These findings were attributed to actions of consequently altered cytosolic Ca2+, [Ca2+]i, on Nav1.4. We extend the latter hypothesis employing cyclopiazonic acid (CPA) challenge, which similarly increases [Ca2+]i, but through contrastingly inhibiting sarcoplasmic reticular (SR) Ca2+-ATPase. Loose patch clamping determined Na+ current (INa) families in intact native murine gastrocnemius skeletal myocytes, minimising artefactual [Ca2+]i perturbations. A bespoke flow system permitted continuous INa comparisons through graded depolarizing steps in identical stable membrane patches before and following solution change. In contrast to the previous studies modifying RyR1 activity, and imposing control solution changes, CPA (0.1 and 1 µM) produced persistent increases in INa within 1–4 min of introduction. CPA pre-treatment additionally abrogated previously reported reductions in INa produced by 0.5 mM caffeine. Plots of peak current against voltage excursion demonstrated that 1 µM CPA increased maximum INa by ~ 30%. It only slightly decreased half-maximal activating voltages (V0.5) and steepness factors (k), by 2 mV and 0.7, in contrast to the V0.5 and k shifts reported with direct RyR1 modification. These paradoxical findings complement previously reported downregulatory effects on Nav1.4 of RyR1-agonist mediated increases in bulk cytosolic [Ca2+]. They implicate possible local tubule-sarcoplasmic triadic domains containing reduced [Ca2+]TSR in the observed upregulation of Nav1.4 function following CPA-induced SR Ca2+ depletion.Skeletal muscle Na These may permit (a) Ca2+ binding to one or more C-terminal EF-like hand motifs1, (b) CaM binding to a C-terminal isoleucine-glutamine (IQ) domain following Ca2+ binding to EF-hand motifs on the CaM itself3, and (c) Ca2+/CaM-binding to a III–IV loop site4. It also possesses (d) sites phosphorylatable by CaM kinase II (CaMKII) itself regulated by Ca2+/CaM5 and (e) a phosphorylation site for phosphokinase C6. One or more of these sites may underly in vitro single-cell patch-clamp reports that elevating [Ca2+]i to ~ 2 µM through rapid photo-release and Ca2+ overspill from neighbouring Ca2+ channels reduced peak Na+ current densities, INa, in Nav1.4-transfected HEK293 cells and skeletal muscle cell lines, effects abrogated by intracellular BAPTA7. These effects were abolished with mutations in either the Ca2+-binding-EF hands on CaM or the Nav1.4 C-terminal IQ domain, implicating CaM binding to the IQ domain in the [Ca2+]i-mediated Nav1.4 modulation8.The skeletal muscle Na2+ perturbations arising from the intrapipette EGTA used in conventional patch clamping. Ryanodine receptor (RyR1) agonists known to gate sarcoplasmic reticular (SR) store Ca2+ release reversibly downregulated INa. These effects of both the Epac activator 8-(4-chlorophenylthio)adenosine 3′,5′-cyclic monophosphate (8-CPT) and RyR1 agonist caffeine were abrogated by the RyR1 antagonist dantrolene10. Dantrolene itself contrastingly produced increases in INa. Murine cardiac muscle showed concordant effects of enhanced SR Ca2+ release either with 8-CPT application11 or in RyR2-P2328S genetic variants12.These findings were extended to native intact working murine skeletal myocytes using loose patch clamp methods that avoided further artefactual intracellular Ca2+]i in such a feedback modification of Nav1.4 function suggested by the latter reports. They now explore effects of cyclopiazonic acid (CPA) challenge. In common with caffeine13 this increases bulk cytosolic [Ca2+]i14. However, rather than enhancing RyR-mediated Ca2+ release, it does so through inhibiting SR Ca2+-ATPase (SERCA). This reduces SR cytosolic Ca2+ uptake thereby depleting SR store Ca2+15. The experiments similarly employed loose patch clamping protocols, but these were further modified to permit pair-wise statistical comparisons of INa in the same established undisrupted patch before and following solution changes delivering the pharmacological challenge. This dealt with any between-patch INa variations reflecting previously reported non-uniform ion channel, including Nav1.4, distributions over the muscle fibre surface16.The present experiments test and extend the resulting hypothesis implicating alterations in TSR themselves influenced by terminal cisternal RyR1-mediated fluxes of SR Ca2+. The latter would be increased by the RyR1 agonists, caffeine and 8-CPT, but decreased by SR Ca2+ depletion produced by the SERCA inhibitor CPA. Such tubular Nav1.4 likely make a substantial contribution to measured INa18, given that T-tubules comprise up to 80% of muscle fibre membrane surface area19.We here report that in contrast to expectations from the previous experiments that modified RyR1-mediated CaINa currents elicited by the pulse protocol, normalized to patch areas giving current densities, initially rapidly increased to attain peak values before showing inactivation decays. This fulfilled expectations from previous studies similarly exploring INa using the loose patch clamp technique16. The peak currents obtained through the range of applied voltage steps were then quantified in current–voltage (I–V) relationships. Figure 2) showed significant variation , confirming previous reports16. Nevertheless, the experimental design separated such between-patch variability from the observed changes associated with the pharmacological challenge under investigation.2+ re-uptake into the SR, in contrast with the RyR1-specific agonist and antagonist agents employed in the previous studies. The CPA was used at 0.1/1.0 µM concentrations, well in excess of the reported 10–20 nM half-maximal concentration, to achieve maximal SERCA inhibition15. When recording from small cells, it is possible to effect rapid solution changes by stepping the interface between parallel continuous perfusing streams20. However this approach was not feasible in this case owing to the size of the tissue, so the entire bath solution was exchanged. The configuration used here nevertheless permitted relatively rapid solution changes by simultaneous removal of the initial whilst adding the new test solution, thereby maintaining bath solution level constant. This prevented vertical forces at the patch site due to changes in buoyancy forces acting on the muscle. This was despite reduction of the bath volume using the obturator insert to ensure completeness of the solution change.To fulfil the latter strategy, the experiments studied ionic currents before and following the solution changes introducing either control solution or solutions containing the test agents, making recordings before and following each solution change, from the same established patch. This permitted the pairwise comparisons of control and test results adopted here. This required maintenance of stable patches before and following the solution changes involving challenge with extracellularly applied cyclopiazonic acid (CPA) or otherwise. The externally applied CPA used here targeted cytosolic SERCA-mediated CaI–V) relationships, with a stability confirmed in comparisons of results obtained before, and 1 min and 4 min following the solution change  solutions containing no CPA were performed to test the effectiveness and validity of this approach. These manoeuvres produced little change in patch seal resistances, which suggested the solution changes did not disrupt patch integrity. Currents obtained from the subsequent patch recordings before Fig. a and folnge Fig. d.Figure 21. Following establishment of the patch seal, a set of recordings using a bath solution containing no drug were then obtained. The solution change was then promptly performed. Further recordings of ionic current were then made in the presence of CPA starting at 1 min post-exposure.In the initial experiments testing the effects of the SERCA inhibitor CPA on the measured currents, the solution exchanger was first loaded with 50 ml of K–H solution containing 0.1 µM CPA and connected to the preparation. This concentration was previously reported to cause significant effects in Langendorff-perfused murine heartsI–V) relationships showed increased inward current magnitudes but otherwise were similar in form. These effects stably persisted at 4 min following the solution change. However, alterations in patch characteristics, particularly the seal resistances, over longer, ~ 16 min, times prompted corroborative studies at higher, 1 µM CPA, concentrations than the 0.1 and 0.15 µM levels used previously in murine preparations21 as these might produce effects at earlier times. These employed less closely graded voltage clamp steps encompassing a smaller range of depolarizations. This permitted recordings to be completed in < 60 s whilst still permitting characterization of the current–voltage relationships derived below.Figure These studies demonstrated similar findings at the CPA concentrations (1 µM) expected to supramaximally inhibit SERCA. They then gave more consistent, greater, increases in the currents observed at 1 min than did 0.1 µM CPA Fig. a,b. Simi2+ stores, complemented previous experiments stimulating SR Ca2+ store release using the RyR1 agonist caffeine. Both agents were reported to increase bulk cytosolic [Ca2+]i14. However, in contrast to the sustained CPA action in increasing INa reported here, caffeine challenge caused decreases in INa. But the latter effects were transient, reflecting its successive, RyR1-mediated, activation followed by inactivation of SR Ca2+ release10. Accordingly, further control experiments challenged CPA-treated muscle preparations with a superimposed addition of 0.5 mM caffeine  Ca+ currents were recorded. Prior to this, the solution exchanger was loaded in readiness with 50 ml of K–H solution containing 1 µM CPA in combination with 0.5 mM caffeine. This permitted the prompt second solution change which thereby additionally introduced caffeine after ~ 10 min of pre-incubation in CPA. Further recordings were then taken from the same patch at 1 and 4 min as before. This challenge with multiple agents targeting Ca2+ homeostasis did not affect patch seal resistances, and recordings of families of Na+ currents remained possible. Furthermore, the findings contrasted with the previous reports in which 0.5 mM caffeine alone markedly decreased Na+ currents. Thus, this additional pre-exposure to CPA if anything, slightly increased the Na+ currents, a change in the opposite direction as that reported when caffeine challenge was applied following SR Ca2+ depletion10.After setting up a preparation in K–H solution with no drug, the solution exchanger introduced 1 µM CPA. Patches were then obtained and a family of Na+ currents and their further analysis. It compares findings obtained prior to solution change with those obtained 1 min following alterations to solutions containing 0.0 µM , 0.1 µM  and 1 µM CPA  respectively. The resulting pairs of current–voltage (I–V) relationships remained consistent in shape, suggesting comparable activation characteristics. The left plots (a–c) show unpaired data showing peak currents from different patches plotted as means and standard errors of the mean (SEM) representing between-patch variations from the replicate experiments in the groups above. The right plots (d–f) illustrate the corresponding data normalized to the corresponding maximum peak current for each individual patch prior to CPA challenge and their SEMs. This analysis separates the between-patch variations in peak currents illustrated in Fig. 16 from the paired within-patch alterations in Na+ current resulting from CPA challenge under examination.Figure As in previous reports, their rising phases for each patch were described by fitting them to a two-state Boltzmann function. Data within a range of voltages from − 40 mV relative to RMP to voltage excursions that produced the greatest magnitude of peak current  were fitted using a scaled Levenberg–Marquardt algorithm to the equation:I, in pA/µm2) were thus related to clamped voltage excursion (V) through the maximum current , voltage excursion at half-maximum current  and a steepness factor . As recordings were taken before and after solution change from a single patch in each case, the results were paired and thus analysed by paired Student’s t-tests. Values of the mean, SEM and p values for each parameter are compared in Table Peak current densities  and 0.1 µM CPA concentrations, 1 µM CPA produced noticeable, statistically significant ~ 30% increases in Imax. In contrast, both 0.1 µM and 1 µM CPA caused only small decreases  in V50 and k, similar to those produced by the control experiments. These changes thus reflected the comparisons in Fig. I–V relationships of similar form differing principally in their maximum currents.Solution changes in the control experiments introducing 0 µM CPA did not result in significant changes in 2+]i-mediated, in vitro, INa downregulation was first demonstrated in skeletal muscle cell lines and Nav1.4-transfected cultured HEK293 cells following rapid photo-release and Ca2+ overspill from neighbouring Ca2+ channels. This were abolished by mutations in the Ca2+-binding-EF hands on CaM or the Nav1.4 C-terminal IQ domain implicating direct or indirect Ca2+ binding to C-terminal functional groups in Nav1.4 8. Subsequent experiments in intact loose patch clamped native murine muscle fibres correspondingly showed reduced or increased INa following respective activation or inhibition of SR Ca2+ release by direct pharmacological, caffeine and 8-CPT-mediated, RyR1 activation, or dantrolene-mediated RyR1 inhibition. Dantrolene pretreatment additionally abrogated the INa downregulatory effects of both 8-CPT and caffeine10.Possible feedback, i14. However, CPA acts by inhibiting sarcoplasmic reticular (SR) Ca2+-ATPase (SERCA)-mediated cytosolic Ca2+ re-uptake22 rather than activating RyR-mediated SR Ca2+ release. This additionally causes SR Ca2+ store depletion that itself can trigger store-operated Ca2+ entry (SOCE) potentially further increasing [Ca2+]i23. In common with the recent studies, experiments were performed on intact native skeletal myocytes. Use of the loose patch clamp technique avoided intracellular perturbations otherwise arising from conventional patch clamp access. Further introduction of novel solution change procedures permitted paired comparisons between families of INa elicited by progressively depolarizing test steps before and following applied solution changes within the same patch, without perturbing the patch seal. These matched comparisons avoided problems arising from previously reported between-patch INa variabilities reflecting non-uniform membrane ion channel distributions in muscle fibres16.The present studies further test and extend the resulting INa recordings were unchanged by solution substitutions when these omitted CPA. However, in contrast to the previous observations in experiments using RyR1 activators10, substituting control for CPA-containing test solutions increased rather than decreased INa. Thus, CPA (1 µM) statistically significantly increased mean maximum currents (Imax) by ~ 31.4%. These effects were demonstrable from 1 min, and stably persisted in patches maintained for at least 4 min, following the solution change. The currents otherwise showed similar time courses, and similar steady-state half-maximum voltages, V0.5, and steepness factors, k.The Imax, accompanied by shifts in V0.5, through similar, 1–10 min intervals following 0.5 mM caffeine challenge, despite the common effects of caffeine and CPA in increasing bulk cytosolic [Ca2+]i. Furthermore, whereas 0.5 mM caffeine had been reported to reduce INa when applied by itself10, we additionally report that CPA pre-treatment abrogated this effect. This parallels previous reports that CPA pre-incubation abrogated the arrhythmogenic effects of caffeine in murine hearts21. Both these effects of CPA thus more closely resembled that of dantrolene, despite its effects in decreasing rather than increasing [Ca2+]i. Dantrolene applied alone had caused significant, 14.9%, increases in Imax10.These findings directly contrast with previously reported 81% decreases in INa implicate correspondingly contrasting effects on the local Ca2+ levels to which Nav1.4 is exposed, despite their similar reported effects on bulk cytosolic [Ca2+]i14. Besides the surface sarcolemma, Nav1.4 occurs in the transverse (T-) tubules18 themselves accounting for ~ 80% of total muscle membrane19, thereby contributing substantial INa. T-tubular membranes come into close proximity to SR terminal cisternal membranes containing the RyR1-Ca2+ release channels, leaving electron-microscopically demonstrable narrow, ~ 15 nm, T-SR gaps permitting the Cav1.1-RyR1 interactions strategic to excitation–contraction coupling26. Although ultimately continuous with remaining cytosol, T-SR gaps form restricted intracellular diffusion spaces close to the RyR1-Ca2+ release sites potentially generating microdomains with local Ca2+ concentrations, [Ca2+]TSR, distinct from the remaining bulk [Ca2+]i, that might modulate T-tubular, even if not surface membrane Nav1.4 function , thought involved in triad junction formation and maintenance was associated with increased myotube diameters and resting [Ca2+]i, and decreased RyR1-mediated Ca2+ release and excitation–contraction coupling gain32.T-tubular as opposed to surface membrane Na15. This involved solution change protocols permitting within-patch comparisons in view of established between-patch variations in maximum Na+ current16. This restricted experiments to each applying a single protocol to any one patch and studying one patch in a single fibre only within each dissected muscle.All experiments were regulated under the Animals (Scientific Procedures) Act 1986 Amendment Regulations 2012 following ethical review and approval by the University of Cambridge Animal Welfare and Ethical Review Body (AWERB). All procedures fell within the scope of Schedule 1 of the UK Animals (Scientific Procedures) Act (1986). The loose patches were made on single fibres within intact muscle preparations studied before and following challenge with extracellularly applied CPA directed at the intracellular SERCA target2, 1.8 CaCl2, 10 glucose and 2.0 Na-pyruvate; pH adjusted to 7.4 using 1 M NaOH) throughout. The skin was reflected to expose gastrocnemius muscle. The overlying biceps femoris muscle was dissected from its superficial surface. The Achilles tendon was ligatured, bisected distally, and gastrocnemius and soleus muscles reflected. Their proximal attachments were freed by bisecting the femur, tibia and fibula, and all other muscles ~ 2 mm above and below the knee joint. The resulting preparation was immediately immersed in KH solution.C57BL6 wild type mice were killed by cervical dislocation  Act 1986) by Home Office licensed personnel and immediately dissected. All exposed tissues were bathed in Krebs–Henseleit (KH) extracellular solution  were pulled by a Flaming/Brown micropipette puller  using a 5-step program to achieve a progressive taper. The tips were visualized under a microscope with a calibrated eyepiece graticule at 250× magnification and scored using a diamond knife mounted perpendicularly to its long axis at a position giving a 30 µm internal diameter. A transverse force applied distal to this site produced a clean perpendicular fracture. The tips were fire-polished using an electrically heated nichrome wire under 400× magnification. The pipettes were bent through 45° at 1 mm from the tip, by heating with a nichrome filament and a transverse displacing force. Internal tip diameters were measured under 1000× magnification. Pipettes with 20–30 µm diameters were selected for use. A recording pipette was inserted into a pipette holder with a chloridized silver wire. This was connected to the headstage of the loose patch amplifier and held at 45° such that the pipette tip contacted the muscle surface perpendicularly Fig. b. KH solThe custom-built loose patch amplifier circuit compensated for voltage drops across the pipette resistance in series with the patch and the current flow through the relatively low seal resistance of the loose patch configuration . Its variable resistances were adjusted to match the voltage drop across the pipette and seal resistances so that the membrane patch was clamped to the command potential and the circuit output corresponded to the current flowing through the patch only Fig. c. Voltagv1.4 inactivation at the RMP. This was then followed by a variable test pulse starting at (RMP − 40) mV and altered in + 10 mV increments with each successive cycle until a test voltage of (RMP + 120) mV was reached  to relieve any residual Nahed Fig. d. The se10. Residual leak was corrected for by the P/4 protocol, whereby four voltage steps of opposite sign and a quarter in magnitude to the test pulse were delivered immediately after it. The control P/4 pulses traversed voltages that would not activate any voltage-gated conductances giving currents that represented leak currents for removal by summation with the test pulse record.Comparison of currents from these two sequences made it possible to check for stability of patch clamp current characteristics. A full set of pulses thus consisted of 34 cycles of such voltage steps, each of which were delivered at 2 s intervals. These permitted a derivation of current–voltage curves reflecting channel activation, an experimental approach that had been independently corroborated in inactivation protocols in previous studies employing the loose patch clamp technique+ current of suitable shape or magnitude, the patching procedure was re-attempted at an adjacent site. Viable patches were tested over a range of clamped voltage steps , half maximal voltage, V50 (mV) and steepness factor, k (mV) were subject to within-patch paired t-test comparisons of values before and following CPA challenge.The currents derived from the pulse protocol applied to each patch were normalized to the estimated patch area calculated using the loose patch pipette diameter to give current densities, and expressed as means ± SEM. Following Shapiro-Wilkes tests for normality, values of currents, 2+]i. Its validation involved added confirmatory test step sequences to confirm recording stability. The added cycles of voltage steps potentially limited the testable range of applied pulse protocols owing to alterations in seal resistances with time following patch establishment. The latter were noticeable at later, 4 min, times at the higher CPA concentrations. However, these did not preclude acquisitions of INa activation curves sufficient for full quantification and comparisons of current–voltage relationships before and following CPA challenge.The present explorations thus developed a novel experimental configuration and simultaneously applied this to demonstrate for the first time within-patch alterations in Nav1.4 properties following hitherto untested pharmacological manipulations of [CaThis study was carried out in compliance with the ARRIVE guidelines. This research has been regulated under the Animals (Scientific Procedures) Act 1986 Amendment Regulations 2012 following ethical review and approval by the University of Cambridge Animal Welfare and Ethical Review Body (AWERB). All procedures were completed by Home Office-licensed personnel and fell within the scope of Schedule 1 of the UK Animals (Scientific Procedures) Act (1986).


We used various inhibitors of Ca2+ channels located on the membrane, including sarcoplasmic/ endoplasmic reticulum Ca2+ATPases , transient receptor potential (TRP) ion channel subfamily member 7 , T-type Ca2+ channels and calcium channel Orai1, to investigate their roles in [Ca2+]i oscillation patterns and mitochondrial membrane potential during oocyte activation by real-time recording. Our results showed that SERCAs, TRPM7 and T-type Ca2+ channels were important for initiation and maintenance of [Ca2+]i oscillations, which was required for mitochondrial membrane potential elevation during oocyte activation, as well as oocyte cytoskeleton stability and subsequent embryo development. Increasing the knowledge of calcium transport may provide a theoretical basis for improving oocyte activation in human assisted reproduction clinics.Oocyte activation inefficiency is one of the reasons for female infertility and Ca According to our previous research, mitochondria, main organelles for energy production in the cell, are involved in producing [Ca2+]i oscillations during oocyte activation [2+ regulates mitochondria activity in cardiomyocytes [2+]i oscillations in the cytoplasm will have similar effects in oocytes. At present, the transporters involved in the regulation of [Ca2+]i oscillations and mitochondria activity have not been fully determined.The importance of Cacognized . There aractions . Ca2+ in+ influx . Hence, tivation . Since Cmyocytes , we thus2+ transporters were introduced to detect the [Ca2+]i oscillation patterns and mitochondria membrane potential dynamic changes, in order to evaluate the role of Ca2+ transporters in oocyte activation. Studying the function and regulatory mechanisms of these transporters will have significant importance for both understanding oocyte activation mechanisms and for improving clinical assisted reproductive technologies (ART).In this study, specific inhibitors for different CaFemale ICR mice at 8 weeks old were purchased from the Beijing Vital River Laboratory Animal Technology Co., Ltd. All mice were handled in accordance with the institutional animal care policies of the Institute of Zoology, Chinese Academy of Sciences. Mice were maintained under a 12-h light and 12-h darkness cycle in a specific pathogen-free stage at the Central Animal Laboratory of the Institute of Zoology, Chinese Academy of Sciences. The Laboratory Animal Care and Use Committee of the Institute of Zoology approved this study .The cytoplasmic spindle formation and F-actin meshwork were investigated by live imaging of F-actin with mRNA encoding b5-tubulin–GFP , 12 and 2+]i oscillations were induced by 10 mM strontium chloride  in Ca2+-free CZB culture medium containing various Ca2+ channel modulators for as long as 4 h. After partheno-activation, embryos were moved into KSOM  for further culture, and pronuclear formation and cleavage were observed.Female mice were injected with 10 IU pregnant mare serum gonadotropin . After 44 to 48 h, the GV oocytes were collected by ovarian mincing. Female mice were injected with 10 IU PMSG followed 44 to 48 h later by injection of 10 IU human chorionic gonadotropin . After 15 h of hCG, ovulated MII oocytes were collected and denuded in 1 mg/ml hyaluronidase. i oscillations were detected using 2 μg/mL Fluo-4 AM  in the partheno-activation solution. Real-time images of [Ca2+]i changes after partheno-activation of oocytes which were co-incubated with various Ca2+ channel modulators were obtained using a time-lapse confocal laser microscope  and recorded at 2 frames per minute. [Ca2+]i intensity was detected using an argon laser. Volocity Software was used to analyze fluorescence intensity.Oocyte [Ca2+]i oscillations regulatory function in mitochondrial activity during oocyte activation. Oocytes were denuded and pre-incubated in M2 culture medium  with 2 μg/mL JC-1 , a cell permeable voltage-sensitive fluorescent mitochondrial dye, for 15 min. In order to reduce the toxicity of JC-1 for long-term observation using laser confocal microscope, we reduced the concentration of JC-1 to 0.5 μg/ml in the activating solution. Briefly, the lower potential mitochondria were imaged as JC-1 monomer emitting in green , and energized high potential mitochondria were imaged in aggregated JC-1 that emits in red  as fluorescence transporters. Oocyte mitochondrial potential dynamic changes after treatment with various Ca2+ channel modulators were recorded after partheno-activation using a time-lapse confocal laser microscope  at identical magnification and gain settings throughout the experiments. A software Volocity was used to analyze fluorescence intensity.In our study, we set out to confirm the i oscillations on mitochondrial dynamic potential.We firstly examined the dose-dependent effects of inhibitors of SERCAs, TRPM7, T-type Ca2+ concentration ([Ca2+]i) oscillations and mitochondrial dynamic potential changes during the activation of oocytes were observed by time-lapse confocal laser microscope . Normal [Ca2+]i oscillations were induced by SrCl2 stimulation   is a major Caha) Fig. , by whic2+]i oscillations . Spindle . Spindle  increases and decreases known as [Ca2+]i oscillations, which encode oocyte activation events, including release from the MII arrest, pronuclear formation and the transition to embryo development [2+ from internal stores and Ca2+ influx from the extracellular matrix induce moderate increases in [Ca2+]i levels. The increase in [Ca2+]i generally lasts about 2 min. As Ca2+ refills back into the ER or causes efflux from the cytoplasm to prepare for the next peak of oscillation, the elevated [Ca2+]i will return to baseline levels, resulting in an average of a ten to twenty minutes resting interval. [Ca2+]i oscillations will last 5–6 h until pronuclear formation takes place i increase pathway appears to be achieved through store-operated Ca2+ entry (SOCE) processes. Ca2+ enters the cytosol from the endoplasmic reticulum (ER), which in turn opens one of the ER channels, sarcoplasmic reticulum/ER Ca-ATPase (SERCAs), to transport Ca2+ back to the ER i oscillations [2+]i and all oocytes died before the end of the activation process (Fig. 2+]i oscillations were suppressed by 1 μM Tha, more than half of the oocytes survived more than 4 h. Under such moderate concentration, the effects of SERCAs on mitochondrial activity can be observed for a long term. Oocyte mitochondrial membrane potential continued to decrease under Ca2+ refilling inhibition with 1 μM Tha incubation. Not only [Ca2+]i oscillations but also mitochondrial activity were suppressed by Tha that induced blocking of ER Ca2+ refilling. Finally, oocytes cannot be activated under SERCAs inhibition.During cell activation, Caganelles ; the cel 1000 μM . The patciencies . In oocyo the ER . Total c from ER . When ERllations . In our 2+ oscillations [2+]i slowly rose, which finally induced oocyte death. Treatment with 1 μM NS-8593 kept the survival rate at 37.5% (Fig. 2+]i oscillations and pronuclear formation were sufficiently suppressed. Under this moderate concentration of 1 μM NS-8593, the effects of TRPM7 on mitochondrial activity can be observed for a long time. Mitochondrial activity did not exhibit the same activation state as the Ctrl in the case of TRPM7 inhibition with NS-8593. Mitochondrial membrane potential continuously decreased when inhibiting the transport of Ca2+ through TRPM7 with 1 μM NS-8593 (Fig. 2+]i oscillation patterns in oocytes, and [Ca2+]i oscillations achieved through TRPM7 is important for mitochondrial activity and oocyte activation.Recently, a member of the TRP channels family, TRPM7, was found to be expressed in mouse GV, MII oocytes and 2-cell embryos . TRPM7 bllations . We appl2+ influx through T-type channels i oscillation pattern and membrane potential changes were caused by complete inhibition of Orai1 or by cytotoxicity induced by the high concentration of 1 mM. The role of Orai1 in the activation of oocytes requires additional evidence to confirm.Orais represents transmembrane proteins that form highly Cachannels . Orais hchannels . Loss-ofchannels  and dysfchannels . SOCE is T-cells . We apploup Fig. . Most of2+]i oscillations are different [2+]i oscillations. The effects of several transporters on the maintenance of [Ca2+]i oscillations were investigated in our study (Fig. 2+]i oscillations. After addition of Mibefradil and NS-8593, the intracellular Ca2+ increased continuously, and the cell death rate was higher than in the Ctrl and other groups. All these results suggest that three Ca2+ transporters, SERCAs, TRPM7 and T-type Ca2+ channels are involved in both the initiation and maintenance of [Ca2+]i oscillations. Interestingly, the addition of Ruthenium Red and other inhibitors into culture of oocytes which had initiated [Ca2+]i oscillations did not significantly influence oocyte activation (Fig. 2+]i oscillations.The mechanisms of initiation and maintenance of i oscillations and mitochondrial activity during oocyte activation, and showed that inhibition of SERCAs, TRPM7 and T-type Ca2+ channels caused disturbances in Ca2+ signaling, mitochondrial activity and subsequent oocyte activation and embryonic development. Once Ca2+ transport is abnormal in oocytes, whether it is at the beginning or halfway, will cause disorders of mitochondrial metabolism, activation of subsequent developmental consequences, finally leading to infertility.In summary, we applied ER-associated Ca


Macadamia has increasing commercial importance in the food, cosmetics, and pharmaceutical industries. However, the toxic compound hydrogen cyanide (HCN) released from the hydrolysis of cyanogenic compounds in Macadamia causes a safety risk. In this study, optimum conditions for the maximum release of HCN from Macadamia were evaluated. Direct headspace analysis of HCN above Macadamia plant parts  was carried out using selected ion flow tube–mass spectrometry (SIFT-MS). The cyanogenic glycoside dhurrin and total cyanide in the extracts were analyzed using HPLC-MS and UV–vis spectrophotometer, respectively. HCN released in the headspace was at a maximum when Macadamia samples were treated with pH 7 buffer solution and heated at 50 °C for 60 min. Correspondingly, treatment of Macadamia samples under these conditions resulted in 93–100% removal of dhurrin and 81–91% removal of total cyanide in the sample extracts. Hydrolysis of cyanogenic glucosides followed a first-order reaction with respect to HCN production where cyanogenesis is principally induced by pH changes initiating enzymatic hydrolysis rather than thermally induced reactions. The effective processing of different Macadamia plant parts is important and beneficial for the safe production and utilization of Macadamia-based products. Macadamia-based commercial products have rapidly increased in recent years. In addition to Macadamia nuts, Macadamia flowers, husks, leaves, and shells are now widely used as a source of functional foods, beverages, and raw materials in cosmetics, feed, and other applications. Abundant antioxidant substances, such as polyphenols, can be extracted from Macadamia skin and husks for utilization in the food and pharmaceutical industries , mol L−1 versus heating time (min) plots for pH 2, 3, 4, 5, 6, and 7. At pH 8 and 9, the 90 min data point had to be excluded. The calculated empirical rates of hydrogen cyanide production (d[HCN]/dt) at different pH , followed by the leaves (513 ± 0.6 vppb), husks ; 256 ± 0.4 vppb, (Oc); 476 ± 0.7 vppb, (A16), and nuts (5.8 ± 0.1 vppb) at p < 0.05. Interestingly, the hydrogen cyanide concentration in Macadamia flowers was approximately 2–3 times higher than the highest concentration of cyanide detected in M. ternifolia cotyledon (~4800–6500 vppb) [Macadamia.00 ppbv) . Previou00 ppbv) . The flo00 ppbv) ,46,47. FMacadamia leaves (513 ± 0.6 vppb) is within the concentration range of cyanide (364–1403 vppb) detected in the leaf tissue of M. ternifolia, M. integrifolia, and M. tetraphylla species during their early to mid-developmental stages (3rd–4th week) [The concentration of hydrogen cyanide in  513 ± 0. ppbv is  513 ± 0. ppbv is th week) ,49,50.Macadamia husks are the fleshy green fibrous pericarp covering the conical or spherical hard brown shell enclosure of Macadamia nuts [Macadamia flowers, there are no available published data reported for the hydrogen cyanide concentration in Macadamia husks for comparison. Moreover, the hydrogen cyanide concentration of husks analyzed from three different Macadamia varieties were significantly different: 21 ± 0.1 vppb for variety 695; 256 ± 0.4 vppb for variety Oc; and 476 ± 0.7 vppb for variety A16  was lower than the reported cyanide concentrations in commercially used seeds (~74 vppb) of M. integrifolia or M. tetraphylla and significantly lower than the concentration detected in M. ternifolia (~4800 vppb), which is considered to be inedible.Seeds of  variety ,43. In tMacadamia plant part extracts with different treatments. Fresh, untreated Macadamia flower (424.4 ± 0.6 mg/L) had the highest amount of dhurrin compared to the fresh, untreated leaves (97.1 ± 0.6 mg/L) (.6 mg/L) A, fresh .6 mg/L) C.Macadamia flower (417.7± 0.8 mg/L), leaves (167 ± 2 mg/L), nuts (67.1 ± 0.6 mg/L), and husks . After full sample treatment using the optimized conditions , significant amounts of dhurrin and cyanide were removed in the analyzed extracts as shown in The cyanide concentration in the extracts have the same trend as the dhurrin concentration. Macadamia flower and leaf samples. On the other hand, treating the Macadamia flower and leaf samples with buffered solution at pH 7 without heating (buffered-only), resulted in significant removal of dhurrin , treatment produced about similar removal effectivity (It is interesting to note that heating samples at 50 °C for 60 min without pH adjustment (heated-only samples) had little to no effect on the removal of dhurrin A or cyan dhurrin  showed t cyanide . Howeverectivity  of dhurrMacadamia samples were 50 °C, 60 min at pH 7. Under these treatment conditions, trace amounts of hydrogen cyanide could still be detected in the headspace directly above the different Macadamia plant part samples using SIFT-MS. The measured hydrogen cyanide in the headspace of the treated samples were 12,535 ± 11 vppb (flower), 513 ± 0.6 vppb (leaves), 6 ± 0.1 vppb (nuts), 476 ± 0.7 vppb (husk A16), 256 ± 0.4 vppb (husk Oc), and 21 ± 0.1 vppb (husk 695). Treatment of Macadamia samples under these optimum conditions produced 93–100% removal of dhurrin and 81–91% removal of total cyanide in the sample extracts. Treatment by pH 7 adjustment (buffered-only) without heating also resulted in an effective removal of dhurrin (86–100%) and total cyanide (88–89%) in Macadamia extract similar to the full, optimized treatment conditions. Heating the samples alone at 50 °C for 60 min without pH adjustment was not effective in the hydrolysis and removal of cyanogenic glycoside dhurrin and total cyanide in Macadamia samples. The varying concentration of generated hydrogen cyanide could be correspondingly attributed to the concentration of cyanogenic glycosides (such as dhurrin) from the different parts of the Macadamia plant and their subsequent hydrolysis to hydrogen cyanide. Cyanogenic glycosides were greatest in Macadamia flowers, followed by the leaves and husks (depending on variety), and lowest in nuts. The results indicate that the hydrolysis of cyanogenic glycosides in Macadamia is predominantly induced by pH changes rather than by heat. This further suggests that the enzymatic hydrolysis involved in cyanogenesis is chiefly pH-directed rather than thermally induced. In addition, the hydrolysis reaction of cyanogenic glycosides could be described as a first-order reaction with respect to the in vitro production of hydrogen cyanide.The optimum conditions for the maximum release of hydrogen cyanide in Macadamia. Moreover, the evaluated optimum conditions for the hydrolysis of dhurrin and removal and release of hydrogen cyanide could be helpful for the effective processing of different parts of Macadamia. Such information provides some guidelines toward the safe production, utilization, and consumption of Macadamia-based products.These results provide further insights into the cyanogenic systems in


FLT3) gene is associated with high risk of relapse and poor clinical outcome upon treatment with conventional chemotherapy. FLT3 inhibitors have been approved for the treatment of this AML subtype but leukaemia relapse remains to be a major cause of treatment failure. Mechanisms of drug resistance have been proposed, including evolution of resistant leukaemic clones; adaptive cellular mechanisms and a protective leukaemic microenvironment. These models have provided important leads that may inform design of clinical trials. Clinically, FLT3 inhibitors in combination with conventional chemotherapy as induction treatment for fit patients; with low-intensity treatment as salvage treatment or induction for unfit patients as well as maintenance treatment with FLT3 inhibitors post HSCT hold promise to improve survival in this AML subtype.Acute myeloid leukaemia (AML) carrying internal tandem duplication (ITD) of Fms-Like Tyrosine kinase 3 ( Acute myeloid leukaemia (AML) is defined pathologically by an abnormal increase in blasts in blood and/or bone marrow (BM). It is a group of heterogeneous diseases with distinct driver events and pathogeneses that may occur at different stages of the haematopoietic hierarchy . SubtypeFLT3) gene: Midostaurin in combination with conventional induction and consolidation chemotherapy in newly diagnosed patients [FLT3-mutated patients [Laboratory studies in leukaemia biology in the past few decades have led to identification of molecular targets and development of novel therapeutic strategies. In the past 3 years, eight therapeutic agents have been approved by the U.S. Food and Drug Administration (FDA) for the treatment of different AML subtypes in different clinical contexts . In partpatients  and giltpatients . HoweverFLT3), first identified to be expressed in normal haematopoietic stem and progenitor cells, is one of the most frequently mutated genes in AML. Internal tandem duplication (ITD) is the commonest genetic abnormality and is associated with leucocytosis at diagnosis and high risk of relapse after conventional chemotherapy, particularly those with high ITD allelic ratio [FLT3-ITD occurs particularly in AML with normal cytogenetics, where it occurs in up to 40% cases, and those with rare t translocation involving DEK/CAN gene fusion, where it occurs in up to 70%–80% [FLT3 mutant AML cases [Fms-like tyrosine kinase 3  and the binding results in dimerization and conformational changes of FLT3 that expose the phosphorylation sites of its TKD. Subsequent auto-phosphorylation of FLT3 leads to binding of adaptor proteins such as SHP2, Grb2 and SRC family kinases, hence activation of downstream signalling kinases including MAPK/ERK, JAK/STAT and PI3K/AKT/mTOR . While bWT cells .FLT3-ITD and TKD in AML and the inferior outcome of this AML subtype provide the basis for developing FLT3 inhibitors. Mechanistically FLT3 inhibitors can be categorised into 2 types. Type I inhibitors bind FLT3 in the active conformation near the activation loop or ATP binding site and are effective against both ITD and mutant TKD as exemplified by midostaurin, sunitinib, lestaurtinib, crenolanib and gilteritinib. Type II inhibitors bind FLT3 in the inactive state near the ATP binding domain, targeting FLT3-ITD but not mutant TKD. Examples include sorafenib, ponatinib and quizartinib. In order of their development, FLT3 inhibitors can be categorised into those of first and second generation. First generation FLT3 inhibitors refer to several multi-kinase inhibitors including lestaurtinib, sunitinib, sorafenib and midostaurin that have been evaluated since early 2000. As monotherapy, sorafenib has been widely used as salvage therapy for R/R FLT3-ITD AML with a rate of combined complete remission (CR) and CR with incomplete haematologic recovery (CRi) at about 16%–46%. Responses were typically transient with median duration of 1–3 months, and sorafenib was perceived at best as a bridging therapy to curative allogeneic HSCT. More recent data showed that sorafenib may be effective as maintenance therapy post HSCT, resulting in improved survival of these patients [FLT3-mutated AML, midostaurin was shown to prolong overall survival [The pathogenetic roles of patients ,15,16. Mpatients . When usFLT3-mutated AML with CR/CRi of 23%–57% and median duration of response of 9–20 weeks (FLT3-mutated AML. In a randomised open-label phase 3 study (QUANTUM-R) [FLT3-mutated AML. Several phase 3 studies were underway including those that evaluate gilteritinib following induction and consolidation chemotherapy  as well as allogeneic HSCT (NCT02997202) in newly diagnosed AML; crenolanib in combination with salvage chemotherapy in R/R FLT3-mutated AML , and crenolanib compared with midostaurin in newly diagnosed FLT3-mutated AML when used in conjunction with conventional chemotherapy (NCT03258931); quizartinib in combination with induction chemotherapy in newly diagnosed FLT3-mutated AML .Second generation FLT3 inhibitors refer to new inhibitors with higher specificity and potency against FLT3, including quizartinib, gilteritinib and crenolanib. They have been tested in clinical trials as monotherapy in R/R 20 weeks . Based oANTUM-R) , quizartFLT3-mutated AML by FLT3 inhibitor monotherapy is leukaemia relapse that often occurs within months after initial remission. In most circumstances, this is related to development of drug resistance. The mechanisms are heterogeneous and may involve emergence of clones that are resistant to FLT3 inhibitors being used; protection of leukaemia cells by BM microenvironment; and adaptation of leukaemia cells to survive FLT3 inhibitors. These mechanisms are reviewed in the following sections.A major limitation in the treatment of FLT3-TKD mutations as a cause of drug resistance to FLT3 inhibitors arose from laboratory studies. In particular, FLT3-TKD mutant clones could be selected from in vitro saturation mutagenesis assay [FLT3-ITD AML cell lines treated with increasing dose of FLT3 inhibitors for 6–7 weeks [FLT3-TKD mutations at relapse. On one hand it has confirmed the on-target effect of FLT3 inhibitors and their anti-leukaemia efficacy, on the other hand it contributes to drug-resistant leukaemia relapse. Mutations may occur at the activation loop  or gate-keeper site , and the frequencies and mutation sites depend on the specific FLT3 inhibitors being used  from R/R FLT3-ITD AML patients who relapsed from FLT3 inhibitors crenolanib [FLT3-ITD clone or new wildtype FLT3 clones. These clones carried mutations of TP53, RAS pathway , IDH1/2, ASXL1 or TET2. The emergence of these mutations, particularly when they occurred in wildtype FLT3 clones, demonstrated FLT3-independent leukaemia cells that were selected under the pressure of FLT3 inhibitors to which they were resistant. Complete loss of FLT3-ITD clone has been reported in up to 30% of cases [Non-enolanib  or gilteenolanib  demonstrof cases ,34.FLT3-ITD AML cells may adapt to FLT3 inhibitors and develop cellular mechanisms that circumvent FLT3 blockade. They include upregulation of FLT3 ligand, change in intracellular acidity and upregulation of other protein kinases. These are discussed as follows:In addition to the emergence of resistant clones, Despite the ligand independence of FLT3-ITD signalling, FLT3L has been shown to confer resistance to FLT3 inhibitors in AML . FLT3L eFLT3-ITD AML samples before sorafenib treatment and at subsequent relapse showed up-regulation of a gene encoding tescalcin (TESC) [Microarray analysis of paired n (TESC) . TESC plFLT3-ITD AML upon development of resistance to FLT3 inhibitors and may play a pathogenetic role. For instance, expression of PIM  kinase, a target of FLT3-ITD signalling, has been shown to increase in primary AML cells at resistance to sorafenib [PIM-2 in MOLM-14 (a FLT3-ITD AML cell line) and FLT3-ITD knock-in mouse model has been shown to confer resistance to quizartinib [FLT3-ITD AML cell lines and primary AML blasts despite inhibition of FLT3 phosphorylation by FLT3 inhibitors, suggesting that at resistance, some AML cells become independent of FLT3 signalling [Upregulation of other oncogenic kinases has been shown in primary orafenib . Overexpzartinib . Also, ezartinib  and its zartinib . Pharmaczartinib . Importazartinib . Furthergnalling ,48. The BM niche has been shown to nurture normal haematopoietic stem cell and progenitor cells and maintain steady state haematopoiesis. Mechanistic studies in mouse marrow demonstrated close interaction between osteoblasts and haematopoietic stem and progenitor cells (HSPC), suggesting a physical niche that provides signals to guide HSPC cell-fate decision . SimilarFLT3-ITD AML cells and MV4-11 (a FLT3-ITD AML cell line) from crenolanib through activation of STAT5 pathway [FLT3-ITD AML cells and promotes resistance to FLT3 inhibitors via activation of downstream MAPK pathway [FLT3-ITD AML patients [FLT3-ITD AML has been shown to highly express CXCR4 [FLT3-ITD transduced Ba/F3 cells (a murine pro-B lymphoid cell line) to the inhibitory effects of sorafenib when co-cultured with protective MS5 stromal cell line [BM stromal cells have been shown to secrete a repertoire of cytokines that protect the blasts from the cytotoxic effect of chemotherapy as well as FLT3 inhibitors. In addition to the FLT3L aforementioned, GM-CSF and IL-3 have been shown to protect primary  pathway . BM stro pathway ,54. FGF2patients ,54 and wss CXCR4 , suggestell line . Activatell line , providiCytochrome P450 3A4 (CYP3A4) is important for the metabolism and elimination of drugs and xenobiotics in human body. It is expressed primarily in hepatocytes and BM stromal cells and for the latter it might inactivate tyrosine kinase inhibitors  in the BFLT3-ITD patients.The mechanisms aforementioned provide the theoretical bases on which clinical trials can be designed to overcome drug resistance to FLT3 inhibitors. However, the precise mechanisms of drug resistance are likely to vary between patients and multiple mechanisms may co-exist, adding to the complexity of trial design. In clinical practice, a number of strategies have been developed to improve the treatment outcome of FLT3-ITD AML. In a Phase III randomised placebo-controlled study, addition of midostaurin to induction and consolidation chemotherapy, followed by maintenance treatment in FLT3-mutated AML was shown to improve overall survival [FLT3-mutated AML. Benefits of sorafenib when added to conventional chemotherapy have also been demonstrated [Until recently, induction chemotherapy, the “7+3” regimen, has been the standard of care for fit patients of all AML subtypes including survival . The resnstrated ,61. Clinnstrated  and giltFLT3-ITD AML cells have been shown to be sensitive to this preparation in vitro [FLT3-ITD remain to be investigated.CPX-351, a liposomal preparation of cytarabine and daunorubicin at a fixed synergistic drug ratio of 5:1, was recently approved as induction therapy in AML with MDS-related changes and therapy-related AML. Patients with this AML subtype receiving CPX-351 showed a superior outcome than those receiving conventional “7+3” . FLT3-ITin vitro . The cliFLT3-ITD AML cells have been shown to exhibit high protein synthesis rate to maintain intracellular level of short-lived oncogenic proteins [ex vivo drug screening using primary AML cells has identified omacetaxine mepesuccinate (OME) as an effective adjunct to FLT3 inhibitors in the treatment of FLT3-ITD AML [FLT3-ITD AML, with a deeper molecular response and extended response duration  among responders [proteins . The add-ITD AML . OME com-ITD AML . It suppsponders . A clinisponders .Furthermore, combination of FLT3 inhibitors including sorafenib, quizartinib and gilteritinib with other low intensity treatment including azacytidine or decitabine, the hypomethylating agents, have been shown to be synergistic in laboratories ,69,70 anFLT3-ITD AML in complete remission after induction chemotherapy or salvage treatments including FLT3 inhibitors [FLT3-ITD cells that may enhance activities of allogeneic T-cells and graft-versus leukaemia effect [FLT3-ITD AML in a phase I study also showed reduced relapse rate [FLT3-ITD AML cells or potentiation on graft-versus-leukaemia effects would have to be further evaluated [HSCT is the mainstay of treatment for hibitors ,75,76,77hibitors . Laborata effect . In RADIa effect . Post-HSpse rate . Whethervaluated .FLT3-ITD AML, leukaemia relapse remains to be a major cause of treatment failure. Incorporation of FLT3 inhibitor to upfront induction and consolidation chemotherapy; combination of FLT3 inhibitor with low intensity regimen as salvage treatment and the use of FLT3 inhibitor as post-HSCT maintenance may improve treatment outcome of this AML subtype. Based on findings of laboratory studies, multiple mechanisms of drug resistance have been proposed and it is likely heterogeneous among individual patients. They have provided the important bases for development of clinical trials and might raise the potential need for treatment personalisation.Despite the availability of effective FLT3 inhibitors in the treatment of


HER2 expression did not predict sensitivity to trastuzumab, with Eso26 xenografts remaining refractory to trastuzumab treatment. Taken together, our study provides strong evidence for an oncogenic role for GRB7 in OAC and suggests that targeting GRB7 may be a potential therapeutic strategy for this cancer. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.Efficacious therapeutic approaches are urgently needed to improve outcomes in patients with oesophageal adenocarcinoma (OAC). However, oncogenic drivers amenable to targeted therapy are limited and their functional characterisation is essential. Among few targeted therapies available, anti‐human epidermal growth factor receptor 2 (HER2) therapy showed only modest benefit for patients with OAC. Herein, we investigated the potential oncogenic role of growth factor receptor bound protein 7 (GRB7), which is reported to be co‐amplified with HER2 (ERBB2) in OAC. GRB7 was highly expressed in 15% of OAC tumours, not all of which could be explained by co‐amplification with HER2, and was associated with a trend for poorer overall survival. Knockdown of GRB7 decreased proliferation and clonogenic survival, and induced apoptosis. Reverse phase protein array (RPPA) analyses revealed a role for PI3K, mammalian target of rapamycin (mTOR), MAPK, and receptor tyrosine kinase signalling in the oncogenic action of GRB7. Furthermore, the GRB7 and HER2 high‐expressing OAC cell line Eso26 showed reduced cell proliferation upon GRB7 knockdown but was insensitive to HER2 inhibition by trastuzumab. Consistent with this, GRB7 knockdown  Cancer of the oesophagus remains the sixth major cause of cancer‐related deaths worldwide . There aIn an era of effective molecular‐targeted therapy in treating solid cancers such as breast , 7 and lHER2 proto‐oncogene is positioned within the 17q12 amplicon and its amplification and overexpression have been frequently associated with gastrointestinal carcinogenesis [HER2, including GRB7 [The ogenesis , 14. Howing GRB7 , 16.GRB7 is an SH2‐domain‐containing adaptor molecule that mediates cellular signalling through interaction with multiple receptor tyrosine kinases and their downstream partners . In thisin vitro cell line models of OAC. Importantly, we demonstrated the therapeutic value of inhibiting GRB7 in OAC xenografts. In summary, our body of work highlights the potential oncogenic role and therapeutic significance of GRB7 in OAC.Herein, for the first time, we report the frequency of GRB7 protein expression levels within an OAC patient cohort and correlation with survival outcome. Furthermore, we assayed the functional effects of manipulating GRB7 expression levels in Use of patient samples in this study was approved by the Human Research Ethics Committee of the Nepean Blue Mountains Local Health District. All patients provided written informed consent prior to recruitment. Tumour microarrays (TMAs) of tumours from an OAC patient cohort were assessed for GRB7 positivity. Information about this patient cohort, including detailed clinico‐pathological data and HER2 status, has been published previously . SufficiTMA sections were obtained from formalin‐fixed, paraffin embedded (FFPE) tissue blocks and stained with H&E for histological features or with anti‐GRB7 antibody  analyses using the PowerPlex® 16 System  and confirmed mycoplasma‐free by PCR analyses .m ON‐TARGETplus non‐targeting pool or siNTC  or GRB7 siRNA  using Lipofectamine RNAiMax solution  according to the manufacturer's instructions. The GRB7 siRNA SMART pool targets exons 4, 5, 11, and 14 of the GRB7 transcript. Target sequences are provided in supplementary material, Table Cells were transfected with 40 nRenilla control (sh Control) were cloned into the doxycycline‐inducible LT3GECIR lentiviral expression vector containing the optimised miR‐E backbone [GRB7‐specific shRNAs  , and viaCells and tissues were lysed and prepared as specified in supplementary material, Supplementary materials and methods. The antibodies used are listed in supplementary material, Table GRB7 was expressed ectopically in FLO‐1, OACP4C, and OE33 cells using the Precision LentiORF pLOC lentiviral vector containing turbo‐GFP as a reporter gene . The ectopic expression of turbo‐RFP in the same vector was used as a control. GFP‐positive FLO‐1, OACP4C, and OE33 cells were sorted by FACS.National Health and Medical Research Council Australian Code of Practice for the Care and Use of Animals for Scientific Purposes and approved by the Peter MacCallum Cancer Centre (PMCC) Animal Experimentation Ethics Committee.All animal experiments were conducted in accordance with the 6 cells were resuspended in 100 μl of 1:1 ice‐cold phosphate‐buffered saline (PBS) and growth factor reduced Matrigel® Matrix  and kept on ice. Cells were injected subcutaneously into the right flanks of 6‐ to 8‐week‐old female NSG mice. When tumours reached 150 mm3 volume , mice were randomised into groups of 5–8 for treatments. Concomitantly with vehicle, doxycycline, and/or trastuzumab treatments, subcutaneous tumour volume was measured with callipers twice per week up to the end of the experiment. All mice were euthanised when subcutaneous tumours reached ≥ 1500 mm3 or at the first signs of ill health or discomfort, apart from six mice that were euthanised earlier at 15, 25 or 30 days to determine GRB7 knockdown efficiency in vivo.NOD‐SCID IL‐2RγKO (NSG) mice were obtained from the Garvan Institute of Medical Research  or bred in‐house. To generate OE19 and Eso26 cell line xenografts (CLXs), 5 × 10n = 3 per treatment). Details of the RPPA protocol are given in supplementary material, Supplementary materials and methods.Protein lysates were prepared from treated tissue culture cells in CLB1 buffer , and protein was quantified using a Pierce™ Coomassie Plus Protein Assay Kit (Thermo Fisher Scientific) . Contingency table analysis with a chi‐squared test was used to compare GRB7 3+, 2+, 1+, and 0 IHC scoring with HER2 positivity (HER2+ tumours). Kaplan–Meier curves were generated for overall survival, defined as the time from diagnosis to death from any cause. Statistics for RPPA protein RFI values between treatment and control samples was obtained with Welch's t‐test. For analyses of the CLX tumour volume over the time course of the treatment, we compared groups of growth curves between different treatments in OE19 and Eso26 xenografts by using a CGGC permutation test. This test performs permutation tests of the differences between groups of the growth curves for the full‐time course of the treatment [p < 0.05 was considered statistically significant.Data were analysed using unpaired reatment , 31. ForGRB7 gene is located within the 17q12 amplicon and is reported to be co‐amplified and overexpressed with HER2 in OAC [p = 0.03) compared with equivocal/low/negative GRB7‐expressing tumours , nearly 70% of high GRB7‐expressing tumours were HER2‐negative. This would suggest that high GRB7 expression was not just the result of co‐amplification with HER2 and that other mechanisms for increasing GRB7 expression exist. Importantly, there was a trend for worse outcomes in patients with high tumour GRB7 expression . s Figure , consistm), only OE19 was found to be sensitive to trastuzumab alone, with maximum efficacy reached at the concentration 0.1 μm (results not shown). Importantly, in OE19 cells, GRB7 knockdown or trastuzumab only treatment significantly decreased cellular proliferation , within Eso26 and OE19 cells upon GRB7 knockdown, demonstrating an essential role of GRB7 signalling in promoting cell survival. Moreover, pro‐apoptotic Bax was increased in Eso26, whereas OE19 cells displayed decreased Rb_P S807;S811, which could contribute to cell cycle arrest and subsequent cell death upon GRB7 knockdown Figure C. Interein vivo.We next examined the consequences of GRB7 overexpression in OAC cell lines with lower endogenous expression of GRB7  and in which GRB7 knockdown had no effect on cell growth. We hypothesised that GRB7 overexpression in these cell lines would activate oncogenic signalling and promote tumour phenotypes. Cells were transduced with lentiviral vectors containing GRB7 or control RFP cDNA. GRB7 overexpression Figure  increasein vivo, we cloned four GRB7‐specific shRNAs into a doxycycline‐inducible lentiviral expression vector  and GRB7 gene copy number (CN) in EAC cell linesClick here for additional data file.


In this review, we highlight the current knowledge regarding TRPC implication in different cellular processes related to ischemia and reperfusion and to heart infarction.Transient receptor potential canonical (TRPC) channels are ubiquitously expressed in excitable and non-excitable cardiac cells where they sense and respond to a wide variety of physical and chemical stimuli. As other TRP channels, TRPC channels may form homo or heterotetrameric ion channels, and they can associate with other membrane receptors and ion channels to regulate intracellular calcium concentration. Dysfunctions of TRPC channels are involved in many types of cardiovascular diseases. Significant increase in the expression of different TRPC isoforms was observed in different animal models of heart infarcts and in vitro experimental models of ischemia and reperfusion. TRPC channel-mediated increase of the intracellular Ca The initial cell-membrane depolarization stimulates sarcolemma L-type Ca2+ channels, prompting a small influx of Ca2+ from the extracellular medium. Ca2+ entry triggers a large release of Ca2+ from the sarcoplasmic reticulum via ryanodine receptors (RyR), resulting in an increase in the intracellular Ca2+ concentration ([Ca2+]i). The rise in [Ca2+]i boosts Ca2+ binding to troponin C, which activates the contractile machinery. After contraction, [Ca2+]i must decrease to allow cell relaxation, which is achieved mainly via two mechanisms: Ca2+ re-uptake by the sarco-endoplasmic reticulum Ca2+ ATPase (SERCA) pump and Ca2+ efflux by the sarcoplasmic Na+/Ca2+ exchanger (NCX) i levels, and cell apoptotic rate in a time-dependent manner in H9C2 cell line. In addition, they suggested TRPC6 as a possible target for cardioprotection in H9C2 cells since the administration of danshensu, an active component of Salvia miltiorrhiza, protects against I/R injury by reducing TRPC6 expression via the c-Jun N-terminal kinases (JNK) signaling pathway i in ECs involving Ca2+ release from intracellular stores and extracellular Ca2+ flux through cation channels, such as TRP channels [2+]i increase in ECs and angiogenesis. Indeed, VEGF- and OAG-induced extracellular signal-regulated kinases (ERK) 1/2 activation and tubulogenesis are significantly suppressed by TRPC3 inhibitor and siRNA in human umbilical vein ECs (HUVEC) [2+]i increase, migration, sprouting, and proliferation, well-known hallmarks of angiogenesis [Angiogenesis relies on new blood vessels forming from pre-existing vessels and the subsequent expansion of the vascular network in the body. Post-ischemic angiogenesis is considered a protective mechanism motivated by the lack of oxygen and blood supply necessary for physiological heart repair after a MI ,85. Angichannels ,94. Therchannels ,96,97. Echannels . It is a (HUVEC) . Meanwhiogenesis . In addiogenesis .Unfortunately, little is known regarding the role of TRPC channels during post-ischemic angiogenesis. In contrast, TRPC channels appear involved in hypoxia-induced angiogenesis ,102. For2+ influx, which activates Ca2+ intracellular signaling pathways, such as calcineurin/NFAT, TRPC channels are suggested as Ca2+ effectors and transducers of hypertrophic genes in the heart. Little is known regarding TRPC channel implication in I/R-induced cardiac hypertrophy. In contrast, there is general agreement regarding the role of TRPC channels in pathological cardiac hypertrophy as a consequence of aortic constriction or under chronic GPCR stimulation using endothelin-1, phenylephrine, or angiotensin-II [2+-dependent factors of transcription and cardiac hypertrophy genes, leading to the same outcome, i.e., HF.An early cardiac hypertrophy and fibrosis are considered compensatory events to the loss of cardiac myocytes, necessary for wound healing and scar formation after heart infarcts. However, prolonged hypertrophy could lead to the development of HF, arrhythmias, and even sudden cardiac death . Since iensin-II ,106,107.ensin-II  revealedensin-II  and the ensin-II  examinedensin-II . Interesensin-II . Altoget2+ handling protein were identified recently using genome-wide transcriptome analysis of infarcted hearts [2+ entry through a direct regulation of TRPC3 and subsequent activation of NFATc3 in human ventricular cardiac fibroblasts, which abrogates myofibroblast differentiation and fibrosis by decreasing collagen secretion. However, the direct contribution of TRPC channels in cardiac fibrosis mediated by ischemia was barely addressed. Different isoforms of TRPC proteins are upregulated in rats showing fibrosis one week post I/R, although their direct role in promoting fibrosis was not examined [2+ entry in adult ventricular myocytes [In the case of fibrosis, multiple well-known markers of fibrosis, hypertrophy, and Cad hearts . TRPC6 id hearts . A recend hearts . On the d hearts  describeexamined . Interesexamined . Moreoveexamined ,106,115.myocytes . At the myocytes . 2+ signaling in cardiac cells. Increasing evidence indicates that TRPC channels contribute to pathophysiological consequences of heart infarction, such as cardiac hypertrophy, fibrosis, and post-ischemic angiogenesis, as summarized in In light of the reviewed studies, TRPC proteins stand out as key ion channels critical for cardiac cell responses under ischemic stress. A clearly defined role for specific TRPC isoforms in cellular events related to ischemic heart diseases still remains elusive, perhaps reflecting the complexity of these channels, the limitations of pharmacological tools, and the lack of specific inhibitors and antibodies. Nevertheless, TRPC channels were extensively studied since they sense and respond to a plethora of endogenous and exogenous stimuli by Ca


Accumulation of calcium in energized mitochondria of pancreatic β-cells is emerging as a crucial process for pancreatic β-cell function. β-cell mitochondria sense and shape calcium signals, linking the metabolism of glucose and other secretagogues to the generation of signals that promote insulin secretion during nutrient stimulation. Here, we describe the role of mitochondrial calcium signaling in pancreatic β-cell function. We report the latest pharmacological and genetic findings, including the first mitochondrial calcium-targeted intervention strategies developed to modulate pancreatic β-cell function and their potential relevance in the context of diabetes. Pancreatic β-cells constitute a small endocrine tissue organized, together with other endocrine cells, in the islets of Langerhans, scattered throughout the exocrine tissue of the pancreas . β-cells2+ represents an innovative approach to modulate β-cell function and to potentially promote beneficial effects for diabetic patients. Thus, dysregulation of Ca2+ signaling has been reported to have profound effects on β-cell performance and to increase the risk of developing diabetes [2+ homeostasis has been proposed to prevent cytokine-mediated β-cell loss in diabetes [2+ homeostasis and β-cell function are substantially linked to mitochondrial function [2+ is an activating signal for insulin secretion, and its requirements for signal-dependent hormone secretion have been highlighted [2+ uniporter (MCU), the transporter that mediates mitochondrial calcium uptake, has been revealed [2+ in modulating pancreatic β-cell signal transduction, opening new perspectives for intervention [In this context, targeting mitochondrial Cadiabetes ,7. Furthdiabetes . In the function . Therefofunction . Diabetefunction . Mitochofunction ,12. Mitohlighted . Recentlrevealed . Geneticrvention ,16,17.2+ in metabolism–secretion coupling in the pancreatic β-cell are available [2+ in pancreatic β-cell signal transduction. We report the latest pharmacological and genetic evidence, including the first intervention strategy targeting mitochondrial Ca2+ in the β-cell.Excellent reviews on the bioenergetic role of mitochondria and mitochondrial Cavailable ,18,19, ivailable . The rolvailable . In thisIn the pancreatic β-cell, metabolism–secretion coupling describes the molecular mechanism linking nutrient sensing and signaling to insulin secretion. This process relates to the consensus model and additional coupling factors (including both triggering and amplifying pathways) of glucose-stimulated insulin secretion ,23. GlucATP channels and promotes plasma membrane depolarization. This opens voltage-gated plasma membrane Ca2+ channels, leading to an increase in cytosolic Ca2+ concentration, which finally triggers insulin exocytosis by activating Ca2+-sensitive granule-resident proteins . In the cytosol, it is metabolized by glycolysis to generate pyruvate, which is taken up by mitochondria. Mitochondrial pyruvate is metabolized by the tricarboxylic acid (TCA) cycle, which generates reducing equivalents NADH (reduced nicotinamide adenine dinucleotide) and FADH2 (reduced flavin adenine dinucleotide), which are substrates of the mitochondrial respiratory chain. Activation of mitochondrial respiration leads to mitochondrial ATP synthesis and thus to an increased cytosolic ATP-to-ADP ratio, which induces the closure of plasma membrane Ktagmin-7 ). The amtagmin-7 ,22.2+ homeostasis in the pancreatic β-cell [2+ concentrations. However, in addition to KATP channels and voltage-dependent Ca2+ channels [2+ channels, which are voltage-independent Ca2+ channels activated upon depletion of the endoplasmic reticulum Ca2+ stores, and transient receptor potential channel 1 (TRPC1), have been indicated to be involved in insulin secretion [2+ channels, Orai1 has been identified as the main protein that conducts the previously described Ca2+ release-activated current (ICRAC). The activity of Orai1 channels is tightly controlled by the endoplasmic reticulum membrane protein stromal interacting molecule 1 (STIM1), which acts as an endoplasmic reticulum Ca2+ sensor and translocates, upon endoplasmic reticulum depletion, to endoplasmic reticulum/plasma membrane regions, where Orai1 is clustered [2+ from the endoplasmic reticulum has been suggested to potentiate glucose-stimulated hormone secretion [2+ leak has been observed in the endoplasmic reticulum, specifically in pancreatic islets and β-cells. This continuous Ca2+ efflux from the endoplasmic reticulum was modulated by GSK3β-dependent phosphorylation of presenilin-1 and promoted mitochondrial activation [2+ homeostasis and thus β-cell function, including insulin granules and other acidic stores  [The coupling between nutrient stimulation and hormone secretion is closely linked to Cac β-cell ,25,26,27channels , other pchannels . In partecretion ,31. For ecretion , and theecretion . Moreovetivation ,35. Addi2+ homeostasis and signal transduction is represented by the mitochondrial network. Therefore, mitochondria are intracellular organelles that take up and release Ca2+, promoting the sensing and shaping of cytosolic Ca2+ signals [2+ signaling in energized mitochondria of the pancreatic β-cell is emerging as a biological process of critical importance to pancreatic β-cell function and is highlighted in the next section.The last player in pancreatic β-cell Ca signals . The rol2+ elevation in triggering insulin secretion in the pancreatic β-cell, it is also accepted that such a rise in itself does not sustain insulin secretion [2+ plays a crucial role as a receiver and generator of the signals essential for metabolism–secretion coupling [2+ in the mitochondrial matrix under physiological conditions, is shedding light on the role of mitochondrial Ca2+ elevation in different tissues, including the pancreatic β-cell  through the ion-impermeable inner mitochondrial membrane [2+ levels are also very close to cytosolic Ca2+ levels, indicating tight regulation of the MCU [2+ uptake when perfused with buffers containing a Ca2+ concentration of only 150 nM [2+ buffers, mitochondrial Ca2+ uptake was more efficient above 2 µM [2+ activation threshold, the MCU of β-cells also behaves as a low affinity, high capacity Ca2+ transport system, with similar properties to those reported in other tissues. In any case, the mitochondrial Ca2+ concentration in glucose-stimulated β-cells was reported to reach only 600–800 nM [2+ events. Purinergic activation and potassium-induced depolarization generate cytosolic Ca2+ transients around one micromolar, with the corresponding mitochondrial Ca2+ rises reaching nearly 5 µM [2+ concentrations in the matrix during physiological activation.The MCU complex is a low-affinity, high-capacity Cand 70 µM ,42,43,44membrane . As reporadient ~0 mV throy 150 nM , which iy 150 nM . This stove 2 µM . These d0–800 nM ,18,49, mrly 5 µM . These r2+ uptake [2+ ions into the mitochondrial matrix, it requires a third subunit known as the essential MCU regulator (EMRE), a 10 kDa protein [2+ uptake [2+ uptake, MCUb exerts a negative effect on mitochondrial Ca2+ uptake [2+ in the intermembrane space exceeds ~0.6 µM, it initiates activation of the MCU complex via the MCU gatekeeper protein paralogs mitochondrial calcium uptake 1 and 2 (MICU1 and MICU2) [2+ binding affinity of ~0.3 µM for MICU1 and a Ca2+ binding affinity of ~0.6 µM for the MICU1–MICU2 dimer [The MCU complex consists of at least six subunits, each of which plays an individual role in orchestrating mitochondrial Ca+ uptake . The MCU+ uptake , has bee+ uptake . In orde protein ,52. EMRE protein . The MCU+ uptake . While t+ uptake . The rat+ uptake ,56,57. Wd MICU2) ,58. MICUd MICU2) . Neitherd MICU2) ,60. In ad MICU2) . Both ga2+ concentration in the intermembrane space for MICU1-regulated MCU activation, resulting in increased mitochondrial Ca2+ uptake [2+ entry into the mitochondria [2+ concentrations and mitochondrial Ca2+ uptake [2+ concentrations leads to increased mitochondrial Ca2+ uptake, while high extra-mitochondrial Ca2+ concentrations are associated with decreased mitochondrial Ca2+ uptake. The final subunit, which has been proposed to contribute to the structure of the MCU complex is a scaffolding factor called mitochondrial calcium uniporter regulator 1 (MCUR1), which can interact with the MCU but not with MICU1 [2+ uptake. Several studies have demonstrated a regulatory effect of MCUR1 on MCU complex activity, noting reduced mitochondrial Ca2+ uptake in the absence of MCUR1 and a decrease in reducing equivalents as well as ATP generation [2+ permeation across the pore, the activation threshold and the cooperativity. Consistent with this hypothesis, it has been reported that in parallel with the distinct relative abundance of MCU components [2+ activation threshold.Genetic ablation of MICU2 lowers the required Ca+ uptake . On the chondria ,62. Stud+ uptake . This suth MICU1 . Howeverneration ,66,67. Aneration . Interesneration , and it mponents , MCU actmponents . AlthougPancreatic β-cells have been demonstrated to express a functional MCU, and some studies have investigated the role of MCU subunits on insulin secretion ,16,17 F.2+ Controls Insulin Secretion; https://doi.org/10.1101/830323 (accessed on 1 February 2021); Vishnu, Hamilton, Bagge, Wernersson, Cowan, Barnard, Sancak, Kamer, Spégel, Fex, Tengholm, Mootha, Nicholls and Mulder). Further studies are expected to consolidate the specific role of the other subunits of the MCU in the pancreatic β-cell  ethane-N, N, N′, N′-tetraacetic acid) [2+ in energy metabolism required for signal-dependent hormone secretion. The discovery of the molecular nature of the mitochondrial Ca2+ uniporter [2+ in pancreatic β-cell function and opened new perspectives for pharmacological and nutritional interventions. In murine pancreatic β-cells, knockdown of MCU showed a strong reduction in mitochondrial Ca2+ uptake, accompanied by impaired ATP production [2+ in metabolism–secretion coupling. Similar results were obtained in pancreatic β-cell lines (INS-1 and INS-1E cells). Genetic depletion of two subunits of the MCU complex (MCU and MICU1) was characterized by reduced hyperpolarization of the inner mitochondrial membrane, diminished mitochondrial Ca2+ transients, impaired respiration rate and ATP levels, and reduced insulin secretion during glucose stimulation [2+ for signal transduction in β-cells and the impact of the MCU on signal-dependent hormone secretion.The importance of mitochondrial Cae matrix  or by loic acid) . The impniporter ,40 then oduction , consistmulation ,16. Take2+ in glucose-induced insulin secretion was finally validated by Georgiadou and collaborators in a mouse model, in which MCU was highly selectively deleted in pancreatic β-cells [2+ uptake, glucose-induced ATP production and insulin secretion were substantially impaired in their animal model in vitro. In the living β-cell-specific MCU-null mice, the first phase of insulin release was also impaired, despite paradoxical improvements in systemic glucose tolerance. Despite the apparent compensatory mechanisms proposed to explain the maintained glucose tolerance in β-cell-specific MCU-null mice (which remain to be established), this study indicated that agents affecting mitochondrial Ca2+ uptake in the pancreatic β-cell may alter insulin secretion and diabetes risk.The crucial role of mitochondrial Ca β-cells . In this2+ to the regulation of insulin secretion is not fully understood. Consistent with the universal role of mitochondrial Ca2+ in the regulation of cellular energetics [2+ for effective ATP generation in pancreatic β-cells is well established [2+ is required for the activation of matrix dehydrogenases involved in pyruvate metabolism and the TCA cycle [2+-activated dehydrogenases form reducing equivalents in the mitochondrial matrix, promoting the reduction of mitochondrial redox pairs. Most notably, studies in Bristol in the 1960s and 1970s led to the recognition that mitochondrial Ca2+ promotes the supply of reducing equivalents in the form of NADH or FADH2 [2+-activated mitochondrial dehydrogenases were specifically described: FAD-glycerol phosphate dehydrogenase , pyruvate dehydrogenase, NAD-isocitrate dehydrogenase and oxoglutarate dehydrogenase . Following early studies with isolated mitochondria, the effects on Ca2+ regulation of mitochondrial metabolism were confirmed in situ [2+ level reached during glucose-induced cell activation H:NAD(P)+ ratio , promoting robust insulin secretion [2+ uptake is a critical event in cellular bioenergetics and thus in the metabolic coupling of insulin, specific buffering or suppression of the mitochondrial Ca2+ increase lowers glucose-induced respiration and ATP synthesis, and impairs second-phase insulin secretion [The molecular characterization of the MCU is shedding light on the role of this transporter in pancreatic β-cell function; however, the precise mechanism and contribution of mitochondrial Caergetics ,71, an eablished . MitochoCA cycle ,72. Mitoor FADH2 ,74,75. Ftion see  was suffP levels . In thispiration . Accordiecretion . Consistecretion ,17.2+ to signal transduction in β-cells is related to the ability of mitochondria to take up and buffer intracellular Ca2+ and to affect the tone and frequency of cytosolic Ca2+ oscillations [2+ has also recently been linked to the regulation of the insulin secretion through local Ca2+ buffering at the sub-plasma membrane level [2+ and β-cell function, the authors proposed that changes in mitochondrial distribution may be important for the generation of the Ca2+ microdomains required for efficient insulin granule release. The precise contribution of mitochondrial Ca2+ rise to the shaping of cytosolic Ca2+ signaling is not completely established in β-cells. However, the aforementioned preprint in bioRxiv, which has not yet been peer-reviewed, , seems to shed some light on this function. Thus, by ablating MICU2 subunits of the MCU complex in insulin-secreting rat and human cell lines as well as in mouse islets, the authors reported reduced glucose-induced mitochondrial Ca2+ elevation and impaired bioenergetics and insulin secretion. In MICU2-deficient cells, although KCl-evoked sub-plasmalemmal Ca2+ increases were more pronounced, the global cytosolic Ca2+ response was surprisingly reduced. The authors concluded that MCU plays a role in stimulated β-cells by regulating net Ca2+ entry across the plasma membrane. They proposed that this was linked to the clearing of sub-plasmalemmal Ca2+ levels by mitochondria located near the plasma membrane.An additional mechanism linking mitochondrial Callations . This rellations . The abine level . In this2+, mitochondrial Ca2+ has also been proposed to modulate β-cell function, linked to the activation of the mitochondrial permeability transition pore (PTP) [2+-dependent, redox-dependent and cyclosporine-A-inhibited permeabilization of the inner mitochondrial membrane [2+-dependence [2+ signaling and the function and fate of pancreatic β-cells.In addition to the regulation of bioenergetics and the modulation of cytosolic Care (PTP) . The PTPmembrane ,82. The membrane ,85,86,87pendence , the modThe molecular identity of the PTP is still a matter of debate ,89,90,912+-induced and thiol cross-linking-dependent, and a mitochondrial Ca2+-independent mitochondrial permeability transition has been reported [In two semi-permeabilized pancreatic β-cell lines (MIN6 and INS-1), the existence of both a mitochondrial Careported . Inhibitreported ,85. In areported . These rreported . The autreported . In addireported .2+-induced PTP may play two distinct roles in the physiology and pathology of the pancreatic β-cell. While activation of the PTP is required to promote insulin secretion upon glucose stimulation [2+ levels, MCU activators are expected to promote insulin secretion; conversely, MCU inhibitors are potential agents to protect β-cells during islet transplantation or nutrient stress in vivo [2+ transport have been synthesized recently [2+ but also various systems in the mitochondria. For instance, the new penetrating inhibitor of the Ca2+ uniporter, DS16570511, has multiple effects on the mitochondria of cerebral cortex cells, while stimulating cellular survival during glutamate overload [2+ transport modulators is crucial to conclusively establish the potential effects of these compounds on pancreatic β-cell function.In summary, the mitochondrial Camulation ,85, PTP mulation ,96. Give in vivo . Importarecently ,99,100. overload . Thus, a2+ elevation reflect the balance between uptake and release mechanisms [2+ uptake in different systems, including the pancreatic β-cell. However, mitochondrial Ca2+ extrusion has also been demonstrated to play a key role in cellular physiopathology and signaling [2+ in the matrix space can lead to mitochondrial Ca2+ overload, followed by opening of the mitochondrial permeability transition pore [2+, mitochondria possess two membrane systems to extrude Ca2+: the mitochondrial Na+/Ca2+ exchanger and the mitochondrial H+/Ca2+ exchanger [During cell stimulation, the amplitude and duration of mitochondrial Cachanisms ,102,103.ignaling ,102,104.ion pore ,89, resuxchanger .+/Ca2+ exchanger was revealed in 2010 and attributed to the protein NCLX [2+/H+ exchanger in the pancreatic β-cell is a more complicated topic. Therefore, the protein LETM1 was proposed as a high-affinity mitochondrial Ca2+/H+ exchanger, capable of driving both the extrusion and uptake of Ca2+ into energized mitochondria at sub-micromolar Ca2+ concentrations [2+ sequestration (but not extrusion), depending on the source and mode of mobilized Ca2+ [2+ sequestration of Ca2+ that entered the INS-1 cells via depolarization-activated L-type Ca2+ channels of the plasma membrane was blunted by diminution of LETM1 expression [2+. The authors concluded that these data point to LETM1 as a high-affinity Ca2+ carrier [+/H+ exchange in the mitochondrial inner membrane [+-induced proton extrusion, whereas mitochondrial Ca2+ efflux was not altered [+/H+ exchanger [2+ fluxes, which can reflect the effects on Na+/H+ exchange activity [2+/H+ exchanger in the pancreatic β-cell remains to be elucidated, and additional studies are needed to reach a conclusion.The molecular identity of the mitochondrial Naein NCLX ; subsequein NCLX . The exitrations ,108. Intzed Ca2+ . Therefopression . However carrier . Howevermembrane ,110. In  altered . Other sxchanger ,112. Moractivity . In summ+/Ca2+ exchanger in the pancreatic β-cell have been reported  [2+-ATPase (SERCA pumps) and ryanodine receptors (RyR) [Prior to the discovery of the molecular identity of the mitochondrial Nansporter . This trnsporter . In contnsporter . The majs, LTCC) , sarcoplrs (RyR) .2+ extrusion [2+ extrusion from β-cells in MIN6 and in primary β-cells after glucose stimulation, and modulates both basal mitochondrial membrane potential and resting calcium levels. In addition, NCLX activity plays a major role in controlling both the rate and amplitude of cytosolic Ca2+ responses [2+ signaling linked to insulin secretion. They proposed that the Ca2+ transport activity mediated by NCLX and its strong effect on increasing cytosolic Ca2+ responses are the primary roles of NCLX in the context of insulin secretion. They suggested a model of mitochondrial Ca2+ efflux that modulates pancreatic β-cell function via NCLX-dependent shaping of the cytosolic glucose-dependent Ca2+ response, which may regulate the rate of insulin secretion. However, further in vivo studies with transgenic NCLX knockout mice are required to determine the precise contribution of mitochondrial Ca2+ extrusion to β-cell bioenergetics, Ca2+ homeostasis and islet physiology.The molecular discovery of NCLX allowed us to highlight the importance of mitochondrial extrusion in different tissues ,104,105,xtrusion ,118. By esponses . Surprisesponses . Thus, t2+ for pancreatic β-cell signal transduction and the possibility of modulating mitochondrial Ca2+ with natural bioactives [2+-targeted nutritional intervention strategy on metabolism–secretion coupling in a model of pancreatic insulin-secreting cells (INS-1E) [2+ elevation, which potentiated insulin secretion. Conversely, the MCU inhibitor mitoxantrone inhibited mitochondrial Ca2+ uptake and prevented both glucose-induced insulin secretion and kaempferol-potentiated effects. Kaempferol-dependent potentiation of insulin secretion was also validated in a model of standardized human pancreatic islets. We concluded that a mitochondrial Ca2+-targeted nutritional intervention activated metabolism–secretion coupling in insulin-secreting cells by modulating mitochondrial Ca2+ uptake.Given the importance of mitochondrial Caoactives , we rece(INS-1E) . We disc(INS-1E)  increase2+ inhibitor mitoxantrone [2+ modulation and β-cell function, it has not yet been definitively proven whether the modulation of mitochondrial Ca2+ by kaempferol is a direct or indirect effect of this polyphenol on the MCU complex.Although pharmacological inhibition of the kaempferol-induced effect obtained by the mitochondrial Caxantrone  indicate2+ rise, which enhanced insulin secretion in both INS-1E cells and mouse islets, and improved glucose tolerance in mice [2+ from the endoplasmic reticulum, thereby improving Ca2+ transfer between the endoplasmic reticulum and mitochondria. This transient mitochondrial Ca2+ increase was accompanied by the activation of two mitochondrial Ca2+-dependent processes (oxidative metabolism and ATP synthase-dependent respiration), which coordinately promoted sustained insulin secretion in the pancreatic β-cell [In another intervention study, we demonstrated that the natural bioactive quinic acid increases glucose-stimulated mitochondrial Ca in mice . In thisc β-cell .2+ in pancreatic β-cells could be used to treat diabetes.Although preclinical and clinical evidence, including interventions in genetic MCU-ablated models, is still needed to validate the efficacy and safety of these phytochemical-based interventions, these studies suggest that bioactive agents that increase mitochondrial Ca2+-targeted intervention strategy for β-cell health and, prospectively, for diabetes treatment. Stimulation of the MCU has been demonstrated to improve β-cell energy metabolism during nutrient stimulation. Such a mechanism could explain how MCU activators might have a beneficial effect on stimulus–secretion coupling in β-cells, leading to improved glucose homeostasis. Encouraging results with the natural bioactive MCU activator kaempferol indicate the potential beneficial effects of the MCU-targeted strategy on pancreatic β-cell function. However, kaempferol is a non-specific activator of mitochondrial Ca2+ rise in pancreatic β-cells. A more specific MCU-targeted pharmacology is expected to improve MCU activation of the pancreatic β-cell, promoting beneficial effects in the context of diabetes treatment.β-cell mitochondria contain a variety of ion channels in both the inner and the outer mitochondrial membrane, with important roles in stimulus–secretion coupling and cell viability . The rol2+ uptake should reveal whether altered insulin secretion is causally related to diabetes progression and could potentially expand the repertoire of therapeutic tools to treat this disease.New studies targeting the proteins that control mitochondrial Ca