Background/Aims: Recently, effective and purified ingredients of traditional Chinese medicine (TCM) were extracted to play crucial roles in the treatment of pulmonary diseases. Our previous research focused on TCM drug screening aimed at abnormal airway muscle contraction during respiratory diseases. Coptisine, an effective ingredient extracted from bitter herbs has shown a series of antioxidant, antibacterial, cardioprotective and neuroprotective pharmacological properties. In the current study, we questioned whether coptisine could also participate in asthma treatment through relaxing abnormal contracted mouse airway smooth muscle (ASM). The present study aimed to characterize the relaxant effects of coptisine on mouse ASM and uncover the underlying molecular mechanisms. Methods: To investigate the role of coptisine on pre-contracted mouse ASM, a series of biological techniques, including force measurement and patch-clamp experiments were employed. Results: Coptisine was found to inhibit high K+ or acetylcholine chloride (ACh)-induced pre-contracted mouse tracheal rings in a dose-dependent manner. Further research demonstrated that the coptisine-induced mouse ASM relaxation was mediated by alteration of calcium mobilization via voltage-dependent L-type Ca2+ channels (VDLCCs) and non-selective cation channels (NSCCs). Conclusion: Our data showed that mouse ASM could be relaxed by coptisine via altering the intracellular Ca2+ concentration through blocking VDLCCs and NSCCs, which suggested that this pharmacological active constituent might be classified as a potential new drug for the treatment of abnormal airway muscle contraction. According to a recent study, asthma and chronic obstructive pulmonary disease (COPD) together threatened 300 million people worldwide [Pulmonary diseases are a series of debilitating, life-threatening respiratory illnesses that have become severe worldwide public health problems and financial burdens . Accordiorldwide . Airway orldwide . The devAs well known, traditional Chinese medicine (TCM) especially a large number of herbal formulations play important roles in pulmonary diseases treatment . HoweverCoptis species [C. teeta and C. chinensis. Coptisine has also been characterized in other plant species such as Coptidis rhizoma, Epimedium sagittatum [Chelidonium majus L. [Coptisine, a protoberberine alkaloid (PBA), is an active constituent which has been identified in a lot of Chinese herbs, especially in  species , such asgittatum , Chelidomajus L.  and etc.majus L. , antioximajus L. , cardiopmajus L. , neuroprmajus L.  antispasmajus L.  properti+ precontracted mouse ASM could be relaxed by coptisine in a concentration-dependent manner. Further research indicated that coptisine exerted its relaxant effects by decreasing intracellular calcium via voltage-dependent L-type Ca2+ channels (VDLCCs) and non-selective cation channels (NSCCs).The purpose of present study was to investigate the roles of coptisine in pulmonary disease treatments with a focus on mouse ASM relaxation. The results showed that acetylcholine chloride (ACh) or high KCoptisine with a purity >0.98 was purchased from ESITE Biotech Co. Ltd.,  and dissolved in dimethyl sulfoxide (DMSO) for use. ACh, gadolinium, pyrazole 3 (Pyr3), nifedipine, niflumic acid (NA) and tetraethylammonium chloride (TEA), KB-R7943 were purchased from Sigma Chemical Co. . All other reagents were of analytical purity and purchased from Sinopharm Chemical Reagent Co. .All the animal experiments were designed and performed as previously described . To be b2 1, CaCl2 2, HEPES 10, glucose 10, pH 7.4) or Li-PSS  without sodium. Each tracheal ring was mounted with a preload of 0.5 g in a 10-ml organ bath containing PSS or Li-PSS gassed continuously with a 95% O2 and 5% CO2 mixture at 37°C. After an initial 60-min equilibration, tracheal rings were given a successive stimulation with either high K+ (80 mM) or ACh (100 μM). To obtain the concentration–response curves, coptisine (0.01–1000 μM) was added cumulatively to the pre-contracted tracheal rings. Particular channel inhibitors including nifedipine (10 μM), Pyr3 (30 μM), gadolinium (30 μM), TEA (10 mM), KB-R7943 (10 μM) and etc were applied in ASM contraction measurements, respectively. To clarify the role of calcium in coptisine-induced contraction, the experiments were carried out in Ca2+-free PSS solution (0 mM Ca2+ and 0.5 mM EGTA).The tension of mouse tracheal ring was measured isometrically as previously described . The mic2PO4 0.44, NaHCO3 4.16, Na2HPO4.12H2O 0.34, HEPES 20, glucose 10, pH 7.1) containing 3 mg/ml papain, 0.15 mg/ml dithioerythritol and 1 mg/ml bovine serum albumin (BSA) at 35°C for 22 min. Then the digested tissues were transferred to ASM dissociation buffer containing 1 mg/ml collagenase H, and 1 mg/ml BSA and were incubated at 35°C for 8 min. The tissues were washed and gently triturated with 1 mg/ml BSA to yield single ASM cells for use in subsequent experiments.Mouse ASM cells were isolated as described previously . Briefly2+ was employed as a charge carrier. The pipette was filled with intracellular solution . ASM cells were patched and held in the bath solution  at −70 mV. Currents were measured following depolarization for 500 ms from −70 to +40 mV in 10 mV increments every 50 ms.The VDLCC currents were measured using an EPC-10 patch-clamp amplifier  as previously described . Ba2+ wa2 1.2, HEPES 10, EGTA 3 and CaCl2 1, pH 7.2). The bath solution  was K+-free PSS. The free Ca2+ concentration was approximately 70 nM as calculated using WEBMAXC STANDARD (http://www.stanford.edu/∼cpatton/webmaxc/webmaxcS.htm). NSCC currents were recorded with a ramp using a perforated whole-cell configuration with a holding potential of −60 mV. The ramp was performed over 500 ms from −80 to +60 mV.For the measurement of NSCC currents, the pipette was filled with solution . P<0.05 was regarded as statistically significant.All data were expressed as the means ± standard deviation (SD). For all analyses, the evaluations were performed with Student’s +-induced smooth muscle contraction was mainly due to the depolarization of cell membrane, opening of VDLCC and influx of extracellular Ca2+, sequentially [+. Our previous study has shown that high K+ could contract mouse tracheal ring gradually [+ was applied to pre-contract ASM in this experiment. As shown in + (80 mM) was completely inhibited by coptisine (0.01–1000 μM) in a dose-dependent manner. According to the dose–contraction curve exhibited in 50) was 45.76 ± 8.54 μM. The IC75 was 194.69 ± 12.38 μM (n=7/7 mice). Comparing with the relaxant characteristic of coptisine on pre-contracted mouse tracheal ring, 316 μM coptisine had no effect on resting mouse tracheal ring , which confirmed that the contraction was induced via the opening of VDLCCs. These results indicated that coptisine inhibited high K+-induced pre-contraction in a dose-dependent manner. Furthermore, the relaxant effect of nifedipine suggested that VDLCCs participated in high K+-induced contraction and also might be involved in coptisine-induced relaxation.Previous research have demonstrated that high Kentially ,21. To eradually  and 80 meal ring C. As sho2+-free solution, high K+-induced contraction did not occur . Meanwhile, high K+ failed to induce a pre-contraction under Ca2+-free conditions in the presence of 200 μM coptisine, and even after Ca2+ restoration, the ASM contraction was not obvious (P>0.05) (n=6/6 mice). It was supposed that VDLCCs has been blocked in coptisine pre-treated mouse tracheal ring, then high K+ could not evoke extracellular Ca2+ influx via blocked VDLCCs. As shown in n=6/6 mice). These data suggested that blocking high K+-induced Ca2+ influx was involved in the relaxant effects of coptisine.The previous study on rat aortic ring indicated that coptisine could attenuate calcium release from the sarcoplasmic reticulum . To furtot occur A, provinoptisine A (n=7/7 (P>0.05) B (n=6/6 I–V) curve of VDLCC was calculated to examine the voltage-dependent property . As shown in n=7/7 mice). In the presence of nifedipine, ACh-induced pre-contraction was also relaxed by 100 μM coptisine (n=6/6 mice). These experiments indicated that besides VDLCCs, NSCCs could also be evoked by ACh and thus might involve in coptisine-induced relaxation.The relaxation of smooth muscle is a complicated electrophysiological process and the collaboration of various ion channels is indispensable ,25. Thus2+ entry during smooth muscle contraction is the NSCC permeable to external calcium ions [2+ entry through NSCCs was studied under ACh-induced pre-contraction. As shown in 2+-free condition, ACh induced a sharp contraction, indicating that ACh could transiently release Ca2+ from intracellular Ca2+ storage. Subsequently, the restoration of 2 mM Ca2+ triggered a steady contraction, which was completely eliminated by 10 μM coptisine (n=7/7 mice). However, in the presence of 10 μM coptisine, ACh failed to raise intracellular calcium. Even with the restoration of 2 mM Ca2+, sustained contraction also did not occur , 30 μM gadolinium (the average relaxation percentage was 20.64 ± 4.87%), and finally almost completely eliminated by 10 μM coptisine (the average relaxation percentage was 43.26 ± 8.90%). Taken together, a critical molecular candidate for NSCC blocked by coptisine seems to be TRPC channels.To further identify the specific components of NSCCs involved in coptisine-blocked Cator Pyr3 ,30 and gtor Pyr3  were emp− channels and K+ channels, nifedipine, NA and TEA were applied respectively. Thus, the residual current was ACh-induced NSCC current. As shown in n=6/6 mice). Three representative ramp current traces at time points a, b and c are shown in To further test whether coptisine has some effects on NSCC currents, whole-cell patch-clamp was employed to measure ACh-induced NSCC currents with or without coptisine . The NSCS and NSCCS, Na+/Ca2+ exchangers (NCX) also play a critical role in the intake of Ca2+ by cells in smooth muscle [2+ influx blocked by coptisine, Li-PSS without sodium was applied instead of PSS to evoke Ca2+ influx via NCX. As shown in the 2+ influx/Na+ outflow’ mode to increase intracellular Ca2+. Following addition of 10 μM coptisine, the contraction was potently attenuated to the base line (Besides VDLCCh muscle . To explase line A–C. Furt+ or ACh in a concentration-dependent way. VDLCCs and NSCCs are two categories of voltage-dependent and receptor-operated channel candidates which are critical for intracellular and extracellular calcium mobilization in ASM contraction [Recently, various studies focused on TCM for a safer and milder treatment of pulmonary disease. A plurality of the investigated natural herbal extracts or single compound exert their relaxant effect on precontracted airway muscle ,8. Coptitraction ,27,35,36traction . To furtIn conclusion, coptisine is an important ingredient of TCM which has shown various medical properties. Through current study, its pharmacological characteristic has been extended to relaxant effect on mouse ASM and the underlying molecular mechanism has been clarified. Further fundamental studies and clinical trials are required to explore more therapeutic properties of TCM including coptisine and explain the underlying molecular mechanism with modern scientific language.+ or ACh could be relaxed by coptisine through blocking VDLCCs and NSCCs then inhibiting calcium influx. Our research work provided evidence that coptisine might have potential therapeutic value for the treatment of pulmonary disease associated with abnormal ASM contraction.In summary, our research indicated that pretreatment of mouse ASM with high K


Toxoplasma gondii infections are common in humans and animals worldwide. Hares and rabbits are important small game species, and their meat is consumed by humans in many countries. Demand for rabbit meat for human consumption is increasing; therefore, toxoplasmosis in rabbits and hares is of epidemiological significance. Viable T. gondii has been isolated from rabbits. The present review summarizes worldwide information on the seroprevalence, parasitological investigations, clinical cases, isolation, and genetic diversity of T. gondii in wild rabbits, free domestic rabbits, hares, and other rabbits from 2010 to 2020. Differences in prevalence, susceptibility, genetic variants, and clinical implications of T. gondii infection in rabbits and hares are discussed. This review will be of interest to biologists, parasitologists, veterinarians, and public health workers. Additional studies are needed to increase our knowledge of genetic variants and the population structure of T. gondii in rabbits and hares and to understand the differences in susceptibility to T. gondii in hares in different areas.Toxoplasmosis is a zoonosis of global distribution, and  Toxoplasma gondii, the only species in the Toxoplasma genus. Toxoplasma can infect virtually every warm-blooded animal, including humans and livestock, as intermediate hosts [Toxoplasma gondii is considered one of the most important foodborne and waterborne parasites of veterinary and medical importance worldwide [Toxoplasmosis is a zoonosis of global distribution caused by the protozoan parasite te hosts . Felids orldwide ,3.T. gondii for humans [Consumption of undercooked or raw meat containing tissue cysts is the primary way of infection of r humans ,4. Humanr humans ,5,6.T. gondii initiates intestinal replication. Bradyzoites and sporozoites, respectively, are released and infect the intestinal epithelium [T. gondii can remain viable in tissues for many years, possibly for the life of the host [T. gondii has evolved strategies to successfully overcome or manipulate the immune system, with mechanisms that include control of host gene transcription and dysregulation of signaling pathways, modulation of cell adhesion and migration, secretion of immunoregulatory cytokines, and apoptosis. Many of these host–pathogen interactions are overseen by parasite effector proteins secreted from the apical secretory organelles, including the rhoptries and dense granules [After being consumed in uncooked or raw meat containing tissue cysts (carnivores) or in feed or drink contaminated with oocysts , ithelium , and theithelium . Immunitthe host . Althouggranules .Lepus) and rabbits (mainly the European rabbit (Oryctolagus cuniculus)) are important small game animals extensively hunted in many countries. Rabbit meat, considered one of the most nutritional white meats, is very popular in many countries [Sylvilagus floridanus), were introduced from North America to some parts of Europe to increase the potential for small game hunting but are now considered an invasive species [Lepus europaeus) is probably the most important game animal in Europe [Lepus tolai), is very popular due to its rich nutritional value [Hares  in Brazil at the beginning of the twentieth century and the same year in a rodent, Ctenodactylus gundi, from Tunisia by Nicolle and Manceaux [Leishmania sp. and was subsequently named Toxoplasma gondii in 1909 by Nicolle and Manceaux [T. gondii antibodies and to standardize serological tests [The isolation of al tests ,16,17.T. gondii via the ingestion of water and plants  containing oocysts excreted by domestic cats or free-ranging felids with which they share the same habitats [T. gondii oocysts [Rabbits and hares are herbivores and are mainly infected with habitats ,18,19. T oocysts ,20.T. gondii for other animals, especially for carnivores, including cats, but also for humans [Infected rabbits and hares can act as a potential source of r humans ,11,21. Hr humans . There ar humans . TherefoT. gondii infection in domestic rabbit from breeding farms and slaughtered animals for human consumption have been included for comparison and to give a complete view of the public health significance of T. gondii infection in these species. The source of rabbit meat  is often not specified in grocery stores or on a restaurant menu. Therefore, we have reviewed information on both domestic and hunted rabbits. The literature cited is from 2010–2020; information before 2009 was reviewed previously [This review provides up-to-date information on the seroprevalence, clinical cases, diagnosis, and genotypes present in wild rabbits, free domestic rabbits, and hares in the last decade 2010–2020). Studies on 0–2020. Seviously .T. gondii have been found in rabbits, mainly in the European rabbit (O. cuniculus), in several surveys worldwide. Data from 2010 to 2020 are summarized in T. gondii in rabbits were reported ranging from 0.9% to 37.5%, with most studies showing seroprevalence levels between 10–15%  was reported in Egypt: 26.7% [T. gondii were detected in 11.3% of farmed rabbits [T. gondii was isolated [The highest prevalence in domestic rabbits  sampled over a 12-year span [S. floridanus), low seroprevalence of T. gondii antibodies (2.1%) was observed in Italy [T. gondii infections than pet rabbits confined indoors due to the possibility of direct contact with the contaminated environment [In free wild rabbits, the most comprehensive survey was from Australia and involved 2114 wild rabbits , the possibility of consuming contaminated vegetables, and cohabitation with cats are the main risk factors that could account for some of these observed differences. Additionally, different serological tests used, types of samples analyzed, cut-off titers, and sample sizes could contribute to these differences as well.T. gondii antibodies in different animal species, including rabbits, but requires conjugate and is not automated. ELISA has the advantage of being automated and allows the testing of many samples, but also needs species-specific conjugates. A commercial ELISA is available for rabbits (see legend T. gondii infection are still necessary [The Indirect fluorescent antibody test (IFAT) is a well-established technique for detecting anti-ecessary .T. gondii antibodies in hares from Europe as well as in two studies from China  was the most studied hare species. Prevalence of anti-T. gondii antibodies ranged from 0% to 21% among studies in different countries (L. europaeus and 4.2% in L. timidus) observed in Finland, concomitantly with cases of fatal toxoplasmosis, might indicate a natural, inherent susceptibility of these hare species to T. gondii at least in those areas [T. gondii infection in the wild in other areas [Lepus granatensis) endemic in Spain, no clinical cases were detected, and a moderate to low seroprevalence (11.4%) was observed [T. gondii infection affected the survival of infected hares, and thus infected juveniles reached the adult stage at lower rates than the non-infected hares or that a short-lived humoral immune response against T. gondii existed in this species [The European brown hare  of 126 domestic rabbits (O. cuniculus) from Zhengzhou, eight of 248 (3.2%) from Luoyang, and two of 96 (2.1%) rabbit brains and hearts from slaughtered rabbits from food markets in China [T. gondii DNA based on a nested PCR targeting the surface antigen 2 (SAG2) gene [T. gondii DNA was also detected in tissues of three  of 144 wild cotton-tailed rabbits (S. floridanus) [T. gondii DNA at higher levels than those shown by serological levels by enzyme-linked immunosorbent assay (ELISA) (10.6%). In this study, differences were observed among breeds with higher prevalence in Flemish giant and chinchilla rabbits compared to crossbreeds and New Zealand white rabbits [T. gondii B1 gene in these pet rabbits [The most frequently used target genes for in China  (Table 4G2) gene . T. gondridanus) . The tisridanus) . Shin etridanus)  found 16 rabbits . On the  rabbits .Toxoplasma gondii DNA has also been detected in hares  [T. gondii using a qPCR targeting the 529 bp repetitive region in the tissues of two of 36 hares killed or found dead (5.5%). One of the infected hares had high levels of parasites in the spleen and lung, a unique finding. Toxoplasma gondii DNA was also detected in the Tolai hare (Lepus tolai) in three different areas of China [T. gondii infection was not found by PCR in the liver of seropositive Iberian hares (L. granatensis) in Spain [L. europaeus in Greece [T. gondii from heart tissues using a bioassay in mice; it should be noted that only three seropositive L. europaeus were bioassayed.in hares . Jokelai. (2020)  detectedof China   for detection of T. gondii- specific antibodies was performed using wild rabbit samples and was compared to an indirect fluorescent antibody test (IFAT) as a reference test [Little is known of the validity of different serological tests for the detection of nce test . The twoT. gondii were 0.89% (3/337) and 0.29% (1/337) for the IgG and IgM ELISAs, respectively. SAG1 and SAG2 were detected as major antigens by the positive rabbit sera in WB analyses and were associated with strong staining observed by IFAT in T. gondii tachyzoites [T. gondii prevalence as detected using nested PCR (16.2%) relative to ELISA (10.6%). The authors suggested that the nested PCR-positive/ELISA-negative rabbits might have harbored prepatent or latent infections with T. gondii. All PCR-positive samples were sequenced and corresponded (99% homology) to partial T. gondii B1 gene sequences.In a study on pet rabbits in Japan, 337 serum samples were tested by different serological methods; initially, IgG and IgM ELISAs were performed, and those samples seropositive by ELISA were further analyzed using a latex agglutination test (LAT), Western blotting (WB), and an indirect immunofluorescence assay (IFAT) . The rathyzoites . In pet hyzoites , whole bL. europaeus), mainly from Scandinavia, where harsh winter conditions are common [T. gondii was diagnosed in a domestic rabbit in Brazil in which T. gondii was identified by immunohistochemistry (IHC) in histological sections of spleen and liver tissue and genotyped [Infections in rabbits are documented to be mainly subclinical . On the e common ,6,40. Toe common . Only twe common . Since tenotyped   in those animals [T. gondii parasites detected in the lungs and spleen, exceeding 1,000,000 parasites/gram. Both studies on L. europaeus hares, in Finland and in the Czech Republic, revealed the genotype to be T. gondii type II clonal lineage [In hares, in a retrospective study of  animals . In anot animals . One har lineage ,40.T. gondii infection in rabbit tissues  revealed mainly granuloma, mononuclear cell infiltrates, and degeneration areas; necrosis was observed in brain and heart tissues [Microscopic lesions of  tissues .T. gondii in Brazil, the spleen was grossly enlarged and had extensive necrosis of parenchyma [T. gondii positive animals were pulmonary congestion, splenomegaly, and granulomatous and necrotic lesions in the liver. As indicated above, in this study, a high number of T. gondii parasites were detected, especially in the lungs and spleen . In other hare studies, the pathomorphological findings consisted of enormous enlargement of the spleen, lung edema, enlarged mesenterial lymph nodes and paleness, an uneven color, and/or hemorrhagic areas in the liver [Splenomegaly seems to be a common finding for rabbits and hares with fatal toxoplasmosis ,40. In arenchyma , as wellrenchyma , the mosToxoplasma gondii load in tissues of asymptomatic animals is low [T. gondii in rabbits and/or hares by IHC in the last decade. Jokelainen et al. (2011) [L. europaeus) and 2.7% (4 of 148) of mountain hares (L. timidus) tested. The liver was chosen because it is typically affected in fatal toxoplasmosis in hares, and it has successfully been used as the organ of choice for IHC. Based on IHC detection of the parasite in liver tissue, 17 of 18 (94%) cases originally diagnosed as fatal toxoplasmosis of hares were retrospectively confirmed, and one new case was found. Only one hare with recorded toxoplasmosis was IHC negative.The s is low , thus ths is low ,40,45 haT. gondii [T. gondii was also demonstrated retrospectively by IHC in histological sections of spleen and liver tissues of one rabbit suffering fatal acute toxoplasmosis in Brazil [In the rabbit that had aborted at a farm in Argentina, disseminated toxoplasmosis was confirmed by IHC testing, PCR, and isolation of viable . gondii   .T. gondii in Brazil [In the case of fatal acute toxoplasmosis in a domestic rabbit in Brazil , genotypn Brazil .T. gondii isolate was archetypal clonal type II and the other a type II variant (W35 = 244). The animal infected with Type II variant W35 had severe disseminated toxoplasmosis, with an estimated 7 million T. gondii per gram of tissue [As indicated above, in the European brown hares, ToxoDB genotype #1  has been reported from clinical samples in Finland  and the f tissue .T. gondii DNA in two studies, one in rabbits and the second in Tolai hares, found genotype #9 [In China, isolation and genotyping of otype #9 ,14; thisT. gondii in domestic and wild rabbits and hares, more studies on the prevalence and clinical and immunological implications of T. gondii infection in rabbits and hares are needed. Additional studies would also help to understand the differences in susceptibility to T. gondii in hares in different areas and to increase our knowledge of genetic variants and the population structure of T. gondii in rabbits and hares. More studies of the specific factors that exacerbate disease in rabbits and hares are also needed.Although in the last decade there have been several studies performed on the epidemiology and clinical features of T. gondii could be present in the organs of seropositive rabbits and hares. Therefore, hunters should be aware of T. gondii infection in clinically healthy rabbits and hares and are advised to take precautions while field dressing game and to freeze or cook rabbit and/or hare meat before human consumption [Viable sumption . In domesumption .


The detailed permeation mechanism of Ca2+ through RyR is still elusive. By using molecular dynamics simulations with a specially designed Ca2+ model, we show that multiple Ca2+ ions accumulate in the upper selectivity filter of RyR1, but only one Ca2+ can occupy and translocate in the narrow pore at a time, assisted by electrostatic repulsion from the Ca2+ within the upper selectivity filter. The Ca2+ is nearly fully hydrated with the first solvation shell intact during the whole permeation process. These results suggest a remote knock-on permeation mechanism and one-at-a-time occupation pattern for the hydrated Ca2+ within the narrow pore, uncovering the basis underlying the high permeability and low selectivity of the RyR channels.Ryanodine receptors (RyR) are ion channels responsible for the release of Ca + and Na+ channels have been extensively studied, the ion permeation mechanism through Ca2+ channels was largely unknown. Here the authors develop a multisite Ca2+ model that can be used in the framework of classical MD simulations to study Ca2+ in a quantitative manner, and use it to investigate the ion permeation mechanism of the ryanodine receptor 1.Although the permeation mechanisms for K The concentration of Ca2+ in the cytoplasm and organelles is precisely controlled by multiple calcium channels, including the voltage-gated calcium channels in cell membranes and the ryanodine receptors (RyR) in the endoplasmic reticulum (ER) membrane. Ca2+ also induces conformational changes of a wide range of Ca2+-interacting proteins, such as calmodulin and Ca2+-activated ion channels, to trigger downstream signal transduction2. Although the pathways of calcium signaling are extensively studied, the molecular interaction details between calcium and proteins have yet to be fully elucidated.As an essential messenger in cells, calcium ions  simulations to provide microscopic and quantitative insights, thereby obtaining the specific functional mechanisms of the relevant proteins2+ model. The polarizable force field is theoretically appealing9, but its implementation and validation still need further work before being widely accepted and utilized for membrane protein simulations. Kohagen et al. proposed to scale the partial charges on the Ca2+ to account for the charge transfer and polarization23. Another strategy is to represent an ion by distributing electrostatic and Lennard-Jones (LJ) interactions on multiple sites, which can introduce a much larger parameter space and therefore make the ion model more tailorable26. Unfortunately, none of the existing Ca2+ models in the non-polarizable classical force field are able to describe the interactions between Ca2+ and protein quantitatively. Therefore, in the present work, we developed a new multi-site Ca2+ model particularly optimized for Ca2+-protein interaction  and dummy (D) atoms, respectively. We further distinguish the LJ interactions of Ca2+ with water and non-water by replacing  with two sets of , the first-peak position of the radial distribution function of water around Ca2+ (R1), and the number of coordinated water molecules in the first solvent shell (NC) have all reached the target values of experiments. In addition, the residence time of water molecules in the first solvation shell (τR) can be optimized to below 100 ps, which was consistent with the previous experimental estimation28, and solved the common problem of Ca2+ being too sticky. We also performed ab initio MD simulations, and observed multiple exchange of water molecules between the first and second solvation shells within 200 ps, providing additional support that the residence time of our model is reasonable  led to a significant overestimation of 150–200 kcal/mol9, while the average binding-energy discrepancies for ten selected proteins was −0.2 kcal/mol for our multi-site model. Therefore, our model is comparable to the quantum mechanics (QM) and polarizable Drude model in calculating the Ca2+-protein binding energies  of pulling a Ca2+ away from its binding site in a calpain. As can be seen in Supplementary Fig. ΔGPMF of ~33 kJ/mol and binding free energy of ~25 kJ/mol, while the default Ca2+ parameters of the CHARMM force field resulted in a much larger ΔGPMF of ~177 kJ/mol and binding free energy of ~168 kJ/mol. The experimental binding affinity is ~35 kJ/mol29, indicating that our Ca2+ model is more accurate in reproducing not only the potential energies but also the free energies in the study of Ca2+-protein interactions.We then further validated the thermodynamic property of our Ca+ or Na+, but fail consistently for Ca2+ ions9, and therefore no calcium permeation was observed in previous MD studies of Ca2+ channels30. We performed MD simulations on the type-1 ryanodine receptor (RyR1), an intracellular calcium release channel required for skeletal muscle contraction, with our Ca2+ model. The open-state structure of RyR1 (PDB ID: 5TAL [10.2210/pdb5TAL/pdb]) was obtained from des Georges et al.’s21 work. The simulation systems consist of the pore domain of RyR1 embedded in a lipid bilayer of 1-Palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine (POPC) and an aqueous solution of either 150 mM Ca2+ or 250 mM K+  for statistical analysis , is presented in Fig. 2+ ions with D4899, E4900, and D4903 residues  was found near residues D4938, E4942, and D4945, which interact with the permeating Ca2+ and may influence the ion permeability as well. This is consistent with a previous experimental study showing that D4938 and D4945 determine the ion flux and selectivity33.We performed an 800-ns simulation without the transmembrane potential to identify the Caues Fig. , which a2+ and monitored from which residues these oxygen atoms were (water or protein). As shown in Fig. + and Na+ channels. As the first solvation shell is around 2.4 Å from the Ca2+ and the radius of a water molecule is usually considered to be 1.4 Å, we consider the pore region with a radius < 4.0 Å to be the narrow pore region that contains the rate-limiting constriction sites. Interestingly, the average number of oxygen atoms coordinated with the Ca2+ was almost constant during the permeation process of Ca2+, as shown in the right panel of Fig. 2+ ions do not need to dehydrate when permeating through the open-state pore, and therefore the first solvation shell was intact in the narrow pore region. On the other hand, in the wide upper selectivity filter, one of the water molecules coordinated with Ca2+ can be replaced by the oxygen from E4900 , and therefore should not be considered as dehydration due to the permeation.We calculated the number of oxygen atoms coordinated with the permeating Ca2+ density, as indicated by the dashed lines in Fig. 2+ ions is shown in Fig. 2+ residing in this narrow pore region, either in the upper or the lower chamber. The probability of both chambers being occupied by Ca2+ was only 2.4% in the trajectories, while the probability of only one chamber being occupied was 68.6%. Therefore, most of the time, only one Ca2+ can occupy the narrow pore region when permeating through the open-state channel. However, this does not mean that other Ca2+ are not involved in the permeation process. In fact, we can see that the exit of the lower Ca2+ from the narrow pore is often accompanied by the (transient) entering of a second Ca2+ , indicating the gate constriction is not the rate-limiting site and Ca2+ will not occupy the G site for a long time before exiting the narrow pore.We calculated the PMF of Ca2+ and protein is of great importance in studying Ca2+-mediated biological processes. Although the multi-site ion model cannot explicitly represent the charge transfer and polarization effect, the simulation results showed that our new model behaves much better than conventional single-point ion models. Moreover, our model is entirely consistent with the currently widely used non-polarizable force fields, such as CHARMM and AMBER, and therefore can be easily used in MD simulations. As shown in the result section, our seven-site Ca2+ model can reproduce the solvation properties of Ca2+, including the hydration energy, the first solvation shell size (first-peak position of RDF), the coordination number, and the residence time of water in the first solvation shell, as well as the Ca2+-protein binding energies in a more quantitative way that is comparable to quantum chemistry calculations. The optimized Ca2+ model was further validated by thermodynamic calculations and then utilized in the simulations of the RyR1 ion channel. In contrast to the conventional Ca2+ model, where ions get stuck in the channel in MD simulations, our new model resulted in the continuous permeation of Ca2+ through the channel, and the calculated conductance is in good agreement with the experimental electrophysiology result. Therefore, we believe this multi-site Ca2+ model can be widely used in simulating many Ca2+-involved bio-systems in addition to ion channels, and our simulations of RyR can provide detailed information about the Ca2+ permeation mechanism.The interaction between Ca+ and Na+ channels with molecular dynamics simulations17, but only rarely studied for Ca2+ channels with conventional ion models34. Very recently, Heinz et al. performed extensive PMF calculations of various cations in the RyR1, and located the key binding sites within RyR1 as well as the monovalent cation permeation pathways30. Miranda et al. used monovalent cations and Cl− to explore the permeability and origin of the high selectivity of cations over anions of RyR234. Both work provided highly valuable insights on the ion permeability and selectivity of RyR channels, but the Ca2+ permeation details remain unclear due to the absence of a reliable Ca2+ model. From our MD simulations with the new multi-site Ca2+ model, the major Ca2+ binding sites in the open-state RyR1 were determined to be near the D4900 and G4894 residues on the luminal side, which is consistent with previous experiments and simulations34, and the rate-limiting step of permeation is found to be the step of passing through the selectivity filter constriction, where Ca2+ ions dwell for a relatively long time before passing through due to the existence of a free energy barrier here  during the Ca2+ permeation in our MD simulations, and this region contains both the lower selectivity filter and cavity. There may be two reasons for this different permeation pattern, one is that the narrow selectivity filter of RyR is shorter compared to that of K+ and Na+ channels  was utilized in this work.All the MD simulations for the ion model parameterization were performed with OpenMM (version 7.0.1)2+ ion in a cubic water box of 3 × 3 × 3 nm3. The TIPS3P water model was used in consistency with the CHARMM force field. In our simulations, NPT ensembles were generated by integrating the Langevin dynamics with a time-step of 2 fs and a collision frequency of 5 ps−1. The temperature was maintained at 298 K, and the pressure was regulated at 1 bar using a Monte Carlo barostat. Water molecules were kept rigid during simulations, and the cutoff of non-bonded interactions was 1 nm. For calculations of the radial distribution function, coordination number, and residence time, a 20-ns trajectory was generated. For hydration free energy calculations, 1-ns trajectories were generated for each of the 14 alchemical states . The hydration free energy was estimated using the python implementation of the multistate Bennett acceptance ratio downloaded from https://github.com/choderalab/pymbar. The properties related to the radial distribution function were calculated using MDTRAJ46.We built an ion-in-water system to optimize the ion properties in the water, which consists of one multi-site Ca2+-Protein systems previously investigated by Li et al.9, and used OpenMM and CHARMM force field to perform the calculations.To optimize the ion-protein interactions, we used the Ca2+ model. One is designated as the protein-fitness function 2+-protein binding energies, p identifies the index among Np (=10) proteins, and f indexes Nf (=21) trajectory snapshots for each protein. The formula for ΔEC = 10 kcal/mol) is added to 9.We define two fitness functions to optimize the parameters of our multi-site Ca2+-protein interactions, we also optimize our model to reproduce its energetical, structural, and dynamic properties in water. Specifically, these properties include the hydration free energy (ΔGh), the first-peak position of radial distribution function (R1), the coordination number (NC), and the residence time of water in the first coordination shell (τR). The water-fitness function is defined ast, e, and w stand for theoretical, experimental, and weighting values, respectively.In addition to Ca47 to determine the target hydration energy of Ca2+ (−1504 kJ/mol) asΔG(CaCl2) is the measured hydration energy of CaCl248, ΔGh(Cl−) the theoretical hydration energy determined from Smith-Dang parameters49 for Cl−, ΔGpress the energy needed to compress one mole of ion gas at 1 atm into a liter, and ΔGsurf the energy change of crossing the air-water interface for 1 mol of ions. Marcus assessed the Ca–O internuclear distances in calcium salt solutions from different studies and concluded that the generally consistent result is 0.242 nm50, which was used as our target value for 51 revealed that the average number of water molecules in the first hydration shell of Ca2+ is close to 7, which we took as the target value for NCe. It is difficult to experimentally determine the residence time of water molecules in the first hydration shell of the calcium ion52, and a nuclear magnetic resonance (NMR) study estimated its value to be <100 ps27. Since the existing Ca2+ models generally overestimate τR, we set We followed the approach of Mamatkulov et al.9 protocol to calculate the binding energies between Ca2+ and proteins. As noted by Li et al., the default single-site model of Ca2+ in the CHARMM C36 force field generally overestimates the Ca2+-protein binding energies by 150–200 kcal/mol with respect to quantum-mechanical binding energies. They selected 10 high-resolution crystal structures of Ca2+-binding enzymatic proteins  that is added to 9.We followed Li et al.’sThe theoretical hydration energy, α = 0.5)53 to annihilate the LJ interaction. MD trajectories (1 ns) of one ion in a cubic water box (3 nm) were used to estimate the alchemical free energy by the method of multistate Bennett acceptance ratio54. For the finite-size correction, we took the formula derived by Hummer et al.55,ξEW is the Wigner potential, εr (=82) is the relative dielectric constant, and L (=3 nm) is the box size.The first term refers to the free energy change corresponding to alchemically switching off the ion-water electrostatic and LJ interactions, and the second term is a correction due to the finite-size simulation box. The alchemical transformation for turning off the electrostatic interaction was performed with 10 intermediate states, during which the charge of ion was sequentially reduced by a tenth of the total charge. After that, it took four uniform intermediate states with a soft-core potential  takes a value 1 if the water molecule j stays in the first hydration shell from tn to tn + t without leaving for any period larger than t*, and takes the value 0 otherwise. Then, nion(t) was fitted with The first-peak position of the radial distribution function 2+-protein binding energies 2+-bound and Ca2+-free configurations. Since our model of Ca2+ has a geometry of octahedron, the total energy of a Ca2+-bound system depends on the orientation of the octahedron. Thus, we first minimized the energy with respect to the orientation of the octahedron and obtained Emin(Ca2+ + Protein), and then calculated the Ca2+-protein binding energy using the formula ΔEMM = Emin(Ca2+ + Protein) − E(Ca2+) − E(Protein).The molecule-mechanical CabCD, QC, εC, σC, εD, σD), i.e., treating interactions with water and protein on an equal footing. The searched parameter space as shown in Supplementary Table 26, and we substantially extended the range of QC, εC, and εD based on our optimization results. However, after systematic optimization, the calculated water properties and protein binding energies were still slightly off from the target values. Therefore, we further split  into . Second, with the parameters of  fixed, the protein-fitness function 2+-protein binding energies. Due to the roughness of εD, which indicated that the landscape of the water-fitness function is much smoother along these two dimensions. Therefore, we selected a couple of combinations of (εD) based on the scanning results and carried out further optimization until the fitness value was sufficiently low. The final optimized parameters are presented in Supplementary Table First, we did a thorough scan and prescreening, using the conventional optimization routines (such as conjugate gradient and basin hopping), random sampling, and sifting the parameter space by the properties described above in sequence. Initially, we focused on six parameters  of the cryo-EM structure of the open-state RyR121  was used as the starting structure. The resolution is not very high, but should be good enough for the present study as the open-state pore of RyR1 is relatively wide and the subtle configurations within the pore may affect the ion permeation to a minor extent, and the side chains are free to move in our MD simulations to sample low-energy local conformations. The OPM58 web server and Membrane Builder in CHARMM-GUI59 were used to build four POPC-RyR1 simulation systems.To study the permeation of Ca31. As K+ exhibits a higher saturation concentration than Ca2+, we used a [Ca2+] of 150 mM and [K+] of 250 mM in our simulations. Six 500-ns trajectories were simulated for the former system, and three 300-ns trajectories for the latter. By counting the number of permeation events (Np) for a trajectory of certain time length (ttraj) under a transmembrane potential of Vtm, the ion conductance Cion was calculated with:Qion is the charge of the permeating ion.A POPC-RyR1 simulation system with ~150 mM of calcium ions and a POPC-RyR1 simulation system with ~250 mM of potassium ions were built to calculate the conductance of the calcium ions and potassium ions with a transmembrane potential of 100 mV. The ion concentrations were chosen to match the experimental conditions under which the conductance was measured2+ model as rigid as possible, which affect the thermodynamics of the Ca2+ to a negligible extent . The position restraints were applied to make sure the open-state conformation was stable in the absence of the large intracellular domain and the caffeine and ATP bound to it, but the atoms other than α carbon, including all the side chains of the transmembrane domain, were fully flexible in our simulations, allowing a certain degree of flexibility of the protein (Supplementary Fig. 60 with a time constant of 1 ps was used to maintain the pressure at 1.0 bar. The Particle-mesh Ewald method61 was used to calculate electrostatics, and the cut-off length of the van der Waals interaction was 1.2 nm.All the simulation systems were first equilibrated with the standard CHARMM-GUI equilibration protocol followed by the production simulations with the harmonic position restraints applied on the Further information on research design is available in the Supplementary InformationReporting SummaryDescription of Additional Supplementary FilesSupplementary Movie 1Supplementary Movie 2Supplementary Movie 3


In the heart, Ca2+ is essential for maintaining normal cardiac function through the generation of the cardiac action potential and its involvement in excitation contraction coupling. As such, the proteins which regulate Ca2+ cycling and signaling play a vital role in maintaining Ca2+ homeostasis. Changes to the expression levels and function of Ca2+-channels, pumps and associated intracellular handling proteins contribute to altered Ca2+ homeostasis in CVD. The remodeling of Ca2+-handling proteins therefore results in impaired Ca2+ cycling, Ca2+ leak from the sarcoplasmic reticulum and reduced Ca2+ clearance, all of which contributes to increased intracellular Ca2+. Currently, approved treatments for targeting Ca2+ handling dysfunction in CVD are focused on Ca2+ channel blockers. However, whilst Ca2+ channel blockers have been successful in the treatment of some arrhythmic disorders, they are not universally prescribed to heart failure patients owing to their ability to depress cardiac function. Despite the progress in CVD treatments, there remains a clear need for novel therapeutic approaches which are able to reverse pathophysiology associated with heart failure and arrhythmias. Given that heart failure and cardiac arrhythmias are closely associated with altered Ca2+ homeostasis, this review will address the molecular changes to proteins associated with both Ca2+-handling and -signaling; their potential as novel therapeutic targets will be discussed in the context of pre-clinical and, where available, clinical data.Diseases of the heart, such as heart failure and cardiac arrhythmias, are a growing socio-economic burden. Calcium (Ca As the focus of this review concerns how Ca2+ handling proteins present a novel therapeutic approach, we discuss the current pharmacological therapies mentioned above in brief, including any known impact they have on Ca2+ handling in the heart.Treatment of HF and cardiac arrhythmias can be surgical (including implantation of a pacemaker or implantable cardioverter defibrillator) and/or pharmacological. Primary drug-based therapies for these disorders include targeting (1) the RAAS signaling system, (2) β-adrenergic hyperactivation, and (3) aberrant ion channel activation (Ca+ current (Ito) and enhance ICaL, impacting AP duration and repolarization. Whilst oxidative stress triggered by RAAS activation is reported to affect cardiac ion currents and several Ca2+ handling proteins .Renin–angiotensin–aldosterone signaling is elevated in patients with HF and reduced ejection fraction   and in aThe maladaptive effects associated with over-activation of the RAAS is one of main focuses of current therapeutics; both ACEI and ARBs are prescribed to patients with HF to help improve cardiac function and survival . Several2+ handling, including LTCC  are generally not prescribed to patients with HFrEF, except for amlodipine. Due to their negative inotropic effects, clinical studies have demonstrated that non-dihydropyridines increase mortality rates and incidence of late-onset HF  include phenylalkylamines and benzothiazepines   . Howeverctively) . There ipy alone .+ channel blockers target the voltage-gated Na+ channel, Nav1.5, which drives the inward Na+ current responsible for the rapid depolarization at the start of the cardiac AP . Class I Na+ channel blockers are further subclassified based on their molecular properties and interaction with the Nav1.5  in the late 1980s studied the effect of Na+ channel blockers on patients with ventricular ectopic activity up to 2 years following a MI. The study revealed a higher rate of death in patients receiving flecainide or encainide compared to the placebo group  and Kv11.1 blockers  have been indicated for the treatment of ventricular tachycardia in patients with structural heart disease or with remote MI, tachyarrhythmias in patients with Wolff-Parkinson-White syndrome, rate control in AF   or ‘reverse’  mode. In a failing cardiomyocyte, NCX1 has been shown to operate in reverse mode which results in Ca2+ entry and a repolarizing current  cardiac arrhythmias, (2) reduced Ca2+ SR content, (3) systolic dysfunction, and (4) altered cardiac energetics  and absent in young patients , induction of cardiac hypertrophy with ANG-II resulted in increased hypertrophy in Trpm4cko mice when compared to controls  have to date been unable to associate SNPs within ATP2B4 with any CVD , and metabolic changes which pre-dispose the development of arrhythmias   has been identified in SAN dysfunction  . Unsurprfunction . In addifunction .2+-mediated. Early afterdepolarizations (EADs) occur during the plateau phase of the cardiac AP and are associated with LQTS and ventricular tachyarrhythmias (associated with HF) (2+ current (ICaL) which drives the EAD upstroke. This can occur due to a reactivation of the current during the repolarization phase caused by a reduction in the outward IK current or by CaMKII-mediated phosphorylation of ICaL (INCX) can contribute to EAD. Spontaneous Ca2+ release events either prior to the completion of repolarization or during the initial phase of EADs can cause an increase in INCX activity which may delay repolarization and allow ICaL to reactivate (Triggered activity refers to voltage oscillations termed afterdepolarizations which are often Cawith HF) . They arwith HF) . A main  of ICaL . In addiactivate .2+ overload driving spontaneous Ca2+ release from the SR  leading to depolarization of the membrane. If the excitation threshold is reached, INa becomes activated and an AP is triggered  typically occur after repolarization and are caused by Cam the SR . Ca2+ reriggered . An incr release . Elevate release . Phospho release . Modulat release .2+ handling can cause cardiac tissue to become vulnerable to re-entry arrhythmias by increasing the dispersion of refractoriness, leading to unidirectional conduction block, and slowing of conduction , AP duration , or contraction amplitude   . Changeslternans .2+ release from the SR can lead to both atrial and ventricular arrhythmias, some primary arrhythmia disorders including CPVT and forms of acquired arrhythmias associated with heart disease (AF during HF)  . To dimi2+ channel  . Dantrol channel . Further channel   is a 1,4-Benzothiazepines derivative initially identified to suppress sudden cardiac cell death associated with Caoverload . Furtheroverload  (Kumagaioverload . PKA hyp(2+ leak . A Phasef JTV519 . Anotherf JTV519 . In a mof JTV519  (Lehnartf JTV519 . The antf JTV519 .+ channel blocker) it is worth noting here the drugs ability to inhibit RyR2 action by reducing the duration of channel opening  where they are thought to contribute to SAN pacemaker activity and atrioventricular conduction , CaMKII inhibition prevented arrhythmias induced by rapid atrial pacing  describes the influx of Ca2+ into the cell when Ca2+ levels within the SR become depleted, a process which has been implicated in arrhythmia development , and via TRPC3-NCX dynamics . The study suggested this may be due to a feedback mechanism from inhibition of TRPM4 since it is known to be activated by Ca2+  have been identified in leukocytes of patients with non-valvular AF . TRPV chfunction . Finallylternans .2+ fluxes achieved via the uptake of Ca2+ through MCU (2+ dynamics, including SR Ca2+ release and LTCC (360, an RR analog, has been shown to be a more specific MCU inhibitor and able to suppress ventricular fibrillation associated with I/R (360 had no effect on left ventricle pressure, at high concentrations RR reduced left ventricle pressure amplitude (It has been suggested that cardiac mitochondria influence arrhythmia development through the regulation of cytoplasmic Caough MCU . Much ofough MCU . Use of ough MCU  (de et aough MCU . Howeverand LTCC . Ru360, with I/R  (de et awith I/R . Both Ruwith I/R . It is wmplitude .2+ plays a critical role in maintaining cardiac physiology and changes to the proteins which are involved in Ca2+ handling underly numerous cardiac diseases. Targeting Ca2+ dysregulation in HF and arrhythmias has long been investigated as a potential therapeutic target and, as is evident from this review, there is considerable current research investigating targeting proteins associated with Ca2+ handling, including RyR2, NCX, SERCA, and TRP channels, as a therapeutic approach for cardiomyopathy, HF and arrhythmias. The pathogenic remodeling of these Ca2+ handling proteins has been shown to differ dependent on the underlying pathological mechanism, suggesting a “personalized” approach to targeting Ca2+ handling proteins may be required for specific cardiac conditions. Furthermore, while several therapeutics targeting Ca2+ have progressed to clinical trials, several are still in early preclinical stages, demonstrating the need for further research in this area. Altogether, targeting Ca2+ handling is a promising approach to developing novel and effective therapies for key cardiovascular disorders.CaEC, CW, and AN contributed conceptually to the manuscript. AN and CW drafted the manuscript. AN contributed the section concerning cardiac hypertrophy and heart failure, and the tables. CW contributed the section regarding cardiac arrhythmias. Both AN and CW contributed to the introduction, the current therapeutic approaches, the discussion, the figures, and the abstract. All authors reviewed the manuscript prior to submission.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Using preclinical models of murine AML, we then demonstrate that absence of CD162 on AML cell surface leads to a significant delay in the onset of leukemia and a significant increase in sensitivity to chemotherapy in vivo associated with a more rapid in vivo proliferation compared to wild-type AML and a lower BM retention. Together, these data reveal for the first time that CD162 is a key AML cell surface receptor involved in AML progression, BM retention and chemo-resistance. These findings highlight specific blockade of AML cell surface CD162 as a potential novel niche-based strategy to improve the efficacy of AML therapy.The interactions of leukemia cells with the bone marrow (BM) microenvironment is critical for disease progression and resistance to treatment. We have recently found that the vascular adhesion molecule E--selectin is a key niche component that directly mediates acute myeloid leukemia (AML) chemo-resistance, revealing E-selectin as a promising therapeutic target. To understand how E-selectin promotes AML survival, we investigated the potential receptors on AML cells involved in E-selectin-mediated chemo-resistance. Using CRISPR-Cas9 gene editing to selectively suppress canonical E-selectin receptors CD44 or P-selectin glycoprotein ligand-1 (PSGL-1/CD162) from human AML cell line KG1a, we show that CD162, but not CD44, is necessary for E-selectin-mediated chemo-resistance  Acute myeloid leukemia (AML) is an aggressive hematological cancer, where immature myeloid blasts accumulate within the bone marrow (BM) and perturb normal hematopoiesis . PrognosWhile most studies have focused on targeting the intrinsic properties of AML cells that lead to treatment resistance, extrinsic factors can also play a significant role in leukemia development and response to treatment. In recent years, the contribution of the BM microenvironment (niche) has been under intense investigation and is a potentially new approach for discovery of novel therapeutic targets .endothelial)-selectin is exclusively expressed on activated endothelial cells, and has been shown to be a key regulator of hematopoietic stem cell (HSC) activity within the BM vascular niche  homing and recruitment to sites of inflammation . Two welA range of other factors have additionally been reported to bind to E-selectin, such as E-selectin ligand-1 (ESL-1) on mouse but not human neutrophils ; L-selecUnlike the other known E-selectin ligands, both CD44 and CD162 receptors have been reported in both mouse and human hematopoietic cells, and have well defined roles in facilitating the rolling of mature leukocytes and/or hematopoietic stem and progenitor cells (HSPCs) on activated E-selectin-expressing vasculature, as well as HSPC homing to the BM . Of noteWe have previously found that when E-selectin becomes expressed at the vascular HSC niche in the BM, it directly awakens otherwise dormant HSC into commitment and differentiation, at the expense of self-renewal . In thesde novo ability of malignant cells to bind biological lectins like E-selectin. Thus, herein we investigate the functional roles of CD44 and CD162 as E-selectin ligands on AML cells in human and preclinical mouse models. We show that CD162, but not CD44, is required for E-selectin-mediated chemo-resistance in vitro and report a novel role for CD162 in AML progression and response to treatment in vivo.Herein, we now explore the hypothesis that AML may utilize a distinct E-selectin receptor repertoire compared to endogenous HSC, or at least that the glycosylation levels of receptor backbones may differ with oncogenic transformation. Indeed, aberrant and overall increased level of glycosylation is recognized as one of the hallmarks of oncogenic transformation , and can+ congenic mice were purchased from the Animal Resource Center . Selplg–/– mice were kindly provided by B. Furie  and had been backcrossed over 10 times into the C57BL/6 background. Mice were housed in Techniplast Greenline IVC caging in a room with controlled temperature (22–26 degrees Celsius) and humidity (30–70%).All experiments on mice were approved by the Animal Ethics committee of the University of Queensland. All mice used were sex- and age-matched between 7–12 weeks. Generally, donors were males, and recipients for long-term survival experiments were females. C57BL/6 and B6.SJKL CD45.1For BM collection, femurs were flushed with a 23G needle in 1 mL ice-cold phosphate buffered saline  containing 2% newborn calf serum . The leukocyte content of the samples was then counted on an AcT Diff Coulter Counter (Beckman Coulter).4Cl, 10 mM NaHCO3, pH 7.4 lysis buffer. Lysed samples were stained with 2 μg/ml 7-aminoactinomycin D (7-AAD) to exclude dead cells. The presence of GFP+ AML cells in the blood was assessed by flow cytometry using the CytoFLEX S analyzer (Beckman Coulter).For test bleeds, heparinized blood was collected from the lateral tail vein and red blood cells were lysed in 0.15 M NHCalculation of whole-body AML blast numbers in BM and blood for Selplg–/– embryos at E13 or E14 were incubated overnight with the irradiated packaging cells in fresh Dulbecco Modified Eagle’s medium  containing 10% fetal calf serum , 10 ng/mL recombinant human (rh) IL-11 (R&D System), 10 ng/mL recombinant murine (rm) thrombopoietin, 50 ng/mL rm stem cell factor (SCF) (both from Peprotech) and 5 μg/mL polybrene (Sigma-Aldrich). The transduced fetal liver cells were then harvested, washed in medium and retro-orbitally injected into 4Gy irradiated C57BL/6 recipients.GPE86 packaging cells producing MLL-AF9-containing expression retroviruses (following transfection with MSCV-MLL-AF9-IRES-GFP plasmid)  were irr+ AML cells in the circulation. Once over 40% of peripheral blood leukocytes were GFP+  mice were euthanized. Femora, tibiae, hips, and spine were removed and crushed in ice-cold PBS with 2% NCS using a mortar and pestle, then filtered through a 40 μm cell strainer. BM KIT+ AML blasts were further enriched from this cell suspension using mouse CD117-conjugated Magnetic Activated Cell Sorting (MACS) beads and “POSSEL” positive selection on auto-MACS  as previously described (Emergence of AML in recipient mice was monitored by flow cytometry from regular tail vein test-bleeds for appearance of GFPescribed .All antibodies used are listed in 2 antibody to human IgM (Jackson Immunoresearch) for 2 h at room temperature, as previously described (2+-dependent), and (ii) a parallel stain with AF647-labeled empty-vector instead of recombinant E-selectin was also used.For analysis of E-selectin binding potential, recombinant E-selectin–human-IgM fusion protein supernatants or matching empty-vector human-IgM as negative control  were precomplexed with AlexaFluor647-conjugated donkey F(ab)escribed . This co+) mice were transplanted with 104 BM GFP+KIT+ leukemic blasts generated from transduced C57BL/6 (CD45.2+) WT or Selplg–/– HSCs. The use of a CD45.1+ recipients were required in this experiment as GFP fluorescence  is lost after fixation. Recipients of WT AML blasts were transplanted ∼4 weeks before euthanasia, and recipients of Selplg–/– AML blasts ∼12 weeks before euthanasia. Both cohorts were euthanized on the same day.B6.SJL mice (CD45.1TM Cell Permeabilization Kit (Thermo Fisher) before staining with monoclonal antibody to Ki67-AF700 then 25 μM Hoescht33342.One million whole BM cells were stained with anti-CD45.2-FITC and anti-KIT-APC. Cells were then fixed and permeabilized using the FIX and PERM® ImageStream®X (EMD Millipore) at 60X magnification. Analysis of receptor co-localization and polarization was performed using IDEAS® software (EMD Millipore).For E-selectin receptors co-localization assays, KG1a cells (from ATCC CCL-246.1) were incubated with the preformed complex of E-selectin–IgM (or matching empty vector) and AF647-labeled antibody to IgM (as described above), in a microfuge tube at 37°C with gentle agitation. After 20 min of incubation, tubes were placed on ice and cells washed in X-VIVO media, before staining with human CD44-PECy7 and CD162-BV421 conjugated antibody. Stained cells were then analyzed for receptor co-localization by imaging flow cytometry with AmnisTissue culture wells (96-well plates) were coated with recombinant human PECAM-1/CD31, P-selectin, E-selectin and CD14 (non-adhesion control) human IgG1 Fc fusion proteins (purchased from R&D Systems) at a concentration of 5 μg/mL in 30 μL of Tris–HCl 20 mM pH 8.6, overnight at 4°C. Non-coated control wells were also included. The next day, unbound proteins were removed and the wells were washed with PBS before being blocked for one hour in X-VIVO media at 37°C. Ten thousand KG1a cells were then added into each well, in a volume of 100 μL of X-VIVO. The plate was incubated for 7 h at 37°C before Ara-C or saline control was added in 20 μL at final concentration of 10 μg/mL, 100 μg/mL, or 1 mg/mL. After 48 h of incubation at 37°C, cell viability was assessed using the CellTox Green Cytotoxicity assay and CellTiter-Glo 2.0 viability assay (both from Promega) on a PHERAstar microplate reader (BMG Labtech) according to the manufacturer’s instructions.Two negative (non-adhesion) controls were included for chemo-sensitivity assay, (1) parallel blocked non-coated wells, and (2) parallel control wells coated with recombinant human CD14 IgG1 Fc fusion protein (as non-adhesion control).CD44 and SELPLG genes from KG1a cells. Design of the single guide (sg) RNAs and optimization were based on a protocol developed by Charles C. Bell in Andrew Perkins group  RNAs sequences with minimal exonic off-target sites. Two sgRNAs were designed for both CD44 and SELPLG (each targeting a different exon), with target sequences as follows: 5′-TCGCTACAGCATCTCTCGGA and 5′-TGGGTTCATAGAAGGGCACG for CD44; 5′-ATCTAGG TACTCATATTCGG and 5′-CTCCGGTCCCGGGCAAGCAG for SELPLG. The sgRNAs were cloned into the pSpCas9(BB)-2A-GFP (PX458) plasmid (Addgene cat# 48138) after Bpil digestion. Purified plasmids were transfected in KG1a cells using the 4D-NucleofectorTM (Lonza) according to the manufacturer’s instructions with the DI-100 setting. At day 6 post nucleotransfection, cell surface staining  into 96-well plates and cultured in MEMα supplemented with 10% FCS and 1X penicillin-streptomycin-glutamine . Around 4 weeks later, several KG1a clones emerged from these single cells and were again tested by flow cytometry for cell surface expression of CD44 and CD162. Clones that displayed >99% cells negative for CD44 or CD162 (as shown in in vitro studies. Control WT KG1a cells were subjected to the same process as transfected KG1a cells except for the omission of sgRNA.Deskgen’s CRISPR guide RNA design toolSelplg–/– AML blasts. Leukemia progression was measured by regular monitoring of mouse wellbeing and blood tests for emergence of GFP+ AML cells. Mice with advanced AML (5–40% GFP+ AML blasts in blood) were administered two high dose rounds of cytarabine  or saline at 24 and 12 h prior to euthanasia. Femurs of each group were collected, pooled and crushed in PBS with 2% FCS. The equivalent of 1% of a femur was transplanted back into cohorts of 4-Gy conditioned C57BL/6 recipients, and mice were then monitored for wellbeing and duration to relapse . Median disease-free survival duration was analyzed by Kaplan Meier survival analysis.Cohorts of 4-Gy conditioned C57BL/6 mice were retro-orbitally transplanted with 30,000 WT or t-test as specified. Data was tested for parametric Gaussian distribution using the D’Agostino-Pearson omnibus normality test. Survival curves were plotted using Kaplan-Meier estimates and compared using a Log-rank (Mantel-Cox) test. All data were plotted as mean ± standard deviation (SD) unless otherwise specified.Prism software version 7 (GraphPad) was used for all statistical analyses. Unless otherwise indicated, one-way ANOVA with Dunnett’s correction was used to compare three or more groups. Statistical differences between two groups were tested by paired or unpaired two-tailed + cell line KG1a. With imaging flow cytometry, we observed that both cell surface CD44 and CD162 localized around the E-selectin-IgM-binding site  software and revealed a significantly higher co-localization score (p = 0.0004) between CD162 and E-selectin compared to CD44 and E-selectin  from KG1a cells . Cell survival was assessed 48 h after addition of various doses of the chemotherapeutic drug cytarabine (from 10 μg/mL to 1 mg/mL). We found that adhesion to E-selectin significantly increased the survival of WT KG1a to cytarabine at all three doses tested compared to CD14-IgG-Fc non-adhesion control (p < 0.0001), as anticipated from our previous findings using murine AML model (SELPLG–/–), E-selectin-mediated chemoresistance was no longer observed  or WT mice with a bicistronic expression vector containing GFP and MLL-AF9 (11q23-rearrangement mimic) fusion oncogene as previously described (+ AML cells were transplanted into WT (C57BL/6) recipients.For escribed . Once esSelplg–/– AML blasts) altered E-selectin binding potential. We found absence of CD162 alone significantly reduced E-selectin binding potential of murine AML blasts when compared to WT AML blasts  (Selplg for E-selectin binding compared to human KG1a.We first confirmed whether the absence of CD162 (in murine  0.0001)  which wa 0.0001) . These dSelplg–/– or matching WT AML blasts to investigate potential roles for CD162 in altering leukemia progression  AML blasts each, there was a marked 3.5-fold (p < 0.0001) increase in median disease-free survival in recipient mice of Selplg–/– AML compared to recipients of WT AML  a slower expansion of AML cells in the host, and/or (ii) a defect in BM retention/homing potential. To investigate the first question , we performed cell cycle analysis (Ki-67 and Hoechst33342 staining) in cohorts of wildtype mice bearing Selplg–/– or WT AML  (Selplg–/– KIT+ blasts actively cycling (phase S-G2-M of cell cycle with genomic DNA > 2n by Hoechst33342 nuclear staining) compared to WT KIT+ AML blasts   compared to hosts of WT AML (2.3 ± 1.26 million circulating AML blasts)  were transplanted into wildtype mice on the same day, but cytarabine treatment started 3 weeks post-transplant for mice recipients of WT AML and at 12 weeks post-transplant in mice that received Selplg–/– AML . Importantly disease will only relapse if these rare LRCs have survived treatment  were administered 24 h high-dose cytarabine or saline vehicle control. Femoral BM cells were then collected for; (i) analysis of residual (surviving) BM AML burden and (ii) transplant of 1% of the femoral content into cohorts of wildtype recipient mice, that were then monitored for duration of survival and AML relapse  leukemic mice. Although this difference was not significant by ANOVA, the side by side difference between WT and Selplg–/– AML after cytarabine treatment was significant by two-tailed t-test (p = 0.0230). These data suggest Selplg–/– AML blasts are significantly more chemo-sensitive than WT AML  when compared to matched vehicle treated control mice, or 55% (17 days) when compared to saline-treated recipients of WT AML. Interestingly, the spleen tumor burden was strongly and similarly reduced in both recipients of WT and Selplg–/– AML at 24 h post-chemotherapy, suggesting that spleen may not be a site where CD162-induced AML blast chemoresistance occurs  donors showed a delayed leukemia onset compared to matching saline-injected WT AML, with a median survival of 22.5 days compared to 15 days, respectively  AML driven by the MLL-AF9 fusion oncogene have delayed leukemia progression compared to WT AML driven by the same fusion oncogene. Selplg–/– AML cells also have reduced retention within and/or homing to the BM. In addition, we show that absence of CD162 from AML led to a marked loss in AML blast quiescence and a strong increase in cycling AML in the BM. This was associated with 2.5-fold increased chemo-sensitivity in Selplg–/– AML blasts compared to WT AML in vivo. These findings may be due to the direct effect of interactions between CD162 and E-selectin, as in our in vitro model we found that E-selectin-mediated chemo-resistance was abrogated when CD162 receptor was absent from the human CD34+ AML cell line KG1a.This study explores the mechanisms by which vascular E-selectin-mediated adhesion promotes chemo-resistance in AML and investigate canonical cell surface receptors potentially involved. We demonstrate that CD162 is a key E-selectin receptor involved in AML progression and resistance to chemotherapy in vitro  did not appear to mediate their chemo-resistance in vitro  similar in vitro . This suSelplg–/– mice we have previously shown that CD162 was redundant for E-selectin-mediated adhesion of murine HSCs  engraftment . This main vitro data suggests that this effect may specifically involve the interaction of CD162 with E-selectin, not P-selectin. Further studies are required to confirm a direct role of CD162/E-selectin interactions in mediating AML progression and chemoresistance.Our study is the first evidence of a direct effect of CD162 in mediating resistance of AML cells to chemotherapy, and our G12D mutation and TP53c.672+1G→A splice donor mutation, but no translocation involving either MLL or AML1 genes  and/or somatic mutations such as FLT3-ITD or NMP1 . While tL1 genes . StudiesInterestingly, despite our findings suggesting a major role for CD162 in mediating E-selectin-induced chemo-resistance, we found that absence of CD162 from human AML cell line KG1a did not significantly reduce E-selectin adhesion. Absence of CD44 from KG1a also did not reduce their binding potential to E-selectin, but deletion of both CD162 and CD44 did result in a significant ∼60% reduction in E-selectin adhesion potential. A recent study also confirmed that inhibition of either CD162 or CD44 on human monocytic leukemia U937 cell line did not alter rolling on E-selectin, whereas knockdown of both CD162 and CD44 increased rolling velocity and reduced U937 adhesion to immobilized E-selectin under flow conditions by about 40% . Collectin vitro. This was unexpected as CD44 has been extensively studied for roles in tumorigenesis (Cd44–/– HSCs for confirmation. However, all four of our attempts failed and we were unable to generate any serially transplantable transduced Cd44–/– AML from murine fetal liver HSPCs, even in situations where parallel transductions on HSPCs from Selplg–/– fetal liver were successful at generating AML. These observations suggest expression of CD44 may be critical in the process of malignant transformation or self-renewal, and are congruent with the recent observation that CD44 is crucial for CML engraftment in mouse models (In the current study we also report no major role for CD44 in E-selectin-mediated chemo-resistance igenesis , metastaigenesis , and cheigenesis , in soliigenesis . We attee models . Anti-CDe models , althouge models . CD44 hae models . Only one models .in vivo. In our in vitro studies, we found that E-selectin adhesion still promoted significant chemo-resistance in an AML cell line even when CD44 had been deleted, suggesting that CD44 may play only minor, or no role in E-selectin-mediated AML chemo-resistance.Overall, although there is a large amount of literature on CD44 in cancer pathophysiology, the specific role of CD44 interaction with E-selectin has been poorly studied In conclusion, our studies have identified CD162 as a key cell surface receptor mediating AML development and resistance to cytotoxic chemotherapy. Blocking the binding of CD162 to E-selectin represents a new potential therapeutic target to delay disease progression, sensitize AML LRCs to chemotherapy and improve overall therapeutic outcomes.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The animal study was reviewed and approved by Animal Experimentation Ethics Committee of the University of Queensland.JE designed the research, performed the experiments, analyzed the data, generated the figures, and wrote the manuscript. JT helped with design of some experiments, analyzed some data, designed the sgRNA for CRISPR gene editing, and commented on the research direction. VB performed experiments and helped with design of some experiments. J-PL participated in the design of the research, commented on research direction, helped analyze some of the data, and critically edited manuscript. IW initiated and supervised the research, analyzed the data, and critically edited manuscript. All authors have discussed and commented on the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Stromal interaction molecule 1 (STIM1) is an important Ca2+ sensor in store-operated Ca2+ entry (SOCE). Both TRPP2 and STIM1 are expressed in endoplasmic reticular membrane and participate in Ca2+ signaling, suggesting a physical interaction and functional synergism.Polycystin-2 (TRPP2) is a CaWe performed co-localization, co-immunoprecipitation, and fluorescence resonance energy transfer assay to identify the interactions of TRPP2 and STIM1 in transfected HEK293 cells and native vascular smooth muscle cells (VSMCs). The function of the TRPP2-STIM1 complex in thapsigargin (TG) or adenosine triphosphate (ATP)-induced SOCE was explored using specific small interfering RNA (siRNA). Further, we created TRPP2 conditional knockout (CKO) mouse to investigate the functional role of TRPP2 in agonist-induced vessel contraction.2+ release and SOCE in HEK293 cells. Inositol triphosphate receptor inhibitor 2-aminoethyl diphenylborinate (2APB) abolished ATP-induced Ca2+ release and SOCE in HEK293 cells. In addition, TRPP2 and STIM1 knockdown significantly inhibited ATP- and TG-induced STIM1 puncta formation and SOCE in VSMCs. Importantly, knockdown of TRPP2 and STIM1 or conditional knockout TRPP2 markedly suppressed agonist-induced mouse aorta contraction.TRPP2 and STIM1 form a complex in transfected HEK293 cells and native VSMCs. Genetic manipulations with TRPP2 siRNA, dominant negative TRPP2 or STIM1 siRNA significantly suppressed ATP and TG-induced intracellular Ca2+ mobilization, SOCE and blood vessel tone.Our data indicate that TRPP2 and STIM1 are physically associated and form a functional complex to regulate agonist-induced intracellular CaVideo abstract The statistical significance was determined using two-tailed Mann-Whitney U test or two-way analysis of variance followed by Games-Howell post hoc tests when more than two treatments were compared. Differences were considered significant with a value of Both TRPP2 and STIM1 are expressed in the ER membrane. To test if TRPP2 interacts with STIM1, C-terminal GFP-tagged TRPP2 (TRPP2-GFP) was co-expressed with N-terminal mCherry-tagged STIM1 (mCherry-STIM1) in HEK293 cells. Double fluorescent imaging showed that TRPP2-GFP colocalized very well with mCherry-STIM1 and ER-DsRed (an ER marker) in the transfected cells  that induces passive ER Ca2+ store depletion (Ca2+ release) via both IP3R-dependent and independent pathways i upon reintroduction of extracellular Ca2+ (Ca2+ entry)  , abolished the ATP-induced Ca2+ release and Ca2+ entry under all conditions tested  causes phospholipase Cβ to convert phosphatidylinositol 4,5-biphosphate into IPglycerol . The IP3rom IP3R . Certainpression . Immunof2+-free solution due to the Ca2+ release from the Ca2+ store , ET-1 (100 nmol/L) and TG (2.5 μmol/L) induced the vessel contraction in the Caore Fig. . SubsequOCE Fig. . Interesion Fig. d, h, m aion Fig. e, i, n.3R antagonist heparin (1 mg/mL), the differences between TRPP2 siRNA or STIM1 siRNA and respective controls were abolished  the opening of TRPP2 will quickly decrease local Ca2+ levels near the channel pore region within the ER lumen; (2) this reduction in local Ca2+ level is sensed by nearby N-terminus of STIM1. Through this association, the local Ca2+ concentration reduction in the ER lumen will activate STIM1 with high efficiency and sensitivity. Santoso et al. reported that TRPP2 competes with STIM1 to associate with IP3R and also found that TRPP2 did not pull down STIM1 in Madin-Darby Canine Kidney (MDCK) epithelial cells i increase. In addition, the vessel tension measurement showed that TRPP2 siRNA and STIM1 siRNAs dramatically decreased the Phe- and ET-1-induced contraction in the Ca2+-free solution and the SOCE-induced contraction. TRPP2 siRNA slightly decreased the TG-induced contraction in the Ca2+-free solution and significantly suppressed the SOCE-induced contraction. STIM1 siRNA significantly decreased the TG-induced contraction in the Ca2+-free solution and the SOCE-induced contraction. It is well known that Phe and ET-1 act on GPCR to produce IP3 which leads to Ca2+ release from IP3R. TG can induce the Ca2+ release via IP3R-dependent and independent pathways [2+ release from IP3R to mediate further Ca2+ release from the ER [2+ release and SOCE.Qian et al. reported that in VSMCs . ATP is te TRPP2 . Here, ws report . As we epathways . TRPP2 im the ER . Therefo2+ release and SOCE. Moreover, heparin treatment using a reversible permeabilization loading procedure to inhibit IP3R abolished the Phe- and ET-1-induced contraction. More importantly, the Phe- and ET-1-induced contractions were significantly reduced in TRPP2 CKO mouse aorta. The data indicate that IP3R-mediated Ca2+ release is an initial factor for the agonist-induced vessel contraction and TRPP2-STIM1 complex may be the downstream of this signal pathway. Taken together, we speculate that TRPP2-STIM1 complex may importantly participate in the agonist-induced Ca2+ release, SOCE and contraction in VSMCs.In a dose-dependent response of mouse aorta, TRPP2 siRNA and STIM1 siRNA strongly suppressed the Phe- and ET-1-induced vessel contraction, which is consistent with the result of the contraction evoked by the Phe- and ET-1-induced Ca2+ release from the ER? Qian’s report and our study support that TRPP2 knockdown decreased the Ca2+ release from the ER [2+ release in MDCK cells [Pkd2+/− mouse [2+-independent [Pkd2+/− arteries [Pkd2+/− mouse.There are two more issues worth discussing. (1) How does TRPP2 affect the Cam the ER . On the CK cells . These c/− mouse . Note thependent  and theyarteries . More im2+ microdomain formed by TRPP2-STIM1 complex plays an important functional role in the agonist-induced Ca2+ release, SOCE and vessel contraction.In summary, we demonstrate that TRPP2 physically associates with STIM1 to form a signal complex in VSMCs, and that this CaAdditional file 1.