In vivo models of cardiac function maintain the complex relationship of cardiomyocytes with other heart cells, as well as the paracrine and mechanoelectrical feedback mechanisms. We aimed at imaging calcium transients simultaneously with heart contraction in zebrafish larvae.Methods: To image calcium in beating hearts, we generated a zebrafish transgenic line expressing the FRET-based ratiometric biosensor Twitch-4. Since emission ratioing canceled out the motion artifacts, we did not use myosin inhibitors or tnnt2a morpholinos to uncouple contraction from changes in calcium levels. We wrote an analysis program to automatically calculate kinetic parameters of the calcium transients. In addition, the ventricular diameter was determined in the fluorescence images providing a real-time measurement of contraction correlated with calcium.Results: Expression of Twitch-4 did not affect the force of contraction, the size of the heart nor the heart rate in 3- and 5-days post-fertilization (dpf) larvae. Comparison of 3 and 5 dpf larvae showed that calcium levels and transient amplitude were larger at 5 dpf, but the fractional shortening did not change. To validate the model, we evaluated the effect of drugs with known effects on cardiomyocytes. Calcium levels and the force of contraction decreased by the L-type calcium channel blocker nifedipine, whereas they increased with the activator Bay-K 8644. Caffeine induced bradycardia, markedly decreased ventricular diastolic calcium levels, increased the size of the calcium transients, and caused an escape rhythm in some larvae.Conclusions: The Tg(myl7:Twitch-4) line provides a physiological approach to image systolic and diastolic calcium levels in the heart of zebrafish larvae. Since the heart is beating, calcium levels and contraction can be correlated. This line will be a useful tool to address pathophysiological mechanisms in diseases like heart failure and arrhythmia, in cardiotoxicity studies and for drug screening. This influx elicits Ca2+ release from the sarcoplasmic reticulum (SR) through ryanodine receptors (RyR). The binding of Ca2+ to myofilaments results in the systolic contraction and ejection of blood. The amplitude and duration of the Ca2+ transient is highly controlled by channels, transporters, and numerous regulatory proteins, and determines the strength of the contraction and overall cardiac performance 2+. Systemic factors like blood volume and peripheral resistance, affecting preload and afterload, also influence the cardiac output.In the heart, electrical excitation triggers a transient Ca2+ dysregulation in the heart is involved in the pathogenesis of inherited and acquired arrhythmias 2+ can occur through several mechanisms: decreased Ca2+ uptake by SR Ca2+ ATPase (SERCA), increased Ca2+ leak from the SR and increased activity of the Na+/ Ca2+ exchanger (NCX1), all reducing SR Ca2+ content 2+ transient and systolic dysfunction 2+ sensitizers (as opposed to Ca2+ mobilizers) as a therapeutic approach to treat congestive heart failure sometimes go beyond their inotropism, acting on other organs with additional benefits for the patients 2+ levels with force is key to evaluate cardiac contractile dysfunction, reserve capacity and the efficiency of therapies. In turn, relaxation and ventricular filling depend on the rate of Ca2+ decay, the sensitivity of myofilaments to Ca2+, the passive mechanical properties of the ventricular walls and the atrio-ventricular pressure difference. Accordingly, altered regulation of diastolic and/or resting Ca2+ seems to be an important factor involved in heart failure with preserved ejection fraction (diastolic dysfunction) 2+, as most methods to measure Ca2+ rely on the fluorescence change in systole relative to diastole. A second reason is that many studies were performed at artificially low pacing rate, when only sarcolemmal fluxes influence diastolic and resting Ca2+Ca2+ in the heart, numerous studies mostly in isolated cardiomyocytes have provided a detailed insight of the Ca2+ handling mechanisms in physiology In vivo models do not allow investigating cellular mechanisms with such precision but preserve the interaction between cardiomyocytes and other cell types in the heart, maintaining their paracrine regulation in vivo models of heart function are complementary to studies in the isolated perfused organ and in cells.Given the pivotal role of Cain vivo animal models in cardiology 2+ release from the SR to contraction in adult zebrafish cardiomyocytes is controversial. Since the ICa,L current density was found to be about 5-fold larger than in pig or human cardiomyocytes 2+ transient is probably less than in mammalians. In another study, only 20% of the action potential-induced Ca2+ transient was mediated by release from SR 2+ release in force generation Zebrafish embryos, larvae and adults are widely used as in vivo disease gene candidates and to unveil the link between genotype and phenotype, for instance, in post-GWAS functional studies in vivo model in early drug discovery and in cardiotoxicity studies since it is amenable to high throughput screening Many cardiomyopathies and arrhythmias have a genetic base. The zebrafish genome has 70% similarity to human 2+ imaging techniques in the heart under physiological conditions and pathological modelling. This approach involves the expression of a genetically encoded Ca2+ indicator (GECI), like the single-fluorophore GCaMPs 2+ dynamics in embryonic hearts 2+ transients, an approach not devoid of limitations due to its photosensitivity and degradation. Importantly, the inhibition of contraction prevents the study of the mechanical parameters outlined above and may potentially affect heart physiology and development. We reasoned that a ratiometric Ca2+ biosensor, by acquiring two wavelength bands simultaneously, could allow assessment of Ca2+ levels in moving hearts, since the ratio is able to cancel out what is common in the two images (motion) while preserving what is different (Ca2+) 2+ levels with motion and contraction during the cardiac cycle, preserving mechano-electrical feedback Zebrafish are advantageous to image cardiac function with optical probes, as the heart develops rapidly while the larva is still translucent 2+ biosensors with different affinity and kinetic properties to validate their use in the zebrafish larva heart in transient expression experiments Opsanus troponin-C-derived biosensor, provided the largest ratio change during the cardiac cycle with good signal-to-noise. In this work, we have established a transgenic line, Tg(myl7:Twitch-4), to characterize the Ca2+ transients and contraction in beating hearts. We validated this model by testing drugs with known effects in the heart at two developmental stages, 3 and 5 dpf.In an earlier report, we screened several ratiometric Ca4, 0.33 mM CaCl2, pH 7.4 in double distilled water) at 28.5 ºC. No methylene blue was added to E3 medium to decrease larva autofluorescence. All animal procedures were carried out in compliance with national and EU regulations .Zebrafish were kept in the Center for Animal Experimentation of the Albacete School of Medicine with a light/dark cycle of 14/10 h. Fertilized zebrafish eggs were obtained following standard procedures and maintained in E3 medium . Injected embryos (F0) were screened by fluorescence in the heart and grown to adulthood. Adult F0 fish were outcrossed to wild-type AB zebrafish to identify founders with insertions in the germline by the cardiac fluorescence in the F1 offspring. To generate the Tg(myl7:Twitch-4) line, fluorescent F1 fish were outcrossed to wild-type AB zebrafish, and F2 embryos expressing Twitch-4 were raised to adulthood and intercrossed to generate the F3 generation. Adult F3 Tg(myl7:Twitch-4) fish were crossed with adult translucent Casper zebrafish  Tg(myl7:ECFP-16aa-EYFP) and F4 Tg(myl7:Twitch-4) heterozygous larvae were used for imaging.The CaNon-anesthetized larvae were embedded in 100 µL of 0.3% low melting point agarose in E3 medium, preheated to 42 ºC, and gelled on 96-well plates with square wells and flat clear bottom  (one larva per well). Larvae at 3 dpf were mounted ventral-side down and 5 dpf larvae right-side down  larvae, and 3 and 5 dpf Tg(myl7:Twitch-4) larvae with a wide-field fluorescence microscope  equipped with a sCMOS camera , controlled by the software Aquacosmos 2.6 . The image acquisition rate was 50 Hz (20 ms integration per image) during 5-10 s; some larvae were imaged at 100 Hz. Larvae under the microscope stage were kept at 28ºC in a chamber incubator . To excite the fluorophores, light from a LED source  was applied continuously for 5-10 s using a 440AF21 nm bandpass filter  and a beamsplitter 455DLRP . A 10x air objective  was used. The donor (CFP) and acceptor (FRET) images were acquired simultaneously with an image splitter , which divided the camera field in two halves corresponding to donor and acceptor emission. Fluorescence emission was separated with beamsplitter  and passed through 483/32 nm and 542/27 nm emission filters . Images were acquired in 16 bits with 2 × 2 binning. With this configuration, the resolution of the images was 1.45 µm × 1.45 µm/pixel. The FRET image corresponds to the cpCitrine174 or EYFP emission (542/27 nm) at the donor excitation (440AF21 nm), and the donor image corresponds to the ECFP emission (483/32 nm) at the donor excitation (440AF21 nm).We acquired fluorescence images of the heart of 3 dpf For drug response experiments, stock solutions of nifedipine (Sigma-Aldrich N7634), (±)-Bay K8644 (Tocris 1544) and ryanodine (Tocris 1329) were made in dimethyl sulfoxide (DMSO) at concentrations of 10, 20 and 25 mM, respectively. A stock solution of caffeine (Sigma-Aldrich N7634) was made in water at a concentration of 50 mM. Drug stocks were diluted in E3 medium at 28 ºC. After recording the basal images, 100 µL of the drugs were added to the wells, reaching a final concentration of 100 µM for nifedipine, (±)-Bay K 8644 and ryanodine, and 3 mM for caffeine. New sets of images were recorded after the incubation with the drugs, as indicated.FRET image/donor image was calculated pixel-by-pixel for each time point. Regions of interest (ROI) were drawn over the atrium and the ventricle walls in diastole. The ratio value for a ROI was calculated by averaging all the pixels' values weighted by the average intensity of donor and FRET channels 2+ rise), decay time (time from 90 to 10% of diastolic Ca2+ decay), rise slope  and decay slope  , we wrote an analysis program in IGOR Pro that automatically detects contraction events  written in the IGOR Pro environment . We corrected for pixel shift between FRET and donor images. The ratio n events  and recoTransmitted light images were analyzed manually in ImageJ s878Tg(myl7:GCaMP) adult zebrafish were outcrossed to wild-type strain and fertilized eggs at 1-cell stage were injected with 2 ng of the morpholino oligomer tnnt2a (5'-CATGTTTGCTCTGATCTGACACGCA-3'). Embryos from 24 hours post-fertilization (hpf) were maintained in 0.003% N-phenylthiourea to prevent pigmentation. Larvae were embedded in 1% low melting point agarose and transferred to an 8-well glass bottom plate . Fluorescence images were acquired at a rate of 200 Hz with a CSU X1 spinning disc confocal microscope  equipped with a Hamamatsu ORCA Flash4.0 sCMOS camera  in 16 bits with 2 x 2 binning. For image analysis, ROIs were drawn in the atrium and in the ventricle to obtain mean intensity values. An exponentially weighted moving average smoothing with a smoothing factor of 0.7 was applied and data was transformed into ΔF/F0 = (Ft - F0)/F0; where Ft is the fluorescence at a given time and F0 is the minimum diastolic fluorescence value. For characterization of the Ca2+ transients, ΔF/F0 data were analyzed with Clampfit 10.7  to determine rise time 10% to 90% and decay time 90% to 10%.Tg(myl7:GFP-Aequorin) was used to compare averaged Ca2+ in 3 and 5 dpf larvae with a luminescence-based method described in Vicente et al The zebrafish line Tg(myl7:Twitch-4) larvae at 3 dpf were euthanized by incubation in 0.3% MS-222 (Sigma-Aldrich A5040) for 5 min. Larvae were embedded in agarose as done for ratiometric imaging and were imaged in an inverted Axio Observer LSM710 confocal microscope  with a PlanApo 20x/0.8 NA objective. Laser excitation was at 488 nm and the emission bandwidth was 520-560 nm.Statistical analysis was done with GraphPad Prism 8  and Igor Pro . The number of independent experiments  and the number of larvae (n) are indicated in each figure. The Shapiro-Wilk test was used to test for normality. Differences between two groups were analyzed using the unpaired Student's t-test for parametric data or the Mann-Whitney test for non-parametric data, as indicated. Comparisons between experimental groups were analyzed by one-way repeated measures ANOVA with Dunnett's multiple comparisons post-test for parametric data or the Friedman test with Dunn's multiple comparisons post-test for non-parametric data, as indicated. Data are shown as the mean ± SD. A p < 0.05 was considered statistically significant and significances are indicated as * for p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns p > 0.05. The Supplementary Data 1 file shows the details of the statistical analysis used throughout.2+ biosensor Twitch-4 belongs to a family of GECIs based on the C-terminal domain of the toadfish Opsanus tau troponin C as the Ca2+-binding domain, with a Kd of 2.8 µM and a decay time constant of 0.5 s determined in vitromyl7 promoter, Tg(myl7:Twitch-4). This promoter drives specific expression in myocardial cells of the atrium, ventricle, and the atrioventricular canal, excluding endocardial and epicardial cells Tg(myl7:Twitch-4) larvae confirmed the expected cytoplasmic localization of the biosensor  larvae, measured by transmitted light, were similar to those of their non-expressing siblings  larvae acquiring simultaneously the fluorescence of the donor ECFP and FRET channels . The fluorescence intensity in both emission channels showed fluctuations, which were positively correlated, increasing during systole, and decreasing during diastole ; in the donor channel the heart movement increased the fluorescence, but high Ca2+ (high FRET) decreased it. Therefore, the relative fluorescence change was larger in the FRET channel than in the donor channel. Ratioing these channels (FRET/donor) cancelled out the effect of motion and augmented the effect of the Ca2+ rise . The ratiometric images and the ratio traces were generated with an intensity weighted method. Thus, pixel average ratios were computed without the need to set intensity thresholds 2+ transients: diastolic and systolic ratio, amplitude (∆R), as well as rise and decay times and slopes  larvae was indeed reporting cardiac Ca2+ fluctuations and to what extent it was affected by motion of the heart. We generated a zebrafish transgenic line that expresses in the heart a FRET construct with no Ca2+-binding domain, composed of an ECFP and an EYFP joined by a flexible linker of 16 amino acid residues Tg(myl7:ECFP-16aa-EYFP) all the fluorescence fluctuations must be entirely due to motion and therefore this artifact should be cancelled in the emission ratio. We observed a small periodic ratio change in Tg(myl7:ECFP-16aa-EYFP) compared to Tg(myl7:Twitch-4)  Figure  and it a) Figure  it was nTg(myl7:Twitch-4) line to characterize cardiac Ca2+ kinetics at two stages of development, 3 and 5 dpf. The ratio levels and amplitude were higher at 5 dpf than at 3 dpf  zebrafish line that we have generated 2+ levels, was higher in the ventricle of 5 dpf larvae than in 3 dpf larvae  was performed on moving hearts, we measured simultaneously ventricular Ca2+, the ventricular diameter and the FS, as a proxy of the contractility. The same FRET channel images used to measure Ca2+ were used to monitor continuously changes in ventricle shape. To this end, we wrote an analysis program that automatically tracks the ventricular wall in the fluorescence images to construct kymograms and obtain the diameter over time (see Methods). Thus, we obtained a continuous recording of ventricle diameter . However, there is some uncertainty in these values due to the kinetics of Twitch-4 (see Discussion). Plotting the FS vs. Ca2+ (Ca2+-contraction curves) showed a loop with a contraction limb and a relaxation limb  line to detect changes in Ca2+ levels we tested the effects of a blocker and an activator of LTCC on Ca2+ kinetics and heart contractility. Since drugs added to the bath have to diffuse through the agarose layer and the skin to reach the internal medium, nifedipine was tested at 1, 10 and 100 µM. It decreased the HR and Ca2+ levels at all doses and we compared the effect of 100 µM nifedipine at 3 and 5 dpf. The atrial and ventricular Ca2+ levels and the HR decreased, particularly at 5 dpf, and the amplitude of the Ca2+ transients decreased more in atrium than in ventricle  larvae  larvae to the inhibition of RyR by incubation with 1, 10 and 100 µM ryanodine. Effects were observed only with 100 µM and were maximal at 2 h. The amplitude of the Ca2+ transients decreased in the ventricle of 3 and 5 dpf larvae compared to larvae treated with DMSO for 2 h , or even it started earlier in the ventricle  and 2 out of 11 (5 dpf) treated with 3 mM caffeine, the normal atrioventricular direction of excitation was altered Figure . The Ca22+ levels in the heart 2+ dynamics parameters in zebrafish larvae stably expressing the GECI Twitch-4.Detailed electrophysiological and mechanobiological studies have been reported in cardiomyocytes mostly derived from rodents and from human induced pluripotent stem cells 2+ concentration changes. The kinetics of GECIs is slower than that of synthetic Ca2+ indicators 2+ affinity and kinetics. Thus, biosensors with decreasing Ca2+ affinity  showed decay time constants of 2.8, 1.5, 0.5 and 0.16 s, respectively 2+ affinity (Kd of 2.8 µM) to minimize kinetics dampening.The response kinetics of a biosensor is essential to analyze dynamic Cain vitro kinetics is difficult to compare between Ca2+ biosensors because measurements in the literature are done in different experimental conditions, like ionic strength and temperature. In addition, the cell milieu differs considerably from the in vitro conditions. The kon of Twitch-4 can be calculated from the published Kd and koff. It is 7 × 105 M-1 s-1, compared to ~107 M-1 s-1 for various GCaMP variants that are considered fast GECIs. The koff of Twitch-4 and GCaMPs is more similar  in vitro parameters, GCaMP fluorescence should report the start of the Ca2+ transient more accurately than Twitch-4 emission ratio. Yet, we show here that Twitch-4 was able to track systolic Ca2+ transients like GCaMP, a widely used biosensor in zebrafish heart studies. Figure in vitro at a lower temperature.The Kd of Twitch-4 determined in vitro (2.8 µM) 2+ levels , so no inference can be made on the diastolic levels across experiments or larvae. It has been argued that this is a reason why relatively little is known about the regulation of diastolic Ca2+ levels in the heart 2+ levels were seen not only during the cardiac cycle or after addition of drugs, but also among larvae. The ratio changes were suggestive of different Ca2+ levels at 3 and 5 dpf, and we confirmed this contention with an independent method based on GFP-aequorin bioluminescence The  Figures  and 6. W2+ changes in beating hearts. These results agree with Tsutsui et al. 2+-dependent changes 2+ with static ROIs, in spite of the lateral movement of the atrium and ventricle in the focal plane and to a lesser extent in the z axis. The contribution of each pixel to the average ratio was weighted by its fluorescence intensity Within the ample toolbox of GECIs tnnt2a morpholino  and its mechanical outcome were gathered in real time at 50 or 100 Hz (fps), analysis of their phase-plane trajectories (Lissajous diagrams) provided a visual snapshot of the changes occurring during development , 0.07 for theophylline, 1- to 2-fold for verapamil and terfenadine, and yet 20-fold for cisapride and haloperidol, highlighting the importance of bioanalysis to interpret results, especially if effects are not observed A limitation in our study and similar reports in zebrafish is that the internal concentration in larvae of the drugs added to the bath is generally unknown. Often, higher concentrations must be used to see effects, compared to 2+ leak of the SR by sensitizing the RyR to the luminal Ca2+ level in vivo, it may act on several targets. The major effects observed with caffeine were a marked decrease in HR, reduced diastolic Ca2+ levels and an increase in the size of the Ca2+ transients , due to inactivation of ICa,L by Ca2+, since it was associated with an increase in the diastolic Ca2+ levels , and the amplitude of the Ca2+ transients increased .Caffeine is known to increase the Cas Figure . These ed Figure . We havein vivo has a complex pharmacology. In the literature there are reports of bradycardia induced by caffeine in humans Caffeine f current) 2+ release from SR that triggers an inward current through the NCX1. The loss of this current specifically in the atrium in KO mice abolished the sinus pacemaker activity and caused an idioventricular escape rhythm, although atrial tissue remained excitable 2+ from the SR of sinoatrial cells in these larvae by caffeine could eliminate the local Ca2+ release, the ensuing inward current through the NCX and sinus pacemaker activity. The characterization of caffeine effects on Ca2+ levels  line offers a physiological, non-invasive approach to image Ca2+ in the heart of zebrafish embryos and larvae, allowing estimation of systolic and diastolic Ca2+ levels, while keeping intact the electromechanical and paracrine regulatory mechanisms. Measurement of changes in contractility is critical for appraisal of cardiac function and this method affords simultaneous registration and correlation of Ca2+ and contraction. In the last years several models of heart failure and cardiomyopathy have been reported in zebrafish, and gene-editing by CRISPR-Cas9 allows to humanize zebrafish models by introducing the normal or mutated human orthologue genes 2+ fluxes are involved and their impact on heart contractility. In addition, cardiotoxicity studies and screening for new drugs could be performed. The applicability of this approach for drug screening is currently limited by the handling and positioning of embryos in agarose in multi-well plates. Automation of this step in vivo data from zebrafish models with the extensive literature in cardiomyocytes and isolated hearts will likely contribute to unravel the mechanisms of heart disease progression and to identify therapeutical targets.The Supplementary figures and movie legends.Click here for additional data file.Supplementary information.Click here for additional data file.Supplementary movie 1.Click here for additional data file.Supplementary movie 2.Click here for additional data file.Supplementary movie 3.Click here for additional data file.Supplementary movie 4.Click here for additional data file.Supplementary movie 5.Click here for additional data file.


RyR2), the sarcoplasmic reticulum (SR) Ca2+ release channel in cardiomyocytes. Pathogenetic mutations lead to gain of function in the channel, causing arrhythmias by promoting diastolic spontaneous Ca2+ release (SCR) from the SR and delayed afterdepolarizations. While the study of Ca2+ dynamics in single cells from murine CPVT models has increased our understanding of the disease pathogenesis, questions remain on the mechanisms triggering the lethal arrhythmias at tissue level. Here, we combined subcellular analysis of Ca2+ signals in isolated cardiomyocytes and in acute thick ventricular slices of RyR2R2474S knock-in mice, electrically paced at different rates (1–5 Hz), to identify arrhythmogenic Ca2+ dynamics, from the sub- to the multicellular perspective. In both models, RyR2R2474S cardiomyocytes had increased propensity to develop SCR upon adrenergic stimulation, which manifested, in the slices, with Ca2+ alternans and synchronous Ca2+ release events in neighboring cardiomyocytes. Analysis of Ca2+ dynamics in multiple cells in the tissue suggests that SCRs beget SCRs in contiguous cells, overcoming the protective electrotonic myocardial coupling, and potentially generating arrhythmia triggering foci. We suggest that intercellular interactions may underscore arrhythmic propensity in CPVT hearts with ‘leaky’ RyR2.Catecholaminergic Polymorphic Ventricular Tachycardia (CPVT) is a familial stress-induced arrhythmia syndrome, mostly caused by mutations in Ryanodine receptor 2 ( Our analyses showed that myocardial slices retained regular cell organization, with intact and morphologically normal cardiac sarcomeres, with the only exception of the cells close to the sectioning surface , during electrical pacing. After 3 min, while the drug diffused into the tissue layers, Ca2+ transient amplitude increased  events, between two consecutive paced transients  vs. WT: 12 (3/24), in %; 6 independent preparations each) . When the pacing rate was increased to 2 Hz, predominantly in the ns each) A. Ca2+ aansients C,D, althRyR2, we focused on the investigation of aberrant diastolic Ca2+ release from the intracellular stores. To this aim, we used a pace-stop protocol in which, after a brief period (5–10 s) of electrical pacing at 2 Hz, pacing was paused, and [Ca2+] declined to levels of unpaced cells  and 25 (WT) cells; p < 0.05), and the number of cells within the slice showing aberrant activity was markedly increased . NE (1 μM) administration decreased the latent period to the generation of SCR in both CPVT and control hearts , possibly reflecting the increased open probability of RyR2RS/wt channels upon phosphorylation.Given the mechanistic link between altered single-channel properties of CPVT mutant ed cells A. After nd waves ,31. In kl hearts C. The in2+ release to be higher in cells contacting a “source” CM, “sinking” the Ca2+-dependent depolarizing current  at the expense of a slight loss of membrane polarity. However, within the average duration of the transient diastolic Ca2+ elevation, occurring in a given cell , the probability that a neighboring one would develop, in turn, secondary diastolic Ca2+ leak was >80%, much higher than the calculated probability of two independent events with the typical frequency of 0.4/s (average SCR/s/cell for any given cell in the slice) to occur. To further analyze such behavior, we calculated the average latency time, after pacing cessation evoked the first Ca2+ release in one of the cells in the field of view, for neighboring cells to develop secondary SCR events. As shown in (n = 16 cells per group). This data suggests that reciprocal influence between neighboring CMs might contribute to the synchronization of diastolic Ca2+ release events within a cluster of neighboring cells in the same tissue region, as shown in the representative plot of Ca2+ fluctuations in regions of interest, corresponding to different cells in the field of view  in young adolescents and athletes. It is well accepted that alterations in intracellular Ca2+ handling, due to functional abnormalities of mutant RyR2 channels, have a role in triggering arrhythmias in CPVT hearts, and Ca2+-dependent delayed after-depolarizations (DAD) have consistently and causally been linked to triggering arrhythmias in various murine models, harboring different CPVT-linked mutations  load, mechanisms not yet uncovered may promote spatial  and temporal synchronization throughout several cells in the tissue to act as effective arrhythmia triggers. Although the previous study by Cerrone and colleagues [2+ release, DADs, and spontaneous APs in response to catecholaminergic stimulation, which makes them equally suited candidates to initiate ventricular arrhythmias in CPVT hearts [In perfect agreement with data obtained in isolated CMs, diastolic Cas et al. , Ca2+ alr groups . It has iggering , and theposed by . While, lleagues  identifiT hearts ,43.2+ load-driven diastolic leak and DAD generation in localized clusters of cells. It is tempting to speculate that the sympathetic network topology may add to the local mechanisms synchronizing triggered activity within a specific myocardial region. We previously demonstrated that sympathetic neurons activate CM β-ARs by releasing NE in a restricted signaling domain confined within the limits of the structured contact site between neuronal varicosity and the coupled CM membrane . In addi2+ waves in a CM were followed within a short time period by Ca2+ waves in neighboring cells. The temporal distance between these events usually fell under a “coupling” interval equal to the mean duration of a traveling wave (around 900 ms). Ca2+ waves can readily travel through the whole cell; however, in multicellular preparations, they generally do not easily propagate through gap junctions from one cell to the next one [2+ diffusion as the main cause of SCRs synchronization. Another study on papillary muscles from a CPVT-associated CASQ2 mutated mouse model suggested the decreased RyR2 refractoriness as a determinant of synchronization between neighboring CMs [RyR2-linked CPVT model [Hence, our finding adds to the existing literature the concept that regenerative SCRs in a single CM may directly and adversely engage the surrounding electrically excitable cells. In the heart slices, we observed that, in a large number of cases, Canext one ,47, exclring CMs . SimilarVT model .2+-activated DADs, the depolarizing current would spread through the whole myocardium if not sufficiently sunk by the surrounding CMs. The current sink is paid with the slight, below-threshold depolarization, which increases SR Ca2+ release and facilitates cell-wide slow Ca2+ wave propagation [Although apparently in contrast with the “source–sink” model for electrotonically coupled CMs, our observation can be explained by considering that, when “source” CMs develop Capagation . The comRyR2 patient mutation, associated with increased channel leakiness, arrhythmogenic Ca2+ waves occur from self-amplifying alterations in local control of Ca2+ dynamics. The same behavior is appreciated in the multicellular myocardial slice model, in which complex multifactorial mechanisms are poised to increase arrhythmogenic Ca2+ wave risk.The results of the current work show that, in neighboring CMs from KI mice harboring a CPVT-linked Some of the results presented in the manuscript stem from the PhD thesis of Dr Giulia Borile.


Nevertheless, the restoration of blood flow to ischemic tissue leads to further damage, namely, myocardial ischemia/reperfusion injury (MIRI). Apoptosis is an essential pathogenic factor in MIRI, and ginsenosides are effective in inhibiting apoptosis and alleviating MIRI. Here, we reviewed published studies on the anti-apoptotic effects of ginsenosides and their mechanisms of action in improving MIRI. Each ginsenoside can regulate multiple pathways to protect the myocardium. Overall, the involved apoptotic pathways include the death receptor signaling pathway, mitochondria signaling pathway, PI3K/Akt signaling pathway, NF-κB signaling pathway, and MAPK signaling pathway. Ginsenosides, with diverse chemical structures, regulate different apoptotic pathways to relieve MIRI. Summarizing the effects and mechanisms of ginsenosides contributes to further mechanism research studies and structure–function relationship research studies, which can help the development of new drugs. Therefore, we expect that this review will highlight the importance of ginsenosides in improving MIRI  Ischemic heart disease (IHD) is characterized by insufficient blood flow to the cardiac tissue . In 2019Panax ginseng  were searched from inception to 21 September 2021 by using the following terms: ginsenoside, myocardial reperfusion injury, etc. This research included and reviewed research studies addressing the anti-apoptosis effects of ginsenosides on MIRI to provide references and suggestions for further research in this field.Myocardial ischemia (MI) is a complex pathological condition resulting from initial restriction of blood supply to the heart , which cCell death occurs widely during pathological processes in multiple diseases and is one of the leading causes of death . ApoptosPanax ginseng, a medicinal plant, belongs to the Araliaceae family and has a long history of usage  . PublishMitochondria play a significant role in adjusting metabolism, generating ROS, and guaranteeing cell activity . Bcl-2 iThe PI3K/Akt signaling pathway is an important pathway by which ginsenosides improve apoptosis. The activation of this pathway promotes angiogenesis, alleviates tissue hypoxia, suppresses cell damage, and improves MC apoptosis . EvidencNF-κB belongs to a family of related transcription factors and participates in the regulation of immune responses, proinflammatory cytokines’ control, and cell death . The RelMAPK is an important signal transducing enzyme that has effects on the regulation of gene expression, cell proliferation, and cell death . MAPK kiIn addition to the above-mentioned apoptotic signaling pathway, other pathways have been reported in previous studies. Li et al. reported that ginsenoside Rb1 improved MIRI by preserving PDH activity and inhibiting SDH activity . Ginsenoin vivo studies and clinical studies are needed.MIRI is functional and organic damage to the heart, which results from restoration of blood flow in ischemic areas . Throughex vivo experiments or animal experiments. Rare clinical evidence has showed that ginsenoside Rb has protective effects on MIRI in patients undergoing mitral valve surgery . Existing studies are in infancy, and more clinical research studies are needed to be designed and conducted to supply further clinical evidence. Thirdly, evidence showed that pyroptosis occurred during the development of MIRI (Currently, multiple studies have explored the anti-apoptotic mechanism of ginsenosides; however, problems still exist, and further studies are needed. Firstly, the research studies about the structure–function relationship of ginsenosides in inhibiting MIRI are still needed to be conducted. The hydroxyl groups and sugar moieties have influences on the pharmacological effects of ginsenosides, which can interact with membrane lipids . Thus, t of MIRI , and pro of MIRI . HoweverOverall, this review of anti-apoptotic mechanisms of ginsenoside in MIRI presents pharmacological mechanisms and lays the foundation for further research studies, hoping to contribute to the development of undiscovered mechanism and new drugs.


Encephalitozoon cuniculi and the apicomplexan protozoa Toxoplasma gondii. Both infections are mainly latent in rabbits but may cause severe or even fatal disease. Although several international studies have reported the seroprevalence of these pathogens in different commercial rabbit populations, similar prevalence studies and risk-factor analyses among family-owned pet rabbits are uncommon and lacking in Scandinavia. We sought to estimate the seroprevalence and possible risk factors for E. cuniculi and T. gondii among Finnish pet rabbits. We used ELISA to measure E. cuniculi IgG seroprevalence of 247 rabbits and modified direct agglutination test for T. gondii seroprevalence of 270 rabbits. Samples were collected as part of the Finnish Pet Rabbit Health Research project. Internet-based questionnaires (n = 231) completed by the rabbit owners were used for risk-factor analysis.Neurological signs, such as head tilt, torticollis, paralysis, and seizures, are common in rabbits. Differential diagnoses include two zoonotic infections caused by the microsporidial fungi E. cuniculi was 29.2% and true seroprevalence of T. gondii 3.9%. Risk factors were analysed only for E. cuniculi due to the low T. gondii seroprevalence. The final multivariable logistic regression model revealed that rabbits spending the whole summer outdoors had a higher risk of being E. cuniculi seropositive than rabbits with limited outdoor access. Additionally, rabbits living in households with only one or two rabbits had higher risk of being E. cuniculi seropositive than those in multi-rabbit households.The apparent seroprevalence of E. cuniculi IgG antibodies, indicating previous exposure to this pathogen. The prevalence is similar to that reported previously in clinically healthy rabbit populations in UK and Korea. While the seroprevalence of T. gondii was low (3.9%), antibodies were detected. Therefore, these zoonotic parasitic infections should be considered as differential diagnoses when treating rabbits.Nearly one third of Finnish pet rabbits participating in this study had  Encephalitozoon cuniculi and Toxoplasma gondii . Values > 2 were classified as positive for E. cuniculi antibodies.We used a commercial enzyme-linked immunosorbent assay  was used for screening of  studies , 35 and  studies , 24, 36. studies . Serum sData were imported from the electronic questionnaire into SPSS statistical program . Several independent variables were recategorized for statistical analyses. Rabbits were divided into two groups by age using 3 years as a cut-off point; in an earlier study we observed that rabbits > 3 years had more health problems than younger rabbits . Breed wMany variables initially had so many categories that only a few observations were made in some of the categories. Therefore, original categories were combined. We defined a household as a multi-rabbit household if it featured three or more rabbits and compared those to households with one or two rabbits. The origin of the rabbits was categorized to those from breeders and of other origin. The use of the rabbits was grouped for pet use and for other use. The living conditions were categorized into house rabbits and non-house rabbits. Access to outdoors was categorized to those spending the entire summer outdoors and to others (access to outdoors daily/weekly/monthly/few times during summer). The frequency of receiving wild plants, salads, vegetables, root vegetables, herbs, fruits, or twigs was divided into three categories  and for regression analyses into two categories  and salads also into ‘given ever or not’.T. gondii and E. cuniculi seropositivity was determined; for E. cuniculi in all variable groups  with all variables were calculated using Fisher’s exact test and unconditional logistic regression analyses (with only one variable in the model at a time). Those with P < 0.2 were entered into multivariable logistic regression. The model selection was performed by backward selection. Significance was considered at α = 0.05 level. Goodness of fit of the model was evaluated with Omnibus and Hosmer-Lemenshow tests. The model prediction was evaluated with percentage accuracy in classification, sensitivity and specificity, and discrimination with area under the curve (AUC) of the receiver operating characteristic (ROC) curve. All two-level interactions between the variables in the final model were tested. Multicollinearity between variables was estimated by Phi coefficient; the limit value for strong correlation was set at 0.5 [Preliminary associations between the outcome . Mixed-breed rabbits were the most common , followed by Dwarf Lops  . Seroprevalence values in different suspected risk-factor subgroups are presented in Table E. cuniculi seropositive than rabbits with limited daily/weekly/monthly/few times during summer access to outdoors. Rabbits in households with one or two rabbits also had four-fold greater odds of being E. cuniculi seropositive than those in multi-rabbit households (Table 2(4) = 20.3, P < 0.0005); the Hosmer-Lemenshow test was not statistically significant (P = 0.123), indicating that the model is not poor fit. The model explained 18.7% (Nagelkerke R2) of the variation in E. cuniculi seropositivity in this study population consisting of asymptomatic pet rabbits, classified 74.3% of rabbits correctly into being seropositive or seronegative, and had 98% specificity but only 16.7% sensitivity. The model had acceptable discrimination based on AUC  [Apparent 0.6–0.8) . Breed wT. gondii was 3.9% . Three out of seven T. gondii-positive rabbits were also E. cuniculi positive  (E. cuniculi test result was missing in one T. gondii-positive rabbit). Mean age of T. gondii-positive rabbits was 2.7 years (range 1.0–6.9). Five were female and two were male. One rabbit was from a rescue centre, two were re-housed from the previous owner, and the remainder were from a breeder. Three rabbits lived inside a house as house rabbits, two in an outbuilding, two outside in a garden, and one rabbit lived in a balcony. All except one rabbit had access to outdoors during summer months; three spent the whole summer outdoors, one had daily outdoor access, two had weekly outdoor access, and one a few times during the summer. There were cats in families of 3/8 T. gondii-positive rabbits. Only one rabbit of these was also E. cuniculi positive.The true seroprevalence of E. cuniculi-positive rabbits were T. gondii seropositive, while 4 out of 177  E. cuniculi-negative rabbits were T. gondii seropositive. Only three rabbits  were seropositive for both pathogens.Three out of seventy  E. cuniculi IgG antibodies. As the sampling was not randomised due to voluntary participation, the prevalence may not represent the prevalence in all Finnish rabbits. This result is consistent with the results of previous studies of asymptomatic pet rabbits in UK and Korea  but wasectively ), asymptectively ; 52.9%, ectively ), and asectively ). The hi  and in  . Severaepublics  listed fT. gondii, risk factors were analysed only for E. cuniculi infections. The final multivariable logistic regression model revealed that rabbits spending the whole summer outdoors had higher risk of being E. cuniculi seropositive than rabbits with limited outdoor access. It is possible that hygiene in the outdoor shelters and hutches are poorer than those inside a house and this may increase the risk for infection. Wild rabbits exist in Southern Finland and may spread E. cuniculi spores via urine to grass and other outdoor vegetation. Pet rabbits spending the whole summer outdoors could potentially get infection from them.Due to the low seroprevalence of E. cuniculi seropositive than rabbits in multi-rabbit households. Hygiene and overall care were expected to be better in the households with one or two rabbits than in multi-rabbit households and would therefore decrease the risk of being seropositive. However, rabbits in multi-rabbit households were significantly younger than rabbits in households with one or two rabbits, which may explain this finding. We analysed IgG antibodies indicating a previously acquired infection. Older rabbits had a longer period to become infected than younger rabbits. It is also possible that the rabbits were infected in utero or postnatally within the first 8 weeks of life while still living in breeding facilities and less frequently later in life if living in conditions with good hygiene. Unfortunately, we did not include a question regarding possible previous 28-day treatment with fenbendazole for E. cuniculi infection in our questionnaire. Routine E. cuniculi treatments with fenbendazole are not generally recommended for asymptomatic rabbits in Finland, but may be more commonly used in multi-rabbit households to prevent outbreaks of clinical encephalitozoonoosis in breeding and hobby rabbits. It is unknown how long rabbits remain E. cuniculi seropositive. Therefore, prospective long-term or even lifetime follow-up studies are necessary to further study this subject.Surprisingly, rabbits in the households with one or two rabbits had higher risk for being E. cuniculi seropositivity. It is possible that rabbits acquire the infection from contaminated hay, water, or the environment instead of these selected diets.Feeding frequency of the selected diets was not associated with increased risk for E. cuniculi seropositive than rabbits of other breeds or younger rabbits. There was high variation in rabbit breeds in our study and most rabbits were of pet rabbit breeds. Therefore, we categorized the rabbits as mixed-breed rabbits, Dwarf Lops, and others. Although breed did not have a significant effect in our study, it was included in the model due to its confounding effect. While the seroprevalence was higher in rabbits > 3 years and older rabbits had a greater risk for E. cuniculi seropositivity in unconditional logistic regression analysis, age did not remain in the final multivariable logistic regression model.Previously, Lonardi et al.  performeT. gondii seroprevalence in our study population was lower than expected, as only 8 out of 270 (3.9%) rabbits were positive. The seroprevalence in Finnish wild rabbits has not yet studied, but the 4.2% seroprevalence was reported in Finnish mountain hares [T. gondii in Japanese cats was 5.4% [T. gondii-positive rabbits in our study were living in a household with cats. However, seven of the T. gondii-positive rabbits had access to outdoors during summertime (data missing in one case), and all except one were fed with outdoor greens at least a few times weekly. Grass and other plants contaminated with cat faeces may have been the source of infection for these rabbits. Sporulated oocysts of T. gondii may survive in the environment for months or even years [T. gondii seropositive than rabbits without cat exposure [The in hares . Toxoplain hares . The obsin hares . A seropin hares ) and in in hares . The serwas 5.4% , which iwas 5.4% ); this cen years . Inverteen years , which men years . This stexposure .T. gondii seroprevalence in E. cuniculi-seropositive rabbits than E. cuniculi-seronegative rabbits. In our study, the low number of T. gondii-seropositive rabbits limited the possibilities to further study these coinfections.Neumayerová et al.  reportedE. cuniculi seroprevalence, a larger sample size would have been necessary due to the slightly higher observed seroprevalence. We also managed to exceed the sample size required for T. gondii seroprevalence and this was a strength of this study. Due to the low seroprevalence of T. gondii, we were unable to perform a risk-factor analysis for T. gondii seroprevalence. As the E. cuniculi seroprevalence was not adjusted with the sensitivity and specificity of the test used, the seroprevalence is presented as apparent seroprevalence. We used owner questionnaires to obtain the rabbits’ background information and some information was therefore missing. Use of owner questionnaires includes a risk of information bias. Although the questionnaire was not validated prior to use, it was used in few other studies previously. We did not have any information about the geographical region of the rabbits wherefore we could not adjust for that in the analyses. As climate conditions during winter in whole Finland are not beneficial for E. cuniculi spores’ stability and T. gondii oocysts’ sporulation, we did not find the information regarding the region as important.Although we collected the minimum planned sample size required for E. cuniculi IgG antibodies, indicating previous exposure to this pathogen. Seroprevalence of T. gondii was low (3.9%). These zoonotic infections should be considered as differential diagnoses when treating rabbits in veterinary clinics.Nearly one-third of Finnish pet rabbits participating in this study were seropositive for


With fluorescence microscopy it was revealed that a decrease in the concentration of extracellular calcium ([Ca2+]ex) results in two types of Ca2+ responses in white adipocytes: Ca2+ oscillations and transient Ca2+ signals. It was found that activation of the connexin half-channels is involved in the generation of Ca2+ oscillations, since the blockers of the connexin hemichannels—carbenoxolone, octanol, proadifen and Gap26—as well as Cx43 gene knockdown led to complete suppression of these signals. The activation of Cx43 in response to the reduction of [Ca2+]ex was confirmed by TIRF microscopy. It was shown that in response to the activation of Cx43, ATP-containing vesicles were released from the adipocytes. This process was suppressed by knockdown of the Cx43 gene and by bafilomycin A1, an inhibitor of vacuolar ATPase. At the level of intracellular signaling, the generation of Ca2+ oscillations in white adipocytes in response to a decrease in [Ca2+]ex occurred due to the mobilization of the Ca2+ ions from the thapsigargin-sensitive Ca2+ pool of IP3R as a result of activation of the purinergic P2Y1 receptors and phosphoinositide signaling pathway. After activation of Cx43 and generation of the Ca2+ oscillations, changes in the expression levels of key genes and their encoding proteins involved in the regulation of lipolysis were observed in white adipocytes. This effect was accompanied by a decrease in the number of adipocytes containing lipid droplets, while inhibition or knockdown of Cx43 led to inhibition of lipolysis and accumulation of lipid droplets. In this study, we investigated the mechanism of Ca2+ oscillation generation in white adipocytes in response to a decrease in the concentration of Ca2+ ions in the external environment and established an interplay between periodic Ca2+ modes and the regulation of the lipolysis/lipogenesis balance.The aim of the study was to investigate the mechanisms of Ca There are two main types of adipose tissue: white and brown White adipose tissue (WAT) stores energy as triglycerides in lipid droplets, whereas brown adipose tissue (BAT) determines adaptive thermogenesis and dissipates energy as heat. Obesity induces a complex remodeling of adipose tissue, which expands to accommodate the excessive caloric intake, with marked changes in its structure and cellular composition, as a result leading to WAT dysfunction . WAT dysWhite adipocytes form fat depots in different parts of the body and are the main type of bone marrow cells; their number increases in various pathological conditions, including radiation therapy . HoweverUnder normal conditions, with a positive caloric balance, free fatty acids (FFA) supplied with blood are stored as triglycerides in mature adipocytes. However, in obesity, “healthy” WAT expansion is achieved by recruiting and differentiating adipose precursor cells ,10. It i2-adrenergic receptors, heterotrimeric Gi protein and αi-subunit, inhibiting adenylate cyclase and cAMP synthesis [2+ ([Ca2+]i): an increase in its concentration leads to a decrease in the accumulation of triglycerides by white adipocytes, and a decrease, conversely, promotes an increase in body weight i in the suppression of lipolysis is also well known and seems to be a complex, poorly studied process i, as a secondary messenger, mediates the action of almost all neurotransmitters and hormones. However, the role of Ca2+ ions in the regulation of the balance between lipolysis and lipogenesis in WAT has not been sufficiently studied, including the implication of Ca2+ oscillations in the control of both processes. Therefore, it is of interest to study the intracellular mechanism of Ca2+ oscillation generation caused by the activation of the Cx43 connexin channels and their impact on the regulation of lipolysis/lipogenesis in white adipocytes.It should be said that an increase in i, repeated application of the Ca2+-free medium was carried out, which led to the release of Ca2+ in 87 ± 21% of adipocytes that responded to the first stimulation. Diminution in the number of cells in response to the repeated stimulation was most likely associated with irreversible desensitization of the calcium-transport systems. Moreover, each of the adipocytes was still able to respond to the Ca2+-free buffer exclusively in the form of Ca2+ responses, which usually occur during the first short-term stimulation; that is, the adipocytes that responded to the first stimulation with an increase in the [Ca2+]i impulse responded with the same type of signal to the repeated stimulation , and the Cx43 type is most abundantly expressed in white fat adipocytes ex  inhibitor, completely inhibited Ca2+ release from white adipocytes after repeated addition of Ca2+-free buffer , compared with the control (2+-free); this may indicate an activation of lipolysis. The addition of a Ca2+-free solution for 60 min in the presence of CBX (100 µM) blocker led to a significant increase in the expression level of 2 out of 5 studied genes, such as Igf2 (by 3.2 times) and Atgl (by 2.9 times)  compared to those observed when only the Ca2+-free solution was used: the number of adipocytes loaded with lipid droplets decreased compared to the control (2+-free solution was added (2+-free). A knockdown of the Gja1 gene suppressed Ca2+ oscillations in white adipocytes in response to application of a Ca2+-free medium and promoted an increase of lipid droplets in white adipocytes (2+-free medium to the Cx43-KD cell cultures suppressed the expression of the Lipe (by 19%) and Sirt1 (by 31%) genes but induced a 5.8-fold increase in Igf2 expression (2+-free medium had on the intracellular accumulation of the lipid droplets (2+-free). The culture of adipocytes stained with OilRed was morphologically similar to the control (Data on changes in gene expression coincide with those on the decrease in the number of adipocytes loaded with lipid droplets (9 times) C. Incuba9 times) F, CBX. Tpression C; this c2+-free medium, we used a method for assessing the area of lipid droplets in relation to the area of cells. For this, all lipid droplets were isolated in each adipocyte using ImageJ and the Analyze Particles Plugin and their areas were determined. The area of the cell itself was also measured. In the control, the area of the lipid droplets on average occupied 60 ± 11% of the area of the entire cell  and 41 ± 20% (2+-free), respectively. In turn, the incubation of control adipocytes with Ca2+-free medium led to a decrease in the cell area to 5 ± 3% (2+-free), and the lipid droplets themselves became significantly smaller in size.White mouse adipocytes in culture are characterized by the presence of a large number of lipid droplets of various sizes A. To rev2+-free medium after 24 h reduces the concentration of glycerol by 68 ± 7%, compared with the control  transcription factor and subsequently downregulates the expression of several antioxidant enzymes and increases oxidative stress in mesenchymal stem cells after adipogenic induction. Therefore, Sirt3 depletion reduces the ability of mesenchymal stem cells to undergo adipogenic differentiation and leads to adipocyte dysfunction .Another important factor, insulin-like growth factor-2 (IGF2), is a growth-promoting polypeptide that has a high degree of structural homology with insulin, a widely expressed peptide hormone in cell division . IGF2 is2+-free medium resulted in suppression of the expression of Igf2 gene encoding insulin-like growth factor-2 (IGF2), which can promote lipolysis. It is known that insulin markedly increases the rate of synthesis and accumulation of triglyceride by 3T3-L1 adipocytes i in endothelial cells [2+]i. There is a hypothesis that in response to a decrease in dietary calcium intake, an increase in the level of parathyroid hormone and 1,25-dihydroxyvitamin D  occurs, which leads to an increase in [Ca2+]i and activates the accumulation of triglycerides by adipocytes  was performed by fluorescent microscopy using Fura-2AM , a ratiometric fluorescent calcium indicator. Cells were loaded with the probe dissolved in Hanks balanced salt solution (HBSS) composed of (mM) 156 NaCl, 3 KCl, 2 MgSO4, 1.25 KH2PO4, 2 CaCl2, 10 glucose, containing 10 mM HEPES, pH 7.4, at a final concentration of 5 µM at 37 °C for 40 min with a subsequent 15 min washout. The coverslip containing the cells loaded with Fura-2 was then mounted in the experimental chamber. During the experiment we used a perfusion system, which enables complete replacement of the cell bathing solution within 30 s. We used an Axiovert 200M-based imaging system  equipped with an HBO100 mercury lamp, AxioCam HSm CCD camera and MAC5000 high-speed excitation filter wheel. Fura-2 fluorescence was excited at two wavelengths using band-pass filters BP 340/30 and BP 387/15; fluorescence was registered in the wavelength range of 465–555 nm. Excitation light intensity was lowered using 25 and 5% neutral density filters in order to prevent phototoxicity. Image frames were acquired at 3 s intervals with a Plan Neofluar 10×/0.3 objective. The time lapse image sequences were analyzed using ImageJ 1.44 . Graphs were plotted using OriginPro 8.0 software Microcal Software Inc., Northampton, MA, USA). Statistical analysis was performed using the same software. Results are presented as the means ± standard error (SE) or as the representative calcium signal of the cells.The measurement of the cytosolic i led to the generation of two types of Ca2+ signals, such as Ca2+ oscillations and Ca2+ transients. Ca2+ oscillations occurred due to the activation of the Cx43 hemichannels and vesicular ATP secretion, leading to paracrine activation of most white adipocytes in vitro. The mechanism of Ca2+ oscillation generation, apparently, is based on the mobilization of Ca2+ ions from the thapsigargin-sensitive endoplasmic reticulum pool via IP3R, and is switched on by the activation of P2Y1 purinoreceptors and G-proteins. Long-term Ca2+ oscillations in adipocytes influence the expression level of the genes involved in the regulation of lipogenesis/lipolysis and altered the balance of both processes in favor of activation of lipolysis, resulting in a diminished lipid droplets number.Thus, in differentiated white adipocytes, the reduced concentration of [Ca


In recent years, studies on early plant defense signaling in response to herbivory have been gaining momentum with the application of genetically encoded GFP-based sensors for real-time monitoring of early signaling events and genetic tools to manipulate ion channels involved in plant-herbivore interactions. In this review, we provide an update on recent developments and advances on early signaling events in plant-herbivore interactions, with an emphasis on the role of ion channels in early plant defense signaling.Plants and insect herbivores are in a relentless battle to outwit each other. Plants have evolved various strategies to detect herbivores and mount an effective defense system against them. These defenses include physical and structural barriers such as spines, trichomes, cuticle, or chemical compounds, including secondary metabolites such as phenolics and terpenes. Plants perceive herbivory by both mechanical and chemical means. Mechanical sensing can occur through the perception of insect biting, piercing, or chewing, while chemical signaling occurs through the perception of various herbivore-derived compounds such as oral secretions (OS) or regurgitant, insect excreta (frass), or oviposition fluids. Interestingly, ion channels or transporters are the first responders for the perception of these mechanical and chemical cues. These transmembrane pore proteins can play an important role in plant defense through the induction of early signaling components such as plasma transmembrane potential (V Abiotic stress includes drought, salinity, extreme temperatures, radiation, floods, and heavy metals, whereas biotic stressors include insect, animal herbivores, and microbial pathogens. Plant and insect-herbivore interactions are among the most significant species interactions found in nature ,2, and ic levels ,7,8,9,10c levels ,24,25,26During herbivory, the damaged areas of the plant need to inform the rest of the plant to keep them ready for the imminent herbivory threat. Therefore, plants need to alert their unaffected parts by sending long-distance signals from the site of damage to various parts of the plant to appraise the threat. Plants respond to diverse stimuli by communicating amongst cells from distinct tissues or organs, a process called systemic signaling . Studies2+ [Considerable progress has been made in understanding this intricate relationship between plants and herbivores with a plethora of field and lab studies. These include studies that have dissected pairwise interactions between a specific herbivore and its host; interactions at species, genus, and community levels with multiple hosts and herbivores; and studies examining plant defense signaling networks through molecular genetics genomics, to name a few ,30. Howe2+ , reactiv2+ ,33,34,35m, Ca2+, and ROS  and Ca2+ flux. Variation in Vm is critical for plant wounding responses [2+ and ROS, versatile secondary messenger, were generated that plants use to sense and transform environmental stimuli into an adaptive intracellular response [2+ concentration, and these spatiotemporal variations have been shown to yield Ca2+ signatures [2, H2O2, and OH−. ROS which has been demonstrated to act as a self-propagating long-distance and fast wound signal [m, Ca2+, and ROS in plant response to herbivory and provide an overview of what is currently known about the role of ion channels in plant-herbivore interactions.There has been considerable research on identifying the factors that are involved in long-distance signaling. Plants can appraise their unaffected parts by extensive network of intracellular regulators, V and ROS ,41. The  and ROS . The proesponses . Finallyresponse . Insect gnatures ,46,47,48d signal . Throughm is an electrical potential of the cell membrane that is maintained via the balance of ion fluxes across the plasma membrane. Vm indicates whether a cell is excited or not. It is responsible for generating action potentials in tissues, muscles, and nerves in animals and plays a crucial role in diverse biological functions such as biological sensing, hearing, cell cycle, proliferation, contractility, and circadian rhythm, to name a few [m to regulate plant cellular functions such as maintaining turgor pressure, osmotic balance, and stomatal closure. There is no net flux of ions through the membrane when in equilibrium, called the resting membrane potential. Changes in the resting membrane potential will occur due to an unbalanced movement of ions, thus leading to Vm being more positive (depolarization) or more negative (hyperpolarization). In general, plants maintain a negative resting membrane potential in the order of −110 to −150 mV [m leads to plant defense responses. The way plants sense insect cues and initiate defense responses has been a point of interest for many years. One hypothesis that has evolved by studying cellular responses following herbivory suggests that the first event following herbivory generates the fluctuation in Vm [Spodoptera littoralis) alter Vm in lima bean (Phaseolus lunatus L.) at increasing distances of 5, 30, and 60 mm from the bite zone. Vm depolarization was observed within the first 15 min of feeding by S. littoralis in the palisade cells. The effect of S. littoralis regurgitant and its components were also tested on Vm in P. lunatus leaf and the results showed that Vm alterations were independent of regurgitate concentration. In addition, they also examined changes in Vm in response to the application of various H2O2 concentrations to mechanically damaged and herbivore-wounded P. lunatus leaves. H2O2 treatment induced a robust Vm that was significantly greater in herbivory-wounded plants than in mechanically injured leaves [The Vme a few . Unlike  −150 mV ,52. It hon in Vm . Maffei on in Vm  has alsod leaves .m alterations in wild-type and plasmodesmata mutated A. thaliana pdko3 lines; plasmodesmata are channels within the plant cell that allow chemicals to pass through, establishing a pathway for cell-to-cell communication. A strong Vm depolarization occurred in wild-type A. thaliana plants within 7 to 8 min after herbivory, but the pdko3 mutant did not exhibit Vm depolarization in response to herbivory or application of OS from S. littoralis. However, Ca2+ elevation was observed in both wild types as well as in pdko3 mutant. This observation ruled out the possibility of Ca2+ channels being involved in Vm depolarization. To dissect the dependence of Vm depolarization on potassium (K+) channels, the K+ channel activity was measured using fluorescent indicator FluxORTM. A significant increase in K+ channel activity was observed in wild-type plants, whereas a complete loss of K+ channel activity was observed in pdko3 plants. This finding also suggests that K+ channels are involved in Vm depolarization and supports the hypothesis that plant cells respond to OS by a Vm-mediated signal transduction pathway.Bricchi et al.  studied m has been known to be induced by the binding of specific components from herbivore OS with the receptors present at the plasma membrane [Ginkgo biloba), a living fossil plant, responds to S. littoralis herbivory by inducing Vm depolarization, which was evident up to 6 h. Another study using A. thaliana also showed that the extent of Vm depolarization was the same for S. littoralis, green peach aphid (Myzus persicae), and the plant pathogenic bacteria Pseudomonas syringe, but the timing of the occurrence of Vm depolarization was different for each of these biotrophs. Moreover, the magnitude of early defense response depends upon the amount of tissue damage by the biotroph. Vm depolarization was rapid upon the attack of chewing herbivore, S. littoralis (30 min to 2 h), as it caused substantial tissue loss, since it consumed large amounts of leaf tissue. On the other hand, less damage was observed by a phloem feeder, M. persicae (4 to 6 h), that delayed the plant defense response since phloem feeders with sucking mouthparts feed on vascular tissues without visible tissue damage as observed with chewing herbivores [2+ and ROS generation are directly tied to Vm when herbivores interact with plants, and Vm is essential for plant defense responses.The fluctuation in Vmembrane . These cmembrane . A studymembrane  showed trbivores . It is a2+ is a ubiquitous signaling molecule in plants. It functions as a secondary messenger in cellular pathways that regulate plant growth and development, cell polarity, cytoskeleton organization, ion transport across membranes, stomatal regulation, root growth, fertilization, nutrient signaling, and plant immunity [2+ signature,” linked with distinct fluctuations in Ca2+ concentration in the cytosol and sometimes in a particular intracellular compartment. Therefore, Ca2+ fluxes, especially oscillations between calcium stores and the cytosol, are important for cell signaling [Caimmunity . Consequignaling ,61,62.2+ concentration is maintained at or below 100 nM; however, the majority of Ca2+ is stored in the apoplast, vacuole, endoplasmic reticulum (ER), and Golgi apparatus. The apoplast serves as the first Ca2+ reservoir of a cell that can store 0.33 mM free resting Ca2+ and the first area that responds to stimuli, while the vacuole serves as the largest Ca2+ pool of a cell that can store up to 0.2–5 mM free resting Ca2+ [In plants, the cytosolic Caing Ca2+ ,63.2+ signature plays an important role in long-distance signal transduction during herbivore attack through which HAEs such as OS, oviposition, and frass is sensed by the cell membrane, and then, a Ca2+ is rapidly propagated in the cytosol and travels throughout the plant to induce defense responses. The shaping of this “Ca2+ signature” during plant-herbivore interactions is achieved through the amplification and integration of Ca2+ signals. The amplification step is mediated via specific ion channels or transporter proteins and enhances Ca2+ fluxes at sites of herbivore attack, whereas the integration step is mediated via Ca2+ sensor proteins, which allow efficient transmission of Ca2+ signals from one cell to another in a tissue or organ. Herbivory induces Ca2+ entry from the apoplast to the cytosol via plasma membrane Ca2+ channels which stimulates Ca2+ signals in the cytosol leading to the amplification of Ca2+ signals. The localized Ca2+ signals from the cytosol are distributed throughout the whole plant. In this way, amplification, and integration of Ca2+ signals constitute two important ways by which “Ca2+ signature” contributes as a signaling molecule during plant-herbivore interactions [The Caractions .2+ signal requires selective Ca2+ sensor proteins that respond to changes in cytosolic Ca2+ levels and encipher the frequency, amplitude, and signal localization of Ca2+ signatures. It is estimated that A. thaliana contains around 250 Ca2+ sensor proteins [2+ dependent protein kinases (CPKs) and the Ca2+ and calmodulin-dependent protein kinase (CCPK) [2+ binding and cause conformational changes in their structure [The amplification of intracellular Caproteins . These cproteins ; (2) theproteins ; and (3)e (CCPK) . All of tructure .Arabidopsis genome has seven calmodulin genes encoding four different isoforms  [A. thaliana signal responsive (AtSR1) proteins [Atsr1 mutants of A. thaliana were sensitive to attack by dark winged fungus gnats (Bradysia impatiens), suggesting the role of CaM as an important sensor in the early stages of the insect-plant attack [2+ binding and act as Ca2+ relays/sensors [A. thaliana upon S. littoralis OS treatment, implying a function in early defense plant signaling [2+ binding domain and a serine/threonine kinase domain into a single protein that performs the fundamental function of converting Ca2+ signals to phosphorylation events [A. thaliana contains 34 CPK family genes that play a role in plant defense responses. CPK 3 and CPK 13 both participate in signaling after Ca2+ influx upon S. littoralis attack through regulation of plant defensin gene (PDF1.2) by phosphorylation of the transcription factor, HsfB2a [cpk3 and cpk13 mutants had much lower transcript levels of the plant defensin gene PDF1.2 in comparison to wild-type plants.CaM functions as a sensor relay protein since it lacks an enzymatic function. The nd CaM7) . CaM/CaMnd CaM7) . A. thalproteins , also knproteins , have bet attack . Along w/sensors . CML and/sensors . CML42 gignaling . CPKs han events ,77. A. t, HsfB2a . The cpk2+ signaling has gained momentum with the advance in Ca2+ imaging techniques. Therefore, it is important to discuss different plant Ca2+ imaging methods, which are widely used in the context of plant-herbivore interactions to observe and record cytosolic Ca2+ concentration in herbivore-infested plants. These techniques include the use of Ca2+ sensing fluorescent dyes and genetically encoded Ca2+ indicators. Various fluorescent Ca2+ sensing dyes, such as Fluo-3, Calcium Orange, etc., have been used to investigate the dynamics of cytosolic Ca2+ signals in plant- herbivore interaction [2+ indicator Ca2+ orange was utilized to identify changes in cytosolic Ca2+ concentrations in P. lunatus following S. littoralis herbivory. The changes in Ca2+ concentration were compared in response to a single wounding (MD) event, continual mechanical damage caused by a robotic worm , and herbivory. After 30 min, a considerable increase in Ca2+ fluorescence was observed due to herbivory in the wounding zone, which persisted for 4 h, but in MD and MW plants, just a faint fluorescence was noticed [2+ sensing dyes have some limitations, including toxicity, fragility, low fluorescence signals, and they cannot be imaged in living plants without permeabilization. To overcome these limitations, researchers have initiated research on the use of genetically encoded Ca2+ indicators. The most widely used Ca2+ imaging method includes genetically encoded Ca2+ indicators, such as GCaMP, Yellow Cameleon (YC) Ca2+-sensors. The Ca2+ sensors were developed from GFP by combining them with calmodulin. These Ca2+ sensors can be expressed in the whole plant and are functional throughout the entire plant. Therefore, it can be used to monitor cytosolic Ca2+ in plants subjected to various herbivore attack conditions [P. rapae caterpillars induced cytosolic Ca2+ responses in the leaves of A. thaliana can be monitored with GCaMP3. This study reported that the increases in cytosolic Ca2+ concentration were associated with ion influx through plasma membrane Ca2+ channels such as GLR3.3/GLR3.6. Another example is Verrillo et al. [2+ induction could be monitored with YC3.60, a YC-based Ca2+ sensor, following application of S. littoralis OS on mechanically damaged A. thaliana leaves. By using these tools, it is now possible to study the dynamics of Ca2+ signaling in plant-herbivore interactions at single-leaf, whole-plant, and whole-plant-insect herbivore attack conditions.In recent years, the research on Caeraction ,79,80,81 noticed . Even thnditions ,82,83,84nditions  showed t2+ level is controlled by the influx of Ca2+ ions from extracellular through apoplastic and vacuolar membranes. Therefore, plant ion channels play an important role in regulating plant development and the perception of many stimuli, including herbivory.Intracellular Ca6 ions per second. Ion channels can control ion fluxes in their target compartment and, thus, modify cellular homeostasis, and are vital in osmoregulation, development, signaling, mobility, and uptake of nutrients by the root and long-distance communication [+ channel, Stelar K+ outward rectifier (SKOR) [+ channels such as the shaker family , tandem pore, and two-pore K+ channels , are responsible for permeation of K+ ion across the plasma membrane and tonoplast membrane. Anion channels include slowly activating anion channels , aluminum-activated malate transporters , chloride channels/transporters , and detoxification efflux carrier . Ligand-gated channels include cyclic nucleotide-gated channel  and glutamate receptor-like channels  [2+-transporting ion channels have been shown to play a role in plant-herbivore interactions, including cyclic nucleotide-gated channels (CNGC19) [Ion channels are macromolecular pores in the membrane that regulate the influx and efflux of ions across the membrane at a rate of 10nication ,86. The r (SKOR) . The lasansport)   , stomataansport) ,91, pollansport) , salt toansport) , and plaansport) , to name(CNGC19) ,96, glut(CNGC19) ,42,97, t(CNGC19) ,84,98, a(CNGC19) ,100.2+ channels, first discovered in retinal photoreceptors and olfactory neurons [A. thaliana, which consists of 20 family members [The cyclic nucleotide-gated channels (CNGCs) are ligand-gated Ca neurons . They pl neurons . These i neurons ,103,104  neurons ,106, the neurons , and sal neurons . These c members .C-termini of the channel  channels are ionotropic glutamate receptor homologs in mammals (iGluRs). The iGluRs have been extensively studied for their central nervous system and have been known to play a vital role in synaptic transmission, learning, and memory [Arabidopsis consists of 20 GLR genes; each subunit hosts a N-terminal domain, two extracellular ligand-binding sites , and transmembrane domains (S1–S4), including a pore region (P) and the C-terminal domain [d-aspartate (NMDA), α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA), and Kainate receptors. Plant GluRs share a high degree of similarity with the NMDA receptors that range from 16 to 63% in the ligand-binding domains and the transmembrane domains [Glutamate receptor-like (GLR) is a non-selective ion channel responsible for permeating Cad memory ,124. It d memory . In pland memory , gravitrd memory , pollen d memory ,129, immd memory ,132,133,d memory . Arabidol domain   var xanthi while the application of lanthanum and Ca2+ chelator, BAPTA, inhibited glutamate-induced Ca2+ increase. This observation suggests that the plant GLR has a role in the modulation of Ca2+ influx that ensures plant defense responses against insect herbivores.Electrophysiological studies have shown the involvement of GLRs in inducing a Carbivores ,140. A s2+ waves from wounded to unwounded sections of the plant. When S. littoralis larvae were allowed to feed on A. thaliana wild-type plants, wound-induced surface potential alterations were detected. However, wounding reduced the surface potential alterations in the four GLR mutants GLR3.1, GLR3.2, GLR3.3, and GLR 3.6. [Pieris rapae) caterpillars in A. thaliana leaf expressing genetically encoded Ca2+ sensor GCaMP3. The cytosolic Ca2+ elevation and subsequent defense gene expression were observed after the application of glutamate and not with other amino acids such as sorbitol. Furthermore, the Ca2+ signals were completely abolished in the GLR3.3/GLR3.6 double mutant in A. thaliana, suggesting that GLR3.3 and GLR3.6 are essential for transmitting Ca2+ signals induced by wounding and herbivory. Another recent study by Shao et al. [2+ elevation and surface wave potential (SWP) in A. thaliana expressing calcium sensor GCaMP6. Additionally, the application of glutamate to the wound site in the root induced an increase in Ca2+ and SWP in all leaves. Interestingly, in the GLR3.3/GLR3.6 double mutant, this wound and glutamate-induced rise in root to shoot Ca2+ was attenuated. This finding suggests that GLR3.3 and GLR3.6 are involved in propagating systemic Ca2+ signaling from leaf to leaf and root to shoot. These results provide evidence for the role of plant GLRs in the modulation of Ca2+ signaling during plant defense responses against insect herbivores.GLR3.3 has been implicated in the transmission of signals in the form of CaGLR 3.6. . This suGLR 3.6.  showed to et al.  demonstr2+ channels [A. thaliana by genome sequencing [N-terminal head and a C-terminal annexin core [2+ binding activity occurs in type II and III binding sites of annexin proteins [N-terminal region than their animal counterparts [N-terminal region that interacts with other proteins.Annexins are the phospholipid-binding proteins and are considered novel mechanosensitive Cachannels ,142. In channels . They archannels ,144. Eigquencing . Plant axin core   herbivory. The study found that annexin 1 was responsible for inducing cytosolic free Ca2+ elevation upon wounding and simulated herbivory in A. thaliana. ANN-1 knock-out and ANN-1 overexpressing lines were employed in this work to evaluate their role in herbivory-mediated Ca2+ signaling. The result showed that in the ANN-1 deletion line, the increase in cytosolic Ca2+ upon herbivory by S. littoralis was impaired, and the larvae gained more weight than those fed on wild-type plants. On the other hand, weight increase was significantly lower in larvae that fed on the ANN-1 overexpressed line compared to the wild type. Additionally, jasmonate accumulation and defense responses were diminished in ANN-1 systemic leaves, demonstrating that ANN-1 is involved in systemic cytosolic Ca2+-dependent jasmonate induction. This finding suggests that ANN-1 modulates plant defenses against herbivore damage through the Ca2+-dependent jasmonate signaling pathway and is required for systemic rather than local defense activation in plants attacked by herbivorous insects.A recent study by Malabarba et al.  reportedA. thaliana (AtTPC1), with 733 amino acids identical to the rat TPC1 sequence [Two pore channels (TPCs) are organellar cation channels that are widely expressed in animals and plants. In animals, they are localized in the endolysosomal membrane, while in plants they reside in the tonoplast of plant vacuoles ,153,154.sequence .2+ and Na+ channels and feature two shaker-like units with six transmembrane domains (S1–S6), each joined by a cytosolic linker containing two Ca2+-binding EF-hands (EF1 and EF2). , the plasma membrane Ca2+ permeable ion channels glutamate receptor-like 3.3 and 3.6 (GLR3.3 and GLR3.6), which are critical regulators of extracellular Ca2+ import into the cytoplasm of plant cells. In addition, this study also revealed that the increase in cytosolic Ca2+ induced TPC1 mediated vacuolar Ca2+ release in response to aphid feeding, suggesting that the TPC1 channel operates in conjunction with the plasma membrane Ca2+ permeable ion channels GLR3.3 and GLR3.6 in mediating cytosolic Ca2+ increase during insect herbivory [Plant and animal TPCs are similar in sequence to voltage-gated Cand EF2). . Voltage opening . The SV 2+ to K+ ,157. Ca2 release ,158. The release  has showt et al.  employederbivory .+-ATPases) are the primary pumps responsible for the generation of a proton gradient across cellular membranes. This electrogenic transporter uses energy from ATP hydrolysis to drive the translocation of protons against their concentration gradient from the cytosol to the external aqueous environment [+-ATPase is located in the plasma membrane (PM) of plant cells. It has been demonstrated that the activation and suppression of the H+-ATPase activity in the plant plasma membrane modulate Vm, resulting in the alteration of PM ion channels and transporters functions [+-ATPase is a single 100 kDa polypeptide and a member of the large family of phosphorylation (P)-type ATPases. It is composed of six transmembrane helices (M1–M6) and a cytoplasmic domain containing phosphorylation (P), nucleotide-binding (N), and actuator (A) domains involved in ATP hydrolysis. The PM H+-ATPase has been implicated in various physiological processes, including cell development, intracellular pH regulation, food uptake, stomatal opening, salt tolerance, and cellular expansion [The proton-pumping ATPases  is involved in the formation of slow wave potentials (SWPs), which are required for long-distance electrical transmission during herbivore-induced plant defense. Fusicoccin, a PM H+-ATPase activator, prolonged the SWP repolarization phase in leaves distal to wounds. The repolarization phase was significantly prolonged in reduced function aha1 mutants, whereas the duration of SWP repolarization was reduced in the presence of a gain-of-function mutant ost2-2D. Additionally, S littoralis larvae performed better on aha1-7 mutants than on wild-type plants. Overall, these observations suggest that the PM H+-ATPase is required for the regulation of the Vm and electrical signal propagation between different parts of a plant during insect herbivory.Plant PM H feeding . A study feeding  demonstri et al.  has reve2−•−), hydrogen peroxide (H2O2•), hydroxyl radical (HO•), peroxynitrite (ONOO), and singlet oxygen (1O2) [2O2. In plants, respiratory burst oxidase homologs (RBOHs) were found to be the key enzymes that catalyze the formation of ROS, which is a key step in plant protection against herbivores [N. attenuate where tobacco hornworm (Manduca sexta) OS enhanced NaRBOHD (N. attenuata NADPH oxidase homolog) on damaged leaves. ROS accumulation was diminished in M. sexta OS treated NaRBOHD-silenced N. attenuata plants without affecting OS-induced gene expression of defense-related genes [Reactive oxygen species (ROS) are highly reactive molecules generated by redox reactions. They are part of several biological processes, such as photorespiration, oxidative phosphorylation, the electron transport chain (ETC), as well as a plant defense against pathogens and herbivores. ROS is produced in the mitochondria, chloroplast, and peroxisomes. There are several forms of ROS like superoxide anion (Oen (1O2) . ROS is rbivores ,171,172.rbivores . The siged genes .A. thaliana [Diuraphis noxia). A strong burst of H2O2, as well as NADPH oxidase activity, was observed in resistant plants 3 h after infestation in comparison to susceptible plants. Treatments of plants with diphenyleneiodonium (DPI), an inhibitor of NADPH oxidase, suppressed the H2O2 production. Elevation in H2O2 levels (47%) was observed by treating resistant wheat plants with a mixture of glucose and glucose oxidase [2O2 plays a role in the defense response against D. noxia infestation.The production of ROS is an inevitable by-product of metabolism in many cell types. Previously, it was assumed that ROS are toxic molecules that cause cellular damage to macromolecules . Still, thaliana . Plants thaliana . ROS prothaliana ,180. The oxidase , suggestPterisvittata) response to herbivory by S. littoralis. The P. vittata plants responded to the attack of S. littoralis by activating peroxidases which produced H2O2. The concentration of H2O2 in leaves was lower in mechanically wounded young leaves than herbivory wounded leaves, suggesting that P. vittata can distinguish between mechanical and herbivory wounding by modulating the amount of ROS production. A study by Shinya et al. [Mythimna loreyi), caused a strong intracellular ROS generation on rice cells, and a similar effect was obtained upon application of synthetically prepared N-linolenoyl-L-Glu, the most abundant FAC present in OS of M. loreyi, indicating that FAC from M. loreyi OS promoted ROS production in rice cells.Studies have shown that ROS serve as early defense signaling molecules in response to herbivore-induced wounding and secretions such as OS and oviposition. Imbiscuso et al.  investiga et al.  demonstrM. sexta oral secretions (OS) induced ROS generation in isolated tomato protoplasts. Interestingly, our study showed that the application of tomato plant-fed (PF) M. sexta OS enhanced ROS generation while artificial diet-fed (DF) OS could not induce ROS in tomato protoplasts, suggesting that the oral secretions of M. sexta play an indispensable role in inducing ROS generation in tomato protoplasts. Our study also showed that the M. sexta PF-OS induced ROS increase was diminished in the presence of a Ca2+ chelator, BAPTA-AM, suggesting that there is a link between Ca2+ and ROS signaling. Several lines of evidence have indicated the existence of a positive feedback mechanism between ROS and Ca2+ production. In a heterologous expression system, treatment with ionomycin, an ionophore that leads to Ca2+ influx into cells, resulting in activation of RHD2 NADPH oxidase (root hair defective 2 reduced nicotinamide adenine dinucleotide phosphate) in root tips of A. thaliana confirming Ca2+ triggered RHD2 NADPH oxidase activity. These observations suggest that Ca2+ acts upstream of ROS production [Recently, our group Gandhi et al.  demonstroduction .2+ binding [2+ dependent modulation [2+ and ROS. A. thaliana mutants lacking certain NADPH oxidases (AtRBOHD and AtRBOHF) do not close their stomata and produce ROS, Ca2+, and Ca2+ channel activation when they are exposed to ABA. Supplementation of H2O2 to guard cells rescues the mutant phenotype, implying that Ca2+ entry proceeds downstream of ROS generation in ABA signaling [Compelling evidence indicates that ROS production by RBOHD is dependent on the Ca binding ,187. RBOdulation . Abscisiignaling ,190.A. thaliana, the production of H2O2 was observed in leaves 72 h after oviposition by cabbage moth (Pieris brassicae) and was recognized by the formation of a reddish-brown precipitate. This result indicates that oviposition can trigger a localized response that resembles the hypersensitive response induced by pathogens [P. brassicae induced generation of H2O2, salicylic acid and defense gene expression in A. thaliana. This study also revealed phosphatidylcholines (PCs) released from eggs is the key signaling molecule that activates gene expression and triggers various defenses in the plants.In athogens . A recenathogens  showed t2DCFDA). Genetically encoded ROS sensors “Ro-GFP” can monitor the cellular redox status at a high spatiotemporal resolution [2DCFDA are easier to use and can measure ROS in real-time with high sensitivity [2DCFDA has gained attention for its potential to measure the ROS levels in real-time on whole plants and as well as plant protoplasts [P. syringae pv. tomato DC 3000 on ROS signaling in H2DCFDA dye sprayed A. thaliana by using whole plant-live imaging. This study suggests that the combination of live-cell imaging and the use of H2DCFDA enables real-time monitoring of ROS in plants in response to various stress and pathogen treatments. This study also utilized an RBOHD (rbohD) knockout, and upon treatment with different stimuli, less ROS generation was observed. In contrast, another cytosolic ascorbate peroxidase 1 (apx) knockout produced more local as well as systemic ROS upon wounding or light stress treatments implying that this mutant had less ROS quenching capacity.While ROS relevance in plant-herbivore interaction is gaining momentum, the detection and characterization of ROS are still a significant bottleneck in this field. The early detection and quantification of ROS can be carried out by either utilizing genetically encoded fluorescent ROS sensors such as redox-sensitive green fluorescence protein (Ro-GFP), or synthetic fluorescent probes, such as 3,3′-diaminobenzidine (DAB) and 2′,7′-dichlorofluorescein diacetate (Hsolution ,198,199.solution  measuredsitivity . DAB hassitivity ,203,204.toplasts ,205,206.2+ and ROS generation, gene expression, and, ultimately, the host plant defense response against herbivores. Transforming plants with biosensors such as GCaMP-Ca2+ and Ro-GFP-ROS sensors can help in the early identification of the plant defense responses. HAEs such as OS, frass, and oviposition could be used to develop strategies for early detection of the impending herbivory. So far, only a handful of Ca2+ permeable channels have been identified that plays a role in plant-herbivore interactions. Further studies are needed to unravel other ion channels that may be contributing to the modulation of Vm, Ca2+, and ROS, the downstream signaling cascade, and, more importantly, the role of these ion channels in triggering a rapid defense response. A deeper understanding of these early signaling events will eventually help us to minimize herbivory by developing pest management strategies based on plant-herbivore monitoring systems. Such knowledge can be instrumental in the design of plants with improved resistance against herbivores. As such, in the future, it will be important to develop effective small-molecule modulators that can inhibit or enhance the early signaling events in plant-herbivore interactions. Such an approach would not only facilitate research on early plant signaling events but also help in developing novel strategies for the development of herbivore-resistant crops.Recent years have witnessed immense progress in identifying the early defense signaling components in plant defense against herbivores, but studies on the molecular identification and characterization of these components are still a work in progress. However, with the advent of state-of-the-art imaging techniques, physiological and biochemical assays, and genomics may help us to understand the early defense signaling events by coordinating the plasma membrane potential changes, ion channels modulation, intracellular Ca