The main SOCE actors are STIM1 and ORAI1. Depletion of the reticular Ca2+ stores induces oligomerization of the luminal Ca2+ sensor STIM1, and the oligomers activate the plasma membrane Ca2+ channel ORAI1 to trigger extracellular Ca2+ entry. Mutations in STIM1 and ORAI1 result in abnormal SOCE and lead to multi-systemic disorders. Recessive loss-of-function mutations are associated with CRAC (Ca2+ release-activated Ca2+) channelopathy, involving immunodeficiency and autoimmunity, muscular hypotonia, ectodermal dysplasia, and mydriasis. In contrast, dominant STIM1 and ORAI1 gain-of-function mutations give rise to tubular aggregate myopathy and Stormorken syndrome (TAM/STRMK), forming a clinical spectrum encompassing muscle weakness, thrombocytopenia, ichthyosis, hyposplenism, short stature, and miosis. Functional studies on patient-derived cells revealed that CRAC channelopathy mutations impair SOCE and extracellular Ca2+ influx, while TAM/STRMK mutations induce excessive Ca2+ entry through SOCE over-activation. In accordance with the opposite pathomechanisms underlying both disorders, CRAC channelopathy and TAM/STRMK patients show mirror phenotypes at the clinical and molecular levels, and the respective animal models recapitulate the skin, bones, immune system, platelet, and muscle anomalies. Here we review and compare the clinical presentations of CRAC channelopathy and TAM/STRMK patients and the histological and molecular findings obtained on human samples and murine models to highlight the mirror phenotypes in different tissues, and to point out potentially undiagnosed anomalies in patients, which may be relevant for disease management and prospective therapeutic approaches.Store-operated Ca Consistently, normal tissue and organ physiology strictly depends on the precise control of Ca2+ entry, storage, and release, while abnormal Ca2+ homeostasis induces various rare and common disorders affecting skeletal muscle, heart, bones, brain, skin, or the immune and hormonal systems , and refilling of the stocks is initiated by store-operated Ca2+ entry (SOCE), a ubiquitous mechanism driven by the concerted action of STIM1 and ORAI1  channel ORAI1  mutations resulting in insufficient SOCE cause CRAC channelopathies characterized by severe combined immunodeficiency (SCID) involving recurrent and chronic infections, autoimmunity, muscular hypotonia, ectodermal dysplasia, anhidrosis, and mydriasis (STIM1 and ORAI1 gain-of-function (GoF) mutations inducing excessive Ca2+ entry through SOCE over-activation were found in patients with tubular aggregate myopathy (TAM) and Stormorken syndrome (STRMK)   have moreover been reported in patients with late-onset muscle weakness and myalgia, forming the mild end of the TAM/STRMK spectrum  . TAM and (STRMK) , and mos (STRMK) . All GoF, I115F)  or in th, I115F) , or in t, I115F) . Missensspectrum .Stim1 or Orai1 or the generation of chimeras through transplantation of hematopoietic –/–Stim1 or –/–Orai1 stem cells results in defective T cell activation and Treg suppression  , the invEye movement relies on the rapid and concerted contraction of six striated extraocular muscles, and ophthalmoplegia including upward gaze paresis , lateral2+-activated chloride channel TMEM16A in the sweat glands, and thereby enables chloride secretion and sweat production  and Stim1 and Stim2 double knockout mice additionally show a reduced suppressive function of Treg and NKT cells and an associated autoimmunity and splenomegaly  and the secretion of alpha granules containing thrombotic factors   , which png (ECC) . The tubr origin . It has gregates . Alternahe triad . Furtherhe triad .Stim1 KO and Orai1 KO Mice die perinatally , lowering the local Ca2+ levels in proximity to the STIM1 EF hands , characterized by muscular hypotonia, mydriasis, ataxia, and intellectual disability  is the major Catrations . In turnate SOCE . Specifiactivity . As calsic signs . A murin in mice . Noteworn the SR . Tubularn the SR . LG-CMS or GMPP8 . All fiv+ influx , and thee fibers . These eSTIM1 and ORAI1 impair SOCE and cause CRAC channelopathy, while GoF mutations in both genes involve SOCE over-activation and result in TAM/STRMK (STIM1 and ORAI1 LoF and GoF mutations at the molecular level, highlighting the importance of strict SOCE regulation for normal tissue physiology. Thrombus formation is impaired in both disorders and enhances the tendency to bleed following injury. This is due to the reduced activation of platelets in CRAC channelopathy (2+ store refill in CRAC channelopathy, or from cytosolic Ca2+ excess disrupting excitation-contraction coupling and/or restraining proper muscle relaxation in TAM/STRMK.LoF mutations in AM/STRMK . In agreAM/STRMK , indicatAM/STRMK . It is ielopathy , and reselopathy . Similar2+ balance and the impact of Ca2+ imbalance on eye, bones, enamel, skin, platelets, spleen, immune system, and skeletal muscle physiology. Patients are usually examined by specialized physicians with a major focus on the principal handicap, and additional phenotypic anomalies might be overlooked, especially in the context of multi-systemic disorders with mild to moderate involvement of specific tissues. In contrast, murine models generally undergo unbiased phenotyping and offer the possibility for a detailed analysis of all organs to provide an overview of the disease. As an example, the complete characterization of the STIM1 R304W TAM/STRMK mouse model unveiled anomalies of the glucose metabolism, hepatic function, and the immune system (2+-related disorders affecting the bones, platelets, spleen, immune system, or skeletal muscle.CRAC channelopathy and TAM/STRMK mouse models recapitulate the main clinical signs of the human disorders, and are valuable and powerful tools to understand the importance of Cae system , which he system , Capgrase system , and mane system  have onlRS-R and JB wrote the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Denio rerio). The goldfish (Carassius auratus) belongs to the same family as the zebrafish and has emerged as an alternative model with which to study cardiac function. Here, we propose to acutely study electrophysiological and systolic Ca2+ signaling in intact goldfish hearts. We assessed the Ca2+ dynamics and the electrophysiological cardiac function of goldfish, zebrafish, and mice models, using pulsed local field fluorescence microscopy, intracellular microelectrodes, and flash photolysis in perfused hearts. We observed goldfish ventricular action potentials (APs) and Ca2+ transients to be significantly longer when compared to the zebrafish. The action potential half duration at 50% (APD50) of goldfish was 370.38 ± 8.8 ms long, and in the zebrafish they were observed to be only 83.9 ± 9.4 ms. Additionally, the half duration of the Ca2+ transients was also longer for goldfish (402.1 ± 4.4 ms) compared to the zebrafish (99.1 ± 2.7 ms). Also, blocking of the L-type Ca2+ channels with nifedipine revealed this current has a major role in defining the amplitude and the duration of goldfish Ca2+ transients. Interestingly, nifedipine flash photolysis experiments in the intact heart identified whether or not the decrease in the amplitude of Ca2+ transients was due to shorter APs. Moreover, an increase in temperature and heart rate had a strong shortening effect on the AP and Ca2+ transients of goldfish hearts. Furthermore, ryanodine (Ry) and thapsigargin (Tg) significantly reduced the amplitude of the Ca2+ transients, induced a prolongation in the APs, and altogether exhibited the degree to which the Ca2+ release from the sarcoplasmic reticulum contributed to the Ca2+ transients. We conclude that the electrophysiological properties and Ca2+ signaling in intact goldfish hearts strongly resembles the endocardial layer of larger mammals.Cardiac physiology of fish models is an emerging field given the ease of genome editing and the development of transgenic models. Several studies have described the cardiac properties of zebrafish ( Danio rerio) has been the model of choice not only for the possibility of performing transgenesis  belong to the same family as zebrafish  assess the role of L-type Ca2+ currents in the excitability of the myocytes as well as its contribution to Ca2+ transients, and (iii) weigh the contribution of SR Ca2+ release to Ca2+ transients and AP repolarization. These goals were tackled using a combination of pulsed local field fluorescence microscopy  evaluate the dependency of the AP and Cacroscopy , sharp mcroscopy , and flacroscopy . The rescroscopy  or dogs croscopy . It is i2+ current is exemplified by our data showing how the influx of Ca2+ through L-type Ca2+ channels in goldfish not only define the AP duration (duration of phase 2), but also is a key trigger in inducing Ca2+ release from the SR. Furthermore, we find that goldfish hearts have a similar AP temperature . Animals were injected intraperitoneally with Na2+ transients were recorded with the pulsed local field fluorescence microscopy (PLFFM) (Ca (PLFFM) . The PLFEpicardial electrical recordings of the APs were obtained using sharp glass microelectrodes filled with 3 M KCl that were connected to a high-input impedance differential amplifier  in hearts paced from 1 to 3 Hz. Glass microelectrodes were fabricated with a micropipette puller  and had a resistance of 10–20 MΩ . Data weA flash photolysis system allows us to fractionally change a specific ionic current by photolyzing . Specifi2+ transients and APs in the same region (the midregion of the left ventricle epicardium). Thus, the data here are presented as the measured times with their standard deviations (SD). To assess electrical changes, AP traces were evaluated at certain repolarization times. Specifically, the time it took for the AP to reach 30, 50, or 90% repolarization was assessed and is referred to as APD30, APD50, or APD90, respectively. To evaluate the kinetics of the recorded Ca2+ transients, recordings were normalized between zero (minimum fluorescence) and one (maximum fluorescence). The kinetics parameters of the Ca2+ transients evaluated were the rise time , half duration , and fall time . Time constants τon and τoff were calculated as τon = RT/2.2 and τoff = FT/2.2  recorded for different measurements (n) on different hearts (N) with the mean ± SD (solid lines). In this paper, we performed 14 experiments with goldfish, 20 experiments with zebrafish, and five experiments with c57BL/6 mice. Statistical significance was tested using a two-sample Kolmogorov–Smirnov test (OriginPro 2020). The difference was significant if the p-value was < 0.01.In whole heart experiments, there are two main causes of variance. The first cause of variance is that two different hearts cannot be completely identical to one another. The second cause of variance is that it is impossible to perform the recordings in the same precise location between multiple different hearts even though we are measuring Ca= FT/2.2 . Each rebulbus arteriosus. The zebrafish heart needs to be cannulated using a 32- to 34-gauge needle, and the goldfish heart can be cannulated on a 27-gauge needle. The use of a larger needle for cannulation is a huge advantage because it allows for larger fluxes during perfusion and will allow for different drugs to be perfused significantly more quickly.Fish hearts have become an interesting model to study both cardiac physiology and pathophysiology. Specifically, zebrafish hearts have come to be a very popular model due to the possibility of developing transgenic animals. However, zebrafish present some experimental limitations when compared to goldfish. The first one is the size of the animal. Goldfish are three times longer and 2.7 times wider than the zebrafish . Additio2+ transients exhibit significantly longer durations , the ventricular APs  and Ca2+urations  than thourations . These f2+ transients recorded from goldfish. The AP parameters were longer for goldfish  compared to the zebrafish .goldfish  having aebrafish . Specifi2+ transients in both goldfish . Interestingly, the kinetic parameters for the Ca2+ transients were faster in zebrafish hearts .Several kinetic parameters, such as the RT, FT, and HD were evaluated the Cagoldfish  and zebrgoldfish . For gol2 + influx is through the L-Type Ca2 + channel. In the experiments presented in 2+ current to the ventricular AP kinetics and the amplitude of the Ca2+ transients. 2+ current with 10 μM nifedipine on the goldfish epicardial AP. All the parameters that define the duration of the AP decreased after the heart was perfused with nifedipine. 30 was reduced from 278.4 ± 12.4 ms to 210.31 ± 12.8 ms, APD50 was reduced from 370.3 ± 8.8 ms to 283.6 ± 8.0 ms, and APD90 was reduced from 481.5 ± 9.5 ms to 389.5 ± 16.9 ms . These results indicate that the L-type Ca2 + channel is partially responsible for the duration of the AP phase 2 (plateau phase).In most vertebrates, the main pathway for Ca2+ transients. The perfusion of the goldfish hearts with 10 μM nifedipine reduced the amplitude of Ca2+ transient by 40% (2+ transient was reduced from 0.84 ± 0.06 (n = 84 measurements) to 0.49 ± 0.04 . To compare the kinetics of the Ca2+ transients before and after nifedipine perfusion, the traces were normalized to its respected maximum amplitude  to 57.7 ± 5.1 ms . This reduction is a factor that can define the RT and the time-to-peak of Ca2+ transients. Consistent with the reduction in the duration of the AP, the HD of the Ca2+ transient was also significantly reduced  and 269.0 ± 12.9 ms for hearts perfused with nifedipine .Nifedipine also had a significant effect on the amplitude and the kinetics of intracellular Cat by 40% . The ampmplitude . The peransients  from 72. reduced . Interes2+ transient amplitude in one of two ways. Either nifedipine reduced the amplitude of the Ca2+ current entering through the L-type Ca2+ channels, or nifedipine reduced the duration of the AP and, as such, shortened the duration of the L-type Ca2+ current. To discriminate between these two hypotheses, we performed experiments in which we first perfused the heart with nifedipine and then used flash photolysis to locally inactivate it. We previously demonstrated , there was an increase in the amplitude of goldfish epicardial Ca2+ transients. 2+ transients and AP upon a photolytic stimulus. Although there was a significant increase in the amplitude of the Ca2+ transients, there was not a measurable effect on the kinetics of the AP. n = 20 measurements, N = 4 hearts). 2+ transients after the photolytic stimulus. The fractional amplitude of the Ca2+ transient increased from 0.97 ± 0.01 before the flash to 1.11 ± 0.02 after the flash .Nifedipine likely mediated the attenuation of the Canstrated  that loc2+ transients. Not surprisingly, an increase in the temperature produced a shortening of the AP duration at 30, 50, and 90% repolarization, opposite that observed in mice . To evaluate the thermodynamics of the AP in greater detail, we decided to calculate the first derivative to obtain the rates of depolarization and repolarization of the AP recordings at both experimental temperatures.Because fish are poikilotherm vertebrates, we decided to assess how temperature could modify both the kinetics of APs and Ca in mice . Figure mice  to 0.33 ± 0.01 . The differences in the kinetics can be better observed in 2+ transient traces were normalized. Interestingly, the Ca2+ transients illustrated in n = 110 measurements at 23°C and n = 119 measurements at 28.5°C). The FT . Once again, to have a better thermodynamic picture of the effect of temperature on Ca2+ transients we calculated Q10.Usually, Catability . One rea smaller , they we smaller . The amp. The FT  was reduansients  diminishonτ) and the time constant for the relaxation (offτ) of the Ca2+ transients. onτ and offτ were calculated asFor these experiments, we first calculated the time constant for activation . A summary of the results is presented in Like with other vertebrates, the fish heart needs to increase its heart rate to cope with environmental and stress conditions. For example, a fish escaping from a predator needs to increase the rate of skeletal muscle action potentials and contractions  in orderansients . Figure 2+ transients from the goldfish heart also present a strong heart-rate dependency. 2+ transients decreased as a function of the heart rate  in a subconductance state and block the SERCA pump, respectively  and decreased the fractional SR Ca2+ release from 1.0 ± 0.05 for the control condition  to 0.45 ± 0.01 , a decrease of approximately 55%. Because the Ca2+ transient recorded in the presence of Ry and Tg mostly represents the influx of Ca2+ across the plasma membrane, it is possible to calculate the gain of the Ca2+ induced Ca2+ release asFor numerous vertebrates, including mammals and birds, the contribution of the SR Caectively . These eGainCICR in the goldfish heart was calculated to be 2.25 ± 0.06. Not surprisingly, mice are also a common model used to study cardiac excitability and Ca2+ transients’ behavior, and this inspired us to compare the goldfish model to the mice model. Similar experiments were performed in mice in order to see how the mice model compared to the goldfish model in terms of SR Ca2+ release contribution. The amplitude of Ca2+ transient dramatically decreased upon perfusion with Ry and Tg  to 0.06 ± 0.007 for hearts perfused with Ry and Tg , a decrease of 90%  to 102.1 ± 4.5 ms in the presence of Ry and Tg . Following a similar trend, perfusion with Ry and Tg prolonged APD50  to 177.9 ± 6.5 ms  and APD90 (n = 180 measurements) to 406.9 ± 19.5 ms (n = 33 measurements).Caincrease  from 72.ed APD50  from 163nd APD90  from 37530 changed (n = 40 measurements) to 347.1 ± 4.9 ms , APD50 (n = 40 measurements) to 409.8 ± 7.9 ms (n = 40 measurements), and APD90  to 448.8 ± 8.9 ms . These results suggest that Ca2+-dependent inactivation of the L-type Ca2+ channel is the most likely mechanism prolonging the AP duration in the absence of Ca2+ release from the SR.The prolongation of the AP duration by Ry and Tg was also observed in goldfish ventricular APs recorded at 23°C.  changed  from 321), APD50  increasend APD90  increaseCarassius auratus) are members of the same phylum (Chordata), class (Actinopterygii), order (Cypriniformes), and family (Cyprinidae) as the zebrafish (Danio rerio). Both species can also regenerate their heart following injury  of a t-tubule network, but the time course of the Ca2+ influx across the plasma membrane and the kinetics of activation of ryanodine receptors.Goldfish   is much ebrafish . Interesmyocytes  is very myocytes  at a simmyocytes . Finallyperature .2+ transients  for goldfish Caansients  are muchansients , but alsansients , 2004 anansients . Intereses A. E.  and at tes A. E. .2+ through the L-type Ca2+ channel is critical in defining the time course of APs and is the main mechanism triggering ventricular Ca2+ release. Results presented in 2+ currents in goldfish excitability and its ability to activate the excitation–contraction coupling process.In most vertebrate species, the influx of Ca2+ currents  than the repolarization  than the FT and the relaxation time constant. This thermodynamic behavior can be attributed to the highly cooperative process of Ca2+ release from the SR, which depends on the intra-SR Ca2+ content , shows a similar profile to those previously observed for mammals  ( as mice . This wa as mice  to the fn (∼40%) .2+ release from the SR promoted by the perfusion of the goldfish hearts with Ry and Tg also modified the repolarization of ventricular AP. 2+-dependent inactivation of L-type Ca2+ channels shortens the action potentials when Ca2+ release is larger but reduced Ca2+-dependent inactivation prolongs the action potential if the Ca2+ release from the sarcoplasmic reticulum is impaired. Interestingly, this effect is contrary to what we observe in mice hearts. Upon coronary perfusion of mice hearts with Ry and Tg, the minuscule amount of Ca2+ released from the SR is unable to activate the Na+– Ca2+ exchanger in the forward mode and, thus, shortens the AP duration. On the other hand, the Na+–Ca2+ exchanger can also prolong the AP for a larger Ca2+ release while in the forward mode by activating an inward Na+ current .MB and AE designed the research, performed the research, analyzed data, and wrote the manuscript. Both authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Extracellular ATP, the levels of which significantly change during epileptic seizures, activates specific receptors leading to an increase of intracellular free Ca2+ concentration ([Ca2+]i). Here, we aimed to functionally characterize human microglia obtained from cortices of subjects with temporal lobe epilepsy, focusing on the Ca2+-mediated response triggered by purinergic signaling.Intracellular Ca2+]i in primary cultures of human microglial cells obtained from surgical specimens. The perforated patch-clamp technique, which preserves the cytoplasmic milieu, was used to measure ATP-evoked Ca2+-dependent whole-cell currents.Fura-2 based fluorescence microscopy was used to measure [Ca2+]i increases depend on Ca2+ entry from the extracellular space and on Ca2+ mobilization from intracellular compartments. Extracellular ATP also induced a transient fivefold potentiation of the total transmembrane current, which was completely abolished when [Ca2+]i increases were prevented by removing external Ca2+ and using an intracellular Ca2+ chelator. TRAM-34, a selective KCa3.1 blocker, significantly reduced the ATP-induced current potentiation but did not abolish it. The removal of external Cl− in the presence of TRAM-34 further lowered the ATP-evoked effect. A direct comparison between the ATP-evoked mean current potentiation and mean Ca2+ transient amplitude revealed a linear correlation. Treatment of microglial cells with LPS for 48 h did not prevent the ATP-induced Ca2+ mobilization but completely abolished the ATP-mediated current potentiation. The absence of the Ca2+-evoked K+ current led to a less sustained ATP-evoked Ca2+ entry, as shown by the faster Ca2+ transient kinetics observed in LPS-treated microglia.In human microglia extracellular ATP evoked [CaCa3.1 channels in human microglia, linking ATP-evoked Ca2+ transients to changes in membrane conductance, with an inflammation-dependent mechanism, and suggests that during brain inflammation the KCa3.1-mediated microglial response to purinergic signaling may be reduced.Our study confirms a functional role for KThe online version contains supplementary material available at 10.1186/s12974-021-02096-0. Ca2+ isecretion , 12. In ductance  which, iductance . Given t as cGMP , possibl as cGMP , 17. To  channel , 19.Ca3.1 activity as a tool to modulate microglia-induced neuroinflammation in epileptic patients i) were measured by time-resolved digital fluorescence microscopy, using an integrated acquisition system (Scientifica), composed of an upright reflected fluorescence microscope (Olympus) and the Optofluor software (Molecular Devices). Cells were incubated with the Ca2+ indicator Fura-2 acetoxylmethylester (1 μM) for 45 min at 37 °C in culture medium. The changes of [Ca2+]i were expressed as R = F340/F380, where F340 and F380 are the fluorescence intensities measured from individual cells at an emission wavelength of 510 nm illuminating the specimen with 340 and 380 nm excitation wavelengths, respectively. Cells were continuously perfused during the experiment. The acquisition frequency was 0.33 Hz.Changes in free intracellular Cat test or one-way ANOVA, as appropriate. Significance for all tests was set at p < 0.05, with statistical power > 0.8. For each experiment, data were pooled from microglial cells obtained from the patients indicated in the corresponding figure legends. The linear regression is calculated by the SigmaPlot software by the least-squares method.Data sampling was performed using the pClamp 10 software (Molecular devices) and the OptoFluor (CAIRN) software. Data analysis was performed using SigmaPlot 14.0 (Systat). All data are expressed as means ± standard error mean and analyzed using paired n=7). The residual cells were contaminating astrocytes, as identified by GFAP staining  thicroglia  and neveicroglia . As expe+]i Fig. . The ATPa2+ Fig. . Under t ATPases , evoked ls Table , confirmfor 48 h , 23. Unddes Fig. e, g but des Fig. .Fig. 1E2+-dependent regulation of ionic conductances in resting and LPS-treated human microglia strictly relies on good preservation of diffusible cytoplasm factors. Thus, whole-cell transmembrane current recordings were performed in the perforated patch-clamp configuration, avoiding cell dialysis [2+]i (1 μM) the current amplitudes recorded from dialyzed cells are much smaller than those recorded from intact cells with high [Ca2+]i and are not significantly different from intact cells at rest . In the absence of external Ca2+ , respectively, with an ATP-induced effect that was significant only in Ca2+-free conditions (1.1±0.2 pA/pF). In Ca2+-free plus BAPTA conditions, the measured current density did not change from the basal value of 0.2±0.1 pA/pF. These results indicate that the ATP-mediated modulation of human microglial transmembrane currents depends on the ability of the cells to mobilize intracellular Ca2+.The study of the Cadialysis . A direcincrease . Under p2+-dependent currents, we first blocked the KCa3.1 channels, which were previously reported to constitute the major K+ conductance in microglia from epileptic patients i changes as well as the current modulation. This molecule evoked Ca2+ transients , buicroglia . We applNeuroinflammation alters the physiological function of the CNS by modifying the activity of both glia and neurons and the molecular pattern of the extracellular milieu, with significant variation of several mediators such as cytokines, chemokines, and neurotransmitters. Neuroinflammation is a key factor in epileptogenesis , 35. In Our results on primary microglia from the human epileptic temporal cortex contribute to better define the complex picture of microglial functions in epileptic patients. Even if, in general, studies on human CNS cells suffer from the scarcity of proper controls, they provide crucial information on the molecular and cellular pathways that could be targeted in human cells.2+ and increased Ca2+ entry i increase is Ca2+ entry through P2X ionotropic receptors. Upregulation of both P2X7 and P2X4 receptors has been observed in microglia following experimental seizures i is an essential step in microglial activation, modulating genetic transcription, motility, secretion, and other processes i increases are inhibited by chelating Ca2+. When we further dissected ionic components contributing to these Ca2+-evoked currents, we found both a Cl− and a K+ component. A Ca2+-dependent Cl− conductance has been recently described in mouse hippocampus i increase. The key role of Ca2+ is confirmed by the fact that a small [Ca2+]i increase due to a different stimulus, the activation of store-activated Ca2+ entry mediated by TRPC3/6 i rise and current potentiation. Indeed, KCa3.1 function was strongly reduced in LPS-treated cells, as revealed using the selective activator NS309 i and potentiates the transmembrane currents in human microglia. Table S1. Patients included in the study.


In addition, crystal structures and thermal melting temperatures revealed a conformational change in the I784F-SPRY1 domain compared to the WT-domain. The novel RyR2-I784F variant in SPRY1 domain causes a leaky channel under non-stress conditions. The presence of several variants affecting Ca2+ handling and cardiac conduction suggests a possible oligogenic origin for the ectopies originating from Purkinje fibres.Idiopathic ventricular fibrillation (IVF) causes sudden death in young adult patients without structural or ischemic heart disease. Most IVF cases are sporadic and some patients present with short-coupled torsade de pointes, the genetics of which are poorly understood. A man who had a first syncope at the age of 35 presented with frequent short-coupled premature ventricular beats with bursts of polymorphic ventricular tachycardia and then died suddenly. By exome sequencing, we identified three rare variants: p.I784F in the SPRY1 of the ryanodine receptor 2 (RyR2), p.A96S in connexin 40 (Cx40), reported to affect electrical coupling and cardiac conduction, and a nonsense p.R244X in the cardiac-specific troponin I-interacting kinase (TNNI3K). We assessed intracellular Ca Current guidelines define IVF as a diagnosis of exclusion in patients who have survived a VF episode without any identifiable structural or metabolic cause, as assessed by clinical evaluation of known cardiac, respiratory, metabolic and toxicological etiologies that may lead to cardiac arrest2. It occurs in 5–10% of patients resuscitated from out‐of‐hospital cardiac arrest without clinical evidence of heart disease4. Most IVF patients are sporadic cases but a subset of patients have a family history of sudden cardiac death, suggestive of genetic origin5. Genetic screening of known genes responsible for arrhythmias led to the identification of only a few variants in a small percentage of cases6. This suggests that these patients are genetically heterogeneous and also that the IVF is likely oligogenic in origin, which could explain the low penetrance in the families. Multiple genetic variants may be causal, as is the case for other channelopathies7.Idiopathic ventricular fibrillation (IVF) is a leading cause of unexplained sudden cardiac death (SCD) in the absence of structural heart disease, particularly in young adults2. It evolves, in most cases, to VF causing SCD. Most scTdP originate from Purkinje fibres9. In the first reported series of 14 patients, 30% of scTdP patients had a family history of sudden death, suggesting a genetic susceptibility to this pathology10.We focused our study on a patient with short-coupled torsade de pointes (scTdP), a ventricular tachycardia with a characteristic rotation of electrical activity around the isoelectric line. Contractions are non-uniform but organized, with a change in amplitude over time, where the first beat is characterized by a very short coupled interval (280–300 ms)2+-blocking agent, reduces arrhythmias in this condition but remains insufficient to eliminate the risk of sudden death11. This suggests that, at least in some patients, ectopies may occur in a context of Ca2+ deregulation. Interestingly, several variants have been identified recently in the ryanodine receptor 2 (RyR2) gene in scTdP patients14.The origin of the scTdP is currently unknown. Nevertheless, verapamil, a Ca2+ release channel located in the cardiomyocyte sarcoplasmic reticulum that governs the Ca2+ release involved in the excitation–contraction coupling (ECC)15. Its gating properties are modulated by numerous accessory proteins, such as the 12.6-kDa FK506-binding protein (FKBP12.6)18. RyR2 mutations can result in severe disease phenotypes, and more than 150 mutations caused catecholaminergic polymorphic ventricular tachycardia (CPVT), and a few have been linked to arrhythmogenic right ventricular cardiomyopathy type 2 and idiopathic ventricular fibrillation19. The previously reported mutations are dominant missense mutations mostly located in specific regions or hotspots in the N-terminal, central, and C-terminal domains including the channel region20. HEK293 cells have been the primary cell type model used to study RYR2 variants22. In cardiomyocytes, we can observe the ECC and also a mechanism called ‘store-overload induced Ca2+ release’ (SOICR), described by the team of SR Wayne Chen as the spontaneous release of Ca2+ through RyR2 which occurs when the sarcoplasmic reticulum store Ca2+ content reaches a critical level23. These events can be observed as sparks in cardiomyocytes and show up as oscillations in the whole cell in HEK293 cells. Sparks can trigger Ca2+ waves which will activate the Na+–Ca2+ exchanger. This activation will lead to an inward Na+ current which induces membrane depolarization and creates delayed after depolarization (DAD). If the amplitude of these DADs is sufficiently high, it may trigger a new action potential and then arrhythmias. It was reported that HEK293 cells expressing RyR2 displayed SOICR in a manner virtually identical to that observed in cardiac cells22.The ryanodine receptor 2, RyR2, is a large tetrameric Ca24. In human cardiomyocytes, the principal connexins are Cx40, Cx43, and Cx45, expressed at different relative levels in a chamber-related manner25. Cx40, also called gap junction protein α 5 (GJA5), is not only the main connexin isoform in atria followed by Cx43, but is also highly expressed in Purkinje fibres27. Because the normal heart rhythm depends in part on the coupling of cardiomyocytes by connexins, any alteration in connexin functions may contribute to arrhythmias25.In the heart, gap junctions (connexins or Cx) are intercellular channels that mediate exchange of ions and small molecules and mediate direct electrical coupling between cardiomyocytes, allowing rapid propagation of action potentials in the atria and ventriclesIn the present study, we showed for the first time that a RyR2 variant localized in the SPRY1 domain affects the channel structure and function, and that, in association with gap junction alteration in Purkinje fibres, it may be responsible for the patient’s tachycardia and ventricular fibrillation.2+ channel blocker (120 mg 3× daily then 240 mg 2× daily) and received an implantable cardioverter defibrillator (ICD)  with salvoes of polymorphic ventricular tachycardia Fig. a. NotewoRYR2 and GJA5 and a nonsense in TNNI3K, which were confirmed by Sanger sequencing  less than 0.1% in genes linked to inherited arrhythmias and cardiomyopathies, and in additional candidate genes overexpressed in Purkinje fibres (see Supplementary Table RYR2 (NM_001035.2) changing an isoleucine to a phenylalanine at position 784, p.Ile784Phe (I784F). This variant, rs794728729, was reported in the Genome Aggregation Database  in two heterozygous subjects, leading to a minor allele frequency (MAF) of 0.0008% in the general population, but not found in GnomAD Genomes. The I784F variant is located in the cytosolic portion of RyR2 within the SPRY1 domain  where most of the previously reported CPVT mutations have been identified . This variant has already been reported as pathogenic in a patient presenting with atrial fibrillation29. An equivalent variant chr1:147231060 GC ⇒ CT (Ala96Ser) is classified pathogenic by UniProt and confirmed using the American College of Medical Genetics (ACMG) classification. The MAF of rs121434557 is 0.0119% in GnomAD Exomes 2.1.1 and 0.0159% in GnomAD Genomes 2.1.A second variant, c.286G>T (NM_181703.3) in exon 2 of TNNI3K, induced the formation of a stop codon at position 244, p.Arg244X (R244X). The MAF of this variant, rs56257331, is 0.0028% in GnomAD Exomes 2.1.1 and 0.00319% in GnomAD Genomes 2.1.A third variant, c.781C>T (NM_015978.2) in exon 8 of -R244X  20 min before Ca2+ imaging. We first evaluated the percentages of oscillating cells and analyzed the data according to two-way ANOVA, the Tukey's post-hoc methodology was then used to identify significant differences between tested conditions while controlling for multiple testing. In Fig. 2+ concentrations and cAMP treatment (for both p < 0.0001). Indeed, the percentages of oscillating cells were increased in RyR2-I784F expressing cells compared to RyR2-WT cells (p = 0.0004). This was observed under basal conditions , as also, in cAMP treated cells  even if the trend was less significant (p = 0.052). Note that the percentages of oscillating cells increased under cAMP treatment, both in RyR2-WT (p = 0.017) and RyR2-I784F (p = 0.0006) expressing cells, whatever the Ca2+ concentrations.We aimed to study whether the RyR2-I784F variant could change the response of the channel to Ca2+, the percentage of oscillating cells was increased up to 5.52-fold in WT cells vs 2.11-fold in I784F cells (p = 0.0067). A difference was also observed at 0.5 mM, with a 1.64-fold increase in WT cells vs 1.24-fold in I784F expressing cells (p = 0.0272). By contrast, no difference was found at 1 mM .Nevertheless, when compared to basal conditions, the response of RyR2-I784F cells to cAMP treatment was lower than that of the RyR2-WT cells. Indeed, at 0.1 mM Ca2+, compared to WT. However, cAMP treatment induces a milder increase in percentages of oscillating cells in I784F cells, especially at low Ca2+ concentrations. These data suggest that the presence of the I784F variant may alter the response of RyR2 to cAMP.These results show that the expression of the I784F variant increases the percentage of oscillating cells under basal conditions at 0.1 mM and 0.5 mM Ca2+ release (SOICR) which can be observed as spontaneous Ca2+ oscillations in transfected HEK293 cells. Using a two-way ANOVA test, we found that Ca2+ oscillation frequencies differed as a function of expressed variant , Ca2+ concentrations (p < 0.0001) and cAMP stimulation (p < 0.0001) with strong relationship between Ca2+ concentration and cAMP treatment  and after cAMP treatment (0.45 ± 0.001 for I784F vs 0.43 ± 0.009 for WT) between the two types of cells, the RyR2-I784F cells displayed a significant increase in oscillation frequencies at 0.5 mM of Ca2+ both under basal conditions and after cAMP treatment . The oscillation frequencies of RyR2-I784F were also significantly enhanced at 1 mM of Ca2+ under basal conditions and after cAMP treatment . Moreover, oscillation frequencies increased after cAMP treatment, both in RyR2-WT and RyR2-I784F (both p < 0.0001) expressing cells, whatever the Ca2+ concentrations.RyR2-I784F expressing cells exhibited an increase in oscillation frequencies compared to RyR2-WT cells under basal conditions (p = 0.0203) and after cAMP treatment (p = 0.0012), especially at higher Ca2+ store. Total Ca2+ release was induced by the addition of 10 mM of caffeine, a known enhancer of the open probability of RyR2. The Ca2+ store content was estimated by measuring the amplitude of caffeine-induced Ca2+ release at 0.5 mM of Ca2+  and cAMP treatment (p < 0.0001). Under basal conditions, the amplitude of caffeine-induced Ca2+ release was significantly reduced by about 20% in RyR2-I784F cells compared to RyR2-WT cells .Next, we aimed to determine whether the increase in oscillation frequencies impacts the total Caa2+ Fig. a,b. Ca2+2+ release was found in WT-RyR2 cells compared to basal conditions . In contrast, no change was observed in RyR2-I784F cells .After cAMP treatment, a significant reduction in caffeine-induced Ca10 helices. In addition, the two flexible loops with no visible electron density in the WT, including in the FKBP-interacting loop, have clear density in the mutant structure  between the WT and I784F structures is only 0.34 Å over 177 Cα atoms, I784F clearly changes the conformation of the loop where the mutation is located. The mutation site faces a hydrophobic pocket formed by loop 663, loop 764 and loop 784. To fit the bulky side chain of Phe784, the main chain of loop 784 shifts away from the pocket by ~ 2.0 Å. This also changes the conformations of several neighboring residues, including V663, E701, E780, N781, F782, N783, D785, L787 and F788  Fig. f, suggesThe origin of ventricular fibrillation in young adults without structural cardiac abnormalities is poorly understood. Here, we identified pathogenic variants in three proteins contributing to cardiac excitability and conduction in a patient who repeatedly developed ventricular tachycardia and scTdP at rest. We showed that the new RyR2 variant in SPRY1 domain affects the channel structure and function and proposed that the cardiac electrical instability of this patient leading to lethal ventricular arrhythmias could be due to the combined effects of at least two of these variants.32. Where a family history of sudden death suggestive of heritable factors is reported, the penetrance is usually partial and low, suggesting an oligogenic inheritance of these types of ventricular arrhythmias7.To date, research into the origin of sudden cardiac death and idiopathic ventricular fibrillation has only identified a limited number of variants in genes encoding ion channels and channel-related proteins. More recently, the importance of gap-junction proteins was evidenced by identification of Cx43 variants in sudden infant death10. DPP6, a protein associated with the I(to) current, was first shown to contribute to IVF and extremely short-coupling interval PVCs by affecting the unique and highly expressed transient outward current in cardiac Purkinje fibres33. Several RyR2 variants were reported in IVF patients with or without scTdP34.ScTdP was first reported in the 1980s, and in 1994 a series of fourteen patients was described with coupling intervals of 240–300 ms initiating TdP at rest; one-third of these patients had a family history of sudden deathRYR2 variant that results in the substitution of isoleucine for phenylalanine at position 784 (RyR2-I784F), located in the SPRY1 domain, outside of the CPVT-associated RyR2 mutational hotspot regions and affecting an evolutionary conserved residue; (2) a missense variant in GJA5 leading to the substitution of alanine at position 96 for a serine (GJA5-A96S), previously reported as pathogenic29; and (3) a nonsense variant in TNNI3K which results in a stop codon at position 244 (TNNI3K-R244X).Following exome sequencing we identified three variants of interest: (1) a novel 2+ release under adrenergic stimulation36. However, the role of RyR2 in the pathogenesis of scTdP, characterized by arrhythmias at rest, in the absence of adrenergic stimulation, is less known. The first identified variant, RyR2-H29D, was found in a case of scTdP at rest; a functional analysis by single channel assays demonstrated that RyR2-H29D caused a leaky channel under non-stress conditions37. More recently, patient-specific human-induced pluripotent stem cell (hiPSC)-derived cardiomyocytes (CMs) with RyR2-H29D confirmed an aberrant sarcoplasmic reticulum (SR) Ca2+ leak through RyR2 under physiological pacing, pro-arrhythmic electrical phenotypes, impaired contractile properties, and abnormal post-translational modifications compared to isogenic controls37. Three other RyR2 variants were identified in scTdP patients, two of which displayed a slight increase in sensitivity to cytosolic Ca2+ (RyR2-V1024I and RyR2-N1551S) and one of which resulted in an almost complete loss of function (RyR2-S4938F)13. Recently, a RyR2-M995V variant was reported in a scTdP patient without functional validation14.RyR2 mutations have largely been associated with CPVT characterized by stress-induced arrhythmias, and associated with an enhanced Ca25, and even if I784 is not directly located at the FKBP binding interface, the conformational change could also alter FKPB12.6 affinity.The novel variant that we identified in the proband, RyR2-I784F, is the first described in the SPRY1 domain of RyR2. To determine the potential pathogenicity of this variant, we determined the thermal stability and the crystal structure of the RyR2 SPRY1 domain containing the I784F variant. The thermal melting experiment showed a strong destabilizing effect of the variant, suggestive of misfolding of SPRY1 domain. The structural analysis revealed that I784F causes a conformational change in a loop located at the interface with SPRY3 and Repeat12. This change might also affect other RyR2 inter-domain interactions and alter channel gating, but it is currently not possible to determine the consequences of this variant on the full RyR2 conformation. In particular, SPRY1 and the SPRY3 domains form part of the FKBP12.6 binding38.If this variant is the first analyzed in the RyR2 SPRY1 domain, a variant in the RyR1-SPRY1 domain was previously identified in a patient with multi-minicore disease and was reported to affect the interaction between SPRY1 and SPRY22+ imaging, we observed a significant increase in Ca2+ oscillation frequency and an increase in the fraction of oscillating cells in presence of different Ca2+ concentration and cAMP treatment in cells transfected with RyR2-I784F compared to WT. In addition, evaluation of the stored Ca2+ by addition of caffeine shows a reduction in the Ca2+ pool. These data suggest a gain of function at rest. The underlying mechanism could be a reduction in the SOICR threshold by an increased luminal Ca2+ sensitivity or a leaky channel due to a conformational change inducing FKBP dissociation. Interestingly, in cAMP-treated cells, which mimics adrenergic conditions, the Ca2+ release was slightly attenuated in RyR2-I784F cells compared to RyR2-WT cells, which is consistent with the occurrence of VT at rest but not under adrenergic stimulation, as well as with the normal stress test of the patient.To assess whether the RyR2-I784F variant affects channel activity, we expressed human RyR2-WT or RyR2-I784F proteins in HEK293 cells. By monitoring spontaneous RyR2 activity using CaThe RyR2-I784F variant was inherited from the proband’s father who never developed syncope or VT prior to the age of 80. Of note, the father remained asymptomatic, and may have been protected by beta-blocker treatment initiated at the age of 40. The paternal grandfather and the paternal great-grandfather died suddenly before the age of 50 and they were probably carriers of the RyR2 variant, which strengthens the likelihood of its intrinsic pathogenicity.39. The first theory, developed by Andrew Marks’ team, suggested that the dissociation of the stabilizing FKBP12.6 from the RyR2 channel would induce Ca2+ leaks during diastole40. This theory could explain the effect of our variant since it is located in SPRY1 close to one of the binding sites of FKPB12.628. Investigation of our variant using crystallography also showed a conformational change in the SPRY3 domain which is another domain involved in FKBP12.6 binding28. These conformational changes might induce a reduced interaction between FKBP12.6 and RyR2, which would lead to an unstable channel that is more prone to leakage. The second theory, developed by Wayne Chen’s team, suggested that the enhanced sensitivity of the channels could be explained by a reduction in the threshold to the luminal Ca2+ triggering the SOICR. A third theory focused on domain unzipping was developed by Yamamoto’s team42. The increase in spontaneous oscillations of the RyR2-I784F channel associated with the reduction in the Ca2+ store could be due to a reduction of the SOICR threshold. The conformational change induced by the variant might alter FKBP binding and/or interdomain stability and thus the channel gating properties, which would be consistent with the abnormal spontaneous activity of RyR2.We found that RyR2-I784F channels have a propensity to more spontaneous oscillations, which could be explained by different mechanisms. There are three main theories to explain RyR2 gain of function variantsAs a conclusion concerning this RyR2 variant, the conformational change could be the only mechanism responsible for the abnormal activity of RyR2, but we cannot exclude the existence of two additive mechanisms.44. The latter reflects functional requirements for conduction velocity which needs to be low in the sinoatrial and atrioventricular nodes and high in the ventricular conduction system. Cx40 and Cx43 play critical roles and their regional ratio is precisely regulated. Cx40 is expressed in atrial cardiomyocytes with Cx43, while Cx43 alone is expressed in working ventricular myocytes. Interestingly, Cx40 is highly expressed in the Purkinje network, with Cx45 at a lower level46 and absence of Cx43 in Purkinje fibres was showed by in situ hybridization47. These data were recently confirmed by single cell transcriptome performed by Goodyer and collaborators who identified Cx40 as the first specific marker of distal Purkinje fibres27. All these studies have been performed in different species, but studies on the human heart led to the same conclusion that Cx40 is Purkinje fibre-specific26. Numerous studies have shown that gap junction channel dysregulation could lead to arrhythmogenic disorders, including cardiac conduction abnormalities in mice lacking Cx4048.Cardiac conduction is mediated by gap junction channels that are formed by connexin (Cx) protein subunits. The mammalian heart shows regional differences both in the connexin expression profile and in the degree of electrical coupling29. Our patient inherited this variant from his mother, who developed late persistent atrial fibrillation and a dilated left atrium. Nevertheless, it is frequent that the same variant can give rise to different phenotypes depending of the genetic and physiological context. In the current study, we postulated that this variant could be a substrate for the development of ectopies from Purkinje fibres and scTdP in association with the RyR2-I784F variant. Of course, identification of other patients with such variants would be needed to strengthen this hypothesis.The Cx40-A96S variant that we identified in the proband has already been characterized in a patient suffering from idiopathic atrial fibrillation, resulting in absent or weak electrical coupling49. Four mutations in this gene have been identified in patients with dilated cardiomyopathy and CCD50. TNNI3K-G526D was identified in a three-generation family of patients with CCD, atrial tachyarrhythmia and dilated cardiomyopathy vulnerability49. TNNI3K-T539A was identified in a young child with conduction abnormalities51. Then, a splicing variant, TNNI3K-c.333 + 2T>C, leading to haploinsufficiency was reported in patients with dilated cardiomyopathy and/or CCD and a history of syncope and sudden death50. More recently, a variant TNNI3K-E768K co-segregating in three large families, displayed enhanced kinase activity in contrast to the two preceding missense variants which were associated with reduced activity52. The pathophysiological consequences of these variants are probably not completely understood. Nevertheless, previous studies in mice demonstrated increased conduction indices and pro-cardiomyopathic effects with increased levels of Tnni3k while mice lacking Tnni3k did not show echocardiographic and electrocardiographic abnormalities, suggesting that it is not essential in this context, at least in mice54. Moreover, there are a relatively high number of TNNI3K nonsense variants in the populations according to ExAC database. In the present study, the proband’s mother was a carrier of the TNNI3K-R244X variant without conduction abnormalities. Altogether, these data suggest that TNNI3K nonsense variants might only be susceptibility factors for conduction abnormalities or arrhythmias but are not causal per se.TNNI3K is a cardiac-specific protein that plays a role in cardiac contractility10. A growing body of evidence suggests that Purkinje fibres play a pivotal role in this rare condition56. A new RyR2 variant I784F in the SPRY1 domain was identified and shown to be pathogenic since it changes the thermal stability of the SPRY1 domain, its structure, and also increases the propensity to spontaneous Ca2+ release in human RyR2-I784F transfected HEK293 cells. Moreover, the patient was a carrier of a pathogenic variant in Cx40, a gap junction protein highly and specifically expressed in Purkinje fibres.Exome sequencing was performed in a patient presenting short-coupled ventricular tachycardia and sudden death at rest, consistent with the diagnosis of scTdP2+ could be responsible for cardiac instability, providing a reentrant path and anchoring reentrant waves, ultimately leading to VF and sudden death.Arrhythmias are multi-factorial in origin, involving interplay between gap-junctional coupling, membrane excitability, and cell and tissue architecture. We hypothesized that the combined presence of variants that decrease the conduction velocity and increase sensitivity to Ca2+ by the RyR2 I784F channel in HEK293 cells compatible with increased propensity for arrhythmias at rest. However, HEK293 cells lack many cardiac-specific proteins, and much remains to be done to fully understand the mechanism of this variant in cardiac cells. In particular, the absence of further enhancement of the Ca2+ release by adrenergic stimulation, as observed for CPVT RyR2 mutations, is an interesting topic to elucidate in order to gain an insight into the underlying mechanisms of scTdP RyR2 variants and this type of ventricular tachycardia originating from Purkinje fibres at rest. Mouse models harboring one or both RyR2 and Cx40 variants are presently unavailable and may not recapitulate the phenotype since extrapolation from mouse to man is limited because the mouse heart cannot accommodate large re-entrant circuits. Further comprehensive and detailed investigations will be required to address these important issues.Our results have shown an enhanced spontaneous release of Ca10. Blood samples obtained after signed informed consent were collected for genetic analyses following the granting of the approval of the local ethics committee of Pitié-Salpêtrière Hospital. The study was conducted according to the principles of the Helsinki Declaration.Clinical evaluation of the proband included 12-lead electrocardiogram (ECG), 24-h Holter ECG, exercise-stress test (EST), echocardiography, and coronary angiography. ScTdP was diagnosed according to previous descriptions17. Base calling was performed using the Real-Time Analysis software sequence pipeline (2.7.6) with default parameters. Sequence reads were mapped to the human genome build (hg19/GRCh37) using Elandv2e  allowing multiseed and gapped alignments.Library preparation, exome capture, sequencing and data analysis were performed by IntegraGen SA . Genomic DNA was captured using Agilent in-solution enrichment methodology  with their biotinylated oligonucleotides probes library , followed by paired-end 75 base pair sequencing on an Illumina HiSeq4000 as previously describedRYR2, digested by SpeI and FspAI, was sub-cloned into pCR-Blunt, and the isoleucine 784 was replaced by a phenylalanine using mutagenesis. Then the mutated SpeI/FspAI fragment of the pCR-Blunt plasmid was amplified by overlapping PCR to be re-inserted into the full-length RYR2 plasmid using a recombination kit .The pcDNA3-eGFP-hRyR2 plasmid containing the human RYR2 sequence (X98330) was kindly provided by Spyros Zissimopoulos (UK). The five mismatches between the X98330 and the RyR2 reference sequences  were first corrected by oligonucleotide directed mutagenesis : proline 1037 was changed to a leucine, and p.RTMRT at position 2785–2789 was replaced by p.WGWRI. To generate the I784F variant, a fragment of h2. Cell transfection was performed using Lipofectamine 3000  according to the manufacturer’s instructions. Briefly, one day before transfection, HEK293 cells were seeded in a 35 mm µ-dish with a glass bottom , and grown to 60–70% confluency. HEK293 cells were transfected with 280 ng of eGFP-hRyR2-WT or eGFP-hRyR2-I784F plasmids.Human embryonic kidney 293 (HEK293) cells were grown in Dulbecco’s Modified Eagle Medium (DMEM)  supplemented with 10% FBS, 100 U/mL penicillin (1%), and 100 μg/mL streptomycin (1%). Cells were cultured in a humidified atmosphere at 37 °C, 5% COTo ensure that the RyR2-WT- and RyR2-I784F expression levels were similar, the protein levels of cell lysates were assessed by immunoblot forty-eight hours after transfection   in Krebs–Ringer HEPES (KRH) buffer  at 37 °C and 5% CO2. Cells were then perfused with KRH buffer containing different Ca2+ concentrations  at room temperature. At the end of each measurement, 10 mM caffeine was added to select only the responsive cells. Single-cell intracellular calcium measurements were performed in Fura-2-loaded cells using a Nikon Eclipse Ti-U inverted fluorescent microscope  equipped with a dual excitation filter wheel (340 nm/380 nm), a 515-nm emission filter and a Hamamatsu ORCA-D2 CCD camera . Calcium traces were obtained by sequential acquisition of one image every 2 s for 3 min using a 20X S-Fluor 0.75 NA (WD = 1 mm) objective. Image acquisition was performed with the HCimage software . Fluorescence intensities in single cells were determined using region of interest (ROI) analysis with ImageJ software . Ca2+ signals were measured in the caffeine-responsive single cells using F340/380 ratios after subtraction of Fura-2 background fluorescence. For experiments with cAMP treatment, cells were pretreated with 250 µM of 8-bromo-cyclic AMP  for 20 min. at room temperature before Ca2+-KRH perfusion.Intracellular Ca2+-containing KRH buffer. After 3 min, 10 mM caffeine was added in order to induce the total Ca2+ store release. The peak amplitude of each caffeine-induced Ca2+ release was determined in each cell using the F340/380 ratio. The total number of cells from six separate experiments were under basal conditions WT n = 1578 and I784F n = 1760, and after cAMP WT n = 957 and I784F n = 1007.Transfected HEK293 cells were loaded with 5 µM of Fura2-AM for 45 min and then perfused with 0.5 mM Ca57. Protein was expressed and purified as previously described for the RyR2-WT-SPRY128. Crystals were grown by hanging drop vapor diffusion at 4 °C. I784F-SPRY1 (10 mg/mL) was crystallized in 0.1 M sodium acetate (pH 4.5) plus 0.8 M sodium. Crystals were transferred to a solution supplemented with 30% isopropyl alcohol and flash-frozen in liquid N2 prior to data collection.The I784F mutation was introduced into the RyR2-SPRY1 domain (650–844) of a mouse construct within a pET28-HMT vector containing an N-terminal His-tag, a MBP-tag and a TEV cleavage site58 and processed using the HKL3000 data processing program59. The I784F structure was solved by molecular replacement in PHENIX using the wild-type mouse RyR2-SPRY1 domain (PDB ID 5C33) as the searching model60. Further model building was performed manually using COOT61. Structure refinement was performed using PHENIX with high-resolution (1.45 Å) native data. Data collection and final refinement statistics are available in Supplementary Table Diffraction data was collected with the Shanghai Synchrotron Radiation Facility (SSRF) beamline BL18U162. Ten ml of protein (1 mg/mL) were diluted to 50 mL with a buffer containing 10 mM sodium phosphate (pH 7.4), 150 mM KCl, 14.3 mM beta-mercaptoethanol and SYPRO orange dye . The melts were obtained using a QuantStudio 6 Flex real-time PCR machine , with the SYBR green filter option. The melting temperatures were held at 10 °C for 3 min., and then increased at a rate of 0.033 °C/s up to 95 °C. The melting temperatures were obtained by taking the midpoint of each transition.The protein melting curves were measured using a fluorescence-based thermal shift assayt test. A p value inferior to 0.05 was considered as significant.Data are expressed as the mean ± SEM. Differential analyses were performed using two-way Analysis of Variance (ANOVA) implemented in the R statistical environment. The Tukey's post-hoc methodology was then used to identify significant differences between tested conditions while controlling for multiple testing. Statistical analysis in Fig. Supplementary Information.


FLT3 are the most common genetic alteration in AML, identified in approximately one third of newly diagnosed patients. FLT3 internal tandem duplication mutations (FLT3-ITD) are associated with increased relapse and inferior overall survival. Multiple small molecule inhibitors of FLT3 signaling have been identified, two of which (midostaurin and gilteritinib) are currently approved in the United States, and many more of which are in clinical trials. Despite significant advances, resistance to FLT3 inhibitors through secondary FLT3 mutations, upregulation of parallel pathways, and extracellular signaling remains an ongoing challenge. Novel therapeutic strategies to overcome resistance, including combining FLT3 inhibitors with other antileukemic agents, development of new FLT3 inhibitors, and FLT3-directed immunotherapy are in active clinical development. Multiple questions regarding FLT3-mutated AML remain. In this review, we highlight several of the current most intriguing controversies in the field including the role of FLT3 inhibitors in maintenance therapy, the role of hematopoietic cell transplantation in FLT3-mutated AML, use of FLT3 inhibitors in FLT3 wild-type disease, significance of non-canonical FLT3 mutations, and finally, emerging concerns regarding clonal evolution.The FLT3 receptor is overexpressed on the majority of acute myeloid leukemia (AML) blasts. Mutations in  FLT3 are the most common genomic alteration in AML, identified in approximately one-third of newly diagnosed adult patients (Acute Myeloid Leukemia (AML) is an aggressive hematologic malignancy characterized by a heterogenous genetic landscape and complex clonal evolution . Fms-likpatients , and arepatients .FLT3 mutations can be subdivided into internal tandem duplicates (ITD), present in approximately 25% of patients, and point mutations in the tyrosine kinase domain (TKD), present in approximately 5%. Both FLT3-ITD and FLT3-TKD mutations are constitutively activating, leading to ligand-independent FLT3 signaling and cellular proliferation  FLT3, the FLT3 juxtamembrane domain inhibits receptor activation; the presence of ITDs disrupts this inhibitory effect, resulting in constitutive activation , generally defined as a FLT3-ITD to FLT3-WT ratio of ≥0.5, is associated with higher disease risk. Low AR (FLT3-ITDlow) is associated with favorable risk in patients with a co-occurrent nucleophosmin 1 (NPM1) mutations and intermediate risk in patients with NPM1-WT. These associations are reflected in the 2017 European LeukemiaNet (ELN) risk stratification of AML  guidelines -based assays and next generation sequencing (NGS). Due to competition from the FLT3-WT allele, the sensitivity of standard PCR-based assays may be lower than NGS-based methods, although this can be overcome using patient-specific PCR primers  has also been used to define FLT3-ITD positivity, primarily in research studies. Unlike the AR, which calculates the ratio of the area under the curve (AUC) of mutant to WT alleles (FLT3-ITD/FLT3-WT), the AF calculates the fraction of mutant alleles as a percentage of all FLT3 alleles (FLT3-ITD/FLT3-WT + FLT3-ITD). For example, a FLT3 AR of 0.5 (0.5 ITD/1.0 WT) would be equal to a FLT3 AR of 0.33 (0.5 ITD/0.5 ITD + 1.0 WT = 0.33 AF). NGS studies are typically reported as VAF while PCR assays typically report AR.The FLT3-ITD has been inherently difficult to detect using NGS. NGS relies on the reconstruction of short (<300 base pair) sequences, and longer length ITDs may not be detected  following treatment. An NGS-based FLT3 assay is now commercially available and is currently used in in an ongoing trial of gilteritinib maintenance therapy following hematopoietic cell transplantation (HCT) (NCT02997202).Historically, detected , 15 althdetected . NGS is FLT3 AR, which can only be determined by PCR. There is currently no standardized methodology or laboratory reference values for AR determination, and the current cut-off of ≥0.5 has not been prospectively validated. In retrospective analyses, higher AR is generally associated with inferior clinical outcomes  AML are summarized in First generation FLT3 inhibitors include sorafenib, midostaurin, lestaurtinib, sunitinib, and tandutinib. These multi-kinase inhibitors target not only FLT3 but other kinases as well, including PKC, SYK, FLK-1, AKT, PKA, KIT, FGR, SRC, PDGFRα/β, and VEGFR 1/2 (midostaurin) and RAF, VEGFR 1/2/3, PDGFRβ, KIT, and RET (sorafenib). The antileukemic effects of these non-specific inhibitors likely derive not only from FLT3 inhibition, but from inhibition of targets in these parallel pathways as well. Similarly, due to their multiple off-target effects, first generation FLT3 inhibitor may also be associated with increased toxicities. In contrast, second generation FLT3 inhibitors are more potent, FLT3-specific and thus have fewer off-target effects at clinically relevant doses. Second generation FLT3 inhibitors include gilteritinib, quizartinib, and crenolanib.FLT3-TKD and FLT3-ITD mutated receptors. Type II inhibitors, including quizartinib and sorafenib, bind to a region adjacent to the ATP-binding pocket and only inhibit the receptor in the inactive conformation. Type II inhibitors are inactive against most FLT3-TKD mutations as these mutations bias the active kinase conformation of FLT3. This distinction is key in understanding mechanisms of FLT3 inhibitor resistance, as discussed later in this review.FLT3 inhibitors can also be subdivided based upon how they interact with the intracellular kinase domain (KD) of the FLT3 receptor. In normal physiology, FLT3 ligand binds to the extracellular domain, causing the FLT3 receptor to dimerize, assume an enzymatically active conformation and subsequently activate downstream signaling. Type I FLT3 inhibitors, which include midostaurin, gilteritinib, lestaurtinib, and crenolanib, bind the receptor in the active conformation, thus inhibiting both FLT3 inhibitors to be studied in AML. Early studies of single-agent midostaurin in R/R AML demonstrated the drug was well-tolerated, but had limited efficacy , with a median OS of 74.7 months in patients receiving midostaurin plus chemotherapy vs 25.6 months in patients receiving placebo plus chemotherapy (p = 0.009)  approval, becoming first agent to achieve FDA approval for AML since the year 2000.Results of midostaurin in combination therapy have been more promising. In the phase III RATIFY trial, midostaurin was evaluated in combination with standard induction and consolidation chemotherapy in adults <60 years with = 0.009) . In 2017FLT3-TKD mutation, significantly larger than that seen in the general population, and perhaps biasing the results toward this less-aggressive disease subtype. In addition, while patients in RATIFY were younger (median age 48), FDA approval was extended to all age groups  rate in all patients and a 93% CR rate in the subset with FLT3-ITD mutant AML . Furthermore, sorafenib demonstrated marked toxicity, presumably due to off-target effects    , there w = 0.23) .FLT3 status demonstrated acceptable toxicity profiles but mixed CR rates, ranging from 10 to 48%  or RFS    . CurrentFLT3 inhibitors are more specific to the FLT3 receptor, exhibit greater potency, and have thus been far more efficacious as single-agent therapy. Both gilteritinib and crenolanib are type I inhibitors, active against both the active and inactive conformation of the FLT3 receptor, while quizartinib is a type II inhibitor, active only against the inactive form.Unlike midostaurin and sorafenib, second-generation FLT3-ITD mutated AML , and a greater proportion of patients were able to proceed to hematopoietic cell transplantation . Total treatment-associated toxicities were similar between the two arms, although 2% of patients receiving quizartinib experienced Grade 3 QTc prolongation. Based on these results, the authors of QuANTUM-R concluded that quizartinib monotherapy should be considered a standard of care option in R/R FLT3-ITD mutated AML  in stratified analysis, a significant survival benefit only when quizartinib was compared against low-intensity therapy (low-dose cytarabine) and not against high-intensity therapy (MEC or FLAG-Ida). The ODAC concluded that, while a modest survival benefit of 6 weeks was still meaningful, especially if more patients were bridged to HCT, ultimately additional data would be needed for quizartinib to be approved in this setting (FLT3-ITD AML (NCT03989713).Despite these positive results, in 2019, both the FDA and the European Medicines Agency (EMA) rejected approval for quizartinib, although regulatory approval was granted in Japan. Although the FDA Oncologic Drug Advisory Committee (ODAC) raised concerns that up to four deaths in the quizartinib arm were attributed to cardiotoxicity, the decision to decline was ultimately due to concerns regarding trial design . These c setting . The phavs placebo in combination with induction and consolidation chemotherapy (NCT02668653). Additional phase I/II studies evaluating quizartinib in combination with frontline cytotoxic chemotherapy are ongoing as well .In newly diagnosed AML, quizartinib is currently being evaluated in the randomized phase III QuANTUM-First trial, which will compare quizartinib FLT-ITD-mutated patients, 17% in FLT3-D835-mutated patients, and 62% in patients with both FLT3-ITD and FLT-D835 mutations  and improvement in OS . While prior use of midostaurin or sorafenib was allowed, as the trial enrolled prior to midostaurin’s approval and widespread use, the majority (88%) of patients had not received a prior FLT3 inhibitor, limiting the ability of this trial to evaluate the ability of gilteritinib to overcome midostaurin resistance; however, results were similar in patients with FLT3-ITD and FLT3-TKD mutated disease .It is unknown whether gilteritinib is similarly beneficial in newly diagnosed AML. Gilteritinib is currently being studied in a phase I study in combination with induction and consolidation chemotherapy in newly diagnosed AML (NCT02236013); interim results indicate this approach is safe and feasible . The phaFLT3-ITD, and FLT3-TKD mutations, including at D835 .Crenolanib also demonstrates promising results in combination with chemotherapy. A phase II trial of crenolanib plus chemotherapy in newly diagnosed 3 months . Crenolatoxicity . A phaseFLT3 and off-target mutations in downstream or parallel signaling pathways. These mutations may develop de novo or via expansion of pre-existing subclones , ASXL1, TP53, TET2, and IDH1/2. These mutations occurred not only in cells expressing the FLT3-mutant allele, but in FLT3-WT cells as well .Upregulation of parallel AXL tyrosine kinase signaling is another mechanism of FLT3 inhibitor resistance. In one study, blasts from a patient with  TP-0903 , 71 and via selection of pre-existing sub-clones harboring survival advantages under the selective pressure of FLT3 inhibition.Together, these studies suggest off-target resistance mechanisms are common to all FLT3 inhibitors and frequently arise via the native FLT3 receptor, even in the presence of FLT3 inhibitors  azacitidine or decitabine. This combination is particularly attractive, both due to the synergistic cytotoxicity in preclinical studies as well as the established tolerability and durable responses of HMAs in older adults , 82.FLT-mutated AML who are unfit for traditional chemotherapy in the frontline setting , with primary endpoints of tolerability and ORR.Multiple phase I/II trials have demonstrated the combination of midostaurin and HMA to be feasible in adults with  setting  and in R setting , 84. AddSorafenib plus HMA have also been shown to be safe and efficacious in single-arm and retrospective studies in both the R/R , 86 and FLT3-ITD AML demonstrated an ORR of 75%, the secondary endpoint for the trial’s phase II portion, including in four/five patients with prior FLT3 inhibitor exposure (FLT3-ITD AML with primary endpoint of OS is currently accruing (NCT02752035) with interim results of secondary endpoints demonstrating CR and ORR rates of 67 and 80%, respectively  and in combination with venetoclax alone in R/R FLT3-ITD AML (NCT03735875).Gilteritinib is currently being studied in combination with venetoclax in R/R AML (NCT03625505). Quizartinib is being evaluated in combination with venetoclax and decitabine in poor-risk newly diagnosed or R/R FLT3-ITD mutant cells in preclinical studies (FLT3-ITD AML (FLT3-ITD AML (NCT01371981) and sorafenib plus bortezomib followed by decitabine in newly diagnosed or R/R AML regardless of FLT3 status (NCT01861314).Proteosome inhibitors, including bortezomib, have demonstrated cytotoxicity toward  studies  and a ph studies . Sorafen-ITD AML . OngoingMultiple agents targeting signaling pathways downstream of FLT3 signaling, including those frequently implicated in FLT3 inhibitor resistance, have been studied in combination therapies.Pim kinase inhibitors inhibit Pim-1, a kinase which promotes FLT3 signaling via positive feedback . In addition, multiple novel dual FLT3/CDK4 inhibitors are in active preclinical development, including AMS-925 (Cyclin-dependent kinase 6 (CKD6) is a key regulator of cell cycle progression, part of a transcriptional complex that promotes leukemogenesis, and is found at the promoter of both M1 genes , 106. A  AMS-925  (Keegan) AMS-925 , and FLX AMS-925 , which rMDM2, has demonstrated synergistic anti-leukemic activity with quizartinib in FLT3-mutant cell lines .Finally, the tumor suppressor p53 is increasingly recognized as a mechanism of FLT3 inhibitor resistance, particularly to crenolanib . Milademll lines  and a phFLT3-mutated disease  and PD-1 ligand (PD-L1) are associated with inferior OS in AML, including in patients with  disease . A phaseIn addition to novel FLT3 inhibitor combinations, multiple novel FLT3 inhibitors are in active preclinical and clinical development (Ponatinib is approved to target BCR-ABL in chronic myelogenous leukemia (CML), but is also a type II FLT3 inhibitor with activity against F691L  has been studied in multiple solid tumors and also demonstrates activity against FLT3, including F691L , with promising preliminary efficacy data , as wellonjugate  . Of note, neither of these trials will directly compare maintenance midostaurin against placebo, which will make it difficult to isolate the true benefit of post-chemotherapy midostaurin maintenance.Currently, both the single-arm AMLSG 16–10 trials, which will also evaluate midostaurin in combination with chemotherapy in older adults, and the phase III HOVON 156 AML trial, which will compare midostaurin Multiple ongoing trials are also evaluating second generation FLT3 inhibitors as post-chemotherapy maintenance. In the HOVON 156 AML trial, patients may receive up to one year of gilteritinib maintenance, although again, this study will not compare gilteritinib against a placebo control. A separate randomized phase III trial will compare gilteritinib vs placebo maintenance for up to two years following induction and consolidation therapy in patients who are not proceeding to HCT accrual (NCT02927262). Similarly, the QuANTUM-First trial will include up to three years of post-chemotherapy quizartinib maintenance and will be placebo-controlled.In the post-HCT setting, midostaurin was evaluated in a phase II trial as single-agent maintenance following midostaurin plus chemotherapy and subsequent HCT. Of the 56% of patients who ultimately received HCT and subsequent maintenance, midostaurin maintenance was associated with improved OS compared to a historical control group; however, given this comparison was relative to historic controls it must be interpreted with caution . Midostavs standard care following HCT. Although preliminary reports suggested that addition of midostaurin could reduce risk of relapse at 18 months post-HCT by 46%, 63% of patients receiving midostaurin required dose modifications and 25% discontinued midostaurin due to toxicity. RADIUS ultimately had inadequate enrollment to detect a statistically significant difference in 18-month relapse-free survival (RFS), the study’s primary endpoint   .FLT3-ITD AML in remission following HCT to maintenance with two years of sorafenib vs placebo ; however, only 9/43 (21%) of patients receiving sorafenib had received a frontline FLT3 inhibitor, so it remains unclear whether the same benefit would be seen in patients who received a FLT3 inhibitor in the frontline setting. Similar to the RADIUS trial, study drug discontinuation was more common in the sorafenib arm (22 vs 5%), although this difference was not significant. Finally, and again similar to the RADIUS trail, SORMAIN did not reach is target accrual and was terminated prematurely.Similarly, post-HCT sorafenib maintenance was shown to be tolerable and potentially efficacious in both retrospective and prospective phase I/II studies, although frequent dose adjustments were needed , 132. Mo placebo . The HR FLT3-mutated AML (NCT03951961) and a phase III trial with compare post-HCT midostaurin vs crenolanib maintenance (NCT03258931).Building on the results of RADIUS and SORMAIN, there the multiple ongoing trails evaluating post-HCT maintenance therapy. The phase II, single-arm MAURITUS trial will evaluate midostaurin maintenance following HCT in MRD-positive vs placebo for FLT3-ITD mutated AML following HCT is ongoing (NCT02997202). Importantly, although prior FLT3 inhibitor treatment is not an inclusion criterion, since enrollment occurred after the widespread use of midostaurin in the frontline setting, the majority of patients enrolled on BMT CTN 1506 will likely have received prior FLT3 inhibitor therapy, answering a key question raised by SORMAIN. In addition, quizartinib was shown to be well-tolerated as single-agent maintenance following HCT in a phase I study (FLT3-mutated AML (NCT02400255).For the second generation FLT3 inhibitors, BMT CTN 1506, a randomized phase III trial of gilteritinib  I study  and thisThe role of FLT3 inhibitor maintenance, while promising, remains unknown, and placebo-controlled randomized trials are necessary to establish the efficacy of this approach. Multiple questions remain, including: How can we identify the patients for which FLT3 inhibitor maintenance is most beneficial? In SORMAIN, the strongest benefit from sorafenib maintenance was in patients with undetectable MRD pre-HCT and detectable MRD post-HCT . NotablyFLT3-mutated AML has been considered adverse risk disease, and patients with FLT3-ITD have been recommended to undergo HCT in the first CR (FLT3-mutated AML such that patients with FLT3-ITDlow (AR < 0.5), normal cytogenetics, and mutated NPM1 are considered low risk, suggesting these patients may have good prognosis without HCT , HCT in first CR was recommended for patients with intermediate or adverse risk al trial . RandomiFLT3-ITD AML was dismal. Historically, while patients with FLT3-ITD responded to induction chemotherapy with similar remission rates as other AML subtypes, patients were more likely to relapse and had inferior OS  and 0.09 for FLT3-ITDhigh/NPM-WT (adverse risk)  . While hFLT3-mutated AML may be changing. Will FLT3 mutations in AML eventually be analogous to HER2 amplification in breast cancer or BCR-ABL1 fusions in acute lymphoblastic leukemia? In both of these cases, the development of targeted therapies has dramatically improved the prognosis of a previously poor-risk subtype, and a similar pattern may evolve with FLT3 as well.Given the multiple FLT3-directed therapies both approved and in development, the prognosis of FLT3 mutations outside of the ITD and D835 regions have been described. These non-canonical (NC) mutations are frequently in exon 14 of the juxtamembrane (JM) domain, where ITDs occur, or in the KD domain adjacent to D835; however NC mutations in other sites, including the extracellular (EC) domain, have been described as well. Select NC mutations identified in the clinical literature are summarized in As sequencing technology improves, FLT3 point mutations and insertions-deletions compared to a 23% total prevalence of all FLT3 mutations (including FLT-ITD and D835). This included 9 JM mutations with activating potential , many which occurred as co-mutations with FLT3-ITD  and two JM  mutations  were maintained at time of relapse , prognostic impact, and role in the FLT3 inhibitor susceptibility and resistance.FLT3 mutations, the FLT3 receptor is over-expressed on leukemic blast regardless of mutational status  AML and may impact prognosis  AML recently completed accrual (NCT01830361).Given that both midostaurin and sorafenib are multikinase inhibitors, it is possible these results are due to inhibition of alternative kinase-dependent pathways. For example, both sorafenib and midostaurin also inhibit KIT, and rognosis , 151. ThFLT3-WT disease has also been demonstrated in second generation FLT3 inhibitors, which are more specific to the FLT3 receptor. While the initial phase I/II study of gilteritinib demonstrated strongest antileukemic effect in patients with FLT3-mutated disease, a 12% ORR was seen in FLT3-WT AML  and chemotherapy +/− quizartinib (NCT04107727), both in patients with newly-diagnosed FLT3-WT AML. In addition, many phase I trials of novel combination therapies, dual FLT3 inhibitors, and biologic agents are not restricted to FLT3-mutant disease.Trials of FLT3 inhibitors in FLT3-mutated AML as FLT3 mutations can be gained with disease progression, and development of a new FLT3-ITD is an independent negative prognostic factor .FLT3-mutated AML, and additional treatment options are needed. This includes increasing the diversity of approved FLT3, investigating FLT3 inhibitors in new combinations and treatment settings, and development of novel agents. Given the diversity in FLT3 inhibitor potency, resistance patterns, and toxicity profiles, ideally the oncologist will have a range of FLT3-directed therapies to choose from, similar to TKI selection in chronic myelogenous leukemia, so that a particular FLT3 inhibitor could be matched to an individual patient’s needs.While this provides a baseline treatment paradigm, survival remains poor in VK and CS both contributed to the writing of this manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.