However, the putative modifications of Ca2+-handling proteins during the progression to heart failure (HF) are not well established. Moreover, the role of apoptosis in SHR is controversial. We investigated intracellular Ca2+, Ca2+-handling proteins and apoptosis in SHR vs. control Wistar rats (W) from 3 to 15 months (mo). Changes associated with the transition to HF , occurred at 15 mo in 38% of SHR (SHRF). In SHRF, twitch and caffeine-induced Ca2+ transients, significantly decreased relative to 6/9 mo and 15 mo without HF signs. This decrease occurred in association with a decrease in the time constant of caffeine-Ca2+ transient decay and an increase in Na+/Ca2+ exchanger (NCX) abundance (p<0.05) with no changes in SERCA2a expression/activity. An increased Ca2+-calmodulin-kinase II activity, associated with an enhancement of apoptosis (TUNEL and Bax/Bcl2) was observed in SHR relative to W from 3 to 15 mo. Conclusions: 1. Apoptosis is an early and persistent event that may contribute to hypertrophic remodeling but would not participate in the contractile impairment of SHRF. 2. The increase in NCX expression/activity, associated with an increase in Ca2+ efflux from the cell, constitutes a primary alteration of Ca2+-handling proteins in the evolution to HF. 3. No changes in SERCA2a expression/activity are observed when HF signs become evident.Spontaneously hypertensive rat (SHR) constitutes a genetic model widely used to study the natural evolution of hypertensive heart disease. Ca Apoptosis. The first one is generally accepted as a signature feature of experimental and human HF During chronic pressure overload, hypertrophy evolves to systolic left ventricular dysfunction and heart failure (HF). Experimental evidence suggests that a critical factor causing cardiac decompensation after pressure overload is the failure to preserve myocardial inotropy. Although the cause of cardiac dysfunction in HF is multifactorial, at the myocyte level two main mechanisms have been alleged to account for the decrease in myocardial contractility: 1. Alterations in Ca2+ handling have been described at very early stages of the disease, even before the appearance of HF 2+ metabolism in SHR hearts, the mechanisms of Ca2+ handling alteration are not clear. Three main abnormalities that may account for Ca2+ mishandling in HF have been suggested: 1. A decrease in the expression of sarco-endoplasmic reticulum Ca2+ ATPase (SERCA2a) relative to its inhibitory protein phospholamban (PLN), which decreases sarcoplasmic reticulum (SR) Ca2+ reuptake. 2. An increased expression of Na+/Ca2+ exchanger (NCX), which enhances Ca2+ extrusion. 3. An hyperphosphorylation of the ryanodine receptor (RyR2), which renders the channel more prone to diastolic spontaneous Ca2+ release events (Ca2+ leak) 2+ load.The spontaneously hypertensive rat (SHR) is a genetic hypertension model widely used to study the natural evolution of hypertensive heart disease similar in many aspects to human essential hypertension. In this model, alterations in Ca2+-calmodulin-dependent kinase (CaMKII) site, Thr17Most reports studying the status of these proteins in SHR hearts are concentrated on a single one of them and/or covered a precise moment in the evolution of this disease. For instance, an increased NCX activity Apoptosis has also been described either as an early phenomenon 2+-handling proteins and their impact on Ca2+ dynamics together with the degree of apoptosis, along the evolution to HF in SHR hearts. This strategy may offer a more realistic interpretation of the progression of the disease and will experimentally support new and rational therapeutic strategies.The main goal of the present study is to provide a sequential and mechanistic insight on the role of CaAll procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals  and approved by the Ethics Committee of the Cardiovascular Research Center, National Research Council  (P20-2010).2+ measurements. At 15 mo, a number of SHR showed HF clinical signs. The criterion to distinguish failing and non-failing animals was the presence of at least two clinical signs  and midwall fractional shortening ≤24.21% and lung weight/tibial length ≥7.71 mg/cm. 100, values which were two standard deviations below or above the mean of the SHR 15 mo group, respectively. The failing group was called SHRF and was considered separately.Male SHR and age-matched normotensive Wistar rats (W) bred and maintained in our animal research facility were studied at 3, 6, 9, 15 months (mo) of age. Blood pressure was monitored weekly in non-anesthetized rats with the standard noninvasive tail cuff method . Before the animals were sacrificed, they were weighed and subjected to echocardiographic examination. The animals were then deeply anesthetized  and after plane three of phase III of anesthesia was reached , heart and lungs were rapidly removed. Heart weight or lung weight-to-tibia length ratios were calculated. Hearts were assigned for biochemical studies, immunohistochemical staining (TUNEL assay), perfusion for contractile measurements and monophasic action potential determination or myocytes isolation for contractile and CaEchocardiogram was performed in each rat under light anesthesia . Cardiac geometry and function were evaluated by 2-dimensional M-mode echocardiography with a 7-MHz linear transducer (ATL G Model 11370 Ultramarc). All measurements, including left ventricular (LV) wall thickness and diastolic dimensions, were performed according to the American Society of Echocardiography method Myocytes were isolated by enzymatic digestion 2+ was measured with an epi-fluorescence system . Briefly, dye-loaded cells were placed in a chamber on the stage of an inverted microscope (Nikon TE 2000-U) and continuously superfused with a HEPES buffered solution at a constant flow of 1 mL/min. Experiments were performed at room temperature (20–22°C) and myocytes were stimulated via two-platinum electrodes on either side of the bath at 0.5 Hz. By conducting experiments under these conditions, isolated myocytes are significantly less stressed, allowing for prolonged stimulation protocols and extended cell viability over time. Fura-2 fluorescence intensity was taken as an index of the intracellular Ca2+.Isolated myocytes were loaded with Fura-2/AM (10 µmol/L for 15 min). Intracellular Ca2+ content was determined by rapidly switching to a HEPES buffered solution containing 25 mM caffeine to cause SR Ca2+ release. The rate constant of decay of caffeine-induced Ca2+ transient (kcaff) was used to estimate the velocity of Ca2+ extrusion by NCX (kNCX). kSERCA, was calculated by subtracting kcaff from the rate constant of decay of systolic Ca2+ transient (kt). These estimations assume that the decay of systolic Ca2+ transient is due to Ca2+ removal from the cytosol by both the SR and surface membrane mechanisms, mainly NCX, whereas the SR does not contribute to the decay of the caffeine response SR Ca2+ leak was studied according to Shannon et al. 2+ in the presence and absence of SR Ca2+ channel blockade by 1 mmol/L tetracaine. After stabilization, to bring the cellular Ca2+ content to a steady state, the stimulation was stopped and the myocytes were exposed to 0 Na+-0 Ca2+ solution (Na+ replaced by choline chloride) for 30 s to block the NCX, so that little or no Ca2+ can enter or leave the resting cell. The difference in diastolic Ca2+ with and without tetracaine was taken as an estimation of SR Ca2+ leak.SR Ca2+ transients were analyzed as the mean value over a 10–12 records for each cell.Fluorescence data were stored for an off-line analysis . CaIsolated hearts from SHR and W were perfused according to the Langendorff technique at constant temperature (37°C) and flow (14 ml) for 20 min, to evaluate contractility, relaxation and epicardial monophasic action potentials 2PO4, 25 NaF, 300 sucrose, 0.1 EDTA plus proteases inhibitor cocktail). Protein was measured by the Bradford method using BSA as standard. Lysates were separated per gel line in 10% SDS polyacrilamide gel 2+-ATPase (SERCA2a) 1∶1000 , Na+/Ca2+ exchanger (NCX) 1∶1000 , phospholamban (PLN) 1∶1000 , Thr17 and Ser16-phosphorylated PLN 1∶1000  , Ryanodine Receptors (RyR2) 1∶1000 , Ser2814 and Ser2809-phosphorylated RyR2 1∶1000 , pCaMKII 1∶1000 . Apoptotic cell death was determined as the ratio of the signals between the pro-apoptotic Bax and the anti-apoptotic Bcl-2. GAPDH 1∶5000  or calsequestrin  1∶2000 signals were used to normalize the signal intensity of the different proteins. Immunoreactivity was visualized by a peroxidase-based chemiluminescence detection kit  using a Chemidoc Imaging system. The signal intensity of the immunoblot bands was quantified using Image J software (NIH).Heart ventricles were freeze-clamped and pulverized. Briefly, 0.1 g of tissue was homogenized in 4 volumes of lysis buffer  positive cells were imaged under a confocal microscope  using a 40x NA 0.95 objective and counted in 20 random fields for each experimental situation using the Olympus cellSens image analysis software v6.3. Results were expressed as percentage of TUNEL positive cells related to total number of cells. DAPI  was used for nuclear staining All data are presented as mean ± SEM. Comparisons within groups were made by unpaired Student’s t test, as appropriate. One-way ANOVA was used for multigroup comparisons. The Tukey post-hoc test was used to examine statistical differences observed with the ANOVA. A value of p<0.05 was taken to indicate statistical significance.Hypertensive heart disease develops progressively in SHR from 3 to 15 mo. Systolic blood pressure and progression of hypertrophy , are shown in 2+ transient dynamics. At 6, 9 and 15 mo there was an increase in the amplitude of Ca2+ transient and SR Ca2+ content (caffeine-induced Ca2+ transient) in SHR vs. W consistent with previous findings 2+ transients with respect to W myocytes, either relative to the same age or to SHR at 3 mo. This different pattern was due to a significant decrease in twitch and caffeine Ca2+ transient amplitude in SHRF myocytes with respect to the previous stage. At 15 mo there was a significant decrease in the time constant of caffeine-induced Ca2+ transient decline in SHR relative to W myocytes that was even more important in SHRF  , did not show any difference between SHR and W myocytes all along the ages studied . However2+ transient decay , was not associated with any significant increase in the rate of Ca2+ transient decay  which, although with a different cellular pattern, also remains until pump failure was reached. 3. An increased expression/activity of NCX, which might underlie, at least in part, the Ca2+ dynamics, Ca2+ handling proteins and cell death in the evolution from hypertrophy to HF. These findings may help to explain the diversity of results obtained when these variables are studied at a given time in the course of the disease. In addition, our results allow us to emphasize that apoptosis and decreased SERCA2a are not sufficient to induce heart function impairment in the SHR model. On the other hand, the increased NCX expression/activity, and the consequent unbalanced NCX/SERCA2a, seems to be a main trigger event in the progression to HF.The results obtained highlight the fact that there is a continuum of different and transitory alterations in CaIt has been shown that very low levels of myocyte apoptosis are observed in patients with dilated cardiomyopathy In addition, our results show an increase in CaMKII activity from 3 mo of age in SHR which persists all along the evolution from hypertrophy to HF. This increase precedes the onset of apoptotic cell death, reinforcing the idea of a causal relationship between both events, as previously described 2+ efflux through an overexpressed NCX at 15 mo in SHRF. The activity of NCX depends not only on the kinetics properties or expression level, but also on the magnitude and change of the driving force. The driving force is a function of intracellular sodium ([Na+]i), [Ca2+]i and transmembrane potential. In HF, the commonly found increased [Na+]i, prolongation of the action potential and altered [Ca2+]i, favor the ratio reverse mode/forward mode of NCX, decreasing Ca2+ efflux 2+ efflux was detected in the current experiments in SHRF which should be secondary to the higher exchanger expression and could explain, at least in part, the decrease in SR Ca2+ load and Ca2+ transient amplitude in the isolated myocytes. Moreover, the finding that no significant changes in NCX expression were observed in 15 mo SHR that did not present HF signs, further suggests the involvement of increased NCX expression in HF progression.A main finding of this study is the significant enhancement in the estimated Ca2+ transporters occurs by different independent signals Moreover the increase in NCX expression appears as an independent phenomenon and not as a compensatory mechanism tending to offset the negative effects on relaxation produced by the decrease in SERCA2a activity described in several HF models. This conclusion is in consonance with previous results in failing human myocardium, suggesting that regulation of the expression of both Ca2+ handling and is responsible for a decompensatory transition to HF under stress conditions Of note, overexpression of NCX in transgenic mice, leads to abnormal CaA decreased expression of SERCA2a has been noted in some experimental models of HF and in the failing human heart 17 site of PLN occurred from 3 mo and persisted all along the evolution of the disease. This phosphorylation may have contributed to the enhancement of SR Ca2+ load observed in isolated myocytes in SHR at 6–15 mo. However it was not associated to any evident relaxant effect as should be expected. A possible explanation for this finding is the prolongation of the action potential that is known to occur in SHR 2+ transient decay that should have produced the phosphorylation of PLN. The lack of significant increase in SR Ca2+ load and Ca2+ transient or the very modest raise in contractility observed in 3 mo SHR myocytes and intact hearts, is more difficult to explain, although it is not a novel finding. Experiments by Boknik et al. In the present experiments a sustained increase in the phosphorylation of Thr16 phosphorylation observed at 3 mo, vanished at 6–15 mo consistent with an early increased sympathetic tone followed by a downregulation of β-adrenergic system, during the course of the hypertensive disease The increase in Ser2808 and Ser2814 sites of RyR2. These results are at odds with previous reports which described an increase in Ser2808 and Ser2814-RyR2 phosphorylation in different HF models 2+ leak at earlier ages can be fully explained by the high SR Ca2+ content.Finally we did not find any significant change in the phosphorylation of Ser2+ handling, Ca2+ handling proteins and cell death, during the progression to HF. These results point to the enhanced expression/activity of the NCX and the consequent increase in the NCX/SERCA2a ratio, as an important mechanism that may contribute to the hypocontractile state typical of HF.In summary, although we are aware of the fact that an increase in NCX has been previously described in HF, these previous studies, particularly in SHR hearts, addressed a single moment in HF evolution and failed to show a causal relationship between the increase in NCX and the impaired contractility. To the best of our knowledge, this is the first study describing the sequence of the changes of three main factors that are critical in determining the contractile state of the heart, namely, CaFile S1File includes Table S1 and Figure S1. Table S1. Echocardiographic parameters of the different experimental groups. Left ventricular end diastolic diameter (LVEDD); Left ventricular end systolic diameter (LVESD); Interventricular septum in systole (IVSs); Interventricular septum in diastole (IVSd); Posterior wall diastolic thickness (PWdt); Posterior wall systolic thickness (PWst). Progression of hypertrophy was observed from 3 mo SHR in IVSs, IVSd, PWdt and PWst. Moreover, 15 mo SHRF showed a highly significant increase in LVESD and IVSD. Values are expressed as mean ± SE. *p<0.05 vs. W. # p<0.05 vs SHR 15 mo. Figure S1. Ex vivo experiments: perfused hearts from Wistar and SHR at 3 mo of age. A) Representative recordings of left ventricular developed pressure (LVDP) and monophasic action potential (MAPs) simultaneously obtained in Langendorff perfused hearts. B) and C) overall results of half relaxation time (t1/2) and monophasic action potential duration at 90% repolarization (MAPD90). D) and E) typical immunoblots and overall results of the phosphorylation of PLN at Ser16 and Thr17 respectively. *p<0.05 with respect to Wistar hearts, n≥3 animals per group.(DOCX)Click here for additional data file.


Bafilomycin A1, which prevents lysosomal Ca2+ uptake by inhibiting H+ pumping into lysosomes, increased the amplitude of the initial Ca2+ signals evoked by carbachol in human embryonic kidney (HEK) cells. Carbachol alone and carbachol in combination with parathyroid hormone (PTH) evoke Ca2+ release from distinct IP3-sensitive Ca2+ stores in HEK cells stably expressing human type 1 PTH receptors. Bafilomycin A1 similarly exaggerated the Ca2+ signals evoked by carbachol or carbachol with PTH, indicating that Ca2+ released from distinct IP3-sensitive Ca2+ stores is sequestered by lysosomes. The Ca2+ signals resulting from store-operated Ca2+ entry, whether evoked by thapsigargin or carbachol, were unaffected by bafilomycin A1. Using Gd3+ (1 mM) to inhibit both Ca2+ entry and Ca2+ extrusion, HEK cells were repetitively stimulated with carbachol to assess the effectiveness of Ca2+ recycling to the ER after IP3-evoked Ca2+ release. Blocking lysosomal Ca2+ uptake with bafilomycin A1 increased the amplitude of each carbachol-evoked Ca2+ signal without affecting the rate of Ca2+ recycling to the ER. This suggests that Ca2+ accumulated by lysosomes is rapidly returned to the ER. We conclude that lysosomes rapidly, reversibly and selectively accumulate the Ca2+ released by IP3 receptors residing within distinct Ca2+ stores, but not the Ca2+ entering cells via receptor-regulated, store-operated Ca2+ entry pathways.Inositol 1,4,5-trisphosphate (IP Furthermore, rapid gating of the Ca2+ channels that initiate most Ca2+ signals and slow diffusion of Ca2+ within the cytosol allow local Ca2+-mediated communication between closely apposed membranes. The mitochondrial uniporter (MCU) 2+ released by IP3Rs when mitochondria are locally exposed to high [Ca2+]i near the mouths of open IP3Rs 3-evoked Ca2+ signals and regulates mitochondrial behaviour. Close apposition of STIM1 in ER membranes to Orai channels in the plasma membrane underlies regulation of the store-operated Ca2+ entry (SOCE) that almost invariably follows depletion of intracellular Ca2+ stores by IP32+]i2+-permeable channels expressed within lysosomal membranes, including two-pore channels (TPCs) 2+ release in response to such stimuli as nicotinic acid adenine dinucleotide phosphate (NAADP) 2+ release from lysosomes can be amplified by Ca2+ release from the ER mediated by Ca2+-activation of either IP3Rs or ryanodine receptors The ER is unique among intracellular organelles in the extent to which it forms intimate associations with other membranes 2+ uptake into lysosomes are not known, although they require the pH gradient established across lysosomal membranes by the V-ATPase that pumps H+ into the lumen of lysosomes 2+ signals evoked by IP3-evoked Ca2+ release from the ER. In our analysis, we demonstrated that dynamic lysosomes are associated with ER and that they selectively accumulate Ca2+ released by IP3Rs. But lysosomes do not sequester Ca2+ entering the cell via SOCE activated pharmacologically by inhibition of the SR/ER Ca2+-ATPase (SERCA) or by buffering of ER luminal Ca2+3-evoked Ca2+ signals and to providing lysosomes with Ca2+ that might regulate their behaviour 3-evoked Ca2+ signals. First, we have argued that receptors, like the endogenous M3 muscarinic receptors of human embryonic kidney (HEK) cells, locally deliver IP3 to IP3Rs within signalling junctions, whereas different ‘extra-junctional’ IP3Rs release Ca2+ from distinct Ca2+ stores in response to lower concentrations of IP3 when their sensitivity is increased by cAMP 2+ released from each of these IP3-sensitive Ca2+ stores? Second, does the SOCE evoked by physiological stimuli (rather than thapsigargin) direct Ca2+ to lysosomes? The answer to this question is important because it addresses whether a significant fraction of the Ca2+ entering cells via SOCE then passes through the ER and IP3Rs before re-entering the cytosol 2+, or is the Ca2+ they accumulate rapidly recycled to sustain refilling of ER Ca2+ stores?The mechanisms responsible for Ca3 receptors of HEK cells with carbachol (CCh) activates PLC. The IP3 produced then evokes Ca2+ release from intracellular stores via IP3Rs  2+ signals evoked by CCh   with bafilomycin A0.2-fold  [30]. PTd by CCh . The incmycin A1 . The sentration) . In thestration) . The simesponses  suggest + uptake .3-evoked Ca2+ release triggers a rapid increase in the luminal pH of lysosomes + for cytosolic Ca2+et al., however, suggest a different interpretation for their results. They argue that Ca2+ release from sea urchin lysosomes increases lysosomal pH, and that IP3-evoked Ca2+ release elicits the same response by locally stimulating formation of NAADP and perhaps also by a direct effect of cytosolic Ca2+ on NAADP-evoked Ca2+ release 3-evoked Ca2+ signals in HEK cells. Firstly, active lysosomes attenuate IP3-evoked Ca2+ signals in HEK cells  is inhibited by thapsigargin  detected 2 min after CCh addition in the absence and presence of extracellular Ca2+ to assess the effects of bafilomycin A1 on CCh-evoked SOCE. The difference between these values (ΔΔ[Ca2+]i  = Δ[Ca2+]i with extracellular Ca2+- Δ[Ca2+]i without extracellular Ca2+) reports the magnitude of the CCh-evoked SOCE  that inhibit both Ca2+ extrusion across the plasma membrane and Ca2+ entry 2+-free HBS were repeatedly stimulated with brief pulses of a maximally effective concentration of CCh (1 mM), demonstrates that the approach is effective, albeit without fully preventing loss of Ca2+ from stimulated cells. The incomplete inhibition of Ca2+ loss by Gd3+ contrasts with a previous analysis of HEK cells where CCh-evoked Ca2+ oscillations persisted for many minutes with undiminished amplitude in Ca2+-free medium supplemented with 1 mM Gd3+2+ oscillations (1–5 µM) 2+-free HBS, cells responded robustly to the first CCh challenge, but not to subsequent challenges , allows Ca2+ to be recycled within the cell and thereby allows the ER to respond to repeated CCh challenges  challenges with CCh in nominally Ca2+-free HBS supplemented with 1 mM Gd3+   . It is,  by Gd3+ . This su1 nor Gd3+ affected the number of cells responding to the initial CCh challenge  and Texas Red , and Ca2+ standard solutions were from Invitrogen . NED-19 was from Enzo Life Sciences . G418 was from Formedium . Cell culture plastics and 96-well plates were from Greiner . Imaging dishes (35-mm diameter with a 7-mm No. 0 glass insert) were from MatTek Corporation  or PAA Laboratories . Carbamyl choline chloride , DMSO, foetal bovine serum (FBS), poly-l-lysine, Pluronic F127 and Triton-X-100 were from Sigma-Aldrich . BAPTA ethane-N,N,N′,N′-tetraacetic acid) was from Molekula . Bafilomycin A1 was from AG scientific . Parathyroid hormone  was from Bachem . Ionomycin was from Merck Eurolab . Thapsigargin was from Alomone Labs .Dulbecco's modified Eagle's/Ham's F-12 (DMEM/F-12), fluo-4-AM, fura-2-AM, dextran-conjugates of Oregon Green  poly-l-lysine.HEK cells and HEK cells stably expressing human type 1 PTH receptors (HEK-PR1 cells) were cultured at 37°C in DMEM/F-12 medium with GlutaMAX-1, FBS (10%) and G418 (800 µg/ml for HEK-PR1 cells) in humidified air with 5% CO2+]i in populations of confluent cells loaded with fluo-4 was measured at intervals of 1.44 s using a fluorescence plate-reader as described previously 2 1.2 mM, CaCl2 1.5 mM, HEPES 11.6 mM and glucose 11.5 mM, pH 7.3). Ca2+ was omitted from nominally Ca2+-free HBS, and replaced by BAPTA (10 mM) in Ca2+-free HBS. Fluorescence (F) was calibrated to [Ca2+]i from [Ca2+]i = KD(F-Fmin)/(Fmax-F), where KD is the dissociation constant of fluo-4 for Ca2+ (345 nM), Fmin and Fmax are the fluorescence signals recorded after treatment of parallel wells with Triton X-100 (0.1% v/v) in HBS supplemented with 10 mM BAPTA or 10 mM CaCl2, respectively. Concentration-effect relationships were fitted to Hill equations using non-linear curve-fitting .i using Ca2+ standard solutions 2+ signals evoked by successive CCh challenges  and Oregon Green  for 12 h at 37°C to allow uptake of the indicators by endocytosis. After a further incubation (4 h) without indicators, the cells were washed with HBS and fluorescence was recorded in HBS at 20°C using an Olympus IX81 microscope with a 60x/1.45 NA objective. Cells were illuminated with a mercury xenon lamp using alternating filter sets: U-MNIBA  and LF561A . Images were captured at 2-s intervals using an EMCCD camera (Andor iXon 897) and analyzed using Cell∧R software . Records were corrected for background fluorescence determined under identical conditions from cells without indicators. Fluorescence changes from defined regions of interest (ROI) are expressed as F/F0, where F0 and F denote the average fluorescence within the ROI at the start of the experiment (F0) and at each time point (F).Almost confluent cultures of HEK cells grown on poly-Figure S12+ rapidly recycles from lysosomes.Responses to repetitive challenges with carbachol reveal that Ca (A) A fraction of the Ca2+ released from the ER via IP3Rs is normally lost to the extracellular space as Ca2+ pumps in the plasma membrane (PM) extrude it from the cytosol. When Ca2+ is present in the extracellular medium, this loss is replenished by store-operated Ca2+ entry (SOCE). Removal of extracellular Ca2+ or blockade of SOCE by Gd3+ prevents this recycling of Ca2+. Lysosomes also sequester Ca2+ released by IP3Rs 2+ is also rapidly recycled via the cytosol to the ER. The experiments shown in 2+ signals evoked by repetitive challenges with CCh  were recorded from HEK cells in Ca2+-free HBS with 1 mM Gd3+ . These raw data were used to produce 2+-free HBS, show that in the absence of high concentrations of Gd3+, cells respond robustly to the first CCh challenge, but not to subsequent challenges.(TIF)Click here for additional data file.


As the main theme of this special issue, CRP not only is an inflammatory marker but also has diverse biological functions associated with different diseases. To investigate CRP's physiologies and their relationship with human pathological significance, it is essential to use appropriate animal models for translational research. The most popular models for the study of CRP are transgenic mice. However, researchers should be careful when extrapolating the findings derived from these animal models. This review will discuss the current concerns on CRP transgenic mice and rabbits. More than 30 epidemiological studies have demonstrated a significant association between elevated serum or plasma CRP concentration and the prevalence of atherosclerotic vascular disease, the risk of recurrent cardiovascular events among those with established disease, or the incidence of first cardiovascular events among those at risk . This stμg/mL levels , 3332, 3Rabbits have been used as an excellent model for the study of human atherosclerosis because their lipoprotein metabolism and cardiovascular system are similar to those of humans . Unlike To elucidate whether high levels of plasma CRP participate in the development of atherosclerosis, our laboratory generated 2 lines of transgenic (Tg) rabbits expressing human CRP (hCRP) transgene in the liver . Plasma Using double balloon-injury models of the femoral arteries in Tg rabbits, we demonstrated that high expression of hCRP led to enhanced thrombosis formation on the injured smooth muscle cell-rich neointima by upregulating tissue factor expression , suggestβ-VLDLs, which may complicate the evaluation of hCRP pathophysiological functions in these models [The major concern about using cholesterol-fed Tg animals is that transgenic proteins  are exogenous to animals and the atherogenic lipoproteins are remnant lipoproteins, so-called e models . To overIn conclusion, rabbits are a suitable model for the investigation of CRP physiology because they resemble humans in many aspects, compared with mice. Cholesterol-fed rabbits along with WHHL rabbits and hCRP transgenic rabbits offer another opportunity to elucidate CRP functions that cannot be conducted in mice. After a decade's effort using these unique models, it is time to draw a conclusion regarding the true role of CRP in atherosclerosis. Is CRP a marker or mediator of atherosclerosis? Do we still need to continue the debate? Should we treat CVD patients who have a high level of CRP? The answers are becoming clearer and clearer: without doubt, plasma CRP levels are indeed increased and CRP is intimately present in the lesions of atherosclerosis. However, the net effect exerted by CRP is not proatherogenic, while we cannot rule out the possibility that CRP is antiatherogenic or participates in other diseases. These experimental observations are also in support of the human studies reported recently –51. Thes


CCAAT/enhancer-binding protein-α (CEBPA) is a critical regulator of myeloid differentiation. Disruption of CEBPA function contributes to the development of acute myeloid leukemia (AML). CEBPA regulates a large number of protein coding genes of which several were shown to contribute to CEBPA function. In this study, we expand the analysis of CEBPA transcriptional targets to the newly identified class of long non-coding RNAs. We show that lncRNAs are a main component of the transcriptional program driven by C/EBPα and that many of these are also induced during granulocytic differentiation of AML cell lines supporting their relevance in proliferation arrest and differentiation.The online version of this article (doi:10.1186/s13045-014-0069-1) contains supplementary material, which is available to authorized users. LncRNAs participate in multiple networks controlling cell differentiation and development , with th−15), coupled with down-regulation of cell cycle genes and upregulation of granulocytic differentiation pathways , confirming the known role of C/EBPα in repressing E2F1 activity  and CEBPD (C/EBPδ) in K562 cells [all-trans retinoic acid (ATRA) [Different AML cell lines are widely used to study the block of differentiation in AML because they can be differentiated in mature and functional myeloid cells by treatment with specific agents. Thus, we analysed the expression of selected lncRNAs in NB4 cells, which are able to undergo granulocytic differentiation by treatment with d (ATRA) . NotablyIn summary, this study shows that lncRNAs are a main component of the transcriptional program driven by C/EBPα. We identified more than 900 lncRNAs regulated by C/EBPα in K562. We confirmed that the majority of these are also induced during granulocytic differentiation of AML cell lines supporting their relevance in proliferation arrest and differentiation. How many of the lncRNAs identified in this study are directly involved in regulating differentiation programmes of AML is an interesting question that warrants further investigations.Moreover, regardless of function, this work indicates that changes in lncRNAs expression might also have diagnostic applications in AML with CEBPA mutations.


Rabbits are also herbivores which efficiently convert fodder to food. They are prolific and converter of plant proteins of little or no use to people as food into high-value animal protein. Rabbit meat is high in protein, low in calories and low in fat and cholesterol contents, being considered as a delicacy and a healthy food product. Feeding rabbits with concentrates is expensive and therefore in order to reduce cost of production, hence the use of rumen content in this study as alternative feedstuff without competition. of different body weight and age where use in this experiment to determine the effects of replacing rumen content with or without enzyme supplementation for groundnut cake. This feeding trial which lasted for 8 weeks was carried out in order to determine the replacement value of groundnut cake with rumen content with or without enzyme in the diet of weaner rabbit. A 3x2 factorial experiment was adopted such that there where three (3) replacement level of rumen content  for groundnut cake by two supplemental level (no enzyme and enzyme supplement).A total of thirty six (36) weaner rabbits p < 0.05) on daily feed intake, rate of weight gain, feed to gain ratio, nitrogen retention, faecal nitrogen, total nitrogen output and nitrogen digestibility. The weight gained by rabbits fed on 0, 25 and 50 % were all comparable (p > 0.05) with weight gained value of 7.62,7.44 and 7.36 g respectively. Similarly there was a significant (p < 0.05) effect of supplement added on the body weight gain of the experimental animals. There was significant effect (p < 0.05) of the diet on the obtained feed to gain ratio. However, there was no significant effect (p > 0.05) of the treatment on urinary nitrogen. Significant (p < 0.05) effects of supplementation was observed on the feed intake, weight gain, feed to gain ratio, faecal nitrogen, nitrogen retention and nitrogen digestibility but there was no significant effects (p > 0.05) of the supplementation on the nitrogen intake. The interaction between the varying levels of rumen content supplementation had significant effects (p < 0.05) on the feacal nitrogen, feed intake and feed to gain ratio but no significant (p < 0.05) effects on interaction of nitrogen intake.The results showed that increased inclusion level of rumen content has significant effects (In conclusion, since the results from this study showed no negative effects on the performance of the experimental animals, the test ingredient can be used as alternative feedstuff at a lower inclusion level so as to reduce production cost and expand rabbit production. Rabbits are also herbivores which efficiently convert fodder to food. They are prolific and converter of plant proteins of little or no use to people as food into high-value animal protein. In an efficient breeding, rabbits convert up to 20 % of the protein consumed in meat, more than for pigs 15–18 %) and cattle (9–12 %) [5–18 % anIn Nigeria, rabbit production is considered a part time venture hence majority of rabbit production are mainly family own with small number of stock. Commercial productions are very rare and so the full potentials of rabbit are yet to be harnessed. Considering the economic and nutritional benefits of rabbit production which has been severally studied and confirmed, rabbit production need to be encouraged even now that Nigeria is in search of economic recovery strategies.The major cost in any livestock production is feed thus feeding cost is a major problem facing rabbit production. Conventional protein feedstuffs such as soya bean meal, groundnut cake, and fish meal are quite expensive. The high cost of these ingredients has resulted in the poor feeding of rabbit. The need to source for alternative feedstuff  so as to reduce high cost of livestock production cannot be overemphasized. Rumen content (RC) is obtained from the abattoir after slaughtering of animal. They are discarded as waste and cannot be consumed by human.There has been a tremendous increase in interest and use of various types of dietary enzymes in swine diets to improve nutrient digestibility in recent years. Enzymes are biologically active proteins that break specific chemical bonds to release nutrients for further digestion and absorption. Enzymes that breakdown proteins are called proteases, lipases breakdown fats, and there are several carbohydrases that breakdown carbohydrates to allow them to be better digested and absorbed as energy sources . Feed stThe experiment was carried out at the rabbitary unit of the department of Animal Science, Faculty of Agriculture, University of Abuja farm, Federal Capital Territory (FCT) Table .Table 1CThirty six weaner rabbits of mixed breeds averaging 0.2 kg- 0.3 kg body weight and aged 5 weeks were purchased from NAPRI . The rabbits on arrival at the experimental site were housed in a metal hutch with wire mesh at the base for easy collection of feaces and urine. The hutches were disinfected before the rabbits were introduced and they were given anti-stress with antibiotics for seven days and allowed to adapt to the environment.The rabbits were randomly allocated to six (6) dietary treatments with two (2) rabbits per replicate and each treatment had three (3) replicates. During the experiment, fed and water were supplied ad-libitum throughout and the performance of the animals were monitored. Quantity of feed consumed was monitored daily for the 8 weeks trial period by taking note of the difference between feed served and feed left after 24 h. The rabbits were also weighed on weekly basis during this period to determine weight gain. The hutches were cleaned on daily basis throughout the experimental period. Feeders and drinkers were made available in each hutch since experimental animals were housed and fed differently.adlibitum diet and water for the eight (8) weeks study period. The six experiment diets had rumen content replacing GNC at 0, 25, 50 % dietary levels.The rabbits were allocated to six (6) dietary treatments in a Completely Randomized Design (CRD) each treatment had three (3) replicates with two rabbits per replicate. Each rabbit was fed th week), the faeces and the urine of the rabbits were collected by the total faeces collection and the urine method and the nitrogen in it was analyzed. The nitrogen digestibility was also calculated.The performance of the rabbits was monitored, the initial live weight of the rabbits were recorded at commencement of the experiment, weekly weight and daily feed intake were recorded, while feed to gain ratio were also recorded and cost implication were calculated. During the last week of the study  . Sample All data obtained were subjected to analysis of variance (ANOVA) using factorial design of Completely Randomized Design (CRD) Model and significant differences between means were separated using Duncan Multiple Range Test .p < 0,05) while rabbit fed on 25 % rumen content had an intake of 50.04 % which is significantly (p < 0.05) higher than 49.79 g consumed by rabbit fed 50 % rumen content. Rabbits fed on enzyme supplemented diet had a higher feed intake value (50.26 g) when compared with rabbits fed on experimental diets without enzyme which had 50.19 g.The results of the growth performance characteristics measured are presented in Table p < 0.05) with weight gained value of 7.62,7.44 and 7.36 g respectively. Similarly there was a significant (p < 0.05) effect of supplementation on the observed body weight gain value obtained by the experimental animals. There was also significant effects (p < 0.05) of the diet on the obtained feed to gain ratio. The rabbits fed 0 % rumen content diets had the best feed to gain ratio of 6.72 g when compared with rabbits on other experimental diets. Rabbits fed experimental diet containing 50 % rumen content had the poorest feed to gain ratio of 6.90 g when compared with rabbits on other experimental diets.The weight gained by rabbits fed on 0, 25 and 50 % were significantly dofferent (p < 0.05) than those fed on other rumen content levels. The experimental diets had no significant effect (p > 0.05) on nitrogen intake between the experimental animals fed enzyme and non enzyme supplemnted diets. The feacal nitrogen of rabbits fed on control diet (2.29 g/rabbit/day) was significantly higher (p < 0.05) than those fed on 25 and 50 % rumen content level that had 2.26 g/rabbit/day and 2.23 g/rabbit/day respectively. Similarly there was no significant (p > 0.05) effect of supplementation no the observed feacal values obtained on the experimental rabbits.The nitrogen intake of 3.58 g/rabbit/day (Table p < 0.05) than those fed on 0 and 25 % rumen content level that has 63.97 and 64.21 % respectively. Similarly there was significant (p < 0.05) effect of supplementation on the observed nitrogen digestibility of the experimental animals.The nitrogen digestibility of 64.96 % in weaner rabbits fed on 50 % rumen content diet was significantly higher (P > 0.05) when rumen content was replaced with soya beans in the diet of broiler finisher.The variation in weight gain per day at different level of rumen content inclusion in the study is in conformity with several reports which indicated that rate of gain reduces with increased amount of crude fibre in the diets of monogastric animals . This reP < 0.05) in feed intake as the level of rumen content increased from 0 to 50 % could be because animal consumes more feed to meet its energy requirement. Also, it may be because rabbit being a pseudo-ruminant tolerates considerable level of fiber in their diet [p < 0.05) better than rabbits fed no supplement. This may be because enzyme increases the intake of feed.The significant increase (eir diet . The sigeir diet  who fed eir diet  stated tThe utilization of feed decreased from 0 to 50 % inclusion level of rumen content. This result is in agreement with the reports of  who repoThe result showed that the nitrogen intake increased as the level of rumen content increased. The difference in the nitrogen intake could be attributed to the difference in the crude protein contents of the diets. This result contradicts the work reported by  who obseThere was significant increase in the nitrogen digested with increase in the level of rumen content. This may be as a result of crude protein content of the diet. This is in contrast with the findings of  who repoThere was significant difference in the nitrogen digestibility as observed with varying levels of rumen content. This may suggest that the crude fibre contents in all the dietary treatment were different and therefore, did affect the digestibility of the rabbits. However, other reports which contradict this result indicated that nitrogen digestibility was lowered with increase in the level of rumen content .In conclusion, since the results from this study showed no negative effects on the performance of the experimental animals, the test ingredient can be used as alternative feedstuff at a lower inclusion level so as to reduce production cost and expand rabbit production.


Because of the difficulties to obtain intact human muscle fibers, human myotubes have been extensively employed for studies of Ca2+-dependent processes in human adult muscle. Despite this, it is unknown whether the Ca2+-handling properties of myotubes adequately represent those of adult muscle fibers.In skeletal muscle, intracellular Ca2+-handling properties of human muscle fibers and myotubes, we developed a model of dissected intact single muscle fibers obtained from human intercostal muscle biopsies. The intracellular Ca2+-handling of human muscle fibers was compared with that of myotubes generated by the differentiation of primary human myoblasts obtained from vastus lateralis muscle biopsies.To enable a comparison of the Ca2+] ([Ca2+]i) transients and force production upon electrical stimulation. In contrast, despite a more mature Ca2+-handling in myotubes than in myoblasts, myotubes lacked fundamental aspects of adult Ca2+-handling and did not contract. These functional differences were explained by discrepancies in the quantity and localization of Ca2+-handling proteins, as well as ultrastructural differences between muscle fibers and myotubes.The intact single muscle fibers all demonstrated strictly regulated cytosolic free [Ca2+]i transients and force production upon electrical stimulation can be obtained from human intercostal muscle biopsies. In contrast, human myotubes lack important aspects of adult Ca2+-handling and are thus an inappropriate model for human adult muscle when studying Ca2+-dependent processes, such as gene expression and metabolic processes.Intact single muscle fibers that display strictly regulated [CaThe online version of this article (doi:10.1186/s13395-015-0050-x) contains supplementary material, which is available to authorized users. Satellite cells are resident stem cells of the skeletal muscle that can be isolated from human muscle biopsies, activated into proliferating myoblasts, and differentiated into multinuclear myotubes ex vivo , 2. Myot2+-handling in myotubes, a notion supported by findings in previous studies investigating the Ca2+-handling properties of human myotubes  ([Ca2+]i) transient amplitude and duration [2+]i transients i transients and force production upon electrical stimulation ex vivo. In contrast, myotubes display only a rudimentary development of Ca2+-handling properties and do not contract. Consistent with this, we identify functionally important differences in the quantity and localization of Ca2+-handling proteins and ultrastructure between human muscle fibers and myotubes.In the present study, we hypothesized that human muscle fibers and myotubes would display major functional differences in intracellular CaSix recreationally active individuals suffering from lung cancer, four males and two females (age 65–76 years), scheduled for lobectomy at the Karolinska University Hospital in Stockholm, Sweden, were recruited for experiments on intercostal muscles. For patient characteristics see Additional file 2 and 5 % CO2 prior to collection and immediately transported to the laboratory. Vastus lateralis muscle biopsies were obtained by the Bergström percutaneous needle biopsy technique i was measured with the fluorescent Ca2+ indicator indo-1 , which was microinjected into the fiber. After injection of indo-1, the fiber was allowed to rest for at least 30 min. Tetanic force was then tested in the injected fibers and at regular intervals throughout the rest of the experiment, and if tetanic force declined by 10 % or more, the data from that fiber were discarded. Indo-1 was excited with light at 360 (±5) nm, and the light emitted at 405 (±5) and 495 (±5) nm was measured with two photomultiplier tubes. The fluorescence ratio of the light emitted at 405 nm to that emitted at 495 nm (R) is monotonically related to [Ca2+]i according to the following equation (Eq. 1) i, and Rmin and Rmax are the ratios at very low and saturating [Ca2+]i, respectively. Tetanic force was measured as the mean over 100 ms where force was maximal.In muscle fibers, i transients of human myotubes and intercostal fibers stimulated with a single 70 Hz, 350 ms train of current pulses. For intercostal muscle fibers, original fluorescent ratios were smoothed one to two times using a seven-point quadratic polynomial to reduce noise prior to calculating [Ca2+]i by Eq. 1. The peak Δ[Ca2+]i/[Ca2+]i0 for intercostal fibers or ΔF/F0 for myotubes, respectively, after the end of the stimulation period was set as the first point of the fit interval. The fit was continued until baseline (defined as <5 % of peak Δ[Ca2+]i/[Ca2+]i0 or ΔF/F0) was reached.The Ca−ΔCT formula.Total RNA was prepared by the Trizol method  and quantified spectrophotometrically by absorbance at 260 nm. One μg of total RNA from each sample was used for reverse transcription into cDNA for a final volume of 20 μl . Real-time PCR  procedures were employed to determine mRNA expression. Probes and primers (TaqMan) for SERCA1 Hs01092295_m1), SERCA2 (Hs00544877_m1), RyR1 (Hs00166991_m1), α1s-DHPR (Hs00163885_m1), myogenin (Hs01072232_m1), MYH7 (Hs01110632_m1), MYH2 (HsHs0040042_m1), MYH1 (Hs00428600_m1), UBB (Hs00430290_m1), UBC (Hs00824723_m1), RPS18 (Hs01375212_g1), GAPDH (4352934E), 18S (4310893E), CTSB (Hs00157194_m1), and HK2 (Hs00606086_m1) were purchased from Applied Biosystems, Life Technologies. Reaction and amplification mixes (10 μl) consisted of the diluted (1:50) cDNA (4.5 μl), TaqMan Fast Universal PCR Master Mix (5.0 μl), and specific primers (0.5 μl). Subsequent cycling protocols were 2 min at 50 °C and 10 min at 90 °C followed by 40 cycles at 95 °C for 15 s and 60 °C for 1 min. To identify the optimal reference gene for normalization, the stability of the following reference genes was analyzed: UBB, UBC, RPS18, GAPDH, 18S, CTSB, and HK2. The geometrical mean of RPS18 and UBB was identified as the most stable reference according to the NormFinder algorithm i and force production. In this way, the events following α-motoneuron activity in vivo can be recapitulated ex vivo [2+]i and compared the resultant [Ca2+]i transients to those in human muscle fibers.In intact muscle fibers, electrically evoked action potentials activate the voltage sensitive Ca ex vivo . We test2+]i and force production  and mean plateau [Ca2+]i was 1.0 μM (±0.2). Mean tetanic force per cross-sectional area generated was 405 kN/m2 (±112). While about half of the myotubes responded to electrical stimulation with a transient increase in fluo-3 signal (reflecting change in [Ca2+]i), none of the myoblasts showed any change in fluo-3 signal upon electrical stimulation  vs. 146 ms (±13), P = 0.002) in myotubes than in muscle fibers.As the i  in fluo-3 signal   but evoked a marked increase (P < 0.001) in fluo-3 signal in myoblasts  Fig. , c. Cellle cells . ATP stists Fig. , c. Figu2+]i and force production in intact single human muscle fibers. This novel model was employed to compare the Ca2+-handling properties of human muscle fibers with that of an existing ex vivo model of human muscle constituted by human myotubes. The major novel findings of the current study are the following: (1) intact single muscle fibers that allow for continuous measurements of [Ca2+]i and force production can be obtained from human intercostal muscle biopsies, (2) marked differences in intracellular Ca2+-handling exist between human muscle fibers and myotubes, and (3) these result from differences in the quantity and localization of Ca2+-handling proteins.In the current study, we demonstrate the first measurements of [Ca2+]i together with specific forces reported here in human muscle fibers are similar to values reported previously in intact single rodent muscle fibers i. This is consistent with previous studies in human myotubes reporting depolarization-induced increases in [Ca2+]i in approximately one third i transients in myotubes than in adult muscle fibers. This finding is in accordance with previous reports of a slow restoration of [Ca2+]i to resting levels in immortalized human myotubes following depolarization [2+]i transient decay. In addition, these differences may help explain the lower stimulation frequencies necessary to attain a fused tetanus in contracting human myotubes in comparison with muscle fibers [2+]i transients differentially affect the cellular signaling initiated by increased [Ca2+]i [2+]i transient decay in myotubes and muscle fibers, as demonstrated in the current study, will result in divergent effects on Ca2+ signaling pathways. Thus, when studying Ca2+-dependent processes such as gene expression and metabolism, myotubes are likely to provide different answers to those given by intact adult muscle fibers.Further evidence of an immature Carization . These ae fibers –14. Impo [Ca2+]i , 42–45, 2+]i was increased either by current pulses or 4-CmC application. This was explained by a low expression of sarcomeric MHCs and a lack of sarcomeric structures in human myotubes when analyzed by electron microscopy. The inability of human myotubes to contract is in accordance with the majority of previous studies and indicates a difference to rodent myotubes, which may contract when grown in culture i in human myoblasts and in the early process of myotube differentiation, with a reduced responsiveness to ATP in later developmental stages of myotube differentiation [2+ release channel RyR1 [2+]i in myogenic cells with a functional SR. The current findings of increased [Ca2+]i upon stimulation with 4-CmC in myotubes therefore indicates a maturation of intracellular Ca2+-handling towards that in adult muscle fibers. Moreover, the present results suggest that myoblasts and myotubes are functionally distinct regarding their Ca2+-handling properties and can reliably be distinguished by their divergent responses to ATP and 4-CmC. This is in line with previous reports of other properties acquired following differentiation of myoblasts into myotubes that are similar to those in adult muscle i transients and force production upon electrical stimulation can be obtained by careful dissection of human intercostal muscle biopsies. In contrast, despite a more mature Ca2+-handling in myotubes than in myoblasts, human myotubes lack important aspects of adult Ca2+-handling. We conclude that human myotubes are an inappropriate model for human adult muscle when studying Ca2+-dependent processes, such as gene expression and metabolic processes. These results provide insights to some important functional differences between adult muscle fibers and myotubes and suggest caution when translating results obtained in human myotubes to muscle fibers.Intact single muscle fibers that display strictly regulated [Ca


A growing body of literature suggests that STIM1 plays a key role in the development of pathological cardiac hypertrophy. However, the precise mechanisms involving STIM-dependent Ca2+ signaling in the heart are not clearly established. Here, we have investigated the STIM1-associated Ca2+ signals in cardiomyocytes and their relevance to pathological cardiac remodeling. We show that mice with inducible, cardiac-restricted, ablation of STIM1 exhibited left ventricular reduced contractility, which was corroborated by impaired single cell contractility. The spatial properties of STIM1-dependent Ca2+ signals determine restricted Ca2+ microdomains that regulate myofilament remodeling and activate spatially segregated pro-hypertrophic factors. Indeed, mice lacking STIM1 showed less adverse structural remodeling in response to pressure overload-induced cardiac hypertrophy. These results highlight how STIM1-dependent Ca2+ microdomains have a major impact on intracellular Ca2+ homeostasis, cytoskeletal remodeling and cellular signaling, even when excitation-contraction coupling is present.In non-excitable cells stromal interaction molecule 1 (STIM1) is a key element in the generation of Ca For instance, in cardiomyocytes, cell contraction involves precise control of local Ca2+ within the T-tubules, a process known as excitation-contraction coupling (ECC)2+ signaling can lead to activation of target genes responsible for the pathological cardiac hypertrophic response that is often induced by injury to the myocardium or chronic cardiac overload. The source of Ca2+ and the spatial distribution of the elements that determine the ECC are well known. Conversely, much debate remains about the source and spatial distribution of the Ca2+ pool that activates Ca2+-sensitive signaling proteins, such as Ca2+-calmodulin–dependent protein kinase (CaMK), calcineurin - nuclear factor of activated T-cells (Cn-NFAT) and the protein kinase C (PKC), each crucial in the conversion to hypertrophy.The ability of Ca2+ channels (SOCC) have attracted considerable attention as a potential source of Ca2+ involved in the activation of transcription factors responsible for the cardiac hypertrophic response22+ signaling42+-binding motif sensing the luminal Ca2+ concentration. When intraluminal ER Ca2+ concentration falls, STIM1 releases Ca2+ from the EF-hand domain oligomerizes and migrates into specialized regions in proximity to the plasma membrane tethering and activating Ca2+ selective channels. Ca2+ -entry through activated SOCC trigger calcium-activated signaling pathways that couple to downstream gene transcription678Recently, store-operated CaSTIM1 expression in the heart shows a dynamic expression profile being up-regulated during fetal development and in the adult heart that is undergoing structural remodeling3in vitro is sufficient to prevent phenylephrine-induced neonatal cardiomyocyte hypertrophy3112+ influx associated with increased NFAT and Ca2+/calmodulin-dependent kinase II (CaMKII) activitySTIM1 knockdown 2+ entry (SOCE) couples with the calcineurin-NFAT axis to convey cytoplasmic Ca2+ signals to the nucleus where it activates the hypertrophic gene transcription program. However, in cardiomyocytes it is not clear how STIM1-dependent Ca2+ signaling co-exists with the more prominent global Ca2+ transients that occur with each heartbeat. Indeed, there is a great paucity of information about the precise details of STIM1-dependent signaling and its spatial distribution in cardiomyocytes. So far, it is not clear whether STIM1 forms Ca2+ microdomains in cardiomyocytes similarly to those reported for non-excitable-cells after Ca2+ store depletion. Importantly, STIM1 has been implicated in the formation of actin stress fibers through RhoA activation1718Current evidence suggests that in non-excitable cells, store operated Ca2+ signals was conducted using an inducible, cardiac specific, STIM1 knockout mouse combined with ex-vivo culture models coupled with live-cell imaging. Our results show that mice with cardiomyocyte-specific STIM1 deletion exhibit a moderate left ventricular dilatation and decreased myocardial contractility. In contrast, STIM1 overexpression reveals formation of subcellular Ca2+ microdomains in proximity of STIM1 clusters, which facilitate myofibrillar protein degradation. Finally, we report that acute STIM1 genetic deletion attenuate pressure overload-induced cardiac hypertrophy by preventing focal adhesion activation and its downstream signaling. Our data reveal previously unidentified STIM1-dependent intracellular Ca2+ microdomains that are required for normal cardiac homeostasis and fibrillar remodeling during hypertrophy.To decipher the role of STIM1 in cardiomyocytes we conducted a comprehensive analysis of the role of STIM1 in the heart. An in-depth characterization of STIM1-dependent Caflox/flox mice and offspring backcrossed until STIM1flox/flox-Cre+/− mice were generated (designated as “mutant” in this study). STIM1WT/WT-Cre+/− was the designated control. This mouse model allows tight gene regulation, however, a careful investigation was conducted to establish the dosage of tamoxifen that maximize Cre-recombinase activity and minimize tamoxifen side effects2123+/− mice 7 days after receiving the last injection of tamoxifen , revealed that Cre-recombinase activity peaks after 3 injections , were crossed with STIM1jections . Since tjections , we used− mice underwent ultrasound interrogation before (day 0) and after (40 days) after tamoxifen treatment. Echocardiographic results were validated postmortem (p < 0.05), which was associated with reduced ejection fraction   . Moreove < 0.05)  and a mo < 0.05) . Left ve < 0.05) .2+]i, we investigated whether STIM1 deletion altered cardiomyocyte contractility and [Ca2+]i cycling in isolated adult cardiomyocytes. Presence of functional SOCE was assessed in fura-2 loaded cells where Ca2+ was pharmacologically depleted from the intracellular stores (see methods for details). Upon re-introduction of extracellular Ca2+, fura-2 ratio signal increased significantly in wild type cardiomyocytes, compared to the smaller fura-2 signal in cardiomyocytes isolated from STIM1 mutant hearts . Consistent with the echocardiographic results indicating reduced myocardial shortening, STIM1 KO cardiomyocytes displayed decreased contractility as determined by reduced sarcomere length to peak amplitude and reduced sarcomere shortening  revealed no difference between control and STIM1 KO cells  mutant, in which the first Ca2+ binding aspartic acid residue in the EF-hand is substituted with alanine. The STIM1-D76A is insensitive to the Ca2+ in the SR lumen mimicking the Ca2+ dissociated state of STIM1, hence, STIM1-D76 oligomerize even in absence of store depletion (store full)19292+]i transients. Cells were grown on a glass coverslip and transfected with STIM1-mCherry. After 48–72 hours post transfection cells were loaded with fluo-4 AM and under these conditions it was possible to image [Ca2+]i transients from one or two STIM1-mCherry expressing cells per selected field (40×) and compare it to the [Ca2+]i transients from non-transfected cells  to impose chronic pressure overload and induce cardiac hypertrophy. Thus, after 5 weeks of transverse aortic constriction (TAC), control mice showed left ventricular hypertrophy as revealed by postmortem morphological analysis . Serial Cardiac remodeling during hypertrophy is accompanied by cytoskeletal binding and phosphorylation of FAK at the Tyr-397 residue with subsequent activation of the extracellular-regulated kinases1/2 (ERK1/2)34Another signaling pathway affecting the growth response in cardiomyocytes, besides the Erks, is the Akt pathway which promotes cell survival and growth in response to extracellular signals372+ signaling relevant to cardiac hypertrophy and ventricular function has largely been overlooked. Using a combination of in-vitro and in-vivo assays we have demonstrated that: i) STIM1 is necessary to maintain optimal cardiac function and plays a critical role in the development of pressure overload-induced cardiac hypertrophy; ii) at cellular level, STIM1 forms intracellular clusters responsible for the generation of spatially restricted Ca2+ microdomains required for cytoskeletal myofilament remodeling and activation of pro-hypertrophic signaling pathways. These results highlight STIM1-dependent Ca2+ microdomains involvement in myocyte growth, cytoskeletal remodeling and cellular signaling, whose mechanism of action coexists with [Ca2+]i fluctuations involved in the excitation-contraction coupling.The precise role of STIM1 and the attendant Ca2+ transients. Specifically, we observed an increased rate of cytosolic Ca2+ clearance in STIM1 KO cells which was associated with an increase of the NCX1protein level. A relationship between STIM1 and NCX1 has also been proposed by other groups39402+ decay, however, the nature of the association between STIM1 and NCX1 and the maintenance of the intracellular Ca2+ homeostasis in cardiomyocytes is unknown and further investigation is required in this matter.The most striking effect on cardiac function resulting from STIM1 deletion was the LV dilatation associated with decreased cardiac contractility, which are consistent with those reported earlier in a constitutive STIM1 knockout mouse modelA key piece of evidence that has emerged from our data is that cytoskeletal elements like vinculin and tubulin were profoundly altered in STIM1 KO cardiomyocytes. Indeed, these cytoarchitectural components play a significant role in the modulation of contraction speed and cytoskeletal mechanics42182+ dependent signaling pathways involved in the hypertrophic gene transcription program35Although cardiomyocytes have lost the ability to migrate, they still retain focal adhesion structures which become macromolecular scaffolds. These scaffolds functionally convert the mechanical forces into the biochemical signals that govern both cytoskeletal remodeling and activation of Ca2+ signaling. In NVCM STIM1 formed clusters without the need of intracellular Ca2+ stores depletion. Similar findings were observed in adult cardiomyocytes. Pre-constituted “punctate” structures have been shown in skeletal muscle48132+ transient we did not observe STIM1 rearrangements, which may be explained by the fact that during Ca2+-induced release not all Ca2+ from the sarcoplasmic reticulum is released into the cytoplasm. Hence, there may be enough stored Ca2+ at all times to prevent further STIM1 oligomerization, these results also suggest that STIM1 is not actively involved in the cardiac ECC.NVCM overexpressing STIM1 provided valuable insight in understanding the relationship between STIM1, myofilaments distribution and sub-cellular Ca2+ concentration. This result is confirmed by a recent published work using a transgenic mouse overexpressing STIM1, the authors reported an increase in cytoplasmic Ca2+ concentration in skeletal and cardiac muscles2+ microdomains isolated from the global Ca2+ transients observed during the excitation-contraction coupling. Remarkably, STIM1-GCaMP6 allowed direct visualization of the Ca2+ “hot-spots” in proximity of the STIM1 clusters, revealing a very high correlation between Ca2+ concentration microdomains and STIM1 clusters. These findings are certainly intriguing and are further supported by studies showing that Ca2+ microdomains are required for selective activation of proteins that drive myofilament disassembly and cytoskeletal remodeling2+ microdomain are instrumental for calpain activation and subsequent myofilament degradation.STIM1 overexpression was associated with high cytoplasmic resting Ca2+ entry that requires complete emptying of intracellular Ca2+ stores. However, whether SOCE is present in cardiomyocytes is still under intense debate. Zhao et al. reported absence of SOCE in adult cardiomyocyteset al. did not observe STIM1 oligomerization upon Ca2+ store depletion (that is behind the pre-existing clusters). However, since Ca2+ dissociation from the STIM1 EF-hand motif is required in order for the cytoplasmic portion to rearrange and oligomerize2+ from the stores STIM1 would not oligomerize. We attempted to induce STIM1 oligomerization by pharmacologically deplete SR Ca2+ store content, unfortunately, upon this treatment cells undergo to contractures introducing significant motion artifacts. To overcome this limitation we transfected the cells with the constitutive active STIM1-D76 mutant, which clearly show STIM1 oligomerization within the clusters.Several laboratories have shown the presence of STIM1 and SOCE in neonatal and adult cardiomyocytes310112+ microdomains. Although it is well established that Orai1 is the essential pore-forming unit of SOCC, an impressive number of proteins have been proposed to interact with STIM1: SERCA54552+ channel5839402+ concentration. Moreover, STIM1 over-expression has been shown to attenuate PMCA-mediated Ca2+ clearance2+ clearance rate. Thus, PMCA can play an active role on the maintenance of the calcium microdomains reported in cardiomyocyte.Since STIM1 itself does not have channel properties, association with other molecular partners is crucial in order to generate Ca2+ signaling associated with STIM1 in both intact tissue and isolated cardiomyocytes. Our data suggest that STIM1 is required for maintaining optimal myocardial function. Further, we provide evidence that STIM1-dependent Ca2+ concentration microdomain are essential component of the focal adhesion complex that acts synergistically to trigger cytoskeletal remodeling and initiate the hypertrophic transcriptional program. In conclusion our data highlight a multicomponent signaling pathway that converge on the importance of Ca2+ microdomains required for downstream signaling activation and myofilament remodeling that are critically involved in the cardiac hypertrophic response.Overall, we have characterized the CaAll animal procedures described in this manuscript were reviewed and approved by the University of Tennessee Health Science Center Institutional Animal Care and Use Committee (IACUC). All methods were carried out in accordance with the approved guidelines.flox/flox) and transgenic α-MHC-MerCreMer mice  were crossed with STIM1flox/flox transgenic mice for at least 7 generations to generate double-transgenic mice (α-MHC-MerCreMer-STIM1flox/flox). Intraperitoneal tamoxifen injections (40 mg/Kg/day) were given to sex and age matched mutant (α-MHC-MerCreMer-STIM1flox/flox) and control mice (α-MHC-MerCreMer-STIMwt/wt). Mice were injected 3 to 4 weeks after birth, at least 28 days of rest post-injection were allowed before mice were used for experiments.Mice homozygous for STIM1-floxed alleles4Neonatal rat cardiomyocytes were isolated from 1–3 days old rat pups and were grown on fibronectin-coated coverslips. Cells were infected with the indicated lentivirus or transfected using lipofectamine 3000 (Life technologies). For immunostaining cells were washed once with ice-cold PBS and fixed in 3.7% with buffered formaldehyde for 10 min. After three washes with PBS, cells were treated for 10 min with 0.2% Triton X-100 in PBS and incubated 30 min with PBS/3% BSA. Cells were washed three times with PBS then incubated with primary antibodies as follows: 1 h with either anti-vinculin  anti-β-tubulin  in PBS, 0.2% BSA. Rhodamine phalloidin (1:200) was used to show actin distribution, while GFP-conjugated anti-GFP antibody (Thermo Scientific) was used against YFP-STIM1. Secondary antibodies were chosen accordingly, cells were incubated with a TRITC-conjugated anti-rabbit secondary antibody (Thermo Scientific), with 5 U/ml of Alexa 488. After a 2-h incubation, excess of secondary antibodies were washed out and coverslips were mounted onto glass slides with anti-fade mounting medium. Confocal microscopy was performed with a Zeiss LSM 510 Pascal or Zeiss LSM710 confocal microscopes. Image-acquisition settings  were strictly maintained constant for all images taken on the same day to allow quantitative, paired comparisons.t test where appropriate , using a significance level of P < 0.05 and P < 0.01.Images from immunoblotting and fluorescence intensity, were evaluated by scanning and measuring the density of the bands . The data were expressed as means ± SEM unless otherwise indicated. The significance of comparison of mean values was determined by ANOVA or unpaired Student For additional methods and details see How to cite this article: Parks, C. et al. STIM1-dependent Ca2+ microdomains are required for myofilament remodeling and signaling in the heart. Sci. Rep.6, 25372; doi: 10.1038/srep25372 (2016).


The mechanisms that regulate intracellular Ca2+ signaling in lymphocytes involve tightly controlled concinnity of multiple ion channels, membrane receptors, and signaling molecules. T cell receptor (TCR) engagement results in depletion of endoplasmic reticulum (ER) Ca2+ stores and subsequent sustained influx of extracellular Ca2+ through Ca2+ release-activated Ca2+ (CRAC) channels in the plasma membrane. This process termed store-operated Ca2+ entry (SOCE) involves the ER Ca2+ sensing molecule, STIM1, and a pore-forming plasma membrane protein, ORAI1. However, several other important Ca2+ channels that are instrumental in T cell function also exist. In this review, we discuss the role of additional Ca2+ channel families expressed on the plasma membrane of T cells that likely contribute to Ca2+ influx following TCR engagement, which include the TRP channels, the NMDA receptors, the P2X receptors, and the IP3 receptors, with a focus on the voltage-dependent Ca2+ (CaV) channels.Elevation of intracellular calcium ion (Ca In T cells, elevation of intracellular Ca2+ levels is a vital event that mediates T cell activation, proliferation, development, differentiation, homeostasis, effector function, and cell death (2) from plasma membrane phospholipids to generate diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3). DAG, along with Ca2+, activates protein kinase C (PKC). IP3 binds to IP3 receptors (IP3R) in the endoplasmic reticulum (ER), leading to the release of Ca2+ from the ER intracellular stores into the cytoplasm. It is proposed that as the Ca2+ concentration in the ER stores decreases, the process termed store-operated Ca2+ entry (SOCE) is triggered, which results in a sustained influx of extracellular Ca2+ through Ca2+ release-activated Ca2+ (CRAC) channels in the plasma membrane  repertoire, are key mediators of immune responses against pathogens. T cell activation occurs when its TCR recognizes cognate antigen presented on major histocompatibility complex (MHC) molecules by an antigen presenting cell, which in turn triggers a series of signaling events including calcium , Ca2+-calmodulin-dependent kinase (CaMK) and its target cyclic AMP-responsive element-binding protein (CREB), and myocyte enhancer factor 2 (MEF2) targeted by both calcineurin and CaMK, NFκB, and Ras/MAPK pathways  channels. Ionotropic glutamate receptors, such as the N-methyl-d-aspartate (NMDA) receptors, function as Ca2+ channels at the plasma membrane of T cells. Ca2+ can also enter the T cell through purinergic P2X receptors, which are channels that become activated once they are bound by their ligand adenosine triphosphate (ATP). IP3 receptors (IP3R) represent another type of Ca2+ channel located at the plasma membrane of T cells. Furthermore, increasing evidence demonstrates the requirement for voltage-dependent Ca2+ (CaV) channels, the focus of this review, in T cell Ca2+ signaling and function  , 26–30.  T cells . Further T cells , 33. ORA T cells , 35. Int numbers . Analogo numbers . It has ficiency , 37, 38.2+-selective (ARC) channels , the vanilloid receptor TRPs (TRPVs), the melastatin TRPs (TRPMs), the mucolipins (TRPMLs), the polycystins (TRPPs), and the ankyrin transmembrane protein 1 (TRPA1) , 48. The through . Several through , 50–52.2+, was shown to be induced by store depletion , hydrogen peroxide (H2O2), ADP-ribose (ADPR), and cyclic ADPR  levels, and subsequent induction of either necrotic or apoptotic cell death in lymphocytes  have been well-characterized in the ER, some evidence demonstrates that they may also exist at the plasma membrane of T lymphocytes  or low voltage-activated (LVA)  . They caed (LVA) .V channels structurally consist of the α1, β, γ, and α2δ subunits, the latter being the result of connective disulfide bonds between the α2 and δ subunits  contains L-type channels; the CaV2 family consists of P/Q-type (CaV2.1), N-type (CaV2.2), and R-type (CaV2.3) channels; and the CaV3 family (CaV3.1–CaV3.3) is also referred to as the T-type channels , drugs that include examples such as the L-type Ca2+ channel activator Bay K 8644 and the inhibitor nifedipine  may result in CaV1.4-mediated Ca2+ influx from outside the cell, allowing the filling of intracellular stores and the initiation of a pro-survival program. This recent data supports the concept that in the absence of CaV1.4, there is a reduction in the influx of extracellular Ca2+ coupled to self/MHC-TCR interaction, resulting in low cytoplasmic Ca2+ levels and depleted Ca2+ ER stores  to interact with the C-terminus of ORAI1 and CaV1.2 channels. STIM1 activates ORAI1 channels, which then open resulting in sustained Ca2+ entry from the extracellular space. On the other hand, STIM1 blocks Ca2+ influx through CaV1.2 and promotes its internalization (V1 channels (Figure V and blocking ORAI1. Further investigation regarding the interplay between different Ca2+ channel families will greatly advance the field of Ca2+ channel research.There is increasing evidence in support of relationships between different types of Cand ORAI1 , 123. Folization , 123. It2+ channels discussed in this review have yet to be fully explored, these channels clearly play important roles in T cell biology and may serve as useful targets for therapeutic drugs. Many drugs already exist for modifying CaV1 channels, for example, isradipine and propofol. Moving forward, drugs that target specific Ca2+ channel splice variants found in lymphocytes may act as superior immunomodulatory agents compared to what are currently available. Pertinent applications would likely include treatment of autoimmune diseases, reduction of transplant rejection risk, and treatment of a wide range of other conditions requiring modulation of the immune system.In conclusion, while the specific functions of the various CaThe 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.


ASXL1, RUNX1, DNMT3A, NPM1, FLT3 and TP53 in 125 AML-MRC patients according to the presence of MLD, cytogenetics and outcome. ASXL1 mutations  were associated with a higher proportion of marrow dysgranulopoiesis  and were mostly found in intermediate cytogenetic AML (23/26) in which they predicted inferior 2-year overall survival . TP53 mutations  were mostly found in complex karyotype AML (26/28) and predicted poor outcome within unfavorable cytogenetic risk AML . In multivariate analysis, the presence of either ASXL1 or TP53 mutation was the only independent factor associated with shorter OS  while MLD, MDS-related cytogenetics and previous MDS history did not influence OS. We conclude that ASXL1 and TP53 mutations identify two molecular subgroups among AML-MRCs, with specific poor prognosis. This could be useful for future diagnostic and prognostic classifications.Acute myeloid leukemias (AML) with myelodysplasia-related changes (AML-MRC) are defined by the presence of multilineage dysplasia (MLD), and/or myelodysplastic syndrome (MDS)-related cytogenetics, and/or previous MDS. The goal of this study was to identify distinct biological and prognostic subgroups based on mutations of  In the WHO 2008 classification, acute myeloid leukemia (AML) with myelodysplasia-related changes (AML-MRC) is defined as a distinct entity by the presence of multilineage dysplasia (MLD), and/or myelodysplastic syndrome (MDS)-related cytogenetics, and/or previously diagnosed MDS or MDS/Myeloproliferative neoplasm (MDS/MPN) . The proASXL1 mutation [Although gene mutations are now a major tool for AML classification into distinct entities with specific prognosis -12, no mmutation . We repoASXL1 , RUNX1 , DNMT3A , NPM1 , FLT3  and TP53 . No mutation was found in 47 (38%) patients (31 with non-complex karyotype NCK-AML and 16 with CK-AML).We studied 149 patients. After morphological review, 24 patients were excluded from the main analysis because of not enough dysplasia to reach the MLD criteria. These patients were analyzed separately . The remTP53 mutations were exclusive from all other mutations and all but two  were found in CK-AML. Mutations in the other genes were almost exclusively found in NCK-AMLs . These mutations were associated with the absence of MDS-related cytogenetics . Other mutations were equally distributed whether MDS-related cytogenetics was present or not . We did not find any correlation between the presence of MLD and other mutations. Median percentages of bone marrow DGP, DEP and DMP in the 94 patients were 64%, 24% and 45% respectively. ASXL1 mutations were associated with higher DGP  but similar DEP  and DMP . There was no difference in percent of morphologic dysplasia according to any other genes . Other gene mutations did not influence the CR rate.Among the 125 AML-MRC patients, only the 67 patients who received intensive induction chemotherapy were considered for the outcome analyses. Among them, 42 achieved CR (63%). Cytogenetic risk group, the presence of MDS-related cytogenetics, previous history of MDS or MDS/MPN, and MLD did not influence the CR rate (data not shown). The presence of an ASXL1 mutation was associated with worse 2-year OS (14%) compared to patients without ASXL1 mutation  than TP53-wild type patients  and UC-AML with mutated TP53  had shorter OS than IC-AML with wild type ASXL1 . Of note, patients with UC-AML with wild type TP53  had similar OS compared to those with intermediate cytogenetic and no ASXL1 mutation .In the unfavorable cytogenetic (UC-AML) group, ASXL1 and TP53 to stratify patient outcome, we found that patients who presented with either ASXL1 or TP53 mutation had worse 2-year OS (15%) than those with both ASXL1 and TP53 wild type  and shorter OS .Using only the mutational status of ASXL1 (17%), 5 in RUNX1 (21%), 1 in DNMT3A (4%), 2 in NPM1 (8%), 4 in FLT3 (17%) and no TP53 mutation. ASXL1 mutated patients had a median percent of DGP of 62% (range: 35-90%) vs. 50% (range: 7-97%) in those with wild type ASXL1.These 24 patients were separately analyzed because they did not reach AML-MRC criteria after morphological review. Indeed, they had no previous history of MDS or MDS/MPN and no MDS-related cytogenetics. Morphological review showed dysplasia that did not reach criteria for MLD. All of them had intermediate cytogenetics and 15 (63%) had normal karyotype . Among tASXL1 and RUNX1 mutations and less DNMT3A, FLT3 and NPM1 mutations than AMLs without criteria of AML-MRC [TP53 mutations and the absence of other mutations. As expected, TP53 mutations predicted a worse outcome among patients with unfavorable cytogenetics, supporting that genetic stratification is useful to predict differential patient outcome although classified in the same cytogenetic risk group. This is in line with previous reports showing the close correlation between TP53 mutation, CK-AML and poor outcome [ASXL1 was the most frequently mutated gene (31%). It was the only mutation associated with the presence of MLD and a higher proportion of DGP in bone marrow. This is in agreement with previous biological reports showing the role of ASXL1 in the appearance of dysplasia and myeloid transformation [ASXL1 mutations and morphological MLD was not complete, because 39% of patients with MLD had ASXL1 mutation and 14% patients without MLD had ASXL1 mutation. Thus, there is no strong association between morphological analyses and the presence of an ASXL1 mutation. Interestingly, ASXL1 mutations were associated with the absence of MDS-related cytogenetics among the NCK-AML, as if ASXL1 mutations and karyotype with MDS-related abnormalities were mutually exclusive. Finally, there was no correlation between ASXL1 mutation and previous history of MDS or MDS/MPN.We previously reported that AMLs with MRC harbor a specific mutational profile with a high proportion of  AML-MRC . In this outcome , 19. In ormation -23. HoweASXL1 mutation could be considered as an independent molecular marker of dysplasia in AML that is not redundant with the criteria defining AML-MRC. This could be helpful in the perspective of developing a molecular classification of AML-MRC. This statement is also supported by the presence of a specific gene expression profile associated with ASXL1-mutated AML [TP53 stratification of outcome in UC-AML, ASXL1 mutations were associated with a shorter OS in intermediate risk patients, with a 2-year OS (14%) close to that observed in unfavorable cytogenetic patients (20%). Different studies that did not specifically focus on AML-MRC also reported this poor outcome associated to ASXL1 mutations [ASXL1 and TP53 mutation, which identified two distinct biological subgroups among AML-MRC, was the only significant predictor of outcome while usual cytogenetic risk classification or the different criteria defining AML-MRC  failed to predict patient outcome in our series. Because of the limited number of patients in some subgroups, these results need to be confirmed in larger studies.Taken together, these results suggest that the presence of an ated AML . Like TPutations , 26. ThuASXL1 and TP53 mutations could classify distinct subgroups of AML-MRC, patients without any of these mutations still represent a heterogeneous group of AML-MRC harboring different morphological, cytogenetic and molecular features. The mutations of DNMT3A and/or NPM1, which are usually found in de novo AML and are mutually exclusive with ASXL1 and TP53 mutations, could identify patients for whom the definition of AML-MRC should be questioned. Falini et al. reported that MLD did not identify distinct biological and clinical entities among NPM1-mutated AML, with overlapping gene expression profiling and similar outcome [ASXL1 mutations might be included in the same subgroup as ASXL1-mutated AML-MRC. This supports the need of redefining AML-MRC upon molecular abnormalities.Although  outcome . In contASXL1 is a key regulator of the polycomb repressive complex 2 (PRC2), it is possible that AML carrying alterations of other genes involved in methylation marks via PRC2 and myeloid transformation such as EZH2 [JARID2 [BAP1 [ASXL1-mutated AML. This could help identify AML-MRC through a common altered molecular pathway and could help develop future targeted treatments. An extensive mutational screening using whole genome or exome sequencing could be useful in this setting to improve the molecular characterization of these unmutated cases.Finally, we did not find any mutation in the 6 genes studied in 47 patients, suggesting the need to identify other molecular markers to stratify these patients. Because  as EZH2 , 28, JAR [JARID2  or BAP1 D2 [BAP1 , 31 coulASXL1 and TP53-mutated AML identify two distinct biological subgroups of AML-MRC with very poor outcome. This molecular characterization could be useful to redefine AML-MRC in a future classification aiming at merging biological characterization and specific prognostic value.We conclude that the criteria defining AML-MRC do not identify distinct clinical and biological subgroups and do not predict outcome of patients with AML-MRC. In contrast, We conducted a retrospective analysis of patients from two French centers. Selection criteria for analyses were: (i) Diagnosis of AML with criteria for AML-MRC according to the WHO classification: previouAML with inv(3)/t, t and t or with favorable risk according to the ELN classification (inv(16)/t, t, t, normal karyotype AML with NPM1 or CEBPA mutations) were excluded as well as therapy-related AML .Smears of bone marrow aspirates made at diagnosis were collected and stained by May-Grünwald-Giemsa. Each smear was retrospectively analyzed by two expert cytologists in both centers. We assessed the percentage of dysplastic cells in each lineage. MLD was assessed according to the WHO classification criteria: at least 50% of dysplastic cells in at least 2 lineages. We established the percentage of dysgranulopoiesis (DGP), dyserythropoiesis (DEP) and dysmegakaryopoiesis (DMP) for each smear.ASXL1 (exon 12), RUNX1 (exon 1-8), DNMT3A (exon 15-23), NPM1 (exon 12), FLT3  and TP53 (exon 4-10).Direct sequencing was done using the Sanger method as previously described . We searChi-square or Fischer tests were used to compare categorical variables. We used non-parametric test (U Mann Whitney) to compare the median percentage of dysplastic cells in each lineage according to mutational status for each gene. As multivariate model, linear regression was used to find correlation between morphologic dysplasia and the presence of mutation. Survivals were calculated using the Kaplan Meier estimator . Time to