To explore the potential use of hESC-CMs as functional cardiomyocytes for drug discovery and cardiotoxicity screening, we examined the effects of bisindolylmaleimide (BIM) (I), which inhibits native cardiac Ca2+ channels, on the Ca2+ channel activity of hESC-CMs. We observed a similar response for the BIM (I)-induced modulation of Ca2+ channels between hESC-CMs and native cardiomyocytes through L-type Ca2+ channel current. These results suggest that hESC-CMs can be useful for evaluation of pharmaceutical efficacy and safety of novel drug candidate in cardiac research.Cardiomyocytes differentiated from human pluripotent stem cells provide promising tools for screening of cardiotoxic drugs. For evaluation of human pluripotent stem cell-derived cardiomyocytes for cardiotoxicity test, in the present study, human embryonic stem cells (hESCs) were differentiated to cardiomyocytes, followed by metabolic selection to enrich the differentiated cardiomyocytes. The highly purified hESC-derived cardiomyocytes (hESC-CMs) expressed several cardiomyocyte-specific markers including cTnT, MLC2a, and α-SA, but not pluripotency markers, such as OCT4 and NANOG. Patch clamp technique and RT-PCR revealed the expression of cardiomyocyte-specific Na In addition, some drugs for cardiovascular diseases carry a risk of damage to the cardiomyocytes, producing abnormal cardiac function, and emphasizing the need for safety assessment of such drugs in clinical trials. Human embryonic stem cell-derived cardiomyocytes (hESC-CMs) are efficient strategies in cardiac research, regenerative therapy, disease modeling, and cardiomyocyte-based models for cardiotoxicity and drug discovery.Cardiovascular disease is a leading cause of mortality and morbidity, and chronic cardiovascular diseases results in major public health burdens. Cardiomyocyte research is important to understand the causes, treatment and prevention of cardiovascular diseases; however, such research poses several critical challenges. Postnatal cardiomyocytes have little or no regenerative capacity2. Since the first attempt in human pluripotent stem cells in 1998, different methods have been proposed including co-culture system, 3D culture, and addition of morphogens (Activin A and BMP4)3. hESC-CMs provide a significant advantage over animal models due to the physiological differences, including beating rate, ion channel expression, and drug toxicity. Besides hESC-CMs exhibit mostly ventricular-like cardiomyocytes, they have been reported to be relatively immature4. In the current study, to generate hESC-CMs for application of drug screening, we adopted hESC-CMs differentiation method utilizing small molecules that modulate the Wnt signaling pathway, in which the differentiated cardiomyocytes showed the high expression of cardiac specific markers  and function 1.To generate hESC-CMs, various research groups developed the methods for cardiomyocyte differentiation and characterized their functional properties+ channels, L- and T-type Ca2+ channels (ICa), transient outward K+ (Ito) channels, ultra-rapid delayed rectifier K+ (IKur) channels, rapid delayed rectifier K+ (IKr) channels, slow delayed rectifier K+ (IKs) channels, inward rectifier K+ (IK1) channels, and ATP-sensitive K+ (KATP) channels6. Overall, ion channels in cardiomyocytes contribute to the generation of action potential shape. Therefore, the detection of functional ion channels and action potential is essential to prove successful differentiation of stem cells into cardiomyocytes.Although there are several cardiac markers to identify successful differentiation of stem cells into cardiomyocytes, specific ion channels expressed in cardiomyocyte can be used as well for this purpose. Cardiomyocytes express tetrodotoxin-insensitive Na2+ channels7, on Ca2+ channels expressed in hESC-CMs.In this study, we explored the differentiation of human embryonic stem cells (hESCs) into cardiomyocytes by detecting cardiac marker proteins and recording cardiac specific ion channels. Furthermore, to prove the pharmaceutical efficacy of hESC-CMs in cardiac research, we investigate the effect of bisindolylmaleimide (BIM) (I), which inhibits native cardiac Ca9, were substantially lower in hESC-CMs than that in adult hLV. To further confirm the immaturity of hESC-CMs, we quantified the ratio of myosin heavy chain (MHC) isoforms, a well-known cardiomyocyte-specific marker, by using the qRT-PCR. In human hearts, there are more β-MHC than α-MHC at all stages; however, it has been reported that there is more α-MHC in fetal than adult hearts11. The hESC-CMs exhibited relatively lower ratio of β-MHC/α-MHC expression than adult hLV  and T-type Ca2+ channel subtype (Cav3.1) were highly increased in hESC-CMs. It has been reported that Cav1.2 and Cav3.1 are associated with cardiac muscle14. These results confirm the functional expression of Ca2+ channels in the hESC-CMs.To record the ent Fig. . We also+ current, we simply applied step depolarizing pulses  with the L-type Ca2+ channel inhibitor Nifedipine (10 µM). As shown in Fig. + currents were observed. However, IK1 currents were not detectable in our recordings. We investigate the expression of representative cardiac molecular subtype, Kv11.1 (IKr) in hESCs and hESC-CMs. Figure K1 currents are deficient compared with native cardiomyocytes.To record the general KK1 channels in the native cardiomyocytes. These results are consistent with the patch clamp result that IK1 currents were not detectable.In order to investigate the electrical activity of differentiated cardiomyocytes, whole-cell patch clamp technique was performed. Figure L-type Ca2+ currents in hESC-CMs, since our previous report suggested that BIM (I) inhibited the L-type Ca2+ currents expressed in native cardiac (ventricular) myocytes7. Application of BIM (I) (1 μM) rapidly reduced the L-type Ca2+ currents within 1 min  (1 μM)  was more sensitive in hESC-CMs (50% inhibition by 1 μM BIM (I)) compared with native cardiomyocytes (47% inhibition by 3 μM BIM (I))7.To explore whether hESC-CMs can be used for drug screening and cardiotoxicity test, we examined the effect of BIM (I), a specific inhibitor of protein kinase C (PKC), on min Fig. . At 0 mVμM) Fig. . AlthougL-type Ca2+ currents, various concentrations of BIM (I)  were applied before measurement of L-type Ca2+ currents. Currents were elicited by one-step depolarizing pulses to 0 mV from a holding potential of −50 mV. As shown in Fig. L-type Ca2+ currents were inhibited by treatment with increasing doses of BIM (I). The dose-response curve was obtained by nonlinear least-squares fit of the Hill equation to the concentration-inhibition data with an IC50 value of 0.71 ± 0.20 μM and a Hill coefficient (n) of 1.16 ± 0.22  on the .22 Fig. . These dL-type Ca2+ channels was due to the inhibition of PKC, we tested the effect of another PKC inhibitor, Chelerythrine on L-type Ca2+ channel. As shown in Fig. L-type Ca2+ current and did not alter the BIM (I)-induced inhibition of L-type Ca2+ channel. The L-type Ca2+ channel current densities were −6.59 ± 0.41 pA/pF in control conditions, −6.09 ± 0.28 pA/pF in the presence of Chelerythrine, and −3.31 ± 0.03 pA/pF in the presence of Chelerythrine together with BIM (I) (1 μM) -induced inhibition of μM) Fig. . These rNa current, Ito current, IKur current, IKr current, IKs current, IK1 current, and ICa current17. In the present study, we demonstrated differentiation of hESCs into functional cardiomyocytes expressing cardiomyocyte-specific markers and ion channels. The hESC-CMs expressed functional ion channels selective for Ca2+, Na+, and K+ ions. The results of the current study may have important implications for the potential applications of the hESC-CMs for drug screening that modulate ion channels.Ion channels are critical for all aspects of cardiac functions, including rhythmicity and contractility, by regulating cardiac action potential. The action potential shape and duration are determined by the sum of inward and outward currents including I2+ channels are key ion channels implicated in cardiac physiology and pharmacology, and largely responsible for the action potential plateau17. Whole-cell Ca2+ transients in pluripotent stem cell-derived cardiomyocytes depend on both Ca2+ influx via L-type Ca2+ channels and intracellular Ca2+ store release, as previously documented in mouse19 and hESC-CMs21. The importance of the L-type Ca2+ current in generating whole-cell Ca2+ transients in these cells was manifested by the elimination of these transients in the absence of external Ca2+ or in the presence of Nifedipine, a selective L-type Ca2+ channel antagonist. A similar requirement for external Ca2+ and the consequent trans-sarcolemmal Ca2+ influx was documented in adult cardiomyocytes24, hESC-CMs21 and mouse ESC-CMs26. We have previously reported that BIM (I), a PKC inhibitor, inhibited L-type Ca2+ channels in rat native ventricular cells with IC50 value of 3.31 µM7. In this study, we obtained the IC50 value of 0.71 µM, therefore, it is likely that the inhibitory effect of BIM (I) on L-type Ca2+ channels in hESC-CMs is more sensitive than that of rat ventricular cells. Although the reason for the higher sensitivity of hESC-CMs to BIM (I) is not clear yet, these results suggest that hESC-CMs can be useful for drug screening based on Ca2+ channel.L-type Ca27. Cell culture conditions and differentiation protocols may influence these action potential properties29. In our results, the action potential recorded in hESC-CMs has a short action potential duration compared with native cardiomyocytes. Moreover, we showed that the L-type Ca2+ channel current recorded in hESC-CMs was smaller than native cardiomyocytes. Ca2+ current is a major inward current maintaining the plateau phase of action potential in native cardiomyocytes, and calcium influx results in cytoplasmic enrichment of Ca2+ and ventricular myocyte contraction23. Therefore, inhibition of Ca2+ current shortens action potential duration and increases the heart rate. The shortening of action potential duration by reducing Ca2+ current amplitude is commonly observed in neonatal cardiomyocytes. The action potential duration and Ca2+ current amplitude of neonatal cardiomyocytes were shorter and smaller than those of adult in guinea-pig ventricular myocytes26. In addition, Ca2+ current amplitude of neonatal cardiomyocytes was small compared with adult cardiomyocytes in rabbit and murine models30. Therefore, it is likely that hESC-CMs used in our experiment possess Ca2+ channel characteristics similar to neonatal cardiomyocytes.Based on the action potential phenotypes recorded in isolated cells, differentiated CMs have been reported to have a heterogeneous population classified as atrial-, nodal-, or ventricular-like33. Although we could not address the detail reasons for the relative short action potential duration compared to the results of other study groups, it may be caused by the small Ca2+ current amplitude. In fact, Ca2+ current recorded in the present study is half of the Ca2+ current that reported in previous papers. For this reason, action potential duration recorded in our group is relatively short compared to other papers  shifted the steady-state inactivation curve toward a more negative potential suggesting that BIM (I) could directly interact with L-type Ca2+ channels7. Considering that L-type Ca2+ channels play an important role in cardiac function by regulating excitation-contraction coupling, action potential duration, and intracellular Ca2+-dependent signaling pathways, application of BIM (I) reduces cardiac contractility, action potential duration, and PKC-dependent signaling pathways. In the present study, we applied BIM (I) on the Ca2+ channels expressed in hESC-CMs to evaluate pharmaceutical efficacy in cardiac research. As similar results with native cardiac Ca2+ channels, BIM (I) efficiently reduced the Ca2+ channel expressed in hESC-CMs suggesting that hESC-CMs will be useful for cardiac functional studies, specifically Ca2+ channels.BIM (I), a specific PKC inhibitor, has known to decrease L-type Ca27. Although the hESC-CMs have several barriers to direct use in clinical field, but hESC-CMs used in this experiment are valuable in the field of cardiac research, specifically cardiotoxicity and the development of new pharmaceuticals. To resolve the problems that the hESC-CMs exhibit the phenotypes of neonatal cardiomyocytes, further experiments are necessary to develop technologies for differentiation of human pluripotent stem cells into functionally matured cardiomyocytes.Overall, the human pluripotent stem cell-derived CMs present a new and rapidly developing technology with exciting applications, and with further refinements it could pave the way for the development of personalized medicine for cardiovascular diseasesDMEM/F12, glucose-free DMEM, RPMI1640, FBS, Penicillin-streptomycin, Collagenase type IV, Dispase, Accutase, B27 supplement, B27 supplement minus insulin, and 0.05% trypsin-EDTA were purchased from Thermo Fisher Scientific . mTeSR1 and mTeSR1 5 × supplement were purchased from STEMCELL Technologies Inc. . Humans ES cell line, H9 (WA09), were purchased from the WiCell Research Institute . Matrigel (#354277) was purchased from Corning Life Sciences . Antibodies against TRA-1-60 (#560884), TRA-1-81 (#560883), SSEA4 (#561565), SSEA3 (#560881) were purchased from BD Biosciences . Antibodies against Oct4 (#AB181557) and Sox2 (#AB59776) were purchased from Abcam . Anti-GAPDH antibody (#MAB374) was purchased from EMD Millipore . Anti-cTnT antibody , anti-MLC2a antibody , and anti-MLC2v antibody  were purchased from the indicated companies. Mitomycin C, Gelatin, Y27632, anti-α-SA antibody (#A7732), and all other unlisted reagents were purchased from Sigma-Aldrich . Adult human left ventricle tissues were obtained as surgical waste from patients undergoing heart transplant operations performed at Pusan National University Yangsan Hospital . Written informed consent was obtained from donors and the protocol was approved by the Institutional Review Board of Pusan National University Hospital.www.cefobio.com, Seoul, Korea). For preparation of feeder, MEFs on passage 5 were treated with 5 ng/ml Mitomycin C for 1.5 h and seeded to 4 × 105 cells/60 mm dish (0.1% gelatin coated) in DMEM containing 10% FBS and penicillin-streptomycin (125 U/ml). Undifferentiated H9 hESC colonies were cultured on Mitomycin C-treated un-mitogenic MEFs feeder layer. H9 hESC colonies were maintained in DMEM/F12 medium containing 1 × L-glutamax, penicillin-streptomycin (125 U/ml), β-mercaptoethanol (60 μM), 1 × NEAA , 20% KSR medium  and bFGF (5 ng/ml)  with daily change of media for 4 days. On the 5th day, H9 hESC colonies were passaged by treatment with dissociation medium (DMEM/F12 containing collagenase type IV(200 μg/ml) and Dispase (1 mg/ml)) for 12 min. After dissociation medium treatment, the colonies were harvested by sedimentation and washed with the maintenance medium twice. The collected hESC colonies were cut into pieces of 50~80 μm in diameter by pipetting and plated onto the MEF feeders with 1:3 or 1:4 split ratio, and then incubated at 37 °C under 5% CO2. The study protocol was approved by the Public Institutional Bioethics Committee designated by the MOHW. All experiments were performed in accordance with relevant guidelines and regulations.Mouse embryonic fibroblasts (MEFs) were purchased from the CEFO Corporation  with a density of 1.4 × 106 cells/well, followed by changing to fresh medium every day for 3 days with 80–90% confluence. To initiate differentiation, the media was changed to RPMI/B27 minus insulin media containing the GSK3-specific inhibitor CHIR99021  for 24 h (day 0), followed by RPMI/B27 minus insulin media for 48 h (day 1–3). Differentiating cells were treated with the Wnt signaling inhibitor IWP2  in RPMI/B27 minus insulin media for 48 h (day 3–5). At day 5 and day 7, culture media was replaced with fresh RPMI/B27 minus insulin media. From day 8, culture media was replaced with RPMI/B27 medium every 3 day up to day 20. Cells were incubated in a humidified incubator at 37 °C under 5% CO2.To differentiate H9 hESCs into cardiomyocytes, hESCs were dissociated to single cells by treatment with Accutase at 37 °C for 7-8 min. Single cell suspension was seeded on Matrigel-coated 6-well culture plate prepared as described39. Differentiating hESCs were dissociated to single cells with 0.05% trypsin-EDTA at 37 °C for 5 min, collected with RPMI supplement with 20% FBS, and seeded on 0.1% gelatin-coated dish with RPMI supplement with B27.In the final phase of cardiomyocyte differentiation, cells were switched to glucose-free DMEM supplemented with Lactate (4 mM) on day 30 as describedTo prepare for single cells, hESCs were dissociated by Accutase treatment and hESC-CMs were dissociated with 0.05% trypsin-EDTA, followed by passing through 40 µm cell strainer. Cells were fixed with 4% paraformaldehyde for 20 min, washed with PBS, and permeabilized with 1% Triton-X-100 in PBS for 10 min. Cells were blocked with 5% BSA for 2 h and incubated with primary antibodies  with 1:1000 dilution and overnight incubation at 4 °C. Secondary antibodies conjugated with fluorescent dyes were incubated with 1:2000 dilutions at 4 °C for 2 h. Fluorescence intensity of stained cells was measured using a CANTO II (BD Biosciences) and analyzed using FlowJo .Total RNA was extracted from 80–90% confluent cultures using TRIzol reagent (Sigma) and reverse transcribed into cDNA using the Reverse Transcription cDNA Kit . cDNA in 1 μL of the reaction mixture was amplified using the Ready-2 × -Go pre-mix PCR kit  and 10 pmol each of sense and antisense primers. The thermal cycle profile was as follows: denaturation at 95 °C for 30 s, annealing at 54 °C for 30 s depending on the primers used, and extension at 72 °C for 30 s. Each PCR reaction was carried out for 25–30 cycles. PCR products were analyzed by 1% agarose gel with ethidium bromide electrophoresis and photographed under UV trans-illumination. The primer sequences for RT-PCR are listed in the Supplementary Table Quantitative RT-PCR was performed using an ABI7500  sequence detection system, using SYBR Green PCR Master Mix  according to the manufacturer’s instructions. Experiments were performed in duplicate, and the data were normalized to expression of GAPDH mRNA. Data were analyzed using the Δ (Δ CT) method and normalized to GAPDH. The primer sequences for q-PCR are listed in the Supplementary Table 3VO4 (1 mM), sodium pyrophosphate (30 mM), β-glycerol phosphate (25 mM), 1% Triton X-100, pH 7.4). The cell lysates were centrifuged for 15 min at 4 °C, and the supernatants were used for western blotting. Lysates were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred onto nitrocellulose membranes, and then stained with 0.1% Ponceau S solution  to ensure equal loading of the samples. After being blocked with 5% non-fat milk for 30 min, the membranes were incubated with primary antibodies overnight, and the bound antibodies were visualized with horseradish peroxidase-conjugated secondary antibodies using the enhanced chemiluminescence Western blotting system .Cells were washed twice with HBSS and then lysed in lysis buffer (Tris-HCl (20 mM), EGTA (1 mM), EDTA(1 mM), NaCl (10 mM), phenylmethylsulfonyl fluoride (0.1 mM), NaUndifferentiated hESCs and hESC-CMs were cultured on Matrigel-coated cover glasses. Cells were fixed with 4% paraformaldehyde for 20 min, washed with PBS, and permeabilized with 1% Triton-X-100 in PBS for 10 min. Cells were blocked with 5% BSA for 2 h and incubated with primary antibodies against hESC-specific markers  or cardiomyocyte-specific markers  with 1:200 dilution at 4 °C for overnight. Secondary antibodies conjugated with fluorescent dyes were stained with 1:500 dilutions at 4 °C for 2 h. Cells were mounted on the slide glass with Vectashield medium (Vector Laboratories) containing 4’,6-diamidino-2-phenylindole (DAPI) for nuclear staining. Images were collected with a confocal microscope system .+ channel recordings, 4–6 kHz for Ca2+ channel recordings, and 15–10 kHz for Na+ channel recordings.Whole cell membrane currents were recorded using the whole-cell patch clamp technique with an EPC-8 amplifier  and an NI-DAQ-7 digital interface . Patch pipettes were pulled from borosilicate capillaries  using a PP-830 puller  to have a 2–3 MΩ when filled with pipette solution. The voltage signals were sampled at a rate of 1–3 kHz for K+ channel recordings contained: NaCl (130 mM), CsCl (15 mM), HEPES (5 mM), NaH2PO4 (0.33 mM), MgCl2 (0.5 mM), CaCl2 (1.8 mM), glucose 16.6, adjusted to pH 7.4 with NaOH. The internal solution for Na+ channel recordings contained: Cs-Aspartate (100 mM), CsCl (25 mM), HEPES (10 mM), EGTA (5 mM), NaCl (5.5 mM), Mg-ATP (4 mM), adjusted to pH 7.25 with CsOH. The extracellular solution for Ca2+ channel recordings contained: NaCl (135 mM), CsCl (10 mM), HEPES (5 mM), NaH2PO4 (0.33 mM), MgCl2 (0.5 mM), CaCl2 (1.8 mM), glucose (16.6 mM), adjusted to pH 7.4 with NaOH. The internal solution for Ca2+ channel recordings contained: CsCl (105 mM), TEA-Cl (22 mM), NaCl (5.5 mM), Mg-ATP(4 mM), EGTA (10 mM), HEPES (10 mM), adjusted to pH 7.25 with CsOH. The extracellular solution for K+ channel recordings contained: NaCl (137 mM), KCl (5.5 mM), HEPES (5 mM), glucose (15 mM), CaCl2 (1.7 mM), MgCl2 (0.7 mM), NaH2PO4 (0.33 mM), adjusted to pH 7.4 with NaOH. The internal solution for K+ channel recordings contained: K-aspartate (110 mM), KCl (27 mM), NaCl (5.5 mM), MgCl2 (1 mM), Mg-ATP (3 mM), HEPES (10 mM), EGTA (10 mM), adjusted to pH 7.2 with KOH. BIM (I) was purchased from Tocris Cookson  and was dissolved in dissolved in dimethyl sulfoxide (DMSO).The extracellular solution for Na50) value and the Hill coefficient (n) as described below:Idrug/Icontrol) at the test potential, and [D] means the drug concentration.Electrophysiological data were analyzed using The Origin 8.0 software . The Hill equation was used to obtain the half-maximal inhibitory (ICt-test, and a value of p < 0.05 was regarded significant.The results are presented as means ± S.E.M. Differences were tested for statistical significance using Student’s Supplementary Movie S1.Supplementary Information


The ability of Ca2+ to function effectively as a regulator of migration requires the generation of temporally complex signals within spatially restricted microdomains. The generation and maintenance of these Ca2+ signals require a specific structural architecture and tightly regulated communication between the extracellular space, intracellular organelles, and cytoplasmic compartments. New insights into how Ca2+ microdomains are shaped by interorganellar Ca2+ communication have shed light on how Ca2+ coordinates cell migration by directing cellular polarization and the rearrangement of structural proteins. Importantly, we are beginning to understand how cancer subverts normal migration through the activity of oncogenes and tumor suppressors that impinge directly on the physiological function or expression levels of Ca2+ signaling proteins. In this review, we present and discuss research at the forefront of interorganellar Ca2+ signaling as it relates to cell migration, metastasis, and cancer progression, with special focus on endoplasmic reticulum-to-mitochondrial Ca2+ transfer.Cell migration is one of the many processes orchestrated by calcium (Ca Similarreviewed , 9 and treviewed , p53 , a regulator of focal adhesion turnover, with the focal adhesion complex -dependent regulation of its phosphorylation status  driven by expression of the SOCE proteins, stromal interaction molecule 1 (STIM1) and Orai1  channels in increasing vimentin expression through the signal transducer and activator of transcription 3 pathway  channel  and cathepsins . Importa channel . Another channel . Activat channel .2+, including the TRP channels and components of STIM/Orai-mediated Ca2+ entry, for reviews see Ref.  and ryanodine receptor Ca2+ release channels deliver Ca2+ to the mitochondria    –62. The eability , 64 thatne GRP75 . From ther (MCU) , 66. Cangulators . Restrucin vitro and suppressed tumor growth in vivo , which is absent in normal colorectal mucosa, is expressed in colorectal carcinoma. Moreover, the expression is greatest at the invasive margin and strongly correlated with metastasis and patient survival . The ability of Drp1 to promote fission is dependent on phosphorylation at serine 616 (S616) and dephosphorylation of serine 637 (S637)  , 113. Cy of S637 , 114, an of S637 . These o of S637 , 116–118 of S637 . Althoug2+ activates several Ca2+-dependent enzymes involved in the tricarboxylic acid (TCA) cycle  cycle . Work byoduction . In a fo in vivo . Althoug in vivo , 122, as in vivo –105.2 and produce ROS. Mitochondrial Ca2+ uptake can increase ROS production at complexes I, III and IV under a variety of conditions. Although the mechanisms are still unclear, a number of possibilities have been proposed, as reviewed previously (2+ can promote the release of ROS accumulated in cristae and intermembrane spaces through Ca2+-dependent increases in matrix volume (Mitochondrial ROS are generated as a consequence of normal respiration. As electrons supplied by the TCA cycle are passed down the electron transport chain, they escape, mostly at complex I and III, to react with Oeviously , 124. Alx volume .2+ uptake (2+ uptake inhibits rather than promotes migration (2+-dependent ROS production is a major mitochondrially derived signal involved in regulating downstream effectors. In one study, increased ER-mitochondrial Ca2+ transfer in non-small cell lung carcinoma cells was caused by MCL1–VDAC interactions that promoted cell migration by increasing mitochondrial ROS production (2+ uptake increased breast cancer cell xenograft growth and metastasis by increased ROS-dependent expression of HIF-1α (2+-dependent ROS production. Nevertheless, many migration effectors are sensitive to ROS signaling (2+ signals, see Ref. (2+-sensitive targets, respectively. The degree of overlap between ROS and Ca2+-sensitive effectors highlights a need to carefully differentiate between ROS and Ca2+-dependent effects when probing the role of ER-mitochondrial Ca2+ transfer.Increased mitochondrial ROS production is a known determinant of tumor growth and metastasis , 127 tha+ uptake –95. Whiligration , 131, phoduction . In anotf HIF-1α . To our ignaling . Perhapssee Ref.  for comp2+ signals regulate cancer cell invasion and metastasis. As we have seen, a picture has emerged in which ER Ca2+ release, mitochondrial Ca2+ uptake, and plasmalemmal Ca2+ influx work together to exquisitely regulate cell motility. This complexity, however, should not dissuade efforts to examine the possibility of therapeutically targeting ER-mitochondrial Ca2+ transfer to affect metastasis. Encouragingly, many of the studies reviewed here have already demonstrated the feasibility of such an approach, showing reduced metastasis in vivo after targeting IP3Rs (2+ transfer.The molecular identification of the SOCE and MCU machinery, the introduction of powerful molecular tools, and the evolution of cancer genetics have all contributed to developing our understanding of how Cang IP3Rs , STIM/Orng IP3Rs , 45, or ng IP3Rs . In adding IP3Rs , 133, asng IP3Rs , would bCW wrote the manuscript and designed and prepared figures.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


B. taurus), lab mice (M. musculus) and lab rats (R. norvegicus) and cardiac muscle calsequestrin from cattle, lab rats and humans. On average, glycosylation of skeletal calsequestrin consisted of two N-acetylglucosamines and one mannose (GlcNAc2Man1), while cardiac calsequestrin had five additional mannoses (GlcNAc2Man6). Skeletal calsequestrin was not phosphorylated, while the C-terminal tails of cardiac calsequestrin contained between zero to two phosphoryls, indicating that phosphorylation of cardiac calsequestrin may be heterogeneous in vivo. Static light scattering experiments showed that the Ca2+-dependent polymerization capabilities of native bovine skeletal calsequestrin are enhanced, relative to the non-glycosylated, recombinant isoform, which our crystallographic studies suggest may be due to glycosylation providing a dynamic “guiderail”-like scaffold for calsequestrin polymerization. Glycosylation likely increases a polymerization/depolymerization response to changing Ca2+ concentrations, and proper glycosylation, in turn, guarantees both effective Ca2+ storage/buffering of the sarcoplasmic reticulum and localization of calsequestrin (Casq) at its target site.Calsequestrin is glycosylated and phosphorylated during its transit to its final destination in the junctional sarcoplasmic reticulum. To determine the significance and universal profile of these post-translational modifications to mammalian calsequestrin, we characterized, via mass spectrometry, the glycosylation and phosphorylation of skeletal muscle calsequestrin from cattle ( The total Asp and Glu content is roughly equal between the two Casq isoforms, and the total Arg and Lys content is highly conserved, totaling 29 in Casq1 and 40 in Casq2 in various animals, which leads to Casq1 having a higher net negative charge than Casq2. In addition, the C-terminal tail of Casq2 contains non-acidic residues, such as Asn, Gly and Ser, while Casq1 contains only acidic residues (Asp and Glu).Calsequestrin (Casq) is considered to be the major Calum (SR)  of both of ~1 mM . Both Ca2+ concentrations increase and depolymerizes as Ca2+ concentrations decrease. Those works in vitro suggest that Casq oligomers should be formed and dismantled in vivo, in response to Ca2+ entry into and release from the SR. When imaged in situ, in muscle cells at rest, structures consistent with the Casq isoforms exist in terminal cisternae (TC) of the SR. These structures have a strikingly different appearance in skeletal and cardiac muscle. In skeletal muscle, they consist of linear polymers that ramify to a three-dimensional dense network that fills the large TC of the skeletal myofibrils. Deep-etch electron microscopy images of the SR terminal cisternae in Nerodia sipedon show that the dense structures that fill the terminal cisternae are indeed linear Casq polymers [2+-dependent polymerization behavior unique to each of the two isoforms [2+ concentrations decrease is instead indirect and has been communicated only recently [When studied in solution, Casq polymerizes as free Capolymers  with brapolymers . In cardisoforms ,7. The disoforms ,9. In surecently .2+ release channels, is seen as having mechanistic significance. Ca2+ ions are proposed to diffuse along the surface of the linear Casq polymer, like current through a wire, guided by the steep Ca2+ gradient, ~104, between the SR and cytoplasm. In an example of diffusion enhancement by the reduction of dimensionality [The arrangement of calsequestrin in linear polymers, tethered to the SR junctional membrane and ending near the luminal mouth of Caionality , such a ionality ,6,12,13.2Man7-5 and GlcNAc2Man9-8, respectively, as well as three phosphorylation sites uniquely in canine Casq2 [2+-binding capacity [2+ homeostasis and signaling.Casq undergoes post-translational modification during its transit to its final destination in the junctional SR ,15. Prevne Casq2 ,15. We pne Casq2 . We alsocapacity ,17. Basecapacity  and phosCasq2 gene [Casq1, such as M87T and D244G, have been identified as possible causes or enablers of malignant hyperthermia and other skeletal muscle diseases, like vacuolar aggregate myopathy and central core disease [Considering the necessity of post-translational modifications for proper function and targeting of Casq, improper post-translational modifications could inhibit the protein’s in vivo functions, with resulting clinical manifestations. One of the diseases caused by dysfunctional Casq2 is catecholaminergic polymorphic ventricular tachycardia type 2 (CPVT2), a potentially lethal hereditary illness caused by mutations to the sq2 gene ,19. One sq2 gene  and is e disease ,22,23. TA first step toward identifying the in vivo effect and pathological link for those post-translational modifications is to determine the exact pattern and effect of glycosylation and phosphorylation in humans and other model animal species. For this purpose, we have characterized the post-translational modifications of the rat and mouse model systems and have compared them to those of native human Casq2 obtained from cardiac muscle from two separate donors. In addition, we analyzed the post-translational modifications and their effects in bovine Casq1 and Casq2, as close models of the corresponding human proteins.B. taurus), lab mice (M. musculus) and lab rats (R. norvegicus) and Casq2 from cattle, lab rats and humans.In order to determine the exact profile of the post-translational modifications to mammalian Casq, we characterized, via mass spectrometry, the glycosylation and phosphorylation of Casq1 from cattle  based on peak height, with the non-glycosylated peptides  also present (2Man2-4 glycoforms (successive +162 m/z peaks) were present in smaller amounts  to GlcNAcMan9 (4728.1 m/z), with no apparent non-glycosylated form. Rat Casq2 ranged from GlcNAc2Man3 (3775.4 m/z) to GlcNAc2Man5 (4099.5 m/z), with the non-glycosylated form present in very small amounts (3893.5 m/z). The two human Casq2s ranged from GlcNAc2Man3 (3783.8 m/z and 3785.6 m/z) to GlcNAc2Man9 (4758.8 and 4757.9 m/z), with the non-glycosylated form present in relatively large amounts for one of them (2891.5 m/z) and relatively small amounts (2893.5 m/z) for the other. The signal-to-noise ratios for most peaks were sufficient to correlate their experimental mass to their expected mass and distinguish them from peaks corresponding to other peptides. The non-glycosylated peptide of rat Casq2 was an exception, as it had a weak signal, but enough signal was present to distinguish it from the background and to calculate its mass from the data.Unlike the preference of Casq1 for GlcNAc heights a. BovineA mixture of non-phosphorylated, mono-phosphorylated and di-phosphorylated C-terminal peptides was present in all bovine, human and rat Casq2 mass spectra b. The no2+ forms of native and recombinant bovine Casq1 crystallized in the absence of added Ca2+ in the crystallization buffer; the high-Ca2+ forms crystallized in the presence of 2.5 mM Ca2+ (17× molar excess of Ca2+ added to solution).To investigate any structural significance/role of glycans or structural change due to the presence of glycans, the crystal structures for both native and recombinant bovine Casq1, which correspond to the glycosylated and nonglycosylated form, respectively, were determined under the same condition. Two structures were closely compared in order to find the structural impact of glycosylation. Calsequestrin crystallized in two forms: the low-Ca2+ native bovine Casq1 crystallized in the P1 space group and contained four molecules in the unit cell  binding 12 Ca2+ ions cooperatively within their respective front-to-front interface and the remaining 40 Ca2+ ions bound non-cooperatively by structural and low-affinity, high-capacity sites.Each monomer in the low-Ca2+ native Casq1, the glycosyl groups on the four molecules in the crystallographic asymmetric unit modeled reliably into the corresponding electron density map as GlcNAc2 for two monomers, GlcNAc2Man1 for another monomer and GlcNAc2Man2 for the remaining monomer. In the high-Ca2+ native Casq1, the glycosyl groups were only modeled reliably up to GlcNAc2, as both the low resolution diffraction data and consequent low contour level of electron density were insufficient for building the remaining mannoses.In the case of low-CaTo identify any possible glycosylation-dependent structural differences between native and recombinant calsequestrin, we superposed the Cα positions of Pro7 through Pro334  had root mean square distances (r.m.s.ds) of 0.44, 0.41 and 0.25 Å, respectively  of high-Caectively a–c. Supeectively d had r.mectively e had r.mmonomers f in the 2+ native Casq1, the glycosyl chain of each monomer appears to stabilize a loop containing Glu350 through Glu354 by establishing a hydrogen bond between the N-acetyl group of the first N-acetylglucosamine and the hydroxyl group of Thr353 , downward against the glycosylation loop, which shifted the glycosylation loop accordingly. The shifted glycosylation loop then shifted α12, with which it interacts, outward towards the solvent. The net effect of the outward shift of α12 in monomer A and the Leu246-mediated, Ca2+-dependent hydrophobic interaction between monomers A and B was a break of the back-to-back symmetry. As a result, the recombinant hexamer was flatter when viewed from the side, in contrast to the bow shape of the native hexamer when viewed from the same angle  and rats (genus Rattus), both of the Murinae subfamily of the family Muridae, are known to have split evolutionarily 11 million years ago [Human evolution diverged from its commonly-used laboratory test analogues, mice, rabbits and rats, to name a few, 61.5 million years ago . Mice  of the glycosylation loop of native Casq1  together2+ concentrations than recombinant bovine Casq1  [2Man8-9 glycoform [In Casq2, our mass spectrometry data show the presence up to the full complement of nine mannoses found among tested animals, a substantially higher mannosylation than in Casq1. It is tempting to speculate that the presence of a longer glycosyl group is associated with the longer and more negatively-charged Casq2 C-terminal tail, as a greater enthalpic contribution is required to overcome the entropic cost for Caraction) ; the optlycoform . Under t2+-binding capacity [2+-dependent responses of Casq2 in cardiac muscle. If so, an improper distribution of Casq2 with zero, one and two phosphoryls should have dire pathological consequences for an organism. In support of this hypothesis, defective or altered canine Casq2 phosphorylation is associated with several muscular complications, including heart failure [2+ ions. Alternatively, Casq2 with phosphorylated C-terminal tails could be preferentially anchored near the RyR channel, as their more ordered conformation would have a higher affinity for continual attachment to triadin and junctin through a mechanism similar to that used by Casq1 [Our mass spectra of the C-terminal peptide of native bovine, human and rat Casq2 showed the presence of up to two phosphorylation sites b, locatecapacity ,27, maki failure ,30,31,32 failure . For exaby Casq1 .2+-dependent polymerization and Ca2+-binding capacity of Casq are modulated by glycosylation and phosphorylation. Those findings make it likely that such post-translational modifications are a tunable mechanism, operating in vivo. As any alteration of the unique characteristics of Casq could affect its Ca2+-binding capacity and the SR function, improper modification can produce pathophysiological consequences [Through this and previous works, we have shown that both Caequences ,17. The 2+-dependent polymer [2+-dependent Casq2 oligomerization and, eventually, SR function [Most CPVT2-related human Casq2 mutations are unable to form a proper Ca polymer . In addifunction ,36,37. WSamples of bovine, mouse and rat cardiac and skeletal muscle were obtained from Hereford cattle , Swiss mice (Washington State University colonies) and Sprague-Dawley rats (Simonsen Laboratories). All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) . Human cardiac muscle was obtained from Seoul National University Hospital of South Korea, under procedures approved by the Institutional Review Board of the Seoul National University Hospital. The heart tissues used in this study were from two patients (recipients) who received heart transplantation. Each heart was harvested after aortic and vena cava clamping along with cardiopulmonary bypass, and immediately, the prepared donor heart was transplanted. The first heart tissue was from a 59-year-old male, diagnosed with “enteropathy-associated T cell lymphoma”. The second heart tissue was from a 62-year-old male, diagnosed with “idiopathic dilated cardiomyopathy”. Both samples were obtained from the left ventricle, which is the site with abundant myocardium. The harvested heart tissues were washed in saline and instantly frozen in a deep freezer at −80 °C.Left ventricular cardiac muscles were used to study bovine and human Casq2, and whole hearts were used to study mouse and rat Casq2. Chuck steak was used for bovine Casq1, whereas back and leg muscles were used to study mouse and rat Casq1.XhoI and NcoI cut sites, following rare codon optimization  and transformed into Rosetta (DE3) pLysS Escherichia coli.The bovine Casq1 gene (GenBank: AB277764.1) was synthesized by GenScript in pET28a(+) with 3, pH 7.4. The homogenates were clarified by centrifugation at 10,000× g for 15 min, followed by centrifugation at 100,000× g for 30 min in a Beckmann Ti 55.2 fixed-angle rotor to harvest the sarcoplasmic reticula. The sarcoplasmic reticulum pellets were suspended in a buffer containing 0.1 M Na2CO3, 10 mM EGTA, 0.5 M KCl and 0.05 g/dL NaN3 and incubated on ice for 10 min to extract Casq. After incubation, the carbonate extracts were centrifuged at 130,000× g for one hour. The carbonate extract supernatant was then loaded onto a phenyl sepharose column  equilibrated with phenyl sepharose wash buffer . After washing the column extensively with phenyl sepharose wash buffer, Casq was eluted using phenyl sepharose wash buffer containing 10 mM CaCl2. The eluates were then buffer exchanged into anion exchange Buffer A  and loaded on to a MonoQ column . Contaminating proteins were eluted from the column using a stepwise gradient from 0% to 25% Buffer B , followed by elution of Casq using a stepwise gradient from 25% to 28.5% Buffer B. The purified proteins were then pooled, concentrated, dialyzed overnight against double reverse osmosis H2O to remove trace Ca2+ and buffer exchanged into buffers suitable for their given purpose.Native Casq1 and Casq2 were extracted from the sarcoplasmic reticulum fractions of homogenized cardiac or muscle tissues from their respective organisms. Briefly, the muscle tissues were homogenized in a buffer containing 20 mM imidazole, 0.3 M sucrose, 0.15 M KCl and 0.05 g/dL NaNRecombinant bovine Casq1 was purified using previously-established protocols . Fractiov/v) trifluoroacetic acid (TFA). The samples were then dried, suspended in water and loaded into a NuTip C8 tip . The tip was washed with 0.1% (v/v) TFA in water, followed by eluting Casq from the tip with 100% acetonitrile. Elutions were then mixed with an equal volume of 10 mg·mL−1 sinapic acid in 50% (v/v) acetonitrile with 0.1% (v/v) TFA, spotted onto a sample plate containing dry matrix and allowed to dry. Phosphopeptides were enriched from the total peptide solution using the NuTip TiO2 + ZrO2 tip (Glygen Corporation) and manufacturer protocols. The eluted peptides were acidified with TFA, dried, suspended in sinapic matrix and spotted onto a sample plate containing dried matrix. The spectra for the peptides were collected using a 4800 MALDI TOF/TOF Analyzer . Mass spectra were collected in positive and negative linear and reflector modes, and the best spectra were chosen based on signal-to-noise ratio. Specifically, the best Casq1 spectra were collected in negative reflector mode, whereas the best Casq2 spectra were collected in linear positive mode, except for the glycosylation of bovine Casq2 and one of the human hearts. The mass of Casq was calibrated internally using the +1 and +2 BSA ions.For the purpose of fragmentation, protein solutions were allowed to react for 2 hours in the dark and at room temperature with a solution consisting of 2 M Cyanogen bromide (CNBr), 10 mM Caesium iodide (CsI) and 70%  containing either 0, 1, 1.5 or 2 mM CaCl2 were injected onto a Yarra 3u SEC-2000 size-exclusion column  equilibrated with the same buffer. The presence of Casq1 was monitored at 280 nm in tandem with a DAWN EOS multi-angle light scattering machine  for measuring scattering intensities.Native and recombinant bovine Casq1 at 50 µM in static-light scattering running buffer  were crystallized using the hanging drop vapor diffusion method at 4 °C. Protein solutions were mixed with an equal volume of crystallization buffer  2-methyl-2,4-pentanediol, pH 7.0), and crystals formed overnight. To obtain the high-Ca2+ native and recombinant bovine Casq1 structures, each protein was incubated with 5 mM Ca2+ prior to mixing with crystallization solution. Crystallographic data were collected at the Advanced Light Source (Beamlines 8.2.1 and 8.2.2) and reduced and scaled using HKL2000. The low-Ca2+ native and recombinant bovine Casq1 structures were solved by molecular replacement PHENIX [2+ recombinant bovine Casq1 structure was solved by molecular replacement using the low-Ca2+ native bovine Casq1 structure. After rebuilding the low-Ca2+ structure to its high-Ca2+ form, a high-Ca2+ recombinant bovine monomer was used to solve the high-Ca2+ native bovine Casq1 structure by molecular replacement. Iterative model adjustment and refinement were completed using COOT [2+ native (PDB ID: 5KN0), low-Ca2+ recombinant (PDB ID: 5KN3), high-Ca2+ native (PDB ID: 5KN2) and high-Ca2+ recombinant (PDB ID: 5KN1) have been deposited in the Protein Data Bank. Refinement statistics are listed in Native and recombinant bovine Casq1 (0.3 mM in 20 mM HEPES, 0.5 M NaCl, 0.05 g/dL NaNt PHENIX  using thing COOT  and PHENing COOT . The cry


Furthermore SOCE could be up-regulated by lithium. The present study explored whether SOCE impacts on neuron apoptosis. Cortical neurons were differentiated from induced pluripotent stem cells generated from fibroblasts of ChAc patients and healthy volunteers. ORAI1 and STIM1 transcript levels and protein abundance were estimated from qRT-PCR and Western blotting, respectively, cytosolic Ca2+-activity ([Ca2+]i) from Fura-2-fluorescence, as well as apoptosis from annexin-V-binding and propidium-iodide uptake determined by flow cytometry. As a result, ORAI1 and STIM1 transcript levels and protein abundance and SOCE were significantly smaller and the percentage apoptotic cells significantly higher in ChAc neurons than in control neurons. Lithium treatment  increased significantly ORAI1 and STIM1 transcript levels and protein abundance, an effect reversed by inhibition of Serum & Glucocorticoid inducible Kinase 1. ORAI1 blocker 2-APB  significantly decreased SOCE, markedly increased apoptosis and abrogated the anti-apoptotic effect of lithium. In conclusion, enhanced neuronal apoptosis in ChAc at least partially results from decreased ORAI1 expression and SOCE, which could be reversed by lithium treatment.Chorea-Acanthocytosis (ChAc), a neurodegenerative disorder, results from loss-of-function-mutations of chorein-encoding gene VPS13A. In tumour cells chorein up-regulates ORAI1, a Ca Loss-of-function-mutations of the chorein encoding gene VPS13A (vacuolar protein sorting-associated protein 13A) lead to chorea-acanthocytosis (ChAc)5, a progressive autosomal recessive neurodegenerative disease with hyperkinetic movements, impaired cognitive functions, myopathy with increased plasma levels of creatine kinase, and erythrocyte acanthocytosis8. The movement and motor disorders of chorea-acanthocytosis include limb chorea, parkinsonism, dystonia, tongue protrusion, dysarthria, dysphagia, tongue and lip biting as well as progressive distal muscle wasting and weakness8.The derangements of motor function are paralleled by cognitive impairment, behavioral changes and epileptic seizures8. The progressive neurodegeneration eventually leads to severe disability and early death8. Gene targeted mice lacking functional chorein display erythrocyte shape changes9, neuronal apoptosis10 and altered behaviour10.Chorein promotes activation of phosphoinositide-3-kinase (PI3K)-p85-subunit and thus participates in the regulation of actin polymerization and cell survival13 and participates in the regulation of diverse functions including dopamine release14, platelet activation13, cytoskeletal architecture15, endothelial cell stiffness12, and tumour cell survival3. Most importantly, chorein is decisive for the survival of neurons and skeletal muscle cells16.Chorein is expressed in a wide variety of tissues2+ activity ([Ca2+]i)18. [Ca2+]i could be increased by Ca2+ release from intracellular stores with subsequent store-operated Ca2+ entry (SOCE) through the pore-forming Ca2+ channel subunits ORAI1, ORAI2 and/or ORAI319. Following store depletion, the ORAI isoforms are activated by the Ca2+ sensing proteins STIM1 and/or STIM222. ORAI1 and SOCE are up-regulated by the PI3K pathway and thus at least in some cell types sensitive to chorein23. Moreover, ORAI1 and SOCE could be up-regulated by lithium24, which has been shown to counteract neurodegeneration27.Regulators of cell survival and cell death include alterations of cytosolic CaThe present study explored whether chorein deficiency and lithium impact on neuronal ORAI1 expression, SOCE and/or cell survival. To this end skin fibroblasts from ChAc patients and age-matched healthy individuals were reprogrammed and differentiated to neurons and ORAI transcript levels, ORAI protein abundance, SOCE and apoptosis determined without or with prior lithium treatment.2+ release activated Ca2+ channel ORAI1 and/or its regulator STIM1, experiments were performed with neurons generated from induced pluripotent stem cells (iPSCs). The iPSCs were generated from skin fibroblasts of healthy individuals (control neurons) and patients with chorea-acanthocytosis (ChAc neurons).In order to test whether the pathophysiology of chorea-acanthocytosis (ChAc) involves deranged neuronal regulation of the CaqRT-PCR was utilized for the quantification of the transcript levels encoding ORAI1 or STIM1. As apparent from Fig. In order to test whether transcription of ORAI1 and STIM1 in ChAc neurons is sensitive to lithium, ChAc neurons were incubated for 24 hours in the absence or presence of 2 mM lithium. The ORAI1 and STIM1 mRNA abundance was subsequently determined using qRT-PCR. As displayed in Fig. Western blotting was employed to test whether the differences of ORAI1 and STIM1 transcript levels between healthy individuals and ChAc patients were paralleled by similar differences in protein abundance. As illustrated in Fig. 2+ entry (SOCE). Fura2 fluorescence was thus employed to quantify the cytosolic Ca2+ concentration ([Ca2+]i). For determination of SOCE, the intracellular stores were emptied by exposure of the cells to the sarco-/endoplasmic reticulum Ca2+-ATPase (SERCA) inhibitor thapsigargin (1 µM) in the absence of extracellular Ca2+. Re-addition of extracellular Ca2+ in the continued presence of thapsigargin resulted in a sharp increase of [Ca2+]i reflecting SOCE. As shown in Fig. 2+ stores with thapsigargin was followed by a transient increase in [Ca2+]i to similar values in control neurons and ChAc neurons. The increase of [Ca2+]i following re-addition of extracellular Ca2+ in the continued presence of thapsigargin was significantly blunted in ChAc neurons as compared to control neurons  (data not shown).Decreased expression of ORAI1 and STIM1 were expected to be paralleled by impaired store operated Caons Fig. . Both, s2+]i was in ChAc neurons similar in the absence or presence of lithium (data not shown). Both, peak and slope of SOCE were, however, significantly higher in lithium treated than in untreated ChAc neurons . SOCE is significantly and markedly down-regulated in neurons lacking functional chorein.The present study uncovers a novel pathophysiological mechanism in chorea-acanthocytosis, i.e. deranged regulation of ORAI1 and STIM1 expression with subsequent impairment of store operated Ca2+ activity ([Ca2+]i)28, resulting from rapid Ca2+ entry due to activation of SOCE followed by inhibition of ORAI1 and rapid decrease of [Ca2+]i due to subsequent Ca2+ extrusion29. The repetitive short pulses of [Ca2+]i activate Ca2+ dependent transcription factors and reorganize the actin filament network without triggering the detrimental consequences of sustained increases of [Ca2+]i31.SOCE may trigger oscillations of cytosolic Ca2+ oscillations influence diverse cellular functions33 including entry into the S and the M phase of the cell cycle34 and cell survival36. Along those lines the ORAI isoforms19, and their regulators STIM 1 or 220 contribute to the orchestration of survival, proliferation, and migration of tumor cells40 and neural stem/progenitor cells41. As shown here, pharmacological inhibition of ORAI1 triggers apoptosis and disrupts the supporting effect of lithium on neuronal cell survival.The Ca2+ oscillations, sustained increases of cytosolic Ca2+ activity have been shown to stimulate apoptosis in a variety of cell types44. Thus, cell survival or death depends on a delicate balance between stimulators and inhibitors of Ca2+ entry.In contrast to Ca45 serum & glucocorticoid inducible kinase SGK147. Along those lines, the effect of lithium is reversed in the presence of SGK1 inhibitor GSK650394. SGK1 is effective by NFκB dependent up-regulation of ORAI1 expression and by inhibition of Nedd4-2 triggered degradation of ORAI1 protein47. Chorein deficiency impairs activation of PI3K3 and is thus expected to disrupt PI3K/SGK1/ NFκB dependent ORAI1 upregulation. The observed signalling may thus well contribute to the anti-apoptotic effect of PI3K which confers survival of a wide variety of cells including cancer cells51 and neurons55.The present study sheds some light on the signalling linking chorein deficiency and ORAI1 protein abundance. Previous observations in other cell types revealed that ORAI1 expression is up-regulated by the PI3K dependent57. Mechanisms involved in the neuroprotective effect of lithium include direct or Akt-mediated inhibition of glycogen synthase kinase GSK-3β, Akt-mediated inhibition of the proapoptotic forkhead box class O transcription factor Foxo3a and murine double minute (MDM), stimulation of production and activity of neuroprotective brain derived neurotrophic factor BDNF, up-regulation of antiapoptotic protein Bcl-2, as well as down-regulation of proapoptotic transcription factor p53, of the proapoptotic proteins Bad and Bax, of glutamate excitotoxicity, of calpain and of oxidative stress58. We show here that the protective effect of lithium is abrogated by Orai1 inhibition. In view of the present observations, it is tempting to speculate that lithium may modify the course of neurodegeneration in part by up-regulating neuronal ORAI1 expression and SOCE thus stimulating proliferation of neuronal progenitor cells and inhibiting neuronal apoptosis.The present observations further uncover an anti-apoptotic effect of lithium in neurons from chorea-acanthocytosis. The effect of lithium apparently requires up-regulation of SOCE. Lithium has previously been shown to counteract neurodegeneration in several diseases such as Huntington’s chorea, Alzheimer’s disease, Parkinson’s disease, amyotrophic lateral sclerosis as well as spinocerebellar ataxias type 1 and type 3In view of the present observations clinical studies appear warranted exploring the effect of lithium treatment on the clinical course of chorea-acanthocytosis. To the extent that neuronal apoptosis is the decisive pathophysiological mechanism leading to this devastating disease, lithium treatment may delay or even halt the deterioration of neuronal function in affected patients.2+ entry leading to compromised neuronal cell survival. Conversely, lithium up-regulates store operated Ca2+ entry and attenuates neuronal apoptosis, an effect abrogated by pharmacological inhibition of ORAI1.In conclusion, lack of Chorein in chorea-acanthocytosis downregulates ORAI1 expression and store operated Ca59, with minor modifications. In brief, 1 × 105 fibroblasts were electroporated  with a total of 1 μg per plasmid carrying the sequences for hOCT4, hSOX2, hKLF4, hL-MYC and hLIN28. After cultivation in fibroblast medium for 1 day, 2 ng/ml FGF-2 (Peprotech) was supplemented to the medium. From day 3 on, cells were cultivated in Essential 8 (E8) medium containing 100 µM NaB (Sigma-Aldrich). iPSC colonies were picked manually after 3–4 weeks and further expanded in Matrigel coated 6-well plates. At passage 7–10, iPSCs were characterized and frozen in E8 medium supplemented with 40% KOSR (Thermo Fisher Scientific), 10% DMSO (Sigma-Aldrich), and 1 µM Y-27632 . Characterization of generated iPSCs included genomic validation via exclusion of plasmid-integration, SNParray analysis for genetic integrity, and resequencing of mutation site, as well as functional validation via confirmation of expression of pluripotency marker, and verification of the in vitro differentiation potential as described previously60.The study has been approved by the Ethical Commission of the University of Tübingen (598/2011). Informed consent was obtained from all participants and/or their legal guardian/s. Human dermal fibroblasts were isolated from ChAc patients (n = 2) and healthy volunteers (n = 3). We confirm that all methods performed, including obtaining of consent, were performed in accordance with the relevant guidelines and regulations as approved by the ethics committee. Dermal fibroblasts were cultivated in fibroblast medium, consisting of DMEM  supplemented with 10% fetal calf serum  and 1% L-Glutamine (Biochrom). Induced pluripotent stem cells (iPSCs) were generated following a protocol published previously61. Briefly, neural induction of iPSCs was achieved by addition of dual SMAD inhibitors (10 µM SB431542 (Sigma-Aldrich) and 500 nM LDN-193189 (Sigma-Aldrich)) to 3N medium. Cells were collected at day 10 and further expanded by cultivation in 3N medium supplemented with 20 ng/ml FGF-2 for 2 days. From day 12 on, cells were cultivated in 3N medium with medium change every other day. Cell cultures were passaged at day 27 and replated appropriately for the specific assay . Where indicated, 2 mM lithium (Sigma-Aldrich) was added to the medium 24 hours prior to measurements. Analysis was performed between day 37 and 41.Cortical neurons were generated as described previously62. Total RNA was extracted in TriFast  according to the manufacturer’s instructions. After DNAse digestion reverse transcription of 2 µg RNA was performed using random hexamers  and SuperScriptIII reverse transcriptase . Real-time polymerase chain reaction (RT-PCR) amplification of the respective genes were set up in a total volume of 20 µl using 40 ng of cDNA, 500 nM forward and reverse primer and 2x GoTaq® qPCR Master Mix  according to the manufacturer’s protocol. Cycling conditions were as follows: initial denaturation at 95 °C for 2 minutes, followed by 40 cycles of 95 °C for 15 seconds, 55 °C for 15 seconds and 68 °C for 20 seconds. For amplification the following primers were used (5′->3′orientation):Transcript levels of ORAI1, STIM1, STIM2, and house keeping Gapdh were determined by RT-PCR as described previouslyGAPDH:fw: TGAGTACGTCGTGGAGTCCAC;rev: GTGCTAAGCAGTTGGTGGTGORAI11:fw: CGTATCTAGAATGCATCCGGAGCC;rev: CAGCCACTATGCCTAGGTCGACTAGCSTIM1:fow: CCTCGGTACCATCCATGTTGTAGCArev: GCGAAAGCTTACGCTAAAATGGTGTCTSTIM2:for: CAAGTTGCCCTGCGCTTTATrev: ATTCACTTTTGCACGCACCGSpecificity of PCR products was confirmed by analysis of a melting curve. Real-time PCR amplifications were performed on a CFX96 Real-Time System  and all experiments were done in duplicate. The house-keeping gene Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was amplified to standardize the amount of sample RNA.62. To this end, cells were centrifuged for 5 minutes at 240 g and 4 °C. The pellet was washed twice with ice cold PBS and suspended in 200 μl ice-cold RIPA lysis buffer  containing Halt Protease and Halt Phosphatase Inhibitor Cocktail . Protein concentration was determined using the Bradford assay . 100 µg of protein were solubilized in sample buffer at 95 °C for 5 min. The proteins were separated by a 10% SDS-PAGE in a Glycine-Tris buffer and electro-transferred onto nitrocellulose membranes for 70 min. After blocking with 5% milk in TBST at room temperature for 1 h, the membranes were incubated with primary anti-ORAI1 antibody , anti STIM1 antibody  and anti-GAPDH antibody  at 4 °C overnight. After washing (TBST), the blots were incubated with secondary anti-rabbit antibody conjugated with horseradish peroxidase  for 1 h at room temperature. Protein bands were detected after additional washes (TBST) with an ECL detection reagent  and quantified with Quantity One Software . To assign the right protein size we used Protein-Marker VI .Protein abundance of ORAI1, STIM1 and GAPDH was determined by Western blotting as described previously2+ concentration ([Ca2+]i), as described previously62, For this purpose cells were loaded with Fura-2/AM  for 20 min at 37 °C. Cells were excited alternatively at 340 nm and 380 nm through an objective (Fluor 40×/1.30 oil) built in an inverted fluorescence microscope . Emitted fluorescence intensity was recorded at 505 nm. Data were acquired using specialized computer software . Cytosolic Ca2+ activity was estimated from the 340 nm/380 nm ratio. SOCE was determined by extracellular Ca2+ removal and subsequent Ca2+ re-addition in the presence of thapsigargin . For quantification of Ca2+ entry, the slope (delta ratio/s) and peak (delta ratio) were calculated following re-addition of Ca2+.Fura-2 fluorescence was taken as a measure of cytosolic Ca4, 2 CaCl2, 2 Na2HPO4, 32 HEPES, 5 glucose, pH 7.4. To reach nominally Ca2+-free conditions, experiments were performed using Ca2+-free Ringer solution containing (in mM): 125 NaCl, 5 KCl, 1.2 MgSO4, 2 Na2HPO4, 32 HEPES, 0.5 EGTA, 5 glucose, pH 7.4.Experiments were performed with Ringer solution containing (in mM): 125 NaCl, 5 KCl, 1.2 MgSO64. To this end, cells were incubated in 100 µl Ringer solution containing 5 mM Ca2+ and 1 µl annexin V-FITC After incubation, the cells were centrifuged at 1000 r.p.m. for 5 minutes. Then, cells were stained with annexin V-FITC  to assess phosphatidylserine exposure and propidium iodide (PI) to estimate cell membrane integrity. To this end, cells were incubated in 100 µl Ringer solution containing 5 mM Ca2+ and 1 µl annexin V-FITC  and 0.075 µl PI stock solution (25 mg/ml PI in PBS) for 15 min at 37 °C. Then, 100 µl of Ringer solution were added and annexin V-FITC as well as PI fluorescence were determined by flow cytometry using a FACS calibur .Propidium iodide  uptake was taken as a measure of cell membrane permeability and annexin V-FITC  binding as a measure of cell membrane scrambling with phosphatidylserine translocation at the cell surface, as described previouslyData are expressed as arithmetic means ± SEM. Statistical analysis was made by unpaired t-test or Mann-Whitney test or analysis of variance (ANOVA), as appropriate. p < 0.05 was considered as statistically significant.Supplementary Information


Cold urticaria and cholinergic urticaria are two distinct entities. The presentation of exclusive cold-induced cholinergic urticaria is very rare. The patient described herein had experienced urticaria in the exclusive setting of exercising in a cold environment. Urticarial testing including laboratory and in-office testing was all negative. The patient has prevented urticaria symptoms with oral antihistamine therapy. Pure cold-induced cholinergic urticaria is rarely described in literature. This form of urticaria has yet to be described in a pediatric patient. Cold urticaria and cholinergic urticaria have been clearly defined as two separate entities. Review of literature shows that the cooccurrence of both as one distinct clinical feature is rare. While there have been a few reported cases of isolated cold-induced urticaria with cholinergic features in adults, we present the first case of pure cold-induced cholinergic urticaria in a pediatric patient.A 16-year-old female presented with complaints of urticaria for the past 18 months. She developed the urticaria and pruritus exclusively in the setting of exercising in a cold environment. Urticaria was never triggered with only cold exposure or exercise in a warm environment. There was no significant contributing family history of disease. She also denied any other associated symptoms such as swelling, difficulty in breathing, dizziness, or any systemic symptoms. The patient's skin lesions were described as erythematous, pinpoint papules which covered the entire body but spared the face.An ice cube test was performed and was negative. All laboratory findings of a complete blood count, basic metabolic panel, tryptase, C-reactive protein, aspartate aminotransferase (AST), alanine aminotransferase (ALT), bilirubin, alkaline phosphatase, chronic urticarial index, and thyroid panel were within normal limits. The patient was initiated on oral hydroxyzine therapy for cold-induced cholinergic urticaria and saw success with symptomatic improvement.The exact mechanisms of both cold and cholinergic urticaria are unclear. The first report of a patient with cold urticaria was described in 1866 . CholineIn cold-induced cholinergic urticaria, Ormerod et al.  proposesOutside of preventing precipitating factors, treatment of cold-induced cholinergic urticaria should initiate with antihistamine therapy. In our case and other descriptions of the disease, antihistamines were used and have generally been successful. In the setting of antihistamine failure, omalizumab has been suggested.The clinical discernment of urticarial disease requires a strict history collection strategy in order to remove triggers accurately. To our knowledge, this is the first pediatric patient with pure cold-induced cholinergic urticaria.


This article comments on:68, 4171–4183Journal Experimental Botany .Hocking B, Conn SJ, Manohar M, Xu B, Athman A, Stancombe MA, Webb AR, Hirschi KD, Gilliham M. 2017. Heterodimerization of Arabidopsis calcium/proton exchangers contributes to regulation of guard cell dynamics and plant defense responses.  But do they do more? Arabidopsis CAX isoforms have heretofore not been known to act in concert, but et al. (2017)Hocking  now present a plethora of evidence suggesting that this underlies plasticity in response to environmental cues. CAX1 and CAX3 may form heteromeric transporters that impact guard cell function/leaf gas exchange, and leaf (mesophyll) cell responses to biotic stress.Vacuolar Ca2+: ‘You can’t live without it, you can’t live with it’ (at too high levels). In fact, you can’t even die (i.e. through apoptosis) without it! Calcium is a ubiquitous signal within cells and its temporally transient elevation within the cytosol is a key activator of numerous signaling events in every known prokaryotic and eukaryotic cell – and it has probably been this way since the earliest cells evolved (2+ (Ca2+cyt) signaling ‘spikes’, cell functions are severely impaired at homeostatic Ca2+cyt above about 100 to 200 nM. Thus, proper cell and organism function, growth, and development require molecular transport systems that maintain low basal Ca2+cyt and also ‘sweep’ away elevated Ca2+cyt after signaling events.Ca evolved . Alongsi2+/H+ antiporters (CAXs) contribute to this vital system by providing a transport pathway for Ca2+ movement from the cytosol to the vacuole lumen against an extreme concentration gradient. This is the paradigm encompassing much of what we currently know about tonoplast-localized CAXs such as CAX1 and CAX3. In fact, we refer here to CAXs as having a ‘sweeping’ function to highlight what could be called a ‘housekeeping’ activity. This term, housekeeping, was also used with reference to CAXs by one of the authors of Hocking et al. in a recent review of CAX involvement in transport and signaling events  of full-length CAX1 and CAX3 resulted in the generation of a functional transporter. Importantly, this CAX transporter had different biochemical properties than that displayed by CAX1 alone (expressed as the truncated ‘sCAX1’ polypeptide). Perhaps, then, native CAX exchangers comprising both CAX1 and CAX3 polypeptides might have altered function in native membranes as compared to dimer transporters made up solely of CAX1 coding sequences. This possibility underlies some of the newly developed conjectures of CAX functional plasticity presented in Hocking et al.One of the technical limitations of CAX research until now has been that in order to demonstrate their transport function , the CAX polypeptide had to be expressed as a truncated protein variant. The CAX N-terminal regulatory region has autoinhibitory activity. Hence, studies were done on truncated translation products of ‘sCAX’ coding sequences  is increased upon perception of the presence of pathogens. Bimolecular fluorescence complementation analysis of CAX1:CAX3 association documented that these CAX isoforms are capable of forming dimers .In conjunction with the transport and biochemical analyses of CAX heterodimers, Hocking in situ using different experimental approaches led the authors to speculate that CAX assembly as heteromeric dimers in the plant could provide some enhanced ability to respond to environmental perturbations. Using several experimental approaches, they surmised that during their protoplast preparation procedures, CAX3 protein generation increased over time. Viewing ‘protoplasting’ as a proxy for general stress responses, this led to speculation that CAX3 expression and, hence, CAX1/CAX3 dimer formation may provide plant cells with a tonoplast Ca2+/H+ antiporter with altered functional properties that provides benefits under a range of stress conditions. The authors noticed that some phenotypes displayed by cax1/cax3 double mutants related to their presence in guard cells, and their function related to facilitating Ca2+ sequestration in the vacuole. A model was developed that linked CAX1–CAX3 function in the guard cell tonoplast to regulation of apoplastic Ca2+ and maintenance of normal stomatal aperture during changes in extracellular Ca2+.These studies, approaching the question of whether CAX polypeptides are capable of, and do, function as heteromeric proteins et al.’s biochemical analysis of the transport properties of CAX1–CAX3 dimers upon expression of the full-length coding sequences together in yeast mutants breaks new ground, and their work represents the first functional analysis of these transporters in the presence of their autoinhibitory domains. However, the technical challenge of characterizing the nature of a possibly heteromeric transport protein as it exists in native plant membranes precludes some definitive conclusions about the molecular basis for the mutant phenotypes. We can be certain from the work of these authors that CAX1 and CAX3 are capable of binding to themselves as well as each other. However, we do not know if native tonoplast membranes (in which both polypeptides are present) have CAX1 and CAX3 homodimers as well as the heterodimer. Future experiments involving immunoprecipitation of native CAX protein complexes and interrogation of the captured proteins with isoform-specific antibodies could resolve this particular issue.Hocking 2+ acting as a cytosolic secondary messenger. Ca2+ signaling is of paramount importance to a myriad range of plant cell responses to environmental, developmental, and physiological cues. However, there is much still undefined at the molecular level regarding how Ca2+cyt elevation acts as a secondary messenger to initiate a specific downstream response. The authors present their analyses of vacuolar Ca2+ sequestration facilitated by antiporters comprising CAX1 and CAX3 as a (perhaps) non-static responder to extracellular events leading to Ca2+cyt signaling. They do not conceptualize their tonoplast Ca2+ sequestration system in the context of what Richard Tsien and colleagues ?Could the CAX Caet al. provide some intriguing evidence that CAX antiporters act in pathogen defense responses: application of flg22  stimulates CAX3 expression in mesophyll cells. Moreover, the flg22 peptide is recognized by a specific plasmalemma receptor, FLS2, and flg22 binding to its cognate receptor initiates an immune signaling cascade that requires Ca2+cyt elevation -localized Ca2+-binding protein STIM1. They functionally interact at physical junctures where the ER and cell membrane are linked in a multi-membrane signaling complex  occurring in local domains of the plasmalemma. CAX proteins are involved in numerous Ca2+ signaling cascades (e.g. the involvement of CAX3 in salinity responses; 2+ signaling events in plants on a specific, individual basis. This conjecture is quite a bit down the line from the work shown in Hocking et al., but their conclusion that CAX antiporters could have different functional properties depending on the specific isoforms making up the dimer protein does raise it as a possibility.Extending the points made by Hocking


Mesenchymal stromal cells (MSC) are fibroblast-like multipotent cells capable of tissue-repair properties. Given the essentiality of tight junctions (TJ) in epithelial integrity, we hypothesized that MSC modulate TJ formation, via the AMP-activated kinase (AMPK) pathway. Liver kinase-2+ switch from 5 μM to 1.8 mM was performed using epithelial Madin-Darby canine kidney (MDCK) cells cultured alone or cocultured with rat bone marrow-derived MSC or preexposed to MSC-conditioned medium. TJ assembly was measured by assessing ZO-1 relocation to cell-cell contacts. Experiments were conducted using MDCK stably expressing short-hairpin-RNA (shRNA) against LKB1 or luciferase . Compound STO-609 (50 μM) was used as CaMKK inhibitor.  The in vitro Ca2+ switch, ZO-1 relocation and phosphorylation/activation of AMPK were significantly higher in MDCK/MSC compared to MDCK. No difference in AMPK phosphorylation was observed between LKB1-shRNA and Luc-shRNA MDCK following Ca2+ switch. Conversely, incubation with STO-609 prior to Ca2+ switch prevented AMPK phosphorylation and ZO-1 relocation. MSC-conditioned medium slightly but significantly increased AMPK activation and accelerated TJ-associated distribution of ZO-1 post Ca2+ switch in comparison to regular medium.  Following Ca MSC modulate the assembly of epithelial TJ, via the CaMKK/AMPK pathway independently of LKB1. Panel B. Representative immunoblotting of phospho-AMP-activated protein kinase (pAMPK) and total AMPK (AMPKt) at baseline (α-MEM), in low-Ca2+ conditions (S-MEM) and following Ca2+ switch using epithelial MDCK cells or mesenchymal stromal cells (MSC) or MDCK/MSC co-cultured cells. Each SDS Page was ran and transferred at the same time and incubated with the same primary and secondary antibodies for the same times. Panel C. Representative immunofluorescence of ZO-1 in MSC cultured alone and MDCK cultured alone. ZO-1 experiments were realized simultaneously using the same reagents for the same time of incubation.


Although extracellular nucleotide, adenosine 5′-triphosphate (ATP), is also known as most potent Ca2+-mobilizing ligand that acts on purinergic receptors, ATP never induces cardiomyocyte hypertrophy. Here we show that ATP-induced production of nitric oxide (NO) negatively regulates hypertrophic signaling mediated by TRPC3/6 channels in NRCMs. Pharmacological inhibition of NO synthase (NOS) potentiated ATP-induced increases in NFAT activity, protein synthesis, and transcriptional activity of brain natriuretic peptide. ATP significantly increased NO production and protein kinase G (PKG) activity compared to angiotensin II and endothelin-1. We found that ATP-induced Ca2+ signaling requires inositol 1,4,5-trisphosphate (IP3) receptor activation. Interestingly, inhibition of TRPC5, but not TRPC6 attenuated ATP-induced activation of Ca2+/NFAT-dependent signaling. As inhibition of TRPC5 attenuates ATP-stimulated NOS activation, these results suggest that NO-cGMP-PKG axis activated by IP3-mediated TRPC5 channels underlies negative regulation of TRPC3/6-dependent hypertrophic signaling induced by ATP stimulation.Cardiac hypertrophy, induced by neurohumoral factors, including angiotensin II and endothelin-1, is a major predisposing factor for heart failure. These ligands can induce hypertrophic growth of neonatal rat cardiomyocytes (NRCMs) mainly through Ca The sus pathway .3-mediated Ca2+ release from Ca2+ store  proteins are now accepted as molecular entities of receptor-activated cation channels . There a2+ store . Studiesertrophy . Inhibitertrophy .2+ signaling pathway to initiate various biological processes. Although ATP activates Gq- PLC-dependent Ca2+ signaling pathway in cardiac cells , has long been recognized as an endogenous ligand to stimulate purinergic signaling . Evidencac cells , it has ac cells .3-dependent Ca2+ signaling and NO production in neonatal rat cardiomyocytes (NRCMs), as well as functional coupling between IP3-responsive TRPC5 channel and endothelial NOS (eNOS) contributes to negative regulation of hypertrophic signaling induced by ATP stimulation.Paracrine modulation of cardiac excitation-contraction coupling (ECC) has recently become a topic of intense interest. Resident heart endothelial cells are well-known physiological paracrine modulators of cardiac myocyte ECC mainly via nitric oxide (NO) and ATP . We prevN-acetyl-DL-penicillamine (SNAP), 1-oleoyl-2-acetyl-sn-glycerol (OAG), ET-1, U73122, 8-Cyclopentyl-1,3-dipropylxanthine (DPCPX) and 2-Aminoethyl diphenylborinate (2-APB) were from Sigma. Ang II was from Peptide Lab. AR-C 118925XX, UTPγS trisodium salt, SCH58261 and MRS1754 were from Tocris Bioscience. Fura 2-AM and Nω-nitro-L-arginine methyl ester hydrochloride (L-NAME) were from Dojindo. Diaminofluorescein-2 diacetate (DAF-2 DA) was from Molecular Probes. Dulbecco’s modified Eagle’s medium (DMEM) was from Wako. Anti-TRPC5, anti-TRPC6 antibodies were from Alomone. Anti-phospho-eNOS (Ser1177), horseradish peroxidase (HRP)-conjugated anti-rabbit IgG and HRP-conjugated anti-mouse IgG were from Santa Cruz Biotechnology. Anti-eNOS antibody was from BD Biosciences. Plasmids encoding NFAT promoter-dependent luciferase and brain natriuretic peptide (BNP) promoter-dependent luciferase, and dual luciferase reporter assay system were from Promega. Collagenase II was from Worthington. Stealth siRNAs for rat TRPC5 and TRPC6, Lipofectamine 2000, and Lipofectamine RNAiMAX reagent were purchased from Invitrogen.4-{amino}-6-methoxyquinazoline (MQ), KT5823, ionomycin and xestospongin C were purchased from Calbiochem. 8-bromo- cyclic guanosine 3′,5′-monophosphate (8-Br-cGMP), S-nitroso-2, 95% air) for 1 h in DMEM containing 10% FBS and 1% penicillin and streptomycin. Floating cells were collected and plated into gelatin-coated culture dishes at a density of around 1.5 × 105 cells/cm2. After 24 h, the culture medium was changed to serum-free DMEM. We confirmed that >90% attached cells were actinin-positive by immunostaining. For TRPC knockdown, NRCMs were transfected with each siRNA (50 nM) using Lipofectamine RNAiMAX for 72 h . DAF-2 DA was excited by 470 ± 20 nm, and the fluorescence images at the wavelength of 525 ± 20 nm were acquired using fluorescence microscopy . The fluorescence intensity of the digital images were analyzed using Image J software. Fold increases in fluorescence were calculated by subtracting fluorescence intensity before stimulation from that after stimulation, which was subsequently divided by that of no-stimulation.Measurement of intracellular Caescribed . After aMeasurement of NFAT-dependent luciferase activity and BNP promoter-dependent luciferase activity was performed as described previously , 2011.6 cells) plated on 6-well dishes were directly harvested with 2 × SDS sample buffer (200 μl). After centrifugation, supernatants (20–40 μl) were fractionated by 8% SDS-polyacrylamide gel and then transferred onto polyvinylidene difluoride membrane. The expression and phosphorylation of endogenous TRPC proteins were detected by anti-TRPC6  and anti-TRPC5 (1:1000) antibodies. We visualized the reactive bands using Western Lightning Plus ECL (PerkinElmer). The optical density of the film was scanned and measured with Scion Image software.NRCMs (1 × 103H]leucine incorporation (3H]leucine (1 μCi/ml) was added to the culture medium and further incubated for 4 h. The incorporated [3H]leucine was measured using a liquid scintillation counter.Measurement of hypertrophic responses was performed by measuring the transcriptional activation of BNP gene as described previously . Proteinporation . After ct-test (for two groups) or analysis of variance followed by Tukey’s post hoc test (for multiple groups). Values of P < 0.05 were considered significant. We made utmost effort to minimize the replicates of animal experiments according to the ethical guideline of 3R .Results are presented as the mean ± SEM. Statistical comparisons were made using Student’s 2+-NFAT signaling in NRCMs, as well as known hypertrophy-inducible ligands, ET-1 . ATP increased NFAT-dependent transcriptional activity more potently than ET-1 and Ang II . In contrast, despite increasing NFAT activity, ATP never increased hypertrophic responses, including protein synthesis determined by [3H]leucine incorporation and transcriptional activation of BNP . These results indicate that ATP-induced increases in [Ca2+]i and NFAT activity are not sufficient to induce hypertrophic responses in NRCMs.We first investigated the effect of ATP on Cads, ET-1  and Ang ds, ET-1 . ATP sho2 receptor stimulation , an inhibitor of NOS, resulted in significant enhancement of ATP-induced sustained NFAT activation to the same extent of that by ET-1 stimulation, while L-NAME had no impact on ET-1-induced NFAT activation. L-NAME also enhanced BNP transcriptional activation in response to ATP stimulation . The enhanced BNP activity was canceled by the treatment with MQ (10 μM), a phosphodiesterase (PDE) 5 selective inhibitor, suggesting the involvement of cGMP-dependent pathway. ATP produced a small transient increase in [Ca2+]i in the absence of extracellular Ca2+ , which was mainly derived from intracellular IP3-responsive Ca2+ store. Replenishing extracellular Ca2+ led to sustained increases in [Ca2+]i, which was derived from Ca2+ influx probably through store-operated Ca2+ channels . Treatment with L-NAME, but not D-NAME , significantly suppressed sustained [Ca2+]i increase . In contrast, although ET-1 stimulation also induced sustained [Ca2+]i increase, L-NAME failed to suppress the ET-1-induced sustained [Ca2+]i increase in NRCMs . These results suggest that ATP induces NO production in NRCMs and negatively regulates Ca2+/NFAT-dependent hypertrophic signaling.We next examined which signal pathway(s) induced by ATP stimulation negatively regulates hypertrophic growth of NRCMs. Previously we reported that mechanical stretch on NRCMs induces ATP release, which leads to NO production through P2Ymulation . As NO hmulation , we inveFigures 3A,B). Heart expresses all three isoforms of NOS . We further measured the phosphorylation of vasodilator-stimulated phosphoprotein (VASP), a substrate of PKG . These results indicate that ATP induces activation of NO-cGMP-PKG pathway in NRCMs.We next examined whether ATP actually induces activation of NO-dependent signaling in NRCMs. Compared to stimulation of NRCMs with ET-1 and Ang II, stimulation with ATP significantly increased fluorescence intensity of DAF-2, an NO-sensitive dye , while significantly reducing ET-1-induced increases in [Ca2+]i and NFAT activity . The ATP-induced Ca2+ response was completely suppressed by the treatment with xestospongin C (20 μM), an IP3 receptor (IP3R) blocker . As eNOS activity is predominantly regulated by the increase in [Ca2+]i as well as its phosphorylation, we further investigated whether IP3-dependent store-operated Ca2+ influx signaling mediates eNOS activation by ATP stimulation. Store-operated Ca2+ influx was evoked by the treatment with ionomycin (1 μM), a Ca2+ ionophore . The ionomycin-induced NO production was completely diminished in the absence of extracellular Ca2+, indicating that store-operated Ca2+ influx is required for ATP-induced NO production in NRCMs. Additionally, inhibition of IP3R or PLC suppressed ATP-induced NO production in NRCMs . These results suggest that ATP-induced Ca2+/NFAT signaling and NO production are not mediated by DAG-activated channels, including TRPC6.We previously reported that TRPC6, rather than TRPC3, predominantly mediates mechanical stretch-induced global i and NFAT activity were not reduced by TRPC5 knockdown . We also found that TRPC5 knockdown markedly reduced ATP-induced NO production , and increased ATP-induced BNP transcriptional activity at a rate similar to ET-1 stimulation . The induction of hypertrophic response by ATP stimulation in TRPC5 knockdown NRCMs were mimicked by the treatment with KT5823 . These results suggest that TRPC5 acts as negative regulator of hypertrophic signaling in NRCMs through eNOS-mediated activation of NO/cGMP/PKG signaling.TRPC5 has been shown to be upregulated in human failing heart, although its physiological role is still not fully understood . TRPC5 ial cells . We thus2R by AR-C 118925XX (10 μM) . Stimulation with UTPγS, a P2Y2/4R-selective ligand, also increased NO production. In addition, U73122 suppressed the ATP-induced Ca2+ response . As AR-C 118925XX has yellow-colored self-fluorescence that entirely overlaps F340 intensity of Fura-2, we could not measure the exact ratiometric changes of fura-2 induced by ATP stimulation in the presence of AR-C118925XX. However, we confirmed that AR-C 118925XX completely suppressed the ATP-induced increase in F340 intensity of Fura-2 (data not shown). These results strongly suggest that P2Y2R-PLC axis predominantly mediates ATP-induced Ca2+ response and NO production in NRCMs.We finally examined which purinergic receptor subtype(s) mediates ATP-induced NO production in NRCMs. As expected, the ATP-induced NO production was significantly suppressed by pharmacological inhibition of PLC by U73122 (1 μM) or P2Y1R-selective antagonist), SCH58261 , and MRS1754  did not significantly suppress ATP-induced NO production . Therefore, adenosine receptors may hardly contribute to ATP-induced anti-hypertrophic NO signaling in NRCMs.Extracellular ATP is rapidly converted into ADP, AMP and adenosine by membrane-bound ectonucleotidases . Adenosi2+ also suppressed ATP-induced Ca2+ response and NO production, some Ca2+ influx pathway(s) may be activated by ATP stimulation. However, the context of store-operated Ca2+ channels (SOCs) are hardly developed in matured cardiomyocytes, and the treatment with 2-APB (10 μM), an inhibitor of SOCs, suppressed Ca2+ response but not NO production induced by ATP stimulation. Thus, IP3-dependent TRPC5-mediated Ca2+ influx pathway, but not other Ca2+ release-activated Ca2+ influx pathway, is involved in ATP-induced Ca2+-dependent NO production in NRCMs.As the removal of extracellular Ca2+/NFAT signaling in cardiac hypertrophy is well established, and local Ca2+ influx through TRPC3/6 channels may be a putative mechanism underlying activation of calcineurin-NFAT signaling pathway in rodent myocardium. We demonstrated that ATP-induced increases in [Ca2+]i and NFAT activity are not sufficient to induce hypertrophic responses in NRCMs. Previous studies revealed that sustained Ca2+ oscillation is more efficient in activating NFAT-dependent hypertrophic gene expression than transient Ca2+ rise induced by hypertrophy-inducible ligands and mechanical stretch  .Although the physiological importance of NO-dependent signaling in heart has been long discussed, the major origin of NO was generally thought to be endothelial cells because of poor expression levels of NOS enzymes in NRCMs. However, local activation of NO signaling in cardiomyocyte induced by sex hormone receptor stimulation has been also attracting attention as a negative regulatory mechanism of cardiac arrhythmia . The rol stretch . We reveertrophy . We founin NRCMs , 2011. WS (iNOS) . In this2+ influx negatively regulates ATP-induced hypertrophic response of NRCMs through activation of NO signaling. Indeed, suppression of TRPC5 resulted in reduction of ATP-induced NFAT activation and NO production, thus promotes hypertrophic response in NRCMs. Although NFAT activation has been long associated with hypertrophic gene expression, we speculate that the transient activation of NFAT mediated by TRPC5-dependent Ca2+ entry is not sufficient to induce hypertrophic gene expression in NRCMs.Although TRPC5 expression is upregulated in pathologic hypertrophied human hearts , its phy2R stimulation in neonatal cardiomyocytes is also applicable to adult cardiomyocytes. As TRPC5-mediated NO signaling requires IP3-mediated Ca2+ release and IP3-mediated Ca2+ signaling is down-regulated in adult cardiomyocytes compared to that in neonatal cardiomyocytes, contribution of TRPC5-eNOS axis might be minor in normal adult cardiomyocytes. Future study using adult cardiomyocytes will be necessary to elucidate the pathophysiological role of TRPC5-eNOS axis in heart.Because of limitation of the study, we could not determine whether the TRPC5-eNOS axis induced by P2Y2+/NFAT-dependent hypertrophic response of NRCMs. Purinergic receptors are well accepted as an attractive therapeutic target of age-related cardiovascular diseases (2R agonists in the prevention of cardiac hypertrophy. Our new finding will provide a new therapeutic strategy for the prevention of pathological cardiac hypertrophy.In summary, we revealed a physiological role of TRPC5 channel in rat cardiomyocytes. TRPC5 functionally couples with eNOS, and activation of TRPC5-mediated NO signaling induced by ATP stimulation negatively regulates Cadiseases , and we MN supervised and conceived the project. CS, KS, and SO designed the experiments and prepared the manuscript. CS, KS, SO, TT, KN, and SM performed the experiments and interpreted the data. MN, AN, and TN-T edited 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. The reviewer AA-T and handling Editor declared their shared affiliation.


Outcomes for those diagnosed with acute myeloid leukemia (AML) remain poor. It has been widely established that persistent residual leukemic burden, often referred to as measurable or minimal residual disease (MRD), after induction therapy or at the time of hematopoietic stem cell transplant (HSCT) is highly predictive for adverse clinical outcomes and can be used to identify patients likely to experience clinically evident relapse. As a result of inherent genetic and molecular heterogeneity in AML, there is no uniform method or protocol for MRD measurement to encompass all cases. Several techniques focusing on identifying recurrent molecular and cytogenetic aberrations or leukemia-associated immunophenotypes have been described, each with their own strengths and weaknesses. Modern technologies enabling the digital quantification and tracking of individual DNA or RNA molecules, next-generation sequencing (NGS) platforms, and high-resolution imaging capabilities are among several new avenues under development to supplement or replace the current standard of flow cytometry. In this review, we outline emerging modalities positioned to enhance MRD detection and discuss factors surrounding their integration into clinical practice.  Acute myeloid leukemia (AML) is a malignancy of blood-forming stem cells in which recurrent genetic mutations and chromosomal aberrations are associated with the clonal expansion of immature myeloid populations ,2. For t10 leukemic cells in their bone marrow  3’-deoxy-3’-fluorothymidine (e marrow . In AML controls . Used asts in CR . AlthougFurther studies must be conducted before imaging technology can be incorporated into standard assessments for AML monitoring. At present, it is unknown which imaging modality would be used, if any, and when the ideal time to insert into practice would be, either before, during, or after treatment. In the era of increasingly sensitive techniques for the detection of MRD, it will be important to consider the timing, combination, and sequence of methods used for the diagnosis and follow-up of AML patients. Ultimately, if imaging is to have utility in AML, it is likely to be in triggering a more sensitive assessment by bone marrow examination in a patient testing negative by peripheral blood assays alone and/or for the detection of EMD.It is increasingly appreciated that MRD testing adds to prognostication in AML beyond established metrics such as patient age and cytogenetics ,69,70. FThe MRD-tracking methodologies described above will likely augment our armamentarium by offering several unique advantages beyond what current techniques provide. As discussed for cases with NPM1 mutations, NGS mitigates the need for DNA sequencing-based mutant identification prior to longitudinal tracking with specific PCR primers and standards. Additionally, multiplexed primer designs can be formulated to capture mutations, fusions, and expression changes. Unlike traditional PCR with limitations surrounding amplification bias and inconsistencies with assay efficiency, ddPCR provides a direct measurement of target molecules rather than an estimate of target sequences based on a relationship to cycling time. Finally, novel imaging modalities have the potential to monitor extramedullary disease not detectable by blood or bone marrow assessment while also potentially informing the timing for invasive bone marrow biopsies. + test result status alone as a clinically actionable predictor of relapse [Several factors, both technical and systemic, currently pose barriers to the integration of such technologies for routine MRD monitoring. The inherent sensitivity and error rates between commercially available NGS platforms can vary markedly, especially for low-frequency variants, and will likely require synchronization . Additio relapse . While d


AML is iewed in , and AMLiewed in . The suriewed in ,10. The Fms-like tyrosine kinase 3 (FLT3), a class III receptor tyrosine kinase, and FLT3 ligand (FL) play an important role in the proliferation, differentiation, and survival of hematopoietic cells . MutatedRegarding other kinase inhibitors, T315, an inhibitor of integrin-linked kinase, has been shown to suppress the proliferation of breast and stomach cancer cells and chronic lymphocytic leukaemia cells. Chiu and collegues  report tThe oncogenic or chromosomal mutations that are present in a patient’s AML cells at diagnosis are important to personalized treatment. The best known is a balanced translocation between chromosomes 15 and 17 [t] resulting in the formation of promyelocytic leukemia (PML) and retinoic acid receptor alpha (RARα) fusion protein . LeukaemVDR) gene by 1,25D and ATRA in blood cells at early stages of their differentiation. ATRA, but not 1,25D, upregulates the expression of VDR in human early-stage blood cells. As to early-stage mouse cells, VDR is upregulated by 1,25D, but not by ATRA. Hence, VDR regulation in humans and mice is different, which is germane to testing combinations of agents for use in differentiation therapy. The findings also bring to attention that the level of expression of VDR protein is low in patients’ AML blasts that do not respond to 1,25D. The level can be upregulated by ATRA treatment and this is relevant to the combined therapeutic use of ATRA and 1,25D. In addition to ATRA, the differentiating agent 1,25-dihydroxyvitamin D  has been reported to have beneficial effects in combination therapy for cancer. The paper by Janik and colleagues  examinesThere is an urgent need for new treatments for AML, which will include the need to identify new molecules to target. A number of recurrent mutations in AML involve genes concerned with regulating the epigenetic landscape . Gain orRUNX1-mutated AML. The response of older patients to treatment with hypomethylating agents and survival were found to be independent of RUNX1 status, leading the authors to conclude that future studies should focus on the prognostic value of RUNX1 mutations relative to co-occuring mutations. Almeida and colleages [The articles by Player et al., Khan et al., and Almeida et al. describe the outcomes from clinical studies. Player and colleagues  examine olleages  examine In summary, there is quite some way to go to extend the success in treating APL to other types of AML. As mentioned above, 90% of older patients die from their disease. In populations of developed countries, cancers are turning into prevalent diseases of the aged. According to epidemiological data, about 80% of cancers are diagnosed in patients older than 55, with the median age at diagnosis above 60 years . This is