Normally, [Ca2+]i is maintained at very low levels but in sickle cells, Ca2+ permeability is increased, especially following deoxygenation and sickling, mediated by a pathway sometimes called Psickle. The molecular identity of Psickle is also unclear but recent work has implicated the mechanosensitive channel, PIEZO1. We used Yoda1, an PIEZO1 agonist, to investigate its role in sickle cells. Yoda1 caused an increase in [Ca2+]i and PS exposure, which was inhibited by its antagonist Dooku1 and the PIEZO1 inhibitor GsMTx4, consistent with functional PIEZO1. However, PS exposure did not necessitate an increase in [Ca2+]i. Two PKC inhibitors were also tested, chelerytherine chloride and calphostin C. Both reduced PS exposure whilst chelerytherine chloride also reduced Yoda1-induced increases in [Ca2+]i. Findings are therefore consistent with the presence of PIEZO1 in sickle cells, able to mediate Ca2+ entry but that PKC was also involved in both Ca2+ entry and PS exposure.Phosphatidylserine (PS) exposure is increased in red cells from sickle cell anaemia (SCA) patients. Externalised PS is prothrombotic and attractive to phagocytes and activated endothelial cells and thus contributes to the anaemic and ischaemic complications of SCA. The mechanism of PS exposure remains uncertain but it can follow increased intracellular Ca PS exposure may thus be associated with red cell death and blockage of the microvasculature. Normally less than 1% red cells are positive for externalised PS. Notwithstanding, increased PS exposure does occur in healthy red cells during their normal ageing process4. In addition, however, red cells from patients with sickle cell anaemia (SCA) show less PS asymmetry than those from normal individuals, such that a high, but variable, proportion (c.1–10%), are positive for exposed PS6. Loss of the asymmetric distribution of this aminophospholipid may thereby contribute to both the chronic anaemia and acute ischaemic episodes characteristic of the morbidity of SCA.As in other cells, the aminophospholipid phosphatidylserine (PS) of red cells is usually located on the inner leaflet of the membrane bilayer. This is important because externalised PS is prothrombotic, and also attractive to macrophages and activated endothelial cells2+-activated scramblase which rapidly moves PS in both directions; and (iii) a floppase which moves PS from inner to outer leaflet7. PS exposure requires both inhibition of the flippase together with activation of the scramblase8. Both of these may be precipitated by increases in intracellular Ca2+ concentration ([Ca2+]i) which under normal circumstances is kept at very low levels, around 30 nM, in comparison with a free plasma Ca2+ at a concentration some five orders of magnitude higher (at about 1.1–1.2 mM). Maintenance of these low levels of [Ca2+]i is dependent upon the activity of a high capacity plasma membrane Ca2+ pump or ATPase (PMCA) coupled with a very low resting permeability to Ca2+9. As well as promoting PS exposure, elevation of Ca2+ is central to a number of other deleterious consequences which compromise red cell survival—including activation of the Ca2+-activated K+ channel (or Gárdos channel10) and of Ca2+-dependent proteases.Movement of PS across the red cell membrane is dependent mainly on three transport systems: (i) an ATP-dependent aminophospholipid translocase (or flippase) which moves PS from outer to inner leaflet using energy provided by ATP hydrolysis; (ii) a Ca11. Replacement of this negatively charged amino acid with a neutral one enables neighbouring molecules of deoxygenated HbS to adhere, forming long, rigid polymers which distort red cell shape, causing the sickling shape change. HbS-containing red cells also have a markedly abnormal cation permeability13. This is especially true for K+. There are three transport systems, which are either absent from or kept at low activity in red cells from normal individuals, but which are upregulated or abnormally active in sickle cells: the KCl cotransporter (KCC), a deoxygenation-induced cation conductance  and the Ca2+-activated K+ channel or Gárdos channel13. These three systems mediate net ion loss, with water following osmotically, thus raising the concentration of intracellular HbS, the abnormal Hb found in sickle cells. These events are relevant to pathogenesis as the lag time to polymerisation of HbS upon deoxygenation is highly dependent on its concentration. A lag inversely proportional to [HbS]15-30 is often quoted14. Small solute loss through these transporters may therefore markedly encourage sickling and a catalogue of damaging sequelae, contributing to the pathogenesis of SCA.Red cells from SCA patients contain a mutated form of Hb, HbS, instead of the normal adult HbA. In HbS, a single base substitution results in the exchange of glutamic acid by valine at the sixth amino acid residue of the β chainsickle, is particularly significant for PS externalisation. The Psickle pathway is activated by HbS deoxygenation, polymerisation and the sickling shape change15, and as well as making the cell leaky to univalent cations, it also mediates entry of Ca2+16. If [Ca2+]i approaches a few hundred nanomolar, the co-ordinated inhibition of the flippase and activation of the scramblase is initiated. It is not surprising therefore that deoxygenation of sickle cells has been associated with increase in PS exposure17, which are both dependent on the presence of extracellular Ca2+ and which may be inhibited by intracellular Ca2+ chelation18.One of these cation permeablities, Psickle and identification of potential therapeutic inhibitors19. Notwithstanding its significance, however, the molecular identity of Psickle still remains uncertain. Various pathways for mediation of Psickle activity have been proposed, ranging from a simple disintegrity of the plasma membrane, to specific protein-mediated pathways including VDAC, NMDA receptors, TRPV channels as well as others20. Latterly attention has focused on PIEZO121. PIEZO1 is a very large, multimeric channel with mechanosensitive properties. It is found in many tissues including red cells in which it has been proposed as a Ca2+ channel. There are two compelling pieces of evidence. Thus several ways of distorting the shape of normal red cells, for example shear stress, activate a Ca2+ entry process. In addition, gain-of-function mutations in PIEZO1 have been found in patients with hereditary xerocytosis24 and stomatocytosis27 with a proposed mechanism of increased Ca2+ entry causing aberrant Gárdos channel activation and therefore K+ loss and dehydration. It is also notable that a common PIEZO1 mutation is present in the African population28 although this mutation did not appear to correlate with Psickle activity29. Psickle has long thought to be unique to red cells from SCA patients, which is odd given that the underlying mutation resides in the β globin gene, and not in any a membrane transport protein. Findings with PIEZO1 suggest that a Psickle entity may therefore be present and functional in red cells of all people.With its proposed central role in SCA pathogenesis, it is understandable that considerable attention has been focused on regulation of P33. These reagents are somewhat problematical in that they may not be specific for PIEZO1 and, at least one of them, Yoda1, has been found have effects via other mechanisms, notably changes in protein phosphorylation34. Notwithstanding, an obvious approach to investigate the involvement of PIEZO1 as a candidate for Psickle was to test them on Ca2+ entry and PS exposure in red cells from SCA patients.Recently, several pharmacological agonists/antagonists of PIEZO1 have been described, including Yoda1, Dooku1 and Jedi12+ channels, but also possibly through a Ca2+-independent action36. Thus various PKC inhibitors reduce PS exposure, and some, but not all, also reduce Ca2+ entry in normal red cells.There have also been several reports implicating an important role for protein kinase C (PKC) in red cell PS exposure, possibly via opening of Ca2+, PIEZO1 and PKC in PS exposure in sickle cells using agonists and inhibitors of PIEZO1, Ca2+ ionophores, and PKC antagonists. Results show that the PIEZO1 agonist Yoda1 stimulated Ca2+ entry and caused PS exposure in almost all red cells from SCA patients. An increase in [Ca2+]i, however, was not a pre-requisite for PS exposure. Rather Yoda1 appeared to act mainly via PKC in a Ca2+-dependent and Ca2+-independent mechanism. These results with PIEZO1 modulators confirm previous reports of the complexity of PS exposure in red cells including sickle cells and emphasise an important caveat that pharmacological reagents are often promiscuous in their effect.In this report, we investigated the roles of CaFluorescein isothiocyanate-conjugated lactadherin (LA-FITC) and phalloidin  came from Haematologic Technologies Inc. , supplied by Cambridge Bioscience . Anti-glycophorin A-PE from Becton Dickinson Biosciences  came from Enzyme Research Laboratories . Bromo-A23187, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) and Fluo-4-AM came from Calbiochem . Yoda1, Dooku1 and GsMTx4 were supplied by Tocris Bioscience , Glixx Laboratories Inc  and AbCam ; and AbCam also supplied the PKC inhibitors chelerytherine chloride and calphostin C. All other chemicals were supplied by Sigma-Aldrich Co. .Blood samples were taken with informed consent from patients homozygous for sickle cell anaemia (SCA), HbSS genotype, using the anticoagulant EDTA. The study was approved by the National Health Service (NHS) National Research Ethics Committee (NREC reference 16/LO/1309). For some experiments, once routine haematological assays had been completed, discarded and anonymised blood was used. All research was conducted with ethical approval and in accordance with the Helsinki Declaration of 1975, as revised in 2008.(HEPES), were either low K+ (LK-HBS) or high K+ (HK-HBS). For LK-HBS, saline comprised (in mM): NaCl 145, KCl 5, MgCl2 0.15, inosine 10 and HEPES 10, . For HK-HBS, salines were similar but with NaCl 54 mM and KCl 90 mM. For Ca2+ titration curves, additions of EGTA (2 mM) and concentrations of total [Ca2+] of 0, 1.35, 1.63, 1.72, 1.81, 1.85, 1.91 and 2.00 mM were used to clamp free extracellular [Ca2+] ([Ca2+]o) at 0, 0.1, 0.2, 0.3, 0.45, 0.6, 1 and 10 µM, respectively. Where required, red cells were permeabilised to Ca2+ with the ionophore bromo-A23187 . For oxidant challenge, a stock solution of tert-butyl hydroperoxide (tBHP) was made in LK-HBS. Exposed PS was labelled with LA-FITC (16 nM) in HK-HBS  containing 1 mM vanadate (LA-FITC binding buffer). To prepare red cells, whole blood was washed four times in HK-HBS (pH 7.4 at RT) to remove plasma and buffy coat. For experiments in which intracellular [Ca2+] ([Ca2+]i) was manipulated, the final two washes contained, in addition, EGTA (1 mM) to remove any contaminant Ca2+. Red cells were stored on ice until required. Haematocrit (Hct) was measured using Drabkin's reagent.The standard salines, buffered with 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid 2+]os, using bromo-A23187 (6 µM) to permeabilise red cells to Ca2+, in the absence or presence of different oxidants or thiol modifiers. The activity of bromo-A23187 was abrogated by adding 0.4 mM Co2+. Red cells were then pelleted and resuspended in HK-HBS containing 1 mM vanadate (pH 7.4 at RT), diluted in LA-FITC binding buffer at 0.01% Hct, and incubated for 15 min in the dark at RT. Red cells were next pelleted , washed once in HK-HBS, resuspended and kept on ice in the dark until flow cytometry analysis. LA-FITC was detected in the FL1 channel of a BD Accuri C6 flow cytometer using logarithmic gain (as for CM-H2-DCF fluorescence). The positive fluorescent gate was set using red cells unlabelled with LA-FITC. For each measurement 10,000 events were gated. PS positive cells were defined as all events falling within the preset FSC, SSC and positive fluorescent gates. See18 for detailed Methodology.To promote lipid scrambling, red cells were incubated in HK-HBS  containing 2 mM EGTA at various i  whilst [Ca2+]i continued to increase up to the maximum concentration used (5 µM). In the absence of extracellular Ca2+, as anticipated, Yoda1 had no effect on [Ca2+]i . These findings are consistent with a Yoda1-induced stimulation of Ca2+ entry across the plasma membrane. As Yoda1, Dooku1 and GsMTx4 are all considered to interact with PIEZO1, these results are compatible with PIEZO1-mediated Ca2+ entry and that the majority of red cells can react to mediate Ca2+ increase.In the presence of extracellular Ca2+ ionophore. As expected from its effects on [Ca2+]I, Yoda1 treatment resulted in increased PS exposure in the majority (about 80%) of the red cell population .In the next series of experiments, the effect of Yoda1 on phosphatidylserine (PS) exposure in red cells from SCA patients was investigated, first in the absence of Ca2+ entry was directly responsible for the lipid scrambling, there was, however, little, or no, inhibitory effect on Yoda1-induced PS exposure when red cells were incubated in Ca2+-free saline  to clamp [Ca2+]i over a range of concentrations from 0 to 1.0 µM. Results compared red cells exposed to Yoda1 at two concentrations, 0.2 and 1.0 µM with those incubated in its absence , PS exposure was also Ca2+ dependent but the concentration dependence was altered to significantly lower values (EC50 0.18). Even in the absence of Ca2+, with 0.2 µM Yoda1 PS exposure reached about 20% (p < 0.05 cf cells without Yoda1). In addition, almost all cells were positive for external PS with Ca2+ of ≥ 0.5 µM (cf about 35% in the absence of Yoda1 at the highest [Ca2+] tested). With the higher concentration of Yoda1 (1.0 µM), PS exposure became Ca2+-independent and all cells were positive for PS over the whole Ca2+ titration, including cells incubated in the absence of Ca2+.The effect of Ca2+ levels. Thus Dooku1 (up to 10 µM) reduced Yoda1-induced PS exposure, in both the presence and absence of Ca2+  had access to phospholipids at both the inner, as well as the outer, leaflet. Results consistent with this hypothesis have been previously obtained using the oxidant tert butyl hydroperoxide37. If FITC-lactadherin had been able to access the inside of the lipid bilayer, positively labelled cells would be present in the absence of PS externalisation. To ascertain whether this possibility had occurred, red cells were exposed to fluorescently-labelled phalloidin  which binds to intracellular actin, but can only gain access to its target if the membrane integrity is disrupted. In a control experiment, as expected, phalloidin-iFluor 647 was unable to label untreated red cells  at concentrations which disrupt the lipid bilayer, red cells stained positively with phalloidin-iFluor 647 due to the membrane damage  in PS exposure36, either via Ca2+ entry through cation channels or via a Ca2+-independent action. The effect of Yoda1 in sickle cells was therefore examined in combination with inhibitors of PKC, chelerytherine chloride which inhibits the active phosphorylation site of PKC, and calphostin C, which is an irreversible inhibitor of the diacylglyceraldehye-binding site.From the above, it would therefore appear that Yoda1 did indeed induce PS exposure, but that neither the presence of extracellular Ca2+]i is shown in Fig. 2+, Yoda1 (3 μM) had no effect on intracellular Ca2+ levels whilst in the presence of extracellular Ca2+, an increase in [Ca2+]I was observed. This rise in Ca2+ was abolished by chelerethyrine chloride (10 µM), such that values were unchanged compared to cells incubated in the absence of Yoda1.The effect of chelerethyrine chloride on Yoda1-induced increase in  ([Ca2+]i) and phosphatidylserine (PS) exposure in red cells from patients with sickle cell anaemia (SCA).The effects of Yoda1, a pharmacological activator of PIEZO1, were investigated on intracellular [Ca2+]i, dependent on the presence of extracellular Ca2+, consistent with its increased entry across the plasma membrane  produced a caveat that some reagents can damage the membrane and allow access of the PS label to inside37. The highest Yoda1 concentrations tested, however, did not allow access of fluorescently-labelled phalloidin . The findings shown in Fig. tBHF  tested, chelerytherine chloride reduced the Yoda1-induced increase in i, indicative of mediation via this enzyme. Using Ca2+ clamping with ionophore, they also show that Ca2+ and Yoda1 interact such that Yoda1 shifts the EC50 for Ca2+-induced PS exposure to lower values. Finally, they also show that high Ca2+ can overcome PKC inhibition (Fig. Using Yoda1-induced PS exposure, the present results clearly indicate that this compound can elicit PS exposure in a dose-dependent manner in the complete absence of Ca2+ Figs. . They shion Fig. b, presum2+ in mediation of PS exposure in red cells from SCA patients.Taken together the present findings provide further evidence for the dual role of PKC and Ca


In this work we have studied the functional consequences of mutations in cardiac troponin T (R131W), cardiac troponin I (K36Q) and α-tropomyosin (E40K) using adenovirally transduced isolated guinea pig left ventricular cardiomyocytes. We find significantly reduced fractional shortening with reduced systolic Ca2+. Contraction and Ca2+ reuptake times were slowed, which contrast with some findings in murine models of myofilament Ca2+ desensitization. We also observe increased sarcoplasmic reticulum (SR) Ca2+ load and smaller fractional SR Ca2+ release. This corresponds to a reduction in SR Ca2+-ATPase activity and increase in sodium-calcium exchanger activity. We also observe dephosphorylation and nuclear translocation of the nuclear factor of activated T cells (NFAT), with concordant RAC-α-serine/threonine protein kinase (Akt) phosphorylation but no change to extracellular signal‐regulated kinase activation in chronically paced cardiomyocytes expressing DCM mutations. These changes in Ca2+ handling and signaling are common to all three mutations, indicating an analogous pathway of disease pathogenesis in thin-filament sarcomeric DCM. Previous work has shown that changes to myofilament Ca2+ sensitivity caused by DCM mutations are qualitatively opposite from hypertrophic cardiomyopathy (HCM) mutations in the same genes. However, we find several common pathways such as increased relaxation times and NFAT activation that are also hallmarks of HCM. This suggests more complex intracellular signaling underpinning DCM, driven by the primary mutation.Dilated cardiomyopathy (DCM) is clinically characterized by dilated ventricular cavities and reduced ejection fraction, leading to heart failure and increased thromboembolic risk. Mutations in thin-filament regulatory proteins can cause DCM and have been shown in vitro to reduce contractility and myofilament CaNEW & NOTEWORTHY Dilated cardiomyopathy (DCM) is a frequently occurring cardiac disorder with a degree of genetic inheritance. We have found that DCM mutations in proteins that regulate the contractile machinery cause alterations to contraction, calcium-handling, and some new signaling pathways that provide stimuli for disease development. We have used guinea pig cells that recapitulate human calcium-handling and introduced the mutations using adenovirus gene transduction to look at the initial triggers of disease before remodeling.https://ajpheart.podbean.com/e/dcm-mutations-alter-intracellular-ca2-and-signaling/.Listen to this article's corresponding podcast at  Clinically, it is associated with chronic heart failure, sudden cardiac death, and cardioembolic stroke. DCM affects roughly 1 in 500 people globally . Approxian (PLN) . In addian (PLN) , actin (an (PLN) , cardiacan (PLN) , 28, 31,an (PLN) , and titan (PLN)  make up an (PLN)  and R131an (PLN) ], TnI [Kan (PLN) ], and α-an (PLN)  and D230an (PLN) ] has sho or α-TM . The pre or α-TM , R141W , as previously described  TnT, R131W TnT, WT TnI, and K36Q TnI, containing an NHescribed , 39. Hig2PO3 (pH 7.3 with NaOH), to the heart at 7 to 8 mL/min and 37°C for 5 min. The left ventricle was dissected and placed on a shaker for a further 10 min. Left ventricular cardiomyocytes (1.5 × 105 cells/mL) were incubated in ACCITT3 culture medium  of recombinant adenovirus to the isolated cells, at which time functional analysis and/or biochemical experiments were performed. We found that as previously described  Act, 1986, incorporating Directive 2010/63/EU of the European Parliament. As previously described , 400-g ae medium  at 37°C escribed , these ez-disk striations (acquired by phase-contrast imaging) at a sampling rate of 100 Hz. Unloaded sarcomere shortening measurements were also taken at 0.5 Hz following loading with 1 μM fura-2 for 5 min simultaneously with Ca2+ transients. This allowed diastolic Ca2+-transient measurements to be more stable at the slower pacing frequency; however, the effect of DCM mutations of contractility parameters was qualitatively comparable across both experimental conditions. Ca2+ transients were acquired using the ratio of fura-2 fluorescence emission at 360/380 nm at a switching rate of 1,000 Hz. All experiments were carried out at 37°C under constant 1 mL/min perfusion of a buffer containing (in mM) 150 NaCl, 10 HEPES, 7 glucose, 1 MgCl, 1 KCl, 0.3 NaH2PO3, 1.8 CaCl2 (pH 7.4 with NaOH) to avoid buffer electrolyte imbalance during electrical pacing. Finally, the ratio of fura-2 fluorescence signal was converted to intracellular Ca2+ concentration ([Ca2+]i) using a Ca2+ calibration kit with Mg2+ (Life Technologies), following the manufacturer’s protocol in a final buffer containing (in mM) 100 KCl, 1 MgCl2, and 30 MOPS (pH 7.2). All experiments were acquired using an IonOptix μstep system, transients were averaged and exported, and parameters were extracted using IonWizard v6.4.11. It was previously observed that the transduction of human flag tagged TnI and TnT does not significantly alter sarcomere shortening and Ca2+ transients compared with uninfected control cardiomyocytes from the same isolation. However, human flag tagged α-TM expression causes an increase in basal sarcomere length and relaxation time  immediately after deactivation of pacing using a separate gravity perfusion system attached to a heated (37°C) perfusion pencil (Digitimer). It was ensured that switching of pacing and caffeine application was instantaneous, by using Clampex software (Axon Instruments) to control each event. Fractional Ca2+ release from the SR was calculated by the division of [Ca2+]i-transient amplitude preceding caffeine application by the SR [Ca2+]i (given by the caffeine-transient amplitude). Sodium-calcium exchanger (NCX) activity was estimated by the τ-decay rate (τ1) following 10-mM caffeine application. Sarcoendoplasmic reticulum ATPase (SERCA2a) activity was calculated by the subtraction of the fast [Ca2+]i-transient τ2 decay rate, which gives the total intracellular Ca2+ reuptake, from the slower τ1 that was previously calculated (Sarcoplasmic reticulum (SR) Caa heated 7°C perfu5 infected cardiomyocytes in cell lysis buffer containing 20 mM imidazole (pH 7.0), 6 M urea, 0.4 M NaCl, 1 mM EDTA, 10% sucrose, 2 mM β-mercaptoethanol, 1% sodium dodecyl sulfate, 1% Triton X-100, 1 mM phenylmethanesulfonyl fluoride (PMSF), 1 mM Nα-p-tosyl-l-arginine methyl ester (TAME), and 1 mM tosyl phenylalanyl chloromethyl ketone (TLCK). To assess recombinant protein expression levels, cardiomyocytes underwent fluorescence-activated cell sorting  to remove any uninfected cells from the suspension before sample preparation. This also served to suspend the cells in a defined volume of PBS per cell, thereby removing the need for a loading control. Samples were blotted using either mouse anti-FLAG-Tag  (Sigma), mouse anti-TnT , rabbit anti-TnI , and mouse anti-α-TM  overnight at 4°C, followed with a horseradish peroxidase (HRP)-conjugated bovine anti-mouse (Santa Cruz) or goat anti-rabbit IgG secondary antibody  at room temperature for 1 h.Western blot samples were prepared from 6 × 105 infected cardiomyocytes were chronically paced at 40 V, 0.5 Hz for 8 h using a C-Pace electrode array (IonOptix) or left as unpaced samples and prepared as above. Membranes were blotted using anti-ERK  or anti-phospho-threonine 202/tyrosine 204-ERK , anti-NFAT-c3 , anti-phosho-serine-185 NFAT , anti-Akt , or anti-phospho-serine 473 Akt , followed by goat anti-rabbit IgG-HRP secondary antibody (Promega). Luminescent bands were developed with ECL select substrate  for 5 min. Images were acquired using a Gel Doc imaging system (Bio-Rad). Antibodies were removed using 10 mL of Restore Western blot stripping solution (Thermo Scientific) at room temperature for 8–10 min. Membranes were reprobed with an anti-GAPDH  that served as a loading control.For assessment of ERK, NFAT, and Akt activation, 6 × 102+ handling proteins and contractile proteins 6 × 105 infected cardiomyocytes were chronically paced at 10 V, 0.5 Hz for 8 h using a C-Pace electrode array (IonOptix) samples and prepared as above or, for PLN samples, were not denatured to allow the assessment of monomer and pentamer ratios. Membranes were blotted with anti-PLN , anti-phosho-Ser16 PLN , anti-phospho-Thr-17 PLN , anti-TnI , anti-phospho-Ser 23/24 , anti-SERCA2a , anti-NCX , and L-type Ca2+ channel , followed by either goat anti-rabbit (Promega), donkey anti-goat (Santa Cruz), or sheep anti-mouse  secondary antibodies. Luminescent bands were developed with Pico PLUS ECL reagent (Thermo Scientific) for 5 min. Images were acquired using a Gel Doc imaging system (Bio-Rad). Antibodies were removed using 10 mL of Restore Western blot stripping solution (Thermo Scientific) at room temperature for 8–10 min. Membranes were reprobed with an anti-GAPDH  that served as a loading control. The intensity of the bands was determined using Image Laboratory software (Bio-Rad).For assessment of CaFlag-tagged thin-filament protein localization was imaged in 48-h transduced cardiomyocytes immobilized in two-well Falcon-chambered cell-culture slides (Fisher scientific) precoated with 40 μg/mL laminin (BD Bioscience) at room temperature for 4 h. Cells were fixed with 4% methanol-free paraformaldehyde (TAAB) for 15 min at room temperature; cells were then skinned and slide blocked simultaneously for 1 h using PBS containing 0.1% Triton X-100 and 5% BSA. Cells were incubated overnight at 4°C with mouse anti-FLAG-tag  and rabbit anti-α-actinin , followed by a further overnight incubation with goat anti-mouse IgG Alexa 564  and goat anti-rabbit IgG Alexa 633 . Slides were mounted with slow-fade diamond with DAPI and a 1.5 thickness coverslip and visualized using a Leica TCS SP5 X confocal microscope with a 63× oil immersion objective.For imaging of NFATc3 following chronic pacing (4 h), cells were immobilized, fixed, and skinned in a round coverslip-bottomed 35-mm culture dish (Matek) using the same conditions as above. Cells were stained with consecutive overnight incubations with first NFAT-c3  primary and then goat anti-mouse IgG Alexa 568 . Finally, the sides of the coverslip were removed using a heated metal scalpel and mounted and imaged as before.n is the total number for each experiment of cells assessed for each WT/DCM mutant comparison. Any cell with sarcomere shortening or Ca2+, amplitudes, or kinetics exceeding two standard deviations from the mean upon analysis were excluded because of phenotypic heterogeneity arising in cultured primary cells. Groups were tested for normality (D’Agostino and Pearson test), and either an unpaired t-test or Mann-Whitney test was performed (GraphPad Prism) to compare WT to mutant for each protein expressed. For Western blot densitometry and immunolocalization, groups were tested for normality (D’Agostin and Pearson test), and either an unpaired t-test or Mann-Whitney test (GraphPad Prism).All cardiomyocyte comparisons were from at least four separate cell isolations with a similar number of cells from each isolation analyzed, and D and E) recombinant TnT, TnI, and α-TM was demonstrated by immunolocalization. Fast Fourier transformation of phase contrast-imaged z-disk striations gave an estimated basal sarcomere length of 1.86 ± 0.01, 1.88 ± 0.01, and 1.90 ± 0.01 µm for WT TnT, TnI, and α-TM, respectively; these were not significantly altered by the presence of DCM mutations in each protein (A–D). DCM mutations, however, produced a consistent reduction in contractile magnitude of 19.8 ± 4.0, 32.6 ± 4.4, and 24.2 ± 6.2% for TnT R131W, TnI K36Q, and α-TM E40K, respectively (T50) of relaxation increasing by 44.2 ± 8.0, 55.2 ± 6.9, and 32.9 ± 6.4 ms for TnT R131W, TnI K36Q, and α-TM E40K, respectively .We performed sarcomere shortening measurements in electrically paced (1 Hz) guinea pig left ventricular cardiomyocytes expressing either DCM mutant or corresponding WT human recombinant TnI, TnT, and α-TM. We first estimated the percent protein replacement of each of the six transductions by densitometry analysis of FACS-sorted cardiomyocytes to enrich the infected cell population in the resultant blots. We found that this enriched the functionally viable rod-shaped cardiomyocytes, as they were transduced and coexpressed GFP preferentially. Blotting using either anti-TnT, anti-TnI, or anti-α-TM A shows t protein , A–D. DCectively E. We alsectively F. In conster T50 G. Wester2+ handling by measuring Ca2+ transients in cardiomyocytes loaded with fura-2 and paced at 0.5 Hz (A–C). Diastolic Ca2+ levels remained unchanged in cardiomyocytes expressing TnT R131W and α-TM E40K compared with WT; however, TnI K36Q showed a 19.1 ± 5.7% reduction in basal Ca2+ levels . We found that fura-2 loading increased diastolic sarcomere length , reduced fractional shortening , and lengthened contraction  and relaxation time  when compared with the same contractile parameters from unloaded cardiomyocytes in 2+ buffering of the chemical Ca2+ dye, which has a Kd of ~150 μM and has been shown to accumulate to ~100 μM levels in various cell types plasmic reticulum Ca2+ ATPase (SERCA2a) . However, the E40K α-TM mutant, but not TnT R131W and TnI K36Q, showed a concordant stabilization of PLN monomer (A–D), driven by a 60.1 ± 12.4% reduction in serine-16 phosphorylation (E and G) and a 98.0 ± 2.3% reduction in threonine-17 phosphorylation (F and H).CaSERCA2a) A. SR Ca2 with WT B. Combinm the SR C. The raectively D. By conectively E. This i monomer , A–D, drrylation , E and Grylation , F and HWe observed the nuclear translocation of the transcription factor NFATc3 by coimmunolocalization with DAPI  in cells transduced with DCM-causing mutations, whereas there was no evidence of NFATc3 in the nucleus compared with cytoplasmic background in WT-transduced cells A. This wA–C). This suggests both this pathway and calcineurin/NFAT activation are driven directly by the expression of DCM mutant troponin and that the mutations affect the distribution of Ca2+ in a resting cardiomyocyte independent of Ca2+ cycling.Short-term expression of DCM mutations also appears to increase the phosphorylation level Akt at Ser-473 irrespective of pacing , A–C. ThD–G). However, no change was observed between DCM-causing mutations and their respective WT-transduced controls in both unpaced (D and E) and paced (D and F) cardiomyocytes.It has been suggested that NFATc3 translocation is a hallmark of both HCM and DCM, whereas ERK activation is increased in HCM and reduced in DCM, thereby differentiating between the distinct remodeling observed clinically . In comp unpaced , D and End paced , D and F2+ handling, and signaling. After 48 h of transgene expression using adenoviral transduction, we observed reduced contractility and peak systolic Ca2+, along with an increase in NCX activity and SR Ca2+ load. Additionally, NFATc3 nuclear translocation was observed and Akt activation was increased in mutant cardiomyocytes compared with cells expressing the corresponding WT protein. These changes were consistent for each of the mutations in three different disease genes. This allows us to suggest that these changes are among the primary mechanisms of disease pathogenesis, in a model free of secondary compensatory molecular alterations.Here we describe the effects of three human DCM mutant proteins  on cardiomyocyte contractility, Ca2+ transient in our model and in humans, whereas murine cardiomyocytes rely almost exclusively on Ca2+-induced Ca2+ release (CICR) from the SR  containing a TnT R173W DCM-causing mutation  Ca2+ affinity using an I61Q mutation found increased Ca2+-transient amplitude . Again, this is at odds with the findings in short-term expression models used in Davis et al.  in the isolated troponin complex do not directly alter Caaffinity . The mecfilament  and affe2+ sensitivity of thin filaments directly, rather they blunted of the Ca2+-sensitizing effect of PKA-dependent phosphorylation. How this phenomenon relates to Ca2+-transient amplitude reduction and to TnC Ca2+ buffering in our model would need to be investigated more fully.Each mutation appears to show subtle differences with respect to their knock-on effect to contractile function. For example, TnT R131W and TnI K36Q increase contraction speed compared with αTM E40K where relaxation is slowed; this could reflect alterations in the precise molecular mechanisms that underlie contractile impairment, for example, the position of each mutation with respect to the underlying actin monomer. Memo et al.  provided2+ levels in certain subcellular pools when the cell is at rest, although there is no significant change to initial overall Ca2+ baselines in our recordings. There may even be potential for mutant troponin to interact directly with signaling molecules and act as cofactors for their activation. There is some evidence for altered myofilament troponin incorporation in induced pluripotent stem cell cardiomyocytes (iPSC) derived from patients with a ΔK210 TnT DCM mutation, where mutant protein was also detected in the nucleus in one third of the cell population  Programme Grant RG/12/16/29939, BHF Centre of Research Excellence Oxford Grant RE/13/1/30181, and BHF Grants PG/18/68/33883 (to A.J.S.) and CH/1992001/6764 (to P.R.).No conflicts of interest, financial or otherwise, are declared by the authors.P.R., Y.H.Z., B.C., H.W., and C.R. conceived and designed research; P.R., A.J.S., S.P., M.M., S.N.R., and Y.H.Z. performed experiments; P.R., A.J.S., S.P., and S.N.R. analyzed data; P.R., A.J.S., S.N.R., and C.R. interpreted results of experiments; P.R. prepared figures; P.R. drafted manuscript; P.R., A.J.S., B.C., and C.R. edited and revised manuscript; P.R., Y.H.Z., B.C., H.W., and C.R. approved final version of manuscript.


Intracerebral hemorrhage (ICH) causes an accumulation of blood in the brain parenchyma that disrupts the normal neurological function of the brain. Despite extensive clinical trials, no medical or surgical therapy has shown to be effective in managing ICH, resulting in a poor prognosis for the patients. Urocortin (UCN) is a 40-amino-acid endogenous neuropeptide that belongs to the corticotropin-releasing hormone (CRH) family. The effect of UCN is activated by binding to two G-protein coupled receptors, CRH-R1 and CRH-R2, which are expressed in brain neurons and glial cells in various brain regions. Current research has shown that UCN exerts neuroprotective effects in ICH models via anti-inflammatory effects, which generally reduced brain edema and reduced blood-brain barrier disruption. These effects gradually help in the improvement of the neurological outcome, and thus, UCN may be a potential therapeutic target in the treatment of ICH. This review summarizes the data published to date on the role of UCN in ICH and the possible protective mechanisms underlined.  Intracranial hemorrhage is defined as bleeding in the brain parenchyma of the intracranial vault and meningeal spaces . IntraceUrocortin (UCN) is a peptide hormone that belongs to the corticotropin-releasing hormone (CRH) family of peptides . UCN wasThe main objective of this article is to summarize the studies regarding the role of UCN with a focus on physiological and pathological conditions of ICH, which may potentiate a new strategy for clinical applications.ICH, characterized by bleeding in the brain parenchyma, is a critical disease that could lead to severe morbidity and mortality worldwide ,11. ICH The Global Burden of Disease 2010 study demonstrated a 47% increase in cases of ICH between 1990 and 2010 worldwide . In the Hypertension is the most common attributable risk factor for ICH . Other mThe pathogenesis of ICH consists of cumulative damages resulted from primary and secondary brain injury  20]..20].The primary mechanism of injury for ICH is caused by the physical destruction of parenchymal tissues by the hematoma mass expansion within 24 to 48 h after the injury . The forThe mechanism of the secondary injury for ICH could be triggered by primary injury , tissue response to the hematoma (inflammation), or clotting factor release (hemoglobin or iron) . The secBinding of thrombin, a clotting cascade to protease-activated receptors 1, initiates central nervous system microglial activation and triggers various immune pathways, leading to apoptosis and necrosis of neurons and astrocytes . High doSince no medical or surgical therapy has shown to be effective in managing ICH, neutrophil-to-lymphocyte ratio (NLR) has been reported as a reliable measure of individual inflammatory status and predictor of clinical outcome . An incrICH is a medical condition that requires urgent management as patients will experience a neurological decline, hematoma expansion, and a decrease in more than 2 points on the Glasgow Coma Scale (GCS) several hours post-injury . The staThe outcome of ICH is affected by several factors, such as low serum magnesium , inflammUrocortin (UCN1 or UCN) belongs to the corticotropin-releasing factor (CRF) family of peptides, which include CRF, urocortin II (UCN2), urocortin III (UCN3), urotensin I, and sauvagine. UCN, first discovered in rat brains in 1995, is a 40-amino-acid peptide that has high sequence homology to urotensin (63% sequence identity) and CRF (45% sequence identity) . UCN2  and CRF receptor type 2 (CRFR2) . UCN binThe key determinant for activation of the CRF receptor is corticotropin-releasing factor-binding protein (CRF-BP), a 37 kD secreted glycoprotein . The expThe activation of CRFR1 has been reported as a key factor that regulates emotional and cognitive outcomes, promoting the recovery of post-ischemic cognitive, depression and social impairment . ThroughUCN2 and UCN3 are expressed in the heart and may have beneficial effects on cardiovascular pathophysiology, particularly heart failure (HF) . In a blStroke is one of the top risk factors that cause loss of neurological function and severe disability. Ten to twenty percent of strokes are caused by intracerebral hemorrhage. Symptoms in humans typically include a sudden and severe headache, dizziness, loss of balance, facial paralysis, hemiparesis, confusion, disorientation, and seizures . RodentsUCNs have been reported as clinically relevant molecules in the pathogenesis of hypertension, inflammatory disorders, depression, or obesity . Most phThe formation of brain edema can be classified into several phases: brain edema progressively increases after 24 h post-ICH, peaks approximately at 3 days post-ICH, and decreases after 9–14 days post-ICH. Clot retraction, inflammation, thrombin activation, erythrocyte lysis, and Hb toxicity-related injury were the reasons contributed to edema formation . To datePreclinical studies showed that human corticotrophin-releasing factor (hCRF) possesses antiedema properties with less toxicity than dexamethasone (a standard drug for brain edema), and thus being proposed as a new treatment alternative for peritumoral brain edema ,103. CorNeuroinflammation is a hallmark in ICH that triggered by the endoplasmic reticulum (ER) stress and proteostasis disruption through early degradation of chaperone GRP78 and IkB protein, leading inflammatory response and increased cytokine expression . InteresMicroglia and astrocytes provide nutritional support which maintains the physiological functions of neurons. UCN1 treatment attenuates ICH-related microglial activation-related neuronal loss . These fBBB, formed by cerebral endothelial cells, astrocytes, and pericytes, creates a barrier to separate peripheral circulation from the brain. Blood components derived from hematomas, such as thrombin, fibrin, and hemoglobin, triggered the disruption of BBB. ICH-induced cytokines also dysregulate the BBB function . Evans bThe preclinical study of UCN had shown novel effects in the central nervous system, specifically in ICH. In the animal study, UCN has been proven as a potent anti-inflammatory agent that can improve neurological function, decrease the injured region, brain edema, and BBB permeability, likely via suppression of microglial activation and inflammatory cytokine production. It has also been shown that a low dose of UCN is more effective in reducing the functional deficits associated with ICH .However, in the clinical study, intra-arterial infusions showed that UCN2 and UCN3 do not show vasodilatation effects from low to high dosage. All the clinical studies are done in a small sample size. Nevertheless, UCN has great therapeutic potential as a target for the development of drug and clinical use. Further investigation with larger clinical sample size, a different formulation of UCN, and pre-clinical pharmacokinetic or pharmacodynamic study should be done to develop UCN as a therapeutic agent in ICH and other traumatic brain injuries.


However, the underlying specific ion channel mechanisms remain largely unknown. Here, we report ryanodine receptor (RyR) channel activity and Ca2+ release both are increased, and association of RyR2 by FK506 binding protein 12.6 (FKBP12.6) is decreased in PASMCs from mice with chronic hypoxia (CH)-induced PH. Smooth muscle cell (SMC)-specific RyR2 knockout (KO) or Rieske iron-sulfur protein (RISP) knockdown inhibits the altered Ca2+ signaling, increased nuclear factor (NF)-κB/cyclin D1 activation and cell proliferation, and CH-induced PH in mice. FKBP12.6 KO or FK506 treatment enhances CH-induced PH, while S107 (a specific stabilizer of RyR2/FKBP12.6 complex) produces an opposite effect. In conclusion, CH causes RISP-dependent ROS generation and FKBP12.6/RyR2 dissociation, leading to PH. RISP inhibition, RyR2/FKBP12.6 complex stabilization and Ca2+ release blockade may be potentially beneficial for the treatment of PH.Ca Pulmonary hypertension is a devastating disease with elevation of pulmonary artery pressure and related to abnormal calcium signalling. Here, the authors show that suppression or stabilization of the calcium channel ryanodine receptor 2 may be a potential treatment approach for this disease. This devastating condition often occurs in respiratory illnesses  and high-altitude residence due primarily to chronic hypoxia (CH)-induced pulmonary artery (PA) constriction and remodeling. These two major cellular responses are principally mediated by an increase in [Ca2+]i in PA smooth muscle cells (PASMCs)4. However, the underlying mechanisms of the increased [Ca2+]i and associated PH remain largely uncertain.Pulmonary hypertension (PH) is a common disease with a short median survival time after diagnosis mostly because of limited specific and effective therapeutic options2+ release from the sarcoplasmic reticulum (SR) through ryanodine receptors (RyRs) is proven to be one of the major routes for the increased [Ca2+]i in PASMCs, and all three subtypes of RyRs  are expressed in this type of cells6. We have shown that RyR2-mediated Ca2+ release serves as an instrumental process in hypoxia-evoked contraction and remodeling9, which are linked to canonical transient receptor potential (TRPC) channel activation and voltage-dependent K+ (Kv) channel inhibition10. These data suggest that RyRs are integral to the development of hypoxic cellular responses in PASMCs. However, whether and how RyR2-mediated Ca2+ signaling exerts its role in the development of PH and the underlying molecular mechanisms are unknown.Ca2+ release from SR in PASMCs12. We have further found that acute hypoxia can induce generation of reactive oxygen species (ROS) and oxidize RyR2/FKBP12.6 complex, leading to their dissociation to increase [Ca2+]i in PASMCs8. It is reported that the dissociation of FKBP12.6 from RyR2 can cause Ca2+ leak from the SR, which may mediate cardiac diseases14. Stabilization of RyR2/FKBP12.6 complex with S107  has been proved to prevent ventricular hypertrophy13, aging-related muscle weakness15, and type-2 diabetes16. However, other reports are debating the role of FKBP12.6 in cardiomyocyte20. To our best knowledge, there is no exploration of the role of RyRs in the development of PH.FK506 binding protein 12.6 (FKBP12.6), an endogenous RyR2 stabilizer, is associated with RyR2 and inhibits Ca2+]i in PASMCs and HPV in isolated pulmonary arteries22. The report by Waypa et al.23 provides further in vivo evidence by using mouse model permitting conditional knockdown of RISP in PASMCs. These mice demonstrate the diminished acute hypoxia-induced ROS production and [Ca2+]i in pulmonary arteries and attenuated acute hypoxia-induced elevation of right ventricular systolic pressure (RVSP). In the current study, we sought to determine whether RISP knockdown in vivo could affect CH-induced PH as well as how it affects the subsequent altered Ca2+ signaling in PASMCs.Rieske iron-sulfur protein (RISP) is a major functional component of mitochondrial complex III, which serves as a critical molecule in the initiation and progression of the hypoxic ROS generation. We have demonstrated that RISP knockdown (KD) abolishes the hypoxic ROS formation in isolated PASMCs, whereas RISP overexpression produces an opposite effect. RISP KD also inhibits the hypoxic increase in i increase in PASMCs, thereby leading to PA vasoconstriction. The increased [Ca2+]i also activates NF-κB/cyclinD1 signaling and provokes proliferation in PASMCs and then results in PA remodeling. This cellular response, together with PA vasoconstriction, produces persistent PH.In this study, we report that CH causes RISP-dependent mitochondrial ROS generation, subsequent FKBP12.6/RyR2 complex dissociation, SR Ca2+ release in PASMCs8. Herein, using [3H]-ryanodine binding assay, we found that the maximal binding (Bmax) of ryanodine to RyR was significantly increased from 0.49 ± 0.06 (pmol/mg protein) in control PASMCs to 0.7 ± 0.08 (pmol/mg protein) in PASMCs from CH mice (P < 0.05), as shown in Fig. Kd) was decreased from 3.1 ± 0.5 in control cells to 1.8 ± 0.2 in CH cells. Similar result was observed in human sample as well  showed a much larger increase in [Ca2+]i in PASMCs from mice exposed to CH than normoxia. The mean [Ca2+]i increase was 442 ± 40 and 800 ± 72 nM, respectively  and smooth muscle cell (SMC)-specific RyR2 gene knockout  mice exposed to normoxia (Nor) and CH. As expected, Western blot and PCR analysis showed the specificity of RyR2 protein KO  protocol14. The ratio of SR Ca2+ leak to SR Ca2+ storage was significantly higher in PASMCs from CH than normoxic animals . Importantly, CH-induced increase in the ratio of RyR-mediated SR Ca2+ leak to SR Ca2+ storage was fully eliminated in PASMCs from RyR2−/− mice relative to WT (control) animals (5.3 ± 1.6% vs 6.5 ± 1.5%), suggesting RyR2 KO was sufficient to reverse the abnormal SR leak in PASMCs from CH mice  was largely enhanced in PASMCs from CH mice, and CH-induced SOCE was almost completely inhibited in cells from RyR2−/− mice   Fig. . Concurr2+ release, contraction (shortening) of PASMCs was augmented in CH mice compared with normoxic animals . As expected, the increased contraction of PASMCs did not occur in RyR2−/− mice following CH . The increased resting [Ca2+]i was fully blocked in RyR2−/− mice , well-known to be a potent endogenous vasoconstrictor and involved in PH−/− mice. We also found that CH increased muscularization degree in muscularized (double elastic lamina > 75%) and partial-muscularized (double elastic lamina < 75%) PAs, but decreased in non-muscularized (only one single elastic lamina) vessels; these effects were blocked in RyR2−/− mice . In contrast, CH caused a large elevation in RVSP in WT littermates, but not in RyR2−/− mice (35.5 ± 2.5 versus 21.5 ± 1.5 mmHg) . As shown in Supplementary Fig. −/− mice -induced contraction of intrapulmonary arteries. As shown in Fig. ice Fig. . Medial ice Fig. . RVSP anHg) Fig. . Similarice Fig. . All theBmax in SR preparations from PASMCs  and RV/LV + S , in mice in vivo significantly attenuated CH-induced increase in TTC Fig. . TTC alsTTC Fig.  and remoTTC Fig. . Moreove11. As such, we investigated whether FKBP12.6 might mediate the essential role of RyR2 in PH due to the dissociation of these two molecules. Co-immunoprecipitation (Co-IP) found that the ratio of FKBP12.6/RyR2 in SR membrane preparations was decreased by ~4 fold in PASM tissues from CH mice. , while there was no difference between FKBP12.6−/− and WT mice under normoxic conditions ; moreover, the similar effects of FK506 on RV weight were observed.Intriguingly, we revealed that RyR2 was mainly expressed in PASMCs, but not in PA endothelial cells (PAECs)  in PASMCs as well as increased PA contractility in CH mice were all abolished . Likewise, CH-evoked muscularization, MWT and SMC proliferation of PAs  were all prevented in CH mice after treatment with S107 -modification is the most commonly used procedure to assess protein oxidation15. To determine RyR2 oxidation, RyR2 protein bound by carbonyls was immunoprecipitated, followed by immunoblotting using anti-DNP antibody. As shown in Fig. P < 0.05, Fig. RISP is a primary mediator of hypoxic ROS generation, and RyR2 oxidation is a major mechanism for RyR2 hyperfunctionsues Fig. . Importaues Fig. . ROS-ind29. As shown in Fig. −/− mice , but had no effect in RyR2−/− mice (25.3 ± 3.0 mmHg). In agreement with the effect on RVSP, PDTC produced a similar effect on RV weights in control, CH and RyR2−/− mice , a low molecular weight thiol compound, is known to inhibit NF-κB activation. As shown in Fig. ice Fig. , but bloice Fig. . Adminisice Fig. . Furtherice Fig. . Collectice Fig. .2+ signaling in PASMCs plays an essential role in the regulation of PA vasoconstriction and remodeling that lead to the development of PH; this well-recognized Ca2+ signaling is precisely generated and regulated by multiple ion channels in the cell. However, it remains unclear which ion channel is most important in the development of PH. Our current study has identified that RyR2 plays an important role in mediating PH. The first evidence in support of this concept is that the activity of RyRs, determined using [3H]-ryanodine binding assay, is significantly increased in PASMCs from mice with CH-induced PH. Our previous studies and others have demonstrated that all three RyR subtypes  are expressed in PASMCs; among them, RyR2 is the most dominantly expressed6, similar to the result in airway SMCs33. A series of our previous in-vitro studies further indicate that all three RyR subtypes are involved in hypoxia-induced Ca2+ and contractile responses in PASMCs, but RyR2 is a very important player9. Indeed, herein we have found that the increased RyR activity and associated Ca2+ signaling are blocked in PASMCs from SMC-specific RyR2−/− mice. We have reported that the caffeine-mediated Ca2+ transients are not affected by IP3 in PASMCs9. Moreover, our previous reports reveal that all three IP3R subtypes  are expressed in PASMCs34; however, only IP3R1 may play an important role in hypoxic Ca2+ and contractile responses34. Apparently, further experiments are needed to disclose the functional role of IP3R1 in the development of hypoxic PH.Ca2+]i are higher in PASMCs from PH mice relative control animals. Our data further reveal that agonist-induced increase [Ca2+]i is enhanced in PASMCS from mice with PH. All these cellular responses are eliminated in PASMCs from RyR2−/− mice, further supporting that RyR2 in PASMCs is hyperfunctional in PH. Interestingly, the SR Ca2+ content is substantially decreased in PH mouse PASMCs. RyR2−/− mice are resistant to CH-induced PH. In support, RyR2 has been reported as a very important player in the hypoxic increase in [Ca2+]i in PASMCs36.In line with the increased RyR2 activity and function, the baseline tension (length) and i are the key processes in the development of PH, like other cardiovascular diseases such as heart failure13 and cerebral arterial disease37. It is known that how the expression of RyR2 changed under pathophysiological condition is controversial38. We have found that the increased RyR2 activity is due to the remodeling of its regulators  rather than the alteration of expression level. Kaβmann et al.39 have reported the successful generation of tamoxifen-inducible SMC-specific RyR2-deficient mice. In their report, the acute hypoxia-induced increase in PA pressure in isolated perfused lungs is enhanced in RyR2−/− mice. Intriguingly, we have found that a significant reduction of acute hypoxia-induced increase in PA pressure in vivo in RyR2−/− mice  shows a protective function in PH possibly by activating BMPR2 signal. A similar result has been observed in this study. Besides, we have found that that FK506 at a high dose (1 mg/kg/d) promotes the development of hypoxic PH. Thus, FK506 produces a biphasic effect on PH. Low dose FK506 is currently used in clinical trials and has shown a promising outcome51. In addition, we have not observed any phenotype of FKBP12.6−/− mouse or effect of FK506 treatment under normoxic condition. Apparently, FKBP12.6 removal cannot induce PH on its own. Accordingly, both FKBP12.6 and RyR2 contribute to the development of hypoxic PH. S107 is known to specifically stabilize FKBPs/RyR complex and inhibit SR Ca2+ leak. Multiple publications indicate that S107 inhibits the development and progression of multiple diseases16. Our study reports that S107 prevents the development of PH through stabilization of FKBP12.6/RyR2 complex. As a benzothiazepine derivative, S107 may display pleiotropic effects, but the findings from the current study unveil that this small molecule at least provides a potential therapeutic target in treating PH. Collectively, our previous investigations and current studies provide strong evidence that inhibition of RyR2 by FKBP12.6 underlies the pathological responses in PAMCS as diagrammed in Fig. A study using FKBP12.6 KO mice has shown that FKBP12.6 is associated with glucose metabolism by its interaction with RyR2 in pancreatic islets2+ signaling becomes hyper-functional in PASMCs are still uncertain, increasing evidence indicates that intracellular ROS play a critical role in mediating the development and progress of PH by regulating multiple ion channels22. We28 and others23 demonstrate that a unique protein in the mitochondrial complex III, namely RISP, serves as an essential molecule in the hypoxic mitochondrial and intracellular ROS generation in PASMCs. In this study, we have further identified that RISP is a key upstream player to FKBP12.6/RyR2 complex and thus has an essential role in CH-induced PH by mediating ROS generation. Indeed, lentiviral shRNA-mediated RISP silencing interrupts the positive feedback between ROS production and Ca2+ release but fails to cause overt deficiency and mitochondrial dysfunction in vivo. It provides a therapeutic drug target in treating PH without a great potential side effect.Although how Ca52. Cell proliferation is regulated by multiple transcriptional factors, among which NF-κB may play a significant role in PH53 and its specific inhibitor PTDC blocks hypoxia-induced PH in rats54. However, whether dysfunctional Ca2+ signal transmit its effect through NF-κB is uncertain. Our previous report indicates the link between TRPC channel and NF-κB in the development of asthma55. Herein, we provide evidence showing that NF-κB and cyclin D1 activation are important for CH-induced PA remodeling and hypertension. These results are consistent with their overall functions in the increased proliferation of PASMCs and associated PH56. Administration of PDTC, a potent inhibitor of NF-κB, fails to affect the upstream Ca2+ signal. RyR2 KO plus PDTC treatment, similar to RyR2 KO or PDTC alone, abolishes hypoxic PH in mice. All these findings further support our concept that NF-κB is a downstream molecular target of RyR2 signaling. It may also need to point out that NF-κB plays diverse and important functions in vivo; thus, it is not an ideal drug target for PH. Molecules modulated by NF-κB and those with more specific expression in PASMCs will be our focus of future study.Regardless of different genetic or pathogenic factors, the pathogenesis of PH can be attributed to increased pulmonary arterial contractility and uncontrolled PASMC proliferation2+ leak and increases [Ca2+]i in PASMCs, thereby leading to subsequent PA remodeling and vasoconstriction. These events may occur due to the mitochondrial RISP-dependent ROS-mediated RyR2 oxidation. Downstream activation of NF-κB/cyclin D1 signaling as a result of the oxidation-evoked RyR2 hyperfunction and Ca2+ singling is directly involved in PASMC proliferation and PA remodeling. The major strength of our study also lies in the evidence that genetic and pharmacological inhibition of RyR2, stabilization of FKBP12.6/RyR2 complex by treatment of S107, and in vivo lentiviral shRNA-mediated knockdown of RISP all can block the development of PH, providing effective strategies to prevent and treat PH in humans.In summary, we have demonstrated the functional importance of FKBP12.6/RyR2 complex in the development of PH in both animals and human samples, in which CH dissociates FKBP12.6/RyR2 complex, causes SR Ca3H]-ryanodine from PerkinElmer Corp; S107  from Cayman Chemical. All animal procedures were approved by the Institutional Animal Care and Use Committee of Albany Medical College. C57BL/6, FKBP12.6−/−, SMC-specific RyR2−/− and their corresponding control (WT) mice with the same background and age were used. The generation and maintenance of FKBP12.6−/− mice were reported in our previous publications57. SMC-specific RyR2−/− mice were produced by breeding ryr2-floxed mice with myh11-cre mice. The ryr2-floxed mice were created by inserting a loxP sequence into the 5′-untranslation region exon 1, the first intron of mouse ryr259; the production of myh11-cre mice was reported60. Forward and reverse primer sequences for genotyping mice were, respectively, 5′-CCAATTTACTGACCGTACACC-3′ and 5′-GTTTCACTA TCCAGGTTACGG-3′ for Cre, 5′-GCACCCTGGGGGCAGCCTTCTCAGC-3′ and 5′-ACATGTGTGCAGGTGTGCGGGTCTG-3′ for WT, 5′-TCCTCGTGCTTTACGGTATCGCCG-3′ and 5′-GCACCCTGGGGGCAGCCTTCTCAGC-3′ for RyR2 heterozygote, and 5′-GAACAGTTCCTCGCCCTTGCTCAT-3′ and 5′-GAGCCCCTAGAACATCCTGGTTAGC-3′ for RyR2 homozygote. Human PA specimens were obtained from the National Disease Research Interchange (NDIR) or Albany Medical Center Hospital and used according to the protocols approved by the Institutional Review Board for the Protection of Human Subjects in Research at Albany Medical College. PH was diagnosed by physical exam, medical history review and various tests. The PA specimens from age-matched subject who did not have any detectable cardiovascular diseases were used control. Written informed consents were obtained from all human subjects.Collagenase, dithiothreitol, dithioerythritol, myxothiazol, papain, TTC, PDTC, tacrolimus (FK506), and SU5416  were purchased from Sigma-Aldrich Corp; anti-FKBP12/12.6 and anti-RyR2 antibodies from ABR Affinity Bio-Reagents Products; p50, α-actin, cyclin D1, IκBα, vWF, and GAPDH antibodies from Santa Cruz Biotech, Inc; p65 antibody from Cell signaling Technology, Inc; Lamin A and anti-Ki67 antibodies from Abcam; Kv1.5, TRPC1, and TRPC6 antibodies from Alomone Labs; fura-2/AM from Molecular Probes; i was made using dual excitation wavelength fluorescence method in our lab32, with TILLvisION digital imaging system (TILL Photonics GmbH). Freshly isolated PASMC with the fluorescent Ca2+ indicator dye fura-2/AM (5 µM) at room temperature for 30 min. The fluorescent dye was alternatively excited at 340 nm and 380 nm, and emitted fluorescence was detected at 510 nm. The background signal was corrected by fluorescence recorded in non-cell regions. In calibration experiments, 340 and 380 nm fluorescence at Ca2+-free and saturating Ca2+ concentrations were determined using nominally Ca2+-free PSS containing 10 mM ionomycin and 10 mM EGTA in PSS containing 10 mM ionomycin and 10 nM Ca2+, respectively.Measurement of [Ca2+ leak in PASMC were measured as following. PASMC were incubated in 1.8 mM Ca2+ PSS until steady state [Ca2+]i was monitored. Then switching to a 0 Na+, 0 Ca2+ with 0.1 mM EGTA in PSS blocked the Ca2+ exchange via Na+/Ca2+ exchanger and L-type Ca2+ channel. Application of TTC (1 mmol/L) was used to antagonize RyR2, followed by administration of caffeine (30 mmol/L) to evaluate SR Ca2+ content. The ratio of TTC-dependent resting [Ca2+]i shift to SR Ca2+ content was SR leak14.SR Ca2 fragments antibody , and FKBP12.6 act as acceptor using anti-rabbit Alexa 555 labeled F(ab)2 antibody. FRET will be measured using LSM-510 laser scanning confocal microscope (Carl Zeiss) and the result will be analyzed using ImageJ with FRET imaging macro plug-in.Fresh isolated PASMC were fixed with 4% paraformaldehyde and permeabilized by 0.5% TritonX-100. The cells were incubated with RyR2 antibody  and FKBP12.6  together overnight. Then RyR2 will act as donor using anti-mouse Alexa 488 labeled Whole/F(ab)13.PA homogenates (200 µg) were used to immunoprecipitate RyR2 with anti-RyR2 antibody (Thermo Scientific). Then add the protein-G (Santa Cruz Biotech) and incubate at 4 °C overnight. The immunoprecipitates were washed and then elute by adding Laemmli sample buffer. After the samples were boiled for 5 min, equal homogenate was separated by SDS-PAGE . Proteins were transferred to PVDF membrane and develop using anti-RyR2 antibody (1:1000), anti-FKBP12.6 antibody (1:800), then visualized by the chemiluminescence detection kit8. According to the manufacturer’s instruction, isolated SR membrane samples (30 µg) were denatured by 5 µl 12% SDS and then derivatize with 10 µl 1 × DNP for 15 min at room temperature. The reaction was stopped by adding 7.5 µl neutralization solution. The proteins were electrophoresed on a SDS-PAGE gel and transfer onto PVDF membrane. The membrane was incubated with an anti-DNP antibody (1:150) for 2 h. Blots were visualized and analyzed. The same membrane was incubated with anti-RyR2 antibody (1:1000) for reblotting.Protein oxidation was detected by reaction with 2,4-dinitrophenyl hydrazine (DNP) using an OxyBlot™ Protein Oxidation Detection Kit 8. The cytosolic and nuclear extract were separated using nuclear extraction kit  according to the manufacturer’s manual. The routine western blot is performed using P50 antibody , p65 antibody . GAPDH  and Lamin A (Abcam) were used as cytosolic and nuclear loading marker respectively.Dissected intrapulmonary PAs were homogenized in buffer containing 0.29 M sucrose and 3 mM imidazole (pH 7.4)65. The concentrated lentiviruses containing shRNAs were diluted to a working titer of 3 × 109 transducing unit (TU)/ml in PBS. Mice were anesthetized by intraperitoneal injection of avertin (250 mg/kg) and placed in a ventral recumbent position on a heating pad with 37 °C. A small incision was made above the neck to expose a jugular vein. A 300 μl solution containing either lentiviral RISP or scrambled shRNAs at 3 × 108 TU/ml was administrated to each individual mouse via an intrajugular vein using 30-gauge needle through pectoral muscle. The injection was manually performed within a 2-min period of time. After that, the injection site was gently pressed by cotton tip for 5 min, skin incision closed by suture, and mice were released from restraining and warmed by heating lamp until they were recovered from anesthesia. After injection for 24 h, mice were exposed to CH or normoxia for 3 weeks.The pGIPZ lentiviral vectors containing RISP shRNA (sequence: CCCTGAATGGGTTATTCTGATA) and scrambled shRNAs were purchased from ThermoScientific OpenBiosystems (Mouse V2Lmm-72156) and were packaged using pCMV-dR8.2 dvpr and pCMV-VSV-G vectors8. Eventually, the enriched SR membrane fraction was obtained by ultra-centrifuge . Each fraction (10 µg) was incubated with each group of [3H]-ryanodine ranged from 0.5–25 nM for 3 h in a binding assay solution (300 µl). The binding mixture was diluted with ice-cold washing buffer and immediately filtered through Millipore Membrane filters presoaked with washing buffer. The radioactivity associated with the filters was determined by liquid scintillation counting. Nonspecific binding was determined in the presence of 50 µM unlabeled ryanodine. All binding assays were executed in duplicate.Isolated PAs were homogenized and concentrated through gradient centrifugation21.Isolated PASMCs, mitochondria  or complex III extract (using immunocapture kits from Mitoscience) with 50 μg protein were added to microplate wells containing respiration buffer. After incubation for 10 min, fluorescence was measured using FlexStation-III spectrophotometer with excitation wavelength of 485 nm and emission wavelength of 532 nm. ROS production was determined by subtracting the fluorescence intensity measured in control wells66. The isolated pulmonary arteries PAs) were washed with PSS (37 °C) and put a piece of thread tightly round one end of PA. Fill the PA with warm collagenase and incubate at 37 °C for 15 min then rinse and massage the artery with 10 ml culture medium. Centrifuge the rinse medium at 300 g for 10 min, discard the supernatant and repeat for three times. Then the PAECs were ready for further studies.The procedure was conducted following the protocol by Seybold et al.2/95% N2) for 1–3 min and then switched back to room air to allow recovery. The change in the RVSP was measured23.When the 1.2F pressure–volume catheter  was introduced for measuring RV pressure with room air, the baseline was recorded. The mice were then ventilated with hypoxic gas 9.Cells were placed on coverslips precoated with fibronectin (40 μg/ml) in phosphate-buffered saline (PBS) for 30 min at room temperature, fixed with 4% paraformaldehyde for 15 min, and permeabilized with 0.2% Triton. After incubated in 2.5% BSA in PBS blocking solution for 30 min, cells incubated with anti-α-actin , anti-RyR2 antibody (1:150) and anti-vWF antibody  for overnight at 4 °C, followed by Alexa488- and Alexa594-conjugated antibodies for 90 min. The staining was examined using LSM510 laser scanning confocal microscope (Carl Zeiss). The nucleus was stained by t test for comparisons before and after treatment in the same sample, unpaired (independent) Student’s t test for 2-sample comparisons, one-way ANOVA followed by Tukey’s multiple-comparison post hoc analysis for comparisons of the means that were classified in two different ways or the mean responses in an experiment with two factors. Values of *P < 0.05 was accepted as statistically significant.All data were analyzed using Origin 8.5. Data are expressed as mean ± standard error. The mean values were obtained from at least three independent experiments. Student’s Further information on research design is available in the Supplementary InformationPeer Review FileReporting Summary


Dear Editor,TP53 mutated AMLs are frequently resistant to intensive treatments and we recently showed that subclonal TP53 mutations confer an equally devastating prognosis [TP53 mutated subclones display characteristics of leukemic stem cells (LSCs) thus contributing to relapse or resistant disease. We, therefore, tested LSC properties of these subclones in a recently developed, highly sensitive humanized bone marrow (BM) ossicle xenotransplantation mouse model [Despite extensive efforts to develop novel therapies, the prognosis of patients with acute myeloid leukemia (AML) is still poor. One of the reasons is the genetic heterogeneity of AML with the majority of patients exhibiting distinct mutational subclones and diverse biological characteristics , 2. TP53se model , 5.null (NSG) mice. These MSCs underwent endochondral ossification leading to the formation of a humanized BM ossicle microenvironment. We transplanted three diagnostic, T cell depleted AML specimens into ossicle-bearing NSG mice either by tail vein or direct intraossicle injection. Two specimens showed subclonal TP53 mutations with a variant allele frequency (VAF) of <20%, one a clonal TP53 mutation serving as control  from healthy donors were expanded in vitro and subcutaneously injected into four sites of immunodeficient NOD/SCID/γof <0.1% .TP53 mutations, CD33+ leukemic cells constituted the predominant cell population within the graft. Interestingly, differentiation into CD19+ B-lymphoid cells was also observed and the subclonal TP53 mutations could be detected in both, the engrafted myeloid and lymphoid compartments  being in line with our previous report on clonal TP53 mutations in AML [TP53 mutations do also face an adverse prognosis since LSCs and pHSCs are considered less vulnerable to cytotoxic therapy ultimately giving rise to relapsed or resistant disease [TP53 aberrations was also shown in murine models exposed to genotoxic stress [All three AML specimens showed engraftment in humanized ossicles as well as mouse BM. However, human cells preferentially engrafted in the humanized ossicle microenvironment Fig. . For botts Figs. ,3. Theses in AML . Stem ce disease . Similarc stress . FinallyESM 1(PDF 2815 kb)


High mobility group box 1 (HMGB1) is a non-histone nuclear protein which has been intensively studied in various physiological and pathological processes including leukemia. Here in this study, we further demonstrated that HMGB1 presents higher expression in the bone marrow mononuclear cells of acute myeloid leukemia (AML) patients compared with the normal controls and contributes to the AML pathogenesis and progression by inhibiting apoptosis, facilitating proliferation, and inducing myeloid differentiation blockade of AML cells. Mechanistic investigation revealed that transforming growth factor beta-induced (TGFBI) acts as a potential downstream target of HMGB1 and lentivirus-mediated knockdown of TGFBI expression impaired phorbol-12-myristate-13-acetate (PMA) and all-trans retinoic acid (ATRA)–induced myeloid differentiation of AML cell lines. On the other hand, chidamide, an orally histone deacetylase inhibitor, decreases HMGB1 expression significantly in AML cells with concomitant upregulation of TGFBI expression, and confers therapeutic effect on AML by inducing cell differentiation, apoptosis and inhibiting cell proliferation. In conclusion, our findings provide additional insights that HMGB1 is a promising therapeutic target of AML, and also present experimental evidence for the clinical application of chidamide as a novel agent in AML therapy by downregulating HMGB1 expression.HMGB1 induces cell proliferation and myeloid differentiation blockade and inhibits apoptosis of AML cells.TGFBI acts as a potential target of HMGB1.Chidamide, a selective HDAC inhibitor, confers promising therapeutic effect for AML via downregulating HMGB1 expression.The online version contains supplementary material available at 10.1007/s00109-020-01998-5. The granulocytic and macrophage-like differentiation and maturation constitute the important part of myeloid differentiation which is a highly orchestrated process regulated by transcriptional factors (such as PU.1 and CCAAT/enhancer-binding protein(C/EBP)α) , non-codHigh mobility group box 1 (HMGB1) belongs to the HMG protein family and is a highly conserved non-histone nuclear protein that acts as a chromatin-binding factor for bending DNA and promoting access to transcriptional protein assemblies on specific DNA targets and contributes to the fine-tuning of transcription in response to rapid environmental changes , 12. AddHere in this study, we demonstrated that HMGB1 presents significantly high expression in AML patients and mediates the effect of facilitating proliferation and apoptotic repression on AML cells. Furthermore, HMGB1 is identified as a negative regulator of myeloid differentiation by influencing the expression of transforming growth factor beta-induced (TGFBI) which is demonstrated to promote phorbol-12-myristate-13-acetate (PMA) and all-trans retinoic acid (ATRA)–induced macrophage-like and granulocytic differentiation of AML cell lines. Chidamide, a histone deacetylase (HDAC) inhibitor, can significantly downregulate HMGB1 expression and confers therapeutic effect on AML by inducing cell differentiation, apoptosis, and inhibiting cell proliferation. These results further demonstrated the important role of HMGB1 in myeloid differentiation and the leukemogenesis of AML, and provide experimental evidence for the clinical application of chidamide in AML therapy by targeting HMGB1 expression.Microarray data of AML expression profile (GSE79605), HMGB1 deletion (GSE18721), and chidamide treatment (GSE104280) were downloaded from GEO DataSets, and the differential genes were screened through bioinformatics analyses and act as clues for subsequent experimental validation.d = 1.077 g/ml] , and CD34+ cells were enriched from MNCs through positive immunomagnetic selection . The characteristics of AML patients used in this study are presented in Table The bone marrow samples of AML patients and iron deficiency anemia (IDA) normal controls were collected from the Hematology Department of the Affiliated Hospital of Jining Medical University. Informed consent to perform the biological studies was obtained from the individuals examined, and the related study was approved by the ethics committees of the hospitals and the Institutional Review Board of Jining Medical University. Mononuclear cell (MNC) fractions were isolated from the samples by Percoll density gradient [2. The macrophage-like differentiation of THP-1 and HL-60 was induced with PMA (Sigma-Aldrich) at final concentration of 10 nM. The granulocytic differentiation of NB4 and HL-60 was induced with ATRA (Sigma-Aldrich) at final concentration of 2 μM. The CD34+ HSPC cells were cultured in IMDM with 30% FBS, 1% bovine serum albumin, 2 mM l-glutamine, 0.05 mM 2-mercaptoethanol, 50 U/ml penicillin, 50 μg/ml streptomycin, 50 ng/ml stem cell factor, and 20 ng/ml IL-3. The granulocytic and macrophage-like differentiation cultures of CD34+ HSPCs were performed as previously described [The following human cell lines were used in this study: THP-1 and HL-60 purchased from cell resource center of Shanghai Institutes for Biological Science, NB4 and 293TN purchased from cell resource center of Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences. THP-1 and NB4 were cultured in PRMI 1640 medium (Hyclone); HL-60 was cultured in Iscove’s Modified Dubecco’s Medium (IMDM) (Hyclone); 293TN was cultured in Dulbecco’s Modified Eagle’s medium (DMEM) (Hyclone). All cultures were supplemented with 10% fetal bovine serum (FBS) (Hyclone), 100 U/ml penicillin, and 100 μg/ml streptomycin  at 37 °C in 5% COescribed : 20 ng/mTotal RNA was extracted from cell samples using TRIzol reagent (Invitrogen) and quantified using the NanoDrop 2000 spectrophotometer . The first strand of cDNA was synthesized using Moloney murine leukemia virus (M-MLV) reverse transcriptase (Invitrogen) according to the manufacturer’s instructions. Oligo (dT) was used as the primer for reverse transcription of mRNA. GAPDH was used as the internal control. qRT-PCR was performed in a Bio-Rad CFX-96 System  using the SYBR Premix (CWBio). The primers used for reverse transcription and qRT-PCR are listed in Table The shRNA sequences for HMGB1 and TGFBI were synthesized, annealed, and inserted into pSIH1-H1-copGFP (System Biosciences). The oligonucleotides used for plasmid construction are listed in Table The recombination lentiviruses for knockdown were produced in 293TN cells using pSIH1-H1-copGFP-based constructs. Lentivirus packaging was performed using the pPACKH1 HIV Lentivector Packaging Kit  according to the manufacturer’s instructions. The culture supernatant containing the virus particles was directly used to infect the leukemia cells in 6-well plates with 5 μg/mL polybrene (Sigma-Aldrich).After lentiviruses infection, the cells were plated in 96-well plate at a density of 10,000 cells/well and incubated in 10% CCK-8  diluted in normal culture medium at 37 °C for 1.5 h. Then, proliferation rates were determined at 0, 24, 48, 72, and 96 h by measuring the absorbance with a microplate reader set at 450 and 630 nm. All experiments were performed in triplicate.The infected leukemia cells were starved in low FBS medium for 48 h and then collected, washed once with PBS, and re-suspended in the 1× binding buffer. Apoptotic cells were stained with APC-annexin V and 7-AAD (Biolegend) at room temperature for 20 min and immediately analyzed by flow cytometry.The infected THP-1 and NB4 cells were induced towards macrophage-like and granulocytic differentiation, respectively, and harvested at 48 h of differentiation. The cells were rinsed twice with PBS, re-suspended in 100 μl PBS, and incubated with APC-conjugated anti-CD14 or anti-CD11b (eBioscience) at 4 °C for 30 min. Then, the cells were washed with 1 ml PBS, re-suspended in 300 μl PBS, and analyzed immediately using an AccuriC6 flow cytometer .The whole cell lysates were harvested and subjected to SDS-PAGE (10% separation gel) and transferred to a polyvinylidene difluoride (PVDF) membrane. Primary antibodies against the following proteins were used: HMGB1 , TGFBI , GAPDH , and actin . Horseradish peroxidase–conjugated secondary antibodies were used (ZSGB-BIO). Signals were detected using an ECL (enhanced chemiluminescence) kit (Millipore).t test (two-tailed) was performed to analyze the data. Statistical significance was set at P < 0.05, as indicated by an asterisk .Student’s To systematically identify key regulator involved in the leukemogenesis of AML, we first performed bioinformatics analysis using the expression profiling data of AML patients (GSE79605) annotated in the GEO DataSets. HMGB1 is defined as one of the significantly differential genes Fig. , and furTo investigate the role of HMGB1 in AML leukemogenesis, we make use of the recombined lentiviruses that express specific shRNA for HMGB1 (lenti-shHMGB1) to infect THP-1, NB4, HL-60, and primary AML MNCs. Western blot analyses revealed that lenti-shHMGB1 infection remarkably decreased HMGB1 expression which significantly inhibits proliferation and facilitate apoptosis of AML cells Fig.  as compa+ HSPCs derived from umbilical cord blood  Fig.  as compaCollectively, HMGB1 is identified as a negative regulator of myeloid differentiation and may participate in the AML development by inducing the differentiation blockade.To reveal the molecular mechanism of HMGB1 regulating myeloid differentiation and AML leukemogenesis, we first performed bioinformatics analyses of the expression profile after HMGB1 deletion (GSE18721). Combined with the expression profile in AML patients (GSE79605), several differentially expressed genes after HMGB1 deletion were selected, and finally, we focused on TGFBI which is a transforming growth factor beta (TGF-β)–induced protein and presents significantly high expression after HMGB1 deletion Fig. . ExperimTo evaluate the effect of TGFBI on the differentiation, we use the recombined lentiviruses that express specific shRNA for TGFBI (lenti-shTGFBI) to infect THP-1 and NB4 cells followed by PMA and ATRA induction, respectively, for 48 h. As expected, lenti-shTGFBI infection of THP-1 cells resulted in significant downregulation of TGFBI expression and impaired the PMA-induced macrophage-like differentiation as revealed by the decreased mRNA expression of differentiation markers (CD11b and CD14) compared with the lenti-ctrl infection Fig. . DecreasTaken together, these results demonstrated that TGFBI could act as a potential downstream target of HMGB1 and positively regulates PMA/ATRA-induced myeloid differentiation of AML cell lines.Our results indicate that targeted inhibition of HMGB1 expression in AML can suppress cell proliferation, induce cell apoptosis, and overcome differentiation blockade, implicating HMGB1 as a promising therapeutic target in AML. Unexpectedly, through bioinformatics analyses, we found that chidamide, a novel orally subtype-selective HDAC inhibitor, whose chemical structure is presented in Fig. AML is a heterogeneous disorder of aggressive hematopoietic disease characterized by malignant proliferation and apoptotic repression of clonal neoplastic cells and also differentiation arrest of myeloid blasts , 21. HMGAs a multi-functional protein, HMGB1 is widely expressed and has been intensively studied in various physiological and pathological processes . HMGB1 iHDACs are enzymes involved in remodeling of chromatin by deacetylating the lysine residues and play important roles in cancer development by regulating the expression and activity of numerous proteins involved in cancer initiation and progression , 31. AbeIn summary, our findings provide additional insights that HMGB1 contributes to the AML pathogenesis and progression by inhibiting apoptosis, facilitating proliferation, and inducing differentiation arrest of AML cells, acting as a promising therapeutic target in AML, and further demonstrating the therapeutic effect of chidamide on AML by reversing the biological processes correlated with the pathogenesis and progression of AML via downregulating HMGB1 expression.ESM 1(DOC 433 kb)


Bone marrow samples were collected from 176 AML patients and 40 healthy donors, and KIF2A expression was measured by real-time quantitative polymerase chain reaction. Treatment response, event-free survival (EFS), and overall survival (OS) were assessed in AML patients. In vitro, KIF2A expression in AML cell lines and CD34+ cells  was measured, and the effect of KIF2A knockdown on AML cell proliferation and apoptosis in HL-60 and KG-1 cells was detected. KIF2A expression was greater in AML patients compared to healthy donors, and receiver operating characteristic curve indicated that KIF2A expression predicted increased AML risk (area under curve: 0.793 (95%CI: 0.724-0.826)). In AML patients, KIF2A expression positively correlated with white blood cells, monosomal karyotype, and high risk stratification. Furthermore, no correlation of KIF2A expression with complete remission or hematopoietic stem cell transplantation was found. Kaplan-Meier curves showed that KIF2A expression was negatively correlated with EFS and OS. In vitro experiments showed that KIF2A was overexpressed in AML cell lines  compared to CD34+ cells, moreover, cell proliferation was reduced but apoptosis was increased by KIF2A knockdown in HL-60 and KG-1 cells. In conclusion, KIF2A showed potential to be a biomarker and treatment target in AML.This study aimed to explore the correlation of kinesin family member 2A (KIF2A) expression with disease risk, clinical characteristics, and prognosis of acute myeloid leukemia (AML), and investigate the effect of KIF2A knockdown on AML cell activities  Acute myeloid leukemia (AML), one of the most malignant hematopoietic system diseases, is characterized by abnormal proliferation, poor cell differentiation, and infiltration of bone marrow, peripheral blood, or other tissues ,2. With in vitro experiments have shown that KIF2A functions as a tumor oncogene in several cancer cell lines  (in vitro was explored.Kinesin superfamily proteins (KIFs) are microtubule-dependent molecular motor proteins presenting adenosine triphosphatase activity . Kinesin tongue) ,10,11. Bde novo AML patients treated at Huashan Hospital were consecutively recruited. The eligibility for inclusion consisted of: i) confirmed diagnosis of primary AML in accordance with the World Health Organization (WHO) Classification of Tumors of Hematopoietic and Lymphoid Tissues (2008); ii) age ≥18 years; and iii) no systematic treatment for AML before enrollment. The exclusion criteria included: i) secondary AML; ii) acute promyelocytic leukemia; iii) accompanied with other malignancies; iii) history of radiotherapy, chemotherapy, or allogeneic stem cell transplantation; iv) infected with human immunodeficiency virus; and v) pregnant or lactating women. During the same period, 40 healthy bone marrow (BM) donors were enrolled as healthy controls when undergoing donation of BM at our hospital.From January 2015 to March 2019, 176 The Ethics Committee of Huashan Hospital approved this study before its initiation. The study was carried out in accordance with the principles expressed in the Declaration of Helsinki. All subjects provided written informed consent before recruitment.Clinical data of AML patients were collected on enrollment, which consisted of demographic characteristics (age and gender), clinical features (French-American-British (FAB) classification, cytogenetics, molecular genetics, and risk stratification), and laboratory test (white blood cells (WBC)). The FAB classification was assessed in accordance with criteria developed by the FAB Cooperative Group , and ris+ cells were further isolated from BMMCs of healthy controls. The relative expression of KIF2A mRNA was determined using real-time quantitative polymerase chain reaction (RT-qPCR).BM samples of AML patients were extracted before initiation of therapy, and the BM samples of healthy controls were collected on BM donation. After collection, BM mononuclear cells (BMMCs) were isolated from BM samples, and CD349/L , platelet count >100×109/L , and independence of red cell transfusions  or therapies of comparable intensity, response assessment was commonly carried out between days 21 and 28 after initiation of therapy. CR was defined as follows : bone marrow blasts <5%, absence of blasts with Auer rods, absence of extramedullary disease, absolute neutrophil count >1.0×10sfusions . After i2 at 37°C.Human AML cell lines KG-1, HL-60, ME-1, and HT-93 were purchased from Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures (Germany) and cultured in 90% Roswell Park Memorial Institute (RPMI) 1640 medium  with 10% fetal bovine serum (Gibco). All cells were contained in a humidified atmosphere of 95% air and 5% COTo detect the expressions of KIF2A mRNA and protein in KG-1, HL-60, ME-1, and HT-93 cell lines, RT-qPCR and western blot were performed, with the healthy donors' BMMCs used as control.5′-CACCGCTGAAGAAGCCAAACTATCGAAATAGTTTGGCTTCTTCAGC-3′; reverse, 5′-AAAAGCTGAAGAAGCCAAACTATTTCGATAGTTTGGCTTCTTCAGC-3′.The knockdown KIF2A plasmid and the knockdown control plasmid were constructed with the use of pRNAT-U6.1/Neo vector . The constructed plasmids were transfected into HL-60 and KG-1 cells using HilyMax , and the shRNA sequences used for KIF2A inhibition were: forward, Notably, we used three shRNAs to knockdown KIF2A, and we chose the one that had the best efficiency for KIF2A knockdown for the following assays. The cells transfected with knockdown KIF2A plasmid were defined as KIF2A-KD group, and the cells transfected with knockdown control plasmid were defined as negative control (NC)-KD group, accordingly. At 24 h after transfection, the mRNA and protein expressions of KIF2A in cells were detected by RT-qPCR and western blot. Cell proliferation and cell apoptosis rate were detected by Cell Counting kit (CCK-8) and Annexin V (AV)/Propidium Iodide (PI) assay. The CCK-8 assay was performed at 0, 24, 48, and 72 h after transfection, and the AV/PI assay was carried out 48 h after transfection.−△△CT formula. GAPDH was used as the internal reference. Primers used were as follows: KIF2A, forward primer: 5′ GCCGAATACATCAAGCAATGGT 3′, reverse primer: 5′ TGCTGGAGGTGGAGGTGTT 3′; GAPDH, forward primer: 5′ GAGTCCACTGGCGTCTTCAC 3′, reverse primer: 5′ ATCTTGAGGCTGTTGTCATACTTCT 3′.For RT-PCR assay, PureZOL RNA isolation reagent  was used to extract the total RNA, and PrimeScript™ RT Master Mix  was used to conduct the cDNA transcription. Subsequently, qPCR was performed by TB Green™ Fast qPCR mix , and the final results were calculated by the 2For western blot assay, total protein was extracted using RIPA buffer , and the protein concentration was determined by the Bicinchoninic Acid Kit for Protein Determination . For protein electrophoresis, NuPAGE™ 4-12% Bis-Tris protein gels  were used, and then proteins were transferred to PVDF membrane , which was further incubated with primary antibody [Rabbit polyclonal to KIF2A  and Rabbit-anti-GAPDH antibody ], and secondary antibody [Goat Anti-Rabbit IgG H&L (HRP) ], in turn. Furthermore, the bands were visualized by Pierce™ECL Plus Western Blotting substrate  and X-ray film .2 at 37°C. Absorbance was read by a microplate reader , which represented cell proliferation.Culture medium was discarded and cells were washed. Then, Cell Counting kit-8  mixed with fresh culture medium was added to the cells. Subsequently, cells were incubated for 2 h under 95% air plus 5% COAt 48 h post-transfection, cells were prepared as suspension. Annexin V-FITC Apoptosis Detection kit  was used to perform AV/PI assay according to the instructions of the manufacturer, and finally, the apoptosis rate was assessed.t-test, chi-squared test, Wilcoxon rank-sum test, or one-way analysis of variance (ANOVA) followed by Dunnett t-test, as appropriate. Receiver operating characteristic (ROC) curve analysis and the derived area under curve (AUC) were used to assess the feasibility of variable to distinguish different subjects. Kaplan-Meier (K-M) curves were plotted to display the EFS and OS of subgroups, and the differences of EFS and OS between subgroups were determined by the log-rank test. Analyses of factors affecting EFS and OS were assessed by univariate and multivariate Cox proportional hazard regression model analyses. All the experiments were carried out in triplicate. P values <0.05 were considered statistically significant.Statistical analyses were carried out using SPSS 21.0 statistical software  and GraphPad Prism 7.02 statistical software . Continuous variables are reported as means±SD or median and inter-quartile range (IQR); categorized variables are reported as count (percentage). Differences were compared with the independent samples One hundred and seventy-six  AML patients were enrolled in our study with a mean age of 44.4±12.2 years and median age of 44.0 (35.0-54.0) years. Further details are shown in https://www.ncbi.nlm.nih.gov/geoprofiles), we found two datasets that exhibited KIF2A expressions in AML patients and normal controls : in detail, one dataset showed that KIF2A expression was increased in the bone marrow samples of pediatric AML patients compared to that of normal controls , and the other one showed that KIF2A expression was similar between the bone samples of AML patients and those of normal controls . These data corroborated our primary observation of higher KIF2A expression in AML patients.KIF2A expression was elevated in AML patients [2.480 (1.232-3.809)] compared to healthy controls [1.162 (0.515-1.878)] (P<0.001) , and ROCKIF2A high expression was correlated with increased WBC level (P=0.036), monosomal karyotype (P=0.022), and high risk stratification in AML patients (P=0.001) . FurtherA total of 142 (80.7%) patients achieved CR while 34 (19.3%) did not achieve CR, and there was a trend of decreased KIF2A expression in CR patients compared to non-CR patients but without statistical significance (P=0.106) . Among tKIF2A high expression was associated with shorter EFS (P<0.001)  and decr9/L (P=0.002), monosomal karyotype (P<0.001), FLT3-ITD mutation (P=0.008), and high risk stratification were associated with shorter EFS; multivariate Cox proportional hazards regression model analysis displayed that WBC >10×109/L (P=0.000) and high risk stratification were independent predictive factors for shorter EFS in AML patients (9/L (P=0.044), monosomal karyotype (P<0.001), NPM1 mutation (P=0.046), and high risk stratification were associated with worse OS and multivariate Cox proportional hazards regression model analysis showed that WBC >10×109/L (P=0.002) and high risk stratification were independent factors predicting worse OS in AML patients , WBC >10×10patients . Univaripatients .+ cells (+ cells (KIF2A mRNA expression was elevated in KG-1 (P<0.05), HL-60 (P<0.001), ME-1 (P<0.01), and HT-93 cell lines (P<0.001) compared to CD34+ cells . KIF2A p  and KIF2In this study, we observed that: 1) KIF2A expression was significantly increased in AML patients compared to healthy controls, and correlated with increased WBC level, presence of monosomal karyotype, as well as high risk stratification in AML patients; 2) KIF2A high expression predicted low EFS and OS in AML patients; and 3) KIF2A was overexpressed in various human AML cell lines and its knockdown repressed cell proliferation but enhanced cell apoptosis in AML cells.KIFs take part in spindle orientation and chromosomal movements during mitosis and cytoskeletal reorganization ,14-17. AEmerging evidence has revealed that KIF2A is overexpressed in tissue samples of several malignancies, such as diffuse large B cell lymphoma (DLBCL), breast cancer, and lung squamous cell carcinoma –9. MoreoSome previous studies have revealed the predictive value of KIF2A in various cancers ,7,8,21. A better understanding on the effect of KIF2A in cancer cells may contribute to explore novel treatment targets. In our study, KIF2A knockdown inhibited cell proliferation and promoted cell apoptosis in both HL-60 and KG-1 cell lines. These data suggested that KIF2A knockdown might serve as an anti-tumor approach by inhibiting cell proliferation and enhancing cell apoptosis in AML cells, which provided indications for further explorations on treatment for AML.Some limitations of our study were: 1) the sample size was relatively low (N=176), thus there might be a relatively low statistical power; 2) the follow-up duration (median: 17.0 months (range 1.0 to 47.0 months)) was relatively short, therefore the predictive value of KIF2A in long-term treatment outcomes was not assessed; 3) as a single-center study, this study might lack wide representativeness; and 4) therapeutic approaches for AML patients with high KIF2A expression were not suggested in this primary research. Additionally, lack of information about downstream targets of KIF2A was a limitation of our study. Further studies should explore the downstream targets of KIF2A.In conclusion, KIF2A high expression predicted increased AML risk, and correlated with elevated WBC level, presence of monosomal karyotype, worse risk stratification, shorter EFS, as well as worse OS. Moreover, its knockdown inhibited cell proliferation but enhanced cell apoptosis in AML. These data further confirmed the oncogene role of KIF2A in AML.