Human TM cells respond to the TRPV4 agonist GSK1016790A with fluctuations in intracellular Ca2+ concentration ([Ca2+]i) and an increase in [Na+]i. [Ca2+]i oscillations coincided with monovalent cation current that was suppressed by BAPTA, Ruthenium Red and the TRPM4  channel inhibitor 9-phenanthrol. TM cells expressed TRPM4 mRNA, protein at the expected 130-150 kDa and showed punctate TRPM4 immunoreactivity at the membrane surface. Genetic silencing of TRPM4 antagonized TRPV4-evoked oscillatory signaling whereas TRPV4 and TRPM4 co-expression in HEK-293 cells reconstituted the oscillations. Membrane potential recordings suggested that TRPM4-dependent oscillations require release of Ca2+ from internal stores. 9-phenanthrol did not affect the outflow facility in mouse eyes and eyes from animals lacking TRPM4 had normal intraocular pressure. Collectively, our results show that TRPV4 activity initiates dynamic calcium signaling in TM cells by stimulating TRPM4 channels and intracellular Ca2+ release. It is possible that TRPV4-TRPM4 interactions downstream from the tensile and compressive impact of intraocular pressure contribute to homeostatic regulation and pathological remodeling within the conventional outflow pathway.Trabecular meshwork (TM) cells are phagocytic cells that employ mechanotransduction to actively regulate intraocular pressure. Similar to macrophages, they express scavenger receptors and participate in antigen presentation within the immunosuppressive milieu of the anterior eye. Changes in pressure deform and compress the TM, altering their control of aqueous humor outflow but it is not known whether transducer activation shapes temporal signaling. The present study combines electrophysiology, histochemistry and functional imaging with gene silencing and heterologous expression to gain insight into Ca IOP is ignaling . TLR4 exignaling . These oignaling  yet the s (SACs) –11 such ntiation –14. Excentiation , 14, 15 2+ release from intracellular stores, receptor-operated and store-operated Ca2+ influx) (The human TRPV4 gene contains 16 exons that encode a protein consisting of a proline-rich (PRD) domain, phosphoinositide binding site, ankyrin repeats and a helix-turn-helix (HTH) linker domain within the N-terminus, six transmembrane domains with the pore between S5-S6, and modulatory sites  in the C-terminus that endow the channel with sensitivity to temperature, metabolites of arachidonic acid, nociceptive and mechanical stimuli . Gain- a influx) , 21–24 iin situ milieu and attendant activation of auxiliary stretch-activated channels (SACs) such as TREK-1 and Piezo1  genes and DEX-induced upregulation of myocilin protein (Primary trabecular meshwork cells (pTM) were isolated from juxtacanalicular and corneoscleral regions of the human donors’ eyes  with no recorded history of eye disease as described , 11, 13. protein .Trpm4 shRNA using Lipofectamin™ 3000 (5 μg per 25 cm2 tissue culture flask). shRNA -transfected cells were identified by green (GFP) or red (mCherry) fluorescence. Knockdown efficiency of TRPM4 shRNA was validated in HEK 293T cells overexpressing GFP-hTRPM4, scrambled shRNA or one of three TRPM4 shRNA constructs. shRNA1 and shRNA2, with target sequences GGACATTGCCCAGAGTGAACT (1218–1238) and GGAAAGACCTGGCGTTCAAGT (1835–1855), showed >65% knockdown efficiency  supplemented with 2% fetal bovine serum (FBS), 100 units/ml penicillin, 100 ug/ml streptomycin, at 37°C and pH 7.4. Cells were transfected with scrambled shRNA (Sc-shRNA) or 2, 2 mM CaCl2, 10 mM HEPES, 10 mM D-glucose, 93 mM mannitol), with final DMSO concentration not exceeding 0.1%.GSK1016790A (GSK101) is a highly specific agonist , 23 and Cells were plated on collagen Type 1-coated glass cover slip 1 day prior to fixation with the 4% PFA for 10 minutes at RT. The samples were blocked with phosphate-buffered saline containing 0.3% Triton X-100 and 5% FBS for 30 minutes at a room temperature (RT). The primary polyclonal rabbit antibody was diluted 1:1000 in PBS (2% BSA and 0.2% Triton X-100) and incubated overnight at 4°C . After r® O.C.T. , and cryosectioned at 12 µm, as described  , 34. 100TM cells were detached from culture flasks by trypsinization and centrifuged at 2000 rpm for 3 minutes. The cell pellet was washed with PBS and lysed in a RIPA Buffer System . Cell lysates were separated by 10% SDS-PAGE followed by electrophoretic transfer to polyvinylidene difluoride membranes . Membranes were blocked with 5% skim milk in PBS containing 0.1% Tween 20 and incubated at 4°C overnight with the TRPM4 antibody, following by 3 washes with PBS containing 0.1% Tween-20, blotted with HRP-conjugated GAPDH antibody for 30 min at room temperature and washed with PBS containing 0.1% Tween 20. The signals were visualized with an enhanced chemiluminescence system .via individual pinch valves . Primary cells were loaded with Fura-2 AM  for 45 min at RT. The cells were perfused with extracellular solution containing (mM): 135 NaCl, 2.5 KCl, 1.5 MgCl2, 1.8 CaCl2, 10 HEPES, 5.6 D-glucose . Epifluorescence imaging was performed on Nikon microscopes using 40x  objectives, 340 nm and 380 nm excitation filters  and a Xenon arc lamp . Fluorescence emission at 510 nm, in response to alternating 340/340 excitation, was captured with cooled EMCCD or CMOS cameras . Data acquisition was controlled by NIS Elements 3.22 software (Nikon). Typically, ~5-10 cells per slide were averaged across ~3-6 slides per experiments, with at least 3 independent experiments . The experiments were performed at room temperature (20 - 22°C).Pharmacological experiments were conducted on a microscope stage in a fast-flow chamber  connected to a gravity-fed perfusion system. The flow rate of the solution was regulated 2+ imaging. Cells were loaded with 3 μM Fura-2 AM (Invitrogen/ThermoFisher) (Kd at RT = 225 nM) for 45 min. ΔR/R (peak F340/F380 ratio – baseline/baseline) was used to quantify the amplitude of Ca2+ signals  for 50 – 60 min in a cell culture incubator at 37°C. Fluorescence was acquired at 484 nm excitation and 520 nm emission . Na+ signals were normalized to the average baseline (F/Fo) obtained at the beginning of the experiments.Intracellular Na2, 2 ethylene glycol-bis(β-aminoethyl ether)-N,N,N’,N”-tetraacetic acid (EGTA), 0.3 Na-GTP (pH 7.3) The pipette solution for recording Ca2+-activated current contained (in mM): 125 Na-gluconate, 10 HEPES, 1 MgCl2, 0.01 CaCl2, 0.3 Na-GTP (pH 7.3). Nominally Ca2+-free pipette solution (standard) contained (in mM): 125 Na-gluconate, 10 HEPES, 1 MgCl2, 5 BAPTA, 0.3 Na-GTP (pH 7.3). The standard extracellular solution contained (in mM): 135 NaCl, 2.5 KCl, 1.5 MgCl2, 1.8 CaCl2, 10 HEPES, 5 D-glucose (pH 7.4). The extracellular solution for recording Ca2+-activated current contained (in mM): 135 Na-gluconate, 10 HEPES, 1 MgCl2, 1.8 CaCl2, 0.3 Na-GTP (pH 7.3). The bathing solution for excised (inside-out) patch clamp experiments contained (mM): 140 NaCl, 2.5 KCl, 1.5 MgCl2, 1.8 Ca2+ or 10 EGTA, 5.6 D-glucose, 10 HEPES . The pipette solution used in excised patch recordings contained 140 NaCl, 2.5 KCl, 1.5 MgCl2, 1.8 CaCl2, 5.6 D-glucose, 10 HEPES . Experiments were performed at room temperature of 21-22°C.Whole cell and single-channel techniques were used to record membrane currents (voltage clamp) or voltage (current clamp) , 35, 36.2+-activated currents was obtained by subtracting voltage ramp-evoked currents traces recorded 40 s after obtaining the whole-cell configuration from preceding and following traces. I-V curves of Ca2+-activated currents were assessed by subtracting currents recorded 40 s after obtaining the whole-cell configuration from steady state currents recorded 4 min after obtaining the whole-cell configuration.Patch clamp data in whole-cell and single-channel configurations was acquired with a Multiclamp 700B amplifier and a Digidata 1550 interface, and controlled by Clampex 10.7 . Cells were held at -100 mV or +100 mV, with currents sampled at 5 kHz, filtered at 2 kHz with an 8-pole Bessel filter and analyzed with Clampfit 10.7 (Molecular Devices) and Origin 8 Pro . Whole-cell currents were elicited by voltage ramps ascending from -100 mV to 100 mV (0.2 V/sec) from the holding potential of 0 mV. The time course of Ca2, 1.5 MgCl2, 10 HEPES, 5.6 D-glucose.Patch clamp combined with fluorescent imaging. Cells were loaded with a calcium indicator Fluo-4 (Invitrogen/ThermoFisher) through a patch pipette. Fluorescence signals in voltage-clamped cells were detected using the excitation filter 484 nm and the emission filter 520 nm . VOCC-mediated current was recorded in the extracellular solution containing as following (mM): 115 NaCl, 2.5 KCl, 20 BaCliPerfusion, a custom-made microfluidic setup  was measured using ic setup , 38. FolP, and flow rate, Q, were calculated over the last 4 minutes of each step, and a power-law relationship of the formOne eye was perfused with 9-PA (25 µM) whilst the contralateral eye was perfused with vehicle (perfusion fluid containing the same concentration of DMSO). IOP was set to 9 mmHg for 1 hour to pressurize and acclimatize the eye to the perfusion environment and to allow sufficient time for 9-PA to reach the outflow tissues. Flow into the eye was then measured over 8 increasing pressure steps from 6.5 to 17 mmHg. Steady state for each step was evaluated when the ratio of the flow rate to pressure changed by less than 0.1 nl/min/mmHg per minute over a 5-minute window . The staQ – P data. The reference outflow facility, rC, represents the value of outflow facility at a reference pressure rP of 8 mmHg, and β characterizes the non-linearity of the Q-P relationship  and homozygous pan-TRPM4 KO mice  were maiescribed . In animhttps://biorxiv.org/cgi/content/short/2021.12.15.472700v1.Student’s paired t-test or two-sample t-test  were applied to estimate statistical significance of results. P < 0.05 was considered statistically significant. Results are presented as the means ± S.E.M. A preprint of this article was submitted at 2+]i and rarely show Ca2+ fluctuations  . Agonist-evoked [Ca2+]i increases peaked within 2-3 min, followed by a gradual decline to a steady plateau at ~27% of the peak   , 13 and the peak 2. A sub2+ signals can reflect activation of voltage-operated mechanisms within the membrane and/or release from internal compartments. Voltage-dependent mechanisms were tested by stimulating the cells in the current-clamp mode. The agonist evoked a ~15 mV shift in the membrane potential  which permeates voltage-operated Ca2+ channels without inactivating the channel pore or stimulating Ca2+ release from ER stores  cells . The increase in Na+ concentration was not associated with an oscillatory component, indicating that TRPV4-induced Na+ influx is distinct from mechanisms that subserve Ca2+ oscillations. We tested the potential involvement TRPM4, a Ca2+-activated channel permeable to monovalent ions that has been implicated in [Ca2+]i oscillations in T cells, cardiomyocytes, and smooth muscle cells (+]i evoked by GSK101 (P < 0.001)  and primary (pTM) cells showed robust expression of TRPM4 and TRPV4 transcripts  labeled 2+-sensitive monovalent current (2+ and Ca2+-activated K+ and Cl- conductances were minimized by replacing extra/intracellular K+ ions (with Na+) and Cl- ions with gluconate (2+-containing solution showed time-dependent increases in inward and outward whole-cell currents. The current-voltage relationship of the Ca2+-dependent current showed modest outward rectification and peaked within ~4 min after obtaining the whole-cell configuration (2+-free solutions or exposed to bath-applied Ruthenium Red (10 μM) (n = 5/5)  or “scrambled” (Sc-GFP) control shRNAs . rC for 9-PA treated eyes was 5.4  nl/min/mmHg and 5.5  nl/min/mmHg for vehicle-treated eyes , indicating that TRPM4 may not be required for steady-state intraocular pressure regulation . Overall, these results suggest that TRPM4 signaling is not required for step-induced fluid drainage across the or for steady-state intraocular pressure regulation.Finally, we investigated whether genetic deletion of TRPM4 channels affects intraocular pressure homeostasis in the mouse eye. TRPM42+ oscillations that are independent of the membrane potential and require expression of TRPM4. Both TRPV4 and TRPM4 were strongly expressed in primary and immortalized TM cells, with functional coupling between the two cation channels revealed by the rise of the Ca2+-activated monovalent current and loss of oscillatory response following TRPM4 knockdown. Our findings identify novel interactions between TRPV4, TRPM4 and intracellular calcium stores and build a new working model that may help increase our understanding of homeostatic and time-dependent signaling within the primary outflow pathway.The goal of this study was to define the properties of calcium homeostasis during sustained stimulation of TRPV4, a transducer of mechanical stimuli that has been implicated in the regulation of conventional outflow and intraocular pressure . We show2+]i represent one of the earliest responses of nonexcitable cells to mechanical stress. Resting human TM cells do not exhibit spontaneous calcium activity whereas sustained stimulation of TRPV4 channels produced three types of time-dependent calcium behavior. (i) The initial increase in [Ca2+]i evoked by GSK101 peaked within 2-4 min and showed slow onset kinetics that was likely shaped by the messenger pathway involving obligatory activation of phospholipase A2 and production of epoxyeicosatrienoic messengers that bind the pocket formed by S2-S3/S4-S5 linker residues  Tatous TM  and presatous TM , 50.+]i; (ii) TRPM4 knockdown in TM cells preserved the GSK101 response but eliminated the oscillatory calcium component, and (iii) Heterologous expression of TRPV4 and TRPM4 channels reconstituted fluctuations in HEK293 cells. TRPM4 expression in TM cells was confirmed by transcriptional profiling and immunohistochemistry, with TRPM4 mRNA levels reaching ~30-70% of TRPV4 mRNA, and immunoblots showing a band at the predicted M.W. As in other cell types  TRPV4-induced fluctuations were suppressed by 9-PA, which also induced a reversible decrease in i oscillations to obligatory voltage-gated Ca2+ influx, TM cells did not show functional voltage-operated calcium channels expression under our experimental conditions. Thus, depolarization had no discernible effect on [Ca2+]i and the transmembrane current, and Ba2+ concentrations that reliably trigger voltage-operated calcium channels-mediated spiking and suppress store release in retinal neurons . A simple model delineating the potential players associated with TRPV4-dependent calcium oscillations is shown in 2+ influx and the IP3R store  subunits that form KATP channels sensitive to [Ca2+]i and intracellular ATP , Stauss-Rankin Foundation, USAMRAA (VR200079) and unrestricted support from Research to Prevent Blindness to the Moran Eye Institute at the University of Utah.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 interestAll claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


In neurons, Ca2+ signaling is crucial for regulating synaptic transmission and for the processes of learning and memory formation. Hence, the dysregulation of intracellular Ca2+ homeostasis results in a broad range of disorders, including cancer and neurodegeneration. A major source for intracellular Ca2+ is the endoplasmic reticulum (ER), which has close contacts with other organelles, including mitochondria. In this review, we focus on the emerging role of Ca2+ signaling at the ER–mitochondrial interface in two different neurodegenerative diseases, namely Alzheimer’s disease and Wolfram syndrome. Both of these diseases share some common hallmarks in the early stages, including alterations in the ER and mitochondrial Ca2+ handling, mitochondrial dysfunction and increased Reactive oxygen species (ROS) production. This indicates that similar mechanisms may underly these two disease pathologies and suggests that both research topics might benefit from complementary research.Calcium ions (Ca The main focus lies on the ER and mitochondrial Ca2+ handling, as well as the close interaction between these organelles. By investigating the Ca2+ impairments, we show that these two very different diseases have more in common than first meets the eye.Neurodegenerative diseases are characterized by the progressive degeneration or loss of neurons, a process that is causative of a wide array of symptoms. In this review, we discuss the important role of Ca2+) are omnipresent in living cells and function as a versatile form of cellular communication, vital for the regulation of numerous cellular processes, including muscle contraction, fertilization, differentiation and metabolism ) (100 nM), compared to the [Ca2+] inside the intracellular Ca2+-storing organelles (250–500 µM) and the extracellular space (>1 mM). The rapid changes in the cytosolic [Ca2+] can relay signals from either extracellular or intracellular stimuli to cellular effectors  can also inhibit ER Ca2+ release, illustrating the bell-curve shaped Ca2+ dependence of ER Ca2+ release  in the ER cause a dissociation of Ca2+ from the Ca2+ sensors’ stromal interaction molecule (STIM1/STIM2)  drive the clustering of STIM2 proteins and consequently augment the SOCE 2+ toolkimin (PV) ,22. Insi GCAP1-3 . These athe IP3R .2+-buffering capacity and intervene in multiple Ca2+-mediated signaling processes  in the microdomains, which can reach more than 10 μM Ca2+, can overcome the inherently low Ca2+ affinity of MCU, ensuring an adequate transfer of Ca2+ into the mitochondrial matrix, even under basal conditions  plays a dual role in regulating cell death  ER depletion, S1R dissociates from BiP and loses its specific MAM localization  and reducing agonist-induced ER–mitochondrial Ca2+ transfer  elevations established by IP3R-mediated Ca2+ release, but not the [Ca2+] elevations caused by other processes, such as SOCE  in AD. Kuchibhotla et al. reported a Ca2+ overload in individual neurites and spines in mice, which was dependent on the proximity to Aβ deposits  was significantly increased in the cultured, cortical neurons from a triple transgenic AD mouse model (3xTg-AD). However, in the absence of extracellular Ca2+, the intracellular Ca2+ levels almost completely returned to normal, suggesting that an influx of extracellular Ca2+ plays a role in the observed intracellular Ca2+ overload. It was further demonstrated that increases in Ca2+ influx via L-type Ca2+ channels and increased IP3R-mediated Ca2+ release are responsible for the elevated cytosolic Ca2+ levels. The observed disturbances in the Ca2+ signaling occur prior to the onset of plaque formation and neurofibrillary tangles, indicating the crucial role of Ca2+ signaling in the early pathophysiology of AD . A. A2+ hombilayers . Of note channel , though elopment . Furthertransfer . A seconradation .2+ homeostasis is still not entirely clear, but most studies seem to point towards a higher resting cytosolic [Ca2+] upon Wfs1 deficiency, possibly through an increased Ca2+ leak from the ER through the Ca2+-release channels  and prevented excessive calpain 2-mediated cell death in the Wfs1-deficient cells [2+] and excessive cell death in the Wfs1-deficient cells  could occur via modulating SERCA activity. Indeed, findings from murine Cisd2 KO fibroblasts [2+] is decreased when lacking Cisd2. Additionally, Cisd2 interacted with SERCA2b [2+] was increased, while the findings in other cell types reported unchanged ER [Ca2+] upon loss of Cisd2  [2+ overfilling of the ER can lead to exaggerated IP3R-mediated Ca2+ releases and thus contributes to elevated cytosolic [Ca2+]. Moreover, a direct link with the IP3R is established in both of the disorders. The mutations in PSEN have been shown to augment the activity of the IP3R  ,157,158.ogy [2+] ,156,162.the IP3R , and botIP3R , or Wfs1- and Cisd2-based therapies (In summary, WS and AD entail common hallmarks in early disease pathology that can be exploited as potential treatment strategies to target both diseases. The possibilities for such therapeutic targets include S1R, SERCA, RyR and the IPherapies . It woul2+ handling, mitochondrial dysfunction and increased ROS production. Disturbances in Ca2+ signaling are central in the early pathophysiology of AD, as well as in WS. This indicates that similar mechanisms may underly these two diseases’ pathologies and suggests that both research topics might benefit from complementary research.AD and WS are both neurodegenerative diseases that display distinct hallmarks and a wide array of symptoms. However, both of the diseases in the early stages share some common hallmarks, including alterations in the ER and mitochondrial Ca


EFhd1 and EFhd2 exhibited Ca2+- and Zn2+-independent actin binding and Ca2+- and Zn2+-dependent actin-bundling activity, and these suggest the possibility that both Ca2+ and Zn2+ act to regulate EFhd1 and EFhd2 in physiological processes.The EF-hands within the crystal structures of the Zn 2+-binding EF-hand motif, are involved in regulating diverse cellular functions. Ca2+ binding induces conformational changes that modulate the activities of EF-hand proteins. Moreover, these proteins occasionally modify their activities by coordinating metals other than Ca2+, including Mg2+, Pb2+ and Zn2+, within their EF-hands. EFhd1 and EFhd2 are homologous EF-hand proteins with similar structures. Although separately localized within cells, both are actin-binding proteins that modulate F-actin rearrangement through Ca2+-independent actin-binding and Ca2+-dependent actin-bundling activity. Although Ca2+ is known to affect the activities of EFhd1 and EFhd2, it is not known whether their actin-related activities are affected by other metals. Here, the crystal structures of the EFhd1 and EFhd2 core domains coordinating Zn2+ ions within their EF-hands are reported. The presence of Zn2+ within EFhd1 and EFhd2 was confirmed by analyzing anomalous signals and the difference between anomalous signals using data collected at the peak positions as well as low-energy remote positions at the Zn K-edge. EFhd1 and EFhd2 were also found to exhibit Zn2+-independent actin-binding and Zn2+-dependent actin-bundling activity. This suggests the actin-related activities of EFhd1 and EFhd2 could be regulated by Zn2+ as well as Ca2+.EF-hand proteins, which contain a Ca All of these phenomena rely on F-actin, a filamentous polymer composed of G-actin monomers, which undergoes assembly, disassembly, severing, branching and bundling mediated by various actin-binding proteins (ABPs) during the course of its activity , which are helix–loop–helix structures, mostly found in pairs, within the hydrophobic core of EF-hand domains  and calcium binding protein (CaBP) by binding with higher affinity than Ca2+ . We demonstrate Zn2+ coordination within the EF-hands of EFhd1 and EFhd2 through analysis of anomalous signals. Lastly, we show that Zn2+ affects the actin-bundling activities of EFhd1 and EFhd2 but not their actin binding, which suggests the possibility that Zn2+ acts to regulate EFhd1 and EFhd2 in both physiological and pathophysiological processes.Here, we report the structures of the mouse EFhd1 and human EFhd2 core domains in the Zn2.2.1.2+-mediated multimerization of EFhd1 and EFhd2  than that of the protein to the EFhd2 proteins to remove native ions and then dialyzed the proteins. Thereafter, ZnCl2 was added to the protein solution to a final concentration of 0.75 mM. In the case of EFhd1Ca, CaCl2 was added to the protein solution directly to a final concentration of 4 mM. We crystallized these proteins and determined the crystal structures of EFhd1Zn, EFhd2Zn and EFhd1Ca at resolutions of 1.72, 2.60 and 2.80 Å, respectively, using molecular replacement methods [Table 1a)]. EFhd1Zn, EFhd2Zn and EFhd1Ca comprised two EF-hands, a ligand mimic (LM) helix at the C-terminus, a PR region at the N-terminus and a C-terminal linker  above 8σ were observed with these EFhd proteins, which suggests metal binding [Table 2b)–1(d)]. Considering our protein preparation protocols, we expected that the map originated from Ca2+ or Zn2+. To identify the coordinating metal, we calculated anomalous difference maps. Because Zn2+ and Ca2+ have different numbers of anomalous electrons at the wavelength the EFhd1 data were collected , the anomalous difference map should be weaker in the Ca2+-bound state than the Zn2+-bound state. In the case of EFhd1Ca, the peak heights in the anomalous difference map were 3.4 and 2.7σ for EF-hand 1 (EF1) and EF-hand 2 (EF2), respectively. EFhd1Zn had peak heights of 23.0 and 14.0σ in the anomalous difference maps for EF1 and EF2, respectively. This suggests the metal coordinated in EFhd1Ca was Ca2+ while that coordinated in EFhd1Zn was Zn2+ [Table 3c) and 1f)].σK-edge (λ = 1.2851 Å/9648 eV), the Ca2+f″ and Zn2+f″ were 0.9 and 0.5 electrons, respectively. For that reason, we cannot rule out the possibility that the anomalous signals for EFhd2Zn originated from Ca2+, because the wavelength for the EFhd2Zn data collection was near the Zn K-edge [Fig. 1g)]. To identify the metal coordinated in EFhd2, we analyzed the difference between anomalous difference maps (ΔAno). We used a single EFhd2Zn crystal to collect datasets at the peak and remote positions of the Zn K-edge, which are termed EFhd2Zn(P) or EFhd2Zn(R), respectively [Table 4]. After calculation of the anomalous difference maps for each dataset, the anomalous difference map for EFhd2Zn(R) was subtracted from that for EFhd2Zn(P) to calculate the ΔAno map. The peak heights of the ΔAno map calculated from EFhd2Zn(P) and EFhd2Zn(R) were 11.1 and 9.3σ in EF1 and EF2, respectively [Figs. 1h) and 1i)]. This demonstrates that the metal coordinated in the EF-hands of EFhd2 was Zn2+.At a wavelength near the Zn Ca, EFhd1Zn and EFhd2Zn, the anomalous difference maps were observed near the α4 of each protein, which is situated at the crystal-packing interface [Figs. 1e)–1(g)]. Analyzing the metal coordination geometry using CheckMyMetal and the MetalPDB server, we expected that those peaks originated from Zn2+  and 1f)]. In the case of EFhd2Zn, the peak height of the ΔAno map was 9.6σ at the crystal-packing interface [Figs. 1h) and 1i)]. Considering both the anomalous signals and the metal coordination geometry, we deemed the metal at the interface to be Zn2+. Collectively, the two EF-hands of EFhd1 and EFhd2 are able to coordinate Zn2+ as well as Ca2+, and Zn2+ could also be coordinated at the crystal-packing interface.Within the crystal structures of EFhd12.2.Zn and EFhd2Zn superimposed well onto each other . Moreover, when we superimposed EFhd1Zn on EFhd1Ca and EFhd2Zn on EFhd2Ca  and 2c)] , 3(Y), 5(Z), 7(−Y), 9(−X) and 12(−Z). Conventionally, one water molecule participates in metal coordination at position 9(−X). These residues coordinate Ca2+ through seven ligands with pentagonal bipyramidal geometry and coordinate Mg2+ through six ligands with octahedral geometry  and 9(−X). The Gly at position 3(Y)  and the Asp at position 9(−X)  are not used to coordinate Zn1 [Figs. 3a) and 3c)]. Therefore, the geometry of the Zn1 coordination formed a distorted pentagonal bipyramid in the two proteins. Only one water coordinated Zn2 at position 9(−X) , which is consistent with the conventional one water coordination at that position [Figs. 3b) and 3d)]. As a result, the geometry of the Zn2 coordination formed the typical pentagonal bipyramid. To compare their EF-hands, we superimposed the structure EF1 or EF2 in EFhd1Zn on EFhd2Zn and found that both superimposed well . In addition, measurement of the average distance between Zn2+ and its coordinating ligands in the EF-hands revealed that the overall average of Zn2+ coordination distances in EFhd1Zn and EFhd2Zn are similar to the average Zn2+–oxygen distance (2.3 ± 0.5 Å) (Table S1 of the supporting information) . In addition, these proteins showed similar geometries in the Ca2+- and Zn2+-bound states. In both proteins EF1 and EF2 formed the distorted or typical pentagonal bipyramid for Ca2+ and Zn2+ coordination [Figs. 4a)–4(d)]. The side-chain topologies of the EF-hand loop were similar between EFhd1Zn and EFhd1Ca and between EFhd2Zn and EFhd2Ca. This suggests the metal coordination geometries of Ca2+ and Zn2+ are similar within EFhd proteins.We next compared the structures of the EF-hands in the Ca2.5.2+ as well as Ca2+ within their EF-hands. Similarly, several other proteins, including Tse3 and calmodulin (CaM), also coordinated Zn2+ within their EF-hand or EF-hand-like motif. In the case of Tse3, an EF-hand-like motif was able to coordinate Ca2+ or Zn2+, and the average coordination distance for Zn2+ is 2.5 Å, which is slightly longer than the average Zn2+–oxygen distance  ]. This shows that the structure and metal coordination geometry of the EF-hand-like motif of Tse3 are comparable to those of EF2 of EFhd proteins but different from those of EF1.EFhd proteins were able to coordinate Zn2+ and Ca2+ within EF-hands . In EF2, seven oxygen atoms, including a water oxygen, coordinated Ca2+ (Ca1) with typical pentagonal bipyramidal geometry. Zn2+ (Zn1) was coordinated within EF2 of CaMZn by six oxygen atoms. Because there is no water in the Zn1 coordination, it formed a distorted pentagonal bipyramidal geometry with a vacancy at position 9(−X) [Fig. 4g)]. In EF3 of CaMCa, Ca2 coordination and geometry were the same as those in EF2 and formed the typical pentagonal bipyramid. Zn2 coordination and geometry in EF3 were also the same as those in EF2 and formed a distorted pentagonal bipyramid with the absence of a water at position 9(−X) [Fig. 4h)]. The average metal coordination distances differed slightly between Ca2+ and Zn2+. The average coordination distances with Ca2+ in CaM EF4, EF2 and EF3 were 2.4, 2.3 and 2.4 Å, respectively; the average coordination distances with Zn2+ in these two EF-hands were 2.3 Å for EF4 and 2.4 Å for both EF2 and EF3 (Table S1). In CaMZn, all of the average coordination distances for Zn2+ were around 2.3 ± 0.5 Å, the average Zn2+–oxygen distance. When we superimposed EF2 of CaMZn and CaMCa or EF3 of CaMZn and CaMCa, the topologies of the alpha helices in CaMZn and CaMCa differed slightly in both cases [Figs. 4g) and 4h)]. Thus, there were no significant structural differences between the Ca2+- and Zn2+-bound EF-hands in EFhd proteins or EF4 of CaM. For EF2 and EF3 of CaM, however, differences between the Ca2+- and Zn2+-bound EF-hands were detected.CaM also coordinates both Znet al., 20012+-bound and Zn2+-bound ALG-2 [ALG-2Ca  and 4j)]. Ca1 was coordinated by six oxygen atoms, including a water oxygen and excluding the S40 oxygen, and assumed a pentagonal bipyramidal geometry. In ALG-2Zn, Zn1 was coordinated by the six oxygen atoms in EF1 with the distorted pentagonal bipyramidal geometry [Fig. 4i)]. Ca2 was also coordinated by six oxygen atoms in EF3 and formed a distorted pentagonal bipyramid. No water molecule was observed. On rare occasions, two Zn2+ ions, Zn2 and Zn3, simultaneously bound to EF3 in ALG-2Zn. Zn2 and Zn3 were coordinated by six and three oxygen atoms, forming a trigonal prism and distorted tetrahedron, respectively. Asp105 which participated in the Ca2+ coordination bound to Zn2 and Zn3 as a bidentate ligand, as did Glu114, and Asp111, which did not participate in Ca2+ coordination, bound to Zn3 [Fig. 4j)]. The average coordination distances for Ca2+ in the two EF-hands were 2.4 and 2.5 Å, respectively; the average coordination distances for Zn2+ in EF1 and EF3 were the same as those for Ca2+, which are slightly longer than the average Zn2+–oxygen distance (Table S1). Therefore, the structures of the EF-hands in ALG-2Zn were similar to those in ALG-2Ca, but the metal coordination of ALG-2Zn showed diverse geometries unlike those of ALG-2Ca. These results show that the EF-hand structures and metal coordination of ALG-2Ca are similar to those of EFhd1Ca and EFhd2Ca, but those of ALG-2Zn differ from those of EFhd1Zn and EFhd2Zn.ALG-2 belongs to the penta-EF-hand (PEF) protein family and contains three metal binding EF-hands  and 5b)]. As previously reported, EFhd1 and EFhd2 bound α- and β-actin independently of Ca2+ , whereas the binding affinity of α-actin was similar for EFhd1 and EFhd2  [Fig. 5c)]. In the presence of Zn2+, EFhd1 and EFhd2 bound to F-actin and showed similar binding affinities for β- and α-actin  [Fig. 5c)]. Thus, both EFhd1 and EFhd2 bind actin in the presence of Ca2+, Zn2+ or EGTA. Consequently, actin binding is both Ca2+- and Zn2+-independent.EFhd1 and EFhd2 both exhibit Ca2+ affects the actin-bundling activities of EFhd1 and EFhd2. Because the subcellular localization of α-actin is in the cytosol and β-actin is in mitochondria, we separately analyzed the actin-bundling activities of EFhd1 and EFhd2 with β-actin and α-actin [Figs. 5d) and 5e)] , Zn2+ contributes to diverse physiological functions , implying that EFhd2 is likely to have Ca2+ bound within resting cells. The binding affinity of EFhd1 for Ca2+ has not been previously reported. We therefore measured the affinity of Ca2+ for EFhd1 using ITC . The mitochondrial [Ca2+] is similar to that in the cytosol, whereas [Zn2+] is in the picomolar range under resting conditions  brains, suggesting a novel amyloid protein may form nucleation centers to induce the formation of tau aggregates  protease cleavage site (Glu–Asn–Leu–Tyr–Phe–Gln/Gly). The amplified EFhd2 core domain was cloned into a modified pET41a vector containing glutathione S-transferase (GST) with a TEV protease cleavage site. Full-length EFhd1 was cloned into a modified pET28a vector (Novagen) with an N-terminal His6-TEV tag. Full-length EFhd2 was cloned into a modified pET28a vector carrying an N-terminal His6 tag.Mouse EFhd1 ΔNTD (residues 69−240) and the human EFhd2 core domain (residues 70−184) were amplified from full-length mouse EFhd1 (residues 1−240) and human EFhd2 (residues 1−240), respectively, using polymerase chain reaction (PCR). The amplified EFhd1 ΔNTD was cloned into a modified pET28a vector (Novagen) containing an N-terminal His4.2.et al., 2021M HEPES-NaOH (pH 7.5), 150 mM NaCl, 0.4 mM PMSF and 14.3 mM β-ME]. The purified protein was concentrated using a 10 K centrifugal filter (Millipore) and stored at −80°C. During purification, the presence of EFhd1 protein was confirmed using SDS–PAGE, and protein degradation was observed following incubation with TEV protease.Protein expression and purification of mouse EFhd1 ΔNTD were performed as reported previously (Mun 4.3.M HEPES-NaOH (pH 7.5), 300 mM NaCl, 0.4 mM PMSF and 14.3 mM β-ME], lysed by sonication and centrifuged at 14 000g for 50 min at 4°C. The supernatant was then subjected to GST-bind agarose (Elpis) affinity chromatography. After washing with the lysis buffer, the target protein was eluted with lysis buffer supplemented with 30 mM glutathione, and the eluted protein was incubated with TEV protease overnight at 4°C to cleave the N-terminal GST-TEV tag. The target protein was further purified through a HiLoad 16/60 Superdex 75 gel-filtration column  pre-equilibrated with the final buffer . To obtain Zn2+-bound EFhd2 protein, the purified protein was treated with a 15-fold excess of EGTA and EDTA for 30 min at 4°C to remove pre-bound metal ions. The protein was then dialyzed in the final buffer for 24 h at 4°C, changing the buffer every 8 h. The dialyzed protein was concentrated using a 10 K centrifugal filter (Millipore) to 9.4 mg ml−1 and treated with 0.75 mM ZnCl2. The resultant Zn2+-bound protein was stored at −80°C.Overall expression of the EFhd2 core domain was similar to that of EFhd1 ΔNTD. Cells transformed with the EFhd2 core domain were harvested by centrifugation, and the cell pellet was suspended in a lysis buffer  were indexed, processed and scaled using the HKL2000 suite  as the template . The σA-weighted mFo − DFc, anomalous difference and ΔAno maps were converted to the CCP4 format using phenix.maps tools , which is more stable than EFhd1 ΔNTD or the full-length protein. For protein expression and purification of EFhd1 (69–200), the affinity chromatography, His6–TEV tag cleavage and gel-filtration steps were same as those used for EFhd1 ΔNTD, except the final buffer contained 20 mM HEPES–NaOH (pH 7.5) and 150 mM NaCl. After purification, EFhd1 (69−200) was treated with a tenfold excess of EGTA and EDTA for 30 min at 4°C to remove pre-bound metal ions. The protein was then dialyzed for 24 h at 4°C in buffer containing 50 mM Tris–HCl (pH 8.5) and 20 mM NaCl. The dialyzed EFhd1 (69–200) was again treated for 30 min at 4°C with a tenfold excess of EGTA and EDTA, after which the protein was again dialyzed for 24 h using the same dialysis buffer, which was refreshed every 8 h. The dialyzed protein was concentrated to 20 µM, and the ligand solution (0.3 mM CaCl2) was prepared in the same buffer. Each EFhd1 (69–200) sample was titrated with 30 injections of ligand (6 µl) in a VP-ITC calorimeter . All measurements were carried out at 20°C, and binding isotherm analysis and fitting were conducted using the Origin software supplied with the calorimeter.To assess the Ca4.10.et al., 2021et al., 2013M CaCl2, 5 mM Tris–HCl (pH 8.0)] to produce actin stock and were polymerized in an actin polymerization buffer  for 1 h at 24°C. Solutions (50 µl) containing polymerized actin (8 µM) were incubated with EFhd1 (12 µM) or EFhd2 (12 µM) for 30 min at 24°C in the presence of 1 mM EGTA, 1 mM CaCl2, or 20 µM ZnCl2. Actin filaments with each protein were pelleted by centrifugation at 100 000g for 2 h at 24°C (for the actin-binding assay). Equal amounts of pellet and supernatant were resolved with SDS–PAGE, and the protein bands were visualized by Coomassie Blue staining. The percentage of each protein in the pellet was quantified with densitometry using ImageJ version 1.53k, and a percentage of pellet histogram was plotted using the OriginPro software  of F-actin (4 µM) and full-length EFhd1 (6 µM) or EFhd2 (6 µM) in the presence of 1 mM EGTA, 20 µM CaCl2 or 20 µM ZnCl2 were allowed to react for 1 h. For grid preparation, 2 µl of reaction mixture were loaded onto C-flat holey gold grids (CF-1.2/1.3-4Au-50) and blotted with filter paper to remove excess sample. The sample-loaded grid was then stained in a solution of 1%(w/v) uranyl acetate. The grids were immersed in the stain solution for 20 min, blotted with filter paper to remove excess stain and air-dried. The samples were imaged using an FEI Tecnai G2 F30 S-Twin transmission electron microscope operated at 300 kV.Muscle and non-muscle actin (Cytoskeleton Inc.) were polymerized in F-actin buffer containing 100 m10.1107/S2052252523001501/jt5066sup1.pdfSupporting figures and table. DOI: 2+-bound EFhd1/Swiprosin-2, 7ygvCaPDB reference: 2+-bound EFhd2/Swiprosin-1, 7ygyZnPDB reference: 2+-bound EFhd1/Swiprosin-2, 7ygwZnPDB reference:


Although there have been clinical cases in which Shenmai injection (SMI) was used to treat cardiovascular diseases (CVDs), there are no pharmacological experiments that investigate the efficacy of the drug 2+) signaling using a microelectrode array (MEA) and a genetically encoded Ca2+ indicator, GCaMP6s, respectively, in human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs).We aimed to systemically evaluate the efficacy and investigate the mechanisms of SMI in modulating electrophysiology and calcium .A MEA system was employed to record field potentials (FPs) in hiPSC-CMs. The QT interval is corrected by the RR interval, the reciprocal of the beating rate. GCaMP6s was used to measure Ca2+ transient frequency was observed.It was found that 0.5%, 1%, and 2% (v/v) SMIs could increase corrected QT (QTc) but did not change other FP parameters. GCaMP6s was successfully applied to measure the chronic function of SMI. The full width at half maximum (FWHM), rise time, and decay time significantly decreased after treatment with SMI for 1 h and 24 h, whereas an increased Ca2+ indicator to measure the chronic effects of TCM. We found that SMI treatment can modulate electrophysiology and calcium signaling and regulate oxidative phosphorylation, cardiac muscle contraction, and the cell cycle pathway in hiPSC-CMs.We first used the Ca •Shenmai injection regulates field potentials in cardiomyocytes.•Shenmai injection regulates calcium signals in cardiomyocytes.•Shenmai injection regulates the transcriptome of cardiomyocytes. Mey.) and Maidong (Ophiopogon japonicus (Thunb.) Ker Gawl.) [Arrhythmia is a common cardiovascular disease (CVD) that manifests as an irregular heart rate . Accordiblockers . For insblockers  and affeblockers  and liveblockers ,7. For pblockers ,9. In reblockers . Containr Gawl.) , Shenmair Gawl.) ,13. Hower Gawl.) .in vitro human cardiomyocyte model that has been widely used to establish disease models and test drug effects [2+ signals, but it is difficult for these dyes to measure the long-term  efficacy of drugs in vitro because of dye leakage and cytotoxicity [2+ indicator, can overcome these drawbacks and has been used for long-term recording of Ca2+ signals [Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are an  effects . Cardiac effects . To noni effects . Thus, M effects . Chemicatoxicity ,22. GCaM signals ,24.2+ indicator (GCaMP6s) for the first time to test the instantaneous and long-term effects of SMI on Ca2+ signals in hiPSC-CMs [In this study, we utilized a MEA system to evaluate the efficacy of Shenmai injection (SMI) on cardiac electrophysiology. Meanwhile, we used a CaiPSC-CMs . In addi22.12, 5 mM KCl, and 10 mM d-glucose buffered with 10 mM HEPES at pH 7.4. Methanol and acetonitrile were purchased from Merck . Formic acid was purchased from West Asia Reagent .HiPSC-CMs were purchased from HELP Regenerative Medicine Technology Co., Ltd. . From Peking Union Medical College , 293T cells were obtained, and the cells were cultured as described previously . SMI was2.22 for 24 h. A microplate reader  was utilized to read the absorbance, and the cell viability was calculated to determine the concentration of SMI that should be used in our subsequent experiments.HiPSC-CMs were seeded in 96-well plates. Media containing 1%, 2%, 4%, and 8% (v/v) SMI were added to the plates and cultured in an incubator at 37 °C with 5% CO2.3GCaMP6s plasmid has been constructed in previous experiments . In a si2.4Cells were seeded in the center of a MEA dish (Multi-Channel Systems (MCS) GmbH, Reutlingen, Germany), and the changes in FP in hiPSC-CMs were recorded after treatments of 0.25%, 0.5%, 1%, and 2% (v/v) SMI. Gradient dosing was applied, and the results were analyzed using Cardio2D + software .2.54 on confocal dishes coated with a plating solution. After 24 h, they were replaced with a cardiomyocyte culture medium, and the medium was changed every other day. The cells were transduced with GCaMP6s lentiviral particles, and a confocal microscope  was used to capture Ca2+ fluorescence in the cytoplasm. Tyrode solution was used to wash the cells before Ca2+ imaging. Intracellular free Ca2+ could bind to GCaMP6s in the hiPSC-CMs, and Ca2+ transients were recorded after SMI treatment for 5 min, 1 h, and 24 h. Peakcaller [2+ signal-related parameters.HiPSC-CMs were seeded with a density of 1 × 10akcaller  was used2.6The effect of SMI on the gene expression of hiPSC-CMs was analyzed using the intelligent analysis platform of BGI . The general process was to first perform quality control on the RNA collected from cell lysis, including the RNA concentration and purity (A260/A280). Reverse transcription was performed if the RNA met the standards, and a library was established. The quality of the constructed library was checked, and subsequent sequencing was performed. The data obtained by sequencing were called raw reads and were also quality-controlled to determine whether they were suitable for subsequent analysis. The data obtained in this step were called clean reads. Step-by-step comparison, quantification, difference analysis, and cluster analysis were then carried out. The gene expression level was quantified by the number of fragments per thousand base fragments (FPKM). Differentially expressed genes (DEGs) were defined as FPKM ≥2, and the false discovery rate (FDR) was ≤0.001.2.7LC-MS was performed on an ultra-high-performance liquid chromatograph system . The general process was to centrifuge SMI at 4 °C and 12,000 rpm for 10 min, and the supernatant was then aspirated and diluted 2–100 times. Afterwards, 10 μL of internal standard (100 μg/mL) was added and passed through a 0.22 μm polytetrafluoroethylene filter for onboard testing. This was followed by metabolite quantitative analysis, that is, calculating the peak area of the metabolite to obtain the relative concentration and percentage of the metabolite in the sample. An analysis platform  and chromatographic column C18 (Zorbax Eclipse C18 (1.8 μm × 2.1 × 100 mm) were then employed for analysis. The mobile phase was composed of A (H2O + 0.1% formic acid) and B (acetonitrile). The flow rate was 0.3 mL/min and the column temperature was 30 °C, while the injection volume was 2 μL and the autosampler temperature was 4 °C. The mobile phase gradient elution program was 5% B (0–2 min), 30% B , 30% B (6–7 min), 78% B , 78% B (12–14 min), 95% B , 95% B (17–20 min), 5% B , and 5% B (21–25 min).2.8P < 0.05 indicated a significant difference. All data results were expressed as mean ± standard error.SPSS was used for the statistical analysis. For data passing the normality and homogeneity tests, a one-way analysis of variance (ANOVA) test was used. Otherwise, a non-parametric test was used. If the data did not conform to a normal distribution, the Kruskal–Wallis test was employed. 3in vitro cell experiments are based on H9C2 cells or rat cardiomyocytes [The effect of SMI on the cardiac functions of hiPSC-CMs has not yet been reported, and most myocytes . Thus, t2+ signaling. We recorded Ca2+ signals 5 min, 1 h, and 24 h after SMI treatment, and the results showed that 2% (v/v) SMI did not change the Ca2+ transient-related parameters at 5 min  SMI upregulated 879 genes and downregulated 971 genes A–C. Diff42+ through voltage-dependent Ca2+ channels. The Ca2+ further activates ryanodine receptors (RyRs) on the membrane of the sarcoplasmic reticulum (SR), which triggers Ca2+ release from the SR into the cytoplasm. This process is known as calcium-induced calcium release [2+ bind to contractile proteins, such as troponin, and cause contraction of the cardiomyocyte. The conversion of the AP into a contractile response is called excitation–contraction coupling [2+ signals to cardiac contractile function, our study was conceptualized to investigate the role of SMI in modulating electrophysiology and calcium signaling in hiPSC-CMs, an in vitro human cardiomyocyte model.Contraction, the most important function of the heart, is a consequence of cardiac electrophysiology and calcium signaling . First,  release . Finallycoupling . The rolcoupling , and Checoupling . Due to 2+ handling can cause instability in AP duration by affecting the repolarization of hiPSC-CMs, but the results of our Ca2+ imaging experiments indicated that SMI did not alter the Ca2+ transients of hiPSC-CMs in short-term  treatment, suggesting that SMI does not cause changes in the QT interval by altering calcium handling. The underlying mechanisms, especially the potential effects of SMI on the activities of various ion channels, deserve further investigation.In existing studies, the range of SMI concentrations used is around 0.25%–5%, but most of the cells under investigation are H9C2. In our study, we tested the effect of SMI with a wider range of concentrations (up to 8%) on the viability of hiPSC-CMs using a CCK-8 assay and found that it did not significantly alter cell viability. Therefore, the minimum effective concentrations of SMI of various doses below 2% were used in field potential recordings. As reflected by the parameters of the field potentials, QT prolongation was observed, which is generally believed to be associated with an increased risk of arrhythmia. However, it is worth noting that the concentrations of SMI used in this study are still higher than the clinical dosage. Abnormal Ca2+ indicator (GCaMP6s) instead of chemical dyes to study the long-term effect of TCM on Ca2+ signaling in cardiomyocytes. GCaMP6s is not likely to leak out of cells compared to chemical dyes, such as Fluo-4 [2+ indicators have been widely used in the literature. For example, Chen et al. used GCaMP6 to measure the long-term activity of neuronal cells [2+-sensing protein, as a molecular probe in ventricular myocytes [2+ indicators to study the long-term effects of TCM on Ca2+ signals, especially when considering the relatively slow onset of action of TCM. We then used GCaMP6s to study the Ca2+ signals in hiPSC-CMs treated with SMIs for a longer period , and our results showed that the FWHMs, rise times, and decay times of Ca2+ transients were significantly decreased, whereas Ca2+ transient frequency was significantly elevated, when the incubation time of the SMI was increased. According to one report, SMI has been reported to enhance energy metabolism while slightly increasing heart rate in patients with heart failure [2+ transient duration. As an indicator of Ca2+ transient duration, FWHM is correlated with the total amount of Ca2+ mobilized during one contraction cycle, which is quantified by the area under the spike of the calcium transient waveform. Thus, long-term SMI treatment reduces the lifespan of Ca2+ transients and promotes heartbeat pulsation.Because of the limited effects of short-term SMI treatment, we, for the first time, used a genetically encoded Cas Fluo-4 . Moreoves Fluo-4 . Thus, gal cells , and Pahmyocytes . Therefo failure , which m2+ plays an important role in regulating cardiomyocyte maturation during early cardiomyocyte development [2+ channels and receptors. This is consistent with the results that Ca2+ transients were altered by SMI treatment for 24 h in hiPSC-CMs. Due to the complex composition of SMI, we dissected its composition using LC-MS, and the compounds identified in SMI have recently been reported to target electrical and calcium signals. For example, ginsenoside has been reported to modulate voltage-gated ion channels by activating Na+/K+ channels [Short-term drug treatment may alter protein activity by, for example, inhibiting calcium channels and phosphorylating receptor proteins, and long-term drug treatment may affect the transcriptome. Thus, we further investigated the effect of 2% SMI on hiPSC-CMs transcriptome using RNAseq. As enriched in KEGG, the signaling pathways related to oxidative phosphorylation and cardiac muscle contraction were affected by SMI. This is consistent with the literature, which indicates that SMI can improve energy metabolism and enhance myocardial contraction and myocardial function. For example, SMI combined with conventional treatment improved energy metabolism in patients with heart failure in a clinical trial , and SMIelopment . It can channels  and to ichannels . These f2+ signals, and contractility could also be studied further [This study has several limitations. First, we only measured the efficacy of SMI at the cellular level, and animal models need to be established in subsequent studies to reflect the efficacy of SMI on changes in heart rate and myocardial function. Second, this article only evaluated the efficacy of SMI based on electrical and Ca further . In addi52+ indicator (GCaMP6s) to study the efficacy of TCM, concluding that the therapeutic effects of SMI might be closely related to its ability to regulate electrical and Ca2+ signals. Multiple pathways are involved, such as the cardiac muscle contraction pathway.We used a MEA system and a genetically encoded CaSL designed the study. ZZ and YXL performed the experiments, analyzed the data, prepared the figures, and drafted the manuscript. MHY, TTY and XY critically reviewed the manuscript. All authors read and approved the final manuscript.10.13039/501100001809National Natural Science Foundation of China (Grant No. 81973698 and 81703942), Young Elite Scientists Sponsorship Program by CACM (Grant No. 2019-QNRC2-B08), Science Fund for Distinguished Young Scholars in BUCM (Grant No. BUCM-2019-JCRC004) and 10.13039/501100004846BUCM research start-up fund (to SL).This study is supported by the All authors report no conflicts of interest.


The activation of KCa2.3 channels induceshyperpolarization,which augments Ca2+ signaling in endothelial cells. Ciliaare specialized Ca2+ signaling compartments. Here, we identifiedcompound 4 that potentiates human KCa2.3 channelsselectively. The subtype selectivity of compound 4 forhuman KCa2.3 over rat KCa2.2a channels relieson an isoleucine residue in the HA/HB helices. Positive modulationof KCa2.3 channels by compound 4 increasedflow-induced Ca2+ signaling and cilia length, while negativemodulation by AP14145 reduced flow-induced Ca2+ signalingand cilia length. These findings were corroborated by the increasedcilia length due to the expression of Ca2+-hypersensitiveKCa2.3_G351D mutant channels and the reduced cilia lengthresulting from the expression of Ca2+-hyposensitive KCa2.3_I438N channels. Collectively, we were able to associatefunctions of KCa2.3 channels and cilia, two crucial componentsin the flow-induced Ca2+ signaling of endothelial cells,with potential implications in vasodilation and ciliopathic hypertension.Small-conductance Ca KCa3.1 channels are often found on the ET cell membraneclose to the endoplasmic reticulum (ER) Ca2+ store.9 Ca2+ release from the ER triggered by acetylcholine orbradykinin receptors may lead to the opening of KCa3.1channels nearby.10 In contrast, KCa2.3 channels seem to co-localize with mechanosensitive or receptor-operatedtransient receptor potential (TRP) cation channels.11 Ca2+ influx through these cation channels may activateKCa2.3 channels. The outflow of K+ can hyperpolarizeET cells, increase the inward electrochemical gradient for Ca2+, and augment the Ca2+ influx, which in turn enhancesnitric oxide (NO) releases.13In blood vessels, K16 Fluid flowthat produces enough drag force on the top of the cells will bendand activate sensory cilia. Transgenic mouse models with cilia mutationsdo not survive at birth, confirming the importance of primary ciliain the physiological processes.20 Primary cilia in vasculatures were once thought tobe vestigial organelles and nonfunctional remnants. It has since beenshown by different laboratories that cilia are mechanosensory organelles.25 Cilia in ET cells sense changes in the fluid shear stress and triggerCa2+ signaling and NO releases.27Non-motile primarycilia are sensory organelles that sense fluidshear stress on the apical membrane of the cells.2+ signalingcompartments.29 Ca2+ influx throughTRPM4, TRPV4, TRPC1, polycystic kidney disease 2 (PKD2), and L-typevoltage-gated Ca2+ (Cav) channels has been consideredthe main Ca2+ source for cilia.29 Ca2+ influx in response to fluid shear stress activatesET KCa2.3 channels.30 In ETcells, KCa2.3 channels functionally couple with Ca2+-permeable PKD211 and TRPV431 channels and exert a positive feedback influenceon intracellular Ca2+ signaling.32 However, it is not clear whether this positive feedback mechanismextends back to the cilia, that is, whether the activation of KCa2.3 channels increases cilia length.Primary cilia have been known as specialized CaCa2.3 and KCa2.2a channels have similaramino acid sequences in their cytoplasmic gates, which makes it difficultto develop subtype-selective positive modulators discriminating thesetwo subtypes. We recently identified the binding site of a prototypeKCa2.2a/KCa2.3 channel modulator, CyPPA.33 We have synthesized a new series of CyPPA analogues.34 Here, we report the identification of a subtype-selectiveKCa2.3 channel modulator, compound 4, thatis ∼21-fold more potent on potentiating human KCa2.3 than rat KCa2.2a channels. The subtype selectivityof compound 4 relies on an I-to-V amino acid residuedifference between KCa2.3 and KCa2.2a channels.The pharmacological activation of KCa2.3 channels by compound 4 increased cilia length, whereas the pharmacological inhibitionof KCa2.3 channels by AP14145 decreased cilia length ina cultured ET cell line, suggesting the critical role of KCa2.3 channels in the regulation of cilia.K22.134 Thepotency of these compounds was measured using inside-out patch clampelectrophysiology recordings with human KCa2.3 channelsheterologously expressed in HEK293 cells. Positive modulators of KCa2 channels require minimal concentration of Ca2+ to be effective.35 Therefore, we measuredthe concentration-dependent responses of the channels to compoundsin the presence of 0.15 μM Ca2+ . To construct the concentration–responsecurves, the current amplitudes at −90 mV in response to variousconcentrations of the compound were normalized to that obtained atthe maximal concentration of the compound. The normalized currentswere plotted as a function of the compound concentrations. CyPPA,NS13001, and our compounds 2m–2n, 2p, 2r–2t, 2v, and 4 concentration-dependently potentiatedthe activity of KCa2.3 channels  of the compounds on KCa2.3 channels, thecurrent amplitudes at −90 mV in response to the compounds werenormalized to that obtained at 10 μM Ca2+ . Toassess the efficacy of the compound, the current amplitudes obtainedat the maximal concentration of the compound were normalized to themaximal KCa2.x/KCa3.1 current in response to10 μM Ca2+. Concentration–response curveswere acquired from multiple patches for each data set. Each curvewas fitted individually, which yielded the EC50 value forthat curve. EC50 values are shown as mean ± SD obtainedfrom multiple patches, and the number of patches is indicated by n.Patchclamp recordings were analyzed using Clampfit 10.5 , and concentration–response curves wereanalyzed in GraphPad Prism 9.0.2 . To construct the concentration-dependent potentiation of channelactivities by the compound, the current amplitudes at −90 mVin response to various concentrations of the compound were normalizedto that obtained at a maximal concentration of the compound. The normalizedcurrents were plotted as a function of the concentrations of the compound.ECt-test wasused for data comparison if there were only two groups. One-way ANOVAand Tukey’s post hoc tests were used for data comparison ofthree or more groups. Post hoc tests were carried out only if F was significant and there was no variance in homogeneity.The Student’s


The inositol 1,4,5-trisphosphate receptor (IP3R), a homo- or heterotetramer of the IP3R1-3 isoforms, amplifies lymphocyte signaling by releasing Ca2+ from endoplasmic reticulum stores following antigen stimulation. Although knockout of all IP3R isoforms in mice causes immunodeficiency, the seeming redundancy of the isoforms is thought to explain the absence of variants in human immunodeficiency. In this study, we identified compound heterozygous variants of ITPR3 (a gene encoding IP3R subtype 3) in two unrelated Caucasian patients presenting with immunodeficiency. To determine whether ITPR3 variants act in a nonredundant manner and disrupt human immune responses, we characterized the Ca2+ signaling capacity, the lymphocyte response, and the clinical phenotype of these patients. We observed disrupted Ca2+ signaling in patient-derived fibroblasts and immune cells, with abnormal proliferation and activation responses following T-cell receptor stimulation. Reconstitution of IP3R3 in IP3R knockout cell lines led to the identification of variants as functional hypomorphs that showed reduced ability to discriminate between homeostatic and induced states, validating a genotype–phenotype link. These results demonstrate a functional link between defective endoplasmic reticulum Ca2+ channels and immunodeficiency and identify IP3Rs as diagnostic targets for patients with specific inborn errors of immunity. These results also extend the known cause of Ca2+-associated immunodeficiency from store-operated entry to impaired Ca2+ mobilization from the endoplasmic reticulum, revealing a broad sensitivity of lymphocytes to genetic defects in Ca2+ signaling.Calcium signaling is essential for lymphocyte activation, with genetic disruptions of store-operated calcium (Ca Iden2+ flux triggered by TCR and BCR engagement is realized via both Ca2+release from intracellular Ca2+ stores and influx from the extracellular compartment in multiple phases. The first step is mediated by the second messenger inositol 1,4,5-trisphosphate (IP3), which is generated upon activation of phospholipase C γ cyt, in the second stage, the ER transmembrane (TM) protein stromal interaction molecule 1 (STIM1) senses lower ER [Ca2+], and via a conformational change directly triggers the opening of plasmalemmal ORAI1 channels cyt triggers downstream signaling, notably the NF-κB and calcineurin/nuclear factor of activated T cells (NFAT) pathways i using Fura2/AM . Briefly, cells were loaded with the ratiometric fluorescent Ca2+ indicator Fura2/AM (1 µM) at RT for 30 min in a modified Krebs solution , and 1.5 mM CaCl2). Cells were allowed to rest for 30 min at RT in the absence of Fura2/AM to allow complete dye de-esterification before analysis was performed with a FlexStation 3 microplate reader . The indicator was alternately excited at 340 and 380 nm, and fluorescence emission at 510 nm was recorded. EGTA was added after 30 s in all conditions to a final concentration of 3 mM to chelate Ca2+ ions in the buffer, and the fluorescence was recorded for 60 s prior to stimulation. The responses to stimuli prepared in Ca2+-free modified Krebs solution containing 3 mM EGTA were acquired for 6 min. Ionomycin and the irreversible sarco-/endoplasmic reticulum Ca2+-ATPase (SERCA) inhibitor thapsigargin were added to final concentrations of 10 µM, and bradykinin was added to a final concentration of 50 nM. All traces are shown as the ratio of both emission wavelengths F340/F380.Fibroblasts were seeded in 96-well plates  and assessed for cyt in response to various stimuli using a ratiometric Ca2+ indicator (Fura2). Stimulation with the Ca2+ ionophore ionomycin and the sarco-/endoplasmic reticulum Ca2+-ATPase (SERCA) inhibitor thapsigargin in the presence of extracellular EGTA, a Ca2+-chelating agent, revealed dysregulated intracellular Ca2+ homeostasis in fibroblasts of P1  agonist bradykinin, we also observed a decreased response in P1, as assessed by the integrated response  as well as the peak amplitude normalized to the respective sample background . By labeling CD4+ T cells from different donors with different fluorochromes before combining the samples, we minimized sample-to-sample variation. The cells were loaded with the ratiometric Ca2+ indicator FuraRed and stimulated with ionomycin or thapsigargin to investigate calcium homeostasis or anti-CD3 and anti-CD28 to assess physiological IP3R-mediated calcium responses. While Ca2+ homeostasis was disrupted in P2 and his parents , although the direct comparison between patient PBMCs was not possible due to HSCT. Ca2+ homeostasis but not signaling defects were also observed in the heterozygous parents of P2.We then studied PBMCs to confirm impaired Caype Fig. . This fiITPR3 variants on calcium signaling in a model allowing direct comparison in the absence of compensatory mechanisms, we used HEK cells that had been engineered to eliminate endogenous expression of all three IP3R subtypes [3R3. Double-knockout of IP3R1 and IP3R2, resulting in a cell line expressing only endogenous IP3R3 (WT), was used as a control with physiological expression levels. WT IP3R3 in HEK-3KO cells was overexpressed (WT OE) to measure the maximum Ca2+ signaling capacity. We generated multiple monoclonal cell lines with transgenic expression of each mutant allele, confirmed the localization of each encoded protein to the ER via immunohistochemistry, and used western blotting to benchmark protein expression levels against endogenous WT expression  and R1850Q  variants. This recessive inheritance may be explained via the location of the variants in the regulatory domain of IPfunction . In thisö et al. . The abs3R3 deficiency and the identification of the R1850Q, F1628L, and R2524C variants as pathogenic, this study provides potential insight into the molecular regulation of IP3R3. Despite advances in the accuracy of electron microscopy techniques that can reveal conformational changes and interactions in specific activation states of IP3Rs [3R3 channel activation in humans [3R1 protein has been suggested to exhibit a crucial function in signal transmission, as mutation into different amino acids markedly reduced channel function [ITPR3 variants and systematic investigation of these mutated receptors may reveal protein interactions and/or conformational changes that are hampered in different cell types, providing better insight into channel regulation.In addition to insights into the differential sensitivity of cell types to IPof IP3Rs , 49, 52,of IP3Rs  and has n humans . In linefunction . FurtherITPR3 as underlying causes for disrupted Ca2+ signaling that results in immunodeficiency. Some clinical characteristics of the patients we describe overlap with those of patients suffering from ORAI1 and STIM1 deficiency, who present with severely reduced or absent store-operated Ca2+ entry in vitro and normal lymphocyte counts but impaired T-cell proliferation following in vitro stimulation [With this study, we add variants in mulation . Comparamulation , 71. Climulation , 73. In mulation , 74, 75,ITPR3 alter physiologically relevant Ca2+ signaling responses, driving defects in immune responses, and are associated with clinical immunodeficiency. Limitations of our work include the limited number of cases, only two patients, and the lack of primary immune cells from P1 due to successful HSCT. Because of the inheritance pattern, we also lacked primary immune cells harboring only the R2524C variant, that would have allowed us to clarify the recessive or dominant nature of this mutation in lymphocytes. The identification of new patients in subsequent studies will be highly informative for identifying the mutational space in ITPR3 associated with immunodeficiency. Additional studies investigating the effect of ITPR3 variants on cellular processes such as apoptosis and autophagy, which are known to be regulated by IP3Rs coupling Ca2+ signaling between the ER and mitochondria, will also be interesting [ITPR3 should be considered as a candidate gene in patients with B- and T-cell defects in association with impaired Ca2+ signaling function, adding variants in ITPR3 as causes of inborn errors of immunity.Here, we provide evidence showing that inherited variants in eresting , 77. AnySupplementary Material


TLN1 gene is a focal adhesion-related protein that binds to various cytoskeletal proteins and plays an important role in cell adhesion and movement. Recent studies have shown that it is overexpressed in prostate cancer, liver cancer, and oral squamous cell carcinoma, and is closely related to tumor progression and metastasis. This study integrated bioinformatics and functional analysis to reveal the prognosis and potential functions of TLN1 in AML. The results showed that the expression level of TLN1 was abnormally increased in AML and localized in the cell membrane and cytoplasm, and TLN1 is a significant prognostic indicator of overall survival (OS). Enrichment analysis of related genes showed that TLN1 is related to neutrophil mediated immunity, neutrophil activation and may regulate important signal pathways in hematological tumors including tyrosine kinase receptor, FLT3 and PIK3/AKT. The PPI network shows that TLN1 and MYH9 may be involved in the process of AML tumors together with PIP5K1C, ROCK1, S100A4, MY01A and WAC. Immune infiltration analysis explains that TLN1 is associated with multiple immune cells and may be an important immune marker in AML. Furthermore, molecular biology experiments confirmed that TLN1 is related to the proliferation, differentiation and cycle of AML cells. Silencing TLN1 can inhibit the proliferation of AML cells and promote differentiation through the Talin1/P-AKT/CREB signaling pathway.The protein Talin1 encoded by the The online version contains supplementary material available at 10.1186/s12885-022-10099-0. Acute myeloid leukemia (AML) is an aggressive hematological tumor characterized by the abnormal proliferation and differentiation of immature myeloid cells a poor prognosis and is prone to drug resistance and recurrence . AccordiTLN1, which is the first adaptor protein to be confirmed to bind to integrin. It’s binding to the intracellular segment of the integrin subunit is the key link for integrin activation . Caspase-3 antibody was purchased from Cell Signalling Technology . Phosphorylated-AKT, CREB, Talin1 and PARP were purchased from Proteintech Technology . Total AKT was purchased from Zen-bioscience . This procedure was repeated 3 times for each protein sample.Collect the cells in logarithmic growth phase, add an appropriate amount of fine RIPA lysis buffer (containing protease inhibitors), and lyse on ice for 30 min. Take 100uL of lysate for Western blot analysis, add 1μg of the corresponding antibody to the remaining lysate, and incubate overnight at 4 °C with slow shaking. Take 10 ul protein A agarose beads, wash 3 times with an appropriate amount of lysis buffer, and centrifuge at 3000 rpm for 3 min each time. Add 10 ul of pretreated protein A agarose beads to the cell lysate incubated with the antibody overnight at 4 °C and incubate with slow shaking for 2-4 h to couple the antibody to the protein A agarose beads. After immunoprecipitation, centrifuge at 3000 rpm for 3 min at 4 °C, centrifuge the agarose beads to the bottom of the tube and remove the supernatant. Wash the agarose beads 3-4 times with 1 ml lysis buffer, and add SDS loading buffer, boiled for 5 minutes, use Western blotting for analysis.The cells were treated with siRNA transfection for 48 hours and fixed overnight with 70% ethanol at − 4 °C. After two times of PBS cleaning, PI staining was used for 30 minutes on the flow cytometer for testing and the Cytexpert software was used to analyze data.P < 0.05 was considered statistically significant.Statistical obtained from TCGA and GTEX were analyzed by R-3.6.1. Other datas were processed using GraphPad Prism 8, Flowjo and Cytexpert software. TLN1 is abnormally highly expressed in 12 tumors including AML, compared with normal tissues  to analyze the expression level of TLN1 in human blood cells, found that the RNA expression of TLN1 showed low cell-type specificity  format are analyzed and compared after log2 conversion. We found that TLN1 is related to poor overall survival (OS) in AML  was 0.721   , the difference is statistically significant (P = 0.009) ). In addition, age (> 60 years), Cytogenetic risk (intermediate and poor) were also poor prognostic factors for AML patients. The multivariate Cox model showed that age (> 60 years), Cytogenetic risk (intermediate and poor), FLT3 mutation and TLN1 were independent poor prognostic factors for AML patients  for statistical analysis of survival data and survminer package [0.4.9 version] for visualization and drawing Kaplan-Meier survival curves. The high expression of 0 versionTLN1, we used LinkedOmics to analyze co-expressed genes of TLN1 in AML [P < 0.05 and |correlation coefficient| > 0.5, we got 285 genes revealed a significant positive association with TLN1, while 165 genes indicated significantly negative associations -7-Oxozeaenol, AZD6482 and BX-795 may provide a therapeutic strategy for targeting TLN1  and 0.71 (p < 0.001). Therefore, we analyzed the expression and prognosis of MYH9 and FLNA in the TCGA AML cohort. The results showed that MYH9 was abnormally highly expressed in AML patients and both of MYH9 and FLNA were associated with poor prognosis , Tem , iDC  etc. , the expression level of ing Fig. B. This mlls Fig. E. We alsTLN1 may be related to the PI3K/AKT signaling pathway. Therefore, we tested the expression levels of AKT and P-AKT after TLN1 silence. Our results showed that interfering with the expression of Talin1 reduced the expression level of P-AKT. Similarly, the expression level of CREB was also downregulated which was a signal molecule downstream of AKT and related to cell proliferation and differentiation . Studies have shown that Talin is an indispensable factor for endothelial cell-mediated angiogenesis [TLN1)was upregulated by 6 times in CML-blast crisis (BC) compared with the CML-chronic phase (CP) stage [TLN1 may play an important role in hematopoietic malignancies.The protein Talin1 encoded by ogenesis . In patiogenesis . In addiP) stage . These rTLN1 in AML. Our results show that the RNA and protein of Talin1 (TLN1) are abnormally highly expressed in AML samples, which may be a prognostic marker of AML patients. Unfortunately, we were unable to analyze the role of TLN1 in event free survival, relapse and disease progression due to the limited clinical information provided by the TCGA data. However, enrichment analysis results show that TLN1 is related to multiple functions of neutrophils and may be involved in multiple important signal pathways in hematopoietic malignancies.In this study, we used a variety of bioinformatics analysis and functional analysis methods to explore the expression, prognostic role and biological function of MYH9) is widely expressed in various tissues and cells. MYH9 is abnormally highly expressed in esophageal squamous cell carcinoma [MYH9 degrades GSK3β protein through TRAF6-induced ubiquitination, enhancing the stem cell characteristics, migration, invasion, growth and sorafenib resistance of HCC cells [MYH9 may be a promising target gene for cancer treatment. Our analysis found that TLN1 strongly correlates with MYH9, and TLN1 may work with MYH9, PIP5K1C, ROCK1, S100A4, MY01A and WAC to promote the growth and invasion of AML cells. Co-immunoprecipitation and immunofluorescence experiments confirmed the interaction between TLN1 and MYH9. However, the mechanism of action between TLN1 and MYH9 needs more experiments to verify.Non-muscle myosin heavy chain9  is a nuclear protein that selectively binds to the cyclic adenylate response element to regulate cell proliferation, differentiation and apoptosis . ResearcTLN1 is a potential prognostic and therapeutic biomarker of AML, and it can affect the immune microenvironment to promote the proliferation of AML cells and inhibit differentiation and regulate multiple signal pathways in hematologic malignancies. However, the regulatory effect of TLN1 on the immune microenvironment and the interaction between TLN1 and MYH9 need to be further confirmed by experimental data.The 13 rod-shaped of Talin1 (R1-R13) domains are entangled into a compact globular structure , 26, andAdditional file 1: Supplementary Figure1. High expression of TLN1 is associated with poor prognosis of Brain Lower Grade Glioma (A), Adrenocortical carcinoma (B), Oral squamous Cell Carcinoma (C), Colon adenocarcinoma, Glioma (D), Cervical squamous cell carcinoma (E) and endocervical adenocarcinoma (F). G: Kaplan–Meier survival curves for TLN1 in AML cohort after excluding M3 patients.Additional file 2: Supplementary Table 1. The gene function notes of GO and KEGG enrichment analysis.Additional file 3.Additional file 4.Additional file 5.Additional file 6.


Pyruvate kinase M2 (PKM2) plays an important role in the metabolism and proliferation of leukemia cells. Here, we show that deubiquitinase JOSD2, a novel tumor suppressor, blocks PKM2 nuclear localization by reducing its K433 acetylation in acute myeloid leukemia (AML). Firstly, we show that JOSD2 is significantly down-regulated in primary AML cells. Reconstitute of JOSD2 in AML cells significantly inhibit cell viability and induce cell apoptosis. Next, PKM2 is identified as a novel interaction protein of JOSD2 by mass spectrometry, co- immunoprecipitation and co-immunofluorescence in HL60 cells. However, JOSD2 does not affect PKM2 protein stability. We then found out that JOSD2 inhibits nuclear localization of PKM2 by reducing its K433 acetylation modification, accompanied by decreased downstream gene expression through non-glycolytic functions. Finally, JOSD2 decreases AML progression in vivo. Taken together, we propose that JOSD2 blocks PKM2 nuclear localization and reduces AML progression.The online version contains supplementary material available at 10.1186/s40164-022-00295-w. To the Editor,Acute myeloid leukemia (AML) is a hematological malignancy characterized by the accumulation of immature myeloid cells with distinct molecular genetic characteristics. Although targeted therapies and immunotherapies, such as BCL2 inhibitors and FLT3 inhibitors, have achieved some clinical efficacy, the overall survival rate for AML remains low. Understanding the pathogenesis of AML is important for the development of new targeted drugs to improve the treatment of AML.The biological functions of Machado-Joseph deubiquitinases (MJDs) in cancer are gradually being revealed , 2. MJDsUsing real-time RT-PCR, we found that JOSD2 and JOSD1 were expressed at lower levels in different types of primary AML cells compared with normal peripheral blood mononuclear cells (PBMCs)  and AML cell lines. Scale bar, 20 μm. b mRNA levels of JOSD2 in AML cell lines compared with normal peripheral blood mononuclear cells. c Western blot analysis of JOSD2 protein expression in bone marrow mononuclear cells from health donors and AML cell lines. Data are presented as mean ± SD, and were analyzed by using One-way ANOVA. ***P < 0.001. Figure S2. Changes of apoptosis and differentiation after overexpression of JOSD2 in AML cells. a Cell apoptosis was determined by flow cytometric analysis of Annexin V and PI staining. b Cell differentiation was determined by flow cytometric analysis of side-scatter profiles (SSC) and the expression of CD11b. Data are presented as mean ± SD, and were analyzed by using the 2-tailed Student t test. **P < 0.01, ***P < 0.001. Figure S3. JOSD2 blocks PKM2 nuclear localization and inhibited the expression of the related genes cyclin D1 and c-myc. a Immunofluorescence assay shows that JOSD2 blocks PKM2 nuclear localization in primary AML (M5) cells. Scale bar, 20 μm. b JOSD2 expression inhibited the transcription levels of cyclin D1 and c-myc, the downstream target genes of PKM2 nuclear localization. Data are presented as mean ± SD, and were analyzed by using the 2-tailed Student t test. ***P < 0.001.


Orai1 proteins have been implicated in platelet’s SOCE. In this study we evaluated the contribution of Orai1 proteins to these processes using washed platelets from adult mice from both genders with platelet-specific deletion of the Orai1 gene  since mice with ubiquitous Orai1 deficiency show early lethality. Platelet aggregation as well as Ca2+ entry and release were measured in vitro following stimulation with collagen, collagen related peptide (CRP), thromboxane A2 analogue U46619, thrombin, ADP and the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) inhibitor thapsigargin, respectively. SOCE and aggregation induced by Thapsigargin up to a concentration of 0.3 µM was abrogated in Orai1-deficient platelets. Receptor-operated Ca2+-entry and/or platelet aggregation induced by CRP, U46619 or thrombin were partially affected by Orai1 deletion depending on the gender. In contrast, ADP-, collagen- and CRP-induced aggregation was comparable in Orai1Plt-KO platelets and control cells over the entire concentration range. Our results reinforce the indispensability of Orai1 proteins for SOCE in murine platelets, contribute to understand its role in agonist-dependent signalling and emphasize the importance to analyse platelets from both genders.Calcium signalling in platelets through store operated Ca Ca2+ peak was measured as maximal Ca2+ concentration within 30 s after either agonist application or Ca2+-addition. To determine the required extracellular Ca2+ concentration to evoke Orai1 dependent Ca2+ entry we tested both 0.3 mM and 3 mM Ca2+ after addition after thapsigargin. Therefore, a concentration of about 2 mM extracellular free-Ca2+ is expected in the presence of 1 mM EGTA  after normality test .Data were analysed and processed with Excel 2010 (Microsoft), Graphpad Prism 8.0 and OriginPro 2019G ver. 9.6.0.172 (OriginLab). Values are shown as mean ± standard deviation (SD), except for original traces of Caflox/flox mice [Plt-KO) and compared them with corresponding litter matched control animals (Orai1Plt-Ctrl) in terms of in vitro platelet aggregation and Ca2+-signalling to determine the contribution of Orai1 proteins after stimulation with different platelet agonists at different concentration , Thromboxane A2 analogue U46619, thrombin and ADP) as well as with the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) inhibitor, thapsigargin. We analysed platelet aggregation in the presence of 2 mM total extracellular Ca2+ concentration; taking into account that our buffer contains albumin, it is assumed that the free Ca2+ is about ~1.2 mM similarly as the physiological free plasma Ca2+ concentration. In the case of intracellular Ca2+ measurements we performed a stepwise Ca2+ re-addition protocol having two extracellular Ca2+ concentrations (>0.1 and ~2 mM). This approach proved to be useful to analyse Orai proteins in other cellular systems [2+ entry since it is possible that very high extracellular concentrations mask the function of Orai1 by recruiting other channels or due to a saturating Ca2+ signal. Deletion of Orai1 proteins leads to perinatal lethality in mice that can be rescued only partially by backcrossing to an outbred mouse strain . To overlox mice  with Pf4 systems ; it impr2+-signalling downstream collagen receptors, especially after activation of Glycoprotein VI (GPVI) receptors by CRP [Plt-KO and Orai1Plt-Ctrl washed platelets from female and male mice after stimulation with collagen  and it was not different in terms of area under the aggregation curve , respectively. These receptors are coupled to different G-proteins enabling regulation of Ca2+-signalling though mechanisms including PLCβ-downstream signalling [2+-signalling. Platelet aggregation induced by 1 µM of the TxA2 receptor agonist analogue U46619 was normal in platelets from Orai1Plt-KO female mice  or ADP. Those agonists activate G-protein coupled receptors (GPCRs) such as protease-activated receptors (PARs), Pgnalling ,48. Consale mice A but sigale mice B. Highertrl mice C,D. We atrl mice . U46619 controls E,F. Howein males E. 2+-entry evoked by 0.1 U/mL in male platelets of Orai1Plt-KO mice was significantly reduced  affected the Store Operated Calcium Entry (SOCE) [2+ signalling in non-excitable cells. We then evaluated the consequence of platelet-restricted Orai1 deletion on SOCE in platelets from both genders. For this, we used the sarco/endoplasmic reticulum (ER) Ca2+-ATPase (SERCA) inhibitor, thapsigargin, which leads to passive depletion of intracellular Ca2+ stores. Thapsigargin applied at a concentration of 0.1 µM induced a near maximal platelet aggregation in Orai1Plt-Ctrl male mice, which was completely abolished in platelet from both genders of Orai1Plt-KO mice  ,37, whic-KO mice A,B. A 3-ale mice C and it ale mice . Furtherblockage . Analysilatelets , indicat-KO mice E,F. 2+ entry (SOCE) and receptor-operated Ca2+ entry (ROCE) mechanisms are central for platelet activation and function. Orai1 proteins have been implicated in platelet’s SOCE and in regulation of arterial thrombosis [2+ signalling. We used physiological agonist at different concentrations such as collagen, collagen related peptide (CRP), thromboxane A2 analogue U46619, thrombin, ADP and the SERCA inhibitor, thapsigargin, and determined the impact of Orai1 deletion in mouse platelets. Because Orai1 global deficient mice die early and to specifically analyse the function of Orai1 in adult platelets, we generated a platelet-specific knockout mouse line (Orai1Plt-KO). Orai1-deficient platelets had strongly reduced thapsigargin-induced SOCE resulting in abrogated aggregation, independent of secondary activation produced TxA2 and secreted ADP. Ca2+-entry and platelet aggregation induced by U46619 or thrombin were partially affected by Orai1 deletion depending on the gender. ADP, collagen and CRP aggregation from Orai1-deficient platelets was comparable with control cells, and only CRP-induced Ca2+-signalling was affected in female platelets at the tested concentrations. Store operated Carombosis . To furtR93W) [−/− mice that show protection of pathological thrombus formation [R93W mice presented reduced SOCE (produced by thapsigargin), impaired agonist-induced increase in intracellular Ca2+ concentration  and normal aggregation triggered by PAR4 peptide, collagen, ADP and convulxin [−/− platelets reported strongly reduced SOCE produced by thapsigargin and showed a reduced increase in CRP-induced Ca2+ transients in the absence of extracellular Ca2+; in addition, apparently Ca2+ transients in the presence of extracellular Ca2+ were reduced after stimulation with thrombin, CRP or ADP, but platelet aggregation after ADP and thrombin were normal. In contrast to the OraiR93W model, Orai1−/− platelets showed reduced GPVI-mediated platelet aggregation when evoked by low dose CRP (0.05 µg/mL) or collagen (1 µg/mL), respectively, but platelet aggregation was not affected at higher concentrations of either agonist [−/− platelets and it was shown to be normal after thrombin, ADP, thromboxane A2 analogue U46619 and collagen stimulation, reduced after low dose CRP stimulation and completely abolished after thapsigargin stimulation [R93W mice [−/− platelets that is reduced response to those agonists [−/− platelets from male mice, similarly as found by Chen and co-workers who used reported reduced aggregation in Orai1−/− platelets after stimulation with 0.5 µM U46619 but not at higher (2 µM) concentrations [−/− studies are compared with ours here, we tested here several concentrations and quantified the response from several independent preparations and from both gender of the animal used. A common point among all reports and us in terms of aggregation is the preserved ADP and thrombin responses [2+ measurements the thapsigargin induced SOCE was strongly reduced, but not completely abolished, in male and female platelets here as well as in platelets from Orai1−/− or OraiR93W mice [2+ entry after thrombin  similarly to others [−/− platelets [−/− platelets by other showed reduction in Ca2+ transients evoked no only by CRP or thrombin but also by combined thrombin/CRP stimulation and determined the presence of residual SOCE [−/−/TRPC6−/− mice had stronger thapsigargin-induced SOCE compared to single Orai1−/− platelets [In previous studies the function of Orai1 in murine platelets was analysed in two models; one model based on the expression of a mutated, inactive form of Orai1 (OraiR93W) , and othormation . Plateleonvulxin . Similaronvulxin  of Orai1 agonist . In anotmulation . When we93W mice , since wagonists ,39. Regaesponses ,37,39, a93W mice ,37,39. Wual SOCE , which w2+-entry is affected by conditions where coagulation components are or not present. From the study of Orai1 deficient platelets Orai1-mediated SOCE specifically regulates (in vitro) GPVI-induced integrin activation and degranulation, and thrombus formation under flow [2+ signalling; however, when the same parameters were observed under conditions allowing coagulation, PS exposure and thrombus formation were preserved in Orai1-deficient platelets, and very interesting a modest reduction in Ca2+ signalling was observed. Even more, the deficiency in Orai1 did not affect GPVI-Induced PS exposure and prothrombinase activity in washed platelets when thrombin is present, but it affected thrombin generation after GPVI stimulation. Altogether and presuming that in washed platelets the coagulation components from plasma are mainly reduced, we assume that our observations can be partly evoked by inhibition of the procoagulant platelet formation and further studies combining approaches like those from the studies mentioned above can be used in the future.The dissection of SOCE in murine platelets revealed how in vitro this Cader flow . Furtherder flow  showed t2+ signalling setup [In human platelets corresponding studies evaluating the contribution of individual Orai subtypes to SOCE is difficult due to the lack of inhibitors with sufficient specificity  of the molecules involved in its regulation like STIM or Orai proteins. However, attempts to study the contribution of SOCE in isolated human platelets have been performed. In one the study using washed human platelets the SOCE evoked by stimulation of thrombin and thapsigargin, respectively, was not affected by an anti-STIM antibody, despite that this antibody had an inhibitory effect on collagen-induced aggregation and thrombus formation on collagen coated capillary under flow . In anotng setup . −/− model [−/− model is based in transplantation of bone marrow into irradiated wild-type mice, and second, the targeting used was gene-trapping, which most probably resulted in a hypomorphic Orai1 allele [R93W) [On the other hand, differences in the observations between our work and the work published by others can be in part attributed to the mouse model type used and the approach used to inactive Orai1 proteins. Two main differences between our approach and those using the Orai1/− model ,38,39 ar1 allele  in compa1 allele . We used1 allele . The Orae [R93W)  differede [R93W) . Importae [R93W) ,57 and le [R93W) ,38,39,552+ entry mechanisms, by further IP3 and diacylglycerol (DAG) formation and or PIP2 depletion [Another influencing factor that need to be considered is that the expression of all Orai1, Orai2 and Orai3 has been reported in both, murine and human platelets ,37,58; tepletion ,18,20,21epletion . TherefoFinally, our results support the concept of Orai1 involvement into SOCE mechanism in murine platelets, contribute to understand its role in agonist-dependent signalling and pointed out the importance to analyse separately platelets from both genders.


Interestingly, emerging evidence indicates that Ca2+ signaling played a key role in bone metastasis, for it not only promotes cancer progression but also mediates osteoclasts and osteoblasts differentiation. Therefore, Ca2+ signaling has emerged as a novel therapeutical target for cancer bone metastasis treatments. Here, the role of Ca2+ channels and Ca2+-binding proteins including calmodulin and Ca2+-sensing receptor in bone metastasis, and the perspective of anti-cancer bone metastasis therapeutics via targeting the Ca2+ signaling pathway are summarized.Bone metastasis is a frequent complication for cancers and an important reason for the mortality in cancer patients. After surviving in bone, cancer cells can cause severe pain, life-threatening hypercalcemia, pathologic fractures, spinal cord compression, and even death. However, the underlying mechanisms of bone metastasis were not clear. The role of calcium (Ca Ca2+/Cars (TFs) . CaM-deprs (TFs) . Treatme2+/CaM-dependent protein kinase kinase α (CaMKKα) and β are the upstream kinases in the CaMK signaling cascade [2+/CaM binding and intramolecular phosphorylation. Activated CaMKKs phosphorylate and activate CaMKI and CaMKIV, adenosine monophosphate (AMP)-activated protein kinase (AMPK), or AKT (PKB). These kinases then phosphorylate downstream proteins, such as cyclic AMP (cAMP) response element-binding protein (CREB), activating transcription factor-1 (ATF-1), CAAT-enhancer-binding protein (C/EBP), and serum response factor (SRF) [2+/CaM for activity, CaMKKβ also can be activated in the absence of Ca2+/CaM. Glycogen synthase kinase 3β (GSK3β) and cyclin-dependent kinase 5 (CDK5) regulate CaMKKβ activities through phosphorylation [2+/CaM complexes for activation, independent of CaMKKs [Ca cascade . CaMKKα or (SRF) . Notablyrylation . Unlike f CaMKKs .CaMKKβ was identified as a downstream target protein of androgen receptor that has been known as prostate cancer bone metastasis enhancer . Androge2+ levels induced by Cx43 activate CaMKII mediating tumor cells’ bone colonization [2+ signaling induces the CaMKII-dependent MeCP2 phosphorylation on serine 421 of MeCP2 and releases MeCP2 from silenced promotors in many cellular contexts [Furthermore, increased cytolytic Canization . Nuclearcontexts , 102. Decontexts , 104, incontexts .2+ via Ca2+ channels and promotes osteoblastic differentiation via Ca2+/CaMKII/extracellular signal-regulated kinase (ERK)/activating protein-1 (AP-1) signaling pathway [2+ levels, inhibits the expression of CaM and CaMKII, and prevents osteoclasts differentiation, providing effective therapy for patients with skeletal involvement from advanced cancers [c-fos gene expression and subsequent AP-1 activation, which can, in turn, drive NFAT2 expression and is involved in osteoclast differentiation and bone remodeling [2+ signaling leading to osteoclastogenesis may provide more specific targets to the treatment regimen for bone metastases, and needs further studies.It has been established that CaMKII is involved in the differentiation of both osteoblasts and osteoclasts. A collagen-binding motif derived from osteopontin induces an influx of extracellular Ca pathway . More im pathway . Zoledro cancers . Furthermodeling . CaMKII modeling . Zoledromodeling . The mol2+-CaM-dependent serine-threonine phosphatase that controls signaling pathways relevant to the migration, invasiveness, and metastatic potency of cancer cells. Increased cytolytic Ca2+ levels activate calcineurin mediating tumor cell bone colonization [2+ oscillation by Ca2+ released from the ER and SOCE promotes CRC bone metastases through the calcineurin/NFATc1/ACP5 axis [Calcineurin is a conserved Canization . Calcinenization . Calcinenization . RANK acCP5 axis . In addiCP5 axis . RegulatCP5 axis . Hence, CP5 axis . MoreoveCP5 axis .Furthermore, calcineurin Aα (CnAα), an isoform of calcineurin, is significantly overexpressed in small cell lung cancer (SCLC) tissues with bone metastasis in contrast to tumor cells where bone metastasis was absent . CnAα isq/11, Gi/o, G12/13, and Gs, facilitating selective regulation of the wide array of cellular effects [2+ and regulates intracellular and extracellular Ca2+ concentrations [CaSR is a GPCR that activates biased signaling in response to ligand stimulation . With di effects . CaSR setrations .2+ homeostasis through its modulation of the parathyroid glands and kidneys, therefore contributing to chondrocytes, osteoblasts, and osteoclasts differentiation, leading to skeletal development and bone turnover [2+ and CaSR in cancers has been identified, promoting tumor cell proliferation, migration, and bone metastasis [2+ levels enhanced migratory potential and proliferation of bone metastasizing primary RCC cells [It has been known that CaSR controls Caturnover . Moreovetastasis . AnotherCC cells .2+ in normal tissue, high Ca2+ concentrations stimulate CaSR to secrete PTHrP in prostate cancer, breast cancer, and lung cancer cells [2+-CaSR-PTHrP axis stimulates the differentiation of osteoclast precursors into mature osteoclast, therefore promoting bone resorption and Ca2+ release [2+ and bone destruction.Parathyroid hormone-related protein (PTHrP) is important for the induction of osteoclasts maturation and differentiation. Unlike suppressed PTHrP secretion by elevated Caer cells –121. Theer cells . This Ca release , initiat2+/CaSR upregulates the expression of cyclin D1, a key component of the cell cycle, to support cancer cell growth, but this upregulation is absent in the nonskeletal metastases [In bone metastatic prostate cells, Catastases . Furthertastases . In lungtastases . These r2+ signaling in tumor cells’ metastasis to the bone have been well established. As a ubiquitous second message, Ca2+ interacts with cancer cells to promote proliferation, migration, and invasion. Moreover, bone has the biggest Ca2+ storage in the human body and Ca2+ signaling mediates osteoclasts and osteoblasts differentiation which can facilitate bone metastasis. Thus, Ca2+ ions’ role in bone metastases is beyond tumor cells alone. It tells a better story along with osteoclasts, osteoblasts, and immune cells. Cancer cells’ colonization in the bone environment depends on the destroyed bone structures and systemic inflammation induced by immune cells. Increased concentrations of intracellular Ca2+ have been proven to contribute to the progress of bone metastasis. However, future scholars should also investigate whether Ca2+ acts as a negative regulator of bone metastases. Numerous Ca2+ channels and Ca2+ signaling pathways have provided us with a plethora of potential therapeutical targets for cancer treatment. However, many Ca2+-associated channels, proteins, and kinases have not been investigated, and for most of the signaling pathways that have been studied, the specific mechanisms in migration, invasion, and metastasis of different types of cancers are only just beginning to be understood. Medications targeting the Ca2+ signaling toolkit are limited. Therefore, a better understanding of the exact molecular functions and mechanisms of Ca2+ signaling in bone metastases is needed and further efforts can focus on the Ca2+ channels, Ca2+-related signaling cascades, and their effects on bone metastases.The process and mechanism of bone metastasis are so complicated that there is no clear therapeutic target. The roles of Ca