Simulation results showed that CaMKII overexpression significantly increased the phosphorylation level of its downstream target proteins, resulting in prolonged AP and smaller calcium transient amplitude, and impaired the Ca2+ cycling stability. These effects were exacerbated by extra reactive oxygen species, which oxidized CaMKII and led to continuous high CaMKII activation in both systolic and diastolic phases. Intracellular Ca2+ depletion and sustained delayed afterdepolarizations (DADs) were observed under co-existing CaMKII overexpression and oxidation, which could be effectively reversed by clamping the phosphorylation level of ryanodine receptor (RyR). We also found that the stability of RyR release highly depended on a delicate balance between the level of RyR phosphorylation and sarcoplasmic reticulum Ca2+ concentration, which was closely related to the genesis of DADs. We concluded that the CaMKII overexpression and oxidation have a synergistic role in increasing the activity of CaMKII, and the unstable RyR may be the key downstream target in the CaMKII arrhythmogenic mechanism. Our simulation provides detailed mechanistic insights into the arrhythmogenic effect of CaMKII overexpression and oxidation, which suggests CaMKII as a promising target in the therapy of atrial fibrillation.This simulation study aims to investigate how the Calcium/calmodulin-dependent protein kinase II (CaMKII) overexpression and oxidation would influence the cardiac electrophysiological behavior and its arrhythmogenic mechanism in atria. A new-built CaMKII oxidation module and a refitted CaMKII overexpression module were integrated into a mouse atrial cell model for analyzing cardiac electrophysiological variations in action potential (AP) characteristics and intracellular Ca The phosphorylation of ryanodine receptor (RyR) by CaMKII can increase the sensitivity of RyR to Ca2+, thereby RyR would have a closer connection with the intracellular concentration of Ca2+ ([Ca2+]i)  for Ca2+, leading to an enhancement of its Ca2+ transportation rate  is the most common persistent arrhythmia, affecting ~33 million of the world's population .In this study, the mouse atrial model constructed by Zhang et al.  was inco4CaM (a CaM binding with 4 Ca2+) and transform to the active state Pb. CaMKII in this state Pb can phosphorylate neighboring subunits in the presence of ATP, transforming them to the state Pt, which process is called the autophosphorylation of CaMKII. Autophosphorylated subunits have long-lasting activity, even if they dissociate with CaMs and further transform to the state Pa. In addition, CaMKII may also bind with Ca2CaM in an environment of more unsaturated CaM (shown as the state Pi to Pb2 and the state Pa to Pt2), waiting for another two Ca2+ for activation. Finally, CaMKII under the state  can be dephosphorylated by protein phosphatase 1 (PP1) to the state , respectively. For the whole model, there are two inactive states  and four active states , and all active states are assumed to have 100% activity. Morotti et al.  and further autophosphorylate adjacent subunits to maintain the activation  model.4CaM but still remain active (state Po). Oxidized CaMKII (state Pot and Po) can be deoxidized by methionine sulfoxide reductases (MsrA) to the inactive state (Pi and Pb), respectively. Each state transition equation can be written as:For modeling CaMKII behaviors under oxidative stress, we built a new four-state Markov chain model . CaMKII other denotes all other transitions with the original model, [ROS], [MsrA], and [Ca4CaM] represent the concentration of ROS, MsrA and Ca4CaM. Details of all parameters in the equation are listed in where Tbi and kib were inherited from the original model  and the Michaelis-Menten constant of MsrA (Km_MsrA) were used as those measured in experiments  and the Michaelis-Menten constant of ROS (Km_ROS), we replicated the Erickson et al. , transient outward potassium current (Ito), inward rectifier potassium current (IK1) and Na+-Ca2+ exchanger current (INCX). Details of modifications on these membrane currents were summarized in In this study, we increased the total concentration of intracellular CaMKII to 6-fold of the WT model for modeling its overexpression as suggested by the previous study  and main membrane currents between the CaMKII-OE and WT model is shown in  by ~50% , consist by ~50% . Physioldy state , which wIn this study, the steady-state protocol was used to investigate the influence of CaMKII overexpression and oxidation on the action potential and intracellular calcium cycling. The cell model was paced for around 5 min (cell time) at 1 Hz until the steady state was reached. The steady state was defined as the situation when differences in ion concentrations between two consecutive beats were lower than one hundred thousandths of the basal level.To further study the effect of abnormal CaMKII at higher heart rates, the widely used burst pacing protocol was applied to induce abnormal cell behaviors. The cell model was first initialized with 1-Hz pacing, then stimulated at 10 Hz for 12 s (burst pacing period). Finally, we resumed the stimulation frequency to 1 Hz to see how myocytes react.2+]SR) and phosphorylation levels. The cell model was first initialized with 1-Hz pacing, then the stimulus ceased and the [Ca2+]SR and CaMKII dependent RyR phosphorylation level were clamped at different levels [ranged from 200 to 800 μM for [Ca2+]SR, and from 10% to 80% for RyR phosphorylation] for 10 s. After that, the clamp on [Ca2+]SR was released and 200 stimuli at 1-Hz were applied to the model, the amplitude of RyR release of the first beat was recorded for corresponding [Ca2+]SR and RyR phosphorylation levels. A similar clamp protocol was also used for obtaining the RyR activation threshold, whereas the differences are: (1) we substituted the ICaL with a manually constructed calcium current (named ICa) in order to control the transmembrane calcium influx; (2) no stimulus current was applied since the RyR could be activated purely by Ca2+ influx. The ICa had a square waveform with a 5-ms duration. The RyR was tested by increasing the amplitude of ICa, until at least 1/4 Ca2+ in the SR was released, where this amplitude of ICa was defined as the RyR activation threshold. The RyR activation threshold was recorded under various levels of [Ca2+]SR (from 200 to 800 μM) and RyR phosphorylation (from 10 to 80%) to produce a RyR activation threshold map.We also carried out a series of clamping simulations to investigate the RyR property, in which we recorded its release amplitude and activation threshold at different sarcoplasmic reticulum (SR) calcium concentration  was also ignorable  during the diastolic phase and about 700 μM during the systolic phase (2+]i and [Ca2+]SR were lower in the CaMKII-OE model (+ ([Na+]i) was 3 mM higher in the CaMKII-OE model than that in the WT model , diastolic CaMKII activity was significantly increased compared with the slow pacing rate while systolic CaMKII activity almost remained the same . In the slightly . On the slightly , therefoiderably , resulti2+ release during the diastolic period. This suddenly increased [Ca2+]i gave rise to an abruptly enhanced forward mode of INCX (Ca2+ extrusion mode), therefore a depolarizing current was formed. However, this instability cannot last since the cell is adaptive to the variation in the pacing rate. In the WT model, only three DADs were seen in the next two cardiac cycles, whereas six DADs were found in the next three cardiac cycles in the CaMKII-OE model, showing the disturbing role of CaMKII overexpression in cell's adaptive ability.When the pacing rate returned to 1 Hz (from 14 to 40 s), DADs appeared in the next several cardiac cycles in both models . This is2+]i, [Ca2+]SR, [Na+]i was observed  was required to induce the SR Ca2+ release. With the increase of [Ca2+]SR and RyR phosphorylation level, this RyR activation threshold gradually decreased, e.g., a calcium current of only 2.9 pA/pF can induce the SR Ca2+ release for condition B [[Ca2+]SR = 450 μM, RyR phosphorylation level = 40%], which indicated that RyR was more easily activated and its stability was impaired. When the [Ca2+]SR and RyR phosphorylation level increased to a certain extent SR = 600 μM, RyR phosphorylation level = 60%, marked as condition C in 2+]SR clamp was released and would not close anymore, i.e., it remained open without any Ca2+ influx stimulus, which means that the RyR channel was extremely unstable.To further study the main factors that related to the instability of RyR, we carried out the clamping experiments to obtain the RyR activation threshold map under different levels of [Ca2+ can release normally (the amplitude of Jrel is over 5 μM/ms) only when the level of [Ca2+]SR and RyR phosphorylation was in a certain range, which we named as the balance region  ROS oxidized CaMKII led to continuous high level of CaMKII activation in both systolic and diastolic phase. The malignant influence of ROS was much more significant with CaMKII overexpression, which considerably disturbed CaT stability and led to persistent DADs that might become the trigger of ectopic activities for AF; (3) The increase in [Na+]i could worsen the cell instability, but this may be a secondary cause. Clamping simulations showed that this instability is highly related to the balance between the RyR phosphorylation and [Ca2+]SR level, which may be the underlying mechanism of the DAD and AF induced by CaMKII hyperactivity.In this study, we built a new CaMKII oxidation module and a refitted CaMKII overexpression module and incorporated them into a mouse atrial model to investigate the arrhythmogenic mechanism of CaMKII overexpression and oxidation. Our simulation results showed that: (1) The overexpression of CaMKII significantly increased the phosphorylation levels of its downstream target proteins, resulting in prolonged AP, increased [Na2+]i. This, on the one hand, brings stronger SR Ca2+ release; on the other hand, lowers the [Ca2+]i threshold to induce the SR Ca2+ release. The phosphorylation of PLB by CaMKII brings a stronger SR Ca2+ reuptake current, leading to a faster Ca2+ reuptake process. Overall, CaMKII accelerates the intracellular calcium cycling (from SR Ca2+ release to reuptake), and has the effect of strengthening cardiac contraction and relaxation, so that contributes to myocyte adaptation to frequency changes.CaMKII can regulate cardiac excitation-contraction coupling by phosphorylating several target proteins including LTCC, RyR, and PLB. Specifically, this process leads to enhanced LTCC, bringing stronger calcium induced calcium release. RyR phosphorylation enhances RyR's sensitivity to i was clamped at a lower level, most of the DADs caused by CaMKII overexpression and oxidative stress could be eliminated  and Heart failure (HF) are closely related. The incidence rate of AF may increase by 6–8 times in patients with HF  lead to more stable and larger CaT amplitude, therefore can reduce the incidence of malignant arrhythmia and improve myocardial mechanical function, which may become an important treatment strategy for AF patients with HF.+ current is an important membrane current that would be greatly affected by the ROS concentration (Bhatnagar, This study inherited limitations from the baseline model as described in Zhang et al. . Apart f2+]SR level, which might be the key mechanism underlying the DAD and AF induced by CaMKII hyperactivity. Finally, we discussed the interrelationship between CaMKII, AF and HF, and CaMKII as a potential target in the treatment for patients with co-existing AF and HF.In conclusion, we investigated the arrhythmogenic mechanism of CaMKII overexpression and oxidation by using a mouse atrial cell model, which incorporated a new-built CaMKII oxidation module and a refitted CaMKII overexpression module. Our results showed that the CaMKII overexpression and oxidation had a synergistic role in increasing the kinase activity of CaMKII, leading to CaMKII hyperactivity in both systolic and diastolic phases, which significantly altered the cardiac electrophysiology, calcium cycling, and stability of RyR. The hyperactivity of CaMKII could also induce sustained DADs. We found that the stability of RyR was highly related to the balance between the RyR phosphorylation and [CaThe original contributions presented in the study are included in the article/WW, SZ, and WS conceived and designed most of the study, performed the simulations and analyses, and wrote most of the manuscript. GL contributed to the figure design and manuscript writing. HZ, YX, and KW supervised the project. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


To synthesize and characterize poly(hydroxybutyrate) (PHB) and norbixin membranes to evaluate them for genotoxicity in rats and wound healing in mice by histological staining.Rattus novegicus ) were divided into three groups (n= 5): 5% PHB/Norbixin membrane introduced into the peritoneum by laparotomy; B – negative control; C – positive control . For the evaluation of biocompatibilty, a cutaneous wound was induced on the back of males mice ( Mus musculus ) divided into two experimental treatment groups: control and membrane that underwent euthanasia after 7 and 14 days treatment. Statistical analysis ware made by One Way Anova post hoc Tukey Test (p<0.05). For the evaluation of genotoxicity, male rats . As for DNA damage, the changes induced in the first 4h were repaired in 24h. In addition, the membrane was effective in abbreviating the inflammatory process and served as a scaffold due to the stimulus to reepithelialization mainly on the 7 days of treatment. The non-genotoxic PHB/Norbixin 5% membrane presented promising results that suggest its effectiveness as a guide for tissue regeneration given its biocompatibility. The annato tree is native to Central and South America. Carotenoids have antioxidant properties and act against free radicals, which biologically favors the evolution of the tissue repair process. Animal studies have revealed the absence of genotoxic, teratogenic or mutagenic effects from these materials17 .Norbixin is a carotenoid pigment extracted from the annatto seed pericarp  and norbixin, can be promising, although there are no experimental studies involving norbixin associated with polyhydroxybutyrate as constituents of biomaterial in the repair of cutaneous wounds or bone regenerationet al .18 synthesized a membrane from poly (hydroxybutyrate) (PHB), norbixin and ethylene glycol and concluded that the micronucleus test suggested that the biomaterial has no biological toxicity as it does not change the incidence of polychromatic erythrocytes. The comet assay revealed an increase in DNA damage in the first 4h, suggested a repair of the same in 24 hours.Moreover, Sousa Bixa orellana L.) to assess its genotoxic potential in rats and wound healing in mice in histological staining.The synthesis of a membrane from a non-synthetic biopolymer (PHB) and a natural antioxidant (norbixin) as a healing biomaterial from renewable resources requires both physico-chemical characterization and biocompatibility tests. The objective of this study was to synthesize and evaluate the physicochemical properties of poly (hydroxybutyrate) membranes containing or not norbixin , Universidade Estadual do Piauí (UESPI) under protocol number 09241/2016, in accordance with the Guiding Principles for the Care and Use of Laboratory Animals.Bixa orellana L .), collected in the city of Teresina-PI, whose registration was carried out at the Herbarium Graziela Barroso -TEPB, at the Federal University of Piauí (nº 31573). The polymer used as matrix of the membranes was PHB, which was supplied by the company PHB Industrial S/A  and was prepared from the fermentation of sucrose by bacteria “ Alcaligenes eutrophus ”. Potassium hydroxide (KOH), ethyl ether, hydrochloric acid (HCl) and chloroform (Synth) all P.A.The norbixin used in this study was extracted from the seeds of Annato ( Bixa orellana L.). Initially, seeds were mixtured with ethyl ether under stirring . After cooling the mixture, HCl was added until a color change precipitate was formed. The precipitate was washed until pH was approximately equal to 4 and then oven dried at 70°C for 24 hours.Norbixin was extracted from annatto seeds .Preparation of membranes: (a) initially the PHB was allowed to rest in chloroform, (b) the PHB held in chloroform was heated to 60°C under stirring. (c) In the preparation of the membrane containing only PHB, after the solution reached room temperature, the mixture remained for 10 min on ultrasound and finally was then poured onto petri dish and allowed to stand for 24 hours. On the 5% PHB/norbixin membrane, as well as on the 10% PHB/norbixin membrane, after step (b), the norbixin pigment previously diluted in chloroform was added to the PHB following the same final process as step (c). The thickness of the membranes was measured using a 0mm-12.7mm digital Micrometer Western ToolFourier Transform Infrared Spectroscopy (FTIR). The membrane spectra were obtained in infrared IRAffinity-1 Shimadzu  with the recording range of waves from 4000 to 400 cm-1. The spectra were performed at a resolution of 16 cm-1 from 45 scans.Thermogravimetric Analysis (TA) and Differential Scanning Calorimetry (DSC) . The thermogravimetric analysis of each sample was performed by using an average mass of 8.0 mg. The analyses were obtained in the temperature range of 30 to 900°C, at a heating rate of 10° C.min-1 under a nitrogen atmosphere, with gas flow of the order of 50mL.min-1. All results were obtained with tests performed on the TGA-51 Shimadzu . Differential Exploration Calorimetry was performed in a DSC-60 Plus Shimadzu , under the same TGA conditions, with an average mass of 6 mg at a temperature ranging from 30 to 500°C.Scanning Electron Microscopy (SEM) . The samples were initially metallized with gold. The images were obtained by using the scanning electron microscope SURPERCAN SSX-550 Shimadzu , to a 15.0 kV voltage, magnification at 2,000x.Rattus novergicus male animals with average weight between 200 and 300g were used simultaneously in both tests. The animals were divided into three groups  as each intervention was performed. The 5% PHB/Norbixin membrane was chosen to perform the experimental procedures because it presented intermediate characteristics between both pure PHB membranes and PHB/Norbixon 10% membranes.The same group of samples composed of 15 In group A, there was no treatment, only a peritoneal laparotomy was performed to simulate the surgical process. In group B, which was exposed to biomaterial, a laparotomy was performed for implantation of the membrane in the peritoneum region. In group C, as a single dose injected of intraperitoneal cyclophosphamide (genotoxic and inducing DNA damage of bone marrow cells) at a concentration of 50 mg/kg per animal. Each experimental group was composed of 05 animals anesthetized with ketamine (1.0 ml/kg) and xylazine (1.1 ml/kg) intramuscularly. The animals were sacrificed 72h after the experiment had started.Micronucleus test . The micronucleus test was carried out by using bone marrow cells collected immediately after each animal was sacrificed. A 1.0 ml syringe filled with fetal bovine serum was introduced into the opening at one end of the femur. The bone marrow material in the fetal bovine serum was resuspended till it got homogeneous. The suspension was centrifuged for 5 minutes at 1000 rpm, and the supernatant was discarded at the end of the procedure by a Pasteur Pipette. Two slides per animal were made.22 .Cells were re-suspended, placed on slides and stained with 10% GIEMSA in phosphate buffer . Then, the cells were observed under an optical microscope (100X), in a short time in a NIKON LABPHOT 2 microscope The comet test. For the comet assay, blood samples were collected from the animal’s tail in two different occasions: 4h post-exposure (acute) and 24h post-exposure (chronic) to the biomaterial. At the end of each exposure (4h or 24h), 40 μL of the blood samples collected from the animal’s tail were transferred to microtubes containing 120 mL of low melting point agarose (1.5%) at 37°C. This mixture was homogenized and transferred to precoated slides with 5% agarose. The slides were then covered with coverslips and placed at 40°C for 30 minutes. The coverslips were removed and the slides were immersed in lysis solution (NaCl (2.5 M), EDTA (100 mM) and 1.2 g TRIS (10 mM), 1% Triton X-100 and 10% DMSO].For DNA denaturation, the slides were placed in an electrophoresis cell containing a buffer solution pH> 13 (300 mM NaOH and 1 mM EDTA), prepared from a stock solution  for 20 min. The electrophoresis was performed at 25 V and 300 mA at a temperature of 4°C for 15 minutes. The slides were then immersed in a neutralization solution .23 .The analysis was performed on an immunofluorescence by Biofocus Stereomicroscope WF10X/22 (x40)  equipped with a 420-490 nm filter and a 520 nm barrier filter. The images were captured with a 5.0 megapixel CCD digital camera for immunofluorescence. DNA damage was assessed by measuring the percentage of DNA in the tail and by the height/length of the tail, which were calculated at 100 nucleoids/ sample . In order to get that, the OpenComet software was usedMus musculus mice, with a mean weight ranging from 20 to 30g, were randomly divided into two groups: control and membrane . Five animals of each group were sacrificed after 7 days and the remaining five mices after 14 days of treatment.In this study, 20 male Induction of experimental injury. The animals were weighed and then anesthetized by intramuscular administration of 10% ketamine hydrochloride at a dose of 0.1 ml for each 100g of body weight associated with the same dose of 2% xylazine hydrochloride. Anesthetic drugs were applied dissociatively, using 1 mL capacity syringes and 8 x 0.30 mm needles. The lesion was induced on the back of the animals, positioned in dorsal decubitus, and began with the tricotomy of the region. To perform the experimental lesion, a surgical instrument (punch) 8 mm in diameter, positioned perpendicular to the back was used. In group membrane, the PHB/Norbixin 5% membrane was implanted.Histological analysis. The mice were submitted to euthanasia by an overdose of sodium thiopental in the concentration of 50 mg/kl. The surgical specimen was removed and after fixation for 24h, the histological technique of routine was processed, including the steps of gradual dehydration, diaphanization, infiltration and embedment in paraffin. The samples were submitted to longitudinal histological cuts, stained with hematoxylin eosin (HE) and analyzed by microscope Olympus CX31 , with x400 magnification. A qualitative histological analysis of inflammatory reaction was made, defined by: reepithelization, granulation tissue, presence of inflammatory cells, fibroblasts, collagen deposition and neovascularization. The criteria of histological analysis followed the protocol of Meirelles et al .24 to facilitate statistical analysis, the following score was created: 0, 1, 2 and 3, where: 0 –absent, 1 – discrete, 2 – moderate, 3 – intense.Data were analyzed using One-Way ANOVA test post hoc Tukey. For all tests was considered a 5% significance level. The results were expressed as mean, or standard deviation of three independent experiments. The result was considered positive if there was a statistically significant increase (p <0.05).Norbixin and PHB have good solubility and processability in chloroform, facilitating the formation of membranes. They presented good consistency with an averge thickness of approximately 400 μm, apparently homogeneous.-1, regarding the OH groups of its two terminal carboxylic acids. The 2921 cm- 1 peak, which has two shoulders at 2985 and 2921 cm-1, is a stretching of the CH2 group, with the shoulders representing symmetrical stretches of both the CH3 and CH groups, respectively. Peaks at 1678, 1563, 1134, 1091, 1034 cm- 1 , represent the terminal carbonyls found in the norbixin structure. The peak at 1611 cm-1, and the peaks in the region between 1000-886 cm-1 are attributed to the double bonds. The methyl groups present in the norbixin chain are represented by the peaks at 1422 and 1364 cm-1 27 .The infrared spectra of norbixin and polyhydroxybutyrate can be seen in 2 group at 2930 cm-1 with two shoulders with spectra at 2977 and 2861 cm-1, depicted the CH3 and CH groups, respectively. Peaks at 1722, 1269, 1224, 1183, 1098, 1049 and 830 cm-1 represent bonds of carbon atoms to oxygen atoms (CO groups). The methyl (CH3) groups show peaks at 1380, 1129 and 974 cm-1 28 .The PHB also has a symmetrical stretching peak of the CHThe spectra of both PHB/Norbixin 5% and 10% membranes compared to the PHB membrane have not showed major modifications concerning the formation and disappearance of PHB peaks   .29 .In In the DSC curve of norbixin, four characteristic thermal processes are observed. Three of them are of exothermic origin - at temperatures of 174, 210 and 466°C   . The fo30 . When compared to the positive control represented by cyclophosphamide, known to be genotoxic, there was a significant difference (p <0.001).Concerning the evaluation of genotoxicity by the comet assay in the group exposed to the 5% PHB/Norbixina membrane, the results indicated an increase in damage in the first 4h of exposure evaluated by the percentage of DNA in the tail   . This rThe different treatment groups in both observation periods of 7 and 14 days are shown in -1 comes up, probably representing the double bonds of norbixin. At 1357 cm-1 one has the definition of a peak, which apparently in the PHB is represented by two small peaks at 1357 and 1347 cm-1. This norbixin peak represents the methyl groups attached laterally to its backbone. It has been found that the addition of 5 and 10% norbixin to the PHB membrane changes the peak intensity at 1722, 1453, 1380, 1269, 1224, 1183, 1129, 1098, 1049, 974 cm-1, suggesting good interaction between the componenets of the membrane.With the addiiton of norbixin to the membranes, a peak at 1613 cm27 . In this region, PHB has a degradation rate of 99.1%; the 5% PHB/Norbixin membrane, 96.9%; and the PHB/ Norbixin 10%, has a 96.3% loss of mass. It is observed that as the norbixin concentration increases in the membranes, the initial temperature of the main degradation decreases, going from 269°C to 243°C.The degradation of both PHB and PHB/Norbixin membranes occurs almost entirely in a single stage, between 243 and 272°C. This result is in accordance with the results found in the literature for polyhydroxybutyrate33 , the norbixin degradation itself starts at 130°C.Norbixin, unlike PHB, has degraded in more than one stage. The first degradation occurred from 25 to 120°C, a loss of 2.0% in its mass probably represented by the release of water. The second degradation band, with approximately 45.2% mass loss, occurred during the range from 145 to 440°C. A slow third degradation, initiated at 440°C, lasts until a temperature above 1000°C is reached. At 1000°C a residual mass of approximately 8.0% remains. Similarly, for to Scootter34 who observed that norbixin does not melt up to a temperature of 300°C. These authors observed that norbixin changes color from 250°C, changing gradually from red color to a darker color. This fact was also reported by Scotter et al .33 who stated that norbixin undergoes carbonization above 290°C, changing from red to a dark color. Probably the endothermic processes found on the norbixin DSC curve are also associated with the decomposition of norbixin.This result has also been reported in the study of Reith and Gielen31 .Regarding the TG and DSC thermal measurements, a thermal behavior of gradual mass decomposition of the membranes was observed in a single stage, which is a typical behavior of PHB. The displacement of the curves to the right of the PHB/Norbixin membranes in relation to the PHB    is asso31 .The membranes presented a homogeneous, non-porous surface, which revealed characteristic spectra of their functional groups and that reflected the adhesion and aggregation of these two components by MEV. The clusters were observed in the Rattus novergicus has not changed.Based on the mentioned results, the micronucleus test suggested that the biomaterial has no biological toxicity inasmuch as the incidence of polychromatic erythrocytes in the blood of Although the results of the comet assay have shown an increase in the DNA damage within the first 4h, they suggested repairing such damage at 24h using the membrane. The results were promising when it comes to the use of norbixin and PHB as biomaterials, though there are few studies in literature that have evaluated both biological effects and properties of norbixin in animal tissues.36 .The in vivo assays used in this research were relevant because they show detailed biological and physiological information and are officially approved and considered as by the Organization for Economic Cooperation and Development (OECD) as part of the security prootcol analysis for genotoxicity assessment40 . Especially in the first days of tissue injury, it was observed that the PHB/Norbixin 5% membrane presented promising results that suggest its effectiveness as a guide for tissue regeneration and abbreviation of acute inflammation.In this study, biomaterial used as scaffold in the repair of cutaneous wounds in mice was effective in both experimental periods, with complete cicatrization of the induced lesion, suggesting the biocompatibility of the biomaterial in the biological system in question. However, it was observed that such scaffold was more effective in the acute phase, that is, in the first 7 days of the lesion after the experiment with the presence of mild inflammatory infiltrate, than at 14 days, because there was a moderate and more intense inflammatory infiltrate. Considering that the literature reports that, in intact tissue, carotenoid pigments are protected from oxidation, the increase of the inflammatory process could be related to the decomposition of norbixin during the observational period of the experimentet al .41 evaluated the therapeutic effect of biomaterial with polymer matrix associated with norbixin compared to laser photobiomodulation , which was associated with or without a polystyrene membrane coated with norbixin and collagen (PSNC) on bone healing in rats with calvarial bone defect. The groups were analyzed after 15 and 30 days treatment. The authors concluded that the PSNC membrane was effective in reducing the inflammatory process and served as a scaffold for bone repair. The laser PBM only showed positive effects on the bone repair process with increased deposition and organization of the newly formed bone. However, the association of laser PBM + membrane failed to improve the bioactive properties of the membrane scaffold.Alves 42 is known in the literature and the promising efficacy of the biomembrane as a guide for tissue regeneration was expected. However, studies that investigated the use of norbixin such as biomaterials are recent18 , since the most common reports still surround their application in the food industry. It is necessary to continue this research line considering the potential for application of the formulated biomaterial, as observed in this study mainly in the first days after tissue injury, necessitating the continuity of this pilot study with longer intervals of observation time, larger samples and increased inflammatory analyses, immunohistochemistry and biochemistry to know the physico-chemical interactions of this biomaterial in an isolated biological system.Use of biomaterials with polymeric matrices in the health fieldHomogeneous, non-porous membranes were obtained, which revealed characteristic spectra of their functional groups and that reflected the adhesion and aggregation of these two components by MEV. In addition, the decrease in temperature was observed as the norbixin content increased, which may optimize its use as biomaterial, since it suggests a better degradation of the PHB/Norbixin membrane regarding the PHB membrane. The micronucleus test and comet assay presented results that corroborate with the 5% PHB/Norbixin membrane suitability for biological purposes and the polyhidroxybutyrate membrane coated with norbixin reduced the inflammatory process and served as a scaffold for wound healing when used in isolation.


Mechanical ventilation (MV) is often a life-saving intervention for patients in respiratory failure. Unfortunately, a common and undesired consequence of prolonged MV is the development of diaphragmatic atrophy and contractile dysfunction. This MV-induced diaphragmatic weakness is commonly labeled “ventilator-induced diaphragm dysfunction” (VIDD). VIDD is an important clinical problem because diaphragmatic weakness is a major risk factor for the failure to wean patients from MV; this inability to remove patients from ventilator support results in prolonged hospitalization and increased morbidity and mortality. Although several processes contribute to the development of VIDD, it is clear that oxidative stress leading to the rapid activation of proteases is a primary contributor. While all major proteolytic systems likely contribute to VIDD, emerging evidence reveals that activation of the calcium-activated protease calpain plays a required role. This review highlights the signaling pathways leading to VIDD with a focus on the cellular events that promote increased cytosolic calcium levels and the subsequent activation of calpain within diaphragm muscle fibers. In particular, we discuss the emerging evidence that increased mitochondrial production of reactive oxygen species promotes oxidation of the ryanodine receptor/calcium release channel, resulting in calcium release from the sarcoplasmic reticulum, accelerated proteolysis, and VIDD. We conclude with a discussion of important and unanswered questions associated with disturbances in calcium homeostasis in diaphragm muscle fibers during prolonged MV. Mechanical ventilation (MV) is often a life-saving intervention for both critically ill patients and patients undergoing surgery. An unwanted side-effect of prolonged MV is the rapid development of inspiratory muscle weakness that occurs due to both diaphragmatic atrophy and contractile dysfunction. Collectively, this syndrome has been labeled ventilator-induced diaphragm dysfunction (VIDD) . VIDD is2+)-activated protease, calpain, plays a central role in MV-induced diaphragmatic atrophy. Indeed, blockade of calpain activation in the diaphragm can markedly reduce both MV-induced diaphragmatic atrophy and contractile dysfunction , resulting in the release of Ca2+ from the SR, calpain activation, and VIDD. In an effort to stimulate future research, we also highlight unanswered questions associated with signaling events.This review provides a summary of the cell signaling events leading to VIDD with a focus on the cellular processes that result in disturbed CaThe observation that prolonged MV results in diaphragmatic wasting was first reported in a retrospective study revealing that diaphragmatic atrophy was present in infants and neonates exposed to prolonged MV . Direct Two primary modes of MV exist: (1) partial support and (2) full support MV. During partial support MV, the ventilator assists during inspiration, but the patients’ inspiratory muscles remain engaged in breathing. During full support MV, the ventilator performs all of the work of breathing, resulting in inactivity of the diaphragm and other inspiratory muscles; compared to partial support, full support MV results in a more rapid rate of VIDD. Indeed, when contrasted with inactivity-induced limb muscle atrophy , full support MV-induced diaphragmatic atrophy is a unique form of skeletal muscle atrophy that occurs extremely rapidly after the onset of MV. For instance, the cross-sectional area (CSA) of diaphragm muscle fibers is reduced by >15% within the first 12–18 h of MV in both rats and humans . In compAs mentioned previously, the diaphragmatic weakness associated with VIDD is a primary risk factor for the failure to wean patients from the ventilator. In this context, weaning is defined as the ability to remove patients from ventilator support and restore spontaneous breathing. The incidence of difficult weaning of patients from MV is variable but can reach >30% or higher in those ventilated for more than 3 days . The ina2+ are required to activate calpains, the next segment provides a brief overview of the regulation of cellular Ca2+ levels.The size of skeletal muscle fibers is controlled by the relative balance between rates of protein synthesis and protein breakdown. It is well established that prolonged MV results in a rapid increase in proteolysis and a decline in protein synthesis in the rodent diaphragm . In rega2+ in the cytosol can be toxic to cells by promoting increased mitochondrial ROS production, protease activation, and myonuclear apoptosis  coupling and a variety of cell signaling pathways. Importantly, high levels of free Capoptosis . Therefo details . Neverth2+ as evidenced by the large difference  between the extracellular and cytosolic Ca2+ concentration , and store operated entry channels (SOC)  .2+ levels are toxic to muscle fibers, the sarcolemma is equipped with ATPases – e.g., plasma membrane Ca2+ ATPases (PMCA) – to pump Ca2+ from the cytosol across the sarcolemma (+/Ca2+ exchanger (NCX) also contributes to Ca2+ exchange across the sarcolemma  SR Ca2+ release channels; (2) SR Ca2+ binding proteins; and (3) Ca2+ ATPase pumps. A brief summary of these classes of SR handling proteins follows.The SR plays an essential role in the management of intracellular Ca2+ release channel in the SR is the ryanodine receptor (RyR). Three isoforms of the RyR  exist in mammals, but RyR1 is the dominant isoform expressed in adult skeletal muscle and is crucial for muscular contraction  . DHPRs atraction .2+ release through the RyR1 is also regulated by several post-translational events along with numerous proteins and small molecules. Cytosolic regulators of RyR1 include ATP, calmodulin II, and protein kinase A (PKA)  . RyR1 ac cytosol .2+ transiently , which allows for the exchange of molecules and ions between the cytosol and mitochondria. However, the mitochondrial inner membrane is impermeable to ions, and therefore, highly specialized channels are required for Ca2+ ions to enter the mitochondrial matrix   . This rue matrix .2+ uptake led to studies revealing that Ca2+ is an important regulator of oxidative phosphorylation. For example, Ca2+ is a positive allosteric activator for several key enzymes that influence flux through the citric acid cycle   . Moreovechondria . In cont stimuli .2+ from the mitochondria in skeletal muscle is the Na+/Ca2+ exchanger and the H+/Ca2+ exchanger located within the inner mitochondrial membrane  can be formed and operates as a conduit for release  and disrupts Ca2+ homeostasis  leading to the disassociation of calstabin1 from RyR1 and Ca2+ leak from the SR. This disturbance in Ca2+ homeostasis provides a required stimulus for calpain activation in the diaphragm leading to accelerated proteolysis and VIDD (Mechanical ventilation-induced diaphragmatic atrophy results from both decreased protein synthesis and accelerated proteolysis. However, MV-induced activation of proteases in the diaphragm play a dominant role in the development of VIDD. Although all major proteolytic systems contribute to this MV-induced increase in proteolysis, evidence suggests that activation of the Caand VIDD .2+ homeostasis in the diaphragm, several important unanswered questions remain. For example, although it is established that an increase in mitochondrial ROS production is a requirement for MV-induced RyR1 leak leading to increased cytosolic levels of free Ca2+, the precise mechanisms responsible for this increased oxidant production remain undefined.Although significant progress has been made toward understanding the mechanisms responsible for the MV-induced disturbance in CaEvidence also exists that accelerated autophagy also promotes an increase in mitochondrial ROS emission in the diaphragm during prolonged MV . However2+ into mitochondria can produce a mitochondrial Ca2+ overload, resulting in significant increases in mitochondria ROS production (2+ in the diaphragm are sufficient to produce mitochondrial Ca2+ overload and stimulate additional mitochondrial ROS production. This is an important issue that warrants future study.Finally, it is generally agreed that an uptake of cytosolic Caoduction . NonetheHH and SP formulated the outline of the review and contributed to writing and editing of the manuscript. Both authors approved the final submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer WN declared a past co-authorship with one of the author SP to the handling editor.


Recently, mechanically induced insulin secretion pathways were also reported, which utilize free cytosolic Ca2+ ions as a direct regulator of exocytosis. In this study, we aimed to investigate intracellular Ca2+ responses on the HIT-T15 pancreatic β-cell line upon low-intensity pulsed ultrasound (LIPUS) stimulation and found that ultrasound induces two distinct types of intracellular Ca2+ oscillation, fast-irregular and slow-periodic, from otherwise resting cells. Both Ca2+ patterns depend on the purinergic signaling activated by the rise of extracellular ATP or ADP concentration upon ultrasound stimulation, which facilitates the release through mechanosensitive hemichannels on the plasma membrane. Further study demonstrated that two subtypes of purinergic receptors, P2X and P2Y, are working in a competitive manner depending on the level of glucose in the cell media. The findings can serve as an essential groundwork providing an underlying mechanism for the development of a new therapeutic approach for diabetic conditions with further validation.In glucose-stimulated insulin secretion (GSIS) of pancreatic β-cells, the rise of free cytosolic Ca Insulin, a peptide hormone secreted by the pancreas, plays a crucial role in maintaining the homeostasis of the blood glucose levels in the human body. It lowers the blood glucose levels by facilitating glucose uptakes in the liver, skeletal muscle, and adipose tissue. Failure to maintain blood glucose levels, also known as hyperglycemia, can give rise to many different severe health conditions, including diabetes. Insulin secretion is stimulated by many different hormones and neurotransmitters such as glucagon-like peptide-1 , but glu+ channels respond to the elevated ATP/ADP ratio in the cytoplasm by closing their channels, resulting in the depolarization of the cell’s membrane potential. This depolarization opens voltage-dependent Ca2+ channels (VDCCS), allowing a transient influx of Ca2+ from extracellular space. In turn, the elevated free cytosolic Ca2+ concentration triggers the release of insulin granules.Over the years, the glucose-stimulated insulin secretion (GSIS) has been extensively studied, and its mechanism is well documented ,4. An in2+ concentration, which mainly originated from the glucose metabolism, synchronize with the pulsatile insulin secretion. Furthermore, it has been known that the insulin secretion occurs in a pulsatile manner , and theATP channel-dependent mechanism, and mechanosensitive mechanisms may also be involved. There are a few hypotheses on mechanosensitive pathways, such as volume-regulated anion channels [2+ plays a fundamental role in all suggested pathways. Recently, Castellanos et al. [2+-dependent insulin release from rat INS 832/13 β-cells upon ultrasound stimulation, suggesting a possible therapeutic intervention of the diabetic condition using mechanical energy-based modality. There have been numerous reports suggesting that GSIS does not rely solely on the Kchannels  or mechachannels . Howevers et al.  reported2+ elevations in cancer cell lines [2+ signaling modulator, which possesses the capability to regulate downstream Ca2+-dependent cellular processes non-invasively and remotely. In the present study, we explored the ultrasound-evoked intracellular Ca2+ dynamics of single cells from the clusters of a clonal HIT-T15 pancreatic β-cell line using a 45-MHz focused ultrasound. The results indicate that ultrasound can induce intracellular Ca2+ oscillations via purinergic signaling. Two distinctive oscillatory Ca2+ patterns, fast-irregular and slow-periodic, were found in the cells stimulated by low-intensity ultrasound, and they have shown a competitive manner depending on the level of glucose. Further study revealed that the fast-irregular mode of Ca2+ oscillation depends on both P2X receptors and L-type voltage-dependent Ca2+ channels, while the slow-periodic mode relies on P2Y receptors and store-operated Ca2+ channels.For the last few years, our group has reported that focused ultrasound can induce intracellular Call lines ,16, humall lines , and humll lines . In the l-glutamine, Fluo-4 AM, and Calcein AM were purchased from Thermo Fisher Scientific . Apyrase, carbenoxolone (CBX), cyclopiazonic acid (CPA), lanthanum (La3+), nifedipine, Pluronic F-127, pyridoxalphosphate-6-azophenyl-2′,4′-disulfonic acid (PPADS), and suramin were purchased from Sigma-Aldrich .Ham’s F12K special (with 1.5 g/L sodium bicarbonate) was obtained from the USC Norris Comprehensive Cancer Center Core . Dialyzed donor equine serum was obtained from Rocky Mountain Biologicals . Heat-inactivated fetal bovine serum (HI-FBS), Penicillin–Streptomycin, l-glutamine, 10% dialyzed donor equine serum, 2.5% HI-FBS, and 1% Penicillin–Streptomycin. The HIT-T15 cells were seeded at a density of 1.3 × 105 cells/cm2 and cultured in 5% CO2 at 37 °C. The flesh complete media was replenished every 2–3 days, and the cells were passaged every week. For this study, only the cells with passage numbers between 20 and 30 were used.The HIT-T15 cell line was purchased from ATCC  and maintained in the complete growth medium with following ingredients: Ham’s F12K with 2 mM 3) transducer was designed and fabricated in house following a protocol previously described [3 was selected because it holds many advantages such as good electromechanical coupling, a low dielectric constant, and high longitudinal sound speed [SPTA: 113.1 mW/cm2) to be in the realm of low-intensity pulsed ultrasound. The cytotoxicity of ultrasound stimulation was examined using a viability dye, calcein AM. No indication of compromised viability was observed up to 60 h after the ultrasound exposure  containing 11.1 mM D(+) glucose was used, but HBSS+ containing 2.8 mM and 5.5 mM D(+) glucose were also used as needed. The HIT-T15 cells on 35 mm culture dish were washed with HBSS+ once and incubated with 2 μM of Fluo-4 AM in room temperature for 30 min for Ca2+ imaging. After the incubation, the dish was washed three times and imaged with an epi-fluorescence inverted microscope . Fluorescence images were acquired either for 30 min at 0.5 frames per second or for 5 min at 1 frame per second.The clonal HIT-T15 cells were seeded on 35 mm culture dishes at a density of 2 × 102+ dynamics  with and without ultrasound exposure. The percentage of responding cells was calculated by the active cells divided by the total number of cells in each image field. In addition, the period of Ca2+ oscillations was measured and compared in the cells, either bathing in 5.5 mM glucose or inhibitors that suppressed the fast-irregular oscillations. Due to the nature of irregular oscillations, the period of oscillations cannot be measured in the fast oscillations.Acquired stacked images were processed with CellProfiler image analysis software  using a 2+ dynamics in HIT-T15 cells using a high K+ (40 mM) extracellular buffer. The high K+ stimulation has been used to depolarize the cell membrane in order to activate VDCCs on the membrane and allow an influx of Ca2+. A sudden increase of intracellular Ca2+ was observed as soon as the imaging solution was replaced by the high K+ buffer  buffer solution. The cells responded to the high glucose with oscillatory Ca2+ signaling  of the ultrasound was fixed at 113.1 mW/cm2  to be in the range of low-intensity and also comparable to our previous reports [To test whether ultrasound stimulation can also evoke intracellular Ca reports ,18. The 2+ oscillations in the HIT-T15 cells (2+ spike less than 20 s. The ultrasound-induced Ca2+ dynamics are comparable to fast-irregular oscillations observed in a stepwise increase in glucose concentration that mimics the transition from fasting to feeding states [Ultrasound induced fast Ca15 cells c. The peg states . 2+ responses were monitored from non-targeted cells (cells located outside of ultrasound beam) throughout all over the image field, suggesting the involvement of intercellular signaling (2+ dynamics before and after ultrasound stimulation were compared as percentages. As illustrated in 2+ dynamics (defined by active cells) was increased by 10-fold, 1.85 ± 1.22% to 21.40 ± 11.08% , in the cells bathed with normal HBSS+ containing 5.5 mM glucose. However, when the cells were bathed in HBSS+ containing 11.1 mM glucose (stimulating concentration), the percent of active cells before ultrasound was 10.60 ± 3.21%, and the number was increased by 2-fold with the ultrasound stimulus to 22.72 ± 5.78% .Although the ultrasound beam was focused on a targeted cluster of cells, only a fraction of cells in the cluster responded. In general, cells on the edges of clusters responded well, while cells on the center did not b. Moreovignaling . To quan2+ responses from otherwise resting cells both in the basal and stimulating level of glucose.Overall, the results indicate that ultrasound can activate Ca2+ spikes with a longer timescale of 30–50 s. To monitor the Ca2+ dynamic for an extended period of time, we increased the imaging duration to 30 min. Surprisingly, another pattern of oscillatory Ca2+ dynamics was found, which has a slow but periodic oscillatory pattern (period: 231 ± 92 s), as shown in 2+ oscillation in a steady-state concentration of glucose [2+ oscillations, and the appearance of each oscillatory pattern depends on the level of glucose to which the HIT-T15 cells were adapted.HIT-T15 cells showed the majority of the fast-irregular oscillations in a bath of 11.1 mM glucose a. Howeve period: 1 ± 92 s, glucose . Aside f2+ responses by facilitating ATP release via mechanosensitive hemichannels based on our previous study [Purinergic signaling plays a regulatory role in the endocrine system. Many different subtypes of purinergic receptors are expressed in secretory cells to meditate hormone release . In the us study . 2+ dynamic , also blocked the ultrasound-induced oscillations, suggesting the involvement of P2 receptors in the pathway  and a single peak appeared upon ultrasound stimulation. As for the source of extracellular ATP, mechanosensitive hemichannels were found to be responsible for the inhibitory effect of CBX , which is known to degrade extracellular ATP. Bathing with apyrase significantly abolished the ultrasound-induced Ca dynamic b,f. A no pathway c,f. InteSC model . It is w2+ oscillations depend on ATP release, the next question was: how do the cells choose one pattern over another? We focused on the fact that two subtypes of purinergic receptors, ionotropic (P2X) and metabotropic (P2Y), have different sensitivity to a range of ATP or ADP concentrations [2+ activities before ultrasound exposure, and delayed Ca2+ responses were observed upon ultrasound stimulation; yet, once it appeared, the slow-periodic oscillatory pattern persisted , which is a selective purinergic P glucose a was coneceptors b,f. This2X receptors work as an ion channel allowing the influx of Ca2+ and Na2+, the rapid oscillatory pattern cannot be explained solely by the action of P2X. Previous studies often suggested the role of L-type VDCC in glucose-induced insulin secretion [2+ oscillation, nifedipine (10 μM) was added to the HBSS+ containing 11.1 mM glucose. When the HIT-T15 cells were pre-bathed with nifedipine, only the periodic Ca2+ oscillations (period: 129 ± 33 s) were observed  than the slow-periodic oscillations observed from 5.5 mM glucose groups , a sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) inhibitor, to deplete Ca2+ storage. As shown in 2+ response was greatly diminished after the first Ca2+ peak, suggesting that the slow-periodic Ca2+ oscillation depends on Ca2+ release from the storage, and without the functioning Ca2+ pump for replenishing the storage, Ca2+ oscillation cannot be sustained. As to confirm the role of P2+ channels (SOCs) on the plasma membrane often serve as a Ca2+ replenishing mechanism. When Ca2+ is released from intracellular storage, such as the endoplasmic reticulum (ER), SOCs are activated and allow Ca2+ entry into the cytoplasm and the storage. SOCs also play a critical role in GSIS [3+, a trivalent cation that inhibits the SOCs, was added while monitoring the ultrasound-induced Ca2+ oscillations to investigate the relevance of SOCs in this pathway. As the SOCs were inhibited by La3+ , the oscillatory Ca2+ response disappeared, as depicted in 2+ from the storage, but SOC itself cannot sustain the oscillatory Ca2+ mobilization.The store-operated Ca in GSIS . La3+, a2+ dynamics in live cells. The HIT-T15 cell line was initially produced by the SV40 transformation of hamster pancreatic islet cells and has been widely used as the β-cell study model, since it secretes insulin in response to a variety of insulin secretagogues such as glucose, glucagon, and methylxanthine [2+ responses via purinergic signaling, and a mechanosensitive hemichannel was found to be responsible for releasing ATP into extracellular space.In the present study, we investigated the cellular responses of the pancreatic HIT-T15 β-cells to 45-MHz low-intensity focused ultrasound by monitoring the ultrasound-induced intracellular Caxanthine . Upon exSATA = 1 W/cm2). In another sequential paper, the authors monitored ultrasound-induced secretary events by carbon fiber amperometry and detected sustained amperometric peaks, which prolong the duration of ultrasound stimulation [2+ by showing that chelating extracellular Ca2+ from the imaging buffer significantly reduced the peaks. However, despite the similarity in the concept, the overall experimental design and stimulation parameters used in our study were mostly different. First, the acoustic output utilized in the present study was considerably lower , which may have resulted in an entirely different Ca2+ mobilizing pathway. More importantly, we aimed attention at spatiotemporal Ca2+ responses using a high-frequency focused ultrasound at 45 MHz, which offered a way to distinguish between directly stimulated cells from their adjacent cells and also allowed us to obtain a better understanding of the underlying mechanism in at the single-cell level. Previously, we have shown that both 3-MHz and 38-MHz ultrasound stimulation can induce Ca2+ responses in cancer cells, indicating that the mechanotransduction pathways reported in this study may not be limited to high-frequency ultrasound [We are not the first to investigate how β-cells respond to ultrasound stimulation. Castellanos et al. ,37 repormulation . In additrasound .SPTA = 113.1 mW/cm2), and acoustic absorption in the cell layer should be minimal, as the absorption correlates with the distance acoustic wave travels [2+ has been extensively studied in pancreatic β-cells, since its dynamic is tightly coupled to the exocytosis of insulin-containing vesicles from single cells, and its physiological significances have been documented [2+ oscillation have been reported in β-cells through different pathways of Ca2+ entry depending on the concentration of glucose or the presence of other stimuli [2+ oscillations in the pancreatic β-cells. It was found that exposure to ultrasound induces Ca2+ dynamics even from the cells that did not respond to the stimulating level of glucose. Note that regardless of the glucose levels to which cells were adapted, the percentages of active cells were consistent after the stimulation . Further investigation with pharmaceutical agents revealed that the two oscillatory Cathan ATP ,46. Consthan ATP , we beli2+ profiles, an instant rise followed by a relatively slow fall and fluctuations of the baseline, which can be often found from Ca2+ mobilization via VDCCs  is required to be more relevant to clinical settings.In summary, ultrasound-mediated intracellular Ca


J147 is an Alzheimer’s disease (AD) drug candidate that was identified using a phenotypic screening platform based upon age-related brain toxicities that are mediated by changes in calcium metabolism. The molecular target for J147 is the α-F1-ATP synthase (ATP5A). J147 has therapeutic efficacy in multiple mouse models of AD and accelerated aging and extends life span in flies. A bioinformatics analysis of gene expression in rapidly aging SAMP8 mice during the last quadrant of their life span shows that J147 has a significant effect on ion transport pathways that are changed with aging, making their expression look more like that of younger animals. The molecular basis of these changes was then investigated in cell culture neurotoxicity assays that were the primary screen in the development of J147. Here we show that J147 and its molecular target, ATP synthase, regulate the maintenance of store-operated calcium entry (SOCE) and cell death during acute neurotoxicity.Calcium dysregulation often underlies pathologies associated with aging and age-associated neurodegenerative diseases. Cells express a unique pattern of Ca The intracellular Ca2+ concentration ([Ca2+]) varies depending on its subcellular location and the maintenance of these Ca2+ stores is critical for the propagation of inter-organelle Ca2+ signals, such as those driving mitochondrial oxidative metabolism3 and the initiation of ER stress responses4. However, perturbations in Ca2+ mobilization can flood the cytoplasm with excess Ca2+, ultimately leading to cell death5. Therefore, a treatment to alleviate this form of acute, Ca2+-dependent toxicity would have significant therapeutic value.Calcium ions 11, a central player in Ca2+ metabolism that can drive Ca2+ flux via changes in the ATP-generated H+ gradient or opening of the mitochondrial permeability transition pore (mPTP).The AD drug candidate J147 was identified using a phenotypic screening platform designed to select drug candidates that protect against toxicities associated with brain aging, some of which involve dysregulated Ca2+-related transport pathways in aged SAMP8 mice. Additionally, we demonstrate that both J147 and its molecular target, ATP synthase, affect Ca2+ mobilization across the cytoplasm, ER, and mitochondrial compartments following acute old age-associated toxic insults by its regulation of store-operated Ca2+ entry (SOCE).Here, we show that J147 reduces age-induced changes in the transcriptome of genes associated with Ca13. Changes in hippocampal gene expression were examined between 9-month-old control mice and 13-month-old mice fed either control diet or J147 for 4 months starting at 9 months of age, a treatment that suppresses the aging phenotype12. Because our screening platform is based in part upon calcium dysregulation, we used gene set enrichment analysis (GSEA) to ask how J147 affected age-associated changes corresponding to Ca2+-related ion channels and transporters.Rapidly aging SAMP8 mice recapitulate much of the progressive, age-associated decline in brain function that is associated with the development of AD in humans, developing both cognitive deficits and pathological hallmarks of dementia by 9 months of age2+ mobilization and SOCE, a specialized form of extracellular-derived Ca2+ influx, including the ATPase sarcoplasmic/endoplasmic reticulum Ca2+ transporter (Atp2a2), stromal interaction molecule 1 (Stim1), and inositol 1,4,5-triphosphate receptor type 3. Significant changes were also detected in genes residing in a variety of subcellular locations, including the plasma membrane, endosomes, golgi apparatus, mitochondria, and those involved in extracellular secretion  pump responsible for controlling extracellular-derived Ca2+ influx. This suggests that ATP synthase, and mitochondria in general, can influence ER-derived Ca2+ signaling nodes  synthesis, resulting in GSH loss and detrimental increases in reactive oxygen species (ROS) and subsequently Ca2+ influx and cell death17. Although ROS accumulation initiates Ca2+ influx, Ca2+ influx also further enhances ROS production14. Importantly, Ca2+ influx is required for cell death17. Thus, we determined the effect of J147 on Ca2+ levels in the cytosolic and mitochondrial compartments after overnight treatment with glutamate. Figure 2+]cyt and [Ca2+]mit in HT22 nerve cells, as measured by Fluo-4 and Rhod-2 Ca2+ indicator dyes specific to the cytoplasm and mitochondria, respectively. J147 not only enhanced cell survival, but also dampened the late-stage cytoplasmic and mitochondrial Ca2+ influx.Ca2+]er depletion leads to the translocation of the ER membrane-resident protein Stim1/2 to the plasma membrane where recruitment of calcium release-activated calcium modulator (Orai) Ca2+ channel proteins promote extracellular-derived Ca2+ influx into the cytosol to replenish ER stores. Dysregulation of SOCE is observed in both aging and AD18. The interaction of ATP synthase and ATP2a2 uncovered through our protein–protein network analysis supports recent reports demonstrating the direct involvement of SOCE proteins in oxytosis/ferroptosis20. Therefore, we asked whether J147 and modulation of ATP synthase activity via siRNA-mediated knockdown, affect SOCE.The maintenance of SOCE is critical for the survival of cells. SOCE-mediated toxicity is triggered when [Ca2+ re-addition method21 that allowed separation of the two phases of Ca2+ mobilization—ER Ca2+ release and extracellular Ca2+ influx. The fluorescent ratiometric Indo-1 Ca2+ indicator dye was used to monitor changes in [Ca2+]cyt resulting from ER Ca2+ release and extracellular-derived influx. First, baseline recordings of [Ca2+]cyt were performed in the absence of extracellular Ca2+. This was followed by the addition of 3 μM thapsigargin to empty ER Ca2+ stores into the cytosol, after which 3 mM CaCl2 was added back to the culture medium to allow for extracellular-derived Ca2+ influx. Figure 2+ increase during the first phase of ER depletion, both J147 and ATP5A knockdown still dampen the extracellular-derived Ca2+ influx in HT22 cells. This suggests that ATP synthase and by extension mitochondria exert control over an ER-regulated Ca2+ influx pathway involving the regulation of extracellular-derived Ca2+ influx that is dampened by J147 and ATP synthase knockdown.SOCE kinetics were measured via flow cytometry using a Ca2+ buffering between the ER and mitochondria may underlie the abilities of J147 and reduced ATP synthase activity to prevent detrimental Ca2+ overload.Thus, alterations in Ca2+ stores that, when used for buffering, can modulate Ca2+-derived signaling events. Ca2+ transfer between the ER and mitochondria can serve as a way to regulate these Ca2+-derived signals22. Up to this point, our data suggest that SOCE can be regulated by the mitochondria as J147 and ATP5A knockdown both dampen SOCE. Therefore, we asked if J147 and ATP synthase had specific effects on inter-organelle Ca2+ transfer between the ER and mitochondria.The ER and the mitochondria serve as intracellular Ca2+ sensors specific to the ER and mitochondria in HT22 cells and performed three-dimensional live-cell imaging to monitor changes in fluorescence in real time . J147 dampened the extent of Ca2+ release from the ER , and thus reduced the subsequent rise in mitochondrial Ca2+ observed in vehicle-treated cells .The inhibition of ER-resident SERCA pumps with thapsigargin revealed a J147-specific effect on ER Ca2+ metabolism, knockdown of ATP5A phenocopied the suppression of ER Ca2+ release during 5 μM thapsigargin treatment . These data show that J147 has an ER-specific effect on Ca2+ maintenance, and point to mitochondrial involvement in the maintenance of ER Ca2+ stores as a way to regulate SOCE.To confirm the target specificity of J147 and its role in Ca2+ release and thus affects [Ca2+]mit and ER–mitochondrial Ca2+ transfer in cells treated with glutamate   Fig. . Upon exses Fig. . In contity Fig. . Importaity Fig. . Thus, t24. This can be mimicked in vitro by treating cells with iodoacetic acid (IAA), an irreversible inhibitor of glyceraldehyde-3-phosphate dehydrogenase, resulting in inhibition of glycolysis and cell death in HT22 cells11. Similar to the effects observed in glutamate and RSL3 toxicities, IAA had a minimal effect on ER Ca2+  occurs in aged neurons, contributing to significant increases in ER–mitochondrial transfer of Ca2+25. Similarly, age-related changes in the expression and/or activity of Ca2+-release/uptake channels have also been reported26. In fact, hippocampal overexpression of FK506-binding protein 12.6/1b (FKBP1b), a negative regulator of RyR Ca2+ release, reverses aging-induced memory impairment, and neuronal Ca2+ dysregulation in rats27. FKBP1b overexpression also counteracted expression changes in 37% of age-dependent genes, emphasizing a vast transcriptional network being affected by the disruption of Ca2+ homeostasis27.Increased Ca2+ metabolism were associated with aging and the development of dementia in the SAMP8 accelerated aging mouse model. RNAseq analysis revealed a specific, age-induced dysregulation of signaling pathways involved in Ca2+ and ion transporters and channels including proteins that regulate Ca2+-flux into and out of the ER during SOCE. Importantly, these changes were prevented by J147 when fed to old, symptomatic mice. By combining a protein–protein interaction network with gene expression data, we uncovered an interaction between ATP synthase and the ER SERCA pump, ATP2a2, implicating ATP synthase involvement in SOCE. We then asked if J147 and ATP synthase knockdown could affect SOCE in cultured nerve cells. Indeed, both dampened SOCE. While ATP synthase has been implicated in Ca2+-efflux from the mitochondria during mPTP formation during cell death, to our knowledge a direct role for ATP synthase in moderating SOCE has not been previously established. Furthermore, this suggests that dampening SOCE is involved in the neuroprotective effects of J147.Initially, we asked if genes involved in Ca2+ exchange between these two organelles. While controlled levels of ER–mitochondrial Ca2+ flux are crucial for driving metabolism28, excessive IP3R3-mediated ER Ca2+ release can lead to mitochondrial Ca2+ overload, opening of the mPTP, cytochrome c release, caspase activation, and apoptotic cell death29. Thus, any alteration in this interface is expected to have pleiotropic effects on cell homeostasis and survival. Therefore, we examined what effect J147 and modulation of ATP synthase activity had on intracellular Ca2+ store maintenance and transfer between the ER and mitochondria by treating cells with thapsigargin to assess intracellular store release. Thapsigargin treatment increased [Ca2+]mit, demonstrating SERCA pump inhibition can increase mitochondrial Ca2+ uptake. J147 and ATP5A knockdown increased retention of ER Ca2+ and minimized mitochondrial influx, indicating that a dampening of ER–mitochondrial Ca2+ transfer is a contributing factor in neuroprotection by J147.Specialized ER–mitochondrial contacts called mitochondrial–ER associated membranes (MAMs) facilitate Ca2+ maintenance, suggesting a conserved Ca2+-mediated cell death pathway involving mitochondrial control over SOCE. Oxytosis/ferroptosis can be induced by glutamate and RSL3, acting at different sites in the cell death pathway. We assessed changes in ER and mitochondrial Ca2+ following glutamate or RSL3 treatment and observed decreases in mitochondrial Ca2+ during both, thus demonstrating that oxytosis and ferroptosis overlap in their convergence on mitochondrial Ca2+ dysregulation. Similarly, energy deprivation via the inhibition of glycolysis induced a decrease in [Ca2+]mit. J147 prevented Ca2+ release from the mitochondria in response to all three insults and prevented the loss of ATP with reduced glycolysis.Our data also show that distinct neurotoxic insults have similar effects on mitochondrial Ca2+-dependent cell death pathway that is a potential drug target for neurotoxic insults associated with aging and disease.To confirm the contribution of SOCE to cell death induced by the three insults, we knocked down the major plasma membrane Orai pore forming proteins that mediate SOCE influx. Interestingly, knockdown of different Orai channels elicited varying levels of protection depending on the toxic insult. Although there is an Orai channel isoform-specific cell survival enhancement for each neurotoxicity model, Orai3 knockdown was most effective overall. Furthermore, increased levels of Orai3 mRNA were observed in both Orai1 and Orai2 knockdown cells, emphasizing its importance among Orai channel subtypes. These data argue that components of SOCE represent a conserved Ca2+-associated pathways occurs during aging, and that administration of J147 to old mice prevents many of these changes. J147 and modulation of its molecular target, ATP synthase, stabilize mitochondrial Ca2+ levels, and minimize SOCE activity to protect cells from multiple, acute neurotoxicities. Since the ER and mitochondria are critical organelles that utilize Ca2+ to drive metabolism and stress responses, identifying drug candidates similar to J147 that target underlying Ca2+-mediated toxicities may be a strategy to combat both aging and age-related neurodegeneration.In summary, gene expression analysis revealed that a dysregulation in Ca30. Cell viability was determined by MTT assays in 96-well plates9 and propidium iodide (PI)  uptake during flow cytometry. Oxytosis and IAA toxicity were performed as previously reported9. HT22 cell survival responses to RSL3  toxicity were measured and performed identically to the oxytosis and IAA toxicity assays. For all of the toxicity assays, HT22 cells were plated at 5 × 103 in 96-well plates, grown overnight, and then treated with the indicated concentrations of the toxins for the indicated times at which point cell death was measured by the MTT assay or PI uptake as indicated in the figure legends. All siRNAs were acquired from Santa Cruz Biotechnology. In experiments assessing the contribution of Orai-Ca2+ channel or ATP synthase proteins in the aforementioned toxicity assays, cells were transfected with siRNA (Santa Cruz Biotechnology) to either Orai1 (sc-76002), Orai2 , Orai3 (sc-76006) or ATP synthase (sc-60228) for 24 h prior to seeding into 96-well plates followed by the MTT assay to assess cell vulnerability as previously described10.Mouse hippocampal HT22 neuronal cells were propagated in DMEM/high glucose with 10% fetal calf serum as previously described12. All experiments were performed in accordance with the US Public Health Service Guide for Care and Use of Laboratory Animals and the protocols were approved by the IACUC at the Salk Institute. Twenty-three 9-month-old female SAMP8 mice were fed with vehicle diet , and 22 9-month-old female SAMP8 mice were fed with J147 . Diet treatment lasted for four months. This dose of J147 was shown to be effective in multiple mouse models of memory, AD and aging12. RNAseq, gene expression, and network analysis was performed as described12. Briefly, SAMP8 count data was re-analyzed by DESeq2 to estimate gene expression changes in different samples. GSEA was performed using WebGestaltR with genes ranked by their -log10 times direction of expression changes (positive for upregulated genes and negative for down-regulated genes). Pathways with adjusted p value < 0.05 were identified as significantly altered (1000 permutation by gene). Log-transformed FPKM values of leading edge genes were used to generate the heatmap using package gplots.The SAMP8 line was acquired from Harlan Laboratories (UK). Tissue preparation and collection were performed as previously described2+ indicator dyes were acquired from Thermofisher Scientific. Fluo-4 (excitation 488 nm (F14201)) and Rhod-2 (excitation/emission 552/581 nm (R1244) ) Ca2+-indicator dyes specific to cytosol and mitochondria, respectively, were exclusively used in a 96-well plate format as previously described11 unless otherwise indicated. Briefly, HT22 cells were plated at 5 × 103 in 96-well plates, grown overnight, and then treated as indicated in the figure legends. The dyes were then added to the cells for 45 min and the fluorescence was measured on a Spectramax M5 plate reader. Indo-1 (I1223) ratiometric Ca2+ indicator dye was used to measure cytosolic Ca2+ during flow cytometry experiments in accordance with the manufacturer’s protocol. Indo-1 maximum emission shifts from ~475 nm (Ca2+-unbound form) to ~400 nm (Ca2+-bound form). All fluorescent Ca2+ sensor plasmids were acquired from Addgene—ER  and mitochondria —and used during flow cytometry and live-cell imaging. The excitation (ex)/emission (em) for pCMV G-CEPIA1er ex/em; 488 nm/512 nm, pCMV CEPIA2mt ex/em: 488 nm/500–550 nm, CMV-mito-LAR-GECO1.2 ex/em: 550–570 nm/590 nm.All Ca3 cells/chamber and maintained in Ca2+-replete media overnight . The following day, the cells were pretreated with 1 μM J147 for 1 h before the medium was removed and the cells were washed 3× with Ca2+-free HBSS without phenol red  supplemented with 0.1% FBS  immediately before the addition of thapsigargin . Baseline fluorescence was recorded for 30 s prior to thapsigargin addition. 488 and 568 nm lasers were used to excite pCMV G-CEPIA1er and CMV-mito-LAR-GECO1.2, respectively. Z-stack images were acquired every 30 s and imaged on a Zeiss LSM 880 rear port laser scanning confocal and Airyscan FAST microscope. Zen Black and Imaris imaging analysis software were used to trace and render cells in 3D to quantitate changes in total cell fluorescence over time.HT22 cells were co-transfected with pCMV G-CEPIA1er and CMV-mito-LAR-GECO1.2 for 48 h prior to seeding into Nunc Lab-Tek II chambered coverglass system  at 1 × 102+ release into the cytosol from internal stores in real time in addition to measuring changes in Ca2+ content in HT22 cells transfected with either fluorescent Ca2+ indicators specific for the ER (pCMV G-CEPIA1er) or mitochondria (CEPIA2mt) during oxytosis, RSL3, and IAA toxicity time course experiments. To measure Ca2+ release from internal stores, HT22 cell were treated with 1 μM J147 or DMSO vehicle control overnight prior to staining with Indo-1. Cells were trypsinized, washed 3× with Ca2+-free HBSS supplemented with 0.1% FBS  and stored on ice. Cells were warmed to 37 °C immediately prior to measurement. Baseline recordings were made for 30–45 s before the addition of 3 μM thapsigargin. Ratiometric acquisition mode and kinetic parameter platform analysis in FlowJo (v10.5.3) were used to measure changes in cytosolic Ca2+ on a per cell basis. In toxicity time course experiments, glutamate (10 mM), RSL3 (500 nM), and IAA (12 μM) were used to assess Ca2+ content across the ER and mitochondrial compartments, while PI uptake was used as an index for cell viability. Indo-1 was used in combination with the Ca2+ re-addition method to assess SOCE13, where baseline measurements were recorded for 30 s followed by the addition of 3 μM thapsigargin to empty ER Ca2+ stores into the cytosol after which 3 mM CaCl2 was added back to allow for extracellular-derived Ca2+ influx.Indo-1 was used to assess CaFurther information on experimental design is available in the Supplementary InformationReporting Summary


Renal angiomyolipomas (AML) are often regarded as benign tumors. This article reports the case of a 47 year old patient with AML associated with a venous invasion of the inferior vena cava.Our 47 year old male patient presented for flank pain and hematuria. CT Scan revealed a 3 cm lobulated low-density lesion in the renal sinus, middle and upper lobes of the right kidney, with evidence of IVC thrombus on angioscan. Right radical nephrectomy and thrombectomy were successfully done with use of cardiopulmonary bypass. On pathology, AML was confirmed.Renal AML are unilateral and sporadic in most cases, with a female predominance. The occurrence of venous invasion in patients with AML is a rare and unique phenomenon. CT scan is the imaging of choice in such cases and surgery remains the optimal treatment.More focus should be put on the ability of AML to invade venous structures. Early imaging and therapeutic planning are necessary for the best outcome in case of vena cava invasion. Collaboration of the urologist with the vascular surgeon can lead to better surgical results. AML consists of three different components in a mixed fashion: fatty tissue, dystrophic blood vessels and smooth muscle: All three components are present in 76% of patients with AML . Venous 2Our 47-year-old man previously diagnosed with right renal AML presented for intermittent right flank pain and gross hematuria. He had no signs or symptoms of tuberous sclerosis complex (TSC). His only relevant past medical history was old peritonitis following an appendectomy.The abdominal US showed a hyperechogenic lesion in the right renal sinus with a thrombus in the right renal vein and IVC. Abdominal computed axial tomography (CAT) scan revealed a 3 cm lobulated low-density lesion in the renal sinus, middle and upper lobes of the right kidney . On abdoConsidering the risk of pulmonary embolism due to IVC thrombus, the decision of right radical nephrectomy and thrombectomy was taken with the patient.The surgery was done under general anesthesia after central and arterial lines insertion, the patient was in dorsal decubitus position. Right subcostal approach with median sternotomy was used . After iPathological analysis of the nephrectomy piece revealed a 5 × 4 × 3 cm tumor with intratumoral hemorrhage extending through the hilum by an 8 cm chaplet like structure with diameters varying between 0.8 and 2 cm. Microscopic examination revealed a stroma composed of mixed layers of mature adipocytes and smooth muscle cells and multiple vascular structures with thick walls and hemorrhagic foci. These characteristics were compatible with that of a benign AML, which was confirmed by immunohistochemistry: positive staining with anti-SMA antibodies  and anti3Renal angiomyolipomas are benign tumors found sporadically in 80% of cases, at an average of 52 years of age, or in associated with tuberous sclerosis complex in the remainder of cases, occurring bilaterally .Uniquely, and opposed to the benign nature of AML, our patient was found with an IVC tumoral invasion. The first case in the literature of a renal AML presenting with a venous thrombus was reported by Kutcher et al. in 1982 and 89 other cases followed, to the best of our knowledge .The majority of cases were female. AML tumors are usually found incidentally on ultrasound, being the primary method of investigation in abdominal pain, but can only visualize the IVC in 60% of cases , thus thStaging an AML thrombus in the venous system is primordial to assess the therapeutical options. A description of the extent of the thrombus in the IVC lumen is necessary to the staging process, and as by the TNM classification, a T3b tumor is below the diaphragm and a T3c tumor is above the diaphragm . HoweverIn conclusion, to our knowledge, only 76 cases of AML with IVC invasion were published so far. Malignant transformation of AML should always be kept in mind especially in case of venous invasion. More focus should be put on the ability of AML to invade venous structures despite its benign characteristics. Early imaging and therapeutic planning are necessary for the best outcome. Collaboration of the urologist and the vascular surgeon is sometimes mandatory for optimal surgical treatment.No conflict of interest.No sponsor for this article.This work is exempt from ethical approval in our institution because of its type.Written informed consent was obtained from the patient forpublication of this case report and accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal on request.Kheir, El Khoury, El Hachem and Abdessater conceived of the presented idea and were encouraged by Tawil to execute it. El Khoury, Akiel, Abdessater, Kheir and Tawil were on the operating field while performing the surgery and all of them participated to the different steps of the surgery but El Khoury and Tawil were the main surgeons. Kheir, El Khoury, Akiel and Abdessater chose the most important figures from the surgery’s video and pathology. El khoury, Abdessater, El Hachem, Akiel and El Khoury contributed to the final version of the manuscript. Tawil supervised the work. Kheir took the lead in writing the manuscript when he found that this case deserve to be published. All authors provided critical feedback and helped shape the manuscript. The 7 authors designed the model and the computational frame-work and analyzed the results. All persons who meet authorship criteria are listed as authors,and all authors certify that they have participated sufficiently in the work to take public responsibility for the content.This is not a first-in-man study so it was not registered.Raghid El Khoury and Nabil Tawil are the guarantors of this work.Not commissioned, externally peer-reviewed.


Due to its ubiquitous nature, Ca2+ is also frequently located in the center of ketamine action, although the precise mechanisms underlying drug’s negative or therapeutic properties remain mysterious for the large part. This review seeks to delineate the relationship between ketamine-triggered imbalance in Ca2+ homeostasis and functional consequences for downstream processes regulating key aspects of neuronal function.Ketamine is a non-competitive antagonist of NMDA (N-methyl-D-aspartate) receptor, which has been in clinical practice for over a half century. Despite recent data suggesting its harmful side effects, such as neuronal loss, synapse dysfunction or disturbed neural network formation, the drug is still applied in veterinary medicine and specialist anesthesia. Several lines of evidence indicate that structural and functional abnormalities in the nervous system caused by ketamine are crosslinked with the imbalanced activity of multiple Ca In neurons, Ca2+ plays a dual role as a charge carrier and ubiquitous second messenger. This dual nature allows for the generation of a broad spectrum of signals, which can be distinguished by spatial and temporal dimensions, amplitude, frequency of oscillations or localization to discrete neuronal compartments i. The overview of Ca2+ homeostasis toolkit in healthy neurons is depicted in The control of intracellular Caartments ,11,12,132+]i can reflect the influx from extracellular milieu through two types of membrane Ca2+ channels: voltage-dependent Ca2+ channels (VDCCs) and ligand-gated ionotropic channels (LGIC) or the release from intracellular reservoirs, mainly endoplasmic reticulum (ER), where Ca2+ concentration is nearly 1000 higher than in cytosol. An increase in i is a major trigger for further Ca2+ release through RyRs in a process called Ca2+-induced Ca2+ release (CICR). The CICR is of paramount importance for shaping Ca2+ signal as it couples the coordinative opening of channel(s) observed as calcium “sparks” or “puffs” to the generation of widespread calcium waves or oscillations i to its basal level following stimulation i by pumping Ca2+ to the cisterns of the ER where it is further sequestered by luminal EF-hand proteins i in large population of neurons. To support it, sustained elevations in basal cytosolic Ca2+ concentration were seen in functionally different regions of rat brain following in vivo ketamine administration (30 mg/kg). Interestingly, a positive correlation between ketamine-induced hyperlocomotion and [Ca2+]i in the cortex and cerebellum was demonstrated i seen already 15 min after washout. In another study, Ali and coworkers, using two-photon calcium imaging, found that ketamine suppressed the activity of somatostatin-expressing interneurons, which are a fraction of GABAergic interneurons, in the medial prefrontal cortex but not in primary motor cortex, suggesting regionally specific effect i rise induced by ketamine. According to one hypothesis, inability to maintain brain Ca2+ homeostasis may be a result of imbalanced expression between calcium “off” and “on” systems. An extended analysis of the expression of genes encoding main players in neuronal Ca2+ handling showed that ketamine predominantly affected the expression of VDCCs and membrane transporters responsible for Ca2+ extrusion  underlying i affecting downstream Ca2+ signaling is largely responsible for a number of neuronal functions. Therefore, it is not surprising that even mild deficits in Ca2+ handling machinery may lead to significant structural and functional deficits in neurons. In some cases, these deficits are temporary and reversible, but in others they may become permanent and trigger irreversible neurodegenerative processes. As reviewed in this article, in vitro and in vivo research using different approach and experimental animal models has indicated that imbalanced Ca2+ homeostasis is tightly associated with ketamine action in the brain. Moreover, a growing body of evidence indicates that Ca2+ is either directly or indirectly engaged in ketamine-related processes, mostly in NMDA and AMPA receptor-driven neurotransmission. This seems apparent as ketamine is a well-known NMDAR blocker. However, several prolonged effects seem to arise from other “post-drug” effects, not necessarily limited to NMDA receptors but involving Ca2+-related, activity-induced structural rearrangements in synaptic connectivity and function. The knowledge of impairments in neuronal Ca2+ upon ketamine administration can sustain the needs for its careful clinical use and validation of animal models of ketamine-induced diseases.Proper control of [Ca


In the central nervous system (CNS), they are involved mainly in Orai-mediated store-operated Ca2+ entry (SOCE). The key molecular components of the SOCE pathway are well-characterized, but the molecular mechanisms that underlie the regulation of this pathway need further investigation. Numerous intracellular target proteins that are located in the plasma membrane, ER, cytoskeleton, and cytoplasm have been reported to play essential roles in concert with STIMs, such as conformational changes in STIMs, their translocation, the stabilization of their interactions with Orai, and the activation of other channels. The present review focuses on numerous regulators, such as Homer, SOCE-associated regulatory factor (SARAF), septin, synaptopodin, golli proteins, partner of STIM1 (POST), and transcription factors and proteasome inhibitors that regulate STIM-Orai interactions in the CNS. Further we describe novel roles of STIMs in mediating Ca2+ influx via other than Orai pathways, including TRPC channels, VGCCs, AMPA and NMDA receptors, and group I metabotropic glutamate receptors. This review also summarizes recent findings on additional molecular targets of STIM proteins including SERCA, IP3Rs, end-binding proteins (EB), presenilin, and CaMKII. Dysregulation of the SOCE-associated toolkit, including STIMs, contributes to the development of neurodegenerative disorders , traumatic brain injury, epilepsy, and stroke. Emerging evidence points to the role of STIM proteins and several of their molecular effectors and regulators in neuronal and glial physiology and pathology, suggesting their potential application for future therapeutic strategies.Stromal interaction molecules (STIMs), including STIM1 and STIM2, are single-pass transmembrane proteins that are located predominantly in the endoplasmic reticulum (ER). They serve as calcium ion (Ca Cytoplasmic Ca2+ level in neurons is regulated in a comprehensive way via the components localized in the plasma membrane (PM) such as ion channels, exchangers, and pumps, as well as the components localized in the mitochondria, endoplasmic reticulum (ER), Golgi apparatus, and nucleus , receptor-operated Ca2+ channels (ROC: α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors [AMPARs] and N-methyl-D-aspartate receptors [NMDARs]), and store-operated channels   and is complemented by store-operated Ca2+ entry (SOCE)  and PM Na+/Ca2+ exchanger (NCX). While NCX affinity to Ca2+ is low, its capacity is high. Contrary, PMCA is characterized by opposite properties  and inositol-1,4,5-trisphosphate 3 (IP3) receptor (IP3R). Ca2+ release through IP3R occurs in response to mGluR activation in the PM. In turn, an elevated level of cytoplasmic Ca2+ is the major trigger for Ca2+ release via RyR in the mechanism known as Ca2+-induced Ca2+ release (CICR) , including STIM1 and STIM2 proteins, that are sensors of Ca2+ levels in the ER  that are formed by Orais or SOCs that consist of Orais and TRPC channels, leading to the activation of these channels  and ISOC  pump.SOCE is based on the influx of CaThe interaction between STIM proteins and Orai1 is widely known to be essential for the proper function of SOCE in non-excitable cells. SOCE is a ubiquitous cell signaling pathway that is also present in many other tissues, including the rodent and human brain , mainly in neurons and glia. Recent data revealed a key role for STIM in several physiological and pathological conditions, including hypoxic/ischemic neuronal injury, traumatic brain injury (TBI), epilepsy, Alzheimer's disease (AD), Parkinson's disease (PD), and Huntington's disease  domain, a non-Ca2+-binding hidden EF-hand (hEF) domain, a sterile α-motif (SAM) with one (for STIM2) or two (for STIM1) N-glycosylation sites, and a transmembrane domain (TMD). The cytosolic C-terminus includes three coiled-coil regions  with a STIM–Orai-activating region (SOAR). The SOAR contains four α-helices  and a KIKKKR sequence, which is required for the activation of Orai1  in dendritic spines and regulates its phosphorylation  mice were better protected against cerebral ischemia  and receptor/ligand-activated Ca2+ channels (AMPARs and NMDARs), couple or engage in an interplay with STIMs in the CNS in a modulatory way as part of SOCE signaling -STIM1 and Orai1 but not those with YFP-STIM2 and Orai1 -N,N,N′,N′-tetraacetic acid (EGTA) triggered the puncta formation of both YFP-STIM1/Orai1 and YFP-STIM2/Orai1. Other results showed that endogenous STIM1 and STIM2 can interact with Orai1, which was observed in a co-immunoprecipitation assay and in situ proximity ligation assay  and thus is thought to be a key regulator of neurogenesis in mammalian cells  in the adult mouse brain , TRPC, and TRPM (TRP channel subfamily M) expression . Additionally, TRPC1-mediated Ca2+ entry activates the neuroprotective AKT pathway. STIM1 or TRPC1 but not TRPC3 silencing increases the UPR. Consistent with these results, Trpc1−/− mice have a higher UPR and lower number of DNs, similar to PD patients. The overexpression of TRPC1 in mice increased DN survival after neurotoxin treatment.TRPC1-mediated SOCE is essential for neuronal survival -derived DNs, similar to native neurons, utilize TRPC1-mediated SOCE , known as transducers of electrical activity that enable Ca2+ influx in response to subthreshold depolarizing stimuli or action potentials  of STIM1 and C-terminus of the Cav1.2 α1 subunit  and two glutamate-binding NR2 (GluN2) subunits  are widely distributed in the CNS and play a key role in synaptic transmission and plasticity. Excessive mGluR activation was shown in acute and chronic neurodegenerative disorders such as PD, AD, and HD -derived NO signaling along with the higher expression of mGluR1, STIM1 expression and cluster density are elevated in PNs  increase the expression of 2+ store depletion, the association between STIM1 and Homer1a decreases, whereas no change occurs in the Homer1a-Orai1 association. These interactions between STIM1, Homer1a, and Orai1 are similar to interactions between STIM1 and Homer1b/c or STIM1, Homer1b/c, and Orai1  is a 339-amino-acid type I transmembrane protein that has exceptionally high transcript levels in neuronal tissues  of Orai1-forming CRAC  improved SOCE  appear to perform antagonistic rather than synergistic functions in Orai channel activation by STIM. This discrepancy may be caused by a different assembly of septins during complex formation and by differences in subsequent filament structure  that are abundantly expressed in immune cells and the CNS  was shown to be significantly lower in STIM1-deficient SH-SY5Y cells, meaning that STIM1 is a positive regulator of ITPR3 gene expression in these cells  are cytoskeleton-organizing components localized in dendrites and axons  is localized between ER stacks of the spine apparatus. This cytosolic actin and α-actinin-binding protein has been shown to be essential for the formation of this organelle  is caused by a dominant inherited mutation of presenilins  and amyloid-β (Aβ) precursor protein  is one of the most important neurogenic transcription factors in the CNS  decreased Sp4 protein levels after depolarization , decreased STIM1 and STIM2 levels in primary rat cortical neurons but did not affect either Orai1 or TRPC1 , as well as the components localized in the mitochondria, ER, Golgi apparatus, and nucleus. Under pathological conditions, Ca2+ homeostasis is dysregulated, with increased cytoplasm, mitochondrial, and changed ER Ca2+ concentration leading to apoptosis  and negative  regulators of STIM that affect STIM expression and structure and its movement to the PM or activation of Orai channels . The maj2+ influx via other molecular channels, including L-type VGCCs, and receptors, such as AMPARs, NMDARs, mGluR1, and mGluR5. The interaction between STIM proteins and their molecular targets can both increase and decrease Ca2+ influx from the extracellular milieu to the cytoplasm. Associations between STIMs and Orai, TRPC, AMPARs, mGluR1, and mGluR5 elevate Ca2+ influx, whereas Ca2+ influx via L-type VGCCs and NMDARs is inhibited by STIMs. Interestingly, maximal SOCE activation occurs under resting conditions, whereas VGCC and NMDAR activation requires cell membrane depolarization. Depolarization activates Ca2+ influx via both NMDARs and L-type VGCCs and decreases Ca2+ content in the ER, thereby enhancing STIM1-mediated SOCE and decreasing Ca2+ influx via VGCCs and NMDARs. Thus, STIM proteins in neurons may regulate Ca2+ influx under both resting and action potential.The main effectors of STIM proteins are Orai and TRPC, which together constitute molecular components of SOCE. The evidence gathered in this review suggests that disturbances of the STIM-Orai- and STIM-TRPC-mediated SOCE pathway contribute to the pathogenesis of diverse neurodegenerative diseases. The STIM-Orai association is also vital for the regulation of neurogenesis in mammalian cells and production of proinflammatory cytokines in astroglia and murine microglia. In turn, the STIM-TRPC interaction is essential for the survival of DNs in animal models of PD. In addition to Orai and TRPC, STIM proteins can also control Ca2+ stores and plasticity of spine heads during LTP. After cell depolarization, the STIM1-POST complex binds to SERCA and keeps it in close proximity to L-type VGCCs to promote ER Ca2+ replenishment during repetitive firing, regulating neuronal excitability and plasticity. The STIM1-POST complex appears to prevent excessive Ca2+ concentrations in the cytoplasm. In turn, the STIM1-IP3R3 interaction regulates basal Ca2+ levels in mitochondria. Interestingly, the dysregulation of both cytosolic and mitochondrial Ca2+ levels contributes to neurodegeneration in AD. In turn, the STIM2-EB3 and STIM2-CaMKII complexes promote the formation of mushroom spines and stabilize them. Targeting these complexes could be a novel way of stabilizing dendritic spines and thus improve memory in AD patients.The relationship between STIM proteins and glutamate receptors is essential for different forms of synaptic plasticity. In primary rat hippocampal neurons, STIM2 promotes the phosphorylation and surface delivery of AMPARs, contributing to LTP. SOCE can be activated by synaptic NMDARs, thus also influencing LTP. Additionally, STIM1 was shown to control the plasticity of L-type VGCC-dependent dendritic spines in PNs and strengthen mGluR1-dependent synaptic transmission, thereby regulating cerebellar motor behavior. Other STIM protein effectors and regulators that reside outside the PM also contribute to synaptic plasticity. Synaptopodin recruits STIM1 to the PSD and regulates the function of Ca2+ influx into the cell. The precise molecular mechanisms of action of STIMs together with these all regulators  also remain to be explored. Such studies will help to elucidate the pathological mechanisms that are involved in the development of various neurodegenerative diseases , affective disorders , chronic pain, oxidative trauma, brain trauma, stroke, and epilepsy. Therefore, it appears that STIM proteins and their modulators/effectors may have potential therapeutic applications for the treatment of these diseases.Studying STIM proteins and their partners in different subcellular compartments enables us to understand a wide range of processes that are regulated by these proteins. Future studies should examine the ways in which these regulators act in concert to modulate STIM activity during CaKS wrote and commented on the manuscript and prepared the figures. JG-B designed the manuscript, contributed to writing it, and prepared the final version. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.