Despite the crucial role of CaV1.3 Ca2+ channels in governing synaptic vesicle fusion, their elementary properties in adult mammals remain unknown. Using near-physiological recording conditions we investigated Ca2+ channel activity in adult gerbil IHCs. We found that Ca2+ channels are partially active at the IHC resting membrane potential (−60 mV). At −20 mV, the large majority (>70%) of Ca2+ channel first openings occurred with an estimated delay of about 50 μs in physiological conditions, with a mean open time of 0.5 ms. Similar to other ribbon synapses, Ca2+ channels in IHCs showed a low mean open probability (0.21 at −20 mV), but this increased significantly (up to 0.91) when Ca2+ channel activity switched to a bursting modality. We propose that IHC Ca2+ channels are sufficiently rapid to transmit fast signals of sound onset and support phase-locking. Short-latency Ca2+ channel opening coupled to multivesicular release would ensure precise and reliable signal transmission at the IHC ribbon synapse.Auditory information transfer to afferent neurons relies on precise triggering of neurotransmitter release at the inner hair cell (IHC) ribbon synapses by Ca The transfer of sound information to the brain relies on the precise release of neurotransmitter from sensory inner hair cell (IHC) ribbon synapses.2+ entry mainly through one type of Ca2+ channel (CaV1.3).Neurotransmitter release from IHCs is triggered by Ca2+ channels open very rapidly following a stimulus with a delay of about 50 μs.In this study we show that in near-physiological conditions Ca2+ channels being very low, they can switch to a burst-like mode during a stimulus, maximizing Ca2+ influx into IHCs.Despite the open probability of the CaThese results help us to better understand how IHCs are able to accomplish high-fidelity signal transfer at individual auditory ribbon synapses.2+ entry in response to cell depolarization during sound-induced hair bundle deflection. The IHC Ca2+ current is carried almost exclusively by CaV1.3 Ca2+ channels  ribbon synapses . IHC neuto sound .2+ channels per synapse with depolarization, with each new channel opening producing an additional vesicle fusion event  Act 1986 and were approved by the University of Sheffield Ethical Review Committee. In Germany, care and use of the animals and the experimental protocol were reviewed and approved by the animal welfare commissioner and the regional board for scientific animal experiments in Tübingen.m): 135 NaCl, 5.8 KCl, 1.3 CaCl2, 0.9 MgCl2, 0.7 NaH2PO4, 5.6 d glucose, 10 Hepes-NaOH, 2 sodium pyruvate, amino acids and vitamins (pH 7.5).Basal-coil IHCs (frequency ∼30 kHz) from the adult gerbil were studied in acutely dissected cochleae  to minimize the fast electrode capacitative transient and filled with the following solution (in mm): 5 CaCl2, 102 CsCl, 10 Hepes-KOH, 15 4-aminopyridine (4 AP), 40 TEA (pH 7.5) containing linopirdine , niflumic acid  and BayK 8644 (5 μm: Sigma). Stock solutions of niflumic acid and BayK 8644 were prepared in DMSO and stored at −20°C . Although the majority of recordings were performed using the above Na+-based extracellular solution, in a few experiments (A and B) the membrane potential of IHCs was zeroed by superfusing a high-K+ extracellular solution (m): 140 KCl, 0.2 CaCl2, 6.2 MgCl2, 0.7 NaH2PO4, 5.6 d-glucose, 15 Hepes-KOH (pH 7.5). Trypsin (0.025–0.05% v/v) was briefly and topically applied onto IHCs (40% of the recordings) prior to patching (For single Caeriments A and B tm): 106 caesium glutamate, 20 CsCl, 3 MgCl2, 1 EGTA-CsOH, 5 Na2ATP, 0.3 Na2GTP, 5 Hepes-CsOH, 10 sodium phosphocreatine (pH 7.3). The calcium current was isolated using extracellular TEA (30 mm), 4–AP (15 mm) and linopirdine (100 μm). Extracellular BayK 8644 (5 μm) and 5 mm Ca2+ were also used to mimic the single-channel recording conditions. Data were filtered at 5 kHz or 10 kHz (8-pole Bessel) and sampled at 50 kHz or 100 kHz. Membrane potentials were corrected for residual series resistance and LJP (−11 mV). All recordings were performed at body temperature (34–37°C).Whole-cell recordings were performed using soda glass capillaries (resistance 2–3 MΩ), coated with surf-wax and filled with  and Ribeye/CtBP2  were detected with Cy3-conjugated  or Alexa Fluor 488-conjugated  secondary antibodies. Images were acquired using a CCD camera and analysed with cellSense Dimension software . The distribution of Ca2+ channels and ribbons were imaged over a distance of several micrometres with the coverage of the entire IHC synaptic region in an image-stack along the z-axis (z-stack) followed by three-dimensional deconvolution as previously described (Gerbil cochleae (P20) were fixed , decalcified and cryosectioned as previously described  and open and closed time histograms. Amplitude distributions were fitted with a Gaussian function. Po was calculated as the fraction of time spent open vs. the total recording time. Sweeps containing two or more Ca2+ channels were excluded from the analysis. The total number of Ca2+ channels per IHC was estimated using:Single CaN is the number of channels, ICa is the peak macroscopic Ca2+ current, i is the single-channel current size and Po the channel open probability. To analyse the single channel open and closed times (-1) with normalization of the number of observations for bin amplitude. The plots obtained were interpolated, using the maximum likelihood method, with the sum of n (two or three) exponential functions , which were all colocalized with synaptic ribbons (CtBP2) (2+ channel activity was extremely low (∼4%).In adult gerbil IHCs, Cac region A, which  (CtBP2) B. Despit2+ channels are encoded by four different pore-forming α1 subunit genes  and are sensitive to 1,4-dihydropyridines, such as the antagonist nifedipine and the agonist BayK 8644. In the mammalian cochlea, IHCs almost exclusively express the CaV1.3 isoform , Ca2+ channels exhibited two distinct opening modes: one characterized by brief and rather infrequent openings  can be seen in the absence of BayK 8644, which increase the duration of all openings, in immature mouse IHCs (I–V) relation was linear in the voltage range investigated with an average slope conductance of 15 pS . The actual patch transmembrane voltage was estimated using the amplitude of the elementary Ca2+ current and extrapolating it from the I–V curves obtained in high-K+ solution  increased with depolarization from 0.01 at about −50 mV to 0.21 at about −20 mV. We found that the maximal Po varied significantly among sweeps, from the lowest value of 0.014 when the channel opened rarely and briefly (gating mode 1) to 0.91 in the presence of prolonged periods of opening (mode 2). The total number of Ca2+ channels present in adult IHCs  is likely to be in the order of 2800. The macroscopic ICa (−197 pA) was measured in adult gerbils using experimental conditions similar to those used for single-channel recordings (see Methods). A higher Po of ∼0.8 and smaller elementary conductance has previously been estimated using fluctuation analysis by calculating the variance and mean of whole-cell tail Ca2+ currents measured at −62 mV from pre-step depolarization to +58 mV  were considered, the Caactivation1= 0.33 ms, τactivation2= 6.08 ms, τinactivation= 92 ms; 300 sweeps from 8 IHCs) from single-channel recordings  using similar experimental conditions . The similarity between the kinetics of the ensemble-average current and that recorded in whole-cell suggests that the cell-attached configuration, and the mechanical perturbation it could induce upon the patched membrane, is unlikely to significantly alter the kinetic behaviour of the Ca2+ channels. Using whole-cell recordings, we also found that the activation kinetics of ICa in 5 mm Ca2+  were about 4 times slower compared to those in 1.3 mm Ca2+ , which is most likely caused by surface screening effects , the weight of which was much larger than the other two components , the first latency time constants can also be expected to become faster. We estimate that τ1, which represents 73% of the total first latency distribution  and three closed  time constants  and an increase in the weight of the shortest close time constant (τc1). A very slow exponential component, with a time constant (τc3) of about 95 ms, was also present in closed-time distributions. Although τc3 was probably underestimated, due to the high probability of long closure events being interrupted at the end of the 500 ms depolarization, it was 12 times greater than the ‘intermediate’ time constant τc2. Moreover, the relative weight of the slowest component was only 2%. Therefore, the average number of ‘short’ closures per sweep exceeded that of ‘long’ closures, indicating that single Ca2+ channel openings had a relatively high probability of being separated by short closings. This implies that Ca2+ channel activity was largely organized in bursts, consisting of sequences of openings separated by short closings, and interrupted by prolonged closures (c2), was 81 ± 72 ms (136 bursts from 101 sweeps: 8 IHCs). Bursting activity greatly increased Po in a sweep. As seen with the high-K+ extracellular solution, bursts of channel openings often appeared in successive sweeps  revealed two or three open . Despite the low mean Po, Ca2+ influx into IHCs through a Ca2+ channel can be maximized by burst activity. We propose that brief single Ca2+ channel openings are sufficient to trigger vesicle release, and the short-latency would ensure reliable and precise signal transmission at the IHC ribbon synapse during high-frequency activity.In this study, using near-physiological experimental conditions, we determined that in adult IHCs the first Ca2+ channels were deemed to be too slow  was ∼0.18 ms in adult gerbil IHCs . This value is much faster than that recorded in immature mouse IHCs . These findings show that Ca2+ channel activation rates are sufficiently rapid to support phase-locking to sound  is the main contributor to the Po, and that in the absence of BayK 8644 the channel Po is expected to be even lower, it is likely that a single-channel opening will provide enough Ca2+ to trigger a vesicle fusion event, which is in partial agreement with previous indirect observations . Thus, our gerbil IHC data indicate that a single Ca2+ channel opening is likely to be able to trigger the simultaneous fusion of multiple vesicles, which supports a similar proposal for bullfrog hair cells (2+ influx (An interesting feature of hair cell ribbon synapses is that vesicle release evokes short-lived EPSCs of variable amplitude (ir cells . In hair2+ channels: Po changed from 0.01 at rest to 0.21 at −20 mV. This agrees with the observation that the frequency of EPSCs increases with IHC depolarization (2+ channel properties: (1) EPSC amplitude does not increase with IHC depolarization, which is normally seen in other synapses  mon2+ influx into IHCs led to the hypothesis that large EPSCs could originate from the incremental recruitment of single Ca2+ channels with depolarization , we propose instead that depolarization increases the chance that Ca2+ channels opening in mode 1 (largely silent) switch to mode 2 (bursting), thus increasing the probability of vesicle-fusion events. This is likely to be true even in the absence of BayK 8644 since CaV1.3 Ca2+ channels show bursting behaviour even without the agonist  at the presynaptic site, or saturation of intracellular Ca2+ buffers, since its volume seems to be restricted by the presence of the ribbon (Pharmacological manipulation of the macroscopic Cachannels . On the


On recognizing osmotic signals, plant cells initiate activation of a widespread signal transduction network that induces second messengers and triggers inducible defense responses. Characteristic early signaling events include Ca2+ influx, protein phosphorylation and generation of reactive oxygen species (ROS). Pharmacological analyses show Ca2+ influx mediated by mechanosensitive Ca2+ channels to influence induction of osmotic signals, including ROS generation. However, molecular bases and regulatory mechanisms for early osmotic signaling events remain poorly elucidated.Mechanosensing and its downstream responses are speculated to involve sensory complexes containing Ca2+-permeable mechanosensitive channels in Arabidopsis (MCAs). OsMCA1 was specifically localized at the plasma membrane. A promoter-reporter assay suggested that OsMCA1 mRNA is widely expressed in seed embryos, proximal and apical regions of shoots, and mesophyll cells of leaves and roots in rice. Ca2+ uptake was enhanced in OsMCA1-overexpressing suspension-cultured cells, suggesting that OsMCA1 is involved in Ca2+ influx across the plasma membrane. Hypo-osmotic shock-induced ROS generation mediated by NADPH oxidases was also enhanced in OsMCA1-overexpressing cells. We also generated and characterized OsMCA1-RNAi transgenic plants and cultured cells; OsMCA1-suppressed plants showed retarded growth and shortened rachises, while OsMCA1-suppressed cells carrying Ca2+-sensitive photoprotein aequorin showed partially impaired changes in cytosolic free Ca2+ concentration ([Ca2+]cyt) induced by hypo-osmotic shock and trinitrophenol, an activator of mechanosensitive channels.We here identified and investigated OsMCA1, the sole rice homolog of putative Ca2+-permeable mechanosensitive channel indicate that OsMCA1 is involved in regulation of plasma membrane Ca2+ influx and ROS generation induced by hypo-osmotic stress in cultured rice cells. These findings shed light on our understanding of mechanical sensing pathways.We have identified a sole MCA ortholog in the rice genome and developed both overexpression and suppression lines. Analyses of cultured cells with altered levels of this putative Ca Electrophysiological and bioinformatic studies have revealed the existence of plasma membrane Ca2+-permeable channels activated by mechanical stimuli, although the structural entity involved and their physiological functions remain largely unknown cyt elevation upon hypo-osmotic shock. However, the direct effects of MCA proteins on osmotic-induced Ca2+ influx through the plasma membrane and the osmotic signaling pathways are little understood.Molecular and electrophysiological studies have shown that han Ca2+ ,14. We hbidopsis ,16, and 2+ influx, protein phosphorylation and generation of reactive oxygen species (ROS) cyt change was partially impaired in the OsMCA1-suppressed cells  recognized by plasma membrane receptors in rice cyt using OsMCA1-overexpressing lines. Real-time RT-PCR analysis revealed that the expression level of aequorin gene in the OsMCA1-overexpressing lines was comparable to the control (data not shown). Thus constitutive overexpression of OsMCA1 does not affect the expression but may affect the stability of aequorin protein or inhibit the aequorin chemiluminescence.We also tried to examine the effect of overexpression of 2+]cyt increase in cultured tobacco cells cyt [Rice cells (cv. Nipponbare) 5 days after subculture were used for measurement of Ca2+]cyt .2O2 produced in extracellular medium, cells (80 mg mL-1) were washed and resuspended in 5 mM MES buffer (pH 7.0) containing 0.5 mM CaCl2, 0.5 mM K2SO4 and with or without 175 mM mannitol . A 25-μL aliquot of medium was mixed in a 96-well microtiter plate with 150 μl 50 mM Tris-HCl (pH 8.0) and 25 μL 0.462 mM luminol in 50 mM Tris-HCl, pH 8.0. Potassium ferricyanide  was added, and H2O2-dependent chemiluminescence was recorded for 15 s using a luminometer .To monitor Ht test; P < 0.05 indicated significance.Statistical significance was determined using an unpaired Student's N, N, N', N'-tetra acetic acid; [Ca2+]cyt: cytosolic free Ca2+ concentration; DIC: differential interference contrast; DPI: diphenylene iodonium; EGTA: ethylene glycol-bis-(2-aminoethylether)-N, N, N', N'-tetra acetic acid; GFP: green fluorescent protein; GUS: β-glucuronidase; H2O2: hydrogen peroxide; MAMP: microbe-associated molecular pattern; MCLA: 2-methyl-6-[p-methoxyphenyl]-3,7-dihydroimidazo pyrazin-3-one; •O2-: superoxide anion radical; MS medium: Murashige and Skoog medium; RACE: rapid amplification of cDNA ends; rlu: relative luminescence units; RNAi: RNA interference; RT: reverse transcriptase; ROS: reactive oxygen species; SHAM: salicylhydroxamic acid; TNP: trinitrophenol.BAPTA: 1,2-bis-(2-aminophenoxy)ethane-2+ uptake experiments in yeast. HH and HS participated in constructing transgenic lines and PCR analyses. YN, KS, HI participated in the design of the study and critically revised the manuscript. All authors read and approved the final manuscript.TK, DN, and YY carried out most of the experiments and data analyses. TK and KK designed the study and wrote the manuscript. DN, YY and MG participated in confocal imaging analyses. MN, TY and KI carried out CaYN Present address: Laboratory of Cell Biology, Institute for Molecular and Cellular Regulation, Gunma University, Maebashi, Gunma 371-8510, Japan.Multiple amino acid sequence alignment of rice OsMCA1 and Arabidopsis MCA1 and MCA2 using Clustal W. Asterisks indicate identical or conserved residues in the whole sequence in the alignment. Colons indicate conserved substitutions. Dots indicate semi-conserved substitutions. Light and dark gray box indicate the PLAC8 motif and coiled-coil motif, respectively. Two putative transmembrane segments (S1 and S2) are underlined.Click here for fileOsMCA1 gene in rice tissuesExpression of the . The expression of OsMCA1 in rice plants was determined by quantitative RT-PCR analysis. Total RNA was extracted from various tissues of rice plants as well as cultured cells. The amount of OsMCA1 mRNA was calculated from the threshold point in the log-linear range of the RT-PCR. The relative OsMCA1 mRNA level in cultured cells was standardized as 1. Data are means ± SD; n = 2-3 independent samples.Click here for fileOsMCA1-suppressed seedlings in MS mediumGrowth phenotype of . Length of roots and shoots of the control line (20-5) and the OsMCA1-suppressed lines (20-2 and 38-5) of 10-days-old seedlings grown on MS medium plate in a growth chamber under long-day conditions . Data are means ± SD; n = 7-10 independent seedlings.Click here for fileEffect of NADPH oxidase inhibitor on hypo-osmotic shock-induced ROS generation. H2O2 concentration in extracellular medium was determined by ferricyanide-catalyzed oxidation of luminol. Diphenylene iodonium  was added to the rice cells 30 min before hypo-osmotic shock treatment. Data are the mean ± SE for five independent experiments for the wild type. ***P < 0.005; significantly different compared with the control.Click here for fileEffect of salicylhydroxamic acid, a peroxidase inhibitor, on hypo-osmotic shock-induced ROS generation. The concentration of •O2- in extracellular medium was measured by MCLA chemiluminescence. Salicylhydroxamic acid  was added to the rice cells 30 min before hypo-osmotic shock treatment. Average values and SE of three independent experiments for the wild type.Click here for fileOsMCA1-overexpression on hypo-osmotic shock-induced ROS generationEffect of . H2O2 concentration in the extracellular medium was determined by ferricyanide-catalyzed oxidation of luminol. As a hypo-osmotic shock, growth medium was replaced by three-fold diluted medium at 0 min. Data are the mean ± SE for four independent experiments for two control lines  and two overexpressor lines  are shown. *P < 0.05, significantly different compared with two control lines (GUS No. 7 and 11).Click here for file


Cellular responses were investigated using real-time 2-photon imaging of lung slices loaded with a Ca2+-sensitive fluorescent indicator (Ca2+ measurements) and phase contrast microscopy (contraction measurements). PVCs displayed global Ca2+ signals and coordinated contraction in response to electrical field stimulation (EFS). The effects of EFS relied on both Ca2+ influx and Ca2+ release, and could be inhibited by nifedipine, ryanodine or caffeine. Moreover, PVCs had a high propensity to show spontaneous Ca2+ signals that arose via stochastic activation of ryanodine receptors (RyRs). The ability of electrical pacing to entrain Ca2+ signals and contractile responses was dramatically influenced by inherent spontaneous Ca2+ activity. In PVCs with relatively low spontaneous Ca2+ activity (<1 Hz), entrainment with electrical pacing was good. However, in PVCs with higher frequencies of spontaneous Ca2+ activity (>1.5 Hz), electrical pacing was less effective; PVCs became unpaced, only partially-paced or displayed alternans. Because spontaneous Ca2+ activity varied between cells, neighboring PVCs often had different responses to electrical pacing. Our data indicate that the ability of PVCs to respond to electrical stimulation depends on their intrinsic Ca2+ cycling properties. Heterogeneous spontaneous Ca2+ activity arising from stochastic RyR opening can disengage them from sinus rhythm and lead to autonomous, pro-arrhythmic activity.The pulmonary vein, which returns oxygenated blood to the left atrium, is ensheathed by a population of unique, myocyte-like cells called pulmonary vein sleeve cells (PVCs). These cells autonomously generate action potentials that propagate into the left atrial chamber and cause arrhythmias resulting in atrial fibrillation; the most common, often sustained, form of cardiac arrhythmia. In mice, PVCs extend along the pulmonary vein into the lungs, and are accessible in a lung slice preparation. We exploited this model to study how aberrant Ca Essentially, membrane depolarisation opens voltage-operated Ca2+ channels (VOCCs) to allow Ca2+ influx into the ‘dyadic’ cleft between the sarcolemma and the sarcoplasmic reticulum . This Ca2+ signal is amplified by Ca2+-induced Ca2+ release via ryanodine receptors (RyRs) on the SR, thus causing a global Ca2+ signal that triggers actin-myosin filament interaction and myocyte contraction 2+ is returned to diastolic levels by Ca2+-ATPases on the sarcolemma and SR, and Na+/Ca2+ exchangers on the sarcolemma Throughout a typical human lifetime, a coordinated ‘cardiac cycle’ of atrial and ventricular contraction is repeated over a billion times 2+ signals as a likely cause 2+ signals occurring during the recovery from a previous AP, or during the quiescent diastolic phase, can depolarise the sarcolemma and potentially trigger an ectopic AP or alter the refractoriness of myocytes relative to their neighbours It is believed that increased ‘automaticity’ (spontaneous depolarisation of myocytes), acute ‘triggered activity’  or ‘re-entry circuits’  contribute to AF. The underlying causes of all these pro-arrhythmic conditions are not fully understood, but substantial evidence has implicated spurious Ca2+ signaling and electrical events can arise within the atrial chambers themselves 2+ signals While spontaneous Cain situ organizational and physiological characteristics of the lung, and are viable for many days. Because the PVCs are disconnected from the left atrial chamber, they no longer receive APs arising from the SA node. This electrical isolation allows the spontaneous Ca2+ signaling capacity of the PVCs to be evident without a background of SA node-evoked events. As required, the PVCs within a lung slice can be activated by application of electric field stimulation (EFS). In the present study, we characterised spontaneous and EFS-induced Ca2+ transients in PVCs to determine whether these two processes use similar signaling mechanisms. Moreover, we explored the hypothesis that the inherent spontaneous Ca2+ signals within PVCs could corrupt their electrical entrainment, and thereby lead to pro-arrhythmic outcomes.The extent of the PVC sheath varies between animal species. Here, we took advantage of the situation in mice, where the PVCs extend from the left atrium to the pulmonary veins within the lung for several branching generations 2. Slices were used within 48 h of preparation, apart from experiments investigating the viability of PVCs. For imaging experiments, lung slices were mounted between 2 cover-glasses separated with silicon grease on a custom-built Perspex support, which allowed for a continuous superfusion of the lung slices with experimental solutions.For the preparation of lung slices 2PO4 0.4 mM, NaCl 137.9 mM, NaH2PO4 0.3 mM, NaHCO34.2 mM, D-Glucose 5.6 mM, MgCl2 0.5 mM, MgSO4 0.4 mM, CaCl2 1.3 mM, HEPES 20 mM), pH 7.4. Ca2+-free HBSS was made by supplementing nominally Ca2+-free sHBSS with 100 µM EGTA. Ryanodine and caged InsP3/PM (Propionoxymethylester) were purchased from Enzo Life Sciences . Unless otherwise mentioned, all other reagents were purchased from Sigma .Experiments were performed in sHBSS , based on an Olympus IX71 microscope, using an oil immersion UApo 340 40x (N.A. 1.3) or PlanApo60x (N.A. 1.42) objective. Lung slices were loaded in the dark with 20 µM Oregon Green BAPTA-1 AM  in presence of 0.1% Pluronic F127 (Life Technologies) and 200 µM sulfobromophthalein in sHBSS at 30°C for 1 hour, followed by a de-esterification period of at least 30 min at 30°C in the presence of sulfobromophthalein. Image acquisition was performed using Video Savant software at 30 images per second. Throughout the experiment, lung slices were perfused with sHBSS using a gravity-fed system. The solution exchange was controlled by Video Savant software. The 2-photon excitation wavelength was set by tuning the laser. The excitation light was reflected using a FF665-Di02-25×36 dichroic , and the emitted fluorescence light was filtered using a short-pass barrier filter FF01-680/SP-25 (Semrock).Imaging experiments were performed on custom-built microscopes A custom-built confocal microscope, based on a Nikon Diaphot 200, with either a 40x Fluor (N.A. 1.3) or 63x Planapo (N.A. 1.4) oil-immersion objective was used for imaging immunofluorescence and di-8-ANNEPS (488 nm excitation for both). Specific staining of the PVC sarcolemma was achieved by incubation of lung slices with 25 µM di-8-ANNEPS  for 30 min. The excitation (488 nm laser line) was reflected using a LPD01-488RU-25×36 dichroic (Semrock), and emitted fluorescence light was filtered using a long-pass LP02-488RU-25 filter (Semrock).2+ transients were evident at 20°C and 37°C . However, electrical pacing, contraction and spontaneous CaImmunostaining was performed in paraformaldehyde-fixed lung slices as previously described EFS  was applied using a Grass Instruments SD9 stimulator and platinum electrodes , placed at the opposite ends of the imaging chamber. The EFS conditions were empirically determined by finding the minimal voltage condition that would reliably pace PVCs with no appreciable run-down of the responses.Statistical analysis was performed in GraphPad Prism 6 , using a Mann-Whitney U-Test (2 datasets) or a Kruskall-Wallis Test followed by a Dunn’s post-hoc analysis (>2 datasets) to test for statistically significant differences by comparing the initial values the first 30 s in sHBSS at the start of the experiment to the values at the indicated time periods. P values <0.05 were considered significant, *P<0.05, **P<0.01, ***P<0.001. Data are shown as mean ± s.e.m.2+ signaling mechanisms within PVCs, lung slices were loaded with the Ca2+-sensitive indicator Oregon Green BAPTA-1 and examined with real-time 2-photon microscopy. PVCs displayed rapid, transient Ca2+ increases in response to electric field stimulation . Disruption of electrical stimulation by stopping EFS . Immunostaining for the cardiac isoforms of RyR (types 2 and 3) revealed a striated distribution of these Ca2+ channels  within the PVCs. These striations were 1.8±0.1 µm apart , similar to the RyR (z-line) distribution in cardiac myocytes i and Dii). Collectively, these data suggest that PVCs structurally resemble rodent atrial myocytes Pulmonary veins with a PVC myocardial sheath are found in mouse lung slices cut from the hilus region, where the pulmonary veins exit the lungs, to a distance approximately half-way through the lung lobe towards the periphery of the lungs . The myoIn addition to their location and morphology, PVCs surrounding pulmonary veins could be readily identified by their prominent spontaneous contractions . The nati–iii; n = 6 slices). Synchronised contraction only occurred during EFS; spontaneous contractile activity resumed with the termination of EFS.In many lung slices, asynchronous spontaneous PVC contractile activity could be coordinated into synchronous contractions by EFS, with a consequent reduction in the lumen size of the pulmonary vein during each contraction i–iii; . During 2+ activity in the absence of EFS (n >250 slices). The spontaneous Ca2+ transients were heterogeneous, and ranged from localized Ca2+ increases (Ca2+ sparks) to Ca2+ waves that propagated along single PVCs . Ca2+ wave propagation occurred with a speed of 70.2±3.5 µm/s, with a mean wave duration of 388.2±18.8 ms . Spontaneous Ca2+ signaling could be imaged within individual PVCs for >15 minutes with little change in activity.The overwhelming majority of PVCs displayed spontaneous Cagle PVCs . The fre2+ signals are illustrated by the line-scan plot in 2+-dependent fluorescence within a pair of PVCs , and propagate bi-directionally to the poles of the cell (arrowheads). The Ca2+ waves stopped at the boundary between cells 1 and 2 (dashed line). Cell 2 had its own intrinsic rhythm, and displayed Ca2+ waves that sometimes temporally coincided with those of cell 1.Typical spontaneous Ca2+ transients are evident in 2+ signals (and triggering contraction) within the PVCs. However, the efficacy of each EFS pulse varied on a pulse-to-pulse basis, and ranged from inducing a whole-cell Ca2+ signal to having no effect. For example, in 2+ signal simultaneously in both cells . The other EFS pulses correlated with one of three types of responses; i) only one cell showed a whole-cell response , ii) one or both cells showed an incomplete response  or iii) neither cell responded . A plausible explanation for variable effects of EFS is that the PVCs enter a refractory period after a Ca2+ rise during which they cannot respond to another Ca2+-releasing stimulus. Therefore, if a cell responds to EFS with a whole-cell response, the subsequent spontaneous Ca2+ signal is inhibited or delayed. Conversely, if a spontaneous Ca2+ wave occurs just prior to an EFS pulse, the cell cannot respond with a Ca2+ transient. In essence, Ca2+ signals evoked by EFS delayed the timing of spontaneous Ca2+ activity and vice versa.EFS-evoked Ca2+ signal and a subsequent spontaneous Ca2+ event was correlated with the frequency of the intrinsic spontaneous Ca2+ activity. The latency was longer in cells with a sparse spontaneous Ca2+ activity (<1 Hz) as compared to cells with more frequent spontaneous Ca2+ activity  and 3 Hz EFS. However, in response to 3 Hz, Cell 1 displayed ‘alternans’ . Conversely, Cell 2 was only partially paced (PP) with 2 Hz EFS (iii), but was fully paced (FP) with 3 Hz EFS without alternans (iii). These observations indicate that while increasing the frequency of EFS could sometimes entrain spontaneous Ca2+ signals, other forms of pro-arrhythmic activity, e.g. alternans, could ensue.We hypothesised that increased EFS frequency might overcome the confounding effects of spontaneous Ca signals . Cell 1  signals ii and 32 Hz EFS iii, butlternans iii. The2+ signaling. It is evident that PVCs were more successfully paced as the EFS frequency increased. However, not all PVCs were paced (i). PVCs that were partially paced were more evident at lower EFS frequencies (ii), whilst higher EFS frequencies were most effective for complete entrainment (iii). However, the higher EFS frequencies were also capable of generating alternans (iv). We suggest that the heterogeneity of EFS-induced Ca2+ signaling arises from the relative timing of an EFS pulse with respect to the intrinsic spontaneous Ca2+ signaling activity; it is easier to fully pace cells which show little spontaneous activity, and more difficult to pace cells with a high frequency of spontaneous activity. In support of this conclusion, we observed that the minimal EFS frequency required for fully paced cells correlated with the intrinsic frequency of spontaneous Ca2+ signals . We suggest that all PVCs have unique periodicities of spontaneous Ca2+ signaling, and this determines the cells’ refractory periods. In some cells, the frequency of spontaneous Ca2+ signaling is so high that EFS is ineffective. It should be noted that whilst there is a clear impact of the frequency of spontaneous Ca2+ signals on the pacing of PVCs, this is not the only factor. We also observed cells with similar frequencies of spontaneous Ca2+ signaling responding differently to the same EFS pulse. This implies heterogeneity in the responsiveness of the cells to EFS in addition to the effects of spontaneous Ca2+ signaling.re paced i. PVCs quencies  Dii, whirainment iii. Howlternans iv. We s signals . Those c2+-free medium caused a progressive reduction in the amplitude and frequency of spontaneous Ca2+ signals . In 58% of the PVCs, the spontaneous Ca2+ activity was fully inhibited within 5 min of Ca2+ removal (mean time to inhibition: 209.5±15.3 s). This inhibition of the spontaneous Ca2+ signals also correlated with the loss of spontaneous PVC contractions. These data indicate i) that spontaneous Ca2+ signals arise via Ca2+ release from intracellular stores, ii) that PVCs have a substantial capacity for recycling Ca2+, and iii) that PVCs require Ca2+ influx to sustain spontaneous Ca2+ activity and contraction.Superfusion of non-paced PVCs with Ca signals , and of 2+ (1.3 mM) did not restore the initial pattern of spontaneous Ca2+ activity, but rather induced an entirely different form of Ca2+ signaling. Within 40 to 60 s of Ca2+ re-addition, PVCs began to display Ca2+ waves that progressively increased in amplitude, frequency and spatial propagation . Moreover, these Ca2+ waves had a longer duration than the original spontaneous activity . Consequently, the cytosolic Ca2+ concentration did not have time to recover between wave fronts, so the diastolic Ca2+ level became elevated. Typically, all the PVCs surrounding a pulmonary vein displayed Ca2+ waves in response to re-addition of extracellular Ca2+ . The Ca2+ waves were associated with strong PVC contractions, but the contractions were not coordinated between cells with the result that the layer of PVCs fibrillated asynchronously. Significantly, the rapid Ca2+ waves and strong contractions persisted for some time after Ca2+ re-addition, and did not return to the original pattern of spontaneous activity within 15 minutes. The spontaneous Ca2+ waves in PVCs under control conditions were predominantly intracellular. The Ca2+ removal/re-addition experiments presented a situation in which intracellular Ca2+ waves frequently turned into intercellular Ca2+ waves  were driven to the fibrillated state that was unresponsive to EFS (ii and Eiii) by removal and re-addition of extracellular Ca2+. Similarly, EFS could not re-synchronize the contractile activity of the PVCs following re-addition of extracellular Ca2+ (i–iv). Re-addition of a lower extracellular Ca2+ concentration (500 µM instead of 1.3 mM), slowed the onset of the rapid Ca2+ waves and contractions, but the PVCs eventually reached a similar state of fibrillation that was unresponsive to EFS.Application of EFS either prior to, or during, Calar Ca2+  Fi–iv. R2+ signaling that is characterized by Ca2+ waves with a longer duration and a persistent elevation of cytosolic Ca2+. Moreover, other maneuvers (see below) that promoted Ca2+ influx or altered Ca2+ homeostasis could also cause PVCs to display a rapid, EFS-resistant, form of Ca2+ signaling.Our data indicate that PVCs can progress to an unrecoverable, EFS-insensitive form of Ca2+ signals continue for some time in the absence of extracellular Ca2+ suggests that these Ca2+ signals depend on Ca2+ release from internal stores. Consistent with this idea, we observed that caffeine  increased the frequency of spontaneous Ca2+ transients and typically caused a sustained elevation of cytosolic Ca2+ (i and Eii). PVCs with EFS showed a similar caffeine-evoked increase in spontaneous Ca2+ activity, and consequent loss of entrainment in 47% of the cells (iii). The effects of caffeine were reversible within 30–60 s (ii and Bii), and the PVCs could be re-entrained by EFS (compare responses between 30 to 60 seconds in i/Di with Cii/Dii). Ryanodine  inhibited the spontaneous Ca2+ signals within ∼150 s  did not appear to play a role in spontaneous Ca2+ responses. Photolytic release of InsP3 did not increase the cytosolic Ca2+ concentration in PVCs . In contrast, airway smooth muscle cells within the same slices showed substantial Ca2+ signals following photolysis of caged InsP3 . Furthermore, 2-aminoethoxydiphenyl borate , did not alter the frequency or spatial properties of spontaneous Ca2+ signaling within PVCs .Inositol 1,4,5-trisphosphate receptors  and stimulated the occurrence of spontaneous events in PVCs that were previously silent (ii and Bii). KCl treatment had a similar effect to that of caffeine and induced rapid Ca2+ waves superimposed on an elevated cytosolic Ca2+ level, which were reversible upon washout (ii). The effects of KCl on PVCs during EFS were similar; KCl induced the progressive development of rapid Ca2+ waves on an elevated Ca2+ baseline to the point that synchronised Ca2+ signals and contractions induced by EFS were prevented . Pacing was lost after 19±1.2 s of KCl application. As with caffeine, the rapid Ca2+ waves evoked by KCl could be reversed upon washout and entrainment re-established 67.1±3.6 s after washout (ii).Depolarising PVCs by elevating the extracellular KCl concentration (from 5.3 mM to 50 mM) progressively increased the frequency of spontaneous Ca washout ii. The  washout ii.2+ signals, nifedipine promoted the re-occurrence of spontaneous Ca2+ events which had been previously suppressed by EFS  had similar effects to nifedipine (data not shown).Nifedipine (100 µM) stopped EFS-induced pacing within 63±26.1 s  and contd by EFS i–Biv. Ie manner . This la2+ signals within PVCs, and examined the effect of spontaneous Ca2+ signaling on the ability of the PVCs to respond to sustained electrical pacing. The use of the lung slice preparation for examining PVCs has the advantages of easy preparation, a cellular architecture resembling the in situ organization, compatibility with microscopy and a viability lasting several days. Spontaneous Ca2+ signals have previously been recorded in PVCs from various species 2+ handling. PVCs display repetitive spontaneous Ca2+ release events, but the frequencies and spatial properties of those events are unique for each cell. Correspondingly, PVCs show heterogeneous responses to electrical pacing such that neighboring cells can become, and remain, desynchronised. Such heterogeneity is known to serve as a basis for cardiac arrhythmias, and this may underlie the generation of arrhythmic events within pulmonary veins.In the present study, we characterised spontaneous and EFS-induced Ca2+ signals are a potent source of arrhythmic activity 2+ signals may be involved PVCs play an important role in the genesis and maintenance of AF 2+ signal during the passage of each AP. At the end of an AP, cytosolic Ca2+ must recover to diastolic levels in order for the cells to reset and be fully responsive to the next stimulation. Typically, there is a diastolic period of hundred(s) of milliseconds between each AP where Ca2+ remains at the resting level of ∼100 nM. Consequently, spontaneous Ca2+ signals may corrupt this normal activity and recovery cycle of cardiac cells in a number of ways 2+ causes the activation of electrogenic ion transporters, such as Na+/Ca2+ exchange or Ca2+-dependent chloride flux that leads to cellular depolarisation. Ca2+ signals occurring during the recovery of an AP, or in the normally quiescent diastolic period, give rise to ‘triggered activity’ such as early- or delayed-after-depolarisations (often denoted ‘EADs’ and ‘DADs’ respectively) 2+ signals can alter gene expression, and lead to deleterious phenotypic remodelling of myocytes Under normal conditions, contractile myocytes and conductive cells within the heart experience a Ca2+ signals even in the presence of electrical pacing. It would be reasonable to expect that EFS could extinguish spontaneous Ca2+ signals by providing regular command pulses, and normalising SR Ca2+ content. Consistent with this idea, we observed that EFS could dampen spontaneous Ca2+ release, in particular in those cells with relatively low spontaneous Ca2+ activity. However, the effectiveness of electrical pacing was determined by the frequency of spontaneous Ca2+ signals. In essence, if spontaneous Ca2+ release occurred close to the EFS frequency then pacing was either partially successful or unsuccessful. Similar observations have been made using model stimulations of spontaneously active atrial myocytes 2+ signals would certainly contribute to heterogeneous electrical responses within a pulmonary vein 2+ release, such as alteration of Ca2+ influx or RyR sensitivity can further decrease the ability of APs to control PVCs and enhance the likelihood of pro-arrhythmic events. Suppressing spontaneous Ca2+ signaling in PVCs may therefore provide a plausible target to prevent the inception of AF. Other studies have also suggested clamping of RyR activity as a putative mechanism for controlling pulmonary vein-induced arrhythmogenesis 2+ influx is necessary for EFS, but also provides a source of Ca2+ for spontaneous Ca2+ signaling. In the present study, we did not characterise all putative Ca2+ influx mechanisms, in particular those activated by removal/re-addition of extracellular Ca2+, but multiple Ca2+ influx pathways may be involved, as in smooth muscle A key finding in the present study was the propensity of PVCs to perpetually display spontaneous Ca2+ signals is unclear. In previous studies of PVCs 3Rs were identified as triggers for spontaneous Ca2+ signaling, but these channels did not play a detectable role within the PVCs used in this study. Since SR Ca2+ content is known to regulate RyR activation 2+ content so that their RyRs open spontaneously. In support of this notion, experiments designed to increase SR Ca2+ content using KCl, or RyR sensitivity using caffeine, increased spontaneous Ca2+ signaling. In addition, it is evident that PVCs have sufficient SR Ca2+ to allow spontaneous Ca2+ signals to occur for several minutes in a Ca2+-free medium. All manoeuvres that provoked PVCs to show more rapid spontaneous events also made the PVCs resistant to electrical pacing. We therefore suggest that if the SR Ca2+ content, Ca2+ influx or RyR sensitivity are sufficiently enhanced, spontaneous Ca2+ activity will be accelerated and the PVCs become insensitive to EFS.The reason why PVCs have a high propensity to show spontaneous CaFigure S1PVCs show spontaneous activity and can be paced by electrical field stimulation (EFS) at 37°C. The line-scans in i–iiiA illustrate the correlation between EFS and contraction in PVCs at 37°C. i)(A and 5 Hz iii)(A EFS caused obvious contraction. Panels i–iiiB illustrate the Ca2+ transients observed in absence of EFS i)(B and 5 Hz iii)(B EFS. (C) The frequency of the spontaneous Ca2+ transients is not significantly different at room temperature  and at 37°C .(TIF)Click here for additional data file.Figure S2Viability of PVCs within a lung slice. The images in iAiii– depict a region of a lung slice with PVCs (a portion of them are outlined) and surrounding cells. The lung slice was incubated with Oregon Green BAPTA-1 AM i)(A and Propidium Iodide (PI) ii)(A to examine dye retention/exclusion. The images are merged in iii) and an inactive cell (sampled from the region indicated by the magenta circle in A). C illustrates that active PVCs maintained a relatively low PI fluorescence over 4 days in culture. In contrast, dead cells (non-identified) within the slices had a relatively higher PI fluorescence. The absolute PI fluorescence sampled from labelled cells did not significantly change over the period of 4 days .(TIF)Click here for additional data file.Figure S32+ transients. (A)Atrial myocytes within atrial slices do not show spontaneous Ca A 2-photon fluorescence image of Oregon Green BAPTA-1-loaded myocytes within an atrial slice. (B) Atrial myocytes do not display Ca2+ transients without EFS. However, these atrial myocytes showed Ca2+ transients in response to 1 Hz (C), 2.5 Hz (D) and 4 Hz (E) EFS. Ca2+ signals were sampled from the region bounded by the dashed box.(TIF)Click here for additional data file.Figure S42+ waves change to intercellular Ca2+ waves after removal and re-addition of extracellular Ca2+.Intracellular Ca(A) A line-scan plot illustrating that intracellular Ca2+ waves  remain within individual cells (the cell boundaries are indicated by the dashed lines) under control conditions. (B) A line scan plot of the same cellular regions as in A following removal and re-addition of extracellular Ca2+ illustrates that the previously constrained Ca2+ intracellular waves are transformed into propagating intercellular Ca2+ waves (experiment similar to that shown in (TIF)Click here for additional data file.Figure S52+ signals in PVCs.Ryanodine inhibits Ca(A) Ca2+ trace and i–Biii). (C) Ca2+ trace and i–Diii).(TIF)Click here for additional data file.Figure S6Effect of a maximal caffeine concentration on PVCs.(A) and (B) show representative traces of Ca2+ signals caused by superfusion of spontaneously active PVCs with 20 mM caffeine. Caffeine increased the spontaneous Ca2+ transient frequency, leading to a maintained plateau of elevated Ca2+.(TIF)Click here for additional data file.Figure S72+ signals, thus revealing spontaneous Ca2+ signals.Nifedipine inhibits EFS-entrained Ca Nifedipine also progressively reduces the frequency of spontaneous Ca2+ signals in unpaced cells. (A) Ca2+ trace and i–Biv) Line-scan analysis illustrating that EFS-induced contraction observed under control conditions i)(C is inhibited by 100 µM nifedipine ii)(C. (D) Ca2+ trace and i, Eii) Quantitation of the declining frequency of the spontaneous Ca2+ signals in nifedipine-treated PVCs .(TIF)Click here for additional data file.Video S1Spontaneous and EFS induced contraction of a pulmonary vein. Phase contrast image of a cross section through a pulmonary vein, showing a small amount of spontaneous contraction around the whole circumference of the vein. After application of EFS a stronger contraction of the pulmonary vein is observed in response to every electric pulse. Image acquisition rate 15 frames per second.(MP4)Click here for additional data file.Video S22+ signals in PVCs.Spontaneous and EFS induced Ca The video shows spontaneous Ca2+ signaling in Oregon-Green BAPTA-1 loaded PVCs. The spontaneous Ca2+ waves either stay intracellular or travel through several PVCs. In response to EFS a simultaneous whole cell Ca2+ increase is seen in all PVCs. The spontaneous activity continues in-between the EFS pulses. Image acquisition rate 30 frames per second.(MP4)Click here for additional data file.Video S32+ signals during a Ca2+ removal and re-addition experiment.Ca The first part of the video shows the spontaneous Ca2+ signals in PVCs before Ca2+ removal from the superfusion medium. The observed Ca2+ waves are predominantly intracellular. The second part of the video shows the absence of spontaneous Ca2+ signals after 5 minutes in Ca2+ free sHBSS (100 µM EGTA). A gradual increase in Ca2+ transients after re-addition of 1.3 mM Ca2+ is shown in the third part of the video. In contrast to the spontaneous activity seen in the first part, most of the Ca2+ waves after Ca2+ re-addition are intercellular Ca2+ waves, travelling though several cells. Strong, uncoordinated contractions develop during the Ca2+ re-addition period. Image acquisition rate 30 frames per second.(MP4)Click here for additional data file.


NPM1, NRAS and FLT3 as compared with AML without erythroid predominance. We conclude that the clinical, cytogenetic, and molecular features of AML with erythroid predominance in the non-therapy-related setting are much closer to those of a high-grade myelodysplastic syndrome than they are to other types of AML.The clinical importance of erythroid predominance in bone marrow of patients with acute myeloid leukemia (AML) is controversial. These cases represent a heterogeneous group of diseases that historically have been classified into different categories. We studied 313 AML patients and specifically compared the clinical, cytogenetic, and molecular features of cases of AML with erythroid predominance, arbitrarily defined as ≥50% erythroid precursors, to AML cases without erythroid predominance. We also assessed 51 patients with a high-grade myelodysplastic syndrome (MDS), refractory anemia with excess blasts (RAEB). All neoplasms were classified according to the World Health Organization classification. With the exception of therapy-related AML/MDS, the presence of erythroid predominance in variously classified categories of AML was associated with a survival advantage. In addition, AML with erythroid predominance had a lower frequency of cytogenetic abnormalities as well as a lower frequency of mutations involving  The definition of myeloid neoplasms with erythroid predominance has been controversial since the inception of this concept about a century ago Previous studies of myeloid neoplasms with erythroid predominance have shown that there are no specific cytogenetic abnormalities that are unique to these diseases In this study we have focused on patients with AML associated with erythroid predominance, to address the clinical characteristics and molecular genetic features that distinguish this group from patients with AML without erythroid predominance. We studied 313 AML patients and compared cases with erythroid predominance to cases in which erythroid precursors were not predominant.We searched the archives of The University of Texas MD Anderson Cancer Center (MDACC) between 2000 and 2010 for cases of AML or MDS with erythroid predominance, defined as erythroid precursors ≥50% of total nucleated bone marrow cells, as determined by counting cells on bone marrow aspirate smears. Available bone marrow slides of cases with erythroid predominance were retrieved and reviewed and these cases were classified using the 2008 WHO system into the following categories: AEL, AML-MRC, t-AML/MDS, or refractory anemia with excess blasts (RAEB). Specifically, AEL cases had both erythroid precursors ≥50% of total nucleated bone marrow cells and myeloblasts ≥20% of non-erythroid cells. Cases were classified as AML-MRC if the bone marrow had myeloblasts ≥20% of total nucleated cells and there was a prior diagnosis of MDS, or MDS-related cytogenetic changes, or substantial dysplasia (≥50% cells in at least two lineages). Cases were classified as t-AML/MDS if the patient had a history of malignant neoplasm that had been treated. In cases of RAEB, the myeloblasts were enumerated as a portion of total nucleated cells. Cases diagnosed as myelodysplastic/myeloproliferative neoplasm (MDS/MPN) were not included in this study. Patients who had received stem cell transplantation were also excluded as the therapy is a confounding factor that affects survival.NPM1, FLT3, RAS, and KIT gene mutations as part of the routine work-up. Some specimens with erythroid predominance were tested specifically for this study if DNA samples were available. For comparison, we also retrieved cases of RAEB and AML without erythroid predominance with mutation data available from 2008 to 2010. As the definition of cases of AML with recurrent cytogenetic abnormalities depends on the presence of the cytogenetic abnormality, and does not discriminate cases with or without erythroid predominance; these cases were not included in this study.A major focus of this study was the molecular characteristics of these neoplasms. In recent years (2008 until the present), virtually all new AML cases at MDACC have been tested for The results of conventional cytogenetics and molecular testing as well as clinical follow-up information were retrieved from the medical records. This retrospective analysis was approved by the Institutional Review Board of MDACC and conducted in accordance with the Declaration of Helsinki. Written consent was given by the patients for their information to be stored in the hospital database and used for research.NPM1 mutations were detected using primers designed to amplify mutational hotspots spanning codons 956–971 of exon 12, followed by capillary electrophoresis as described previously FLT3 internal tandem duplication (FLT3-ITD) and tyrosine kinase domain codon 835/836 point mutations (FLT3-D835) were detected by a fluorescent-based multiplex PCR assay followed by capillary electrophoresis.FLT3-D835 point mutation analysis, PCR products were digested with EcoRV before capillary electrophoresis.The K-RAS and N-RAS mutations at codons 12,13 and 61 were tested using PCR followed by pyrosequencing as described previously KIT gene were detected by using Sanger sequencing P-values were <0.05 in a two-tailed test. The probability of overall survival (OS) was estimated with the Kaplan-Meier method. The log-rank test was used to compare risk factor categories in survival analysis. Multivariate analysis was performed by Cox proportional regression model to examine the relationship between OS and patient characteristics. The significant factors were identified by Wald backward stepwise elimination.Fisher’s exact test or the Chi-square test was applied to categorical variables. The Mann-Whitney test was used for numerical comparisons between groups. Differences among groups were considered significant if The study group included 313 cases of AML and 51 cases of RAEB that met the inclusion criteria for this study. A total of 106 cases were associated with ≥50% erythroid precursors. Each case was reviewed and reclassified according to the 2008 WHO classification scheme. The cases with erythroid predominance included 59 cases of AEL, 24 cases of AML-MRC, 23 cases of t-AML/MDS, and 23 cases of RAEB. In the 59 cases of AEL, 43 (73%) cases had myeloblasts ≤20% of total nucleated cells but ≥20% non-erythroid cells. The remaining 16 (27%) cases of AEL had myeloblasts ≥20% of total nucleated cells, but were not associated with other features necessary to be classified as AML-MRC . All other cases in this study were not associated with erythroid predominance and included 57 cases of AML-MRC, 150 cases of t-AML/MDS, and 28 cases of RAEB.P = 0.042) in the bone marrow.P = 0.012), as compared with other groups (P = 0.001). A monosomal karyotype was observed in a subset of cases in all AML groups: AEL, 25 (42%), AML-MRC, 27 (33%), and t-AML/MDS, 79 (46%), with no statistically significant difference (P = 0.173). When all AML cases were grouped according to the presence or absence of erythroid predominance, patients with erythroid predominance had a lower frequency of cytogenetic abnormalities (P = 0.004), and tended to have a lower cytogenetic risk (P = 0.061) (P = 0.011) within the t-AML/MDS group and similarly within the AML-MRC group (P = 0.054)  criteria for AML r groups , especia= 0.061) . These f= 0.054) .P = 0.768, or P = 0.587 in non-therapy related setting). This was true even though a complex karyotype was less frequent in the RAEB group (P = 0.045) (P = 0.006). A monosomal karyotype was observed in 14 (27%) RAEB cases, with no statistical significance of erythroid predominance (P = 0.353). The cytogenetic risk of patients with AML versus RAEB was difficult to compare as the latter is classified using the International Prognostic Scoring System (IPSS).Overall, the frequency of a normal karyotype in patients with AML associated with erythroid predominance was comparable to that of RAEB patients (= 0.045) . By contNPM1 mutations (P = 0.010). Particularly, in patients with a normal karyotype, the NPM1 mutation frequency in cases without erythroid predominance was 44%, in keeping with the reported 50–60% reported frequency in literature NPM1 mutation. This patient was a 63-year-old Hispanic man. Bone marrow aspirate smears showed 74% erythroid precursors with dysplastic features and 10% myeloblasts, representing 38% of all non-erythroid elements. The patient had a normal karyotype and no other genes tested were mutated.The gene mutation frequencies in patients with myeloid neoplasms with or without erythroid predominance are summarized in RAS mutations than AML cases with <50% erythroid precursors . FLT3 mutations also appear to be less frequent in AML with erythroid predominance, but this did not achieve statistical significance . KIT mutations were not identified in any of the patient groups in this study.Similarly, patients with AML with erythroid predominance had a lower frequency of In aggregate, the mutation frequencies detected in patients with AML associated with erythroid predominance were comparable to those of patients with RAEB , in partP = 0.003; P = 0.382; P = 0.032; P = 0.320; P = 0.909) groups.We stratified the study cohort by the presence or absence of erythroid predominance and compared clinical outcomes. Overall, patients with myeloid neoplasms associated with erythroid predominance had significant better overall survival than patients without erythroid predominance (P = 0.013), unfavorable risk karyotype (P = 0.019), age (P = 0.002), and the diagnosis of t-AML/MDS (P = 0.027) were independent factors for predicting inferior overall survival. Bone marrow myeloblast count as a percentage of total nucleated cells was a borderline hazard (P = 0.064) for overall survival. In contrast, the presence of erythroid predominance, and mutation status of genes tested did not significantly predict overall survival.To identify independent predictive factors for patient survival, we performed multivariate analysis in AML patients . A complWe performed this study in order to better understand the clinical, cytogenetic, and prognostic features of patients with AML associated with erythroid predominance. In particular, we defined erythroid predominance as ≥50% bone marrow nucleated cells, as has been done by others, and we compared patients with AML with erythroid predominance to patients with AML without erythroid predominance. We have shown that the presence of erythroid predominance confers a better overall survival in various AML patient groups, with the exception of the therapy-related setting. We further show that patients with AML associated with erythroid predominance have a cytogenetic and molecular profile that differs from patients with AML without erythroid predominance, and more in keeping with patients with high-grade MDS.FLT3, RAS and KIT, are infrequent in AML associated with erythroid predominance NPM1 mutations have been shown to occur at an early stage in stem cells, and NPM1 mutation is known to affect myeloblast differentiation and play an important role in leukemogenesis. The frequency of NPM1 mutation reported in AML is highly variable in the literature, ranging from 4% to 100%, presumably attributable, at least in part, to case selection NPM1 mutation is extremely low in all types of AML with erythroid predominance, including t-AML, AML-MRC, or AML with a normal karyotype. JAK2 gene mutations are known to be very common in polycythemia vera, a myeloproliferative neoplasm characterized by expanded erythropoiesis. Although not specifically assessed in this study, others have shown that JAK2 mutation is rare in AEL Gene mutations in AML pathogenesis have been divided by other into two types; class I and class II. The former affect proliferation whereas the latter affect differentiation. The results of this study, as well as those of previously published studies, suggest that class I mutations that affect myeloblast proliferation, such as GAT1, RSP14, EVI1 and TELErythropoiesis in these cases is prominent, often associated with significant morphologic dysplasia and severe macrocytic anemia, indicating ineffective erythropoiesis, similar to that observed in MDS. In MDS, although not entirely clear, the possible mechanisms involved in ineffective erythropoiesis may be related to mutations or epigenetic dysregulation of a number of important genes, such as Historically, patients with AEL have been thought to have a poor prognosis. However, the definition and classification of AEL have been changed over the years, and particularly so in the most recent WHO classification scheme. Many cases of AML/MDS with erythroid predominance are now classified under other well-defined categories, such as t-AML/MDS, AML with recurrent cytogenetic abnormalities or AML-MRC. In this study, based on the findings in this patient cohort, we question the utility of some aspects of the 2008 WHO classification for the sub-categorization of myeloid neoplasms associated with ≥50% erythroid precursors. The data in this study show that the outcome of patients with therapy-related AML/MDS is generally poor, regardless of the presence or absence of erythroid predominance. Furthermore, the WHO diagnostic categories of RAEB, AEL and AML-MRC do not appear to have prognostic significance in this study, as has been shown by others FLT3 mutation rate of 5.7% found in AML cases with erythroid predominance is much closer to that seen in patients with RAEB than patients with AML without erythroid predominance One plausible explanation for the survival advantage of patients with AML associated with erythroid predominance is the overall lower absolute blast count in this group of diseases. Based on the definition, in the presence of ≥50% erythroid precursors, the myeloblast percentage is calculated from the non-erythroid cells, which results in a significantly lower absolute number of myeloblasts in this group of AML cases. However, this explanation is probably too simplistic, or is only a part of the explanation, because the myeloblast count, either enumerated as a percentage of total cells or non-erythroid cells, does not have an impact on clinical outcomes within this group of diseases In summary, the findings in this study provide further evidence that AML associated with erythroid predominance represents a group of diseases with clinical and molecular genetic features that differ from other types of AML. The data presented here suggest that cases of AML with erythroid predominance are clinically, cytogenetically, and at the molecular level much closer to RAEB than they are to cases of AML without erythroid predominance.


Studies in cardiomyocytes have shown that diabetes results in impaired [Ca2+]i homeostasis due to altered sarcoplasmic reticulum Ca2+ ATPase (SERCA) and sodium-calcium exchanger (NCX) activity. Importantly, altered calcium homeostasis may also be involved in diabetes-associated endothelial dysfunction, including impaired endothelium-dependent relaxation and a diminished capacity to generate nitric oxide (NO), elevated cell adhesion molecules, and decreased angiogenic growth factors. However, the effect of diabetes on Ca2+ regulatory mechanisms in cardiac endothelial cells (CECs) remains unknown. The objective of this study was to determine the effect of diabetes on [Ca2+]i homeostasis in CECs in the rat model (streptozotocin-induced) of DCM. DCM-associated cardiac fibrosis was confirmed using picrosirius red staining of the myocardium. CECs isolated from the myocardium of diabetic and wild-type rats were loaded with Fura-2, and UTP-evoked [Ca2+]i transients were compared under various combinations of SERCA, sarcoplasmic reticulum Ca2+ ATPase (PMCA) and NCX inhibitors. Diabetes resulted in significant alterations in SERCA and NCX activities in CECs during [Ca2+]i sequestration and efflux, respectively, while no difference in PMCA activity between diabetic and wild-type cells was observed. These results improve our understanding of how diabetes affects calcium regulation in CECs, and may contribute to the development of new therapies for DCM treatment.Diabetic cardiomyopathy (DCM) is a diabetic complication, which results in myocardial dysfunction independent of other etiological factors. Abnormal intracellular calcium ([Ca Diabetes mellitus is a growing epidemic in the United States that afflicts 8.3% of the total US population 2+) homeostasis that may precede clinically manifested DCM-related cardiac dysfunction 2+ regulatory mechanisms are a hallmark of cardiomyopathy and heart failure in human patients 2+ concentration ([Ca2+]i) in cardiomyocytes increases due to Ca2+ entry via Ca2+ channels and inositol trisphosphate (IP3) mediated Ca2+ release from sarco/endoplasmic reticulum (SER). Cardiac relaxation is subsequently initiated by the Ca2+ decay process, which is governed by Ca2+ sequestration and Ca2+ efflux mechanisms. During Ca2+ sequestration, the sarcoplasmic reticulum Ca2+ ATPase pumps (SERCA) re-sequester the Ca2+ from the cytosol back to the SER. Similarly the Ca2+ efflux mechanism extrudes Ca2+ via plasma membrane Ca2+ ATPase pumps (PMCA), and sodium- calcium exchangers (NCX) Studies in animal models have shown that diabetes results in abnormal calcium  are instrumental in normal myocardial function, including endocardium interactions, myocardial capillaries, vascular permeability and cardiac tissue remodeling. Therefore, this information is essential for a better understanding of mechanisms for the impaired cardiac function in DCM and for the development of new therapies to treat this condition.It is well established that endothelium plays an essential role in vascular function, including nitric oxide (NO) -mediated modulation of vascular tone, control of adhesion between the vessel and cells circulating in the blood, and regulation of local cell and vessel growth via a variety of growth factors. Calcium is an important regulator of endothelial function that can mediate each of these pathways, including signaling via NO 2+ homeostasis in CECs in a rat model (streptozotocin-induced) of DCM. Uridine-5′-triphosphate (UTP) -evoked [Ca2+]i transients were compared between diabetic and wild-type CECs under various combinations of SERCA, PMCA and NCX inhibitors. Our results demonstrate that diabetes results in altered Ca2+ homeostasis in endothelial cells via decreases in SERCA and NCX activities during Ca2+ sequestration and Ca2+ efflux, respectively.The objective of this study was to determine the effect of diabetes on intracellular Ca2Y2 receptor agonist) evoked a [Ca2+]i transient which decayed to basal level .The objective of these experiments was to establish the baseline for normal (wild-type) CEC response to SERCA, NCX and PMCA inhibitors. Consistent with previous studies al level . The decsigargin , p<0.05.2+ decay time  consistent with previous reports As expected, in the presence of NCX inhibitor (benzamil), the Cacay time  was signcay time , p<0.05.ted CECs , p<0.05.2+]i peak amplitude in wild-type CECs remained similar in the presence of SERCA and PMCA inhibitors as compared to no inhibitor controls. However, cells treated with NCX inhibitor showed a higher [Ca2+]i peak as compared to no inhibitor controls  and NCX (benzamil) inhibitors. Under these conditions, thapsigargin induced a slow and long-lasting Ca2+ transient in wild-type CECs. Diabetic CECs showed a similar response, with [Ca2+]i decay time similar to that of wild-type CECs  and NCX (benzamil) inhibitors, the [Ca2+]i decay time in diabetic CECs was significantly increased and the peak amplitude was significantly decreased as compared to wild-type CECs (2+]i transients evoked in the presence of extracellular Ca2+ (2 mM CaCl2 added) were similar, with significantly increased [Ca2+]i decay time and decreased peak amplitude in diabetic CECs as compared to wild-type  to exclude the contributions of SERCA-mediated Caild-type , p<0.05,2+]i transients were evoked by UTP in the presence of SERCA and PMCA inhibitors  to exclude contributions of SERCA-mediated Ca2+ sequestration and PMCA-mediated Ca2+ efflux. Interestingly, the [Ca2+]i decay time was significantly increased in diabetic CECs as compared to wild-type , PMCA (carboxyeosin) and NCX (benzamil) inhibitors. Under these conditions, the [Ca2+]i decay in wild-type CECs was completely blocked  to extrude Ca2+ out of the cell 2+ influx when the intracellular Na+ concentration is high enough to drive the NCX. Our findings of a decrease in NCX activity (with no changes in PMCA activity) in diabetic CECs suggest that the ability of diabetic CECs to maintain Ca2+ homeostasis is compromised, which may contribute to abnormal cardiac function. This reduced ability of NCX to extrude Ca2+ may also be related to the reduced NCX protein levels as shown by the immunofluorescent staining analysis of diabetic CECs. Our results of altered NCX activity and reduced protein expression levels in diabetic CECs are consistent with previous studies in cardiomyocytes 2+ efflux via NCX, while resulting in altered Ca2+ efflux via PMCA. Our results also suggest that in diabetic CECs, PMCA effectively extrudes Ca2+ in the presence of SERCA and NCX inhibitors.Our results show that NCX activity during Ca2+ is primarily extruded by the PMCA. Overall, our findings suggest that abnormal endothelium-dependent relaxation and diminished capacity to generate NO in diabetes 2+ transients due to impaired NCX and SERCA, which can reduce activation of eNOS and NO production by endothelial cells 2+ transients in diabetic cells may affect ICAM-1-coupled cytoskeletal rearrangements and endothelial cell interactions with inflammatory cells Our results show that while diabetic conditions result in impaired function of SERCA and NCX, there are no differences in PMCA function and protein expression between diabetic and wild-type cells. These results are consistent with those by Hill et al. In this study, we specifically focused on the effects of DCM on intracellular calcium regulatory mechanisms , and therefore, the experiments were performed in the absence of extracellular calcium (unless otherwise stated). However, in order to elucidate the mechanisms for DCM-induced alterations in overall calcium signaling, information regarding the effects of diabetic conditions on store operated calcium entry is important as well, and will be the subject of future studies.2+ homeostasis has been identified as a hallmark abnormality for this disorder 2+ dependent signaling pathways .In this study, a rat model of type I (streptozotocin-induced) diabetes and non-atherogenic diabetic cardiomyopathy 2+ experiments, CECs were trypsinized and seeded on multi-well glass coverslips  for 24 hours.Cardiac cells were isolated from the myocardium of diabetic and wild-type rats as described previously 2+ influx during [Ca2+]i transient, experiments were performed in a Ca2+ free medium , 0.5% bovine serum albumin , 2.5 mM probenecid , pH 7.45) and 0.5 mM EGTA , unless otherwise stated. Wild-type and diabetic CECs were loaded with 5 μM Ca2+ sensitive indicator Fura-2 AM  for 40 minutes at room temperature. In some experiments, extracellular Ca2+ was incorporated by adding 2 mM CaCl2 in the Ca2+ free medium without EGTA. [Ca2+]i transients were evoked in CECs by adding 10 µM UTP . UTP has been shown to act on P-type purinergic receptors (P2Y2), which leads to a G protein-mediated activation of phospholipase C, followed by inositol 1,4,5-trisphosphate (IP3) formation and the release of Ca2+ from IP3 sensitive Ca2+ stores (mainly from SER) 2+) excitation wavelengths were recorded at 510 nm emission after every 250 msec for at least 20 minutes. For each time point, F340/F380 ratiometric analysis was performed by determining the ratio of 340 nm and the corresponding 380 nm image . Regions of interest outlining cell boundaries on the ratio image were defined, and the variations in fluorescent intensity of selected cells were tabulated th point.To exclude the contribution of extracellular Ca2+]i transients, wild-type CECs were treated with 1 μM thapsigargin , a selective SERCA inhibitor 2+ sequestration mechanism studies (SERCA activity), [Ca2+]i transients were first evoked using 1 μM thapsigargin, followed by 10 μM UTP in a Ca2+ free medium. During these experiments, the Ca2+ efflux was inhibited using PMCA and NCX inhibitors at all times. To examine the Ca2+ efflux mechanism (PMCA and NCX activities), diabetic and wild-type CECs were treated with NCX inhibitor (benzamil) or PMCA inhibitor (carboxyeosin). These experiments were performed under the following conditions: a) SERCA activity was inhibited at all times (thapsigargin present in the medium), b) NCX activity was inhibited to examine PMCA activity, and c) PMCA activity was inhibited to examine NCX activity. In these experiments, UTP was used to evoke [Ca2+]i transients.To assess the activities of SERCA, PMCA and NCX during the decay of i decayed from 80% to 20% of its peak amplitude (presented as t80–20). The results are expressed as mean ± SD. One way ANOVA  was used to determine the effect of diabetes on [Ca2+]i decay kinetics and protein expression levels in CECs. A p<0.05 was considered to be statistically significant. All experiments were repeated at least 3 times; at least 70 diabetic/wild-type CECs were analyzed per treatment.The decay time of agonist-evoked Ca


Although the DNA double-strand break (DSB) is defined as a rupture in the double-stranded DNA molecule that can occur without chemical modification in any of the constituent building blocks, it is recognized that this form is restricted to enzyme-induced DSBs. DSBs generated by physical or chemical agents can include at the break site a spectrum of base alterations (lesions). The nature and number of such chemical alterations define the complexity of the DSB and are considered putative determinants for repair pathway choice and the probability that errors will occur during this processing. As the pathways engaged in DSB processing show distinct and frequently inherent propensities for errors, pathway choice also defines the error-levels cells opt to accept. Here, we present a classification of DSBs on the basis of increasing complexity and discuss how complexity may affect processing, as well as how it may cause lethal or carcinogenic processing errors. By critically analyzing the characteristics of DSB repair pathways, we suggest that all repair pathways can in principle remove lesions clustering at the DSB but are likely to fail when they encounter clusters of DSBs that cause a local form of chromothripsis. In the same framework, we also analyze the rational of DSB repair pathway choice. The defining feature of a double-strand break (DSB) as DNA lesion is the associated disruption of molecular continuity. The DSB severs in two fragments a linear DNA molecule and linearizes a circular molecule by disrupting the sugar–phosphate backbone on both strands and at sites located directly opposite each other—or just a few nucleotides apart (up to ∼10 bp).DSBs, by affecting both DNA strands, compromise the fundamental principle used for the repair of lesions confined to one DNA strand: the possibility to use the complementary, undamaged strand as template to restore sequence in the damaged strand. Indeed, excision-based repair pathways, such as base excision repair (BER), nucleotide excision repair and mismatch repair, use the undamaged strand as template to restore the DNA molecule after removal (excision) of the damaged, or mismatched, segment .This feature of the DSB allows the inference that its repair will be difficult, inherently inefficient and slow. However, comparison of the DSB repair kinetics with the kinetics measured for the repair of forms of DNA lesions only affecting one DNA strand provides a surprising outcome. Thus, CHO cells repair DSBs markedly faster than base damage or ultraviolet (UV)-induced lesions . Only thThe apparently effortless removal notwithstanding, DSBs remain biologically highly dangerous DNA lesions. Indeed, among DNA lesions, DSBs have the highest per lesion probability of causing numerous adverse biological effects including cell death, mutation, as well as transformation to a carcinogenic state.The severity of the DSB as DNA lesion is evolutionarily ingrained into cellular function. This is convincingly demonstrated by the evolutionarily conserved, highly elaborate and complex network of responses cells mount, when detecting a DSB. The so called ‘DNA damage response (DDR)’ , originaThe DDR is triggered not only by accidental DSBs randomly generated in the genome by exogenous agents such as ionizing radiation (IR) and certain chemicals, or during DNA replication stress , but alsIt may seem surprising why a lesion that can be processed by the cell efficiently and for which the cell devotes extensive resources still remains highly dangerous and linked to severe adverse biological consequences. Extensive work carried out over the past several decades converges to the idea that the adverse consequences of DSBs mainly result from errors or accidents in their processing. Indeed, there is evidence that the probability of processing errors is for DSBs much higher than for lesions confined to one DNA strand .de novo additions of nucleotides are also possible  ,17–20, aExperimental evidence for all three error scenarios is abundant and typical examples are shown in The causes of the aforementioned described types of erroneous DSB processing events warrant discussion. Of particular importance and relevance is, without any doubt, the identification and characterization of parameters determining the probability of their occurrence. Available information on the mechanisms underpinning DSB processing allows the definition of three main sources of DSB processing errors:1. Inherent limitations of repair pathways engaged in DSB processing. As we briefly describe later in the text, multiple pathways process DSBs, and each shows distinct and frequently inherent propensities for errors. Notably, and possibly unexpectedly, the propensity for errors can vary dramatically among repair pathways. It follows that depending on the repair pathway choice made for a particular DSB, the associated risk for errors will vary accordingly. These limitations are compounded by the specifics of the 3D organization of the genome that define the form of possible errors (e.g. translocations) on the basis of proximity between interacting regions and possibly other parameters as well .2. The nature of the initiating DSB. Although the DSB is defined as a rupture in the double-stranded DNA molecule that can occur without chemical modification in any of the constituent DNA building blocks, it is recognized that this form of DSB is relatively rare and restricted to certain biologically induced DSBs (see later in the text). DSBs generated by physical or chemical agents can include with the DNA rupture a spectrum of chemical alterations (DNA lesions) in the neighboring bases. It is now widely considered that chemical alterations accompanying the DSB may be determinants of the form of DSB processing chosen by the cell and the probability that errors will occur during this processing. The term complexity is frequently used to describe some of these characteristics of the DSB ,30.3. The localization of the DSB in chromatin. The term ‘location’ can refer to the condensation status of chromatin at the site of the DSB, with two extremes: localization in euchromatin or in heterochromatin ,32. HoweIn the following sections, we discuss possible sources of DSB processing errors and accidents. We first focus on structural aspects of the DSB, define levels of DSB complexity and discuss how DSB complexity may interfere with processing to cause errors. Subsequently, we describe briefly repair pathways engaged in DSB processing and discuss how processing errors such as those summarized in Here, we attempt a classification of DSBs on the basis of increasing complexity and analyze how the specific characteristics of each class affect the processing requirements and the probability of processing errors. To facilitate presentation and subsequent discussion, we will define categories (types) comprising DSBs with progressively increasing complexity.Classic examples of DSBs are those generated by restriction endonucleases (RE) ,35. ThisRE generates the simplest possible form of DSB, as they disrupt the continuity of the DNA molecule without chemically altering any of its constituent moieties, i.e. lesions in the form of sugar or base modifications are not introduced. We will term here this form of DSB type 1, T1-DSB, to distinguish it from more complex forms that are described later in the text A. Notablin vitro, and blunt-ended DSBs are generally more difficult to ligate than DSBs with protruding matching ends  or length of protruding ends have been shown to affect the efficiency of DSB processing ing ends ,38,39.RE are frequently used as model reagents to generate DSBs at specific sites of a DNA molecule and to analyze the associated cellular responses. This approach has gained ground with the introduction of rare cutting RE and the I-SceI homing endonuclease for which the recognition sequence (18 bp) is not present in mammalian cells but can be introduced according to a pre-conceived design using molecular biology approaches . These s, which require transfection and expression, or at a minimum intracellular translocation, of the I-SceI, or other endonuclease. Moreover, the enzyme remains functional in the cell nucleus for periods that are difficult to accurately define or precisely limit.When DSBs are induced by physical or chemical agents, the alterations generated in the DNA are more complex. Among physical agents inducing DSBs, IR takes a prominent place. This is because IR, at low doses, is present in the environment and frequently used in diagnostic medicine. At higher doses, IR is used for the treatment of human diseases like cancer and inflammation ,47. Rece•OH produced by the radiolysis of adjacent water), a wide spectrum of lesions including sugar and base damages each of which outnumbers DSBs by ∼20:1 ,53•OH prby ∼20:1 . We will2O2, an oxidative agent that is also produced intracellularly as byproduct of the cellular metabolism . DSBs can subsequently form through the processing of a base lesion opposite an unrepaired SSB, or through the parallel processing on both DNA strands of base damage D 30,70–. There iAlthough the extremely fast processing of SSBs and the particularly slow processing of base damage  reduce tIndirect DSBs forming by the simultaneous disruption of the phosphodiester bond at base damage sites in opposite DNA strands or with the combination of BER activity with a SSB at the opposite strand, form yet another level of complexity that integrates the parameter time post-irradiation in the induction process, and which we will, therefore, term here type 4 DSBs, T4-DSBs. A discriminating characteristic of a T4-DSB is that as it forms, proteins participating in SSBR and/or BER may already be engaged at or near the ends of the resulting DSB, which may impair its recognition and processing by the cell.There is evidence that IR induces, in addition to sugar lesions promptly disrupting the sugar–phosphate backbone (prompt DSBs), also lesions doing so after temperature-dependent chemical processing (delayed DSBs) . These tUntil recently, it was believed that in mammalian cells evolution of such lesions to DSBs only occurs when DNA is incubated after irradiation at high, non-physiological temperatures  . HoweverThis process represents yet another way for generating indirectly DSBs within a CDS, which we will term here type 5 DSBs, T5-DSBs, to distinguish them from the other categories described above and also later in the text E. Like TAs an additional level of increasing DSB complexity, we consider here clusters of DSBs, where the individual DSBs can in principle belong to any of the aforementioned defined types. This form of DNA damage disrupts the continuity of the DNA in the same general way as simpler forms of DSBs do. However, by involving several DSBs in close proximity (DSB clusters), it severely undermines local chromatin stability and thus overall processing in a location- and composition-dependent manner. On the basis of its constitution, this form of damage can also be considered as a form of highly local chromothripsis—a phenomenon whereby as of yet undefined processes cause extensive local genomic fragmentation (thripsis), which invokes inaccurate rejoining that feeds carcinogenesis .DSB clustering as a source of small DNA fragments in irradiated cells and a cause of irreversible radiation effects has been considered by several investigators ; ‘Bundesministerium für Wirtschaft und Technologie’ . Funding for open access charge: Bundesministerium für Bildung und Forschung Germany.‘Conflict of interest statement. None declared.


Voltage-dependent Ca2+ channel (VDC) and Na+/Ca2+ exchanger (NCX) system are the main pathways of intracellular Ca2+ overload in pathological conditions. Here, we review the potential underlying mechanisms involved in CI-AKI and discuss the role of NCX-mediated intracellular Ca2+ overload in the contrast media-induced renal tubular cell injury and renal hemodynamic disorder.The precise mechanisms underlying contrast-induced acute kidney injury (CI-AKI) are not well understood. Intracellular Ca These findings suggested that intracellular Ca2+ overload plays an important role in contrast-induced renal hemodynamic disorder. Besides changes in calcium physiology, contrast-induced vasoconstriction might also be a result of a direct effect on vascular smooth muscle [Contrast-induced acute kidney injury (CI-AKI) is the third leading cause of hospital-acquired acute renal failure accounting for 10–12% of all causes of hospital-acquired renal failure . In patih muscle  or from h muscle  and endoh muscle  productiIt must be pointed out that, under normal circumstances, the contrast-induced renal hemodynamic disorder was not enough to induce CI-AKI based on the facts that humans as well as experimental animals without risk factors do not usually exhibit CI-AKI following contrast media injection. This is because, under physiological state, the renal circulation is subjected to autoregulation which is associated with neural, hormonal, paracrine, and autocrine influences. Injured autoregulation of microcirculation might be the cause that all kinds of risk factors such as preexisting renal impairment, diabetes mellitus, and hypercholesterolemia,   make the kidney vulnerable to iodinated contrast media.Renal tubular cells apoptosis is a key mechanism of CI-AKI. Studies have shown that contrast media can induce renal tubular epithelial cell apoptosis via ROS (reactive oxygen species) pathway, JNK/p38 stress kinase pathway, and intrinsic apoptotic pathways –20 and c2+ overload in ischemic kidney and CI-AKI have not been fully elucidated, there is evidence indicating that increased cytosolic Ca2+ may be an important mediator of epithelial cell apoptosis and necrosis [2+ overload induced by contrast media might not be completely suppressed by CCB. So we cannot conclude based on these clinical data that intracellular calcium overload was not associated with CI-AKI because VDC is not the only pathway that induces Ca2+ influx. There is the possibility that other channels besides VDC may also be involved in the contrast-induced intracellular calcium overload. Our recent study [2+ and reactive oxygen species and KB-R7943, inhibitor of the reverse mode of NCX, attenuated the contrast media-induced renal tubular epithelial cell apoptosis by suppressing the intracellular Ca2+ overload and reducing oxidative stress, which suggested that intracellular Ca2+ overload via the NCX system is also involved in contrast-induced renal tubular apoptosis.Intracellular calcium overload is considered to be a key factor in ischemic cell injury and CI-AKI . Studiesnecrosis . So, thenecrosis –13. Howenecrosis . Only onnecrosis , 27. Thent study  has show+ for 1 Ca2+, depending on the electrochemical gradients of the substrate ions [2+ outside the cell using the Na+ concentration gradient across the cell membrane to keep a low intracellular Ca2+ level, which is referred to as the forward-mode operation of the exchanger. In pathological conditions, NCX can reversely extrude Na+ for Ca2+ influx and result in intracellular Ca2+ overload, which is referred to as the reverse mode or calcium influx mode of NCX. In the normal kidney, NCX plays an important role in the active calcium transport in distal convoluted tubules [+ for Ca2+ influx and results in intracellular Ca2+ overload and tubular epithelial cell injury [NCX is a bidirectional plasma membrane transporter that catalyzes the exchange of 3 or 4 Naate ions , 29 and ate ions ; and NCXate ions , 32. Und tubules . In the l injury , 35.2+. The inhibitor of reverse mode of NCX, KB-R7943, can alleviate contrast-induced renal tubular apoptosis through suppressing the increase of intracellular Ca2+ and subsequent ROS overproduction and p38 activation. These data demonstrate that intracellular Ca2+ overload via the reverse mode of NCX system is involved in contrast-induced renal tubular epithelial cell apoptosis.It has been verified that contrast media can induce renal tubular epithelial cell apoptosis via ROS pathway, JNK/p38 pathway, and intrinsic apoptosis pathway , 20. Our2+ overload via the reverse mode of NCX, followed by renal ET-1 overproduction and renal vasoconstriction, plays an important role in the pathogenesis of CI-AKI.Recent animal model experiments , 37 also2+, ROS overproduction, p38 MAPK activation, and Caspase-3 overexpression and the findings [+ for Ca2+ influx and results in increased intracellular Ca2+. The increased intracellular Ca2+ can stimulate Ca2+ release from the mitochondrial and endoplasmic reticulum and result in intracellular Ca2+ overload [2+ overload via the reverse mode of NCX and VDC induced by contrast media in the renal tubular epithelial cell can result in ROS overproduction and oxidative stress. Increased ROS and intracellular Ca2+ can induce upregulation of p38 and p-p38 MAPK expression [+, which is caused by inhibition of Na+/K+-ATPase activity because of decreased ATP production [+/H+ exchange because of intracellular acidosis [2+ influx. The increased intracellular Ca2+ stimulates Ca2+ release from the mitochondrial and endoplasmic reticulum and results in intracellular Ca2+ overload, which induced ROS overproduction and oxidative stress. Increased ROS and intracellular Ca2+ activate p38 MAPK. On one hand, p38 MAPK activates intrinsic apoptotic pathways such as bcl-2, bax, and caspase-3 and induces renal tubular epithelial cell apoptosis, which is the underlying cause of contrast-induced direct renal tubular toxicity. On the other hand, activated p38 MAPK also results in renal ET-1 overproduction, followed by renal vasoconstriction and renal ischemia, which is one of the underlying causes of contrast-induced renal hemodynamic abnormalities. ET-1 overproduction and renal ischemia can cause depletion of ATP and development of intracellular acidosis, which can result in accumulation of intracellular Na+ and further activate the reversion of the mode of NCX and subsequently cause Ca2+ influx and ET-1 overproduction, forming a vicious cycle.Based on the findings , 22, 36 findings , 37 thatoduction  and actiacidosis , can als2+ overload via the reverse mode of NCX1 and VDC, followed by ROS overproduction, p38 MAPK activation, and ET-1 overproduction, plays an important role in the contrast-induced renal hemodynamic disorder and renal tubular epithelial cell apoptosis, which suggests that, in clinical practice, CCB should be recommended to patients with hypertension who are undergoing radiographic examination or therapy requiring contrast media and that selective inhibitors of NCX1 may be beneficial in the prevention and treatment of CI-AKI in humans.In summary, Ca