NFAT1 is stimulated by sub-plasmalemmal Ca2+ microdomains, whereas NFAT4 additionally requires Ca2+ mobilization from the inner nuclear envelope by nuclear InsP3 receptors. NFAT1 is rephosphorylated (deactivated) more slowly than NFAT4 in both cytoplasm and nucleus, enabling a more prolonged activation phase. Oscillations in cytoplasmic Ca2+, long considered the physiological form of Ca2+ signaling, play no role in activating either NFAT protein. Instead, effective sustained physiological activation of NFAT4 is tightly linked to oscillations in nuclear Ca2+. Our results show how gene expression can be controlled by coincident yet geographically distinct Ca2+ signals, generated by a freely diffusible InsP3 message.Excitation-transcription coupling, linking stimulation at the cell surface to changes in nuclear gene expression, is conserved throughout eukaryotes. How closely related coexpressed transcription factors are differentially activated remains unclear. Here, we show that two Ca  •2+ entry, whereas NFAT4 also needs nuclear Ca2+NFAT1 is activated by local Ca•2+ increases via InsP3 receptors located on the inner nuclear membraneNuclear Ca•NFAT1 and NFAT4 show very different rephosphorylation (deactivation) kinetics•Slow deactivation of NFAT1 affords a form of short-term memory to gene expression 2+ microdomains near CRAC channels, whereas NFAT4 additionally requires nuclear Ca2+. Kar et al. now show that nuclear Ca2+ is raised by InsP3 receptors on the inner nuclear membrane. The dependence of NFAT isoforms on distinct sub-cellular Ca2+ signals regulates gene expression in cell- and stimulus-specific contexts.NFAT1 and NFAT4 are widely coexpressed transcription factors. NFAT1 is activated by Ca In resting cells, NFAT proteins are extensively phosphorylated and thereby trapped within the cytoplasm. Upon stimulation of cell-surface receptors that increase cytoplasmic Ca2+, the proteins are dephosphorylated by Ca2+-calmodulin-activated calcineurin, the target for immunosuppressants cyclosporine A and tacrolimus. Dephosphorylation of cytoplasmic NFAT exposes a lysine-rich nuclear localization sequence, enabling the transcription factor to migrate into the nucleus, in combination with the protein importin β  channels activate NFAT1 without the need for a rise in either bulk cytoplasmic or nuclear Ca2+  evokes repetitive baseline cytoplasmic Ca2+ oscillations that are similar to those widely observed in other cells types and in response to different phospholipase C-coupled agonists  receptors embedded in the endoplasmic reticulum (with a contribution from Ca2+ entry through CRAC channels), which then invades the nucleus  receptors with the pro-inflammatory molecule leukotriene Cagonists . In prinModel 1; A, or by Model 2; A.2+ buffering should prevent NFAT4 activation by cysLT1 receptor stimulation because this manipulation would reduce the rate and extent of Ca2+ diffusion from the cytoplasm into the nucleus. By contrast, the expectation from Model 2 is that NFAT4 activation will be unaffected by changes in cytoplasmic Ca2+ buffering capacity. To test these predictions, we overexpressed the cytoplasmic Ca2+ buffer parvalbumin that had been genetically manipulated to express only in the cytoplasm through addition of a nuclear export sequence (PV-NES)  . Althoug(PV-NES) B and 1C (PV-NES) . PV-NES of NFAT4 B and 1C.of NFAT4 B and 1C.igration B and 1C.2+ signals, we measured bulk cytoplasmic Ca2+ with the fluorescent Ca2+ indicator dye Fura-2. The Ca2+ response was significantly decreased by the presence of PV-NES following stimulation with thapsigargin  or nuclear localization sequence (GCaMP3-NLS), to report Ca2+ in the cytoplasm or nucleus, respectively. In control cells expressing GCaMP3-NES, thapsigargin activated a robust Ca2+ signal due to Ca2+ influx, but the response was reduced by ∼60% following expression of PV-NES (2+ oscillations to LTC4 ran down more quickly (2+ rise induced by thapsigargin (measured with GCaMP3-NLS) was also substantially reduced by PV-NES . Simultaneous measurements of nuclear Ca2+ using GCaMP3-NLS and nuclear accumulation of NFAT4-cherry showed that oscillatory nuclear Ca2+ signals induced by LTC4 .Caker BTP2 . Similar2+ influx contributes little to the size or decay rate of each cytoplasmic Ca2+ oscillation evoked by LTC4  for the same isoform in HEK293 cells. Hence, the kinetics of NFAT1 export varies between different cell types, perhaps reflecting the activities of nuclear kinases.In the Ar-5 T cell clone, significant rephosphorylation of NFAT1 occurs within 15 min , which i−1 , which i2+ coupled with addition of cyclosporine A in the continued presence of leptomycin B led to rapid rephosphorylation of nuclear NFAT4 . NFAT4 was rephosphorylated quickly, with a half-time of ∼4 min  gene, which contains the GGAA core nucleotides for an NFAT binding site within the promoter . Collectively, the data indicate that NFAT-driven gene expression in different cell types can be differentially regulated by nuclear Ca2+. Moreover, the same gene (IL-5) can be differentially controlled by nuclear Ca2+, depending on the NFAT isoform present.Although NFAT and c-pression , had no 3, which lowers the endoplasmic reticulum Ca2+ content sufficiently for CRAC channels in the plasma membrane to open, a step necessary and sufficient for NFAT1 activation  . Regions2+ measurements, using GCaMP3 constructs, were carried out at room temperature by using the IMAGO charge-coupled device camera-based system from TILL Photonics , at 0.5 Hz. Fura-2 experiments  and for 1 hr with anti-mouse IgG −15 nm gold complex . All antisera were diluted in 0.1 M phosphate buffer containing 0.1% egg albumin (pH 7.2). As a control the primary antibody was replaced with non-immune sera. After immunolabeling, sections were lightly counterstained with lead citrate and uranyl acetate and examined with a JEOL transmission electron microscope , and images were collected with a Gatan Orius digital camera (Gatan).Briefly, cells were fixed with 3% paraformaldehyde/0.05% glutaraldehyde in 0.1 M phosphate buffer for 10 min, stained with uranyl acetate (2% w/v in distilled water), dehydrated through increasing concentrations of methanol (70%–100%), and embedded in LR Gold (London Resin Company). Ultrathin sections (50–80 nm) were prepared by use of a Reichert Ultracut S ultramicrotome (Leica), mounted on 200-mesh nickel grids, incubated at room temperature for 2 hr with a monoclonal anti-InsP3 receptor type I antibody (Abcam), used at 1:100 dilution. Primary antibody was then visualized with Alexa Fluor 488 goat anti-mouse secondary antibody (1:500)HEK293 cells were fixed with paraformaldehyde (4%) at room temperature and permeabilized with PBS/Triton X-100 (0.5%). Cells were then stained overnight with anti-InsP3 type I receptor , anti-phospho Ser177 NFAT1 , anti-phospho Ser165 NFAT4 , and IL-5  were used. Secondary antibodies for rabbit or mouse were used at 1:5,000 or 1:3,000 dilutions. The membranes were then washed with PBST again and incubated with peroxidase-linked anti-rabbit IgG from Santa Cruz Biotechnology or anti-mouse IgG from BD Bioscience for 1 hr at room temperature. After washing with PBST, the bands were detected by an enhanced chemiluminescence (ECL) plus western blotting detection system (Amersham Biosciences). Blots were analyzed by UN-Scan software. Nuclear and cytoplasmic extracts were separated as before  were analyzed by SDS-PAGE on a 10% or 12% (IL-5) gel. Membranes were blocked with 5% nonfat dry milk in PBS plus 0.1% Tween 20 (PBST) buffer for 1 hr at room temperature . For phos before  and asse2+ for 20 min; see relevant figure legends) was measured and the region (same dimensions and position) was transposed to adjacent lanes. Data are presented relative to the intensity of the dephosphorylated state. Gel analysis was carried out on blots from at least two independent experiments.Gel shifts were quantified in ImageJ. Intensity of the region corresponding to the dephosphorylated state , and then kept in an incubator at 37°C for 6–8 hr. Cells were then washed with PBS, and total RNA was extracted using an RNeasy Mini Kit (QIAGEN). RNA was quantified spectrophotometrically by absorbance at 260 nm. Total RNA (1 μg) was reverse transcribed using the iScriptTM cDNA Synthesis Kit (Bio-Rad), according to the manufacturer’s instructions. Two-microliter samples (cDNA), from RT-PCR, were subjected to polymerase chain reaction (PCR) with the specific primers and GoTaq Green master mix (Promega). The mRNA levels were normalized to GAPDH for 16-HBE cell and β-actin for RBL-1 cells.IL-5 (human): Forward-5′-CTGAGGATTCCTGTTCCTGT-3′; Reverse-5′-CAACTTTCTATTATCCACTC-3′GAPDH (human): Forward-5′-GGTATCGTGGAAGGACTCAT-3′; Reverse-5′-CCACCCTGTTGCTGTAGCCAAATTC-3′IL-5 (rat): Forward-5′-AGACCGGTCATACATGCACA-3′; Reverse-5′-TCGTCTCATTGCTCGTCAAC-3′β-actin (rat): Forward- 5′-TTGTAACCAACTGGGACGATATG-3′; Reverse- 5′-GATCTTGATCTTCATGGTGCTAGG-3′16-HBE cells transfected with cys LT1 receptors or wild-type RBL-1 cells were stimulated with thapsigargin or LTCThe details of mathematical modeling are explained in the ∗p < 0.05 and ∗∗p < 0.01.Data are presented as the mean ± SEM. Statistical significance was determined by using Student’s t test. P.K. and A.B.P. designed experiments. P.K. carried out the experiments. Electron microscopy was conducted by H.C.C. Analyses of data were performed by P.K. and A.B.P. Discussion of the model and how to identify various rate constants involved P.K., G.R.M., and A.B.P. The modeling was executed by G.R.M. A.B.P. wrote the manuscript.


Both MSCs and primary AML cells show constitutive release of several soluble mediators, and the mediator repertoires of the two cell types are partly overlapping. The two cell populations were cocultured on transwell plates, and MSC effects on AML cells mediated through the local cytokine/soluble mediator network could thus be evaluated. The presence of normal MSCs had an antiapoptotic and growth-enhancing effect on primary human AML cells when investigating a group of 51 unselected AML patients; this was associated with increased phosphorylation of mTOR and its downstream targets, and the effect was independent of cytogenetic or molecular-genetic abnormalities. The MSCs also supported the long-term proliferation of the AML cells. A subset of the patients also showed an altered cytokine network with supra-additive levels for several cytokines. The presence of cytokine-neutralizing antibodies or receptor inhibitors demonstrated that AML cells derived from different patients were heterogeneous with regard to effects of various cytokines on AML cell proliferation or regulation of apoptosis. We conclude that even though the effects of single cytokines derived from bone marrow MSCs on human AML cells differ among patients, the final cytokine-mediated effects of the MSCs during coculture is growth enhancement and inhibition of apoptosis.Acute myeloid leukemia (AML) is a bone marrow malignancy, and various bone marrow stromal cells seem to support leukemogenesis, including osteoblasts and endothelial cells. We have investigated how normal bone marrow mesenchymal stem cells (MSCs) support the  FMS-related tyrosine kinase 3 internal tandem repeats  and mutations in nucleophosmin  are the most prominent (Acute myeloid leukemia (AML) is an aggressive malignancy that mainly affects the elderly; the disease is characterized by bone marrow infiltration of immature leukemic blasts , and it rominent .Mesenchymal stem or stromal cells (MSCs) are capable of self-renewal and differentiation into osteoblasts, chondrocytes, or adipocytes , the mosIn this study, we have therefore characterized the cytokine-mediated crosstalk between AML cells and normal bone marrow MSCs. Due to the heterogeneity of the disease, we have investigated a large group of unselected patients. Our studies suggest that MSC-derived cytokines have antiapoptotic effects and support AML cell proliferation for most patients, but the molecular mechanisms causing these effects differ among patients.de novo AML  and samples collected after written informed consent. AML blasts from peripheral blood were derived from 51 consecutive/unselected patients admitted to our department for AML therapy . A majority of 36 patients had Acute myeloid leukemia cells were isolated from peripheral blood of patients with levels of circulating blasts by density gradient separation . The cells were stored in liquid nitrogen until use .The following neutralizing antibodies and receptor antagonists  were used at the following concentrations: (i) 100 ng/mL of affinity purified polyclonal antibodies (goat IgG) against the vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), basic fibroblastic growth factor (bFGF), and IL-6. At this concentration, the antibodies will block ≥50% of receptor binding to VEGF (80 ng/mL), HGF (8 ng/mL), bFGF (0.1 ng/mL), and IL-6 (5 ng/mL), thus concentrations higher than the levels in our MSC cultures ; (ii) 3 l-glutamine; cells were trypsinized and used for the experiments in passages three or four. Our previous studies of global gene expression profiles of in vitro expanded MSCs showed no evidence for differentiation of such expanded MSCs  were purchased from Lonza . According to the distributor’s information, the cells were obtained in passage two and showed the ability to differentiate into the mesenchymal lineages. All cells tested negative for mycoplasma, bacteria, and fungi. The MSCs were expanded in complete mesenchymal stem cell growth medium , which contains 10% fetal bovine serum (FBS) and 4 mM ded MSCs .4 cells/well) were seeded in the lower chamber in complete MSCGM™ medium (1 mL/well). After 3 days of culture  the medium was exchanged and subsequently 1 × 106 AML cells were added in 0.5 mL medium to the upper chamber separated from the MSCs by a semipermeable membrane (0.4 µm pore size). The cells were cultured for 3 days, in which the MSCs did not reach confluence.Cultures were prepared in transwell plates . The MSCs  was added to the upper wells and the cells were incubated for another day. The nuclear 3H-thymidine incorporation was then measured by liquid scintillation counting as described in detail previously  and fluorescein isothiocyanate-conjugated Annexin V antibodies  as described in detail previously . Briefly6 primary AML cells were added to each well. AML cell viability was analyzed 20 h later before the MSCs reached confluence.We also cultured primary AML cells from 10 patients in direct contact with MSCs in 6-well tissue culture plates; 20,000 MSCs were precultured for 3 days before 1 × 10Supernatants were harvested from the lower chambers and stored at −80°C. The following cytokine levels were determined by Luminex analyses or enzyme-linked immunosorbent assay (ELISA) (R&D Systems): (i) the chemokines CCL2-5 and CXCL1/5/8/10/11, (ii) the interleukins IL-1β/1RA/6/10/33, (iii) the matrix metalloproteinases MMP-1 and -2, (iv) tumor necrosis factor-α (TNFα), and (v) granulocyte colony-stimulating factor (G-CSF), granulocyte macrophage colony-stimulating factor (GM-CSF), HGF, bFGF, VEGF, and soluble angiopoietin 1 (Ang-1) receptor Tie-2.in vitro culture.Cocultures of MSCs with primary AML cells were prepared as described above. The blasts were transferred weekly to new upper chambers of transwell cocultures where fresh MSCs had been seeded in the lower chambers 3 days in advance. The medium was exchanged twice a week throughout the 3 weeks of culture period in transwell cocultures. After 3 weeks, the AML cells were harvested and subsequently seeded in the colony-formation assay to estimate the number of colony-forming units (CFU). We used two different colony-formation assays; MethoCult™ H4434 Classic and H4534 Classic without erythropoietin medium . The cells were seeded in duplicate in 24-well plates with 0.5 mL medium/well. Colonies containing more than 30 cells were scored using an inverted microscope after 14 days of ® 647 Mouse anti-PDPK1 (pS241), Alexa Fluor® 647 Mouse anti-PKCα (pT497), Alexa Fluor® 647 Mouse anti-Akt (pS473), phycoerythrin (PE) Mouse Anti-Akt (pT308), PE Mouse Anti-mTOR (pS2448), Alexa Fluor® 647 Mouse anti-4E-BP1 (pT36/pT45), Alexa Fluor® 647 Mouse anti-elF4E (pS209), Alexa Fluor® 647 Mouse anti-S6 (pS244), PE Mouse anti-S6 (pS240), V450 Mouse anti-S6 (pS235/pS236) Alexa Fluor® 647 Mouse anti-PKCα (pT497) , and mTOR (7C10) Rabbit mAb (Alexa Fluor® 647 Conjugate) .The following antibodies were used for analysis of protein phosphorylation: Alexa FluorMSC24539 was cultured as described above and the conditioned medium harvested before the cells reached confluence.5 cells/sample) before staining. All staining panels included the same live dead discriminator FITC Mouse anti-Cleaved PARP  and Alexa Fluor® 647 Mouse anti-Cleaved PARP , as well as one blank sample. Flow cytometric analysis of protein phosphorylation was performed as described in detail previously  for 30 min before being directly fixed in 1.5% paraformaldehyde and thereafter permeabilized with methanol. The cells were then rehydrated by adding 2 mL phosphate-buffered saline (PBS), gently resuspended, and then centrifuged. The cell pellet was washed twice with 2 mL PBS and resuspended in 150 µL PBS + 0.1% BSA (Sigma-Aldrich). The washed cells were blocked with immunoglobulin  and 1% BSA, and split evenly into separate tubes .The percentage of phosphorylated compared to total H2AX was detected using a cell-based ELISA kit . The assay was performed according to the manufacturer’s instruction for suspension cells; 5 × 10U-tests were used to compare different groups. The χ2 test was used to analyze categorized data and the Kendall’s tau-b test for correlation analyses. P-values <0.05 were regarded as statistically significant. Supervised hierarchical clustering was performed using the J-Express 2012 software . The data were log(10) transformed prior to clustering.The statistical analyses were performed with the IBM Statistical Package for the Social Sciences (SPSS) version 23 . The Wilcoxon signed-rank test was used to compare paired samples, whereas Kruskal–Wallis and Mann–Whitney p ≤ 0.001, Wilcoxon’s signed-rank test), even though the AML cells showed undetectable proliferation for two patients, both when cultured in medium alone and in the presence of all three MSC donor cells. The other 16 patients showed increased proliferation for at least one of the three MSCs. The median increase in 3H-thymidine incorporation corresponded to approximately fivefold increase for each of the three donors.Primary AML cells from 18 consecutive patients were cocultured with normal MSCs from the three different donors  in transwell plates. AML cell proliferation was determined after 3 days of culture. AML cell proliferation was significantly increased by coculture with MSCs compared with the corresponding control cultures with AML cells alone . MSCs were pre-cultured for 3 days before AML cells were added and leukemic cell viability assayed after 3 days of coculture by flow cytometric analysis. The effect of all three human bone marrow MSCs was investigated . The results are summarized in Table S1 in Supplementary Material and presented in detail in Figure in vitro coculture (variation range 0.4–84.7% viability). When comparing the overall results, the fraction of viable cells was significantly increased  after coculture in the presence of all three MSCs compared with the corresponding MSC-free controls, and the median fraction of viable cells was approximately doubled for each of the three MSC donors.Primary AML cells show spontaneous or stress-induced apoptosis during  culture . We prepp < 0.00001, Wilcoxon’s signed-rank test). The growth enhancement showed no significant association with AML cell differentiation , karyotype , or Flt3/NPM1 mutations (data not shown). Finally, cells from 11 patients showed detectable proliferation neither in medium alone nor in cocultures with MSC24539. For five additional patients, the presence of MSCs showed no or minimal growth enhancement, corresponding to less than 20% alteration and an absolute change of less than 2,000 cpm in the presence of MSCs. These 16 patients differed significantly from the other 35 patients with regard to cell differentiation as CD34− cells showed weaker proliferation in the presence of MSCs than CD34+ cells  (data not shown).Because the proliferation and viability results were consistent among MSC donors, the comparison of MSC effects for a larger group of 51 consecutive patients was performed only for MSC24539. MSC-induced enhancement of AML cell proliferation in transwell cocultures was highly significant also when analyzing the overall results for this larger group of patients . This antiapoptotic effect showed no significant association with AML cell differentiation, karyotype, or Flt3/NPM1 mutations (data not shown). However, proliferation <1,000 cpm in the presence of MSCs seemed to be associated with weaker antiapoptotic effects of MSCs; i.e., 8/21 patients with viability increase <10% points and 0/20 patients with an increase >20% points showed undetectable proliferation in cocultures .The same 51 patients were also tested in transwell cocultures with regard to a MSC-associated antiapoptotic effect; viability analyzed by the Annexin V-PI assay was then compared for AML cells cultured in medium alone or cocultured with MSC24539 for 3 days. The presence of MSCs increased AML cell viability significantly also when testing this extended patient group  after MSC coculture was seen also for these 16 patients with non-proliferating AML cells, and a >10% point increase was seen for eight of these patients (see above). Thus, the MSC effect on AML cell viability is not only caused by the increased proliferation but also by additional effects possibly affecting the balance between pro- and antiapoptotic signaling.Because primary AML cells derived from 16 patients showed undetectable proliferation during coculture with MSCs, we also analyzed separately the effect of MSCs on AML cell viability for these patients. However, when analyzing the overall results, a highly significant increase in AML cells viability . These results show that there is heterogeneity among patients with regard to the proapoptotic effect of cytarabine, and decreased AML cell viability was seen only for a subset of patients when testing cytarabine at concentrations 12.5–500 nM and with a large overlap between the effects of 50 and 500 nM. At the same time, the drug had a clear antiproliferative effect even at 50 nM and at higher concentrations no detectable cytokine-dependent proliferation could be detected (data not shown). Finally, the concentrations 50–500 nM correspond to levels reached in vivo and 50 nM is close to the steady-state concentrations seen during conventional AML induction treatment and higher than the levels reached during low-dose subcutaneous cytarabine treatment  pilot experiments, which showed that cytarabine at 50 nM decreased AML cell viability for a marked subset of patients when using our reatment ; both threatment , 28.p = 0.045). Finally, increased AML cell viability was seen for all 10 patients in MSC cocultures, both in presence and absence of 50 nM cytarabine.Primary AML cells were cultured in transwell cultures for 20 h either alone or in coculture with MSC24539, and cultures with/without MSCs were prepared with and without 50 nM cytarabine , i.e., H2AX phosphorylation seems to be a marker of spontaneous in vitro apoptosis during culture of AML cells alone. However, significantly increased H2AX phosphorylation was observed after coculture with MSC-conditioned medium compared with leukemic cells cultured in medium alone  when comparing the overall results for CCL3, CCL4 (p = 0.043), CXCL1, CXCL5, CXCL8, CXCL10, CXCL11 (p = 0.014), IL-1β, IL-6, IL-10, TNFα (p = 0.037), bFGF, G-CSF, GM-CSF, MMP-1, and MMP-2. Thus, even though individual differences among patients are maintained during coculture, several mediators show increased levels in cocultures and supra-additive levels are common.We determined the supernatant levels for 23 soluble mediators in transwell cocultures of MSC24539 and AML cells derived from all 51 patients Table . First, 2 likelihood analyses) characterized by a significantly higher number of patients  showing detectable proliferation when cultured in the FBS-containing MSC medium alone without MSCs and also a higher number of patients  with high AML cell proliferation exceeding >20,000 cpm in the cocultures. The supra-additive subset also showed a higher fraction of patients with cell viability >50% after coculture with MSCs . Finally, monocytic differentiation (FAB-M4/M5) was also more common among these patients . However, even though there was an association between supra-additive cytokine levels and high AML cell viability/proliferation in the cocultures, these supra-additive levels do not simply reflect increased proliferation/viability because supra-additive levels would then be expected for all the mediators and not only for a subset as we observed.To further investigate the patient heterogeneity, we performed a cluster analysis Figure . For eacNormal karyotype. The patients were heterogeneous with regard to karyotype. The only subset being large enough for statistical analysis was the 15 patients with normal karyotype, and for this subset, anti-IL-6 had a significant antiproliferative effect  of borderline significance.Patients with and without NPM1 mutations. NPM1 insertions were detected for 13 of the 32 patients, and all of them had normal karyotype. As would then be expected, anti-IL-6 had an antiproliferative effect for NPM1-mutated patients, but the statistical comparison reached only borderline significance . The same was true for anti-bFGF, which had an antiproliferative effect for patients with NPM1-wt . Because several patients showed a relatively strong effect of chemokine-targeting pharmacological intervention we used statistical analysis based on categorized data when comparing NPM1-mutated and NPM1-wt patients. A significant alteration of AML cell proliferation was then defined as a difference having an absolute value of >2,000 cpm and in addition being >20% of the corresponding control. NPM1-wt was then associated with increased and NPM1 mutation with decreased proliferation for all four chemokine receptor antagonists: AMD3100 , J113863 , BX513 , and BX471 . Thus, growth-modulating effects of several chemokines differed between NPM1-wt and NPM1-mutated patients.FAB classification, CD34 expression, Flt3 mutations. The effects of cytokine targeting showed significant associations neither with differentiation nor with Flt3 mutations (data not shown).To further study the cytokine-mediated crosstalk between MSCs and AML cells, we investigated the effects of cytokine-neutralizing antibodies and receptor antagonists on AML cell proliferation and viability in transwell cocultures with MSC24539. We then tested inhibitors of cytokines released by MSCs and being able to modulate AML cell proliferation –35, inclNPM1 mutations. Based on this analysis, our patients could be classified into three different groups ; these three groups did not significantly differ with regard to constitutive release of supra-additive coculture levels for IL-6, bFGF, or chemokines. Thus, these differences among patients are not caused by differences in cytokine release during coculture. Based on our overall results, we conclude that AML patients are heterogeneous also with regard to the effect  of single cytokines on AML cell proliferation in the presence of normal bone marrow MSCs, but despite this heterogeneity the final/overall cytokine-mediated MSC effect is increased AML cell proliferation for the majority of patients.We also did a clustering analysis of the overall results Figure  showing p < 0.0001), the CCR1/CCR3 inhibitor J113863 (p = 0.009), and the CCR1 antagonist BX417 (p = 0.015). Statistical analyses based on categorized data  showed associations between the CXCR4 mediated antiproliferative effect and monocytic AML cell differentiation (p = 0.009) and favorable karyotype (p = 0.025). These observations suggest that CCR1-, CXCR4-, and possibly CCR3-mediated signaling is important for the regulation of MSC proliferation in cocultures, and these results further illustrate that this bidirectional cytokine-mediated crosstalk between MSCs and AML cells affect not only the AML cells but also the MSCs.We further investigated the effects on MSC24539 proliferation of the same antibodies and antagonists as described above during coculture with AML cells from the same 32 patients. Anti-bFGF, anti-IL-6, and the CCR1 antagonist BX513 had only minimal effects (data not shown). In contrast, a significant antiproliferative effect was seen for the CXCR4 inhibitor AMD3100  cytarabine levels reached during in vivo chemotherapy (50 nM), and in addition, a concentration corresponding to the steady-state levels during conventional cytarabine treatment with 100–200 mg/m2. A reduction of cell viability corresponding to more than 5% was seen only for approximately half of the patients when testing 50 nM cytarabine in the initial dose-response experiments in transwell cocultures, and in direct-contact cocultures. Both our experiments with transwell cocultures and cytarabine but especially the direct-contact experiments with 500 nM cytarabine showed that MSCs can counteract the proapoptotic effect of cytarabine, and at least for certain patients the cytokine-mediated crosstalk contributes to this effect. However, further studies are required to characterize and explain this patient heterogeneity and to clarify whether this variation is seen also for other chemotherapeutic agents.Primary AML cells show spontaneous or stress-induced apoptosis during The local cytokine network was also altered during AML-MSC coculture; the levels of three mediators were relatively low , whereas especially CXCL1/5, IL-1β/10, TNFα, MMP-1, G-CSF, and GM-CSF showed supra-additive effects in cocultures. A strong supra-additive effect for a subset of the mediators was especially seen for a patient subset also characterized by monocytic differentiation, high cell viability in coculture, and strong AML cell proliferation both when cultured in medium alone and in the cocultures with MSCs. The constitutive mediator release by AML cells cultured alone also differed considerably among patients, and despite the supra-additive effect for several cytokines, there was still a significant correlation for all but one mediator between the levels for AML cells cultured alone and in cocultures. Thus, individual differences between patients with regard to constitutive cytokine release by the AML cells are also maintained during coculture with MSCs.We characterized the cytokine network in MSC/AML cell transwell cocultures in a previous study that also included a characterization of the effect of the AML-MSC crosstalk on the MSCs . The stuWe investigated possible molecular mechanisms behind the growth-enhancing and antiapoptotic effect of MSCs on primary human AML cells. These experiments included leukemic cells from 10 patients. The PI3K–Akt–mTOR pathway is often constitutively activated in AML cells and is important for cell survival, proliferation and metabolism , 47. We p = 0.005). However, this increase of H2AX phosphorylation combined with increased cell viability suggests that the increased H2AX phosphorylation is not an effect of increased apoptosis. In our opinion, the most likely explanation is a reversal of the AML-associated decrease in the DNA damage response; this may be caused by IL-6 that is present at high levels in MSC-conditioned medium  angiogenic growth factors like VEGF , 54, bFGNPM1-wt cells and this is consistent with previous observations of AML cells cultured alone  agents. Both AML cells and MSCs show constitutive release of several ligands for these receptors, suggesting that autocrine or paracrine loops involving these receptors are important for the regulation of MSC proliferation . This hyin vitro culture and most of these patients show only low release (Systemic plasma levels of both IL-6, bFGF, and several chemokines have been investigated in human AML. IL-6 levels are increased in patients with untreated AML and high levels seem to be associated with decreased survival . In cont release , increasOur present study shows that even though AML is a heterogeneous disease and the response of primary AML cells to the various MSC-derived cytokines during MSC-AML cell coculture differs among patients, the final effect of MSC-derived cytokines on primary AML cells is increased proliferation and viability. Our overall results suggest that therapeutic targeting of the cytokine-mediated AML-supporting effects probably as MSC-directed strategies which inhibit the release of several cytokines, or alternatively receptor blocking could be tried in selected patients.ØB designed the study and wrote the paper. IN performed the phosphoflow experiments and analyzed the results. AB performed the other experiments, analyzed the results, and wrote the paper.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.


RyR2‐P2328S hearts to reduced action potential (AP) conduction velocities (CV), reflecting acute functional inhibition and/or reduced expression of sodium channels. We explored for acute effects of direct exchange protein directly activated by cAMP (Epac)‐mediated ryanodine receptor‐2 (RyR2) activation on arrhythmic substrate and CV. Monophasic action potential (MAP) recordings demonstrated that initial steady (8 Hz) extrinsic pacing elicited ventricular tachycardia (VT) in 0 of 18 Langendorff‐perfused wild‐type mouse ventricles before pharmacological intervention. The Epac activator 8‐CPT (8‐(4‐chlorophenylthio)‐2′‐O‐methyladenosine‐3′,5′‐cyclic monophosphate) (VT in 1 of 7 hearts), and the RyR2 blocker dantrolene, either alone (0 of 11) or with 8‐CPT (0 of 9) did not then increase VT incidence (P>.05). Both progressively increased pacing rates and programmed extrasystolic (S2) stimuli similarly produced no VT in untreated hearts (n=20 and n=9 respectively). 8‐CPT challenge then increased VT incidences . However, dantrolene, whether alone (0 of 10 and 1 of 13) or combined with 8‐CPT (0 of 10 and 0 of 13) did not increase VT incidence relative to those observed in untreated hearts (P>.05). 8‐CPT but not dantrolene, whether alone or combined with 8‐CPT, correspondingly increased AP latencies (1.14±0.04 (n=7), 1.04±0.03 (n=10), 1.09±0.05 (n=8) relative to respective control values). In contrast, AP durations, conditions for 2:1 conduction block and ventricular effective refractory periods remained unchanged throughout. We thus demonstrate for the first time that acute RyR2 activation reversibly induces VT in specific association with reduced CV.Recent papers have attributed arrhythmic substrate in murine  This was attributed to acute effects of altered intracellular Ca2+ on Na+ channel (Nav1.5) functionv1.5 membrane expression.Abnormal Ca2+ homeostasis could influence CV and introduce arrhythmic substrate in wild‐type hearts expressing normal RyR2, as opposed to RyR2‐P2328S hearts. They complement recent studies that had modified Ca2+ homeostasis through exchange protein directly activated by cAMP (Epac) activation of RyR2‐Ca2+ release channels2+ spark frequencies in adult rat cardiac myocytes,2+‐dependent Ca2+ release after isoproterenol treatment,2+ release in mouse ventricular cardiomyocytes.The present experiments assessed whether acute manipulations of CaO‐methyladenosine‐3′,5′‐cyclic monophosphate),2+ release, decrease the frequency and duration of aberrant Ca2+ sparks in cultures of myocytes modelling CPVT,2+‐induced‐Ca2+‐release from the sarcoplasmic reticulum in myocytes from failing but not control rabbit hearts.2+‐mediated ventricular arrhythmias.The present experiments therefore explored for arrhythmic susceptibility in relationship to alterations in conduction latency as a measure of CV, and to VERP and APD. It did so under conditions both of Epac activation by 8‐CPT (8‐pCPT‐2′‐O‐Me‐cAMP: 8‐(4‐chlorophenylthio)‐2′‐2in vivo patterns of electrical activity in the heart. Recordings during the initial regular 8 Hz pacing stimuli, simulating in vivo steady state resting heart rates, corresponding to a basic cycle length (BCL) of 125 ms ; Figure 90), and permitted quantifications of the frequency of spontaneous VT. No arrhythmia was observed with this initial pacing in control experiments (0 of 18). Challenge by 8‐CPT, dantrolene or dantrolene in combination with 8‐CPT all did not increase the incidence of arrhythmia .Results were first compared between hearts studied under control conditions  increases in the incidence of arrhythmia with challenge by 8‐CPT. Although dantrolene administered alone had no significant impact on incidence of VT, when applied with 8‐CPT, dantrolene reversed the arrhythmic effects of 8‐CPT  (Table Table 5) Table .Figure P<.05) but not by dantrolene alone (P>.05) significantly increased arrhythmic incidence but that dantrolene reversed these arrhythmic effects of 8‐CPT   Table .90 , VERPs , and BCL values at the onset of 2:1 block during incremental pacing (41.00±4.54 ms (n=4)). Values of APD90 and VERPs had also been measured by the earlier studyDeterminations of control AP parameters prior to 8‐CPT challenge gave control values of latency , APD90, BCL at onset of 2:1 block, and VERP were statistically assessed for significant differences. It was seen that 8‐CPT significantly increased AP conduction latency . However, it had no effect on the AP recovery parameters of APD90, BCL at onset of 2:1 block, and VERP . In contrast, dantrolene alone did not affect either the AP activation or recovery parameters . However, results obtained following addition of 8‐CPT combined with dantrolene demonstrated that dantrolene reversed the effect of 8‐CPT on latency. It then yielded latencies indistinguishable from the control values  and preserved normal AP recovery parameters .Table + channel protein expression reported with genetic modifications of RyR2.Together these findings suggest actions of Epac‐mediated RyR2 activation by 8‐CPT both increases the incidence of arrhythmia following extrasystolic, and incremental pacing and reduces CV without affecting AP recovery properties. The RyR2 blocker dantrolene did not produce such changes. However, it reversed both the pro‐arrhythmic actions of 8‐CPT, and its actions on CV. The findings are thus consistent with acute pro‐arrhythmic actions of RyR2 receptor modulation on both CV and arrhythmia, adding to previous studies implicating chronic alterations in Na32+ release channel on arrhythmogenesis and its relationship to alterations in AP conduction velocity (CV) in isolated perfused wild‐type (WT) murine hearts. Previous studies had reported that: (i) The RyR2‐P2328S modification, associated with human catecholaminergic polymorphic ventricular tachycardia (CPVT) resulted in increased arrhythmic tendency and increased RyR2‐mediated Ca2+ release in murine ventricles.2+ homeostasis also appeared to modify the Na+ current with consequences for CV. The latter could involve (a) a long term downregulation of cardiac Na+ channel (Nav1.5) expression that was observed in both atrialRyR2‐P228S myocytes.v1.5, Nav1.5 mRNA and total Nav1.5 protein following challenge by the Ca2+ channel blocker verapamil and the Ca2+ ionophore calcimycin respectively.2+ resulting from enhanced RyR2‐mediated Ca2+ release might modify Na+ channel function. Nav1.5 thus contains two potentially directly Ca2+‐sensitive regions: (i) a Ca2+‐binding EF‐hand motifv1.5 C‐terminal region.2+‐mediated modulation of Na+ channel function. Thus, acute increases and decreases in Na+ current densities followed increases or decreases in intracellular [Ca2+]. This was demonstrated in patch‐clamped cultured neonatal rat myocytes using buffering by BAPTA ethane‐N,N,N′,N′‐tetraacetic acid tetrakis‐acetoxymethyl ester) and caffeine administration respectively.2+ may indirectly affect Nav1.5 through the action of calmodulin kinase II (CamKII) on multiple phosphorylation sites on the channel.This study explored the effects of acute interventions involving the ryanodine receptor (RyR2)‐Ca2+ channel activation through the phosphokinase A (PKA)‐independent EpacO‐methyladenosine‐3′,5′‐cyclic monophosphate) as Epac agonist. Despite being a cAMP (3′5‐cyclic adenosine monophosphate) analogue, the 2‐hydroxyl group substitution on its cAMP ribose moiety disrupts its interaction with PKA whilst permitting binding to Epac. This gives 8‐CPT a 300‐fold selectivity for Epac relative to PKA at the low, 1 μmol/L, concentrations employed here.The present experiments explored the effects of acute RyR2‐Ca2+ release2+ waves resulting from Ca2+‐induced Ca2+ release.2+ homestasis following its interaction with and opening of the RyR2‐Ca2+‐release channel.Epac is implicated in adrenergically‐mediated cardiac arrhythmogenesis.Secondly, the arrhythmic effects of 8‐CPT challenge were accompanied by a slowed CV as reflected in changes in AP latencies. However, it did not affect the AP recovery properties of APD and VERP.2+ leak, decreasing the incidence and duration of Ca2+ sparks in cultures of cardiomyocytes modelling both CPVT2+ release particularly under conditions of increased channel open probability.Thirdly, pharmacological manipulations using the RyR2 antagonist dantrolene corroborated these findings. Multiple studies have demonstrated that dantrolene reduces RyR2‐mediated Ca2+ homeostasis accompanying interventions increasing RyR2‐mediated sarcoplasmic reticulum Ca2+ release. They complement previous observations of a longer term reduction in Nav1.5 expression following chronic alterations in Ca2+ homeostasis associated with the RyR2‐P2328S mutation.Together these findings suggest that reversible arrhythmic effects related to slowed AP propagation but unchanged AP recovery can follow acute modifications of Ca43 [25 mmol/L], KCl [4 mmol/L], MgCl2 [1 mmol/L], KH2PO4 [1.2 mmol/L], CaCl2 [1.8 mmol/L], glucose [10 mmol/L] and sodium pyruvate [1.8 mmol/L]); the solution was bubbled through with 95% O2 and 5% CO2 .All animals used for experiments were of the WT sv129 background. A total of 20 male and female, between the ages of 5 and 7 months, were used for the experiments. Mice were kept in animal house facilities at 21°C and exposed to 12 hour dark : light cycle each day, with free access to sterile rodent chow, drinking water, and bedding at all times. All animals were killed by cervical dislocation by Home Office‐licensed personnel. Procedures used conformed to Schedule 1 of the UK Animals (Scientific Procedures) Act (1986), and to Institutional Ethics Committee Guidelines. Procuration of animals, their husbandry and all experiments also conformed to the ‘European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes’ . The hearts were isolated from the animal by bilateral sternotomy and placed upon ice‐cold Krebs‐Henseleit (KH) solution  was used to fix the cannula into place. Thereafter, the heart was quickly placed within the perfusion system. KH solution used to perfuse the heart was first passed through a 200 μm followed by a 5 μm filter, and warmed to 37°C by a water jacket and circulator. A peristaltic pump  was used to deliver the perfusion fluid at a constant rate of 2.3 mL/min. After commencement of continuous perfusion, hearts were investigated for spontaneous contractions and confirmatory signs of viability.Perfused hearts were extrinsically paced using a bipolar Ag/AgCl stimulating electrode placed on the right ventricular epicardium. The stimulus voltage on the constant voltage stimulator  was set to deliver stimuli at double the threshold voltage. The isolated Langendorff‐perfused WT murine hearts were paced at 8 Hz for at least 15 minutes before beginning MAP recordings from the left ventricular epicardium, midway between ventricular apex and base, using a MAP electrode . The relative dimensions of the MAP electrodes and of the murine ventricles permitted only a single channel of MAP recording at any given time The recorded data was amplified with a NL100AK head stage pre‐amplifier, followed by a NL104A amplifier (Digitimer). The signal was then band pass filtered  and digitised . All MAP traces were recorded electronically, and the waveforms analyzed using Spike2 software (Cambridge Electronic Design).The control procedure used KH solution alone without any added pharmacological agents.O‐methyladenosine‐3′,5′‐cyclic monophosphate; BIOLOG Life Science Institute, Bremen, Germany) was made into a stock of 1 mmol/L with dimethyl sulfoxide . When needed for use, the stock was then diluted in KH solution to make up a final concentration of 1 μmol/L 8‐CPT, in accord with that previously used in the earlier study.8‐CPT (8‐pCPT‐2′‐O‐Me‐cAMP: 8‐(4‐chlorophenylthio)‐2′‐Dantrolene sodium  was dissolved in DMSO to make a 5 mmol/L stock (stored at 4°C). The final concentration of 5 μmol/L of dantrolene used in experiments were obtained by dilution of the stock with KH solution. Following control recordings, the pharmacological manoeuvres involved applications of either 8‐CPT alone (1 μmol/L) followed by treatment with both 8‐CPT and dantrolene (1 μmol/L and 5 μmol/L respectively), or dantrolene alone (5 μmol/L) followed by treatment with both drugs. During these procedures, hearts were paced at 8 Hz for at least 3 minutes after the estimated time of drug arrival at the heart before recordings were made.Experiments were carried out under the following conditions, defined by the perfusate used for the heart.n. For categorical data, Fisher's exact test was used to test for statistical significance between two data sets, each with two possible outcomes, the presence or absence of VT. Changes in the continuous parameters of AP latency, AP duration at 90% recovery (APD90), basic cycle length (BCL) at the onset of 2:1 block, and VERPs were tested using Student's paired t‐test. Statistical significance obtained from the t‐test was defined as P<.05.All data are expressed as mean±SEM; where time is the variable, data is presented in milliseconds (ms). The number of hearts from which data is derived from for each condition is designated None declared.


This review focuses on the role of this response in cancer. We discuss evidence that during transformation, the UPRmt may play an essential role in the maintenance of the integrity of the mitochondria in the face of increased oxidative stress. However, the role of the UPRmt in other diseases is also emerging and is therefore also briefly discussed.Upon accumulation of misfolded proteins in the mitochondria, the mitochondrial unfolded protein response (UPR The proper folding of proteins is fundamental to cellular life. Perturbations to this process promote the formation of protein aggregates and underlie a number of human pathologies.ER, which communicates the proteotoxic stress to the nucleus  to be discovered also implicates CHOP , which cause oxidation of proteins and their misfolding.The elevation in ROS in cancer cells combined with the Warburg effect, which refers to the elevation in glycolysis for the production of ATP, have led to the misconception that cancer cells have defective mitochondria. In contrast, it is now recognized that most cancer cells continue to require oxidative phosphorylation. This observation has led to the idea of the reverse-Warburg effect, the recognition of oxidative tumors, and metabolic flexibility [for a recent review ]. ROS coOur group has previously sought to understand the mechanisms by which misfolded proteins in the IMS of the mitochondria are managed and resolved in the context of cancer cells. Using a mutant form of endonuclease G (EndoG), which misfolds and forms protein aggregates in the IMS, we first reported that the proteasome in the cytosol and the protease OMI in the IMS cooperate to limit the accumulation of misfolded proteins in the IMS . We propmt was originally identified in mammalian cells using the overexpression of mitochondrial matrix localized misfolded OTCΔ  positive breast cancer cells, accumulation of misfolded proteins in the IMS did not activate the CHOP axis. While confirming in this model system that expression of OTCΔ in the matrix activates CHOP, we found that IMS stress activates AKT, which then phosphorylates the ERα to promote its transcriptional activity in a ligand-independent fashion     . We foun (SIRT3) . We repo (SIRT3) . In theslifespan .mt induces the elimination of irreversibly damaged mitochondria through the process of mitophagy (mt.In addition to the antioxidant machinery, we reported that the SIRT3 axis of the UPRitophagy . As for mt has been found to be activated by other, more indirect, stressors. Notably, inhibition of the ETC, inhibition of mitochondrial translation, or inhibition of matrix chaperones can also activate the UPRmt. The use of these alternative stimuli has led to the identification of additional axes. First, in hematopoietic stem cells, SIRT7 has been shown to negatively regulate NRF1 activity and induce CHOP target genes—Hsp60, Hsp10, and ClpP (Figure While the identification of the CHOP axis by the Hoogenraad group and the ERα and SIRT3 axes by our group was obtained causing accumulation of misfolded proteins directly in the matrix or the IMS, the UPRP Figure . The tramt using EndoG and OTCΔ overexpression as tools to induce stress in the IMS and matrix, respectively, we next aimed at validating these axes under more physiological conditions.Following the identification of ERα and SIRT3 axes of the UPRmt. Further, in this model, the absence of the SOD1G93A cytosolic aggregates, which we postulate may mitigate the ability of the UPRmt to protect the integrity of the mitochondria, may explain the longer survival in the IMS targeted model of SOD1G93A.The field of neurodegeneration has long appreciated the importance of misfolded proteins, as it has been identified as a common mechanism in a number of human neurodegenerative disorders, such as Alzheimer’s disease, Huntington’s disease, Parkinson’s disease, and ALS , 45. Whimt in both the untargeted and IMS-targeted mouse models of SOD1G93A familial ALS (mt in a disease relevant model in vivo but it also raises the distinct possibility that sex differences observed in several neurodegenerative diseases may be related to the ERα status of the affected tissue. We will actively pursue this possibility in the future.To test this hypothesis, we recently validated the activation of the ERα axis of the UPRlial ALS . Intereslial ALS . Furtherlial ALS , 49. Thimt, in reality the accumulation of misfolded proteins is likely to be present in both the matrix and the IMS as ROS is produced on both sides of the inner-membrane simultaneously. Further, since our hypothesis is that activation of the UPRmt will increase mitochondrial fitness and adaptation to stress, we reasoned that the activation of UPRmt could be linked to a more aggressive cancer phenotype, such as increased invasion capacity. We therefore investigated whether the SIRT3 and CHOP axes of the UPRmt may be linked to the metastatic potential of breast cancer cells.While the use of EndoG and OTCΔ was instrumental in the discovery of several players of the UPRmt could distinguish metastatic from non-metastatic cells in a panel of breast cancer cell lines (mt, increased invasion. When SOD2 was inhibited by shRNA, the invasion of normally invasive cells was significantly reduced. Additionally, through the analysis of a collection of cybrids—cells lines with a common nuclear genome, but different mitochondrial genomes—we demonstrated that mitochondrial disease patient-derived mtDNA mutations influence the levels of mitochondrial stress and subsequently the levels of activation of the SIRT3 axis of the UPRmt. Activation of the SIRT3 axis of the UPRmt correlated with invasion capacity of the cybrids, further emphasizing the link between this pathway and metastasis. Importantly, activation of the SIRT3 axis of the UPRmt was seen in primary breast cancer patients and high expression, using SOD2 as a marker, was significantly associated with worse disease-free survival (mt, in metastatic lesions when compared with primary lesions. These patient data strongly support our hypothesis that activation of the SIRT3 axis of the UPRmt increases the invasiveness and metastatic potential of cancer cells. In contrast the CHOP axis, monitored using hsp60 as a marker, did not distinguish the metastatic form the non-metastatic cells. Rather, hsp60 was found to be elevated very early after oncogene induction (mt engaged in mounting a protective response to mitochondrial stress may increase over disease progression.We found that markers of activation of the SIRT3 axis of the UPRll lines . When ovsurvival . Additionduction . This fimt is a complex transcriptional pathway that expands well beyond the activation of proteases and chaperones of the matrix. We propose that the IMS plays a central role in initiating this pathway. One critical question that remains to be answered is what are the sensors/transducers of the UPRmt. Proteins able to translocate from the mitochondria to the nucleus, such as ATF5 (mt or that signaling cascades such as the one recently described, initiated by Lyn kinase in the IMS by ROS (mt. Additional in vivo validation of the UPRmt in mammalian systems in the context of cancer and other pathologies is an important future direction for the new pathway. Further effort should be made to mechanistically integrate the currently known axes of the UPRmt as there is undoubtedly complex orchestration of multiple responses activated in response to mitochondrial stress. Notably, the name mitochondrial stress response has been recently proposed (Psedumonas aeruginosa, which causes mitochondrial dysfunction and leads to UPRmt activation in C. elegans and the transcriptional upregulation of innate immunity genes (mt to integrated mitochondrial stress response to more fully capture the numerous retrograde signaling cascades that are activated in response to changes in mitochondrial homeostasis, including proteostasis.In light of the recent findings discussed in this review, it becomes increasingly clear that the UPR as ATF5 , are pri as ATF5 –55, whic as ATF5 , may be S by ROS , may alsproposed . Howeverty genes . TherefoTK has contributed to the writing of this review and made the figure. GM has contributed to the writing of this review. DG is the corresponding author and has contributed to the writing and finalized this review.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.


An individual with CPVT harboring a novel mutation in the type 2 cardiac ryanodine receptor (RyR2) was identified whose persistent ventricular arrhythmias during β-blockade with nadolol were abolished during flecainide treatment. iPSC-CMs generated from this patient and two control individuals expressed comparable levels of excitation-contraction genes, but assessment of the sarcoplasmic reticulum Ca2+ leak and load relationship revealed intracellular Ca2+ homeostasis was altered in the CPVT iPSC-CMs. β-adrenergic stimulation potentiated spontaneous Ca2+ waves and unduly frequent, large and prolonged Ca2+ sparks in CPVT compared with control iPSC-CMs, validating the disease phenotype. Pursuant to the patient's in vivo responses, nadolol treatment during β-adrenergic stimulation achieved negligible reduction of Ca2+ wave frequency and failed to rescue Ca2+ spark defects in CPVT iPSC-CMs. In contrast, flecainide reduced both frequency and amplitude of Ca2+ waves and restored the frequency, width and duration of Ca2+ sparks to baseline levels. By recapitulating the improved response of an individual with CPVT to flecainide compared with β-blocker therapy in vitro, these data provide new evidence that iPSC-CMs can capture basic components of patient-specific drug responses.Although β-blockers can be used to eliminate stress-induced ventricular arrhythmias in patients with catecholaminergic polymorphic ventricular tachycardia (CPVT), this treatment is unsuccessful in ∼25% of cases. Induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) generated from these patients have potential for use in investigating the phenomenon, but it remains unknown whether they can recapitulate patient-specific drug responses to β-blockers. This study assessed whether the inadequacy of β-blocker therapy in an individual can be observed  Editors' choice: Clinically observed drug response differentials to β-blocker and flecainide treatment in catecholaminergic polymorphic ventricular tachycardia can be modeled in vitro using patient-derived iPSC-cardiomyocytes. Evidence suggests that the majority of RyR2 mutations promote catecholamine-driven spontaneous Ca2+ release from the SR during diastole by reducing the threshold for store overload-induced Ca2+ release  and triggered arrhythmias in CPVT (Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a life-threatening inherited arrhythmia that predisposes young individuals with structurally normal hearts to cardiac arrest. The autosomal dominant form of CPVT is linked to mutations in the gene encoding the type 2 ryanodine receptor (RyR2) , an ion  release . In turn in CPVT .in vitro patient-specific drug response differentials to in vivo clinical data. A notable proof-of-principle study for this paradigm demonstrated that CPVT patient-derived iPSC-CMs can replicate in vivo individual drug responses to dantrolene in a mutation-specific manner  are the foundation of pharmacological CPVT therapy. However, for unknown reasons, ∼25% of CPVT patients are inadequately protected by β-blockers . In the c manner . Howeverin vitro in CPVT iPSC-CMs. To this end, iPSC lines were derived from an individual with CPVT harboring a novel RyR2-L3741P mutation whose ventricular ectopy was not abolished by the widely prescribed β-blocker nadolol, but was resolved with flecainide. As controls, iPSC lines were derived from two unrelated healthy individuals with no history of cardiac disease. iPSC-CMs differentiated from the two CPVT lines displayed Ca2+-handling defects generally associated with CPVT CMs, including increased spontaneous Ca2+ release during β-AR stimulation and reduced SR Ca2+ content compared with control cells. Consistent with the patient's in vivo outcomes, flecainide proved more effective than nadolol in reducing potentially arrhythmogenic Ca2+ release in iPSC-CMs derived from the individual during β-AR agonism. Further investigation of the therapeutic effects of flecainide on CPVT CMs following β-AR stimulation showed that it successfully improved Ca2+ homeostasis and mitigated electrical instability by reducing the incidence of DADs and asymmetrical beat periods.The present study sought to determine whether a patient-specific response to therapeutic β-blockade can be observed These results support the hypothesis that iPSC-CMs can capture key components of patient-specific drug responses, and imply that CM-specific factors play a role in determining a patient's receptiveness to β-blocker therapy.The pedigree of the 12-year-old male individual with CPVT (III-2) selected for this study shows several affected family members demonstrating an autosomal dominant inheritance pattern of the syndrome A. GenotyFig. S1). Control and CPVT iPSCs were positive for pluripotency markers , and two control lines (control A and control B) were derived from two unrelated healthy adult males with distinct genetic backgrounds. All four lines displayed typical iPSC colony morphology and normal 46, XY karyotypes  and CPVT (n=32) CMs. Representative traces illustrate the protocol for assessing the SR Ca2+ leak/load relationship  in NT solution were significantly ∼33% lower in CPVT CMs compared with control CMs (2+ leak in CPVT CMs was significantly greater (∼35%) than in control CMs  compared with control  displayed extemporaneous Ca2+ oscillations, whereas all control CMs remained quiescent , and transients exhibiting ‘other’, undefined abnormalities. Line-scans were categorized as ‘normal’ if the Ca2+ transients therein had mostly consistent amplitudes and beat periods, typical cardiac transient morphology (i.e. rapid upstroke and decay kinetics), and no notable instances of spontaneous Ca2+ release between transients  and frequency  of SCW (P<0.0001) and frequency . Although cells treated with nadolol during β-agonism also showed reductions in the amplitude  and frequency  of SCW, the drug was less effective than flecainide. Flecainide successfully rescued Ca2+ cycling during β-agonism, as there were no significant differences in SCW amplitude or frequency in flecainide-treated cells compared with baseline  and ∼150% higher (P<0.01), respectively, in nadolol-treated cells compared with baseline  of SCW D. Comparbaseline D. In conbaseline D. Confocbaseline E.2+ release at the elementary level in control and CPVT CMs, spontaneous Ca2+ sparks were analyzed across all treatment groups for several key parameters: full width at half maximum (FWHM), full duration at half maximum (FDHM), amplitude (ΔF/F0), and spark frequency (events per 100 µm per second)  A,B.Fig.2+ spark parameters were observed between control lines A and B or between CPVT lines A and B. Therefore, data from control A and control B and from CPVT-A and CPVT-B were combined for comparisons between conditions. At baseline, the mean FWHM and amplitude of Ca2+ sparks were comparable between control and CPVT CMs; however, FDHM and spark frequency were ∼27% and ∼33% higher, respectively, in CPVT CMs than in control. All spark properties in CPVT CMs were significantly increased by β-AR stimulation with isoproterenol, but amplitude did not increase in control CMs. In both control and CPVT CMs, flecainide treatment restored Ca2+ spark amplitude, FWHM and FDHM to pre-stimulation levels, showing no significant differences from baseline. Notably, spark frequency in CPVT CMs during flecainide treatment was significantly ∼10% lower than at baseline, representing levels similar to control CMs at baseline. With the exception of amplitude, nadolol treatment failed to restore spark parameters; spark frequency, FWHM, and FDHM remained significantly ∼13%, ∼14% and ∼12% higher, respectively, than at baseline.No significant differences in Ca2+ sparks typically occurred randomly throughout the cell regardless of treatment  and CPVT CMs (r2=0.137)  A,B, indiTo test the anti-arrhythmic potential of flecainide in CPVT CMs during β-AR stimulation, electrophysiological experiments were performed using microelectrode arrays (MEAs) as previously described . ExtraceModified Poincaré plots of beat period and corrected field potential duration (FPDcF) in CPVT CMs show low beat-to-beat variability in both parameters at baseline A. Isopro2+ release and enhances electrical stability in CPVT CMs during β-AR stimulation, SR Ca2+ content and RyR2-mediated diastolic Ca2+ leak were assessed in CPVT CMs following isoproterenol exposure (n=14) and flecainide treatment (n=16). Representative traces illustrate the protocol for assessing the SR Ca2+ leak/load relationship for both treatment groups  were not significantly different between treatment groups  of the untreated CPVT CMs, but only three out of 16 flecainide-treated CPVT CMs (∼19%). In addition to increased incidence, the oscillations in isoproterenol-treated cells were larger in amplitude and more frequent than in flecainide-treated CPVT CMs  differentials. It is possible that attenuated β-blocker efficacy is RyR2 mutation-specific, as KCNQ1 mutation-specific responses to β-blocker therapy have been observed in type I long QT syndrome, another inherited arrhythmogenic channelopathy  competence is diminished in RyR2-L3741P CPVT CMs. Measurement of SR Ca2+ load and RyR2-mediated diastolic Ca2+ leak revealed mean SR Ca2+ load was significantly decreased in CPVT CMs, concomitant with significantly increased diastolic Ca2+ leak and enhanced fractional Ca2+ release. Moreover, in the absence of transsarcolemmal fluxes , only CPVT CMs displayed Ca2+ oscillations. These oscillations ostensibly denote undue RyR2 channel activation during diastole, given that they were abolished upon RyR2 inhibition with tetracaine. As these properties have been associated with electrical instability and increased RyR2 sensitivity to luminal Ca2+  of RyR2s  indirectly mitigate electrical instability through NCX  with 1 µg each of Addgene plasmids 27077 (pCXLE-hOCT3/4-shp53-F), 27078 (pCXLE-hSK), and 27080 (pCXLE-hUL) using the Amaxa Nucleofector II/2b Device (program U-023). Transfected cells were cultured in fibroblast medium containing 10 µM ROCK inhibitor Y27632 (Stemgent) for one day. For the next 6 days, fibroblast medium was changed daily. On post-transfection day 6, the cells were dissociated and 1×105 cells were re-plated onto irradiated mouse embryonic fibroblasts (MEF), and cultured in human pluripotent stem cell (hPSC) medium  supplemented with 8 ng/ml human basic fibroblast growth factor  and Human iPS Reprogramming Boost Supplement II (EMD Millipore). All medium components were obtained from Thermo Fisher unless otherwise specified. The boost supplement was withdrawn on day 11. On post-transfection day 14, iPSC colonies were picked and re-plated at one colony per well. iPSCs were then cultured in ‘feeder-free’ conditions as previously described  were reprogrammed into iPSC lines using integration-free episomal vectors as previously described . Low-pasescribed . Controlescribed .Tables S1 and S2. Cells were imaged with a phase contrast and fluorescence AxioVert A1 inverted microscope (Zeiss) equipped with AxioCam digital camera system (Zeiss). Images were exported using AxioVision LE (Zeiss) and merging was performed in Adobe Photoshop.Adherent cells were rinsed with cold 1× PBS and fixed with 2% paraformaldehyde solution for 10-15 min at room temperature (RT), permeabilized with ice-cold 100% ethanol for 5 min, rinsed again with 1× PBS, and blocked overnight at 4°C with 5% normal goat serum . Cells were incubated for 2 h at RT with primary antibodies, then rinsed three times with 1× PBS to remove excess antibody. Cells were incubated with fluorescently conjugated secondary antibodies for 1 h at RT in the dark. Cells were washed three more times with 1× PBS. Nuclear counterstaining was performed using Vectashield mounting media with DAPI (Vector Laboratories). For more information on the antibodies used, please refer to The embryoid body (EB) assay for pluripotency was performed as previously described . UndiffeT values (corrected for GAPDH) as previously described . cDNAs were prepared from individual 1 µg RNA samples using the SuperScript VILO cDNA Synthesis Kit (Life Technologies), and incubations were performed using a C1000 touch thermal cycler (Bio-Rad). Gene expression levels were quantified using the 7500 Real-Time PCR System (Applied Biosystems). PCR amplifications were performed in skirted 96-well PCR plates (GeneMate) with iTaq Universal SYBR Green PCR Supermix (Bio-Rad). For each sample, mRNA levels were normalized to GAPDH mRNA levels. Data for each gene are presented in color-coded heatmaps of inverse ΔCescribed  on a sca2+ load and RyR2-mediated diastolic Ca2+ leakage were assayed using fluo-4 fluorescence and the Shannon–Bers technique (2+ dye fluo-4 AM (Life Technologies) to load the indicator into the cytosol. Following incubation, the indicator-containing medium was removed; cells were washed once, and incubated in cell culture medium for an additional 30 min at 37°C to allow for de-esterification of the indicator. Recordings were captured using an epifluorescence microscope (Olympus® IX51) equipped with the IonOptix calcium and contractility system. Cells were bathed in 37°C NT solution  and field-stimulated at 1 Hz  for at least 20 s to bring the intracellular Ca2+ content to a steady state. Once achieved, stimulation was turned off, and the bathing superfusate was rapidly switched to a 0 Na+, 0 Ca2+ Tyrode buffer  for 10 s to abolish transsarcolemmal Ca2+ fluxes through the Na+/Ca2+ exchanger (NCX). Tetracaine and caffeine solutions were added dropwise using a temperature-controlled needle directly above each cell. RyR2 channels were inhibited by adding a 1 mM tetracaine  solution dropwise for ∼10 s. At this time, tetracaine was stopped, and SR Ca2+ stores were depleted by adding a 30 mM caffeine  solution dropwise for ∼10 s. At this time, caffeine was stopped, and the bathing solution was rapidly switched from 0 Na+, 0 Ca2+ Tyrode buffer back to NT solution. Data files were exported and monotonic transient analysis was performed with IonWizard® 6.5 software.Cardiomyocyte SR Caechnique . Cardiom2+ transient amplitude was defined as ΔFtrans/F0, diastolic, where ΔFtrans is the change in signal between the peak fluorescence and the minimum diastolic fluorescence . The tetracaine-induced drop in fluorescence was defined as ΔFtet/F0, diastolic, where ΔFtet is the change in signal between the diastolic fluorescence preceding the addition of tetracaine , and the minimum fluorescence after tetracaine  under 0 Na+, 0 Ca2+ conditions. The caffeine-induced Ca2+ transient amplitude was defined as ΔFcaff/F0, leak, where ΔFcaff is the difference between peak fluorescence after caffeine and the minimum fluorescence preceding the addition of caffeine  under 0 Na+, 0 Ca2+ conditions. Fractional Ca2+ release was defined as the ratio of the action potential-induced Ca2+ transient amplitude to the caffeine-induced Ca2+ transient amplitude /. For a diagram that assists interpretation of these formulas, refer to Fig. S2.When cells were field stimulated at 1 Hz in NT solution, the action potential-induced Ca® 10.0 software (Molecular Devices).Spontaneously beating cardiomyocytes (30±2 days old) differentiated from control and CPVT iPSC lines were incubated with 10 μM of fluo-4 AM  at 37°C in culture medium, then transferred to an inverted laser confocal scanning microscope (Olympus FV1000) equipped with FluoView software (Olympus), where they were perfused with NT solution. For each pharmacological agent, cells were allowed to perfuse for 10 min with drug-containing NT before initiating recording. Fluo-4 was excited by the 488 nm line of an argon laser and emitted fluorescence was captured at >505 nm. Recordings of fluo-4 fluorescence were acquired in line-scan mode, where line-scans were preferentially positioned in the center of the cell (as opposed to the periphery) along the longitudinal axis . Regions0). Estimates of intracellular Ca2+ are presented as changes in ΔF/F0, where ΔF=F−F0. Isoproterenol (100 nM), nadolol (10 µM), and flecainide (10 µM) dosages (Sigma) and incubation time (10 min) were initially selected according to previous literature reports . The nadolol dosage was chosen as the concentration that returned the beating frequency approximately to baseline levels. The flecainide dosage was limited to 10 µM, as the two higher concentrations tested (25 µM and 50 µM) caused both control and CPVT cells to arrest beating.As differentials in fluo-4 loading efficiency between cell lines can influence observed fluorescence intensities, loading and acquisition conditions were kept as consistent as possible, and all absolute fluorescence (F) measurements were normalized to inherent basal (i.e. background) fluorescence  and baseline (i.e. no treatment), not for differences between CPVT and control CMs. Kernel density estimates of Ca2+ spark parameter distributions were generated for each condition using JMP from Statistical Analysis Software.Quantification of Car plugin  for Imagvelopers , a detecn against that of beat n–1 for 60 beats. Coefficients of variation (CV) for beat period and FPDcF were calculated for each cell aggregate as the standard deviation (s.d.) of each parameter recorded in 60 s divided by the mean (x), multiplied by 100. Large CV values are indicative of beat irregularity and potential arrhythmia.Microelectrode array (MEA) recordings were performed using the 64-channel Muse MEA system (Axion Biosystems). M64-GL (SU-8/Pt) MEA chambers (Axion Biosystems) were coated with matrigel (1:30 dilution) overnight before being seeded with 200 µl containing 6-12 CPVT iPSC-CM cell aggregates (day 20±5) per array. Spontaneously beating CPVT CMs were recorded at baseline and following 5 min incubation with 100 nM isoproterenol, and later 10 µM flecainide. Recording times were at least 1 min per sample per condition, and chambers were maintained at 37°C during recording. Analog field potential signals were acquired through a 0.1 Hz high-pass filter and a 2 kHz low-pass filter at a sampling frequency of 12.5 kHz. Data files were exported and analyzed using Axion Integrated Studio (AxIS) 2.3 software. Field potential parameters were analyzed, and waveforms were evaluated for the presence of DADs. Low-amplitude depolarizations occurring after completion of repolarization were considered DADs. Beat period (BP) was defined as the time in seconds between two successive depolarization spikes. Field potential duration (FPD) was defined as the time in milliseconds between a depolarization spike and the subsequent T-wave, and was corrected for beating rate using Fridericia's formula:  . Modifie  were gen2+ leak and load, CPVT CMs were incubated for 10 min with 100 nM isoproterenol in NT solution at 37°C. After 10 min, the coverslips pre-treated with isoproterenol were perfused with NT solution for an additional 5 min before initiating the SR Ca2+ leak/load protocol. To assess the ability of flecainide to improve Ca2+ homeostasis in CPVT CMs during β-AR stimulation, cells were again pre-treated for 10 min with 100 nM isoproterenol, but were then perfused with 10 µM flecainide (rather than NT solution) for an additional 5 min before beginning the protocol.To determine the effects of β-agonism on SR Cat-test. As no significant differences were observed, data from cell lines control A and control B and from CPVT-A and CPVT-B were combined, where necessary, for comparisons between conditions. For SR Ca2+ leak/load, significance was assessed by two-sample test. For Ca2+ spark analyses, spontaneous Ca2+ wave analyses, and MEA analyses, significance was assessed using one-way ANOVA, and post-hoc individual comparisons for each variable were performed using Tukey's HSD test.Across all data sets, significant differences between control lines A and B and between CPVT lines A and B were assessed by two-sample


FUS‐ERG fusion gene. The basophilic nature of leukemia blasts was demonstrated by cytomorphology, toluidine blue metachromasia, mature basophil‐associated antigen expression, and characteristic granules under electron microscopy. The molecular link between t/FUS‐ERG and basophilic differentiation remains unclear.We herein report a rare case of acute basophilic leukemia with t generating the  Acute basophilic leukemia (ABL) is listed as a separate entity in the acute myeloid leukemia (AML), not otherwise specified category in the 2008 WHO classification, comprising <1% of all cases of AML FUS (FUS RNA binding protein) at 16p11 and ERG  at 21q22, encoding the FUS‐ERG chimeric protein FUS‐ERG to human umbilical cord blood cells, showing altered myeloid and arrested erythroid differentiation and marked increases in the proliferative and self‐renewal capacities of transduced myeloid progenitors The t16;21) translocation was initially described in a patient with AML characterized by a large number of abnormal eosinophils in BM ;21/A 65‐year‐old man was admitted to the Hematology Department of our hospital with a diagnosis of acute leukemia. Two weeks before, he developed swelling in the right lower leg that extended to the ankle and foot. He presented to the Orthopedic Department, and a blood examination revealed marked leukocytosis. There was no surface lymphadenopathy or splenomegaly. He did not have any symptoms suggestive of the excess release of histamine.9/L, containing 91.2% blasts, and platelet count 31 × 109/L. His lactate dehydrogenase level was 1850 U/L, C‐reactive protein level 1.7 mg/dL, and fibrin degradation products 6.6 μg/mL. The mRNA level of the Wilms’ tumor‐1 gene was 2.8 × 105 copies/μg RNA.His hemoglobin level was 10.1 g/dL, white cell count 73.77 × 10Bone marrow showed 30~40% cellularity containing 95.0% blasts. Leukemia blasts in PB and BM were of medium size and had round or indented nuclei with dispersed chromatin. Nucleoli were inconspicuous. Approximately, 60% of PB and 70% of BM blasts had variable amounts of a light‐blue cytoplasm containing coarse basophilic granules Fig. A–C, and +, CD33+, CD34+, and CD117+, but lacked HLA‐DR expression  revealed that leukemia blasts showed an immature myeloid cell phenotype, that is CD13ion Fig. A. Approxion Fig. B. Other μm in diameter, and the nuclear‐cytoplasmic ratio ranged between 0.5 and 0.9. Cells had round, indented, or irregular nuclei with slightly condensed chromatin and small to medium‐sized nucleoli. In the cytoplasm, variable amounts of mitochondria, rough endoplasmic reticulum (ER), and Golgi apparatus were found among the cells. Twenty to 30% of leukemia blasts contained granules that were 0.3–1.2 mm in diameter with a variable appearance, representing immature basophils tp11;q22) chromosome, while the reciprocal der(16)t was deleted. The karyotype according to the ISCN (2013) ;q22 chroier Fig. B 12. The 10 Fig. C.FUS‐ERG fusion mRNA fell to below the level of detection. The patient then underwent hematopoietic stem cell transplantation from his daughter, who was HLA‐A locus mismatched in the graft‐versus‐host direction and HLA‐C and DR loci‐mismatched in the host‐versus‐graft direction. Although the course of the transplant was uneventful, his leukemia briefly relapsed on day 56 of transplantation. A reduction in the dosage and withdrawal of tacrolimus led to the elimination of circulating leukemia blasts; however, he developed severe graft‐versus‐host disease involving the entire gastrointestinal tract. He finally died of Pseudomonas aeruginosa septicemia on day 419 of the initial presentation.Although the initial treatment regimen consisting of idarubicin and cytarabine failed to eradicate leukemia cells, MEC  salvage treatment led to a hematological response. Cellulitis of the leg resolved during the induction treatment. After three cycles of MEC, FUS‐ERG fusion gene. Basophilic features were clearly demonstrated by (1) cytomorphological differentiation from agranular blasts to mature basophils, (2) toluidine blue metachromasia, (3) the expression of mature basophil‐associated antigens, that is CD123 and CD203c, and (4) the presence of characteristic granules under electron microscopy.We herein described a patient with a rare type of AML characterized by the marked basophilic differentiation of leukemia blasts and the t/MYB at 6q23 and GATA1 at Xp11 IKAROS alterations are associated with disease acceleration and basophil expansion in CML RUNX1‐RUNX1T1DEK‐NUP214 is observed in AML with or without monocytic features and 44–62% of cases show >2% basophilia in PB and BM ETV6Chromosomal translocations in AML with the proliferation of basophils are heterogeneous. t is recurrently observed in male infants with ABL, and this translocation generates the fusion gene between FUS‐ERG–transduced human cord blood cells FUS‐ERG and basophilic differentiation in leukemia blasts remains to be elucidated.t16;21) has been identified in AML with any French American British (FAB) subtype of AML other than FAB‐M3, and the disease may be preceded by myelodysplastic syndrome. In rare cases, this translocation has been observed in acute lymphoblastic leukemia, acute megakaryoblastic leukemia, and the blast crisis of CML 1‐AML cells with all‐trans retinoic acid led to reductions in the viability and differentiation of these cells Due to the paucity of ABL and AML with t, no guidelines for the treatment of this particular patient were available. Although leukemia was refractory to the idarubicin and cytarabine induction treatment, MEC salvage treatment led to remission at the molecular level. However, FUS‐ERG fusion gene. MH: performed flow cytometric analysis. HO: was responsible for writing and reviewing the manuscript, literature review, and overall revision of the manuscript.YT: main author, was responsible for writing and reviewing the manuscript, literature review, and provided direct care to the patient. YN: was responsible for writing and reviewing the manuscript. DS: performed electron microscopic analysis. CK: performed cytogenetic analysis. KT: performed cytomorphological analysis. KF: performed RT‐PCR and direct sequencing of The authors declare that there is no conflict of interest regarding the publication of this paper.


Although effective at preventing cardiovascular disease, statin use is associated with muscle weakness, myopathies and, occasionally, fatal rhabdomyolysis. As simvastatin, a commonly prescribed statin, promotes Ca2+ release.RyR1 and RyR2 single‐channel behaviour was investigated after incorporation of sheep cardiac or mouse skeletal SR into planar phospholipid bilayers under voltage‐clamp conditions. LC‐MS was used to monitor the kinetics of interconversion of simvastatin between hydroxy‐acid and lactone forms during these experiments. Cardiac and skeletal myocytes were permeabilised to examine simvastatin modulation of SR Ca2+ spark frequency towards higher values in skeletal fibres. In contrast, simvastatin reduced RyR2 Po and shifted the distribution of spark frequency towards lower values in ventricular cardiomyocytes. The lactone pro‐drug form of simvastatin (inactive at HMG‐CoA reductase) also activated RyR1, suggesting that the HMG‐CoA inhibitor pharmacophore was not responsible for RyR1 activation.Hydroxy acid simvastatin (active at HMG‐CoA reductase) significantly and reversibly increased RyR1 open probability (Po) and shifted the distribution of Ca2+ release and thus may contribute to its reported adverse effects on skeletal muscle. The ability of low concentrations of simvastatin to reduce RyR2 Po may also protect against Ca2+‐dependent arrhythmias and sudden cardiac death.Simvastatin interacts with RyR1 to increase SR Ca AFatrial fibrillationAICAR5‐aminoimidazole‐4‐carboxamide ribonucleotideCCDcentral core diseaseFDBflexor digitorum brevisHMG‐CoA3‐hydroxy‐3‐methylglutaryl CoALog Dpartition coefficientMHmalignant hyperthermiaPoopen probabilityRyRryanodine receptorSim‐Hsimvastatin hydroxy acidSim‐Lsimvastatin lactoneSRsarcoplasmic reticulumTransluminal3‐hydroxy‐3‐methylglutaryl‐CoA (HMG‐CoA) reductase, the rate‐limiting enzyme in the synthesis of cholesterol. They are the most widely prescribed medication for the treatment of hypercholesterolaemia and prevention of cardiovascular disease. Furthermore, statin use is increasing with recent reductions in the cardiovascular risk threshold for statin prescription  vesicles , central core disease (CCD) and exercise‐induced rhabdomyolysis  fibres, as well as increased heat production and muscle contractures  in situ in single isolated, permeabilised rat skeletal muscle cells.In view of the known association of ‘leaky’ RyR1 channels with severe muscle myopathies, we therefore investigated if simvastatin exerts a direct effect on the gating and conducting properties of the native skeletal RyR1 channel complex incorporated into planar phospholipid bilayers under voltage‐clamp conditions. We also examined if simvastatin could alter the spontaneous SR CaRyR2 in cardiac muscle and RyR3 is widely expressed in various tissues but often at low levels , which was withdrawn from the market in 2001, is also the most lipophilic statin that has been used clinically  does not  reduced the Po of RyR2. Sim‐H also tended to reduce the frequency of Ca2+ sparks in isolated permeabilised ventricular cardiomyocytes. It will be important to investigate the effects of other prescribed statins on cardiac and skeletal RyR channel function as the effects described in this paper may contribute to the unwanted muscular side effects or to, as yet, uncharacterized effects on cardiac function.Our results show RyR isoform‐specific effects of simvastatin. We demonstrated that ≥1 μM Sim‐H significantly increases the Po of RyR1 in a cytosolic Caet al., 3H]‐ryanodine binding; sheep hearts were used for RyR2 single‐channel experiments and [3H]‐ryanodine binding; rat skeletal fibres and cardiac myocytes were used for analysis of Ca2+ sparks in permeabilised cells. All rodent work was carried out in accordance with the Directive 2010/63/EU of the European Parliament with local approval of the Animal Research Committee according to the regulations on animal experimentation at the University of Oxford (mouse) and the Animal Welfare and Ethical Review board at the University of Leeds (rat). Rats were housed in a conventional unit, in filter top cages with Aspen bedding on a 12 h light/dark cycle with free access to food and water. Mice were housed in a specific pathogen free unit in individually ventilated cage on a 12 h light/dark cycle with free access to food and water. Sheep hearts were obtained from an abattoir. To comply with the ‘three Rs’ principles, we prepared SR membranes from animals that were killed for other purposes wherever possible . Our choice of tissue was based on the similarity of RyR proteins between species and designed to minimize animal use without compromising the integrity of the data. Identity between mouse (E9PZQ0.1) and rat (F1LMY4.1) RyR1 sequences is 96%. RyR2 sequences are also 96% identical between sheep (XP_014959831.1) and rat (B0LPN4).Animal studies are reported in compliance with the ARRIVE guidelines (Kilkenny Heavy sarcoplasmic reticulum (HSR) membrane vesicles were prepared from mouse skeletal muscle or sheep hearts as described previously 2 (free [Ca2+] approximately 50 mM) on the trans  side of the bilayer at 21°C. The trans chamber was voltage‐clamped at ground. The compound to be investigated was added to the cytosolic chamber. The free [Ca2+] and pH of the solutions were maintained constant during the experiment and were determined using a Ca2+ electrode  and a Ross‐type pH electrode  as previously described . The addition of Sim‐H or Sim‐L did not alter the free [Ca2+] or pH of solutions.RyR1 or RyR2 channels were incorporated into planar phospholipid bilayers as previously described : KCl 100, HEPES 25, EGTA 0.35, MgCl2 5.72, Na2ATP 5, Na2‐creatine phosphate 10, CaCl2 0.05, pH 7. Permeabilised FDB fibres and ventricular myocytes were loaded with Fluo‐3 pentapotassium salt (25 or 10 μM respectively) and perfused with 10 μM Sim‐H for 5 min. Confocal images were acquired with a Nikon Eclipse TE300 inverted microscope equipped with a confocal scanhead, Bio‐Rad MicroRadiance 2000 and a ×60 water‐immersion objective. Fluo‐3 pentapotassium was excited with the 488 nm line of a 20 mW coherent sapphire laser, and the fluorescence emitted was measured at >515 nm. Images were acquired in line scan mode (every 6 ms). Ca2+ sparks were identified and analysed with ImageJ software (NIH) using the Sparkmaster plugin  fibres  buffer, respectively, and sampling by LC‐MS was begun immediately. Samples were kept at room temperature for the duration of the analysis. Analyses were performed using a Thermo Exactive mass spectrometer equipped with Waters Acquity liquid chromatography system. Instrument control was performed using Thermo Xcalibur Software. Electrospray source conditions were adjusted to maximize sensitivity; 3 μL of each sample was injected onto the system. The column was a 2.1 × 50 mm (3 μm) ACE equivalence C18. A mixed eluent gradient was employed and is described in Supporting Information Sim‐H and Sim‐L were prepared as 10 mM solutions in water and DMSO, respectively, and stored at −78°C, and warmed to room temperature immediately prior to injection; 1 μL of each solution was added to 1 mL of water or Tris/HEPES  was purchased from CalBioTech (567021). Simvastatin lactone (Sim‐L) was purchased from Sigma‐Aldrich . All other chemicals were purchased from Sigma‐Aldrich  or VWR  unless stated otherwise. Water was deionized , and all solutions used in single‐channel experiments were filtered through a membrane with a 0.45 μm diameter pore .http://www.guidetopharmacology.org, the common portal for data from the IUPHAR/BPS Guide to PHARMACOLOGY .The effects of the hydroxy acid form of simvastatin  after channel activation by 10 μM Sim‐H. A typical experiment is shown in Figure 2+], the response to 10 μM Sim‐H can be observed. Subsequent lowering of the cytosolic [Ca2+] to sub‐activating concentrations  completely abolished any channel openings (Po = 0). Thus, the 10 μM Sim‐H is absolutely dependent on the presence of activating levels of cytosolic Ca2+ in order to raise RyR1 Po. The mean data are shown in Figure 2+ (like Sim‐H) also activates RyR1 predominantly by increasing frequency of opening  on RyR1 channel gating. Sim‐L also significantly activated RyR1 channels Figure A, B, suget al.,It has been reported previously that the interconversion of statins has a strong pH dependence (Taha 3H]‐ryanodine binding to sheep cardiac heavy SR membrane vesicles was inhibited by low concentrations of Sim‐H (0.5 μM) and that this effect was reversed for concentrations ≥10 μM  significantly decreased Po while 10 μM produced no observable effect Figure A, B. TheM Figure D.2+ sparks, spontaneous releases of Ca2+ from a cluster of RyR channels, were measured in confocal linescan mode. Sim‐H appeared to exert opposing effects on Ca2+ spark frequency in skeletal fibres and cardiac myocytes. Representative linescan images illustrate typical Ca2+ spark frequency observed in skeletal fibres .Next, we wanted to investigate whether effects of Sim‐H on RyR1 and RyR2 would be replicated in a more physiological setting. Skeletal muscle fibres and ventricular myocytes from the rat were permeabilised by saponin  and perfused acutely with 10 μM Sim‐H for 5 min. Cas Figure A and cars Figure D in the n Figure B and cumn Figure C plots. 2+ sparks towards higher frequencies in permeabilised skeletal fibres consistent with activation of RyR1 in situ. The ability of simvastatin to activate RyR1 may be common to other statins and could explain why cerivastatin, a compound removed from the market due to reports of rhabdomyolysis, was shown to trigger Ca2+ release from isolated skeletal SR vesicles  produces a [Ca2+]i transient which is comparable to that of caffeine and inhibited by pretreatment of fibres with ryanodine  are natural products or derived from natural products and contain a trans‐decalin‐ring motif, while the more recently developed, fully synthetic, type 2 statins  contain a pyrrole core   have been shown to lead to accumulation of simvastatin in tissues at levels in excess of this   that significantly activates RyR1. Higher concentrations then reverse the inhibition of RyR2 indicating that there may be a high affinity inactivation site and a lower affinity activation site on RyR2. The distribution of Ca2+‐ leak. This is important since a significant proportion of statin users are already predisposed to Ca2+‐dependent arrhythmias and sudden cardiac death  to cause skeletal muscle side effects but could also contribute to the reported beneficial antiarrhythmic properties of statins.In summary, we demonstrated that simvastatin can act both as an activator of RyR1 and an inhibitor of RyR2, results that were observed at the single‐channel level and confirmed by measuring Ca3H]‐ryanodine binding experiments. K.W. and A.W. isolated SR vesicles. C.L. and J.R.W. performed LC‐MS experiments, and C.L. analysed the results. S.L., Z.Y., E.S. and S.C. performed and analysed cellular Ca2+ spark experiments. R.S., E.V., C.L. and S.C. prepared the manuscript. All authors discussed the results and critiqued the manuscript for intellectual content.R.S., E.V., C.L., S.C., S.L. and A.J.R. conceived and designed the study, E.V. and C.L. performed single‐channel experiments and analysed data. K.W. and C.L. performed and analysed [The authors declare no conflicts of interest.Declaration acknowledges that this paper adheres to the principles for transparent reporting and scientific rigour of preclinical research recommended by funding agencies, publishers and other organisations engaged with supporting research.This Table S1 The effects of Sim‐H on lifetime parameters.Table S2 The effects of Sim‐H on lifetime parameters at low Ca2 +.Table S3 LC‐MS analysis of statin samples.Click here for additional data file.