How Epac activates STOCs is unknown.The vasodilator‐induced second messenger cAMP can relax vascular smooth muscle via its effector, exchange protein directly activated by cAMP (Epac), by activating spontaneous transient outward currents (STOCs) that hyperpolarize the cell membrane and reduce voltage‐dependent CaIn the present study, we map the pathway by which Epac increases STOC activity in contractile vascular smooth muscle and show that a critical step is the activation of CaMKII.To our knowledge, this is the first report of CaMKII activation triggering cellular activity known to induce vasorelaxation.2+ sparks activate plasma membrane Ca2+‐activated K+ (BKCa) channels, evoking spontaneous transient outward currents (STOCs) that hyperpolarize the cell and reduce voltage‐dependent Ca2+ entry. In the present study, we investigate the mechanism by which Epac increases STOC activity. We show that the selective Epac activator 8‐(4‐chloro‐phenylthio)‐2′‐O‐methyladenosine‐3′, 5‐cyclic monophosphate‐AM (8‐pCPT‐AM) induces autophosphorylation (activation) of calcium/calmodulin‐dependent kinase 2 (CaMKII) and also that inhibition of CaMKII abolishes 8‐pCPT‐AM‐induced increases in STOC activity. Epac‐induced CaMKII activation is probably initiated by inositol 1,4,5‐trisphosphate (IP3)‐mobilized Ca2+: 8‐pCPT‐AM fails to induce CaMKII activation following intracellular Ca2+ store depletion and inhibition of IP3 receptors blocks both 8‐pCPT‐AM‐mediated CaMKII phosphorylation and STOC activity. 8‐pCPT‐AM does not directly activate BKCa channels, but STOCs cannot be generated by 8‐pCPT‐AM in the presence of ryanodine. Furthermore, exposure to 8‐pCPT‐AM significantly slows the initial rate of [Ca2+]i rise induced by the RyR activator caffeine without significantly affecting the caffeine‐induced Ca2+ transient amplitude, a measure of Ca2+ store content. We conclude that Epac‐mediated STOC activity (i) occurs via activation of CaMKII and (ii) is driven by changes in the underlying behaviour of RyR channels. To our knowledge, this is the first report of CaMKII initiating cellular activity linked to vasorelaxation and suggests novel roles for this Ca2+ and redox‐sensing enzyme in the regulation of vascular tone and blood flow.Activation of the major cAMP effector, exchange protein directly activated by cAMP (Epac), induces vascular smooth muscle relaxation by increasing the activity of ryanodine (RyR)‐sensitive release channels on the peripheral sarcoplasmic reticulum. Resultant Ca 2+ and redox‐sensing enzyme Ca2+/calmodulin‐dependent kinase 2 (CaMKII) is a crucial and well‐established signalling molecule in the heart and brain.The Ca2+ signals and oxidative stress, surprisingly little is known about the role of CaMKII.In vascular smooth muscle, which controls blood flow by contracting and relaxing in response to complex Ca2+ influx. How Epac activates STOCs is unknown.The vasodilator‐induced second messenger cAMP can relax vascular smooth muscle via its effector, exchange protein directly activated by cAMP (Epac), by activating spontaneous transient outward currents (STOCs) that hyperpolarize the cell membrane and reduce voltage‐dependent CaIn the present study, we map the pathway by which Epac increases STOC activity in contractile vascular smooth muscle and show that a critical step is the activation of CaMKII.To our knowledge, this is the first report of CaMKII activation triggering cellular activity known to induce vasorelaxation. Cav1.2 channels provide the main Ca2+ influx pathway in vascular smooth muscle and, consequently, membrane hyperpolarization lowers global intracellular Ca2+ and induces muscle relaxation  and the generation of STOCs is thus a central mechanism that opposes arterial constriction induced by intravascular pressure or vasoactive transmitters. Indeed, inhibition of Ca2+ spark activity elicits contraction in most vascular beds, emphasizing the functional significance of this negative‐feedback pathway  on the peripheral SR. RyRs respond to changes in both cytosolic and luminal SR Ca2+ levels . Epacs are guanine nucleotide exchange factors (GEFs) for the small Ras‐related G proteins Rap1 and Rap2 , we have recently shown that Epac increases Ca2+ spark and STOC activity to induce membrane hyperpolarization and relaxation of vascular smooth muscle ‐dependent phosphorylation of both RyRs and phospholamban, a small pentameric protein complex that controls the activity of the SR Ca2+ ATPase (SERCA)  Act 1986 and was approved by the University of Liverpool and Queens University, Belfast local ethics committees.Tissues were obtained from adult male Wistar rats  and from wild‐type and CaMKIIδ and CaMKIIγ knockout mice . Wild‐type mice were littermates of the homozygous knockouts. All animals were killed by a rising concentration of COO‐methyladenosine‐3′,5′‐cyclic monophosphate‐AM , Fura‐2‐pentapotassium . All other chemicals were purchased from Sigma‐Aldrich .The chemicals employed in the present study were KN‐93, NS11021 , 8‐(4‐chlorophenylthio)‐2′‐et al. 2+ buffer containing (mm): 134 NaCl, 6 KCl, 0.42 Na2HPO4, 0.44 NaH2PO4, 10 Hepes, 10 glucose, 1 MgCl2 and 0.1 CaCl2 (pH 7.4) at 35°C for 10 min. After incubation, branches were moved to the first stage digestion buffer consisting of low Ca2+ buffer containing papain (1.4 mg ml−1), 4‐dithioerythritol (0.9 mg ml−1) and bovine serum albumin 0.9 mg ml−1 for 8–10 min at 35°C. Branches were then washed three times in pre‐warmed low Ca2+ buffer and transferred to second stage digestion buffer consisting of low Ca2+ buffer containing hyaluronidase (0.9 mg ml−1), collagenase (1.4 mg ml−1) and bovine serum albumin (0.9 mg ml−1) at 35°C. The incubation period for second stage digestion varied from between 8 and 12 min. Branches were subsequently transferred to fresh low Ca2+ buffer and carefully washed before dispersion of cells by gentle trituration of the tissue through a heat‐polished glass pipette. RMASMCs were stored in low Ca2+ buffer on ice and used in experiments between 1 and 8 h after digestion.Rat mesenteric arterial smooth muscle cells (RMASMCs) were isolated by enzymatic digestion of first‐order branches of the rat superior mesenteric artery as described previously : 140 KCl, 3 MgCl2, 0.2 EGTA, 10 Hepes and 3 Na2ATP (pH 7.2). Extracellular recording solutions contained (mm): 6 KCl, 134 NaCl, 1 MgCl2, 2 CaCl2 and 10 Hepes (pH 7.4). STOCs were recorded at a holding potential of −20 mV and defined as events deviating from the baseline by a factor of 4 standard deviations above baseline noise. Autoinhibitory‐2‐related peptide (1 μm) was placed in the intracellular solution and allowed to dialyse into the cell for 5 min before the start of recording. In experiments to assess direct effects of 8‐pCPT‐AM on BKCa channel activity, the pipette‐filling solution contained 300 nm free calcium as calculated by Maxchelator Ca‐Mg‐ATP‐EGTA Calculator, version 1.0, using constants from NIST database #46, version 8 (http://maxchelator.stanford.edu).STOCs were recorded from single freshly isolated RMASMCs using the whole‐cell configuration with an Axopatch 200B amplifier  as described previously  145 NaCl, 6 KCl, 1 MgCl2, 0.1 CaCl2, 10 Hepes and 10 glucose (pH 7.4) and placed in a perspex perfusion chamber. Background fluorescence counts for dual excitation wavelengths were taken after establishing a tight seal but before achieving the whole‐cell clamp configuration. On achieving the whole‐cell configuration, the cell was dialysed with pipette‐filling solution containing (in mm) 145 CsCl, 3 MgCl2, 3 Na2ATP, 10 Hepes and 0.05 fura‐2 pentapotassium (pH 7.2 with CsOH). The cell membrane potential was held at −60 mV, and normal Ca2+ extracellular solution, where CaCl2 was raised to 3 mm, was superfused. The cell was illuminated alternately with 340 and 380 nm light (bandpass 5 nm) using a PTI DeltaRAM Illuminator  and the emission signal was detected at 510 nm (40 nm bandpass). Temporal changes in photon counts were measured, backgrounds subtracted, and the complete ratio obtained at 50 Hz. Fluorescence signals and cell membrane potentials were acquired using FelixGX (Horiba Scientific). RyRs were activated by rapid application of 5 mm caffeine using a U tube superfusion system  or 8‐pCPT‐AM (5 μm), thereafter caffeine transients were continuously triggered at regular intervals. To convert the fluorescence ratio to [Ca2+]i, in vitro calibration was carried out using a range of EGTA‐buffered intracellular solutions containing a desired free Ca2+ calculated with MAXchelator. Fluorescence ratios in the absence of added CaCl2 (Rmin) and in the presence of saturating Ca2+ (Rmax) were decreased by 15% to account for viscosity  and the rate of Ca2+ increase was determined as a half‐time taken to reach a [Ca2+]i of 200 nm. For each parameter, the value of the third Ca2+ transient after switching to the test solution was normalized against that of the Ca2+ transient immediately before solution change.Transient Cam): 20 Tris‐HCl; 250 NaCl, 3 EDTA, 3 EGTA, 0.5% (v/v) Triton‐X 100 (pH 7.6), 1% (v/v) protease inhibitor cocktail (Sigma‐Aldrich) and 1X PhosSTOP Phosphatase Inhibitor Cocktail Tablets . Resultant lysates were placed on ice for 10 min then centrifuged at 16 100 g for 10 min at 4°C. Supernatants were removed and mixed 1:3 (v/v) with 4 × SDS sample buffer, before heating to 98°C for 10 min. Lysates for phospholamban immunoblots were prepared by lysis in either radioimmunoprecipitation assay (RIPA) buffer or 1 × SDS sample buffer to maximize phosphorylation preservation and increase protein solubilization. RIPA lysates were then mixed 1:3 with 4 × sample buffer as with Triton X‐100 based lysates. Samples were kept at −20°C until use. Proteins within the lysates were resolved by SDS‐PAGE on either 5%, 10% or 15% polyacrylamide‐Tris‐glycine gels (dependent on the protein of interest) and transferred electrophoretically onto nitrocellulose membranes . When immunoblotting for phospholamban, proteins were transferred to 0.22 μm polyvinylidene fluoride membrane. Immunoblotting was performed as described previously , 1% SDS.First‐ or second‐order branches of rat superior mesenteric arteries (five to eight branches) were homogenized in a pre‐cooled hand‐held homogenizer containing 100–500 μl of ice‐cold lysis buffer comprising (m286: dilution 1:1000); phospholamban (dilution 1:500), Rap 1A/B (dilution 1:1000) were from Cell Signaling Technology . CaMKII (pan: dilution 1:2500) was from Abcam . Smooth muscle α actin  was from Sigma‐Aldrich. The secondary antibodies used were anti‐mouse IgG (H+L) HRP conjugated polyclonal antibody and anti‐rabbit IgG (H+L) HRP conjugated polyclonal antibody .For immunoblotting, the primary antibodies used were: phospho‐CaMKII (TS‐transferase (GST) fused to the Rap binding domain (RBD) of Ral guanine nucleotide dissociation stimulator , was expressed from the pGEX4T3‐GST‐RalGDS‐RBD plasmid, kindly donated by Professor Johannes Bos , in the BL21 strain of Escherichia coli as described previously  at room temperature for 15 min. Then, 1 μl of EDTA (25 mmol l−1) was added and the reaction heated at 65°C for 10 min. First‐strand cDNA was synthesized using SuperScript® III reverse transcriptase (Invitrogen) in accordance with the manufacturer's instructions. The purity and concentration of the resulting cDNA template was determined by measuring absorbance at 260 and 280 nm using a NanoDrop spectrophotometer (Thermo Fisher Scientific Inc.).Total RNA was extracted and purified from first‐ or second‐order branches of rat superior mesenteric arteries using an RNeasy Mini Kit®  in accordance with the manufacturer's instructions. Extracted RNA was treated with DNase I by incubating 8 μl of total RNA, 1 μl of 10 × DNase I buffer and 1 μl of DNase I (1 U μlTouchdown PCR was carried out using HotStarTaq Master Mix Plus (Qiagen) in accordance with the manufacturer's instructions. Primers used to amplify specific CaMKII isoforms were: δ: Forward (5′‐GCATGTGGCGTCATCCTCTA‐3′) Reverse (5′‐TCCTTTACCCCATCCGGTT‐3′); γ: Forward (5′‐CATCCACCAGCATGACATCG‐3′) Reverse (5′‐CTTTCCTCAAGACCTCAGG‐3′). δ splice variants: Forward (5′‐CGGAAATTGAAGGGTGCCATC‐3′) Reverse (5′‐CCCTACCAGGTGTACGTGAG‐3′); γ splice variants: Forward (5′‐GAAGGGTGCCATCCTCACAA‐3′) Reverse (5′‐GTTACCAAGGGCTTCTGGCT‐3′). Following an initial 5 min denaturing step at 95°C, each reaction went through a touchdown PCR protocol for 15 cycles: 94°C for 1 min, annealing temperature  for 30 s, extension at 72°C for 1 min. This was followed by 25 cycles at the lowest annealing temperature. Final extension was at 72°C for 5 min. Products were analysed by running on a 3% agarose gel containing Midori Green  for ∼1 h at 80 V. Bands were excised under ultraviolet light and products purified using a QIAquick Gel Extraction kit (Qiagen) in accordance with the manufacturer's instructions. Products were verified by sequencing .post hoc test, or Student's t test for simple comparisons; levels of significance were P < 0.05 (*) and P < 0.01 (**). For electrophysiological and calcium fluorometry recordings, n is the number of cells recorded from. Cells were from at least three different animals. For immunoblots, n is the number of experimental repeats. These repeats were on different days and with tissue taken from different animals.Results are expressed as the mean ± SEM. Intergroup differences were analysed using repeated measures ANOVA followed by Newman–Keuls A). Sequencing of these products revealed them to be γ predicted isoforms X18 and X16, and δ isoforms 2 and/or 3. Consistent with the expression of multiple CaMKII isoforms, immunoblots of arterial lysates using pan‐specific CaMKII antibodies produced three/four distinct immunoreactive bands . To assess the effect of Epac activation on CaMKII activity, we incubated arteries in the selective Epac activator 8‐pCPT‐2′‐O‐Me‐cAMP‐AM  or vehicle control (DMSO). As a positive control, we also incubated arteries in the Ca2+ ionophore A23187 (12.5 μm), which would be expected to globally raise intracellular Ca2+ and activate CaMKII. Immunoblot analysis of 8‐pCPT‐AM‐treated arterial lysates indicated phosphorylation of CaMKII at Thr286/7, an autophosphorylation site that indicates CaMKII activation, following 8‐pCPT‐AM exposure (n = 3) . Of the three/four immunoreactive bands detected by pan‐specific anti‐CaMKII, one band (second from bottom) strongly bound phospho‐CaMKII antibodies against Thr286/7 following 8‐pCPT‐AM treatment. Exposure to A23187 induced phosphorylation of all immunoreactive bands detected by pan‐specific anti‐CaMKII .CaMKII is encoded by four closely related genes  with alternative splicing producing multiple variants . In arterial lysates obtained from CaMKIIδ knockout animals, the lowest molecular weight band was absent. By contrast, in CaMKIIγ knockout lysates, the lower band remained but the higher molecular weight bands were missing . This suggests that Epac activation predominantly induces phosphorylation of CaMKII γ variants in vascular tissue. Whole arterial lysates contain a combination of different cell types and we thus undertook electrophysiological experiments on single isolated mesenteric myocytes to directly assess the effects of Epac‐mediated CaMKII activation in vascular smooth muscle.To identify the CaMKII isoform/s differentially phosphorylated by Epac activation, we used immunoblotting to analyse arterial tissue obtained from mice in which the gene encoding either CaMKIIδ or CaMKIIγ had been globally knocked out  reversed the increase in STOC frequency  induced by 8‐pCPT‐AM  . This was not mimicked by application of KN‐92 (500 nm), an inactive KN‐93 analogue (n = 3) . Because KN‐93 has cellular effects other than inhibition of CaMKII , a highly specific peptide inhibitor of CaMKII that corresponds to its autoinhibitory domain . Indeed, there was a significant reduction in STOC frequency following 8‐pCPT‐AM addition from 1.08 ± 0.23 s−1 under basal conditions to 0.59 ± 0.12 s−1 in the presence of AIP  .In whole‐cell recordings from single, freshly isolated RMASMCs, application of the CaMKII inhibitor KN‐93  (100 μm), an IP3 receptor (IP3R) inhibitor. 2‐APB application following STOC activation with 8‐pCPT‐AM caused an initial rapid increase in STOC frequency followed by a decline to levels significantly below those measured in 8‐pCPT‐AM alone  . These data suggest that Epac‐induced CaMKII activation may be triggered by Ca2+ release from intracellular stores via IP3Rs. To directly link the effects of 2‐APB to CaMKII activity, we stimulated rat mesenteric arteries with 8‐pCPT‐AM (5 μm) in the presence and absence of 2‐APB (100 μm) before homogenizing the tissue and quantifying the changes in CaMKII phosphorylation by immunoblot analysis using phospho‐specific antibodies against the CaMKII Thr286/7 autophosphorylation site. In the presence of 2‐APB, 8‐pCPT‐AM was unable to induce phosphorylation of Thr286/7, indicating that Epac‐induced Ca2+ efflux through IP3Rs is a probable mechanism that initiates CaMKII activity (n = 3) . Consistent with the idea that store Ca2+ is needed to activate CaMKII, pretreatment with thapsigargin (500 nm), which depletes intracellular Ca2+ stores by blocking Ca2+ uptake via SERCA, also abolished 8‐pCPT‐AM‐induced phosphorylation of CaMKII at Thr286/7 (n = 3) .Because CaMKII is activated by the binding of Cals) Fig. A and B. 3) Fig. C. Consi 3) Fig. D.3Rs, inhibition of the IP3‐generating enzyme phospholipase C (PLC) with U73122 (2 μm) significantly reduced the ability of 8‐pCPT‐AM to induce sustained activation of Rap1  and Ca2+ (300 nm) included in the pipette‐filling solution, application of 8‐pCPT‐AM (5  μm) failed to increase BKCa channel activity (n = 6) . Under these conditions, Ca2+ release via RyRs will be blocked but BKCa channels will still be available for activation. This is demonstrated by the subsequent addition of the selective BKCa channel activator NS11021 (10 μm), which induced a large outward current . Consistent with the idea that Epac‐mediated changes in STOC activity rely on underlying changes in the activity of RyRs, application of ryanodine (15 μm) to the superfusing bath solution reversed the increases in STOC activity induced by the application of 8‐pCPT‐AM  . Similarly, in cells pre‐incubated in ryanodine (15 μm), subsequent application of 8‐pCPT‐AM does not increase STOC activity (n = 3) .We have previously shown that selective activation of Epac with 8‐pCPT‐AM in the presence of PKA inhibition increases the frequency of Cavity n =   at 60 s intervals triggered a train of Ca2+ release transients . At the point indicated by the bar above the trace, the extracellular superfusing solution was switched to test solutions containing either vehicle control (DMSO) or 8‐pCPT‐AM (5 μm). In both cases, the amplitude of the caffeine‐induced Ca2+ transients decreased during the course of the experiment, suggesting that the SR Ca2+ is being slowly depleted. Epac activation with 8‐pCPT‐AM had no significant effect on Ca2+ transient amplitude during the course of the experiment  . There was however a significant shortening of the relative duration of the transient following Epac activation  .Repeated 100 ms application of caffeine  was almost indistinguishable before and after DMSO application, despite the later Ca2+ transients being smaller in amplitude . The initial rate of Ca2+ rise was, however, considerably slower after application of 8‐pCPT‐AM (5 μm) . The relative initial rate of Ca2+ rise was 1.17 ± 0.07 and 2.00 ± 0.41 for DMSO and 8‐pCPT‐AM treated cells respectively  . These results suggest that Epac activation has no significant effect upon store content, although it may affect the kinetic behaviour of the RyR release channels.During the experiments, it was noted that the initial upward deflection of the Caude Fig. B, left.μm) Fig. B, right17, another key CaMKII target in the heart. Interestingly, we did see 8‐pCPT‐AM‐mediated changes with respect to both phospholamban mobility within gels and susceptibility to detergent extraction from membranes, which may indicate protein modification following Epac activation  Epac activation has little effect on intracellular Ca2+ store content but affects caffeine‐induced store release, possibly as a result of changes in the kinetic behaviour or synchronous opening of RyRs. We do not consider that the observed increase in STOC activity is attributable to Epac‐activated CaMKII directly phosphorylating and activating BKCa channels. We see no significant BKCa channel activation by 8‐pCPT‐AM under conditions where RyR channels are blocked, even though BKCa channels are still susceptible to activation by NS11021.Activation of Epac has previously been shown to induce smooth muscle relaxation by increasing the frequency of localized Caet al. et al. et al. et al. et al. The main CaMKII holoenzymes found in vascular smooth muscle form from splice products from γ and δ genes Singer, . ConsistBecause these experiments were conducted in whole arteries, we cannot fully distinguish whether the isoforms we detect are in smooth muscle cells, endothelial cells or innervating nerves. However, our electrophysiological experiments have all been conducted in single isolated smooth muscle cells and clearly show that inhibition of CaMKII activity by KN‐93 or AIP, a highly specific peptide inhibitor of CaMKII, blocks the ability of Epac to generate STOC activity.2+‐sensing enzyme, agents that elevate intracellular Ca2+ should activate CaMKII and, indeed, in large blood vessels, CaMKII is activated in response to contractile stimuli and this activation maintained throughout tonic contraction , which may indicate a constant Ca2+ leak from a common Ca2+ store via IP3Rs which, when blocked, alters store content and RyR activity. We see some evidence of basal phosphorylation of CaMKII in immunoblots, which may be consistent with this idea.Our data suggest that CaMKII activity in vascular smooth muscle is triggered by Epac‐induced CaCa channels in response to localized Ca2+ release from RyRs on the subjacent SR. Changes in STOC frequency or amplitude thus give a direct indication of changes in underlying behaviour of RyRs. To increase STOC activity, Epac may affect RyR behaviour directly via enhancement of Ca2+ sensitivity, or indirectly by increasing the content of the RyR‐accessible Ca2+ store. We thus measured the effect of Epac activation on store content and the rate of Ca2+ clearance from the cytosol by inducing transient Ca2+ release from the SR by pulse applications of the RyR activator caffeine in voltage clamped and fura‐2‐dialysed myocytes. Epac activation significantly slowed the initial global rise in [Ca2+]i in response to caffeine but had no effect on the relative transient amplitude. This suggests that depleted SR Ca2+ content is probably not the reason for the sluggish Ca2+ rise in 8‐pCPT‐AM treated cells. A possible explanation for these observations is that the rapid and synchronized opening of RyRs is compromised by Epac activation. CaMKII probably induces changes in RyR activity through phosphorylation of the release channel itself or associated regulatory proteins . As with RyR, we could not reliably detect phosphorylation of the SERCA pump regulator phospholamban at Thr17, another key CaMKII target in the heart. Interestingly, we did see 8‐pCPT‐AM‐mediated changes in both phospholamban mobility within gels and susceptibility to detergent extraction from membranes, which may indicate protein modification following Epac activation  to ESAH.


The glutathione transferases (GSTs) are a family of phase II detoxification enzymes with additional functions including the selective inhibition of RyR2, with therapeutic implications. The C-terminal half of GSTM2 (GSTM2C) is essential for RyR2 inhibition, and mutations F157A and Y160A within GSTM2C prevent the inhibitory action. Our objective in this investigation was to determine whether GSTM2C can enter cultured rat neonatal ventricular cardiomyocytes and influence contractility. We show that oregon green-tagged GSTM2C (at 1 μM) is internalized into the myocytes and it reduces spontaneous contraction frequency and myocyte shortening. Field stimulation of myocytes evoked contraction in the same percentage of myocytes treated either with media alone or media plus 15 μM GSTM2C. Myocyte shortening during contraction was significantly reduced by exposure to 15 μM GSTM2C, but not 5 and 10 μM GSTM2C and was unaffected by exposure to 15 μM of the mutants Y160A or F157A. The amplitude of the Ca2+ transient in the 15 μM GSTM2C - treated myocytes was significantly decreased, the rise time was significantly longer and the decay time was significantly shorter than in control myocytes. The Ca2+ transient was not altered by exposure to Y160A or F157A. The results are consistent with GSTM2C entering the myocytes and inhibiting RyR2, in a manner that indicates a possible therapeutic potential for treatment of arrhythmia in the neonatal heart.The cardiac ryanodine receptor (RyR2) is an intracellular ion channel that regulates Ca Thechannels , 12, 16.2+ as a result of excess Ca2+ leak through RyR2. Thus the ability of GSTM2C to reduce the spontaneous activity in neonatal cardiomyocytes suggests that the drug may also be effective in reducing spontaneous activity leading arrhythmia in the neonatal heart.Arrhythmia in neonatal cardiomyocytes can be generated in a similar manner to spontaneous activity in adult cardiomyocytes in that delayed after depolarisations (DADs) are generated when the NCX is activated during diastole by an increase in cytoplasmic Ca2+ transients in electrically stimulated myocytes was examined in order to avoid the individual variability seen in spontaneously active cells and to simulate regular pacing of myocytes in the intact heart. Following exposure to GSTM2C the degree of myocyte shortening was reduced presumably as a result of the smaller Ca2+ transient. That the rate of rise of the Ca2+ transient was slower after GSTM2C treatment can be explained by the known actions of GSTM2C in specifically blocking RyR2 and thus reducing the rate of Ca2+ release in response to the action potential. On the other hand, the decay of the Ca2+ transient indicates the rate of removal of Ca2+ from the cytoplasm and is effectively set by the rate of Ca2+ extrusion and uptake into the SR, minus any Ca2+ leak back into the cytoplasm through RyR2. This inhibition of RyR2 leak would increase the rate of Ca2+ uptake as we observed as back flux into cytoplasm would be reduced as mentioned in the Results. The slower release rate combined with the faster removal rate would account for the reduced amplitude of the Ca2+ transient.The effects of GSTM2C on contractility and Ca2+ transient amplitude could also be caused by a reduced Ca2+ load in the SR with GSTM2C. A reduced Ca2+ load occurs with increased Ca2+ leak through RyR2 or with reduced SERCA2 (sarcoplasmic/endoplasmic reticulum Ca2+ ATPase) activity. However we know that RyR2 activity is inhibited by GSTM2C [2+ load would be reduced in the neonatal myocytes. Indeed the enhanced uptake rate during Ca2+ removal from the cytoplasm during the Ca2+ transients is consistent with inhibition of RyR2 in both preparations. Further evidence that Ca2+ load in the SR is not reduced, and that there was no greater than normal Ca2+ extrusion across the surface membrane is provided by the fact that the amplitude of the Ca2+ transient did not decline during the AP train in the presence of GSTM2C any more than it did under control conditions. If Ca2+ uptake was reduced, the Ca2+ load in the SR would decline during the stimulus train and result in a rapid decline in the amplitude of the Ca2+ transients.A smaller Cay GSTM2C , but thay GSTM2C  in adult2+ transients in intact cells. This suggests that the effects of GSTM2C on myocytes is at least partly a result of GSTM2C interactions with RyR2. We show that there is little effect on the ability of field stimulation to evoke contractions, indicating that the action potential generation mechanism was not altered by GSTM2C. We did not specifically examine the effects of the constructs on other factors such as SERCA or NCX activity. However the fact that the specific inhibition of the cardiac RyR2 depends on GSTM2C binding to a cardiac RyR2-specific sequence in the divergent region 3 of RyR2 [The concentration dependence of the effects of GSTM2C, and the lack of an effect of F157A and Y160A, was similar on RyR2 in isolated cardiac SR and on myocyte contractility and Ca2+ during diastole in both neonatal and adult heart. It is possible that a reduction in the amplitude of the Ca2+ transient and fiber shortening in both neonatal (reported here) and in adult myocytes [As mentioned above, the ability of GSTM2C to reduce spontaneous contractions in the neonatal cardiomyocytes suggests that it may be effective in blocking arrhythmia due to NCX activation by high cytosolic Camyocytes , might rmyocytes  and we smyocytes . The fac2+ transient and contraction in myocytes electrically stimulated with a brief train of pulses and in continuous self-paced activity in intact tissue. It is likely that altered systolic Ca2+ release may not be maintained in continually active cells where an auto-correcting mechanism can maintain optimal systolic Ca2+ transient amplitude [2+ leak from the SR can cause the DADS that lead to arrhythmia. We did not see Ca2+ transient auto-correction in acutely isolated adult myocytes [In addition, we predict that GSTM2C may not have the same effects on the Camplitude . In contmyocytes , where t2+ release and contraction is very similar to that reported for adult cardiomyocytes [2+ release from the SR through RyR2 in both juvenile and adult conditions. The study shows for the first time that the drug is equally effective in cultured adult and neonatal cardiomyocytes.We show here that GSTM2C is translocated into the cytoplasm of neonatal cardiomyocytes and is effective in reducing spontaneous contractility and that its actions on field stimulated Camyocytes . All resmyocytes . The res


The GI mucosal bicarbonate secretion is the main mechanism of mucosal protection. Our research team has been working in this field and provided solid evidence for the important role of Ca2+ signaling in the regulation of GI epithelial secretion and the underlying molecular mechanisms. In this review, we attempt to systemically review the current status of our knowledge on the role of Ca2+ signaling in the regulation of intestinal bicarbonate secretion and in the upper GI epithelial protection. We expect that novel targets could be identified for drug development to better protect GI mucosa and treat mucosal injury with the advance in this filed.The cytosolic calcium ([Ca Under physiological conditions, the function of Ca2+ as a cellular messenger is based on the presence of a concentration gradient between intracellular Ca2+ ([Ca2+]i) and extracellular Ca2+ cyt. A major determinant of these variations is based on functionally distinct calcium channels and exchangers. Ca2+ release from intracellular stores is mediated by ryanodine receptor (RyR) and inositol triphosphate receptor (IP3R) channels. RyRs are activated by a rise in [Ca2+]i, i.e., Ca2+-induced Ca2+ release (CICR) [Cytosolic free Capoptosis . Therefoceptor IPR channel2+]cyt also plays critical roles in the regulation of many biological functions in the digestive system [2+]i is also an important factor that can accelerate GI epithelial wound repair. Bicarbonate (HCO3−) secretion in the intestinal mucosa is the main protective mechanism for the intestinal mucosal barrier. The ability of the duodenal mucosa to secrete mucus and bicarbonate combined with epithelial cell proliferation and migration constitutes an important protective mechanism for the intestinal mucosa.i2+ signaling in this regulation. Our research team has been working in this field to provide solid evidence for an important regulatory role of Ca2+ signaling and the underlying mechanisms. Therefore, in this review, we attempt to systemically review the current status of knowledge of the roles of calcium signaling in the regulation of intestinal bicarbonate secretion and upper GI protection.The cAMP and cGMP signaling pathways play important roles in the regulation of intestinal bicarbonate secretion, and the details of the underlying mechanisms are well understood. Several good reviews have discussed this topic. However, little is known about the role of CaThe proximal portion of the duodenal lumen often attains acidity approaching a pH of 2 –9, but t3− is secreted in higher amounts by the duodenum than by areas such as the stomach and small intestine.Epithelial cells in the gastrointestinal tract remain in close proximity to one another by closing the top of the spaces between cells. In contrast to gastric epithelial cells, duodenal epithelial cells are commonly referred to as a “leaky” epithelium due to the high penetration of ions between the cells via electrolyte passive transport. The GI epithelium covers the largest surface area in the body. Because they are exposed to the external environment, skin cells are subjected to potentially destructive factors of exogenous and endogenous origin. Duodenal mucosal bicarbonate secretion is recognized as the main defense mechanism for the intermittent expulsion of gastric hydrochloric acid. HCO3– is secreted by the duodenal mucosa in the presence of hydrochloric acid and pepsin and intermittently exits the gastric duodenum to protect people from the pulsed key role. One unique function of the duodenal epithelium in the small intestine is the secretion of bicarbonate with a higher velocity, which enables it to reach the more distant parts of the mucosa. HCO3− is secreted in response to major physiological stimuli, such as the presence of duodenal acid. Acid-induced HCO3−responsive medullary nerve pathways are involved in the release of vasoactive intestinal peptide (VIP), acetylcholine (ACh), and E-type prostaglandins (PGs) from mucosal epithelial cells i [2+]i activates the sarco/endoplasmic Ca2+ ATPase (SERCA) and plasma membrane Ca2+ ATPase (PMCA) pumps to restore [Ca2+]i to basal levels i . Finallyl levels .2+ signals in epithelial cells are highly polarized. This polarization is caused by the expression of the polylactic secretion receptor and Ca2+ signal transduction protein. For instance, polarized expression of Ca2+-signaling proteins, such as IP3Rs cyt to the target transporter can regulate its function, as has been demonstrated in Ca2+ activation of Cl- channel (CaCC) [2+]i can regulate the function of target Ca2+ signaling proteins, such as calmodulin and Ca2+-calmodulin-dependent protein kinase (CamKs). Finally, Ca2+ evokes receptor-mediated Ca2+ signaling agonists that can regulate membrane transporters through the production of by-products, such as diacylglycerol (DAG) and IP3. Protein kinase C (PKC) mediates DAG activation or IP3-induced release of the IP3-binding protein with IP3 (IRBIT) from IP3Rs, which has been shown to regulate the number of epithelial transporters  in real time i may mediate PGE2 activation via PLC/IP3 upstream of the EP1 receptor. Additionally, in vitro studies of gastric epithelial cells have reported that PGE2 is released by PLC inhibitors, suggesting that an increase in [Ca2+]i in response to damage enhances PGE2 production via the late maintenance cycle, which is expected to stimulate repair while maintaining high Ca2+ levels i increases, which most likely results from the active Ca2+ efflux from surviving epithelial cells as a result of epithelial cell repair.PMCA1 has been reported to be essential for the routine maintenance of intracellular Ca2+ homeostasis, whereas PMCA4 performs specialized physiological functions [2+ following injury [2+ concentrations at the site of injury.The key early observation was that the chelating activity of extracellular Caunctions . PMCA1 ig injury . Since t2+. Ca2+ is the most important signaling molecule involved in epithelial restitution; beyond HCO3– secretion, the promotion of intestinal epithelial restitution becomes the primary barrier against epithelial damage. We can apply these new perspectives to drug development; however, whether the activation of receptors that have recently been implicated in stimulating epithelial HCO3– secretion may be feasible and whether this approach will provide therapeutic benefits are unknown. The development of small molecules targeting CFTR, regulatory proteins, or stimulatory receptors suggest that such strategies may become available in the future.In conventional signaling models, most physiological changes are triggered by intracellular second messengers, such as cAMP, cGMP, and Ca


As a consequence, the t-tubules play a role in recovery (autoregulation) from reduced, but not increased, sarcoplasmic reticulum Ca2+ release.This study shows that in contrast to the rat, mouse ventricular Na 2+ influx and efflux regulates the Ca2+ load of cardiac myocytes, a process known as autoregulation. Previous work has shown that Ca2+ influx, via L-type Ca2+ current (ICa), and efflux, via the Na+/Ca2+ exchanger (NCX), occur predominantly at t-tubules; however, the role of t-tubules in autoregulation is unknown. Therefore, we investigated the sarcolemmal distribution of ICa and NCX current (INCX), and autoregulation, in mouse ventricular myocytes using whole cell voltage-clamp and simultaneous Ca2+ measurements in intact and detubulated (DT) cells. In contrast to the rat, INCX was located predominantly at the surface membrane, and the hysteresis between INCX and Ca2+ observed in intact myocytes was preserved after detubulation. Immunostaining showed both NCX and ryanodine receptors (RyRs) at the t-tubules and surface membrane, consistent with colocalization of NCX and RyRs at both sites. Unlike INCX, ICa was found predominantly in the t-tubules. Recovery of the Ca2+ transient amplitude to steady state (autoregulation) after application of 200 µM or 10 mM caffeine was slower in DT cells than in intact cells. However, during application of 200 µM caffeine to increase sarcoplasmic reticulum (SR) Ca2+ release, DT and intact cells recovered at the same rate. It appears likely that this asymmetric response to changes in SR Ca2+ release is a consequence of the distribution of ICa, which is reduced in DT cells and is required to refill the SR after depletion, and NCX, which is little affected by detubulation, remaining available to remove Ca2+ when SR Ca2+ release is increased.The balance of CaNEW & NOTEWORTHY This study shows that in contrast to the rat, mouse ventricular Na+/Ca2+ exchange current density is lower in the t-tubules than in the surface sarcolemma and Ca2+ current is predominantly located in the t-tubules. As a consequence, the t-tubules play a role in recovery (autoregulation) from reduced, but not increased, sarcoplasmic reticulum Ca2+ release. Contraction is initiated by Ca2+ influx [Ca2+ current (ICa)] through L-type Ca2+ channels (LTCCs); this activates ryanodine receptors (RyRs) in the adjacent sarcoplasmic reticulum (SR) membrane to cause Ca2+ release from the SR [Ca2+-induced Ca2+ release (CICR)]. ICa, RyRs, and thus CICR occur predominantly at t-tubules  and sarcolemmal Ca2+ ATPase, also play an important role  and its stimulation by Ca2+ released from the SR have been reported to occur predominantly at the t-tubules  for ~15 min. The ventricles were then removed and shaken in enzyme solution for 4–6 min before being filtered and centrifuged. Cells were resuspended in isolation solution (pH 7.4) plus 0.1 mM CaCl2 and stored for 2–8 h before use on the day of isolation. Detubulation (DT), the physical and functional uncoupling of the t-tubules from the surface membrane, was achieved using formamide-induced osmotic shock, as previously described, by incubating cells with 1.5 M formamide for 2 min before centrifugation and resuspending the cells in Tyrode solution  and killed by cervical dislocation. The heart was excised and washed in isolation solution supplemented with 0.1 mM CaCl2PO4, 4.2 HEPES, 10 glucose, 1.4 MgCl2, 20 taurine, and 10 creatine (pH 7.6 using NaOH). Tyrode solution used for experiments contained (in mM) 133 NaCl, 1 MgSO4, 1 CaCl2, 1 Na2HPO4, 10 d-glucose, and 10 HEPES (pH 7.4 using NaOH) plus 5 CsCl to inhibit K+ currents. The pipette solution contained (in mM) 100 CsCl, 20 TEACl, 10 NaCl, 0.5 MgCl2, 5 MgATP, 10 HEPES, 0.4 GTP-Tris (pH 7.2 using CsOH), and 0.1 pentapotassium salt of the fluorescent Ca2+ indicator fluo-4 .All reagents were obtained from Sigma-Aldrich  unless otherwise specified. The isolation solution contained (in mM) 130 NaCl, 5.4 KCl, 0.4 NaHMyocytes were placed in a chamber mounted on a Diaphot inverted microscope . Membrane currents and cell capacitance were recorded using the whole cell patch-clamp technique using an Axopatch 200B patch-clamp amplifier, a Digidata 1440A analog-to-digital converter, and pClamp 10 software , which was also used for data acquisition (at 2 kHz) and analysis. Pipette tip resistances were typically 1.2–2.0 MΩ when filled with pipette solution. All experiments were performed at room temperature.2+ influx and efflux, and thus Ca2+ balance, during a Ca2+ transient, holding potential was set to −80 mV; a 500-ms ramp to −40 mV was used to inactivate Na+ current followed by step depolarization to 0 mV for 100 ms to activate ICa at a frequency of 1 Hz. ICa was measured as the difference between peak inward current and current at the end of the pulse to 0 mV, and the integral was taken as a measure of Ca2+ influx. Inactivation of ICa was quantified by measuring the time to 50% inactivation (T50%). The current representing Ca2+ removed by NCX after the step depolarization  was measured by fitting a single-exponential function to 350 ms of the current trace starting 20 ms after repolarization from 0 to −80 mV and extrapolating back to when the membrane was repolarized. The integral of the exponential was taken as a measure of Ca2+ efflux during the Ca2+ transient , and, thus, current density, between the surface and t-tubular membranes was calculated from measurements in intact (whole cell) and DT (surface membrane only) myocytes, as previously described . The rate of decay of Ca2+ transients was obtained by fitting single exponential functions to the declining phase of the ICa- and caffeine-induced Ca2+ transients.Fluo-4 fluorescence was excited at 450–488 nm, and emitted fluorescence was collected at wavelengths <560 nm. Fluorescence was normalized to fluorescence just before application of caffeine  or NCX  overnight. Cells were then incubated in Alexa fluor 488-conjugated anti-mouse secondary antibody before being mounted with ProLong Gold. Cells were imaged on an LSM 880 confocal microscope (Zeiss) in Airyscan “super-resolution” mode, with a 1.4 numerical aperture ×63 oil-immersion objective.Staining at the cell surface and in the center of the cell was determined from a binary cell image obtained using Otsu’s method . The perStaining was quantified as follows:ICa and INCX densities) and the subsequent statistical analysis were calculated using propagation of errors from the constituent measurements . The limit of statistical confidence was P < 0.05. All statistical tests were performed on the number of cells. Sample sizes (n numbers) are given as c/h, where c is the number of cells used from h number of hearts.Data are expressed as means ± SE. Errors of derived variables  was decreased from 10% to 8% . The relative changes obtained experimentally in DT cells were used to model the data in these cells: ICa was decreased from 10 to 4.1, fractional SR Ca2+ release (f) was decreased from 0.7 to 0.287, and r was maintained at 8%, the value measured in DT cells. Changes in INCX relative to steady state obtained from the experimental data were incorporated for intact and DT myocytes.The mathematical model of autoregulation described by Eisner et al.  was usedn = 28/13) to 128 ± 6 pF , with no change in cell volume, calculated as previously described previously (3) [54 ± 4 pl (intact) vs. 58 ± 4 pl (DT)]. After correction for incomplete DT, this suggests that 41% of the cell membrane is t-tubular.DT significantly decreased cell capacitance from 181 ± 9  of the caffeine-induced Ca2+ transient . Thus, ~25% of total INCX appears to occur in the t-tubules, consistent with the small, although statistically nonsignificant, decrease in mean kCaff on DT . However, the loop was shifted in DT cells, with a greater INCX density for a given Ca2+, consistent with a similar Ca2+ release but greater INCX density at the cell surface. These data are consistent with the majority of NCX being located at the surface sarcolemma in the mouse and the location of INCX determining the hysteresis, which arises at the site of highest INCX density: the t-tubules in the rat and the surface sarcolemma in the mouse.Previous work in rat ventricular myocytes has shown that when caffeine is applied to release Cantration . Hysterese cells , 41. BecINCX. P < 0.0001, Bonferroni post hoc test) but similar in the cell center, although both proteins had a greater density at the cell edge than at the cell center . However, although the images were obtained using Airyscan “super-resolution” to minimize the contribution of out of focus light, there may be more surface membrane than t-tubular membrane in the optical field, since the former is likely to be present in the full depth of the field. This could lead to a higher apparent NCX density at the cell surface, although its effect on measured RyR density is unclear. However, although quantification is difficult, these data show that Ca2+ release sites and NCX are present at the cell edge, consistent with the observed distribution of INCX and the hysteresis observed in DT myocytes.Immunohistochemistry was used to investigate the distribution of NCX and RyR. INCX distribution appears to be different in mouse and rat myocytes, we also investigated the distribution of ICa in these cells. ICa and the elicited Ca2+ transients in intact , as in the rat  of caffeine, and during and after application of a low concentration (200 µM) of caffeine to sensitize CICR  and DT (i) myocytes. Recovery was accompanied by a decrease in the amplitude and integral of ICa, and an increase in the amplitude and integral of INCX in both intact (ii) and DT (ii) myocytes. However, steady-state Ca2+ transient amplitude was significantly smaller in DT cells (P < 0.0001), consistent with reduced ICa and loss of t-tubules, and the half-time (t1/2) to reach steady state was significantly longer ; thus, the rate of recovery due to SR refilling was slower in DT myocytes .h intact 4A,i and ) and DT B,i myocyh intact A,ii and ) and DT B,ii myocmyocytes C. Figuremyocytes D and E. ll types F, reflecDT cells F and G, 2+ influx and efflux was calculated to compare Ca2+ balance in intact and DT cells , similar to the recovery after 10 mM caffeine.To test this idea further, Cao intact A and DT o intact B myocyteICa in intact and DT myocytes  reduced in DT cells, the overall balance was not significantly different , as observed experimentally (cf. t1/2: 8.8 stimuli in intact cells vs. 12.8 stimuli in DT cells), consistent with the data from the experiments  decrease in the rate of decay of the caffeine-induced Ca2+ transient after DT in the mouse, in contrast to the marked decrease observed in the rat , suggesting a similar INCX density in the surface sarcolemma and t-tubules of guinea pig myocytes . The staining data support this idea: RyRs were observed not only in striations in the cell interior, consistent with localization at the t-tubules, but also at the surface membrane in clusters at the mouth of t-tubules, close to NCX, which, in agreement with the INCX measurements, was also observed at both the t-tubules and cell surface. These data suggest that RyRs, and thus Ca2+ release, occur at the t-tubules and cell surface and thus that it is the distribution of INCX that determines the site of the hysteresis in these cells. These data also suggest that the site of origin of arrhythmogenic delayed afterdepolarizations, which are caused by activation of INCX by SR Ca2+ release, will depend on the location of INCX and Ca2+ release and may thus be different in different species.It has been suggested that the hysteresis between myocytes , suggest2+ transient amplitude following altered SR Ca2+ release is associated with reciprocal changes in Ca2+ influx via ICa and Ca2+ efflux via NCX.The present data show that, in agreement with previous studies in other species , 39, rec2+ transient amplitude result in changes in Ca2+ influx through Ca2+-dependent inactivation of ICa. In rabbit cells, Ca2+ influx can increase by 50–60% in the absence of SR Ca2+ release , while the hysteresis between INCX and cytosolic Ca2+ showed a larger current for a given Ca2+ in DT myocytes, with no loss of hysteresis, consistent with the greater density of INCX in the surface membrane and similar Ca2+ release at the surface and t-tubular membranes. Thus, the observed changes are consistent with the observed distribution of INCX, with no evidence for altered regulation.In contrast to 2+ content, as assessed by releasing SR Ca2+ using 10 mM caffeine, but did result in a smaller and slower voltage-stimulated Ca2+ transient, due to decreased ICa and thus CICR and loss of synchronization of SR Ca2+ release. The smaller Ca2+ transient, in turn, decreased INCX, even though it was present at the surface sarcolemma.As in previous studies , 14, DT 2+ transient amplitude to steady state when SR Ca2+ release was increased using low-dose caffeine, recovery was slower from a decreased SR Ca2+ content after caffeine-induced SR Ca2+ depletion or on washout of 200 µM caffeine. This asymmetric response to changes in SR Ca2+ release in DT cells was not due to the nonlinear response of fluo-4 to Ca2+ because it was still present after the conversion of fluorescence to Ca2+ (not shown), an idea supported by the model. It may, however, be explained by the differential distribution of ICa and INCX. In this case, when an increase in SR Ca2+ release occurs, Ca2+ efflux via NCX is the main mechanism to decrease Ca2+ transient amplitude and return the cell to steady state; thus, in DT mouse cells, in which the majority of INCX occurs at the surface, recovery is similar in intact and DT myocytes and is not affected by the loss of ICa. However, when SR Ca2+ release is decreased, Ca2+ influx via ICa is necessary to refill the SR and increase Ca2+ transient amplitude to steady state and is thus essential for recovery. Since steady-state SR Ca2+ content is similar in intact and DT myocytes but Ca2+ influx via ICa is smaller in DT cells, more stimuli are required to refill the SR to steady state, so that recovery is slower in DT than in intact cells, even though the majority of NCX is present and quantitatively shows greater changes over time than ICa. Slowing of recovery from caffeine has also been reported after DT of rat myocytes , an idea supported by the lack of Ca2+ release in the center of DT myocytes (2+ and have a small volume, they are also unlikely to take up Ca, and previous work inhibiting surface NCX in DT rat myocytes during application of caffeine showed that only ~2% of Ca2+ removal occurs into non-SR sinks within the cell (2+ cycling or autoregulation, an idea supported by the effects of DT on the Ca2+ transient and autoregulation.Although t-tubules uncoupled from the surface membrane by DT may reseal within the cell and, in principle, sequester and release Ca2+ , 24, it myocytes . Becausethe cell , which i2+ release, because of the high t-tubular ICa density, but play little role in the recovery from increased Ca2+ release. These data also suggest that the response of a particular species to such changes will depend on the distribution of ICa and INCX between the t-tubules and surface sarcolemma. Although the distribution of Ca2+ fluxes in human myocytes is unknown, the current work shows that the distribution of ICa and INCX determines cell function and enhances our understanding of how this occurs. Thus, when human data become available, it should be possible to better predict the consequent changes in cell function in both physiological conditions and after t-tubule disruption and changes in the distribution of ICa and INCX, which have been reported in heart failure (Thus, it appears that the t-tubules play an important role in autoregulation in mouse myocytes during recovery from decreased SR Ca failure  and whicThis work was supported by British Heart Foundation Grants PG/14/65/31055 and RG/12/10/29802.No conflicts of interest, financial or otherwise, are declared by the authors.H.C.G. performed experiments; H.C.G. and C.H.K. analyzed data; H.C.G., S.M.B., A.F.J., and C.H.O. interpreted results of experiments; H.C.G. prepared figures; H.C.G. and C.H.O. drafted manuscript; H.C.G., C.H.K., S.M.B., A.F.J., and C.H.O. edited and revised manuscript; A.F.J. and C.H.O. conceived and designed research; A.F.J. and C.H.O. approved final version of manuscript.


In this study, we generated several FRET sensors for divalent cations based on cTnC flanked by a donor fluorescent protein (CFP) and an acceptor fluorescent protein (YFP). The sensors report Ca2+ and Mg2+ binding, and relay global structural information about the structural relationship between cTnC’s N- and C-domains. The sensors were first characterized using end point titrations to decipher the response to Ca2+ binding in the presence or absence of Mg2+. The sensor that exhibited the largest responses in end point titrations, CTV-TnC,  was characterized more extensively. Most of the divalent cation-dependent FRET signal originates from the high affinity C-terminal EF hands. CTV-TnC reconstitutes into skinned fiber preparations indicating proper assembly of troponin complex, with only ~0.2 pCa unit rightward shift of Ca2+-sensitive force development compared to WT-cTnC. Affinity of CTV-TnC for divalent cations is in agreement with known values for WT-cTnC. Analytical ultracentrifugation indicates that CTV-TnC undergoes compaction as divalent cations bind. C-terminal sites induce ion-specific (Ca2+ versus Mg2+) conformational changes in cTnC. Our data also provide support for the presence of additional, non-EF-hand sites on cTnC for Mg2+ binding. In conclusion, we successfully generated a novel FRET-Ca2+ sensor based on full length cTnC with a variety of cellular applications. Our sensor reveals global structural information about cTnC upon divalent cation binding.Cardiac troponin C (cTnC) is a key effector in cardiac muscle excitation-contraction coupling as the Ca Its tertiary structure consists of two globular domains which are connected by a central helical linker. The C-domains of the cardiac and skeletal isoforms of TnC contain two EF-hand, divalent cation-binding sites (sites III and IV) that can bind either Ca2+ with a high affinity (2 x 107 M1) or Mg2+ at lower affinity (3.5 x 103 M-1) total to achieve 2mM [Mg2+] free. Small aliquots of Ca or Mg were added stepwise during these titrations, and free Ca2+ and Mg2+ concentrations were calculated as described above for fiber solutions  or [Mg2+]. Four replicate measurements were made (N = 4). FR data  were fit using a double Hill equation when Ca2+ was titrated in the presence or absence of Mg2+ to simultaneously evaluate both high and low affinity EF hands:Peak fluorescence values for donor , and FR  was calc2+ was titrated, FR data  were fit to a single Hill equation:When Mg2+ or Mg2+.The binding affinities were estimated by using:R (2+. For panels (A) and (C) of 2+/EGTA , 400μM Ca2+ (light grey bars), 3mM EDTA or Mg2+/EGTA . The average of all FR values at the different λex for each divalent cation condition was calculated; error bars represent SE. Panels (B) and (D) of R = FR(Ca2+) - FR(Ca2+ free) calculated at each λex, and averaged across all λex. FR(Ca2+) was obtained at each λex for the Ca2+ saturated condition (400μM Ca2+); FR(Ca2+ free) was calculated at each λex as the average of the initial condition  and the final condition . The signal reported, indicative of FRET, is the FRET ratio (FR)  . The advx varies .R in both the absence and presence of Mg2+ is small (R) was observed upon Ca2+ addition  and final (3mM EGTA/3mM Mg2+) states are Ca2+ free, but are expected to have Mg2+ bound at the C-terminus (~2Mg2+ state) where FR ~0.3  results in a modest increase in FR of amplitude 0.05, presumably indicating a slight structural change associated with Ca2+ binding at N-terminal site II  have a higher FR ~0.4  in the presence of Mg2+  between the fluorophores and cTnC . TurNg-T2+ state . Both co of Mg2+ . This reR and FR in all conditions tested. CTV-TnC and TurcpV-TnC have the largest ΔFR in the absence of Mg2+ , 3mM EGTA/3mM Mg2+ (~2Mg2+-TnC) and 3mM EDTA (apo-TnC). The sedimentation coefficient (s) measured for CTV-TnC varies with condition , s increases to 4.33 + 0.05 S, and f/f0 is smaller (~1.9) indicating some compaction of the protein. When it is fully saturated with Ca2+ (3Ca2+-TnC), s is the largest with s = 4.45 + 0.05 S, and f/f0 is the smallest (~1.7) indicating the molecule adopts the most compact structure of these three states. AUC  in skinned fiber preparations after endogenous TnC extraction and following reconstitution with either WT-cTnC or CTV-TnC, normalized (%) to the maximum Ca2+ activated isometric force prior to extraction of endogenous TnC. The measurements for each TnC in 2+-activated force  measured during steady-state activation over the range of pCa values which yielded a full Ca2+ titration. The average pCa50 for fibers reconstituted with WT-cTnC was 5.74 + 0.01 and 5.53 + 0.02 for CTV-TnC reconstituted fibers, resulting in a rightward shift of 0.21 pCa units with the FRET construct. The Hill coefficient n was 1.37 + 0.05 for fibers reconstituted with WT-cTnC and 1.25 + 0.06 for fibers reconstituted with CTV-TnC. We conclude that the FRET construct CTV-TnC can functionally reconstitute into troponin complex in thin filaments within the sarcomere lattice of fibers, and can regulate activation of cardiac muscle in a Ca2+ dependent manner with a slight de-sensitization to Ca2+.Skinned fiber force measurements were used to test the functionality of cardiac TnC as part of the CTV-TnC protein modified by addition of two fluorophores. We compared the Cace ~70%; . Fig 6B R, we evaluated a series of mutants where one or more Ca2+ binding site on TnC was abolished . We performed two types of end point titration: one was performed with Ca2+  decreased the signal almost by half , after correction with the triple mutant FR signal, show that all three sites contribute to the signal, with the largest from site III and nearly equal but opposite in amplitude contributions from sites II and IV. The lower portion of the table shows the sum of all three EF hand signals after correction (FR = 0.204) is similar to the signal resulting from the CTV-TnC (FR = 0.217).Mutation of site III (D104A) abolished the Ca by half . The douR values. For validation of our model, the predicted sum of all 3 sites in the case of Ca2+ , or the sum of 2 sites in the case of Mg2+ (binding to sites III and IV) is given in the second to last row. This prediction can be compared to the FR signal obtained directly from CTV-TnC WT in the end point titration with Ca2+ or Mg2+ (last row).Contributions of each site were estimated from the data in 2+ binding by each construct and its effect on the FRET signal  after subtraction of the triple mutant FR signal. The sum of the contributions of sites III and IV to the overall signal was calculated (0.193) for comparison with the signal from CTV-TnC WT (0.169)  vs Mg2+ (0.079). Site IV differs only slightly with Ca2+ (0.100) vs Mg2+ (0.114). The analysis in 2+-dependent FRET signal in our study. Finally, Ca2+ binding at site II results in a negative contribution (-0.120), reducing the overall signal. In terms of conformational changes, a positive signal could be interpreted as a compaction of TnC bringing the ends and the fluorophores closer together, whereas a negative signal could mean an opening of TnC or a rotation of the N- and C-domains relative to one another, resulting in an increase in the distance of the ends and the fluorophores; it could also mean a change in orientation of the two fluorophores relative to one another, changing their dipole coupling, rendering FRET less optimal [Surprisingly, there was a small but statistically significant increase in Fexpected . This rey others . Potter y others  have sug optimal .2+ and 3mM Ca2+. Each protein’s sedimentation coefficient was compared under these 3 states, apo-state, ~2Mg2+ state and 3Ca2+ state, respectively. s values for all mutants as can be seen in the first row of s between the apo-state and the 3Ca2+ state, indicating that the negative FRET signal induced by Ca2+ binding to the N-terminus alone  than for Mg2+ (4.036 + 0.080). Similarly, data for mutant D104A CTV show that site IV confers a larger FRET signal in the presence of Mg2+ compared to Ca2+, consistent with a larger sedimentation coefficient (4.107 + 0.031) and (4.070 + 0.012) respectively. These AUC results are in agreement with the end point titration experiments summarized in 2+ versus Ca2+ at sites III and IV affect FRET in a different manner, different global conformations detected by AUC are induced in cTnC. In addition, for mutant D104-140A with only site II active, Mg2+ induces a change in sedimentation coefficient compared to apo and Ca2+-bound TnC  led to dissociation of TnC from thin filaments, which can be explained by a conformational change of TnC. Taken together, these observations could account for the change in AUC sedimentation coefficient upon non-physiological Mg2+ binding even in the absence of active sites III and IV.AUC measurements were performed using mutant proteins D104A CTV, D140A CTV, and D104-140A CTV to correlate the observed changes in FRET with global conformation associated with each of the EF hands upon divalent cation binding. Similarly to CTV-TnC described above, each mutant was prepared in 3 different buffer conditions: 3mM EDTA, 3mM EGTA/3mM Mgus alone , Table 1us alone . The ~2Mound TnC . This wa2+ in the absence of Mg2+  results in decrease of FR. Based on the end-point titration data in 2+ revealed a biphasic response with larger overall amplitude, although the biphasic response and assignment of FRET changes to C- and N-domain sites are consistent with our analysis of the EF hand mutant data , we assayed the assembled complex by non-denaturing gel electrophoresis and size exclusion chromatography (data not shown). Data in pCa50 1 of 7.81 and pCa50 2 of 5.69  whereas pCa50 for fibers reconstituted with WT-cTnC was 5.74 + 0.01 (Ka = 5.5 x 105 M-1). The rightward shift in Ca2+ responsiveness, although non-negligible in physiological terms, is similar to that observed with a number of single amino acid changes in troponin or tropomyosin, and is smaller than the most extreme shifts in pCa50 associated with single residue mutants [2+ and Mg2+  crystal and solution structures for TnC; ii) cardiac and skeletal TnC both for TnC; iii) in the Tn core domain crystal structures. Structural studies show that TnC structure appears to be dependent not only on Casubunits ,19,62. Ia et al.  show thaulations  that sug of Ca2+ ,61.2+ (2+ affinity binding of Ka = 1.5 x 107 M-1 (pCa50 = 7.17), and lower Ca2+ affinity binding site of Ka = 3 x 105 M-1 (pCa50 = 5.47) (R (at low Ca2+) was elevated in the presence of 2mM free Mg2+, as expected ; the relatively large error in this regression parameter estimate can be attributed to the small amplitude of change in FR  for the high affinity sites, and Ka = 4.9 x 105 M-1 (pCa50 = 5.69) for the low affinity site   . Even thexpected , we coulal sites  inset; on et al. , and as  of Ca2+ ,64 that  of Ca2+ . The appFmax1 in . As partity site . The sol studies , althougociation . Finally = 3.28)  consisteurements  for cardfunction .2+ compared to Mg2+ , derived from experiments with mutant D140A CTV (site IV inactivated). In addition, AUC data for the same mutant show a larger s in the presence of Ca2+ compared to Mg2+  , and in terms of structural changes induced upon binding divalent cations. Sites III and IV are considered as structural sites because of their fundamental role in anchoring TnC to the thin filament, while the regulation of muscle function occurs at site II, which has been the primary target of most studies. A key, novel finding in our study is that each of these sites makes an essentially independent contribution to the global conformation to TnC upon binding divalent cations . We muta to Mg2+ . We concctively) . ConcurrI and IV .pCa50 2) and nH 2 ~1 are consistent with Ca2+ binding at site II of cTnC, and were associated with a decrease in FR (negative ΔFR or Fmax2)  Figs . While tEF-hands . In contII alone . Decrease et al.  showed tdynamics  and obsedynamics , suggest2+ sensors  have been used to follow cellular Ca2+ dynamics in live cells. CTV-TnC presents great potential to be used as a Ca2+ sensor, as part of the family of GECI’s. The advantage of GECIs includes their ability to be targeted to specific organelles in a cell, and could also be potentially delivered by injection of the protein directly into cells/tissues. Additionally, TnC is only expressed in muscle tissues and therefore is less likely to interfere with the function of most cells, as opposed to calmodulin (used in some GECI’s) which may deregulate signaling [We have demonstrated that CTV-TnC responds to divalent cation binding through a change in global conformation, allowing energy transfer. Caignaling . We ackn2+ sensor is of particular interest to muscle research. To our knowledge, this construct is the first GECI FRET reporter of divalent cation binding to full length cardiac TnC while retaining its function to bind and activate the myofilament, albeit with a relatively small, non-negligible Ca2+ desensitization. This allows our sensor to have numerous potential applications such as live myocyte imaging, and could also be used to study thin filament dynamics since it was designed to be a single molecule reporter [Furthermore, our Careporter . While areporter , and ons


JAK2 V617F-positive MPN was diagnosed after seven years in remission. Retrospective evaluation of the JAK2 V617F detected a low allele burden at AML diagnosis and following one course of induction chemotherapy. This putative chemoresistant clone subsequently expanded over the intervening seven years, resulting in a hematologically overt MPN. As AML relapse has not occurred, the MPN may have arose in a separate initiating cell from that of the AML. Alternatively, both malignancies possibly evolved from a common precursor defined by a predisposition mutation with divergent evolution into MPN through acquisition of the JAK2 V617F and AML through acquisition of different mutations. This case emphasizes the protracted time frame from acquisition of a disease-driving mutation to overt MPN and further underscores the clonal complexity in MPN evolution.Although transformation of the myeloproliferative neoplasms (MPNs) to acute myeloid leukemia (AML) is well documented, development of an MPN in patients previously treated for, and in remission from, AML is exceedingly rare. A case is described in which a patient was successfully treated for AML and in whom a  JAK2 V617F and CALR exon nine mutations are absent in a significant number of MPN patients evolving to AML implicating the transformation of a common myeloid progenitor at a stage before acquisition of the MPN driver mutation or evolution from a distinct initiating precursor from that harboring the JAK2 V617F or CALR mutation [JAK2 V617F-positive MPN on the background of AML is exceedingly rare. Nearly all those cases reported have occurred in AML patients in long-term remission after chemotherapy or autologous stem cell transplantation and where retrospective genotyping of the AML presentation material was performed, demonstrating the absence of the JAK2 V617F mutation [JAK2 V617F-positive MPN when in remission of AML for seven years and in whom retrospective molecular investigation was able to unravel the evolutionary trajectory of the MPN.The clonal evolution of a myeloproliferative neoplasm (MPN) into acute myeloid leukemia (AML) is complex: both the mutation . Furthermutation . In contmutation –8. Molecmutation . A case 9/L, and a white cell count of 96.4 × 109/L with a large population of blasts. On physical examination, there was no palpable liver or spleen. Peripheral blood immunophenotyping showed two blast populations: one population (20%) positive for HLA-DR, CD117, CD33, and CD13 and a second population (48%) positive for HLA-DR, CD33, CD13, CD14, and CD15. The bone marrow aspirate was hypercellular with the vast majority of cells either myeloblasts or monoblasts, all consistent with a diagnosis of acute myeloid leukemia (AML) of myelomonocytic type . The bone marrow trephine biopsy at this time showed no evidence of blasts but remained hypercellular with some abnormal megakaryocyte forms showing focal clustering  was noted in October 2013 and an eosinophilia (0.4 × 109/L) was noted in January 2017, slowly rising to 18.9 × 109/L neutrophils and 0.6 × 109/L eosinophils. Qualitative molecular analysis of peripheral blood in July 2017 showed no evidence of BCR-ABL1 transcripts but detected the presence of the JAK2 V617F mutation. Subsequent bone marrow aspirate and biopsy in August 2017 (seven years after completion of AML therapy) revealed hypercellularity with increased myelopoiesis undergoing normal maturation. Megakaryocytes were increased in number with some immature forms being seen and an increase in mature myeloid cells to erythroid cells  and trisomy 8 cytogenetic abnormalities, with myelodysplastic changes, and a high allele burden [Molecular analysis of AML derived from a ectively . The che7F clone  remainedmia vera . This phiagnosis . Whole-eiagnosis . Moreovee burden , 16, feaJAK2 V617F driver mutation to development of a hematologically and clinically overt MPN accentuating the different kinetics of development of MPN and AML and suggesting that initiation of the MPN may have been an earlier event relative to that of the AML. Studies have shown that this process may take several years and have attempted to quantify the annual increase in JAK2 V617F allele burden [A protracted time course is clearly evident from acquisition of the initial e burden .JAK2 V617F, as these may not represent residual disease but a latent, coexisting hematological malignancy.Finally, given the expanding focus in minimal residual disease-directed therapy for AML patients , caution


FLT3) occur in approximately 25% of children and adults with AML and are associated with particularly poor prognoses. Identification and development of targeted FLT3 inhibitors represents a major precision medicine paradigm shift in the treatment of patients with AML. While further development of many first-generation FLT3 inhibitors was hampered by limited potency and significant toxicity due to effects upon other kinases, the more selective second- and third-generation FLT3 inhibitors have demonstrated excellent tolerability and remarkable efficacy in the relapsed/refractory and now de novo FLT3-mutated AML settings. While these newest and most promising inhibitors have largely been studied in the adult population, pediatric investigation of FLT3 inhibitors with chemotherapy is relatively recently ongoing or planned. Successful development of FLT3 inhibitor-based therapies will be essential to improve outcomes in children with this high-risk subtype of AML.Acute myeloid leukemia (AML) is the second most common leukemia of childhood and is associated with high rates of chemotherapy resistance and relapse. Clinical outcomes for children with AML treated with maximally intensive multi-agent chemotherapy lag far behind those of children with the more common acute lymphoblastic leukemia, demonstrating continued need for new therapeutic approaches to decrease relapse risk and improve long-term survival. Mutations in the FMS-like tyrosine kinase-3 receptor gene ( It iectively . RelapseFMS-like tyrosine kinase-3 FLT3; CD135) is a 993 amino acid single transmembrane type III receptor tyrosine kinase in the same family as the structurally similar stem cell growth factor receptor c-KIT CD117), colony-stimulating factor-1 receptor , and platelet-derived growth factor receptor (PDGFR) . FLT3 ha35 is a 9, colony-FLT3 is overexpressed in most cases of B-lymphoblastic leukemia and AML and in a smaller percentage of T-lineage ALL and chronic myeloid leukemia (CML) in blast crisis  internal tandem duplication (FLT3–ITD) mutations, which are 3–400 bp in-frame duplications located in the juxtamembrane domain and (2) activating point mutations, which are found in the tyrosine kinase domain (FLT3–TKD) and most often involve residue D835  is usually based on cytogenetic risk stratification and transplant eligibility status . With thutations –32. Chilutations . Higher utations , 33. In utations , 34, demFLT3-mutated AML has been investigated for the past decade with promising results of several studies recently reported. This review discusses the current landscape of and future potential for clinical testing of FLT3 inhibitors in adults and children with FLT3-mutated AML  are small molecules that inhibit the enzymatic activity of tyrosine kinases and block downstream signaling activation. Treatment of adults with CML with the SRC/ABL inhibitor imatinib is one of the major early successes of modern precision medicine , 36. ImaML Table .FLT3-wild-type AML (de novo FLT3–ITD or FLT3–TKD AML. Addition of midostaurin to chemotherapy significantly improved median EFS (8.2 versus 3.0 months) and OS (74.7 versus 25.6 months) compared to patients treated with chemotherapy and placebo (de novo FLT3-mutated AML (Midostaurin (PKC412) is a first-generation oral FLT3 inhibitor initially named due to its inhibitory effects upon protein kinase C. Midostaurin was subsequently recognized as a promiscuous kinase inhibitor with strong inhibitory effects also against the vascular endothelial growth factor receptor (VEGFR), PDGFRα and β, spleen tyrosine kinase (SYK), c-KIT, and FLT3 . Midostatype AML , 63. Howtype AML . These s placebo . The pre placebo , 64. BasFLT3-mutated AML or KMT2A-rearranged ALL (which overexpresses wild-type FLT3). The trial closed early due to inadequate accrual. While the number of subjects studied was too small for more rigorous analysis, 5 of 15 patients with AML and 3 of 13 patients with KMT2A-rearranged ALL had partial or complete responses with midostaurin monotherapy with OS of 3.7 (AML) and 1.4 months (ALL), respectively  assays demonstrated that few patients achieved sustained FLT3 inhibition (Lestaurtinib (CEP-701) is another first-generation multi-kinase inhibitor with activity against FLT3, Janus kinase 2, and tropomyosin receptor kinase A. One randomized trial tested lestaurtinib administration after induction chemotherapy in adult patients with AML in first relapse and demonstrated no survival benefit with lestaurtinib addition versus chemotherapy only . Howeverhibition , limitinhibition , 66. Thehibition . Howeverhibition .FLT3-overexpressing KMT2A-rearranged ALL. Despite very promising preclinical data, the randomized COG phase 3 trial AALL0631 (NCT00557193) failed to demonstrate benefit of lestaurtinib addition to chemotherapy in infants with KMT2A-rearranged ALL, although achievement of sustained FLT3 inhibition as measured by PIA assays was variable among patients and across therapy phases  in children and adolescents and young adults (AYAs) <30 years of age with relapsed/refractory AML. These patients were induced with cytarabine and idarubicin, then treated with lestaurtinib. The trial closed after the safety phase demonstrated tolerable combination dosing, but without completion of efficacy phase testing . In the y phases .FLT3-mutated AML demonstrated safety and tolerability with minimal toxicity  is another first-generation pan-kinase inhibitor with activity against Raf, c-KIT, PDGFR, VEGFR, and FLT3 . Sorafentoxicity , 73. Subobserved . The sucobserved .de novo FLT3–ITD AML. The study was amended to include a 1-year sorafenib maintenance phase post-HSCT based upon smaller studies reporting potential efficacy of this post-transplant strategy to minimize relapse risk  first studied sorafenib monotherapy in children with relapsed/refractory AML, then in combination with clofarabine and cytarabine. Five of 12 enrolled patients had FLT3–ITD AML. Responses were observed in most patients, including all five FLT3–ITD patients, with complete remission (CR) in four patients, CR with incomplete count recovery (CRi) in two patients, and a partial response in one patient . A concopse risk . ResultsFLT3-mutated AML with reported similar efficacy as sorafenib (Sunitinib (SU11248), another multi-kinase inhibitor with activity against FLT3, has been studied in adult patients with orafenib . Toleraborafenib , 77. Sunorafenib .in vivo potency than first-generation inhibitors  is the first drug specifically designed as a FLT3 inhibitor and has 10–50 times greater hibitors , 79, 80.hibitors . Initialhibitors , and subhibitors , 82. Othhibitors , 84.KMT2A-R ALL. Quizartinib was well-tolerated without dose-limiting toxicity, and correlative PIA assays demonstrated near-complete pharmacodynamic inhibition of FLT3 at all tested doses (KMT2A-rearranged ALL (The Therapeutic Advances in Childhood Leukemia/Lymphoma (TACL) consortium conducted a phase 1 trial of quizartinib with cytarabine and etoposide in 17 children (ages 1 month to 21 years) with relapsed/refractory AML or ed doses . All sevnged ALL . These dPonatinib (AP24534) is a third-generation multi-kinase inhibitor with activity against BCR–ABL and FLT3. Ponatinib is currently FDA-approved for treatment of adults with TKI-resistant CML or Ph+ ALL . In precFLT3-mutated AML with encouraging activity (Crenolanib (CP-868596) was originally designed as a PDGFR inhibitor, although later studies also demonstrated its potency as a FLT3 inhibitor. Due to its short half-life, crenolanib requires thrice-daily dosing. Early data suggest that this third-generation TKI has robust activity against both FLT3–ITD and FLT3–TKD mutations, including those that confer resistance to quizartinib , 91, 92.activity , and comactivity .via a phase 1 trial conducted at SJCRH in children with central nervous system gliomas, which have activated PDGFR signaling  (Table FLT3-mutated AML. A pediatric development program for gilteritinib is planned.Gilteritinib ASP2215), the newest third-generation oral FLT3 inhibitor, is the most potent and selective FLT3 inhibitor developed to date with moderate additional activity against the AXL kinase. In preclinical studies, gilteritinib has it c-KIT , therebyit c-KIT . A firstit c-KIT , 60. Gil5, the neFLT3-mutated AML is a particularly high-risk leukemia subtype in both adults and children, and the potential for selective FLT3 inhibitors to decrease relapse risk and improve cure rates is alluring. Initial trials of first-generation FLT3 inhibitors have validated FLT3 as a viable therapeutic target in AML and expedited FDA approval of midostaurin for adults with FLT3-mutated AML is a major recent achievement (Despite maximal therapeutic intensification and significant improvements in supportive care, more than one-third of children with AML still die from leukemia or associated complications. ievement . Howeverievement .It is probable, perhaps certain, that children with FLT3 signaling-driven AML will similarly benefit from addition of FLT3 inhibition to chemotherapy. The newest and more selective inhibitors, quizartinib, crenolanib, and gilteritinib, have demonstrated very promising activity in adults with relapsed/refractory and newly diagnosed AML, but have been minimally or not studied to date in children. These agents merit broader clinical investigation in pediatrics. Emerging data from sorafenib maintenance studies also demonstrate the potential importance of such therapeutic strategies in the post-HSCT setting , 98. In FLT3-mutated AML is ongoing or on the imminent horizon. Such important clinical investigation is critical to improve remission and decrease relapse in this highest-risk population of children with AML, potentially also reducing the significant toxicities associated with salvage therapy.Acquisition of resistance mutations following FLT3 inhibitor therapy remains a major source of treatment failure, although the incidence of such mutations in children with AML is not fully known. It is plausible that combining FLT3 inhibitors with chemotherapy may decrease the incidence of resistance mutations that occur with inhibitor monotherapy, analogous to lower mutation rates often observed in children with Ph+ ALL treated with TKI and chemotherapy , 102. HoANS and SKT wrote and edited the manuscript. Both authors approved the final version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Here we have addressed this question, using mass spectrometry combined with a disulfide searching algorithm DBond. We found that the binding of Ca2+ to hSCGN promotes the dimerization of hSCGN via the formation of a Cys193-Cys193 disulfide bond. Hydrogen/deuterium exchange mass spectrometry (HDX-MS) and molecular dynamics studies revealed that Ca2+ binding to the EF-hands of hSCGN induces significant structural changes that affect the solvent exposure of N-terminal region, and hence the redox sensitivity of the Cys193 residue. These redox sensitivity changes were confirmed using biotinylated methyl-3-nitro-4-(piperidin-1-ylsulfonyl) benzoate (NPSB-B), a chemical probe that specifically labels reactive cysteine sulfhydryls. Furthermore, we found that wild type hSCGN overexpression promotes insulin secretion in pancreatic β cells, while C193S-hSCGN inhibits it. These findings suggest that insulin secretion in pancreatic cells is regulated by Ca2+ and ROS signaling through Ca2+-induced structural changes promoting dimerization of hSCGN.Secretagogin (SCGN), a hexa EF-hand calcium binding protein, plays key roles in insulin secretion in pancreatic β-cells. It is not yet understood how the binding of Ca Scgn, originally cloned from pancreatic β cells1, is expressed selectively in pancreatic β cells and neuroendocrine cells1, as well as at distinct neurosecretory loci in the brain3. SCGN controls stress hormone release2 and olfactory neuron differentiation in Alzheimer’s disease4. Recently, we reported that SCGN also plays crucial roles in insulin secretion in pancreatic β cells by interacting with vesicle fusion and trafficking proteins5 or with actin cytoskeleton6. We also showed that SCGN regulates focal adhesion by regulating the actin cytoskeleton during insulin secretion. SCGN physically interacts with actin and this interaction is increased by H2O2 and glucose stimulation, indicating that Ca2+ and reactive oxygen species (ROS) regulate the interaction between SCGN and actin. ROS responsive insulin secretion was diminished by silencing SCGN which further indicates that SCGN mediates Ca2+ and ROS signaling in insulin secretion.Secretagogin (SCGN) is a Ca2+ and ROS are known to play key roles in glucose-stimulated insulin secretion (GSIS) by regulating intracellular signaling and nutrient sensing, coupling glucose metabolism to insulin secretion11. Subtle changes in Ca2+ signaling affect β-cell physiology and the pathogenesis of diabetes. Ca2+ signaling is mainly mediated by Ca2+ binding proteins, one of which is SCGN12. SCGN has a low Ca2+ affinity and functions as a Ca2+ sensor protein, controling protein-protein interactions and cellular signaling via conformational changes14. ROS induce oxidative modifications, including reversible disulfide bonds, and structural and functional changes that modulate the signaling activity of proteins15. Dimerization of re-folded recombinant SCGN, possibly due to oxidation, has been reported16, raising the possibility of physiological redox regulation of SCGN. Recently it was reported that SCGN is a redox sensitive Ca2+ sensor, and that this dimerization and Ca2+ binding increase the stability of SCGN18. However, the structural regulation and functional implication of Ca2+ binding and dimer formation of SCGN remain to be elucidated.In pancreatic β cells, intracellular CaDanio rerio SCGN monomer (apo-SCGN) has been determined, but not that of SCGN in the presence of Ca2+ 19. Hydrogen/deuterium exchange monitored by mass spectrometry (HDX-MS) provides a complementary method for investigating structural change to x-ray crystallography. In previous work employing HDX-MS, we identified structural changes in the NDP kinase protein (Nm23-H1) caused by stepwise oxidation20. Here we report on HDX-MS studies on the structural changes in human SCGN (hSCGN) to see how its Ca2+ binding might affect its structure and impact upon insulin secretion. We validated these findings by molecular modeling of Ca2+ bound hSCGN. Our findings indicate that Ca2+ binding promotes the formation of stable dimers of hSCGN in the presence of ROS, and suggest that this dimerization is necessary for SCGN to promote insulin secretion.An x-ray structure of the calcium-free ViaFECT transfection reagent: Promega , Effectene transfection reagent: QIAGEN , Insulin ELISA kit: ALPCO , BCA protein assay: Thermo Scientific . For expression in mammalian cells, the constructs encoding pcDNA3.1 Human SCGN WT, mutants C193S, C253S, C269S and ΔEF1 (a.a.: Δ1-52) were generated by cloning and mutation using the pcDNA3.1 myc his (−) A vector and an SCGN cDNA clone obtained from human cDNA library as a template. All plasmid constructs were confirmed by DNA sequencing.Scgn was provided from Dr. Ludwig Wagner . Mutant hSCGN C193S, C253S and C269S were generated using the Quik-Change®II site-directed mutagenesis kit (Agilent Technologies) according to the manufacturer’s protocol using pGEX-1λT-Scgn as a template. The mutagenesis oligonucleotides used were the following: for mutation C193S, sense primer 5′-TTCTCCAATTTAAAATGGATGCTAGTTCTACTGAAGAAAGGAAAAGG-3′, antisense primer 5′-CCTTTTCCTTTCTTCAGTAGAACTAGCATCCATTTTAAATTGGAGAA-3′; for mutation C253S, sense primer 5′-ATTCTCCTGCGTCACAGCGACGTGAACAAGG-3′, and antisense primer 5′-CCTTGTTCACGTCGCTGTGACGCAGGAGAAT-3′; for mutation C269S, sense primer 5′-AGTCTGAGCTGGCTTTGAGTCTTGGGCTGAAAATC-3′, and antisense primer 5′-GATTTTCAGCCCAAGAC TCAAAGCCAGCTCAGACT-3′ (mutated codons are underlined). Construct integrity was validated by DNA sequencing.pGEX-1λT-hScgn gene was expressed in BL21 (DE3) E. coli cells. The bacteria were cultured in LB medium at 37 °C, and recombinant fusion protein production was induced with 0.5 mM isopropyl-β-D-thiogalactopyranoside (IPTG). After 4 h of additional incubation at 37°C, the cells were harvested and lysed by vortexing with lysozyme containing hSCGN purification buffer  on ice. The soluble protein fraction was recovered by centrifugation at 60,000 g for 1 h at 4°C. The GST-fused recombinant proteins in the supernatant were purified by chromatography on a glutathione-agarose column followed by washing, and equilibration by hSCGN elution buffer . To cleave hSCGN from the GST-hSCGN, the beads were incubated overnight at room temperature with thrombin in SCGN elution buffer. After 24 h, the purified hSCGN was eluted and quantity was measured.A pGEX-1λT plasmid carrying the human 2-95% air. NIT-1 insulinoma cells were maintained in 5.6 mM glucose DMEM supplemented with FBS and antibiotics as above. For transient overexpression of specific proteins in HeLa cells, cells were transfected using Effectene and analyzed at 24 h post transfection. For NIT-1 cells, the cells were transfected using ViaFECT and analyzed at 48 h post transfection according to the manufacturer’s recommendations.HeLa cells were purchased from ATCC and cultured in EMEM supplemented with 10% fetal bovine serum (FBS), 100 μg/mL streptomycin, 100 units/mL penicillin G at 37 °C in an atmosphere of 5% CO6. Tandem MS (MS/MS) spectra were matched against amino acid sequences in the SwissProt human database  using DBond (https://prix.hanyang.ac.kr).The peptides resulting from trypsin-digested protein in non-reducing gel were resuspended in 10% acetonitrile containing 0.1% formic acid and analyzed using nanoAcquity™ UPLC™/ESI/MS  as described previously21. Briefly 2 μg of recombinant hSCGN was pre-incubated with/without Ca2+ at room temperature for 15 min followed by incubation with NPSB-B  at room temperature for 2 h. Proteins were separated by 12% SDS PAGE and transferred to PVDF membranes. Labeled proteins were detected by streptavidin-HRP. The amount of total loaded protein was detected by Coomassie staining.Methods for the labeling of reactive cysteine residues by NPSB-B were previously reported2O, pH 7.4) and incubated at 25°C for10, 60, 300, 1800, or 10800 s. The deuterium labeling reaction was quenched by 2.5 mM tris (2-carboxyethyl) phosphine (TCEP), formic acid, pH 2.3. For protein digestion, 1 μg of porcine pepsin was added to each quenched protein sample and incubated on ice for 3 min before injection. Peptic peptides were desalted on C18 trap column cartridge  and gradient eluted from 8% CH3CN to 40% CH3CN, 0.1% formic acid on 100 µm i.d. × 100 mm analytical column, 1.7 µm particle size, BEH130 C18,  for 7 min. The trap, analytical column and all tubing were immersed in an ice bath to minimize deuterium back-exchange. Gradient chromatography was performed at a flow rate 0.5 μL/min and was sprayed on line to a nanoAcquityTM/ESI/MS (SYNAPTTM HDMSTM) . The extent of deuterium incorporation was monitored by the increase in mass of the isotope distribution for each identified peptide, and calculated using Microsoft Excel. The theoretical maximum deuterium incorporation value was calculated for each peptide based on the number of exchangeable amides20. Each experiment was performed in triplicate. Deuterated peptides were calculated by DynamX v3.1 µL hSCGN (1 mg/mL) was diluted 19-fold with labeling buffer  was used to generate homology models of hSCGN. The 3D structure of SCGN from Danio Rerio (PDB ID: 2BE4)19 which has high sequence identity to hSCGN (~70%) was used as a template. A Ca2+-bound hSCGN structure was manually constructed with COOT22. Six Ca2+ ions were added to each potent EF-hand loop ) containing 0.1% BSA. After glucose starvation, the cells were stimulated at 37 °C for 25 min with HBSS containing 16.8 mM D-glucose. Secreted insulin was normalized to cellular total protein concentration, measured by a BCA protein assay performed according to the manufacturer’s instructions.Insulin secreted into the medium was measured using Mouse Ultrasensitive Insulin ELISA kit (ALPCO) following the manufacturer’s protocol. NIT-1 cells were starved at 37°C for 2 h with glucose-free HBSS  equilibrated with PBS. The column was eluted at a flow rate of 0.5 mL/min, and proteins were fractionated using FPLC (AKTA Explorer) into 15 fractions of 0.5 mL each. Proteins used as molecular weight markers were albumin (66.4 kDa) and α-lactalbumin (14.2 kDa).450 µg of protein was loaded on a SuperdexMDA-MB 231 cells were lysed with lysis buffer  containing protease inhibitors for 30 min on ice. The cell lysates were then centrifuged at 12,000 rpm for 15 min at 4°C. The supernatant was incubated with anti-SCGN antibody for 2 h and then with protein G Sepharose beads for 1 h at 4°C. The beads were washed three times with 1 mL of lysis buffer and additionally twice with 1 mL of lysis buffer without detergent. The immune complex was solubilized in SDS gel sample buffer, separated on SDS-PAGE, and visualized by silver staining or Western analysis.2, and 100 μM Na3VO4), and a mixture of protease inhibitors (Roche) for 15 min on ice and disrupted using 32-gauge syringes. Crude nuclear fractions were pelleted by 5000 rpm for 10 min centrifugation at 4 °C, and supernatants were separated by 40,000 rpm for 1 h centrifugation at 4°C to cytosol (supernatants) and membrane (pellets).Cells (70–80% confluent in 100 mm dishes) were washed and collected by scraping into ice-cold PBS, pelleted by centrifugation at 4°C, resuspended in hypotonic buffer . Samples were pretreated with EGTA and DTT to diminish dimerization due the possible effects of trace amounts of Ca2+ and air during the purification procedures. In the presence of Ca2+, H2O2 produced stable disulfide-linked dimers, while in the absence of Ca2+, less dimerization and more non-specific high molecular weight species were observed , which showed SCGN mRNA was not detected in HeLa cells. When HeLa cells transfected with hSCGN were exposed to various concentrations of H2O2, the overexpressed hSCGN was found to dimerize in a dose- and time-dependent manner , in a H2O2 dose-dependent manner. This indicates that the availability of reactive Cys-SH residues can be affected by low concentrations of H2O2. To elucidate which cysteine residue is reactive and whether it is regulated by Ca2+, we examined NPSB-B labeling of various cysteine mutants. The cysteine reactivity of hSCGN WT, C253S and C269S was reduced by Ca2+ treatment; in contrast, the C193S mutant could not be labeled with NPSB-B  simulations of hSCGN with and without six Ca2+ ions  were calculated with respect to the backbone coordinates of the starting structures, with and without six Ca2+ ions  plot  and conformational changes at 2 mM CaCl2 have been clearly shown in the HDX experiments. Our experiments showed that EF1 and EF2 bind to Ca2+ at high calcium concentration due to their low calcium affinity. And our studies on MD simulation with six instead of four Ca2+ ions are in agreement with the above. Since there is no other calcium ion binding site in the N-terminal domain, the only plausible explanation of the conformational change around EF1 and EF2 was the binding of calcium ions in these EF motifs. Therefore, HDX experiment proves that hSCGN binds 6 Ca2+ in high Ca2+ concentration.Rogstam 30. The Ca2+-bound dimeric form clearly showed that the dimeric interface is well formed and that the N-terminal domain is located on the same side as Cys193 , which has the stable conformation of purified protein. We examined the effect of truncating EF1 on Ca2+ triggered dimer formation. As shown in Fig. 2+, while H2O2 induced dimerization was not affected by EF1 deletion. This is consistent with the hypothesis that a conformation change in the N-terminal region mediates the Ca2+-induced dimerization of hSCGN. To further test these results in in vivo, we examined hSCGN dimer formation in HeLa cells. HeLa cells were transfected with hSCGN WT or ΔEF1 and were pre-incubated with ionomycin for 5 min to raise the intracellular Ca2+ concentration, followed by oxidative stimulation with H2O2 for 30 min. Dimer formation in each case was determined by Western analysis under non-reducing conditions. As shown in Fig. 2O2 treatments was significantly higher in cells overexpressing hSCGN WT than the ΔEF1 mutant.Since Ca2+ binding , we examined insulin secretion in NIT-1 cells, a mouse insulinoma cell line that expresses SCGN endogenously33. We first assessed the effect of Ca2+ and oxidative stress on SCGN dimerization in NIT-1 cells. We treated NIT-1 cells transfected with SCGN WT and C193 mutant with ionomycin, followed by H2O2 at doses that have been previously determined to be the physiologically relevant and optimal for NIT-1 cells6. WT hSCGN was found to be dimerized and oligomerized by treatment with ionomycin plus H2O2, while C193S hSCGN did not  in pancreatic β-cells. NIT-1 cells transiently transfected with control vector, hSCGN WT and C193S mutant were starved for 2 h, followed by stimulation with glucose for 30 min and assay of secreted insulin. Figure Since glucose stimulation promotes intracellular Ca6. hSCGN interacts with actin and this interaction was increased by H2O2 and glucose stimulation, indicating that Ca2+ and ROS signaling regulate the interaction between SCGN and actin. To determine whether C193S hSCGN interferes with actin binding, we performed immunoprecipitation with hSCGN WT and C193S mutant. Since NIT-1 cells are not suitable for immunoprecipitation with ectopic hSCGN because of their high endogenous expression of SCGN, we used MDA-MB-231 cells, which express only trace amounts of hSCGN. We found that the C193S mutant did not interact with the actin cytoskeleton, whereas hSCGN WT did (Fig. We previously reported that hSCGN regulates insulin secretion through interaction with the actin cytoskeletondid Fig. . These r2O2 treatment enhanced the co-localization with actin cytoskeleton in plasma membrane6. The dimerization of SCGN has a modulatory role in insulin secretion and further oligomerization of SCGN occurs only inside cells in response to H2O2 and ionomycin (Figs 34. It is also possible that glucose stimulation increases the intracellular Ca2+ concentration and acutely dimerizes Ca2+-bound SCGN by ROS generation, with the dimerized SCGN forming oligomers that bind with the actin cytoskeleton to enhance insulin secretion. However, cellular function of oligomers remains to be further elucidated.We previously reported that SCGN regulates GSIS via ROS signaling and Hcin Figs  and 5A. 2+ binding, followed by SCGN dimerization by cysteine oxidation in response to ROS. Moreover, these changes appear to be important for GSIS. The elevation of intracellular Ca2+ concentration in glucose-stimulated pancreatic β-cells is hypothesized to cause conformational changes in inactive apo-SCGN via Ca2+ binding by inducing N-terminal flexibility. This is followed by allosteric regulation of Cys193 reactivity and stabilization of C-terminal disulfide bonds between C253 and C269 that prevents non-specific dimerization and induces an N-terminal conformation change to form Cys193-linked dimers more efficiently. This study identifies the molecular crosstalk between Ca2+ binding and oxidation in the formation of functionally-important disulfide dimers, crucial for the biological function of SCGN in insulin secretion in pancreatic β-cells.In summary, this study identifies a novel regulatory mechanism in the regulation of insulin secretion in pancreatic β-cells by SCGN that involves stepwise structural changes brought on by CaSupplementary Information