Myostatin (MSTN) is a negative regulator of muscle mass, related to muscle growth and differentiation. The knockout of MSTN with the desired phenotype of double muscle has been successfully generated in mice, goats, pigs and cattle, but not in rabbits. In this study, the MSTN knockout (KO) rabbits were generated by co-injection of Cas9 mRNA and sgRNA into zygotes. The typical phenotype of double muscle with hyperplasia or hypertrophy of muscle fiber was observed in MSTN KO rabbits. Furthermore, a similar phenotype was found in the F1 generation, suggesting that the mutation of MSTN could be stably inherited in the MSTN KO rabbits. In summary, we have successfully generated MSTN KO rabbits using CRISPR/Cas9 system with high efficiency, which is a reliable and effective animal model for the study of muscle development and related diseases.CRISPR/Cas9 has been widely used in generating site-specific genetically modified animal models.  Gene editing technologies have been developed to study the function of genes, generate genetically modified animal models for biomedical research and improve animal traits in agriculture. CRISPR/Cas9 system consists of a single guide RNA (sgRNA) and a human codon-optimized Cas9 nuclease, which can induce targeted mutations by nonhomologous end joining (NHEJ). Co-injection of Cas9 mRNA and sgRNA into zygotes has been used as an efficient tool to generate gene-targeted animal models in miceRabbits are a promising animal model for biomedical research, as they show more similarities to human beings in terms of physiology and anatomy than mice and rats, and require low cost maintenance and short pregnancy period compared to pigs and monkeys6Myostatin (MSTN) is a member of the transforming growth factor beta (TGF-β) superfamily, which acts as a negative regulator of muscle growth8MSTN in cattleMSTN KO sheepMSTN KO rabbits for the study of muscle development and improvement of animal traits for agriculture in the future.MSTN KO rabbits, the in vitro transcribed mRNA encoding for Cas9 and sgRNA targeting the MSTN gene, was microinjected into the cytoplasm of rabbit pronuclear-stage embryos. We demonstrated the high efficiency of CRISPR/Cas9 system-mediated gene editing and the desired phenotype of double muscle was obtained in the MSTN KO rabbits.Here, in order to generate MSTN in rabbits, two sgRNAs targeting the CDS of the rabbit MSTN gene were designed using the CRISPR/Cas9 online design tool (http://tools.genomeengineering.org) (MSTN was found in 10 tested blastocysts (83.3%), in which 3 blastocysts (25%) displayed biallelic mutations, suggesting that the two sgRNAs-directed CRISPR/Cas9 system was an efficient tool for disrupting the rabbit MSTN gene in zygotes.In order to disrupt the function of ing.org) . To deteMSTN KO rabbits, a total of 158 injected zygotes were transferred into the oviducts of 4 surrogate rabbits. After a full-term gestation, the 4 surrogate mothers gave birth to 20 live pups successfully  counterparts  for each sgRNA were predicted by the CRISPR design online tool. All POTS were PCR amplified and subjected to T7E1 cleavage and Sanger sequence. The results showed that no mutation was detected in these POTS, indicating that the Cas9/sgRNA system did not induce undesirable off-target effect in the  rabbits .MSTN KO could be stably transmitted to the offspring, the female founder F01–7 was mated with male rabbit F01–5. T-cloning sequence analysis and T7E1 cleavage assay demonstrated that 7 out of 9 newborn F1 rabbits carried MSTN mutations. In these pups, the F12, F15, F16, F18, F19 were monoallelic, while F13, F17 were biallelic MSTN KO rabbits  were recorded weekly. As shown in MSTN+/− rabbits were obviously heavier than WT rabbits after 6 weeks of age. In addition, the typical double-muscled phenotype was also found in MSTN+/− rabbits at 2 months of age  was substantially larger than in WT rabbits (1055.81 ± 257.57 μm2)  rabbits, compared to the WT rabbits (1318.51 ± 261.48um2). Furthermore, the average fiber density in gluteus maximus from the MSTN+/− rabbits  was significantly higher than in the WT rabbits (211.37 ± 19.89)  . Additio± 19.89) .MSTN KO rabbits is due to fiber hypertrophy. Whereas, it seems that the increased muscle mass of gluteus maximus in the MSTN KO rabbits is due to both fiber hyperplasia and hypertrophy.These results therefore suggest that the enlarged tongue in the MSTN has been modified in various species such as miceMSTN KO rabbits by microinjection of Cas9/sgRNA into one-cell stage embryos, demonstrating that this system could be used to efficiently generate gene knockout rabbits. Moreover, fragment deletions were frequently detected between the two sgRNAs targeting sites in MSTN KO rabbits, suggesting that the dual sgRNA-directed CRISPR/Cas9 system could provide an efficient tool for gene knockout in the mammal genomeIn recent years, with the aim to study muscle development and increase meat production, MSTN KO rabbits. We believe that it might be due to the low concentration of Cas9/sgRNA, which degraded immediately after targeting the aimed gene. In addition, the strict match of seed sequences (8–12 bases close to PAM) is a critical factor for ensuring the site-specific cleavage of CRISPR/Cas9 systemOff-target mutation has been frequently reported in Cas9-mediated gene editing system1718MSTN KO livestock, the calving difficulty induced by enlarged body size of fetus is a big problem in these MSTN-mutant animalsMSTN-deficient animals exhibit disorders similar to large offspring syndrome (LOS)8MSTN KO rabbits showed a typical double-muscled phenotype and a dramatically increased body weight, there was no significantly difference in body size and weight at birth compared to the WT controls. In addition, the MSTN KO rabbits appears normal, healthy, and without reproduction problems, indicating that MSTN KO rabbits generated by CRISPR/Cas9 system are suitable for the study of muscle development and related diseases.Although the increased muscle mass makes it attractive to create MSTN mutations are characterized by increased muscle mass, which results from a combination of increased muscle fiber size and numberMSTN−/− pigsMSTN KO miceMSTN+/− rabbits. Therefore we believe that the increased muscle phenotype in the MSTN KO rabbits is attributed to hyperplasia and/or hypertrophy of muscle fibers. Besides, the MSTN-regulated muscle proliferation and differentiation are also affected by expression levels of MSTN mRNA, the health state and the individual developmental stage of the organism.Animals with MSTN KO rabbits were efficiently generated by microinjection Cas9/sgRNA mixture into the pronuclear-stage embryos, and the typical double-muscled phenotype was obtained in the founder and offspring MSTN KO rabbits. These MSTN KO rabbits can be a promising tool for studying muscle development and improving economically important traits in livestock.In conclusion, the All experiments involving animals were conducted according to the guidelines for the animal care and use of laboratory animals established by the Animal Care Center and Use Committee of Jilin University. All experimental protocols were approved by the Ethics Committee of Jilin University. New Zealand rabbits were housed under standard laboratory conditions of a 12-h light and 12-h dark cycle in individual cages, and were fed twice a day with commercial rabbit basic diet  and water ad libitum. The sexually mature rabbits were used for embryo collection and embryo transfer recipients.NotI and in vitro transcribed to mRNA using the mMessage mMachine SP6 Kit (Ambion). Then the product was purified with the RNeasy Mini Kit (Qiagen) according to the manufacturer’s instructions.The 3× FLAG-NLS-SpCas9-NLS vector (Addgene ID 48137) was linearized with in vitro transcription of sgRNA, the pair of complementary 20-nt DNA oligos were annealed to generate a double-stranded DNA fragment, and then sub-cloned into the BbsI-linearized pUC57-T7-gRNA vector (Addgene ID 51306). The sequence of two sgRNAs is shown in in vitro transcription of sgRNA were amplified using T7 primers (T7-F: 5′-GAAATTAATACGACTCACTATA-3′ and T7-R: 5′-AAAAAAAGCACCGA CTCGGTGCCAC-3′). Then in vitro transcription was performed using T7 RNA Synthesis Kit (Ambion) and the synthesized mRNA was purified using miRNeasy Mini Kit (Qiagen) according to the manufacturer’s instructions. The concentration and quality of the synthesized mRNA were determined by Nandrop 2000 and agarose gel electrophoresis, respectively.To prepare the vector of 2O. A mixture of in vitro transcribed sgRNA (40 ng/μL) and Cas9 mRNA (180 ng/μL) was injected into the cytoplasm of pronuclear stage embryos. The injected embryos were transferred to embryo culture medium for 30–60 min culture at 38.5 °C, 5% carbon dioxide and humidity conditions. Then approximately 30–50 injected embryos were transferred into the oviduct of the recipient mother.Zygotes were collected through surgical oviduct flushing from donors after superovulation treatment and natural mating, as described previouslyTo test the mutation patterns, the injected embryos developed to blastocyst stage were collected and the genomic DNA was extracted with embryo lysis containing 1% NP40 at 50 °C for 20 minutes and 90 °C for 5 minutes in BIO-RAD PCR machine. The genomic DNA from ear punch tissues of newborn pups was isolated using the TIANamp Genomic DNA Kit . Then the target site was PCR amplified using the primers as follows: F 5′GGAGCAAGAGCCAATCATAGA 3′ and R 5′ TGAGGCTGTGAAGGCATAAG 3′. The PCR products were subjected to T7EI assay and T-cloning sequence.The T7EI assay was performed as described previouslyhttp://tools.genomeengineering.org). The top 5 POTS were selected for each sgRNA according to ranking scores. The PCR primers used in this study are shown in The POTS of the two sgRNAs were predicted using the CRISPR design tool (MSTN KO and WT rabbits (euthanized at 2 months of age) of F1 generation were homogenized and lysed in RIPA buffer supplemented with 2.5 μL/mL protease inhibitor cocktail (Roche) on ice for 30 min. The protein concentrations were determined by Bradford method (Bio-Rad). 35 μg of protein sample was subjected to a 5% stacking/12% separating SDS-polyacrylamide gel. The antibodies used in this study include: anti-MSTN polyclonal antibody (Abcam) and goat anti-rabbit IgG conjugated with horseradish peroxidase (HRP) . The β-actin antibody (Santa Cruz) was used as an internal control.Samples from gluteus maximus tissue in MSTN KO and WT rabbits (euthanized at 2 months old) of F1 generation were fixed with 4% paraformaldehyde, then embedded in paraffin wax and slide sectioned. The sections were stained with hematoxylin and eosin (H&E) and analyzed by microscope (Nikon ts100).The tissues of tongue and gluteus maximus from MSTN+/− rabbits of F1 and four WT counterparts controls were bred under the same conditions and used in this study. All rabbits were weaned at 30 days of age and housed in individual cages of the same litter. The body weight was recorded weekly from 1–8 weeks. At 2 months of age, the rabbits were anesthetized with isofluorane, following the injection of beuthanasia (1 mL/4 kg). Then the carcasses were dissected and the heart, tongue, gluteus maximus and vastus lateralis of the MSTN KO and WT groups were weighed and recorded separately.Four p value < 0.05 was considered statistically significant. *p < 0.05, **p < 0.01, ***p < 0.001.Data was statistically analyzed by Graphpad prism software (T test) and a How to cite this article: Lv, Q. et al. Efficient Generation of Myostatin Gene Mutated Rabbit by CRISPR/Cas9. Sci. Rep.6, 25029; doi: 10.1038/srep25029 (2016).


Reduced Ca2+ transient amplitude due to decreased sarcoplasmic reticulum Ca2+ content is a common finding in heart failure models. However, heart failure models also show increased propensity for diastolic Ca2+ release events which occur when sarcoplasmic reticulum Ca2+ content exceeds a certain threshold level. Such Ca2+ release events can initiate arrhythmias. In this study we aimed to investigate if both of these aspects of altered Ca2+ homeostasis could be found in left ventricular cardiomyocytes from rats with different states of cardiac function six weeks after myocardial infarction when compared to sham-operated controls. Video edge-detection, whole-cell Ca2+ imaging and confocal line-scan imaging were used to investigate cardiomyocyte contractile properties, Ca2+ transients and Ca2+ waves. In baseline conditions, i.e. without beta-adrenoceptor stimulation, cardiomyocytes from rats with large myocardial infarction, but without heart failure, did not differ from sham-operated animals in any of these aspects of cellular function. However, when exposed to beta-adrenoceptor stimulation, cardiomyocytes from both non-failing and failing rat hearts showed decreased sarcoplasmic reticulum Ca2+ content, decreased Ca2+ transient amplitude, and increased frequency of Ca2+ waves. These results are in line with a decreased threshold for diastolic Ca2+ release established by other studies. In the present study, factors that might contribute to a lower threshold for diastolic Ca2+ release were increased THR286 phosphorylation of Ca2+/calmodulin-dependent protein kinase II and increased protein phosphatase 1 abundance. In conclusion, this study demonstrates both decreased sarcoplasmic reticulum Ca2+ content and increased propensity for diastolic Ca2+ release events in ventricular cardiomyocytes from rats with heart failure after myocardial infarction, and that these phenomena are also found in rats with large myocardial infarctions without heart failure development. Importantly, beta-adrenoceptor stimulation is necessary to reveal these perturbations in Ca2+ handling after a myocardial infarction.Abnormal cellular Ca Alteredmyocytes . This isa2+ leak . Howeverhythmias , 10. Theld level , 12. Thikey role . Althougkey role –16, withkinase A , 17–19.2+ homeostasis related to contractility or abnormal Ca2+ release events associated with arrhythmias. Fewer studies show that these features really coexist in clinically relevant models of heart failure. Experimental studies of heart failure are mostly based on animal models of end-stage heart failure, and rarely on clinically relevant intermediate forms of the disease. In addition, descriptions of heart failure after myocardial infarction, the most common cause of heart failure, are mostly based on comparisons to sham-operated animals, rather than the equally clinically relevant post-infarction animals without heart failure. The aim of this study was therefore to investigate whether increased diastolic Ca2+ release events and reduced SR Ca2+ content could be found in ventricular cardiomyocytes from a well-characterized rat model of myocardial infarction from which two groups with distinct phenotypes can be distinguished, i.e. rats with developing heart failure and rats with myocardial infarction but without heart failure.The accumulated insight in the processes underlying contractile dysfunction and arrhythmias in heart failure come from a number of models, both in terms of species and interventions to induce heart failure. However, most studies of heart failure have focused on either CaAnimal experiments were approved by the Norwegian National Animal Research Authority (FOTS ID 4173 and 6577), which conforms to the National Institute of Health guidelines  and European Directive 2010/63/EU. Two animals per cage were housed in a temperature-regulated room with a 12:12 h light-dark cycle, and unlimited access to food and water. Euthanasia was performed by cardiac excision or cervical dislocation during anesthesia with 4.5% isofluorane inhalation. Criteria for humane endpoints was a) acute decompensated heart failure , b) postoperative wound infection  and c) major disruption of the surgical wound.2 and 4.5% isoflurane (Abbott Laboratories), and subsequently ventilated through endotracheal intubation on a Zoovent ventilator  with 68% N2O, 29% O2, and 2% isoflurane. Thoracotomy was performed on the left side, the heart was exteriorized and the left coronary artery was ligated by a silk suture inducing a large myocardial infarction as described elsewhere [Male Wistar rats (~300 g) were anesthetized in a chamber with a mixture of Olsewhere . The samLVOT x πraorta2 .Echocardiography was performed with a Vevo 2100  on anesthetized rats, six weeks after coronary artery ligation. Data were collected from parasternal long and short axis images of the left ventricle and left atrium, Doppler signals in the mitral, pulmonary and aortic valves and tissue velocity in the left ventricular posterior wall as previously described [Post-myocardial infarction rats were stratified into two groups: rats with developing congestive heart failure (CHF) and rats with large myocardial infarction (> 30%) but without heart failure development (MI). The inclusion criteria for MI were lung weight under 2.5 g and left atrial diameter (LAD) under 5.0 mm, and for CHF lung weight over 2.5 g and LAD over 5.0 mm . The shasurgical procedure. A large thoracotomy was carried out; 200 IU heparin was administered intravenously through the hepatic vein before the heart was excised and immediately put in ice-cold saline . The heart and lungs were weighed. The heart was then mounted on a modified Langendorff setup with cannulation of the aorta, and retrogradely perfused with ~30 ml 37°C oxygenated solution A: 130 mM NaCl, 25 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), 22 mM Glucose monohydrate, 5.4 mM KCl, 0.5 mM MgCl2, 0.4 mM NaH2PO4 (pH 7.4). The heart was then perfused with 37°C oxygenated solution A supplemented with ~200 U/ml collagenase Type-II  and 0.08 mM Ca2+ for 20 min. An incision was made in viable myocardium to assure that the border zone proximal to the infarction was excluded. In Sham, cardiomyocytes were harvested from both the septum and the left ventricular free wall. The viable left ventricle free wall and septum were chopped into small pieces and gently stirred in solution A supplemented with 15 μM bovine serum albumin (Sigma Aldrich) and ~100 U/mL deoxyribonuclease I . This mixture was transferred to a centrifuge tube through a Nitex mesh with 255 μm openings (Sefar). Cardiomyocytes sedimented by gravity were transferred to solution A supplemented with 0.1 mM Ca2+ and 15 μM bovine serum albumin. After subsequent sedimentations the cardiomyocytes were transferred to solution A supplemented with 0.2 mM Ca2+ and 15 μM bovine serum albumin, and finally 0.5 mM Ca2+ and 7.5 μM bovine serum albumin. Cardiomyocytes were stored at room temperature and used within 10 h of isolation.Six weeks after coronary artery ligation, animals were weighed, intubated and ventilated as described under 2, 1 mM CaCl2, 5.5 glucose, 0.4 mM NaH2PO4, 5 mM HEPES . Another 1 min was allowed for de-esterification as field-stimulation was applied with platinum electrodes at ~20% above the individual threshold for contraction for 30 s at 1 Hz followed by a period of 20 s without stimulation. For measurements of SR Ca2+ content, stimulation was applied for another 30 s before electrical stimulation was stopped with a simultaneous switch to solution B supplemented with 10 mM caffeine (Sigma Aldrich). The protocol was repeated after 100 s exposure to 20 nM isoprenaline (Norwegian Pharmacy Association) added to solution B and 1 Hz electrical stimulation. Ca2+ transients, Ca2+ waves, Ca2+ sparks and caffeine-induced Ca2+ release were recorded using line-scan imaging  and whole-cell Ca2+ imaging (PhotoMed PTI microscope Photometer D-104G). Only cells without visible Ca2+ waves for 10 s before electrical stimulation, with preserved striation and no blebs were included. Cell shortening was recorded in Clampex software  with a Nikon Diaphoto-TMD inverted microscope and a video edge detector .Cardiomyocytes were loaded with 5 μM fluo-4 AM (Molecular Probes), 10 min for line-scan images and 15 min for whole-cell fluorescence, and plated on laminin-coated coverslips mounted in a perfusion chamber. To allow sufficient time for the cell to attach to the cover slip and the de-esterification of the fluorescent probes, superfusion was only started after five minutes. Cardiomyocytes were superfused with solution B: 140 mM NaCl, 5.4 KCl, 0.5 mM MgClCell isolation and the viable left ventricle including ventricular septum was rapidly frozen using liquid nitrogen. Frozen ventricular tissue was homogenized in buffer . Protease inhibitor, phosphatase inhibitor  and sodium dodecyl sulfate  was added. Micro Bicinchoninic Acid Protein Assay Kit  was used to quantify protein concentrations. The following antibodies were used . Ca2+ sparks were analyzed using ImageJ  and CaSparks  [2+ wave velocities were analyzed using ImageJ. Data were expressed as mean ± standard error of the mean. Two-tailed distribution t-tests were performed in Microsoft Excel. Due to a skewed distribution of Ca2+ spark frequencies, these data were analyzed with a Poisson test using R software . p<0.05 was considered statistically significant.CaGermany) . Ca2+ waPost-myocardial infarction rats were stratified to CHF and MI groups based on echocardiographic criteria and lung weight. Representative echocardiographic images are shown in 2+ handling and contractile properties were first studied in baseline conditions . In these conditions, fractional shortening was increased in CHF myocytes compared to Sham . The Ca2+ transient amplitude response to ISO was also decreased in MI compared to Sham .CHF exhibited decreased response to ISO when compared to Sham in terms of Ca2+ handling (2+-ATPase (SERCA) abundance was reduced in CHF compared to Sham  compared to Sham Click here for additional data file.


In this study, a new pathway for bitter tastant-induced relaxation was defined and investigated. We found nifedipine-insensitive and bitter tastant chloroquine-sensitive relaxation in epithelium-denuded mouse tracheal rings (TRs) precontracted with acetylcholine (ACH). In the presence of nifedipine (10 µM), ACH induced cytosolic Ca2+ elevation and cell shortening in single airway smooth muscle cells (ASMCs), and these changes were inhibited by chloroquine. In TRs, ACH triggered a transient contraction under Ca2+-free conditions, and, following a restoration of Ca2+, a strong contraction occurred, which was inhibited by chloroquine. Moreover, the ACH-activated whole-cell and single channel currents of non-selective cation channels (NSCCs) were blocked by chloroquine. Pyrazole 3 (Pyr3), an inhibitor of transient receptor potential C3 (TRPC3) channels, partially inhibited ACH-induced contraction, intracellular Ca2+ elevation, and NSCC currents. These results demonstrate that NSCCs play a role in bitter tastant-induced relaxation in precontracted airway smooth muscle.Bitter tastants can induce relaxation in precontracted airway smooth muscle by activating big-conductance potassium channels (BKs) or by inactivating voltage-dependent L-type Ca In 1867, Schofield RH discovered “taste-goblets” in cat and dog tongues 2+ through the Gβγ protein-PLCβ-IP3-IP3R pathway. This Ca2+ increase then activates BKs, resulting in membrane hyperpolarization and partial relaxation 2+ channels (VDLCCs) mediated by G proteins TAS2Rs have recently been found to be expressed in airway smooth muscle cells and bitter taste stimuli can affect airway muscle force +, and K+) and divalent  cations with relatively poor discrimination. Thus, the activation of NSCCs results in Ca2+ influx-inducing contraction in muscle. In this study, we found that, in addition to VDLCCs, these NSCCs also play a role in bitter tastant-induced relaxation in precontracted airway smooth muscle.NSCCs represent a family of ion channels that generally conduct mono- . The other reagents were purchased from Sigma  and Tocris Bioscience . Niflumic acid, fluo-4 AM, and fura-2 AM were dissolved in DMSO, and other agonists and antagonists were dissolved in physiological saline solution (PSS). In single cell experiments, the reagents were locally delivered onto the cells through a 200 ad libitum access to water and food.Mature BALB/c male mice were purchased from Hubei Provincial Center for Disease Control and Prevention, Wuhan, China. Mice were housed under controlled temperatures (21–23°C) and light conditions (lights on 8∶00–20∶00) with This study was performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All experiments were approved by the Institutional Animal Care and Use Committee at the South-Central University for Nationalities (Permit number: 2012-QHL-1). Mice were sacrificed by intraperitoneal injection of sodium pentobarbital (150 mg/kg) and tissues were then taken.2, 2 CaCl2, 10 HEPES, and 10 glucose (pH = 7.4). The epithelium-denuded TRs were prepared and mounted in a 10-mL organ bath chamber with a preload of 0.5 g. After a 60-min equilibration, the TRs were precontracted with ACH (10−4 M), washed, and rested for 3 times. Following an additional 30 min rest, the experiments were started.Muscle force was measured as previously described 2+ physiological saline solution (LCPSS) containing (mM) 135 NaCl, 5 KCl, 1 MgSO4, 10 glucose, 10 HEPES, and 0.1 CaCl2 (pH = 7.4). The epithelium-denuded trachealis tissues were minced and incubated for 20 min at 37°C in LCPSS containing 1 mg/mL papain, 0.5 mg/mL dithioerythritol, and 1 mg/mL bovine serum albumin (BSA). The partially digested tissues were transferred to LCPSS containing 1 mg/mL collagenase H, 1 mg/mL dithiothreitol, and 1 mg/mL BSA at 37°C for 20 min. The tissues were then washed 3 times and triturated in LCPSS to yield single ASMCs.Single mouse ASMCs were enzymatically isolated as previously described 2+ was measured and analyzed as previously described 2/95% O2-bubbled PSS for 10 min. The excitation was provided at 488 nm and the emitted fluorescence of fluo-4 AM was imaged through a 505 nm filter. XY images were acquired and fluorescence intensity was analyzed using Zen 2010 software .Intracellular Ca2+ was measured using fura-2 AM as previously described 2+ concentration.Intracellular CaTo measure changes in cell length 2, 10 HEPES, 3 EGTA, and 1 CaCl2 (pH adjusted to 7.2 with Tris) 2+ concentration was approximately 70 nM, calculated using WEBMAXC (www.stanford.edu/~cpatton/webmaxc/webmaxcS.htm). The pipette solution contained 300 µg/mL nystatin for perforation of the membrane of smooth muscle cells. The bath solution was PSS as described above, but with K+ omitted. We added 10 mM tetraethylammonium (TEA) chloride, 100 µM niflumic acid, and 10 µM nifedipine to the bath solution to block the K+, Cl−, and VDLCC currents, respectively, to further purify NSCC currents.The ion channel currents were measured using an EPC-10 patch-clamp amplifier  Single NSCC currents were recorded using an outside-out approach. The single channel currents at −90 mV were acquired at a digitization rate of 4 kHz and filtered at 1 kHz. Events were detected and all-point amplitude histograms were plotted using the Clampfit 9 software . The histograms were fitted using the Gaussian distribution function, and amplitudes of single channels were obtained and used to calculate the single channel conductance . The pipette and bath solutions were similar as that previously used for single NSCC recordings with outside-out technique t-test using Origin 9.0 software . Differences with p<0.05 were considered significant.The results are expressed as mean ± SEM. Comparisons between two groups were performed with Student’s We first observed relaxation following blockade of VDLCCs in this study. TRs were contracted with ACH (100 µM). When contraction reached a steady-state, nifedipine, a selective blocker of VDLCCs, was cumulatively added to the organ chambers, which resulted in a series of relaxations . When ni10−2.5) chloroquine. These results further indicate that a VDLCC-independent pathway plays a pivotal role in chloroquine-induced relaxation.To further confirm the existence of nifedipine-insensitive component, we conducted the following experiments. We used 10 µM nifedipine , ACH was added, which triggered a transient contraction through intracellular Ca2+ release. When the contraction reached a steady-state, 2 mM Ca2+ was restored in the organ chamber and a strong and sustained contraction subsequently occurred. Following the addition of chloroquine (3 mM), the contraction was inhibited by 104.9±3.0% (8 rings/8 mice), and the contraction was reversed following washout  receptors, resulting in intracellular Ca2+ increase To define the chloroquine-inhibited CaIn order to further explore the mechanism of chloroquine-induced inhibition of NSCCs, we observed the effect of chloroquine on single NSCC activity. Single NSCC currents were recorded at −90 mV using the outside-out configuration, which were completely blocked by 1 mM chloroquine . NSCC co2+ measurements using fura-2 AM dye. As shown in 2+ increase, which was partially inhibited by Pyr3 (10 µM), and the Pyr3-resistant component was blocked by chloroquine (1 mM). The Ca2+ levels at the time points indicated by number 1, 2, and 3 were analyzed as shown in To further define the role of NSCCs, we used pyrazole 3  to inhibit TRPC3 channels To further support these findings, we recorded ACH-activated NSCC currents. We found that the currents were partly blocked by Pyr3 (10 µM) and the remaining components were inhibited by chloroquine (1 mM). Pyr3 and chloroquine decreased the currents from −14.74±1.66 pA to −6.59±0.65 pA and −0.19±0.07 pA (n = 6 cells from 4 mice), respectively . These rIn the present study, our data demonstrated that chloroquine can induce relaxation in precontracted airway smooth muscle through inhibition of VDLCCs and NSCCs. The mechanism of inhibition on NSCCs is due to direct blockade by chloroquine.2+ influx to increase intracellular Ca2+, triggering a contraction in airway smooth muscle M receptor agonists can induce the activation of both VDLCCs and NSCCs, which in turn lead to Ca2+ decrease. Thus, we found that chloroquine inhibited the ACH-induced Ca2+ rise and corresponding cell shortening (2+ decrease was due to chloroquine blocking Ca2+ influx, as chloroquine can reversibly block Ca2+ influx-induced contraction (One cause of relaxation is intracellular Caortening . The Ca2traction .2+ influx. Previous studies have demonstrated that the activation of M receptor results in the activation of NSCCs in ASMCs. The activated NSCCs in turn mediate Ca2+ influx, leading to a rise in cytosolic Ca2+We then defined which pathway was responsible for the Ca2+ measurement (Since TRPC1-7 are NSCCs, we used Pyr3, a blocker of TRPC3, to test whether these channels are involved in chloroquine-induced relaxation surement  and wholsurement ; howeverβγ signaling pathway-mediated VDLCC inactivation plays a pivotal role in chloroquine-induced relaxation It has previously been reported that TAS2R-GIn summary, chloroquine can induce relaxation in precontracted mouse airway smooth muscle through inactivation of VDLCCs and NSCCs. The inactivation of NSCCs is a result of direct blockade on the channels. Therefore, both VDLCCs and NSCCs should be blocked when treating airway hyperresponsiveness.


A literature review showed that several ion channel proteins are likely responsible for regulation of the Ca2+ uptake in spermatozoa. Therefore, manipulation of the functions of channel proteins is closely related to Ca2+ influx, ultimately affecting male fertility. Recently, it has been shown that, together with different physiological stimuli, protein-protein interaction also modifies the Ca2+ influx mechanism in spermatozoa. Modern proteomic analyses have identified several sperm proteins, and, therefore, these findings might provide further insight into understanding the Ca2+ influx, protein functions, and regulation of fertility. The objective of this review was to synthesize the published findings on the Ca2+ influx mechanism in mammalian spermatozoa and its implications for the regulation of male fertility in the context of sperm proteins. Finally, Pathway Studio (9.0) was used to catalog the sperm proteins that regulate the Ca2+ influx signaling by using the information available from the PubMed database following a MedScan Reader (5.0) search.Freshly ejaculated spermatozoa are incapable or poorly capable of fertilizing an oocyte. The fertilization aptness of spermatozoa depends on the appropriate and time-dependent acquisition of hyperactivation, chemotaxis, capacitation, and the acrosome reaction, where calcium (Ca Several studies have hypothesized that elevation of sperm intracellular Ca2+ ([Ca2+]i)/Ca2+ influx regulates motility, hyperactivation, chemotaxis, capacitation, and the acrosome reaction and facilitates the spermatozoa reaching and fertilizing of an oocyte i regulates the release of Ca2+ into the cell. Therefore, how Ca2+ crosses into cells through the sperm plasma membrane is a matter of paramount importance. In eukaryotic cells, the Ca2+ influx occurs through specific Ca2+ permeable ion channel proteins located on the plasma membrane i in spermatozoa are induced by several psychological stimuli such as cyclic nucleotides  i and sperm hyperactivation. This study provided insight that the Ca2+ influx in spermatozoa is partially regulated by UQCRC2 protein. Kwon et al. [2+]i was observed in (−) DIDS conditions, while [pH]i significantly increased in (−) DIDS, regardless of Ca2+. Simultaneously, a significantly elevated [pH]i was observed in (+) Ca2+. This study provides strong evidence that the modulation of Ca2+ influx by VDACs is pH-dependent, which is consistent with the result of a previous study by Ho and Suarez [2+]i by regulating the function of arginine vasopressin in mice spermatozoa. However, it remains to be clarified as to why spermatozoa motility is decreased even in increased [Ca2+]i conditions.It has recently been found by our laboratory that treatment of mouse spermatozoa with nutlin-3a, a small molecule antagonist of the mouse double minute 2 repressor, potentially downregulates the functions of the ubiquinol-cytochrome-c reductase complex component UQCRC2 and correlated with significantly reduced i i , 66.2+ regulated capacitation and the acrosome reaction have been known for the last two decades, the molecular basis of this process is still poorly understood. For capacitation, the cholesterol influx initially stimulates the elevation of [Ca2+]i and bicarbonate into the spermatozoa and finally activates PKA and tyrosine phosphorylation, respectively, via the production of the cAMP [2+]i influx to proceed further, and they are believed to be activated by PKC through the opening of the calcium channels. Interestingly, PKA together with a secondary messenger, inositol trisphosphate, activates calcium channels localized in the outer acrosomal membrane and increases the calcium concentration in the cytosol. Further increase of cytosolic Ca2+ influx occurs through a store-operated calcium entry mechanism in the plasma membrane, resulting in further depletion of Ca2+ in the acrosome [Although the biochemical phenomenon of Cathe cAMP –68. In athe cAMP . Spermatacrosome .2+]i decreases because acrosome-reacted spermatozoa release a substantial amount of Ca2+ from their inner cell layers i, ATP, and actin polymerization. Removal of the actin polymerization node from this aforesaid network causes disorganization of the network topography and affects capacitation, and this has been confirmed by zona pellucida-induced capacitation and the acrosomal reaction in an in vitro demonstration i in eggs and is mediated via inositol 1,4,5-trisphosphate (InsP3) receptors  remain a matter to be elucidated.A large number of Caentified , 43 thatproteins  and the 2+ influx mechanism in spermatozoa is regulated by several physical stimuli, although the underlying mechanism is less clearly defined. Protein-protein interactions also potentially regulate the Ca2+ uptake mechanism in spermatozoa. Although recently applied proteomic approaches have identified several sperm-specific proteins, their functions in Ca2+ regulation and interaction with channel proteins are unclear. Therefore, future research should target this topic to provide a robust understanding of Ca2+ and male fertility in both humans and other animal species.A literature review found that the Ca


We systematically analyzed different aspects of intra- and inter-cellular Ca2+ signaling in the human LECs, which are attached to surgically isolated lens capsule (LC), obtained during cataract surgery. We monitored the temporal and spatial changes in intracellular Ca2+ concentration after stimulation with acetylcholine by means of Fura-2 fluorescence captured with an inverted microscope. In our analysis we compared the features of Ca2+ signals in individual cells, synchronized activations, spatio-temporal grouping and the nature of intercellular communication between LECs. The latter was assessed by using the methodologies of the complex network theory. Our results point out that at the level of individual cells there are no significant differences when comparing the features of the signals with regard either to the type or the stage of the cataract. On the other hand, noticeable differences are observed at the multicellular level, despite inter-capsule variability. LCs associated with more developed cataracts were found to exhibit a slower collective response to stimulation, a less pronounced spatio-temporal clustering of LECs with similar signaling characteristics. The reconstructed intercellular networks were found to be sparser and more segregated than in LCs associated with mild cataracts. Moreover, we show that spontaneously active LECs often operate in localized groups with quite well aligned Ca2+ activity. The presence of spontaneous activity was also found to affect the stimulated Ca2+ responses of individual cells. Our findings indicate that the cataract progression entails the impairment of intercellular signaling thereby suggesting the functional importance of altered Ca2+ signaling of LECs in cataractogenesis.In this work we investigated how modifications of the Ca Next we focus on the collective behavior of LECs and examine the global responsiveness to stimulation, synchronization behavior and the spatio-temporal grouping of LECs. Finally, we assess the nature of intercellular communication by means of graph-theoretical approaches. Our results show that at the level of individual LECs there are no significant differences when comparing the features of Ca2+ signaling with regard either to the type or the stage of the cataract. On the contrary, a less orchestrated intercellular Ca2+ signaling between LECs associated with more developed cataracts is identified. We discuss our findings in the context of the role of intercellular communication for ensuring a normal functioning of multicellular systems and how its malfunctioning is associated with different pathologies.The aim of this study is to investigate Ca2+ signaling in the human anterior lens epithelium, build from LECs, whereby we compare different signaling characteristics with regard to the type of the cataract–C and N, and the degree of cataract progression. The latter encompasses the comparison between mild, C&N, and moderate to severe, C2-4&N2-4, cataracts, irrespective of the type of cataract. In our analysis we analyze altogether 12 different C (2619 cells) and 13 different N cataract (2963 cells) LCs. In the group of mild, C&N, and moderate to severe, C2-4&N2-4, cataract LCs we have 15 (3964 cells) and 10 (1618 cells) samples, respectively. The experimental protocol consisted of the time before stimulation, the time of stimulation with ACh, and the subsequent recovery time.We systematically analyzed different aspects of intra- and inter-cellular Ca2+]i, which was followed by a decrease towards the stationary state, as exemplified in 2+ signals we defined three parameters of the temporal characteristics of the signal: tres−the time at which a particular cell started to respond with a rise in [Ca2+]i after stimulation with ACh; tmax−the time at which the highest [Ca2+]i of a particular cell was achieved; thalf−the time at which [Ca2+]i of a particular cell decreased to 50% of its maximal value , within a given small time-interval that was being slid along the recorded time series. Values of Ravg(T) close to 0 indicate rather low correlations between [Ca2+]i signals in LECs at the given point in time, whereas on the other hand, values close to 1 signify well synchronized dynamics. Results in Ravg(T), as a function of time (red line with dots), and the corresponding average signal of all LECs (black line). It can be observed that the level of correlation between LECs either prior to or after the response to ACh is rather low. On the other hand, within the activation/deactivation phases Ravg(T) is very high. This is a result of rather simultaneous increases/decreases of [Ca2+]i in all LECs. The decrease in Ravg(T) in the region of maximal ACh-induced [Ca2+]i is attributed to the time-delays between individual signals. In other words, while in some cells [Ca2+]i begins to decrease, in other cells, which probably responded to Ach stimulation with a delay, [Ca2+]i is still increasing. Hence, in this situation average correlation is lower. Furthermore, small increases of Ravg(T) in the period prior to Ach-induced [Ca2+]i transients (between 50 s and 100 s) are a consequence of spontaneous activity in the locally synchronized clusters of LECs. Namely, spontaneously active LECs are frequently found to operate in localized groups with a concurrent [Ca2+]i activity, as exemplified in For the quantification of the temporal evolution of correlations in Caysis see . In partRavg(I), whereby the average correlation between all LECs at a given distance is calculated over the whole recorded time series of the [Ca2+]i activity. The results in Ravg(I) is monotonically decreasing function of distance, indicating an obvious tendency that nearby cells are better correlated than the remote ones. Noteworthy, the values of correlations are very high, even at large intercellular distances. This, however, is not a consequence of cell-to-cell interactions, but is rather associated with the fact that several cells respond to the stimulation simultaneously, i.e. in a short time interval, irrespective of both, the way of intercellular communication and their locations. Yet, the obviously decreasing trend in For a more detailed insight into the synchronization behavior we calculate the average correlation as a function of physical distances, tres, characterizing the onsets of [Ca2+]i increases in individual LECs, varied significantly from cell to cell. In order to characterize the responsiveness of a given LC we show in 2+]i in a given LEC falls below 50% of its maximal value , the cell is considered as deactivated. In tihalf, of the first deactivated cell on. For the description of the responsiveness with a single parameter, which enables a straightforward comparison between different LCs, we calculate the activation speed, SA, and the deactivation speed, SDA, which are defined by the slopes between 20/80% and 80/20% of activated/deactivated cells, respectively   is found to increase with the distance, I. This demonstrates that LECs are not arranged randomly with respect to their activity, but they tend to form clusters with simultaneous activations or similar signal durations. However, different slopes of the curves can be noticed, which indicates the differences in the extent of the spatio-temporal grouping. Evaluation of the results with respect of the type of the cataract  see . The subcataract  reveals cataract  reveals 2+ waves. Accordingly, only the cells being located less than 30 μm apart can be considered as directly connected. The extracted networks can be subsequently directly used for the evaluation of the features of intercellular communication. Four typical lens epithelium networks corresponding to different types and degrees of the cataract are shown in To assess the nature of intercellular communication between LECs in more details, we use the theoretical framework, which was primarily developed in the field of complex networks. In particular, we construct the so-called activation networks on the basis of simultaneous activations of LECs see . A spatikavg, which reflects the level of connectedness within the tissue. The global efficiency, EG, reflects the network’s functional integration such as the traffic capacity. The next measure is the average clustering coefficient, Cavg, that characterizes the cliquishness of a network by indicating how concentrated the connections around a typical cell are. Higher values of Cavg correspond to clustered connections around individual LECs. Finally, we compute the relative number of communities, nic,/Ni, that is a measure of the network’s segregation. If in the i-th LC there are relatively many different communities, this indicates that the network is very compartmentalized and segregated. Results are presented in To quantify the properties of the activation networks and to compare their characteristics with respect to the type or the degree of the cataract, we calculate several network metrics see . We calc2+ signaling patterns in LECs. We were particularly interested in the comparison of the Ca2+ signaling characteristics with regard to the type of the cataract and the degree of cataract progression. With regard to the cataract type, C and N, we detected no significant differences in any herein examined aspect of Ca2+ signaling. A rather high inter-capsule variability in [Ca2+]i responses was present. Noteworthy, Gupta et al. [2+]i levels are always found high in LECs, irrespective of the type of the cataract. This might be an indication of an impaired Ca2+ homeostasis in both types of cataracts. Intracellular Ca2+ homeostasis might be broken down possibly due to Ca2+ influxes exceeding the ability of LECs to remove [Ca2+]i from the cytosol. It is not clear, whether this is due to upregulated Ca2+ influx, impaired ability to remove excess [Ca2+]i or due to a combination of both processes. This is the field to be studied further.In the present paper we analyzed the intra- and inter-cellular Caa et al.  also rep2+ signaling in LECs associated with mild, C&N, and moderate to severe, C2-4&N2-4, cataracts, irrespective of the type of cataract. Again, no significant differences were detected by comparing the Ca2+ signal characteristics in individual LECs. On the other hand, significant differences were observed on the multicellular level. First, cells associated with less developed cataracts were found to have better orchestrated and faster global response to stimulation by agonist ACh. They have higher activation speeds meaning that LECs are activated faster and more simultaneously once the first cell is activated. This could be related with the responsiveness of the receptors, which is potentially decreased in more developed cataracts. On the other hand, there are no differences in the activation and the deactivation times of individual cells either with regard to the type or the degree of the cataract, suggesting that the intracellular processes involved both in activation and deactivation phases of [Ca2+]i response of more developed cataracts do not differ from those in less developed cataracts. The parameter thalf is likely related to the functionality of the Ca2+ removal mechanisms and our results suggest that they are not related with the degree of cataract. Second, we showed that with increasing levels of pathology, the tendency of nearby cells having similar characteristics regarding their times of activation or their durations of the Ca2+ signal, decreases. This implies the impairment of intercellular communication mechanisms and also suggests the existence of intercellular communication which ensures more synchronized responses between adjacent LECs in less developed cataract. To further address this issue we additionally described the connectivity patterns and signals propagation between LECs by means of graph-theoretical approaches. Our results show that the extracted intercellular networks are much sparser and more segregated in more developed than in mild cataracts. It appears evident that intercellular communication between LECs is affected by the severity of the cataract, which results in less integrated networks within more developed cataract stages. In mild cataracts the average network degree and the average network clustering were significantly larger, and the fraction of communities in the activation networks was smaller than in more developed cataracts. In other words, at higher degrees of cataract pathology, larger segregation occurs, resulting in less unified and less effective intercellular communication within the lens epithelium. The fact that LECs of C&N cataracts are better interlinked and their [Ca2+]i response after ACh is faster and more synchronized than C2-4&N2-4 cataracts suggests that in more developed cataracts lens epithelium has functionally more damaged LECs, probably due to the loss of gap junctional coupling. This could have been expected, but we have clarified that by our present quantitative analysis.Since no significant differences were observed between C and N cataracts, we compared the Catact, in comparison to spontaneously non-active cells. This finding might suggest that spontaneously active LECs are damaged and have impaired intracellular mechanisms of Ca2+ increase regulation. Moreover, the larger activation times might also be related with the fact that after Ca2+ release from the endoplasmic reticulum in spontaneously active LECs a series of processes takes place, including the pumping of Ca2+ back into the internal stores. Consequently, the LECs might therefore respond with time delay due to the refractory period, in which they are less susceptible. We also showed that spontaneously active LECs usually form clusters, which together with the finding that spontaneously active LECs respond slower to ACh, suggest the existence of the regions of the human lens epithelium that are functionally damaged. The presence of spontaneously active cells influences significantly only the average activation times of individual cells, whereas the responsiveness of the whole LC is not affected. The fact that LECs’ spontaneous activity occurs irrespective of the cataract pathology might be an indication that these LECs’ functional changes are an additional disturbance of Ca2+ homeostasis, which could not be directly correlated with the type and/or the degree of cataract.We investigated also the extent and the role of spontaneous LECs activity. No significant correlation between the spontaneous activity and the type or the degree of cataract was detected. Rather large variability in the portion of spontaneously active LECs was observed. However, further analyses have revealed that individual spontaneously active LECs have on average higher activation times, Δ2+]i, LECs are not able to regulate Ca2+ homeostasis well anymore, and as a consequence, cataractogenesis is facilitated. Although the LEC is equipped with machinery to combat with cataractogenic insults, any alteration in the lens epithelium may proceed further in the remaining part of the lens and may lead to cataract [2+ levels by regulating the activity of Ca2+- pumps and Ca2+-channels as well as the inhibition of Ca2+-dependent enzymes is necessary for its homeostasis and can help in prevention of cataract [2+]i low as well as the mechanisms responsible for its increase i in cultured bovine LECs i response, according to the data by Collison et al. i, while the 360/380 nm ratio allowed visualization of the Ca2+ concentration changes in the cytoplasm. Image acquisition, timing and filterwheel operation were all controlled by WinFluor software via a PCI6229 interface card . The criteria for selecting the region for imaging were the presence of adherent cells and good cell morphology both assessed by observation of transilluminated and 360 nm fluorescencent images. Individual image frames were acquired every 500 ms resulting in frame cycles being 1 s long (2 wavelengths). All offline analyses were done with custom made scripts written in C/C++ and Python.Image acquisition was done with the 12-bit cooled CCD camera SensiCam . The software used for the acquisition was WinFluor . Objectives used were: 40x/0,75 Plan-NeoFluar and 63x/1,25 oil Plan-NeoFluar . The light source used was XBO-75W  Xe arc lamp. The light intensity was attenuated when necessary with grey filters with optical densities 0.5, 1 and 2 . The excitation filters used, mounted on a Lambda LS-10 filterwheel , were 360 and 380 nm . Excitation with the 360 nm filter (close to the Fura-2 isosbestic point) allowed observation of the cells’ morphology and of the changes in the concentration of the dye, irrespective of changes in [Ca+2]i dynamics of individual LECs from the experimentally recorded data sets were measured off-line by exporting the 360/380 nm signal ratio for all ROIs using ImageJ software i signaling in individual LECs we defined three characteristic times . To this purpose we first constructed the correlation matrix R(T), with the ij-th element Rij(T) representing the correlation coefficient among the recorded [Ca+2]i signals ui(t) and uj(t) within a time window T ± Δt. The correlation coefficient Rij(T) was thus computed as:i-th and j-th cell and their corresponding standard deviation are given by t ∈ . The temporal average correlation in the given time window Ravg(T) is then computed as:For the characterization of dynamical correlations in , and calculate the corresponding average correlation coefficient Ravg(I).Furthermore, to capture the spatial aspects of correlations between LECs, we computed the distance matrix tires,, and their activation/deactivation times, Δtact/Δtdeact, we calculate the average temporal difference between these three characteristic times as a function of the distance I:N(ΔI) is the number of cells within the distance interval, ΔI, from the i-th cell, and the inner sum calculates corresponding time differences between the i-th and the j-th cell, whereby j(ΔI) runs through all the cells within a given distance interval ΔI. The subscript x refers to different times of interest with regard to τi and τj signify tres of the i-th and j-th cell, respectively). Similarly, tact and tdeact (see τx(I) is an increasing function of I, the cells are not arranged randomly with respect to their intracellular signaling, but tend to form clusters with simultaneous responses or similar signal durations. To overpass the variability expressed by different average activation times in different LCs (see τx(I) in individual LCs, so that the smallest value identified at any distance, is set to one. Afterwards, the values in different LCs are averaged, in accordance to the type or degree of the cataract.To quantify the grouping of cells with respect to their simultaneous responses to stimulation, eact see . If Δτx LCs see , we normi-th and j-th LECs are considered as connected, if they are simultaneously activated, i.e. |tires, − tjres,| ≤ 1 s. To prevent the creation of connections that are established due to simultaneous responses in different parts of the LC, we incorporate a spatial constrain that limits the edge formation only among cell pairs which are less than 30 μm apart. In this manner we extract the connectivity patterns that reflect the intercellular communication between LECs.For the characterization of the intercellular communication among LECs within a LC, we make use of graph theoretical approaches and construct a so-called activation network. The construction of the network is based on finding LECs pairs that responded to external stimulation with ACh within a narrow time interval. In particular, the kavg. This measure is defined as the averaged sum over all individual degrees, ki, i.e. number of connections of individual cells. The second measure is the global efficiency of the network, EG. This network feature is computed as averaged sum over the inverse shortest path lengths among individual cell pair. The shortest path length between the i-th and j-th lij cell equals the shortest geodetic distance between them or the smallest number of edges which separates them. Thereby, EG characterizes the network’s functional integration that reflects the effectiveness of information exchange and synchronizability. The third implemented measure is the average clustering coefficient, Cavg, which quantifies the level of clustered connectivity and functional segregation. To compute this network feature we implemented the algorithm introduced by Watts & Strogats norm, and B deactivation times, norm, between spontaneously active and non-active cells. In the normalization process all values were divided by the average Δtact or Δtdeact in the given LC in order to ensure a reliable comparison between cells in different LCs. LCs without spontaneously active cells were excluded from the analysis. Altogether 3391 spontaneously non-active and 506 spontaneously active cells were included in the analysis. The asterisk indicate statistically significant differences in activation times, whereas on the other hand significant differences in deactivation times with regard to spontaneous activity could not be detected. Values within individual groups are represented by means of the box charts diagrams in which the boxes determinate the 25th and 75th percentiles, the whiskers denote the 5th and 95th percentiles, the crosses stand for the minimal and maximal values, the line within the box signifies the median, and the small square stands for the average.Comparison of: (TIF)Click here for additional data file.S2 FigA Positions of LECs with marked spontaneously active cells  in the LC. Positions of 6 spontaneously active LECs, for which traces are presented in the B panel (colored black rings). B Temporal traces of the normalized [Ca2+]i dynamics, u′(t), for 6 selected spontaneously active LECs, as indicated by the coloring in panel A. It can be observed that the majority of spontaneously active cells is organized into localized small groups that exhibit rather aligned spontaneous [Ca2+]i responses.(TIF)Click here for additional data file.S3 FigSA, and deactivation, SDA, speeds of LCs. Values within individual groups are represented by means of the box charts diagrams in which boxes determine the 25th and 75th percentiles, the whiskers denote the minimal and the maximal values, the line within the box signifies the median, and the small square stands for the average.The one-way ANOVA test did not detect significant differences between the two groups of LCs, i.e. the group with spontaneously active LECs and the group without. The results indicate that the spontaneous activity does not have a significant impact on the activation, (TIF)Click here for additional data file.


ACTN3 gene. α-Actinin-3 deficiency is detrimental to sprint performance in elite athletes and beneficial to endurance activities. In the human genome, it is very difficult to find single-gene loss-of-function variants that bear signatures of positive selection, yet intriguingly, the ACTN3 null variant has undergone strong positive selection during recent evolution, appearing to provide a survival advantage where food resources are scarce and climate is cold. We have previously demonstrated that α-actinin-3 deficiency in the Actn3 KO mouse results in a shift in fast-twitch fibres towards oxidative metabolism, which would be more “energy efficient” in famine, and beneficial to endurance performance. Prolonged exposure to cold can also induce changes in skeletal muscle similar to those observed with endurance training, and changes in Ca2+ handling by the sarcoplasmic reticulum (SR) are a key factor underlying these adaptations. On this basis, we explored the effects of α-actinin-3 deficiency on Ca2+ kinetics in single flexor digitorum brevis muscle fibres from Actn3 KO mice, using the Ca2+-sensitive dye fura-2. Compared to wild-type, fibres of Actn3 KO mice showed: (i) an increased rate of decay of the twitch transient; (ii) a fourfold increase in the rate of SR Ca2+ leak; (iii) a threefold increase in the rate of SR Ca2+ pumping; and (iv) enhanced maintenance of tetanic Ca2+ during fatigue. The SR Ca2+ pump, SERCA1, and the Ca2+-binding proteins, calsequestrin and sarcalumenin, showed markedly increased expression in muscles of KO mice. Together, these changes in Ca2+ handling in the absence of α-actinin-3 are consistent with cold acclimatisation and thermogenesis, and offer an additional explanation for the positive selection of the ACTN3 577X null allele in populations living in cold environments during recent evolution.Over 1.5 billion people lack the skeletal muscle fast-twitch fibre protein α-actinin-3 due to homozygosity for a common null polymorphism (R577X) in the  ACTN3, popularly known as “the gene for speed.” In 1.5 billion people, a certain variation in the genetic sequence of their ACTN3 gene causes their muscles to produce no α-actinin-3 protein at all. These people have no muscle disease; however, in elite athletes, a lack of α-actinin-3 seems to be beneficial for endurance activities and detrimental to sprinting activities. Intriguingly, α-actinin-3 deficiency varies in frequency across the globe, being most common in European and Asian populations and least common in African populations. During recent human evolution, there appears to have been strong positive selection for α-actinin-3 deficiency in places where food resources are relatively scarce and climate is cold. We have previously demonstrated that α-actinin-3 deficiency in the Actn3 knockout (KO) mouse causes a shift towards more “energy efficient” forms of muscle metabolism which would enhance survival in times of famine, and benefit endurance performance. Our present study, using single muscle fibres from Actn3 KO mice, demonstrates changes in calcium handling that would adapt muscles to cold environments and provide a survival advantage in cold climates.α-Actinin-3 is a protein found inside the muscles of most people around the world. It is encoded by a gene called  ACTN3 gene i (free myoplasmic Ca2+ concentration) and increased fatigue resistance. Mechanistically, changes in Ca2+ handling by the SR are a key factor underlying these adaptations. Our aim in this study, therefore, is to investigate the Ca2+-handling characteristics of single fibres from Actn3 KO mouse muscle, to see if there are any features consistent with cold acclimatisation. We examine the Ca2+ transients in fast glycolytic fibres from the flexor digitorum brevis (FDB) muscle of untrained, non-cold-exposed Actn3 KO mice, and provide the first evidence of a heat-generating mechanism that could enhance survival in cold environments and promote the positive selection of the 577X null allele in certain populations.Bruton et al.  have sho2+ handling by α-actinin-3-deficient muscle fibres, we examined Ca2+ kinetics of individual twitches in single FDB fibres from WT and Actn3 KO mice. 2+ transients elicited by a single action potential. Actn3 KO fibres in the time taken to rise from 20% to 80% of maximum [Ca2+]i .As an overall indicator of potential alterations in Ca [Ca2+]i B. Howevst phase C and slst phase D of [Ca2+ re-uptake by the sarcoplasmic reticulum (SR) is one of the main contributors to the decline of [Ca2+]i following fibre stimulation [2+ re-uptake could underlie the faster [Ca2+]i decay rates of Actn3 KO fibres observed in 2+ re-uptake, we derived SR pump function curves from the slow phases of [Ca2+]i decay in the twitch transients from 2+ pump under steady-state conditions i and the rate of [Ca2+]i decline during the slow phase of [Ca2+]i decay. It is clear that for any level of [Ca2+]i, the rate of [Ca2+]i decline is higher in KO than in WT. The rate of [Ca2+]i decline is a balance between the rate of SR Ca2+ pumping and the rate of Ca2+ leak from the SR i. Pi also slows the Ca2+ pump, thus reducing the rate of decay of the tetanic transient i, force and relaxation rate during repetitive stimulation of FDB fibres. This was attributed to an increased capacity for oxidative metabolism due to increased mitochondrial enzyme activity i of about 30 nM following cold exposure i, possibly because the increased Ca2+ leak from the SR was accompanied by an increased rate of Ca2+ pumping, whereas in cold-exposed mice the rate of Ca2+ pumping actually decreased [2+]i might be contributing to increased calcineurin activity, and this would need to be investigated in future studies using full intracellular dye calibrations in WT and Actn3 KO fibres i transient type i was used to correct for this. The fibre was stimulated using a bipolar stimulating electrode placed close to the neuromuscular junction, which was visible in the light microscope as a corrugated oval on the fibre. The fibre was stimulated with pulses of 1 ms duration from 1 to 100 Hz. Shortening in response to action potential activation of the fibres was minimal. In some experiments, fibres were immobilised by application of the selective inhibitor of the ATPase activity of skeletal muscle myosin; 4-Methyl-N-(phenylmethyl)benzenesulfonamide (BTS) 25μm to the bath; in this case, the [Ca2+]i transients were not significantly different to those before application of BTS, indicating minimal interference from movement artefacts. During the experiments, the isolated fibres were superfused (1 mL/min) with Krebs solution maintained at room temperature (22°C–24°C) and aerated with 95% O2-5% CO2.The fibres were placed onto glass coverslips for fluorescence microscopy and became firmly attached. Individual muscle fibres were viewed with a 40 UV-F objective on a Nikon TE300 inverted microscope equipped a xenon light source. Fibres with diameter ent type . An intrthe cell . After fthe cell . For the2+]i using our previously determined in vivo calibration curve measured in isolated fibre segments from mouse extensor digitorum longus muscle i values. We used this correction in calculating the rise times reported in 2+]i was calculated from the raw [Ca2+]i using the following equation i decay were measured by fitting exponential equations to the decay phases of the twitch and tetanic transients. The decay kinetics of the twitch transient  were determined at selected time points. Then the following SR pump function equation was fitted to these values [A reflects the rate of Ca2+ uptake by the SR pump, L represents the rate of Ca2+ leak from the SR and N is a power term indicating the cooperativity of Ca2+ binding by the SR pump [et. al. [N to a value of 4 to facilitate the comparison of A and L between fibers of WT and Actn3 KO mice. Other investigators have obtained N values close to 4 when allowed to be freely fit [N = 4.27 ± 0.09 when allowed to be freely fit.A double-exponential function was fitted to the decay phase of the twitch transient, as described above. Then, from the slow phase of the fitted curve, the values of [Cae values –22:−ddt SR pump . Followieely fit ,22, and For high-speed acquisition of transmitted illumination images during shortening of intact single FDB fibres electrically field-stimulated with a single supramaximal voltage pulse of 0.3 ms duration and 10 V amplitude, a CMOS PCO1200hs high-speed camera  was mounted to the camera side-port of the Olympus inverted microscope. The Peltier-cooled camera was connected to a PC for acquisition control and data storage. Single fibres approximately covered a 520×160 pixel area when visualised through a ×20 objective which allowed frame rates for shortening sequences to push up to ≈4,200 frames per second. Recordings were synchronised with the induction of a single twitch and image read-out and storage from the ring-buffer of the camera was performed offline. For offline analysis of each experiment, an image sequence of approx. 1,000 to 1,700 frames per fibre were analysed using a modification of a previously written processing algorithm in IDL language environment . This alActn3 KO FDB and QUAD muscle samples as determined using the Pierce BCA assay kit (Thermo Scientific) and EDL muscles using Stain Free gel technology (BioRad). A total of 4 to 20μg of total protein was loaded per sample and separated by SDS—PAGE on 4–12% pre-cast mini-gels (Life Technologies) or 4–15% Criterion Stain Free gels (BioRad), transferred to polyvinylidene fluoride (Millipore) or nitrocellulose membranes (BioRad) blocked with 5% skim milk/1× tris buffered saline (TBS)/0.1% Tween-20, then probed with indicated antibodies overnight and developed with ECL chemiluminescent reagents (Amersham Biosciences and Thermoscientific). Images were collected using Image Lab software (BioRad) for EDL blots or X-ray film for FDB and QUAD analyses. Primary antibodies for immunoblotting include; EDL muscle lysates: anti-α-actinin-3 , anti-calsequestrin VIIID12 , anti-sarcoplasmic reticulum ATPase1 ), anti-ryanodine receptor 1 , anti-dihydropyridine receptor , anti-Parvalbumin , with secondary goat-anti-mouse IgG-horse radish peroxidase , goat anti-rabbit IgG HRP  and rabbit anti goat IgG HRP . FDB and QUAD muscle lysates: anti-α-actinin-3 , SERCA1 , Sarcalumenin , Parvalbumin , and α-sarcomeric actin . Secondary antibodies used were sheep anti-mouse IgG-HRP conjugates  and donkey anti-rabbit IgG-HRP conjugates  [Immunoblotting for selected proteins was performed using equally loaded WT and lthcare) .t-tests at a significance level of 5%. All statistical tests and curve fitting were performed using a standard statistical software package .Data are presented as Mean ± S.E.M.. Unless otherwise stated, all statistical tests are two-tailed


Bletilla striata into microspheres as a novel embolic agent for decades. The aim of this study was to evaluate the biocompatibility of Bletilla striata microspheres (BSMs). After a thermal test of BSMs in vitro, the cell biocompatibility of BSMs was investigated in mouse fibroblasts and human umbilical vein endothelial cells using the methyl tetrazolium (MTT) assay. In addition, blood biocompatibility was evaluated. In vivo intramuscular implantation and renal artery embolization in rabbits with BSMs were used to examine the inflammatory response. The experimental rabbits did not develop any fever symptoms after injection of BSMs, and BSMs exhibited no cytotoxicity in cultured mouse fibroblasts and human umbilical vein endothelial cells. Additionally, BSMs exhibited high compatibility with red blood cells and no hemolysis activity. Intramuscular implantation with BSMs resulted in a gradually lessened mild inflammatory reaction that disappeared after eight weeks. The occlusion of small renal vessels was associated with a mild perivascular inflammatory reaction without significant renal and liver function damage. In conclusion, we believe that BSMs exhibit high biocompatibility and are a promising embolic agent.We have prepared Chinese traditional herb  In the last decade, various embolic agents have been developed to further improve the safety and efficacy of embolization procedures. In particular, the development or refinement of spherical embolic particles has remarkably increased the spectrum of interventional radiology. Spherically shaped particles were introduced into the armamentarium of embolization techniques to overcome the disadvantages of irregularly shaped particles, such as the promotion of catheter clogging, incomplete occlusion of the target vessels, and unpredictable behavior . Convers Bletilla striata (BS) polysaccharide (BSP) extracted from the traditional Chinese medicine BS is the polysaccharide macromolecule glucomannan [ Bletilla striata microspheres (BSMs) of different sizes. The biocompatibility of BSMs was observed both in vitro and in vivo, the findings of which may help us to examine the feasibility of applying BSMs as potential embolic agents.The primary component ofcomannan , which icomannan , 5. In tcomannan , 7 has sBletilla striata polysaccharide was prepared by ethanol precipitation following 60°C water extraction, deproteinization using the Sevage method, petroleum ether  defatting, and activated carbon  bleaching. The polysaccharide was further isolated by ion-exchange chromatography on a DE-52 column  and gel filtration on a Sephadex G-100 column , and purified BS polysaccharide was obtained. BSMs were prepared according to the modified method of emulsion-condensation-chemical cross-linking [60Co-γ exposure for disposal.-linking . The micμm, 100–200 μm, 200–300 μm, 300–400 μm, 400–500 μm, 500–700 μm, 700–900 μm, 900–1200 μm, and 1200–1400 μm in diameter, and then they were placed into bottles based on the sphere size, packaging, and sterilization. BSMs dispersed well in normal saline and Omnipaque . The settling times in these 2 solutions were long enough for syringe drawing. The microspheres went well through 5F catheter in both normal saline and Omnipaque solution. There was no aggregating and deformation. Microsphere size less than 400 µm could pass through 3F SP microcatheter  and was not deforming and aggregating. For microspheres with diameter larger than 400 µm, it was difficult to pass through a 3F SP microcatheter. They would aggregate, swell, and occlude the microcatheter. The characteristics of BSMs in vitro met the requirements for interventional approaches that appeared in other manuscripts.Scan-electromicroscope  showed that the BSMs were regular and uniform in size and without aggregation. Small holes were noted on the surface of microspheres . The micOur Animal Care and Use Committee approved all of the experiments (SCXK2011-0011), and eighty-eight animals were used in this study. New Zealand rabbits, both male and female, weighing 2.0–2.5 kg, were obtained from the Laboratory Animal Center of Tongji Medical College, Huazhong University of Science and Technology. After the surgery, analgesic promethazine  was injected intramuscularly for pain control. All the animals were returned to their cages after surgery, kept warm, and monitored in the animal laboratory twice in one day. Humane endpoints were used and animals were euthanized prior to the end of our experiments according to AVMA Guidelines on Euthanasia, 2007, American Veterinary Medical Association.A thermal test was conducted by the thermal inspection method specified in Chinese Pharmacopoeia (2010 version). Warm BSMs in physiological saline solution (PSS) at approximately 38°C were injected into ear veins of the selected three New Zealand rabbits within 15 min after taking their baseline temperature; their temperatures were then taken 6 times in 30-min intervals. Subsequently, the temperature increment of the experimental rabbits was obtained by subtracting the normal temperature from the highest temperature. Another five rabbits were used for the above experiments in the same test method again if the temperature of one experimental rabbit rose by 0.6°C or higher or if the temperatures of all experimental rabbits rose by 1.3°C or higher.Result Determination. The thermal tests of the rabbit samples were considered in accordance with regulation when the temperature increment of each experimental rabbit was lower than 0.6°C, the total increment of the three rabbits was lower than 1.3°C in the first test, and the total temperature increment of eight experimental rabbits was 3.5°C or lower in the first and second tests. The thermal tests of the rabbit samples were considered in discordance with regulation when the temperature of more than one rabbit rose by 0.6°C or higher in the three rabbits for the first test, the temperature of more than one rabbit rose by 0.6°C in the five rabbits for the second test, or the total increment of the eight rabbits was higher than 3.5°C in the first and second tests. The negative temperature increment values were considered 0°C.µL BSM suspension was added per well of a 96-well plate. Mouse fibroblasts and human umbilical vein endothelial cells in the log growth phase were prepared for cytotoxicity experiments after trypsin digestion. Two hundred microliters of the cell suspension at a density of 1–10 × 104 was seeded into 96-well plates and mixed with BSMs. Three parallel wells were set for each sample. The plates were placed in a humidity culture chamber at 37°C and 5% CO2. Cell viability was observed using the methyl tetrazolium (MTT) method 1 h, 4 h, 24 h, and 72 h after incubation. Blank medium without BSMs was set as the negative control. Fifty microliters of fresh MTT solution (5 mg/mL) was added to each well and incubated for 4 hours. Next, 200 µL of dimethyl sulfoxide (DMSO) was added to each well and shaken throughout the evening using a flat shaking table. Finally, the optical density (OD) was determined using a microplate reader (the detection wave length is 570 nm). Cells treated with blank medium without BSMs were set as the control group. The inhibition ratio was calculated according to the following formula: inhibition ratio =  × 100%.Five milligrams of sterilized BSMs was diluted in 1 mL of culture medium, and a 30-Blood was obtained from one New Zealand rabbit by cardiac puncture before euthanasia using intravenous injections of 30 mg/kg of sodium pentobarbital. Defibrinated blood was prepared after filtering the whole blood with sterile absorbent gauze. One hundred fifty milligrams of BSMs, which was diluted with 10 mL of physiological saline, was added to 0.1 mL of defibrinated rabbit blood and stored at 37°C for 24 h. Determination standard was solution layering without hemolysis. If there is no layering of the solution by gross observation, then it indicated occurrence of hemolysis, lastly observed erythrocyte state with light microscope after Wrights staining of erythrocyte at the vascular bottom.Five milliliters of whole blood (heparin anticoagulation) and 5 mL of 2% erythrocyte-physiological saline suspension were obtained using the method mentioned above and then added to the test tubes. Finally 100 mg of BSMs was added to the test tubes separately and mixed evenly, and the samples were taken after storing the test tubes at 4°C in the fridge for 6 h and 24 h, respectively, to observe erythrocyte morphology with a light microscope after Wrights staining.Twelve New Zealand rabbits were used in the present intramuscular implantation study. Under anesthesia and sterile conditions, two implanted points were selected at the same distance from the spine, and a pocket was made in the gluteal muscle at a depth of 1 cm. Polyvinyl alcohols  and BSMs of 1 mg were implanted on each side. Finally, the fascia, muscle, and skin layer were sutured. One week, 4 weeks, and 8 weeks after implantation, 4 rabbits were sacrificed at each time point, and the implant sites were excised and removed with the surrounding muscle tissue. The samples were fixed in 10% formaldehyde, embedded in paraffin, sectioned, and stained using the hematoxylin-eosin (HE) method.µm) or PVA with the same size as the control group. There were 36 rabbits each in the experimental group and control group, with four rabbits for each of nine periods to observe liver and kidney functions. Renal tissue slices were embedded in paraffin and subjected to vascular fiber Masson staining. Then, renal target vascular wall changes and the inflammatory response of surrounding cells were observed after embolization.Seventy-two New Zealand rabbits, regardless of gender, were subjected to embolization, after anesthesia, on the right renal artery with BSMs . After fasting the rabbits for 12 hours, all of the experimental rabbits were subjected to anesthesia and were fixed on a wooden plate in the supine position. For the procedure, the right common femoral artery was surgically cut, a 4-F catheter sheath  was introduced, and a 4-Fr Cobra visceral catheter (Terumo) was used to select the abdominal aorta through which digital subtraction angiography (DSA) was performed. The trunk of the renal artery was selected using a 4-Fr Cobra-type catheter, and renal arteriography was performed by manual injection of 2 mL of Omnipaque, diluted to 50%. The renal artery was embolized immediately after the administration of the mixture of embolic agents. At each time point after embolization, angiography and pathological examination of kidneys as well as liver and renal function tests were performed. All of the animals were returned to their cages after surgery, kept warm, and monitored in the animal laboratory until they recovered from anesthesia. They were monitored after the operation and given analgesics if they showed any sign of physical distress. None of the animals died during that phase of the protocol.P value of less than 0.05. Nonparametric Wilcoxon signed-rank test was applied for paired data (intragroup comparison). For the comparison of unpaired data (intergroup comparison: after embolization with PVA versus BSM), the nonparametric Mann-Whitney U test was applied.SPSS 17.0 statistical software was adopted for analyses, the measurement data are expressed as the mean ± SD, and the difference was regarded as statistically significant with a The rectal temperatures of the three rabbits were 38.5°C, 38.6°C, and 38.5°C before BSM injection and slightly increased after injection. The temperature increment of the rabbits was not higher than 0.6°C, and the total temperature increment of the three rabbits was not higher than 1.3°C refer to , indicatTwo normal cell lines Figures –4, mousRabbit erythrocytes were treated with BSMs for 24 h. The red blood cells were all sunk down and the supernatant was colorless and transparent. Consequently, no erythrocyte lysis was observed, and no abnormalities of red blood cell morphology were observed by light microscopy after Wright staining . ErythroExperimental rabbits observed grossly were free from deterioration, exudation, formation of granulation tissue, adhesion of surrounding muscle tissue, and significant differences at both sides in the part planted at the first, fourth, and eighth week. The implant sites were free from necrosis and deterioration after muscle implantation during the study period. As shown in Figures P > 0.05) during the corresponding period.All of the experimental rabbits presented normally after embolization and during the observation period. As shown in The inflammatory response concerning the renal artery of the two groups was similar, mainly for the exudation of neutrophilic granulocytes at the early stage and then for the exudation of lymphocytes and neutrophilic granulocytes during the first week. PVA granule vascular injury was not obvious during the second week, was aggravated during the fourth week, and was alleviated after the sixth week; however, BSM vascular injury was not very obvious Figures .The biocompatibility of medical material arises from complex biological, physical, and chemical reactions after the mutual effect of medical biological materials and the human body, primarily including blood compatibility and tissue compatibility . Blood cPyrogen is the general term of substances that can make homeothermic temperature rise abnormally in a microorganism compound consisting of phospholipid, lipopolysaccharide, and protein . This exCurrently, cell toxicity tests significantly affect biocompatibility evaluations . Cells mThe pathology and healing response of organisms to implantation is an important component in the evaluation of the biocompatibility of BSMs and in the detection of cell toxicity . Wound hThe vascular inflammatory response and organ functional change of the target organ to the embolization material for interventional therapy are important indexes to evaluate the compatibility of the material and organ . A stronBSMs caused neither hemolysis nor cytotoxicity. The results of the implantation test and transcatheter renal embolization indicated that BSMs had good tissue biocompatibility and no adverse effects, such as hepatotoxicity and renal toxicity, indicating their promising potential for use as a highly biocompatible embolic agent.


Portulaca oleracea (PO) has been widely used as traditional medicine because of its pharmacological activities. However, the effects of PO on osteoclasts that modulate bone homeostasis are still elusive.2+ mobilization, nuclear factor of activated T-cell c1 (NFATc1) amplification, tartrate-resistant acid phosphatase-positive (TRAP+) multinucleated cell (MNC) formation, and cytotoxicity.In this study, we examined the effects of PO ethanol extract (POEE) on receptor activator of nuclear factor-κB ligand (RANKL)-mediated Ca2+ oscillations by inhibition of Ca2+ release from internal Ca2+ stores, resulting in reduction of NFATc1 amplification. Notably, POEE attenuated RANKL-mediated cytotoxicity and cleavage of polyadenosine 5′-diphosphate-ribose polymerase (PARP), resulted in enhanced formation of TRAP+ MNCs.Our results demonstrated that POEE suppressed RANKL-induced Cain vitro effects of POEE on RANKL-mediated osteoclastogenesis and suggest the possible use of PO in treating bone disorders, such as osteopetrosis.These results present  Portulaca oleracea (PO), also known as verdolaga and pigweed, has been widely used not only as food, but also as traditional medicine treating insect bites, bacillary dysentery, diarrhea, and hemorrhoids. The chemical constituents of PO have been repeatedly reported to have diverse pharmacological activities. For instance, polysaccharides and betacyanins isolated from PO have antifatigue effects and improve cognition deficits in mice, respectively i) oscillations modulate NFATc1 activity [2+]i oscillations sequentially activates Ca2+/calmodulin-dependent protein kinase, calcineurin, and NFATc1. Activated NFATc1 accumulates inside the cell nucleus, eliciting the induction of gene expression necessary for osteoclastogenesis. On the other hand, RANKL is also known to inhibit cell proliferation and induce apoptosis through a tumor necrosis factor receptor-associated factor 6 (TRAF6)-dependent but NF-κB-independent mechanism i was determined using the Ca2+-sensitive fluorescent dye Fura-2, as described previously i measurement. The cells on a cover glass were transferred to HEPES buffer containing 10 mmol/L HEPES, 140 mmol/L NaCl, 5 mmol/L KCl, 1 mmol/L MgCl2, 1 mmol/L CaCl2, and 10 mmol/L glucose, adjusted to pH 7.4 and 310 mOsm, and then loaded with Fura-2 fluorescent Ca2+ indicator for 50 min at RT and placed in a chamber connected to a perfusion system. Cells were briefly washed out with regular HEPES buffer and continuously perfused with HEPES buffer without RANKL and M-CSF. Each of the indicated chemicals was diluted in HEPES buffer or Ca2+-free HEPES buffer  and perfused for the designated length of time. Under continuous perfusion with regular HEPES buffer (37 °C), intracellular fluorescence was excited at dual wavelengths (340 and 380 nm) and emitted fluorescence (510 nm) was captured using a charge-coupled device (CCD) camera. Captured images were digitized and analyzed using MetaFluor software, with data expressed as the ratio of fluorescence intensities (F340/F380).[Caeviously . Brieflyt-test. Data are presented as mean ± SEM of the stated number of observations obtained from the indicated number of independent experiments. P-values less than 0.05 were considered statistically significant .Results were analyzed using Student’s 2-tailed Glechoma hederacea ethanol extract elicited a transient increase in [Ca2+]i in BMMs, which sequentially suppressed RANKL-mediated [Ca2+]i oscillations, NFATc1 activation, and formation of TRAP-positive (TRAP+) MNCs [2+]i oscillations. Isolated BMMs were plated on cover glass, and [Ca2+]i responses were measured after 2 d of RANKL stimulation. Figure 2+ peaks, but higher concentrations of POEE (50 and 100 μg/mL) immediately abolished RANKL-induced [Ca2+]i oscillations. Notably, abolished RANKL-induced [Ca2+]i oscillations were not rescued by removal of POEE. This negative effect of POEE on RANKL-induced [Ca2+]i oscillations strongly suggests that POEE may inactivate NFATc1. To examine the effect of POEE on NFATc1 activation, BMMs were simultaneously treated with 50 μg/mL of POEE (the minimum concentration that immediately abolished RANKL-induced [Ca2+]i oscillations) and RANKL, and cultured for the indicated time. Figure 2+]i oscillations after 24 h of stimulation with RANKL is critical to activate NFATc1 and trigger the late-stage of osteoclastogenesis i oscillations require orchestration of Ca2+ mobilization from internal and external Ca2+ stores i oscillations [2+]i oscillations led us to further investigate Ca2+ mobilization responses following treatment with POEE. To confirm the effect of POEE on Ca2+ influx through SOCCs, internal Ca2+ stores were depleted by CPA in the absence of extracellular Ca2+, and extracellular Ca2+ influx through SOCCs was then determined by restoring extracellular Ca2+ with or without POEE . Interestingly, one study reported that both low and high concentrations of ATP mobilized [Ca2+]i, but low concentrations of ATP did not induce NF-κB localization into the nucleus, which strongly regulates osteoclast formation i oscillations and NFATc1 activation suggests that POEE may suppress cell differentiation into osteoclasts, whereas altered internal Ca2+ mobilization in response to a low concentration of ATP suggests that POEE may work in a different way. To confirm this, we performed in vitro osteoclast formation assays with and without POEE. Isolated BMMs were stimulated with RANKL and simultaneously treated with POEE in a concentration-dependent manner. Figs. 2+]i oscillations and NFATc1 activation, POEE simultaneously activates other signal pathways resulting in the enhancement of MNC formation. Since Takayanagi et al. suggested that NFATc1 is the master regulator of RANKL-induced osteoclastogenesis i oscillations by inhibiting internal Ca2+ mobilization, which is known as a critical factor for osteoclastogenesis, whereas Fig. in vitro osteoclast formation assays were performed with and without POEE. After various incubation periods, cytotoxicity was measured by evaluating the amount of glucose-6-phosphate dehydrogenase (G6PD) released in the culture medium. Figure Figures 2+ - Ca2+/calmodulin-dependent kinase – calcineurin – NFATc1 signal pathway, which induces differentiation-related gene expression i oscillations and NFATc1 activation but also markedly enhances RANKL-mediated osteoclastogenesis by attenuating RANKL-induced cytotoxicity. Our findings suggest that the RANKL-induced cytotoxicity caused by Ca2+ release from internal Ca2+ store is attenuated by POEE, which resulted in enhanced RANKL-mediated osteoclastogenesis.Our results demonstrate that POEE has dual and opposite effects on RANKL-mediated osteoclastogenesis. POEE not only inhibits the generation of RANKL-induced [Ca