Therefore, we determined the association of differences in cellular Ca2+ dynamics and collagen secretion/deposition between hVFs from failing and normal (control) hearts. Histology of left ventricle sections (Masson trichrome) confirmed excessive fibrosis in HF versus normal. In vitro, hVFs from HF showed increased secretion/deposition of soluble collagen in 48 h of culture compared with control . However, collagen gene expressions  were not different. Ca2+ imaging (fluo-3) of isolated hVFs showed no difference in the thapsigargin-induced intracellular Ca2+ release capacity (control 16±1.4% vs HF 17±1.1%); however, Ca2+ influx via store-operated Ca2+ entry/Ca2+ release-activated channels (SOCE/CRAC) was significantly (P≤0.05) greater in HF-hVFs (47±3%) compared with non-failing (35±5%). Immunoblotting for ICRAC channel components showed increased ORAI1 expression in HF-hVFs compared with normal without any difference in STIM1 expression. The Pearson's correlation coefficient for co-localization of STIM1/ORAI1 was significantly (P<0.01) greater in HF (0.5±0.01) than control (0.4±0.01) hVFs. The increase in collagen secretion of HF versus control hVFs was eliminated by incubation of hVFs with YM58483 (10 µM), a selective ICRAC inhibitor, for 48 h . In conclusion, hVFs from HF have increased collagen secretion capacity versus non-failing hearts and this is related to increase in Ca2+ entry via SOCE and enhanced expression of ORAI, the pore-forming subunit. Therapeutic inhibition of SOCE may reduce the progression of cardiac fibrosis/HF.Excessive cardiac fibrosis, characterized by increased collagen-rich extracellular matrix (ECM) deposition, is a major predisposing factor for mechanical and electrical dysfunction in heart failure (HF). The human ventricular fibroblast (hVF) remodeling mechanisms that cause excessive collagen deposition in HF are unclear, although reports suggest a role for intracellular free Ca Summary: The excessive collagen secretory phenotype found in failing human hearts is associated with ventricular fibroblast remodeling, caused by an elevated influx of intracellular calcium via SOC channels. Fibrosi2+ . Hence, 2+ release and store-operated Ca2+ entry (SOCE) are the major intracellular Ca2+ sources in nonexcitable cells, including fibroblasts, any possible upregulation of either source in the failing heart could contribute to excessive fibroblast proliferation, activation, and ECM deposition. Therefore, the objective of this study was to determine the differences in Ca2+ dynamics and collagen secretion/deposition between human ventricular fibroblasts (hVF) from failing and normal hearts. Specifically, we investigated the extent of intracellular Ca2+ release; store-operated channels (SOC) mediated Ca2+ influx, particularly the contribution of the ICRAC channel to regulation of intracellular free Ca2+ in fibroblasts; the expression of the pore-forming ORAI1 and the store Ca2+ sensor, STIM1; magnitude of collagen secretion/deposition; and whether selective inhibition of the ICRAC channel could mitigate the altered collagen secretion/deposition, if any, in ventricular fibroblasts from HF patients.Since intracellular CaP<0.05; control n=3, HF n=7).We confirmed that human HF is associated with increased ventricular fibrosis. Left ventricle (LV) sections from non-failing and failing hearts were stained with Masson's trichrome to determine the secreted fibrillar collagens as a marker of fibrosis . As show6 vs 58.5±8.8 µg/106 cells, P<0.05; control n=4, HF n=9; The capacity of fibroblasts to synthesize and secrete precursors of fibrillar collagen, the major structural protein of the ECM, changes with cardiac remodeling . BecauseCOL1A1 and COL1A2 gene expressions was performed and compared between the hVF-HF and control groups. As displayed in COL1A1 and COL1A2 between human failing heart and non-failing heart ventricular fibroblasts.The increased collagen secretion in hVF-HF could be the result of enhanced transcription of the collagen gene, translation to protein, or secretion of synthesized protein. To assess whether hVF-HFs have greater collagen secretion due to changes in transcription of collagen genes, real time-polymerase chain reaction (RT-PCR) for 2+ – was assessed, as Ca2+ is an important second messenger that is involved in many of the fibroblast functions, including secretion and other receptor-mediated signal transduction mechanisms  induced by the Ca2+ ionophore ionomycin (2 µM), as shown in 2+ release capacity between control and hVF-HF  greater in the hVF-HF group  than the control group  . While there was no significant difference in STIM1 protein expression  than the control group  . Therefo P=0.08; D.Fig. 52+ sensor, STIM1. Confocal microscopic images and double immunolabeling of ORAI1 (green) and STIM1 (red) from the control and hVF-HF groups are shown in n=63 cells from three different patients) compared to the control group . The overall percentage of inhibition by YM58483 of the amount of collagen secreted/deposited in the absence of the drug was about 5% in the control fibroblasts and 17% in failing heart fibroblasts, indicating a greater role of ICRAC-mediated Ca2+ influx in enhanced collagen secretion by failing heart hVFs.To determine if elevated ORAI1 expression and SOCE in hVF-HFs were responsible for greater collagen secretion, we assessed the impact of selective pharmacological inhibition of Inhibitor , elimina2+ influx via SOCE in isolated fibroblasts, (4) augmented expression of the ORAI1 channel, the pore-forming component of the ICRAC channel, (5) no change in the store Ca2+ sensor, STIM1 expression, (6) increased co-localization of STIM1/ORAI1, and (7) ICRAC channels as a mediator of enhanced collagen secretion, which can be suppressed by pharmacological channel inhibition.The overall findings of this study in human failing hearts compared with non-failing hearts are: (1) enhanced cardiac fibrosis, (2) increased collagen secretion by isolated fibroblasts, (3) elevated CaHeart failure in the elderly is one of the leading causes of morbidity, mortality, hospitalization, and increased healthcare utilization . Most of2+ influx are through SOCE pathways that are activated by depletion of intracellular calcium stores, such as within the endoplasmic reticulum. ICRAC (Ca2+-release-activated channel)  that is used to deplete endoplasmic reticulum calcium with a resultant elevation in cytoplasmic calcium. This is unlike studies in failing heart cardiomyocytes, in which altered Ca2+ levels caused by reduced sarcoplasmic reticulum Ca2+ uptake was evident because of de-phosphorylation of phospholamban   is one she cells . HVFs frholamban . In our  failure , suggestLA cells . WhetherLA cells . The greLA cells , and traLA cells . Indeed,CRAC channel is associated with cellular calcium signaling that has been well characterized in cells involved in inflammation and immunity, pulmonary artery smooth muscle cells from patients and animal models with pulmonary arterial hypertension  implantation was used to extract hVFs that were grown as primary culture. Patients with New York Heart Association class III to IV heart failure who underwent either cardiac transplantation or LVAD implantation were studied. Vendor-authenticated HVFs isolated from trauma victims free of any structural heart disease were used as a control . The study was approved by the Institutional Review Board and adhered to the Health Insurance Portability and Accountability Act (HIPAA) and the institution's patient privacy and security guidelines. Informed consent was obtained from all subjects. The study conformed to the principles of the Declaration of Helsinki. mm culture dish in the tissue culture hood and cut into 1 mm blocks, transferred to a 25 cm2 TPP tissue culture flask , washed thrice with DPBS, twice with FM-b (ScienCell) containing penicillin/streptomycin, spread evenly into 20-30 blocks per flask and cultured in 5 ml FM-2 media (ScienCell) containing 5% fetal bovine serum (FBS) and penicillin/streptomycin. The flask was inverted, with the bottom (surface with tissue) up, in the incubator  and blocks were allowed to adhere for about 4 h. After 2-3 h, the flasks were turned to the normal orientation. The media was changed every 2 days. In 2 weeks, cardiac fibroblasts migrated from the ventricular tissue explants and reached 70% confluency. The cells were trypsinized and transferred to 150 cm2 TPP tissue culture flasks (MidSci) with FM-2 media (ScienCell) containing 5% FBS and penicillin/streptomycin and grown to 70% confluency. These initial cultures were split, and passages 2 and 3 were stored in liquid nitrogen until experiments were conducted.Cardiac tissues were transferred from the operating theater in Dulbecco's phosphate-buffered saline (DPBS) on ice. The ventricular tissue block was carefully cleaned to remove all non-myocardial portions in a 60http://rsbweb.nih.gov/ij/), blue-stained areas and non-stained myocardial areas from each section were determined using color-based thresholding. The percentage of total fibrosis area was calculated as the summed blue-stained areas divided by total myocardial area.Pieces of LV tissue were washed in phosphate-buffered saline (PBS) fixed overnight in 4% paraformaldehyde, and embedded in paraffin by standard protocols . Followi4 cells/cm2 in T-25 flasks in FM-2 media (ScienCell) containing 5% FBS. After 24 h, the media was replaced with fresh media with or without YM-58483 (10 µM), a specific blocker of ICRAC channel (Ventricular fibroblasts (passage 3 or 4) from both failing and non-failing hearts were plated separately at 10 channel . After 44 cells/cm2 in collagen-coated MatTek dishes  in FM-2 media containing 5% FBS. After 24 h, the media was replaced with serum-free fresh media. After 18-24 h of serum-starvation, the cells were incubated in FM2 media containing fluo-3 dye (2 µM) for 30 min. Cells were washed with Ca2+-free DPBS twice and kept in Ca2+-free Krebs-Ringers Henseleit (KRH) solution that contained (in mM): NaCl, 116; KCl, 4; MgCl2, 1; glucose, 25 and HEPES, 10 (pH 7.4). Under confocal microscopy , in Ca2+-free KRH, thapsigargin (2.5 µM) was added to release the Ca2+ from intracellular stores and deplete the store. CaCl2 (2 mM) was then added extracellularly to facilitate Ca2+ influx via the store-operated channels that opened because of Ca2+ store depletion. Ionomycin (2 µM), a Ca2+ ionophore, was added to determine the maximum possible Ca2+ fluorescence in the selected region of interest, which was used for normalization of the test Ca2+-induced fluorescence. Peak fluorescent intensities following thapsigargin, CaCl2, or ionomycin were captured and average values from at least two sets of cells per patient or control were obtained for analysis. Data were analyzed as the percentage of ionomycin response for each cell.Ventricular fibroblasts from both failing and non-failing hearts were plated at 10Standard western blotting protocols were followed per the manufacturer's protocol. Equal amounts of proteins were loaded and electrophoresis was performed at 110 V for 2 h. The separated proteins were transferred to PVDF membrane (7 min iBlot) and probed for STIM1 and ORAI1 proteins using STIM1 (D88E10) monoclonal antibody  and monoclonal Anti-ORAI1 antibody , respectively. All samples were subjected to immunoblotting simultaneously; this was repeated at least twice.COL1A1 (NM_000088.3) R: 5′-AGCCTCTCCATCTTTGCCAGCA-3′, F: 5′-GATTCCCTGGACCTAAAGGTGC-3′; COL1A2 (NM_000089.3) R: 5′-ATCACCACGACTTCCAGCAGGA-3′, F: 5′-CCTGGTGCTAAAGGAGAAAGAGG-3′; STIM1 (NM_003156) R: 5′-CTGTCACCTCGCTCAGTGCTTG-3′, F: 5′-CACTCTTTGGCACCTTCCACGT-3′; ORAI1 (NM_032790) R: 5′-GACCGAGTTGAGATTGTGC-3′, F: 5′-GAGTTACTCCGAGGTGATGA-3′; B2M (NM_004048) R: 5′-CCAATCCAAATGCGGCATCTTCA-3′, F: 5′-CCACTGAAAAAGATGAGTATGCCT-3′. The cycling conditions were 50°C for 2 min, 95°C for 10 min, followed by 40 cycles at 95°C for 15 s, 1 min at 60°C, and 72°C for 40 s. Melt curve analysis was performed by an additional dissociation step of 1 cycle at 95°C for 15 s and ramping data collection at 60°C for 1 min and 95°C for 15 s. Ct values were calculated using Applied Biosystems 7300 system software v1.4.0. Data were normalized against the β-2-microglobulin (B2M) reference gene. Relative expression values were obtained by normalizing Ct values of the tested genes with Ct values of the reference gene, using the ΔCt method.Total RNA was isolated from fibroblasts using Trizol Reagent (Thermo Fisher Scientific) according to the manufacturer's instructions. RNA concentrations were evaluated spectrophotometrically using the NanoQuant on the Tecan Infinite M200 plate reader . Total RNA (300 ng) was used for reverse transcription using the miScript RT II kit with the supplied HiFlex buffer (Qiagen). qPCR was performed on the ABI 7300 Real-time PCR System , using the Power SYBR Green PCR Master Mix (Thermo Fisher Scientific) and 10 ng diluted cDNA per well. The following primers were used: 2+ depletion, hVFs were treated with thapsigargin (2.5 µM) for 15 min. HVFs were then fixed in 4% paraformaldehyde at 4°C for 20 min and permeabilized with 0.2% (v/v) Triton X-100 in PBS for 45 min and blocked with 10% (v/v) donkey serum (Sigma) containing 0.2% (v/v) Triton X-100 for 45 min. After rinsing in HBSS, the samples were incubated with primary antibodies – rabbit monoclonal anti-STIM1 (D88E10) antibody , and mouse monoclonal anti-ORAI1 antibody  – and diluted (1:200) in 1% donkey serum with 0.2% Triton X-100/PBS overnight at 4°C. Samples were washed with DPBS (no Ca2+/Mg+) three times and incubated with secondary antibodies conjugated with Alexa Fluor 594 (anti-rabbit IgG) or Alexa Fluor 488 (anti-mouse IgG) in 1% (w/v) donkey serum with 0.2% (v/v) Triton X-100 in PBS. After washing three times with DPBS, two drops of NucBlue Fixed cell stain  were added to stain the nucleus for 5 min at room temperature. After washing again with DPBS twice and HBSS (with Ca2+/Mg+), confocal images were acquired with the Olympus confocal microscopy system.HVFs were plated in collagen-coated MatTek dishes in FM-2 media containing 5% FBS. After 18-24 h, media was replaced and incubated for 2 h. To initiate store-Cat-test. P≤0.05 was considered statistically significant. Sample sizes for Ca2+-imaging and collagen-secretion assays were calculated using University of California – San Francisco Clinical & Translational Science Institute calculators (http://www.sample-size.net/sample-size-means) with 80% statistical power.Comparisons of mean values between control and hVF-HF groups were performed by unpaired


Ca2+ extrusion is operated by a wealth of transporters, such as Ca2+ pumps and Ca2+/H+ antiporters, which often require a rise in Ca2+ concentration to be activated. Here, we report a regulatory fine-tuning mechanism of the Arabidopsis thaliana plasma membrane-localized Ca2+-ATPase isoform ACA8 that is mediated by calcineurin B-like protein (CBL) and CBL-interacting protein kinase (CIPK) complexes. We show that two CIPKs (CIPK9 and CIPK14) are able to interact with ACA8 in vivo and phosphorylate it in vitro. Transient co-overexpression of ACA8 with CIPK9 and the plasma membrane Ca2+ sensor CBL1 in tobacco leaf cells influences nuclear Ca2+ dynamics, specifically reducing the height of the second peak of the wound-induced Ca2+ transient. Stimulus-induced Ca2+ transients in mature leaves and seedlings of an aca8 T-DNA insertion line exhibit altered dynamics when compared with the wild type. Altogether our results identify ACA8 as a prominent in vivo regulator of cellular Ca2+ dynamics and reveal the existence of a Ca2+-dependent CBL–CIPK-mediated regulatory feedback mechanism, which crucially functions in the termination of Ca2+ signals.Ca Ca2+ sensor proteins decode and relay the information contained in the Ca2+ signature into phosphorylation cascades, modification of enzymes activity, and alteration of gene expression, thus allowing the organism to set up the proper cellular response  . Physicagnatures . Each cedynamics . The pre2+ pumps are high-affinity, low turnover pumps that mediate Ca2+ extrusion from the cytoplasm and have been found at the PM and at the membranes of the main intracellular Ca2+ stores  indicated that the N-terminus can be phosphorylated by this class of protein kinases on Ser19 and Ser22  assay experiments that the identified CIPKs interact with full-length ACA8 1M–I116)] of ACA8 inserted in the Escherichia coli expression vector pET15b  of the first 116 amino acids ATP. Reactions were stopped by addition of 20 mM EDTA, solubilized by addition of Laemmli buffer  are able to interact with ACA8, supporting the evidence that targeting of CIPKs depends on their interaction not only with CBLs, but also with their putative targets (e.g. channels or transporters).As a rule, CIPKs are cytoplasmic proteins and their final cellular localization is promoted by interaction with other partners, primarily CBLs , F, H wainations . This lae signal , cannot in vitro transcription/translation system, already described as a successful method to produce suitable amount of active CIPKs  followed by a second slower, smaller, and more sustained peak  for the presence of fluorescence originating from the differently expressed proteins in order to confirm their efficient co-expression  on wounding-induced Ca2+ transient which was essentially the same in all conditions (P<0.05) lower than that of the control , while greatly decreasing the number of cells in which it was >30% of the first peak  by targeting a larger amount of CIPK9 to the PM, CBL1 would improve its interaction with ACA8; and/or (ii) it could stimulate CIPK9 kinase activity. In fact, it has been shown that CBLs are able to stimulate the activity of several isoforms of CIPK  or with the full set of proteins (ACA8::YC-YN::CIPK9 and CBL1::OFP)  did not nditions , E. The  control . When NU control . This ef control . The altirst one . Comparerst peak . These r.6 alone , F–H. We.6 alone , inset.  the CNT  probablyin vivo accelerates the recovery phase of the first peak and greatly decreases the height of the second peak of the nuclear wounding-induced Ca2+ signature that mirrors that generated in the cytosol. Overall, the timing of these effects is congruent with the time necessary for the formation of the complex between ACA8, CIPK9, and CBL1, and for phosphoregulation of the pump to take place. Noticeably, S/D mutation of Ser27, which is localized in a consensus motif for SnRK and is at least one of the amino acid targets of phosphorylation by CIPK9 (data not shown), results in a partially deregulated ACA8 with faster kinetics of interaction with CaM . Both Ca2+ peaks, typical of the response, were significantly modified, being higher in aca8 plants compared with the wt . Besides, although largely co-expressed with the ACA10 isoform in the plant body, ACA8 represents the most expressed PM-localized ACA in the root tip region , that is strictly correlated to wounding, being released during physical damage, and known to induce defined cytosolic Ca2+ signatures in root tip cells  to basal [Ca2+]cyt when compared with the wt. This alteration influenced the time required to reach basal [Ca2+]cyt levels, with aca8 that readjusted significantly later . This resulted in a more sustained [Ca2+]cyt transient in mutant seedling roots compared with the wt.Since experiments were carried out with mature plants, we tried also to analyse cytosolic Cadecrease . Compariand aca8 , F, a rentration . As presntration , aca8 ex2+ kinetics between the wt and the single mutant aca8.In contrast to what was previously reported by using the biosensor AEQ  and, in agreement with the data obtained by transient expression of ACA8 and its regulatory circuit in al cells , highlig2+-dependent phosphorylation events involved in activity modulation of target proteins to specifically transduce stimuli into accurate physiological responses  two isoforms of CIPK, namely CIPK9 and CIPK14, are able to interact with ACA8 in vitro and in vivo and phosphorylate ACA8 in vitro; and (ii) ACA8 phosphoregulation by the CBL1–CIPK9 complex shapes the cytosolic Ca2+ transients induced by mechanical wounding of leaves in vivo.Despite the functional overlap of PM-localized Ca2+-dependent CBL1–CIPK9–target regulatory circuit in tobacco. This allowed us to evaluate the function of a CBL–CIPK complexes in planta and to uncover how phosphorylation-mediated fine tuning of a Ca2+ pump crucially controls the appropriate formation of cytoplasmic Ca2+ signatures.Previous studies have demonstrated the ability of CBL–CIPK complexes to interact with and regulate several targets in heterologous expression systems such as frog oocytes, yeast, or HEK cells (2+-dependent regulatory network for the fine tuning of intracellular Ca2+ homeostasis. Fine tuning of spatial, temporal, and concentration parameters of Ca2+ signatures is probably fundamental for the ability of plants to elicit adequate responses to environmental cues that lead to growth adaptation.As a target of activation by CaM (JXB online.Supplementary data are available at Fig. S1. Subcellular localization of the NUP::YC3.6 probe in a representative tobacco epidermal leaf cell.2+ transients in leaves of N. benthamiana plants transiently transformed with NES::YC3.6 or NUP::YC3.6.Fig. S2. Comparison of wounding-induced CaArabidopsis thaliana.Fig. S3. Expression levels of PM-localized ACA isoforms in roots of 2+ ratio images of a N. benthamiana leaf subjected to woundingVideo S1. Series of nuclear Casupplementary_Figures_S1_S3Click here for additional data file.supplementary_video_S1Click here for additional data file.


The mitochondria and the endoplasmic reticulum (ER) are connected via “mitochondria-associated ER membranes” (MAMs) where ER–mitochondria Ca2+ transfer occurs, impacting the mitochondrial biology related to several aspects of cellular survival, autophagy, metabolism, cell death sensitivity, and metastasis, all cancer hallmarks. Cancer cells appear addicted to these constitutive ER–mitochondrial Ca2+ fluxes for their survival, since they drive the tricarboxylic acid cycle and the production of mitochondrial substrates needed for nucleoside synthesis and proper cell cycle progression. In addition to this, the mitochondrial Ca2+ uniporter and mitochondrial Ca2+ have been linked to hypoxia-inducible factor 1α signaling, enabling metastasis and invasion processes, but they can also contribute to cellular senescence induced by oncogenes and replication. Finally, proper ER–mitochondrial Ca2+ transfer seems to be a key event in the cell death response of cancer cells exposed to chemotherapeutics. In this review, we discuss the emerging role of ER–mitochondrial Ca2+ fluxes underlying these cancer-related features.Calcium ions (Ca In aerobic conditions, pyruvate is transported into the mitochondria and metabolized to CO2 through the tricarboxylic acid (TCA) cycle. The TCA cycle is coupled to oxidative phosphorylation (OXPHOS), which is a pathway for the production of large amounts of ATP. In contrast, in anaerobic conditions, pyruvate is fermented to lactate, a process often referred to as anaerobic glycolysis, which is less energy effective. Nevertheless, proliferative cells exhibit enhanced glycolysis, producing high levels of lactate, even in the presence of O2 (aerobic glycolysis) ], plays a pivotal role in the regulation of mitochondrial metabolism and bioenergetics  with SERCA2b  and ryanodine receptors (RyRs). IP3Rs, which are activated by the second messenger IP3, are ubiquitously expressed in virtually all human cell types β/γ, an enzyme activated in response to hormones, neurotransmitters, and antibodies. IP3R activity can be suppressed by compounds like xestospongin B  (3Rs at the ER side and the Ca2+-permeable channels voltage-dependent anion channel type 1 (VDAC1) at the mitochondrial side . The functional properties of the MCU are tightly regulated by a growing list of interacting proteins, which enable a tight control over the Ca2+ levels in the mitochondrial matrix (The efficient Cas (MAMs) –31. Seveial side , 33. Thel matrix . These Ml matrix –44.3-evoked Ca2+ signals were efficiently transferred into the mitochondria even when IP3-induced cytosolic Ca2+ concentration ([Ca2+]cyt) rises were relatively small . This indicated that local [Ca2+]cyt can be very high in the proximity of IP3R channels, which is then sensed by the mitochondria, leading to mitochondrial Ca2+ accumulation. These findings were underpinned by perimitochondrial [Ca2+] measurements, showing that the local [Ca2+] was about 20-fold higher than global [Ca2+]cyt, allowing a “quasi-synaptic” transmission of the Ca2+ signal from the ER into the mitochondrial matrix , which physically links IP3Rs to VDAC1 within the MAMs  will dampen ER–mitochondrial Ca2+ transfer, while increasing [Ca2+]ER will augment ER–mitochondrial Ca2+ transfer. As a consequence, changes in the ER Ca2+-store content will eventually impact the level of Ca2+ transfer from the ER to the mitochondria and thus eventually cell death and survival decisions. A lowering of the ER Ca2+-store content has been shown to serve as a survival mechanism exploited by several pro-survival proteins and oncogenes, including Bax inhibitor-1 (BI-1), antiapoptotic Bcl-2 proteins, and Ras  and subsequent increase in autophagic flux , an upstream activator of AMPK. Thus, low [Ca2+]ER can suppress autophagy by diminishing [Ca2+]cyt. It was proposed that antiapoptotic Bcl-2, by lowering the ER Ca2+ levels, could suppress cytosolic Ca2+ signals evoked by various pharmacological and physiological agents, thereby counteracting the activation of CaMKKβ-controlled autophagy  in the mitochondrial matrix, resulting in the opening of the mPTP . Th. Th2+ onher hand , 72. Indcy Table .2+ signaling via calcineurin-mediated dephosphorylation of Drp1  and Mfn-2, and mitochondrial fission, mainly mediated by the cytosolic soluble dynamin-related protein 1 (Drp1) , 97. Mit of Drp1 . Mitocho+ uptake , 100. Hy+ uptake .3R-driven Ca2+ signals and subsequent ER–mitochondrial Ca2+ transfer impact cell death and survival processes. Inhibition of IP3Rs and thus spontaneous Ca2+ signals lead to reduced mitochondrial bioenergetics and increased autophagy, allowing cell survival , which may be due to differences in the produced breakdown products and their usage in metabolic and biosynthetic pathways . T. T2+ fluescribed . The G1-escribed , cells wescribed  and an aescribed . In lighfor CDKs . HoweverS arrest . Thus, r2+ fluxes, ER–mitochondrial contact sites and Ca2+-signaling events might be altered to favor cancer cell survival. This concept is supported by another recent study. TMX1, a redox-sensitive oxidoreductase that is enriched in the MAMs in a palmitoylation-dependent manner, was shown to regulate mitochondrial bioenergetics and in vivo tumor growth by controlling ER–mitochondrial Ca2+ signaling  require further research.In addition to the addiction of some cancer cells to constitutive ER–mitochondrial Caignaling , 76. Upod others , loss ofynthesis , 104. Ho2+ transfers will not only impact mitochondrial bioenergetics but also cancer cell senescence and sensitivity toward chemotherapeutic drugs  but could also lead to cell-death resistance, as has been shown recently for tumor cells lacking PML (2+ uptake. As a consequence, FATE1 upregulation, like in adrenocortical carcinoma cells, results in cell-death resistance not only in response to pro-apoptotic stimuli that impinge on ER–mitochondrial Ca2+ signaling but also in response to mitotane, a chemotherapeutic drug clinically used in the treatment of patients with adrenocortical cancer. Moreover, FATE1 expression is also inversely correlated with the overall survival of adrenocortical cancer patients (2+ transfer will favor cell-death therapies (2+ contact sites (2+ overload and subsequent cell death (2+ transfer and the contact sites (2+-dependent step is resveratrol (2+ uptake of cancer cells, while normal cells remained unaffected. Similarly to other phenols, resveratrol inhibits ATP synthase and impairs ATP production, thereby decreasing mitochondrial [ATP] without affecting cytosolic [ATP] (2+ through IP3Rs and augmenting mitochondrial uptake (Table 2+ uptake in cancer and in normal cells in the presence of resveratrol was attributed to the enhanced and more stable MAMs in cancer cells, which facilitate the ER–mitochondrial Ca2+ transfer (via a mechanism that requires cytosolic Ca2+ and the presence of IP3Rs. In this study, resveratrol triggered a depletion of the ER in intact cells independently of IP3Rs, but not in permeabilized cells where Ca2+ stores are loaded by application of ATP, arguing against a direct inhibition of SERCA by resveratrol. Thus, these findings may relate to an in cellulo decline in SERCA activity due to a decline in ATP (The adequate ER–mitochondrial Caignaling , 113. Upll death . In additransfer . Thus, dking PML  or FATE1king PML . FATE1 iking PML . Recentlpatients . Oppositherapies . Interesherapies , 115 couct sites . Specifill death . Moreovect sites . Anotherveratrol . This naic [ATP] . This retransfer . In addie in ATP .2+ fluxes impact several cancer hallmarks, including mitochondrial metabolism, autophagy, apoptosis resistance, and metastasis. It is very likely that different tumor stages require different levels of ER–mitochondrial Ca2+ flux for instance to ensure cell survival at early stages, promote invasion at intermediate stages and tumor growth at late stages. Moreover, different oncogenes and tumor suppressors exert their part of their function at the MAMs by impacting Ca2+-transport systems.Endoplasmic reticulum–mitochondrial Ca2+ transfers. Thus, suppressing these basal ongoing ER–mitochondrial Ca2+ fluxes represent a therapeutic strategy to target tumor cells thereby suppressing their survival, invasion and growth.An emerging concept is that cancer cells become addicted to constitutive ER–mitochondrial Ca2+ fluxes appear instrumental for proper therapeutic responses to chemotherapeutic drugs, since an adequate ER–mitochondrial Ca2+ transfer is important for their cell death-inducing properties. Hence, enhancing ER–mitochondrial Ca2+ transfer may provide an attractive strategy to overcome cell death resistance of certain types of cancer toward chemotherapeutics.In contrast to this, ER–mitochondrial Ca2+ transfers hold therapeutic potential, dependent on the clinical stage of the tumor and the applied anticancer strategy. However, a major challenge will be to limit these effects to cancer cells, as obviously ER–mitochondrial Ca2+ fluxes also underlie the survival of healthy cells. Nevertheless, the presence, composition, and properties of ER–mitochondrial contact sites in healthy versus cancer cells and the dependence of these cells on these sites for cell survival may be strikingly different, creating a therapeutic window for the selective targeting of cancer cells while sparing healthy cells.Hence, it is expected that both dampening and boosting ER–mitochondrial CaGB and RR drafted the manuscript. HI, MK, RR, and GB wrote parts of the manuscripts. HI, RR, and GB prepared figures. All the authors have read and approved the final version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer, IS, and handling editor declared their shared affiliation, and the handling editor states that the process nevertheless met the standards of a fair and objective review.


In many cases, researchers resort to tools that were optimized in animal cells. However, the obviously large evolutionary distance between plants and animals implies that there is a good chance observed effects may not be specific to the intended plant target. Here, we provide an overview of pharmacological strategies that are commonly used to activate or inhibit plant Ca2+ signalling. We focus on highlighting modes of action where possible, and warn for potential pitfalls. Together, this review aims at guiding plant researchers through the Ca2+ pharmacology swamp.Calcium is one of the most pleiotropic second messengers in all living organisms. However, signalling specificity is encoded via spatio-temporally regulated signatures that act with surgical precision to elicit highly specific cellular responses. How this is brought about remains a big challenge in the plant field, in part due to a lack of specific tools to manipulate/interrogate the plant Ca In its most simple form, the sensing of Ca2+ elevation signals a breach of membrane integrity. This is true for the simplest Prokaryotes and the most complex, multicellular Eukaryotes. This basic signalling function is a by-product of the cell’s need to avoid high intracellular Ca2+ levels, as this can interfere with the primary metabolism due to the propensity of Ca2+ to form insoluble precipitates with phosphates. While still being an unwanted guest in high concentrations, evolution has integrated Ca2+ signalling into complex signalling cascades that fine-tune many, if not all cellular processes.The ion Ca2+ binding motif cyt that are induced by extracellular ATP and ADP via their ATPase and GTPase activities cyt increase 1 (OSCA1.1) and Calcium Permeable Stress-gated cation Channel1 (CSC1/OSCA1.2) were identified as hyperosmolality-gated Ca2+-permeable channels cyt is restored to its resting levels. While Ca2+ channels are responsible for the fast influx of Ca2+ into the cytosol after detection of specific stimuli, the active efflux of Ca2+ out of the cytosol after a Ca2+ signalling event is regulated by three classes of Ca2+ transporters: P-type Ca2+-ATPases, Ca2+/H+ antiporters (CAX) and possibly cation/Ca2+ exchangers (CCXs).When a Ca2+-ATPases are Ca2+ efflux pumps that are directly energized by ATP and belong to the second subclass of phosphorylated (P)-type ATPases (PII-ATPases), a family of ion pumps that are ubiquitous in all life forms. In plants, they can be further subdivided in two groups: PIIA- and PIIB-ATPases cyt increases triggered by the pollen ligand SP11/SCR (S-locus protein 11) [2+ currents that are induced by microbe-induced molecular patterns (MAMPs) were blocked by several animal iGluR antagonists [Physcomitrella patens, PpGLR1 currents could be inhibited by CNQX and AP5 [2+ currents in planar lipid bilayers, corroborating a direct effect on GLR-function [2+ signals reflect the inhibition of GLR3.4 and related GLRs [Several popular competitive antagonists of iGluRs, namely DNQX, CNQX, and AP5, were able to inhibit pollen tube growth in tobacco seedlings . In Arabtein 11) . Furtheragonists . Besidesagonists ,124,125) and AP5 . The hyp and AP5 . This obfunction . Therefoted GLRs ,124.2+-binding protein in eukaryotic organisms. The high degree of sequence conservation makes that many of these CaM antagonists are also active in plants. They can interfere with canonical CaMs, probably several CaM-likes and CPKs, which have a CaM-based auto-inhibitory domain.A popular inhibitor target is CaM, the most important intracellular Ca2+-dependent manner and block its ability to activate enzymes [The oldest and most commonly used CaM antagonists include the phenothiazines  and chlorpromazine), which strongly interact with CaM in a Ca enzymes ,130. The enzymes ,273,274. enzymes ,276). To enzymes ,278) and enzymes , making  enzymes .2+ dependent protein kinases (CPKs) in which the autoinhibitory domain consists of a CaM moiety. Via such a vast array of potential targets, CaM antagonists elicit strong pleiotropic effects in plants. This ranges from secretion [Funaria [2+ and protein import [Besides several CaM isoforms, higher plants also contain calmodulin-like (CML) proteins and Caecretion , mitoticecretion , auxin tecretion , gravitrecretion , red andecretion , inhibit[Funaria , plant g[Funaria , and pern import ,287. Addn import ,129 whicn import ,290,291.n import .2+ influx mechanisms, it is sometimes desirable to induce or facilitate the influx of Ca2+ ions across membranes. For this purpose, several methods are available. One approach is the use of Ca2+ ionophores that directly facilitate the transport of Ca2+ across the plasma membrane. Two commonly used Ca2+ ionophores are A23187  and ionomycin.Instead of inhibiting Ca2+, it also functions as an efficient Ca2+ ionophore and has thus been used extensively to increase intracellular Ca2+ concentrations in intact cells . Ho. Ho2+, it cells  are potent inhibitors of ACAs, [2+/Heavy metal pump AtHMA1 [2+ homeostasis. Eosin Y affects ACA activity with 10,000 fold greater affinity than that of H+ ATPases [+ [2+ ATPases may affect the intracellular pH. However, this proton current is independent of the concentration gradient [2+ ATPase activity cannot be modulated via the extracellular pH.All Caof ACAs, ,139,141,of ACAs, ). Thapsip AtHMA1 , but notp AtHMA1 , suggest ATPases ), Eosin Pases [+ . This imgradient , implyin2+ signalling research has seen great progress over the last years. However, while great insights and understanding have been gained about the Ca2+ signalling components responsible for decoding and translating Ca2+ signatures, such as calmodulins, CMLs, CPKs, and CBL/CIPK complexes, our knowledge about the machinery responsible for generating these Ca2+ signatures severely lags behind. Many putative Ca2+-permeable channels have been identified in plants, including CNGCs, GLRs, Annexins, MSCCs, OSCAs, and TPC1, our knowledge of the physiological roles of these channels in Ca2+ signalling is still limited. While Ca2+ signalling mutants form promising tools for identifying more Ca2+-permeable channels in plants, this strategy is not without its drawbacks. Indeed, Ca2+-permeable channel families in plants often consist of many different structurally similar members, which can share significant functional redundancy between each other, thus hindering genetic studies on these channels. Therefore, the use of agonists and antagonists that specifically interact with Ca2+-permeable channels or other Ca2+-components forms an interesting alternative for studying Ca2+-signalling processes in plants. The currently most frequently used Ca2+ signalling blockers, such as L-type Ca2+ channel blockers, GLR antagonists and CaM antagonists, are mostly derived from the animal field. However, since the Ca2+ signalling machinery differs significantly between the plant and animal kingdoms, these inhibitors often have undesirable off-target effects or unknown targets in planta. Therefore, an obvious strategy to obtain specific Ca2+ channel inhibitors is to start an unbiased de novo chemical screen for inhibitors directly in plant systems. A suppressor screen of cpr22 (AtCNGC11/12)-induced seedling lethality yielded 13 chemicals that partially restored seedling growth [2+ channel blockers, dibucaine erythrosine B and diethylstilbestrol. However, it was not tested if these inhibitors affected the Ca2+ channelling activity of the hyperactive CNGC-based CPR22 channel [2+-permeable channels, in combination with chemical compound screen approaches could lead to the identification of such plant specific Ca2+-signalling inhibitors, leading to the generation of a more robust toolset for studying plant Ca2+-signalling pathways in the future.Plant Cag growth , which i channel . Another


Although the underlying causes for T‐tubular electrical failure are still unknown, our findings suggest that they are likely to be related to local ultrastructural alterations. Here, we first review the experimental approach that allowed us to observe and dissect the consequences of TATS electrical dysfunction and then propose two different strategies to unveil the reasons for T‐tubular electrical failures. The first strategy consists in a correlative approach, in which the failing T‐tubule identified with the RAMP microscope is then imaged with electron microscopy. The second approach exploits the diffusion of molecules within TATS to gain insights into the local TATS structure, even without a thorough reconstruction of the tubular network. Although challenging, the local electrical failure occurring at single T‐tubules is a fundamental question that needs to be addressed and could provide novel insights in cardiac pathophysiology.Electrical excitability is an essential feature of cardiomyocytes and the homogenous propagation of the action potential is guaranteed by a complex network of membrane invaginations called the transverse‐axial tubular system (TATS). TATS structural remodelling is a hallmark of cardiac diseases and we demonstrated that this can be accompanied by electrical defects at single T‐tubular level. Using a random‐access multi‐photon (RAMP) microscope, we found that pathological T‐tubules can fail to conduct action potentials, which delays local Ca AODacousto‐optic deflectorAPaction potentialCICRcalcium induced calcium releasecTnTcardiac troponin TDHPRdihydropyridine receptorECCexcitation–contraction couplingEMelectron microscopyHCMhypertrophic cardiomyopathyHFheart failureNCX+–Ca2+ exchangerNaRAMPrandom access multi‐photonRyR2ryanodine receptor 2SERCA2+‐ATPasesarcoplasmic reticulum CaSRsarcoplasmic reticulumSSsurface sarcolemmaTATStransverse‐axial tubular systemTPFtwo‐photon fluorescenceTTT‐tubuleVSDvoltage‐sensitive dyeet al. et al. et al. 2+ release from the sarcoplasmic reticulum, and eventually the contraction of myofilaments . During the plateau of each AP, Ca2+ enters the cell through depolarization‐activated Ca2+ channels, known as dihydropyridine receptors (DHPRs). Ca2+ binds to the ryanodine receptor 2 (RyR2) inducing a massive release of Ca2+ from the sarcoplasmic reticulum (SR) . The combination of external Ca2+ influx and release from the SR raises the free intracellular Ca2+ concentration allowing Ca2+ to bind to troponin C and switch on the contractile machinery. The SR Ca2+‐ATPase (SERCA) and sarcolemmal Na+–Ca2+ exchanger (NCX) lower the intracellular Ca2+ concentration allowing muscle relaxation  is distributed in the TATS. Alternatively, currents distribution has been studied by selectively disrupting the T‐tubular network from SS. This technique, named detubulation, has been described and extensively validated in isolated ventricular myocytes  or T‐tubules  have identified pathological alterations of the TATS for the first time  did not show any voltage variation while a stimulated AP normally occurs on the surface sarcolemma and neighbour T‐tubules;2.the presence of local spontaneous depolarizations that occur only in AP‐failing T‐tubules and do not propagate to the whole sarcolemma.A number of studies have speculated that the AP at the T‐tubules is longer than at the SS in ventricular cardiomyocytes based on differences in channel density and electrochemical cation gradients between the two membrane domains . In HF, Ca2+ transients are globally delayed compared to control, but the electrical abnormalities further impair the local Ca2+ release. In fact, Ca2+ transients in correspondence with electrically failing T‐tubules show a significant additional delay likely to be due to a propagative Ca2+‐cascade from the neighbouring functioning sites . Moreover, we observed that local spontaneous depolarizations occasionally trigger local Ca2+ release . V‐sparks may represent a novel pro‐arrhythmogenic phenomenon in HF setting. Finally, electrical defects can locally blunt β‐adrenergic signalling in HF , while TATS morphological alterations were minimal , expressing a human mutated cardiac troponin T  that is associated with high risk of sudden cardiac death in patients  and the dispersion of the current across the membrane (rm) =V0e−xλ where λ is the membrane space or length constant, x is the distance from the site where AP is originated (the surface sarcolemma), and V0 is action potential amplitude at x=0. The length constant λ can be calculated as λ=rm/r TATS  and a theoretical estimation indicates it is ≅240μm , the cardiomyocyte responds in an active manner by opening voltage‐gated ion channels, producing an all‐or‐none response in the form of an AP. Yet, membrane potential variations elicit also passive (electrotonic) responses of the cellular membrane that do not need the opening of gated ion channels. Considering the AP propagation through the TATS, the extent of passive responses is only limited by the voltage drop due to the current flowing down the TATS (m) Fig. . The chaBased on the considerations above, the underlying causes for electrical failure at the level of single T‐tubules seem to relate to ultrastructural alterations such as local strictures of the T‐tubular lumen. Strictures may produce a local drop of conductivity at the tubular level and thus a sudden interruption of electrical propagation; this alteration could also represent the earlier event of T‐tubular detachment.Here, we propose two very different approaches to address this issue: (i) a correlative study, and (ii) an assessment of the diffusion of the TATS.correlative microscopy. Correlative microscopy has been widely used for a variety of investigations  is probably the first‐choice tool for the fine exploration of biological structures. In fact, the spatial resolution of EM is superior to optical microscopy and can extend to 1 nm, assuring a proper reconstruction of the detailed T‐tubular ultrastructure. However, EM provides a very limited field of view and, most importantly, requires fixed samples, which undermines the possibility of studying the electrical function of T‐tubules. Consequently, a combined strategy could be employed: first, the failing T‐tubule can be located by using the RAMP microscope and then, the ultrastructure of that particular failing T‐tubule could be accurately outlined exploiting the nanometric resolution of the EM. The integration of multiple systems for a multi‐level study of the same biological event is called D'can be defined for T‐tubules as D′=Dσπr TT 2, where σ is the superficial density of T‐tubules and rTT is the T‐tubular radius, i.e. the ratio between the T‐tubular cross‐sectional area and the total area of the cellular surface. We hypothesize that diseased cardiomyocytes characterized by failing T‐tubules display local structural alterations that suddenly affect the passive voltage drop of AP as well as the availability of space for molecules to diffuse into the TATS lumen. Thus, studying the diffusion of fluorescent molecules from extracellular space to the TATS could provide information regarding the geometry of the system itself.A totally different approach for studying T‐tubular ultrastructure could exploit the diffusion within the TATS. Diffusion refers to the process by which mass is transferred from a region of high concentration to a region of low concentration. Molecules that are impermeable to the membrane can diffuse in the extracellular space including the TATS lumen. Of course, the TATS represents a restrictive diffusion space and the smaller the accessible section the slower is the diffusion process. A diffusion coefficient et al. In conclusion, we propose, here, two conceptually different strategies to investigate the biophysical reasons underlying T‐tubular electrical defects. The development of non‐invasive ultrafast technologies for imaging and optically controlling cardiac function , by the Italian Ministry for Education, University and Research in the framework of the Flagship Project NANOMAX, by the Italian Ministry of Health (WFR GR‐2011‐02350583), by Telethon–Italy (GGP13162), by Ente Cassa di Risparmio di Firenze (private foundation), and by Regione Toscana .


Currents through L-type Ca2+ channels were expected to be dominant; however, action potentials were not eliminated in nominally Ca2+-free bathing solution or by addition of L-type Ca2+ channel blocker nifedipine (10 μM). Instead, Na+ channel blocker tetrodotoxin  reduced the maximal velocity of the upstroke at low, but not at normal (2 mM), Ca2+ in the bath. The magnitude of TTX-sensitive currents recorded with 140 mM Na+ was about 20 pA/pF. TTX-sensitive currents decreased five-fold when Ca2+ increased from 2 to 10 mM. The currents reduced three-fold in the presence of 10 mM caffeine, but remained unaltered by 1 mM of isobutylmethylxanthine (IBMX). In addition to L-type Ca2+ currents (15 pA/pF in 20 mM Ca2+), we also found Ca2+ currents that are resistant to 10 μM nifedipine (5 pA/pF in 20 mM Ca2+). Based on their biophysical properties, these Ca2+ currents are likely to be through voltage-gated T-type Ca2+ channels. Our results suggest that Na+ and at least two types (T- and L-) of Ca2+ voltage-gated channels contribute to depolarization of smooth muscle cells in skeletal muscle arterioles. Voltage-gated Na+ channels appear to be under a tight control by Ca2+ signaling.Voltage-dependent inward currents responsible for the depolarizing phase of action potentials were characterized in smooth muscle cells of 4 Since specific blockers of voltage-gated Ca2+ channels suppress both the upstroke and the after-depolarizing components of action potentials, Ca2+ channels are thought to be the main pathway for the depolarizing current. Several studies have recorded currents through dihydropyridine-sensitive Ca2+ channels (CaV1.2) in the arteries of various vascular beds ) measured by Ca2+-selective electrode was 2–5 μM. The extracellular solutions 10Ca, 20Ca, and 20Ba contained (in mM): 150 NaCl, 10 HEPES, and corresponding concentration of either CaCl2 or BaCl2. The intracellular solution for whole-cell voltage-clamp experiments contained (in mM): 20 CsCl, 110 aspartic acid, 5 Mg-ATP, 4.5 CaCl2 ([Ca2+free] ≈ 100 nM), 10 HEPES, and 10 EGTA. The pipette solution for perforated patch-clamp experiments contained (in mM): 150 KCl and 10 HEPES. All solutions were at pH = 7.3, 310–315 mOsm/kg and room temperature (RT). All chemicals, unless specified, were purchased from Sigma, USA.The dissection solution contained (in mM): 140 NaCl, 4 KCl, 1.5 MgClMice with C57BL6/C57BL10 genetic backgrounds were used in this study. Animals were sacrificed for the purposes of other studies –36 and tThe study conforms to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health . The Newark IACUC of Rutgers University is Fully Accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International . Rutgers University has an Animal Welfare Assurance on file with the Public Health Service (PHS) Office of Laboratory Animal Welfare (OLAW). The Assurance number is D16-00098 (A3158-01). IACUC protocol for studies the tissue was harvested from (post mortem) is 05072D0109. The Institutional Animal Care and Use Committee is constituted in accordance with the AWA and the PHS Policy. IACUC approval was not required according to PHS policy for the use of dead animals or parts of animals which were not sacrificed for the purpose of obtaining or using their tissues or other materials, and for which there was no project-specific antemortem manipulation prior to sacrificing them.Fourth-order arterioles were enzymatically isolated from total of 42 animals  of either sex. First, semitendinosus and biceps femoris muscles were excised from the animal and placed in the dissection solution. Muscles were cleaned of fat and connective tissue, chopped into small pieces and transferred into 0.35% collagenase  dissolved in the dissection solution. The tissue was shaken at 130 rpm, 37C°, for 45–70 min depending on the animal’s weight. After digestion, the tissue was centrifuged for 45 sec at 1,000 rpm and gently washed with dissection solution containing 10% of fetal bovine serum to stop enzymatic reaction. Approximately 0.2 mL of digested tissue was added to the electrophysiology perfusion chamber mounted on an inverted microscope and individual, isolated vessel were identified  using the normal distribution function and conventional estimates of standard deviation of the difference. In t-test. Curve fitting was done by a nonlinear least-squares routine of Sigma Plot .Measured values are presented as sample average ± SEM. The statistical significance of the difference in averages was established using the two-tailed + channel blocker TTX  extracellular solution, consistent with the idea that both Ca2+ and Na+ ions contribute to the depolarizing current. Importantly, TTX had much stronger effect in 0Ca, rather than in 2Ca, solution . The slower current was likely to be through Ca2+ channels, as its magnitude increased with [Ca2+] . Averaged peak current–voltage relationships are shown in peak = 20 ± 2 A/F (n = 6) with 2 mM Ca2+ in the bath. In the absence of TTX, peak current amplitudes declined with increase in [Ca2+], n = 6. However, peak current amplitudes recorded in the presence of TTX increased with [Ca2+], indicating that the TTX-resistant current was entirely through Ca2+-selective channels. The maximal density of TTX-sensitive current dropped to Ipeak = 4 ± 1 A/F when extracellular Ca2+ was changed from 2 mM (n = 6) to 10 mM (n = 4).To dissect inward ionic currents, we used the ruptured whole-cell voltage-clamp with cesium-based pipette solution designed to block outward K2+ ions partially block TTX-sensitive Na+ channels . Caffeine is known to have multiple effects on blood vessels [2+ release channels of the sarcoplasmic reticulum and hence elevates intracellular bulk [Ca2+] well above 0.5 μM during the first 2–3 minutes of application. In contrast to vessel constriction induced by K+ depolarization, caffeine induces only a small transient contraction by accelerating Ca2+ re-uptake into the sarcoplasmic reticulum [+ currents are affected by 1 mM IBMX. IBMX blocks various phosphodiesterases with IC50 of 1–50 μM [2+] in small mesenteric arteries of rats ),, portal NaV1.8, ), and va,  shifts the voltage-dependence of their activation to more positive voltages [2+-dependent down-regulation of Na+ channels might be via the Protein Kinase C (PKC) pathway. It has been shown that activation of PKC decreases peak sodium currents through brain NaV1.2 and skeletal muscle NaV1.4 channels by up to 80% [TTX-sensitive Nalmodulin –62. Ca2+irection . NaV1.3 voltages . Therefop to 80% , 65.2+ currents in normal- and patho-physiology of various vascular beds is under intense investigation. Two separate voltage-dependent Ca2+ currents have been found in smooth muscle cells of resistance arteries in cerebral [2+ currents were recorded in single smooth muscle cells isolated from resistance arteries of hamster cremaster muscle [2+ currents in arterioles of leg muscles of mice is expected since both CaV3.1 and CaV3.2 T-type channels are expressed at the messenger RNA level in rat cremaster arterioles [2+ did not exhibit specificity in blockage of T-type currents. Application of 40 μM of Ni2+ to the bath, which should block primarily CaV3.2 channels [V3.1 and CaV3.2 channels were present. It is not clear why T-type Ca2+ currents were not found in hamster cremaster [The exact role of T-type voltage-gated Cacerebral , 13, 63,cerebral  and mesecerebral –68 vascur muscle . Our finterioles . In our channels , had onlremaster . It is premaster  is that 2+ influx through T-type Ca2+ channels contributes to vascular contractility [V3.2 T-type channel die from a constitutive spasm of coronary arteries because of impaired relaxation of smooth muscle cells [2+ channel is essential for relaxation of smooth muscle cells.Several groups have proposed that Caactility , 70. Howle cells , 71. Alt2+ due to constitutively active L-type Ca2+ channels (sparklets) have been proposed to be primary for both the near-membrane and global Ca2+ control of smooth muscle cell contractility [2+ due to L-type Ca2+ channels was found to be from –40 to –15 mV, peaking at –30 mV [2+ channels is from –90 to –30 mV [2+ channels in smooth muscle cells of skeletal muscle arterioles activate at least 20 mV more positively than in neurons, their activation occurs at more negative voltages in comparison with L-type Ca2+ channels  and Ca2+-dependent stabilization of negative voltages . Ca2+ influx through the low-voltage activated T-type Ca2+ channels might serve to inhibit Na+ channels and thus prevent the occurrence of action potential spikes.To summarize, we developed a methodology that allows for the isolation and study of murine skeletal muscle arterioles in ex-vivo preparation. We confirmed that smooth muscle cells of skeletal muscle arterioles express L-type voltage-gated CaS1 FigThe tail currents elicited after activating step to 30 mV had significant slow component (tracings a). The slow component was absent if the conditioning that inactivates currents at –20 mV preceded the activating step (tracings b). Similar observations were done on three cells (n = 3).(TIF)Click here for additional data file.S2 Figx-axis by intervals proportional to the pre-pulse voltage (indicated). Before averaging (n = 3), amplitudes of the tail current in each cell were normalized to their value for activation step to 50 mV.Test voltage protocol is shown at the top. Traces of representative currents for different pre-pulse are shifted along the (TIF)Click here for additional data file.S3 FigRecording conditions: gramicidin-perforated patch-clamp with 2Ca bathing solution and 150 mM KCl in the pipette. Similar rhythmic activity was observed in six out of 81 cells studied. Most cells had stable membrane potential (n = 6).(TIF)Click here for additional data file.S4 FigA, T-type Ca2+ currents were recorded in 2Ca solution in the presence of 1 μm TTX and 10 μm Nifedipine, with and without 40 μm Ni2+. B, Averaged peak current-voltage relationships (n = 5). Application of 40 μm Ni2+ did not have a significant effect on the magnitude of T-type Ca2+ currents.(TIF)Click here for additional data file.


Background: Hypertrophic cardiomyopathy (HCM) is the most common inherited cardiac muscle disease with left ventricular hypertrophy, interstitial fibrosis and diastolic dysfunction. Increased myofilament Ca2+ sensitivity could be the underlying cause of diastolic dysfunction. Epigallocatechin-3-gallate (EGCg), a catechin found in green tea, has been reported to decrease myofilament Ca2+ sensitivity in HCM models with troponin mutations. However, whether this is also the case for HCM-associated thick filament mutations is not known. Therefore, we evaluated whether EGCg affects the behavior of cardiomyocytes and myofilaments of an HCM mouse model carrying a gene mutation in cardiac myosin-binding protein C and exhibiting both increased myofilament Ca2+ sensitivity and diastolic dysfunction.Methods and Results: Acute effects of EGCg were tested on fractional sarcomere shortening and Ca2+ transients in intact ventricular myocytes and on force-Ca2+ relationship of skinned ventricular muscle strips isolated from Mybpc3-targeted knock-in (KI) and wild-type (WT) mice. Fractional sarcomere shortening and Ca2+ transients were analyzed at 37°C under 1-Hz pacing in the absence or presence of EGCg (1.8 μM). At baseline and in the absence of Fura-2, KI cardiomyocytes displayed lower diastolic sarcomere length, higher fractional sarcomere shortening, longer time to peak shortening and time to 50% relengthening than WT cardiomyocytes. In WT and KI neither diastolic sarcomere length nor fractional sarcomere shortening were influenced by EGCg treatment, but relaxation time was reduced, to a greater extent in KI cells. EGCg shortened time to peak Ca2+ and Ca2+ transient decay in Fura-2-loaded WT and KI cardiomyocytes. EGCg did not influence phosphorylation of phospholamban. In skinned cardiac muscle strips, EGCg (30 μM) decreased Ca2+ sensitivity in both groups.Conclusion: EGCg hastened relaxation and Ca2+ transient decay to a larger extent in KI than in WT cardiomyocytes. This effect could be partially explained by myofilament Ca2+ desensitization. MYH7 (encoding β-myosin-heavy chain) and MYBPC3 (encoding cardiac myosin-binding protein C), which constitute about 80% of known mutations. Besides a typical hypertrophy of the left ventricle, patients often present a normal or increased ejection fraction, but a compromised diastolic function with an incomplete relaxation and increased filling pressures  is the most common cardiac genetic disease, with more than 1400 different mutations in genes encoding primarily sarcomeric proteins  developed by us and others . Additionally, myofilaments with increased sensitivity to Ca2+ may act as Ca2+ buffers, prolonging the export of Ca2+ and relaxation time which could contribute to diastolic dysfunction and arrhythmias  mutant  and cTnT mutants in a reconstituted acto-myosin system  mice.Epigallocatechin-3-gallate (EGCg), a major component of green tea, has been suggested to be effective against cardiovascular diseases. Proposed mechanisms were anti-oxidative, anti-inflammatory, vasorelaxant, and positive inotropic effects  perfusion system. After retrograde perfusion with Ca2+-free buffer solution  for 6.5 min, hearts were digested with 0.075 mg/ml Liberase TM  dissolved in buffer solution containing 12.5 μM CaCl2 for 7–8 min. Ventricles were disconnected from the atria and minced with forceps to dissociate single cardiomyocytes. Afterwards Ca2+ was introduced stepwise up to a concentration of 1 mM.Cardiomyocytes were isolated from WT and KI mouse heart ventricles as previously described  as described . When the cells showed stable contraction amplitude, contractile function was recorded. Subsequently, the perfusion was switched to buffer containing different EGCg concentrations  and contractile function was recorded again.For contractile analysis only rod-shaped myocytes without membrane blebs, hypercontractile zones, and spontaneous activity showing a stable contraction amplitude and rhythm at 1-Hz pacing frequency (4 ms long 10 V pulses) and 37°C were recorded. Sarcomere shortening and Ca2+ relationships, trabeculae were prepared from the left ventricular endocardial surface of WT and KI mice as reported before . Triton X-100 permeabilized strips of the left ventricle of WT and KI mouse hearts were mounted between a force transducer and a length controller. Trabeculae were stretched above slack length until they developed force in activating solution (pCa 4.5) at 15°C. Subsequently they were exposed to increasing Ca2+ concentrations from pCa 9 to pCa 4.5 in EGTA-buffer. Force development was measured in each pCa solution. Measurements were repeated in the presence of 30 μM EGCg after 5 min preincubation in relaxing solution , and with one-way ANOVA, followed by Bonferroni's post-test as indicated in the figure legends , as indicated in the figure legends. Concentration response curves were fitted to the data points and force-pCa relationship comparison was done by using extra sum-of-squares F-test . A value of P < 0.05 was considered statistically significant.Data were expressed as mean±SEM. Comparisons were performed by paired or unpaired Student's Mybpc3 KI mice, which showed lower diastolic sarcomere length and higher twitch amplitude than WT cardiomyocytes . The positive inotropic effect of EGCg occurred within 5 min of exposure and was reversible by washout (loss of effect after 5 min). The highest concentration of EGCg that did not alter myocyte contractions was 1.8 μM  increased sarcomere shortening . Plotting the sarcomere length against the F340/380 ratio of Fura-2 in WT cells showed that with 1.8 μM EGCg, the descending phase of the relation between the F340/380 ratio and the sarcomere length was shifted to the right, and the relaxed state of the sarcomere length in diastole was reached at higher F340/380 ratios, resulting in smaller loops . Since other groups had reported that EGCg concentrations below 30 μM had no effect on Ca2+ sensitivity we also used 30 μM  mutant  and cTnT mutants in a reconstituted acto-myosin system , as reported with 10 nM  vesicles isolated from rabbit left ventricles  The Ca2+-desensitizing effect of EGCg on the permeabilized trabeculae was only detected at 30 μM, which is higher than the concentrations used in the intact myocyte experiments. This discrepancy has also been observed before  The 2+ transient decay in Mybpc3 WT and KI cardiomyocytes, which seems to be partly due to Ca2+ desensitization of the myofilaments. We show for the first time that EGCg is also effective in a thick filament mutation mouse model. In support of other studies  and the Deutsche Stiftung für Herzforschung (F/28/12).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.


Despite technological advances, the prognosis and survival of acute myeloid leukemia (AML) adult patients remain low, compared with other hematologic malignancies. Some antigens detected by immunophenotyping may soon play a significant role in the pathophysiologic, prognostic, and overall survival (OS) rate of AML patients. Therefore, we conducted a systematic review and meta-analysis of PubMed, Scopus, Science Direct, Web of Science, and the Cochrane Library (using PRISMA guidelines). We analyzed 11 studies and 13 antigens, detected through the immunophenotyping of 639 patients. From them, twelve exhibited a negative impact with AML prognosis. The meta-analysis demonstrated a high expression of AML markers, which have been associated with a decrease in survival over 10 months  and over 20 months . Knowing that the expression of immunophenotypic markers, which are not used on a routine basis, might be able to influence disease behavior, looks promising. However, they have been associated with a poor prognosis as well as a decrease in survival. This may allow for different chemotherapeutical protocols, including future studies for new therapeutic targets. The genetic heterogeneity of this group of hematological malignancies makes it impractical to perform initial analyses that can encompass different genes involved in AML. This makes diagnoses difficult, which can negatively influence therapeutic strategy4. Even with major improvements in the understanding and treatment of AML over the past several years, few advances have been made in the outcomes and survival of AML patients6. Complete remission is expected for approximately 60‒70% of adults with AML after the induction phase of treatment, but only about 25% survive three or more years with the possibility of being cured7. The 5-year relative survival rate for patients from birth to 19 years has been reported to be 62.8%, but declines to 5.4%, for patients older than 65 years8.Acute myeloid leukemia (AML) is an aggressive hematological malignancy characterized by clonal proliferation of immature myeloid cells at various stages of maturation10. AML presents highly heterogeneous immunophenotypic profiles, which is probably due to genetic diversity. Leukemia-associated phenotypic markers (LAIPS) are useful to discriminate between normal/reactive immature myeloid precursors from leukemic cells and is commonly used in minimal residual disease (MRD) studies. In addition, LAIPS are also associated with molecular alterations with well-recognized prognostic implications (such as CD19 expression in AML with RUNX1-RUNX1T1)12.Multiparameter flow cytometry (MFC) immunophenotyping provides relevant information for AML diagnosis, classification, and monitoring. MFC allows identification, quantification, and lineage assessment of abnormal blast cells and disease classification according to the maturation stage to be made13. Improving the accuracy of prognostic assessment of AML may allow the treatment to be more specific and risk-adapted, increase the probability of cure, and minimize treatment-related morbidity and mortality14.Previous studies show that a great number of distinct antigens affect AML prognosis and prediction. Nevertheless, difficulties are still found in performing accurate risk stratification for diagnoses based on immunophenotypic featuresSince there are no clear recommendations and no consistent approaches to the use of new markers in the immunophenotypic panels for AML evaluations as well as for the markers’ influence on determining prognosis and survival in clinical practice, a systemic review was done. In this review, we aimed to identify relevant publications about the influence of these new monoclonal antibodies used as immunophenotypic markers in the prognosis and survival of AML patients.15.A systematic review was performed based on a scientific research protocol describing the aims and methods used. Within the limitations of the research in this area, this synthesis was performed according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statementAre there new immunophenotypic markers that may aid in the prognosis and survival of acute myeloid leukemia”?The question of this systematic literature review was: “The literature search was conducted using Pubmed, Science Direct, Web of Science, Scopus and Cochrane Library databases for articles published from 2012 to 2015. In addition, the reference lists of relevant papers were searched for additional AML studies.The following search terms consisted of a range of pertinent terms: 1) antigens CD (MeSH); 2) antigens, differentiation (MeSH); 3) biological Markers (MeSH); 4) tumor markers, 5) biological (MeSH) and prognosis (MeSH); 6) survival rate (MeSH); 7) survival analysis (MeSH) and leukemia myeloid, acute (MeSH); or 8) acute leukemia.16); (5) articles assessing AML cases; and (6) articles with available abstract and full text.The articles found in the search were compared with the previously defined inclusion criteria to determine the relevance of the study: (1) papers published from 2012 to 2015; (2) articles published in English, Spanish, and Portuguese; (3) articles that used immunophenotyping in their methodologies; (4) articles assessing monoclonal antibodies not included in the consensus diagnostic panels for AML  assessment scale, which consisted of 22 itemsFrom the included studies, information regarding several parameters was obtained: (1) journal of publication; (2) The Journal Citation Reports (JCR) impact factor; (3) location; (4) study design; (5) aim of the study; (6) number of samples analyzed; (7) AML classification; (8) most incident subtype; (9) immunophenotypic marker; (10) prognostic value; (11) first induction treatment protocol (12) follow up; (13) survival; (14) limitations; and (15) STROBE scores. For inaccessible or incomplete full texts, authors were contacted for additional information.A meta-analysis of the relative risks related to the probability of survival at 10 to 20 months was performed. For analysis purposes, low expression of the immunophenotypic marker was considered as absent, and high expression was considered as present.18. A contingency table was constructed  classification for AML, six used the French-American-British (FAB) classification, and three used both FAB and WHO to classify the AML subtypes30. The most reported subtype in the articles was FAB M2 followed by FAB M4.An overview of the characteristics of the 11 studies included in the final analysis is summarized in Tables 27. The Journal Citation Reports (JCR) impact factor found in the search ranged from 1.826 to 22.26825. There were only two pediatric studies, and the others focused on adults30. The expression of 13 different antigens was evaluated in all articles (Table 30. The most common chemotherapy regimen was anthracycline-based induction therapy (3 + 7), although other alternative treatments were chosen by some groups.Most studies were performed in countries with high scientific and technologic development, including the United States (US), Germany, and Japanes Table . All 11 30.The methodological quality of observational studies conducted with the STROBE tool showed that eight articles scored >90% revealing a high methodological quality of the included studies. One article scored 19 points, which was equivalent to 86.4%, four scored 20 points, which was equivalent to 91%, and four scored 21 points, which was equivalent to 95.4%27. All five cohort articles showed a decreased survival related to the high expression of the immunophenotypic markers.Most articles showed that the expression of the evaluated antigens has a negative impact on prognosis of AML. Only one article showed that CD263 has a positive value on prognosis, and two articles showed that the values of CD90 and ILT3 were not determined+/CD38− cells) and the CD34+/CD38+ compartments of AML cells. They showed that LSC expressed a higher amount of CD82 than CD34+/CD38+ AML cells; these findings suggested that overexpression of CD82 may render LSC able to adhere to the bone marrow (BM) niche where it appears to regulate maintenance of leukemia stem cells within the BM niche. In addition, they showed that down-regulation of CD82 in LSC may stimulate mobilization of these cells from the BM niche to PB and sensitize them to chemotherapeutic agents24.Two articles by the same authors discussed CD82, a member of the tetraspanin superfamily that was originally identified as an accessory molecule in T-cell activation and in nonimmune cells in integrin-mediated cell adhesion to the extracellular matrix. Both articles evaluated the expression of CD82 in the self-renewing leukemia stem cell (LSC) compartments  and showed that CD135 was expressed in 82% of all cases. There was no association of CD135 expression with the FLT3 internal tandem duplication mutation, a molecular abnormality associated with unfavorable AML prognosis. Nevertheless, co-expression of CD135 and CD177 was associated with a decrease in event free survival (EFS) and overall survival (OS) in multivariable analysis in both age groups30.CD135 (FLT3) is a tyrosine kinase receptor that has a significant role in leukemogenesis. Sharawat et al. investigated the expression of CXCR4 (CD184) in AML. CXCR4 is a receptor for stromal-derived factor 1 (SDF1) that plays a very important role in hematopoiesis development and organization of immune system. After analyzing whole blasts and CD34+ cells in 142 adult non-M3 AML cases, the authors used mean fluorescence intensity (MFI) and showed a correlation between high CXCR4 expression and decrease in EFS and OS in addition to an unfavorable prognosis. In addition, CXCR4 expression was associated with high leukemic burden, as estimated by DHL level and white blood cell and peripheral blast cell counts29.Mannelli et al. evaluated the expression of this antigen in 30 AML cases and 30 acute lymphoblastic leukemia patients and observed that CD133 was expressed in 56.6% of AML (n = 17). The authors observed an important correlation between the expression of CD133 and the survival of AML patients and demonstrated a decrease in OS with an increase in CD133 expression. As for the prognostic value, it was concluded that CD133 expression was highly associated with poor prognosis in AML patients22.CD133 is a novel five transmembrane molecule expressed on primitive normal hematopoietic progenitors. Tolba et al. assessed the expression of the death receptors, including TRAILR1, 2, and 3 , TNFR1 (CD120a), and FAS (CD95). CD262 is involved in induction of apoptosis in lymphoid cells. CD263 is a marker of neutrophilic granulocytes that participates in apoptosis regulation and inhibition of cell death through competitive binding activity. TNFR1 is expressed on monocytes, lymphocytes, and granulocytes, and is involved in cytotoxicity mediation. The authors concluded that CD262 and TNFR1 expressions showed significantly shorter OS, earlier disease onset, and higher probability of relapse in AML patients. Conversely, CD263 expression exhibited an enhanced OS. As for prognostic value, high expressions of CD262 and TNFR1 were found to be associated with unfavorable prognostic groups, and CD263 was found in cases with favorable risk19.In 46 AML patients, Schmohl et al. evaluated one member of the immunoglobulin-like transcripts (ILT3 expression) in normal and leukemic myeloid precursors in 20 healthy individuals and 37 AML cases. ILT3 is a member of the large family of ILT molecules, leukocyte Ig-like receptors (LIRs), and monocyte/macrophage Ig-like receptors (MIRs). The authors showed that ILT3 was expressed in all cases of AML displaying monocytic differentiation but not in the AML subtypes M1/M2 and M3. It was shown that expression of ILT3 by leukemic cells contribute to the inhibition of tumor specific T cell responses. As for the prognostic impact, frequent cytogenetic abnormalities observed in AML patients with ILT3+ were those associated with intermediate prognosis, but it’s potential value as a prognostic marker, particularly in cytogenetically normal AML, remains to be determined27.Dobrowolska et al. evaluated hMICL expression in one article in 93 AML patients. hMICL is a heavily glycosylated transmembrane C-type lectin with an unknown function. Cryopreserved mononuclear cells and bone marrow samples were used in the analysis. They showed that hMICL was found to be restricted to the CD45low/SSClow population of AML cells. The authors suggest that no loss of hMICL expression in AML patients may be an early indicator of relapse. In addition, hMICL preserved fluorescence intensity at relapse, suggesting that this antigen could be a tool for minimal residual disease quantification by flow cytometry in AML28.Larsen et al. evaluated the expression of these two antigens in two distinct primitive cell population compartments from bone marrow. CD34+ CD38− cells (enriched for HSC) and CD34+ CD38+ cells (enriched for HPC) obtained from 12 pediatric AML patients were used for the analysis. The results showed that CD90 showed slight incremental increases in patients who reached remission and did not relapse. As for CD96, the authors describe greater expression in relapse and higher levels in AML cells than in normal bone marrow cells before and after chemotherapy26.CD90, a cell-surface glycoprotein, seems to be involved in proliferation and expansion processes, and CD96 is a member of the immunoglobulin superfamily. In one article, Chaves-Gonzales 30. The expression of these markers are considered independent events, since their pathogenic significance are not necessarily interconnected within certain pathways. The survival analysis over 10 months showed a great heterogeneity with a significant Cochran Q test  and the Higgins I² test showed a result of 83.33%, indicating high heterogeneity and suggesting an impediment to achieving the meta-analysis. Despite that impediment, the tests were still performed showing a meta-analytical estimate of no significant risk (1.49). The funnel plot and regression testing for evaluation of publication bias showed a large asymmetry   = 1.0512; p = 0.7889), and the Higgins I² test showed a result of 0% indicating no heterogeneity. The same results were found in relation to the markers collected at 20 months, both in relation to Q Cochran test (Q (df = 4) = 2.6083; p = 0.6254) and the Higgins I² test (0%). It was decided then, to perform the two meta-analyses using the fixed effects model.The meta-analytical estimate represented by the relative risks of survival rate at 10 months Fig.  was 2.55After resetting the final models, especially the 10-month survival, the funnel plot and the regression test for analysis of the asymmetry were used to assess the potential for publication bias. Both tests showed that publication bias was not significantly associated with both the 10 and 20 month survival   and new analytical strategies. However, the discovery of new immunophenotypic markers for AML diagnosis was limited, and immunophenotypic panels have remained similar over the last twenty years. Therefore, there is still a need for new markers for leukemic myeloid cells to be included in clinical routines to increase the value of MFC not only as a diagnostic but also prognostic tool for monitoring of MRD and for development of drugs for targeted therapy5. The inhibitory receptor ILT3 is a highly sensitive and specific marker for both diagnosis and monitoring of AML with monocytic differentiation; ILT3 as a marker is particularly useful in the differential diagnosis of AML with monocytic differentiation and microgranular acute promyelocytic leukemia, two leukemia subtypes that require different treatment strategies27. Another marker, hMICL, expressed significantly higher fluorescence intensity compared to normal bone marrow, suggesting suitability of this antigen as a pan-AML marker28. Regarding cell adhesion, CD82 has been shown to have a high expression in AML cells suggesting an increase of adhesion to the BM niche. On the other hand, down-regulation of CD82 in AML cells may stimulate circulation of these cells into peripheral blood23.In this systematic review, we observed that some markers could be used for AML diagnosis and during the follow up. For example, the high expression of CD87 is an indicator of morphologically and antigenically poorly differentiated disease, especially in the M4 and M5 subtypes5. CXCR4 could be also included due to its high expression on the surface of the entire leukemic population and demonstration of its influence on the overall CR rate and as poor prognostic factor for DFS and OS29. Another interesting study described the association between high expression of CD93 and loss of CDKN2B (p15Ink4b) expression; a cell-cycle inhibitor gene that has been shown to play a prominent role in leukemia pathogenesis and correlate with poor prognosis25.Many prognostic factors have been established in the past few decades in AML, including age, cytogenetic abnormalities, white blood cell count, serum lactate dehydrogenase, and the presence of antecedent hematologic disorder. This systematic review has shown that the high expression of CD87 on peripheral blood blasts was associated with relapse and poor prognosis and could be incorporated into the initial diagnostic work-up of AML patients29. Another study showed that FLT3 receptor (CD135) was associated with the FLT3 Internal tandem duplication (ITD) mutation, which confers a poor prognosis30. Since gene mutations are of utmost importance in deciding the patients’ risk-stratification, the knowledge of this association can bring advancement on treatment decision and give even more precision to it, raising the chances of cure. From this preliminary information, it is possible to envision future studies involving the connection of these two types of markers to predict prognosis and survival of AML.The demonstration that the high expression of these immunophenotypic markers influence not only the survival of AML patients but also the prognosis, brings up some questions. Since immunomarkers are signaling those important prognostic data and it is known that molecular mutation can also have influence on the disease pathways regarding both survival and prognostic features, how these two information can be combined on determine better accurate prognostic factors, and what association could be found between them? We could observe that this data are rarely combined on the articles included on this systematic review and meta-analysis. It was evidenced that majority of patients that expressed high CXCR4 also presented the NPM1 mutated gene, what gives it a worst prognosis5. One method to attempt to increase these patients’ survival could be a more specific chemotherapeutic protocol for this heterogeneous group of diseases. It is also necessary to identify in advance those patients who have greater resistance to treatment, higher relapse rates, and lower survival. We performed a meta-analysis using the fixed effects model. Four new immunophenotypic markers, CD133, CD135, TRAIL2 (CD262), and TNFR1 (CD120a) were demonstrated and showed correlation with lower survival at 10 and 20 months. Thus, these antigens can be used as early markers in combination with other prognostic factors for risk stratification of relapse. CD133 antigen appears to be expressed restrictively in the more immature cell population, and examination of the articles has made it possible to observe that expression of the CD133 in acute leukemia could be correlated with an immature phenotype of the myeloid blasts and highly associated with poor prognosis5. Proliferation regulators (such as tyrosine kinase receptors) play an important role in the pathogenesis of acute myeloid leukemia. The high expression of CD135 was associated with lower EFS and OS30. As verified by this study, the expression of death receptors is typically associated with the apoptotic regulation of leukemic blasts that demonstrated a significant association of TRAILR2 expression on blasts from patients in adverse risk groups and showed a negative impact on overall survival. Results concerning TNFR1 showed that this receptor is for the immune-modulating cytokine TNF. TNFR1 may play a role in initiation and proliferation of AML blasts due to its high expression, which appears to be related to a lower survival in AML patients19. Technically, this meta-analysis demonstrated that meta-analytical estimates, represented by the relative survival risk at 10 and 20 months, were both significant when the risk of survival in the absence and presence of the markers was analyzed. Notably, in our analysis, there was no heterogeneity among the cohort studies, which implied that the present method to combine the results from these studies was reasonable. Thus, the conclusions from this analysis should be credible. However, due to the relatively limited number of cases included in these studies, further analyses of larger series of patients are needed to confirm these preliminary observations.Despite the advancement of therapeutic progress, the overall survival of AML patients remains low27. CD93, an AML with MLL rearrangements marker expressed on leukemia stem cells (LSC), may also be a useful therapeutic target candidate for anti-LSC therapy and prognostic marker for quantitation of minimal residual disease25. hMICL is a pan-AML marker uniformly present on cells. It can be poorly immunophenotypically lineage-characterized and difficult to monitor for residual disease and development of drugs for targeted therapy28. The glycoprotein CD82 could be an attractive target for LSC eradication, due to its important role in regulation of the AML cell survival and their adhesion to bone marrow microenvironment23.Minimal residual disease is a term used to describe detection of subclinical levels of leukemia using multiparameter flow cytometry or molecular-based approaches. Employing the MFC for minimal residual disease detection appears to be a reliable method for obtaining rapid and objective patient remission status, provide early end points in clinical trials, and to inform patient management of a patient’s status. Emerging evidence indicates that MRD detection in patients with AML is also associated with poor prognosis, and early therapeutic interventions may be of clinical benefit. In addition, other studies have reported on the progression of new drug development that target specific areas of the leukemia cells; this is important since the current treatment can help, but does not always cure, AML patients. In this review, we observed that some antigens can be useful both as a marker for MRD and potential therapeutic target. ILT3 is an antigen expressed in AML displaying monocytic differentiation that supports differentiation and subsequent AML diagnosis of AML. It may also be a candidate marker for MRD detection in AML patients due to its high sensitivity, specificity, and stable expression. This antigen also can be a target for therapy in AML with monocytic differentiation as a result of inhibition of ILT3 signaling with specific antibodies or antagonists, which may render ILT3+ AML cells more susceptible to differentiation agents and anti-tumor T cell responses8. It was notable that some data discrepancies were found as in the case with hMICL, in which the author associated its high hMICL expression with relapse, but its role in the prognosis was not clear. More data are necessary to establish the extent that hMICL-based immunophenotyping can detect treatment failure, which is common in most AML patients. The same results were observed for CD90 and CD96, in which the author also shows an association of its high expression with relapse, but does not associate either CD90 or 96 with prognosis. CD96, especially, does not demonstrate high fluorescence in the first diagnosed sample, only in the normal bone marrow sample.There are some limitations that should be noted in our systematic review and meta-analysis. One of the most important is the lack of actual patient data included in the analysis. Despite that, it is known that AML is a disease with low incidence. In the US, the estimate for 2024 predicts that leukemia, including AML, will represent 3% of all cancers in male and femalesRegarding the limitations of our systematic review and meta-analysis, survival information was extracted from survival curves and not from a mortality table. Secondly, positive and negative expression were considering basing on the MFI of each study Third, the criteria to determine the positive or negative antigen expression varied across the included studies. Fourth, from the six immunophenotypic markers included in the meta-analysis, two were removed as they caused heterogeneity between the studies, which may have been due to some methodological differences. Fifth, we only searched for articles published in English, Spanish, and Portuguese and may have missed relevant publications in other languages. Finally, some studies included in the qualitative synthesis also evaluated M3 AML patients, a subgroup recognized for its excellent prognosis. However, only two studies included in the meta-analysis contained M3 AML cases, both with a low number of patients (<10%, Table Despite the limitations listed above, the present analysis revealed the prognostic value of new antigen expression in AML. Although the high expression of CD133, CD135, TRAIL2 and TNFR1 was not significant in most of the individual studies regarding the occurrence of the outcome, when they were collected in the meta-analysis a high correlation was observed with poor prognosis and low DFS and OS. However, further prospective studies with larger sample sizes are required to include these new immunomarkers in MFC routine of acute myeloid leukemia.Supplementary Informationpdf


Understanding how this occurs has been the focus of numerous studies including many that use models. However, CaMKII is notoriously difficult to simulate in detail because of its multi-subunit nature, which causes a combinatorial explosion in the number of species that must be modeled. To study the Ca2+-calmodulin-CaMKII reaction network with detailed kinetics while including the effect of diffusion, we have customized an existing stochastic particle-based simulator, Smoldyn, to manage the problem of combinatorial explosion. With this new method, spatial and temporal aspects of the signaling network can be studied without compromising biochemical details. We used this new method to examine how calmodulin molecules, both partially loaded and fully loaded with Ca2+, choose pathways to interact with and activate CaMKII under various Ca2+ input conditions. We found that the dependence of CaMKII phosphorylation on Ca2+ signal frequency is intrinsic to the network kinetics and the activation pattern can be modulated by the relative amount of Ca2+ to calmodulin and by the rate of Ca2+ diffusion. Depending on whether Ca2+ influx is saturating or not, calmodulin molecules could choose different routes within the network to activate CaMKII subunits, resulting in different frequency dependence patterns. In addition, the size of the holoenzyme produces a subtle effect on CaMKII activation. The more extended the subunits are organized, the easier for calmodulin molecules to access and activate the subunits. The findings suggest that particular intracellular environmental factors such as crowding and calmodulin availability can play an important role in decoding Ca2+ signals and can give rise to distinct CaMKII activation patterns in dendritic spines, Ca2+ channel nanodomains and cytoplasm.Calcium/calmodulin-dependent protein kinase II (CaMKII) holoenzymes play a critical role in decoding Ca 2+ signals are commonly used by cells for various types of activities. In neurons, Ca2+ can regulate gene expression and dendritic spine enlargement, strengthen synaptic connectivity, and promote neural growth or even death. One important Ca2+ binding protein is calmodulin, which has a wide range of downstream effectors that relay and interpret Ca2+ signals. These downstream proteins have different calmodulin binding kinetics and can interact with calmodulin differently depending on Ca2+ signaling patterns and in particular, on signaling frequencies. In this study, we focus on one particular pathway that is thought to be important for learning and memory and involves calmodulin and the effector protein, Ca2+/calmodulin-dependent protein kinase II (CaMKII). We have developed a novel computational method that avoids the problem of combinatorial explosion inherent with modeling this pathway, and the method allows us to study the pathway in more detail than conventional methods. Experiments have shown that CaMKII activity is sensitive to Ca2+ signal frequency and our models demonstrate how this frequency dependence relies on the amount of Ca2+ input, calmodulin availability and the Ca2+ diffusion rate.Ca More links to the relevant online repositories are in Please follow the GitHub link ((GZ)Click here for additional data file.S1 FigWe see that because geometry is a box, reflective and periodic boundary conditions give comparable results. (A) Phosphorylated CaMKII subunits from 10 trials with reflective boundary conditions and periodic boundary conditions (B) The same as in A but for CaM-bound CaMKII subunits.(TIFF)Click here for additional data file.S2 FigHoloenzymes has 2, 3, 6, or 12 subunits. Fully loaded CaM saturated CaMKII. The percentage of phosphorylated subunits are plotted over time.(TIFF)Click here for additional data file.S3 Fig(A) Bound CaMKII subunits simulated separately using the spatial Gillespie algorithm (red line) and modified Smoldyn (blue line) (B) The same as in A but for phosphorylated CaMKII subunits.(TIFF)Click here for additional data file.S4 Fig(A) Probability of release measured experimentally  using hi(TIFF)Click here for additional data file.S5 Fig2+ influx.Soma is colored in blue and dendritic spines are marked in black. Probabilistic synaptic input as in (TIFF)Click here for additional data file.S6 FigThis is a rearrangement of the same data shown in (TIFF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.