Herein, we exploited single-cell Ca2+ imaging to assess the signal transduction pathways whereby histamine generates intracellular Ca2+ signals in the human fetal lung fibroblast cell line, WI-38. WI-38 fibroblasts were loaded with the Ca2+-sensitive fluorophore, FURA-2/AM, and challenged with histamine in the absence and presence of specific pharmacological inhibitors to dissect the Ca2+ release/entry pathways responsible for the onset of the Ca2+ response. Histamine elicited complex intracellular Ca2+ signatures in WI-38 fibroblasts throughout a concentration range spanning between 1 µM and 1 mM. In accord, the Ca2+ response to histamine adopted four main temporal patterns, which were, respectively, termed peak, peak-oscillations, peak-plateau-oscillations, and peak-plateau. Histamine-evoked intracellular Ca2+ signals were abolished by pyrilamine, which selectively blocks H1R, and significantly reduced by ranitidine, which selectively inhibits H2R. Conversely, the pharmacological blockade of H3R and H4R did not affect the complex increase in [Ca2+]i evoked by histamine in WI-38 fibroblasts. In agreement with these findings, histamine-induced intracellular Ca2+ signals were initiated by intracellular Ca2+ release from the endoplasmic reticulum through inositol-1,4,5-trisphosphate (InsP3) receptors (InsP3R) and sustained by store-operated Ca2+ channels (SOCs). Conversely, L-type voltage-operated Ca2+ channels did not support histamine-induced extracellular Ca2+ entry. A preliminary transcriptomic analysis confirmed that WI-38 human lung fibroblasts express all the three InsP3R isoforms as well as STIM2 and Orai3, which represent the molecular components of SOCs. The pharmacological blockade of InsP3 and SOC, therefore, could represent an alternative strategy to prevent the pernicious effects of histamine on lung fibroblasts in asthmatic patients.Histamine is an inflammatory mediator that can be released from mast cells to induce airway remodeling and cause persistent airflow limitation in asthma. In addition to stimulating airway smooth muscle cell constriction and hyperplasia, histamine promotes pulmonary remodeling by inducing fibroblast proliferation, contraction, and migration. It has long been known that histamine receptor 1 (H1R) mediates the effects of histamine on human pulmonary fibroblasts through an increase in intracellular Ca Asthma is a heterogeneous disease, generally characterized by chronic inflammation of the airways, defined by a clinical history of respiratory symptoms, such as wheezing, shortness of breath, chest tightness, and cough that vary in intensity and frequency, along with variable expiratory airflow . About 3The complex network of inflammatory responses in the pathophysiology of asthma involves the release of inflammatory mediators, such as cytokines, chemokines, proteases, and histamine . HistamiIn vitro studies demonstrated that histamine stimulated lung fibroblast collagen synthesis (2+ concentration ([Ca2+]i) in human valvular myofibrobasts , while the extracellular Ca2+ concentration is 1000-fold higher (> 1 mM) and the Ca2+ concentration in the primary intracellular Ca2+ store, the endoplasmic reticulum (ER), fluctuates between 100–800 µM , H1R, which activates phospholipase C (PLC). PLC hydrolyzes a membrane phospholipid, phosphatidylinositol 4, 5 bisphosphate (PIP2), to produce inositol-1, 4, 5-trisphosphate (InsP3). InsP3 triggers Ca2+ mobilization from the ER through InsP3 receptors (InsP3R) that are located on the ER membrane and can in turn activate adjacent ryanodine receptors (RyR) through the process of Ca2+-induced Ca2+ release (CICR)  (2+ channels (SOC), which are the main Ca2+ entry pathway in non-excitable cells. In the ER, Stromal Interaction Molecules (STIM1/2) act as sensors of ER Ca2+ concentration that, after a reduction in intraluminal Ca2+, multimerize and translocate towards peripheral ER cisternae to functionally interact with the Ca2+ permeable Orai channels on the plasma membrane (2+ has been termed store-operated Ca2+ entry (SOCE) and mediates agonist-induced Ca2+ influx in human fibroblasts isolated from several tissues, including lungs (An elevation in i also in human lung fibroblasts  and cultured to 75% confluence in DMEM (Dulbecco’s Modified Eagle Medium) culture medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin at 37°C in an atmosphere of 95% O2 and 5% CO2. Fibroblasts from passages 5–10 were seeded on coverslips for 24 h and then incubated for 48 h in medium devoid of serum.Human fetal lung fibroblast cell lines were purchased from American Type Culture Collection, Collection WI-38 , Ca2+ was replaced with 2 mM NaCl and 0.5 mM EGTA was added as a Ca2+ chelator. Osmolarity was measured with an osmometer  solutions were adjusted to pH 7.4 with NaOH.Physiological saline solution (PSS) with the following composition (in mM) 150 NaCl, 6 KCL, 1.5 CaCl2 1 MgCl2, 10 glucose, 10 HEPES was used for this study. To obtain Ca2+ measurement in fibroblasts has been previously described , the filter rotating wheel, as well as to draw the regions of interest of the fluorescent signal to be measured, a customized software, previously validated, running in LINUX environment, was used. The signal measurement was captured every 3 s and the images obtained were stored on a hard disk and subsequently converted into 340/380 ratio images using ImageJ software . An increase in 340/380 is indicative of an elevation in [Ca2+]i  gel agarose, stained with ethidium bromide, and acquired with the iBrightTM CL1000 Imaging System . The molecular weight of the PCR products was compared with the DNA molecular weight marker VIII .Total RNA was isolated from fibroblasts using QIAzol Lysis Reagent , and reverse transcription was performed as described in . ReverseFor the acquisition of fluorescence values, ImageJ software was used, and Origin Pro 2021 and GraphPad Prism 8.0 were used for graphing and statistical analysis of the results.p ≤ 0.05) were statistically analyzed by the nonparametric Mann-Whitney test for two groups and Kruskal–Wallis for more than two groups. For normal data, an unpaired Student t-test for two groups and ANOVA for more than two groups were used. A value of p ≤ 0.05 was considered statistically significant.Data were expressed as mean ± standard error (SE). Non-Gaussian data, identified by the D'Agostino and Pearson omnibus normality test (Y is the response (relative to the Ca2+ transient amplitude), [Histamine] is the histamine concentration and the mean maximal effective concentration (50EC) is the [Histamine] that induced 50% of the maximal response.Histamine concentration-response data were adjusted by the following 2+]i was measured simultaneously in several individual fibroblasts from the same population. A 22.5% of WI-38 fibroblasts displayed spontaneous Ca2+ oscillations, as also reported in human cardiac fibroblasts ; the second in a peak followed by cyclic increases and decreases in [Ca2+]i, termed peak-oscillations  are shown in 2+]i at very low concentrations, such as 100 and 300 nM. The Ca2+ response to histamine appeared at 1–3 µM; at these concentrations, the Ca2+ signal arising in most WI-38 cells displayed a single peak in response to agonist stimulation. However, at 10 µM histamine, the Ca2+ peak was followed by a short train of consecutive Ca2+ oscillations. The number of oscillations over 60 min of histamine application was increased at histamine concentrations ranging from 30 µM up to 1 mM. The non-cumulative concentration-response curve of histamine-induced elevation in [Ca2+]i is depicted in 2+ response (peak). The maximum increase in the peak amplitude was observed at concentrations higher than 300 µM, whereas raising histamine concentration up to 1 mM did not significantly augment the magnitude of the response. Slight stimulation occurred at 3 µM, while no effect was detectable at concentrations lower than 1 µM (100 nM and 300 nM). The concentration of histamine required to produce a half maximal response (EC50), which was calculated by fitting the concentration-response curve as described in Materials and Methods, was 4.96 μM  was calculated  to the same fibroblast leads to receptor desensitization.Homologous desensitization is a feature of GqPCRs, including H1R . Figure plitudes , as wellplitudes , were si2+ signal in WI-38 human lung fibroblasts, specific HR antagonists were used: for H1Rs pyrilamine (100 μM), for H2Rs ranitidine (50 μM), for H3R clobenpropit (50 μM) and, finally, for the H4R receptor, NJ7777120 (10 μM). After 30 min preincubation and in the continuous presence of the histaminergic antagonists, histamine (300 µM) was applied as indicated by the green bars. In order to confirm cell viability, arachidonic acid (AA) 50 µM was applied in cells in which histamine failed to induce an increase in [Ca2+]i  and presence of the different antihistaminergic receptors antagonists  are summarized. These results indicate that, for the [Ca2+]i elevation to take place in the lung fibroblast cell line, WI-38, the activation of mainly H1R and, to a lesser extent, H2R is necessary, whereas H3R and H4R seem to play no role.In order to elucidate the HR subtype through which histamine triggers an intracellular Ca [Ca2+]i . Pharmac+ signal , H2R blo+ signal , while H+ signal  and H4R + signal  had no s2+ signaling. H3R and H4R are canonically coupled to Gαi/o proteins  was not statistically different from untreated cells  U73122 (10 μM), a selective inhibitor of PLC  prevented the Ca2+ response to histamine  U733r of PLC ; and 3) r of PLC . The res+ signal , whereasresponse . Likewisistamine . These r2+ stores contributes to histamine-evoked Ca2+ signals upon PLC activation, we evaluated the involvement of InsP3R, which provides the main pathway for ER Ca2+ release in fibroblasts    and a sp, 10 μM) . The resr nickel  or nifedr nickel , respect2+ entry pathway sustaining the Ca2+ response to chemical stimuli in human lung fibroblasts  and Gd3+ (10 μM) abrogated the plateau and Ca2+ oscillations in 79 and 40 cells exposed to La3+ and Gd3+, respectively  subfamily, i.e., TRPC1, TRPC3, TRPC4, TRPC5, and TRPC6  peak, 2) peak and oscillations, 3) peak plateau and oscillations, and 4) peak and plateau. Spatial and temporal heterogeneity of the Ca2+ signature can be a hallmark of the Ca2+ response evoked by both mechanical  transient, by repetitive Ca2+ oscillations, c) a sustained Ca2+ plateau or d) a sustained plateau overlapped by repetitive Ca2+ oscillations  in human microvascular endothelial cells   . It is ldulation . The prilate RyR  or inhib RyR  peak-oscillations, 64.7%, and mode 3) peak-plateau-oscillations, 13.39%, entail the occurrence of intracellular Ca2+ oscillations. In agreement with this observation, STIM2 and Orai3 can enhance the percentage of cells showing intracellular Ca2+ oscillations upon GqPCR stimulation (2+, InsP3R3 may provide a constant release of Ca2+ that stimulates InsP3R1 and InsP3R2 to rhythmically release ER stored Ca2+, whereas SOCE maintains the Ca2+ response by ensuring ER Ca2+ refilling (2+ transients transition into a sustained plateau either when InsP3R3 expression increases (2+ release through InsP3R3 is enhanced by the tight coupling with the ER-embedded protein, Jaw1 (3R isoform, as well as cell-to-cell variability in their expression, subcellular distribution, or posttranslational regulation, could contribute to pattern a heterogenous array of Ca2+ signatures in WI-38 adult lung fibroblast (2+ waves because our experiments were performed in fibroblasts devoid of serum for 48 h, which causes cell cycle arrest in G0 phase (2+ dynamics is not due to cell cycle asynchrony (A preliminary qRT-PCR analysis of the Caroblasts , in whicllations . Intriguepletion , which mence see  but not roblasts . Conversroblasts . Quite s+ influx . In agreroblasts . It is wmulation . Furtherefilling . Howeverncreases  or when in, Jaw1 . Therefobroblast . ConversG0 phase . Similarynchrony .2+]i in the widely employed human pulmonary fibroblast cell line, WI-38. The Ca2+ signal is mainly triggered by H1R and can adopt multiple signatures, the most common of which encompasses intracellular Ca2+ oscillations, which have long been known to stimulate gene expression, proliferation, contraction and migration in human pulmonary fibroblasts (2+ response to histamine is triggered by ER Ca2+ release through InsP3R and maintained over time by SOCE activation. These data suggest that the Ca2+ handling machinery could provide an alternative molecular target to prevent the pernicious effects of histamine on lung fibroblasts in asthmatic patients, as recently suggested also for pulmonary hypertension (Streptococcus pneumoniae-induced lung injury (2+ signaling in lung fibroblasts. A limitation of the present study is that we did not use lung fibroblasts from an asthmatic model or from patients with asthma. Future work will have to compare the effect generated by histamine in lung fibroblasts from normal airways and in lung fibroblasts from asthmatic airways.In conclusion, the present investigation showed that histamine induces a dose-dependent increase in [Caroblasts . The Ca2rtension , Streptog injury , and astg injury . Much re


However, research on stimulus-specific Ca2+ signals has mostly been pursued in Arabidopsis thaliana, while in other species these events are little investigated .Plants are continuously exposed to changing environmental conditions and biotic attacks that affect plant growth. In crops, the inability to respond appropriately to stress has strong detrimental effects on agricultural production and yield. Ca2+ reporter-encoding gene APOAEQUORIN into the crop species barley (Hordeum vulgare). Measurements of the dynamic changes in [Ca2+]cyt in response to various stimuli such as NaCl, mannitol, H2O2, and flagellin 22 (flg22) revealed the occurrence of dose- as well as tissue-dependent [Ca2+]cyt transients. Moreover, the Ca2+ signatures were unique for each stimulus, suggesting the involvement of different Ca2+ signalling components in the corresponding stress response. Alongside, the barley Ca2+ signatures were compared to those produced by the phylogenetically distant model plant Arabidopsis. Notable differences in temporal kinetics and dose responses were observed, implying species-specific differences in stress response mechanisms. The plasma membrane Ca2+ channel blocker La3+ strongly inhibited the [Ca2+]cyt response to all tested stimuli, indicating a critical role of extracellular Ca2+ in the induction of stress-associated Ca2+ signatures in barley. Moreover, by analysing spatio-temporal dynamics of the [Ca2+]cyt transients along the developmental gradient of the barley leaf blade we demonstrate that different parts of the barley leaf show quantitative differences in [Ca2+]cyt transients in response to NaCl and H2O2. There were only marginal differences in the response to flg22, indicative of developmental stage-dependent Ca2+ responses specifically to NaCl and H2O2.In this study, we introduced the Ca2+ signals with stimulus-specific kinetics in the crop species barley, as well as quantitative differences along the barley leaf blade. A number of notable differences to the model plants Arabidopsis may be linked to different stimulus sensitivity. These transgenic barley reporter lines thus present a valuable tool to further analyse mechanisms of Ca2+ signalling in this crop and to gain insights into the variation of Ca2+-dependent stress responses between stress-susceptible and -resistant species.This study reveals tissue-specific CaThe online version contains supplementary material available at 10.1186/s12870-022-03820-5. Arabidopsis thaliana has been extensively studied to decipher the molecular basis of stress tolerance; however, the study of more stress-tolerant species is more likely to yield relevant information.Plants as sessile organisms are exposed to constantly changing conditions in their environment that require short-term acclimation responses enabling them to fulfil their lifecycle. Thus, it is crucial to understand mechanisms of stress response to maintain and improve crop production, especially in the face of increasing global climate changes . InteresHordeum vulgare L.) is well-adapted to a wide range of environmental conditions and has a relatively high tolerance to drought and salinity compared to other crops and Arabidopsis cyt) are maintained in the nanomolar range [2+]cyt with stimulus-specific spatio-temporal parameters such as frequency, amplitude and duration, defined as “Ca2+ signatures” [2+]cyt is one of the earliest signalling events in plants challenged by biotic elicitors cyt result from the influx of Ca2+ via Ca2+-permeable channels, either from intracellular Ca2+ stores or across the plasma membrane from the apoplast. These elevations in [Ca2+]cyt are in turn counteracted by the activity of Ca2+ transporters or ATPases extruding Ca2+ out of the cytosol to restore the low basal level of [Ca2+]cyt. In Arabidopsis, multiple families of Ca2+-permeable channels, transporters and pumps have been identified, each with several members cyt signals in this crop. The transgenic plants exposed to different abiotic stimuli  and the PAMP flg22 showed increases in [Ca2+]cyt in a dose- and tissue-dependent manner. The spatio-temporal patterns of the cytosolic Ca2+ dynamics (Ca2+ signature) were unique for each stimulus. Furthermore, we revealed a conspicuous spatial heterogeneity of Ca2+ responses to some stimuli along the blade of barley leaves, indicating a dependence on the developmental stage of the cells. Compared to Arabidopsis notable differences in the barley Ca2+ signatures were observed. The comparison of Ca2+ signatures between barley and Arabidopsis, phylogenetically distant species with different stress tolerance, highlights the diversity in Ca2+ signals in higher plants and might help to reveal novel aspects in stress response mechanisms.In this study, the stable transformation of barley plants with 2+]cyt changes in barley, transgenic lines expressing the genetically encoded Ca2+ sensor APOAEQUORIN under the control of the ZmUBI1 promoter were developed and named Hv-AEQcyt. Four independent transgenic lines , based on the barley cultivar Golden Promise, were selected. To estimate the abundance of APOAEQUORIN in these lines, the aequorin-based luminescence was recorded in excised leaf tips (5 mm) after reconstitution with coelenterazine by injecting a discharge solution. Upon application of the discharge solution, all transgenic lines showed an increase in luminescence, with line #18 showing the highest intensity  varied depending on NaCl concentration and tissue. Already 50 mM NaCl induced obvious increases in [Ca2+]cyt in both tissues, which was more pronounced in the root  to the base (S5) in case of NaCl application   pathway has been suggested to be essential for salt tolerance cyt in the roots of barley and Arabidopsis, whereas leaves of both species did not show any response  and 1,2-diacylglycerol cyt transients were mainly observed in leaves in both species  the coding region of APOAEQUORIN was amplified from the vector pBIN19-AEQ cyt measurements in barley, while entire shoots or roots of five-day-old seedlings were used for Arabidopsis. Tissues were reconstituted for 16 hours in the dark in 2.5 μM coelenterazine solution . After reconstitution, the coelenterazine solution was replaced by in ddH2O, and tissues were allowed to recover for one hour in light before measurements. For inhibitor treatments, leaf or root tips of barley were incubated for one hour in 1 mM lanthanum chloride (LaCl3) in ddH2O after reconstitution. All measurements were performed in 96-well plates  in a plate luminometer (Mithras LB940). Luminescence was detected for 90 seconds with an integration time of 1 sec to record the baseline before the injection of an equal volume of a 2-fold-concentrated solution of H2O2, NaCl, mannitol, or flg22. After injection, changes in luminescence were recorded for another 600 sec  or 1200 sec (flg22). After injection of discharge solution  luminescence was recorded for another 300 sec with the same integration time. [Ca2+]cyt was calculated as described [2+]cyt, the mean [Ca2+]cyt derived from 10 sec of baseline prior to treatment was subtracted from the maximum [Ca2+]cyt obtained after injection. All experiments were repeated at least twice with similar results.Unless otherwise stated, 5 mm sections from the tip of the leaf and the primary root of three-day-old Hv-AEQescribed  with a bcyt plants were mounted on a Petri dish using double faced adhesive tape and APOAEQUORIN was reconstituted by spraying leaves with 10 μM coelenterazine in 0.01% Tween 20 and subsequent incubation for six hours in the dark. Aequorin imaging was performed according to [2 in 10% ethanol. Ca2+-dependent light emission was analysed with the IFS32 software (Photek) by drawing defined regions of interest (ROIs) and normalized by calculating L/Lmax  for each ROI. All experiments were repeated at least twice with similar results.For luminescence imaging, intact Hv-AEQrding to  using a All experiments were repeated at least twice. The data on which the graphs and bar plots in figures are based are shown in the Additional Data Files 2-5. Statistical analyses were performed using Sigma Plot 13.0 (Systat Software).Additional file 1. Figures S1to Figures S9Additional file 2. Table S1Additional file 3.Table S2Additional file 4.Table S3Additional file 5.Table S4


We therefore visualised rapid-scanning confocal fluo4-[Ca2+]i dynamics and morphology of cardiomyocytes in Langendorff-perfused rat hearts following saponin-membrane permeabilisation. Our data demonstrate that 0.4% saponin-treated myocytes immediately exhibited high-frequency Ca2+ waves (131.3 waves/min/cell) with asynchronous, oscillatory contractions having a mean propagation velocity of 117.8 μm/s. These waves slowly decreased in frequency, developed a prolonged decay phase, and disappeared in 10 min resulting in high-static, fluo4-fluorescence intensity. The myocytes showing these waves displayed contraction bands, i.e., band-like actin-fibre aggregates with disruption of sarcomeric α-actinin. The contraction bands were not attenuated by the abolition of Ca2+ waves under pretreatment with ryanodine plus thapsigargin, but were partially attenuated by the calpain inhibitor MDL28170, while mechanical arrest of the myocytes by 2,3-butanedione monoxime completely attenuated contraction-band formation. The depletion of adenosine 5′-triphosphate by the mitochondrial electron uncoupler carbonyl cyanide 4-trifluoromethoxy phenylhydrazone also attenuated Ca2+ waves and contraction bands. Overall, saponin-induced myocyte [Ca2+]i overload provokes agonal Ca2+ waves and contraction bands. Contraction bands are not the direct consequence of the waves but are caused by cross-bridge interactions of the myocytes under calpain-mediated proteolysis.Although irreversible cardiomyocyte injury provokes intracellular Ca The myocytes suffering from [Ca2+]i overload present with Ca2+ waves, i.e., spontaneous propagating rises in the [Ca2+]i concentration, which accompany localised contractions6. Our previous work demonstrated that myocytes in Langendorff-perfused rat hearts exhibit three different patterns of Ca2+ waves, with each pattern depending on the degree of [Ca2+]i overload7. Of these, extremely high-frequency Ca2+ waves, called “agonal waves”, have been regarded as the representative [Ca2+]i dynamics in irreversibly injured myocytes, e.g., myocytes subjected to cryoinjury8 or Ca2+-paradox injury9. We have also observed that irreversible membrane injury inflicted on cardiomyocytes by saponin initiates extremely high-frequency Ca2+ waves, which are comparable to those induced by Ca2+-paradox injury9. However, the precise spatiotemporal [Ca2+]i dynamics under the irreversible [Ca2+]i overload and their effect on the cellular morphology leading to their eventual cell death remain unknown. To address these issues, we sought to visualise the myocyte [Ca2+]i dynamics induced by saponin in the Langendorff-perfused rat heart. We chose saponin because this chemical simply permeabilises the sarcolemmal membrane10, and the resultant entry of Ca2+ through the membrane pores provides definitive, reproducible induction of irreversible [Ca2+]i overload compared with other injury models. For example, the cryoinjured model exhibits diverse and complex myocyte [Ca2+]i dynamics, including high-frequency Ca2+ waves with or without diminution by Ca2+ transients and high-static [Ca2+]i, an indication of cell death8; therefore, their morphological relevance is minimal. The Ca2+-paradox model, which has been widely studied as an alternative model of ischaemia‒reperfusion injury, shows detachment of cell‒cell junctions with hypercontracture of myocytes due to cadherin inhibition induced by Ca2+ depletion9.Intracellular Ca2+ waves contribute to the formation of contraction bands, a morphological pattern associated with reperfusion injuries following ischaemia12. We evaluated this theory using high-speed imaging of the subepicardial myocyte [Ca2+]i dynamics in saponin-perfused rat hearts via rapid-scanning confocal microscopy together with confocal fluorescence imaging of myocyte morphology to address whether and how the Ca2+ waves contribute to the formation of contraction bands. Some of the results have been published as an abstract13.Here, we postulate that saponin-induced Ca2+]i dynamics of the subepicardial myocardium before and during saponin perfusion. In the absence of saponin, individual myocytes exhibited spatiotemporally uniform Ca2+ transients during systole and no abnormal increases in [Ca2+]i concentration during diastole as shown in the sequential X–Y fluo4-fluorescence images and corresponding X-t images  progressively decreased in amplitude and widened, eventually leading to ventricular asystole. In addition, the myocytes producing the high-frequency Ca2+ waves were unresponsive to electrical stimulation of the heart, and the properties of the Ca2+ waves were not influenced by external electrical stimulation  of 117.8 ± 23.2 μm/s (n = 60 waves), revealing fixed patterns of intracellular propagation  in the heart. In the absence of saponin, the cell membrane and T-tubules were clearly visualised by di-4-ANEPPS but no nuclear fluorescence of PI was identified. In contrast, after saponin treatment, the cell membrane and T-tubules became ambiguous, and the di-4-ANEPPS dye was redistributed into the cytosol and the PI fluorescence into the nuclei    (n = 185 waves)  similar to those under continued saponin perfusion  and the reuptake of Ca2+ into the SR via Ca2+-ATPase (SERCA). This is because [Ca2+]i overload following membrane permeabilisation can lead to the depletion of cellular ATP reserves by opening of the mitochondrial permeability transition pore (mPTP)14. Given this, we evaluated sequential changes in the mitochondrial membrane potential using a tetramethylrhodamine methyl ester (TMRM) fluorescence-based assay16  and thapsigargin (5 μM)17, under which conditions myocytes barely showed Ca2+ transients or Ca2+ waves  at 20 mMves Fig. a. Quanti24, myocytes showed no Ca2+transients or Ca2+ waves  at 10 μM, which depletes intracellular ATPves Fig. a, consisves Fig. b.Figure 2+ waves and coexisting contraction bands within the myocytes of perfused rat hearts following saponin-permeabilised membrane injury. The novel points of this study are as follows: (i) the high-frequency Ca2+ waves provoked by irreversible injury show progressive alterations during the progress toward cell death, and (ii) the simultaneous contraction band formation is not the direct consequence of the Ca2+ waves but might be mediated by cross-bridge interactions and calpain-mediated proteolysis as a result of [Ca2+]i overload.The present study demonstrates the high-frequency Ca2+ waves here were similar to those of our previous studies, which allowed us to identify these responses as “agonal” Ca2+ waves8. These waves present as asynchronous, ripple-like wavefronts with oscillatory contractions within individual myocytes induced by an irreversible [Ca2+]i overload. These properties of the Ca2+ waves are likely to be due to the enhanced release of Ca2+ from the SR and the SERCA-mediated reuptake of Ca2+ into the SR in an oscillatory fashion25 and the unresponsiveness to electrical stimulation might be due to gap-junctional uncoupling via [Ca2+]i overload27.The observed Ca2+ waves were not static but changed with time. Our understanding is that the initiation and subsequent alterations of the behaviours of agonal waves depend on both the concentrations of saponin and [Ca2+]i and the time after saponin membrane permeabilisation: the higher the concentrations of saponin and [Ca2+]i are, the more rapidly the agonal waves and subsequent transition to cell death develop showing high-static fluo4 fluorescence intensity.Our data revealed that the properties of the saponin-induced agonal Ca30. Such alterations are likely to produce gradual declines in [Ca2+]i cycling and possible depletion of ATP facilitated by the opening of the mPTP14 in response to progressive [Ca2+]i overload. In practice, [Ca2+]i overload in the saponin-treated myocytes was accompanied by progressive mitochondrial damage as indicated by the progressive, sporadic loss of TMRM fluorescence intensity in individual myocytes  of these agonal waves. In this regard, the reduction in wave frequency may result in the lengthening of the Ca2+ restitution kinetics in the SR34, but this would not adequately account for the extensive prolongation of T1/2 observed in this study. Instead, we assume that the dissipation of cellular ATP may be responsible for these increased T1/2 values, as they markedly increased after 6 min of saponin exposure . All of the animal experiments described in this study were conducted between December 2020 and August 2022 in accordance with the Guide for the Care and Use of Laboratory Animals  following approval by the Animal Research Committee at Kyoto Prefectural University of Medicine . Our experiments used male Wistar rats (9–12 weeks old) weighing 200–300 g (n = 57 hearts). These animals were placed under deep general anaesthesia using intraperitoneal injection with 0.1 mg/kg medetomidine, 3.0 mg/kg midazolam, and 5.0 mg/kg butorphanol, and then injected with heparin (2 U/g body weight) via the inferior vena cava before their hearts were quickly removed and then perfused using the Langendorff protocol and HEPES-buffered Tyrode’s solution supplemented with 137 mM NaCl, 4 mM KCl, 1 mM MgCl2, 0.33 mM NaH2PO4, 1.2 mM CaCl2, 10 mM HEPES and 10 mM glucose  for approximately 3 min. Once the blood was removed each heart was loaded with the Ca2+-indicator fluo4-AM  for 15 min at room temperature (23–25 °C) in accordance with the efficient conditions for fluo4 loading as previously described9. Then the heart was perfused with Tyrode’s solution containing probenecid (0.1 mg/ml) for 5 min at 37 °C to augment the activity of esterase for de-esterification of the acetoxy methyl ester (AM) form of fluo4. The fluo4-loaded heart was then used in all subsequent experiments and was maintained under constant perfusion with HEPES-buffered, 0.3-mM Ca2+-containing Tyrode’s solution (5 mL/min) by a microtube pump  at 30 °C using a tube heater  for efficient visualisation of [Ca2+]i dynamics based on our experiences: higher temperatures up to 37 °C show a tendency to diminish the fluo4 fluorescence intensity. Organ ECGs were recorded using silver wires (0.5-mm diameter) placed at the bottom of a customised chamber.This study was performed in accordance with the ARRIVE  guidelines 2.0 (2+ Tyrode’s solution before and during the application of saponin (0.4%). These images were obtained from the subepicardial myocardium of the fluo4-loaded rat hearts and high-speed, multipoint-scanning confocal microscopy facilitated by an upright microscope  and a spinning disc-type confocal unit CSU-21  under a glass coverslip (170-μm thickness) (n = 19 hearts). We prevented “coverslip hypoxia”46 of the heart by carefully placing the coverslip to the heart and limiting the observation period for each image to 30 s before the coverslip was removed, and the tissues were allowed to perfuse normally between imaging runs. The fluo4-fluorescence signals were detected using an image intensifier  and a charge-coupled device (CCD) camera . All images were captured using a 20× objective lens , and some experiments included electrical stimulation of the left ventricular apex using silver wires (0.5 mm in diameter). We also obtained a series of confocal images of tetramethylrhodamine methyl ester (TMRM)  fluorescence in the subepicardial myocardium using rapid-scanning confocal microscopy with excitation and emission wavelengths of 563 nm and > 665 nm, respectively. TMRM (100 nM) loading of the heart was performed by 15-min perfusion at room temperature (23–25 °C) followed by washing for 5 min prior to imaging.We collected a series of spatiotemporal images of the fluo4 fluorescence intensity  in the subepicardial myocardium of the heart using constant Langendorff perfusion with 0.3 mM CaSaponin  was dissolved in Tyrode’s solution with adequate ultrasonic agitation for 30 min. In addition, several experiments required the hearts to be perfused with Tyrode’s solution supplemented with various reagents including ryanodine , thapsigargin , MDL28170 , 2,3-butanedione monoxime (BDM)  or carbonyl cyanide 4-trifluoromethoxy phenylhydrazone (FCCP) , for specific pharmacological interventions.For confocal fluorescence imaging of the structural proteins for myocyte morphology, the hearts were fixed using Langendorff perfusion with 2% paraformaldehyde for 30 min (n = 29 hearts). We used Alexa Fluor 633 phalloidin  for actin histochemistry. For sarcomeric α-actinin immunohistochemistry, incubation with a primary antibody  for 48 h at 4 °C was followed by incubation with a secondary antibody for 48 h  prior to visualisation at room temperature. Confocal fluorescence images of both phalloidin and α-actinin were obtained using samples from the subepicardial surface of the heart using an FV-1000 confocal microscope . The excitation wavelengths for phalloidin and sarcomeric α-actinin were 633 nm and 488 nm, respectively, and the emission wavelengths were > 650 nm and between 500 and 600 nm, respectively. To confirm membrane permeabilisation by saponin, we perfused in advance the membrane dye di-4-ANEPPS at 5 μM  and the membrane-impermeable DNA dye propidium iodide (PI) at 7.5 μM  for 5 min and subsequent washout for another 5 min for confocal imaging (n = 3 hearts).prop) of the Ca2+ waves was calculated from the slope of the X-t image  of the Ca2+ wave was obtained from the plot profiles of the X-t images . X-t images were obtained by plotting the fluorescence intensity along the longitudinal axis of each myocyte in the X–Y image movies and the propagation velocity (VSupplementary Figures.Supplementary Video 1.Supplementary Video 2.


To identify the early adaptive changes in energy supply prior to failure, we utilised a rat model of pulmonary artery hypertension to investigate RV cardiomyocytes during compensated hypertrophy in comparison to their normotensive controls. Mitochondrial Ca2+ fluxes were increased in hypertrophied cardiomyocytes, but no difference was found in oxidative phosphorylation between the groups. This suggests that the larger mitochondrial Ca2+ transients are a compensatory mechanism to match ATP supply to the increased energy demands of hypertrophic cardiomyocytes.In pulmonary artery hypertension, right ventricular (RV) afterload is increased, which requires the cardiomyocytes to contract with greater force against the additional pulmonary artery pressure. In response, RV cardiomyocytes increase contractile protein content to maintain greater workload, consuming larger amounts of energy (supplied by the mitochondria) on a beat-to-beat basis. Failing hearts have been described as an “engine out of fuel”, but it is unclear how the mitochondria match ATP supply to demand in hypertrophic hearts prior to failure. Therefore, our aims were (i) to measure beat-to-beat mitochondrial Ca2+ handling that might impact mitochondrial function in the compensatory phase of RV hypertrophy. Our aims, therefore, were (i) to measure beat-to-beat mitochondrial Ca2+ fluxes, and (ii) to determine mitochondrial abundance and function in non-failing, hypertrophic cardiomyocytes. Male Wistar rats were injected with either saline (CON) or monocrotaline (MCT) to induce pulmonary artery hypertension and RV hypertrophy after four weeks. Cytosolic Ca2+ ([Ca2+]cyto) transients were obtained in isolated right ventricular (RV) cardiomyocytes, and mitochondrial Ca2+ ([Ca2+]mito) was recorded in separate RV cardiomyocytes. The distribution and abundance of key proteins was determined using confocal and stimulated emission depletion (STED) microscopy. The RV mitochondrial function was also assessed in RV homogenates using oxygraphy. The MCT cardiomyocytes had increased area, larger [Ca2+]cyto transients, increased Ca2+ store content, and faster trans-sarcolemmal Ca2+ extrusion relative to CON. The MCT cardiomyocytes also had larger [Ca2+]mito transients. STED images detected increased mitochondrial protein abundance (TOM20 clusters per μm2) in MCT, yet no difference was found when comparing mitochondrial respiration and membrane potential between the groups. We suggest that the larger [Ca2+]mito transients compensate to match ATP supply to the increased energy demands of hypertrophic cardiomyocytes.Pulmonary artery hypertension causes right ventricular hypertrophy which rapidly progresses to heart failure with underlying cardiac mitochondrial dysfunction. Prior to failure, there are alterations in cytosolic Ca On the matching . Nonethe2+ fluxes remain poorly understood, primarily due to the challenges of measuring mitochondrial Ca2+ in intact muscle preparations  fluxes. Myocytes were subjected to 1 Hz stimulation until steady state was achieved. At this point, both the flow and the stimulation were switched off, and a 20 mM bolus of caffeine (Sigma-Aldrich) in Tyrode’s solution was applied to the bath to determine Ca2+ store content and sarcolemmal NCX activity. Superfusion with caffeine-free Tyrode’s solution and stimulation at 1 Hz were then re-commenced. The response to β-adrenergic stimulation with 1 mM Ca2+ Tyrode’s solution containing 1 µM isoproterenol  was then determined.Cytosolic Ca2+ measurements were taken from dhRhod-2 loaded myocytes using the same spectrofluorometric system described above. Cells were field-stimulated at 0.1, 0.5, and 1 Hz (room temperature) and continuously superfused with 1.5 mM [Ca2+] Tyrode’s solution containing 1 µM isoproterenol and 150 µM spermine . Myocytes were illuminated with a 542 ± 10 nm excitation wavelength and emitted fluorescence was collected at 581 nm (±10 nm) from whole cells as a measure of mitochondrial Ca2+ fluxes.Mitochondrial CaFor details on fixation and labelling of isolated cardiomyocytes and tissue sections, refer to the Ψ) were measured in a separate cohort of saline and monocrotaline-injected rats . Following rat euthanasia and cardiac excision, an RV sample was processed for high-resolution respirometry coupled to fluorometry with an Oxygraph-O2K™ respirometer . Approximately 20 mg of tissue was homogenised for 10 s in 25 volumes of ice-cold respiration buffer : MgCl2.6H2O (3), K-lactobionate (60), taurine (20), KH2PO4 (10), HEPES (20) and sucrose (110), with essential fatty acid free bovine serum albumin  and adjusted to pH 7.1 with KOH at 37 °C.). RV homogenate (1 mg mL−1) was added in duplicate to each chamber of the oxygraph containing 2 mL of RB prior to the addition of 2 µM safranine-O. ΔΨ was measured by following safranine-O fluorescence using a filter set for excitation/emission wavelengths of 495/587 nm in the oxygraph fluorometers [Ψ. Oxidative phosphorylation (OXPHOS) was then initiated by the addition of 0.1 mM ADP along with 5 mM glucose and 2 U mL−1 of hexokinase to keep mitochondria in a phosphorylating state [2 was added to each chamber followed by 0.25 mM titrations to achieve free [Ca2+] from 0.39 µM to 52 µM . Then, 2 µM carbonyl cyanide p- (trifluoro-methoxy) phenyl-hydrazone (FCCP) was added to achieve a zero-membrane potential for safranine-O calibration. To calculate the ΔΨ, the Nernst equation (Equation (3)) was used as previously described cyto transients for CON RV myocytes  vs. before ISO , whereas MCT myocytes showed no change in [Ca2+]cyto amplitude in response to ISO (1.67 ± 0.16 a.u.) relative to pre-ISO (1.51 ± 0.16 a.u.). However, MCT RV myocytes had larger baseline [Ca2+]cyto transients (1.58 ± 0.46 a.u.) relative to CON RV myocytes  before the introduction of ISO. 2+]cyto transients had faster maximum rate-of-rise in response to ISO (0.09 ± 0.01 a.u. ms−1) compared to pre-ISO . MCT RV myocytes showed no change in maximum rate-of-rise in response to ISO; yet the maximum rate-of-rise before ISO was faster for MCT cardiomyocytes (0.10 ± 0.11 a.u. ms−1) in comparison to CON (0.06 ± 0.09 a.u. ms−1). In addition, neither CON nor MCT RV myocytes showed a change in the time-to-peak fluorescence in response to ISO (p < 0.01), whereas ISO had no effect on the time constant of decay of MCT RV cardiomyocytes.Cytosolic Cae to ISO C. ISO ree to ISO D from th2+ store content was assessed by addition of a 20 mM caffeine bolus to the solution bathing the CON and MCT RV cardiomyocytes. 2+]cyto transient recorded from a single CON RV myocyte. The amplitude of the caffeine transient (340/380 ratio) in the absence of stimulation gave a measure of total [Ca2+]SR, while the time constant of the caffeine [Ca2+]cyto transient decay in the continued presence of caffeine gave a measure of trans-sarcolemmal Ca2+ efflux. 2+]cyto transients (0.78 ± 0.06 a.u.) in comparison to the CON RV myocytes . The MCT RV myocytes also showed a decreased time constant of decay (p < 0.05).SR Ca2+ transients (2+ transient ([Ca2+]mito) amplitude . 2+]mito and time-to-peak fluorescence in MCT RV myocytes relative to CON RV myocytes at any stimulation frequency. The MCT RV myocytes had a time-to-peak fluorescence of 0.201 ± 0.015 s at 0.1 Hz, and 0.206 ± 0.021 s at 0.5 Hz, relative to the CON RV myocytes, which had a time-to-peak fluorescence of 0.165 ± 0.011 s at 0.1 Hz and 0.166 ± 0.010 at 0.5 Hz , with no difference in the mitochondrial area between CON (37.2 ± 1.0%) and MCT  per myocyte. The ratio of the mitochondrial area relative to the F-actin area was therefore less for MCT RV myocytes (p < 0.001), whereas mitochondria and F-actin occupied equal areas of the myocytes in CON sections (p = 0.11).The area of cardiomyocyte occupied by mitochondria relative to the area occupied by the myofilaments was compared in longitudinal sections from healthy and hypertrophic RV tissue sections. 2) relative to CON RV myocytes . Despite smaller TOM20 cluster sizes, the MCT RV myocytes had an increased number of TOM20 clusters per µm2 (2 regions) vs. CON RV myocytes . The MCT RV myocytes also showed smaller RyR2 cluster size (2) in comparison to CON  but with no difference in the number of RyR2 clusters per 1 µm2 between groups (p = 0.2).Mitochondrial and RyR2 distribution were also compared in longitudinal RV tissue sections between groups using confocal and STED microscopy . The lar2 consumption and mitochondrial membrane potential were recorded in RV homogenates in the oxygraph. The driving force for ATP production via oxidative phosphorylation is from the electrochemical potential across the inner mitochondrial membrane, the majority of which is made up of the mitochondrial membrane potential (ΔΨ). This was measured using ΔΨ fluorescence indicator safranine-O with increasing free [Ca2+]. Ψ is developed which was partially depolarised following the addition of ADP, with hexokinase and glucose, which regenerates ADP to reach a steady-state ΔΨ. An initial titration of 0.3 mM CaCl2 (free [Ca2+] of 0.39 μM) depolarises the ΔΨ and stimulates the O2 flux; however, further titrations of CaCl2 result in the inhibition of O2, and ΔΨ repolarises until opening of the mitochondrial permeability transition pore (mPTP) and depolarisation of the ΔΨ. This can be accelerated by the addition of FCCP. Mitochondrial respiration was not different between CON and MCT RV homogenates for all respiratory states before or after the addition of CaCl2  stimulatory effect of respiration. The developed ΔΨ did not differ between the groups and the addition of ADP and CaCl2 similarly depolarised the ΔΨ.To test for any difference in mitochondrial function between CON and MCT hearts, concurrent measurements of Oof CaCl2 B. After 2+ were studied in hypertrophic cardiomyocytes using the monocrotaline (MCT) rat model of pulmonary artery hypertension (PAH) and their saline-injected controls (CON). Morphometric data confirmed that MCT rats four weeks post-injection were not in heart failure, and there was no difference in heart weight to body weight between CON and MCT animals on the day of experimentation. This was unexpected as the hypertrophic RV was expected to increase the mass of the whole heart. However, LV atrophy has been previously reported in MCT myocardium cyto in CON RV myocytes (2+]cyto transients in the absence of ISO relative to CON (2+]cyto (2+ transient kinetics during ISO (maximum rate-of-rise (2+]cyto transient amplitudes from MCT in comparison to CON (in the absence of ISO) is unlikely to be a result of endogenous β-AR activation in isolated cells stored in the physiological buffer following isolation. Instead, we suggest that the diminished MCT response to ISO might be a result of the SR Ca2+ stores already being at capacity prior to β-AR stimulation. A decreased response to ISO can also be due to the distribution of β-ARs, whereby spatially diverse cAMP diffusion occurs depending on the receptor subtype that is activated. For example, activation of β1-AR induces cell-wide cAMP diffusion and PKA activation, whereas the β2-AR subtype has been shown to be present specifically in the T-tubules, inducing a more localised cAMP diffusion/PKA activation cyto  were una-of-rise B and tim-of-rise D). Overativation . MCT-indtivation ,27, whicn  concentration to the diastolic level cyto, such as at the peak of the [Ca2+]cyto transient during EC coupling mito) amplitudes at 0.1 Hz and 0.5 Hz stimulation in comparison to CON RV cardiomyocytes  it is likely that any uptake would be through other uptake pathways that are not fully resolved  triggers opening of the mPTP and irreversible depolarisation of the ΔΨ, causing cardiomyocyte death mito uptake and increased mitochondrial abundance in hypertrophic cardiomyocytes. We conclude that this provides a potential mechanism by which hypertrophied cells compensate to match ATP supply to the increased energetic demands of additional contractile protein and bigger Ca2+ transients, prior to progression to heart failure.Overall, MCT RV cardiomyocytes showed increased cytosolic Ca


In this review, we first discuss the structure of the Sec61 complex and focus on the pharmacology and regulation of the Sec61 complex as a Ca2+-leak channel. Subsequently, we will pay particular attention to pathologies that are linked to Sec61 mutations, such as plasma cell deficiency and congenital neutropenia. Finally, we will explore the relevance of the Sec61 complex as a Ca2+-leak channel in various pathophysiological  and pathological  settings.The heterotrimeric Sec61 protein complex forms the functional core of the so-called translocon that forms an aqueous channel in the endoplasmic reticulum (ER). The primary role of the Sec61 complex is to allow protein import in the ER during translation. Surprisingly, a completely different function in intracellular Ca While the RyR is expressed in a predominant way in only a limited number of tissues, especially skeletal and cardiac muscle as well as the brain  that are activated by IP3 and that additionally are exquisitely regulated by Ca2+ itself, by phosphorylation/dephosphorylation processes and by multiple protein-protein interactions (The endoplasmic reticulum (ER) forms the largest intracellular Ca signals . Membershe brain , the IP3xpressed . The latractions .2+-leak channels that, depending on the cell type and the intracellular milieu, modulate the basal permeability of the ER for Ca2+  pumps can no longer compensate for the Ca2+ leakage, thereby leading to decreased filling of the ER Ca2+ store. Second, if the latter occurs or if the endogenous leak activity is anyway exceeding the capacity of the SERCA pumps, the setpoint for the ER Ca2+ concentration ([Ca2+]ER) will lower and thus directly or indirectly lead to decreased IP3-dependent Ca2+ release. Finally, highly active Ca2+-leak channels may themselves produce small Ca2+ signals interfering in a positive or negative way with intracellular Ca2+ signaling.Less well understood, though physiologically at least even important, are the so-called Cafor Ca2+ . These Cple ways . First, 2+-leak channels form a large though very heterogenous group of proteins that share as their only common characteristic, their potency to increase the permeability of the ER membrane for Ca2+ (2+ handling such as IP3R or RyR. Dysfunctions can originate from (excessive) post-translational modifications, protease-mediated cleavages or mutations. Ca2+ signaling events such as Ca2+ puffs and sparks can also be considered to result from Ca2+-leak activity, although the channels involved  are not dysfunctional, and their activity strongly depend on the local environment, especially [Ca2+]cyt and [Ca2+]ER . Its primary function is of course related to protein import in the ER during translation , allowing the proteins to cross or insert into the ER membrane  is one of the major ER chaperones that assists nascent proteins in the folding process  leading to a decrease in protein synthesis and an increase in chaperone expression . In paraThe transient nature of the many interactions at the Sec61 complex level made it difficult to determine its structure. Even in the case of the Sec61 complex core, the composition, architecture, and mechanism of operation remain difficult to determine. However, technical advances, particularly in the field of cryo-electron microscopy, have made it recently possible to increase the understanding of the heterotrimeric Sec61 complex.in vivo , TRAP and OST . A seconin vivo .The main feature of the ER Sec61 complex is the existence of an aqueous pore across the membrane. Thanks to experiments using puromycin treatment, the Sec61 complex has been seen as a channel gated by the ribosome. Indeed, these ER transmembrane pores were detected by conductivity measurements, and the dependence of these channels on puromycin suggested that the nascent polypeptide chain must be released from the ribosome to allow the transmembrane passage of ions . Since tSince the lumen of the ER, and not the cytoplasm, forms a continuum with the Sec61 complex pore upon protein translocation, the ribosome itself must form the permeability barrier by strongly binding the cytoplasmic surface of the Sec61 complex and sealing the pore from there .et al. , P (peptidyl) and E (exit). These three sites are located at the junction between the ribosomal subunit and the tRNA . Indeed, leakage .in vitro system , we determined that puromycin induced Ca2+ leakage through the Sec61 complex independently of IP3Rs or RyRs  to enhance Ca2+ release. Altogether, these data highlight the role of the Sec61 complex as a major Ca2+-leak channel in the LNCaP cell model. In addition, it is well known that ER Ca2+ depletion can activate SOCs (store-operated channels). Interestingly, we have also demonstrated that Ca2+ release that occurs via the Sec61 complex activates SOCs. This was the first characterization of store-operated Ca2+ entry triggered by passive Ca2+-leak channels  in human primary macrophages. Altogether, this work clearly indicates that the Sec61 complex is not only an ER Ca2+-leak channel but also acts, at least in macrophages, as a regulator of ER Ca2+ content and of the [Ca2+]cyt at resting state.In addition to these studies, we should mention the interesting work of Layhadi et al. . They hi2+-leak channel in several cell types. Nevertheless, the Ca2+-leakage rate depends on the dynamics of plug binding/dissociating the pore. An important question, therefore, is which factors will control Ca2+ leakage?Taken together, various studies have demonstrated that the Sec61 complex is a functional ER Ca2+-leak channel would in the long run form a hazard for the cell as both ER Ca2+ levels and cytosolic Ca2+ levels control many aspects of cell function, including the occurrence of ER stress and/or the induction of cell death  are particularly prone to experience increased Ca2+ leakage out of the ER, potentially leading to apoptosis .In conclusion, BiP, the luminal plug of the Sec61 complex, might attach and detach from the pore with particular dynamics determining the Ca2+-binding protein. It contains four EF-hand motifs with an affinity for Ca2+ in the physiological range (Kd’s between 0.5 and 5 µM), making CaM ideally suited as an intracellular Ca2+ sensor  connected by a short intraluminal loop .2+ leakage out of the ER and reduced cell viability while increased Sec62 expression correlated with protection against thapsigargin-induced apoptosis  in the C terminus of Sec62, increased thapsigargin-induced Ca2+ leakage out of the ER. Taking into account that Sec62 silencing and treatment with CaM antagonists lead to the same phenotype, the authors propose a model in which Sec62 binding to Sec61 is relieved by Ca2+, allowing Ca2+/CaM to occupy its binding site and thus to inhibit ER Ca2+ leakage through the Sec61 complex. Obviously, another possible mechanism would involve the recruitment of BiP at the luminal side, subsequent to Sec62/Sec63 binding.Analysis of various prostate cancer cell lines demonstrated that reduced Sec62 protein levels lead to larger Capoptosis . These rf Sec61α . Moreove2+ leakage by multiple Ca2+-dependent proteins may indicate the importance of keeping Ca2+ leakage under tight control, or may indicate that depending on the situation, e.g., the presence or absence of ER stress, the cell relies on a different mechanism for controlling Ca2+ leakage out of the ER.Taken together, the regulation of Sec61 complex-mediated Ca2+ handling. Interestingly, however, a number of those mutations were determined to impact ER Ca2+ leakage in mice  is a group of diseases of various origins that are generally characterized by impaired B-cell differentiation/function, resulting in low levels of antibody production and, consequently, recurrent infections. Two mutations in SEC61A1, a heterozygous missense mutation (V85D) and a nonsense mutation (E381*), have been linked to CVID . SEC61A12+ store compared to wild-type SEC61A1. This was similar to the depletion observed when expressing in the same cells SEC61A1-V67G or SEC61A1-T185A, two heterozygous missense mutations already shown to result in autosomal-dominant tubulo-interstitial kidney disease (ADTKD)  forms another genetically heterogeneous group characterized by differentiation arrest in the formation of granulocytes. Mutations in over 20 genes are already involved in this pathology, while for many patients the responsible mutation has not yet been characterized. In a recent study, the point mutation Q92R in SEC61A1 was identified in a patient with severe congenital neutropenia . Express (ADTKD) . AdditioInterestingly, the ADTKD patients expressing the V67G mutation also displayed some neutropenia . Similar2+ leakage  are thus far all located in or near TMD2  should always be used in these types of studies. Since the effect of one molecule blocks that of the other, their use allows us to rule out any non-specific action that would impede the correct interpretation of the results. The study by Hammadi et al. , leading to cell death  protocols, mimicking I/R at the cellular level. In these cells, the Sec61 complex was first shown to function as a Ca2+-leak channel. In cardiomyocytes, it is well known that during excitation-contraction coupling, type 2 RyRs are involved in Ca2+ release via a Ca2+-induced Ca2+ release mechanism. Interestingly, Sec61 complex activation mobilizes a RyR-independent Ca2+ pool that affects neither contraction nor RyR-dependent Ca2+ stores (2+ stores: a puromycin-sensitive Ca2+ pool where Sec61 complexes are located (probably the ER) and a caffeine-sensitive Ca2+ pool containing RyR channels, located in the SR. Pharmacological pre-activation of the Sec61 complex with puromycin induces a preventive ER Ca2+ release from RyR-independent stores that consequently decreases the rate of Ca2+ increase in the cytosol as well as mitochondrial Ca2+ overload and mPTP opening during hypoxia. These data explain how puromycin, applied before H/R (pre-conditioning), significantly reduces cell death. In vivo cardioprotective experiments show that pharmacological modulation of the Sec61 complex (pre-conditioning) protects the mouse heart from I/R injury and reduces infarct size after I/R logical settings.From the above it thus appears that the Sec61 complex plays an important role in intracellular Caside see  and by Cside see . These r2+ content results from the dynamic equilibrium existing between Ca2+ release from the ER and its reuptake by SERCA pumps. The activity of both the Ca2+-release channels and SERCAs is finely regulated and depends to a large degree on [Ca2+]cyt and [Ca2+]ER  incapacitates Ca2+ reuptake, uncovering the  activity of the ER Ca2+-leak channels. How does Ca2+ leakage via the Sec61 complex now fit in ER Ca2+ homeostasis? To answer this question, we can consider two scenarios.As mentioned above, the ER Ca[Ca2+]ER , so that2+ pool. This is, for example, the case in the human prostate cancer cell line LNCaP. In this cellular model, it has been shown that the acute activation of the Sec61 complex by puromycin induces partial emptying of a non-compartmentalized Ca2+ store. The rate of Ca2+ release is slower than that of IP3R- or RyR-mediated Ca2+ release  and SERCA reuptake activity. One element to consider is that during the acute action of puromycin (or emetine), only the Sec61 complexes involved in translation are sensitive to puromycin. Complexes that do not participate in translation remain impermeable to Ca2+. During chronic puromycin or emetine perfusion, their concentration should therefore be chosen so that it does modulate the ER Ca2+ levels without any effect on translation  or gluco-lipotoxicity (T2D)), it will be to promote cell survival. In the case of cancer, it would therefore likely be necessary to use Ca2+-leakage inducers, which could be used in combination with existing therapies to act preferentially on cancer cells rather than on healthy cells. We assume that a small dose of these molecules could already tip the scales towards cell death. In the case of T2D or stunned myocardium, however, it is likely that longer-term treatments would be more effective. Conversely, in the framework of cardiac I/R in a mouse model, a puromycin bolus has already shown its efficacy, both in vivo and in vitro.From all these studies, what therapeutic conclusions, based on pharmacological modulation of Ca2+ leakage in additional pathological situations. Since the basis of neurodegenerative diseases lies in the accumulation of unfolded proteins and Ca2+ dysregulation, neuroprotective strategies involving the regulation of Ca2+-leak channels in general and the Sec61 complex in particular will probably emerge. Obvious possible candidates include neurodegenerative diseases such as Parkinson’s disease, Alzheimer’s disease, Huntington’s disease, and Creutzfeld-Jacob disease. The role of intracellular Ca2+ in general and of ER Ca2+ in particular logical role.Taken together, these findings strengthen the evidence that Sec61 complex-mediated Ca


A promising approach is to augment the anti‐tumour immune response in these patients; most cancers do not activate immune effector cells because they express immunosuppressive ligands. We have previously shown that CD200 (an immunosuppressive ligand) is overexpressed in AML and confers an inferior overall survival compared to CD200low/neg patients. Here we show that a fully human anti‐CD200 antibody (TTI‐CD200) can block the interaction of CD200 with its receptor and restore AML immune responses  High AML patients exhibited reduced Natural Killer (NK) and T cell immune responses in comparison to CD200Low patients, indicating that CD200 is a potential immunotherapeutic target in this disease. Our results also suggested that CD200 can be a contributing factor responsible for AML immune evasion and therapy relapse.Acute myeloid leukaemia (AML) is a highly heterogeneous disease in which leukaemic stem cell (LSC) persistence is considered to be the primary cause of relapse.+), by co‐culturing them with normal human NK cells . As expected, diminished expression of the NK degranulation marker CD107a was observed for K562‐CD200+ compared to K562‐CD200‐ cells , compared to isotype control , which are central to adaptive immune responses in AML.High AML patients. As expected, an increase in frequency of IFN‐γ‐producing CD4+ Th1 cells was observed in the presence of TTI‐CD200  (NSG) mice was required. Cytokine Induced Killer (CIK) cells are an adoptive immune cellular therapy which had shown promising pre‐clinical and clinical efficacy in AML, without causing graft‐versus‐host‐like disease which is normally observed with adoptive transferred human T cells in immuno‐deficient mice.+CD3+ and CD56‐CD3+). CD200R expression was detectable in these populations at day 10 of culture . SB, MH, AFG assisted with patient AML blast isolation. SM and SJC provided resources and edited the paper. SK, RLD and AT secured funding and contributed to experimental design, data analysis and co‐wrote the manuscript.NR designed and performed Figure S1. TI‐CD200 antibody inhibits CD200‐mediated suppression with nanomolar potency. The NK‐sensitive target cell C1R expressing cell surface CD200 was incubated with titrated TTI‐CD200 antibody or isotype control for one hour and then co‐cultured with a human NK.High AML cells in vitro.Figure S2. TTI‐CD200 improves immune responses in CD200Figure S3. Expression of CD200 receptor on CIK cells.in vitro.Figure S4. TTI‐CD200 treatment enhances CIK‐mediated lysis in AML cells Figure S5. CD200‐blocking increases CIK‐mediated cell lysis in AML blasts.Click here for additional data file.


The sizable transient changes in Ca2+ concentrations are necessary for the activation of signaling pathways, which rely on Ca2+ as a second messenger, including those involved with force generation, fiber type distribution and hypertrophy. However, prolonged elevations in intracellular Ca2+ can result in the unwanted activation of Ca2+ signaling pathways that cause muscle damage, dysfunction, and disease. Muscle employs several calcium handling and calcium transport proteins that function to rapidly return Ca2+ concentrations back to resting levels following contraction. This review will detail our current understanding of calcium handling during the decay phase of intracellular calcium transients in healthy skeletal and cardiac muscle. We will also discuss how impairments in Ca2+ transport can occur and how mishandling of Ca2+ can lead to the pathogenesis and/or progression of skeletal muscle myopathies and cardiomyopathies.In healthy muscle, the rapid release of calcium ions (Ca Innately, Ca2+ has flexibility in its bonding angles and lengths, allowing for an array of potential ligation patterns [2+-binding molecules [2+ signaling becomes apparent as it can be regulated by the location and expression of the Ca2+-binding proteins and the kinetics of the Ca2+-binding site.Calcium  can fluctuate from resting concentrations of 100 nM to values above 1000 nM during tetanus [2+]cyt is elevated above resting concentrations, Ca2+ interacts with two categories of Ca2+ binding molecules: buffers and sensors [2+ sensors elicit a downstream signal when binding occurs. The temporal range of Ca2+ signaling can be as brief as seconds but may also exist on the timescale of days [2+ signaling is effectively diminished when [Ca2+]cyt reverts back to resting concentrations. One strategy for increasing the rate of [Ca2+]cyt decay is through Ca2+ buffering. Buffers act to sequester Ca2+ without being directly incorporated into molecular signaling. The presence of Ca2+ buffers allows for the regulation of Ca2+ diffusion, which can indirectly affect molecular signaling pathways cyt is mediated primarily through the movement of Ca2+ across phospholipid membranes [2+]cyt is through Ca2+ sequestration in the sarcoplasmic reticulum (SR), a membrane bound organelle which surrounds the contractile myofilaments cyt but also in the regulation of the energy expenditure of the cell [2+ is also extruded across the plasma membrane to effectively reduce [Ca2+]cyt during the cardiac cycle cyt can result in the unwanted activation of proteolytic and apoptotic pathways, leading to muscle damage, dysfunction, and even disease. In this review, we discuss the role of Ca2+ transport in the maintenance of healthy muscle as well as the role it can have in the genesis and exacerbation of pathological states.Although Caembranes . Within ilaments . Ca2+ isdrolyzed ,10. SERCthe cell . Within ac cycle . The rol2+ release cyt [2+]cyt results in the immediate activation of SERCA, which acts to pump Ca2+ back into the SR. However, with the continuance of high frequency neural signaling the rate of Ca2+ outflow is greater than the ability for SERCA to re-sequester Ca2+. Consequently, [Ca2+]cyt rises within the myofiber and binds to troponin C resulting in the movement of tropomyosin and thus uncovering the myosin binding site on the thin filaments cyt begins.The RyR is a CaCa2+]cyt . Within ilaments ,17. Withilaments ,19. Musc2+]cyt to a peak is quickly followed by a decay, all within milliseconds [2+]cyt has a negative exponential relationship in which the initial rate of decay is rapid but as [Ca2+]cyt approaches pre-stimulation resting concentrations the rate of calcium uptake is attenuated. Within skeletal muscle tissue, the characteristics of a Ca2+ transient varies across different types of muscle fibers. Baylor and Hollingworth (2003) compared the ICTs of slow fibers from soleus tissue and fast fibers from EDL muscle [2+]cyt during Ca2+ release in the slow and fast fibre types; however, the peak [Ca2+]cyt amplitude was two times greater and the ICT half duration was ∼1.6 times shorter in fast EDL fibers compared with the slow soleus fibers cyt, PLN dissociates from SERCA and does not affect Vmax cyt is elevated, CAMKII will detect these elevations and respond by phosphorylating PLN, thus relieving its inhibitory effect on SERCA and allowing for faster calcium clearance cyt. MLN, on the other hand, is believed to be another inhibitory regulator of SERCA cyt rises due to SR Ca2+ release, the membrane potential effect becomes negligible, and the direction of the exchanger now changes to the forward mode with Ca2+ being extruded from the cytosol [+] and [Ca2+] can impact the direction of the exchanger, under physiological conditions, the direction is largely driven by the [Ca2+]cyt  to potentially help to overcome Na+-dependent inactivation by increasing the electrostatic potential [+ levels and works independent of the [Na+]cyt levels and (2) secondary or slow blockade that is dependent on [Na+]cyt levels, which enhance the affinity of NCX for H+ cyt during repeated muscle contractions when [Ca2+]cyt is very high cyt [2+ at an optimal ratio of 2 mol Ca2+:1 mol PV [2+ at resting [Ca2+]cyt (<100 nM) [2+ is dependent on the rate of dissociation of Mg2+ from the binding sites [2+]cyt, which is why it is often considered a slow Ca2+ buffer cyt ,111,112.Ca2+]cyt . In contCa2+]cyt . FurtherCa2+]cyt . PV is a1 mol PV ,116. The<100 nM) . The ratng sites . Consequ+ buffer . The binl muscle ,120. Howl muscle . Taken t2+ buffering, there is evidence showing that Ca2+ uptake through the mitochondrial Ca2+ uniporter is important for the maintenance of myocyte homeostasis [2+ uptake in cardiomyocytes does not alter [Ca2+]cyt transients or cardiac contractility, there is evidence that mitochondria can modulate [Ca2+]cyt in fast-twitch skeletal muscle fibers under certain conditions cyt. The first theory posits the lack of dystrophin results in transient tearing of the sarcolemma membrane during contractions. The membrane damage could allow for an influx of extracellular Ca2+ into the myofiber [2+ influx into the myofiber [2+]cyt can increase proteolytic activity and the production of ROS [2+ dysregulation [2+]cyt alone are enough to promote a shift toward a DMD like phenotype [2+]cyt by overexpressing SERCA in dystrophic tissue, which ameliorated the pathology cyt and decreased muscular degeneration and fibrosis cyt is decreased in these cells [2+]cyt cyt ,186.2+ release with HF could be due to defective coupling between the RyR and DHPR, reduced SR Ca2+ content could also contribute. This was assessed in one study using a canine model of HF by assessing the CICR gain, which is the amount of Ca2+ released from the SR for a given level of current density from the DHPR channel and ε, which is the effectiveness of coupling between DHPR activation and SR Ca2+ release cyt and inducing relaxation of the myocardium during diastole. Several studies have shown that SERCA Ca2+ uptake is reduced in failing hearts from both human and animal models cyt, it can actually increase the probability of arrhythmogenic events. In failing hearts, with increasing frequency of stimulation, the intracellular Na+ ([Na+]cyt) levels rise [+/H+ exchanger on the sarcolemma [+/K+ ATPase activity [2+]cyt and SERCA uptake of Ca2+ into the SR would also increase leading to SR Ca2+ overload cyt during E-C coupling must be rapidly lowered to re-establish resting concentrations and induce muscle relaxation, which is primarily accomplished by SERCA-mediated Ca2+ transport into the SR. During short contractions of fast-twitch rodent and amphibian myofibers, the cytosolic Ca2+ buffer PV also contributes to muscle relaxation by rapidly lowering [Ca2+]cyt. In the heart, Ca2+ extrusion from cardiomyocytes through the NCX contributes significantly to ventricular relaxation, and the PMCA and MCU also contribute to a very minor extent. Impairments in Ca2+ transport and dysfunctional Ca2+ homeostasis has consistently been shown to be involved with the pathogenesis and/or the progression of multiple myopathies within skeletal muscle and in HF. A better understanding of the function and regulation of muscle Ca2+ transport proteins can allow for greater insights into skeletal and cardiac muscle physiology and disease. Furthermore, this body of work can lead to novel approaches in the treatment of diseases involving Ca2+ dysregulation.Maintenance of Ca


For effective transmission of excitation in neuromuscular junctions, the postsynaptic response amplitude must exceed a critical level of depolarization to trigger action potential spreading along the muscle-fiber membrane. At the presynaptic level, the end-plate potential amplitude depends not only on the acetylcholine quanta number released from the nerve terminals in response to the nerve impulse but also on a degree of synchronicity of quanta releases. The time course of stimulus-phasic synchronous quanta secretion is modulated by many extra- and intracellular factors. One of the pathways to regulate the neurosecretion kinetics of acetylcholine quanta is an activation of presynaptic autoreceptors. This review discusses the contribution of acetylcholine presynaptic receptors to the control of the kinetics of evoked acetylcholine release from nerve terminals at the neuromuscular junctions. The timing characteristics of neurotransmitter release is nowadays considered an essential factor determining the plasticity and efficacy of synaptic transmission. Following the activation of motoneuron, acetylcholine (ACh) released from the presynaptic nerve terminals initiates a depolarization of the muscle end plate, which triggers an action potential causing muscle-fiber contraction. Vesicular or “quantal” hypothesis of ACh release introduced by B. Katz presumes that the nerve impulse evokes the release of tens (warm-blooded) to hundreds (cold-blooded) of ACh quanta from the motor nerve terminals at the neuromuscular junction ,2. This Computational analysis of the synaptic delay distribution  at the frog neuromuscular junction showed that it can be represented as the sum of normal and exponential distributions, and this suggests a two-stage mechanism of early and late periods of phasic synchronous secretion . InitialThe time course of ACh quanta release depends differently on the motor nerve stimulation pattern and on calcium ion entry into the nerve terminals. Under physiological calcium levels, ACh quanta, which form multiquantal postsynaptic response, are also released with different latencies. Their distribution is obtained by different computational methods from multiquantal end-plate currents and spontaneous miniature end-plate currents ,14,15,16Before the consideration of the role of presynaptic ACh receptors (AChRs) in the modulation of the time course of quantal release, we would like to point out the significance of the evoked ACh release kinetics for neuromuscular transmission. Certainly, the main factor defining the efficiency and reliability of the neurotransmission is the number of ACh quanta released in response to a nerve action potential, i.e., quantal content. This parameter determinates the peak amplitude of the multiquantal end-plate potential (EPP) and if the amplitude exceeds a critical level of depolarization, then the EPP induces the muscle-fiber action potential, and eventually the muscle-fiber contraction occurs. In the late 1960s, Stevens introduced the stochastic approach for the kinetics analysis of quanta released following an impulse at motor nerves . In the Neuromuscular synapses of vertebrates contain presynaptic AchRs, which implement a modulatory action of ACh on its own release. Despite a long history of studies showing the effects of both AChR activators and blockers on quantal ACh secretion ,26,27,28Our investigations have shown that exogenous ACh and its nonhydrolyzable analog carbacholine affect not only the quantal content but also the synaptic delay dispersion of the uniquantal end plate currents (EPCs) at the frog neuromuscular junction ,35. Both2+ concentration, a typical for fast motor units’ high-frequency firing), a modification of the ACh time-course release might be one of the general presynaptic mechanisms that contributes to synaptic plasticity during motor-neuron activity in vivo. Since ACh and carbacholine can activate both nAChRs and mAChRs, pharmacological analysis using specific agonists and antagonists is necessary to determine the type of cholinergic receptors that modulate the kinetics of neurosecretion.Under physiological conditions, neuromuscular junction operates in a rhythmic activity mode with transmission of frequency from 10 to 100 Hz. Endogenous ACh released from nerve terminals can affect presynaptic AChRs, causing changes in kinetics of ACh quanta secretion. Our study on the kinetics of ACh quanta release during rhythmic stimulation in frog synapses showed that the motor-nerve stimulation at 10–100 Hz increased the rise time of the EPCs . The higThere are a large amount of data about the effects of nAChRs agonists and antagonists on the ACh quanta number released in response to nerve stimulus ,25,26. NMuch more information is available about the contribution of presynaptic muscarinic receptors that modulate the evoked secretion of ACh quanta ,28. Diffcutaneous pectoris muscle [Immunohistochemical staining with specific antibodies enables the detection of distinct receptor subtypes in synaptic regions. Santafe et al. revealed the immunoexpression of all five (M1-M5) currently identified mAChR subtypes in the rat neuromuscular junction ,33. Tsens muscle . The div2+/high Mg2+ solution at the frog neuromuscular junction. Selective antagonists of M1, M2, and M4 mAChRs have prevented the synchronizing action of muscarine on ACh secretion [Activation of mAChRs by muscarine, which is an agonist for all mAChRs, induced the shortening distribution of “real” synaptic delays of EPCs recorded in a low Caecretion .2+ ions’ input into the nerve terminals [There are also data on the influence of mAChR inactivation on the kinetics of ACh secretion. Slutsky et al. showed that methoctramine, a selective M2/M4 antagonist of mAChRs, caused longer synaptic delays under a low temperature and slowed down the rise time of the EPCs at the frog neuromuscular junction ,43. Howeerminals ,39,43.At a later date, it was shown that muscarine decreased the quantal content of the multiquantal EPCs and synchronized the quanta secretion . The depThese data are different from I. Slutsky’s observation that the blockade of mAChRs led to desynchronization of the release of ACh quanta at the rodent’s neuromuscular junctions . In the 2+ sensor, is located near to presynaptic voltage-gated Ca2+ channels [The molecular mechanisms for regulation of quantal release time course are not fully elucidated. The ACh release synchronicity depends on the state of the vesicle exocytosis machinery. The critical factors for quanta release are the accessibility of synaptic vesicles from the ready releasable pool, the activity of exocytosis proteins, and the proximity of the synaptic vesicle to the active zone, where vesicular protein synaptotagmin I/II, fast Cachannels ,45.Based on the current experimental data, the several most probable stages of mechanisms controlling synaptic-vesicle exocytosis can be identified. These steps, individually or in combination, may be involved in the regulation of the kinetics of evoked ACh secretion:2+ entry into the axoplasm. It is known that an increase in the concentration of Ca2+ in the nerve terminal initiates a fusion of synaptic vesicles with the presynaptic membrane. Therefore, a higher Ca2+ should accelerate the rate of the release reaction and result in short synaptic delays [2+ ions both in the extracellular environment and in the axoplasm [2+-dependent pathways. A high level of the ACh-release asynchronicity was observed at the developing neuromuscular junctions [2+ channels, which trigger exocytosis. Mathematical analysis of quanta-release kinetics showed that under low Ca2+ input and fixed endogenous Ca2+ buffers , a more asynchronous ACh quanta release should occur. If in this system a mobile Ca2+ buffer (with a high diffusion capacity) is added, the dispersion of synaptic delays decreases and ACh release becomes more synchronous [2+ profile near the active zone can change the synaptic delay dispersion in the dependence on either extracellular Ca2+ concentration or intracellular Ca2+ buffer activity [2+ entry leads to the rise in the peak rate of vesicle exocytosis and ACh release becomes more synchronous [2+ dynamics within the active-zone regions is the most potent mechanism for regulation of the synaptic delays and quanta secretion synchronicity. It is important to note that the presynaptic AChRs activation induce intracellular Ca2+ modulation [(i) The synchronization of neurosecretion from motor-nerve terminals depends on Cac delays ,47,48. Taxoplasm ,47,49. Oaxoplasm , temperaaxoplasm ,50, as waxoplasm , can infunctions , charactchronous . This coactivity . The augdulation .2+ concentration in the vicinity of the active zone can be changed by several mechanisms: (a) direct Ca2+ influx through the nAChRs; (b) Ca2+ input via voltage-gated Ca2+ channels that can be activated due to the nAChR-mediated depolarization of the presynaptic membrane; (c) Ca2+-induced Ca2+ release from the endoplasmic reticulum [2+ described above, one would expect an increase in synchronicity of the ACh quanta secretion upon nicotinic AChR activation. However, we have observed an increase in the dispersion of synaptic delays under nicotine treatment [(ii) Nicotinic AChRs are ligand-gated cation channels. Following the activation of the presynaptic nAChRs, Caeticulum . Accordireatment .2+-induced Ca2+ release and/or voltage-gated Ca2+ channels. Ca2+ input through nAChRs can positively modulate the activity of L-type Ca2+ channels in the neuromuscular junctions [2+ channel is responsible for a higher degree of secretion asynchrony in the neuromuscular synapses of newborn rats [2+ influx through L-type channels can regulate a number of signaling enzymes as well as ion channels, which finally affects the state of exocytotic machinery.As an alternative option for the nAChR participation in the regulation of the secretion kinetics, we have to take into account that nAChRs can be differentially coupled to Caunctions ,55. Thisorn rats ,56 as weorn rats . Ca2+ in2+ entering through both the ion channels of the AChRs and voltage-gated Ca2+ channels located near to the AChRs. The computer simulation showed that an increase in the distance between the voltage-gated Ca2+ channels and synaptic vesicles or the removal of a significant number of Ca2+ channels from the active zone led to a less pronounced increase in synaptic delay, i.e., to a lesser degree of asynchrony of the ACh secretion. Moreover, the synaptic delay is sensitive to alterations in the spatiotemporal Ca2+ dynamics near the active zone at the time of release. These alterations can be caused by manipulations of the density and localization of voltage-gated Ca2+ channels or the concentration of Ca2+ buffering proteins [One is clear that synaptic delay is dependent on the interaction between Caproteins  as well proteins . Thus, d2+ level or widening the presynaptic action potential [It should be noted that there is a relatively new hypothesis about the participation of postsynaptic nAChRs in modulating both the quantal content and time course of ACh secretion. This mechanism is related to homeostatic synaptic plasticity . A partiotential .2+-regulated exocytosis. In turn, SNARE proteins, synaptotagmin, and the Ca2+ channels can be involved in the regulation of ACh release kinetics [(iii) mAChRs are metabotropic, G protein-coupled receptors. As we described above, their subtypes regulate both the quantal content and the neurotransmitter release kinetics. mAChRs can interact with the exocytotic machinery, which includes the proteins implicated in the control of fast Cakinetics ,61.2+ [2+ influx and activity of intracellular enzymes, particularly PKC and PKA [M2 and M4 subtypes of mAChRs are coupled to the Gi/o proteins and inhibit an activity of adenylyl cyclase. Early, it was shown that an increase in intracellular cAMP concentration results in the synchronization of ACh quanta release . M2 and 2+ . mAChRs  and PKA ,64. In n and PKA . Activat and PKA .2+ channels–PKC ensemble [A detailed investigation by J. Tomas’s laboratory showed that the M1 and M2 types of mAChRs can induce activation of PDK1, a master kinase that may control other kinases. M1-receptor stimulation increased the interaction of PKCs with the presynaptic membrane and promoted phosphorylation of Munc18-1, SNAP-25, and MARCKS. In contrast, M2 downregulated PKCε through a PKA-dependent pathway, which caused the inhibition of Munc18-1 synthesis and PKC phosphorylation. These results show the existence of a balance between mAChRs, which can regulate the presynaptic PKC and their action on ACh release through the SNARE-SM mechanism  or L-typensemble . Thus, tThe accumulated data indicate that the changes in kinetics of ACh release from the motor-nerve terminals may be mediated by the various types of presynaptic AChRs and complex downstream signaling mechanisms. The kinetics of the changes in quanta release during in vivo activity should be taken into account as a pathway underlying the plasticity in chemical synapses. In addition, the contribution of presynaptic autoreceptors for ACh to shaping the time course of neurosecretion can serve as an additional feedback mechanism for adaptation of neurotransmitter release to the pattern of the nerve activity.2+-induced desynchronization as well as oxidative burst can be greatly augmented by Zn2+, which can aggravate damage of motoneurons in ALS [DTNBP1 encoding dysbindin protein. The decreased abundance of dysbindin causes a desynchronization of neurotransmitter secretion, which reduces the amplitude of evoked postsynaptic response and increases its duration [2+ channels, which is involved in the development of muscle weakness [Changes in the time course of phasic secretion were observed in a wide range of pathological conditions. A marked enhancement of the asynchronous ACh release in combination with decreased amplitude of the EPPs was found in the neuromuscular junctions of model mice with spinal muscular atrophy . Similars in ALS . Increass in ALS . The pros in ALS . It has duration . High asweakness .Taken together, these data give a reason to believe that searching for methods that can affect the time course of phasic synchronous release can lead to the creation of new therapies aimed at overcoming the consequences of synaptic defects .


AZD7442—a combination of 2 human, extended–half-life, SARS-CoV-2–neutralizing monoclonal antibodies (mAbs) (tixagevimab/cilgavimab)—has received US Food and Drug Administration emergency use authorization for COVID-19 prevention in immunocompromised individuals. We evaluated the effect of AZD7442 in prevention and treatment settings in Syrian hamsters challenged with SARS-CoV-2.5 plaque-forming units) in prevention; OR control mAb (5 mg) or AZD7442 (0.5–5 mg) 1 day after IN SARS-CoV-2 challenge in treatment. The impact of AZD7442 on lung viral RNA and pathology and AZD7442 serum levels was assessed on Days 3 and 7 post infection. Body weight was recorded daily through Day 7.Hamsters received intraperitoneal isotype control mAb (2 mg) or AZD7442 (0.002–2 mg) 1 day before intranasal (IN) SARS-CoV-2 challenge  levels were seen with doses ≥0.2 mg AZD7442. Concomitantly, increased serum levels of AZD7442 were observed. By Day 7, infectious virus titer and sgmRNA fell below the level of detection (LOD) at all doses tested. Moreover, AZD7442 at doses ≥0.2 mg protected hamsters from weight loss versus controls. Lung pathology scores  were generally dose dependent, with mean scores of < 2 for AZD7442 versus 10 for controls, indicating less SARS-CoV-2–induced inflammation and alveolar damage in hamsters given AZD7442. Lower AZD7442 doses were associated with mean pathology scores similar to controls. With AZD7442 treatment, infectious virus titers were below the LOD at Day 3 post infection and at Day 7 for sgmRNA, for all doses tested. Mean lung pathology score was <2 for AZD7442 versus 12 for controls. AZD7442 doses ≥0.5 mg protected against weight loss relative to controls.In a SARS-CoV-2 challenge model, AZD7442 administered as prevention or treatment led to significantly lower lung viral loads and improved lung pathology, without weight loss. There was also no evidence that AZD7442 mediated antibody-dependent enhancement of disease or infection.Yueh-Ming Loo, PhD, AstraZeneca: Employee|AstraZeneca: Stocks/Bonds Patrick M. McTamney, PhD, AstraZeneca: Employee Richard Roque, B.S., AstraZeneca: Employee|AstraZeneca: Stocks/Bonds Yingyun Cai, PhD, AstraZeneca: Employee|AstraZeneca: Stocks/Bonds Mark T. Esser, PhD, AstraZeneca: Employee|AstraZeneca: Stocks/Bonds.