However, the molecular identity of the transport system remains elusive. Previous studies have demonstrated that isolated SVs exhibit two distinct Ca2+ transport systems depending on extra-vesicular (cytosolic) pH; one is mediated by a high affinity Ca2+ transporter which is active at neutral pH and the other is mediated by a low affinity Ca2+/H+ antiporter which is maximally active at alkaline pH of 8.5. In addition, synaptic vesicle glycoprotein 2 s (SV2s), a major SV component, have been proposed to contribute to Ca2+ clearance from the presynaptic cytoplasm. Here, we show that at physiological pH, the plasma membrane Ca2+ ATPases (PMCAs) are responsible for both the Ca2+/H+ exchange activity and Ca2+ uptake into SVs. The Ca2+/H+ exchange activity monitored by acidification assay exhibited high affinity for Ca2+ (Km ~ 400 nM) and characteristic divalent cation selectivity for the PMCAs. Both activities were remarkably reduced by PMCA blockers, but not by a blocker of the ATPase that transfers Ca2+ from the cytosol to the lumen of sarcoplasmic endoplasmic reticulum (SERCA) at physiological pH. Furthermore, we rule out the contribution of SV2s, putative Ca2+ transporters on SVs, since both Ca2+/H+ exchange activity and Ca2+ transport were unaffected in isolated vesicles derived from SV2-deficient brains. Finally, using a PMCA1-pHluorin construct that enabled us to monitor cellular distribution and recycling properties in living neurons, we demonstrated that PMCA1-pHluorin localized to intracellular acidic compartments and recycled at presynaptic terminals in an activity-dependent manner. Collectively, our results imply that vesicular PMCAs may play pivotal roles in both presynaptic Ca2+ homeostasis and the modulation of H+ gradient in SVs.Ca The V-ATPase generates both a pH gradient (ΔpH) and a membrane potential  across the SV membrane. Depending on the chemical properties of neurotransmitters and the intrinsic characteristics of vesicular transporters, the dependence of neurotransmitter uptake on either driving force varies among the neurotransmitters. In principle, the uptake of cationic transmitters such as monoamines and acetylcholine depends primarily on ΔpH, whereas the uptake of anionic transmitters such as glutamate and ATP depends more on ΔΨ, although pivotal roles of protons in glutamate transport have also been suggested4. The uptake of zwitter-ionic transmitters such as GABA and glycine depends equally on both ΔpH and ΔΨ . Therefore, a balance between ΔpH and ΔΨ across individual SVs differentially regulate transport rates and storage of neurotransmitters in SVs1.Synaptic vesicles (SV) are storage organelles for neurotransmitters and play a key role in synaptic transmission. Vesicular transport of classical neurotransmitters including glutamate, GABA/glycine, biogenic monoamines, acetylcholine, and adenosine triphosphate (ATP) is mediated by the respective vesicular transporters for these neurotransmitters. Vesicular transport is driven by a proton electrochemical gradient generated by the vacuolar-type HΨ is regulated by the permeability of the SV membrane to various charged ions and their concentration gradients6. In fact, SVs contain various transporters and/or channels for ions that directly or indirectly modulate H+ flux across SVs. For instance, it has been demonstrated that SVs exhibit a Cl− conductance that serves a shunting current for H+, resulting in an increase in net proton influx and thus a generation of larger ΔpH8. In the case of SVs that store glutamate, the vesicular glutamate transporters (VGLUTs) are proposed to confer the Cl− flux10 . The contribution of the chloride channel (CLC) family has been proposed, especially for SVs that transport other neurotransmitters, but their physiological relevance remains controversial15. More recently, cation/H+ exchange activity conferred by either Na+/H+ exchangers (NHEs)16 or VGLUTs10 (in the case of glutamate-containing vesicles) were shown to decrease ΔpH and consequently facilitate ΔΨ. In stark contrast, the contribution of divalent cations such as Ca2+ and Zn2+ has received less attention. To date, the mechanism and molecular entity of the transporting molecule(s) remain elusive.The balance between ΔpH and ΔTorpedo marmorata revealed that SVs exhibited an ATP-dependent active Ca2+ transport activity19. Consistent with this, a transient increase of Ca2+ in the SV lumen was observed after stimulation at the cholinergic synapses of the electric organ of Torpedo marmorata20, suggesting that SVs can function as a Ca2+ store at presynaptic terminals in a physiological context. Efforts to decipher the Ca2+ transport protein(s) in mammalian SVs have revealed that at least two distinct mechanisms may exist: vanadate-sensitive P-type Calcium ATPases, which are fully active at neutral pH, and a molecularly unidentified Ca2+/H+ antiporter, which functions maximally at alkaline pH of 8.523. These two transport systems exhibit substantial differences in their affinities for Ca2+. While Km of the vanadate-sensitive Ca2+ ATPases lies in the order of several hundred nM, Km of the Ca2+/H+ antiporter exceeds 300 µM, which far surpasses physiological Ca2+ concentrations at presynaptic terminals24. Moreover, these two transport systems exhibit characteristic dependence on Ca2+ concentrations. Notably, Ca2+ ATPase activity increases with Ca2+ concentration in the medium, reaching a maximum at ~25 µM, whereas its activity is inhibited at higher Ca2+ concentrations and is completely abolished at ~500 µM Ca2+ 22.Classical studies using SV fraction isolated from the electric organ of 2+ transport systems, it has long been conjectured that one of the major SV proteins, SV2s (synaptic vesicle glycoprotein 2s), may function as a Ca2+ transporter in SVs25, because the genetic deletion of SV2s revealed synaptic phenotypes that could be well-explained by the increase in cytoplasmic [Ca2+]27 (but see28). Nevertheless, the relative contributions of these proteins to Ca2+ transport into SVs has not been elucidated, and whether/how the activities of these proteins may affect ΔµH+ to influence neurotransmitter uptake remains unclear.Aside from these two SV Ca2+ transporting protein(s) in SVs and how their activity influences ΔµH+ of SVs. Using a fast Ca2+ chelator BAPTA, instead of EGTA that was previously used to control free Ca2+ concentrations in the buffer29, we show that SVs isolated from rodent brains exhibit high affinity Ca2+/H+ exchange activity even at neutral pH that would attenuate ΔpH in SVs. Biochemical properties of the exchanger such as the affinity for Ca2+, ion selectivity, and sensitivity to specific inhibitors resemble those of the plasma membrane Ca2+ ATPases (PMCAs). Furthermore, we ruled out a contribution of SV2s to Ca2+ uptake into SVs by analyzing vesicle fractions from SV2-deficient brains. We further demonstrated using a pH-sensitive fluorescent protein (pHluorin)30 as a reporter that PMCA1, an isoform of PMCA expressed in the mammalian nervous system, indeed resides mainly on SVs and is capable of recycling at presynaptic terminals in an activity-dependent manner. Given the predominant role of PMCAs in regulating Ca2+ homeostasis at presynaptic terminals31, our results imply that vesicular PMCAs play dual roles at presynaptic terminals in Ca2+ homeostasis and in modulating ΔµH+ in SVs via Ca2+/H+ antiport activity.In this study, we aimed to clarify the molecular identity of the Ca2+/H+ antiport activity relied essentially on an acidification assay in which fluorescence quenching of acridine orange due to V-ATPase-dependent acidification was reversed by addition of Ca2+ 23. This activity was characterized by its low affinity (Km > 200 µM) and operated at pH 8.5. However, chelation of Ca2+ by EGTA would result in rapid liberation of H+ from EGTA, and the ability of Ca2+ chelation by EGTA depends critically on pH. As such, addition of Ca2+ in the presence of EGTA during the measurement may have caused a change in pH, raising the possibility that the affinity and pH dependence may have been disturbed. Therefore, we decided to use BAPTA whose KD is similar to that of EGTA, but the ability to chelate Ca2+ is much less affected by pH changes. First, we tested the effect of Ca2+ in the presence of BAPTA at neutral pH of 7.2, and compared with that in the presence of EGTA  under the same conditions as those in Fig. 10) was estimated to be ~1.6 . To this end, we first estimated free Ca2+ concentrations in our assay buffers using fura-2 as a Ca2+ indicator and found it to be ~1 µM . After complete acidification of SVs in the presence of 100 mM KCl was achieved, we added a series of Ca2+ concentrations ranging from 0 to 75 µM that were equivalent to 3.63 to 6410 nM free [Ca2+] in the assay medium. With the ranges of Ca2+ added, the rate of alkalization increased as a function of free Ca2+ concentrations 34. However, contributions of these ATPases to SV acidity due to a H+ coupling have not been directly investigated. Furthermore, according to synaptic phenotypes related to changes in presynaptic Ca2+ concentrations and in presynaptic calcium buffering in SV2-deficient neurons27, it has been hypothesized that SV2s may be involved in Ca2+ transport into SVs. We therefore sought to clarify which protein was responsible for the observed Ca2+/H+ exchange activity in SVs at neutral pH.As described in the Introduction, there are several candidates responsible for Ca2+/H+ exchange, we generated SV2A/2B- and SV2B/2C-double knockout (DKO) mice using the CRISPR/Cas9 system. We then tested if Ca2+/H+ exchange activity was affected. Western blot analysis of LP2 fractions purified from SV2A/2B-DKO or SV2B/2C-DKO mice using isoform-specific antibodies confirmed efficient gene knockout by the CRISPR/Cas9 system . Again, LEV did not reduce Ca2+-induced alkalization  is an established second-generation antiepileptic drug, whose molecular target is SV2Ange Fig.  in SVs fion Fig. . Taken t2+ ATPases, including PMCAs and SERCA. Among the available inhibitors, 500 µM vanadate, a PMCA blocker, potently inhibited Ca2+-induced alkalization of SVs  to background levels measured in the absence of ATP  was not significantly different from that derived from wild-type mice  was conjugated to the first luminal loop of PMCA1  that was similar to the reported values for PMCAs44, whereas previously reported Ca2+/H+ exchange activity at alkaline pH was characterized as a low affinity Ca2+/H+ exchanger (K0.5 ~ 217 µM)22. Third, although PMCAs have been characterized as residing in the plasma membrane, our imaging analysis revealed that PMCAs were potentially capable of recycling in an activity-dependent manner at presynaptic terminals. This observation was further substantiated by the fact that recycling of PMCA1-SEP depended exclusively on the presence of synaptobrevin/VAMP2 on the same vesicles, which is essential for stimulus-dependent exocytosis of SVs45.In this study, we characterized the biochemical properties of Ca+ ATPases, vanadate-sensitive P-type ATPases constitute the major ATPases in cholinergic SVs from Torpedo electric organs46 as well as clathrin-coated vesicles from bovine brains47. Although PMCAs, which are P-type ATPases, have been characterized as plasma membrane residents, our results demonstrate that PMCAs constitute the major ATPase on recycling SVs as well. This is also consistent with proteomic analyses that identified PMCAs within SV fractions48. Thus, besides the main role of PMCAs in excluding Ca2+ from the presynaptic cytosol to the extracellular space at the plasma membrane, vesicular PMCAs may also contribute to Ca2+ clearance from the presynaptic cytosol. Furthermore, since Ca2+ transport into SVs is coupled to H+ efflux, vesicular PMCAs may be involved in the regulation of ΔµH+ of SVs, which drives neurotransmitter uptake into SVs. Indeed, external (cytosolic) Ca2+ strongly inhibited dopamine uptake into isolated SV fraction49, which is reasonable given that ΔpH, the driving force for dopamine uptake, is attenuated by Ca2+/H+ exchange. In contrast, glutamate uptake, which is predominantly driven by ΔΨ, was only marginally facilitated under the condition where ΔΨ predominated in the presence of 17 mM Cl−49. This subtle effect by Ca2+ on glutamate transport indicates that PMCAs on SVs are electro-neutral pumps (Ca2+:H+  = 1:2), which is consistent with observations in snail neurons43. Alternatively, the fact that glutamate transport in the presence of Ca2+ retained a typical biphasic Cl− dependency49 (note that increasing Cl− concentrations build up greater ΔpH) may indicate an important contribution of protons (or vesicular pH) on VGLUT function4. Further investigations will be needed to clarify how Ca2+/H+ exchangers in conjunction with vesicular Cl− carriers, either VGLUTs or the ClC-type Cl− transporters, affect glutamate transport into SVs.Our results are highly consistent with biochemical evidence that, in addition to V-type H31. Our imaging analysis indicated that a substantial portion of vesicular PMCAs are inserted into the plasma membrane by exocytosis and retained there until endocytosis is completed, indicating that the total PMCA expression on the plasma membrane increases, especially during high activity. Although the contribution of the vesicular PMCAs that are inserted into the plasma membrane to acidification of the cytoplasm seems to be negligible31, it remains to be determined to what extent Ca2+ extrusion from the cytoplasm is accelerated by the vesicular PMCAs that translocate into the plasma membrane during high neural activity.The plasma membrane PMCAs are responsible for transient acidification of the cytoplasm during sustained stimulation at mouse motor nerve terminals, which may regulate endocytosis50. PMCA1 and PMCA4 are expressed ubiquitously, whereas PMCA2 and PMCA3 are expressed predominantly in the central nervous system. Furthermore, each isoform has multiple splice variants, comprising more than 30 spliced isoforms, although little is known about the functional significance of multiple splice variants50. In this study, we show that one of the PMCAs, the full length PMCA1  fused to pHluorin at its luminal region, is sorted preferentially to recycling SVs rather than to the plasma membrane at presynaptic terminals, and its distribution and recycling behaviors are very similar to those of the SV marker, synaptophysin  have been identified in PMCA genes that are associated with neuronal disorders such as autism and deafness53, it will be crucial to establish how these SNPs affect the function and distribution of PMCAs in neurons to elucidate the mechanisms underlying these diseases.In summary, our present results collectively suggest that Ca54. Briefly, whole brains from C57BL/6 mice were homogenized in homogenization buffer . The homogenate was centrifuged fro 10 min at 1,000 × g, the resulting supernatant (S1) was centrifuged for 15 min at 12,000 × g. The resulting pellet (P2) was washed with homogenization buffer and re-centrifuged for 15 min at 13,000 × g to obtain crude synaptosomes (P2′). To release SVs from the synaptosomes, P2′ fraction was subjected to an osmotic shock by the addition of 9 volume of ice-cold water and the subsequent homogenization. The resulting suspension was centrifuged for 20 min at 33,000 × g. After centrifugation, the supernatant  was centrifuged for 2 hours at 260,000 × g. The final membrane pellet (LP2) was resuspended in standard acidification buffer , and stored at −80 °C until use. Essentially, all fluorometric assays, unless indicated otherwise, were performed with this fraction. For determination of apparent affinity of Ca2+/H+ exchange activity and radioactive45 Ca2+ uptake, LP2 obtained from Wistar rat brains was further purified by sucrose gradient centrifugation (50–800 mM continuous gradient). After centrifugation at 65,000 × gmax for 4 hours, turbid materials visible in the middle of the gradient (in the range of 200 to 400 mM sucrose) were pooled and sedimented by centrifugation at 260,000 × gmax for 90 min. The resulting pellet (SV) was resuspended in acidification buffer and stored at −80 °C until use.Crude synaptic vesicle fraction (LP2) was isolated from mouse whole brains with slight modifications2+ binding with BAPTA ([CaBAPTA]), concentration of Mg2+ binding with BAPTA ([MgBAPTA]), concentration of Mg2+ binding with ATP ([MgATP]) and that of Ca2+ binding with ATP ([CaATP]) as follows.Free calcium concentration was calculated by solving simultaneous equations in four unknowns: concentration of Ca2+]T, [Mg2+]T, [BAPTA]T and [ATP]T are the total concentrations of each substance. [Ca2+]T is calculated from an equation 2+]add and [Ca2+]ctm are concentration of added CaCl2 and of contaminated Ca2+ determined routinely by fura-2 assay, respectively. K′CaBAPTA, K′MgBAPTA, K′CaATP and K′MgATP are the overall apparent association constants. Microsoft Excel Solver was used to solve the equations.[CaK′s  were converted from the absolute association constants, Ks, which were determined for standard conditions (see details in Marks and Maxfield55). Supplementary Table ΔH values for BAPTA and ATP. ΔH values were necessary for the conversions from the absolute association constants to the overall apparent association constants.To set up the equations above, the overall apparent association constants, 3. Changes in AO fluorescence  were monitored in a Hitachi F2500 fluorometer  at 32 °C, unless otherwise stated3. Typically, 20 µg of LP2 or SV fraction was preincubated in 1 mL of assay buffer  with varying composition of 5 mM K-glutamate, 3 mM or 100 mM KCl, 50 µM EGTA, and 50 µM BAPTA as indicated in the figures or figure legends. After a stable baseline was achieved , 2 mM ATP was added to start acidification. Various concentrations of CaCl2 or 50 µM other divalent cations were added at 10 min where indicated. At the end of recordings, a V-ATPase inhibitor, bafilomycin A1 (500 nM) was added to ensure that quenching of AO was due to proton translocation by the V-ATPase. For Figs 10) for the Ca2+-dependent AO de-quenching, acidification assays were performed at two different temperatures. The Q10 was calculated from an equation:1 and τ2 are time constants of recovery phases of acridine orange fluorescence after the addition of Ca2+ at temperature T1 and T2. τ1 and τ2 were obtained by a first order exponential fitting using a Solver function in Excel software. T1 and T2 (T2 > T1) are 32.3 and 37.3 °C, respectively.Acidification measurements were performed according to previous publications using acridine orange  as a ΔpH reporter2+ concentrations in some experimental solution were measured using fura-2  as a Ca2+ reporter. Changes in fura-2 fluorescence  were monitored in a Hitachi F2500 fluorometer  at 32 °C. 1 ml of an assay buffer  was preincubated for 10 min. The preincubation was followed by adding 300, 600, 900, 1200 and 1500 nM total exogenous CaCl2 each with 30 sec intervals. At the end of recordings, 50 μM BAPTA was added to chelate exogenous and contaminated Ca2+.Caad libitum. Animals were kept in an SPF facility with a 12-hour light and 12-hour dark cycle. The ambient temperature was maintained around 21 °C with a relative humidity of 50%. ICR mice (12 to 20 weeks old) were used as recipients. A combination anesthetic  was used for surgery. The anesthetics were administered to recipient mice by intraperitoneal injection. All animal experiments were approved by the Institutional Animal Care and Use Committee of the RIKEN Kobe branch  and the Institutional Animal Care and Use Committee of Doshisha University.C57BL/6NJcl mice were purchased from CLEA, Japan. ICR mice were purchased from SLC, Japan. All mice were given food and water 2 incubator at 37 °C. Cas9 mRNA (100 ng/µL) and six gRNAs  were co-injected into the cytoplasm of fertilized eggs in M2 medium (Merck Millipore) at room temperature. Details of the cytoplasmic injection procedure have been described previously56. After microinjection, the injected embryos were cultured for 1 hr in KSOM medium (Merck Millipore) in a 5% CO2 incubator at 37 °C, then 15–30 embryos were transferred to the oviducts of recipient ICR female mice.C57BL/6N females (4–6 weeks old) were superovulated and mated with C57BL/6N males. Fertilized eggs were collected from the ampulla of the oviduct of plugged C57BL/6N females by micro-dissection and kept in KSOM medium (Merck Millipore) in a 5% CO57. Briefly, Cas9 mRNA and sgRNAs were synthesized according to the protocol reported previously57. All gRNAs were selected from pre-made design in Database (http://crispr.riken.jp). Cas9 mRNA (100 ng/µL) and six gRNAs  were injected into the cytoplasm of fertilized eggs of C57BL/6NJcl mice. For SV2A/2B DKO, six gRNA targets were used . For SV2B/2C DKO, six gRNA targets were used .Double gene knockout (DKO) mice of SV2A/2B and SV2B/2C were generated by the triple-target CRISPR method45Ca2+ dissolved in sucrose buffer containing ATP (2 mM), vanadate (500 µM), eosinY (10 µM), cyclopiazonic acid (15 µM) and levetiracetam (30 µM) when indicated. All incubations contained 0.125% (v/v) DMSO and 0.167% (v/v) ethanol in a final volume of 150 µL. After incubation for 10 min at 32 °C, the reaction was stopped by addition of 3 mL of ice-cold buffer, followed by filtration through nitrocellulose filters. The incubation tubes were washed out with 3 mL of ice-cold buffer, and the wash out buffer was poured through the filter. The filters were washed out twice with 2.5 mL of ice-cold buffer. Radioactivity retained on filters was measured by liquid scintillation counting using an ALOKA LSC-6100 liquid scintillation counter .The concentrations of SV or LP2 fractions were adjusted to 0.2 mg/mL in sucrose buffer. 100 µL aliquots were preincubated for 10 min at 32 °C. The reaction was started by addition of 10 µM  SDS-PAGE was used to separate 20 µg of LP2 fractions from wild-type or SV2-DKO mice. Proteins were transferred to a PVDF membrane. The resulting blots were probed with isoform specific antibodies for SV2s  or with a pan-SV2 monoclonal antibody . For detection, the appropriate secondary antibodies conjugated to horseradish peroxidase were used. After washing steps, the horseradish peroxidase was detected by enhanced chemiluminescence using a commercially available kit . As loading controls, anti-synaptophysin monoclonal antibody  was used.StuI site of pCR-Blunt vector (Thermo Fisher Scientific). A DNA fragment encoding the N-terminal region of PMCA1 (a.a. 1–139) with linker sequence (STSGGSGGTGGS) and a fragment of super-ecliptic pHluorin (SEP) amplified from SypHy plasmid59 (a kind gift from Leon Lagnado) were amplified by PCR and cloned into an EcoRV site of pcDNA3.1(+) using In-Fusion Cloning Kit (Clontech). A DNA fragment encoding the C-terminal region of PMCA1  was PCR-amplified and cloned into a NotI site of pcDNA3.1(+) which contained the N-terminal region of PMCA1 and SEP using In-Fusion Cloning Kit. To construct Syntaxin1a-SEP, a full length mouse Syntaxin-1a (accession no. NM_016801.3) without the stop codon and a fragment of SEP were amplified by PCR and simultaneously cloned into a NheI/EcoRV site of pcDNA3.1(+). A DNA fragment encoding SypHy59 was amplified by PCR and subcloned into a NheI/XbaI site of pcDNA3.1(+).To construct PMCA1-SEP, a full length mouse PMCA1 (accession no. NM_001359506.1) was amplified by PCR from mouse adult brain cDNA generated using SuperScript RT-PCR system (Invitrogen) and subcloned into a 60 with slight modifications. Briefly, hippocampi were dissected and were incubated with papain  for 20 min at 37 °C. After digestion, hippocampal cells were plated onto poly-D-lysine-coated coverslips in 24- or 12-well plates  at a density of 20,000 cells/cm2 and kept in a 5% CO2 humidified incubator. At 2–4 days in vitro (DIV), 40 μM FUDR (Sigma) and 100 μM uridine (Sigma) were added to inhibit the growth of glial cells. One-fifth of the culture medium was replaced with fresh medium every 2–4 days. Cultures were transfected with plasmids encoding either PMCA1-SEP, SypHy, or Syntaxin-1a-SEP at 5–7 DIV using CalPhosTM mammalian transfection kit (Clontech) in accordance with a calcium phosphate transfection method which is optimized for neuronal cultures61, and were subjected to experiments at 12–14 DIV. Animals for the primary neuron cultures were treated according to our institutional guidelines for the care and use of animals (Doshisha University).Primary hippocampal cultures were prepared from embryonic day 16 ICR mice as described previouslyAt DIV12, neural cells were fixed with 4% (wt/vol) paraformaldehyde in phosphate buffer (Wako) for 10 min at room temperature (RT). After washing with phosphate buffered saline (PBS), the neurons were permeabilized with PBS containing 0.1% Triton X-100 for 20 min at RT, and incubated with PBS containing 10% (vol/vol) fetal bovine serum (FBS) and 0.1% Triton X-100 for 30 min at RT. The cells were incubated with rabbit polyclonal anti-GFP antiserum  and mouse monoclonal anti-synaptophysin antibody  (both were kind gifts from Reinhard Jahn) for 1 hour at RT. The cells were rinsed three times with PBS, and further incubated with Alexa-488-conjugated anti-rabbit IgG  and Alexa-555-conjugated anti-mouse IgG  for 30 min at RT. After washing steps, Alexa-488 or Alexa-555 fluorescence was acquired with 470/22 nm excitation and 514/30 nm emission filters or 556-to 570-nm excitation and 600- to 650-nm emission filters, respectively.2, 1 MgCl2, 0.02 CNQX, and 0.025 D-APV (pH 7.4). A solution containing 50 mM NH4Cl (pH 7.4) was applied directly onto the area of interest with a combination of a fast flow exchange microperfusion device and a bulb controller, both of which were controlled by Clampex 10.2. To estimate luminal pH and surface fraction of the SEP probes, a MES-buffered solution at pH 5.5 and 50 mM NH4Cl (pH 7.4) were successively applied to cultured neurons as described previously35. Briefly, SEP fluorescence during acid quenching (FQ) and during subsequent NH4Cl application (FNH4Cl) was measured. The observed fluorescence in a given terminals is thought to be the sum of the fluorescence derived from the surface fraction of the probes (S) that experiences extracellular pH and from the vesicular fraction (1 − S) that is exposed to luminal pH (pHv). The fluorescence during the baseline (F0) and the fluorescence during FQ are expressed aspH 5.5 and FpHv are the total fluorescence values predicted when all probe molecules in a terminal are exposed to pH 5.5 and pHv, respectively. By solving Equations : 140 NaCl, 2.4 KCl, 10 HEPES, 10 glucose, 2 CaClpH 5.5/FpH 7.4 was given as follows:Ka value of SEP estimated previously by using SypHy was 7.153.According to the Henderson-Hasselbalch equation, Fv) was then calculated as follows:Finally, SV pH (pHFluorescence imaging was carried out at room temperature (~27 °C) on an inverted microscope (Olympus) equipped with a 60× (1.35 NA) oil immersion objective and 75 W Xenon lamp. Images (1024 × 1024 pixels) were acquired with a scientific cMOS camera  with 100 ms exposure time under the control of MetaMorph software (Molecular Devices). Fluorescence of SypHy, PMCA1-SEP or Syntaxin-1a-SEP was imaged with 470/22 nm excitation and 514/30 nm emission filters.4Cl was applied at the end of recordings as described above.For live imaging, images were acquired in time-lapse mode at 1 Hz under control of MetaMorph software. Field stimulation was delivered via bipolar platinum electrodes with 1 msec constant voltage pulses (50 V). A solution containing 50 mM NHAll experiments in this study were carried out under the approval of Doshisha University Animal Committee and Recombinant DNA Experiments Committee, and of the Institutional Animal Care and Use Committee of the RIKEN Kobe branch . All experiments were performed in accordance with the guidelines and regulations of the respective institutions.Supplementary materials


Herein, we further investigated our observations and comprehensively analyzed cell differentiation in primary AML blasts after treatment with ATRA, AZA, and PGZ ex vivo. The drug combination was found to significantly inhibit cell growth as well as to induce cell differentiation in about half of primary AML blasts samples independent of leukemia subtype. Notably and in comparison to ATRA/AZA/PGZ triple-treatment, effects on cell growth and myeloid differentiation with ATRA monotherapy was much less efficient. Morphological signs of myeloid cell differentiation were further confirmed on a functional basis by demonstrating increased production of reactive oxygen species as well as enhanced phagocytic activity in AML blasts treated with ATRA/AZA/PGZ. In conclusion, we show that biomodulatory treatment with ATRA/AZA/PGZ can induce phenotypical and functional differentiation of primary AML blasts into neutrophil like cells, which aside from its antileukemic activity may lower neutropenia associated infection rates in elderly AML patients in vivo. Clinical impact of the ATRA/AZA/PGZ treatment regimen is currently further investigated in a randomized clinical trial in chemorefractory AML patients (NCT02942758).Effective and tolerable salvage therapies for elderly patients with chemorefractory acute myeloid leukemia (AML) are limited and usually do not change the poor clinical outcome. We recently described in several chemorefractory elderly AML patients that a novel biomodulatory treatment regimen consisting of low-dose azacitidine (AZA) in combination with PPARγ agonist pioglitazone (PGZ) and all- Clinical outcome of elderly AML patients refractory to intensive chemotherapy is poor, especially when allogeneic stem cell transplantation is not feasible due to comorbidities. Therefore, there is an urgent clinical need for alternative treatment regimens, which are effective and tolerable.We recently described a small series of elderly AML patients treated with a novel biomodulatory drug combination consisting of low-dose AZA, the peroxisome proliferator-activated receptor γ agonist PGZ, and ATRA . Beside Induction of cell differentiation is well-described for APL treated with ATRA alone or in combination with ATO . HoweverNeutropenia associated bacterial and fungal infections are major complications in AML patients and have a significant impact on the clinical course  as well as on the outcome of patients . InductiAML cell line HL-60 was provided by Dr. M. Rehli, University Hospital Regensburg. MV4-11 and MOLM-13 cell lines were kindly provided by Dr. M. Hudecek, University Hospital Würzburg. All cell lines were routinely tested for mycoplasma contamination. Primary AML blasts were isolated from peripheral blood of patients who showed more than 70% leukemia blasts within total white blood cells at primary diagnosis. Blood samples were drawn before start of leukemia therapy after written informed consent in accordance with the Declaration of Helsinki and upon approval of the study by the ethics committee of the University Regensburg. HL-60 cells were cultured in RPMI 1640 medium  supplemented with 10% FCS , 2% Penicillin-Streptomycin (Life Technologies), and 10 mM HEPES buffer (Life Technologies).5 cells/mL in 24 well-plates. Half of medium supplemented with cytokines was replaced on days 4, 7, and 11. ATRA, AZA, and PGZ were dissolved in DMSO. Where indicated, single agent DMSO , ATRA , AZA , PGZ , or midostaurin  or drug combinations were added on day 0 of culture. Due to its short half-life AZA was added daily until day 7. Medium control contained 10% HS, SCF, and G-CSF. AML blasts were analyzed on day 14 of culture.Primary AML blasts were thawed and cultured in AIM-V medium (Life Technologies) supplemented with 10% HS, 50 ng/mL stem-cell factor  and 50 ng/mL granulocyte-colony stimulating factor  at a density of 5 × 10Flow cytometry was performed on FACSCalibur  and analyzed using FlowJo V10 software . Relative fluorescence intensities were calculated from the MFI value of the relevant staining divided by the MFI value of the medium control sample. Fluorochrome-labeled monoclonal antibodies were anti-human CD11b (ICRF44), CD15 (HI98), CD33 (WM53), CD34 (581), CD80 (L307.4), CD83 (HB15e), CD86 (2331), CD117 (YB5.B8), CD274 (MIH1), HLA-class I (G46-2.6), HLA-DR (G46-6), and Annexin V .The fraction of U937 cells presenting apoptotic nuclei, among the total cell population was calculated by counting at least 300 cells in at least three different randomly selected fluorescence microscopic fields, after staining cells with the cell-permeable DNA-specific dye Hoechst 33342 directly added to the culture medium at the final concentration of 10 μg/mL . Apoptos4 primary AML cells on microscope slides  using a Shandon Cytospin 3 Cytocentrifuge  and air drying for 5 min. Subsequently, cytospin slides were stained for 3 min with May-Grünwald  and afterward for 13 min with 25% Giemsa solution (Merck). For each condition, 200 cells were analyzed for cellular morphology and counted by two individuals independently using a Leica DMLB light microscope type 020-519.511  and Color Video Camera model MC-3289 .For morphological analysis of myeloid cell differentiation, cytospins were prepared by centrifugation  of 5 × 106 in 0.5 mL AIM-V medium supplemented with 10% HS and incubated with 1 mg/mL NBT (Sigma-Aldrich) and 200 ng/mL PMA (Sigma-Aldrich) at 37°C for 90 min. Cells were then harvested and cytospins were prepared as described above. Numbers of formazan colored cells were counted for each condition in a blinded fashion by two individuals independently.For NBT assay , AML celE. coli bacteria were incubated with 0.25 μg enhanced GFP plasmid DNA  was used for statistical analysis. Friedman and Dunn’s multiple comparisons test was used for significance testing . Differences were considered statistically significant for in vitro. To avoid cytotoxic effects by AZA, we used a very low dose of AZA (0.1 μM) which was at least 10-fold lower than described in other studies   studies . We obse studies  on day 7 studies . Further studies . Again,  studies . In clea studies .We then tested single agent and combined ATRA, AZA, and PGZ treatment on pro-monocytic U937 cells. While ATRA remains the more effective agent, AZA and PGZ display also a certain level of activity, and mostly, they both enhanced ATRA action, as shown by results from analysis of cell growth Figure  and apopp = 0.04) or DMSO control  during 14 days of culture  and significantly lower in DMSO controls   Figure . Most no) Figure . Of note) Figure .We further evaluated whether ATRA/AZA/PGZ therapy promotes cell differentiation in primary AML blasts. Morphological assessment of individual AML samples on day 14 of culture demonstrated signs of differentiation as can be seen from increased and brightened cytoplasm, granular formation and altered nuclear morphology in AML cells during ATRA/AZA/PGZ therapy . Again and although not significant, effects with ATRA mono-treatment were less potent than those observed with ATRA/AZA/PGZ   remained unaffected Figure . Overall) Figure .To further dissect the role of each drug, we tested single , double , and triple combination (ATRA/AZA/PGZ) with sample AML101 Figure . As obseIn addition to morphological changes we investigated immunophenotypical changes of differentiation markers . Figure p = 0.0009)  through the generation of ROS , which i) Figure . Althoug) Figure . Most no) Figure .E. coli in ATRA/AZA/PGZ treated samples (median 112.0%)  and comparable to ATRA treated cells   Figure . In cont) Figure . Again, + AML cell lines MV4-11 , we analyzed the effects of the multikinase inhibitor midostaurin (MIDO) as single agent as well as in combination with ATRA, AZA, and PGZ on FLT3-ITD1 Figure  and MOLM1 Figure . In line1 Figure , ATRA moin vitro. Differentiated granulocyte like cells were characterized by morphological cell differentiation, expression of the myeloid differentiation markers, ROS production and most notably phagocytic activity. In addition, these observations parallel our previous data in patients with refractory AML, in which ATRA/AZA/PGZ treatment induced considerable increases of peripheral leukocyte counts while neutrophils still expressed molecular aberrations of the blast population  has been shown to unlock the ATRA-induced therapeutic response in non-APL AML , it mighn ≤ 1 μM . Howevern ≤ 1 μM , which wIn vivo, administration of PPARγ agonists additionally induced BM adipogenesis, which rescued healthy hematopoietic maturation while repressing leukemia growth  have also been suggested as potential modulators in myeloid leukemia by activating the transcriptional activity of target genes that control apoptosis and differentiation  as well a growth . TogetheFrom our experiments, we cannot draw conclusions about the contribution of every single drug to the observed effects, mostly because we observed much heterogeneity in the response in various patients. Heterogeneity is confirmed by comparison of two cell lines HL-60 and U937, which show different response to AZA and PGZ. More studies will be thus required to understand the molecular determinants responsible for these differences. Detailed analyses of sample AML101 suggests that the combination ATRA/AZA might be similarly effective compared to the triple combination ATRA/AZA/PGZ Figure . Howeverin vitro. A potential clinical benefit aside from anti-leukemic activity might be a lower rate of AML- and therapy-related neutropenia and lower incidences of severe infections. These promising data with ATRA/AZA/PGZ as well as our small case series (We show herein for the first time that biomodulatory treatment of primary AML blasts with ATRA/AZA/PGZ leads to phenotypical and functional differentiation of leukemic cells into neutrophil like cells in a significant fraction of patient samples e series  will nowSK designed and performed the experiments, analyzed the data, and wrote the manuscript. TS contributed to the design of experiments, performed the experiments, and analyzed the data. EB and CM performed the experiments. BH supplied human samples. LG, WH, and AR discussed the data and contributed to write the manuscript. ST contributed to the design of experiments, analyzed the data, and wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Isocitrate dehydrogenase (IDH) is a key enzyme involved in the conversion of isocitrate to α-ketoglutarate (α-KG) in the tricarboxylic acid (TCA) cycle. IDH mutation produces a neomorphic enzyme, which can lead to the abnormal accumulation of R-2-HG and promotes leukemogenesis. IDH mutation occurs in 20% of acute myeloid leukemia (AML) patients, mainly including IDH1 R132, IDH2 R140, and IDH2 R172. Different mutant isoforms have different prognostic values. In recent years, IDH inhibitors have shown good clinical response in AML patients. Hence, enasidenib and ivosidenib, the IDH2 and IDH1 inhibitors developed by Agios Pharmaceuticals, have been approved by the Food and Drug Administration on 1 August 2017 and 20 July 2018 for the treatment of adult relapsed or refractory (R/R) AML with IDH2 and IDH1 mutations, respectively. IDH inhibitor monotherapy for R/R AML is efficacious and safe; however, there are problems, such as primary or acquired resistance. Clinical trials of IDH inhibitors combined with hypomethylating agents or standard chemotherapy for the treatment of R/R AML or newly diagnosed AML, as well as in post hematopoietic stem cell transplantation as maintenance therapy, are ongoing. This article summarizes the use of IDH inhibitors in AML with IDH mutations. Approximately 40–45% of young and 10–20% of elderly AML patients treated with standard therapy will be cured. There are no more than 10% of relapsed or refractory (R/R) AML , and theIDH mutations occur in the active catalytic sites of the arginine residues resulting in a neomorphic enzyme activity converting α-KG to the oncometabolite R-2-hydroxyglutarate (R-2-HG) , 20. TheAGI-6780 is the first small selectively allosteric inhibitor of IDH2 R140Q mutation developed by Agios Pharmaceuticals in 2013. It could reverse the abnormal methylation of histones and DNA, and reduce the 2-HG level that is caused by the mutant IDH2 R140Q in TF-1 cell lines , as wellEnasidenib (Idhifa®) is an oral selectively allosteric inhibitor of IDH2 mutation developed by Celgene Corporation under a global exclusive license from Agios Pharmaceuticals. It shows more powerful inhibitory effects on IDH2 R172K rather than IDH1 R140Q  mutation, although it has inhibitory effects on both the mutations . FDA hasEnasidenib could reduce the serum total 2-HG level by more than 90%, reverse abnormal epigenetic changes, and induce differentiation of mutant IDH2 AML cells both in vitro and in murine xenograft models. Furthermore, it shows no cytotoxicity . The resEnasidenib combined with standard chemotherapy or the hypomethylating agent (HMA) azacytidine is well tolerated and effective in AML patients with IDH2 mutation. The initial results of an ongoing clinical trial of enasidenib combined with the standard induction chemotherapy (NCT02632708) in the treatment of newly diagnosed AML patients with IDH2 mutations showed that the ORR was 62%, the CR rate was 50%, and the less 30-days and 60-days mortality rate were 5 and 8%, respectively , 59. AzaDifferent IDH1 inhibitors are effective against different types of IDH1 mutation. The ongoing clinical trials of IDH1 inhibitors in adult AML are shown in Table The unacceptable pharmacodynamic and pharmacokinetic properties have limited the clinical applications of AGI-5198, and AG-120 was the drug developed after due optimization . IvosideThe approval of ivosidenib by the FDA was based on a phase 1/2 clinical trial of ivosidenib monotherapy in adult R/R AML with IDH1 mutations (NCT02074839). The trial concluded that ivosidenib monotherapy was safe and effective and it could reduce or even eliminate variant allele frequency (VAF) so that some patients could achieve a deep and lasting remission. In this clinical trial, the ORR was 41.6% with a median response time of 6.5 months, and the CR rate was 21.6% with a median duration time of 9.3 months. The patients who reached molecular remission had a longer CR duration than those who did not. The CR + CRi (CR with incomplete hematologic recovery) rate was 30.4%, thus leading to 35% of the patients being independent of blood transfusion. Ivosidenib induce AML cell differentiation and seems no cytotoxicity. Adverse events occurred in 99% patients treated with ivosidenib. Mainly included diarrhea (31%), leukocytosis (30%), febrile neutropenia (29%), nausea (28%), fatigue (26%), dyspnea (25%), prolonged QT interval (25%), peripheral edema (22%), anemia (22%), fever (21%) and cough (21%). There were no adverse events related deaths, although the treatment-related grade ≥ 3 adverse events occurred in 21% of patients, and the most common were prolonged QT interval, DS and leukocytosis, which were also the most common reasons for drug withdrawal. Treatment with hydroxyurea or leukapheresis could be initiated for leukocytosis. IDH-DS occurred in about 11% of the patients with a median onset time of 29 days, and could be cured by temporary withdrawal of the drug and adding glucocorticoids and diuretics , 62, 63.The ongoing clinical trial of ivosidenib combined with standard therapy or azacytidine for the treatment of adult R/R AML showed promising results. Ivosidenib combined with standard therapy was safe and effective in both primary and secondary AML (NCT02632708), although the ORR was higher in the former (86% vs. 44%) . The CR A global phase 3 clinical trial evaluating the efficacy and safety of ivosidenib combined with azacytidine vs. placebo combined with azacytidine in newly diagnosed AML patients with IDH1 mutations who are not suitable for intense chemotherapy is ongoing (NCT03173248). A phase 1/2 clinical trial of ivosidenib combined with venetoclax with or without azacytidine is also ongoing (NCT03471260).BAY1436032 is an orally administered allosteric inhibitor of all the IDH1 mutation proteins. It can pass through the blood-brain barrier . BAY1436FT-2102 is an inhibitor of the mutated IDH1. A phase 1/2 clinical trial of FT-2102 monotherapy and combined therapy with azacytidine or cytarabine for the treatment of adult R/R AML is ongoing (NCT02719574). The phase 1 results indicated the efficacy and safety of the drug. The ORR was 33% and the CR rate was 14%, and there were no deaths related to FT-2102. The main adverse reactions were thrombocytopenia, febrile neutropenia, anemia, pneumonia, etc. There were no deaths related to FT-2102. Like for other IDH inhibitors, IDH-DS was reported, but it could be cured by withdrawing the drug and adding dexamethasone and hydroxyurea. Based on the phase 1 data, FT-2102 at a dose of 150 mg twice daily has been administered in the ongoing phase 2 clinical trial . In addiIDH305 is a selective allosteric inhibitor of IDH1 mutation. It is an optimized product of IDH889. Preclinical studies have indicated that it could reduce the 2-HG level and induce differentiation of AML cells . A phaseThe other IDH1 inhibitors include AGI-5198, ML309 (AGI-5027), GSK 321, and DC_H31. AGI-5198 is a selective IDH1 R132H inhibitor , The phaDeveloped by Agios Pharmaceuticals, AG-881 is a pan-IDH inhibitor that shows inhibitory effects to both IDH1 and IDH2 mutations. The mutated IDH1 and IDH2 bind to AG-881 at different residues in a similar way due to different allosteric inhibitory regions. AGI-881 combined with IDH1 R132H and IDH2 R172K acts faster than IDH2 R140. The drug could easily pass through the blood-brain barrier and induce myeloid differentiation, thereby making it a more promising IDH inhibitor , 74. A pAbnormal accumulation of R-2-HG inhibits cytochrome c oxidase (COX) producing an environment similar to hypoxia, and thus, activates the apoptotic precursor proteins BAX and BAK through the BCL-2 homologous effector. The mitochondrial outer membrane becomes transparent and ultimately apoptosis is promoted. However, the anti-apoptotic gene BCL-2 could antagonize BAX and BAK, thereby preventing the increased permeability of the mitochondrial outer membrane . This pr13C-tracing studies have shown that the carbon atoms of 2-HG in AML cells with IDH mutations are mainly derived from glutamine or glutamate. Glutamine is converted to glutamate catalyzed by glutaminase, and then GLUD catalyzes the oxidative decarboxylation of glutamate to produce α-KG. Finally, α-KG is converted to 2-HG in the mutated IDH cells [DH cells , 82. TheDH cells . In the In IDH mutant cells, the abnormal accumulation of R-2-HG could down-regulate the levels of HIF-1α by activating proline 4-hydroxylase EgIN as a result of inhibition of cell differentiation. This may be one of the factors driving leukemogenesis , 84–86. There were some problems in the clinical applications of the IDH inhibitors in AML patients with mutations, such as primary and acquired resistance, although IDH inhibitors could provide in these patients an average survival time of 18–21 months . Among pThe mechanisms of acquired resistance remain unclear. One study found that in R/R AML patients with IDH2 R140Q mutation treated with enasidenib, there are second site IDH2 mutations in the trans-state [Q316E mutation (at glutamine 316 position) and I319M mutation (at isoleucine 319 position) when they had acquired resistance . AnotherEnasidenib and ivosidenib have been approved by the FDA for the treatment of adult R/R AML with mutant IDH2 and mutant IDH1, respectively, due to their high treatment response and absence of cytotoxicity in clinical trials , 49. How


Maintaining intracellular Ca2+ homeostasis is crucial for the ability to generate this signal. This review will describe the current knowledge of the mechanisms involved in uptake and transport of calcium as well as cellular homeostasis and signal generation, describing known genes involved and discussing possible implications the plant’s nutritional status with regard to calcium might have on immunity.Calcium is an essential element needed for growth and development of plants under both non-stressed and stress conditions. It thereby fulfills a dual function, being not only an important factor for cell wall and membrane stability, but also serving as a second messenger in many developmental and physiological processes, including the response of plants to biotic stress. The perception of non-self hereby induces an influx of calcium ions (Ca Calcium is an essential macronutrient in plants, with concentrations in the shoot ranging from 0.1 to over 5% of dry wt . It therTo fulfill the structural role, calcium has to be available for the plant in sufficient amounts. In general, calcium deficiency as a result of low soil availability is not very common . Deficie2+ concentration thereby serve as a signal, which is decoded into downstream responses  or host-derived damage-associated molecular patterns (DAMPs) are recognized by surface-localized pattern-recognition receptors (PRRs), initiating a signaling cascade leading to induction of downstream responses such as defense gene expression, stomatal closure, and callose deposition at the infection site. The Ca cascade . To evade in ETI .2+ signals and discusses possible links.This review summarizes our current knowledge of the role calcium plays in the plant, with a focus on structural aspects, nutrition, and immunity. It describes known genes involved in its uptake and in the generation of Ca2+ thereby is cross-linking negatively charged carboxyl groups of de-esterified pectin in the middle lamella. In addition to cell walls, Ca2+ also stabilizes cell membranes through the interaction with phospholipids  and hyperpolarization-activated channels (HACCs)  , 2008. I (HACCs) . However2+ concentrations in the cytosol need to be in the submicromolar range to ensure the ability of the cell to generate a Ca2+ signal. Symplastic movement therefore would interfere with the signaling function of calcium. A common model suggests that Ca2+ moves apoplastically from the epidermis through the cortex until it reaches the casparian strip of the endodermis . While CaM, CMLs, CBLs, and CDPKs contain EF-hand Ca2+-binding motifs, CIPKs are activated by the interaction with CBLs , and HAK5 (high-affinity K+ transporter 5) in Arabidopsis are both regulated via the same Ca2+ decoding complex consisting of CBL1/9 and CIPK23 , which transports iron, but also zinc, manganese, cobalt, and cadmium. CIPK23 in this case has been shown to phosphorylate IRT1 upon binding of non-iron metals, which subsequently leads to its degradation and thus prevents unfavorable concentrations of other metals in the cytosol   . This phso CHL1) . Despite2+ into the cytosol, the production of reactive oxygen species (ROS), MAPK signaling, and expression of defense genes as well as late responses such as callose deposition and stomatal closure. Prominent examples for PAMPs or DAMPs are the bacterial flagellin, or its synthetic analog flg22, which in Arabidopsis is recognized by the receptor FLS2 (FLAGELLIN SENSING 2)    or the eNSING 2) , which iNSING 2) , 2010. T1 (WAK1) . Cell wa2+ constitutes one of the earliest responses in the induced cascades. The channels mediating the Ca2+ influx are so far mostly unknown. Mainly two families of channels have been investigated in this context, CNGCs and GLRs. For example, mutants in the AtCNGC2 locus were entitled “defense, no death” (dnd1) according to their phenotype, which showed no HR but still resistance to avirulent Pseudomonas syringae as well as a range of virulent pathogens  as well as defense gene expression such as PR1  production required for HR   . Anotherignaling .2+ in as well as export out of the cell and those that decode Ca2+ signals into downstream responses. Some genes have already been identified and their role in specific processes described; however, most of the proteins mediating Ca2+ uptake and distribution as well as signal generation and decoding are still unknown. Also, despite few examples, surprisingly little is known about how the involvement of calcium in one plant aspect is affecting another and whether the same or different genes are involved in two processes, as, for example, those taking up Ca2+ as a nutrient in roots and those responsible for influx during signal generation. Future research will shed more light on the different functions of this important nutrient and how they are interconnected.Calcium is an essential element in plants. It serves as a constituent of cell walls and membranes and thus contributes to the structure of cells and the upholding of physical barriers against pathogens. In accordance with this structural role, plants deficient in calcium have been shown to be more susceptible to pathogens and exogenous calcium supply in turn has been shown to improve the plant’s resistance. However, in recent years, it has emerged that the main function of calcium is to serve as a second messenger. It does so in signaling events connected to a vast variety of physiological, developmental, and environmental cues, among them the signaling of other nutrients as well as pathogen attack. Research is now concentrating on identifying the proteins mediating influx of CaThe author confirms being the sole contributor of this work and has approved it for publication.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The 6‐Gin decreased the ICa‐L of normal and ischemic ventricular myocytes by 58.17 ± 1.05% and 55.22 ± 1.34%, respectively. 6‐Gin decreased ICa‐L in a concentration‐dependent manner with a half‐maximal inhibitory concentration (IC50) of 31.25 μmol/L. At 300 μmol/L, 6‐Gin reduced the cell shortening by 48.87 ± 5.44% and the transients by 42.5 ± 9.79%. The results indicate that the molecular mechanisms underlying the cardio‐protective effects of 6‐Gin may because of a decreasing of intracellular Ca2+ via the inhibition of ICa‐L and contractility in rat cardiomyocytes.Ginger has been widely used as a flavor, food, and traditional medicine for centuries. 6‐Gingerol (6‐Gin) is the active components of ginger and offers some beneficial effects on cardiovascular diseases. Here, the effects of 6‐Gin on L‐type Ca Ginger has also been used medicinally for indigestion, vomiting, arthritis, fever, pains, cramps, etc  or sarcoplasmic reticulum (SR) , which are essential to cardiac excitability and excitation–contraction coupling  to create the “spark” triggering contraction. Contractility is related to intracellular Ca2+ ([Ca2+]i) and the sensitivity of myofilaments to Ca2+. There is ample evidence that excess Ca2+ can produce pathological changes in cardiac tissue , contractility, and Ca2+ transients in isolated rat ventricular myocytes via the patch‐clamp technique and the Ion Optix system. It further explored the possible cellular mechanism of 6‐Gin for the management of ischemic cardiac diseases.Recent reports have detailed the cardio‐protective effect of 6‐Gin against ischemia–reperfusion injury in rats . Type Ⅱcollagenase was bought from Worthington Biochemical Corporation (USA). VER was from Hefeng Pharmaceutical Co., Ltd. (China). Other chemicals and reagents were acquired from Sigma (USA) and were of analytical grade.2.2Male Sprague‐Dawley rats (180–220 g) were from the National Experimental Animal Center of Hebei, National Science Council. They were housed in cages at a constant temperature of 25 ± 1°C and supplied with food and water .2.32+‐free Tyrode solution for 4 min and Ca2+‐free Tyrode's solution containing CaCl2 (34 μmol/L) and collagenase (500 mg/L) for 15–20 min via Langendorff equipment. The hearts were then washed with Tyrode's solution after the digestion. The freshly dissociated cells were stored in Kreb's buffer solution.Rat ventricular myocytes were isolated via Mitra and Morad  to induce cardiac ischemia . The patch electrodes were pulled with a pipette puller . By recording the ICa‐L, the external solution contained (in mmol/L) TEACl 140, MgCl2 2, CaCl2 1.8, glucose 10, and HEPES 10, and the pH was adjusted to 7.4 with CsOH. The intracellular pipette solution contained (in mmol/L) CsCl 120, tetraethylammonium chloride (TEACL) 20, HEPES 10, Mg‐ATP 5, and EGTA 10, and the pH was adjusted to 7.2 with CsOH. Drugs were dissolved in Tyrode's solution.The Ca2.5The contractions of ventricular myocytes were recorded with a video‐based edge‐detection system . Cells were placed on the stage of inverted microscope, and contractility was induced at a frequency of 0.5 Hz. Clear myocytes were selected to measure contractions.2.62+]i dynamics and associated myocyte contractile function. Ventricular myocyte was loaded with the fluorescent dye in the dark and measured with a fluorescence system (Ion Optix). The contractility of the myocytes was stimulated with a 0.5 Hz field.Fura‐2/AM (1 mmol/L) was fitted with a 340 or 380 nm optical filter and used to study ventricular myocyte i. Myocardial contractility was trigged mainly by cytosolic calcium ions entry through calcium channels  act by changing the mode of channel opening from long‐duration to shorts. Thus, CCAs lower the rate at which Ca2+ enters via the LTCCs. VER is a CCA and interfered with the calcium‐dependent processes.Alternatively, CaCa‐L (Figure 50 of 31.25 μmol/L in cardiomyocytes. Figure Ca‐L did not change. Furthermore, the contractility and Ca2+ transients were inhibited by 6‐Gin (Figures Ca‐L in ischemic ventricular myocytes (Figure 2+]i via decreasing the extracellular Ca2+ influx. Excitation–contraction coupling in all cardiac cells requires Ca2+ influx, therefore the inhibitory effects of 6‐Gin on contractility may through the reduction on Ca2+ influx. Collectively, these results detail the cardio‐protective effects of 6‐Gin on rat ventricular myocytes as well as and its cellular mechanism.Our data suggest that 6‐Gin reduces the IL Figure  in a con Figures . Also, 6s Figure . Our dat52+ transients and contractility of cardiomyocytes. This is mainly via inhibition of the L‐type Ca2+. This restricts Ca2+ flow into the ventricle myocytes and decreases [Ca2+]i. The findings of the present study provide new perspectives for further research on pharmacology of 6‐Gin as a possible candidate for the treatment of cardiovascular diseases.These results clearly indicate that 6‐Gin inhibits the CaThe authors declare no conflict of interest.All animal care and experimental protocols were ethically reviewed and approved by the Ethics Committee of Hebei University of Chinese Medicine.


Ca2+ homeostasis in neurons and cardiomyocytes is not only crucial for the cellular wellbeing but more importantly to generate action potential in both neurons and cardiomyocytes. By challenging Ca2+ homeostasis in CGNs, and in adult and neonatal cardiomyocytes of FRDA models, we have assessed the impact of frataxin decrease on both neuronal and cardiac physiopathology. Interestingly, we have found that Ca2+ homeostasis is altered both cell types. CGNs showed a Ca2+ mishandling under depolarizing conditions and this was also reflected in the endoplasmic reticulum (ER) content. In cardiomyocytes we found that the sarcoplasmic reticulum (SR) Ca2+ content was pathologically reduced, and that mitochondrial Ca2+ uptake was impaired. This phenomenon is due to the excess of oxidative stress under FRDA like conditions and the consequent aberrant modulation of key players at the SR/ER and mitochondrial level that usually restore the Ca2+ homeostasis. Our findings demonstrate that in both neurons and cardiomyocytes the decreased Ca2+ level within the stores has a comparable detrimental impact in their physiology. In cardiomyocytes, we found that ryanodine receptors (RyRs) may be leaking and expel more Ca2+ out from the SR. At the same time mitochondrial uptake was altered and we found that Vitamin E can restore this defect. Moreover, Vitamin E protects from cell death induced by hypoxia-reperfusion injury, revealing novel properties of Vitamin E as potential therapeutic tool for FRDA cardiomyopathy.Friedreich’s Ataxia (FRDA) is a neurodegenerative disorder, characterized by degeneration of dorsal root ganglia, cerebellum and cardiomyopathy. Heart failure is one of the most common causes of death for FRDA patients. Deficiency of frataxin, a small mitochondrial protein, is responsible for all clinical and morphological manifestations of FRDA. The focus of our study was to investigate the unexplored Ca These eechanism . Ca2+ miotective . CGNs arotective . We have2+ homeostasis. In particular, it has been associated with reduced peak systolic Ca2+ levels, slowed rates of Ca2+ relaxation and elevated diastolic Ca2+ levels in diseases such as hereditary haemolytic anemia and other iron-overload conditions  operating under a system of Ca2+-induced Ca2+ release (CICR), where the presence of Ca2+ itself induces further Ca2+ release from SR stores, generating a Ca2+ wave that contributes to cardiac action potential  pumps contain many free cysteine (Cys) residues that are sensitive to oxidation  on FRDA patients showed a beneficial effect on mitochondrial bioenergetic monitored directly in the cardiomyocytes and muscle cells with magnetic resonance spectroscopy (31P-MRS)  injury to understand how cells were coping during H/R damage. The beneficial effect of Vitamin E improved cell viability in cardiomyocytes stressed with H/R challenge. Our results confirm that Vitamin E has a protective effect on FRDA like cardiomyocytes. Therefore, this compound could be proposed as a therapy for FRDA patients.In addition to the pathology of the central and peripheral nervous system, a hypertrophic cardiomyopathy is commonly seen at the beginning of the disease, and this progressive cardiomyopathy is considered one of the most common causes of death in FRDA . Iron-innditions , which c of FRDA , howeverdiseases , this stotential . Thus, Cxidation . Oxidati . At that31P-MRS) . We obseD-lysine (1 mg/ml), glass coverslips. Cells were maintained in Neurobasal with 2% of B27 (Invitrogen) and 2 mM L-glutamine, 1% of penicillin/streptomycin (Sigma Aldrich) and 25 mM KCL . To avo2PO, 5.5 D-Glucose, 5.4 KCl, 0.4 MgSO4, pH adjusted to 7.35. In 100 mL isolation buffer were added: 60 mg collagenase type II CLS2 , 25 mg pancreatin from porcine pancreas (Sigma). After digestion and cardiomyocytes extraction process the cells were plated in Dulbecco’s Modified Eagles Medium supplemented with 15% fetal calf serum and 1% peniccilin /streptomicyn, and maintained in incubator at 37°C, 5% CO2.Primary cultures of neonatal cardiomyocytes were obtained from 6 day-old YG8R and Y47R mice . CardiomL-glutamine. Prior plating flasks were coated with a solution containing 0.02% gelatin and 5 μg/ml fibronectin. H9c2 cells were maintained in DMEM with 10% FBS and 2 mM L-glutamine. Cells were harvested with 0.25% trypsin-EDTA (Sigma Aldrich), then inactivated with complete media and plated on 25 mm glass coverslips or directly into a 6 multi-wells plate. All cell lines were maintained in a humidified incubator at 37°C and at 5% CO2.HL-1  and H9c2  were cultured. HL-1 cells were maintained in Claycomb media  with 10%To obtain frataxin knock down (FxnKD) cells were tranfected with Lipofectamine 2000, accordingly to the manufacturer instruction. Fxn-siRNA was co transfected with YFP-tagged plasmid (pcDNA3-YFP). Mm_Fxn_1 FlexiTube siRNA was used as the predesigned siRNA against mouse frataxin  in HL-1 cells and Rat_Fxn FlexiTube siRNA  was used in H9c2 cells. Negative control (Scr) was achieved with AllStars Negative Control siRNA  and Rat_Fxn FlexiTube siRNA was used in H9c2 cells.Cells were loaded with dihydroethidium  in HBSS (Sigma), which measures superoxide in the cytosol. This fluorophore was either used ratiometrically by exciting the unoxidised form in blue (364 nm) and the oxidized form in red (543 nm) or by measuring only the oxidized form. The ratio (543/364 nm) was applied for each cell measurement. Rates were then calculated. Mitochondrial ROS were measured with 1 μM CM-H2Xros (Thermo scientific) loaded for 20 min prior the beginning of the experiments.To measure lipid peroxidation, cells were loaded with 10 μM C11 BODIPY (581/591) for 10 min prior the beginning of the experiments. The dye was excited at 488 nm (oxidized form) and 561 nm (un-oxidized form) , detecte2+ and 5 μM X-Rhod1 AM (562 nm) to measure mitochondrial Ca2+ levels, in HBSS (Sigma). Dyes were incubated for 20 min and washed three times prior experiments. For all the experiments with Thapsigargin HBSS was removed prior the beginning of the recordings and replaced with a Ca2+ free saline. Fluorescence measurements were obtained either using a cooled charge-coupled device (CCD) camera  or 710 Zeiss Confocal system.Transfected cells were loaded with 5 μM Fura-2-AM (340/380 nm) or 5 μM Fluo4-AM (488 nm) to measure cytosolic Cam depolarization was detected as an increase in fluorescence (de-quenching). At the end of each experiment 1 μM Carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone (FCCP) was applied. FCCP is protonophore that dissipate the potential completely. TMRM was visualized using an excitation wavelength of 561 nm with a long pass filter using 710 Zeiss Confocal microscope.Tetramethyl rhodamine, methyl ester (TMRM) is a cationic, cell-permeant fluorescent dye readily sequestered by healthy mitochondria. To measure small differences we have used a quenching mode. Cells were incubated 20 min with 500 nM TMRM and washed out. ΔΨTo visualize the FxnKD we have used immunofluorescence . BrieflyD-glucose was added and cells were kept in a humidified incubator at 37°C and at 5% CO2, for 2 h (Reoxigenation). 200 μM Vitamin E was added 30 minutes before the H/R treatment, in the samples indicated. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay was performed in order to detect apoptotic cell death accordingly to the manufacture instructions (Roche).After 48 h from transfection the culture media was replaced with HBSS (Sigma) without glucose and cells placed in a hypoxic chamber saturated with Argon for 1 h (Hypoxia). Afterward, 5.5 mM U test and ANOVA tests were applied when appropriate and the point of minimum acceptable statistical significance was taken to be 0.05 with Bonferroni correction. Representative averages were taken from n > 3 independent experiments.Statistical analysis was performed with Excel, Origin 9  and GraphPad5 software . Results are expressed as means ± SEM or SDM. Mann–Whitney Figure 1 shows the loading of the dye (CM-H2Xros) in CGNs and the kinetic curves and rates generated by Control and YG8R neurons . By measuring lipid peroxidation using C11 BODIPY (581/591), which fluoresces at two wavelengths  we performed the ratio and calculated the rates. This the presence of overall oxidative stress in this model, as YG8R neurons showed a significant increase in rate compared to control . For our study on cardiomyocytes we choose two cell lines, representative of both adult and juvenile phenotypes. HL-1 cells (murine adult cardiomyocytes) and H9c2 cells  were co-transfected with an empty vector expressing YFP and scramble siRNA (Scr) (as control) or mFxn siRNA to achieve FxnKD. The efficiency of the FxnKD was assessed via immunocytochemistry and we found that the level of frataxin was significantly reduced when compared to control cells . To assess the oxidative stress in our cardiac models, we measured cytosolic and mROS. By loading the cells with dihydroethidium (Het), a fluorophore which becomes fluorescent in red upon oxidation, sensing ROS within the cell, we measured cytosolic ROS and found already a significant increase in resting condition when compared Scr to FxnKD, in HL-1 cells . The curves in Figure 2A show the ratio of kinetic traces obtained from recording the blue (un-oxidized) and red (oxidized) fluorescence over time . The rate of fluorescence increase calculated over time revealed a significant increment in FxnKD . We then detected mROS generation , using CM-H2Xros. Our data show that FxnKD cells have an increased level of mROS . We have repeated the experiments on H9c2 cells (recording only the oxidative form of Het) and found similar results. Figures 2F,G show an increase of cytosolic ROS . Also mROS were increased in this model . These results demonstrate that by reducing the level of frataxin expression there is an overall increase of ROS both in the cytosol and the mitochondria. Having assessed the oxidative stress in our FRDA-like models we then wondered whether this could affect Ca2+ homeostasis.It is well established that frataxin decrease causes an elevation of ROS in many cell types . Therefo2+ homeostasis. By challenging CGNs with a depolarizing injury we have assessed the Ca2+ response in control and YG8R. Cells were loaded with Fluo4-AM  and challenged with 30 mM KCL . The Ca2+ response to high KCL in YG8R CGNs was higher than the Control and more importantly there was no recovery to Ca2+ resting levels. The amplitude of this response was calculated in percentage to control and YG8R cells showed a significant difference .Mitochondrial dysfunction and oxidative stress present in CGNs of FRDA-like model contribute to neuronal death , and the2+ enters the plasma membrane increasing the cytosolic levels but then it is quickly sequestered by the internal stores to restore the Ca2+ homeostasis and neurons go back to resting condition, ready for the next action potential. FRDA-like neurons showed an impairment on restore the resting state and a decreased level of ER Ca2+. Indeed, when YG8R cells were challenged with Thapsigargin the response was smaller . The amplitude of the response was much lower in YG8R than the control  and also by adding Ca2+ there was a little response. These results could suggest that the sarcolemmal ATPase (SERCA) was impaired. Further investigation much be carried out to verify this possibility.During depolarization Ca2+ homeostasis  response. Cells were loaded with 5 μM Fura2-AM and images recorded every second. Interestingly the response to caffeine was remarkably smaller in FxnKD HL-1 cells compared to control . This result could indicate that RyRs were potentiated to extrude more Ca2+ than what was necessary to restore the Ca2+ homeostasis and/or that SERCA pumps were not efficiently pumping Ca2+ into the SR. By looking at the second phase of the Ca2+ response, in HL-1 cells, we observed that the CICR rhythmic signal was different in the Scr compared to FxnKD . The frequency and the amplitude of the signal were higher in FxnKD than the controls implying a potentiation of the RyRs which then stabilizes above baseline, reflecting an energetic collapse of the system. At the same time the decreased amplitude of the caffeine response could underline a mild impairment at the level of SERCA pumps. Similarly, the caffeine response was reduced in transfected H9c2 cells ; however, there were no signs of oscillatory events, perhaps due to the different developmental stage. The detrimental effect of Ca2+ signaling could be explained by the exacerbation of the oxidative stress in the FRDA like cells, as RyRs and SERCA have cysteines that are affected by the variation of the oxidative environment  increases their probability of being open . The decrease of [Ca2+]i in response to Thapsigargin was also observed in H9c2 cells . To corroborate our data obtained on cell lines we monitored [Ca2+]i also in primary cardiomyocytes of YG8R mice. The results showed yet again a significant decrease in Ca2+ content of the SR .It is well known that oxidative stress may cause alteration at many levels in Caeostasis . In ordeironment . Perhapsing open  and alsoing open . We haveFigures 6A,B). In particular by comparing FxnKD cells untreated and pre-treated with dantrolene we found a significant increase of [Ca2+]i represented in the scatter plot showing the amplitude of Thapsigargin response . These results show that RyR2s were not inhibited and perhaps activated, causing the decrease of Ca2+ in the SR, which was restored with dantrolene. Potentiation of RyR2s causes an excessive release of Ca2+ from the SR.By pre-treating cardiomyocytes with 20 μM dantrolene, an inhibitor of RyRs, we assessed whether the receptors were already inhibited or not. The results revealed that the response to Thapsigargin was higher in HL-1 cells pre-treated with dantrolene than without (m) during caffeine administration. We found that in both cell lines the response to caffeine was combined to a depolarization of ΔΨm in control cells and that was significantly reduced in FxnKD . This implied that instead of a physiological mitochondrial Ca2+ [Ca2+]m load, which can happen when the cytosolic Ca2+ is low, we were experiencing perhaps a decreased Ca2+ uptake from the mitochondria.To investigate the role of mitochondria in this process we looked at the mitochondrial membrane potential . By pre-incubating Vitamin E, as an antioxidant, we found that we could recover cells with the calcium re-uptake. These results led us to conclude that the effect of oxidative stress is not only limited to the SR but also to the MCU uniporter involved in Ca2+ uptake (2+ store depletion and associated contractile dysfunction are characteristics of systolic heart failure (HF) that are thought to be the consequence of depressed SERCA function, leaky RyRs and increased expression of NCX, mechanisms that might be implicated in the cellular pathogenesis of FRDA .To this point we have challenged our cardiomyocytes with hypoxia reperfusion (H/R) to stress the cells and cause cell death. By investigating whether the H/R was pushing further the FRDA compromised phenotype or not we have also wondered if Vitamin E could protect the cells from apoptosis looking at TUNEL experiments. The results showed that apoptosis was increased in FxnKD cells compared to Scr and that could be prevented by Vitamin E administration  and Ligand gated Ca2+ channels (LGCCs) and/or via its release from the ER. Then Ca2+ should be taken up by the internal stores and the homeostasis restored. By challenging CGNs with a depolarising insult, FRDA-like neurons could not restore their resting potential, leaving the membrane depolarised until death. This could be ascribed to a possible hyperactivity of either Inositol trisphosphate receptor (IP3Rs) or RyRs but also from an inhibition of the SERCA pumps impeding the Ca2+ to enter the ER and restore its cytosolic homeostasis. At the same time Ca2+ homeostasis in cardiac myocytes results from the integrated function of transsarcolemmal influx and efflux pathways modulated by membrane potential and from intracellular Ca2+ uptake and release predominantly by SR. Thus, modification of Ca2+ homeostasis may cause significant alterations in contraction and relaxation of the heart and cardiac failure  increases its open probability  that are thought to be the consequence of depressed SERCA function, leaky RyRs and increased expression of NCX, and this could be also the mechanism involved in FRDA (SR Ca in FRDA .2+ ([Ca2+]m) uptake is partially inhibited. This could be due to the effect of oxidative stress, not only limited to the SR, but also to the mitochondria and in particular to the mitochondrial calcium uniporter 1 (MCU) involved in [Ca2+]m uptake  to induce cell death, and we have found that Vitamin E was protective for FxnKD cells. Vitamin E has been proposed as possible treatment for FRDA and cardiac abnormalities several years ago but never followed up due to lack of natural history on FRDA patients with this secondary effect to the pathology (2+) and oxidative stress potentiating RyRs (2+ homeostasis has been studied in other FRDA cellular models such as neuronal cell lines and DRG, where the authors found Ca2+ homeostasis alteration (2+ homeostasis; those results could indirectly confirm that the major problem of Ca2+ homeostasis disruption in our cardiac FRDA-like models is generated by defective RyRs (On the other side we have observed that mitochondrial Cam uptake . This waathology . Vitaminathology . Patientathology . In physing RyRs . Oxidatiteration . In cardive RyRs .2+ content in FRDA-like neurons and cardiomyocytes. In neurons this could be due to SERCA pumps inhibition which needs to be further investigated. Although a similar phenomenon could happen also in cardiomyocytes, we have proven that there could be a problem perhaps because of RyRs potentiation bringing out a greater portion of Ca2+ from the SR. The effect of dantrolene, an inhibitor of RyRs, proved that the receptors were not inhibited as the SR Ca2+ was nearly restored. Mitochondrial Ca2+ uptake was found inhibited, similarly to other FRDA-like models (2+ in FRDA-like cardiomyocytes could be exploited to provide novel therapeutic strategies.Our findings have shown a decreased ER/SR Cae models  and Vitae models  in otherRA designed the study, conducted and designed the experiments, analyzed the results, and wrote the manuscript. AB contributed to performing the experiments. MM contributed to performing the experiments and interpreting the data and editing the manuscript. SP contributed to interpreting the data and editing the manuscript. PG contributed to design the study and to write the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Ca2+ overload in SR increased resting Ca2+ efflux through the type II inositol 1,4,5-trisphosphate (IP3) receptors (InsP3R) as well as ryanodine receptors (RyRs), which finally triggered massive Ca2+ release through activation of RyRs via local Ca2+ accumulation in the vicinity of RyRs. The new PVC model exhibited a resting potential of −68 mV. Under NA effects, repetitive Ca2+ release from SR triggered spontaneous action potentials (APs) by evoking transient depolarizations (TDs) through Na+/Ca2+ exchanger (APTDs). Marked and variable latencies initiating APTDs could be explained by the time courses of the α1- and β1-adrenergic influence on the regulation of intracellular Ca2+ content and random occurrences of spontaneous TD activating the first APTD. Positive and negative feedback relations were clarified under APTD generation.Cardiomyocytes and myocardial sleeves dissociated from pulmonary veins (PVs) potentially generate ectopic automaticity in response to noradrenaline (NA), and thereby trigger atrial fibrillation. We developed a mathematical model of rat PV cardiomyocytes (PVC) based on experimental data that incorporates the microscopic framework of the local control theory of Ca Several types of atrial fibrillation may be attributed to the ectopic activity of myocardial cells in the sleeves of pulmonary vein cardiomyocytes (PVCs) under augmented sympathetic stimulation ,3,4,5,6.3) receptors (InsP3Rs) with type II ryanodine receptors (RyRs). The Ca2+ release through InsP3Rs from the sarcoplasmic reticulum (SR) was suggested to directly evoke a larger Ca2+ release via RyRs to initiate Ca2+ sparks. If multiple Ca2+ sparks overlap others, the resultant Ca2+ transient can trigger the temporal depolarization (TD) of the resting membrane through the Na+/Ca2+ exchanger (NCX), thereby triggering action potentials (APTDs). Co-immunostaining of atrial myocytes with antibodies against type II RyRs and type II InsP3Rs revealed that they were localized on the SR membrane facing the subsarcolemmal space. This coupling of InsP3R with RyR has scarcely been observed in the free running network of SR, where RyRs are expressed at a lower density. Thus, the excitation–contraction (E–C) coupling in atrial myocytes may be activated if InsP3R is activated by the α1-AR stimulation. Spontaneous TD might occur at the junction of the SR terminal cisterna with the cell surface membrane in atrial myocytes.The arrhythmogenic influencing of the stimulation of the α1-adrenergic receptor (AR) has been extensively studied in atrial cardiac myocytes ,11. Thes3R expression is much lower than that in the atrial myocytes  in our PVC model, we examined steady-state solutions in the minimal model of intracellular Ca2+ dynamics. The constituents of this minimal model are described in 2+. To this minimal model, we applied bifurcation analyses, which have been successfully used to identify equilibrium points and/or the stable limit cycle in the mathematical whole cell or reduced models jnc = 0.178 mM was observed. When pOInsP3R increased, stable limit cycles were evoked between pOInsP3R 0.0047 and 0.067. Increasing pOInsP3R induced stable limit cycles at a Catot level at which the system was stable without InsP3R activation, and vice versa, increasing Catot induced stable limit cycles even when there was no InsP3R activation in this system. As is evident from the lower panels in tot or pOInsP3R.Firstly, we fixed the total Ca2+] fluctuations in the bulk cytosol space  was examined by conducting numerical time integration of the same minimal model  at −68 mV, which was similar to that observed in experiments (>−75 mV). This less negative resting potential, compared to ventricular cells, is due to a much lower density of the inward rectifier potassium current (IK1) distribution compared with ventricular myocytes. Okamoto et al. revealed a hyperpolarization-activated Cl– current (IClh) at a voltage range more negative than the resting membrane potential (Vrest)  and 0.15 μM [IP3]. Although the cycle length was markedly different, in both cases, the time course of Vm was flat during diastole except for a small slow diastolic depolarization of a few mV observed over ~300 ms after the preceding AP (2+ flux (JCa) from SR via RyR (passive leak component (Jleak_SR) and active release component (Jrel_SR)), and via InsP3R (JInsP3R) recovered in parallel with the replenishment of [Ca2+]SRrl within the following 400–500 ms because of the Ca2+ diffusion from SR uptake sites. The total continuous Ca2+ efflux (Jrel_SR) finally evoked the final full Ca2+ release as indicated by the rapid fall of [Ca2+]SRrl through an increase in [Ca2+]jnc.The intracellular Caeding AP A,C. The IK1 conductance temporarily by six-fold  every time when APTD was triggered, whereas the second peak disappeared when TD failed to trigger APTD.Note that the downward deflection of . For a reference level of Catot, a representative 3.86 and 3.59 femtomole levels will be indicated in graphs of plotting time courses of Catot in the following section.The threshold Catot is the key factor for the initiation of the APTD in the present PVC model. Therefore, activation of the individual target components LCC, SERCA, and the Na+/K+ pump via β1-AR stimulation were examined by modifying Catot at a saturating concentration of ISO (0.2 μM). In tot evoked by the full member activation of β1-AR stimulation  was compared with different combinations of target activation. The increase in Catot evoked by activating LCC plus SERCA was slightly larger than the control response. The separated Na+/K+ pump activation  was intact (0.15 μM + DS), the initial decreasing phase of Catot was interrupted by the time-dependent desensitization.The findings in both ic [IP3] B, which tot induced by activating InsP3R (α1-AR) might counteract the β1-AR activation, which promotes the APTD burst by increasing Catot through ICaL and SERCA activation (tot rapidly decreased (by ~7%) after the start of AR stimulation. In this simulation, the control Catot level was set slightly lower than the threshold of initiating APTD  in the absence of AR stimulation. Factors involved in this response are shown in InsP3R) of InsP3R (green) quickly increased from 0.00027 to a peak of 0.00385 and then decayed to 0.00039 due to the desensitization of the α1-AR receptor activation. The Catot followed a time course similar to pOInsP3R , and Na+/K+ pump  are biphasic because of delayed fractional (30%) desensitization of β1-AR. The discharge of APTD evoked a full CICR, and thereby [Ca2+]SRrl decreased to a minimum level  of the spontaneous APTD discharge even after the desensitization of α1-AR. The AP burst was initiated only when the Catot was further increased by augmenting the β1-AR stimulation. In such case, a train of APTDs started at a given delay, different from the experimental finding of large variation in the 0–10 min latency cyt was relieved by the extrusion of Ca2+ via NCX. Thus, the APTD burst occurred intermittently (not shown). Note, [Na+]cyt was decreased via augmentation of the Na+/K+ pump by the β1-AR stimulation when the repetitive APTDs were absent  but at different amplitudes of ICaL. In the control run  [2+ extrusion through NCX is augmented by this transient increase in [Ca2+] and thereby a temporal transient membrane depolarization is evoked due to the electrogenic stoichiomety of Na+/Ca2+ exchange  within this jnc in the Hinch formalism of CaRU SRrl at the terminal cysterna through excitation-contraction (EC) coupling. Then, the [Ca2+]SRrl gradually recovers through the Ca2+ diffusion from the Ca2+ uptake site of SR. If Ca2+ leaks through RyRs and InsP3R are added to this core cycle, any Ca2+ accumulation in jnc potentially triggers the next event of Ca2+ release via CaRU. The bifurcation analysis  depressed through α1-AR stimulation has not yet been clarified. Alternatively, the desensitization of α1-AR might partially occur and some fraction remains intact. If so, the pharmacologic blockade of α1-AR might decrease the frequency of repetitive APTD generation as indicated by the relationship between InsP3R conductance and the frequency  cleft depicted in green. Each CaRU is separated from the others to support the local control of CICR, but a moderate cooperativity of multiple CaRUs is conserved by a local Ca2+ domain called jnc, which allows a temporal accumulation of released Ca2+ as firstly described in the HuVEC model  after the Ca2+ release, and their distribution to each compartment is given in the 2+ release, [Ca2+]SRrl is depleted to halt the Ca2+ release through the couplons.The major compartment of SR is jnc, iz, and blk, and their activation evokes TD. If the peak of transient depolarization crosses the activation voltage threshold of INa, APTD is triggered. A series of AP discharge occurs when Catot within the cell is increased above a threshold level. In the present study, the extent of Ca2+ overload was adjusted simply by magnifying the hypothetical ICabg assumed on the surface membrane. Simulating various mechanisms of the Ca2+ overload was beyond the scope of the present study. The myofilament contraction model of Negroni and Lascano (2008) was adopted to calculate the free Ca2+ concentration in the blk.NCX is distributed to 2+]nd is determined in different combinations of LCC and couplons under the assumption of instantaneous Ca2+ distribution SRrl. In the case of LCC, a single LCC is assumed so that the conductance GL represents the limiting conductance of LCC, but is an exponential function of the membrane potential Vm as described by Equation (1). Note, Lf and Rf also provide proportional coefficients for [Ca2+]nd in respect to [Ca2+ ]o in the extra-cellular solution and [Ca2+]SRrl, respectively.The opening of LCC or a couplon is approximately represented by the parameters 2+]nd is the sole factor in determining the spontaneous Ca2+ release via couplon. Then, [Ca2+]jnc is calculated from the total amount of Ca2+ within the space ([Catot]jnc), which is determined by the four Ca2+ fluxes as represented by Equation (4).jncV is the volume of jnc, and JCa,m, JCa-couplon, JCa-InsP3R, and JCa,jnciz are membrane Ca2+ fluxes in the jnc space, Ca2+ release through couplon and InsP3R, and Ca2+ diffusion between the jnc and iz, respectively. Under a Ca2+-overload condition, the JCa_rel through the basal openings of the couplon or activated InsP3R increases in proportion to the level of [Ca2+]SRrl above the threshold level. The amplitude of Ca2+flux is determined by Equations (5) and (6).An increase in -dependence in nd or other [Ca2+]. We assumed that 75% of ICaL was connected to jnc, and 15% to iz, and the rest to blk. A slightly smaller LK (LK = 0.00154 mM) was used for the at PVC cell model to increase the Ca2+ sensitivity.In rat PVC cells, the amplitude of tionship ,48. No otionship . Okamotoar cells . In the IKr tail current has been recorded on the jump from the positive potential to the holding potential in the voltage clamp experiment in rat PVCs  using a concentration [IP3](t), and an inactivation (i) parameter of the α1-AR at time t. To obtain [IP3 ], equal to the control 0.015 mM after the desensitization, a lower limit of 0.1 was applied to the range of We failed to find an α1-AR model to incorporate in the PVC model. In this study, the time-dependent activation and desensitization of the receptor was simply represented by changes in the active second messenger [IP3](t) at time t is also given by an exponential function:The  during the Ca2+ transient. The resting level of [IP3] was set to 0.015 μM according to Cooling et al. [JInsP3R was virtually zero. The [IP3] was increased 10-fold (0.15 μM) during the stimulation of α1-AR. The Ca2+ flux via InsP3R channel was calculated using Equation (21).In the cardiac myocytes, the type II homologous isomer is the major component of InsP3R  and is e3R . Thus, iII model . The infg et al.  so that IKbg was set at 20%–30% with a time constant of 120 s to obtain the experimental time course of depolarization.Doisne et al. observed a slow continuous depolarization of ~+21 mV during a 15 min application of NA to the rat PV tissue, which was larger than the +12 mV depolarization in the atrial tissue . Similar2+]jnc fluctuates over a range slightly lower than the threshold of full CICR activation, the membrane potential may vary randomly. However, in the conventional numerical integration of d[Ca2+]/dt, the average time course of [Ca2+]jnc only changes smoothly and continuously. In simulation, the random activation of a couplon could be technically evoked by introducing a random function in the present Hinch format of CICR. For practical purposes, the magnitude of [Ca2+]jnc was multiplied two-fold at a probability of 0.0007 when the random function implemented in the VB system provided a value larger than (1 – 0.0007) at every step of numerical integration. This additional fluctuation randomly induced a full-blown CICR shown in 2+]jnc is only used to determine the opening rate of a couplon, but does not interfere with the mass conservation of Ca2+ dynamics.If [Cam of 115.3 pF was assumed in the PVC model according to the measurements of Okamoto et al. [m as well as the intracellular ion concentrations were calculated.A Co et al. . The AP The concentration changes of intracellular ions were calculated by the numerical integration of the membrane fluxes across the surface membrane as well as the SR membrane.tot (attomole) was composed of various compartments as described by Equation (38).The model equations were all translated into an ordinary differential equation (ODE) file. Initial values were obtained using a numerical simulation program. Then equilibrium points were calculated using xppaut . When Hotot is converted to femtomole from attomole.In the bifurcation diagram and the main text, the unit of Ca


The total number of scholarly publications grows day by day, making it necessary to explore and use simple yet effective ways to expose their metadata. Schema.org supports adding structured metadata to web pages via markup, making it easier for data providers but also for search engines to provide the right search results. Bioschemas is based on the standards of schema.org, providing new types, properties and guidelines for metadata, i.e., providing metadata profiles tailored to the Life Sciences domain. Here we present our proposed contribution to Bioschemas (from the project “Biotea”), which supports metadata contributions for scholarly publications via profiles and web components. Biotea comprises a semantic model to represent publications together with annotated elements recognized from the scientific text; our Biotea model has been mapped to schema.org following Bioschemas standards. Availability: Data and software used are available at https://doi.org/10.5281/zenodo.2595281.The high and sustained growth rate in scholarly publications requires ever more efficient ways to identify the most relevant documents for the own research work and increasingly, the scientific text is exploited to guide this search. This requires Named Entity Recognition and Text-Mining approaches, but also effective ways to expose identified data as structured metadata and to interlink to data and metadata with external sources such as ontologies and fact repositories, which is still a challenging research domain. The Journal Article Tag Suite (JATS) serves as the preferred (semi)structured approach for publishers to provide machine-readable access to scholarly articles . AlthougSchema.org is a collaborative initiative offering a simple yet effective way to add structured metadata to web pages via markup: making pages more findable and improving search results as search engines can identify better the clues what a page is about. Schema.org also eases the ways to interlink related resources; for instance, a movie can be linked to its actors, directors or similar movies. Such interlinking of web resources can boost interoperability, allowing search engines to move from mainly presenting search only to providing comprehensive summaries. Scholarly literature repositories such as Zenodo and EuropePMC have recognized the potential of schema.org and currently support markup on their pages. However, they mostly limit to metadata, i.e., authors, title, abstract and journal, leaving aside any entity of interest from the life science domain that could be recognized from the text. Despite its popularity, the high diversity of properties in schema.org makes it still difficult for not well prepared data scientists to adopt such markup. Bioschemas, a community and collaborative project, focuses on integrating Life Science types to schema.org and, at the same time, makes its adoption easier via profiles tailored to the Life Science domain . A profiBiotea proposes a conceptual model to represent scholarly publications as Linked Data ,4, coverhttp://bioschemas.org/groups/Biotea2Bioschemas). These profiles corresponding to journal, volume, issue and scholarly article are reasonably stable as they correspond to regular metadata and data that have been well-established for publications, e.g., title, authors, abstract, journal, publisher, pages, dates and citations. Biotea supports two ways to represent annotations, one based on the Annotation Ontology [Biotea profiles proposed to Bioschemas (as a draft) include journal, volume, issue, scholarly article and semantic scholarly annotation  with the Findability, Accessibility, Interoperability and Reusability (FAIR) principles .https://github.com/ricardoaat/bioschemas-gocrawlit).Web components make it easy to add customized elements to web pages so they can easily be embedded into any web page. In order to parse and render scholarly publication metadata as well as semantic annotations from their text, we have created two web components. Our web components take their input, parse it and render it following the corresponding Bioschemas markup. The markup is added at the end of the web page as a new element, such element corresponds to a script with the MIME-type “application-json”. We have chosen this asynchronous approach as it makes it easier for web page providers to optimize the end-user content, leaving any Biotea-Bioschemas machine-readable bits to the end. Rendering the markup as JSON-LD also provides readable HTML, as it keeps the content separated from the markup. A possible drawback in this approach is the uncertainty regarding the capacity of crawlers to support asynchronous loading of the structured metadata. Asynchronous loading is nowadays a common approach on web page development and therefore should be supported by main search engines when it comes to structured metadata. Bioschemas has already recognized this and therefore has initiated efforts to contribute with a crawler capable to deal with delayed loading (https://www.ncbi.nlm.nih.gov/pmc/tools/oai/). It converts such a response into a JSON object and then selects elements of interest such as title authors, publication data and abstract and map them to the Biotea-Bioschemas profiles. For our second web component, biotea-bioschemas-annotations, we use PubAnnotation [https://bioportal.bioontology.org/), as ontologies are updated, so could annotations using such ontologies, meaning that a different set of annotations for the same article could be recovered at different times. In order to avoid this, we have created a Biotea project in PubAnnotation so we can host there a set of frozen annotations used to provide the corresponding Bioschemas markup.The Biotea-bioschemas-metadata web component requires as input a JATS XML response from the PMC-OA Interface service . Although it is possible to use our web components with any PMC-OA article or any PubAnnotation PMC hosted annotation set, we have limited our showcase to those 2,596 articles currently hosted at PubAnnotation. All of the articles are included in a sitemap, making it easier for search engines to index the content. In http://biotea.github.io/bioschemas?pmc=2628047.As a proof of concept, we have uploaded 2,596 full text articles, annotated their abstracts with 13 ontologies from Bioportal, covering gene, protein, drug, disease and symptom areas, for a total of 894,926 annotations, and exposed them via PubAnnotation. Our web components can be seen in action in our Biotea-Bioschemas main page (https://scigraph.springernature.com. Schema.org and Bioschemas are, however, easier to adopt as adding markup to HTML does not require such a robust infrastructure as ontology-based approaches do. As a future work, our initial step on realizing a Bioschemas knowledge graph potential would be extracting author-expertise networks. From there, we hope more developments will come, exploring more complex associations such as target-disease.By mapping Biotea to schema.org following the standards proposed by Bioschemas, we are preparing the basis towards literature-based knowledge graphs based on Bioschemas. At this point, it remains open what types of inferences based on such a graph could be drawn, under the consideration that the semantics given by schema.org is less strict in comparison to ontology-based approaches such as