N-retinylidene-N-retinylethanolamine  in the retinal pigment epithelium (RPE) is a major cause of visual impairment in the elderly. Photooxidation of A2E results in retinal pigment epithelium degeneration followed by that of associated photoreceptors. Present treatments rely on nutrient supplementation with antioxidants. 9’-cis-Norbixin  was prepared from Bixa orellana seeds. It was first evaluated in primary cultures of porcine retinal pigment epithelium cells challenged with A2E and illuminated with blue light, and it provided an improved photo-protection as compared with lutein or zeaxanthin. In Abca4-/-Rdh8-/- mice (a model of dry AMD), intravitreally-injected norbixin maintained the electroretinogram and protected photoreceptors against light damage. In a standard rat blue-light model of photodamage, norbixin was at least equally as active as phenyl-N-tert-butylnitrone, a free radical spin-trap. Chronic experiments performed with Abca4-/-Rdh8-/- mice treated orally for 3 months with norbixin showed a reduced A2E accumulation in the retina. Norbixin appears promising for developing an oral treatment of macular degeneration. A drug candidate (BIO201) with 9’-cis-norbixin as the active principle ingredient is under development, and its potential will be assessed in a forthcoming clinical trial.The accumulation of  In developed countries, AMD is the major cause of blindness in the elderly . Dry AMDPhotoreceptor death in the central part of the retina (the macula) is responsible for the loss of high-resolution colour vision. It follows the death of the retinal pigment epithelium (RPE) cells , which htrans-retinal molecules with phosphatidyl-ethanolamine [in vitro [A2E and its isomers are formed by the reaction of two nolamine . A2E is nolamine  and impanolamine ,7; A2E cnolamine , increasin vitro ,9 and poin vitro .in vitro [High A2E concentrations increase oxidative stress in RPE cells in vitro . In the in vitro ,12. It gin vitro ,14,15 anin vitro ,17,18,19in vitro . A2E phoin vitro  and actiin vitro . The toxin vitro .Given the direct involvement of A2E in the pathology, several strategies have been considered for designing treatments, either by preventing the formation/accumulation of this molecule, or by counteracting/reducing its deleterious effects ,26,27,28reducing A2E formation by retinylamine, a visual pigment cycle inhibitor , by redupromoting A2E removal by intravitreal injection of cyclodextrins ,33 or byreducing A2E oxidation by feeding natural antioxidants  ,37,38 orcounteracting some A2E direct effects, such as treatments for a re-acidification of lysosomes ,41, or tcounteracting long-term consequences of A2E accumulation (drusen formation) by using inhibitors/antibodies to complement alternative pathway .Despite promising results, none of these approaches has resulted up to now in a recognized efficient treatment of dry AMD. Current treatments have a limited efficacy and and rely on nutritional formulae containing zinc, various antioxidants (vitamins C and E) and carotenoids, whose components have been tested either individually or as various mixtures over several years (AREDS 1 and 2 studies). These studies have indeed established the protective role of zinc and, for a part of the population tested, of carotenoids ,45.The rationale for using specific carotenoids  relies on the fact that they are naturally present in the macula. As with other antioxidants, their biological activity is not limited to an antioxidant effect ,47. Theyin vitro tests with RPE primary cultures from porcine retina and a set of in vivo experiments to assess the efficiency of these molecules, as described below.We previously observed the photo-protective activity of two diapocarotenoids, bixin and norbixin , for skiAbca4-/-Rdh8-/- [Abca4+/+Rdh8+/+ mice both carrying the Rpe65-Leu450 mutation and the rd8 mutation in the Crb1 gene were used. Abca4+/+Rdh8+/+ mice were derived from backcrossing Abca4-/-Rdh8-/- and C57Bl/6N mice . Pigmented C57BL/6J mice (25–30 g) were provided by Harlan Laboratories  and used in pharmacokinetics studies. Male Sprague-Dawley rats (240–320 g) were provided by Charles River  and used for BLD. Animals were housed under 12-hour on/off cyclic normal lighting.For blue-light-induced retinal damage (BLD) studies pigmented Rdh8-/-   and were approved by the French Ministry of Agriculture (OGM agreement 6193) and by the Committee on the Ethics of Animal Experiments of the French Ministry of Research. All efforts were made to minimize suffering.All usual chemicals were from Sigma . Reagents for cell culture were from Thermo Fisher Scientific . Ketamine, xylasine, tropicamide and oxybuprocaine chlorhydrate were from Centravet . Optimal cutting temperature compound and other reagents for histology were from Roth Sochiel .cis-Bixin  extracted from Bixa orellana seeds was purchased from Aica-Color . 9’-cis-Norbixin was prepared from bixin upon alkaline hydrolysis according to Santos et al. [cis isomer) showed a HPLC purity of 97% as confirmed by 1H-nuclear magnetic resonance .Products for the synthesis of A2E or analogues  were supplied by Sigma. Reference carotenoids—lutein, zeaxanthin, crocetin—were from Extrasynthèse . 9’-s et al. . Bixin  emitting at 470 nm . Just before illumination, the culture medium was replaced by a modified DMEM without any photosensitizer and with 2% FCS. 24 hours after blue-light irradiation, all cell nuclei were stained with Hoechst 33342 and nuclei of dead cells with ethidium homodimer 2, fixed with paraformaldehyde  and 9 pictures per well were captured using a fluorescence microscope (Nikon TiE) equipped with a CoolSNAP HQ2 camera and driven by Metamorph Premier On-Line program. Quantification of live cells was performed using Metamorph Premier Off-Line and a home-made program by subtraction of dead cells from all cells.Pig eyes were obtained from a local slaughterhouse and transported to the laboratory in ice-cold Ringer solution. After removal of the anterior segment of the eye, the vitreous and neural retina were separated from the RPE and removed. The eyecup was washed twice with phosphate buffer saline (PBS), filled with trypsin (0.25% in PBS) and incubated at 37°C for 1.5h. RPE cells were harvested by gently pipetting, centrifuged to remove trypsin and re-suspended in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 20% (v/v) foetal-calf serum (DMEM20%FCS) and 0.1% gentamycin. Cells were seeded into 60 mm diameter Petri dishes, cultured in an atmosphere of 5% COCell treatments were performed as followed. All the drugs used in these experiments were prepared as stock solutions in DMSO. Drugs tested for their protective effect were added to the culture medium 48 hours before illumination.For experiments aimed at measuring A2E in RPE in culture cells were seeded in 12-well plates and the treatments were performed as before see , but theAbca4-/-Rdh8-/- mice aged 7 weeks were used for intravitreal injections. Twenty-four hours before BLD, one group received an intravitreal injection of 2 μL of 500 μM norbixin (in 0.3% DMSO) in one eye. The vitreous volume of the mouse eye being 5.3 μL [Two groups of 14 g 5.3 μL  we estim-tert-butylnitrone , a potent free-radical trapping agent, or an equivalent volume of vehicle  30 min prior to light damage and 2, 4 and 6 hours after the beginning of the exposure.Rats were randomly assigned to non-induced control or light-exposed groups (n = 6 per group). Rats were injected intraperitoneally with either norbixin , phenyl-NAbca4-/-Rdh8-/- mice, aged 2 months, received norbixin orally in drinking water for 3 months. Since the product is poorly soluble in water when the pH is neutral, an exact amount of 50 mg was weighed into 15 mL Falcon tubes and first dissolved in 2 mL DMSO; then 2 mL Tween 80 were added and the mixture was transferred in feeding bottles and adjusted up to 100 mL with water. The mice absorbed ca. 5 mL water daily, corresponding to a dose of 2.5 mg norbixin/day. Abca4-/-Rdh8-/- mice aged two and five months were used as non-treated controls. After 3 months of supplementation, mice were euthanized and eyes were removed for A2E measurements.In order to test the preventive action of norbixin, two groups of 10 A custom-made light damage device equipped with fluorescent lamps (Phillips TL-D 36W/18) with UV filter was used to induce BLD in mice . All manipulations with the animals were performed in dim red light. Pupils were dilated with 0.5% tropicamide eye solution before illumination. Mice, previously maintained in a 12-hour light (≈ 10 lux)/ 12-hour dark cycle environment for two weeks, were dark-adapted for 24 hours. Rats were dark-adapted for 36 hours. Light damage was induced at 4000 lux for 1 hour for mice and 6 hours for rats. Following exposure to light damage, animals were placed in the dark for 24 h and then returned to the dim cyclic light environment for 7 days.-2 were averaged. Amplitudes of a- and b-waves of mixed rod and cone response were determined.ERG recordings were performed with the Electroretinograph Toennies Multiliner Vision designed for rodents. ERG was performed one week after BLD in mice and rats. After overnight dark adaptation, mice and rats were anesthetized with ketamine (100 mg/kg) and xylazine (10 mg/kg). Eye drops were used to dilate the pupils (0.5% tropicamide) and anesthetize the cornea (0.4% oxybuprocaine chlorhydrate). Body temperature was maintained at 37°C using a circulating hot-water heating pad. Corneal electrodes  were placed on the corneal surface of each eye. Eye gel  was used to maintain good contact and corneal moisture. Needle electrodes placed subcutaneously in cheeks served as reference and a needle electrode placed in the back served as earth. The ERG was recorded from both eyes simultaneously after placing the animal into the Ganzfeld bowl. Five responses to light stimulus at 10 cd.s.mAfter ERG, animals were euthanized and eyes were enucleated and dissected to remove the cornea and lens. For mice, eyes were fixed in 4% paraformaldehyde/5% sucrose (in PBS) for one hour at 4°C. The eye cups were then cryoprotected by successive bathing in 5% sucrose (1h), 10% sucrose (1h) and 20% sucrose (overnight), embedded in optimal cutting temperature compound, and cryosections (10 μm) were prepared using Superfrost® Plus slides and stored at -20°C until analysis. Sections were stained with Hoechst 33342 to label nuclei and were scanned using a nanozoomer  with fluorescence imaging modules. Photoreceptor nuclei were quantified at 200 μm intervals superior and inferior to the edge of the optic nerve head along the vertical meridian using the NDP.view software.Rat eyes were fixed with Hollande’s Bouin fixative solution for at least 48 hours at room temperature. The cornea and lens were removed from the eyeballs. The fixed tissues were then embedded in paraffin. Sections (5 to 7 μm thick) were performed along the vertical meridian and stained with Trichrome-Masson. Digitized images of fourteen locations (seven in the superior retina 250–3250 μm above the optic disc and seven in the inferior retina 250–3250 μm below the optic disc) were obtained for each section using a Digital Imaging system . The number of photoreceptor cell nuclei in each image was counted on each photograph.N-retinylidene-N-retinylethanolamine) was synthesized by Orga-link  as described before [trans-retinal, ethanolamine and acetic acid were mixed in absolute ethanol in darkness at room temperature over 7 days. The crude product was purified by preparative HPLC in the dark to isolate A2E with a purity of 98% as determined by HPLC. A2E (20 mM in DMSO under argon) was stored at -20°C.A2E  was synthesized using propylamine instead of ethanolamine and retinal according to the described procedure ,57. BrieMass spectrometry provides accurate and sensitive quantification of A2E . HPLC-MS2O  and the internal standard (A2E-propylamine) was added to each sample. The calibration curve of A2E was performed in ACN/H2O  and under these conditions, with 10 μL injections, the limit of quantification (LOQ) was 50 nM.The concentration of A2E in RPE cells or culture media was determined with HPLC-MS/MS method described above. Samples were diluted with ACN or ACN/H3/MeOH  (0.5 mL) with homogenizer (Precellys-24) during 2 cycles (30 s) at 6500 rpm. The internal standard (A2E-propylamine) was added and the organic layer was extracted. The homogenate was then extracted two times with CHCl3/CH2Cl2 (0.5 mL). The combined organic extracts were dried in vacuo without heating . Then they were dissolved in 100 μL DMSO/MeOH  and transferred to microtitre plates. The calibration curve of A2E was prepared in CHCl3/MeOH  and dried in vacuo without heating , then dissolved in 100 μL DMSO/MeOH . Under these conditions, with an injection volume of 10 μL, the limit of quantification (LOQ) was 10 nM.A2E present in eyes was determined with the HPLC-MS/MS method described above. Each eye was homogenized in CHClUpon oxidation, A2E can fix between 1 and 9 oxygen atoms, each corresponding to an increase of molecular mass of 16 units [per os  or intra-peritoneally . In the second set, only norbixin was used and was dissolved in 10%DMSO/20%Tetraglycol/70%H2O for both p.o. (50 mg/kg) and i.p. (5 mg/kg) administrations. Blood samples were collected after 0.25, 0.5, 1, 3, 6, 8 and 24 hours, centrifuged, and plasma samples were kept at -20°C until analysis. Eyes were dissected after 1, 3, 6 and 24 hours and immediately frozen. Bixin, norbixin and their metabolites were analyzed by HPLC-MS/MS.Two sets of pharmacokinetic studies were performed using C57BL/6J mice using bixin and/or norbixin. In the first set, bixin or norbixin were administered either HPLC analysis was performed on an Agilent 1200 with DAD. Bixin and norbixin were eluted from a reverse-phase C18 column  with the following gradient of acetonitrile in water (containing 0.1% formic acid): 0–90% (1.5 min), 90% (1 min), (flow-rate: 0.5 mL/min) and they were monitored at 460 nm. For quantification of bixin and norbixin, a calibration curve was performed under the same conditions of the matrix of samples, with various amounts of bixin and norbixin (10–50000 ng/mL).ca. 2.5 pmol). Eye samples were treated with the same protocol as for A2E measurements (see above).Plasma samples (30 μL) from different animals, and methanol (100 μL) were distributed in a 96-well microtitre plate, mixed for 10 min and precipitated. The microtitre plate was frozen at -20°C for 30 min and then centrifuged. The hydro-alcoholic phase was removed from each well and transferred into another microtitre plate for LC-MS/MS analysis. Under these conditions, with 20 μL injections, the limit of quantification (LOQ) was 50 ng/mL  operated in MRM positive-ion mode. HPLC used a reverse-phase C18-column  eluted with the following gradient of acetonitrile in water (containing 0.1% formic acid): 60–95% (2.5 min), 95% (2 min), (flow-rate: 0.3 mL/min). Norbixin and its isomers or metabolites (= glucuronides) were monitored at 460 nm and MRM mode with precursor ion/product ion ratio (381.1/144.9).For statistical analyses, Student’s t-Test or one-way ANOVA followed by Dunnett’s or Tukey’s tests were performed using Prism 5  depending of the sample size.in vivo we developed an acute model of phototoxicity using porcine primary RPE cells loaded with 30 μM A2E and illuminated with blue light . As shown in in vivo experiments.Pharmacokinetic studies performed with bixin and norbixin in mice showed that (1) bixin was much less bioavailable than norbixin and (2) bixin was efficiently converted into norbixin owing to esterase activities in both intestine and plasma . The ovecis-norbixin underwent a significant isomerization (trans- and one di-cis-isomer(s), on the basis of their HPLC behaviour [ca. 50% of the total norbixin 6 hours after administration. In addition, two more polar compounds were also detected in the Multiple Reaction Monitoring (MRM) mode. They were shown to have a molecular mass of 566 a.m.u., and they disappeared upon glucuronidase treatment. These two peaks are thus expected to be monoglucuronides of norbixin.A second set of pharmacokinetic studies with norbixin was performed using chromatographic analyses with greater resolution, which showed that the administered 9’-rization  to probaehaviour ; their fca. 20 μM after 15 min and remained above 5 μM for at least 6 hours after administration. Eye uptake was rather low (1–2 ng/eye), but some norbixin (1 ng/eye) was still found unchanged 24 hours after a single administration, together with glucuronides.In the second experiment, the oral bioavailability of norbixin was close to 96% (data not shown). Plasma norbixin levels peaked at in vivo, we developed a model of BLD using 7-week-old −/− Rdh8−/−Abca4 mice, an animal model of dry AMD [Abca4+/+Rdh8+/+ mice . At this concentration we observed a slight, but not statistically significant, effect on ERG or photoreceptor survival (data not shown). In a second set of experiments 2 μL of 500 μM norbixin were injected in order to reach an intravitreal concentration of 130 μM. Mice were then kept in the dark until BLD. Single-flash ERGs were recorded under scotopic conditions one week later. Mice injected with dimethylsulfoxide (DMSO) alone served as negative controls. As seen in To evaluate the therapeutic effect of norbixin, we used the BLD model described above and vs vehicle), whereas 50 mg/kg of PBN induced a 50% protection. The decrease in b-wave amplitude was also significantly reduced by norbixin and PBN with a maximum of 57% protection in rats treated by 100 mg/kg norbixin. To confirm the protective effect of norbixin, morphological evaluation of the retina was performed. The number of photoreceptor cell nuclei was measured along the retina and values were plotted as a function of the distance from the optic nerve  was injected intraperitoneally prior to BLD and 2, 4 and 6 hours after the beginning of the exposure. PBN (50 mg/kg), a potent free-radical trapping agent known to protect the retina in this model ,62 was uic nerve . After lic nerve . For theic nerve .−/− Rdh8−/−Abca4 mice are characterized amongst other things by an elevation of A2E in RPE cells owing to an abnormal functioning of the visual cycle . In order to see whether chronic treatment with norbixin could reduce A2E accumulation in these mice, we supplemented their water-beverage during 3 months before removing eyes for A2E quantification (vs 24 pmol/eye) showing a rapid increase over 3 months. In norbixin-treated mice A2E quantity represented only 55% of that found in vehicle-treated mice (67 pmol/eye vs 122 pmol/eye).fication . A2E conPorcine RPE cells were treated with A2E and various concentrations  of norbixin, according with the protocol described in in vivo, and, upon chronic oral administration, it reduced A2E accumulation in a mouse model of dry AMD.The present study demonstrated the high photoprotective effect of norbixin and bixin at low concentration on primary cultures of porcine RPE cells exposed to A2E and blue light. Norbixin administrated by intravitreal (120 μM) or intraperitoneal  injections protected the retina from an acute blue-light damage in two different models in situ RPE cells, as they do not express the same cellular receptors [RPE cell cultures are widely used for AMD studies for testing drug candidates or analysing the molecular mechanisms of AMD . Either eceptors ,65.In the present study, RPE cells cultivated in the presence of A2E, and then exposed to blue light provided a reproducible model of RPE phototoxicity. Schütt et al. showed a significant loss of cell viability by 72 hours after light exposure of A2E-treated RPE cells . Sparrow2O2, tBuOOH) rather than A2E exposure, and the authors measure either cell survival or the production of ROS or of malondialdehyde (MDA). We may however wonder whether these different models are equivalent. Indeed, vitamin C was efficient in protecting against tBuOOH, but not against H2O2 [2O2 or tBuOOH [Most in vitro studies consider RPE cells protection against an oxidative stress . In the absence of cells, 85% of lutein and 64% of zeaxanthin bound to plastic and could be removed with alcohol, versus 8% for bixin and norbixin. In the presence of RPE cells, binding to plastic was reduced (58% for lutein and 51% for zeaxanthin). This certainly contributes to lowering the efficiency of xanthophylls, although this does not suffice to explain the difference with bixin/norbixin.cis isomer) undergoes significant isomerization and glucuronidation .In the rat blue-light model. norbixin proved at least as efficient as PBN . Similarin vitro [Bixin  is also able to protect retinal ganglion cells (RGC) against an endoplasmic reticulum stress induced by intravitreal injections of tunicamycin, as well as tunicamycin-treated RGC cells in vitro . Similarin vitro .Chronic oral supplementation with norbixin resulted in a reduced accumulation of A2E in mice eyes. There is a general consensus that reducing A2E formation and/or accumulation is a worthwhile target for pharmacological interventions against AMD. Although severe light restriction reduces A2E formation in animal models , this aptrans-retinal [Ccl2-/-Cx3cr1-/- mice fed for 3 months with AREDS2 formula containing lutein and zeaxanthin had lower concentrations of A2E compared to the non-supplemented group [Blocking the visual cycle to limit the density of visual pigment and in turn production of trans-retinal and A2E was also investigated. Several inhibitors of the visual cycle have been tested, but they cause night-blindness, which may be uncomfortable for the patients, and retinoid-based compounds can cause significant systemic effects and teratogenicity ,81,82. O-retinal , or mole-retinal  or remov-retinal ,32 have -retinal . Ramkumaed group . How lutA2E accumulation has deleterious effects that may proceed directly, owing to its cationic detergent properties, or indirectly after through its photo-oxidation by-products. A2E impairs lysosome and proteasome activity  and initin vitro (Norbixin reduced A2E accumulation by RPE cells in vitro . WhetherOne possible protection mechanism involves the activation of Nrf2 and the subsequent production of phase II detoxification enzymes (e.g. superoxide dismutase SOD). Such enzymes protect the retina against oxidative stress, and animals where SOD gene is inactivated develop AMD-like signs of retinal degeneration ,88. TherPrimary target(s) should possibly be considered among PPAR receptors, which accept a great variety of ligands and could play a key role in the prevention of AMD . In othecis-norbixin as active principle ingredient is under development, and its potential will be assessed in a forthcoming multicentre clinical trial.Norbixin appears promising for developing an oral macular degeneration treatment. This molecule is already used in human food as a natural dye and its lack of toxicity established in many assays (e.g. ). A drug


Moreover, activation of 5-HTR3 and 5-HTR4 both significantly inhibited the opening of mPTP. Our results provided the first evidence that 5-HTR as a GPCR and an ion channel, functionally expressed in mitochondria and participated in the mitochondria function and regulation to maintain homeostasis of mitochondrial [Ca2+], ROS, and ATP generation efficiency in cardiomyocytes in response to stress and O2 tension.5-HT has been reported to possess significant effects on cardiac activities, but activation of 5-HTR on the cell membrane failed to illustrate the controversial cardiac reaction. Because 5-HT constantly comes across the cell membrane via 5-HT transporter (5-HTT) into the cytoplasm, whether 5-HTR is functional present on the cellular organelles is unknown. Here we show 5-HTR3 and 5-HTR4 were located in cardiac mitochondria, and regulated mitochondrial activities and cellular functions. Knock down 5-HTR3 and 5-HTR4 in neonatal cardiomyocytes resulted in significant increase of cell damage in response to hypoxia, and also led to alternation in heart beating. Activation of 5-HTR4 attenuated mitochondrial Ca Despite modulation of serotonin at synapses receives a major attention in challenging depression, cardiovascular pathogenesis has also been addressed to associate with serotonin. Alternation in the heart rates2+ flux plays important roles in many other cellular activities, including cytoplasmic Ca2+ signals and activation of cell death pathways2+ was uptaken through a Ca2+ -selective ion channel- mitochondrial Ca2+ uniporter (MCU) driven by voltage across the inner mitochondrial membrane (IMM)2+ concentration leads to mitochondrial Ca2+ accumulation and myocardial injury. This accumulation is achieved by activation of mitochondrial Ca2+ uniporter during ischemia. The mitochondrial Ca2+ overload results in opening a nonspecific pore in the inner mitochondrial membrane, mitochondrial permeability transition pore (mPTP), which is permeable to small molecules. Opening of mPTP usually occurs at early reperfusion. The cell death mediators such as apoptosis inducing factor (AIF) and cytochrome c are released to cytosol followed by mPTP openingHeart is one of the most active tissues because its main function is to continuously pump blood to other tissues. This continuous work requires a stable operation to provide energy in a relatively constant speed. It is well known that mitochondrion is a membrane-enclosed organelle, which generates most of the cell’s supply of energy in the form of adenosine triphosphate (ATP)The previous researches has shown the evidence that 5-HT transporter (5-HTT) is expressed in cardiac cells, and the environmental 5-HT concentration affects the heart cells132+ transient was used to represent the myocyte contraction. Cardiomyocytes of which 5-HTR3 and 5-HTR4 were knocked down by siRNA (37 ± 2.23 min−1) exhibited an increase of 59.14% in the contraction frequency comparing with the control group (23.25 ± 1.49 min−1) . 5-HTR3 and 5-HTR4 were shown to be localized on the membrane of cardiomyocyte mitochondria, by co-immunostaining with an inner-mitochondria membrane marker, COX VI . These c9 min−1) .ATP, mostly produced in mitochondria, decreased rapidly in cytoplasm after a cell injury or oxygen depletion. As indicated in Lactate dehydrogenase (LDH), a biomarker of cellular damage, was measured under the condition of normoxia and hypoxia. In normoxic condition, 5-HTR3 and 5-HTR4 knock-down cardiomyocytes showed a slight increase (6.76%) in the release of LDH into the culture medium . After c2+ could stimulate mitochondrial Ca2+ uptake (MCU), thus stimulate mitochondrial bioenergetics (NADH production) to generate ATP2+ uptake respectively (2+ uptake in the mitochondria was mediated by a ruthenium red (RuR)-sensitive mechanism   serves as the driving force of mitochondrial Ca (Rh123) , suggest2+ uptake, specific agonists and antagonists were further employed. Zacopride, a 5-HTR4 agonist2+ uptake by 45.1677%  were measured because they display the efficiency of the movement of electrons along the electron transport chain and the coupling of this movement to the production of ATP by oxidative phosphorylation. State 3 respiration rates were determined by the addition of ADP, and state 4 respiration was measured in the presence of adequate substrate but without addition of ADP. Mitochondrial rate of respiration was measured using an Ochloride . As show5-HTR4 was activated by different 5-HTR4 agonists respectively, including zacopride, ML 10302 Hydrochloride and BIMU . RCRs of2+ pulse is a widely accepted method to open mPTP, resulting in mitochondrial swollen. The swollen of mitochondria could be reflected by the decrease of relative UV-light absorbance. CsA, a common mPTP blocker, is therefore shown to prevent this swollen. Either 2 uM CsA or 5-HT3R agonists including 5-HT, m-Chlorophenylbiguanide hydrochloride and 1-Phenylbiguanide hydrochloride prevented mitochondrial swelling followed by a Ca2+ pulse of 200 uM for 60 s , exhibited a similar effect as CsA in preventing 200 uM Caswelling . Moreove2+ homeostasis is critical in both physiological and pathological functioning of the heart, resulting in the rate of energy production20212022232+ uptake in our study. 5-HT, as the general agonist for all sub categories of 5-HTR, inhibited mitochondria Ca2+ uptake and O2 consumption under normoxia, but enhanced mitochondria Ca2+ uptake and O2 consumption under hypoxia. Key steps of mitochondrial metabolism in ATP production are Ca2+ dependent2 tension. Additionally, ROS is also recognized as an important signal molecule2 consumption. By the way, it is well documented that hypoxia induces the elevation of intracellular Ca2+ via a variety of mechanisms262+ release from internal store2+] takes place followed by a cytosolic [Ca2+] increase induced by hypoxia in cardiomyocytes292+ uptake under hypoxia effectively buffers this elevation in intracellular Ca2+, and it limits the damage caused by Ca2+, especially in ischemia-reperfusion.5-HT actively comes across the cell membrane via 5-HT transporter (5-HTT) into the cytoplasm2+] in mitochondria buffering is well known to be involved in arrhythmia, the mechanisms are not clear342+ rapidly and buffer cytosolic [Ca2+] during E-C coupling, which supports the theory that fluxes in cytosolic [Ca2+] directly lead the increases in mitochondrial [Ca2+]. In 2006, Maack reported his findings that the role of mitochondrial Ca2+ buffering in cardiac myocytes during contraction is to match the energy/ATP production with demand. Our data supported their conclusion. As shown in 2+] increased is a signal to couple energy production in mitochondria when the lack in ATP supply, the larger frequency of cytosolic [Ca2+] transient in 5-HTR3, 5-HTR4 knock-down cardiomyocytes might be due to the maintenance of ATP level in cells. The alternations in frequency of cytosolic [Ca2+] transient accompanies with alternations in heart beating, further relating to arrhythmia.Due to mitochondrial and nuclear genes mutations, arrhythmia is often associated with patients of mitochondrial diseases31322+ concentration and lowers the risk of Ca2+ overload by reducing Ca2+ influx into mitochondria. 5-HTR3 is the only a ligand-gated ion channels in all 5-HTR families. It is noticed that 5-HTR3 exhibited only augment effect in mitochondria Ca2+ uptake while under hypoxia, suggesting the other second messengers are required in addition to activation of 5-HTR3.5-HTR3 and 5-HTR4 are both functionally located on the mitochondrial membrane and exhibit distinct effects on the mitochondrial function. 5-HTR4 is a type of G-protein coupled receptors via Gαs subunits to generate cAMP as the second messenger2 and substrate e.g. ischemia causes the dysfunction and even damage of cardiomyocytes. There is therefore a constant status for cardiomyocytes to keep homeostasis of mitochondrial [Ca2+], ROS, and ATP generation efficiency. Served as “guards”, 5-HTR3 and 5-HTR4 together protected cardiomyocytes by increasing the mitochondrial Ca2+ uptake when facing anoxia, which kept [Ca2+] homeostasis in the cytoplasm. At the same time, they cooperated to reduce the mPTP opening caused by increased [Ca2+] in mitochondria. As a result, the damage of ischemia/reperfusion injury on cardiomyocytes was minimized. 5-HT via its receptors 5HTR3 and 5-HTR4 on the mitochondria membrane contributes to this homeostasis. Our data contributes to a better understanding in how 5-HT, as a neurotransmitter and a potent activator in the blood stream, participates in the regulation of cardiac behaviors and how cardiomyocytes preform in response to O2 tension by using 5-HT in mediating its mitochondrial activities to pass through the stress.The inhibition of mPTP opening induced by 5-HTR3 and 5-HTR4 could be physiologically important to protect mitochondria from loss of mitochondrial membrane integrity, and ability of generation of ATP. In addition, it could also reduce the pro-apoptotic proteins released from mitochondria and protect the heart from apoptosis. Heart is a high energy demanding organ. Shortage in OAll of the animal experiments were approved by the Ethics Review Board for Animal Studies of IMM, Peking University. All methods were performed in accordance with the relevant guidelines and regulations.2+ uptake36Cardiac mitochondria were isolated from BALB/c mice by using a differential centrifugation method that retains mitochondrial structure and functions such as respiration and Caet al. with modificationsMitoplast preparation was previously introduced by Kirichok 2PO4, 2.5 mM MgCl2, 0.5 mM EDTA, 1% BSA, pH 7.4 adjusted with KOH). The oxygen consumption rate was measured using an O2-sensing electrode -mitocell miniature respirometer MT 200  at the room temperature. After recording state 4 oxygen consumption, 100 mM ADP was added to the chamber to induce state 3 respirations using microliter fixed-needle syringes.Mitochondrial suspensions were diluted to a total volume of 1 ml in measurement buffer . Mitochondrial membrane potential was measured using Rhodamine 123 . Isolated mitochondria were suspended in potassium chloride (KCl) media  containing 5 mM succinate. Freshly prepared mitochondria were incubated with 5 uM Fluo-8 or Rhodmine123 for 15 min at room temperature, and observed using Nikon ECLIPSE Ti-u. After recording the baseline of fluorescence intensity, the mitochondria were treated with 10 uM Ca2+ or 5 uM FCCP . Both Fluo-8 and Rhodmine were excited at 488 and emissions were collected at 505–503 nm.Mitochondrial Calcium concentration  transients were measured in Tyrode’s Buffer at ~35 °C, using a laser Laser Spinning Disk Confocal Microscope for Live Cell .Cells were loaded with 2 uM Quest Fluo-8TM, AM . The spontaneous sytocolic [CaIsolated heart mitochondria were lysed in 0.1 N HCl solution and cAMP was measured using a competitive binding ELISA  according to the manufacturer’s protocol. For screening of pharmacologic response of 5-HTR4, mitochondria were pre-treated with 10 uM Serotonin hydrochloride , 10 uM BIMU , and 100 uM zacopride respectively .et al. with modificationsChannel function was measured at room temperature by patch-clamp electrophysiology of mitoplast in the single-channel as previously introduced by Kirichok How to cite this article: Wang, Q. et al. 5-HTR3 and 5-HTR4 located on the mitochondrial membrane and functionally regulated mitochondrial functions. Sci. Rep. 6, 37336; doi: 10.1038/srep37336 (2016).Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Experimental measurements of Ca2+ and inositol trisphosphate (IP3) concentrations in HSY cells, a human salivary duct cell line, show that when the cells are stimulated with adenosine triphosphate (ATP) or carbachol (CCh), they exhibit coupled oscillations with Ca2+ spike peaks preceding IP3 spike peaks. Based on these data, we construct a mathematical model of coupled Ca2+ and IP3 oscillations in HSY cells and perform model simulations of three different experimental settings to forecast Ca2+ responses. The model predicts that when Ca2+ influx from the extracellular space is removed, oscillations gradually slow down until they stop. The model simulation of applying a pulse of IP3 predicts that photolysis of caged IP3 causes a transient increase in the frequency of the Ca2+ oscillations. Lastly, when Ca2+-dependent activation of PLC is inhibited, we see an increase in the oscillation frequency and a decrease in the amplitude. These model predictions are confirmed by experimental data. We conclude that, although concentrations of Ca2+ and IP3 oscillate, Ca2+ oscillations in HSY cells are the result of modulation of the IP3 receptor by intracellular Ca2+, and that the period is modulated by the accompanying IP3 oscillations.Saliva is an essential part of activities such as speaking, masticating and swallowing. Enzymes in salivary fluid protect teeth and gums from infectious diseases, and also initiate the digestion process. Intracellular calcium (Ca 2+ and IP3 oscillations in HSY cells, a salivary ductal cell line from human parotid. The model reproduces the experimental data that exhibit coupled oscillations of [Ca2+] and [IP3] with the peak of each Ca2+ spike being followed by the peak of an IP3 spike. Recently, it was conjectured that IP3 oscillations in HSY cells are not necessary for Ca2+ oscillations. We corroborate this statement with our model and show that Ca2+ oscillations can occur without oscillating [IP3]. Further to this, based on our model simulation, we hypothesise that IP3 oscillations in HSY cells may affect the frequency of Ca2+ oscillations. Indeed, experimental data verify that oscillating [IP3] lengthens the period of Ca2+ oscillations.We construct a mathematical model of Ca Saliva secretion and regulation are vital for a range of activities, but can be compromised in a number of ways. Radiation therapy for head and/or neck cancer often causes salivary gland hypo-function, which can lead to xerostomia, commonly known as dry mouth , 2. PatiThere are three main salivary glands: parotid, sublingual and submandibular. The parotid glands are the largest pair, and each gland is structured like a bunch of grapes, with a network of ducts and a cluster of acinar cells on the ends. Generally, studies of saliva formation have focused on the understanding of acinar cells, as ductal cells are not the primary source of saliva secretion. However, Baum et al.  presente2+]) are important in both intracellular and intercellular signalling  and the functions of cells involved in the secretion and regulation of saliva.It is well established that changes in intracellular calcium concentration  and carbachol (CCh), they exhibit oscillations in [Ca2+] , 23. How2+ oscillations in HSY cells: release and re-uptake of Ca2+ from the endoplasmic reticulum (ER), and fluxes across the plasma membrane. The release of Ca2+ from the ER is initiated by inositol -trisphosphate (IP3) binding to IP3 receptors (IPR) on the ER membrane, which opens the receptor  allowing the flow of Ca2+ out of the ER. When [Ca2+] is high, Ca2+ is pumped back into the ER or removed from the cell across the plasma membrane.There are two major pathways that govern Ca2+ oscillations in duct cells. Although parotid duct cells differ from HSY cells in many respects, much more is known about the Ca2+ dynamics of HSY cells, which makes them a better candidate for modeling work. In this paper, we present a mathematical model that reproduces Ca2+ oscillations in HSY cells. In order to verify the model, we compare model simulations with experimental data, and make and test predictions. Our aim is to understand the mechanisms underlying Ca2+ oscillations in HSY cells, and how IP3 dynamics affects the oscillations.We aim to use the studies in HSY cells to try to understand better the possible mechanisms that control Ca2, 0.41 mM MgSO4, 0.49 mM MgCl2, 0.34 mM Na2HPO4, 0.44 mM KH2PO4, 5.5 mM glucose, 20 mM Hepes-NaOH (pH 7.4). Intracellular-like medium (ICM) contained 125 mM KCl, 19 mM NaCl, 10 mM Hepes-KOH (pH 7.3), 1mM EGTA, and appropriate concentrations of CaCl2 (330 μM CaCl2 for 50 nM free Ca2+).Hanks’ balanced salt solution with Hepes (HBSS-H) contained 137 mM NaCl, 5.4 mM KCl, 1.3 mM CaClμg/ml streptomycin, as previously described. These cells were grown in fibronectin-coated experimental chambers consisting of plastic cylinders (7 mm in diameter) glued to round glass coverslips. For monitoring changes in intracellular [Ca2+], cells were incubated with 2 μM Fluo-3 acetoxymethyl ester  in HBSS-H HBSS-H containing 1% bovine serum albumin (BSA) for 30 min at room temperature.HSY-EA1 cells were cultured in Dulbecco’s Eagle’s medium nutrient mixture F-12 Ham (Sigma) supplemented with 10% newborn calf serum, 2 mM glutamine, and 100 U/ml penicillin and 100 Experiments were carried out on a TE2000 inverted fluorescence microscope  with dual-source illumination system, in which two different light sources, a 150 W xenon arc light source U7773  and a 100 W mercury light source (Nikon) were equipped. An experimental chamber was placed on the microscope, and fluorescence images were captured using a imaging system consisting of a C9100-13 EM-CCD camera (Hamamatsu photonics) and were analysed with AQUACOSMOS 2.6 software (Hamamatsu photonics).3-induced Ca2+ oscillations were examined with cell-permeable caged IP3, iso-InsP3/PM(caged), . In this experiments, cells were incubated with cell-permeable caged IP3 (2–10 μM) and Fluo-3 (2 μM) in HBSS-H containing 1% BSA for 30 min at room temperature. During monitoring Ca2+ responses with 490 nm light from the Nikon mercury light source, cells were also illuminated with 400 nm light that was derived from U7773 for the photolysis of cell-permeable caged IP3. These illumination lights were reflected by a 500 nm long pass dichroic beamsplitter, and exposed the chamber through a Nikon Plan Fluor 40x  or 60x objective oil immersion lens . In this experiment, 400 nm light was continuously exposed to a small area including several cells, and the strength of the light was controlled with the grating monochromator in U7773 and neutral density filters, so that the Ca2+ oscillations was induced in a constant frequency. The emitted light of Fluo-3 can passed the dichroic beamsplitter and 535 nm emotion filter, and detected by the EM-CCD camera. ATP, CCh, and U73122 were directly added to the chamber during the measurements.IP2+ dynamics of various cell types. The details of these models are different, as each aims to describe the precise dynamics in a certain cell, but they share a primary goal of explaining how Ca2+ dynamics is regulated. For recent reviews of computational models of Ca2+ oscillations, see Dupont et al. ) driven by cross-coupling between Ca2+ and IP3. In the former hypothesis, Ca2+ activation and inhibition of the IPR regulate the periodic opening of the receptors, giving rise to Ca2+ oscillations in the absence of oscillating [IP3]  and [IP3] , which in turn stimulates the IPR to release Ca2+ from the ER. However, Ca2+ can also be involved in the degradation of IP3, by activating IP3 3-kinase (IP3K), the enzyme that degrades IP3 to IP4. This forms a negative feedback on the Ca2+ release through the IPR. Other negative feedback processes may involve Ca2+ inhibition of the IPR or suppressed production of IP3 by protein kinase C (PKC)  , 27. Cyt oocytes , hepatoc oocytes , and air oocytes . The modnd [IP3] , 32. As  C (PKC) . The mod3 biosensors has enabled the monitoring of changes in [IP3] during Ca2+ oscillations, which led to the discovery of concurrently oscillating [Ca2+] and [IP3] in some cells  at the mouth of an open channel (denoted by Cp); thus there is no direct interplay between the open probability of the receptors and the cytosolic Ca2+. The idea of the microdomain around the mouth of an IP3 receptor, or around a cluster of IPR, was first introduced by Swillens et al. [2+-binding sites. Assuming that the receptor activity is modulated by the [Ca2+] in the microdomain, they were able to reproduce experimental observations where increments of IP3 induce repeated transient release of Ca2+. Dupont and Swillens further showed that the model could account for Ca2+ oscillations as well . Thus, the total Ca2+ influx across the plasma membrane is:Jpm is the Ca2+ efflux through the PMCA, and we follow  at the pore. The expressions for the V’s, a’s and k’s areA crucial part of the model is our choice of the model for the IPR. We use the model of Cao et al. , which io et al. .2+ feedback on the opening and closing kinetics of IPR is the main driving force behind Ca2+ oscillations. Intracellular Ca2+ can increase or decrease the open probability of an IPR by binding to its different binding sites. In this case, time-dependent Ca2+ feedback on IPR is necessary to generate Ca2+ oscillations. Conversely, Class II models assume that Ca2+ oscillations are the result of Ca2+ feedback on the production and degradation of IP3. As a positive feedback, Ca2+ can activate phospholipase C (PLC), and increase the rate of IP3 production. Ca2+ can also have a negative feedback on IP3 by activating IP3 3-kinase (IP3K) to remove IP3. The increases and decreases in [IP3] are coupled with the open probability of the IPR, leading to oscillations in [Ca2+]. Whereas a Class II model requires oscillating [IP3] in order to give rise to Ca2+ oscillations, a Class I model does not, and hence Ca2+ oscillations can occur even at a constant [IP3].As mentioned before, Class I models assume that Ca3 to monitor changes in the [Ca2+] in the IP3-sensitive store. The addition of IP3 caused subsequent release of Ca2+ from the store, followed by re-uptake of Ca2+ via SERCA pumps. This Ca2+ release and re-uptake were repeatedly observed in the absence of Ca2+ buffer (EGTA) but disappeared in the presence of EGTA. Their result suggests that Ca2+ oscillations in HSY cells are mainly generated by feedback effects of Ca2+ on IPR dynamics. It was also hypothesised in Tanimura et al.  and [IP3] peaks, in which [Ca2+] peaks are followed by [IP3] peaks  is expressed with a Hill function  also increases IP3 production rate, leading to a positive feedback loop. We assume a constant degradation rate of IP3,rdeg is the rate of decay. Thus, the ODE for [IP3] is,The production of IPs of PLC . There is of PLC  proposedmembrane . The cel agonist . These rion rate . The Ca2function , 50. We ε = 0 or 1 determines whether the model is a closed-cell model or an open-cell model. We nondimensionalised the system (with ε = 1), not only to make the system dimensionless, but, more importantly, to identify the timescale of each variable. The nondimensionalisesd system is included in the Cb evolves on the fastest timescale, while Ct evolves on the slowest. The other variables have timescales between the fastest and the slowest.Based on the above, we write the full HSY cell calcium model with six ODEs,ε = 0, the model transforms into a closed-cell model, as there is no change in the total intracellular [Ca2+] (dCt/dt = 0). In order to confirm that Ca2+ oscillations in HSY cells indeed arise from cycles of Ca2+ fluxes in and out of the ER, we need to ensure that the closed-cell model, ε = 0, can still produce Ca2+ oscillations. Then the model should be able to reproduce the Ca2+-free experiments in . They also showed that addition of the SERCA pump inhibitor thapsigargin abolishes CCh-induced Ca2+ oscillations. These experiments suggest that HSY cells do not require cycles of Ca2+ fluxes across the plasma membrane in order to generate Ca2+ oscillations. Thus, Ca2+ oscillations in HSY cells are primarily dependent on the periodic release and re-uptake of Ca2+ in the ER. Secondly, the experiments also suggest that the change in the total intracellular [Ca2+] is relatively slow, such that it does not much affect the whole cell behaviour. If the fluxes across the plasma membrane were fast, removing external Ca2+ would have resulted in termination of Ca2+ oscillations, with rapid loss of intracellular Ca2+.When ments in  and 56]ε = 0, tho et al.  examined2+ oscillations. We model ATP application by increasing ν, the concentration of applied agonist. With the parameter values in Tables 2+] and [IP3] at ν = 15 μM . Physiologically, a closed cell does not have influx or efflux of Ca2+ across the plasma membrane, and thus maintains a constant intracellular [Ca2+]. For the purpose of this model simulation, Ct is no longer treated as a variable, but rather as a constant parameter. Thus, the closed-cell model forms a five-dimensional system, Ct. Preliminary analysis of the closed-cell model revealed that in order to generate Ca2+ oscillations in the closed-cell model, ν and Ct need to balance each other so that the system is within its oscillatory regime. In other words, even if the system is stimulated with large ν, the system cannot produce oscillations if Ct is too low. In order to generate Ca2+ oscillations from the closed-cell model stimulated with ν = 15 μM, we need to set the parameter Ct to a value between 65 μM and 78 μM.As discussed in the previous section, experimental results suggest that plasma membrane fluxes are not necessary to produce CaSY cells , 56. We Ct = 68 μM. At t = 0 s, the system is at its steady state without any stimulation; agonist is then applied with ν = 15 μM for t > 0. This shows that the closed-cell model can exhibit Ca2+ oscillations, which agrees with the experimental data that suggest Ca2+ oscillations in HSY cells do not require fluxes across the plasma membrane.3 oscillations in HSY cells do not seem to be essential in generating Ca2+ oscillations. We hypothesise that the main mechanism for generating Ca2+ oscillations in HSY cells is Ca2+ feedback on IPR. We aim to validate this hypothesis through proposing model predictions, and comparing them to experimental data.Thus, our model suggests that IP2+-free medium experiment in Tojyo et al.  in a HSY cell that was placed in Ca2+-free solution is shown in 2+ in the extracellular domain, as the cells were surrounded by Ca2+-containing medium. CCh was used to stimulate the cells and induce Ca2+ oscillations. After some time, the cells were perfused with Ca2+-free solution, to wash away all the extracellular Ca2+. As we predicted from the model, Ca2+ spikes persisted for a while, before they eventually stopped. 26 out of 40 oscillating cells showed similar responses. In particular, the model predicts that it takes about 20 minutes for the oscillations to disappear, which is confirmed by the representative response in 2+ across the plasma membrane, until eventually intracellular [Ca2+] was too low to allow another Ca2+ spike. Also, in the absence of the external Ca2+, each interspike interval was longer than the previous one, as predicted by the model.A representative experimental trace of [Ca3] in the cytosol affects the open probability of the IPR, and hence governs the majority of Ca2+ release from the ER. In order to investigate the response of intracellular [Ca2+] to an increase of [IP3], experimentalists often use caged IP3, which is biologically inactive, and then release IP3 by flashing UV light. This photoreleased IP3, which has the same function as IP3, binds to the IPR and opens them. However, photoreleased IP3 metabolises more slowly than IP3, and therefore stays longer in a cell  slowly accumulates to its steady state concentration, μM. Once there is enough photoreleased IP3 and Ca2+, the system starts generating Ca2+ spikes (at t ≈ 350 s). At t = 600 s, ψ2 is decreased to 0.4 μM s–1 which halves the coupling strength between Ca2+ and IP3, and VS is decreased to 9.5 μM s–1. The model predicts that the effects of PLC inhibition are a decrease in the Ca2+ spike amplitude and an increase in the oscillation frequency. The frequency increase in the model simulation shown in t = 900 s, ψ2 and VS are further decreased to 0 μM s–1 and 9 μM s–1, respectively, to amplify the effects of applying a PLC inhibitor, and eliminate Ca2+ feedback on PLC. As a results, the oscillations are almost destroyed. ψ2 and VS. It is clear that diminishing Ca2+ feedback on PLC alone induces a substantial decrease in the oscillation period. When only VS is decreased, the oscillations get slower, but the impact on the oscillation period is greater when both ψ2 and VS are decreased.2+ response in an HSY cell to the PLC inhibitor U73122. 13 out of 21 oscillating cells showed qualitatively similar responses. The cells were treated with caged IP3, and were then exposed to low UV light continuously. As a result, photoreleased IP3 generated Ca2+ oscillations. The cells were then subjected to 2 μM of U73122 to inhibit PLC. Subsequently, the dose of U73122 was increased to 5 μM to enhance the effect of U73122. The result shows that impeding PLC decreased the amplitude of the Ca2+ spikes, while increasing their frequency. When a higher concentration of U73122 was applied, the oscillations were abolished. These results verify the model prediction. μM of U73122, measured from 13 cells. On average, the cells exhibited a 50% increase in the oscillation frequency.The experimental test of this model prediction is shown in 3, while PLC activity is being inhibited. ε = 1, ψ2 = 0 μM s–1, and VS = 9 μM s–1 to model the addition of U73122. At t = 200 s, ks_plc is increased to 0.00056 μM s–1 to describe a constant increase in Ps, which mimics the application of continuous UV light after the inhibition of PLC. As a result, the system starts generating Ca2+ spikes at around t = 500 s. The model predicts that photolysis of caged IP3 can elicit Ca2+ oscillations in HSY cells, even under inhibited PLC activity.With this model, we can also investigate the effects of the photolysis of caged IP3. Then they were applied with U73122, in order to inhibit any PLC activities. Subsequently, they were continuously exposed to UV light for the photolysis of caged IP3 at a constant rate. As a result, 13 cells exhibited a series of Ca2+ spikes, which indicates that Ca2+ oscillations can be generated from photoreleased IP3, without any contribution from PLC activities. The data show that the oscillations emerged as soon as the photolysis took place, whereas in the model simulation, it takes about 5 mins before the first oscillation is generated. This discrepancy is addressed in more detail in the Discussion.This model prediction was tested through experiments, with the result shown in 2+ feedback on PLC. From t = 0 s, the system is stimulated with ks_plc = 0.0006 μM s–1, which induces Ca2+ oscillations at around t = 300 s. At t = 600s, ψ2 is decreased from 0.8 μM s–1 to 0 μM s–1, to simulate the complete inhibition of positive Ca2+ feedback on PLC. In this simulation ψ2 is decreased to 0 μM s–1 while all the other parameters are kept the same. Setting ψ2 = 0 μM s–1 essentially removes the coupling between Ca2+ and IP3. Without any PLC activation from Ca2+ or external agonist, [IP3] decreases to 0 μM. This allows the total cytosolic [IP3], which is the sum of [IP3] and photoreleased [IP3], to relax to its steady state, μM; see 2+ and IP3 is completely eliminated, the oscillation frequency increases by about 24%; see As U73122 inhibits both PLC and SERCA, it is difficult to discern the effects of inhibiting PLC only, independent of SERCA activities. However, it can be studied through model simulations. 2+ oscillations in HSY cells, a human salivary duct cell line. The basic structure of the model follows the schematic diagram shown in 3 is dependent on ν, the concentration of applied agonist, and cytosolic [Ca2+]. As shown in 2+ feedback on the production of IP3 as a Hill function, where the parameter Kplc is the PLC activation constant. The parameters in Tables 2+] and [IP3] in HSY cells, with IP3 spike peaks preceded by Ca2+ spike peaks  elimination of extracellular Ca2+, 2) photolysis of caged IP3, and 3) inhibition of PLC. Experiments were conducted to test the model predictions. Firstly, the model predicted that when external Ca2+ is removed, intracellular [Ca2+] keeps oscillating but with slowly decreasing frequency. The second model simulation predicted that a pulse of photoreleased [IP3] accelerates Ca2+ oscillations. In the third simulation, we wanted to investigate the role of the coupling between Ca2+ and IP3, particularly whether it is essential for Ca2+ oscillations in HSY cells. We generated Ca2+ oscillations using photoreleased IP3, which induced IP3 oscillations through positive Ca2+ feedback on IP3. We then removed Ca2+ activation on the production of IP3, while photoreleased IP3 stayed unaffected. The model simulation showed that the Ca2+ oscillations persist at a constant concentration of photoreleased IP3, in the absence of oscillating [IP3]. The result corroborates a hypothesis of Tanimura et al.  and [IP3]. In other words, Ca2+ spikes in the model represent global and simultaneous increase and decrease in the cytosolic [Ca2+]. Practically, this is an incorrect assumption, as it does not take into account that the cytosol is spatially inhomogeneous. When the ER starts to release Ca2+, the [Ca2+] near a dense group of IPR clusters is unlikely to be the same as in some other parts of the cytosol, where the clusters are sparse, particularly since Ca2+ diffusion is relatively slow. Mathematically, we can incorporate spatial dimensions in our ODE model by extending it to a partial differential equation (PDE) model, and reproduce Ca2+ waves across the domain. With a PDE model, we can specify the spatial distribution of the receptor clusters, and hence we can include coupled reactions between the neighbouring clusters. For instance, a small Ca2+ release from a cluster can trigger a series of releases from the adjacent clusters, and consequently, lead to global Ca2+ waves. In order to build a Ca2+ model with PDEs, we need high resolution images of intracellular Ca2+ dynamics, so that we can observe Ca2+ waves and accurately analyse the wave speed, direction, and amplitude. At this stage, no such data are available from HSY cells, making it difficult to construct a quantitative version of such a model. Thus, although it is possible that spatial aspects might explain some of the quantitative differences between the present model and the data, we cannot be entirely confident in the ability of a spatial model to do so.The model consists of a set of ODEs, and hence assumes homogeneous cytosolic  from the peak, as mitochondria release Ca2+ back into the cytosol during the decay phase of the spike. However, there is not enough information about the regulation of mitochondria in HSY cells for us to implement it in our model.Numerous studies have reported the possibility of mitochondria serving as a Ca+ buffer –67. Give2+ oscillations in airway smooth muscle cells, which exhibit fast oscillations, with periods on the order of a few seconds. Although many of the parameters of the IPR model were determined by fitting to single-channel data, not all of the transition rates could be determined from the data. In particular, λh42, the rate at which h42 responds to changes in [Ca2+], was not determined by the single-channel data, and thus L and H were chosen by Cao et al. so as to give a model that could reproduce the fast Ca2+ oscillations in airway smooth muscle cells. However, there is no reason to believe that these parameters  should be the same in HSY cells. Thus, based on the slow timescale of Ca2+ oscillations in HSY cells, we assume that L and H are smaller in HSY cells than in airway smooth muscle cells. Extensive efforts to reproduce the long periods seen in HSY cells by varying other model parameters were unsuccessful; although the period can be changed a small amount by changes in other parameters, changes in L and H are by far the most effective at doing so.The original IPR model of Cao et al.  was used2+ oscillations can be most effectively manipulated by changing how fast the rate of IPR activation by Ca2+ responds to changes in [Ca2+]. However, we have not tested this prediction directly, and so this proposed mechanism for generating slow oscillations in HSY cells remain hypothetical.In effect, we are predicting that the period of Ca2+-activated PLC on Ca2+ oscillations in HSY cells, a PLC inhibitor, U73122, was applied to the cells. For the purpose of this experiment, the cells were pre-treated with caged IP3, so that their Ca2+ oscillations could be initiated by photoreleased IP3, in a PLC-independent manner. According to our model simulations, it takes a certain amount of time (about 5 mins) to trigger Ca2+ oscillations from the slowly increasing photoreleased [IP3]; see Figs 3 almost immediately induces Ca2+ spikes, as shown in Figs 3. Our model assumes that the continuous photolysis of caged IP3 would initially give a constant increase in photoreleased [IP3], and hence would be equivalent to having constant rates of photoreleased IP3 production and degradation. However, the actual biological process may be quite different from our model assumption. For instance, at the beginning of the photolysis, there could be a sharp increase in photoreleased [IP3], followed by a decrease to a saturated level. This type of reaction would explain the immediate Ca2+ responses to the continuous photolysis of caged IP3.In order to study the effects of Ca3 with the correct timescale, our model accurately predicts that the photolysis can trigger Ca2+ oscillations, even when PLC is inhibited. Furthermore, it is not our main purpose to model the accurate biological process of the continuous photolysis. Thus, we are not concerned about the time that it takes for the model to generate Ca2+ oscillations with constant rates of photoreleased IP3 production and degradation.Although there is still room for improvement in modeling the continuous photolysis of caged IP2+] and [IP3] in HSY cells, which suggests the inclusion of positive and/or negative feedback in the model. Specifically, peaks of IP3 spikes being preceded by those of Ca2+ spikes strongly suggests the presence of positive Ca2+ feedback on the formation of IP3. Dupont et al. , and there would be a latency before the next Ca2+ spike as [IP3] would need to build up to a certain level to activate the IPR again. We studied a case where both positive and negative feedback coexist in the model. For this case, both production and degradation rates of IP3 were modeled as functions of [Ca2+]. The main conflict between the model with positive and negative feedback and the data from [2+] and [IP3] peaks. In fact, the model generated coupled oscillations with a IP3 spike peak occurring just before a Ca2+ spike peak, which is not the case for HSY cells. For this reason, we decided not to include negative feedback in the model. However, we note that the phase shift in peak order could be relevant to other cellular systems in which Ca2+ feedback on the degradation of IP3 leads to a system with negative feedback, such as the models of Meyer and Stryer , followed by a slow increase until the next Ca2+ spike. Politi et al. also found that both positive and negative Ca2+ feedback on IP3 regulation extend the range of oscillation frequencies.Positive and negative Cai et al.  and Gaspi et al. , both th3 buffer in models with positive or negative Ca2+ feedback on IP3. In the absence of buffer, the models exhibited agonist-induced Ca2+ oscillations. When an IP3 buffer was added to the negative feedback model, it responded with decreased oscillation frequency and increased latency before the first spike. The oscillations persisted even at high concentrations of IP3 buffer. In contrast, when IP3 buffer was added to the positive feedback model, the oscillations were abolished. In this model, the Ca2+ flux across the plasma membrane was assumed to be relatively small compared to the flux across the ER membrane, and hence was neglected from the model. However, when the model had substantial Ca2+ fluxes across the plasma membrane, the inclusion of IP3 buffer slowed Ca2+ oscillations. They concluded that positive feedback of Ca2+ on the production of IP3 is essential for the generation of long-period, baseline-separated Ca2+ oscillations and waves. We have not tested the effects of introducing an IP3 buffer in our model. However, the results of our current work is similar to the findings of Gaspers et al., whereby positive Ca2+ feedback on IP3 regulation in HSY cells is shown to induce Ca2+ oscillations with lower frequency.Gaspers et al. compared the effects of introducing an IP2+ oscillations in hepatocytes, primarily induced by photolysis of caged IP3. One of their experiments involved pre-treating hepatocytes with U73122, followed by applying UV light. They observed that uncaging of IP3 can elicit oscillatory Ca2+ behaviours even after PLC is inhibited. Based on their experimental results, they concluded that Ca2+ oscillations induced by uncaging of IP3 in hepatocytes  do not require PLC activities, and are generated from CICR. Also, their data indicated that positive Ca2+ feedback on PLC is not a key player in Ca2+ oscillations elicited by photolysis of caged IP3. Our experiments and model simulations of intracellular Ca2+ dynamics in HSY cells lead to observations that are parallel to those of Bartlett et al., where both HSY cells and hepatocytes show oscillatory Ca2+ responses to photolysis of caged IP3, that seem to be independent of PLC activation. We then further investigated the contribution of Ca2+ activation on PLC to caged IP3-induced oscillations. The model simulations revealed that although positive Ca2+ feedback on PLC may not be necessary to trigger the oscillations in HSY cells, it has some effects on the oscillation frequency. It would be interesting to test whether Ca2+ activation on PLC in hepatocytes has the same effects on the oscillations generated from photolysis of caged IP3, without any external stimulation.Bartlett et al.  investig2+ oscillations without Ca2+ feedback on PLC. Mathematically, this can be achieved by decreasing the parameter ψ2 from 0.8 μM s–1 to a smaller value, so that the role of Ca2+ on PLC is minimised. However, once cells are stimulated with external agonist, we cannot selectively inhibit Ca2+ feedback on PLC, as U73122 inhibits overall PLC, including agonist-activated PLC. It was necessary for the model to have Ca2+ oscillations in the absence of external agonist, as a simple decrease of ψ2 alone would prevent the formation of IP3, thus preventing any oscillatory activity. We thus simulated the situation where ψ2 is decreased at the same time as IP3 is photoreleased by continuous application of low-level UV light. This lets us predict the Ca2+ responses under the conditions where PLC is not being stimulated by Ca2+, but with photoreleased IP3 in the background as a primary stimulus of the oscillations.We wanted to study Ca3 and exposed to continuous UV light, the concentration of photoreleased IP3 is expected to reach a steady state, due to the constant rates of production and degradation. During Ca2+ oscillations, additional IP3 is introduced on top of the baseline level of photoreleased IP3. Thus, the average concentration of IP3 during Ca2+ oscillations is lower when Ca2+-activated PLC is eliminated. In many cell types, it is generally the case that, within an appropriate range, higher concentrations of IP3 lead to faster oscillations. However, the model simulation of inhibiting PLC while simultaneously photo-releasing IP3, showed an increase in oscillation frequency. Although this result seems somewhat counterintuitive from a biophysical point of view, it is in fact consistent with the theory of oscillations that are generated by positive feedback. Tsai et al. [When HSY cells are pre-treated with caged IPi et al.  studied 2+ oscillations in the model with different ψ2 values , having larger ψ2 generates slower oscillations. For this computation, the value of parameter VS was kept the same (VS = 10 μM s–1), to confirm that the elimination of the cross-coupling between Ca2+ and IP3 is responsible for the increase in oscillation frequency, without any input from modified SERCA parameters. The maximum oscillation period with ψ2 = 0.8 μM s–1 was about 2 minutes, whereas the maximum period with ψ2 = 0 μM s–1 was 1.5 minutes. This result agrees with what was found in Politi et al. [2+ feedback on PLC caused oscillations to have shorter periods. We treat Ca2+- and agonist-stimulated PLC as separate activities, which is different from the work of Politi et al., where Ca2+ activation is applied to agonist-induced PLC. However, our results resemble the findings of Politi et al., whereby the cross-coupling between Ca2+ and IP3 is shown to increase the range of oscillation frequencies. This indicates that Ca2+ activation on PLC in some cells, whether it is agonist-dependent or independent, may modulate oscillation frequency. Interestingly, the experimental data showed a similar result Click here for additional data file.S1 DatasetThe experimental data are included as an Excel file. The name of each sheet within the file corresponds to the experimental setting. Column A’s show the number of frame and Column B’s show the corresponding time in minutes. Column D’s show the condition that was applied to the cells.(ZIP)Click here for additional data file.


Angiotensin-(1-9) [Ang-(1-9)] is a novel peptide of the counter-regulatory axis of the renin-angiotensin-aldosterone system previously demonstrated to have therapeutic potential in hypertensive cardiomyopathy when administered via osmotic mini-pump. Here, we investigate whether gene transfer of Ang-(1-9) is cardioprotective in a murine model of myocardial infarction (MI).The authors evaluated effects of Ang-(1-9) gene therapy on myocardial structural and functional remodeling post-infarction.C57BL/6 mice underwent permanent left anterior descending coronary artery ligation and cardiac function was assessed using echocardiography for 8 weeks followed by a terminal measurement of left ventricular pressure volume loops. Ang-(1-9) was delivered by adeno-associated viral vector via single tail vein injection immediately following induction of MI. Direct effects of Ang-(1-9) on cardiomyocyte excitation/contraction coupling and cardiac contraction were evaluated in isolated mouse and human cardiomyocytes and in an ex vivo Langendorff-perfused whole-heart model.Gene delivery of Ang-(1-9) reduced sudden cardiac death post-MI. Pressure volume measurements revealed complete restoration of end-systolic pressure, ejection fraction, end-systolic volume, and the end-diastolic pressure volume relationship by Ang-(1-9) treatment. Stroke volume and cardiac output were significantly increased versus sham. Histological analysis revealed only mild effects on cardiac hypertrophy and fibrosis, but a significant increase in scar thickness. Direct assessment of Ang-(1-9) on isolated cardiomyocytes demonstrated a positive inotropic effect via increasing calcium transient amplitude and contractility. Ang-(1-9) increased contraction in the Langendorff model through a protein kinase A–dependent mechanism.Our novel findings showed that Ang-(1-9) gene therapy preserved left ventricular systolic function post-MI, restoring cardiac function. Furthermore, Ang-(1-9) directly affected cardiomyocyte calcium handling through a protein kinase A–dependent mechanism. These data emphasized Ang-(1-9) gene therapy as a potential new strategy in the context of MI.  The renin-angiotensin-aldosterone system (RAAS) maintains cardiovascular homeostasis through angiotensin II (Ang II). Clinically, angiotensin-converting enzyme (ACE) inhibitors or angiotensin receptor blockers are mainstay treatments for hypertension and heart failure (HF). Following myocardial infarction (MI), RAAS inhibition stabilizes adverse cardiac remodeling and function and limits progression to HF.A natural counter-regulatory axis of the RAAS exists, centered on ACE2, an ACE homologue that metabolizes Ang II to angiotensin-1-7) [Ang-(1-7)] -7  reduces adverse cardiovascular remodeling in rat models of hypertension and MI following peptide administration via osmotic mini-pump  i2+]o HEPES superfusate containing 1 μmol/l Ang-(1-9). Calcium transients and contractility in human-induced pluripotent stem cell-derived cardiomyocytes  were measured in the optical platform CellOPTIQ  in cells loaded with 3 μmol/l Fura-4F-AM. Calcium transients were obtained from the 360/380 ratio and contraction was assessed using a high-resolution camera coupled to CellOPTIQ. Male adult Wistar rats were sacrificed, hearts excised, and Langendorff perfused at 37°C and constant flow (10 ml/min) Detailed methods are presented in the t test for direct comparisons and 1-way analysis of variance with Tukey’s post-test for multiple comparison were performed. Echocardiography was analyzed using repeated measures analysis of variance with Tukey’s post-test. Statistical significance was demonstrated with a p < 0.05.Data are represented as mean ± SE of the mean (SEM). Paired Student 11 viral genomes (vg) AAV9 demonstrated robust cardiac transduction Previously, tail vein delivery of 1 × 10Serial echocardiography was performed  and a simax) remained significantly reduced to 78.5% of sham  following AAVAng-(1-9) delivery (Eight-week PV loop measurements in MI/AAVAng-(1-9) revealed significant attenuation of the decreased systolic indexes observed in MI and MI/AAVGFP . AAVAng- of sham   ratios were significantly increased in all MI groups to 121%, 118%, and 125% of sham for MI, MI/AAVGFP (p < 0.05), and MI/AAVAng-(1-9) (p < 0.01), respectively . Cardiom2R expression in MI/AAVAng-(1-9) was observed, while the angiotensin type 1 receptors were significantly decreased in all MI groups  handling, in particular sarcoplasmic reticulum (SR)–mediated Ca2+ release, is the major determinant of cardiomyocyte contractility. Therefore, characteristics of SR-mediated Ca2+ release and uptake (Ca2+ transients) were determined in murine cardiomyocytes acutely exposed to soluble Ang-(1-9) peptide. Ang-(1-9) significantly increased Ca2+ transient amplitude : 933.7 ± 107.0 nmol/l; p <0.05)  (data not shown), suggesting no change in rate of Ca2+ removal from the cytosol through SR uptake via SERCA or the sodium calcium exchanger. To determine SR Ca2+ content, a major determinant of Ca2+ transient amplitude 2+ into the cytosol. The caffeine-induced Ca2+-transient amplitude in Ang-(1-9)–incubated cardiomyocytes was significantly increased compared to control, indicating an increased SR Ca2+ content : 1,535.2 ± 188.8 nmol/l; p < 0.05)  was unaltered by Ang-(1-9), supporting the conclusion that Ang-(1-9) does not alter cardiomyocyte Ca2+ extrusion . To assess this, cardiomyocytes were continuously perfused with Ang-(1-9) and a 10 mmol/l bolus of caffeine applied for 10 s after 15 min followed by 2 min of steady state measurements while cells were stimulated. The amplitude of the first Ca2+ transient after caffeine was taken as an index of Ca2+ influx via the L-type Ca2+ channel 2+-transient amplitude versus controls   ; an obsep <0.05) . In parap <0.05) . The rat < 0.05) . SERCA-mxtrusion . One pos < 0.05) .max, confirming a positive inotropic response to Ang-(1-9) (2+ channel following application of Ang-(1-7) To assess whether the positive inotropy observed in isolated cardiomyocytes translated to whole heart contractile function, hearts isolated according to the Langendorff model were perfused with Ang-(1-9). After 4 min of perfusion, Ang-(1-9) induced a significant increase in developed pressure with a concomitant elevation in dP/dtng-(1-9) . Since p2+ and contraction were measured before (baseline) and after 15 min incubation with different Ang-(1-9) concentrations. A dose-dependent increase in Ca2+ transient and contraction amplitudes was observed within concentrations 0.5 μmol/l to 2 μmol/l (data not shown) with no effect on parameters such as calcium transient upstroke, rate of decline, or contraction/relaxation times. Ca2+ transient amplitude and contraction following incubation with 1 μmol/l Ang-(1-9) compared to control cells was measured and a 210 ± 10% change from baseline in Ca2+ transient amplitude for cells incubated with 1 μmol/l Ang-(1-9) was observed, an effect significantly different from control cells (98 ± 13% change from baseline)  . A paralaseline) .2+ handling by increasing SR Ca2+ content and Ca2+ transient amplitude (Our study focused on an innovative gene therapy approach to deliver Ang-(1-9) directly to the heart to assess therapeutic effects and mechanisms of action in a murine MI model. AAV9-mediated delivery of Ang-(1-9) reduced acute rupture and mildly affected cardiac hypertrophy and fibrosis, but preserved LV systolic function, even at 8 weeks post-MI . The effmplitude .While rupture rates in MI and MI/AAVGFP groups were consistent with previous studies AAVAng-1-9) delivery significantly reduced fibrosis, although not specifically in the LV, suggesting this did not significantly contribute to Ang-(1-9)’s inotropic effect. The reduced fibrosis  aligned with previous studies where osmotic mini-pump delivery attenuated cardiac fibrosis  deliveryAAVAng-(1-9)–transduced hearts consistently had greater contraction and blood ejection, evidenced by dramatically increased CO and stroke volume and normalized EF, showing that regardless of MI-induced dilation, function was maintained. This contrasted with a previous study assessing osmotic mini-pump-mediated Ang-(1-9) delivery on cardiac function in rats post-MI that showed significantly reduced LV dimensions and volumes and reported reduced wall thickness, but no change in LV systolic function 2R gene expression. AT2R expression is reported to increase acutely following MI 2R is associated with cardioprotective effects Additionally, AAVAng-(1-9) delivery significantly increased myocardial AT2+ transient amplitude leading to increased contraction, and possibly explained via increased L-type Ca2+ influx paralleled by increased SR Ca2+ content. Although a direct inotropic effect of Ang-(1-9) has not been reported previously, when Ang-(1-7) is applied intracellularly to cardiomyocytes, PKA is activated, leading to increased L-type Ca2+ channel activity 2+ transient amplitude and intracellular Ang II is reported to increase Ca2+ transient amplitude via modulating L-type Ca2+ current and releasing SR Ca2+2+ handling proteins, including the L-type Ca2+ channel, are regulated by PKA-mediated pathways To gain further insight into potential mechanisms underlying the positive inotropic effects of Ang-(1-9), excitation contraction coupling was studied in isolated murine cardiomyocytes  and the whole rat heart and hiPSC-CMs. We demonstrated a direct inotropic effect of Ang-(1-9), mediated through increasing CaOur studies were performed in a murine model of permanent LAD ligation and future studies in larger animal models following ischemic reperfusion would be helpful to inform translation of the gene therapy. Furthermore, the inotropic effects studied in isolated cardiomyocytes were performed via peptide perfusion and further work to isolate cardiomyocytes from hearts infused in vivo with the gene therapy combined with use of patch clamping would enable full dissection of the inotropic effects of Ang-(1-9). Nonetheless, the current studies strongly support a beneficial effect of cardiac Ang-(1-9) gene therapy in the setting of MI.PerspectivesCOMPETENCY IN MEDICAL KNOWLEDGE: In a preclinical model of MI, gene therapy with Ang-(1-9) preserved systolic function by mediating a direct positive inotropic effect on cardiomyocytes.TRANSLATIONAL OUTLOOK: Further work is needed to assess whether Ang-(1-9) gene delivery in other large animal models of myocardial infarction preserves systolic function and prevents heart failure.This study suggested that gene therapy to augment Ang-(1-9) levels in the heart produces clear benefit in a murine MI model. Our data supported the notion that administration of the counter-regulatory RAAS peptide Ang-(1-9) via translational gene therapy is a novel and promising approach in heart disease that preserves cardiac systolic function post-MI and is maintained in a sustained manner.


A well‐known example of this is the control of neurotransmitter release by Ca2+ entry through voltage‐gated Ca2+ channels, generating short‐lived local nano‐domains of high Ca2+ concentration ([Ca2+]i)   and is supported by a Medical Research Council Programme grant (MR/J002771/1).


The aim of our study is to present the benefit of using the transvers acetabular ligament for intraoperative determination of the anteversion of acetabular component. Twenty-one total hip arthroplasties were performed. The transverse acetabular ligament was identified and used as a guide to position the acetabular component. The mean anteversion angle was 16.9. None of the patients studied sustained a postoperative dislocation during this short follow-up period. We conclude from this preliminary study that the transverse acetabular ligament can aid positioning of the acetabular component of a THR. It defines the version of the acetabular component without the need for external instrumentation, and is independent of the position of the patient. Level of Evidence IV; Prospective Study. Acetabular cup positioning has a significant effect on the out- come of total hip arthroplasty (THA). It affects dislocation rate, component impingement, edge loading, accelerated bearing surface wear and osteolysis and/or loosening.A retrospective study was conducted between January 2013 and January 2015. It included 21 total hip arthroplasties performed by two senior surgeons. Ten patients were operated for hip osteoarthritis, eight for femoral neck fracture, and three for coxitis. Each patient had a postoperative anteroposterior (AP) radiograph of the pelvis, which was used to measure the abduction angle of the acetabular cup from lines drawn through the face of the cup and the inter-teardrop line. A standardized crosstable true lateral view of the hip was taken with the patient supine on the radiographic table with the contralateral lower extremity flexed as much as possible to eliminate lumbar lordosis and place the pelvis in a standard position. The anteversion angle of the acetabular component was measured according to the technique of Woo.Minopen posterior approach was used.  The piriThe Hospital Ethics Committee approved the study, and all patients signed a Free and Informed Consent form before the surgery.The transverse acetabular ligament was identified and exposed in all cases. Radiological results are shown in -,--,-,Positioning of the acetabular component in THA has been shown to affect the range of motion of the hip, wear of the polyethylene, and the rate of pelvic osteolysis, being of paramount importance in minimizing the risk of dislocation.Archbold et al.,In order to assess the influence of acetabular component alignment relative to TAL and the posterior labrum as soft-tissue landmarks, Kalteis et al.In our study, we had no case of hip dislocation. The TAL was easily identified in all patients. However, a larger and comparative study is needed to confirm this results.We conclude from this preliminary study that the transverse acetabular ligament can assist to position the acetabular component in THR. It defines the degree of version of the acetabular component without the need for external instrumentation, and it is independent of the patient's position.


Upon excitation, it releases Ca2+ into the cytosol, inducing contraction of myofibrils. During relaxation, it takes up cytosolic Ca2+ to terminate the contraction. During exercise, Ca2+ is cycled between the cytosol and the SR through a system by which the Ca2+ pool in the SR is restored by uptake of extracellular Ca2+ via a specific channel on the plasma membrane. This channel is called the store-operated Ca2+ channel or the Ca2+ release-activated Ca2+ channel. It is activated by depletion of the Ca2+ store in the SR by coordination of two main molecules: stromal interaction molecule 1 (STIM1) and calcium release-activated calcium channel protein 1 (ORAI1). Recently, myopathies with a dominant mutation in these genes have been reported and the pathogenic mechanism of such diseases have been proposed. This review overviews the calcium signaling in skeletal muscles and role of store-operated Ca2+ entry in calcium homeostasis. Finally, we discuss the phenotypes and the pathomechanism of myopathies caused by mutations in the STIM1 and ORAI1 genes.Calcium is a crucial mediator of cell signaling in skeletal muscles for basic cellular functions and specific functions, including contraction, fiber-type differentiation and energy production. The sarcoplasmic reticulum (SR) is an organelle that provides a large supply of intracellular Ca The. Thel-ty–200 nM)  and it h2+ from the SR via RyR1 channels has versatile functions and is necessary for force generation primarily by interactions between myosin and actin filaments. Upon Ca2+ release from the SR, two Ca2+ molecules bind troponin C, pulling the troponin–tropomyosin complex aside to allow myosin heads to bind to actin filaments  have been identified in patients with Brody disease, resulting in the failure of SERCA activity to keep up with the Ca2+ release during repetitive stimulation . H. HORAI1 trations . The Pro2+ overload than the STIM1 EF-hand mutant or the wild type; therefore a broad phenotype spectrum in Stormorken syndrome is seen [What turns the phenotypes into TAM, alone, or Stormorken syndrome remains unclear. An explanation has been elucidated from the difference in the fast CDI between the STIM1 CC1 mutation of Arg304Trp and the EF-hand mutation of Asp76Ala. STIM1 Asp76Ala showed a fast CDI similar to wild type STIM1, whereas Arg304Trp mediated a reduced fast CDI. These data suggest that prolonged opening of the CRAC channel in the STIM1 Arg304Trp mutant mediates more severe cytosolic Ca is seen .The progression of TAM and the contribution of genetic factors to its variability need to be further delineated.2+ is essential for skeletal muscles. Multiple roles in the regulation of contraction, metabolism, and plasticity [2+ release from the SR and SOCE. However, the detailed explanation of the differentiation into a variety of phenotypes by the STIM1 and ORAI1 mutations is unclear. The cause of the phenotypical differences between TAM alone and Stormorken syndrome, and the distribution of involved muscles in TAM remains unknown. Recently-found molecules modulating STIM1 and ORAI1 interactions, such as Septin [2+ signaling in addition to the components of SOCE and CRAC channels and development of therapeutic applications for TAM genes are expected.Caasticity  are coors Septin , SOCE-ass Septin , CRAC res Septin , and STIs Septin , increas


In a previous study, we demonstrated that the depletion of Ca2+ stores in odontoblasts activated store-operated Ca2+ entry (SOCE), a Ca2+ influx pathway. However, the precise biophysical and pharmacological properties of SOCE in odontoblasts have remained unclear. In the present study, we examined the functional expression and pharmacological properties of Ca2+ release-activated Ca2+ (CRAC) channels that mediate SOCE and evaluated the alkali sensitivity of SOCE in rat odontoblasts. In the absence of extracellular Ca2+, treatment with thapsigargin (TG), a sarco/endoplasmic reticulum Ca2+-ATPase inhibitor, induced an increase in [Ca2+]i. After [Ca2+]i returned to near-resting levels, the subsequent application of 2.5 mM extracellular Ca2+ resulted in an increase in [Ca2+]i which is a typical of SOCE activation. Additionally, application of 2-methylthioadenosine diphosphate trisodium salt (2-MeSADP), a P2Y1,12,13 receptor agonist, or carbachol (CCh), a muscarinic cholinergic receptor agonist, in the absence of extracellular Ca2+, induced a transient increase in [Ca2+]i. The subsequent addition of extracellular Ca2+ resulted in significantly higher [Ca2+]i in 2-MeSADP- or CCh-treated odontoblasts than in untreated cells. SOCE, that is activated by addition of extracellular Ca2+ in the TG pretreated odontoblasts was then suppressed by Synta66, BTP2, or lanthanum, which are CRAC channel inhibitors. Treatment with an alkaline solution enhanced SOCE, while treatment with HC030031, a TRPA1 channel antagonist, inhibited it. The amplitude of SOCE at pH 9 in the presence of HC030031 was higher than that at pH 7.4 in the absence of HC030031. These findings indicate that CRAC channel-mediated alkali-sensitive SOCE occurs in odontoblasts. SOCE is mediated by P2Y and muscarinic-cholinergic receptors, which are activated by endogenous ligands in odontoblasts.Odontoblasts play a crucial role in dentin formation and sensory transduction following the application of stimuli to the dentin surface. Various exogenous and endogenous stimuli elicit an increase in the intracellular free calcium concentration ([Ca The stismitters . The relsmitters . In addismitters .2+ signaling is mediated by two closely related components: external stimuli-evoked Ca2+ influx from the extracellular medium and Ca2+ release from intracellular Ca2+ stores. Ca2+ influx is caused by the activation of TRP channels following the generation of external stimuli-induced hydrodynamic forces inside the dentinal tubes  receptors or ryanodine receptors in response to G-protein coupled receptor (GPCR) activation or depolarization  subtypes 1, and 3 (2+–ATPase (PMCA)  (refilling)   in the dfilling) .2+ entry (SOCE) has been well described as a Ca2+ entry pathway in the plasma membrane that is activated by the depletion of IP3- and/or ryanodine-sensitive Ca2+ stores  channels. The best-characterized SOC channels are Ca2+ release-activated Ca2+ (CRAC) channels composed of the pore-forming subunit Orai1, Orai2, or Orai3 , a Ca2+ store calcium-sensor, and causes translocation of STIM1 to the plasma membrane. In the plasma membrane, STIM1 interacts directly with Orai1, resulting in the activation of CRAC channels  Act, 1986. This study was approved by the Ethics Committee of our institute .2+]i measurement, enzymatically treated and isolated odontoblasts from the dental pulp slice were plated onto a culture dish, immersed in alpha-minimum essential medium  including 10% fetal bovine serum and 5% horse serum, and maintained at 37°C in a 5% CO2 incubator. The primary cultured odontoblasts from the dental pulp slice were used for [Ca2+]i measurements; we stably measured [Ca2+]i increases within 24 h of isolation. Cells were confirmed to be odontoblasts in a previous study with the odontoblast markers dentin matrix protein-1, dentin sialoprotein, and nestin within 24 h of isolation  using a previously described method . Brieflysolation .2+]i was defined using the fluorescence ratio (RF340/F380) of F340 to F380 (F340/F380), and is described as F/F0 units; the RF340/F380 value (F) was normalized to the resting value (F0). The F/F0 baseline was configured at 1.0. All experiments were performed at room temperature (30 ± 1.0°C).Cells in dental pulp slices were loaded with 10 μM fura-2-acetoxymethyl ester   and 0.1%2, 0.5 MgCl2, 10 HEPES, 10 glucose, and 12 NaHCO3 (pH 7.4 Tris) was used as the standard extracellular solution. To prepare high pH (pH 9) extracellular solutions, 12 mM NaHCO3 in Krebs solution was replaced by 10 mM (for pH 9) NaOH. This replacement did not affect extracellular free Ca2+ concentrations in the test solution. Synta66 was obtained from AOBIOUS INC. . BTP2 was obtained from Santa Cruz Biotechnology . HC030031, 2-Methylthioadenosine diphosphate, and DHPG were obtained from Tocris Bioscience . All other reagents were obtained from Sigma Chemical Co. . Stock solutions of lanthanum chloride, carbachol, and DHPG were prepared in ultra-pure water . All other stock solutions were prepared in dimethyl sulfoxide. Stock solutions were diluted to the appropriate concentration with Krebs solution (pH 7.4 or 9) before use.Krebs solution containing (in mM) 136 NaCl, 5 KCl, 0 or 2.5 CaClN observations, where N shows the number of independent experiments. The Wilcoxon test or Mann–Whitney test were used to evaluate the non-parametric statistical significance. A P-value < 0.05 was considered significant. Statistical analysis was performed using GraphPad Prism 7.0 .Data are represented as the mean ± standard error (SE) of the mean of 2+, application of 10 μM thapsigargin (TG), an inhibitor of sarco/endoplasmic reticulum Ca2+-ATPase  . The transient increases in [Ca2+]i are caused by the release of Ca2+ from intracellular Ca2+ stores. After [Ca2+]i returned to the near-resting levels, subsequent application of 2.5 mM extracellular Ca2+ increased [Ca2+]i  to a peak value of 1.32 ± 0.04 F/F0 units (N = 9) .In the absence of extracellular Ca+-ATPase , induced2+ influx by store depletion. In the absence of extracellular Ca2+, application of 50 nM 2-methylthioadenosine diphosphate (2-MeSADP), a P2Y1,12,13 receptor agonist  . Carbachol (CCh) (100 μM), a muscarinic-cholinergic receptor agonist  , while application of 100 μM DHPG, an agonist of group I metabotropic glutamate receptors  . These transient [Ca2+]i increases are elicited by the Ca2+ release from intracellular Ca2+ stores. After [Ca2+]i returned to near-resting levels following each application of 50 nM 2-MeSADP, 100 μM CCh, and 100 μM DHPG, subsequent addition of 2.5 mM extracellular Ca2+ increased [Ca2+]i . The peak values following application of 2.5 mM extracellular Ca2+ with 50 nM 2-MeSADP were 1.72 ± 0.04 F/F0 units (N = 6) , while those with 100 μM CCh were 1.38 ± 0.05 F/F0 units (N = 5) . After pretreatment of 2-MeSADP, and CCh, the Ca2+ influx induced by subsequent application of 2.5 mM extracellular Ca2+ was significantly larger than that without pretreatment; the values of Ca2+ influx without any pretreatment were 1.23 ± 0.01 F/F0 units (N = 5) . However, there were no significant differences in the Ca2+ increases (that was elicited by subsequent application of 2.5 mM extracellular Ca2+) between with DHPG pretreatment  and without any pretreatment .PLC-coupled receptors, P2Y , muscari agonist , increas agonist , evoked eceptors , induced2+ influx activated by subsequent application of 2.5 mM extracellular Ca2+ after store depletion, we investigated the effects of CRAC channel inhibitors, synta66 . The increases in [Ca2+]i were significantly suppressed in the presence of 10 μM synta66 to 1.20 ± 0.03 F/F0 units (N = 6) . In addition, when the cells were subjected to preincubation with 1 μM BTP2 for 60 min at 37°C, the [Ca2+]i increases following Ca2+ store depletion by TG pretreatment were inhibited to 1.08 ± 0.01 F/F0 units (N = 10)  compared to those without BTP2 (1.32 ± 0.04 F/F0 units (N = 9).To identify the pathway of Ca synta66  and BTP2 synta66 , on the 2+, application of 2.5 mM extracellular Ca2+ increased [Ca2+]i to a peak value of 1.32 ± 0.02 F/F0 units (N = 7), and an application of 100 μM lanthanum (La3+), a non-specific CRAC channel inhibitor  . After La3+-induced suppression of SOCE, removal of La3+ resulted in a slow return of SOCE activity over several minutes .After store depletion by pretreatment with 10 μM TG in the absence of extracellular Canhibitor , caused 2+, subsequent application of alkaline solution (pH 9) with 2.5 mM extracellular Ca2+ enhanced SOCE to a peak value of 2.17 ± 0.1 F/F0 units (N = 5) , while the peak value of [Ca2+]i increase by application of standard (pH 7.4) extracellular solution with extracellular 2.5 mM Ca2+ was 1.49 ± 0.03 F/F0 units (N = 14) . Odontoblasts express alkali-sensitive TRPA1 channels  . The SOCE evoked by subsequent application of 2.5 mM extracellular Ca2+ with 100 μM HC030031 at pH 9 were larger than those without HC030031 at pH 7.4 (N = 14) .We examined the effects of extracellular alkalization on SOCE in odontoblasts. After store depletion by continuous treatment with 10 μM TG in the absence of extracellular Cachannels . To remotagonist . HC030032+ induced Ca2+ influx (SOCE). The Ca2+ influx was suppressed by CRAC channel inhibitors, synta66, BTP2 and lanthanum. The activation of P2Y and muscarinic-cholinergic receptors triggered SOCE. However, the activation of group I metabotropic glutamate receptors did not elicit SOCE. After store depletion, alkaline solution containing Ca2+ enhanced SOCE under TRPA1 channel inhibition, compared to that using the extracellular solution with Ca2+ (pH 7.4) without TRPA1 inhibition. These results indicate that, in odontoblasts, store depletion activates CRAC channel-mediated SOCE, which is promoted in an alkaline environment. The results are in line with previous results showing the expression of Orai1, subunits of CRAC channels, by immunohistochemical analysis in odontoblasts  channels have been also proposed as possible candidates for the channel proteins mediating SOCE . Among techanism , TRPC1 cchannels . It has channels . If odon2+, P2Y, muscarinic-cholinergic, and group I metabotropic glutamate receptor agonists increased [Ca2+]i in odontoblasts. The results demonstrate the expression of these receptors in odontoblasts, and are in line with previous reports , we demonstrated SOCE mediated by CRAC channels in odontoblasts. SOCE is activated by PLC-coupled receptors in odontoblasts. Endogenous ADP, released from odontoblasts in the dental pulp in response to cellular deformation or cellular damage, as well as muscarinic-cholinergic agonist from intradental parasympathetic neurons, evoked SOCE in odontoblasts. SOCE was enhanced by an alkaline environment and may play important roles in accelerating cellular functions, such as high-pH sensitive tertiary/reactionary dentin formation, following alkaline stimuli applied to dentin. In addition, alkaline stimuli activate TRPA1 channels in odontoblasts and evoke Ca2+ influx via TRPA1 channels. [Ca2+]i increases due to TRPA1 channel-mediated Ca2+ influx, which is closely involved in dentin formation under both physiological and high pH conditions (2+ release from intracellular Ca2+ stores via ryanodine and/or IP3 receptors. During dental treatments, the use of dental materials, such as calcium hydroxide or MTA, results in a high pH/Ca2+ extracellular environment. This external environment activates Ca2+ signaling mediated by SOCE, TRPA1 channels and alkali sensitive-metabotropic receptors, which can sense the increased pH in odontoblasts, resulting in the induction of dentinogenesis.In conclusion (see nditions . Alkalinnditions , and theMK, KN, MT, and YS were responsible for the conception and design of the experiments. MK, KN, AH, SO, KS, MT, and YS were responsible for the acquisition, analysis, and interpretation of data. MK, KN, and YS were responsible for drafting and critically revising the intellectual content of the article. YS was responsible for final approval of the version to be submitted/published. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.