A mathematical compartmental closed-cell model of Ca2+ dynamics was developed that accounts for ER-mitochondria Ca2+ microdomains as the µd compartment , Ca2+ influx to/efflux from each compartment and Ca2+ buffering. Varying the distribution of functional receptors in MAMs vs. the rest of ER/mitochondrial membranes, a parameter called the channel connectivity coefficient (to the µd), allowed for generation of [Ca2+]Cytoscillations driven by distinct mechanisms at various levels of IP3 stimulation. Oscillations could be initiated by the transient opening of IP3 receptors facing either the cytosol or the µd, and subsequent refilling of the respective compartment by Ca2+ efflux from the ER and/or the mitochondria. Only under conditions where the µd became the oscillation-driving compartment, silencing the Mitochondrial Ca2+ Uniporter led to oscillation inhibition. Thus, the model predicts that alternative mechanisms can yield [Ca2+]Cyt oscillations in non-excitable cells, and, under certain conditions, the ER-mitochondria µd can play a regulatory role.Upon inositol trisphosphate (IP By imparting frequency- and/or amplitude-dependent information to Ca2+-sensitive kinases and phosphatases, [Ca2+]Cyt oscillations are known to regulate gene expression and, thus, cell function and survival9. Experimental evidence demonstrated that the mitochondria (second largest Ca2+ store), via Ca2+ uptake from and release to the ER, actively participate in shaping and modulating [Ca2+]Cyt oscillations in different cell types12. The evidence was supported by the discovery that the ER and mitochondria form functional complexes called Mitochondria-Associated ER Membranes (MAMs)14. During cell stimulation, Ca2+ concentrations in the ER-mitochondria microdomains ([Ca2+]μd) can reach up to 10-fold higher values than in the bulk cytosol, enough to activate the Mitochondrial Ca2+ Uniporter (MCU) and allow for Ca2+ uptake17. Since the inositol trisphosphate receptor (IP3R) is regulated by Ca2+ in a biphasic manner (stimulatory at low levels/inhibitory at high levels)18 and functional IP3Rs localize preferentially in MAMs20, local Ca2+ uptake by mitochondria at the microdomains has the potential to either increase ER Ca2+ release by alleviating the Ca2+-dependent IP3R inactivation22 or decrease ER Ca2+ release by preventing the positive feedback of Ca2+ on IP3R 23. Mitochondrial Ca2+ release to the cytosol  via the mitochondrial Na+/Ca2+ exchanger (mNCX) was shown to refill the ER via the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA)25, and could also affect the ER Ca2+ release by supporting either the Ca2+-dependent IP3R activation (and CICR)10 or the Ca2+-dependent IP3R inhibition21.In non-excitable cells exposed to submaximal agonist levels, inositol 1,4,5-trisphosphate , while disregarding the presence of plasmalemmal ion channels (closed-cell), and expressed the dependence of [Ca2+]Cyt on fluxes across the ER and mitochondrial membranes and on binding to cytosolic buffer proteins, but the ER-mitochondria Ca2+ exchange took place only via the cytosol29. A direct flux was later introduced between ER and mitochondria, and, in that case, the MCU flux depended on the ER Ca2+ concentration ([Ca2+]ER)30. More recently, a report accounted for the Ca2+ microdomains between IP3R-MCU31: In their closed-cell model, MCU was exposed to [Ca2+]μd, which was calculated based on hemispherical symmetry of Ca2+ diffusion from a point source, a cluster of IP3Rs. MCU flux, [Ca2+]Cyt oscillation amplitude, and [Ca2+]Cyt oscillation frequency varied with the diffusion distance between the cluster of IP3Rs and MCU31.Due to the complexity of underlying mechanisms and often conflicting experimental findings, computational modeling has been employed to provide insight into the subcellular processes involved in [Ca2+]Cyt increases in vascular endothelial cells (ECs), a non-excitable cell type, are typically the result of activation of paracrine signaling due to binding of an extracellular agonist  to Gq protein-coupled receptors (GPCR), stimulation of IP3 production, release of Ca2+ from the ER, and, upon ER emptying, Ca2+ influx from the extracellular space via store-operated Ca2+ channels34. However, experimental evidence suggests that non-excitable cells, including ECs, when exposed to submaximal agonist levels  exhibit sustained [Ca2+]Cyt oscillations, at least for short times, without dependence on Ca2+ from the extracellular space (isolating the cell membrane and preventing Ca2+ exchange with the extracellular space was achieved by repeating the experiments in Ca2+-free medium supplemented with La3+)35. Understanding [Ca2+]Cyt dynamics in ECs is essential, because EC [Ca2+]Cyt responses, besides their role in gene expression, are responsible for the production of nitric oxide (NO) and prostanoids, which lead to smooth muscle cell relaxation and vasodilation38. Fluid mechanical shear stress exerted on cultured ECs is known to cause increases in IP3, due to release of endogenous ATP, and subsequently [Ca2+]Cyt mobilization and NO production41. [Ca2+]Cyt oscillations were present in ECs of both blood-perfused mouse cremaster muscle arterioles in situ and en face rat, carotid or mesenteric, arteries perfused in saline43. Importantly, our earlier experimental work demonstrated a critical role for the ER-mitochondria Ca2+ exchange (and the MCU) in regulating the [Ca2+]Cyt oscillations in cultured ECs exposed to shear stress44. The present study on [Ca2+]Cyt oscillations applies to non-excitable cells, including ECs, exposed to low/intermediate chemical agonist concentrations, as well as to ECs exposed to physiological fluid shear stress.[Ca2+ dynamics in a closed cell model. To further analyze the role of the µd in [Ca2+] dynamics, we introduced a parameter, termed the connectivity coefficient, for IP3R, MCU, SERCA and mNCX channels. This parameter indicates the fraction of each channel facing the µd. Our approach using connectivity coefficients allowed us to investigate the role of the µd on a spectrum from total isolation to its contiguity with the cytosol. This approach revealed conditions at which sustained oscillations occur and helped identify which subcellular compartment, cytosol or µd, is driving the oscillations under each condition. Interestingly, inclusion of the µd resulted in the presence of two distinct oscillatory regions in the bifurcation diagram of the system with respect to different IP3 levels. Oscillations in these two regions differed mechanistically, and also behaved differently when either the activity of MCU channels was altered or the ER-mitochondria distance  was varied. Global sensitivity analysis of model outputs indicated that the model can produce robust and physiological [Ca2+]Cyt oscillations over a wide range of parameter values. In summary, the modeling framework in the present study provides important insight into the intricacies of Ca2+ transport between the µd and each of the other Ca2+ compartments, as well as the effect of these processes on the global Ca2+ response. This information is currently limited and/or difficult to obtain experimentally, although it is critical for an accurate representation of Ca2+ signaling in non-excitable cells, including ECs.More specifically, our study aims to elucidate the role of the ER-mitochondria µd compartment in Ca2+ dynamics in each compartment is governed by a balance of Ca2+ fluxes, leaks, and buffering processes. Since the µd is in reality a part of the Cyt, several µd parameters  were kept the same as for the Cyt. Temporal changes in [Ca2+] in each compartment is represented using the following ordinary differential equations:i is flux of Ca2+ ions , and Voli is the compartment volume . Subscript µd in fluxes through the IP3R, MCU, SERCA, and mNCX channels denotes that the channel is facing the µd; if no subscript, then the channel is facing the Cyt. Leak fluxes (Jleak) are represented as leak from superscript to subscript compartment. All fluxes are defined with respect to VolCyt; thus, Eqs The cell model Fig.  contains45. The buffering factor for each compartment is described as:i is the buffering factor, BPi is the concentration of buffering proteins, and Ki is the buffer rate constant ratio .Buffering in each compartment is accounted for using a fast buffering approximation3R channels is defined as:IP3R represents the maximum total flux through IP3R channels (in s−1)31. PoIP3R and 3R channels facing the Cyt and µd, respectively  of ~200. Thus, the volume of the µd compartment was estimated based on the SA of a single mitochondrion, their total number (N), and the ER-Mt distance (D):We assumed that each mitochondrial object is a sphere with 20% of its surface area (SA) in close proximity to the ER53 with uniform distribution was used to select 250,000 random parameter sets. The model was solved for each set and the partial rank correlation coefficient (PRCC) analysis was performed to identify parameters that exerted statistically significant positive or negative influences on [Ca2+]Cyt oscillations. For all the analyses, 95% confidence interval was chosen for statistical significance.Robustness of the model was assessed using a global sensitivity analysis of model parameters. Each parameter of the model was allowed to vary within a lower and upper bound .A graphical user interface (GUI)-enabled MATLAB implementation of the model is available online . Inclusion of the Mt compartment (red curve) resulted in a slight decrease in the predicted oscillatory amplitude, while the [IP3] window for oscillations (0.1–2 µM) and the corresponding 3 (region α) while oscillations with larger amplitudes are predicted at intermediate levels of stimulation (region β). The presence of the µd reduced the IP3/Ca2+ concentration range for oscillations and the oscillation amplitudes at a given level of stimulation (compare black curve with red and blue ones). Temporal traces of 3] in regions α, β, and γ, are shown in Fig. 3], respectively, while panel D (region γ) showed damped oscillations with elevated 3 stimulation. The amplitude of oscillations is lower (up to hundreds of nM) and the frequency is higher (~4 oscillations/min) at low levels of stimulation (region α)  Fig.  compared α) Fig. . These d2+] in the subcellular compartments  before and after a step increase in IP3 levels were superimposed onto the simulated 2+ oscillations showed slightly shorter period (higher frequency) in region α compared to region β. While resting Ca2+ levels prior to IP3 stimulation remained the same for both regions, IP3 stimulation resulted in higher [Ca2+]Mt and lower [Ca2+]ER in region α compared to β. The shuttling of Ca2+ content between different compartments upon stimulation is better depicted in Fig. 2+ content (free and buffered). Following IP3 stimulation, ER Ca2+ is channeled to both the Cyt and the µd. At low level of IP3 stimulation (region α), the mitochondria pick up a larger portion of Ca2+ released by the ER through the µd compared to the portion picked up by the Cyt , this portion is reduced, as more of ER-released Ca2+ is picked up by the Cyt  in the generation of Ca2+ oscillations. Simulations were performed under control conditions and after clamping the Ca2+ flux through each pool of IP3Rs, those facing the Cyt or those facing the µd, to its time-averaged value over a cycle (effectively abolishing the periodic opening of IP3R and maintaining the same average Ca2+ efflux from the ER). When the IP3R flux into the Cyt was clamped (JIP3R Clamped), 3R flux into the µd (JIP3Rµd Clamped) resulted in loss of oscillations in region α  and intermediate (region β) levels of IP3 stimulation. To better compare Ca2+ traces in compartments with significantly different Ca2+ content, we opted to normalize the ranges that concentrations span in each compartment  to be between 0 and 1. The slopes of Ca2+ traces in Fig. 2+ flux is inward in that compartment , whereas negative slopes (below the dashed line) indicate an outward net Ca2+ flux from the respective compartment . Figure 2+] peaks in each compartment, and also provides information on how Ca2+ is shuttled between compartments to enable them to refill and sustain oscillatory activity. Upon stimulation, oscillatory activity is initiated when [Ca2+] in either Cyt or µd increases to sufficient levels, causing the IP3Rs facing these compartments to open, resulting in a Ca2+ burst/spike , the oscillation-driving compartment  during the interspike interval varies depending on the level of IP3 stimulation, thus, yielding different modes of oscillatory activity. During the interspike interval of 3] stimulation  changes at low and intermediate levels of IP3 stimulation. Oscillation frequency was highly sensitive to changes in VMCU in the μd-driven oscillations  is critical for 2+ shuttling between the ER and the Cyt . Figure MCU and [IP3]. The heat map highlights conditions  where oscillations occur and could be decomposed into two areas by clamping either the Cyt-facing , high CMCU values are required for oscillations and the frequency depends on CMCU , oscillations appear independently of the MCU presence in the μd (CMCU = 0) and oscillation frequency did not change substantially with respect to CMCU and remained at ~3 oscillations/min ; in fact, oscillations were sustained at all distances and connectivity values except when CIP3R > ~0.75.Variations in ER-Mt distance D alter the Volµd (Eq. ) and, th µM Fig. , increash D Fig. . These l2+]Cyt oscillations. The PRCC value  indicates the relative magnitude of the influence of the parameter (only parameters with PRCC > 0.05 are shown). The sign indicates whether the parameter is positively or negatively correlated to output. For example, CIP3R is negatively correlated to the amplitude and frequency of oscillations owing to a reduction of the fraction of IP3R efflux that enters the Cyt. D is also negatively correlated to the amplitude and frequency of oscillations, whereas the level of IP3 is positively correlated. Overall, Fig. 3R, MCU and SERCA channel activities, have differential effects on the peak amplitude and frequency of [Ca2+]Cyt oscillations. Figure 2+ amplitude in each compartment . In Fig. 2+]Mt and increased [Ca2+]Cyt and [Ca2+]ER slopes). In Fig. 2+]Cyt up to the critical level. Thus, mechanistically-distinct modes of oscillations can be generated based on the contribution of different model components.Simulations using our control parameter values revealed two distinct oscillatory mechanisms under low and intermediate stimulation conditions. At low [IP2+]Cyt oscillatory activity in non-excitable cells, such as vascular ECs, when exposed to submaximal agonist stimulation or fluid shear stress. A mathematical model of IP3-induced Ca2+ mobilization in non-excitable cells was developed that accounts for all major subcellular Ca2+ compartments , their respective Ca2+ channels and buffering, and also considers explicitly the ER-Mt Ca2+ microdomains as a distinct Ca2+ pool (µd). The model was able to produce physiologically relevant [Ca2+] in all compartments, at rest and following stimulation, and exhibited robust oscillations. Simulations highlighted the role of the mitochondria as regulators of [Ca2+]Cyt oscillatory activity and the importance of the ER-Mt µd in the process.In the present study, we investigate the mechanisms underlying Cyt oscillatory dynamics and uncover the critical regulatory role of Mt in [Ca2+]Cyt oscillations, in agreement with experimental observations12.Mitochondria are able to uptake and store significant amounts of Caet al.59 previously explored the role of the ER-lysosome µd in driving [Ca2+]Cyt dynamics in stimulated fibroblasts by varying the distribution of two-pore Ca2+ channels on lysosomes. They showed that the distribution and density of the Ca2+ channels on lysosomes that face the ER-lysosome µd regulate the IP3R-dependent [Ca2+]Cyt oscillations59. To the best of our knowledge, the only mathematical study that has explored the role of the ER-Mt µd prior to our study is the one by Qi et al.31, where they assumed Ca2+ diffusion from a point source, a cluster of IP3Rs to the MCU, and varied the distance between the ER and Mt compartments  and generation of physiologically relevant [Ca2+]Cyt oscillations, and abnormally high [Ca2+]Cyt arises when the distance is greater than the optimal one31. In contrast, in the present model, we incorporated an explicit µd compartment with a variable volume which depended on the ER-Mt distance  is as follows: A small increase in [IP3] activates the IP3R causing ER Ca2+ release into the µd and Cyt. The elevation in [Ca2+] in each of these compartments increases the IP3R open probability causing further release of Ca2+ from the ER . High receptor density in the small µd volume results in a substantial increase in [Ca2+]μd, which further activates the µd-facing IP3Rs and results in a Ca2+ burst and the initiation of oscillations . As a result, [Ca2+]Cyt increases slower and cytosolic oscillations are delayed compared to those in the µd  decreases. For the next oscillatory cycle to occur, this compartment needs to be replenished, so the IP3Rs can be reopened by sufficient [Ca2+] levels. Simulations suggest that fluxes from the ER  was refilled by Mt  cells exposed to the same treatment61. Our findings in region α corroborate these observations: At certain levels of low [IP3] (<0.25 µM), the oscillation frequency was shown to decrease with either decreasing VMCU .The model predictions on the effect of MCU KD on the oscillation frequency are in agreement with experimental findings: There is ample evidence in the literature that in non-excitable cells stimulated by low/intermediate levels of agonists, e.g. 1–10 µM histamine, as well as in ECs exposed to physiological levels of shear stress, MCU KD decreases the frequency of  traces in Wacquier et al. 61 and in Pecze et al. 7 suggest that the Cyt is mainly refilled by the Mt during the interspike interval, while, in Qi et al. 31, the Cyt is refilled by the ER. The current model consolidates these previous findings and shows that, depending on the choice of parameter values that regulate Ca2+ fluxes between compartments (including the µd), we can achieve different feeding mechanisms to maintain [Ca2+]Cyt oscillations. Different parameter value sets produce oscillations where the Cyt is fed by only the ER, only the Mt, or a combination of both 10. The experimental work by Ishii et al.10, where they transduced HeLa cells with retroviruses expressing fluorescence Ca2+ indicators targeted to the Cyt, ER and Mt, clearly showed that, during histamine (10 µM) stimulation, [Ca2+]Cyt and [Ca2+]Mt oscillate almost in phase with [Ca2+]Cyt leading, while [Ca2+]ER is in anti-phase with them, and the feeder compartment is the Mt  studies on Caoth Fig. . Furtheroth Fig. . Our fin2+ influx from the extracellular space is not necessary for initiating and maintaining [Ca2+]Cyt oscillations 35. Furthermore, the present model builds on earlier work, in particular, the models by Qi et al.31, Wacquier et al.61, and Pecze et al.7, all of which considered a closed cell and demonstrated the generation of physiological [Ca2+]Cyt oscillations based on IP3R regulation by Ca2+ and on ER-Mt Ca2+ transport. However, neglecting transmembrane fluxes may lead to overestimation of Ca2+ levels during oscillations, i.e., The present work has certain limitations: In the model we assumed a closed system, based on experimental data showing that Ca3R, MCU, SERCA and mNCX channels in the MAMs by assigning values between 0–1 to their respective connectivity coefficients. However, the model was still able to provide insights on how varying the above channel activities in the MAMs, at different levels of IP3, influences the role of the µd in regulating Ca2+ signaling and ultimately cell function. Quantitative experimental information on the connectivity coefficients of Mt and ER channels is currently lacking in the literature. Immunofluorescence studies have detected IP3R and SERCA in MAMs and a number of studies found the IP3Rs to be MAM-enriched in certain cell types, but local channel activity levels were not assessed quantitatively (reviewed in14). More recently, De La Fuente et al.63 used size exclusion chromatography to verify the presence of MCU-EMRE complexes  in the mitochondria associated with the sarcoplasmic reticulum (SR) fraction of heart homogenate, followed by 45Ca2+ isotope uptake assays to measure MCU-mediated Ca2+ uptake activity. They found a 2-fold increase in MCU activity in SR-associated mitochondria compared to non SR-associated ones, and concluded that MCU “hot spots” exist in cardiomyocyte MAMs63. Furthermore, at the current stage, our model does not differentiate among the IP3R isoforms, i.e. IP3R1, 2, and 3. IP3 sensitivity, Ca2+ gating (i.e. feedback inhibition), distribution, and other properties are different among these isoforms, and their relative abundance varies in different cell types. IP3R isoforms were recently found to differentially regulate ER-Mt contacts and the local Ca2+ fluxes54. Their role in Ca2+ dynamics can be accounted for in future modeling studies.Our model did not consider the spatial distribution of subcellular organelles and it adopted a simplified approach regarding the presence of functional IPMCU on a constant inner mitochondrial membrane voltage (Ψ). This assumption of a constant Ψ is based on experimental evidence that Mt uptake does not significantly alter Ψ48. More detailed modeling of Ψ kinetics, as demonstrated in Wacquier et al.61, would be an appropriate addition in future modeling efforts. Last, the model utilized several assumptions regarding the shape and number of mitochondria in a non-excitable cell, as well as in the percentage of Mt surface area that faces the µd, in order to relate the Volµd with the ER-Mt distance D and assess the importance of D  in Ca2+ oscillations. In conclusion, additional experiments combined with mathematical modeling are needed to better understand how the µd regulates global Ca2+ signaling.The present model also used an explicit dependence of V2+ dynamics in a prototypical non-excitable cell that includes a Ca2+ µd between ER and Mt. Model results showed robust oscillatory activity using physiologically relevant parameter values, and alternative oscillatory modes were identified depending on stimulus strength and the relative distribution of Ca2+ channels facing each compartment. This enabled us to consolidate previous theoretical and experimental findings and suggested conditions where the mitochondria play a critical regulatory role, rather than simply modulating oscillatory activity, upon stimulation. We provided evidence that the microenvironment between the ER and Mt can play a critical role in Ca2+ dynamics by modulating the activity of IP3Rs, allowing shuttling of Ca2+ between the ER and Mt, and creating oscillations sensitive to the mitochondrial function.In summary, we developed a compartmental closed-cell mathematical model of CaSupplementary figure and table


One-quarter of systemic symptoms associated with chronic spontaneous urticaria (CSU) are related to gastrointestinal complaints (GICs).To investigate the prevalence and features of urticaria-overlapping GICs.In this retrospective cross-sectional survey, 1426 consecutive outpatients were observed at our University Department. Only patients suffering from urticaria or GICs with a complete diagnostic work-up including serum total IgE level (Tot-IgE), differential blood count and urticaria activity score (UAS), were evaluated.Among different GICs, gastroesophageal reflux disease (GERD) was the most frequent syndrome observed . The prevalence of overlap syndrome for urticaria and GERD was 5.9% (95%CI: 4.7–7.2). In urticaria-patients, the prevalence of GERD was four-fold higher than in patients without hives . UAS was significantly higher in urticaria and GERD overlap syndromes vs. isolated urticarias. In patients with GERD or acute/chronic urticaria or overlap syndrome, Tot-IgE and eosinophil blood count (EBC) differed significantly, with a stepwise increase in their values; from the subgroup of patients with GERD only, to that with overlap of CSU to GERD. Prevalence values for urticaria overlapping with GERD were three- and two-fold higher in CSU and in long-duration GERD cases respectively compared to acute urticaria or short-duration GERD cases. Similar to Th2 pathology models, CSU and GERD overlap syndrome was significantly and independently associated with Total-IgE ≥100IU/ml or EBC ≥250/mmc compared to CSU or GERD. Endoscopic/bioptic findings of non-erosive reflux disease (NERD) or Barrett’s esophagus (BE) were more frequent in chronic overlap syndrome than in GERD-patients.GERD was the most frequent GIC in patients with urticaria. Overlap syndrome was more frequent among patients with CSU, where this syndrome was associated with higher values of UAS, Tot-IgE, EBC and frequencies of NERD and BE. These results suggest that overlap syndrome is frequently a chronic syndrome with a Th2-like profile. Urticaria is a common and heterogeneous skin disorder characterised by a mast cell-driven vascular reaction causing wheals, itch and /or angioedema in response to several either identifiable infectious, allergic, physical, chemical and psychological stimuli or other unidentifiable stimuli –6 Due toTC) in humans [Because human skin MCs are stabilised by anti-IgE agents , omalizun humans . Furthern humans , 23. In n humans  that, dun humans  and abiln humans , may plan humans . With ren humans . Prelimin humans . Moreoven humans  and sharn humans , 30, 31,n humans . On thisWe examined all patients affected by urticaria and/or GICs among a sample of 1426 newly observed consecutive patients who attended the Allergy and Clinical Immunology Section of our University Multispecialty Department from October 2013 to December 2014.Diagnosis of urticaria and/or GICs was confirmed by a clinical allergist/immunologist or a gastroenterologist based at the Department where the study was conducted. Among GICs, GERD was suspected on the basis of a weekly/daily recurrence of heartburn and/or reflux  and confAlthough missing in all ASU cases, results of gastrointestinal endoscopic examination were available for all patients with clinically predictable GERD and used to confirm and grade it . DiagnosIn addition to physical examination, basic demographic data and current Urticaria Activity Score (UAS) during clinical observation, all patients completed our relatively extensive diagnostic work-up, to avoid any potential diagnostic bias. Work-up included a complete blood count with differential count and main liver function tests, the determination of erythrocyte sedimentation rate, C-reactive protein levels, urine routine examination, stool examination for parasites, dosage of total IgE level  and the presence/absence of infection by HP as determined by 13C-Urea breath/HP, stool antigen test or endoscopic biopsy. Our data of interest were demographic data, eosinophil blood count (EBC), total IgE serum level (Tot-IgE), the presence/absence of H. Pylori and current UAS.All patients with urticaria we observed were receiving treatment with different dosages of different non-sedating antihistamines and they consented to the temporary suspension of drugs during the week of urticaria activity determination. For this purpose, patients used the simple and validated UAS that assesses daily pruritus and number of hives; each component of the UAS was scored on a scale of 0 to 3; the 2 scores are added together for a daily total of 0 to 6 which were added up (sum of each day) over a week, providing the UAS7 .Demographic, clinical and main biological data of patients were presented as absolute frequencies with percentage for categorical variables or median values with interquartile range for continuous variables. Differences among sub-groups of patients were evaluated by bivariate analysis using the Chi-squared test for categorical variables, and the non-parametric Wilcoxon rank sum test or Kruskall-Wallis test respectively, for comparison of continuous variables between two and three or more sub-groups. Some continuous variables  were also analysed after recoding into categories of sufficient size to allow for stratification. The results of the Chi-squared test performed for differences among sub-groups after recoding were completely consistent with those derived from the non-parametric tests run for continuous variables before categorization.We calculated the prevalence of urticaria, GICs and their overlap syndromes in the whole sample and by sub-groups of patients also considering their 95% confidence interval (CI). The association between syndrome of urticaria overlapping GERD with urticaria duration (chronic vs. acute) and between overlap syndrome and GERD duration (> 12 months vs. ≤ 12 months) was evaluated using a multivariate log-binomial model which was controlled for potential confounding effect due to sex, age and presence of HP. The same model was used to evaluate the independent association of chronic urticaria and GERD overlap syndrome with Tot-IgE, EBC and their interaction in both CSU and GERD patients. The strength of associations were described by the adjusted prevalence ratios (PR) and their 95% CI. All p-values less than 0.05 were considered statistically significant. All analysis was performed using Stata/MP version 1.3 .From the entire sample (n = 1426), 290 patients reported GICs . A total of 191 patients were suffering from urticaria  of which 71 were from ASU  and 120 from CSU . Among GICS, GERD was the most frequent syndrome , followed by irritable bowel syndrome (IBS)  and dyspepsia . However, overlap with IBS and/or dyspepsia was present in 150  patients suffering from GERD. The overlap of urticaria with GERD was observed in 84 patients  while overlap of urticaria with other GICs was slightly less frequent .The results shown in Levels of serum total IgE and EBC in patients affected from non-associated diseases  or urticaria and GERD overlap syndromes are presented in After adjusting for sex, age, and HP co-infection, the prevalence of urticaria and GERD overlap syndrome was three-fold higher in CSU compared to ASU patients , while overlap syndrome-prevalence was two-fold higher in long-duration GERD compared to short-duration cases of GERD  .Moreover, in both CSU and GERD patients the CSU and GERD overlap syndrome was significantly and independently associated with increased levels of Tot-IgE (≥ 100 IU/ml) (p = 0.002) or EBC (≥ 250 cells/mmc) (p = 0.035) without a significant interaction between these two biological parameters .Finally, endoscopic and bioptic data revealed a significantly higher prevalence of NERD in patients suffering from CSU and GERD overlap syndrome (61.4%) compared to those only suffering from GERD (40.6%) as well as an increased prevalence of short BE in the former group compared to the estimated general epidemiological forecast among patients with GERD  Table 7Table 7.To our knowledge, this is the first study evaluating the prevalence of GERD in patients with chronic and acute urticaria. Our analysis revealed that GERD was confirmed in 94% of all cases from endoscopic findings. The overall prevalence of urticaria and GERD overlap syndrome was approximately 6% and patients affected from acute or chronic variants of this syndrome presented a grade of urticaria severity that was more intense than that observed in patients suffering only from ASU or CSU respectively. Patients with a chronic urticaria overlapping a GERD were significantly more frequent than those with overlap of acute urticaria and showed Tot-IgE levels and EBCs that were more elevated than in patients without the overlapping syndrome. Finally, in cases of CSU overlapped to GERD there was a higher prevalence of NERD and, although not statistically significant, , an unusual high frequency of Barrett’s esophagus compared to that observed in patients suffering only from GERD.The overall prevalence of urticaria was in line with that estimated in the general population  althoughHowever, the observed prevalence values of these syndromes in urticaria patients were more than three-fold higher than those in patients without urticaria. Interestingly, patients with CSU had a risk of overlap with GERD that was about three-fold that in patients with ASU and, even after adjusting for differences in age, sex and HP co-infection distributions between these two groups, it was strongly associated with relatively high levels of Tot-IgE and EBC. However, although overlap of IBS and GERD as well as other functional gastrointestinal syndromes is very frequent in the general population –48, the Although the association between mucosal neurogenic inflammation of GERD and cutaneous neurogenic inflammation of CSU as well as those between serum SP levels and both neurogenic inflammations are currently unknown, preferential activation by SP of human skin MCs via MRGPRX2 , and theTC subset, the main actor of CSU history, largely depends on its multiple and not yet fully clarified functions by activation of FcεRI-IgE axis, among others also by auto-allergens [TC are able to exhibit a selective uptake of IgE by their positioning and by extending a cell process across the vessel wall to capture intraluminal IgE [Eosinophilic esophagitis, GERD and other chronic dysfunctional gastrointestinal diseases have recently been revisited as possible expression of atopy/food allergy –55. Actullergens , 59, 60.inal IgE .Unfortunately, in GERD or other chronic gastrointestinal syndromes as well as more and more in CSU where there are often altered skin responses, gastrointestinal synthesis of food-specific IgE may be difficult to be detected in the blood or skin due to their minute levels that could well be below the sensitivity threshold of diagnostic methods we currently use , 63.TC and induce a further activation of these cells [Corroborating findings from other CSU reports , also inse cells , 25. Impse cells . With rese cells , 68.TC in the submucosa, as well as MCTC in the skin, where SP would act initially via MRGPRX2 present on intravascular processes of these cells [On this basis, we speculate that in the presence of a longstanding GERD, the slightly elevated serum levels of SP as well as low levels of IgE initially produced in the gut could progressively activate gut MCs and the small subset of MCse cells , 61. These cells –72 not ose cells , 74. SimS1 DatasetLegend symbols: GERD = gastroesophageal reflux disease; EBC = Eosinophils blood count; Tot IgE = serum total IgE level; HP = Helicobacter Pylori infection; *URT-DUR = duration of urticaria (weeks); **GERD-DUR = duration of gastroesophageal reflux disease before urticaria onset (months); UAS7 = urticaria activity score according to UAS7; IBS = irritable bowel syndrome; ***Clinical diagnoses: ASU = acute spontaneous urticaria; CSU = chronic spontaneous urticaria; acute/chronic UGOS = acute/chronic Urticaria and GERD Overlap Syndrome; IBD = inflammatory bowel diseases; ****Endoscopic study: endoscopy/bioptic data of GERD according to Los Angeles Classification (L.A.)–grade A, B, C, D (L.A. A/B/C/D); NERD = Non erosive disease; S-BE = Short-Barrett’s esophagus; bioptic data of celiac disease (CD) according to the modified Marsh–Oberhuber classification–type 1–4 (Marsh 1–4).(XLSX)Click here for additional data file.


Two different Ca2+-sensitive proteins, STIM1 and extended synaptotagmin-1 (E-syt1), are activated during this process. Due to the lack of live cell super-resolution imaging, how MCSs are dynamically regulated by STIM1 and E-syt1 coordinately during ER Ca2+ store depletion and replenishment remain unknown. With home-built super-resolution microscopes that provide superior axial and lateral resolution in live cells, we revealed that extracellular Ca2+ influx via SOCE activated E-syt1s to move towards the PM by ~12 nm. Unexpectedly, activated E-syt1s did not constitute the MCSs per se, but re-arranged neighboring ER structures into ring-shaped MCSs (230~280 nm in diameter) enclosing E-syt1 puncta, which helped to stabilize MCSs and accelerate local ER Ca2+ replenishment. Overall, we have demonstrated different roles of STIM1 and E-syt1 in MCS formation regulation, SOCE activation and ER Ca2+ store replenishment.In many non-excitable cells, the depletion of endoplasmic reticulum (ER) Ca Upon depletion of Ca2+ stores, STIM1 on the ER membrane changes configuration to form transient MCSs via its interaction with Orai1 on the PM2, which opens Ca2+ release-activated Ca2+ (CRAC) channels to mediate the store-operated Ca2+ entry (SOCE)3. Subsequently, Ca2+ influx via SOCE activates E-syt1 to dock ER onto the PM that’s enriched with phosphatidylinositol 4,5-bisphosphate (PIP2), culminating in the formation of new ER-PM contacts6. E-syt1-MCSs appear partially co-localized with STIM1 in live HeLa cells upon ER Ca2+ store depletion and replenishment5, and catalyze phospholipid shuttling between ER and PM that mediates the sustained activation of the receptor-induced Ca2+ signaling pathway6 and the slow Ca2+-dependent inactivation of the CRAC current7. However, arguing against a collaborative role of E-syt1 and STIM1 in inducing MCSs, knockdown of E-syt1 failed to reduce SOCE4, distinct from the essential role of STIM1 in the activation of SOCE9. Under an electron microscope, the ER membrane in the MCSs formed by E-syt1 is ~15 nm away from PM, while the ER membrane in the MCSs formed by STIM1 are further away from the PM (~22 nm)10, despite no dynamic information is available. Conventional live fluorescence imaging techniques, on the other hand, do not have sufficient spatiotemporal resolution to resolve MCSs that have an average size of ~100 nm in the lateral direction11. Although super-resolution imaging techniques were used to explore the structure of MCSs12, the prerequisite need for excessive photons has prevented the unequivocal visualization of the in vivo assembly and disassembly of ER-PM MCSs in live cells.Various cellular organelles communicate via MCSs that mediate important cellular processes, such as intracellular signaling, metabolism, metabolite trafficking and organelle division13, which allows to recover dynamic with a 3D volume from images obtained at continuously varied incident angles, and another high-NA total internal reflection fluorescence structured illumination microscopy (TIRF-SIM) with the ~sub 90 nm lateral resolution at minimal photon budgets14. Using the VA-TIRFM system, we performed live-cell imaging of HEK293 cells expressing ER membrane-located red fluorescent marker mCherry-Sec 61β at different incident angles under the TIRF mode15. The raw images were then reconstructed using a previously developed algorithm16 and rendered in Amira  to enable three-dimensional structure visualization  system that enables live-cell super-axial-resolution volumetric imaging2+-ATPase (SERCA) inhibitor, to deplete the ER Ca2+ store in a Ca2+-free bath solution, followed by store replenishment via SOCE by switching to a bath solution containing 1.26 mM CaCl25. Upon ER Ca2+ store depletion, STIM1 gradually aggregated to induce new ER-PM MCSs formation, as indicated by the increased number and size of the STIM1-EGFP and mCherry-Sec 61β, which remarkably co-clustered within 50 nm beneath the PM in the raw images, and the images rendered by volumetric imaging  with similar kinetics, while the STIM1 fluorescence in the middle layer (200–250 nm beneath the PM) remained nearly unchanged . Assuming an exponential decay of TIRFM excitation determined by the illumination angle, such changes of fluorescence intensity corresponded to an axial movement of 12 ± 2 nm , consistent with previous reports10. Therefore, MCSs mediated by STIM1 or E-syt1 differ in formation kinetics, morphology and mechanism.E-syt1-mediated ER-PM MCSs were already partially appeared under resting conditions and the depletion of the ER Caion Fig. . NeverthCSs Fig. , which oCSs Fig. . The shaCSs Fig. . To quanged Fig. . In cont PM Fig. . Altoget2+ store depletion and replenishment in HEK293 cells co-transfected with both STIM1-EGFP and SNAPf-E-syt1. Under regular TIRFM, STIM1 and E-syt1 mostly co-localized regardless of whether the cells were in resting state, or after store depletion or subsequent SOCE activation  were smaller than the Sec 61β and KDEL rings    Fig. . Using tm2) Fig. . The extm2) Fig. . Therefo2+ regulation. We either loaded HEK293 cells with the fluorescent calcium indicator Cal-520 or transfected cells with the gene-encoded ER Ca2+ indicator G-CEPIA1er18. When compared to control cells, overexpression of STIM1 alone potentiates maximal Ca2+ transients evoked by SOCE, but did not affect tBuBHQ-stimulated ER Ca2+ store release or rate of ER Ca2+ uptake . The mechanism was not known in the previous study4, but could be explained by the possible coupling between E-syt1 and SERCA, i.e. E-syt1 may contribute to ER Ca2+ store replenishment through regulating SERCA. Following the knockdown of E-syt1, pulsed histamine stimulation at 1 pulse/min triggered the damping of cytosolic Ca2+ transients over time , which was hypothesized to be due to defective PIP2 replenishment6. As E-syt1 is important for the refilling of ER Ca2+ store  without phenol red (Life Technologies) supplemented with 10% (vol/vol) fetal bovine serum (FBS), 100 U/ml penicillin and 100 U/ml streptomycin. A2058 (obtained from ATCC) cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum . The cells were grown at 37 °C in a humidified atmosphere of 5% CO2 and 95% air. The transient transfection was performed using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. After the transfection, the cells were trypsinized and seeded onto poly-L-lysine-coated customized high refractive index coverslips.HEK293 cells were originally purchased from ATCC, and have been used in several previous papers2 for 30 min, washed three times with fresh medium and incubated for another 30 min before imaging.The cells transfected with SNAPf-E-syt1 were labeled with 3 µM cell-permeable far-red SNAP-Cell 647-SiR  at 37 °C and 5% COTo clean the custom-made coverslips  for the live-cell imaging, we immersed the coverslips in 10% Alconox (Sigma-Aldrich), followed by sonication for 20 min. After 8 rinses in deionized water, the coverslips were sonicated in acetone for 15 min and then sonicated again in 1 M KOH for 20 min. Finally, we extensively washed the coverslips with deionized water, followed by sonication for 10–15 min. The clean coverslips were stored in 95% ethanol at 4 °C.tBuBHQ was purchased from Alomone Labs. Primary antibodies: anti-STIM1 (ProteinTech) rabbit polyclonal antibody (11565-1-AP); anti-Esyt1 (Sigma-Aldrich) rabbit polyclonal antibody (HPA016858); mouse monoclonal anti-tubulin antibody (Sigma-Aldrich). Secondary Antibody: goat anti-rabbit IgG (H + L) cross-adsorbed secondary antibody, Alexa Fluor® 647 conjugate (A-21244); anti-rabbit-HRP (Santa Cruz).4, mCherry-Sec 61β27 and G-CEPIA1er18 were obtained from De Camilli lab, Voeltz lab and Lino labs through Addgene. SNAPf-E-syt1 was generated by inserting the E-syt1 gene into a pSNAPf vector . All plasmid constructs used in this study were prepared according to standard molecular cloning approaches and validated by DNA sequencing.The plasmids EGFP-E-syt1Human melanoma A2058 cells transfected with mSec 61-mCherry were fixed in 4% paraformaldehyde in PBS for 1 hour at room temperature, permeabilized in 0.2% Triton X-100 buffer for 20 minutes, and then blocked in PBS containing 0.2% goat serum. For immunofluorescence staining, cells were stained with primary antibody 1:200 at room temperature overnight. Slices were washed twice and incubated with secondary antibody before mounting with Antifade.Equal amounts of proteins were loaded and separated on SDS-PAGE . Protein bands were transferred from the SDS-PAGE to a nitrocellulose membrane which were probed with primary and then secondary antibodies. Secondary antibodies were incubated for 1 h at room temperature. Chemiluminescence was visualized and exposed to film after washing.2+-free Hank’s Balanced Salt Solution . Under resting conditions, 150 frames of the images were taken at 5.28 Hz using VA-TIRFM or high-NA TIRF-SIM. Then, these cells were treated with 100 μm tBuBHQ for 5 min, and 150 frames at 5.28 Hz were obtained. After washing the tBuBHQ, we began to perform 200-frame imaging (5.28 Hz), and during the imaging, the incubation solution was changed to Ca2+-containing HBSS  by perfusion. We selected representative frames under each condition for presentation in the figures.To monitor MCSs dynamics, cells transfected with SNAPf-E-syt1/mCherry-Sec 61β/STIM1-EGFP or with G-CEPIA1er were initially bathed in Ca2+, the cells were loaded with Cal-520-AM (5 μM) for 20 min at 37 °C. After three washes, we incubated the cells for another 20 min in fresh DMEM without phenol red at 37 °C. To image the whole-cell ER Ca2+, the cells were transfected with pCMV G-CEPIA1er. Then, these two cell types were transferred to Ca2+-free HBSS for time-lapse imaging at 2 Hz using wide-field microscopy. The entire imaging process included 1 min under resting conditions, 5 min of incubation with tBuBHQ, ~3 min of recovery after washing the tBuBHQ and another 5 min after changing to Ca2+-containing HBSS.To image the cytosolic Ca15.The VA-TIRFM instrument was built on a commercial inverted Olympus IX81 microscope equipped with a TIRF objective . Combined illumination solid state lasers  controlled by an Acousto-Optical Tunable Filter  were projected onto the active area of a digital micro-mirror device  to generate different orientations and angles. The emitted fluorescence was captured by an EMCCD camera . An OptoSplit  placed between the microscope and camera allowed us to image two spectral channels side-by-side on a single CCD chip. The entire system was controlled by Andor iQ commercial software. Typically, 10 raw images with incident angles of 48.4, 48.5, 48.6, 48.7, 48.9, 49.3, 49.8, 50.9, 52.8 and 60.9 degree were recorded at a frequency of 0.8 s/vol, which corresponded to TIRF penetration depths of 832.5, 488.1, 379.3, 321.0, 256.4, 195.4, 158.3, 119.6, 90.8 and 55.3 nm beneath the cell surface, respectively16. The accurate separation of fluorescence objects 40~50 nm axially apart has been validated with tilted coverslips coated with fluorescent beads under different incident angles of TIRF illumination, and also by the three-dimensional actin architecture and dynamics movements of TfnR-phluorin/Rab11A-mCherry labeled vesicles. Using the same technology, we have also visualized invagination of clathrin-coated pits in axial direction in our previous paper15, which also validated the method in dealing with biological samples.With rapidly changing incident angles, the ensemble fluorescence intensities of molecules within the ER-PM contacts along axial-axis were measured multiple times. Having known different exponential dependences of fluorescence excitation at different excitation incident angles, we could solve the problem of the amount and the spatial distribution of fluorescent molecules within the volume of TIRF illumination. For the reconstruction of VA-TIRF data, we have used a well-established algorithm14. It was built on a commercial inverted fluorescence microscope  equipped with a TIRF objective , a multiband bandpass filter set  and a continuous autofocus system to maintain focus over time. We used two lasers  as light sources and an acoustic optical tunable filter  to combine, switch, and adjust the illumination power of the lasers. We used a ferroelectric liquid crystal on a silicon spatial light modulator  to generate the structure illumination patterns, which were formed by the interference of the ±1 order of the diffraction beam from the SLM. Then, the light passed through another lens  and a tube lens  to focus on the back focal plane of the objective lens, which was interfered by the image plane after passing the objective lens. Emitted fluorescence collected by the same objective passed through a dichroic mirror (DM), an emission filter and another tube lens. Finally, the emitted fluorescence was split by an image splitter  before being captured by a sCMOS camera . The time-lapse images were acquired using Hamamatsu’s image acquisition and analysis software suite HCImage.We have recently published the detail of our high-NA TIRF-SIM elsewherei at incident angle θ decays exponentially with distance z away from the interface,For an incident laser with a wavelength λI0 is the intensity at the interface, d is the penetration depth,n1, n2 are the refractive indices of glass and the media at the interface, respectively.z1, z0 away from interface is,The relative increases in fluorescence intensity So,Specifically, we imaged under the TIRF illumination at incident beam angle The particle size distribution, fluorescence intensity quantification and post-reconstruction image processing were primarily performed using Fiji software. The 3D segmentation and the rendering of ER-PM junctions were performed by the Amira (Thermo Fisher Scientific). Fluorescence images of ER marker were firstly imported into Amira, and then automatically segmented into labeled field by balanced histogram thresholding approaches with segmentation editor tool of Amira. Finally, we manually edited the features slice-by-slice and used the SurfaceGen module to generate the 3D rendered volume for data visualization and analysis.28. Then we manually applied a threshold a bit lower to generate a mask to ensure all junction structures were included. After manually frame-by-frame confirmation to discard incorrect regions of interest, we used the weighted centroid method to calculate their localizations down to sub-pixel resolution. Both ring-shape and puncta-shape ER structures behave similarly under the TIRF microscope. The different localizations of individual ER structure at different time points were jointly connected using a multiple spots tracking method as reported previously29, which produced the trajectory map of all ER structures in time.Although super-resolution TIRF-SIM could resolve puncta or ring-shaped structures, we still used the corresponding conventional TIRF image stacks to determine their sub-pixel localizations. First, we removed the background from obtained raw image stack with the à trous wavelet transform algorithmThe schematic diagram was prepared using Illustrator (Adobe). The reconstruction of the images acquired by TIRF-SIM was processed using customized MATLAB software. The data were plotted using IGOR Pro software (WaveMetrics).Video S1Video S2Video S3Video S4Video S5Supplementary Information


We show that MT stabilization alters ER morphology, favoring an enrichment in ER sheets at the expense of tubules. Stabilizing MTs has a negative impact on the process of SOCE and results in a reduced ER Ca2+ content, affecting the flight ability of the flies. Restoring proper MT organization by administering the MT-destabilizing drug vinblastine, chronically or acutely, rescues ER morphology, SOCE and flight ability, indicating that MT dynamics impairment is responsible for all the phenotypes observed.The endoplasmic reticulum (ER) extends as a network of interconnected tubules and sheet-like structures in eukaryotic cells. ER tubules dynamically change their morphology and position within the cells in response to physiological stimuli and these network rearrangements depend on the microtubule (MT) cytoskeleton. Store-operated calcium entry (SOCE) relies on the repositioning of ER tubules to form specific ER-plasma membrane junctions. Indeed, the tips of polymerizing MTs are supposed to provide the anchor for ER tubules to move toward the plasma membrane, however the precise role of the cytoskeleton during SOCE has not been conclusively clarified. Here we exploit an  It extends as a single membrane-bound entity composed of interconnecting sheets and tubules spreading all over the cell. Although the ER can form a reticular network independently of cytoskeletal structures  coordinates a variety of cellular processes, such as synthesis, modification, quality control and transport of proteins, as well as lipid metabolism and CaMTs are composed of tubulin polymers and constitute essential components of the cytoskeleton. In neurons, they are critical in order to support long-range motor-driven cargo transport within neuronal processes and play fundamental roles in polarity, axon differentiation and growth  is contacted by ER tubules in a process called store-operated Ca2+ entry (SOCE) , by which a plus end-attached ER tubule extends together with a MT growing end  containing 1 mM Ca2+, then motor neuron cell bodies were imaged on a Leica TCS SP5 II confocal microscope equipped with a HCX PL APO lambda blue 63x/1.40-0.60 Oil objective, using a 488 nm laser.Brains and ventral ganglia from third instar larvae expressing BiP-sf-GFP-ER alone or together with spastinFor the quantification of ER distribution along nerves, BiP-sf-GFP-ER fluorescence was measured in regions located near the ganglion, along the axon, and at the end of the larval body. Mean fluorescence was calculated using ImageJ software.K467R under the control of the motoneuron promoter D42-Gal4. GRS Full Sample Purification Kit  was used according to the manufacturer's instructions. The protein pellet was solubilized in 80 μL of RIPA Buffer , supplemented with proteases and phosphatases inhibitors mixtures  and 3 M urea. Insoluble particles were spun down at 10,000 g for 5 min. Proteins were separated by SDS-PAGE, transferred into nitrocellulose membranes  and probed using the following antibodies: anti-GFP , 1:1000; anti-ACT (beta-actin) , 1:2500. The intensity of the bands was analyzed using ImageJ software.Proteins were extracted from 15 flies expressing BiP-sf-GFP-ER alone or together with spastinLarval neurons were dissociated as previously reported  on an inverted microscope (Nikon Ti-E). Fluorescence illumination was achieved by 50–75W Lamp (USHIO UXLS50A) and alternating excitation wavelengths (340/380 nm) were obtained by a monochromator (Optoscan CAIRN-Research) controlled by NIS-ELEMENTS AR (Nikon) software. A neutral density filter, ND4  and a FF-409-DiO3 Dichroic (Semrock) were used in the excitation pathway. The emitted fluorescence was collected using a 510/84 nm (Semrock) filter. Images were acquired every 1 s, with 100 ms exposure time at each wavelength, by a Zyla-CMOS 4.2-P  controlled by the same software. During the experiment, cells plated on coverslips were mounted into an open-topped chamber and maintained in an extracellular-like medium containing the following:Neuronal cells were incubated with fura-2/AM (1 μM), pluronic F-127 (0.02%), and sulfinpyrazone (200 μM) for 20 min at room temperature (RT) in a M1 buffer (see below) and then in a fresh solution without the Ca+-based) medium: 30 mM HEPES, 150 mM NaCl, 5 mM KCl, 1 mM MgCl2, 35 mM sucrose, 5 glucose, pH 7.2 with NaOH at RT;(1) M1  K2+ content evaluation, cells were firstly perfused with M1 containing 1 mM CaCl2; after addition of 500 μM EGTA, cells were stimulated by addition of ionomycin (10 μM) or cyclopiazonic acid . In the second case, for residual Ca2+ evaluation, cells were further stimulated with addition of ionomycin (10 μM). For SOCE activation experiments, cells were pre-treated with the irreversible SERCA inhibitor thapsigargin (100 nM) for 10 min in a Ca2+-free, EGTA (500 μM)-containing M1; cells were then perfused with the same medium without the SERCA inhibitor and challenged with CaCl2 (2 or 5 mM). Where indicated, M1 (Na+-based) medium was substituted with K+-based medium.For store CaFor acute vinblastine treatment, the drug (1 μM) was added in each solution and step of the experimental protocol, from fura-2/AM cell loading to cell stimulations. Neurons dissociated from larvae exposed to chronic vinblastine treatment, were similarly treated.2+ imaging experiments was performed using the NIS-Elements software. F340 and F380 images were subtracted of background signals and proper regions of interest (ROIs) were selected on each imaged cell. The ratio of the emitted fluorescence intensities (R = F340/F380) was calculated for each ROI, normalized to the value measured before stimulus addition, or at the Ca2+-free status, and averaged offline. Data were analyzed using Microsoft Excel and Graphpad Prism 8 to calculate areas under the curves (AUC).Off-line analysis of Can = number of cells, unless otherwise specified). Statistical analyses were performed using unpaired Student's t-test. Analyses of differences between fly populations were made using chi-square independent proportion analysis. Both tests were applied with a confidence interval of 95% .Fura-2 traces represent average values of 100 to 1,000 cells collected in 3–10 independent experiments. Average values are expressed as mean ± standard error of the mean . The amino acid substitution, located in the AAA ATPase domain, corresponds to the pathogenic mutation K388R in the human spastin protein, known to produce a dominant-negative effect , or from controls  . A large cytosolic Ca2+ concentration ([Ca2+]c) increase, followed by a sustained plateau, due to Ca2+ influx across the PM, was observed in control neurons  , loaded  neurons . The eff2+ entry, thus potentially affecting the extent of SOCE , causing the collapse of the membrane potential. A higher concentration of CaCl2 (5 mM) was applied, after emptying stores, to obtain an appreciable Ca2+ influx even under a reduced electrical gradient. Under such depolarizing conditions, the effect of spastinK467R expression on SOCE was similar to that found in the standard Na+-containing medium , indicating the relative low abundance of this type of Ca2+ stores in these cells. This result indicates that Ca2+ content of intracellular stores is diminished in cells expressing the spastinK467R mutation. To determine whether the observed reduction was due to a specific partial depletion of the ER Ca2+ store, dissociated neurons were treated with the SERCA inhibitor CPA, thus inducing the passive release of Ca2+ from the organelle, resulting in a transient increase in [Ca2+]c  on neurons dissociated from spastinK467R larvae raised in the absence of drug in the food. Application of vinblastine for 40 min before SOCE activation and visualization was able to induce a partial rescue in the extent of Ca2+ entry, compared to untreated spastinK467R-expressing neurons  is sufficient to rescue SOCE and ER Ca2+ content. Within this time-window, transcriptional activation is unlikely to occur, suggesting that the level of Ca2+ handling proteins, and in particular of the SOCE machinery, is expected to be unaltered and the defects observed are directly ascribable to a MT dynamics impairment.Beneficial effects from treatments with vinblastine have been reported in flies expressing spastinTubulin represents about 4% of total cellular proteins in many cultured cells, however it reaches 25% in the brain (Zhai and Borisy, The datasets generated for this study are available on request to the corresponding author.DP conceived the work. NV and RN performed the experiments. NV and NR analyzed the results. PP and AD contributed to the interpretation of the results. DP wrote the manuscript. DP and PP secured funding. All the authors revised the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Therefore, the investigation of the functional roles of ion channels in cancer development may identify novel approaches for determining tumor prognosis. Malignant mesothelioma is an aggressive cancer that develops from the serosal surface of the body, strictly related to asbestos exposure. The treatment of malignant mesothelioma is complex and the survival outcomes, rather than the overall survival data are, to date, disappointedly daunting. Nevertheless, conventional chemotherapy is almost ineffective. The alteration in the expression and/or activity of Ca2+ permeable ion channels seems to be characteristic of mesothelioma cells. In this review, we explore the involvement of the Ca2+toolkit in this disease. Moreover, the established sensitivity of some Ca2+channels to selective pharmacological modulators makes them interesting targets for mesothelioma cancer therapy.Calcium ions (Ca Cancer cells embody characteristics that allow them to survive beyond their normal life span and to proliferate abnormally, presenting defects in the mechanisms underlying cellular growth, migration, and death .2+) play a pivotal role in these processes, acting as principal signalling agent and the expression of Ca2+ channel transcripts have been highlighted as a potential biomarker in the growing number of cancers i in unstimulated cells is around 100 nM, whereas the concentration is higher in the extracellular space and in the intracellular Ca2+ stores, ranging from 1 to 2 mM. Although the [Ca2+]i is maintained very low, small variations, due to intracellular release from endogenous stores, or to extracellular Ca2+ entry may induce local or global Ca2+ signals, thereby activating specific Ca2+-dependent downstream pathways i. As KCNMA1 contributes to the resting membrane potential, this effect seems to be due to an incomplete membrane depolarization and greater activity of Ca2+ channels i dysregulation in mesothelioma cells by stimulation of T-type Ca2+ channels.Moreover, as a confirmation of the role of T-type channels, another work  has demo2+ channels as a way to inhibit cancer cell growth and increase cancer cell death i may play an important role in acquired drug resistance [2+ signal between neoplastic and non-neoplastic phenotypes, coupled with the use of high throughput and/or advanced Ca2+ imaging approaches, might further support the translation of deregulated Ca2+ signalling assessment into the development of targeted tumour therapy [The important role of Casistance . Models  therapy .2+ permeable ion channels seem to be a distinguishing sign of mesothelioma cells. The clearly recognised sensitivity of some Ca2+ channels to selective pharmacological modulators makes them attractive targets for mesothelioma management. Additional studies are essential to explain the finest therapeutic strategy for mesothelioma as well as the involvement of the Ca2+ toolkit in this disease. Moreover, the changes in the expression and/or activity of Ca


This study aimed to understand the roles of this axis in podocyte apoptosis and explore potential targets for podocyte protection.The mechanism of podocyte apoptosis is not fully understood. In addition, the role of the inositol 1,4,5-triphosphate receptor (IP3R, Grp75, VDAC1, and MCU and mitochondrial Ca2+ were analyzed during Adriamycin- or angiotensin II-induced apoptosis in cultured mouse podocytes. The interaction between IP3R, Grp75, and VDAC1 was investigated using co-immunoprecipitation experiments. The effects of IP3R, Grp75, and MCU agonists and antagonists on mitochondrial Ca2+ and apoptosis were investigated in cultured podocytes. The podocyte-protective effects of an MCU inhibitor were further investigated in rats with Adriamycin-induced nephropathy.The expression of IP3R, Grp75, VDAC1 and MCU, enhanced interaction among the IP3R-Grp75-VDAC1 complex, mitochondrial Ca2+ overload, and increased active caspase-3 levels were confirmed during Adriamycin- or angiotensin II-induced mouse podocyte apoptosis. Agonists of this axis facilitated mitochondrial Ca2+ overload and podocyte apoptosis, whereas specific antagonists against IP3R, Grp75, or MCU prevented mitochondrial Ca2+ overload and podocyte apoptosis. A specific MCU inhibitor prevented Adriamycin-induced proteinuria and podocyte foot process effacement in rats.Increased expression of IP3R-Grp75-VDAC1-MCU calcium regulation axis mediated podocyte apoptosis by facilitating mitochondrial Ca2+ overload. Antagonists that inhibit Ca2+ transfer from ER to mitochondria protected mouse podocytes from apoptosis. An MCU inhibitor protected podocytes and decreased proteinuria in rats with Adriamycin-induced nephropathy. Therefore, antagonists to this pathway have promise as novel podocyte-protective drugs.This study identified a novel pathway in which the IP Several patients with nephrotic syndrome are resistant to current therapies such as corticosteroid, calcineurin inhibitors, and mycophenolic acid, and they are at a higher risk of developing end stage renal disease . Among poverload .2+ functions as a second messenger and plays a central role mediating apoptosis. Ca2+ homeostasis is mainly regulated by membrane Ca2+ channels and intracellular Ca2+ stores; ER, mitochondria, lysosomes, endosomes and Golgi apparatus are the main Ca2+ stores. Among the Ca2+ stores, the transfer of Ca2+ from the ER to mitochondria via close contacts between the two organelles named, ER-mitochondria coupling, plays a critical role in maintaining intracellular Ca2+ homeostasis. The coupling occurs via mitochondria-associated membranes (MAMs), which account for ~ 20% of the mitochondrial surface. A single yeast cell contains around 100 couplings. Under normal physiological conditions, the cytosolic Ca2+ concentration is ~ 100 nM, the ER Ca2+ concentration is ~ 1000 μM, and the mitochondrial Ca2+ concentration is ~ 1000 nM [2+ transfer from the ER to the mitochondria is required for mitochondrial ATP production and maintaining cell survival, but excessive Ca2+ entrance into the mitochondria can lead to mitochondrial Ca2+ overload and trigger opening of the mitochondrial permeability transition pore, release of pro-apoptotic factors such as cytochrome c, caspase activation, and apoptosis. This pathway of mitochondrial Ca2+-dependent cell death is crucial in a plethora of cell types [Ca 1000 nM . Ca2+ trll types –8.2+ transfer from the ER to mitochondria is the inositol 1,4,5 triphosphate receptor (IP3R)/glucose-regulated protein 75 (Grp75)/voltage dependent anion channel 1 (VDAC1) complex, which is located at sites of ER-mitochondrial coupling. IP3R is the Ca2+ release channel located at the ER membrane, VDAC1 is the channel located at the outer mitochondrial membrane, and Grp75 is a bridging protein that interacts physically with IP3R and VDAC1. The Ca2+ released from the ER forms Ca2+ hot spots between the ER and the mitochondria, enters the mitochondrial intermembrane space, and then finally enters the mitochondrial matrix via the mitochondrial calcium uniporter (MCU) [2+ transfer, we call IP3R-Grp75-VDAC1-MCU the intracellular calcium regulation axis.The molecular basis of Ca3R-Grp75-VDAC1-MCU axis in the mechanism of podocyte apoptosis. In the current study, the roles of the IP3R-Grp75-VDAC1-MCU calcium regulation axis in podocyte apoptosis were investigated in cultured mouse podocytes exposed to Adriamycin (ADR) and angiotensin II (Ang II) in vitro. Further studies on proteinuria regulation and the protective effects of calcium regulation axis antagonists on podocytes were performed in an ADR nephropathy rat model.Little is known about the roles of the IPMouse podocytes  were cultured as reported previously . Briefly6 cells/ml. Then, a 100 μl cell suspension was transferred to a 5-ml culture tube. The podocytes were stained with 5 μl FITC-conjugated annexin V and 5 μl propidium iodide  for 15 min at room temperature in the dark and analyzed by flow cytometry  within 1 h to analyze apoptosis [Cells were washed twice with cold PBS and then resuspended in 1× binding buffer  at a concentration of 1 × 105 cells/well for 24 h, treated with different drugs or transfection and used for mitochondrial Ca2+ detection. Mitochondrial Ca2+ was labeled with Rhod-2 AM  [2+ level of cells in each well.Podocytes were cultured in 96 well plates at 1 × 10Germany) . The worWestern blotting was used to detect active caspase-3 . Rabbit 3R , rabbit monoclonal anti-Grp75 , mouse monoclonal anti-VDAC1 , rabbit monoclonal anti-MCU , and mouse monoclonal anti-GAPDH antibodies were used. Western blotting and semi-quantitation were performed as the method described above.For western blot analysis, rabbit polyclonal anti-IP). The lysates were transferred to a micro centrifuge tube and centrifuged at ~ 13,000×g for 10 min to pellet the cell debris. Then, the supernatant was transferred to a new tube for protein concentration determination and further analysis. Co-IP was performed using a Thermo Scientific Pierce Co-IP Kit  according to the manufacturer’s protocols. Anti-Grp75 antibody was used as the bait antibody to capture mitochondria-ER coupling proteins. Rabbit monoclonal anti-Grp75 antibody  was first immobilized using AminoLink Plus Coupling Resin . Then, the resin was washed and incubated with lysate overnight. After incubation, the resin was washed again and proteins were eluted using Elution Buffer . Normal rabbit IgG without antigenicity provided with the kit was used as a negative control to detect nonspecific binding. The control was treated in the same way as the Co-IP samples, including incubation with the Grp75 antibody. After Co-IP, the proteins pulled down by anti-Grp75 antibodies were analyzed by western blotting [Podocytes lysates  were collected using ice-cold IP lysis buffer  was used at a concentration of 10 μM diluted in ultra-pure water to stimulate IP3R in cultured mouse podocytes for 24 h. Spermine  was used at a concentration of 20 μM, diluted in ultra-pure water, to stimulate MCU in cultured mouse podocytes for 2 h.D-myo-inositol 1,4,5-triphosphate tripotassium salt  [2+ uptake, respectively. Podocytes were pre-treated with the above inhibitors for 60 min before treatment with ADR or Ang II, respectively. Specific siRNA targeting the bridging protein Grp75 and a non-targeted negative control siRNA were synthesized by Invitrogen. Podocytes were plated in six-well plates and treated with 100 pmol/well siRNA duplexes using 10 μl RNAiMAX reagent (Invitrogen) according to the manufacturer’s protocol. After 8–12 h, the media were changed according to the status of cell growth at 40–50% confluence. The podocytes were collected for further experiments 24 h after transfection.The IP, Sigma)  and the , Sigma)  were usen = 6), RR control , ADR group , and ADR plus RR . The rats were fed a standard diet, and water was given ad libitum; they were maintained using alternating 12-h cycles of light and dark. After acclimatization for 48 h, the rats in ADR and ADR + RR group received a tail vein injection of 0.8 mg/100 g bodyweight ADR in sterile water (the 0 time point). Immediately after ADR injection, rats in the ADR + RR group were administered RR at a dose of 2.5 mg/kg/d by intraperitoneal injection for 14 days. The rats in Ctl and RR groups received normal saline and RR injections for 14 days, respectively. All rats were sacrificed at the 6-week time point and the kidneys were harvested. Twenty-four hours urine was collected from each rat at 0, 2 weeks, 4 weeks, and 6 weeks using metabolic cages.All protocols were approved by the Institutional Animal Care and Use Committee of Peking University First Hospital (Number: 11400700229305). Ruthenium red  was used as a specific inhibitor of MCU. Thirty-two male Sprague Dawley rats weighing 80–100 g were randomly divided into four groups: normal saline control . Ultrathin sections of renal cortex were made for electron microscopy using a method reported previously . Averaget-tests were used to compare differences between two groups. One-way ANOVA was used to compare differences among more than two groups. P < 0.05 was used to define statistical significance.SPSS20.0 statistical analysis software was used for all analyses. All data are presented as means ± SD. Unpaired Student’s P = 0.000, n = 3) or Ang II  increased significantly  or Ang II  increased significantly  or Ang II  increased significantly  was used to chelate extracellular Ca2+. However, compared with the podocytes treated with ADR only  or Ang II only , the increase in mitochondrial Ca2+ induced by ADR or Ang II was not prevented by additional treatment with EDTA , Grp75 , VDAC1  and MCU  was observed during ADR-induced podocyte apoptosis , Grp75 , VDAC1  and MCU  was also observed during Ang II-induced podocyte apoptosis , VDAC1 , and Grp75  pulled down by anti-Grp75 antibodies was increased significantly during ADR-induced podocyte apoptosis , VDAC1 , and Grp75  pulled down by anti-Grp75 antibodies was also increased significantly during Ang II-induced podocyte apoptosis  in mouse podocytes, which was partially prevented by treatment with the Ca2+ chelator EDTA , which was not prevented by EDTA , there were increased levels of urinary proteins in rats in the ADR group (n = 10) at 2 (P = 0.000), 4 (P = 0.000), and 6-weeks (P = 0.000), but RR alone (n = 6) did not induce proteinuria. Compared with the ADR group, urinary protein levels in rats in the ADR + RR group (n = 6) were decreased significantly at 2 (P = 0.000), 4 (P = 0.000), and 6 (P = 0.017) weeks  and RR  groups, podocyte foot process width was increased significantly in rats in the ADR group . Compared with the ADR group, podocyte foot process width was decreased significantly in rats in the ADR + RR group (1452.68 ± 115.36 nm, P = 0.000, n = 6, Fig. All the rats in the Ctl, RR and ADR + RR group were included in electron microscopic analysis. Considering that 6 rats are enough for quantitation analysis, 6 rats from the ADR group were randomly used for electron microscopic analysis. Compared with the Ctl (504.63 ± 30.70 nm, 2+ homeostasis, which is regulated by membrane Ca2+ channels, such as TRPC6 and TRPC5, is a key mediator of podocyte apoptosis or injury [2+ transfer from the ER to mitochondria by the IP3R-Grp75-VDAC1-MCU axis, located at sites of ER-mitochondria coupling, will lead to mitochondrial Ca2+ overload, opening of mitochondrial permeability transition pore, the release of pro-apoptotic factors such as cytochrome c, caspase activation, and apoptosis in many different cells and diseases including neurodegeneration, metabolic diseases, and cancer [Podocytes are core target cells for proteinuria control . The mecd cancer –8. Howev3R-Grp75-VDAC1-MCU calcium regulation axis during podocyte apoptosis. First, we explored whether this calcium regulation axis affects mitochondrial Ca2+ and apoptosis in podocytes by using IP3 to specifically stimulate Ca2+ release from IP3R located at the ER membrane and Spermine to specifically activate the mitochondria calcium uniporter. As expected, our results revealed that both agonists significantly increased mitochondrial Ca2+ levels and apoptosis rate in mouse podocytes. Second, to assess whether the IP3R-Grp75-VDAC1-MCU calcium regulation axis plays a role in podocyte apoptosis, we used the common apoptosis-inducing drugs ADR and Ang II. Western blotting identified significantly increased levels of IP3R, Grp75, VDAC1, and MCU during ADR- or Ang II-induced podocyte apoptosis. Next, Co-IP experiments revealed that the interaction among the IP3R, Grp75, and VDAC1 complex was enhanced, which suggests that these drugs may increase ER-mitochondria coupling during podocyte apoptosis. As previously described [2+ transfer from the ER to mitochondria occurs; therefore, more coupling might lead to more Ca2+ transfer to the mitochondrial matrix. Indeed, significantly increased mitochondrial Ca2+ levels accompanied by increased active caspase-3 levels were found during ADR- and Ang II-induced mouse podocyte apoptosis. Together, these data revealed that the increased expression of IP3R, Grp75, VDAC1 and MCU, enhanced interaction of IP3R-Grp75-VDAC1, increased mitochondrial Ca2+ and active caspase-3 were observed during ADR- and Ang II-induced mouse podocyte apoptosis.For the first time, this study investigated the roles of the IP3R-Grp75-VDAC1-MCU axis on mitochondrial Ca2+ overload and apoptosis in mouse podocytes, three different antagonists were used to inhibit different proteins in the axis. XeC is a specific IP3R inhibitor that decreases Ca2+ release from the ER. Si-Grp75 was used to knockdown Grp75 and hence decouple the IP3R-Grp75-VDAC1 axis [2+ entry into the mitochondrial matrix. The current results clearly demonstrated that these three antagonists all prevented ADR- and Ang II-induced mitochondrial Ca2+ overload, increased active caspase-3 levels, and mouse podocyte apoptosis. These results confirmed that an enhanced IP3R-Grp75-VDAC1-MCU axis mediates mouse podocyte apoptosis by facilitating Ca2+ transfer from the ER to mitochondrial and mitochondrial Ca2+ overload. Therefore, antagonists of the IP3R-Grp75-VDAC1-MCU axis could prevent ADR- and Ang II-induced mitochondrial Ca2+ overload and apoptosis in mouse podocytes.To demonstrate the possible causative effects of an enhanced IPAC1 axis . RU360 i2+ regulation axis in podocyte apoptosis. During palmitic acid-induced podocyte apoptosis, Yuan et al. [2+ and cytochrome c levels. They also showed that inhibiting MCU prevented mitochondrial Ca2+ uptake and the mouse podocyte apoptosis induced by palmitic acid. To our knowledge, no studies have investigated the roles of Grp 75 and VDAC1 in podocyte apoptosis. Gong et al. [3R-Grp75-VDAC1-MCU calcium axis in podocytes are needed.Little is known about the roles of this Can et al.  found thg et al.  compared2+ to mitochondrial matrix can protect against proteinuria. Since MCU is the final channel that regulates the entry of Ca2+ into the mitochondrial matrix, the MCU-specific inhibitor RR was used to treat ADR nephropathy rats. The dose of RR was selected based on a previous report in a subarachnoid hemorrhage rat model [Finally, an ADR-induced nephropathy model was used to assess whether inhibiting the transfer of Caat model . Interes2+ transfer from the ER to mitochondria via the IP3R-Grp75-VDAC1-MCU axis located at sites of ER- mitochondria coupling. This study also revealed that increased expression of the IP3R-Grp75-VDAC1-MCU calcium regulation axis proteins mediated podocyte apoptosis, possibly by facilitating mitochondrial Ca2+ overload. In addition, antagonists to the axis could protect podocytes from ADR- and Ang II-induced apoptosis. Blocking the entry of Ca2+ into mitochondria could control proteinuria in the ADR nephropathy rat model. Further investigation into the pathway might facilitate the development of new drugs for podocyte-specific protection and proteinuria control.This study identified a novel pathway mediating podocyte apoptosis and proteinuria involving Ca


Results showed that the body and heart weight of rabbits decreased significantly after suffering HF when compared with those in the control group. However, they gradually recovered after CCM application. The CCM significantly decreased collagen volume fraction in myocardial histology of HF rabbits, indicating that CCM therapy attenuated myocardial fibrosis and collagen deposition. The levels of Akt, FOXO3, Beclin, mTOR, GSK-3β, and TORC2 were significantly downregulated, but Pi3k concentration was greatly upregulated after CCM utilization. Based on these findings, it was concluded that CCM could elicit positive effects on HF therapy, which was potentially due to the variation in the Pi3k/Akt signaling pathway.The Akt plays an important role in regulating cardiac growth, myocardial angiogenesis, and cell death in cardiac myocytes. However, there are few studies to focus on the responses of the Akt pathway to cardiac contractility modulation (CCM) in a chronic heart failure (HF) model. In this study, the effects of CCM on the treatment of HF in a rabbit model were investigated. Thirty six-month-old rabbits were randomly separated into control, HF, and CCM groups. The rabbits in HF and CCM groups were pressure uploaded, which can cause an aortic constriction. Then, CCM was gradually injected to the myocardium of rabbits in the CCM group, and this process lasted for four weeks with six hours per day. Rabbit body weight, heart weight, and heart beating rates were recorded during the experiment. To assess the CCM impacts, rabbit myocardial histology was examined as well. Additionally, western blot analysis was employed to measure the protein levels of Akt, FOXO3, Beclin, Pi3k, mTOR, GSK-3 Chronic heart failure (CHF) is the terminal stage of various structural and functional cardiac diseases . This diIt is well known that the pathological cardiac dysfunction is contributed to myocardial hypertrophy and cell apoptosis , which iβ, and TORC2 expression in cardiac myocytes were investigated.Cardiac contractility modulation (CCM) is a series of nonexcitatory signals which can be applied during the absolute refractory period to enhance the strength of left ventricular contraction without increasing myocardial oxygen consumption ,12. The Thirty six-month-old healthy New Zealand white rabbits (2.5–3.5 kg) were obtained from the Experimental Animal Center of Hebei Medical University (HMU) and housed with 12-hour dark/light cycle for free access to food. This study complied with standards for the Care and Use of Experimental Animals of HMU and the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health. All experimental procedures were approved by the Ethics Review of Lab Animal Use Application of Hebei Medical University.The rabbits were randomly separated into three groups, namely, control, HF, and CCM groups, and each group contained 10 rabbits. The rabbits in the control group only received thoracotomy. The HF is characterized by the transition from an initial compensatory response to decompensation, which can be partially mimicked by transverse aortic constriction in rodent models . TherefoThe rabbits in HF and CCM groups were anesthetized via the ear vein with 3% sodium pentobarbital at 1 ml/kg body weight, and their thoracic cavities were opened. Subsequently, their ascending aorta was dissected until the aorta circumference was constricted up to 60%. After 12 weeks, symptoms of heart failure occurred in the rabbits, including appetite reduction, breathing acceleration, and fewer activities. The HF model was thought to be successfully established while the left ventricular ejection fraction reached 40%.Before the experiment, an electrode was used to deliver CCM. Specifically, one end of the electrode was sutured to the left ventricular anterior wall of rabbits, and the other end was punctured subcutaneously to the neck. Then, a cardiac stimulator  was used to deliver CCM signals to the heart by sensed R-wave at the absolute refractory period. These signals consisted of biphasic square-wave pulses with phase duration of 2 ms, stimulus amplitude of 7 V, and 30 ms delay after R-wave sensing . The CCMμm thickness) were slightly cut from the paraffin blocks and stained with 1% hematoxylin and eosin (H&E) and Masson's trichrome for 10 min at 37°C for histopathological analysis of myocardial fibrosis [At the end of the experiment, all rabbits were anesthetized with 0.5 mg/g tribromoethanol. Then, the hearts were fixed in 4% paraformaldehyde solution before storage for 10 min at −20°C. The hearts were sectioned transversely across myocardial papillary muscle before completely covered by paraffin. Myocardial histologic sections . After blocking with 5% fat-free milk at room temperature for 2 h, the membrane was incubated overnight at 4°C with primary antibody raised in mice against Akt (1 : 1000), FOXO3 (1 : 1000), Beclin (1 : 1000), Actin (1 : 1000), pi3k (1 : 1000), mTOR (1 : 1000), p-Akt , GSK-3β (1 : 1000), and TORC2 (1 : 1000). All antibodies were purchased from Cell Signaling Technology (USA). Then, the membranes were incubated with HRP-conjugated secondary antibodies  at room temperature for two hours. The immunoreactive bands were visualized using the enhanced chemiluminescence kit  in a western blotting detection system . Developed films were scanned and Image-ProPlus 5.1 was used for quantitative analysis. In this experiment, β-actin  was used as the standard and the results were expressed as density values normalized to β-actin.The total protein was obtained from leaf ventricular myocardial tissues after extraction with the RIPA lysis buffer . Protein concentration was measured by the Pierce™ BCA Protein Assay Kit . A total of 60 p < 0.05.Data were presented as mean ± standard deviation. The main effect of treatment was analyzed by using Mixed proc in the SAS software , and the differences among groups were analyzed using the LSD test. For the heart rate, the sampling dates were treated as a repeated measurement. Shapiro–Wilk was used to test the normality assumption on the residuals of each model, while residual plots were used to verify the homogeneity of variances. When the normality assumption was not met, response variables were transformed using the Box-Cox transformation. All analyses were performed using the Mixed procedure of SAS  at a significant level of At the end of this experiment, 27 rabbits survived , which met the criteria of HF. Their weight, including body and heart weight, significantly decreased for HF rabbits when compared with those in the control group and they progressively increased after CCM application . The CCMRegarding the heart tissue section photomicrographs of different treatments stained with H&E, we observed the slightly disordered arrangement of myocardial cells, partial myocardial necrosis, and fibrous tissue proliferation and inflammatory cells infiltration in the HF group while compared with those in the control group. These pathological variations were partly recovered after the CCM application .To assess the changes in cardiac interstitial fibrosis, histological sections of the heart were stained with Masson's trichrome, and the CVF were also examined. Heart tissue in the HF group rabbits presented a massive and intensive collagen accumulation when compared with those in the control group . But, thIn this study, three isoforms of Akt, including Akt1, Akt2, and Akt3, were investigated and they showed similar tendency after CCM application . Resultsβ, and TORC2 expressions were sharply upregulated in the HF group and then significantly downregulated after CCM was used . Activation of mTOR stimulates protein translation through its effects on p70 ribosomal S6 kinase (p70 S6K) and eukaryotic translation initiation factor 4E binding protein-1 (eIF4E-BP) [β [β, and TORC2 expression in myocardial cells in rabbits while compared with those in the HF group   and inhiIF4E-BP) . HoweverE-BP) [β  stimulatE-BP) [β  by diminE-BP) [β . AKT (SeE-BP) [β , which mHF group . Based o


To evaluate possible role of this interaction we studied effects of Li+ on NMDA-elicited whole-cell currents and Ca2+ responses of rat cortical neurons in vitro before and after cholesterol extraction by methyl-β-cyclodextrin (MβCD).The plasma membrane Na+ for Na+ in the external solution caused a concentration-dependent decrease of steady-state NMDAR currents from 440 ± 71 pA to 111 ± 29 pA in 140 mM Na+ and 140 mM Li+, respectively. The Li+ inhibition of NMDAR currents disappeared in the absence of Ca2+ in the external solution (Ca2+-free), suggesting that Li+ enhanced Ca2+-dependent NMDAR desensitization. Whereas the cholesterol extraction with MβCD induced a decrease of NMDAR currents to 136 ± 32 pA in 140 mM Na+ and 46 ± 15 pA in 140 mM Li+, the IC50 values for the Li+ inhibition were similar (about 44 mM Li+) before and after this procedure. In the Ca2+-free Na+ solution the steady-state NMDAR currents after the cholesterol extraction were 47 ± 6% of control values. Apparently this amplitude decrease was not Ca2+-dependent. In the Na+ solution containing 1 mM Ca2+ the Ca2+-dependent NMDAR desensitization was greater when cholesterol was extracted. Obviously, this procedure promoted its development. In agreement, Li+ and KB-R7943, an inhibitor of NCX, both considerably reduced NMDA-activated Ca2+ responses. The cholesterol extraction itself caused a decrease of NMDA-activated Ca2+ responses and, in addition, abolished the effects of Li+ and KB-R7943. The cholesterol loading into the plasma membrane caused a recovery of the KB-R7943 effects.Substitution Li2+-dependent NMDAR desensitization. Most likely, this is determined by a tight functional interaction of NCX and NMDAR molecules because of their co-localization in membrane lipid rafts. The destruction of these rafts is accompanied by an enhancement of NMDAR desensitization and a loss of NCX-selective agent effects on NMDARs.Taken together our data suggest that NCXs downregulate the Ca N-methyl-d-aspartate activated glutamate receptors (NMDARs) are ligand gated ion channels which naturally transfer currents determined by Na+, K+ and Ca2+ permeation. High permeability of NMDARs to Ca2+ makes them involved in synaptic plasticity ethyl] isothiourea methanesulfonate) or by the substitution of Li+ for Na+ in the external physiological solution considerably enhances the Ca2+-dependent desensitization of NMDARs  of 13 nM. The pH was adjusted to 7.4 with CsOH. Measured osmolarities of the external bathing solution and the patch-pipette solution were 310 and 300 mOsm, respectively. Patch pipettes (2–4 MΩ) were pulled from 1.5-mm (outer diameter) borosilicate standard wall capillaries with inner filament . In whole-cell configuration the series resistances did not exceed 10 MΩ. Holding membrane voltage (Vm) was corrected for the liquid junction potential between the Na+-containing external bathing solution and the Cs+-containing pipette solution of − 15 mV.Whole-cell currents were recorded on rat cortical neurons in primary culture (10–15 days in vitro) by patch clamp technique using a MultiClamp 700B amplifier with Digidata 1440A acquisition system. Details of recording and fast perfusion system were described previously . Unless Cells were loaded with Fluo-3 AM  using conventional protocols as described previously . Coversll-glycine (Gly). KB-R7943 (2-[4-[(4-nitrophenyl)methoxy]phenyl]ethyl ester, methanesulfonate, 10 µM) application or proportional substitution of Li+ for Na+ in the external bathing solution were used to inhibit NCX. Methyl-β-cyclodextrin  application for 5 min was used to destruct lipid rafts by extracting cholesterol from the plasma membrane. The complex of cholesterol with methyl-β-cyclodextrin  as a donor of cholesterol was applied for 30 min to restore the cholesterol content of the plasma membrane. All compounds were from Sigma-Aldrich, St. Louis, MO, USA or Tocris Bioscience, UK.Functional activity of NMDARs requires binding of both glutamate and a co-agonist, glycine. Unless otherwise stated, to activate NMDARs we applied 100 µM NMDA with 10 µM t-test were used for statistical analysis. Number of experiments is indicated by n throughout. In the patch-clamp experiments n represents a number of recorded neurons. In the Ca2+-imaging experiments n represents a number of used culture coverslips. From every coverslip a single mean value obtained from many cells was utilized for statistics. The data were considered as significantly different based on a confidence level of 0.05. Current measurements were plotted using ClampFit 10.2 (Molecular Devices). The IC50  and Hill coefficient (h) for inhibition of NMDA-evoked currents with Li+ were estimated by fitting of concentration–response curves with the Hill equation, I = Imin + (Imax − Imin)/(1 + [Li+]h/IC50h), where the Imax and Imin are the current of maximal and minimal amplitudes elicited by NMDA at different [Li+].Quantitative data are expressed as mean ± SEM. ANOVA and Bonferroni multiple comparison methods as well as Student’s two-tailed