Arabidopsis using IZEs as explants. We characterized the process at the light and scanning electron microscope level and studied several specific aspects such as WUS expression, callose deposition, and principally Ca2+ dynamics during the first stages of the process of embryogenesis induction, by confocal FRET analysis with an Arabidopsis line expressing a cameleon calcium sensor. We also performed a pharmacological study with a series of chemicals know to alter calcium homeostasis , the calcium–calmodulin interaction , and callose deposition (2-deoxy-D-glucose). We showed that, after determination of the cotiledonary protrusions as embryogenic regions, a finger-like appendix may emerge from the shoot apical region and somatic embryos are produced from the WUS-expressing cells of the appendix tip. Ca2+ levels increase and callose is deposited in the cells of the regions where somatic embryos will be formed, thereby constituting early markers of the embryogenic regions. We also found that Ca2+ homeostasis in this system is strictly maintained and cannot be altered to modulate embryo production, as shown for other systems. Together, these results contribute to a better knowledge and understanding of the process of induction of somatic embryos in this system.In this work, we studied the induction of somatic embryogenesis in  Daucus carota date from 1958 [Plant embryos are biological structures that aim to give rise to a new individual. Zygotic embryos are formed upon double fertilization and further zygote development, but this is not the only way to produce plant embryos. Embryos can also be produced artificially from immature male gametophytes, female gametes, or from vegetative (somatic) cells under certain in vitro conditions ,2. Somatrom 1958 ,4. More rom 1958 . TherefoArabidopsis thaliana, somatic embryogenesis can be in vitro induced from protoplasts [Arabidopsis uses immature zygotic embryos (IZEs) at the late cotyledonary stage as explants [WUSCHEL (WUS), a homeodomain protein characterized by its role in the shoot apical meristem maintenance, promotes the occurrence of somatic embryogenesis [WUS [BABYBOOM [LEC2 [SERK [WOX2 (a WUS-related homeobox gene), has been used to determine the spatio-temporal patterns of this process.Somatic embryos are produced either directly from the explant or indirectly through an intermediate callus phase. In toplasts , root extoplasts , shoot atoplasts , shoot atoplasts , or germtoplasts . Howeverexplants . It was explants ,14. Longexplants . All othexplants ,15. Histexplants . The estexplants ,16. Specexplants ,18. Specogenesis . The expsis [WUS , and of BABYBOOM , LEC2 [1OM [LEC2 , SERK [2C2 [SERK , and WOX2+ is one of the most important secondary messengers in plant cell signal transduction processes, controlling gene expression upon binding to calmodulin (CaM) or other Ca2+-sensing proteins [2+ in plant cells is usually kept very low, within the range of 50–100 nM, being higher in the different cell compartments considered as Ca2+ reservoirs, namely the vacuole, the ER, and the apoplast [2+ as a secondary messenger is based on the generation of concentration gradients and transient increases of cytosolic concentrations [2+ that increases acting as a rapid activator of embryo identity genes. Indeed, a number of studies in different species, such as Musa [Cocos nucifera [Hevea brasiliensis [Daucus carota [2+ supplementation of in vitro media enhances somatic embryogenesis, therefore pointing to an important role of this secondary signal as inducer of somatic embryogenesis. Other studies, however, suggest that increased Ca2+ levels are not positive to increase production of somatic embryos [2+ dynamics during the first stages of this morphogenic process would give us valuable information about the factors governing the induction of somatic embryogenesis and, in particular, to decipher the role of Ca2+ in this process.Although the last decade has witnessed significant advances in the elucidation of the genetic networks and the epigenetic mechanisms operating for the formation of somatic embryos ,17, the proteins , and furapoplast ,24. Due trations . Thus, a as Musa , Cocos nnucifera , Hevea biliensis , and Daus carota , among o embryos ,31,32,332+ as a molecular signal, some tools have been developed to monitor Ca2+ levels in a broad range of biological systems. In Arabidopsis, the study of Ca2+ dynamics can be greatly facilitated by the use of cameleon lines. Cameleon constructs are based on the principle of fluorescence resonance energy transfer (FRET) that occurs between two fluorophores when they become spatially closer. This happens when the linker protein, usually CaM in the case of cameleons, binds to Ca2+ and undergoes a conformational change that approaches the donor fluorophore to the acceptor. Hence, when the donor is excited, it transfers a certain amount of energy to the acceptor, which becomes excited itself, emitting fluorescence. Krebs et al. [Arabidopsis lines that express the cameleon construct specifically in certain cell regions, such as the cytoplasm or the plasma membrane, using cyan fluorescent protein (CFP) as a donor fluorophore and yellow fluorescent protein (YFP) as an acceptor. They are therefore excellent Ca2+ sensors. However, these lines have not yet been used to study Ca2+ dynamics during somatic embryogenesis.Due to the universal role of Cas et al.  developeArabidopsis as a model to study somatic embryogenesis [2+ in this process, it is surprising that, to the best of our knowledge, no studies of Ca2+ distribution or chemical modulation during somatic embryogenesis have been published in Arabidopsis. In this work, we studied Ca2+ dynamics during somatic embryogenesis from Arabidopsis IZEs. Using wild type, WUS-reporter and cameleon-transformed lines, we performed a multidisciplinary study including a characterization of the different stages through scanning electron microscopy (SEM), confocal microscopy for WUS-reporter expression and FRET-based Ca2+ imaging in cameleon lines, and a pharmacological study where different chemicals known to interfere with Ca2+ homeostasis and signaling were applied to embryogenic cultures. Our results shed light on the different origins of the IZE-derived somatic embryos, the embryogenic nature of the newly proliferating tissues, the distribution of Ca2+ during the initial stages of embryogenesis induction, and the role of Ca2+ and CaM modulation in this process.Despite the importance of ogenesis  and the Arabidopsis seeds transformed with the cameleon YC3.6-Bar construct to isolate and in vitro culture IZEs at the cotyledonary stage , callose was synthesized in the cell walls of cells of the cotyledon protrusions as they grow and switch towards embryogenesis . In our 2+ during the process of induction of somatic embryogenesis in our Arabidopsis cameleon lines  and compared the embryo production with that of control cultures with 1.02 mM CaCl2, the standard CaCl2 concentration of the induction medium  in the egg and central cells, associated with pollen tube burst and the discharge of sperm cells. Then, there is a second, prolonged Ca2+ increase exclusive for the egg cell, associated with successful egg fertilization [2+ signature should also be observed as a trigger for embryogenesis initiation. We were not able to identify the first, short Ca2+ transient peak at the onset of somatic embryogenesis, most likely due to its short duration, and principally because it is associated to pollen tube discharge [2+ increase in the embryogenic regions that initiated in the protoderm and extended to the inner cells of the cotyledon nodes and the shoot apical meristem appendix . For thike Musa , Cocos nnucifera , Hevea biliensis , or Daucs carota , but notum album , Pinus ps patula , Coffea anephora , and AraWUS is a transcription factor defined as a master regulator in plant growth signaling due to its key role in the regulation of both embryogenic and meristematic stem cells [Arabidopsis plants and zygotic embryos, WUS is typically expressed in the central zone of the shoot apical meristem [WUS is also expressed in the newly induced embryogenic cells even before they transform into embryos [WUS an early marker of the developmental transition from vegetative to embryogenic development. We used Arabidopsis WUS lines to check for the induction of primary somatic embryos from IZEs. Early WUS expression in the protrusion-producing protodermal and subprotodermal cells of the cotyledon node  wild type and transgenic lines for expressing the YC3.6-Bar cameleon construct [WUS expression, we used the A. thaliana transgenic line pCLV3:GFP-ER_pWUS:DsRED-N7 (NASC ID: N23895), kindly provided by Dr. Cristina Ferrándiz . This WUS-reporter line expresses the DsRED fluorescent protein under the control of the WUS promoter.We used onstruct  carryingArabidopsis plants were used as donors of explants. Siliques were harvested and surface-sterilized for 30 s in 70% ethanol and 20 min in 10% commercial bleach, followed by three rinses in sterile distilled water. Under a binocular microscope, siliques were dissected to isolate the immature seeds. IZEs with fully developed, bent, and green cotyledons were used as explants. They were rescued by carefully removing the seed coat and the endosperm, and in vitro cultured as previously described [Eight week-old silique-producing escribed . IZEs weArabidopsis IZEs cultured in vitro during 3, 5, 7, and 14 days in solid E5 medium. Samples were fixed in Karnovsky fixative as previously described [For scanning electron microscopy, we processed samples of escribed  for 5 h cameleon and WUS-reporter lines were observed in a Zeiss 780 Axio Observer  confocal laser scanning microscope. First, IZEs were cultured in solid E5 medium for five days. For callose staining, samples were then stained with 0.1% aniline blue in phosphate buffer for 1 h [cameleon and WUS-reporter lines, cultured IZEs were transferred to a microscope slide and mounted in 50 µL of 1.5% liquid low melting point agarose . Samples were immediately covered with a cover glass, solidified at room temperature, and observed. For dsRED visualization of WUS-reporter lines, samples were excited at 561 nm and the 563–618 nm emission was recorded. For FRET visualization of cameleon lines, samples were excited at 405 nm and emission was recorded between 490–570 nm. CFP was excited at 405 nm and emission was recorded between 440–488 nm. YFP was excited at 514 nm and emission was recorded between 518–570 nm. Image treatment and calculation of fluorescence emission ratios were performed as previously described [Callose staining and  for 1 h  and obseescribed . For ima2+ levels were defined as very low, low, moderate, high, or very high according to the colorimetric scale based on the FRET ratio images. Very low Ca2+ levels corresponded to dark blue colors, low levels to light blue, moderate to green–yellow, high to orange–red, and very high levels to white color. Image analysis was performed using the FIJI software v 1.53t [For all cases, Ca v 1.53t .2+ levels, we used CaCl2, the Ca2+ ionophore A23187, inositol 1,4,5-trisphosphate (InsP3), ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA), chlorpromazine, and N-(6-Aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (W-7). To inhibit callose deposition, 2-deoxy-D-glucose was used. Stocks of CaCl2, InsP3, EGTA, chlorpromazine, and 2-deoxy-D-glucose were prepared in sterile water. Stocks of ionophore A23187 and W-7 were prepared in DMSO following manufacturer instructions. Different concentrations of each compound were applied to IZE in vitro cultures, as described in Results. Control plates were prepared with the corresponding solvent concentration. Fifteen days after initiation, the percentage of embryogenic IZEs out of the total was calculated by field counting IZEs in microscopic images of culture dishes [To modulate the intracellular Cae dishes .2+ chemical modulation were analyzed performing an ANOVA and LSD test (p ≤ 0.05). For non-homocedastic samples, data were transformed with the arcsine transformation or the square root of the arcsine transformation to stabilize variance.Statistical analysis was performed using StatGraphics. The results of Ca


We assessed their performance and effects on cardiomyocyte contractility, determining fluorescent signal-to-noise ratios and sarcomere shortening in primary ventricular myocytes from adult mouse and guinea pig, and in human iPSC-derived cardiomyocytes. Chemical Ca2+ dyes displayed dose-dependent contractile impairment in all cell types, and we observed a negative correlation between contraction and fluorescence signal-to-noise ratio, particularly for fura2 and Fluo-4. R-GECO had no effect on sarcomere shortening. BAPTA-based dyes, but not R-GECO, inhibited in vitro acto-myosin ATPase activity. The presence of fura2 accentuated or diminished changes in contractility and Ca2+ handling caused by small molecule modulators of contractility and intracellular ionic homeostasis , but this was not observed when using R-GECO in adult guinea pig left ventricular cardiomyocytes. Ca2+ handling studies are necessary for cardiotoxicity assessments of small molecules intended for clinical use. Caution should be exercised when interpreting small molecule studies assessing contractile effects and Ca2+ transients derived from BAPTA-like chemical Ca2+ dyes in cellular assays, a common platform for cardiac toxicology testing and mechanistic investigation of cardiac disease physiology and treatment.We compared commonly used BAPTA-derived chemical Ca  •2+ dyes inhibit contractility in all single cardiomyocytes and reduce force production in loaded cardiac fibres.Chemical Ca•2+ dyes inhibit the acto-myosin ATPase with an IC50 ∼5μM, competing with the myosin inhibitor blebbistatin (IC50 ∼1 μM).Ca•2+ transient visibility in single cells is challenging even at [dye] ∼20 μM.Ca•2+ transients at ∼1 μM.R-GECO doesn't impair cellular contractility or acto-myosin ATPase activity, generating clear Ca•In small molecule assays compound effects of dye plus drug are apparent and mask the drug effect. After cardiomyocyte depolarization, extracellular Ca2+ enters through voltage gated L-type Ca2+ channels held near ryanodine receptors (RyR) in the intracellular membranes of the sarcoplasmic reticulum (SR). RyR channels open triggering an amplifying Ca2+-induced Ca2+ release (CICR) from the SR before contraction [2+ diffuses into the myofilament, binds to troponin C, and activates myosin-ATPase [2+ also modulates contractile function via myosin regulatory light chain (RLC) phosphorylation by Ca2+/calmodulin kinase (CaMKII) positioned on thick myosin filaments [2+ is taken back up into the SR by the sarco-endoplasmic reticulum Ca2+ ATPase (SERC2a) and removed from the cell by the plasma membrane Ca2+ ATPase (PCMA) or the sodium calcium exchanger (NCX) [ Catraction . Ca2+ diilaments . During r (NCX)  , reducin2+ levels impact cardiac function in disease states like atrial fibrillation, heart failure and cardiomyopathy [2+ buffering also affects the regulatory system [2+ binds to TnC [2+ handling and cycling in the cardiomyocyte [2+ sensitivity and/or contractility [2+ affinity hold potential as therapies for various cardiac conditions [Altered Camyopathy . Myofilay system . During s to TnC , and modomyocyte . Inheritactility . Small mnditions .2+ handling in cardiovascular research often uses fluorescent chemical Ca2+ dyes, such as fura2, Fluo-3/4, and Rhod-2, derived from 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA), a selective Ca2+ chelator discovered by Roger Tsien in 1980 [2+ cation, making them polar, hence early studies required microinjection [2+ binding motif, which is then cleaved by intracellular esterases to reveal an active Ca2+ indicator unable to diffuse out of the cell. Over 2000 primary cardiac research publications have used these dyes [Studying Ca in 1980 . These dnjection . Howeverese dyes , includi2+ this was attributed to Ca2+ buffering - if Ca2+ is bound to the dye, it is not available to participate in contraction. Dose-dependent reductions in contractile parameters remain with fluorescent BAPTA derivatives such as fura2 [2+ transient obtained by chemical dye were noted in cardiac tissue compared to the genetically encoded calcium indicator of the time aqueorin [2+ transient is different. In rat ventricular trabecular preparations, significant reductions in force production were only avoidable at very low fura2 concentrations  [2+ handling are inevitable at higher concentrations [Early use of such compounds in contractile cell models revealed concerns regarding confounding effects “attenuating” contractility with ∼mM range BAPTA concentrations . As thesas fura2 . Importaaqueorin , suggeste level) . Changestrations .2+ buffering may be over simplified. Other routes to contractile impairment are known, including the breakdown of the AM-ester and photolytic by-product formation [2+ binder EGTA-AM, which has similar Ca2+ buffering activity [2+ handling via myofilament Ca2+ buffering [2+ sensor, R-GECO, introduced by adenovirus, in various cardiomyocyte models [2+ transients revealing HCM variant myofilament buffering directly [Attributing the contractile impairment observed when using BAPTA and its fluorescent derivatives on Caormation , and othactivity . Decreasactivity . Such efuffering ,18, but e models . This hadirectly .2+ dyes [2+ indicator R-GECO [2+ indicator using fura2 and R-GECO as exemplars. This study provides a comparison of the suitability of BAPTA-based calcium dyes and R-GECO for evaluating drugs designed to alter contractility.Due to significant improvements in optical instrument sensitivity over the past 30 years, which may allow reduced dye concentrations, and the emergence of new model systems (particularly the stem-cell derived cardiomyocyte), we wished to answer the question whether historical shortcomings of chemical Ca2+ dyes  remain rr R-GECO  in mouser R-GECO , levosimr R-GECO , and fler R-GECO  give dif2An expanded methods section is available in the Supplementary Material.2.13 media for 48 h. Freshly isolated mouse or cultured guinea pig cardiomyocytes were treated with 0.2, 1 or 5 μM fura2, Fluo-4, Rhod-2, or SBFI-AM for 5 min at room temperature followed by a 30-min wash period to allow for complete AM ester de-esterification. Mouse / guinea pig cardiomyocytes perfused with Krebs solution containing 1.4 / 1.8 mM CaCl2 and paced at 1 / 0.5 Hz respectively using an IonOptix μStep system. Sarcomere shortening was acquired from fast Fourier transform averaging of sarcomeric striations, Ca2+ transients were obtained using photomultiplier fluorescence light emission using and ex365/380 em510 or ex488 em525 filters for fura2 or Fluo-4 respectively. To compare sarcomere shortening and fura2 Ca2+ transients in the presence of either mavacamten (250 nM), levosimendan (10 μM) or flecainide (0.5 μM), 200 μl of cultured (48 Hours) cardiomyocytes were incubated in either drug or DMSO vehicle for 5 min. At least 3 cell preparations were analysed for each DMSO/drug comparison. No differences or clustering was observed between each cell preparation assessed.Adult left ventricular cardiomyocytes were isolated from wild type 12–16 week old C57Bl6 mice by retrograde perfusion of liberase as previously described . Adult l2.22. These were applied in low calcium (200 μM) Tyrode-HEPES buffer for 5 min, followed by three 5-min sequential washes with low, medium (300 μM) and high (420 μM) calcium buffer at 37 °C. Imaging was performed with an Olympus IX81 inverted microscope  with an Andor iXon Ultra 897 EMCCD camera . Cell videos were captured at 37 °C at 50 frames per second under electrical pacing at 1 Hz, at 485 / 20–25 nm excitation and 525 / 50 nm emission with at 495 nm dichroic mirror. Videos were processed and parameters extracted with SarcTrack [2+ transients were recorded for Rhod-2, Fluo-4. Resultant raw traces were extracted and analysed with CalTrack software [Wild-type iPSC cardiomyocytes with a titin N-terminal enhanced green fluorescent protein (eGFP) tag were kept in culture to passage 35 before differentiation with Wnt pathway modulation with small molecule inhibitors and cultured in in RPMI 1670 / B27 media minus insulin for 7 days. Insulin was added back to the media for 9–11 days until the establishment of spontaneous beating as previously described . Cardiomsoftware . The sig2.3Rabbit skeletal myosin S1 ATPase activity was measured in the presence of either isolated rabbit skeletal F-actin or thin filaments reconstituted with actin, human Ala-Ser-tropomyosin, and bovine cardiac troponin as previously described . Assay m2.4Cardiac muscle fibres were taken from 10 week old C57Bl6 mice, demembranated and attached to a 1400A Permeabilized Fibre System from Aurora Scientific as previously described . Followi2.52+ transients were acquired from all cell types using 560/25 nm excitation, 620/60 nm emission filters with a 565 nm dichroic mirror and 40 fps video capture imaging at 37 °C using the microscope equipment described in Recombinant adenovirus expressing R-GECO was generated by Welgen Inc. and used to transduce adult guinea pig or iPSC cardiomyocytes at a multiplicity of infection of 444 or 10 for each cell type respectively. Contractility measurements used either IonOptix or SarcTrack for adult or iPSC cardiomyocytes as described in 2.6t-test, or non-parametric Mann-Whitney test for two data sets or One-way ANOVA or non-parametric Kruskal-Wallis test with Tukey post hoc analysis for multiple comparisons where multiple data sets are compared to a single control.All data were assessed for normality using a D'Agostino & Pearson test and, where appropriate, were compared using students 33.1n = 52, p < 0.0001) and 87.1 ± 12.3%  compared with control in mouse and guinea pig cardiomyocytes, respectively. SBFI, a Na+ indicator used as a control for the toxic by-products of AM-de-esterification, had no significant effect on fractional shortening in mouse cardiomyocytes and only a limited effect in guinea pig cardiomyocytes. While we observed dose-dependent lengthening of the sarcomere with each Ca2+ dye tested, only higher concentrations of Fluo-4 and Rhod-2 slowed cellular relaxation of mouse cardiomyocytes, but in contrast, relaxation of guinea pig cells was slowed by all dyes . Signal-to-noise ratios of fura2 and Fluo-4 fluorescence increased with dose  compared to untreated controls  at 5 μM. The signal-to-noise ratio was assessed across multiple doses of Fluo-4 and Rhod-2 (Patient-derived induced pluripotent stem cell (iPSC) or gene-edited human embryonic stem cell-derived cardiomyocytes can be used for human disease modelling and therapeutic screening. Although iPSC cardiomyocytes respond to electrical pacing and provide readouts of contractility and calcium cycling , their scontrols A and B. d Rhod-2 C-F. In c3.32+ dye (5 μM) using bovine cardiac isoforms of actin, myosin, and troponin complex, along with human recombinant Ala-Ser α-tropomyosin where the additional di-amino acid modification mimics endogenous N-terminal acetylation [2+, Na+) at 5 μM concentrations. All dyes reduced ATPase rates, with fura2-AM, Fluo-4-AM, Rhod-2, and SBFI-AM causing reductions of 15.3 ± 0.8%, 14.4 ± 1.1%, 15.8 ± 0.5%, and 16.4 ± 1.1%, respectively   A.Fig. 4C2+ by the thin filament regulatory components or directly via actin/myosin, we repeated the assay without the thin filament regulatory proteins. Chemical dyes directly inhibited acto-myosin ATPase in all cases, with reductions of 32.4 ± 1.5% for fura2-AM, 25.4 ± 1.7% for Fluo-4-AM, 27.5 ± 1.8% for Rhod-2-AM, and 25.7 ± 1.88% with SBFI-AM  . Intracellular fura2 levels were determined in guinea pig cardiomyocytes to ensure that the in vitro dye concentrations reflected the intracellular dye concentrations achieved in intact cell studies. We found that intracellular fura2 levels were 7 to 20-fold higher than the exogenous cell concentration treated (5 and 1 μM respectively) after 5 min of dye exposure . This suggests that cellular accumulation of dye may expose myofilaments to concentrations higher than those tested in vitro.To determine if the inhibition mechanism was due to competition for Ca 0.0001) B. The AT2+ dyes on acto-myosin inhibition to a known inhibitor (blebbistatin) [50 for blebbistatin was 0.88 ± 0.04 μM, and 4.92 ± 0.62 μM for fura2-AM (50) concentration of blebbistatin. Progressive prevention of blebbistatin inhibition between 0.2 and 2.5 μM fura2 was seen, followed by a plateau (To compare the effect of Caistatin) , we perffura2-AM C. To det plateau D.n = 7, Δ-Force = −49.6 ± 9.3 and − 80.2 ± 10.3 mN.mm2 p < 0.05 and < 0.001 respectively)  at an isometrically stretched sarcomere length of 2.2 μm we tested if dye inhibition of acto-myosin interaction was preserved in a co-operative system under load. Sequential increase in fura2 to concentrations of 0.2, 1.0 and 5.0 μM reduced the force reading acquired E. Both 1ctively) E, F.3.42+ with advantages of subcellular targeting, affinity and spectral variants that complement the chemical dye toolset [2+ indicator R-GECO is a red spectral variant of a circularly permuted fluorescent reporter protein (mApple) engineered to contain a classic Ca2+ binding domain  [2+ transients [Proteins have been developed to measure Ca toolset . The genlin/M13) . It can lin/M13) , R-GECO ansients ,20.2+ indicators . Additionally, there was no difference in cardiomyocyte contractility between guinea pig cardiomyocytes transduced with R-GECO and those infected with a control adenovirus expressing GFP, or when compared to uninfected control cells , ruling out significant contractile effects induced by adenoviral infection itself. We observed MOI-dependent expression variability of genetically encoded indicators following viral transduction, typically 0.2–2 μM range , with an estimated average expression of ∼1 μM in guinea pig cardiomyocytes [50 at maximal (pCa 4.5), or minimal (pCa 8.5), activated ATPase activity in the presence of R-GECO  for cells loaded with 5 μM Fluo-4 , indicative of the stronger Ca2+ affinity of the dye as expected. The kinetics and fluorescent intensity of Fluo-4 was unaffected by the presence of R-GECO . Collectively, our results suggest that R-GECO reports the Ca2+ transient with better SNR, less dose dependency, less contractile impairment, and less Ca2+ buffering than chemical dyes in the studied cardiomyocyte models.Finally, we performed dual indicator Ca imaging , where c3.52+ indicators, which cause contractile impairment  could be significant for the development of novel therapeutics that work at the myofilament [2+ signal is uncertain, but could lead to safe compounds being rejected, or harmful compounds being accepted.Concerns about the use of chemical dyes as Cafilament . There i2+ indicators  for evaluating the effects of three representative drugs that affect both contractility and Ca2+ transients. These drugs included mavacamten [2+ transient and cause negative inotropy [To investigate this potential issue, we tested the suitability of both chemical and genetic Cavacamten , which svacamten , which ivacamten , which hinotropy .2+ reporter (p < 0.001) and decreased T50 relaxation time (15.1 ± 5.4 msec p < 0.05) without attenuating fractional shortening   increased fractional shortening in all experimental groups, but only decreased basal sarcomere length in fura2-treated cells (−2.64 ± 0.75%) giving the false impression of hypercontraction, rather than the relaxation shown in the control or R-GECO transduced cardiomyocyte (+3.23 ± 0.95% and + 2.06 ± 0.85% respectively) (p < 0.01) and contraction and relaxation times increase (by 28.4 ± 11.2% p < 0.05 and 24.5 ± 6.9% p < 0.01 respectively) (Flecainide (0.5 μM) appears to have no contractile effect in the presence of fura2, yet in control and R-GECO treated cells fractional shortening is reduced ) of ∼5.8 msec at 37 °C and pH 7.4 . Although association and dissociation rates are intrinsically slower than fura2  [2+ signal readouts are not constrained by the properties of the probe itself, making it a viable alternative to chemical Ca2+ dyes for safety and efficacy studies of drugs.At 3 μM Ca7.8 s−1)  over the2+ levels, which cannot be assessed by the intensiometric R-GECO  increased Ca2+ transients  , than virally transduced GECIs ). Indeed historical literature [50 .We found that BAPTA-derived indicators cause contractile impairment in cardiac cell models due to multiple components not present in genetically encoded sensors. A major aspect is Caterature  shows thture [50 C, equiva2+ handling and membrane potential [The toxic by-products of chemical dye loading are the AM-ester breakdown products such as formaldehyde and acetic acid which affect protein conformation  and loweotential . The resotential  can be e2+ buffering [50 for blebbistatin was 0.88 ± 0.04 μM, and 4.92 ± 0.62 μM for fura2-AM . Blebbfilament . As acto50) estimate [As dyes accumulate irreversibly once de-esterified ; intraceestimate . We were2+ measurements are a common technique in research, and cardiac Ca2+ handling is a central aspect of toxicology testing in industry [2+ handling, we identified fura2 masked the effect of the drug on both contractility and Ca2+ transients compared to an untreated or R-GECO transduced control. The cultured guinea pig cardiomyocyte may be a more extreme example of this issue  should be done to determine the minimal concentration that optical systems can handle to generate clear Ca2+ signals. Electrical pacing of cardiomyocytes can be used to standardize beat frequency and duration for averaging of multiple transients, but photo-bleaching with intensiometric indicators may limit the efficacy of this approach .Researchers measuring Ca2+ indicator it is possible the Ca2+ transient captures more than just the intended perturbation to the system. For this reason, we prefer genetically encoded Ca2+ sensors such as R-GECO or GCamp6 [2+ dye  use can be routinely avoided. Recent publications investigating contractility and Ca2+ simultaneously in iPSC derived engineered heart tissue and 2D iPSC cardiomyocyte cultures with genetically encoded probes GCaMP6 and R-GECO indicate that momentum may be shifting towards this [Changes in contractility should be corroborated by finding similar results in cardiomyocytes that have not been treated with any indicator. If contractility is different in the presence of the Car GCamp6  in cardirds this ,53.2+ indicators successfully used in adult cardiomyocytes have been almost exclusively intensiometric. This prevents measurement of changes in absolute Ca2+ concentration which may be altered by drug or disease as these alterations are missed by indicators such as R-GECO and Fluo-4. Ratiometric dyes such as fura2 or indo1 more accurately calibrate Ca2+ concentration. Whilst ratiometric genetically encoded sensors have been developed i.e. YC3.6, Ratiometric Pericam [2+ transients without impairment of contractility. Improvements in optical sensitivity of next generation microscopy equipment, coupled with improved techniques to culture and mature iPSC cardiomyocytes or maintain higher quality isolated adult cells in culture could further help achieve this goal.To date, the genetically encoded Ca Pericam , REX-GEC Pericam , their uPR and MJD conceptualized the study. PR, CR, MJD, AJS, CT, YP designed and interpreted the experiments. PR and AJS, YP performed the chemical dye experiments. PR and AJS made recombinant R-GECO and thin filament proteins. PR, AJS, MAG performed the characterisation of the GECO's. CNB, Y-FC, FAB, performed pilot experiments in iPSC cardiomyocytes. XZ, YAA performed impedance assay measurements. JS, isolated mouse cardiomyocytes. PR, AB, Y-JW performed force fibre experiments. PR and AJS performed guinea pig and mouse cardiomyocyte experiments. YP, VS and CT performed iPSC cardiomyocyte culture and SarcTrack assessments. AJS, CT and YP provided analytic tools. AJS, YP, and PR performed all analysis. PR wrote the 1st draft. MJD, CR, PR, AJS, CT, HW edited the manuscript and all authors provided comments on this manuscript.No competing interests declared.


Lower limb robotic exoskeletons are often studied in the context of steady-state treadmill walking in laboratory environments. However, the end goal of these devices is often adoption into our everyday lives. To move outside of the laboratory, there is a need to study exoskeletons in real world, complex environments. One way to study the human-machine interaction is to look at how the exoskeleton affects the user’s gait. In this study we assessed changes in gait spatiotemporal variability when using a robotic ankle exoskeleton under proportional myoelectric control both inside on a treadmill and outside overground. We hypothesized that walking with the exoskeletons would not lead to significant changes in variability inside on a treadmill or outside compared to not using the exoskeletons. In addition, we hypothesized that walking outside would lead to higher variability both with and without the exoskeletons compared to treadmill walking. In support of our hypothesis, we found significantly higher coefficients of variation of stride length, stance time, and swing time when walking outside both with and without the exoskeleton. We found a significantly higher variability when using the exoskeletons inside on the treadmill, but we did not see significantly higher variability when walking outside overground. The value of this study to the literature is that it emphasizes the importance of studying exoskeletons in the environment in which they are meant to be used. By looking at only indoor gait spatiotemporal measures, we may have assumed that the exoskeletons led to higher variability which may be unsafe for certain target populations. In the context of the literature, we show that variability due to robotic ankle exoskeletons under proportional myoelectric control does not elicit different changes in stride time variability than previously found in other daily living tasks . Lower limb robotic ankle exoskeletons can aid rehabilitation and augment healthy human performance, but are not yet widely adopted into daily use in the general population –4. To imGait variability is an important measure that is often ignored in the training and use of robotic lower limb exoskeletons during human locomotion. There is extensive literature on gait variability for healthy individuals and individuals with gait deficits. Researchers use gait measures, such as stride length, stride time, stance time, cadence, and swing time and the variability of those measures as indicators of healthy, typical gait –17. HealThe purpose of this study was to determine the effect of proportional myoelectric controlled robotic ankle exoskeletons on gait variability while walking on a treadmill indoors and overground outside. Previous research on human walking indicates that gait variability is lower while walking on a treadmill compared to walking overground ,14,31–33We recruited 12 participants  with the following characteristics, mean (s.d.): height: 1.73 (0.075) m; mass: 69.3 10.2) kg; age 22.6 (8.66) yrs. The standard deviation for the age of our participants is high because of a single 50-year-old participant. None of that participant’s data, besides their age, were outliers from the group data and therefore was kept for all analyses. Participants were recruited between June and December 2022 for this study using flyers around the University of Florida campus as approved by the Institutional Review Board. For treadmill walking participants walked at an average speed of 1.14 (0.12) m/s. For the outdoor conditions participants walked at an average speed of 1.2 (0.05) m/s while walking with the exoskeletons and 1.17 (0.07) m/s when walking without the exoskeletons. Participants reported no previous neurological or musculoskeletal conditions. Each participant was right-handed and had no previous experience walking with a robotic ankle exoskeleton prior to the first day of training for this study. Each participant read and signed a written informed consent form approved by the University of Florida Institutional Review Board (IRB#201801218). Authors were aware of the identity of participants during data collection, but the data themselves were deidentified during analysis. We expected sufficient statistical power from 12 participants based on our previous research .2 kg; ag,36,39. Pnd order Butterworth, cutoff frequency 50 Hz), full wave rectified, and low-pass filtered . We then multiplied the resulting signal by a participant specific gain determined while walking with the exoskeletons unpowered to get the controlling motor current . A proportional myoelectric controller with an input of the user’s soleus muscle provided the input current to the motor . The sol current . ParticiParticipants completed 4 days of testing with at least 48 hours between data collection days to allow for motor learning consolidation  Fig 1)..Fig 1). We found heel strikes and toe offs from IMUs on the lateral longitudinal arch of the feet. We found the peaks in vertical acceleration surrounding the near zero velocity region that indicates stance phase similar to what is described in previous research . We ensuWe used 2-way MANOVAs to assess differences in means and coefficient of variations in gait parameters between factors. The first used factors of Day (1 and 4) and Condition , the second used factors of Location (Treadmill and Overground) and Condition (Exoskeleton and No Exoskeleton). We did post-hoc Tukey-HSD when statistical differences were found across the Exoskeleton, Unpowered, and No Exoskeleton conditions. We did the calculations for the Day and Condition factors first, once we determined there was not a significant difference due to day, we used the data from Day 4 for statistical analyses between indoor and outdoor. We assigned a significance value of α = 0.05 for both tests. For EMG RMS values we either did 1-way ANOVAs with condition being the factor for each muscle on each day, or for the outdoor condition we performed a t-test because there were only 2 conditions. We used SPSS for statistical analysis.Consistent with our hypothesis there were significant differences in mean and variability of gait spatiotemporal measures when walking outside over ground compared to inside on a treadmill. The coefficient of variation while walking outside compared to walking on a treadmill was significantly higher for stance time (p = 0.044), swing time (p = 0.002), and stride length (p<0.001) . In partThe stride length and stance time were significantly shorter outside compared to inside on a treadmill (p<0.001), and the swing time was significantly longer outside compared to inside on a treadmill (p<0.001) . There wThere were no significant differences in stride time, stance time, stride length, or swing time between the no exoskeleton, unpowered, and exoskeleton conditions or between day 1 and day 4 .Walking with the exoskeleton inside on the treadmill resulted in higher coefficients of variation compared to not wearing the exoskeletons in stride time (p<0.001), stride length (p<0.001), stance time (p = 0.033), and swing time (p = 0.005) with no significance due to day. In addition, walking with the exoskeletons powered resulted in significantly higher coefficients of variation compared to walking with the exoskeletons unpowered in stride length (p = 0.031), stride time (p = 0.015), and swing time (p = 0.039). There were no significant differences in coefficients of variation between the no exoskeleton and unpowered conditions. Contrary to our hypothesis we did not find significant differences in the measurements between day 1 and day 4 . On day There were non-significant differences in the EMG activity across conditions. We present the Soleus as a representative muscle because of its part in the control mechanism . On the As hypothesized, we found significantly higher coefficients of variation of stride length, stance time, and swing time while walking outside both with and without the exoskeletons compared to walking inside on a treadmill. There was an 119% difference in stride length variability when walking with the exoskeletons outside compared to walking with them on the treadmill. There was a 162% difference in stride length variability when walking without the exoskeletons outside compared to on the treadmill. This was the largest increase in variability we found. Contrary to the findings in other studies without exoskeletons, we saw an average of 29% shorter stride lengths walking outside compared to on the treadmill ,47. The Contrary to our hypothesis, participants had significantly higher coefficients of variation while walking with the exoskeletons compared to without the exoskeletons when walking on the treadmill. Stride time, stride length, swing time, and stance time all had greater variability with the exoskeleton on the treadmill compared to not using the exoskeleton on the treadmill. These data suggest that there was more exploration in walking strategies by the participants when they had the exoskeletons on the treadmill. However, there was no difference in walking with and without the exoskeletons outside. The fact that when walking overground, stride length and swing time variability when using the exoskeletons  compared to not using them  was similar suggests that the variability due to terrain differences (treadmill vs. outside) has a larger effect than the variability due to exoskeleton assistance. This is further supported by the non-significant changes in muscle activity when walking with and without the exoskeleton overground on the treadmill . There wFuture studies should include tests of indoor overground walking, uneven treadmill walking, as well as incline and decline treadmill walking in addition to the conditions studied in this paper. Having indoor and outdoor conditions on flat, incline, decline and uneven terrains will give a more encompassing view of how people adapt their gait to exoskeletons. A comparison of these exoskeletons on a self-paced treadmill may give more insight into how much of the variability we observed on the treadmill was due to the constrained speed rather than exploration using the exoskeletons. Other papers saw similar stride time variability but higher stride length variability when walking on a self-paced treadmill than we saw in this study ,55. ExplWe tested the hypotheses that individuals using electromechanical ankle exoskeletons under proportional myoelectric control would: 1) have higher variability in gait spatiotemporal measures when walking outside both with and without the exoskeletons compared to walking on a treadmill, and 2) have similar amounts of variability when walking with the exoskeletons compared to without the exoskeletons. In support of our first hypothesis, participants had larger coefficients of variation in stride length, stance time and swing time when walking outside overground compared to on the treadmill. In partial support of our second hypothesis there were no differences in variability when using the exoskeletons outside compared to not using the exoskeletons outside. However, when walking on the treadmill participants had larger coefficients of variation when using the exoskeletons than when not using the exoskeletons. Based on our results, only studying gait variability on a treadmill may lead to the conclusion that exoskeletons elicit significant changes in variability. Walking overground revealed that the exoskeletons do not lead to significant changes in gait spatiotemporal measures or their variability in the real-world. When using exoskeletons intended for use in assistance or rehabilitation the devices should work with the user to adapt to changes in the environment to create a relationship of trust between the human and the machine, rather than adding to the uncertainty of movements. In the context of the literature, we show that variability due to robotic ankle exoskeletons under proportional myoelectric control does not elicit different changes in stride time variability than previously found in other daily living tasks .


The internal tandem duplication of the juxtamembrane domain of the FMS-like tyrosine kinase 3 (FLT3-ITD) is the most common genetic change in acute myeloid leukemia (AML), and about 30% of all AMLs harbor a FLT3-ITD mutation. Even though FLT3 inhibitors have displayed encouraging effects in FLT3-ITD-mutated AML, the extent of the clinical response to these compounds is cut short due to the rapid development of drug resistance. Evidence has shown that FLT3-ITD triggered activation of oxidative stress signaling may exert a pivotal role in drug resistance. The downstream pathways of FLT3-ITD, including STAT5, PI3K/AKT, and RAS/MAPK, are considered to be major oxidative stress signaling pathways. These downstream pathways can inhibit apoptosis and promote proliferation and survival by regulating apoptosis-related genes and promoting the generation of reactive oxygen species (ROS) through NADPH oxidase (NOX) or other mechanisms. Appropriate levels of ROS may promote proliferation, but high levels of ROS can lead to oxidative damage to the DNA and increase genomic instability. In addition, post-translational modifications of FLT3-ITD and changes in its subcellular localization can affect downstream signaling which may also be one of the mechanisms leading to drug resistance. In this review, we summarized the research progress on NOX mediated oxidative stress signaling and its relationship with drug resistance in FLT3-ITD AML, and discuss the possible new targets in FLT3-ITD signal blocking to reverse drug resistance in FLT3-ITD-mutated AML. FLT3-ITD-triggered activation of oxidative stress signaling may play a critical role in drug resistance in AML.Post-translational modification of FLT3-ITD and changes in its subcellular localization can affect downstream signaling which may be one of the mechanisms leading to drug resistance in AML.What is the exact mechanism underlying the alteration of downstream signaling of the FLT3-ITD oncogene?What is the exact relationship between post-translational modifications, subcellular localization, and FLT3-ITD oncogene signaling?How to efficiently and persistently block FLT3-ITD oncogene signaling?+ stem/progenitor cells [Abnormal signals from receptor tyrosine kinases (RTKs) are often discovered to exert a pivotal role in tumor initiation and progression. FLT3 protein belongs to the type III RTKs family and is solely expressed in normal bone marrow CD34or cells , 2. The or cells . Currentor cells . Accumulor cells –10.Several publications have delineated that the FLT3-ITD mutation is closely linked with an increased ROS production in AML cells , 12. As Recently, numerous studies have shown that the post-translational modifications that involve FLT3-ITD and its localization in cells can lead to changes in downstream signaling which may also be another mechanism leading to TKI drug resistance. Therefore, effective targeting of FLT3-ITD in the personalized approach to AML management necessitates an in-depth understanding of the underlying links between FLT3-ITD localization, activity, and downstream signaling.The FLT3 gene is located on chromosome 13q12 in humans. This gene encodes a protein consisting of 993 amino acids, whose structure is made up by an “N-terminal extracellular domain, a transmembrane region, a juxta-membrane domain, and a C-terminal cytoplasmic tyrosine kinase domain” . The FLTvia western blotting analysis. One 150 kDa receptor represents a complex glycosylated mature form, and the other 130 kDa receptor represents an underglycosylated immature form with a mannose-rich structure. The 130-kDa forms were predominantly detected in cells expressing FLT3-ITD, whereas 150 kDa forms were found to have a higher proportion in the cells expressing FLT3-WT [Schmidt-Arras et al. highlighted the existence of two forms of the FLT3 receptor  FLT3-WT . Multipl FLT3-WT , 20. Evi FLT3-WT .Currently, the mechanism by which activated mutations lead to altered FLT3-ITD subcellular localization is unclear. Besides being possibly related to the glycosylation status, recent studies have found that palmitoylation and phosphorylation may be two other key factors regulating FLT3-ITD intracellular localization. According to Lv et al., the S-palmitoylation of FLT3-ITD is responsible for the inhibition of FLT3-ITD’s surface expression, and the application of the palmitoylation inhibitor 2-bromopalmitate (2-BP) can decrease the ER retention of FLT3-ITD and promote its transportation to Golgi and cell surfaces . SimilarAML is a group of hematologic malignancies with high heterogeneity which is also extended to their metabolic profiles –26. It w2−) from extracellular O2 [ROS are an important by-product of aerobic metabolism. Excessive production of ROS is frequently observed in FLT3-ITD AML and is demonstrated closely associated with the pro-survival mechanism of AML cells –29. In elular O2 . NOX inclular O2 , 31.+ hematopoietic progenitor cells [via STAT5 mediation. Furthermore, it facilitates the inactivation of one of its negative regulators, that is, the protein-tyrosine phosphatase density-enhanced phosphatase-1 (DEP-1), by reversible oxidation of its catalytic cysteine residue. NOX4 knockdown reduces ROS concentrations, restores the activity of DEP-1, and reduces FLT3-ITD-driven transformation [Among all NOX isoforms, NOX1, NOX2 and NOX4 are expressed by human CD34or cells –32. Amonor cells , 33. Salor cells . LikewisRecently, Moloney et al. reported that PM-located FLT3-ITD can promote NOX4 activation by activating AKT, thereby inducing the production of pro-survival ROS. This process depends on the AKT activation and only PM- located FLT3-ITD can activate AKT. Therefore, it is believed that only FLT3-ITD at the PM is carcinogenic, and targeting PM-localized FLT3-ITD may prove beneficial in the prevention of downstream ROS-driven carcinogenesis . In anotvia regulation of apoptosis-related genes, thereby leading to the initiation and development of AML [The downstream signaling pathways of the FLT3-ITD oncogene, e.g., PI3K/AKT, JAK/STAT, and Ras/ERK, are representative signaling pathways that are abnormally activated in AML. The aberrant FLT3-ITD oncogene signal constitutively activates the above signaling pathways, which subsequently inhibit cell apoptosis, promote cell proliferation and survival t of AML –37.Recently, several studies have highlighted that the intracellular disposition of FLT3-ITD may determine which downstream pro-survival signals are activated constitutively by FLT3-ITD. In particular, the PM-localized FLT3-ITD strongly activates the mitogen-activated protein kinase (MAPK) and PI3K pathways with diminished STAT5 phosphorylation . Golgi-lRas/ERK, PI3K/AKT and JAK/STAT are the main oxidative stress signaling pathways and can induce pro-survival ROS production through different mechanisms, such as inhibiting the activity of forkhead O box (FOXO) or enhancing NOX expression , 39. RecMounting evidence have shown that, secondary mutations caused by accumulation of DNA damage may critically contribute to the development of drug resistance –45. HoweROS represent a group of oxygen-containing and highly reactive molecules which serve as important second messengers involved in a variety of signal transduction that regulates cell growth, proliferation, and differentiation of cells . It is nLike other cell types, leukemia cells also have well-established and complex mechanisms for maintaining their redox balance, and the level of pro-survival ROS is finely regulated in AML. It was reported that in FLT3-ITD AML, elevated ROS concentrations induced by oncogene signaling lead to compensatory enhancement of antioxidant capacity that protecting leukemia cells from oxidative damage, thereby bringing leukemia cells survival benefits , 50. An A commonly accepted chemoresistance mechanism is the oncogenic kinases-driven DNA damage repair activity enhancement which favors the survival of malignant cells under genotoxic stress . RecentlMAPK-mediated cycle arrest and/or apoptotic signaling [Previous studies have shown that suppression of apoptosis signals can also help AML cells maintain survival under oxidative stress. Hole et al. reported that AML cells can escape the oxidative damage by inhibiting p38ignaling . Besidesignaling , 57, wheignaling , 58–60. ignaling –65.MAPK and induction of mitochondrial ROS [As the FLT3-ITD oncogene signal is a crucial player in the development of AML, it has emerged as a relevant target in the management of this malignancy. To date, several FLT3 inhibitors have been developed, tested in randomized clinical trials, i.e., midostaurin, gilteritinib, quizartinib, and other TKIs, and approved for clinical application. On this basis, the development of TKIs with multi-target inhibition has been continuously strengthened to improve their efficiency and durability on mutant FLT3 kinases Table . Furtherrial ROS . Downregrial ROS .Table 2Nvia preventing advanced glycosylation of the receptor, reducing FLT3-ITD cell surface expression, inhibiting the activation of MAPK and AKT, and reducing the TKI drug resistances while leading to the apoptosis of FLT3-ITD-mutated AML cells [via glycosylation inhibition, as well as FLT3-ITD cell surface expression and signaling. Moreover, the co-administration of FLT3-ITD inhibitors and tunicamycin enhanced cell death synergistically, in particular in FLT3-ITD-mutated AML cells [Protein post-translational modifications (PTMs) are important in the regulation of protein function. As mentioned above, PTMs such as glycosylation and palmitoylation not only affect the subcellular localization of the FLT3-ITD protein, but also lead to alterations in downstream pathways, which may further lead to drug resistance. Therefore, targeting the PTMs of FLT3-ITD protein to overcome drug resistance of AML cells has become a hot topic of current research. According to Williams et al., a lipophilic statin, i.e., fluvastatin, can decrease the mutant FLT3’s kinase activity ML cells . The antML cells  and tuniML cells  have alsML cells . RecentlML cells .Blockage of intracellular transport routes of FLT3-ITD has also been shown to effectively inhibit oncogenic signals. Yamawaki et al. reported that the use of 2-methylcoprophilinamide (M-COPA) or brefeldin A to inhibit FLT3-ITD’s export from the ER or the use of monensin to inhibit FLT3-ITD’s export from the Golgi apparatus can notably decrease signals of tyrosine phosphorylation in AML . In addiThe strategy of blocking the downstream signal of FLT3-ITD has also attracted much attention. For example, the oncogenic serine/threonine kinase Pim-1 is transcriptionally upregulated downstream of FLT3-ITD and stabilizes and phosphorylates FLT3-ITD, thus promoting the signaling of FLT3-ITD in a positive feedback loop in AML cells . BaldwinThe FLT3-ITD mutation is one of the most important gene mutations in AML, and it is also a crucial indicator of poor prognosis in this blood cancer. Current second-generation TKIs, such as sorafenib, gilteritinib, crenolanib, and quizartinib, are highly specific and potent, and often lead to favorable outcomes when employed in the early treatment of FLT3-ITD-mutated AML. However, the efficacy of TKIs is often challenged by the development of resistance caused by a myriad of factors, e.g., secondary mutations or signaling compensations. Therefore, most of these drugs fail to achieve the expected efficacy.Recently, a global untargeted metabolomics and stable isotope-labeling mass spectrometry analysis was applied to evaluate sorafenib resistance in FLT3-ITD-mutated leukemic cells . It was The use of antioxidants during leukemia therapy is also currently a hot topic. It was recently reported that N-acetyl-cysteine, a natural product, was able to reduce ROS generation in FLT3-ITD-mutated AML and contributed to the prevention of aberrant interchromosomal homologous recombinations which are involved in leukemia survival and progression . Theoret


Previous studies have shown that Cd2+ induced cytotoxicity by disrupting the intracellular Ca2+ homeostasis that is physically regulated by the endoplasmic reticulum (ER) Ca2+ store. However, the molecular mechanism of ER Ca2+ homeostasis in Cd2+-induced nephrotoxicity remains unclear. In this study, our results firstly revealed that the activation of calcium-sensing receptor (CaSR) by NPS R-467 could protect against Cd2+ exposure-induced cytotoxicity of mouse renal tubular cells (mRTEC) by restoring ER Ca2+ homeostasis through the ER Ca2+ reuptake channel sarco/endoplasmic reticulum Ca2+-ATPase (SERCA). Cd2+-induced ER stress and cell apoptosis were effectively abrogated by SERCA agonist CDN1163 and SERCA2 overexpression. In addition, in vivo, and in vitro results proved that Cd2+ reduced the expressions of SERCA2 and its activity regulator phosphorylation phospholamban (p-PLB) in renal tubular cells. Cd2+-induced SERCA2 degradation was suppressed by the treatment of proteasome inhibitor MG132, which suggested that Cd2+ reduced SERCA2 protein stability by promoting the proteasomal protein degradation pathway. These results suggested that SERCA2 played pivotal roles in Cd2+-induced ER Ca2+ imbalance and stress to contribute to apoptosis of renal tubular cells, and the proteasomal pathway was involved in regulating SERCA2 stability. Our results proposed a new therapeutic approach targeting SERCA2 and associated proteasome that might protect against Cd2+-induced cytotoxicity and renal injury.Cadmium (Cd Previous studies have tried to explore the potential mechanism of Cd2+-induced cytotoxicity of the renal proximal tubule cells, which will help find potential intoxication targets and explore novel renoprotective strategies [2+-induced cytotoxicity remain unclear.Cadmium (Cd), as a widely distributed environmental pollutant, is unavoidably absorbed by humans through environmental and occupational exposures . After irategies . However2+-disrupted intracellular calcium (Ca2+) homeostasis induced autophagy inhibition, promoted oxidative stress, and thereby mediated cell apoptosis [2+, Pb2+) exposure induces subcellular Ca2+ redistribution by increasing Ca2+ release from the endoplasmic reticulum (ER) Ca2+ stored in primary rat proximal tubular cells [2+ increased cytosolic Ca2+ levels by activating phospholipase C (PLC)-inositol 1, 4, 5-trisphosphate receptor (IP3R), which is one of the main Ca2+ release channels of ER [2+ release channels, ER also has Ca2+ reuptake channel sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) to maintain ER Ca2+ store level. In NIH 3T3 fibroblast cells, Cd2+ exposure decreased SERCA activity and inhibited the reuptake of Ca2+ from the cytoplasm back into the ER, which caused the disruption of ER Ca2+ homeostasis and cell apoptosis [2+-induced apoptosis of renal tubular cells have not been clarified.Cdpoptosis ,5,6,7,8.ar cells ,10,11. Ils of ER . In addipoptosis . However2+ sensing receptor (CaSR) [2+-disrupted intracellular Ca2+ homeostasis and protect against Cd2+-induced kidney apoptosis and injury [2+-modulated Ca2+ homeostasis and Cd2+-induced ER stress and apoptosis in renal tubular cells and explore the underlying mechanism of Cd2+-targeted SERCA regulation. SERCA activity and expression were significantly increased in human embryonic kidney cells HEK-293 with gain-of-function variants of the Car (CaSR) . It suggd injury ,15. Ther2+-induced nephrotoxicity originated from the damage of renal tubular cells. As shown in 2+ treatment increased the expression of kidney injury biomarker, kidney injury molecule-1 (KIM-1) in mRTEC (2+ treatment induced apoptosis of mRTEC cells in a dose-dependent manner (2+ (5 μM)-induced apoptosis was effectively reversed by R-467 pretreatment  treatment alone (2+ release channel IP3R)  E,F. Alth minutes G,H. Thes2+ level is dependent on extracellular Ca2+, we detected the change of cytosolic Ca2+ level after the short-term application of Cd2+ in Ca2+ free medium. The result of Flou-3/AM showed that the Ca2+ levels increased after short-term Cd2+ treatment (0.1 h) in both Ca2+-free HBSS and RMPI 1640 medium (2+ could return to resting level (0.5–4 h)  A, which  for 4 h B. It sug2+ store in the cells and maintains intracellular Ca2+ homeostasis. As shown in 2+ content as it is specially co-localized or surrounded with ER-trackers in mRTEC (2+ treatment (4 h) did not alter the ER Ca2+ content (2+ treatment (24 h) would reduce the ER Ca2+ content , and a Ca2+ reflux channel SERCA. The balance between these three Ca2+ channels is crucial to maintain the dynamic balance of ER Ca2+ [2+ channels on the plasma membrane, such as transient receptor potential vanilloid (TRPV) channels, could maintain ER Ca2+ homeostasis and protect against ER stress-induced apoptosis [2+ competites the TRPV5 and TRPV6 channels to block the Ca2+ uptake [2+ release channel on the ER is mainly IP3R [3R, Cd2+ induced Ca2+ release from the ER and caused Ca2+ redistribution, further resulting in primary rat proximal tubular cell apoptosis [2+ increased intracellular Ca2+ levels by activating the PLC-IP3R pathway [2+-disrupted cytosolic Ca2+ homeostasis and protect against Cd2+-induced kidney apoptosis and injury [2+-induced ER Ca2+ loss was effectively reversed by CaSR activator R-467 and SERCA agonist CDN1163. It suggests that Cd2+-disrupted ER Ca2+ homeostasis was modulated by the SERCA pump.The ER is a sophisticated organelle controlling Ca ER Ca2+ ,22. In apoptosis ,24. Howe+ uptake ,26,27. Inly IP3R ,29. Thro pathway . Moreoved injury ,15. The 2+-induced cytotoxicity. Cd2+-decreased SERCA activity causes the disruption of ER Ca2+ homeostasis and apoptosis in NIH 3T3 fibroblast cells [2+ induced apoptosis by inhibiting Ca2+-ATPase activity [2+-increased triglyceride levels [2+ transport rate from the cytoplasm into ER decreases, which is reversed by the phosphorylation of PLB (p-PLB) [2+ reduced the protein levels of p-PLB both in mouse renal tubular cells and kidney tissues. It suggests that Cd2+ induces ER stress, and apoptosis of renal tubular cells might be by inhibiting SERCA activity.It suggests that SERCA activity plays an important role in Cdst cells . In the activity . In HepGe levels . The SER (p-PLB) . Here, o2+ depletion, which triggers apoptotic pathways in diabetic nephropathic mice [2+ in vascular smooth muscle, which might benefit the treatment of vasodilation in human intrarenal arteries [2+ reduced the protein level of SERCA2 and upregulated the expression of ER stress and apoptosis biomarkers, which was reversed by CaSR activator R-467 and SERCA agonist CDN1163, as well as overexpression of SERCA2. It suggests that Cd2+-inhibited expression of SERCA2 causes ER stress and apoptosis in renal tubular cells.It is supposed that dysregulation of SERCA2 could cause kidney diseases. Reducing SERCA2-dependent ER stress could alleviate podocyte injury in diabetic nephropathy mice . Upregulhic mice . Activatarteries . By attearteries . The res2+ inhibited autophagy flux by increasing Ca2+ released from ER through the IP3R channel [2+ channels IP3R and SERCA, the ER can transfer the cytosolic Ca2+ to the lysosomes to affect the lysosomal acid environment, an essential element for autophagy flux [3R inhibitor 2-APB could restore the Cd2+-disrupted lysosomal acidic environment, which was also dysregulated by SERCA inhibitor TG [2+-induced p62 accumulation could not be reversed by R-467 and SERCA agonist CDN1163. It indicates that Cd2+ inhibits autophagy flux and might simultaneously increase IP3R-mediated Ca2+ release and suppress SERCA-dependent Ca2+ reflux.Our previous study suggested that Cd channel . With thagy flux ,37. SERCagy flux . In livebitor TG . Here, o2+-induced SERCA2 degradation, which was obviously reversed by proteasomal inhibitor MG132. It suggests that Cd2+ induced SERCA2 degradation by promoting the proteasome pathway. The ubiquitin-proteasome system (UPS) is a main protein degradation process tightly related to ER stress [2+ decreased SERCA2 stability by stimulating the proteasomal pathway in renal tubular cells.Moreover, the lysosomal acidity inhibitor CQ could not inhibit CdR stress . ER streR stress  and reduR stress . Our res2+ exposure induces apoptosis of renal tubular cells mRTEC due to disrupting ER Ca2+ homeostasis by suppressing SERCA2 expression and activity. SERCA2 agonist and overexpression of SERCA2 could abate Cd2+-induced ER stress and cell apoptosis. In addition, Cd2+ reduced SERCA2 protein stability by promoting proteasomal degradation. Therefore, SERCA2 might be a potential new target to protect against Cd2+-induced cytotoxicity and renal injury.The present study demonstrates that Cd3R inhibitor 2-APB, SERCA inhibitor thapsigargin (TG), and SERCA agonist CDN1163 were purchased from Tocris Biosciences . Fura-2/AM, Fluo-3/AM, and Mag-Fluo-4/AM were purchased from Thermo Fisher Scientific ; Calcimimetics NPS R-467 was purchased from NPS Pharmaceuticals .Cadmium chloride, cycloheximide (CHX), and proteasome inhibitor MG132 were purchased from Sigma . IPn = 5, around 25 g) were assigned to Cd2+ exposure group and control group (5 in each group), performed at least three times. CdCl2 was dissolved in distilled water and then added into Specific Pathogen Free (SPF) grade diets to final concentration of 1000 ppm Cd2+. Mice were continually fed on the Cd2+ contaminated diet for 28 days. To determine the effect of NPS R-467, the mice were fed on the 1000 ppm Cd2+ contaminated diet and tail vein injection with NPS R-467 (10 μM) in 20 μL PBS every day for 28 days. The mice were raised at 18~22 °C, with 50–60% humidity, natural daylighting, and tidy housing surrounding. All mice were euthanized by cervical dislocation at time point, and the kidney tissues were taken out for immunofluorescence staining. All animal procedures complied with the guidelines approved by the Ethical Committee for the Use of Laboratory Animals of Jiangsu University (SYXK 2018-0053). As in our previous studies, 10 male ICR mice .Mouse renal tubular epithelial cells (mRTECs) obtained from American Type Culture Collection  were cultured and maintained in RPMI 1640 medium (pH 7.4) supplemented with 10% fetal bovine serum (FBS)  and 1% penicillin/streptomycin in 5% COAfter being treated for 24 h, the cell viability was measured by the MTT -2,5-diphenyl-2-H- tetrazolium bromide) assay . In brief, cells were incubated in 100 μL MTT solution (0.5 mg/mL in RPMI 1640 medium) in 96-well plate for 4 h before the end of incubation. The supernatant was discarded, and 100 μL DMSO  was added to dissolve the colored product (formazan). The absorbance was measured at 540 nm (690 nm as reference) using a Synergy H4 Hybrid Multi-Mode Microplate Reader .2  or CdCl2 (5 μM) + R-467 (1 μM) for 24 h, 1 × 106 cells were re-suspended in binding buffer containing Annexin V-FITC (0.025%) and/or PI (20 μg/mL) and incubated in darkness at room temperature for 3 and 10 min, respectively. Then, the cells were immediately analyzed by a FACScan flow cytometer .The apoptosis rate was determined using an Annexin V-FITC and propidium iodide (PI) double staining kit  according to the manufacturer’s instructions. Briefly, after being treated with CdClThe cell total RNA was isolated using Trizol . All RNA isolations were performed as directed by the manufacturer. Gel electrophoresis and ethidium bromide staining confirmed the purity and integrity of the samples. Quantification of RNA was based on spectrophotometric analysis at 260/280 nm. cDNA was made from 10 μg total RNA using an RNA-to-cDNA kit . Real-time PCRs were carried out by ABI7300 system using SYBR Green master mix . Gene-specific primers for Mouse-SERCA2-F: GGCATCTTTGGGGAGAACGA, Mouse SERCA2-R: CCATCACCTGTCATGGCTGT, Mouse-βActin-F: AAGCTGTGCTATGTTGCTCTA, Mouse-βActin-R: GTTTCATGGAT GCCACAGGA. The occurrence of primer-dimers and secondary products was inspected using melting curve analysis and agarose gel electrophoresis. Control amplification was done either without reverse transcriptase or without RNA. The relative expression ratio of a target gene was calculated according to their threshold cycle Ct values.2+ (5 μM) for 24 h. In some experiments, cells were either untreated or pre-treated with regulators, i.e., R-467 (1 μM), CQ (20 μM), and CDN1163 (10 μM) for 1 h, followed by treatment with Cd2+ (5 μM) for 24 h, or TG (0.1 μM) and MG132 (10 μM) for 6 h. The cells were lysed in RIPA buffer on ice for 30 min. After centrifugation at 13,000× g for 15 min at 4 °C, the supernatant was collected, and the total protein concentration was determined by BCA . The protein lysates containing 40 μg total cellular protein in RIPA buffer were subjected to electrophoresis on 8–12% polyacrylamide gels. The gels were then blotted onto PVDF membranes . Western blotting was conducted using mouse monoclonal antibody C/EBP homologous protein (CHOP)  or rabbit monoclonal antibodies against phosphorylation phospholamban (p-PLB) , Ubiquitin , SERCA2, Binding immunoglobulin Protein (BiP), Protein Disulphide Isomerase (PDI), Inositol-requiring enzyme 1 (IRE-1α), endoplasmic reticulum oxidoreductase 1 (ERO1), p62, cleaved caspase-3  (1:1000), followed by incubation with horseradish peroxidase-conjugated goat anti-rabbit antibody (1:4000). Specific bands were visualized using ECL chemiluminescent reagent . The blots were then washed in PBST and re-probed with rabbit anti-actin  (1:1000). The cells were treated with Cd2+ (5 μM) for 24 h before protein extraction.SERCA2-GFP overexpression plasmid (VB190422-1024rhe), which contains full length of mouse SERCA2 expression cassette, was constructed by VectorBuilder . Transfection was performed on 60–70% confluent mRTEC cells according to the manufacturer’s instruction of Lipofectamine 2000 . Transfection mixture (per well in 6-well culture plates) consisted of 1.5 μL Lipofectamine 2000, 1.5 μg ultra-pure plasmid SERCA2-GFP, and 900 μL optimum reduced serum-free medium. Cells were washed once with optimum reduced serum-free medium, the transfection mixture was added and incubated for 6 h, and then the cell medium was replaced by fresh completed medium without antibiotics. After being cultured for 48 h, cells were treated with or without Cd2+ for 24 h and cycloheximide  for 0, 3, and 6 h. Total protein was extracted from those treated cells, and the amount of SERCA2 was estimated by Western blotting using anti-SERCA2 antibodies, which were used to determine the half-life of those proteins. Quantification of the relative protein levels of SERCA2 was performed using the software Image J 1.54c.The cells were incubated with 5 μM Cd2 (5 μM) with or without R-467 (1 μM) for 24 h. The cells were fixed for 30 min in 4% Formaldehyde . Then, the cells were permeabilized with 0.1% Triton X-100  in PBS for 20 min. Paraformaldehyde-fixed mice spleen sections were dewaxed, rehydrated in graded ethanol, and rinsed in PBS + 0.1% Tween 20 (PBST). The staining procedure involved incubation of the cells or tissue sections with 3% normal goat serum in PBST to reduce non-specific staining, followed by overnight incubation at 4 °C with rabbit polyclonal antibodies against SERCA2 , p-PLB  KIM-1  antibodies. Then, the cell or sides were incubated with Alexa Fluor 488 goat anti-rabbit IgG  for 1 h at room temperature. The cells or tissues were stained by the DAPI  for several minutes and were examined by Olympus IX73 microscopy . The cells or slides were washed for 3 × 15 min in PBST after each antiserum application. To detect the expression of SERCA2 and p-PLB in mRTEC, the cells were treated with CdCl2+ on intracellular Ca2+ levels in renal cells, mRTEC cells were seeded at a density of 5 × 103 cells/well in 96-well plates. Next day, the cells were loaded with 1 μM Fura-2/AM in the RMPI 1640 medium (phenol red free) for 30 min at 37 °C in the dark. After dye loading, the cells were washed twice with the medium. Adding the medium with CdCl2 (5 μM), R-467 (1 μM) or CdCl2 (5 μM) + R-467 (1 μM), or pretreated with TG (0.1 μM), 2-APB (50 μM). Then, the fluorescent intensity was recorded with excitation at 348 nm and emission at 380 nm using a Synergy H4 Hybrid Multi-Mode Microplate Reader .To detect the transient effect of Cd2+ on cytosolic Ca2+ levels, the cells were treated with CdCl2 (5 μM) for 0–4 h. For long-term effect of ER Ca2+ content, the cells were treated with CdCl2 (5 μM) for 24 h, or pre-treated with regulators, i.e., R-467 (1 μM), CDN1163 (10 μM) for 1 h, followed by treatment with Cd2+ (5 μM) for 24 h, or TG (0.1 μM) for 6 h. Then, the cells were washed twice with RMPI 1640 medium (phenol red free), followed by loading with 1 μM Mag-Fluo-4/AM  in the RMPI 1640 medium (phenol red free) for 30 min at 37 °C in the dark. After dye loading, the cells were washed twice with the medium. Then, the fluorescent intensity was recorded with excitation at 488 nm and emission at 535 nm using a Synergy H4 Hybrid Multi-Mode Microplate Reader . Moreover, cells were loaded with ER-specific dye ER-tracker red  and/or Mag-Fluo-4-AM to indicate that Mag-Fluo-4 was selectively labeled on ER. The co-localization of Mag-Fluo-4 with ER-tracker red was observed by Leica TCS SP5 confocal microscope . For measurement of the short-term effect of Cdt-test or one-way analysis of variance (ANOVA) followed by Duncan’s multiple range test by IBM SPSS Statistics 27.0.1 software as described in figure legends. Groups were considered significantly different if * p < 0.05.Chemical treatments were performed in triplicate in each experiment, and every experiment was repeated at least three times. All data are represented as means ± standard deviation (SD). Statistical significance was assessed with Student’s


Measurement of cellular Ca2+ is routinely performed using fluorescence microscopy with biological indicators. Analysis of deterministic signals is reasonably straightforward as relevant data can be discriminated based on the timing of cellular responses. However, analysis of stochastic, slower oscillatory events, as well as rapid subcellular Ca2+ responses, takes considerable time and effort which often includes visual analysis by trained investigators, especially when studying signals arising from cells embedded in complex tissues. The purpose of the current study was to determine if full-frame time-series and line-scan image analysis workflow of Fluo-4 generated Ca2+ fluorescence data from vascular myocytes could be automated without introducing errors. This evaluation was addressed by re-analyzing a published “gold standard” full-frame time-series dataset through visual analysis of Ca2+ signals from recordings made in pulmonary arterial myocytes of en face arterial preparations. We applied a combination of data driven and statistical approaches with comparisons to our published data to assess the fidelity of the various approaches. Regions of interest with Ca2+ oscillations were detected automatically post hoc using the LCPro plug-in for ImageJ. Oscillatory signals were separated based on event durations between 4 and 40 s. These data were filtered based on cutoffs obtained from multiple methods and compared to the published manually curated “gold standard” dataset. Subcellular focal and rapid Ca2+ “spark” events from line-scan recordings were examined using SparkLab 5.8, which is a custom automated detection and analysis program. After filtering, the number of true positives, false positives, and false negatives were calculated through comparisons to visually derived “gold standard” datasets. Positive predictive value, sensitivity, and false discovery rates were calculated. There were very few significant differences between the automated and manually curated results with respect to quality of the oscillatory and Ca2+ spark events, and there were no systematic biases in the data curation or filtering techniques. The lack of statistical difference in event quality between manual data curation and statistically derived critical cutoff techniques leads us to believe that automated analysis techniques can be reliably used to analyze spatial and temporal aspects to Ca2+ imaging data, which will improve experiment workflow.Intracellular Ca Measurement of cellular Ca2+ is routinely performed using fluorescent microscopy techniques with biological indicators. Classic approaches rely on ratiometric fluorescent indicators including Fura-2 and Indo-1 that were originally designed roughly 40 years ago i was measured in pulmonary arterial myocytes in situ, using an en face preparation with the Ca2+ sensitive dye Fluo-4 AM  and a Zeiss 710 NLO laser scanning confocal imaging workstation  with an inverted microscope (Zeiss Axio Observer) using procedures and processes as described [2+ oscillatory activity in individual cells, which was followed by 30–50 line scans of 18.9 s that were made to measure subcellular Ca2+ spark activity, with 1 line being made in each cell.[Ca2+ oscillations were detected automatically post hoc using the LCPro plug-in for ImageJ, with analysis following the workflow of 2+ responses due to the activation of ryanodine receptors, which are commonly known as Ca2+ sparks [2+ events based on the rise time (between 0.78 and 30.0 s), decay (between 0.78 and 36.72 s), amplitude (between 1.25 and 3.7 F/F0), area (between 6.74 and 108.5 F*s/F0), and duration (between 4.7 and 40 s) of the response. All outlier fencing methods were based on the first and third quartiles  and the interquartile range (IQR). The lower inner fence was Q1—(n × IQR), while the upper outer fence was Q3 + (n × IQR), where n represents 1.5, 3.0, and 5.0. Percentiles, quartiles, and interquartile ranges were obtained via Prism 9.1 . After filtering the data set, each method was compared by determining the number of true positives, false positives, and false negatives and calculating positive predictive value (PPV), sensitivity and the false discovery rate (FDR). The false negative rate was calculated by determining the number of true positive events eliminated from the LCPro derived dataset by each filtering technique under the presumption that LCPro accurately identifies all Ca2+ oscillations. Further statistical analysis was also performed, as mentioned below.As described, regions of interest with Ca+ sparks ,37. Data2+ sparks were recorded in pulmonary arterial myocytes as previously described [2+ spark events and characterized Ca2+ spark morphology and spatial–temporal features of the events. Analysis worked toward the goal of optimizing the threshold used for Ca2+ spark detection and generating critical cutoffs for automated analysis. We initially evaluated data mining techniques based on critical evaluation of over 20,000 Ca2+ spark events that were published in previous studies performed in pulmonary [2+ spark responses above background noise using various thresholds for spark detection including 0.5, 0.6, 0.7, 1.0, and 1.5 above background noise. Notably, our previous projects using various versions of Sparklab have used a threshold of 0.5 for the detection of Ca2+ sparks [2+ spark events were manually observed in the line-scan traces, and comparisons were made to automated detection at various thresholds. From these data, the number of true positives, false positives, and false negatives were determined. This allowed for the calculation of the PPV, sensitivity, and FDR. The resultant analysis of Ca2+ spark events for event amplitude full width half maximum (FWHM), full duration at half maximum (FDHM), and the time constant for decay (Tau) were then filtered using our datamined criteria of amplitude (Lower and Upper), FWHM (Lower amd upper), FDHM (Lower and Upper), and Tau (Lower and Upper), and statistical comparisons made.Caescribed . Processulmonary ,37, uterulmonary , and basulmonary . However+ sparks ,14,38, wThe current studies were based on an analysis of existing data as described in ; thus, n2+ oscillatory signals are presented as violin plots with the median and 25 and 75% quartiles, while Ca2+ spark events are displayed as individual data points with mean and standard deviation. Data were evaluated for normality prior to any comparative statistical analysis. The specific test used is denoted in the figure legend or table including a Kruskal–Wallis One Way ANOVA with a Dunn’s multiple comparison test. p < 0.05 was considered statistically significant, unless otherwise noted. The number of studies performed are provided as the number of events, arteries, animals, or scans examined. The exact details are provided along with each figure. The percentage of cells firing with Ca2+ sparks was computed as the number of individual line scans with events relative to the total number of line scans, with statistical differences examined by Fisher’s exact test.All time-series recordings were graphed and statistical analyses performed with Prism 9.1. Summarized data for the Ca


The endoplasmic reticulum (ER) helps regulate beta-cell cytosolic Ca2+, and ER stress can lead to ER Ca2+ reduction, beta-cell dysfunction, and an increased risk of type 2 diabetes. However, the mechanistic effects of ER stress on individual calcium channels are not well understood. To determine the effects of tunicamycin-induced ER stress on ER inositol 1,4,5-triphosphate receptors (IP3Rs) and ryanodine receptors (RyRs) and their involvement in subsequent Ca2+ dysregulation, we treated INS-1 832/13 cells and primary mouse islets with ER stress inducer tunicamycin (TM). We showed TM treatment increased RyR1 mRNA without affecting RyR2 mRNA and decreased both IP3R1 and IP3R3 mRNA. Furthermore, we found stress reduced ER Ca2+ levels, triggered oscillations in cytosolic Ca2+ under subthreshold glucose conditions, and increased apoptosis and that these changes were prevented by cotreatment with the RyR1 inhibitor dantrolene. In addition, we demonstrated silencing RyR1-suppressed TM-induced subthreshold cytosolic Ca2+ oscillations, but silencing RyR2 did not affect these oscillations. In contrast, inhibiting IP3Rs with xestospongin-C failed to suppress the TM-induced cytosolic Ca2+ oscillations and did not protect beta cells from TM-induced apoptosis although xestospongin-C inclusion did prevent ER Ca2+ reduction. Taken together, these results show changes in RyR1 play a critical role in ER stress-induced Ca2+ dysfunction and beta-cell apoptosis.Pancreatic beta cells maintain glucose homeostasis by secreting pulses of insulin in response to a rise in plasma glucose. Pulsatile insulin secretion occurs as a result of glucose-induced oscillations in beta-cell cytosolic Ca In pancreatic beta cells, Ca2+ triggers insulin secretion to maintain postprandial blood glucose (2+ is sequestered within the endoplasmic reticulum (ER), the organelle where the synthesis and folding of secretory proteins occurs, along with lipid synthesis  is associated with ER stress and apoptosis and accompanies many pathologies  and Ca2+ release by inositol 1,4,5-trisphosphate receptors (IP3Rs) and ryanodine receptors (RyRs)  . Both IPnditions , 14, 15.nditions ; IP3R1 inditions . RyRs arand RyR3 , with Ryand RyR3 . Beta-ceablished .2+ handling and the production of cytosolic Ca2+ ([Ca2+]cyto) oscillations. We used tunicamycin (TM) to experimentally induce ER stress in the rat insulin-secreting INS-1 832/13 beta-cell line, as well as isolated mouse islets. TM activates the UPR by inhibiting N-acetylglucosamine phosphotransferase, leading to protein misfolding in the ER  and led to the appearance of [Ca2+]cyto oscillations in parallel with oscillations in membrane potential    . In respper)   and dantC, 1 μM) , to blocC, 1 μM) , 23, 24,C, 1 μM) .A–C, TM treatment decreased IP3R1 and IP3R3 mRNAs, while the IP3R2 transcript remained unchanged after a 16 h exposure to TM in INS-1 832/13 cells. We also found that RyR1 mRNA was increased by TM treatment, while the level of RyR2 transcript was unchanged (D and E). While we wished to measure RyRs and IP3Rs at the protein level, our efforts to do so were hampered by the lack of commercially available, working antibodies for these ER Ca2+ channel proteins.We first analyzed the effect of TM on the expression of each of the receptor isoforms. As shown in nchanged , D and E2+ (2+ probe D4ER was transiently expressed in islets and INS-1 832/13 cells with adenovirus. Islets and INS-1 832/13 cells were then treated with vehicle control (DMSO), TM, XeC, Dan, TM+XeC, or TM+Dan for 16 h, and [Ca2+]ER was measured using a recording solution containing 5 mM glucose. Representative traces show [Ca2+]ER  in islets and the effect of the reversible SERCA blocker cyclopiazonic acid  reduced ER Ca2+, as expected . Steady-state [Ca2+]ER is measured in 5 mM glucose in islets (B and C) and in INS-1 832/13 cells (D and E) in the absence of CPA. Exposing islets to TM for 16 h significantly reduced steady-state [Ca2+]ER compared to controls, and the presence of either XeC or Dan with TM prevented ER Ca2+ loss. Interestingly, including Dan with TM slightly increased [Ca2+]ER over control levels but had no effect in the controls, perhaps because ER Ca2+ leak through RyRs was low in the controls . Figure 2F shows representative ER Ca2+ responses to CPA in 5 mM glucose in control or TM-treated islets, and we saw that TM-treated islets had lower ER Ca2+ both before and after CPA acute application compared to control. The change in D4ER arbitrary units was calculated by subtracting the unit in steady state at the end of the recordings after CPA application from the unit at the beginning of the recordings before CPA application. We found that the drop in ER Ca2+ was significantly smaller after TM exposure than control .To test whether blocking IP3Rs or RyRs prevented the TM-mediated reduction of ER Ca2+ , the ER expected   (2+]cyto (2+ oscillations in 5 mM glucose (2+ oscillations in stressed islets (A and C); however, TM-triggered oscillations were suppressed when Dan was present (<5% oscillating islets) (B and D), suggesting RyRs but not IP3Rs were involved in their genesis. In Dan or XeC-treated islets, as for DMSO, little or no oscillatory Ca2+ activity was observed in the absence of TM , TM, XeC, Dan, TM+XeC, or TM+Dan for 16 h before recording cyto oscillations in 5 mM glucose, we transfected RyR1 siRNA in INS-1 832/13 cells, resulting in a 50% decrease of RyR1 mRNA (A). Decreasing RyR1 expression may increase RyR2 mRNA levels, but not significantly (B). IP3R1 mRNA was found to be elevated (C). Transfected cells were recorded using imaging buffer containing 5 mM glucose (D). After 16 h of TM treatment, the percentage of cells displaying [Ca2+]cyto oscillations was decreased in RyR1-knockdown cells (∼10%) compared to siCon cells (∼40%) (E). RyR1 knockdown did not affect the Ca2+ activity of unstressed control cells (D and E).To confirm that RyR1 played a role in the production of TM-triggered cyto oscillations in 5 mM glucose, we silenced RyR2 using siRNA in INS-1 832/13 cells and achieved a ∼50% reduction in RyR2 mRNA (A). Neither RyR1 or IP3R1 mRNA was changed after RyR2 knockdown (B and C). The cytosolic Ca2+ levels of the transfected cells were recorded using imaging buffer containing 5 mM glucose (D). The percentage of cells displaying [Ca2+]cyto oscillations in response to 16 h of TM treatment in this case was unaffected by silencing RyR2 (E).RyR2, rather than RyR1, is the predominant RyR isoform of beta cell in terms of abundance . To studyR2 mRNA A. Neithenockdown , B and C glucose D. The peing RyR2 E.Figure XBP1 reached its peak at this time , ATF4 (B and E), or CHOP (C and F), which we observed when these inhibitors were included with TM. Thus, blocking IP3Rs or RyR1 failed to prevent UPR activation in TM-treated beta cells.Activation of the UPR occurs in response to TM in many cell types, including beta cells , 20, 30.tal XBP1 , A and DD), ATF4 , B and E or CHOP , C and FATF4 and CHOP expression in RyR1, RyR2, or RyR1+2 knockdown cells and observed that neither ATF4 nor CHOP upregulation was reversed by silencing these isoforms individually or collectively (A and B). To further confirm this, we also examined the effect of blocking RyRs on UPR activation using ryanodine (Ry), a classic nonspecific RyRs blocker .To confirm that blocking RyRs does not affect TM-triggered UPR activation, we assessed ectively , A and Bctivator ). As shonockdown , A and B2+ oscillations, and the inclusion of Dan lowered the percentage to 10% (A and B). In addition, we saw increased ratio of spliced XBP1/total XBP1, ATF4, and CHOP mRNA after a 6 h treatment that was not affected by Dan (C–E) or Ry. Although Ry seemed to suppress TM-induced upregulation of XBP1s/XBP1t ratio and CHOP mRNA, the difference was not statistically significant (F and G).We also treated islets with a much lower dose of TM (300 nM) for 24 h and verified if such low dose of TM could trigger UPR activation. We observed that 90% of TM-treated islets exhibited subthreshold Cae to 10% , A and Bd by Dan , C–E or nificant , F and G2+]cyto oscillations (CHOP mRNA levels (A) in INS-1 832/13 cells. We then measured RyR isoforms and IP3R1 mRNA levels in the cells and observed an upregulation of RyR1 mRNA after high glucose culturing, although not significant (p = 0.0832) (B), and unchanged RyR2 (C) or IP3R1 mRNA levels (D). We next tested whether XeC or Dan affected [Ca2+]cyto oscillations (A). Dan significantly reduced the percentage of islets exhibiting oscillations seen following HG treatment from ∼70% to ∼20% (D), and XeC decreased it to ∼30% (average).We previously reported that culturing mouse islets in high glucose  for 16 h induced subthreshold cyto oscillations in TG -treated mouse islets (F), while XeC did not show much affect (I).Thapsigargin (TG) is another widely used ER stress chemical inducer . We preve islets F, while h affect I.Programmed cell death or apoptosis has been shown to occur in beta cells in response to prolonged ER stress , 34, 35.db/db mice, an animal model of type 2 diabetes. These mice harbor a mutation in the leptin receptor and have been shown to exhibit a left shift in their glucose sensitivity over time (db/db mice compared to control mice (db/db mice rapidly gained weight (34.73 ± 4.996 g SD) compared to their heterozygous controls (19.30 ± 4.073 g SD) and developed hyperglycemia , while db/+ mice maintained normal blood sugar . After isolation, islets from the mice were treated with control DMSO or Dan for 16 h before [Ca2+]cyto measurements were carried out. As shown in Figure 11A, islets from db/+ mice treated with vehicle lacked oscillations in 5 mM glucose, and as expected, exhibited normal oscillations in response to 11 mM glucose; these responses were unaffected by pretreatment with Dan. In contrast, db/db mice exhibited oscillations in 5 mM glucose and plateaus in 11 mM glucose. In these islets, the inclusion of Dan abolished the oscillations and decreased the percentage of oscillating islets observed from ∼70% to 25% in 5 mM glucose (B), without affecting the plateaus seen in 11 mM glucose (A).To test the possible relevance of these results to diabetes, we extended our study to islets isolated from ver time  and uprerol mice , 39. We  glucose B, withou glucose A.Figure 2+]cyto oscillations and increased insulin secretion observed in ER-stressed beta cells in subthreshold glucose are triggered by ER Ca2+ reduction and SOCE activation   , 40. How2+]ER in 5 mM glucose, blocking SERCA with CPA further decreased basal [Ca2+]ER (F), but to a lesser degree compared to control (G) as [Ca2+]ER was lower in TM-treated beta cells (B and C). While blocking IP3Rs with XeC also prevented a loss in [Ca2+]ER in TM-treated islets (B), the IP3R blocker XeC failed to inhibit TM-induced subthreshold [Ca2+]cyto oscillations (C) or beta-cell apoptosis (A). On the other hand, blocking RyR1 with Dan suppressed the TM-induced subthreshold [Ca2+]cyto oscillations (D) and beta-cell apoptosis (B) in addition to preventing the reduction in [Ca2+]ER induced by TM (C). Similar results were reported in a study of Dan as a potential treatment for Wolfram syndrome, a rare autosomal recessive disorder that is associated with childhood-onset diabetes mellitus as well as sensory and neurological deficits . Moreover, Dan inclusion  also inhibited TG-induced cell death in neural progenitor cells derived from the induced pluripotent stem cells of a Wolfram syndrome patient , we also exposed islets to glucotoxic  or GLT conditions  to mimic the milieu beta cells are exposed to in T2D; these treatments also trigger ER stress (A) . In addition, we observed that subthreshold [Ca2+]cyto oscillations were prevented by Dan in islets from db/db mice . W. WC), we/db mice . These det al. showed that blocking RyRs with ryanodine prevented TM-induced sub-threshold [Ca2+] oscillations and cell apoptosis and suggested RyR2 to be the mediator due to its abundance in beta cells , in accordance with results we obtained using Dan or ryanodine (D–F and C and D). These data taken collectively suggest that [Ca2+]ER reduction in our system may be downstream of UPR activation by TM, although we are not ruling out other mechanisms, such as reducing SERCA activity (Yamamoto ta cells . As ryanta cells ), we sil did not  and 5. M did not , A and B D–F and , C and Dactivity , 44.2+ depletion to ER stressors nonetheless had differential effects on beta-cell apoptosis and SOCE activation/subthreshold [Ca2+]cyto oscillations seems paradoxical, if ER Ca2+ depletion is indeed causative. However, it may be that the two classes of ER Ca2+ channels have different spatial distributions such that RyRs due to their localization within the cell are capable of selectively activating ER Ca2+ release in the vicinity of the SOCE mechanism. Thus, these receptors may not be uniformly distributed within the cell but selectively and dynamically organized into specific subcellular domains (2+ release-activated Ca2+ channel protein 1 (ORAI1) are the molecular subunits of SOCE channels. STIM1 resides in the ER membrane where it monitors the level of Ca2+ in the ER lumen (2+ entry from the extracellular space into the cytosol (SOCE)  , 41. In than Dan .2+, individual portions of the ER may be too small or poorly sampled by D4ER to resolve differences in these very small and highly localized subdomains. For example, there may be portions of the ER Ca2+ reticular network where RyR1 is dominantly expressed over RyR2 or IP3Rs and which is in closer proximity to SOCE. Therefore, although inhibiting IP3Rs prevented a global ER Ca2+ loss in response to TM, SOCE activation was not inhibited because ER Ca2+ was still low in a STIM1 localized subcompartment of the ER. On the other hand, RyR1, despite its relatively lower abundance in beta-cells, is selectively localized near STIM1 and ORAI1, making the SOCE compartments very sensitive to Ca2+ release by RyR1 localized to this ER Ca2+ pool. Resolving the spatial localization of these molecules within the ER will require higher resolution imaging approaches than that used here, ones that can quantitatively and accurately assay the subcellular localization of RyR1, IP3Rs, STIM1, and ORAI1 under various ER stress conditions.For this scenario to hold, however, we posit that while D4ER indeed measures global ER Ca2+ loss and downstream alterations in beta-cell function and viability. Combining these new data with existing knowledge of RyRs and IP3Rs suggests that RyR1 might be a potentially useful therapeutic target for treatment during the onset or progression of type 2 diabetes.In summary, the present report demonstrates that RyR1 is a critical player in ER stress-induced ER CaTM, CPA, TG, and XeC were obtained from Cayman Chemical, Ann Arbor, MI. Dan was from Sigma-Aldrich, St Louis, MO. Ryanodine (Ry) was from Abcam, Waltham, MA. RNeasy mini kit for RNA extraction was from Qiagen, Germantown, MD. Superscript RT II was from Invitrogen, Carlsbad, CA. SYBR Green PCR master mix was from Applied Biosystems, Bedford, MA. Primers for qRT-PCR were from Integrated DNA Technologies, Coralville, Iowa. Small interfering RNAs (siRNAs) were from ThermoFisher scientific, Waltham, MA. The catalog and lot numbers of these materials can be found in db/db mice (BKS.Cg-Dock7m+/+Leprdb/J) and female heterozygous mice at 5 to 7 weeks of age, as described  and female escribed . Animal escribed .2 humidified atmosphere and used for experiments after reaching ∼70% confluency. INS-1 832/13 cells were transfected with RyR1-specific siRNA, RyR2-specific siRNA, or negative control siRNA using lipofectamine RNAiMAX reagent as described in the manufacturer’s protocol (Invitrogen). Transfection was assessed by qPCR.INS-1 832/13 cells were grown in standard RPMI 1640 medium as described above in 6-well plates at 37 °C in a 5% COTotal RNA was extracted from INS-1 832/13 cells and reverse-transcribed to cDNA as described . qPCR wa2, 5 KCl, 2 MgCl2, 10 HEPES, and 5 glucose. Similarly, INS-1 832/13 cells seeded on glass coverslips were loaded with Fura-2/AM (2.5 μM) for 30 min in standard RPMI 1640 medium containing 11 mM glucose. Coverslips containing INS-1 832/13 cells were transferred to the perfusion chamber and imaged in imaging buffer. Ratiometric Fura-2/AM imaging was performed using 340/380 nm excitation and collecting 502 nm emission, as previously described (Islets were loaded with Fura-2/AM (2.5 μM) for 45 min in RPMI medium including 5 mM glucose. Islets were then transferred to a 1 ml perfusion chamber containing imaging buffer for 6 min, followed by perfusion at ∼1 ml/min. Imaging buffer contained (in mM): 140 NaCl, 3 CaClescribed . Fluores2+]ER was measured using an ER-localized FRET biosensor D4ER . Dz was included to keep the KATP channel in its open state to prevent oscillatory Ca2+ activity and improve the signal/noise ratio and stability of the ER Ca2+ recordings. FRET ratios were acquired using Metafluor software, plotted using Prism, and mean values were calculated using Excel.[Casor D4ER . The samINS-1 832/13 cells were harvested and prepared for sub-G1 apoptosis assay as described . The pert test (Prism) with hypothetical value set for 1.0 or student’s t test when comparing two groups as indicated in the figure legends. Differences between two or more groups were analyzed using ordinary one-way ANOVA or two-way ANOVA (Prism) as specified in the figure legends with post hoc multiple comparisons by Tukey’s procedure. Values of p < 0.05 were considered statistically significant.Data were expressed as means ± SD and analyzed using one sample All data are contained within the manuscript.This article contains The authors declare that they have no conflicts of interest with the contents of this article.