HSC niche is the inability to maintain and expand murine HSC cultures over extended periods of time . FurtherDicer 1, a gene coding for an RNA-processing endonuclease in bone progenitor cells, resulted in the development of a MDS-like phenotype in mice that occasionally progressed into AML [Ptpn11 in bone marrow osteoblasts and mesenchymal cells have been implicated in leukemogenesis [The role of the HSC niche in AML development has been speculated for decades ,28. Acquinto AML . Similarogenesis ,36. Addiogenesis ,37,38. Hogenesis ,40,41. Aogenesis ,28.+ HSCs during leukapheresis for stem cell transplantation, VLA-4 could also play a role in chemotherapy resistance and LSC survival [FLT3-ITD mutated blasts and VLA-4. In vitro data suggest that upregulation of integrins reduced the sensitivity of FLT3-ITD-mutant cells to sorafenib via the phosphatidylinositol 3-kinase (PI3K)/Akt/glycogen synthase kinase-3 beta (GSK3β) pathway leading to enhanced β-catenin activation [The mechanisms by which leukemic blast mobilization from and homing to the bone marrow occurs remain incompletely defined. HSCs express and interact with bone marrow stromal cells via various cell adhesion molecules that belong to the integrin receptor superfamily such as VLA-4, VLA-5, and LFA-1 which act as receptors for fibronectin and intracellular adhesion molecule-1 (ICAM-1) ,43. Othesurvival ,47. The survival . As a prsurvival ,50. Supptivation . AdditioRUNX1-RUNX1T1 fusion gene leading to an enhanced migration into the peripheral blood [FLT3-ITD, NPM1, DNMT3A, NRAS, CEBPA, and TET2 mutations and the presence of hyperleukocytosis was independently associated with an adverse prognosis [NPM1 and FLT3 mutations and AML M4 and M5 subtypes by FAB classification; all of which have been previously associated with a higher risk of hyperleukocytosis [Under physiologic conditions mobilization of HSCs is regulated by cytokines, circadian rhythm, and hormonal signals ,53. Seveal blood . Furtherrognosis . Howeverocytosis ,56,57. TAdhesion of leukocytes to the endothelium is a prerequisite for the recruitment of leukocytes to sites of inflammation and is mediated by the interaction between selectins  expressed on endothelial cells and leukocytes and integrins on leukocytes as well as several cell adhesion molecules  ,61. The Leukostasis has been described as a life-threatening complication of hyperleukocytosis in AML for decades. It has been noted that the extent of hyperleukocytosis in AML does not necessarily correlate with the likelihood of developing leukostasis which suggests specific molecular interactions between AML blasts and endothelial cells leading to leukostasis ,62,63. TGiven the high mortality associated with leukostasis and the need for urgent cytoreduction in the absence of a diagnostic gold standard, predictive scores to supplement clinical assessment have been developed . AcknowlAML blasts have been shown to express various cell adhesion molecules such as CD11, CD49, PSGL-1, and L-selectin with significant differences between various subtypes of AML blasts ,19,43,67Besides adhesion of leukemic blasts to the endothelium, other mechanisms of tissue damage in leukostasis include cytokine- and matrix-metalloproteinase (MMP)-mediated endothelial damage, leukemic blast extravasation into tissues and local hypoxia among others  9,73,7474,75. ThOvert disseminated intravascular coagulation (DIC) has been reported in up to 32% of patients with non-APL AML ,77,78. DRisk factors for the development of DIC in non-APL AML patients include an elevated WBC count, high levels of C-reactive protein, CD13- and HLA-DR-negativity, and 11q23 abnormalities ,77. BecaAML patients with hyperleukocytosis are at risk for spontaneous TLS and even more so after the initiation of cytoreductive therapy. TLS is characterized by hyperuricemia, hyperphosphatemia, hypocalcemia, and hyperkalemia because of the rapid destruction of tumor cells which can be complicated by renal failure due to urate crystal deposition in the kidneys and cardiac arrhythmias . Based oReports on the incidence of TLS in AML patients vary substantially because of the various definitions and implementation of preventive measures with rates of TLS of 3–17% having been reported ,92,93. HSeveral scoring systems have been proposed to predict the likelihood of developing TLS in patients receiving induction chemotherapy ,95. HoweHyperleukocytosis, especially if associated with leukostasis, constitutes a hematologic emergency that warrants urgent cytoreduction. Options for cytoreduction include mechanical removal of leukemic cells via leukapheresis and pharmacologic cytoreduction with hydroxyurea or intensive chemotherapy ,16. BecaFLT3-ITD mutations has been associated with a higher incidence of hyperleukocytosis and those agents could therefore be a valuable adjunct in those patients [Since 2017 the treatment landscape of AML has significantly expanded with the approval of nine novel agents in various settings in the United States ,99. Amonpatients . Additiopatients ,105.MLL-AF9 fusion oncogene that AML blasts exhibit an increased E-selectin-binding potential that enabled retention of AML blasts within the bone marrow and could contribute to chemotherapy resistance [Sele−/−) further supporting the mechanism of action of GMI-1271 via E-selectin inhibition [Unfortunately, randomized clinical trials specifically enrolling patients with leukostasis are exceedingly challenging to conduct given the rarity and acuity of the clinical presentation. However, targeting the interaction between LSC and selectins on endothelial cells is an area of active research as upregulation of E-selectin on endothelial cells has been associated with LSC survival and chemotherapy resistance ,111. Inhsistance . Furthersistance . Similarhibition . Additiohibition ,112. In hibition . This lehibition . With a hibition . Importahibition ,115. Simhibition ,117,118.hibition ,119. Howhibition . AlthougAcute lung injury secondary to leukostasis is a common complication in AML patients presenting with hyperleukocytosis and especially in patients with acute monocytic leukemia (AML FAB-M5) ,121,122.FLT3-ITD mutations has been associated with a higher incidence of hyperleukocytosis, those agents can be a valuable adjunct in those patients. Additionally, advances in supportive care and aggressive management of TLS and DIC with uric acid-lowering agents such as rasburicase, renal replacement therapy, and transfusion of blood products have led to a decrease in the mortality rate during AML induction although specific data from patients with hyperleukocytosis and leukostasis are missing [Finally, advances in targeted therapies with the approval of the FLT3 inhibitors gilteritinib and for R/R-AML and midostaurin in the frontline setting in addition to cytarabine-anthracycline induction chemotherapy, respectively, have enabled a personalized approach to the treatment of AML patients ,104. As  missing ,124.Hyperleukocytosis in AML is associated with excess mortality and higher rates of leukostasis, DIC, and TLS compared to patients without hyperleukocytosis. On a molecular level the processes leading to hyperleukocytosis and leukostasis are still incompletely understood but the several key targets mediating the interaction between the leukemic blast and endothelial cells in the peripheral circulation continue to be identified. Additionally, translational studies investigating the interaction of AML blasts and the bone marrow microenvironment could lead to a better understanding of the processes underlying leukostasis. However, one needs to be mindful of the limitations of extrapolating those results to the clinical setting. Nonetheless, an understanding of these molecular mechanisms is crucial for the rational development of novel targeted agents with GMI-1271 (uproleselan) and plerixafor currently being studied in clinical trials. Because of the rarity and emergent nature of this presentation, clinical trials specifically enrolling AML patients with hyperleukocytosis have proven to be very difficult to conduct and patients with hyperleukocytosis have either been excluded from recent randomized trials or results have not been reported separately for this high-risk patient population. Especially regarding the management of leukostasis, the evidence base is limited and mixed; although leukapheresis has not been shown to be associated with improved survival in several recent retrospective studies and meta-analyses. While awaiting novel therapies, cytoreduction with intensive chemotherapy, leukapheresis, and/or hydroxyurea as well as excellent supportive care of TLS and DIC remain the mainstay of therapy and physicians need to be mindful of the gaps in the evidence base for these interventions.


Nanoparticles were characterized by XRD, TEM, SEM, HR-TEM, FTIR, Raman, Thermogravimetry, inductively coupled plasma spectroscopy, thermoanalysis, dynamic light scattering, electrophoretic mobility, and fluorescence spectroscopy. They displayed ill-defined isometric morphologies with sizes ≤50 nm, hydration number n ~ 0.9, tailored Ca2+ content (0.42–8.11 wt%), and long luminescent lifetimes (800–2600 µs). Their relative luminescence intensities in solid state are neither affected by Ca2+, citrate content, nor by maturation time for Ca2+ doping concentration in solution below 0.07 M Ca2+. Only at this doping concentration does the maturation time strongly affect this property, decreasing it. In aqueous suspensions, neither pH nor ionic strength nor temperature affect their luminescence properties. All the nanoparticles displayed high cytocompatibility on two human carcinoma cell lines and cell viability correlated positively with the amount of doping Ca2+. Thus, these nanocrystals represent promising new luminescent nanoprobes for potential biomedical applications and, if coupled with targeting and therapeutic moieties, they could be effective tools for theranostics.Luminescent lanthanide-containing biocompatible nanosystems represent promising candidates as nanoplatforms for bioimaging applications. Herein, citrate-functionalized calcium-doped terbium phosphate hydrate nanophosphors of the rhabdophane type were prepared at different synthesis times and different Ca Indeed, the growth morphology of a nanocrystal results from the interplay between the growth rates of its outermost crystallographic planes and the growth-inhibiting/promoting effect of the additives. The raphdophane structure of the LnPOtructure ,20,57. C2+ doping the cit-Ca+:TbPO4·nH2O structure, we conclude that Ca2+has limited inhibiting/promoting effect on the growth of these nanocrystals, against that of citrate, in contrast to what found in cit-Ca2+:EuPO4·nH2O  direction, and leading to the formation of nanocrystals with lower aspect ratios than those formed in additive-free solutions. As we obtain basically the same crystal morphology irrespective of the amount of CaPO4·nH2O . On the PO4·nH2O . Moreoveapatites .2+ content when x is lower than 0.07 M Ca2+, as occurred on cit- Ca2+:EuPO4·nH2O, while the R.L.I. at x = 0.07 M Ca2+ is high at short maturation time (due to the amorphous nature of the samples) and low at high maturation time . Concerning the luminescence properties in aqueous dispersion, neither pH nor ionic strength nor temperature affect their luminescence properties at physiological range.Finally, the luminescence study of these nanoparticles in solid phase indicate that R.L.I. is affected by neither the maturation time nor the Ca2+:TbPO4·nH2O nanocrystals free of vaterite or with negligible amount of this phase, with enough and exploitable luminescent properties (R.L.I and luminescent lifetimes) and higher stability of their aqueous suspensions at the pH range of biological interest (5–7.4), are when using Ca2+ doping concentration x = 0.01 M and x = 0.03 M, at any of the maturation times from 4 to 96 h.According to all the above considerations the optimal conditions to synthesize cytocompatible cit-CaSumming up, all the above properties are positive relative to the applications of these nanoparticles as luminescent labeling agents and highlight the potential of the thermal decomplexing synthetic method to prepare this kind of nanophosphors.2+:TbPO4·nH2O nanocrystals of the hexagonal rhabdophane phase, with n ~ 0.9 and tailored Ca2+ content between 0.42 and 8.11 wt% were successfully obtained by thermal decomplexing of Ca2+/Tb3+/citrate/phosphate/carbonate solutions. These nanocrystals of about 29–37 nm are mainly monodomains and display basically isometric ill-defined morphologies, with Ca2+ having limited inhibiting/promoting effect on the growth of these nanoparticles, but enhancing the amount of citrate adsorbed on the surface in dose-depending manner. All the nanoparticles displayed high cytocompatibility on two human carcinoma cell lines and cell viability correlated positively with the amount of doping Ca2+.Cit-Ca2+doping concentration. Their relative luminescence intensities in solid state (around 200 units) are affected by neither Ca2+, nor citrate content, nor maturation time for Ca2+ doping concentrations below 0.07 M Ca2+. Only at this doping concentration the maturation time strongly affects R.L.I, decreasing it. At low maturation times  it is high (around 650 units). However, at 96 h, it strongly decreases up to ~40. This finding was related to the amorphous nature of the precipitates obtained at 4 and 24 h, whereas at 96 h, the precipitates were crystalline. In addition, this sample also contains vaterite and a higher percent weight of citrate than the samples obtained with lower doping concentrations of Ca2+, and therefore it presents a lower percent weight of emissive material. In aqueous suspensions, neither pH nor ionic strength nor the temperature affect their luminescence properties.Luminescence properties of the nanocrystals reveal that luminescent lifetimes increase (between ~800 and ~2600 µs) by increasing the Ca2+:TbPO4·nH2O nanocrystals free of vaterite or with negligible amount of this phase, with enough and exploitable luminescent properties (R.L.I and luminescent lifetimes) and higher stability of their aqueous suspensions at the pH range of biological interest (5–7.4), are when using Ca2+ doping concentration x = 0.01 M and x = 0.03 M, at any of the maturation times from 4 to 96 h.According to all the above considerations, the optimal conditions to synthesize cytocompatible cit-CaSumming up, the above properties are encouraging relative to the potential bioimaging applications of the nanocrystals. They can thus represent promising new luminescent nanoprobes for such applications and, if coupled with targeting and therapeutic moieties, they could be effective tools for theranostics.


Acute myeloid leukemia (AML) disrupts the generation of normal blood cells, predisposing patients to hemorrhage, anemia, and infections. Differentiation and proliferation of residual normal hematopoietic stem and progenitor cells (HSPCs) are impeded in AML-infiltrated bone marrow (BM). The underlying mechanisms and interactions of residual hematopoietic stem cells (HSCs) within the leukemic niche are poorly understood, especially in the human context. To mimic AML infiltration and dissect the cellular crosstalk in human BM, we established humanized ex vivo and in vivo niche models comprising AML cells, normal HSPCs, and mesenchymal stromal cells (MSCs). Both models replicated the suppression of phenotypically defined HSPC differentiation without affecting their viability. As occurs in AML patients, the majority of HSPCs were quiescent and showed enrichment of functional HSCs. HSPC suppression was largely dependent on secreted factors produced by transcriptionally remodeled MSCs. Secretome analysis and functional validation revealed MSC-derived stanniocalcin 1 (STC1) and its transcriptional regulator HIF-1α as limiting factors for HSPC proliferation. Abrogation of either STC1 or HIF-1α alleviated HSPC suppression by AML. This study provides a humanized model to study the crosstalk among HSPCs, leukemia, and their MSC niche, and a molecular mechanism whereby AML impairs normal hematopoiesis by remodeling the mesenchymal niche.  The hematopoietic system is a conglomerate of multiple lineages with the hematopoietic stem cell (HSC) as common origin . Under nDespite having an extensive vascular network, the BM niche is characterized by low oxygen pressure (or hypoxia) . HIFs foIt was shown in mouse AML models that AML cells extensively reorganize the cellular, physical, and transcriptional niche architecture to facilitate their own expansion –27 and pp19, p21, and Egr1–3 (Our previous work on AML patient samples and patient-derived xenograft (PDX) models demonstrated an enrichment of HSCs at the expense of less-primitive progenitors among the remaining normal hematopoietic cells in the BM . Consistd Egr1–3 . These oThe molecular mechanisms governing the suppression of normal hematopoiesis are poorly understood but could provide insight into HSC regulation. Several lines of evidence from murine studies suggest an indirect mechanism via a dysregulated BM niche , 35, butIn this study, we modified 2 established fully humanized hematopoietic niche systems on the basis of MSC coculture to investigate the multidirectional crosstalk among AML, HSPCs, and the microenvironment. HSPCs recapitulated reversible proliferation and differentiation inhibition by AML cells, which was linked to transcriptional and secretome alterations of the stromal niche. Further investigation and functional validation identified stanniocalcin 1 (STC1) and its transcriptional regulator HIF-1α as niche-specific negative regulators of HSPC proliferation.+ cells cultured with healthy donor MSCs alone  or together with AML cell lines (+AML cell lines) for 4 days  (https://doi.org/10.1172/JCI133187DS1) showed a significant increase in CTVbright progenitors and HSPCs in groups with AML cells, indicating that these cells undergo fewer divisions  . Similarivisions . These d+ cells in the +AML groups adopted a more immature HSPC phenotype (CD34+CD38–) than CD34+ cells alone, indicating a potential differentiation defect   in CD34ary AML) . Similarary AML) , suggestary AML) . Howevernificant . These d+ cells alone or with AML cell lines or AML patient samples, and maintained in NSG-S mice for up to 8 weeks (+) from leukemic cells, we either lentivirally transduced CD34+ cells to express GFP  but not with CB CD34+ cells  .We confirmed a 3- to 4-fold increase in 4+ cells . STC1 le4 ng/mL) . STC1 mRTo validate the presence of STC1 in primary AML samples, we determined the level of STC1 in BM plasma of 27 primary AML samples at diagnosis and 21 samples at the time of remission . The lev+ cell–MSC coculture  in the CB-derived CD34ture see . rSTC1 fture see  without ture see . Gene setors Myc  and E2F tors Myc  and mTORtors Myc , and shotors Myc . In agretors Myc , and ledtors Myc  and a hi including STC1, we compared MSCs cultured under normoxia and hypoxia and MSCs cocultured with CB-derived CD34+ cells or AML in normoxic condition. Interestingly, coculture of MSCs with AML induced a transcriptional response similar to that of MSCs under hypoxic conditions   , which w+ cells) . These dSevere cytopenias are a prominent clinical feature of AML independent of blast percentage in the BM, and major contributors to morbidity and mortality in patients with leukemia . Data frIn this study, we generated 2 humanized mesenchymal niche model systems, and in agreement with our previous report on AML patient samples , we obseWe show that HSPC suppression is multifactorial and potentiated via the mesenchymal niche. Indeed, factors secreted from the MSCs in contact with leukemic cells play major roles in HSPC malfunction, although for certain AML samples, direct and contact-dependent effects were also observed.Using a transcriptomic approach, we aimed to identify common niche-derived factors and pathways. Besides STC1, we detected previously annotated regulators of HSPCs, but we focused on STC1 for its potent role in solid cancers –74 and aOur functional and transcriptomic analysis suggested that addition of recombinant hSTC1 retained HSPC quiescence and stemness. Further analysis of the role of MSC-secreted STC1 factor in human HSC regulation might provide additional insights into adult HSC biology in the context of disease, stress, and aging.We further confirmed elevated levels of STC1 in BM and PB plasma of AML compared with healthy control samples and demonstrated that STC1 levels decreased during remission. It might be of interest to analyze the potential correlation between STC1 level in AML samples and cytopenic markers in a larger patient cohort.Upregulation of hypoxia-regulated factors occurred rapidly in MSCs when cocultured with AML, but this is not maintained after leukemia cells are withdrawn from the culture, potentially explaining why STC1 and HIF-1α have not been identified in AML patient–derived MSCs , 35. HypIn summary, our study provides not only humanized models to study the crosstalk among HSPCs, leukemia, and their MSC niche, but also a mechanistic insight into the reversible suppression of HSCs in leukemic hosts, via remodeling of the MSC niche. Clinically, HIF-1α inhibition or more specifically STC1 neutralization represents a possible route to promote residual normal hematopoiesis and thus alleviate cytopenia in AML. This may be particularly relevant for older adults not determined to be tolerant of intensive therapy or for patients with adverse cytogenetic factors for whom chemotherapy is unlikely to produce a long-lasting response.Further information can be found in 2 (or 3% O2 for hypoxia conditions). The murine stroma cell line (MS-5) was purchased from LGC Standards. For some experiments, AML cell lines were transduced with lentivirus containing EGFP or a iCASP9  profiling using the PowerPlex 16HS system (Promega). All cells were grown as suggested by the suppliers at 20% Oa iCASP9 .AML patient samples (PBMCs and/or BM) were collected at diagnosis from patients at St Bartholomew’s Hospital, London. Plasma (from PB or BM) was obtained from patients at diagnosis or remission at the Royal Marsden Hospital. Details of patient samples are provided in Umbilical CB (UCB) samples were obtained after normal full-term births. Mononuclear cells (MNCs) from 2 to 5 UCB collections were pooled and purified by Ficoll-Paque density centrifugation , followed by ammonium chloride red cell lysis. Density-separated CB MNCs were magnetically sorted for CD34 positivity via the EasySep Human CD34 Positive Selection Kit (STEMCELL Technologies) according to the manufacturer’s instructions.–, hCD31–, hCD90+, hCD73+, and hCD105+.Primary hMSCs were purchased (Lonza) or provided by Christine Dosquet  from human BM obtained during orthopedic surgery. hMSCs were grown in α-MEM and 10% hMSC-specific FBS (Gibco-Life Technologies) and used at low passages . A total of 3 samples were used throughout this study. The phenotype of hMSCs was hCD454/cm2 MSCs were seeded in tissue culture–treated plates  containing 285 μL/cm2 α-MEM supplemented with 10% MSC-FBS and 100 U/mL penicillin/streptomycin . On day 2, UCB CD34+ cells alone or together with AML were added to the hMSC culture at 0.4 × 105 to 1 × 105 CD34+ cells/mL and 1 × 105 to 8 × 105 cells/mL (AML patient samples), or 2 × 105 cells/mL (AML cell line) were added in MyeloCult H5100 (STEMCELL Technologies). Fresh media top-up was performed after 4 days. AML cell lines were lentivirally transduced to express GFP to distinguish non-leukemic CB CD34+ cells from AML cell lines.All culture of HSPCs/AML patient samples was carried out on primary hMSCs unless stated otherwise. On day 0, 2 × 10+ cells were cocultured with AML cell lines and had to be re-isolated for functional analysis, 0.4-μm polycarbonate Transwell inserts (Corning) were used to separate AML cell lines from MSCs and CD34+ cells. Cells in the well were grown in 500 μL media or 200 μL for the Transwell. For comparison between direct contact and Transwell, CD34+ cells were grown in the Transwell and MSCs and AML cell lines at the bottom of the well for 4 days.When CD346 to 2 × 106 cells/mL. Subsequently, the medium was replaced with MyeloCult H5100 containing 20 ng/mL SCF. UCB CD34+ cells were sorted based on HLA-A2 mismatching between UCB and AML samples.For AML patient samples, AML cells were thawed and allowed to recover for 48 hours in the MyeloCult H5100 supplemented with IL-3, G-CSF, and TPO  at densities of 0.5 × 10For STC1 neutralization experiments, 1 μg/mL blocking antibody (Santa Cruz Biotechnology) or equivalent species IgG was added at the beginning of the experiment.+ HSPCs were seeded at 0.04 × 105 to 0.1 × 105/mL.MSCs were cocultured for 5 days with AML iCasp9 cell lines, then treated with 5 nM of AP1903 (Insight Biotechnology). Twenty-four hours after the addition of AP1903, all apoptotic and dead AML cells were washed away, and CD346/mL in PBS with 5 μM CTV or CFSE (Thermo Fisher Scientific) and incubated for 10 minutes at 37°C. Then, cells were washed twice, and the pellet was resuspended in the desired medium.Cells were suspended at 1 × 10For cell cycle analysis, cells were fixed and permeabilized with a Fix/Perm kit according to the manufacturer’s instructions and stained with 2 μL anti–Ki-67–APC (BD Biosciences). Staining was performed overnight at 4°C. Cells were washed in 2% FBS/PBS and resuspended in 300 μL of 2% FBS/PBS with 1 μg/mL DAPI.For EdU analysis, 200 μL of 10 mM EdU-PBS solution was injected i.p. 16 hours before the mice were culled. After scaffold harvest and digestion, the cells were fixed and permeabilized according to the Click-iT Plus EdU Alexa Fluor 647 flow cytometry assay kit instructions (Thermo Fisher Scientific). After fixation, cells were stored overnight at 4°C to allow any remaining PFA to evaporate. The next day, cells were stained in Alexa Fluor 647 Click-iT staining cocktail for 30 minutes at room temperature and subsequently analyzed by flow cytometry. As negative control, an aliquot of cells was stained with Click-iT staining cocktail without Alexa Fluor 647 picolyl azide.g for 5 minutes. For plasma samples, cells were removed from plasma by centrifugation for 10 minutes at 1000–2000 g using a refrigerated centrifuge. The supernatant was further centrifuged for 15 minutes at 2000 g to deplete platelets in the plasma sample.STC1 measurements in the media were conducted according to the manufacturer’s instructions using the STC1 ELISA kit (R&D Systems) in cell culture supernatant. Cell culture supernatant was processed by centrifugation at 300 −/− (NSG) mice were originally a gift from Leonard Shultz  and since then have been bred at the Francis Crick Institute Biological Resource facility.NOD/SCID/IL2rγ137Cs source (IBL 637 Gamma Irradiator).Seven days before transplantation, mice were put on acidified water. Twenty-four hours before transplantation, 8- to 12-week-old mice were sublethally irradiated with 3.75 Gy from a + cells and for secondary engraftment, 2 × 106 purified hCD45+ cells (isolated with a human/mouse chimera isolation kit from STEMCELL Technologies) were injected intravenously per mouse.For primary engraftment, 30,000–45,000 purified CD34Engraftment of human cells in the murine BM was assessed 12 weeks after injection. Mice were sacrificed by cervical dislocation and tibia, femur, and ilium dissected bilaterally. BM was harvested by crushing with mortar and pestle, and each mouse was processed individually. Remaining erythrocytes were lysed with 2–3 mL ammonium chloride for 3 minutes at room temperature and remaining MNCs pelleted in 2% FBS/PBS before analysis by FACS.6 cells/mL, and 100 μL (1 × 105 cells) was injected into each scaffold. The scaffolds were cultured in Nunclon Sphera low-attachment 24-well plates (Thermo Fisher Scientific) and MSCs allowed to adhere for 3–5 hours before the addition of media. Scaffolds were then cultured for 3–5 days to allow MSCs to expand. HSPCs (1 × 105 CD34+), AML cell lines (0.1 × 105); and AML patient samples  were injected into these MSC-seeded scaffolds; and medium was replaced with MyeloCult H5100 supplemented with cytokines  when AML patient samples were used. After seeding of the cells in the scaffold, the scaffolds were implanted subcutaneously into nonconditioned NSG mice (maximum 4 scaffolds/mouse).All the experimental procedures were performed as described by Abarrategi et al. . BrieflyAt different end points, scaffolds were harvested and digested in an HBSS digestion solution containing 2% FBS, DNAse I (10 μg/mL), collagenase (1 mg/mL), dispase II (5 mg/mL), and heparin (20 U/mL) at 37°C for 1.5 hours. The reaction was stopped by addition of 3 times the volume of MSC media, and cells were washed twice before resuspension of the pellet and analysis of the cells by FACS.+ cells. Microarray data were analyzed using limma and affy packages in the R environment (R version 3.5.1). Raw CELL files were uploaded with the ReadAffy function, and robust multiarray averaging (RMA) normalization was then performed. A differentially expression statistical analysis was consequently performed in which we set up the experimental design, including conditions and batch effect as factors. Differentially expressed genes were then obtained by contrasting the different conditions using the makeContrasts function. Given the linear model fit, the statistics were computed by empirical Bayes moderation of the standard errors with the eBayes function.Microarray analysis was performed on MS-5 cells cocultured for 5 days with 5 AML cells lines, 2 primary patient samples, or CB CD34+CD38– cells cultured for 48 hours with or without rSTC1. Raw reads were quality and adapter trimmed using cutadapt 1.9.1 . Reagents and resources used in this article are described in P value less than 0.05 was considered significant. Normal distribution was assessed by D’Agostino-Pearson normality test for groups with 8 or more and Shapiro-Wilk test for 3–7 samples. For data sets composed of heterogeneous groups (AML cell lines or patient samples combined into 1 group), nonparametric tests were performed. For 2 paired groups, we used Wilcoxon’s matched-pairs signed-rank test; for 2 unpaired groups, Mann-Whitney U test; for 3 or more paired groups, Friedman’s test with Dunn’s test corrected for multiple comparisons. For 3 or more unpaired groups, we used Kruskal-Wallis with Dunn’s test corrected for multiple comparisons, except for the CD34+-alone group and +AML cell lines/AML patient samples group ex vivo, which were compared with Mann Whitney U test with Bonferroni’s correction for multiple comparisons. For data sets composed of homogeneous groups , parametric tests were performed. For 2 paired groups, we used paired 2-tailed Student’s t test; for 2 unpaired groups, 2-tailed Student’s t test; for 2 or more unpaired groups, ANOVA. Multiple comparisons were corrected with Dunn’s test for Kruskal-Wallis and Friedman’s tests, Holm-Šidák for multiple Student’s t tests and Mann-Whitney U tests, and Tukey’s for ANOVA.Results are shown as mean ± SEM unless stated otherwise. Box-and-whisker plots show bounds from 25th to 75th percentile, median line, and whiskers ranging from smallest to largest values All statistical analyses were performed using GraphPad Prism software. A AML samples, healthy BM samples, and UCB samples were collected after obtaining written informed consent. This study was approved by the East London and the City Research Ethics Committee in accordance with the Declaration of Helsinki (REC 06/Q0604/110). Human BM MSCs were provided by Christine Dosquet (Paris Diderot University) from human BM obtained during orthopedic surgery . All animal experiments were performed at the Francis Crick Institute in accordance with UK Home Office and Crick guidelines and were undertaken under Home Office project license PLL 70/8904.AW, AH, KRP, and DB conceptualized the study. AW, KRP, AA, and AH developed the methodology. AW, AH, MGA, FMM, NB, AA, KRP, and DB analyzed and interpreted results. AW and DB wrote the original draft of the manuscript. AW, JG, JF, DT, and DB reviewed and edited the manuscript. JF, DT, and DB acquired funding. DB, DT, and JG provided resources. AH, KRP, and DB supervised the study.


SOCS2 and CALCRL and of the downregulation of MTSS1 and KDM6A in therapy resistance and/or stemness of AML. These findings spur the assumption that functional investigations of genes consistently altered at recurrence of AML have the potential to promote the development of novel targeted drugs that may help to improve the outcome of this currently often fatal disease.Relapse is associated with therapy resistance and is a major cause of death in acute myeloid leukemia (AML). It is thought to result from the accretion of therapy-refractory leukemic stem cells. Genetic and transcriptional changes that are recurrently gained at relapse are likely to contribute to the increased stemness and decreased therapy responsiveness at this disease stage. Despite the recent approval of several targeted drugs, chemotherapy with cytosine arabinoside and anthracyclines is still the mainstay of AML therapy. Accordingly, a number of studies have investigated genetic and gene expression changes between diagnosis and relapse of patients subjected to such treatment. Genetic alterations recurrently acquired at relapse were identified, but were restricted to small proportions of patients, and their functional characterization is still largely pending. In contrast, the expression of a substantial number of genes was altered consistently between diagnosis and recurrence of AML. Recent studies corroborated the roles of the upregulation of  In acute myeloid leukemia (AML), excessive proliferation of partially differentiated or undifferentiated blasts supersedes the production of normal blood cells, thereby causing infections, anemia, bleeding, and, in untreated patients, death within months. The annual incidence of this aggressive hematopoietic malignancy is ~5/100,000, and the median age at diagnosis ~70 years . SimilarLike other malignant diseases, AML is caused by genetic and molecular alterations. These occur in hematopoietic stem or progenitor cells, transforming them into LSCs ,7, and iall-trans-retinoic acid, which, since the late 1980s, has greatly improved the outcome of patients with rearrangements of the retinoic acid receptor a gene [The first molecularly targeted drug in AML was r a gene . In the r a gene ,13. Never a gene .One plausible hypothesis explaining therapy resistance at relapse is that it is caused by genetic, epigenetic, and/or transcriptional alterations newly appearing at this disease stage . These cFLT3 internal tandem duplications already present in a heterozygous state at diagnosis), and deletions or mutations of the tumor suppressor gene TP53. All of these changes were found only in low single-digit percentages of patients [these criteria were deletions of the long arms of chromosomes 5 and 7, UPD(13q)  [PRDM16 [A related line of research addressed the potential contribution of epigenetic and gene expression changes to relapse-related disease properties. The methylation density of the CpG islands (CGIs) located in the regulatory regions of certain preselected genes was significantly increased in relapsed versus diagnostic AML samples . FurtherERG , MYCN [2RG [MYCN , MSI2 [2CN [MSI2 , WT1 [26CN [MSI2 , DNMT3B  [DNMT3B , ADGRG1 s GPR56) , and PRD [PRDM16 . AdditioSOCS2, IL2RA, NPDC1, and PHGDH. Its elevated expression was an independent prognostic parameter for poor overall survival in several independent AML cohorts [SOCS2 and NPDC1 were also upregulated at relapse of AML [SOCS2, a component of the JAK−STAT signaling pathway. This pathway is aberrantly activated in several tumor types [SOCS genes are transcriptionally induced by JAK−STAT signaling [SOCS2 was downregulated in several cancer types [SOCS2 promoted proliferation, anchorage-independent growth, apoptosis resistance, and in vivo tumor growth of prostate and colon cancer cell lines [Several gene expression signatures associated with poor outcome in AML have been established, but most of these were either based on preselected genes and/or contained too many genes to be readily applicable to clinical practice ,31−38. W cohorts . Interese of AML , supportor types . In AML,or types . SOCS geignaling . They enignaling . Based oer types . On the er types ,43. Morell lines −44, testSocs2 was highly expressed in HSCs and required for stress hematopoiesis [SOCS2 expression was significantly increased in patients with chronic myeloid leukemia (CML) in blast crisis as compared with chronic phase patients and healthy controls [SOCS2 was upregulated as compared with healthy controls [SOCS2 in this disease.In the healthy murine hematopoietic system, opoiesis . SOCS2 econtrols . In AML,controls , and SOCcontrols , yet litSOCS2 enhanced the proliferation of the malignant human myeloid cell lines U-937 and HL-60 [Socs2 was strongly elevated in LCs from both an Flt3-ITD/Npm1c- and an MLL-AF9-driven mouse model of AML as compared with normal murine hematopoietic cells [Socs2 in Flt3-ITD/Npm1c-driven murine AML using short hairpin RNAs (shRNAs) led to a rapid and complete loss of shSocs2-, but not of shCtrltransduced LCs in culture. Even though this precluded the use of these cells for downstream experiments, it impressively illustrated an essential role of Socs2 in the proliferation and/or survival of Flt3-ITD/Npm1c-driven LCs [MLL-AF9 model, knockdown of Socs2 also inhibited LC proliferation in vitro, yet sufficient cell numbers for transplantation experiments could be recovered. SOCS2 depletion increased disease latency and promoted the myeloid differentiation of LCs in vivo. Moreover, it reduced the abundance and quiescence of immunophenotypically defined LSCs, as well as LSC activity as determined through a serial replating assay [In our studies, experimental expression of nd HL-60   population, impaired hematopoiesis under various stress conditions [ADM was expressed in hematopoietic cells ,66,67 annditions . Most renditions .CALCRL expression and therapy resistance in patients with AML, and the functions of CALCRL in normal HSCs as well as in other malignancies, we set out to investigate a possible role for CALCRL in AML. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) confirmed the expression, and the upregulation at relapse, of CALCRL in primary human AML cells. Interestingly, among the RAMP genes, only RAMP1 mRNA was detectable in all analyzed specimens, whereas RAMP2 and RAMP3 transcripts were measurable only in 0 and 25% of the samples, respectively, suggesting that CALCRL acts mainly as a CGRP, rather than as an ADM, receptor in AML [CALCRL and RAMP1 [CALCRL was upregulated in LSC-enriched versus LSC-depleted AML cell populations and in LSC-versus HSC-enriched cell populations and was an independent prognostic parameter for poor outcome in several AML cohorts [CALCRL- and RAMP1-expressing human AML cell lines to araC and daunorubicin, and this effect was counteracted both by shRNA-mediated downregulation of CALCRL and by receptor inhibition through the truncated peptide, CGRP(8−37), or the small molecule olcegepant [MLL-AF9-driven congenic AML mouse model, in vivo treatment with olcegepant reduced leukemic burden in BM and spleen, promoted myeloid differentiation, reduced the abundance and quiescence of an immuno-phenotypically defined LSC-enriched cell population [Ramp1 impeded stress hematopoiesis in mice [Given the association between high r in AML . Treatmend RAMP1   decreased proliferation, increased apoptosis, and enhanced cellular sensitivity to araC and idarubicin -mediated knockout and shRNA-mediated knockdown of MTSS1 in three different human myeloid cell lines increased their resistance to araC and daunorubicin, possibly via an increased DNA damage response [MTSS1 expression, a robotic screen of more than 100 drugs under development or approved for oncological indications was performed. However, none of these substances exhibited increased effectivity toward MTSS1 knockout cells. Rather, nine additional drugs representative of various classes, including, for example, the tyrosine kinase inhibitor regorafenib and the antimitotic vincristine, were less effective toward MTSS1 knockout versus control cells [Supporting a contribution of response . To uncool cells  , and MECOM [Genomewide gene expression profiling provided further evidence for central roles of nd MECOM .Mtss1 expression on AML aggressiveness in vivo, an MLL-AF9-driven mouse model of AML was used. Short hairpin RNA-mediated knockdown of Mtss1 in this model decreased disease latency to less than half. Furthermore, it strongly increased leukemic burden in BM and decreased the maturation of myeloid LCs. Mtss1-depleted cells also proliferated faster than control cells ex vivo and were more resistant to the anthracyclines daunorubicin and doxorubicin [To validate the effect of reduced orubicin  −specific demethylase, and carries somatic loss-of-function mutations in various types of cancer. KDM6A was also mutated in some patients with AML at the time of diagnosis, and the variant allele frequency of these mutations increased at relapse [KDM6A mRNA at relapse was observed in 46% of patients with AML, but 37% exhibited upregulation of this transcript [KDM6A at diagnosis were associated with shorter overall survival. Together, these data suggest a role for KDM6A mutation or downregulation in primary and relapse-associated therapy resistance. Indeed, in PDX from one patient each with wild-type and mutated KDM6A, the mutation was associated with increased resistance to araC ex vivo and to araC + liposomal daunorubicin in vivo [KDM6A), those with KDM6A mutations were more resistant to araC. Knockdown or knockout of KDM6A in AML cell lines decreased their sensitivity to araC, and, albeit less consistently, to daunorubicin and 6-thioguanine. Conversely, re-expression or experimental overexpression of KDM6A increased araC responsiveness [Following a rationale related to ours, Stief et al.  identifi relapse . Downreganscript  (Figure  in vivo  (Figure siveness  (Figure siveness .KDM6A may be another contributor to chemotherapy resistance, particularly toward the key drug araC, in AML. The consequences of KDM6A upregulation at relapse, observed in more than a third of patients, remain to be clarified.Taken together, mutation or downregulation of Relapse is frequent, generally associated with therapy resistance, and a major cause of death in AML. Molecular and genetic alterations promoting leukemia cell survival under therapy may either newly emerge or pre-exist in a subclone at diagnosis and are selected for during treatment. Changes that are newly acquired, or strongly enriched, at relapse are thus likely to contribute to therapy resistance and other properties of this disease stage. Certain mutations were recurrently gained at relapse of AML, but each of them was found only in a small proportion of patients ,16. AML In addition to genetic changes, changes in gene expression contribute to leukemogenesis, are of prognostic value, and may represent therapeutic targets in AML ,13,31,96SOCS2, CALCRL, MTSS1, and KDM6A in therapy resistance and/or stemness of AML. CALCRL may be of particular interest, because small molecule inhibitors targeting it have already been developed [The relapse signature established by Hackl et al. contained several genes previously implicated in key aspects of AML. Recent research additionally revealed roles for the deregulation of eveloped ,76.Future studies should address the functional contributions of additional genes to relapse-related disease properties. Furthermore, single-cell expression analyses at the mRNA and protein levels will provide important insights into the heterogeneity of AML cell populations with respect to the expression of these genes. Gene expression patterns in AML LSCs, and their possible changes between diagnosis and relapse, will be of particular interest. For any resulting potential novel drugs, it will be pivotal to identify susceptible patient populations and to establish standardized procedures allowing their identification in clinical routine. Also, escape mechanisms will need to be examined. It is still early days in the investigation of molecular and genetic drivers of AML relapse. Much remains to be learned, but this research holds great promise to lead to the development of novel targeted drugs with the potential to improve outcome of a presently still often fatal malignancy.


To examine whether CaMKII‐dependent RyR2 phosphorylation mediates diastolic Ca2+ release and apoptosis in sepsis, we evaluated apoptosis in mutant mice hearts that have the CaMKII phosphorylation site of RyR2 (Serine 2814) mutated to Alanine (S2814A). S2814A CASP mice did not show increased apoptosis. Consistent with RyR2 phosphorylation‐dependent enhancement in diastolic SR Ca2+ release leading to mitochondrial Ca2+ overload, mitochondrial Ca2+ retention capacity was reduced in mitochondria isolated from WT‐CASP compared to Sham and this reduction was absent in mitochondria from CASP S2814A or dantrolene‐treated mice. We conclude that in sepsis, CaMKII‐dependent RyR2 phosphorylation results in diastolic Ca2+ release from SR which leads to mitochondrial Ca2+ overload and apoptosis.Sepsis is associated with cardiac dysfunction, which is at least in part due to cardiomyocyte apoptosis. However, the underlying mechanisms are far from being understood. Using the colon ascendens stent peritonitis mouse model of sepsis (CASP), we examined the subcellular mechanisms that mediate sepsis‐induced apoptosis. Wild‐type (WT) CASP mice hearts showed an increase in apoptosis respect to WT‐Sham. CASP transgenic mice expressing a CaMKII inhibitory peptide (AC3‐I) were protected against sepsis‐induced apoptosis. Dantrolene, used to reduce ryanodine receptor (RyR) diastolic sarcoplasmic reticulum (SR) Ca This SR Ca2+ leak reduces SR Ca2+ content and thus systolic Ca2+ release, mediating at least in part the contractile dysfunction associated with sepsis.2+ leak which is then taken up by the mitochondria, leading to mitochondrial Ca2+ overload which triggers the apoptotic cascade.2+ leak mediates apoptosis in sepsis.Ca2+ leak leading to mitochondrial Ca2+ overload and apoptosis. Importantly, we further show that the RyR2 plays a critical role in sepsis‐induced cell death and that agents that reduce RyR2 open probability (RyR2 stabilizers) are effective in preventing apoptosis, suggesting a potential therapeutic advantage of these compounds to reduce apoptosis and contractile dysfunction associated with sepsis.The results presented herein, using a colon ascendant stent peritonitis (CASP) model of sepsis that closely mimics the clinical course of diffuse peritonitis with early and steadily increasing systemic infection and inflammation,2All procedures followed during this investigation were approved by Animal Welfare Committee of La Plata School of Medicine (University of La Plata), following the Guide for the Care and Use of Laboratory Animals published by the National Research Council, National Academy Press, Washington DC 2010 and/or European Union Directive for Animal Experiments 2010/63/EU. Three‐month‐old male wild‐type (WT) C57BL/6 mice and transgenic (TG) mice with cardiomyocyte‐delimited transgenic expression of either a CaMKII inhibitory peptide (AC3‐I) or a scrambled control peptide (AC3‐C) or mutant mice where the CaMKII‐dependent phosphorylation site on the RyR2 (site Serine 2814) is mutated to Alanine (S2814A) were used. All genetically modified mice were generated on a C57BL/6 background.2.1Colon ascendens stent peritonitis (CASP) surgery was performed to induce sepsis as previously described.2.2Myocytes were isolated by enzymatic digestion.2.3g of centrifugation to discard unbroken tissue and debris, the supernatant was centrifuged at 8000 × g for 10 minutes to sediment the mitochondria. The mitochondrial pellet was washed twice with IS and the last one with suspension solution  at 8000 × g for 5 minutes each. The residue was washed and re‐suspended in SS. The mitochondrial protein concentration was evaluated by the Bradford methodIsolated hearts were washed and homogenized in ice‐cold isolation solution (IS) consisting of 75 mmol/L sucrose, 225 mmol/L mannitol and 0.01 mmol/L EGTA neutralized with Trizma buffer at pH 7.4. After tissue pieces were settled, the entire supernatant was discarded and fresh IS was added and the mixture was transferred to a hand homogenizer. Proteinase  was added just before starting the homogenization procedure. The homogenate was carefully transferred after each step to a polycarbonate centrifuge tube. After 5 minutes of 750 × 2.4Mitochondrial Cytochrome C levels and the ratio between pro‐ and anti‐apoptotic proteins Bax and Bcl‐2, respectively, were used as an index of apoptosis  fluorescence quenching in a buffer containing (in mM): 120 KCl, 20 MOPS, 10 Tris‐HCl and 5 KH2PO4 adjusted to pH = 7.4 containing RH‐123 0.1 μmol/L. Isolated mitochondria loaded with RH‐123 were excited at 503 nm, and fluorescence emission was detected at 527 nm.2.72+ before the irreversible opening of the mitochondrial permeability transition pore (mPTP) occurs was monitored by following the changes in the fluorescence of the Ca2+ sensitive indicator, Calcium Green‐5N.2+. After each addition, the fluorescence in the medium increases until the mitochondria start to take up Ca2+ and then it decreases. When mitochondria are sufficiently loaded with Ca2+, the opening of the mPTP occurs and the release of the mentioned ion in the medium increases again the fluorescence.The ability of mitochondria to retain exogenous Ca2+ required to trigger a massive Ca2+ release by isolated cardiac mitochondria.2+ accumulation and expressed as nmol CaCl2 per mg of mitochondrial proteins.Calcium retention capacity (CRC) was defined here as the amount of Ca2.8t test, Mann‐Whitney test and one‐way ANOVA were applied accordingly using GraphPad Prism 5.0 . The data are presented as means ± SEM. The level of significance was set at P < .05.The normal distribution of the data was corroborated with the Kolmogorov‐Smirnov test; unpaired Student's 33.1Apoptosis was assessed by four independent methods, in heart homogenates, cardiomyocytes and cardiac mitochondria isolated from colon ascendens stent peritonitis operated mice (CASP) and from Sham‐operated controls. Heart homogenates, cardiomyocytes and cardiac mitochondria were prepared 24 hours after surgery when contractile dysfunction is already installed.3.2We recently reported that CaMKII mediates at least part of the contractile dysfunction associated with sepsis, and several laboratories including our own have provided clear evidence that CaMKII is involved in triggering apoptosis in several disease models.3.32+ handling. Nevertheless, abnormal Ca2+ handling and Ca2+ leak via the RyR2 have recently been shown by us and others to be key determinants of cardiomyocyte apoptosis under different pathological conditions.2+ leak favouring contractile dysfunction.2+ leak, is functionally involved in sepsis‐induced cell death. Enhanced Ca2+ leak from the SR has been shown to promote mitochondrial Ca2+ overload, resulting in the opening of the mitochondrial permeability transition pore (mPTP) and activation of the apoptotic cascade.2+ retaining capacity (CRC) of mitochondria isolated from Sham and CASP wild‐type mice. For this purpose, we performed experiments using Calcium Green‐5N to measure extramitochondrial Ca2+ levels  mouse model that closely mimics the clinical situation of abdominal sepsis, we investigated the mechanisms involved in CaMKII‐dependent apoptosis.Our previous study demonstrates that CaMKII‐dependent SR‐CaConsistent with previous reports, we confirmed that the CASP model of sepsis promotes cardiac apoptosis, as evidenced by an enhanced ratio between pro‐ and anti‐apoptotic proteins, Bax and Bcl2, respectively (Bax/Bcl2), a decrease in mitochondrial cytochrome C levels, a significant decrease in mitochondrial membrane potential and by an increase in TUNEL staining. These results showing altered mitochondrial homeostasis further suggest that cardiac myocyte cell death, in sepsis, is mediated at least in part, by the mitochondrial pathway of apoptosis.4.12+ leak which in turn reduces SR Ca2+ content and contractility in the CASP model of sepsis.Oxidative stress has been shown to be enhanced in sepsis, and experimental evidence demonstrates that antioxidants can prevent cardiac dysfunction associated with sepsis.4.22+ release and mitochondrial Ca2+ uptake, resulting in mitochondrial Ca2+ overload and opening of the mPTP.2+ leak and mitochondrial Ca2+ elevation, associated with exacerbated cell death in transgenic mice lacking phospholamban and overexpressing CaMKIIαδc.2+ leak which would be taken up by the mitochondria provoking mitochondrial Ca2+ overload. To test this hypothesis, we used myocytes isolated from transgenic mice with RyR2 site Serine 2814 mutated to Alanine (S2814A) and therefore not phosphorylatable by CaMKII. We observed that S2814A CASP mice were protected from sepsis‐induced apoptosis ; Investigation (lead); Methodology ; Writing‐original draft (lead). Juan Ignacio Burgos: Formal analysis ; Investigation ; Methodology . Alejandro Ciocci Pardo: Formal analysis ; Investigation ; Methodology . Luisa González Arbelaez: Formal analysis ; Investigation ; Methodology . Susana Mosca: Investigation ; Supervision . Martín Vila Petroff: Funding acquisition (lead); Investigation (lead); Writing‐original draft (lead); Writing‐review & editing (lead).Supplementary MaterialClick here for additional data file.