Apowerful controller of Ca2+ waves is the cytoplasmic H+concentration ([H+]i), which fluctuates spatially and temporallyin conditions such as myocardial ischaemia/reperfusion. H+-control ofCa2+ waves is poorly understood. We have therefore investigated how[H+]i co-ordinates their initiation and frequency.Spontaneous Ca2+ waves were imaged (fluo-3) in rat isolated ventricularmyocytes, subjected to modest Ca2+-overload. Whole-cell intracellularacidosis (induced by acetate-superfusion) stimulated wave frequency. Pharmacologicallyblocking sarcolemmal Na+/H+ exchange (NHE1) prevented thisstimulation, unveiling inhibition by H+. Acidosis also increasedCa2+ wave velocity. Restricting acidosis to one end of a myocyte, using amicrofluidic device, inhibited Ca2+ waves in the acidic zone (consistent withryanodine receptor inhibition), but stimulated wave emergence elsewhere in the cell.This remote stimulation was absent when NHE1 was selectively inhibited in the acidiczone. Remote stimulation depended on a locally evoked, NHE1-driven rise of[Na+]i that spread rapidly downstream.Spontaneous Ca2+ waves via inhibitory 2+-loading through modulation of sarcolemmalNa+/Ca2+ exchange activity). During spatial[H+]i-heterogeneity, 2+-signalling in the same myocyte. If the principle of remoteH+-stimulation of Ca2+ waves also applies in multicellularmyocardium, it raises the possibility of electrical disturbances being driven remotelyby adjacent ischaemic areas, which are known to be intensely acidic.Acidosis influences Ca One factor that may trigger Ca2+ wavesis a fall of pHi, although wave initiation has been variously reported to bestimulated or inhibited by acidosis.,2+ waves are also promoted by other factors, in particular by elevations of[Na+]i that occur when the Na+/K+ pump isinhibited.+]i rise occurs when the sarcolemmalNa+/H+ exchanger (NHE1) is stimulated by acidosis.2+ waves to changes of pHi.An aberrant form of Ca+ ions are powerful modulators of cell function. In ventricularmyocytes they are generated metabolically, but maintained at low cytoplasmic levels, most commonly via+]i of about 30 nM occur physiologically , while much larger increases of about 400 nM  occur during myocardial ischaemia.+]i elevations represent a form ofintracellular H+ signalling. Prominent among the targets for such signalling areCa2+ handling proteins, namely the ryanodine receptor (RyR),2+ ATPase (SERCA),,+/Ca2+ exchanger (NCX),+ interaction with theseproteins is predominantly inhibitory, an elevation of [H+]i canenhance the electrically evoked calcium transient (CaT),+ activation of NHE1. The resulting rise of [Na+]islows Ca2+ efflux on sarcolemmal NCX, which boosts sarcoplasmic reticulum (SR)Ca2+ loading via SERCA, thereby increasing CaT amplitude.,2+ signalling impacts on the generation ofarrhythmogenic Ca2+ waves has yet to be examined.Intracellular Hspatial interactions among [H+]i,[Na+]i and [Ca2+]i in ventricular myocytes hasbeen highlighted in recent work.+ mobility is low,+]i microdomains can form.+]i, by locallystimulating NHE1, elevates + diffuses rapidly in cytoplasm,,2+ waves are similarly controlled through interacting spatial The importance of 2+ waves to pHi, we havemanipulated intracellular pH, Na+, and Ca2+ in rat ventricularmyocytes. Our data indicate that H+ ions exert direct inhibitory and indirectexcitatory effects on Ca2+ waves. The excitatory effect is mediated viaintracellular Na+. When excitation is induced by a localised acidic microdomain,it is typically expressed remotely in non-acidic regions of the cell, driven by fast2+ waves.In order to investigate the coupling of CaDetailed methods are available in the Supplementary material online.All procedures were performed in accordance with UK Home Office and local guidelines.Ventricular myocytes were isolated from 46 male Sprague-Dawley rats, as previouslydescribed,ipH: myocytes were loaded with carboxy-seminaphthorhodofluor-1 at room temperature. Cells were imaged confocally on a Leica SP5inverted microscope. Intracellular calibration of cSNARF-1 was performed in separateexperiments using nigericin.2+ waves and sparksCa: myocytes were AM-loaded with fluo-3 (18μM). Cells were superfused and quiescent cells were imaged in linescan(xt) mode along the cell’s longitudinal axis at 400 lines per second.Ca2+ sparks were imaged in solution containing 1 mM free Ca2+.Switching the superfusate to one containing raised (5 mM) free Ca2+ induced amodest calcium overload that triggers Ca2+ waves.i gradient was induced using a microfluidic device consisting of asquare-bore double-barrelled micropipette, which released two parallel microstreams ofsolution perpendicular to the cell.2 plus10 mM sucrose to help visualize the interstream boundary; the other microstream contained80 mM acetate with 5 mM free Ca2+. The position of the microstream boundaryacross the cell was recorded in xy before and after each linescanexperiment. The smooth pHi gradient induced is illustrated in Figure.A pHt-test or nested ANOVA, depending on the experimental protocol.Where relevant, the Holm correction for multiple testing was applied. Significance isdenoted as * for P < 0.05, ** forP < 0.01, *** forP < 0.001.Data are expressed as mean ± SE. Statistical significance was tested using pairedStudent’s Wave propagation velocity was calculated from linescan images using the angle ofincidence of the wavefront, relative to the longitudinal axis of the cell.2+ sparks were normalized using a custom-built Matlabmacro, followed by spark frequency analysis using an algorithm developed by Konget al.Linescan recordings of Ca2+ waves, by first imposingwhole-cell changes of pHi, at constant pHo. Individual ventricularmyocytes, when subjected to modest Ca2+ overload , with a frequency of 18.7 ± 2.5 min−1. At times thesewaves converted into whole-cell CaTs , induced by theCa2+-driven transient inward current (Iti)triggering an action potential.,i 7.3 to 6.6), induced by superfusion of 80 mM acetate,initially reduced wave frequency . After 30 s, wave frequency then progressivelyincreased above control levels . The increase could be graded by varyingthe magnitude of intracellular acidosis . The increase was attributable to an N,N-dimethyl) amiloride , a high-affinity NHE1 inhibitor. Under these conditions, reducing pHi from7.3 to 6.6 now produced a decrease in Ca2+ waves , which was also graded with the severity ofacidosis . Abolition of2+ waves was confirmed with astructurally different NHE1 inhibitor, cariporide, which targets NHE1 more specificallyamong NHE isoforms than DMAFigure S2C, D. Thus, for the pHi range from 7.3 to 6.6,raising [H+]i exerts opposing effects on Ca2+ wavefrequency; an NHE1-independent inhibition, and a delayed, NHE1-dependentstimulation.We explored the +-evoked stimulation of Ca2+ waves is accompanied by aDMA-sensitive rise of [Na+]i , consistent withenhanced Na+ influx on NHE1. The [Na+]i rise leads, via aslowing of Ca2+ efflux on sarcolemmal NCX, to retention of intracellularCa2+ 7 and, ultimately, toincreased Ca2+ wave frequency. The [Na+]i measurementshave been extracted from our previously published work, measured under comparableexperimental conditions+]i-rise was recorded in 1 mM[Ca2+]o, whereas current experiments were conducted in 5 mM[Ca2+]o, we confirmed that the rise was not influenced by thedifferent [Ca2+]o levels; see Figure S6). For clarity, [Na+]i data have beenreplotted in Figures  and . The data emphasize that preventing theH+-evoked rise of [Na+]i with DMA, prevents wavestimulation. Stimulation is thus dependent on the rise of [Na+]i,while acid-induced wave inhibition, observed in the presence of an NHE1 inhibitor, isattributable directly to the rise of [H+]i. N.B.since the rise of [Na+]i is completely prevented by inhibiting NHE,we do not anticipate a role for H+-induced inhibition of theNa+/K+ pump under these conditions.H2+ wave stimulation has been quantified in Figure, whichplots, on logarithmic axes, wave frequency vs. [Na+]i . Wave frequency rises steeply with[Na+]i . In contrast, wave frequency in the absence of NHE1activity decreased with a rise of [H+]i, displayinga more shallow H+-dependence . The net result for whole-cellacidosis on wave frequency will be a combination of these stimulatory and inhibitorycomponents. At steady-state, the combination is net stimulatory ,reflecting the steep positive dependence of wave frequency on[Na+]i.Ca+]i-dependenceof Ca2+ wave frequency, this does not exclude an additional role formitochondrial NCX (mNCX). Elevating [Na+]i has been proposed topromote mitochondrial Ca2+ efflux on mNCX,2+ efflux may therefore supplementthe domain of cytoplasmic Ca2+ that eventually enhances Ca2+ wavefrequency. To test this hypothesis, myocytes were first subjected to intracellularacidosis in the presence of the sarcolemmal NCX inhibitor, SN-6. The drug fully inhibitedthe H+-evoked stimulation of Ca2+ wave frequency , indicatinga necessary role for the sarcolemmal exchanger. In contrast, selective inhibition of mNCXwith CGP-37157 had no effect on H+-evoked waves , indicatingthat mNCX is unlikely to be supplementing Ca2+ wave stimulation. Indeed, it ismore likely that mitochondria attenuate Ca2+ waves. This is supported by theobservation that when the mitochondrial uniporter (MCU), which mediates Ca2+uptake, was blocked by pre-incubation with ruthenium-360 (Ru360), followed by inhibitionof mNCX with CGP-37157, then H+-induced Ca2+ wave frequency wasenhanced by 70% . The result suggests that mitochondrialCa2+ uptake normally plays a protective role during intracellular acidosis,by sequestering some of the intracellular Ca2+-overload.Although sarcolemmal NCX is likely to mediate the i influenced other features, notably, Ca2+ wavepropagation velocity (vprop), amplitude, and time-course. Raising[H+]i increased wave velocity, both in the presence and absence ofNHE1 activity . As shown in Figure , velocity rose with a fall ofpHi from 7.7 to 6.65 , increasing by ∼40% over the range. According to a recentmodel of Ca2+ wave propagation,+-dependent reduction in cytoplasmic Ca2+ buffering, for example,by troponin C and other proteins, as well as by cytoplasmic histidyl-dipeptides.2+buffering may reflect a reduction in the Ca2+ binding constant(kon), or an increase in the unbinding constant (koff). Increasing[H+]i also increased resting [Ca2+]i .In addition to CaFigure ). This generated a stable end-to-end pHi gradient of ∼0.6units .Acidosis was imposed locally at one end of an isolated myocyte using a dualmicro-superfusion device positioned at right-angles to the cell (see Methods). Onemicrostream contained 80 mM acetate while the other contained acetate-free Tyrode(i gradient), no significantdifference was observed in Ca2+ wave frequency between ROIs positioned atopposite ends of the cell . In contrast, after imposing the pHi gradient,Ca2+ waves were initiated, and became clustered in the non-acidic end of thecell, even though this region was many sarcomere-lengths away from the acidic zone. Thus,over the first 2 min of an imposed pHi gradient, Ca2+ waves wererarely initiated in the acidic zone, but their frequency increased up to five-fold in thenon-acidic zone . Selective pharmacological inhibition (DMA) of NHE1 activityin the non-acidic zone did not prevent the local increase in Ca2+ wavefrequency , indicatingthat the relevant NHE1 activity driving wave initiation was in the acidic zone. This wastested further by selectively superfusing DMA over the acidic end of the myocyte. Althoughthe pHi gradient itself was unaffected by this manoeuvre, the stimulation ofCa2+ waves in the non-acidic zone was now greatly attenuated . Thus, locally enhancing NHE1activity remotely stimulates Ca2+ waves.Under control conditions  there isselective stimulation and clustering of Ca2+ waves in downstreamnon-acidic zones.In summary, during uniform acidosis, all regions of a myocyte are equally likely tosupport a Ca+ ions affect multiple proteins involved in Ca2+ signalling,the mechanism by which Ca2+ waves are inhibited in the acidic microdomain, butremotely stimulated in the non-acidic microdomain, is not immediately obvious. Onepossibility is that H+ ions locally decrease RyR open probability(Po), which could account for inhibition of wave initiation. A decreasedPo may also enhance SR Ca2+ retention.2+ load then diffused rapidly into non-acidicregions of the SR lumen.Since H2+ spark frequency was measured. Under resting conditions, sparkfrequency at either end of the myocyte was not different. However, when apHi gradient was imposed, Ca2+ spark frequency in the acidicmicrodomain decreased by 72 ± 11% , while that in the non-acidic microdomain was not significantlyaltered . This phenomenon was insensitive to NHE1 inhibition. The results argue for local H+ inhibition of RyRs,but also indicate that any resulting local rise in luminal SR [Ca2+] does notaffect the Po of remote RyRs.To explore whether the spatial effects of acidosis on wave frequency are all due to RyRinhibition, Ca2+ waves of regional RyR inhibition, tetracaine (anRyR antagonist) was applied to one half of a myocyte to simulate the inhibitory effect ofH+ ions, while maintaining pHi uniformly at resting levelsthroughout the cell. Figure  shows that Ca2+ wave initiation frequency observed withlocally applied tetracaine was very similar to that observed with local acidosis and NHE1inhibition . In both cases, Ca2+ wave frequency wassuppressed in the tetracaine/acetate-exposed region of the cell, and did not significantlychange in the downstream (unexposed) region. These findings confirm that, while local RyRblockade by H+ ions or tetracaine can explain local (upstream) inhibition ofCa2+ waves, it cannot explain wave stimulation in downstream regions. Thislatter effect requires the local, upstream activity of NHE1.To verify the effect on Ca2+ waves, other than initiationfrequency, also map onto pHi heterogeneity, Ca2+ wave velocity(vprop), amplitude and time-course were measured in the acidic and non-acidicmicrodomains. For the few Ca2+ waves that successfully propagated from thenon-acidic to the acidic zones of the cell, their velocity increased in the acidic zone. A similar trend was observed when DMA was superfused over theacidic end of the cell, indicating that the faster propagation was independent of NHE1activity .To investigate whether properties of Ca0 of the Ca2+ wave was also significantly higher in theacidic microdomain compared with the non-acidic microdomain , while wave relaxation was significantly slower in theacidic microdomain . It was also noted thatCa2+ waves frequently failed to propagate within the acidic microdomain(irrespective of the initiation site), unlike the negligible failure rate observed undercontrol conditions . Taken together, our results show that multiple properties ofthe Ca2+ wave  are all subservient to the local pHi.Spatial non-uniformity of pHi thus induces dramatic heterogeneity ofCa2+ waves.The peak F/Fi gradients can be established in multicellularcardiac structures, as well as in isolated cells, dual microperfusion was used to delivera microstream containing 40 mM lactate in parallel to a lactate-free microstream over aconfluent monolayer of neonatal rat ventricular myocytes. Under control conditions (i.e.uniform exposure to lactate-free solution), no pHi heterogeneity was observedwhen imaging the cSNARF-1 fluorescence ratio . However, when the monolayer wasregionally superfused with 40 mM lactate , a stable pHi gradient of ∼0.3 units formed in the regionof the solution boundary, of width ∼100 µm, equivalent to several neonatal cell-lengths. Cytoplasmic acidification inlactate-exposed cells activates NHE1 locally, which produces a rise in[Na+]i, detected using SBFI fluorescence.Figure+ ions then resulted in a smooth gradient ofelevated [Na+]i, ∼2.5 mM in magnitude, co-located with thepHi gradient but extending up to 100 µm beyond the acidiczone. Note that, although SBFI fluorescence is modestly pH-sensitive,i gradient, because the shapes of thepHi and + signalling can be stimulated downstream of a localisedacidic domain.To explore whether spatial pH2+waves. While this is consistent with earlier phenomenological descriptions,+]i, [Na+]i, andCa2+ waves, and demonstrated antagonistic control by + and Na+ diffuseat different rates in myocyte cytoplasm, the control of Ca2+ wave generationbecomes a complex spatio-temporal process. A key finding is that a localised source ofintracellular acid inhibits Ca2+ waves in the immediate vicinity, but remotelystimulates them in distal, non-acidic regions, an effect mediated via the diffusiblemessenger, We have demonstrated that acidosis powerfully stimulates arrhythmogenic Ca2+ wave initiation depends on a balancebetween the stimulatory effects of H+-activated Na+ influx viapHi regulatory transporters such as NHE1, and the inhibitory effects ofH+ ions on Ca2+ handling proteins, notably RyRs. Wave frequency issteeply stimulated by a rise of [Na+]i compared with a more shallowinhibition by [H+]i. Thus, when NHE1 flux is enhanced,[Na+]i rises, and waves can be profoundly stimulated. When,however, NHE1 flux is tempered, inhibitory effects of H+ can dominate, thusreducing Ca2+ wave frequency. This latter inhibitory effect has been suggestedto be cardioprotective.During uniform acidosis, spontaneous Ca2+ wavesuppression,+]i. In our own work, we have clearlyidentified both +-dependentinhibition of Ca2+ waves.A previous report concluded that intracellular acidosis caused only Ca+]i couples toCa2+ waves via the modulation of sarcolemmal NCX, since the increase in wavefrequency was prevented by pharmacological inhibition of this transporter. In principle,mitochondrial NCX could also make a contribution, however inhibiting mNCX provided noevidence for this. Instead, we confirm a cardioprotective role for mitochondria inbuffering excess cytoplasmic Ca2+.We further demonstrate that increased i raises luminal SR Ca2+ within theacidic zone . Elevated luminalCa2+ might then diffuse throughout the whole SR, enhancing Po forRyRs in non-acidic regions,2+ wave generation. Measurements of SRCa2+ diffusivity, however, are in the range of 9 to 60 µm2/s,,2+ waves are already well established within 1 min of imposing a localacidosis, the contribution from diffusive re-distribution of luminal SR Ca2+can, at best, only partially account for our observed results. Additionally, there-balancing of flux between SERCA activity and passive leak would tend to return SRCa2+ to physiological levels in regions away from the acidic source.One explanation for remote Ca+ ions, initially imported by NHE1 into the acidic microdomain, is thesignal that raises SR [Ca2+] throughout the cell. This global stimulation byNa+ is then superimposed with local inhibitory effects of H+ inthe acidic microdomain, leading to Ca2+ wave suppression in acidic zones, butwave initiation and clustering in downstream, non-acidic zones. Our recent measurements ofhigh cytoplasmic Na+ diffusivity (∼680 μm2/s)2+ wave responses.An alternative explanation is that rapid downstream diffusion of cytoplasmicNaFigure 8), based on the [Na+]i- and[H+]i-dependencies of Ca2+ wave frequency. This was then used to predict the frequency of Ca2+ wavesduring imposition of a local acid load . In the simulation, if[Na+]i is restrained at resting levels of ∼8 mM , the model predicts that wave frequency is unchanged in areas of normalpHi, but inhibited in the acidic microdomain. If [Na+]iis allowed to rise uniformly to around 20 mM, comparable to that observed under whole-cellacidosis , the model predicts Ca2+ wavestimulation globally. Thus, regardless of the pHi gradient, a significantintermediate [Na+]i-rise to 12 mM. This rise iscomparable to that measured experimentally during imposition of the pHigradient+]i-rise is near-uniform throughout thecell2+ waves in the acidicmicrodomain and stimulation in the more alkaline microdomain .Quantitative analysis therefore supports the mechanistic interpretation of our data.To test the quantitative feasibility of the above hypothesis, a simple model wasconstructed . The question is whether myocardialpHi gradients will remotely trigger Ca2+ wave initiation in a waycomparable to that observed in single myocytes. In the electrically coupled myocardium,because 2/s2/s- dependent inhibition ofaberrant Ca+-signalling within the ischaemic zone should also be spatiallyrestricted, coupled with a wider spatial stimulation by Na+ions, as illustrated schematically in Figure . Propagation of Ca2+ waves throughmyocardial gap junctions has a high failure rate,,44while junctional H+ permeation is slow and requires chaperoning bycarrier-molecules.+ ions readily permeate gap junctions,i.+]i that is fuelled by Na+ diffusion from moreacidic (ischaemic) zones . The elevated [Na+]i maycontribute to the Ca2+ waves and arrhythmias that have been observedconsistently at borderzones.,Accumulation of metabolic weak acids such as CO,2+waves are most likely to be arrhythmogenic under conditions of local heterogeneityi non-uniformity in the myocardium2+ waves. The arrhythmogenic risk will also be increased by the enhancedwave propagation velocity induced by acidosis. While many factors, other thanH+ ions, also contribute to arrhythmogenic activity in ischaemia,+-stimulation of Ca2+ waves, established here in our single cellstudies, now merits experimental evaluation in the regionally ischaemic myocardium.Recent studies+ ions inhibit Ca2+waves, via inhibition of Ca2+-handling proteins such as RyRs, but they alsostimulate waves by activating Na+ influx on NHE1. The steep dependence ofCa2+ wave initiation on [Na+]i often results in overallstimulation. When spatial pHi heterogeneity is introduced into a myocyte, we findthat acidic zones remotely trigger Ca2+ waves in non-acidic zones. This isexplained by the different-sized spatial domains for elevated inhibitory [H+] andstimulatory [Na+], predicted by their different ionic diffusivities. As well asproviding a stimulus for aberrant Ca2+ signalling in acidic myocytes, suchspatial signalling may also have relevance in regional myocardial ischaemia, where theformation of pHi gradients across groups of myocytes is likely. RemoteH+-triggering of Ca2+ waves emphasizes the importance ofNa+ as a spatial messenger in the heart.We have shown that, in ventricular myocytes, HCardiovascular Research online.Conflict of interest: none declared.This work was supported by the British Heart Foundation ; the Wellcome Trust (PhDStudentship to RDVJ and EM); and the Royal Society (University Research Fellowship toPS).Supplementary DataClick here for additional data file.


Scientific Reports6: Article number: 28177; 10.1038/srep28177 published online: 07052016; updated: 10102016.In this Article the legend for Figure 5 is incomplete.2+ in cytosol was measured using Fura-2 and Ca2+ in ER was measured using FRET-based probe D1ER cameleon in the basal state and after thapsigargin stimulation (100 nM for 1000 sec). Representative Ca2+ measurements in cytosol and ER (left panel). Quantitative ER Ca2+ levels of the average value prior to thapsigargin stimulation  and the minimum value after addition of thapsigargin (Tg(+)) (right panel). Data are means ± SEM, n = 43 for control, n = 50 for IRS1KO, and n = 40 for IRS2KO β-cells. *P < 0.05, **P < 0.01”.“CaShould read:2+ in cytosol was measured using Fura-2 and Ca2+ in ER was measured using FRET-based probe D1ER cameleon in the basal state and after thapsigargin stimulation (100 nM for 1000 sec). Representative Ca2+ measurements in cytosol and ER (left panel). Quantitative ER Ca2+ levels of the average value prior to thapsigargin stimulation  and the minimum value after addition of thapsigargin (Tg(+)) (right panel). Data are means ± SEM, n = 43 for control, n = 50 for IRS1KO, and n = 40 for IRS2KO β-cells. *P < 0.05, **P < 0.01. (b) Immunoblot of SERCA2 and β-actin in control β-cells, IRS1KO β-cells, and IRS2KO β-cells incubated with vehicle or thapsigargin (100 nM) for 8 h. Data are means ± SEM, n = 4. *P < 0.05”.“(a) Ca


Here, we found that GDF11 induced intracellular Ca2+ increases in neonatal rat cardiomyocytes and that this response was blocked by chelating the intracellular Ca2+ with BAPTA-AM or by pretreatment with inhibitors of the inositol 1,4,5-trisphosphate (IP3) pathway. Moreover, GDF11 increased the phosphorylation levels and luciferase activity of Smad2/3 in a concentration-dependent manner, and the inhibition of IP3-dependent Ca2+ release abolished GDF11-induced Smad2/3 activity. To assess whether GDF11 exerted antihypertrophic effects by modulating Ca2+ signaling, cardiomyocytes were exposed to hypertrophic agents (100 nM testosterone or 50 μM phenylephrine) for 24 h. Both treatments increased cardiomyocyte size and [3H]-leucine incorporation, and these responses were significantly blunted by pretreatment with GDF11 over 24 h. Moreover, downregulation of Smad2 and Smad3 with siRNA was accompanied by inhibition of the antihypertrophic effects of GDF11. These results suggest that GDF11 modulates Ca2+ signaling and the Smad2/3 pathway to prevent cardiomyocyte hypertrophy.Growth differentiation factor 11 (GDF11), a member of the transforming growth factor-β family, has been shown to act as a negative regulator in cardiac hypertrophy. Ca Growth differentiation factor 11 (GDF11) is a member of the transforming growth factor-β (TGF-β) superfamily and has various roles in the cardiovascular system . Circula2+ signals are important effectors that facilitate normal cardiac function. In cardiomyocytes, Ca2+ plays major roles in excitation-contraction coupling and as a second messenger for numerous agonist-mediated Ca2+-dependent pathways. Moreover, Ca2+ has been shown to have critical roles in diverse cellular events, including differentiation, metabolism, apoptosis, and hypertrophy -leucine . The cells were lysed, treated with 10% trichloroacetic acid, and centrifuged at 15,000× g rpm. The resulting pellet was dissolved in NaOH (0.2 M). Radioactivity of the cells was evaluated using a LS&500 liquid scintillation counter  to obtain the radioactive counts per minute (CPM). The CPM for each experimental condition in triplicate was obtained using a scintillation counter and normalized to the concentration of proteins in each sample.Cardiomyocytes were seeded onto 24-well plates at a density of 4 × 10Cardiomyocytes were pretreated with GDF11 (10 nM) for 24 h and stimulated with testosterone (100 nM) or phenylephrine (50 µM) for 48 h. To perform cell size measurements, the cells were washed with PBS buffer and incubated for 30 min with Cell Tracker Green . Next, the cells were washed with PBS and fixed with 4% paraformaldehyde for 15 min at room temperature. A Zeiss–Colibri epifluorescence microscope (Zeiss) and ImageJ software (NIH) were used to determine cardiomyocyte surface area. Five observation fields were selected randomly on each cover slip, and 5–8 cells within each observation field were selected for determination of the mean cardiomyocyte surface area according to the image analysis system.t tests, and comparisons between several means were analyzed by one-way analysis of variance, followed by Tukey’s post-hoc test using GraphPad Prism 6 . Differences with a P value of less than 0.05 were considered statistically significant.All data are presented as mean ± standard error of the mean (SEM). Comparisons of two groups were analyzed by Student’s 2+ during cardiomyocyte hypertrophy, little is known about the involvement of Ca2+ in antihypertrophic pathways. In the present study, we determined that GDF11 induces intracellular Ca2+ increase mediated through the IP3 pathway and activates Smad2/3 to modulate cardiomyocyte hypertrophy. It will be interesting to study the mechanisms through which Ca2+ is involved in the regulation of Smad2/3 activity and to determine the sources of Ca2+ mediated by GDF11 in cardiomyocytes. Additionally, further studies are needed to determine whether GDF11 prevents hypertrophy through a Smad2/3-dependent pathway or whether this protein can activate other antihypertrophic pathways (such as the glycogen synthase kinase-3β pathway) or inhibit prohypertrophic pathways (such as the nuclear factor of activated T cells pathway). Improving our understanding of these pathways will be useful to elucidate the mechanisms of GDF11 in cardiomyocytes, increase our knowledge of the signaling pathway of TGF-β family members, and characterize the regulation of anti- and prohypertrophic pathways in the heart.In contrast to the well-established role of Ca


Here we report the development of a zebrafish transgenic line, Tg(igfbp5a:GFP), which faithfully reports the mitotic action of IGF1R-PI3K-Akt-Tor signaling in epithelial cells in real-time. This platform is well suited for high-throughput assays and real-time cell cycle analysis. Using this platform, the dynamics of epithelial cell proliferation in response to low [Ca2+] stress and the distinct roles of Torc1 and Torc2 were elucidated. The availability of Tg(igfbp5a:GFP) line provides a whole organism platform for phenotype-based discovery of novel players and inhibitors in the IIS-PI3K-Akt-Tor signaling pathway.Aberrant regulation of the insulin-like growth factor (IGF)/insulin (IIS)-PI3K-AKT-TOR signaling pathway is linked to major human diseases, and key components of this pathway are targets for therapeutic intervention. Current assays are molecular target- or cell culture-based platforms. Due to the great  In vertebrates, the ancestral IIS pathway has diverged into the insulin and IGF signaling pathways5. The biological actions of IGFs and insulin are mainly mediated through the IGF1 receptor (IGF1R) and insulin receptor (InsR), respectively. A simplified view is that the insulin-InsR pathway is critical in regulating metabolism, while the IGFs-IGF1R pathway plays a more important role in regulating growth and longevity5. In many cell types, IGFs and insulin are strong activators of the phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)-Akt pathway8. Ligand binding activates the respective receptor, which in turn phosphorylates an insulin receptor substrate (IRS). The tyrosine phosphorylated IRS binds to and activates PI3K9. PI3K increases PIP3, while PTEN opposes this reaction. Elevated PIP3 recruits Akt to the plasma membrane where it is phosphorylated at T308 by PDK1. Target of Rapamycin (TOR), a serine/threonine kinase, exists in the rapamycin-sensitive TORC1 complex and the rapamycin-insensitive TORC2 complex10. While TORC2 phosphorylates Akt at S473 and increases Akt activity11, TORC1 activity is under the regulation of Akt. Akt-mediated phosphorylation of TSC2 inhibits its GAP activity, allowing GTP-bound RHEB to stimulate TORC1 activity12. Additionally, Akt phosphorylates PRAS40, which relieves its inhibition of TORC1 catalytic activity13.The evolutionarily ancient insulin-like growth factor (IGF)/insulin (IIS) hormonal pathway plays key roles in regulating development, growth, metabolism, and aging. The IIS system is also implicated in stem cell biology, neurodegenerative diseases, diabetes, and cancers14. Hyper-activation of PI3K, AKT, and/or mTOR contributes to the initiation and progression of tumors15. In fact, the IIS-PI3K-AKT is one of the most frequently mutated signaling pathways in several types of cancer4. For instance, multiple mutations have been identified in genes belonging to this pathway in colon cancers, including mutations that increase IGF2 gene expression, increase PIK3CA and PIK3R1 activities, and reduce PTEN expression16. The IRS2 gene is also frequently gained in colon cancer17 and has been proposed as a potential colorectal cancer “driver” oncogene18. In glioblastoma, the class IA PI3K p110α subunit gene is the most mutated gene19. Mutations in the TSC gene have been shown to lead to a tuberous sclerosis complex, which exhibits as benign lesions and increases the risk of renal cell carcinoma14. As such, major components of the IIS-PI3K-Akt pathway have targeted as points of therapeutic intervention. A number of assays have been developed, and potent inhibitors for IGF1R/InsR, PI3K, AKT, PTEN, and mTOR have been discovered21. Most, if not all, available assays are molecular target- or cell culture-based platforms. We now understand that there are tremendous complexities in the IIS-PI3K-AKT-mTOR signaling pathway in vivo, ranging from crosstalk with other hormones/growth factors, the availability of local nutrients and oxygen, to various feedback mechanisms. IGFs in particular are bound to a family of high-affinity IGF binding proteins (IGFBPs) in the extracellular environments. The IGF/IGFBP complexes prolong the half-lives of IGFs and buffer the potential hypoglycemic effects of high concentrations of IGFs in circulation. Locally expressed IGFBPs alter IGF availability and biological activity by modulating their interaction with the IGF1R22. These complexities cannot be captured by a molecular target- or cell culture-based assay. The existence of various redundant and compensatory mechanisms in an organism cannot be assayed in vitro, either.The IIS-PI3K-AKT-TOR signaling pathway is dysregulated in many human diseases, including growth disorders, diabetes, and obesity23. Zebrafish are vertebrates and share a high degree of genetic and anatomical homology with humans. Zebrafish spawn and produce large numbers of eggs daily. It is easy to subject the tiny, free living, and transparent embryos and larvae to various doses of chemicals and to observe and score morphological and physiological changes. The chemicals can be added conditionally during a specific developmental stage(s) and/or at different concentrations, thus adding a temporal dimension and making this a tunable system23. With the rapid advances in genetic tools such as CRISPR/Cas9 genome editing technology and Tol2 transposon-BAC transgenesis, it is feasible to genetically engineer mutant and transgenic zebrafish lines that can mimic certain human disease conditions24. Recent adoption of automated screening platforms has further increased the power of the zebrafish model in chemical biology/chemical genomics screens23.Zebrafish is an ideal model organism for whole organism-based chemical biology screens+-K+-ATPase-rich (NaR) cells, under low [Ca2+] stress25. Zebrafish live in aquatic environments and must import Ca2+ from the surrounding water using NaR cells to maintain their calcium homeostasis26. These cells are functionally equivalent to the Ca2+-transporting epithelial cells in human intestine and kidney and contain all the molecular players for transcellular Ca2+ transport, including Trpv5/6, an epithelial Ca2+ channel27. In the adult stage NaR cells are located in the gills (a major osmoregulatory organ in adult fish) and intestine. In the embryonic and larval stages, however, they are located on the surface of the yolk sac skin26, making them easily accessible for drug treatment and observation. When exposed to a low [Ca2+] environment, NaR cells undergo robust and unrestrained proliferation. This is entirely due to the activation of IGF1R-PI3K-Akt signaling in these cells25. This has provided an excellent inducible system to investigate the regulatory mechanisms of IGF1R-PI3K-Akt-Tor pathway in an in vivo whole animal setting. However, visualization of NaR cells by in situ hybridization and measuring their number manually is not only labor intensive, but also prevent real time analysis of the NaR cell proliferative response.In a recent study, we have shown that the IIS-PI3K-Akt signaling pathway is activated and is required in the proliferation of a group of epithelial cells, known as Na2+] stress and the distinct roles of Torc1 and Torc2 in this process were elucidated.In this study, we have developed a stable zebrafish transgenic line by labeling NaR cell with GFP. These transgenic larvae faithfully report the action of IGF1R-PI3K-Akt signaling and are well suited for high-throughput and real-time cell cycle analysis. Using this platform, the dynamics of NaR cell proliferation in response to low  on igfbp5a and trpv5/6 expression were not examined and it is unclear whether the low [Ca2+] effects are reversible. To answer these questions, wild type zebrafish embryos were raised in embryo rearing solutions containing various concentrations of [Ca2+] from 0 to 120 hpf. Compared to the larvae raised in normal [Ca2+] (0.2 mM) and high [Ca2+] (2 mM) solution, those raised in 0.02 and 0.001 mM [Ca2+] solutions had many more igfbp5a mRNA- and trpv5/6 mRNA-expressing NaR cells  cells, which was labeled by atp6v1al mRNA expression  were 3.5-fold greater than the N group   resulted in a 6.3-fold increase in the trpv5/6 mRNA levels  significantly reduced the trpv5/6 mRNA levels  Fig. . The levoup Fig. . Switchiels Fig. , while iels Fig.  or the Nels Fig. . Conversels Fig.  but had els Fig.  or NaR cels Fig. . Therefo2+] stress in different developmental stages. Low [Ca2+] treatment during the embryonic and early larval stage  significantly increased the trpv5/6 mRNA levels , which contains 113 kb upstream and 60 kb downstream of igfbp5a   larvae, GFP-expressing cells were found on the yolk sac skin and gills in a salt-and-pepper pattern  larvae were transferred to the low [Ca2+] solution from 72 hpf to 120 hpf. Low [Ca2+] treatment markedly increased the number and size of GFP-labeled cells  faithfully reports low [Ca2+]-induced NaR cell proliferation, and the NaR cells can be determined by automated measurement of GFP fluorescence intensity.Next, lls Fig. . Compareoup Fig. , a robusoup Fig. . At the oup Fig. . We alsooup Fig. . Correlaoup Fig. . These d2+] group at 120 hpf, while only 3.4% NaR cells was in M/G2 phase in the normal [Ca2+] group  larvae were transferred to normal and low [Ca2+] solution, NaR cells were isolated by FACS-based cell sorting. qPCR analysis showed that igf1ra, igf1rb, insra, and insrb mRNA were all detected and their levels did not change under low [Ca2+] stress  larvae  of pS6-positive cells in the low [Ca2+] group were NaR cells, while none of the pS6-positive cells (0/44) in the normal [Ca2+] group were NaR cells  larvae were treated with different concentrations of rapamycin, which preferentially inhibits Torc132. Rapamycin inhibited low [Ca2+]-induced NaR cell proliferation at concentrations as low as 0.1 μM  larvae faithfully recapitulate NaR cell development and low [Ca2+]-induced NaR cell proliferation. Real-time imaging revealed that newborn NaR cells are mainly derived from pre-existing NaR cells. NaR cells undergo at least two cell divisions during the 48 h treatment period. While the first cell division is completed around 24 h after the treatment, the second is completed 12 h later. These data suggest that low [Ca2+] stress promotes NaR cell proliferation by re-activating a mitotic program in pre-exiting NaR cells. Importantly, the low [Ca2+]-induced NaR cell proliferation is dependent on IGF1R-PI3K-Akt-Tor signaling activation. The Tg(igfbp5a:GFP) larvae can be readily adapted for automated high-throughput analysis because changes in NaR cell number can be determined by measuring GFP fluorescence.In this study, we have generated a stable transgenic zebrafish line expressing GFP in NaR cells under the control of the igf1r genes and two insr genes due to a teleost linage-specific genome duplication30. The results of this study showed that all 4 genes are expressed in NaR cells but their levels did not change under low [Ca2+] stress. The low [Ca2+]-induced pAkt signaling and NaR cell proliferation are likely mediated through the IGF1Rs. This notion is supported by the following facts: (1) two structurally distinct and selective IGF1R inhibitors blocked low [Ca2+]-induced pAkt signaling and NaR cell proliferation; (2) knocking down of Igfbp5a abolishes low [Ca2+]-induced Akt signaling in NaR cells25. Studies in mammalian systems have shown that IGFBP5 has several modes of action. IGFBP5 can modulate local IGF activities by inhibiting or enhancing the IGF activities or can even have ligand-independent action35. In differentiating mouse myoblasts, for example, IGFBP5 is located on the cell surface35. The cell surface-associated IGFBP5 binds to IGF-2 and brings IGF-2 into close proximity of the IGF1R, thereby enhancing IGF1R-mediated PI3K-Akt signaling activity35. In NaR cells, locally expressed Igfbp5a may act in a similar fashion to facilitate the IGF ligand–receptor interaction. Future studies will be needed to elucidate the specific mechanism(s) by which Igfbp5a promote IGF signaling in NaR cells.In the zebrafish genome, there are two 2+]-induced NaR cell proliferation. These effects are unlikely due to any potential indirect effects on calcium signaling because we have shown that BAPTA-AM, an intracellular Ca2+ chelator, did not affect the low [Ca2+]-induced NaR cell proliferation25. Likewise, addition of the L-type calcium channel blocker verapamil and two calmodulin antagonists  had no effect25. However, we cannot exclude the possibility that one or more of these IGF1R, PI3K, and Akt inhibitors may indirectly alter intracellular calcium levels in NaR cells. Future in vivo calcium imaging studies will be needed to clarify this issue.In this study, we have shown that several structurally distinct inhibitors targeting PI3K and Akt all blocked low [Ca2+] stress activates Tor signaling in NaR cells specifically and that Tor signaling is required for low [Ca2+]-induced NaR cell proliferation. Although there were considerable levels of pS6-positive cells on the yolk sac under normal [Ca2+] condition, these cells were not NaR cells since they do not express igfbp5a mRNA. Low [Ca2+] stress caused a marked increase in pS6-positive cells in the yolk sac region, and 98% of these pS6-positive cells in the low [Ca2+] group were NaR cells. This conclusion is also in good agreement with our previous study showing that low [Ca2+] stress activates causes PI3K-Akt signaling in NaR cells exclusively25. Addition of Torin1, AZD8055, and rapamycin all abolished NaR cell proliferation under low [Ca2+]. TOR is the catalytic subunit of TORC1 and TORC210. TORC1, consisting of TOR, Raptor, MLST8, PRAS40, and DEPTOR, regulates protein synthesis36. TORC2 is composed of TOR, RICTOR, mSIN1, DEPTOR, TTI1 and TEL2. It phosphorylates Akt/PKB at S473, which in turn leads to Akt phosphorylation by PDK1 at T308 and results in full Akt activation. The activated Akt then activates TORC136. Using Tg(igfbp5a:GFP) larvae, we showed that Torc2 contributes to NaR cell proliferation by phosphorylating Akt, while Torc1 plays a more direct role in NaR cell proliferation under low [Ca2+] stress. This conclusion is supported by the following lines of evidence: (1) treatment of zebrafish larvae with Torin1 or AZD8055 blocked low [Ca2+] stress-induced increase in pAkt at S473. Torin1 and AZD8055 treatment also abolished low [Ca2+] stress-induced NaR cell proliferation; (2) rapamycin inhibited low [Ca2+] stress-induced NaR cells at concentrations as low as 0.1 μM. At this concentration, rapamycin completely abolished the pS6 signal, while it did not affect pAkt signaling.A new finding made in this study is that low [Ca2+] environments, many fishes, including zebrafish, show elevated Ca2+ transport capacity37. This enhanced Ca2+ uptake capability is critical for maintaining Ca2+ homeostasis and is achieved by elevated trpv5/6 expression and increased NaR cell number37. The signaling mechanisms underlying these two adaptive responses are poorly understood. Our results suggest that the IIS-PI3K-Akt-Tor signaling plays a critical role in the increase in NaR cell number. The mitogenic action of IGF1R-PI3K-Akt-Tor signaling in NaR cells is likely due to its ability in regulating cell cycle regulators. It has been shown in mammalian cells that the PI3K-Akt-mTOR pathway elevates cyclin D1 level by increasing its translation through mTOR38 and by enhancing its stability and translocation into the nucleus through inhibition of GSK3B40. Akt can also down-regulate CDKN1B through FOXO141. Activation of the IGF1R-PI3K-Akt-Tor pathway, however, does not play a major role in the low [Ca2+]-induced up-regulation of trpv5/6 expression in NaR cells. This conclusion is based on the following lines of evidence: (1) low [Ca2+] stress resulted in a similar degree of increases in igfbp5a mRNA levels and NaR cell number, but the increase in trpv5/6 mRNA levels was more than 10-fold higher; (2) although low [Ca2+] treatment induces trpv5/6 expression in both embryonic and larval stages, it only stimulates NaR cell proliferation and igfbp5a expression in the larva stage; (3) when switching from the normal [Ca2+] to the low [Ca2+] solution, the levels of trpv5/6 increased much quicker compared to those in igfbp5a mRNA and NaR cell numbers; finally (4) inhibition of Tor signaling abolished the increased igfbp5a mRNA expression and NaR cell proliferation, while it had no effect on the elevated trpv5/6 expression under low [Ca2+] stress. These findings indicate that the elevated trpv5/6 mRNA levels are due to the increased NaR cell number as well as an increased trpv5/6 expression per cell, and the latter change occurs faster. The mechanism(s) underlying low [Ca2+] stress-induced rapid increase in trpv5/6 gene expression in NaR cells are yet to be elucidated. 1,25-(OH)2D3 has been shown to increase TRPV5 and/or TRPV6 expression at the transcriptional levels in mammals43, likely through the vitamin D receptor response elements (VDREs) on the gene promoter45. In zebrafish, knockdown of VDRa decreased the trpv5/6 mRNA expression46. It will be of interest to determine the possible role of vitamin D in regulating trpv5/6 expression in zebrafish NaR cells.When subjected to low [Ca2+]-induced epithelial cell proliferation was determined, and the in vivo roles of Torc1 and Torc2 in epithelial cell proliferation elucidated. Because of their tiny size, optical clarity, and since NaR cells are located on the yolk sac skin and readily take up chemical compounds, these Tg(igfbp5a:GFP) larvae can be readily used for chemical biology screens to identify novel players and inhibitors of the IGF1R-PI3K-Akt-Tor pathway.In summary, we have developed a whole organism and phenotype-based platform that faithfully reports the mitotic action of IGF1R-PI3K-Akt-Tor signaling in epithelial cells in real-time. This platform is well suited for high-throughput analysis and real-time cell cycle analysis. Using this platform, the dynamics of low  was prepared as previously reported25. All experiments were conducted in accordance with the guidelines approved by the Institutional Committee on the Use and Care of Animals, University of Michigan at Ann Arbor.Zebrafish were maintained at 28 °C under a light/dark cycle of 14/10 hours and fed twice daily. Fertilized eggs were obtained by natural breeding. They were raised and staged as described previouslyBAC(igfbp5a:GFP) construct was generated following Suster et al.48. Briefly, a BAC clone (DKEYP-56B7), which contains zebrafish igfbp5a genome DNA, was selected from the DanioKeypilot library . DKEYP-56B7 DNA was transformed into SW102 cells and verified by restriction enzyme digestion. Next, the iTol2 cassette and GFP-Kanamycin resistance gene cassette were recombined into the BAC. The primers used to amplify the GFP cassette sequence are: igfbp5a_GFP_fw, 5′-GTTTTGCCATTTCAAAGCTGGTGAAATAGGTGTTCTACAGTAGGACGATGGTGAGCAAGGGCGAGGAGCTGTTC-3′ and igfbp5a_frt-kan-rev, 5′-GTTTACTTTTGTCCCATATAAAACAAATACTACAAGTCAATAAAACATACCCGCGTGTAGGCTGGAGCTGCTTC-3′. The resulted BAC(igfbp5a:GFP) was validated by PCR using the following primers: igfbp5a-5UTR-fw, 5′-GAACGCTGTTCGCTTGAT-3′ and GFP-rev, 5′-GCTGAACTTGTGGCCGTTTACG-3′.The BAC(igfbp5a:GFP) DNA and Tol2 mRNA were mixed and injected into 1-cell stage zebrafish embryos. The injected embryos were placed in normal [Ca2+] embryo rearing solution and kept at 28.5 °C. The F0 embryos were screened at 72 hpf by checking GFP fluorescence. GFP-positive F0 embryos were raised and crossed with wild type fish to obtain F1 individuals. The F1 heterozygous fish were identified by GFP fluorescence, and genotyped by ADL-PCR. The F1 heterozygous adult fish carrying same integration site were in-crossed to obtain F2 individuals. F2 homozygous fish were identified by crossing with wild type fish and checking the ratio of GFP-positive offspring.The 48. Briefly, the adaptor was made by annealing ASS primer (5′-GATCACCTGCCCCCGCTT-3′) and ALS primer (5′-CTAATACGACTCACTATAGGGCTCGAGCGGCCGCGGGGGCAGGT-3′). The genomic DNA was extracted and digested by Sal 1 at 37 °C for 0.5 h. The adaptor was ligated to the digested genomic DNA. The ligation product was used as a template in the first round PCR using primer Ap1 (5′-GGATCCTAATACGACTCACTATAGGG-3′) and primer 150R-out (5′-AATACTCAAGTACAATTTTA-3′). The PCR product (1:10 dilution) was used as a template for the second round of PCR. Primer Ap2 (5′-CACTATAGGGCTCGAGCGG-3′) and 100R-out (5′-AGATTCTAGCCAGATACT-3′) were used in the second round of PCR. The final PCR product was sequenced at the University of Michigan DNA Sequencing Core Facility.The ADL-PCR was performed following a published protocolin situ hybridization was performed as previously reported47. Double-color in situ hybridization and immunofluorescence co-staining were conducted as previously described25. Briefly, igfbp5a mRNA and trpv5/6 mRNA were detected using a digoxigenin (DIG)-labeled antisense riboprobe. After incubation in an anti-DIG-POD antibody (Perkin Elmer. MA), larvae were stained in Alexa 488-tyramide substrate. After staining, they were washed in 1xPBST and incubated with an anti-GFP antibody overnight at 4 °C. Next, they were stained with a Cy3 conjugated Goat anti-Rabbit IgG antibody. Fluorescent images were acquired using a Leica TCS SP5 confocal microscope with the Leica LAS AF software.Single color β-actin or 18S RNA level. The following primers were used .Total RNA was isolated from embryos using TRIzol reagent and reverse-transcribed to cDNA using M-MLV reverse transcriptase . qPCR was carried out using iQ SYBR Green Supermix (Bio-Rad) with an iCycler iQ Multicolor real-time PCR detection system . The efficiency and specificity of the qPCR were verified by the standard curve and denaturing curve analyses. The expression level of a particular gene transcript was calculated based on the standard curve and normalized to Tg(igfbp5a:GFP) larvae were transferred to 0.2 or 0.001 mM [Ca2+] water for at 72 hpf and sampled at 120 hpf. Wild type larvae of the same stage were used as negative controls. 40 larvae in each group were digested with Liberase TM (0.28 Wünsch units/ml in HBSS) for 1 h at 28.5 °C. The digestion reaction was stopped by adding fetal calf serum (5% v/v). The dissociated cells were collected by centrifugation, washed three times in HBSS, and stained with Vybrant® DyeCycle™ Violet (1:1000) for 1 h. They were then subjected to Flow Cytometry Analysis in an Attune Acoustic Focusing Cytometer . A two-parameter dot-plot of VL1 (DyeCycle™ Violet) area (VL1-A) versus VL1 width (VL1-W) was constructed to exclude unstained cells and cell clumps. A two-parameter dot-plot of FSC-A versus BL1 height (GFP) (BL1-H) was constructed to gate GFP-positive cells. A single-parameter VL1-area histogram was constructed to illustrate relative DNA content.For Flow cytometry analysis, Tg(igfbp5a:GFP) larvae (72 hpf) were transferred to 0.2 mM or 0.001 mM [Ca2+] water for 18 h. Single cell suspension was made as described above. NaR cells were sorted using FACSAria cell sorter . Wild type larvae with the same treatment were used as negative control to set the sorting gate of GFP fluorescent signal. The gate was set so that less than 0.2% of cells from wild type were GFP positive cells (GFP+). The sorted cells  were directly collected into 75 μL cell lysis buffer provided with RNeasy Micro kit  followed by RNA isolation. 22 ng total RNA was reverse transcribed using SuperScript III Reverse Transcriptase. RNaseOUT™ Recombinant Ribonuclease Inhibitor was added into the reverse transcript system to protect RNA from degradation. qPCR was carried out as described above.For FACS sorting, 200 Tg(igfbp5a:GFP) larvae (72 hpf) were immobilized using low melting point (LMP) agarose gel. Mounting solution was prepared by dissolving 0.5% (w/v) LMP agarose in normal [Ca2+] or low [Ca2+] solutions containing Tricaine and PTU. The immobilized larvae were placed in a petri dish and submerged with normal [Ca2+] or low [Ca2+] embryo rearing solution containing Tricaine and PTU. Lateral views were taken hourly from 72 to 120 hpf using a Leica MZ16F stereo microscope  equipped with a QICAM 12-bit Mono Fast 1394 Cooled camera . QCapture Pro Version 6.0.0 software was used. The time-lapse video was generated using Windows Movie Maker (http://www.windows-movie-maker.org/).p < 0.05 or greater. Correlation analysis was carried out by fitting the data to the equation Y = X to calculate r2 using GraphPad Prism 6.Values are presented as mean ± standard error of the mean (SEM). Statistical significance among experimental groups was determined using one-way ANOVA followed by Tukey’s multiple comparison test. Statistical tests were performed using GraphPad Prism 6 software , and significance was defined as Supplementary informationSupplementary Video


To minimize effects of experimental manipulations on astroglial physiology, here we monitored Ca2+ in cells connected via gap junctions to astrocytes loaded whole‐cell with cytosolic indicators in area CA1 of acute hippocampal slices. Aiming at high sensitivity of [Ca2+] measurements, we also employed life‐time imaging of the Ca2+ indicator Oregon Green BAPTA‐1. We found that dopamine triggered a dose‐dependent, bidirectional Ca2+ response in stratum radiatum astroglia, a jagged elevation accompanied and followed by below‐baseline decreases. The elevation depended on D1/D2 receptors and engaged intracellular Ca2+ storage and removal whereas the dopamine‐induced [Ca2+] decrease involved D2 receptors only and was sensitive to Ca2+ channel blockade. In contrast, the stratum lacunosum moleculare astroglia generated higher‐threshold dopamine‐induced Ca2+ responses which did not depend on dopamine receptors and were uncoupled from the prominent inhibitory action of dopamine on local perforant path synapses. Our findings thus suggest that a single neurotransmitter—dopamine—could either elevate or decrease astrocyte [Ca2+] depending on the receptors involved, that such actions are specific to the regional neural circuitry and that they may be causally uncoupled from dopamine actions on local synapses. The results also indicate that [Ca2+] elevations commonly detected in astroglia can represent the variety of distinct mechanisms acting on the microscopic scale. GLIA 2017;65:447–459Whilst astrocytes in culture invariably respond to dopamine with cytosolic Ca ACAdenylyl cyclasecAMPCyclic adenosine monophosphateDADopamineFLIMFluorescence life‐time imagingGJCGap‐junction connectedKMSPotassium methyl‐sulphonatePACPatched astrocyte cellsPKAProtein kinase APLCPhospholipase C+ channels . Among the prominent downstream actions of DA on neurons are the altered NMDA receptor sensitivity  is a neuromodulator which exerts powerful behavioral effects and thus has been implicated in numerous psychiatric conditions. However, the exact mechanisms by which DA acts on brain circuits remain poorly understood, partly because of the exceedingly wide range of its effectors , the question arises whether such astrocytic signals could mediate effects of DA on local neural circuitry in organized tissue. We therefore set out first to establish whether astrocytes in situ respond to DA with an intracellular Ca2+ change—and if so, by what mechanism—and second to ascertain whether such a response could mediate previously reported DA‐induced modulation of excitatory synaptic transmission in the hippocampus.Astrocytes have increasingly been implicated in the modulation of the synaptic efficacy and its use‐dependent changes Rusakov, ). Since 2 7, NaH2PO4 1.25, CaCl2 0.5, Ascorbic acid 1.3, Sodium Pyruvate 3, and glucose 6 (osmolarity 304–312 mOsM), continuously bubbled with 95% O2/5% CO2. The whole hippocampus was dissected, placed in an agar block and transverse slices were prepared using a Leica VT 1200S slicer. Slices were then transferred to slicing solution kept at 34°C for 15 min before being transferred to an interface or an immersion chamber containing Ringer solution comprised of (in mM): NaCl 119, KCl 2.5, MgSO4 1.3, NaH2PO4 1, NaHCO3 26, CaCl2 2, glucose 10 (osmolarity 298‐302 mOsM). Ringer was kept at room temperature, and continuously bubbled with 95% O2/5% CO2. Slices were rested in Ringer for at least 45 minutes before recording. All experimental protocols were carried out in full compliance with UK guidelines on animal experimentation. For recordings, slices were transferred to a submersion‐type recording chamber and perfused with Ringer solution saturated with 95%O2/5%CO2. Experiments were carried out at 32–33°C unless specified otherwise.All experiments were carried out in accordance with the national and international rules and regulations for animal experimentation including EU Directive 2010/63/EU of 22 September 2010. Acute hippocampal slices, 350 μm thick, were prepared from 3‐4‐week‐old male Sprague–Dawley rats. Animals were anaesthetized to death with a lethal intraperitoneal injection of Sodium Pentobarbitol. The skull was opened up and the brain excised into an ice‐cold slicing solution containing (in mM): NaCl 50, sucrose 105, KCl 2.5, MgClFor imaging astrocytes loaded with sulforhodamine101 (SR101) and Fluo‐4‐AM, slices were allowed to rest for 30 minutes in Ringer solution, then incubated in Ringer containing 10 μM SR101 for 10 minutes at 35°C. Slices were then washed three times in plain Ringer and then incubated in Ringer containing 5μM SR101, 5μM Fluo‐4‐AM and 0.04% pluronic acid for 40 minutes at 35°C. Slices were then washed three times in Ringer, rested in Ringer at room temperature for 30 minutes and were then ready for recording.2+] monitoring, two fluorescent dyes were added to the intracellular solution, Alexa Fluor 594 (50 μM) as a morphological marker and Fluo‐4 (200 μM) as a Ca2+ indicator. For every patched astrocyte, the membrane current‐voltage (I‐V) relationship was plotted by stepped injection of current through the patch pipette, at 200 pA steps. Positive electrophysiological identification of a passive protoplasmic astrocyte required a linear I‐V relationship and a low input resistance (<10 MΩ) as well as a resting membrane potential lower than −80 mV  were recorded through glass micropipettes of 1‐2 MΩ resistance. For Schaffer collateral stimulation, axonal fibers were stimulated with a bipolar electrode from a Digitimer DS3 constant current stimulator box. The stimulating electrode was placed in Two setups were used to record fluorescent intensity images; either a modified Bio‐Rad Radiance 2000 on an Olympus BW50 microscope with a 40x objective, or a Fluoview FV1000 MPE microscope with a 25x objective. Both were optically linked to a separate femtosecond pulse TiSapphire MaiTai laser (SpectraPhysics) set to emit at 800 nm. Laser power was kept between 5 and 8 mW for bulk‐loaded Fluo‐4‐AM imaging, 2–5 mW for patch‐pipette‐loaded Fluo‐4 fluorescence imaging (as measured under the objective at 800 nM). Alexa Fluor 594 normally equilibrated across the astrocyte tree within 10–15 min  astrocytes. The procedure usually required low input resistance and ∼30 min for dye diffusion through the syncytium, the procedures similar to standard one‐cell patch‐clamp experiments. For these recordings a recording configuration was chosen to allow simultaneous recording from both the patched cell processes and the GJC somata. In a subset of experiments, only GJC somata could be recorded from. Recordings were only made when the diffusion of the dye from the pipette into the syncytium had equilibrated, as measured by the intensity of the reference dye, Alexa Fluor 594. If significant (>10%) fluctuations in Alexa fluorescence intensity in GJCs were witnessed, the recording was discarded.F/F0.Regions of interest (ROIs) were divided into four categories based on the morphology in the Alexa 594 channel: Patched astrocyte (PAC) soma; PAC process; and GJC soma. Only visible structures with clear astrocyte morphology were chosen as ROIs. An area of each image showing no fluorescence from the morphological marker was chosen to give a measure of average background fluorescence. For each ROI the average pixel fluorescence was taken for each frame and the average background fluorescence for that frame was subtracted . In control trials and where indicated, Fluo‐4 signal  was normalized to the morphological marker signal  to correct for fluorescence changes due to tissue movement (giving G/R) and laser power fluctuation. Fluo‐4 signals presented here have been normalized to the average fluorescence from each ROI in the baseline phase of each recording, to give Δ2+ , individual cells are referred to as a statistical unit, to follow the practice of real‐time single‐cell physiology studies.2+ fluctuations. All recorded cells were routinely added to the statistical sample: the a priori rejection criterion was the baseline fluorescence approaching the microscope detection threshold (thus affecting ΔF/F measures) or being too weak for FLIM acquisition. Astrocytes showing any significant (>20%) experiment‐wise trend in baseline fluorescence were also discarded. Samples were tested with the Shapiro‐Wilks test for normality; for comparing populations with a normal distribution, the Student's t‐test was used, otherwise, the Mann‐Whitney test was used.Physiological viability of imaged astrocytes was routinely checked by observing their spontaneous Castratum radiatum of the hippocampal CA1 region, we held astrocytes in whole‐cell mode loading them with the morphological tracer Alexa 594 and Ca2+ indicator Fluo‐4, as detailed previously  to validate these observations using the imaging method which is highly sensitive to nanomolar [Ca2+] but not to the possible concomitants of fluorescence intensity readout (such as cell volume changes or focus drift).To confirm this basic observation with an alternative imaging method, we labeled astroglia in bulk by slice incubation with cell‐permeable Fluo‐4 AM , mGluR5 blocker MPEP (1 μM), mGluR5 blocker LY367385 (100 μM), mGluR2/3 blocker LY341495 (500 nM) and GABAB receptor blocker CGP (1 μM), separately or in a cocktail. This receptor blockade did not appear to affect DA‐induced Ca2+ elevations in either bulk‐loaded or GJC astroglia whereas such elevations were completely blocked by the mixture of the D1/5 receptor blocker SCH23390 (20 μM) and D2/3 receptor blocker sulpiride (50 μM), or alternatively by sulpiride alone (20 μM)  appeared to halve the DA‐induced Ca2+ rise  leading to lipid peroxidation and downstream PLC‐mediated activation of the IP3 pathway  with selegiline (20 μM) has been reported to inhibit DA‐induced Caine Fig. G,H. Thus2+ imaging in GJC cells detected that the D1/5 receptor blockade with SCH23390, while abolishing DA‐induced [Ca2+] elevations, unmasked DA‐induced [Ca2+] decreases in these cells  completely abolished the DA‐evoked Ca2+ response in experiments with paired application of DA . Applied in bath, CPA induced a prominent Ca2+ elevation , which occluded any effects of DA applied subsequently  immediately after the first DA stimulus, to test whether the thapsigargin‐induced depletion and subsequent blockade of Ca2+ stores, as documented earlier in neuronal axons  and removal play a major role in generating DA‐induced Ca2+ elevations in our experiments.It has long been known that IPer 2‐APB 00 μM com2+ elevations depend on diffusion of the IP3 receptor ligand IP3 across the cell volume , we used a genetic animal model that produced an IP3 'sponge' protein complex targeted to astroglia. This IP3 ‘sponge’ exhibits 1000‐fold greater affinity to IP3 than the native IP3(1) receptor  of OGB‐1, which is highly sensitive to free nanomolar Ca2+ but not to the dye concentration, photobleaching, or light scattering and absorption in tissue  decreases in astroglial [Caner Fig. I. To val2+] Fig. A,B. A poted Fig. C. Intrigone Fig. C, consis2+] . It was earlier suggested that D2/3 receptor activation can reduce Ca2+ mobilization through modulation of L‐type VGCCs in rat nucleus accumbens neurons of the rat   which will dialyze rapidly with the pipette solution, probably washing out small endogenous signaling molecules that could be vital for DA‐induced effects.Here we report that DA can trigger increases and decreases in intracellular astrocytic calcium concentration in acute brain slices. Astrocytes 2+], which were consistent with the previous findings in cultured astroglia  can decrease Ca2+ in chromaffin cells through PLC inhibition  and Ca2+ entry into the cell  (Otmakhova and Lisman, 2+ rises in SLM astroglia while eliminating the DA‐induced local synaptic inhibition, whereas the latter was unaffected by Ca2+ clamp in SLM astroglia. Interestingly, the DA receptor independence of DA‐induced Ca2+ rises in SLM astrocytes resembles that in cultured astroglia (Vaarmann et al., 2+ elevations in astroglia, which are often associated with significant effects on local synaptic function may be consequential to multiple and diverse cellular cascades which do not necessarily lead to similar physiological consequences (Bazargani and Attwell, Unlike the prominent Ca Hewett, . This hi3‐sponge mice; D.A.R. and C.H. narrated the study, and A.J. and D.A.R. wrote the paper.A.J. planned and carried out experiments and data analyses; O.T. and K.Z. designed and carried out FLIM experiments; D.A.R, L.B. and C.H. contributed to experiment planning, selected experiments and data analyses; A.S. contributed to experiments and analyses in IP


Intracellular variation in free Ca2+ concentration is among the earliest events following the plant perception of environmental change. These Ca2+ variations differ in their spatio-temporal properties according to the nature, strength and duration of the stimulus. However, their conversion into biological responses requires Ca2+ sensors for decoding and relaying. The occurrence in plants of calmodulin (CaM) but also of other sets of plant-specific Ca2+ sensors such as calmodulin-like proteins (CMLs), Ca2+-dependent protein kinases (CDPKs) and calcineurin B-like proteins (CBLs) indicate that plants possess specific tools and machineries to convert Ca2+ signals into appropriate responses. Here, we focus on recent progress made in monitoring the generation of Ca2+ signals at the whole plant or cell level and their long distance propagation during biotic interactions. The contribution of CaM/CMLs and CDPKs in plant immune responses mounted against bacteria, fungi, viruses and insects are also presented.Calcium (Ca These aesponses ,5,6,7. Tnfestans , whereasimmunity .2+, a ubiquitous and versatile second messenger [2+ message needs to be decoded and relayed in order to activate the appropriate cell response and this is carried out by Ca2+-binding proteins termed Ca2+ sensors [2+ changes at cellular and tissue levels  are proposed to carry information and are denoted as the Ca2+ signature [2+ signature encodes a first layer of specificity and will be considered first, with a particular emphasis on how methods to monitor Ca2+ signatures have evolved and brought new information. Ca2+-binding proteins and their downstream targets provide a second layer of specificity. Most Ca2+-binding proteins are characterized by the presence in their sequence of the canonical Ca2+-binding motif called the EF-hand [Arabidopsis thaliana encodes at least 250 EF-hand-containing proteins [2+ sensors do not have homologs in others organisms [2+ sensors [2+ sensors are classified into four major groups: the calcineurin B-like (CBL), the Ca2+-dependent protein kinases (CDPK), the calmodulin (CaM) group and its closely related group, the Calmodulin-like protein (CML) family [Arabidopsis) that are not present in animals, as is also the case for CBLs and CDPKs  [2+ sensors remain unknown but recent studies have pointed out the roles for some of them in physiological processes associated with development, abiotic plant stress responses and plant immunity [In this review, we will focus on the importance of Caessenger , in plan sensors . The comignature ,14  family ,19. Calm) family . Plants ctively) ,22. To dimmunity ,24,25. H2+ is a universal second messenger, and increases in cytosolic Ca2+ concentration are among the earliest signalling events occurring in plants challenged with mutualistic or pathogenic partners. If we consider plant-pathogen interactions, the plant immune system is schematically organized as a two-tiered system composed of Pathogen-Associated Molecular Patterns (PAMP)-Triggered Immunity (PTI) and Effector-Triggered Immunity (ETI) [2+ signature. For instance, PTI activation involves Ca2+ transients that returns to basal levels within minutes [2+ increase that can last for hours [2+ channel blocker lanthanum strongly impairs the immune responses associated with both types of plant immunity [2+ signals and their decoders at the centre of immune signalling pathways.Free Caty (ETI) . Activatty (ETI)  while ac minutes  whereas or hours . The genimmunity ,30, ther2+ elevations during immune signalling are critical for the control of gene reprogramming which is required to mount the adequate responses . Such widely acknowledged importance of these Ca2+ variations in plant signalling has been made possible through the emergence in the early 1990s of tools allowing the monitoring of the dynamics of Ca2+ changes in plant cells. The pioneering work of Trewavas’s group and his collaborators demonstrated the occurrence of Ca2+ variations in plant cells using the fluorescent Fluo3 Ca2+ indicator and caged Ca2+ or IP3 as triggers [2+ probe aequorin [2+ controls plant adaptive responses. In the seminal paper reporting the use of aequorin in plants, Knight et al. established for the first time that biotic stimuli consisting of various yeast preparations were able, like abiotic stimuli, to trigger Ca2+ signals in tobacco seedlings [2+ cation should be taken into consideration as an important second messenger in studies dealing with plant-microbe or plant-pest interactions. Due to the numerous studies available in the literature, we will focus in this section, more specifically on several well-documented examples illustrating how exploiting the diversity of Ca2+ probes has contributed to increasing our knowledge on the role of Ca2+ during plant immunity, symbiosis and response to herbivory attacks.Catriggers . His groaequorin . The useeedlings . This re2+ sensors [2+ response followed by a delayed oxidative burst [2+ response. This result was among the first to suggest a possible role of Ca2+ transients in the ROS-mediated defence signalling pathway [2+ increases and defence responses using chitotetraose, a fungal cell wall component and β-glucans. A sustained high Ca2+ concentration response was observed in the case of β-glucans while this was not the case with the chitotetraose [2+ increase generated after β-glucan application was linked to the increase of a major soybean phytolalexin glyceollin [Phytophtora sojae-derived peptide (Pep 13) elicited a biphasic Ca2+ variation due to an influx of extracellular Ca2+ in parsley cells stably expressing aequorin. Only the sustained Ca2+ variation reaching a concentration around 250–300 nM could be associated with ROS and phytoalexin production [Nicotiana plumbaginifolia cells stably expressing cytosolic aequorin challenged with the Phytophtora cryptogea elicitin “cryptogein” allowed us to demonstrate that oligogalacturonic acids and cryptogein each elicit a very specific cytosolic Ca2+ response [2+ response appeared as a biphasic Ca2+ increase with a first transient peak lasting 10 min followed by a sustained increase. Suppression of this sustained Ca2+ phase using the Ca2+ channel blocker lanthanum chloride just after the first Ca2+ transient, resulted in the suppression of MAPK activation, accumulation of PAL and hsr203j immune-related gene transcripts and induced cell death [Chandra and Low pointed out the advantages of the aequorin probe versus other available Ca sensors . Using ave burst ,35. Usin pathway . At the tetraose . The susyceollin . One yeaoduction . Interesoduction . Similarresponse . After all death .2+ variations, for example in lima bean leaves incubated with the fluorescent Ca2+ probe Fluo3-AM or in soybean cell cultures expressing cytosolic aequorin. Spodoptera littoralis larvae bites elicited a Ca2+ response on the margin of the leaves within a delimited region of 30 to 200 µm from the bite zone whereas several components from the larvae regurgitate such as linolenoyl-L-Glutamine or volicitin were able to elicit Ca2+ transients in soybean cells [2+ transients are correlated with early membrane depolarization/hyperpolarization events that are known to be linked to plant defence responses through systemin synthesis, ROS and jasmonate signalling  or compounds derived from the activity of pathogens on plant cell walls, the Damage-Associated Molecular Patterns (DAMPs) ,40. Simian cells . These Cview see ).2+ variations reinforced the concept of the Ca2+ signature [2+ variation is defined by its form, amplitude, frequency, duration, spatial localization and the Ca2+ pool involved. All these parameters are tightly linked to the nature and strength of the stimulus perceived by the cell. Subsequent studies performed on guard cells largely corroborated this concept. Artificial modulation of the frequency, transient number of spikes, duration and amplitude of Ca2+ oscillations were found to control the degree of long-term steady-state stomata closure [2+ signature concept come from work performed with Medicago truncatula plants expressing the Ca2+ probe cameleon YC2.1. Using a pharmacological approach modulating Ca2+ homeostasis, the authors were able to demonstrate a link between the bacterial Nod factor-induced Ca2+ oscillations and the activation of some selected nodulation markers genes. For example, the induction of the ENOD11 nodulin gene required about 30 consecutive Ca2+ spikes [These few examples showing that different elicitors trigger different Caignature ,14. This closure . Other f+ spikes .2+ variations in plant immunity were performed with elicitors and cultured cells. Although convenient, this model cannot inform us about the downstream events activated during genuine plant-microorganism or plant-pest interactions, such as cell-to-cell communication leading to long distance and systemic signalling. Pathogen elicitors only mimic the recognition step when surface motifs of pathogens are recognized by pattern recognition receptors (PRRs) of the host [2+ signalling in whole plants challenged with a microorganism  or insect, in a non-destructive manner. One of the first examples of such studies, was the monitoring of Ca2+ response in Arabidopsis leaves stably expressing aequorin under control of the 35S promoter challenged either with the compatible strain of Pseudomonas syringae pv. tomato DC3000 or with incompatible strains carrying the avirulence genes avrRPM1 or avrB, both detected by the resistance Arabidopsis RPM1 protein [2+ peak lasting about 10 min and peaking between 8–12 min whereas the incompatible strains avrRPM1 and avrB induced an additional delayed peak giving a maximal increase at around 105 ± 10 and 137 ± 7 min, respectively [2+ response. In addition, Arabidopsis rpm1 mutant failed to generate the second Ca2+ peak and the HR. Thus, the aequorin technology allowed, in this specific case, to make a clear correlation between the delayed Ca2+ peak and the gene for gene interactions responsible for the HR response.Most of the initial studies on Cathe host . In addithe host . Therefothe host ,48. Aequthe host . It has  protein . In thisectively . This se2+ spikes [2+ probe, this group demonstrated two years later that spatiotemporal Ca2+ spiking studies were helpful in understanding the regulation of nod gene expression. Indeed, they showed that strains differing by their ability to produce Nod factor could be differentiated by the Ca2+ oscillations they induce. Thus, two strains derived from the same parent displayed large differences in the kinetics of Ca2+ spiking; Rm1021 triggered a robust Ca2+ spiking after a lag phase of 10–15 min in more than 50% of the root hair cells whereas Rm2011 did not [2+ probes [2+ responses at the organ and cell level during symbiotic relationships with rhizobia and mycorrhizal fungi. Kosuta et al. [2+ oscillations. They demonstrated that the two symbiotic pathways require both DMI1 and DMI2 for both the establishment of functional symbiosis and also for the generation of Ca2+ oscillations. This result indicates that Ca2+ oscillations are also required for plant colonization by rhizobia or mycorrhizal fungi. Interestingly, the symbiotic fungus induced a Ca2+ oscillation that differs from the Nod-factor-induced Ca2+ oscillations by its amplitude and its periodicity [2+ probe demonstrated a regular nuclear Ca2+ spiking in Medicago root hairs challenged with Nod Factors [In symbiotic interaction studies, the use of Oregon-dextran dye injected in root hair cells by iontophoresis demonstrated that two important genes in the rhizobia symbiosis were involved upstream of the Ca+ spikes . Using t did not . The new+ probes  facilitaa et al.  were abliodicity . In the  Factors .2+ waves, but most of these studies were performed in response to abiotic stimuli and were exploiting either cameleon derivatives such as the YCNano-65 FRET sensor [2+ waves propagating from the root to the shoot were reported in response to salt [2+ waves and that the Two-Pore Channel (TPC1) vacuolar plant Ca2+ channel appeared to be a main player in this spreading [2+ responses have come from plant–insect interactions [2+ signals on the neighbouring leaves. Interestingly, TPC1 appeared to be involved in the systemic propagation of the Ca2+ wave but not to be involved in the generation of the primary Ca2+ transient observed at the feeding point [2+ sensors has made it possible to evaluate the spatio-temporal specificity of immune responses as well as the direction in which the PAMP-induced Ca2+ waves propagate [2+ sensor, Keinath et al. studied Ca2+ responses induced by Pathogen-Associated Molecular Patterns (PAMPS) such as flagellin and chitin [2+ variations in cultured cells when Ca2+ changes are monitored with aequorin, was also able to induce Ca2+ variations in roots. They localized these variations to the elongation zone where callose deposition takes place and immune-responsive genes are upregulated supporting a clear correlation between Ca2+ changes and defence responses [The whole plant approach allowed researchers to detect long-range CaT sensor ,56 or BiT sensor . Using t to salt . A similpreading . To our ractions . Woundinng point . The recropagate . Using Rd chitin . They shesponses .2+ signalling and illustrate how monitoring Ca2+ signals at the cell tissue or whole plant level and the use of alternative Ca2+ probes from the available panel can be very helpful to decipher both immune and symbiotic responses.Overall, these few examples highlight the complexity of Ca2+ signals previously described  and calmodulin-like proteins (CMLs), Ca2+-dependent protein kinases (CDPKs) and calcineurin B-Like proteins (CBLs) . Genome analysis of the model plant A. thaliana revealed the presence of 3 distinct CaM isoforms (encoded by seven genes) [CaM is the prototype of a Can genes) ,66. Untin genes) . During n genes) ,68,69. Wn genes) ,68,70,71n genes) ,72, in mA. thaliana infected cells is enhanced by up-regulation of the expression of ICS1 (Isochorismate synthase 1) and EDS1 (Enhanced Disease Susceptibility 1) genes and the expression of ICS1 and EDS1 is positively and negatively controlled by CBP60g and CAMTA3/AtSR1, respectively, two CaM-binding TFs [CaMBPs with predicted or demonstrated functions during plant immunity have been reported ,24. For ding TFs ,74,75.cbp60g knock-out mutant exhibits defects in pathogen-induced SA accumulation and shows enhanced susceptibility to the Pseudomonas syringae phytopathogenic bacteria [cbp60g mutant. This shows the importance of CaM in regulating CBP60g function and its contribution to plant immunity [cbp60a mutations reduced P. syringae growth in planta, indicating that CBP60a acts as a negative regulator of immunity whereas CPB60g acts as a positive regulator [2+-CaM/CML complex in the activation of immune responses.CBP60g belongs to a plant-specific DNA-binding protein family comprising eight members in Arabidopsis ,76,77. Tbacteria . A variaimmunity . The rolegulator . Altoget2+ and CaM. Indeed, the CaM-binding Transcription Activator (CAMTA) family certainly constitutes one of the most important Ca2+/CaM-regulated TF families in plants [Arabidopsis thaliana Signal Responsive 1), in plant defence responses against pathogens [camta3/Atsr1 Knock-Out mutants display elevated SA levels, high expression of EDS1 and constitutive defence responses. CaM binding is crucial for the function of CAMTA3 in the control of SA production and defence [camta3 mutants are more resistant to P. syringae, Botrytis cinerea and Sclerotinia sclerotiorum [Trichophusia) [The CBP60 transcriptional regulator family is not the only one regulated by Can plants ,80. CAMTn plants . One of athogens  and its athogens . Arabido defence . Surprisrotiorum ,83 but aophusia) ,82. In aophusia) ,85.Xanthomonas oryzae [2+ signatures are integrated in this transcriptional pathway. Efforts are being made in this perspective and dynamic mathematical models incorporating several parameters are being developed with the aim of predicting regulation networks according to the generated Ca2+ signatures [Other studies revealed additional roles for CAMTA3/AtSR1 in defence. For instance, AtCAMTA3 negatively regulates plant immunity following PAMP recognition as well as non-host resistance to s oryzae . CAMTA3 s oryzae . More res oryzae . To dategnatures ,88.2+-dependent manner [Although the CaM contribution in plant immunity was mainly revealed by the identification of CaMBP, it is now clear how CaM plays a pivotal role in the fine tuning of immune responses by acting either as a positive or a negative regulator of defence responses. Identification of the whole set of CaMBPs is certainly not complete and other CaM-binding TFs including several members of TGA, WRKY, MYB and NAC families also contribute to plant immunity either positively or negatively ,88,89. Ft manner  but the A. thaliana) [2+ ions and do not possess other known functional domains [CMLs vary according to plant developmental stages, tissues and environmental stimuli, indicating that each CML may have a specific role in plants [In addition to the presence of the typical CaMs, plants also possess a broad range of divergent forms of CaM called CaM-like proteins  ,19. Like domains ,19. Whern plants . Indeed,n plants ,90,91,92CaM/CML gene expression or loss of a CML function in mutated plants affect plant defence responses to various pathogens. The first report involving CMLs in plant defence came from gain-of-function experiments by overexpressing CMLs. The over-expression of soybean CMLs  in tobacco confers enhanced resistance to a wide spectrum of virulent and avirulent pathogens, including bacteria, fungi and viruses [Arabidopsis, SCaM-5 confers enhanced resistance to Pseudomonas syringae infection [Phytophtora sojae and to two necrotrophic fungal pathogens (Alternaria tenuissima and Phomopsis longicolla) supporting the idea that CaM/CMLs do take part in plant immune responses [Although we cannot rule out functional redundancy between members of the CaM/CML family, accumulating evidence indicate that deregulation of individual  viruses . When confection  whereas nfection . Althougesponses .APR134 in tomato suppresses the hypersensitive response (HR), whereas overexpression of the APR134 orthologue from Arabidopsis CML43 stimulates the HR in response to an avirulent strain of P. syringae [cml24 knockout in Arabidopsis impairs the HR response and reduces nitric oxide production following PAMP recognition [P. syringae [cml9 mutants and CML9 overexpressing lines exhibit enhanced or reduced susceptibility to virulent strains of P. syringae, respectively [P. syringae but compared to CML9, the enhanced resistance observed in CML8 overexpressing lines relies mainly on SA-dependent responses [amiRNA and CML41 overexpressing lines, Xu et al. reported that CML41 acts as a positive regulator of defence against Pseudomonas syringae [Loss-of- function genetic approaches also demonstrated roles in plant immunity for several CMLs. For instance, silencing syringae . Similarognition . Gain- asyringae ,99,100. syringae  and latesyringae . The cmlectively . These pectively . CML8 alesponses . Indeed,esponses . It was esponses . CML41 isyringae .2+ signalling is not only required for defence mechanisms upon microbial pathogen attack but also in response to herbivores [Spodoptera littoralis which is correlated with the up-regulation of jasmonic acid-responsive genes and to an accumulation of aliphatic glucosinolates [S. littoralis [cml37 Knock-Out mutant exhibits an enhanced susceptibility to herbivory which is correlated to a lower level of the bioactive form of jasmonate  known to be crucial in plant defence coordination against insects [Carbivores ,105,106.inolates . In contttoralis . Indeed, insects . These d insects .2+ influx [Among the range of processes regulated by CaM/CMLs, CMLs are also involved in the suppression of post-transcriptional gene silencing (PTGS), a regulatory mechanism targeting mRNA content . Plant v+ influx .PSEUDO-RESPONSE REGULATOR 2), a plant specific transcription factor was also described to interact in planta with CML9 but not with the typical CaM [A. thaliana, PRR2 was shown to act as a positive regulator of plant immunity through SA-dependent responses [To date, most of the data describing the involvement of CMLs in different cellular processes associated with plant physiology remain descriptive and new research is now required to decipher the molecular mechanisms controlled by these CMLs. The identification of CML-interacting partners will be crucial to clarify how these CMLs exert their action at the molecular level in plant immunity. For example, CML9, previously described to positively regulate plant immunity, was reported to interact with transcription factors such as the WRKYs and the TGAs, two classes of transcription factors known to play key roles in the regulation of defence processes . PRR2  and the CML (SCaM-4) physically interact with MYB2, a TF that regulates the expression of salt- and dehydration-responsive genes in Arabidopsis [2+ in these interactions? Efforts are now made to answer these questions and to better understand the contribution of these sensors in Ca2+ signalling with a particular interest on plant specific CMLs.In many cases, the regulation of these TFs by CML9 or by other CMLs remain presumptive. The next challenge will be to elucidate if all CMLs really act as Cabidopsis . Howeverbidopsis . Althougbidopsis  but do t2+ sensors referred to as Ca2+ dependent protein kinases or CDPKs. Found only in green algae, land plants and unicellular protists, CDPKs are important Ca2+ decoders and relays in plant defence signalling against various types of pathogens  [2+ decoders since they combine into a single module Ca2+ sensing and downstream signal propagation capabilities. They are composed of a variable N-terminal part, a kinase domain and an activation domain. The activation domain contains an auto-inhibitory pseudo-substrate linked to a CaM-like domain typically containing four EF-hands [2+ binding by the CaM-like domain, a conformational change displaces the pseudo-substrate from the kinase to allow for downstream phosphorylation events. The dynamic range of Ca2+ concentrations that activate a given CDPK therefore depends on the CaM-like domain affinity for Ca2+. This likely accounts in part for the specific decoding of different Ca2+ variations. Of note, some CDPKs do not show strict Ca2+-dependent kinase activity. For example, Arabidopsis thaliana CPK7, 8, 13, and CPK30 showed more than half-maximal in vitro syntide-2 phosphorylation activity at very low Ca2+ concentrations (1 to 10 nM), with little to no additional kinase activity at higher Ca2+ concentrations [2+-independence in vivo or specific Ca2+ requirements depending on substrates remains to be clarified [Beside CaMs and CMLs, plants also possess another class of Ca review: ). Their ersicum) . The famersicum) ,118. In EF-hands ,120. Upotrations ,122. Whelarified .Arabidopsis CPK4, 5, 6 and 11 positively regulate defence gene expression upon bacterial flagellin perception [Pseudomonas syringae [PDF1.2 defence gene induction after wounding by the caterpillar Sodoptera littoralis [Magnaporthe oryzae [Functional analyses in planta revealed that CDPKs are important components of the plant immune system. Acting alongside and in synergy with the Mitogen Activated Protein Kinases (MAPKs)-dependent signalling cascade, rception . CPK4, 5syringae . CPK5, bsyringae ,123. A msyringae . Throughttoralis . These pe oryzae . This ene oryzae .Nicotiana sp. showed that CDPK2 and CDPK3 are required for the PCD response triggered after perception of the Cladosporium fulvum race-specific Avr4 or Avr9 elicitors [Arabidopsis with the demonstration that CPK1 and 2 control the onset of HR upon challenge with avirulent Pseudomonas syringae and subsequent NLR-mediated effector recognition [2+ inputs to be in an active state. Expression of CPK5-VK in Arabidopsis leaf protoplasts leads to cell death and this requires kinase activity [Phytophthora infestans. As a trade-off however, plants became more susceptible to the necrotrophic fungus Alternaria solani [per se to provoke PCD since not all CDPKs tested under their auto-active configuration induce PCD [In addition to the control of defence gene induction, another important aspect of CDPK function is the control of plant cell death in response to pathogens. Early work on CDPKs in licitors . This piognition . In lineactivity . Similaractivity . This laa solani . This coa solani ,129. Actduce PCD ,129. Theduce PCD , it is tduce PCD . The exicdpk mutants remain necessary tools and high-order cdpk mutants are often required to observe a phenotype of interest such as pathogen susceptibility [exo70B1 autoimmune phenotype uncovered several cpk5 loss-of-function alleles [exo70B1-mediated cell death and enhanced resistance to Golovinomyces cichoracearum phenotypes were lost in the absence of a functional CPK5, but not of CPK4, 6, or 11 [exo70B1-mediated autoimmunity also requires the atypical truncated NLR resistance gene TN2 [Arabidopsis, remain open questions.As already, mentioned, functional redundancy can be problematic when studying particular gene functions within a large family. Although gain-of-function auto-active CDPKs proved of immense value in this context, tibility ,127. In  alleles . The exo6, or 11 . This adgene TN2 . A tripagene TN2 . WhetherMagnaporthe grisae strains [B. graminis f. sp. hordei) [Arabidopsis, perception of several PAMPs by their cognate receptors requires the activity of Somatic Embryogenesis Receptor Kinases 3 (SERK3), a plasma membrane localized receptor and co-regulator also referred to as BAK1 (BRI1-Associated Kinase 1) [bak1–5 loss-of-function allele [cpk28 mutants. The loss of CPK28 reverted the impaired ROS production phenotype observed in bak1–5 after PAMP treatments [2+ influx resulting from PRR activation first participates in ROS production through CPK5 activity, before CPK28 dampens immune outputs. In line with this hypothesis, in vitro assays determined that CPK28 is indeed Ca2+ responsive [2+/CaM, however, this interaction negatively affects its kinase activity in vitro [2+ therefore seems to have a dual role in regulating CPK28 activity, thereby, adding another layer of complexity that has yet to be resolved.In addition to these positive roles, CDPKs also perform negative functions in plant defence. For example, overexpression of OsCPK12 increased rice susceptibility to avirulent and virulent  strains  while in hordei) . In Arabinase 1) . In a fon allele , Monaghaeatments . CPK28 beatments . The seaeatments . It is tsponsive . Interesin vitro . Ca2+ th2+-binding proteins whose functions in plant physiology start to be unravelled. Far from being exhaustive, we have summarized here some examples that describe the importance of certain CaM/CMLs and CDPKs in plant immunity [2+-regulated events.Globally, plants have a wide repertoire of Caimmunity ,18. The 2+, Ca2+ sensors and their targets are positioned among the first actors setting up the plant response to biotic interactions. One of the challenges of the coming years will be to understand and expect plant behaviour in a changing environment, with the long-term goal to breed stress-tolerant crops for agriculture. However, although Ca2+ signals are the very first step in the signaling pathway, it appears that their manipulation to improve plant resistance could be very challenging. Indeed, calcium signals result from the intricate interaction between pathways and components involved in both their generation and their dissipation such as calcium channels, exchangers or calcium pumps [Increasing data support that Caum pumps . Neverthum pumps ,141 or tum pumps ,143. Suc2+ sensors or their targets that decode Ca2+ messages. To date, studies about CaM/CML and CDPKs focus mainly on their interaction with plant target proteins. However, recent and promising data indicate that Ca2+ signalling components can be themselves the direct targets of pathogen effectors. In the context of plant immunity, studies on these specifically targeted plant calcium sensors by pathogen effectors might provide a new way for breeding plant resistance. Indeed, it was recently demonstrated that plant CaM is required by the HopE1 effector from Pseudomonas syringae to target MAP65, a microtubule-associated protein, to reduce plant immune responses [Xanthomonas AvrBsT effector, able to acetylate ACIP1, a microtubule-associated protein in Arabidopsis and required for both PTI and ETI, possesses a CaM-binding region. AvrBsT is the first member of the YopJ family known to suppress effector-triggered plant immunity [2+-dependent manner and that a mutation of the AvrBsT CaM-binding domain alters or delays the hypersensitive response phenotype . Whereaing 2016 ; these n2+ signalling and biotic stress responses. We point out that CMLs and CDPKs can exhibit a dual function as either positive and/or negative regulators in plant biotic stresses. Moreover, many other data highlight the contribution of Ca2+ and Ca2+ decoding processes following abiotic stress perception [2+, Ca2+ sensors and their respective targets can be at the crossroads of various signalling pathways and that they could be good candidates to act as central integrators involved in the fine tuning of plant physiological responses to pathogens under fluctuating environmental conditions. As stated in the introduction, it is obvious that Ca2+ signalling is not acting as a unique contributor in plant stress responses and we cannot rule out crosstalk between Ca2+ and ROS, as well as other second messengers. The next mid and long-term challenges should better characterize the interplay between these signalling pathways.In this review, we focused on Carception ,63,148. rception . Therefo