Nascent contraction initiated at around E8.0 and was associated with sarcomeric assembly and rapid Ca2+ transients, underpinned by sequential expression of the Na+-Ca2+ exchanger (NCX1) and L-type Ca2+ channel (LTCC). Pharmacological inhibition of NCX1 and LTCC revealed rapid development of Ca2+ handling in the early heart and an essential early role for NCX1 in establishing SACOs through to the initiation of beating. NCX1 blockade impacted on CaMKII signalling to down-regulate cardiac gene expression, leading to impaired differentiation and failed crescent maturation.The mammalian heartbeat is thought to begin just prior to the linear heart tube stage of development. How the initial contractions are established and the downstream consequences of the earliest contractile function on cardiac differentiation and morphogenesis have not been described. Using high-resolution live imaging of mouse embryos, we observed randomly distributed spontaneous asynchronous CaDOI:http://dx.doi.org/10.7554/eLife.17113.001 The heart is the first organ to form and to begin working in an embryo during pregnancy. It must begin pumping early to supply oxygen and nutrients to the developing embryo. Coordinated contractions of specialised muscle cells in the heart, called cardiomyocytes, generate the force needed to pump blood. The flow of calcium ions into and out of the cardiomyocytes triggers these heartbeats. In addition to triggering heart contractions, calcium ions also act as a messenger that drives changes in which genes are active in the cardiomyocytes and how these cells behave.Scientists commonly think of the first heartbeat as occurring after a tube-like structure forms in the embryo that will eventually develop into the heart. However, it is not yet clear how the first heartbeat starts or how the initial heartbeats affect further heart development.Tyser, Miranda et al. now show that the first heartbeat actually occurs much earlier in embryonic development than widely appreciated. In the experiments, videos of live mouse embryos showed that prior to the first heartbeat the flow of calcium ions between different cardiomyocytes is not synchronised. However, as the heart grows these calcium flows become coordinated leading to the first heartbeat. The heartbeats also become faster as the heart grows. Using drugs to block the movement of calcium ions, Tyser, Miranda et al. also show that a protein called NCX1 is required to trigger the calcium flows prior to the first heartbeat. Moreover, the experiments revealed that these early heartbeats help drive the growth of cardiomyocytes and shape the developing heart.Together, the experiments show that the first heartbeats are essential for normal heart development. Future studies are needed to determine what controls the speed of the first heartbeats, and what organises the calcium flows that trigger the first heartbeat. Such studies may help scientists better understand birth defects of the heart, and may suggest strategies to rebuild hearts that have been damaged by a heart attack or other injury.DOI:http://dx.doi.org/10.7554/eLife.17113.002 The CC subsequently expands and migrates to the midline whereupon, between E8.25 and E8.5, the two sides of the CC fuse and form the linear heart tube (LHT)  in a process termed Ca2+ induced Ca2+ release (CICR) , ryanodine receptors , the sarco (endo) plasmic reticulum Ca2+ ATPase  and the Na+/Ca2+ exchanger . Targeted disruption of genes encoding ECC proteins in mice has shown that contractile activity of immature cardiomyocytes does not require ECC. Embryonic cardiomyocytes have a less developed SR and T-tubule system as well as an increased requirement for sarcolemmal Ca2+ flux  . ECC rele (CICR) . Increasa2+ flux  and whila2+ flux , the expa2+ flux . Using im the SR . In conttraction . Whilst to 70 hr . Thus th2+ transients during the earliest manifestation of murine heart development well before any indication of spontaneous cardiac contractions. We employed the use of multiple pharmacological inhibitors to address the contribution of the NCX1 and LTCC Ca2+ channels during this process and reveal an essential early role for NCX1-dependent Ca2+ handling on downstream cardiac differentiation and morphogenesis.We report here, for the first time, high-resolution live imaging of CaIt is commonly stated that initiation of contraction begins with the formation of the LHT , and whiMyom1 (encoding Myom), Actn2 (encoding α-actinin) and Tnnt2 (encoding cTnT) , a protein of the Z-line; Myomesin (Myom), a protein of the M-line and cardiac Troponin T (cTnT), the Tropomyosin binding subunit of the troponin complex . At stagng cTnT) . Myom1,  the LHT .2+ flux, therefore, to investigate the earliest manifestations of Ca2+ handling within the cardiac crescent, we loaded embryos with the fluorescent Ca2+ indicator Cal-520 followed by live imaging using confocal microscopy. From stage 1 crescents until late stage 3, lateral propagation of Ca2+ transients was observed across the embryonic midline (2+ oscillations (SACOs) were observed within the forming crescent, in the absence of contractile activity and sarcomeric banding of cTnT -derived model of cardiomyocyte development to complement the embryo studies and provide further insight into the stages of cardiac contraction coincident with cardiomyocyte specification and differentiation. While ESC-derived cardiogenesis is aligned with stages of mesoderm induction, pre-cardiac mesoderm, cardiomyocyte specification and differentiation based on temporal patterns of gene expression , Cacna1c and Cacna1d , Iptr2 (encoding InsP3 type 2 channels) and Ryr2 (encoding RyR2) that collectively are key components of SR Ca2+ regulation , whilst Cacna1c revealed a modest but non-significant increase of 2.5 fold  and Cacna1d a significant 4-fold increase . From stage 1 onwards Slc8a1, Cacna1c and Cacna1d expression continued to significantly increase until stage 3 . Between stage 3 and the LHT, expression of Slc8a1 and Cacna1c was maintained whilst Cacna1d significantly decreased , expression of Atp2a2 only increased 1.7 fold  and Iptr2 did not significantly change  and adulthood  with NCX inhibitors (CB-DMB and KB-R7943) or LTCC inhibitor (Nifedipine), affected the embryos in a stage dependent-manner  followed shortly afterwards by complete loss of Ca2+ signal (2+ transient generation (2+ efflux from the cell) and reverse (Ca2+ influx into the cell) modes, our data suggested that NCX1 in the early cardiac crescent was functioning in reverse mode, as KB-R7943, that is reported to specifically inhibit reverse mode NCX1 function  this wagnalling , suggest2+ via NCX1, we cultured ESCs in media containing reduced concentrations of Ca2+ (0.1 mM and 1.0 mM) relative to the normal level of 1.8 mM  as with CB-DMB treatment  which, whilst previously associated with pathological cardiac hypertrophy, contribute here towards  cardiomyocyte differentiation  in the presence of DMSO, CB-DMB or Nifedipine for 12 hr . Whilst  embryos . Equallyf CB-DMB , whereasf CB-DMB . These d2+-handling and downstream changes in gene expression and morphology. Using a staging method based on morphological landmarks (stages 0–3), we characterized in detail the in vivo progression of physiological activity during early heart development. The stage series defined correlates with gradual expression of several cardiac related genes and sarcomere assembly. We observed spontaneous asynchronous Ca2+ oscillations (SACOs) at stage 0 in the developing cardiac mesoderm, before any detectable cardiac contractions. These transients appeared sporadically in individual cells within the forming cardiac crescent and did not appear to be synchronized. At stage 1, periodic Ca2+ transients began to be propagated laterally through the cardiac crescent, and traversed the midline of the crescent where there were no visible contractions. This was a rapid and dynamic process occurring through stages 0–3 that involved sarcolemmal ion channel and exchanger function. NCX1 was exclusively required for SACOs at stage 0, whereas both NCX1 and LTCC appeared to play equally important roles at stage 1 and 2. NCX1 was no longer required from stage 3 onwards when the LTCC channels maintained Ca2+ transients. We observed no contribution of the SR in regulating Ca2+ transients within the cardiac crescent, as assessed by simultaneous blockade of RyR2 and InsP3. However, consistent with previous studies  in order to up-regulate genes necessary for further differentiation and morphogenesis. This would explain why SACOs are present much earlier than complete sarcomere assembly. An alternative hypothesis is that SACOs are a by-product of cells that are already committed to specific cardiac lineages and, therefore, arise with the expression of specific channels required for future function. This could explain the variation in duration and frequencies of SACOs observed within the same embryo. At this point, quite how SACOs become synchronised transients is unknown. We speculate that release of a Ca2+-dependent signal from a 'pacemaker' cell may entrain neighbours to have synchronised transient periodicity. To this end we observed highly variable Ca2+ periodicity but also regions containing cells of similar periodicity  within the forming cardiac crescent was a surprising finding that, to the best of our knowledge, has not previously been reported in any type of excitable cell. The specific role of SACOs is currently unknown and we hypothesise that they are required in cells that need to optimally activate Caiodicity . Since b2+ handling is assigned to sequential roles for the NCX and LTCC channels, whereby NCX has been assumed to compensate, at least in part, for the rudimentary  SR in the developing mouse heart , and it has been previously demonstrated that these three exchangers share similar physiological properties (tre) zebrafish which have a mutation in NCX1 leading to absence of normal calcium transients and cardiac fibrillation , to regulate Ca2+ efflux while NCX1 is working in reverse mode. Due to the slow nature of SACOs, oscillations in energy production along with adenosine triphosphate levels could also be involved in SACO generation, especially with reduced SR function. Whilst SR inhibition did not prevent stage 3 Ca2+ transients, we have not tested the involvement of SR function at stage 0 and, therefore, cannot fully exclude that periodic Ca2+ releases from the SR are involved in the generation of SACOs. The latter has been reported in cultured E8.5–9 embryonic cardiomyocytes as a pace making mechanism  or forward mode NCX1 function (inhibition of Ca2+ efflux leading to Ca2+ overload), imbalance of Ca2+ homeostasis or off-target effects. Although the existence of the NCX1 acting in reverse mode is contentious, and the precise function of KB-R7943 is still debated in the field, it is difficult to otherwise explain how inhibition of NCX1 can block SACOs. That said, regardless of mode of action, the observation that at stage 0 SACOs were abolished with both CB-DMB and KB-R7943 treatment, but persisted when treated with nifedipine, suggests that NCX1, and not the LTCC, plays a major role in Ca2+ transient generation within the early cardiac crescent.Parallel analyses on ESC-derived cardiomyocytes revealed that lowering the concentration of Caactivity , replicaechanism  at later2+ entry, as demonstrated by nifedipine-induced inhibition of beating at later stages in both the embryo and ESC-derived cardiomyocytes. NCX1 expression was further maintained at high levels during more advanced stages (stage 0-LHT), suggesting a later role in ensuring Ca2+ removal via its forward mode of action. Of note, the early versus late roles for mammalian NCX1 and LTCC, respectively, are further supported by studies on tre zebrafish, whereby sarcomeric assembly defects in developing cardiomyocytes following loss of NCX1 function are not recapitulated by mutations in LTCC  Act 1986 and approved by the local Biological Services Ethical Review Process. To obtain wild-type embryos C57BL/6 males (in house) were crossed with CD1 females . All mice were maintained in a 12-hr light-dark cycle. Noon of the day finding a vaginal plug was designated E0.5. In order to dissect the embryos the pregnant females were culled by cervical dislocation in accordance with the schedule one of the Animal Scientific Procedures Act. Embryos of the appropriate stage were dissected in M2 medium .Progressive crescent stages were defined based on morphological criteria: the length  and the maximum height  of the cardiac crescent was measured for each embryo. Embryos were considered to be at the LHT stage once both sides of the cardiac crescent were completely folded and fused. The ratio between width and maximum height measurements (µm) was used to categorize the embryos from stage 0 through to stage 3 .2+ imaging was performed as previously described, with some adaptations (2 + Air. Images were acquired at 10 frames per second (fps) for up to 20 s. For all experiments involving Ca2+ imaging, embryos were loaded with 8 μM of Cal-520 by incubating the embryos in 50% CMRL + 50% Knockout Serum Replacement with the dye for 15 min at 37°C and an atmosphere of 5% CO2 + Air. The embryos were then transferred to fresh media in a MatTek dish  without the dye and imaged. To image Ca2+ transients in embryoid bodies (EBs), ESCs were cultured in hanging drops in MatTek dishes for four days under differentiation conditions. At day 7, the cells were loaded with 8 μM Cal-520 for 30 min in media without serum at room temperature and then transferred into media with serum and cultured in the presence of the dye for 10 min at 37°C and an atmosphere of 5% CO2 + Air. All Ca2+ imaging was performed with a Zeiss 710 LSM fitted with an environmental chamber to maintain the embryos at 37°C at 5% CO2. Embryos were imaged with a 20× air objective (0.6 NA) with a single optical section every 97 ms (~10 frames per second). Images were captured at 256 × 256 pixel dimensions, with a 2× line step and no averaging to increase the scan speed. For acute inhibition of Ca2+ transients in embryos (stage 1 to LHT) and EBs, Ca2+ transients were first imaged at baseline, drug containing media was then added and imaging was performed at 5, 15 and 30 min post drug treatment. Inhibition was defined as the complete cessation as well as confinement of Ca2+ transients to small regions of the cardiac crescent. For inhibitor experiments involving stage 0 embryos, imaging was carried out at baseline, 5 and 15 min post treatment. For all the experiments involving calcium imaging of embryo the overlying yolk sac endoderm had to be dissected out in order to allow proper drug and dye penetration, as well as better visualisation of the cardiac mesoderm. For experiments involving embryos Nifedipine  was used at a final concentration of 10 μM, CB-DMB  at 20 μM, KB-R7943  at 30 μM, Ryanodine  at 100 μM and 2-APB at 200 μM, all diluted in DMSO . We found that most drugs had to be used at a higher concentration than previously used in studies involving isolated cells, presumably due to penetration difficulties inherent with using whole embryos. Control experiments for Nifedipine, CB-DMB and KB-R7943 were performed with 0.002% DMSO, and for experiments involving dual inhibition with Ryanodine + 2-APB with 0.6% DMSO. All experiments involving embryos were repeated with at least three different litters (6–10 embryos). Both embryo and ESC experiments were performed on at least three independent days.Live imaging of embryos, including Captations . Briefly2+ Air. Only embryos at E7.5, prior to cardiac crescent and head fold formation were cultured. For these experiments embryos were cultured in the presence of either in 10 μM Nifedipine, 3 μM CB-DMB or 0.0003% DMSO. Experiments for immunostaining were performed on three independent days, while experiments for qRT-PCR were performed on four different days for Nifedipine treatments and five days for CB-DMB treatments.Embryos were cultured in the presence of drug (CB-DMB or nifedipine) for 12 hr, from E7.5 to E8.0 using a rolling culture system. Embryos were cultured in a mix of 50% CMRL and 50% Knockout Serum Replacement at 37°C and with an atmosphere of 5% CONkx2.5-GFP (Eomes-GFP (2+ concentrations was achieved using Ca2+ free DMEM (Gibco) with Ca2+ being added back to the desired concentration . For immunostaining, ESCs were cultured on 0.1% gelatin coated glass coverslips before being fixed with 3.7% PFA for 30 min on ice. For RNA isolation tissue samples were dissociated for 2 min using 0.25% trypsin-EDTA at 37°C prior to snap freezing.Cardiomyocyte differentiation from ESCs was carried out as previously described using an x2.5-GFP  and Eomeomes-GFP  cell linomes-GFP . ApproxiDissected embryos were fixed for 1 hr at room temperature with 4% PFA in PBS. The embryos were then washed 3x in PBT-0.1% (PBS with 0.1% Triton X-100) for 15 min, permeabilised in PBT-0.25% for 40 min and washed again 3x in PBT-0.1%. The embryos were transferred to blocking solution  overnight (o/n) at 4°C. Primary antibodies  were thein vivo timecourse, GAPDH & HPRT; ES cell differentiation, GAPDH & 18 s  were either designed using Primer-BLAST  or obtained from PrimerBank  or previous publications , whilst for drug treated experiments fold change was calculated relative to control (DMSO) samples. Non-template controls were performed by replacing cDNA with water, to test for non-specific amplification.RNA extraction of whole embryos, embryonic hearts, head folds and ESC samples was performed using an RNeasy Micro Kit  according to manufacturer’s instructions: briefly, homogenisation was carried out with a 21G needle and the extract run through an on-column DNase I treatment. For P0 and adult heart samples, RNA extraction was performed using Trizol , according to the manufacturer’s instructions, additional DNase I  treatment on Trizol-extracted RNAs was carried out to eliminate genomic DNA contamination. In order to collect enough RNA for isolated cardiac crescent and head fold development, each biological replicate was composed of 10 individual cardiac crescents or head folds. For both types of extraction, RNA pellets were dissolved in RNase-free water and the RNA quality and quantity determined by Nanodrop readings at 260, 280 and 230 nm wavelengths. cDNA was generated from 1 μg of RNA using random primers and SuperScript III polymerase (Invitrogen). The expression of mRNAs for genes of interest , togetheH & 18 s  were meaProtein was extracted on ice using direct lysis of cells with NP-40 extraction buffer ) and 1x protease and phosphatase inhibitor cocktail . Lysates were span at 10,000 rpm for 20 min at 4°C and supernatant collected. An aliquot was taken for protein quantification using a DC protein assay . Supernatants were prepared for SDS-PAGE with the addition of 4x Laemmli sample buffer and boiling at 95°C for 5 min. Western blotting was performed using standard SDS-PAGE methods using HRP-conjugated secondary antibodies and enhanced chemiluminescence detection .All data involving beat rate and qPCR gene levels was compared using one-way ANOVA followed by a Tukey test for multiple comparisons. In cases where the raw data failed to map to a normal distribution with consistent variance, we applied Taylor’s law to choose the best transformation for the data. All data to be analysed passed the Shapiro-Wilk normality test and Bartlett test for homogeneous variances. To compare the number of affected embryos upon acute treatments, a Freeman-Halton extension of Fisher exact probability test was applied due to a smaller number of samples. To compare the effect of different treatments on the number of beating EBs, due to a large number of samples, a Chi Square test with a Bonferroni correction for multiple comparisons was performed instead. Principal Component Analysis (PCA) was carried out to directly compare temporal gene expression data from EBs with that derived from embryos; each biological replicate for embryonic stage was composed of 5–6 embryos and for EBs was composed of 10–80 EBs, depending on the day of differentiation. The data from each sample was normalized by assessing the ratio with the maximum value of all samples. A log transformation was applied to the normalized data and the principal components were calculated using R environment. The 3D representation of the PCA was constructed by plotting the 3 first components given that these explained more that 95% of the observed variance. A hierarchical clustering analysis was performed using Ward’s minimum variance method as a more precise clustering algorithm to divide the samples in different groups .2+ signal was subtracted from all frames of a given time-lapse using ImageJ. To obtain the profiles for Ca2+ transients, regions of interest were plotted using the ratio between observed fluorescent and minimum fluorescent (F/F0) after background subtraction.A variant of absolute image filter was used to visualize and plot measurements of cardiac contractions in the developing cardiac crescent as described elsewhere . Briefly In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. Your full submission has been evaluated by Janet Rossant (Senior editor) and three peer reviewers, as well as a member of our Board of Reviewing Editors, and the decision was reached after discussions between the reviewers. Based on our discussions and the individual reviews below, we regret to inform you that your work will not be considered further for publication in eLife.Thank you for choosing to send your work entitled "Calcium handling precedes cardiac differentiation to initiate the first heart beat" for consideration at We appreciate the importance of this study on the establishment of calcium gradients and its implications for cardiogenesis. We also appreciate the technical advances in this paper which presents a multidisciplinary approach and includes functional studies. However, a number of points raised by the reviewers indicate problems with the paper as it stands. Notably new mechanistic insights are weak. There is a lack of data on LTCC Cav1.3. The mechanistic link between early NCX-1 dependent asyncronous activity and maturation of electrical activity and excitation contraction coupling is lacking – blocking experiments in embryo culture are inadequate. There is also a question about novelty. The Introduction is weak and does not adequately cover current available papers on the subject. Reconsideration of experimental approaches in the context of earlier studies should lead to the design of further experiments such as genetic ablation of key components or electrical measurements in embryo culture.eLife for return of a revised manuscript. However, we would encourage you carry out further experiments and if you then feel that you have addressed all the issues, eLife remains open to receiving a revised manuscript in the future for consideration as a new submission.We consider that the issues raised by the reviewers cannot be addressed in the short time frame required by Reviewer #1:The authors present an interesting study demonstrating calcium-mediated contractile activity in the mouse cardiac crescent earlier than previously thought. The study is strengthened by the use of both embryonic data and ESC-derived cardiomyocyte data. The experiments follow a logical progression with the data and figures presented clearly. I recommend accepting the manuscript with revision and additional experiments.While concise scientific communication is appreciated, the Introduction is a bit too concise (one paragraph composed of three sentences). The manuscript would benefit from additional background and justification for the current study.Given the strength of the assertion that this study is a definitive analysis of early crescent contractility, the sarcomere immunostaining should be more thorough and expanded to include other sarcomeric proteins that spatiotemporally indicate sarcomere formation. Although the current data is supportive of the conclusions presented, cTnT alone is not sufficient in this regard. At minimum, immunostaining for α-actinin, a Z-disc protein would much improve and strengthen this data. Further, inclusion of staining for myomesin, an M-band protein would be much more definitive especially if double stained showing alternating actinin/myomesin.The gene expression studies should be complemented by whole mount in situ hybridization. This is especially important for More information is needed to evaluate the gene expression studies. At minimum, primer details, amplicon details, replicates  and PCR parameters should be reported. In the absence of validating the reference genes for qPCR, what is the rationale for choosing HPRT for in vivo samples and GAPDH for ESCs? How is fold change calculated? Is it in relation to E7.5 for embryos and D0 for ESCs? The following references should be helpful in this regard.Johnson G, et al. Methods Mol Biol. 2014;1160:5-17.Bustin SA, et al.. Nat Methods. 2013 Nov;10(11):1063-7.Bustin SA, et al. BMC Mol Biol. 2010 Sep 21;11:74.Reviewer #2:The manuscript addresses the earliest stages of electrical activity in the mammalian heart and ES cell-derived EBs and in particular the relative requirements of the Na/Ca exchanger NCX1 and L-type Ca channel (LTCC). The main implications of the work are when Ca transients are established and how this relates to cardiogenesis. In general, the work is well written and the data interesting and relevant to the above issues. The functional and multidisciplinary approach to cardiac development is a strength. For the most part the experiments are carefully performed and results make a strong contribution.1) The novelty of the findings are diminished somewhat by prior publications on genetic deletion of NCX and LTCC genes in mice and fish. The novelty of this study lies in the detailed assessment of beating, Ca transients, and relative requirements for NCX1 and LTCCs. I recommend that some of the material relegated to the Discussion come forward to the Introduction to make clear the state of the field including the broad findings relating to NCX1 and LTCC pharmacological and genetic inhibition in the context of embryogenesis, and the novelty of the present findings in a summary paragraph. Background studies should cover what has been observed in the chick embryo wrt beating and pacemaker function, and electrophysiology.2) The text is not rigorous in defining the dose of drugs used and the duration of use in each experiment. Different doses and treatments are mentioned. In addressing the relative requirements for NCX1 and LTCCs, dose response data would seem important as different drugs may have different Ki etc. It seems important to address this formally throughout or to do specific experiments that incorporate dose responses.3) Furthermore, rationale is not given for differing doses between experiments e.g. for CB-DMB is used at 20 μm in experiments shown in 4) The experimental design in the experiment described at the end of page 8 onwards does not allow the stated conclusions to be drawn. First, duration of drug treatment is unknown. Second, the design of the sequential experiment is informative but does not allow the authors to conclude that the earlier NCX-1-dependent waves are essential for subsequent differentiation and beating.2 transients in the developing cardiac crescent”. the effects on GFP from Nkx2-5GFP should be quantified, preferably by FACS.5) In the last paragraph of the subsection “NCX1 is essential for the initiation of Ca2 transients in the developing cardiac crescent”. This is a key experiment that addressed an important theme for the paper; the response of the cardiac network to inhibition of NCX1 and LTCC was poorly analysed . Effects could be secondary.6. In the last paragraph of the subsection “NCX1 is essential for the initiation of Ca2 via NCX1 and downstream CAMKII signalling promotes cardiomyocyte differentiation”; inhibition of beating in low Ca is not surprising. How do the results show that Ca is required for maturation and differentiation?7) In the subsection “Inward Ca8) In the subsection “NCX1 is essential for cardiac crescent formation during development”. For such an important point, actin staining of embryos treated with inhibitors and PCR are insufficient to draw the broad conclusion that there is a lack of crescent cells. This experiment needs far more development. What happens to the pre-cardiac cells and cardiac progenitors over time needs to be analysed with appropriate markers?Reviewer #3:2 imaging, gene expression analysis and pharmacological blocking during culture of mouse embryos and embryonic stem cells. The main result is that the sodium-calcium-exchanger (NCX) is important for cardiomyocyte development and function because pharmacological block of NCX reduces Ca2 transients and beating as well as lowers expression of cardiac genes and delays cardiac crescent formation.The manuscript "Calcium handling precedes cardiac differentiation to initiate the first heart beat" by Tyser et al. tries to address the importance of calcium cycling for cardiac crescent formation in the embryonic heart by Ca2 transients from the cardiac crescent prior to beating, the study remains descriptional and the proposed mechanism is based on over interpretation of results and assumptions including drug specificity of long term drug application.Although the manuscript presents major technical advancements in this field including mouse embryo culture and recording of CaMost importantly the authors have not measured or discussed the involvement of voltage signals in cardiomyocytes and several other known facts on the physiology of embryonic heart cells or on the pacemaking mechanism in the embryonic heart.2 Oscillations, a Potential Pacemaking Mechanism in Early Embryonic Heart Cells" Sasse P et al. 2007).The authors write "NCX1, have not previously been implicated in the initiation event of cardiac contraction, nor investigated at the earliest stages of heart development coincident with the onset of beating" and in the Abstract "how the initial contractions are established have not been described". This is incorrect. It is well known that in the embryonic heart cell, a periodic intracellular calcium oscillation (IP3-dependent) is driving pacemaking and translated into voltage fluctuations specifically by the NCX (forward mode of the electrogenic pump). These fluctuations can induce action potentials that synchronize the cardiac tissue and therefore NCX is essential for cardiac development . This has not been shown for embryonic cardiomyocytes and is highly unlikely because of the lack of Na channels or Na driven action potentials at this stage.The main conclusion of the blocker experiments is that NCX reverse mode is important for Ca2 exchange current by KB-R7943 in guinea-pig cardiac myocytes" by Kimura, J et al. 1999) show that this specificity is not existent and other papers show that this is highly depending on intracellular Na concentrations and drug dosage used. Thus without electrophysiological analysis, it remains unclear, if the inhibitory effect on beating and cardiac crescent formation after block of NCX by KB-R or CB-DMB is due to effects on of Ca2 oscillations, pacemaking, translemmal Ca2 entry or exit, membrane potential fluctuation, synchronization by action potentials, imbalance of Ca2 homeostasis or off-target effects that occur especially when applied long term.The (confusing) idea of a reverse mode NCX comes from the literature that state that at low dosages (EC50 in the low µM range) KB-R is preferential blocking a reverse mode. Although oocyte experiments with non-physiological intracellular Na concentrations suggest this, other experiments on cardiomyocytes . In addition to cite these earlier work in introduction it should be better pointed out, why these earlier studies are not sufficient and the current study is required .Also the first paragraph of the section "A role for the Sodium-Calcium exchanger…" belongs to the Introduction. Please describe the well-known ECC in the adult heart a bit less but introduce more the known facts on ECC and the importance of NCX for pacemaking in the embryonic heart (see above).Because of the low resolution of the supplied PDF file, the cardiac maturation and cross striation at the different crescent sages is difficult to review. Maybe providing inserts to highlight the specific patterns  would help. Is cross striation completely absent in stage 0?2 transients at stage 0 is fascinating. Maybe better call them "spontaneous asynchronous Ca2 oscillations" also to highlight the difference semantically to the  coupled transients at stage 1 . What is the average frequency and is there a correlation between frequency and TTP (maybe more interesting to show than TTP vs TT1/2M which lacks a conclusion)? Is there evidence that transients are coupled between individual cells (from the video this seems to be sometimes the case). Although not in the scope of the authors´ manuscript, recording of membrane potential is essential to understand the nature of cell-cell coupling of these fascinating data.The finding of CaUnfortunately, Cacna1d (Cav1.3), which is the dominant isoform of LTCC in the early embryo  was not analyzed and therefore the expression data of Cacna1c does not suggest a minor role of LTCC, but just highlights the well-known isoform switch  Calcium Channel Gene in the Mouse"). The analysis of other LTCC genes or western blot at different stages from heart tissue might help in this regard.The establishment of an embryo culture for investigation of cardiac crescent or heart tube formation as well as for gene expression analysis is a definite plus and a main methodological advancement of this paper. Very nice! However, the fact that "Embryos cultured in CB-DMB were delayed in terms of cardiac crescent formation" is not supported by the one actin image shown  although it is one of the main messages of the paper. Repeating of experiments, extensive histological analysis by specific crescent staining, and quantitation is suggested. Also a time course analysis of crescent formation with and without CB-DMB and KBR would enhance the paper. How many hours are the cardiac crescent forming delayed under NCX blockage?2 transients and remaining Ca2 oscillations .Was the data in In the Discussion the authors have claimed to use "two independent pharmacological channel blockers CB-DMB and KB-R7943, in embryo cultures", but only show CB-DMB data in Figure 6. Please also show the KB-R data on embryonic cultures.The authors describe “sequential addition of CB-DMB to nifedipine-treated embryos after 15 mins, subsequently blocked the transients that were refractory to LTCC inhibition (not shown)." Please provide the data and statistics and explain the mechanism. What is the percentage of transients that were not blocked by LTCC inhibition? Was this observed at all stages or is this stage specific?[Editors’ note: what now follows is the decision letter after the authors submitted for further consideration.]eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Janet Rossant as the Senior Editor. The reviewers have opted to remain anonymous.Thank you for submitting your article "Calcium handling precedes cardiac differentiation to initiate the first heartbeat" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.2 oscillations (SACO) in the cardiac crescent and that these are essential for cardiac gene expression and subsequent development. However, interpretation of the results and the proposal that reverse mode NCX, allowing Ca2 to enter the cell, is involved in the mechanism are still problematic.The manuscript resubmitted after revision is much improved and is now under review with a view to publication of these interesting findings. The authors show that the sodium calcium exchanger (NCX) early, at what they define as stage 0, is essential for generating spontaneous asynchronous Ca2 channels (VDCC), which interestingly at stage 0 has less effect on SACO, in contrast to later stages when it blocks activity. CB-DMB, which blocks NCX, affects SACO at stage 0, but it also blocks Ca2 exit mode. In the manuscript KB-R7943 is claimed to specifically block reverse mode NCX but it was not used at stage 0. This result should be shown. The finding that this inhibitor blocks activity at stages 1 and 2 is not so important because nifedipine does this too. In their interpretation the authors should be aware of controversy about the action of KB-R7943.Firstly, in the experiments with inhibitors, the stage 0 experiments are critical. The authors use nifedipine to block voltage dependent Ca2 cycling frequency and amplitude and how often are single uncoupled  to enable the reader to follow the analysis and statistics. Line 310: What does "0.87 0.083, mean SEM" refer to? How are cells without SACO identified if they do not show Ca2 imaging traces. A DMSO control for stage 3 is missing.The novel 2 transients and reduction of Ca2 transient amplitude. Also please provide time courses and report the delay of drug action .In general, in the text it is important to clarify what is meant by inhibition and partial inhibition.2 entry though NCX is the basis for the SACO, the mechanism of how this could be periodic and lead to oscillations in Ca2 is unclear. The authors are correct that NCX can  lead to Ca2 entry, but once this occurs, the electrogenic nature of NCX (~3 Na for 1 Ca2 ) will again hyperpolarize the cell and export Na until the equilibrium potential is reached and Ca2 flux ceases. Thus it is not possible that the NCX can generate periodic Ca2 oscillations alone. Reverse mode only occurs if periodic action potentials are depolarizing the cell but then cells should be synchronized (no SACO). Maybe Na levels are cycling or the Na/K-ATPase is involved? This could be clarified by testing whether SACO at stage 0 are blocked by RyR/2-APB or by SERCA blockers (Thapsigargin/CPA). These mechanistic considerations should be taken into account in the authors' Discussion.Although the pharmacological block using CB-DMB shown in 2 cycling mechanism and introduce notes of caution in their Discussion. The authors are encouraged to attempt electrophysiological analysis since without this it remains unclear if the inhibitory effect on beating and cardiac crescent formation of NCX by KB-R7945 or CB-DMB is due to effects on SACO, translemmal NCX reverse mode Ca2 entry (mechanism unclear) or NCX forward mode Ca2 exit , imbalance of Ca2 homeostasis or off-target effects. These analyses as well as straight forward siRNA experiments, proposed in the first reviews prior to resubmission, should be feasible with the elegant embryo culture technology set up by the authors.On the mechanistic front, as it stands the paper does not permit definitive conclusions; the authors must be less dogmatic about the SACO/CaA second aspect which requires further revision is the gene expression data. This should be normalised relative to housekeeping control genes. Normalising to E7.75 when most genes are not expressed and there is mainly noise is not informative, preventing meaningful comparison between genes. For instance, in the current presentation the time course of NCX and Cav1.3 seems to be superimposable. The data should be represented as dCT  and then the meaningful differences in time course/relative levels discussed.eLife.Thank you for resubmitting your work entitled "Calcium handling precedes cardiac differentiation to initiate the first heartbeat" for further consideration at Before publication, the following remaining concerns of one reviewer will need to be addressed:2 -handling in the cardiac crescent” ("variable frequencies and durations"). From the authors comment in the rebuttal and from 2 transients at later stages (1) Please provide information on the frequency, duration and regularity of SACO at stage 0 as requested and as stated in the subsection “The onset of Car stages  the SACO2 rises slowly and reaches the peak between 0.5 and 10 s. Cells with slow Ca2 influx also showed similar slow Ca2 efflux (2) Please describe the meaning of 2 efflux ."3) Because of the very slow nature of SACO, also oscillations in energy production and ATP levels can be involved (in fact the most likely explanation of SR is not involved). Please add this to the Discussion (fourth paragraph).2 releases from the SR are involved in the generation of SACO as reported before as a pacemaking mechanism in early embryonic cardiomyocytes (cite adequate references).4) The authors state that Ryanodine 2-APB only affected embryos that had already undergone cardiac looping but the earliest stage tested was stage 3. Although requested, the effect of Ryanodine 2-APB on SACO was not analyzed. Thus it seems to be fair to include in the revised Discussion on the SACO mechanism (fourth paragraph) something like: "Because we have not tested the involvement of SR function at stage 0, we cannot fully exclude that periodic Ca [Editors’ note: the author responses to the previous round of peer review follow.]We appreciate the importance of this study on the establishment of calcium gradients and its implications for cardiogenesis. We also appreciate the technical advances in this paper which presents a multidisciplinary approach and includes functional studies. However, a number of points raised by the reviewers indicate problems with the paper as it stands. Notably new mechanistic insights are weak.in vitro analyses of isolated cells . In revision, we have now added additional experiments using the inhibitors Ryanodine and 2-APB to target the sarcoplasmic reticulum (SR) as a source of Ca2 and whilst we found these inhibited cardiac function at later stages of heart development (in turned embryos at ~E8.5), there was no effect at the earliest onset of Ca2 handling and contractile function. This new data suggests that mechanistically the SR is not required for the initiation of spontaneous asynchronous calcium oscillations (SACOs) or Ca2 transients within the cardiac crescent. A requirement for SR function develops at later stages and further supports the role of NCX in initiating the earliest Ca2 influx and the generation of SACOs.Regarding the editorial summary on mechanistic insight, our study is the first to identify the onset of cardiac function and physiology within the embryo at significantly earlier stages of heart development (during formation of the cardiac crescent) than previously described. Prior studies have focused on linear heart tube looping (E8.5) stages and have relied extensively on In this study we present the following:We attribute a mechanism for the onset of Ca2 handling and cardiac function at significantly earlier stages of heart development than previously appreciated;in vitro studies;Our data from culture and imaging of live embryos is representative of the in vivo setting and avoids caveats associated with isolated cell preparations and We exclude an essential role for the SR and reveal that sarcolemmal Ca2flux via NCX, and then sequentially LTCCs, predominates within the early developing heart as distinct from adult modes of Ca2 -handling;NCX alone is required for the generation of SACOs prior to the initiation of contraction.There is a lack of data on LTCC Cav1.3.2 handling. Application of Nifedipine at stage 0, did not inhibit SACOs in the forming cardiac crescent, excluding an early role for both Cav1.2 and Cav1.3.We have now included detailed expression profiling of the LTCC subunit Cav1.3, in the revised manuscript. However, whilst this is of interest, our use of Nifedipine blocks all LTCC isoforms, inhibiting both Cav1.2 and Cav1.3 and therefore, is a viable reagent to assess a general role for LTCCs in early CaThe mechanistic link between early NCX-1 dependent asyncronous activity and maturation of electrical activity and excitation contraction coupling is lacking – blocking experiments in embryo culture are inadequate. There is also a question about novelty.2 -handling and contractile function feed back onto cardiomyocyte differentiation/maturation and early cardiac morphogenesis functioning.Whilst we are interested in understanding how SACOs mature into propagating synchronized transients, this was not the focus in this study and beyond the scope of the current manuscript. The focus on the study was detailing how cardiac function is first initiated and the downstream consequences of this in terms of how CaThe Introduction is weak and does not adequately cover current available papers on the subject. Reconsideration of experimental approaches in the context of earlier studies should lead to the design of further experiments such as genetic ablation of key components or electrical measurements in embryo culture.The Introduction has been re-written to add more detail, citing the existing studies on the development of cardiac function and more comprehensively addressing those that have established the current dogma of contraction occurring at the linear heart tube stage.Reviewer #1:The authors present an interesting study demonstrating calcium-mediated contractile activity in the mouse cardiac crescent earlier than previously thought. The study is strengthened by the use of both embryonic data and ESC-derived cardiomyocyte data. The experiments follow a logical progression with the data and figures presented clearly. I recommend accepting the manuscript with revision and additional experiments.We thank the reviewer for the positive comments and suggestion to accept the manuscript with revision and additional experiments. We have now addressed the major points below with further detailed experimental analyses and significant new data.While concise scientific communication is appreciated, the Introduction is a bit too concise (one paragraph composed of three sentences). The manuscript would benefit from additional background and justification for the current study.We have now expanded the Introduction to cover more background and justification for this study.Given the strength of the assertion that this study is a definitive analysis of early crescent contractility, the sarcomere immunostaining should be more thorough and expanded to include other sarcomeric proteins that spatiotemporally indicate sarcomere formation. Although the current data is supportive of the conclusions presented, cTnT alone is not sufficient in this regard. At minimum, immunostaining for α-actinin, a Z-disc protein would much improve and strengthen this data. Further, inclusion of staining for myomesin, an M-band protein would be much more definitive especially if double stained showing alternating actinin/myomesin.We thank the reviewer for the suggestions and have now included a developmental timecourse of sarcomeric α-actinin and Myomesin immunostaining, which has allowed us to more conclusively define sarcomere formation  and PCR parameters should be reported. In the absence of validating the reference genes for qPCR, what is the rationale for choosing HPRT for in vivo samples and GAPDH for ESCs? How is fold change calculated? Is it in relation to E7.5 for embryos and D0 for ESCs? The following references should be helpful in this regard.Johnson G, et al. Methods Mol Biol. 2014;1160:5-17.Bustin SA, et al.. Nat Methods. 2013 Nov;10(11):1063-7.Bustin SA, et al. BMC Mol Biol. 2010 Sep 21;11:74.We have now included further details to more comprehensively evaluate our gene expression studies . The main implications of the work are when Ca transients are established and how this relates to cardiogenesis. In general, the work is well written and the data interesting and relevant to the above issues. The functional and multidisciplinary approach to cardiac development is a strength. For the most part the experiments are carefully performed and results make a strong contribution.We thank the reviewer for their positive comments and for highlighting both the strength of using a multidisciplinary approach to address cardiac development as well as the view that these results make a strong contribution to the field.1) The novelty of the findings are diminished somewhat by prior publications on genetic deletion of NCX and LTCC genes in mice and fish. The novelty of this study lies in the detailed assessment of beating, Ca transients, and relative requirements for NCX1 and LTCCs. I recommend that some of the material relegated to the Discussion come forward to the Introduction to make clear the state of the field including the broad findings relating to NCX1 and LTCC pharmacological and genetic inhibition in the context of embryogenesis, and the novelty of the present findings in a summary paragraph. Background studies should cover what has been observed in the chick embryo wrt beating and pacemaker function, and electrophysiology.2 -handing and contraction given the varied lethality. There is also insufficient evidence in these prior studies to assess whether complete KO has occurred and/or whether compensation is occurring via NCX1 splice variants, other NCX isoforms or alternative mechanisms of Ca2 influx. By using pharmacological inhibition (with two independent specific NCX-inhibitors) at defined stages of development, we are able to rule out any compensatory effects arising from germ-line genetic ablation and moreover, simultaneously target multiple isoforms to converge on a role for NCX during the onset of cardiac function.We thank the reviewer for highlighting the novelty of our manuscript and have rewritten the introduction accordingly. In terms of this paper’s findings being diminished by prior publications on genetic deletion models in mice and fish, it is important to note that genetic loss of function for NCX1 has produced a wide array of embryonic heart phenotypes, despite the fact that all the published studies targeted the same exon of NCX1. The hearts of the various NCX1 mutants range from arresting at the linear heart tube stage to completed looping morphogenesis and there is no insight into a putative early role in establishing Ca2) The text is not rigorous in defining the dose of drugs used and the duration of use in each experiment. Different doses and treatments are mentioned. In addressing the relative requirements for NCX1 and LTCCs, dose response data would seem important as different drugs may have different Ki etc. It seems important to address this formally throughout or to do specific experiments that incorporate dose responses.in vitro cell-based assays. We tended to use slightly higher concentrations, albeit within the same µm range, due to penetration issues when using whole embryos. It is reassuring to note that whilst NCX inhibitors blocked transients at early stages, the effect was lost at later stages, thus excluding non-specific effects such as impaired cell survival, etc.We appreciate the problems with using pharmacological inhibition and hope that the revised manuscript is more thorough in defining the doses used. Drugs were selected based on previous publications which also defined the optimal working concentrations as effective when applied to 3) Furthermore, rationale is not given for differing doses between experiments e.g. for CB-DMB is used at 20 μm in experiments shown in 2 measurements. Unfortunately, we are unable to definitively measure the concentration of drug to which the tissue was actually exposed.We apologise for the confusion; 1mM was an error, which has now been corrected. The highest concentration of CB-DMB used was 20 µM in the acute inhibitor experiments. The reason why higher concentrations were used when working with whole embryos is due to the penetration issues, associated with performing in vivo embryo Ca4) The experimental design in the experiment described at the end of page 8 onwards does not allow the stated conclusions to be drawn. First, duration of drug treatment is unknown. Second, the design of the sequential experiment is informative but does not allow the authors to conclude that the earlier NCX-1-dependent waves are essential for subsequent differentiation and beating.2 transients within the forming cardiac crescent are dependent upon NCX1”) to address this issue. From these new experiments we are able to definitively conclude that NCX1-dependent waves are required for the generation of SACOs which precedes LTCC function. Drug treatment was maintained for 15 mins and assessed at both 5 mins and 15 mins as now stated in the aforementioned subsection and the revised Methods.We thank the reviewer for pointing this out and have included new experiments in revised 2 transients in the developing cardiac crescent”. the effects on GFP from Nkx2-5GFP should be quantified, preferably by FACS.5) In the last paragraph of the subsection “NCX1 is essential for the initiation of Ca2 channel blockade inhibits ESC differentiation into cardiomyocytes”). We have carried out independent sets of experiments using two entirely distinct ESC lines and observed the same phenotypes.We have now quantified Nkx2.5-GFP by assessing the percentage of GFP area across multiple images taken from different experiments . Effects could be secondary.6. In the last paragraph of the subsection “NCX1 is essential for the initiation of CaSlc8a1, Cacna1c and CamKIId. We initially focused on a single end point when control cardiomyocyte would have been expected to be at their most differentiated. In revision, we have now also included a time course of ESC differentiation with and without CB-DMB, to more carefully analyse the effect of inhibition. This new data reveals that inhibition of NCX occurs during the initiation of cardiomyocyte differentiation between day 4 and 7 and supports our previous conclusions  In the subsection “Inward Ca2 , as stated in the Methods, therefore a reduced percentage of beating embryoid bodies would represent a decrease in cardiomyocyte formation and would not be just a physiological consequence of low Ca2 .Percentage of beating embryoid body analysis was performed in media containing 1.8 mM Ca8) In the subsection “NCX1 is essential for cardiac crescent formation during development”. For such an important point, actin staining of embryos treated with inhibitors and PCR are insufficient to draw the broad conclusion that there is a lack of crescent cells. This experiment needs far more development. What happens to the pre-cardiac cells and cardiac progenitors over time needs to be analysed with appropriate markers?We have now repeated these experiments for multiple embryos, using immunohistochemistry for the contractile protein cTnT as a marker of differentiation and the transcription factor Nkx2.5 as a marker of cardiac progenitors within the crescent  is important for cardiomyocyte development and function because pharmacological block of NCX reduces Ca2 transients and beating as well as lowers expression of cardiac genes and delays cardiac crescent formation.The manuscript "Calcium handling precedes cardiac differentiation to initiate the first heart beat" by Tyser et al. tries to address the importance of calcium cycling for cardiac crescent formation in the embryonic heart by Ca2 transients from the cardiac crescent prior to beating, the study remains descriptional and the proposed mechanism is based on over interpretation of results and assumptions including drug specificity of long term drug application.Although the manuscript presents major technical advancements in this field including mouse embryo culture and recording of Ca2 transients from the cardiac crescent prior to beating. We have had to include detailed descriptions of what we observe at the very earliest stages of cardiac crescent formation, Ca2 handling and contractility given this has not been described before and is attributed to our advanced live imaging of mouse embryos. The pharmacological blockade of NCX1 and LTCCs provides mechanistic insight into the requirement for the exchanger, acting in reverse mode, to initiate Ca2 influx and the appearance of SACOs prior to full transients and contractility within the crescent. Any issue with drug specificity is significantly reduced by our use of two independent inhibitors for NCX and the fact that we observe consistent results across independent models of mouse embryos and ESC-cardiomyocyte differentiation.We are grateful for the positive comment regarding major technical advancements in this field including mouse embryo culture and recording of CaMost importantly the authors have not measured or discussed the involvement of voltage signals in cardiomyocytes and several other known facts on the physiology of embryonic heart cells or on the pacemaking mechanism in the embryonic heart.2 handling and asynchronous oscillations (without pacing) as precedes contractile function. We have not made any statements regarding voltage signals, as we have not developed the whole tissue approach to record them in early embryos and we respectfully suggest this is beyond the scope of the current manuscript.Pace-making appears not to apply to stages earlier than the looping heart tube, consistent with our focus on the initiation of Ca2 Oscillations, a Potential Pacemaking Mechanism in Early Embryonic Heart Cells" Sasse P et al. 2007).The authors write "NCX1, have not previously been implicated in the initiation event of cardiac contraction, nor investigated at the earliest stages of heart development coincident with the onset of beating" and in the Abstract "how the initial contractions are established have not been described". This is incorrect. It is well known that in the embryonic heart cell, a periodic intracellular calcium oscillation (IP3-dependent) is driving pacemaking and translated into voltage fluctuations specifically by the NCX (forward mode of the electrogenic pump). These fluctuations can induce action potentials that synchronize the cardiac tissue and therefore NCX is essential for cardiac development  and moreover, have examined these cells in isolation from the embryo. We have done additional experiments with IP3 and RyR inhibitors and indeed, we have confirmed their findings of a later requirement of IP3 signalling (that serves as a positive control for our inhibitors), but have also made the novel finding that during the earlier stages that constitute the main focus of our manuscript, SR Ca2 entry and gene regulation but how this should induce periodic beating or pacemaking has not been addressed or discussed. NCX and can only work in reverse mode when intracellular Na concentration is excessively high during an action potential . This has not been shown for embryonic cardiomyocytes and is highly unlikely because of the lack of Na channels or Na driven action potentials at this stage.The main conclusion of the blocker experiments is that NCX reverse mode is important for Ca2 influx, the establishment of Spontaneous Asynchronous Ca2 Oscillations (SACOs) prior to beating, and how this impacts on cardiomyocyte differentiation and crescent morphogenesis. The presence of SACOs precedes pacemaker activity and synchronisation which, are outside the focus and scope of this manuscript.We are focusing on the earliest onset of Ca2 and Na in addition to the membrane potential . The conditions that favour reverse mode NCX1 activity include less depolarised membrane potentials. Reversal potential, for NCX1 is around -26mV. In terms of NCX1 function this means that, if the membrane potential was lower than -26mV, “forward mode” activity (Ca2 efflux) would occur, however at membrane potentials above -26mV “reverse-mode” activity (Ca2 influx) would predominate. Resting membrane potential of immature cardiomyocytes is known to be significantly higher than the adult (-85mV). Embryonic cardiomyocytes at around E10.5 have been shown to have a resting membrane potential of around -40mV , with less differentiated cardiomyocytes potentially having an even greater than -40mV membrane potential, increasing the likelihood and potential functional relevance of “reverse mode” NCX1 activity as well as making it more susceptible to changes in the concentration of intracellular Ca2 and Na. Thus NCX1 function within the stage 0 cardiac crescent could be responsible for both the Ca2 influx and efflux, correlating with the slow dynamics of stage 0 Ca2 transients, potentially regulating Ca2 homeostasis until contraction is initiated.NCX has been shown to act in both forward and reverse mode in embryonic cardiomyocytes  and the directionality of NCX1 activity is dependent on the concentration gradient of intra- and extra-cellular CaWe have qualified our interpretation of NCX1 acting in reverse mode in the revised text:2 influx at this early stage.”“While the existence of the NCX reverse mode is contentious, the observation that at stage 0 SACOs were abolished with CB-DMB treatment but persisted when treated with nifedipine suggests that NCX and not the LTCC plays a major role in Ca2 exchange current by KB-R7943 in guinea-pig cardiac myocytes" by Kimura, J et al. 1999) show that this specificity is not existent and other papers show that this is highly depending on intracellular Na concentrations and drug dosage used. Thus without electrophysiological analysis, it remains unclear, if the inhibitory effect on beating and cardiac crescent formation after block of NCX by KB-R or CB-DMB is due to effects on of Ca2 oscillations, pacemaking, translemmal Ca2 entry or exit, membrane potential fluctuation, synchronization by action potentials, imbalance of Ca2 homeostasis or off-target effects that occur especially when applied long term.The (confusing) idea of a reverse mode NCX comes from the literature that state that at low dosages (EC50 in the low µM range) KB-R is preferential blocking a reverse mode. Although oocyte experiments with non-physiological intracellular Na concentrations suggest this, other experiments on cardiomyocytes . To this end we have revised the manuscript to reflect this (see response to comment above).In summary, the presented data, although in part novel and interesting, together with the known literature on embryonic cardiomyocytes and on the physiology of ECC, LTCC and NCX function does not allow to conclude a mechanism.We thank the reviewer for the positive comments on our data being novel and interesting. Given the extensive new data and our revised interpretation we believe we have now converged on a mechanism whereby NCX1 is required for the establishment of SACOs within the earliest crescent stages as a pre-requisite for cardiomyocyte differentiation and crescent morphogenesis.Specific major points:The Introduction is too short and only very general. Specifically, several known facts on initiation of the first heart beat and the role of calcium gene regulation during embryonic heart development should be cited. For instance, several earlier publications have tried to address "the important question of when and how contractile activity of cardiomyocytes is first initiated during development" (see above). In addition to cite these earlier work in introduction it should be better pointed out, why these earlier studies are not sufficient and the current study is required .Also the first paragraph of the section "A role for the Sodium-Calcium exchanger…" belongs to the Introduction. Please describe the well-known ECC in the adult heart a bit less but introduce more the known facts on ECC and the importance of NCX for pacemaking in the embryonic heart (see above).We appreciate the reviewer’s suggestions and hope that the revised manuscript now incorporates more thoroughly previous literature (Introduction) as well as clearly stating the novelty of the studies presented herein.Because of the low resolution of the supplied PDF file, the cardiac maturation and cross striation at the different crescent sages is difficult to review. Maybe providing inserts to highlight the specific patterns  would help. Is cross striation completely absent in stage 0?We have now looked in more detail at cardiac maturation and sarcolemmal development within the developing cardiac crescent. In the revised manuscript we have now included analyses of two sarcomere related proteins (myomesin and sarcomeric α-actinin) as well as cardiac troponin T. In addition, we have performed qRT-PCR to assess the expression of these genes during crescent development. In both the revised 2 transients at stage 0 is fascinating. Maybe better call them "spontaneous asynchronous Ca2 oscillations" also to highlight the difference semantically to the  coupled transients at stage 1 . What is the average frequency and is there a correlation between frequency and TTP (maybe more interesting to show than TTP vs TT1/2M which lacks a conclusion)? Is there evidence that transients are coupled between individual cells (from the video this seems to be sometimes the case). Although not in the scope of the authors´ manuscript, recording of membrane potential is essential to understand the nature of cell-cell coupling of these fascinating data.The finding of Ca2 oscillations" (SACOs) to describe Ca2 “transients” at stage 0. We have now edited the figure containing characterisation of stage 0 transients and no longer include the data relating to TTP and TT1/2M. The revised We thank the reviewer for their positive comments and have taken on-board the suggested terminology of "spontaneous asynchronous CaAfter analysing the qRT-PCR data in multiple different ways we do not find an increase in noise when comparing at time point 0. Within the ESC model, there is variation in the extent of differentiation between cultures, which becomes greater the longer the differentiation time-course, therefore comparing to the latest stage (e.g. D14), as befits higher expression values, incurs a greater variation between experiments. This same applies to the longer-term embryo culture experiments.Unfortunately, Cacna1d (Cav1.3), which is the dominant isoform of LTCC in the early embryo  was not analyzed and therefore the expression data of Cacna1c does not suggest a minor role of LTCC, but just highlights the well-known isoform switch  Calcium Channel Gene in the Mouse"). The analysis of other LTCC genes or western blot at different stages from heart tissue might help in this regard.Cacna1c). The previous suggestion of a “minor role” for LTCC was based on the fact that the NCX inhibitor (CB-DMB) had a significant inhibitory effect on SACOs as compared to LTCC inhibition (Nifedipine) or control (DMSO). This is now further reinforced in the revised 2 handling.We thank the reviewer for this suggestion and have now investigated the expression of Cav1.3  although it is one of the main messages of the paper. Repeating of experiments, extensive histological analysis by specific crescent staining, and quantitation is suggested. Also a time course analysis of crescent formation with and without CB-DMB and KBR would enhance the paper. How many hours are the cardiac crescent forming delayed under NCX blockage?We thank the reviewer for their positive feedback on the embryo culture experiments. As suggested, we have further expanded these experiments on multiple cultured embryos per treatment  and performed immunostaining for Nkx2.5 to mark cardiac progenitors within the forming crescent and cardiac troponin T to mark the more differentiated cardiomyocytes. These experiments revealed a significant decrease in the number of cTnT cells in CB-DMB-treated embryos in comparison to control or Nifedipine treatment revised  and clea2 transients and remaining Ca2 oscillations .Was the data in 2 imaging was used with differential interference contrast (DIC) microscopy to obtain bright-field images. Imaging was conducted at 5 minutes after drug application to allow for diffusion and penetration of the embryo. Unfortunately, our current in vivo imaging setup does not allow us to image instantaneously after drug application, meaning we cannot currently observe acute changes in Ca2 dynamics which would be informative. Attempts to continuously image a single embryo impacted on viability and cardiac function due to excessive photo- damage. Instead we have focused on imaging multiple embryos  and the fact that during later stages the inhibitors seem to have the same effect, we focused on CB-DMB to reduce the number of animals used for the experiments and to enable comparisons to be drawn with the parallel experiments conducted in ESC- cardiomyocyte cultures.The authors describe “sequential addition of CB-DMB to nifedipine-treated embryos after 15 mins, subsequently blocked the transients that were refractory to LTCC inhibition (not shown)." Please provide the data and statistics and explain the mechanism. What is the percentage of transients that were not blocked by LTCC inhibition? Was this observed at all stages or is this stage specific?These sequential inhibitor experiments were initially carried out, but have since been superseded by a more rigorous focus on the independent inhibitor studies, in light of the evident early versus later roles for NCX1 versus LTCCs. We have since edited the manuscript to remove the sequential inhibitor data.[Editors’ note: the author responses to the re-review follow.]2 oscillations (SACO) in the cardiac crescent and that these are essential for cardiac gene expression and subsequent development. However, interpretation of the results and the proposal that reverse mode NCX, allowing Ca2 to enter the cell, is involved in the mechanism are still problematic.The manuscript resubmitted after revision is much improved and is now under review with a view to publication of these interesting findings. The authors show that the sodium calcium exchanger (NCX) early, at what they define as stage 0, is essential for generating spontaneous asynchronous CaWe thank the reviewers and editors for the assessment that the manuscript is much improved following the first round of revisions and for the progression towards review to publish our “interesting findings”. We have now toned down our interpretations on mechanism of reverse NCX1 function in the revised text as requested. In addition, we now include an additional set of KB-R7943 inhibitor experiments on stage 0 embryos and have re-normalised all of our real time qPCR gene expression data in line with the reviewer requests. Please find a detailed point-for-point response to the remaining issues detailed below.2 channels (VDCC), which interestingly at stage 0 has less effect on SACO, in contrast to later stages when it blocks activity. CB-DMB, which blocks NCX, affects SACO at stage 0, but it also blocks Ca2 exit mode. In the manuscript KB-R7943 is claimed to specifically block reverse mode NCX but it was not used at stage 0. This result should be shown. The finding that this inhibitor blocks activity at stages 1 and 2 is not so important because nifedipine does this too. In their interpretation the authors should be aware of controversy about the action of KB-R7943.Firstly, in the experiments with inhibitors, the stage 0 experiments are critical. The authors use nifedipine to block voltage dependent CaWe have now performed follow-up experiments using KB-R7943 at stage 0, and have included this data in the revised 2 cycling frequency and amplitude and how often are single uncoupled  and the time to ½ maximum Ca2 signal (Ca2 efflux)  as well as more data on the Ca efflux) . In gene2 signals? How is the variance of cells with SACO per embryo? ANOVA comparisons are required to take these variations in control embryos into account.The experiments on blocking SACO with CB-DMB in contrast to nifedipine lead to one of the most important findings, however the analysis and statistics are very limited. Please provide absolute numbers of cells with SACO and original data  to enable the reader to follow the analysis and statistics. Line 310: What does " 0.87 0.083, mean SEM" refer to? How are cells without SACO identified if they do not show Ca2 transients within the forming cardiac crescent are dependent upon NCX1”, last paragraph). We have also included a supplementary table of the original data analyses to document absolute number of SACOs observed before and after treatment, ratio of SACOs maintained after treatment, percentage inhibition, area containing SACOs and length of imaging . The term ‘turned’ is well accepted within the mouse embryology field and refers to a specific morphogenetic event when the entire embryo turns to assume a more conventional ‘foetal position’ within the yolk sac, curled up around its ventral aspect. The missing statistics, original Ca2 imaging traces and a DMSO control for stage 3 have now been included in a new We apologise that the later staging was confusing. This data was collected using embryos in which the heart had begun to loop, which is much later than the LHT stage and is why we cannot use our new staging method (applies to stages up to LHT). Stages later than the LHT represent a positive control for the Sarcoplasmic Reticulum inhibitor experiments, which we found had no effect at the earlier stage, .In general, in the text it is important to clarify what is meant by inhibition and partial inhibition.2 transients – and hence we have performed a Freeman-Halton extension of the Fisher exact probability test as the correct statistical analysis for this data type. We have also edited the text so that all inhibition data is based on Ca2 transient propagation as it is a clearer to assess the effect of pharmacological blockade. The Ca2 imaging data has now been reanalysed and we have revised 2 transient amplitude given the method for imaging Ca2 transients in intact tissue does not allow for ratiometric measurements.We have now included an explicit definition of what is meant by% inhibition in the Methods section  and have removed the term partial inhibition as we appreciate this was misleading. The reason for no error bars is that our data is binary either – there were or were not Ca2 entry though NCX is the basis for the SACO, the mechanism of how this could be periodic and lead to oscillations in Ca2 is unclear. The authors are correct that NCX can  lead to Ca2 entry, but once this occurs, the electrogenic nature of NCX (~3 Na for 1 Ca2 ) will again hyperpolarize the cell and export Na until the equilibrium potential is reached and Ca2 flux ceases. Thus it is not possible that the NCX can generate periodic Ca2 oscillations alone. Reverse mode only occurs if periodic action potentials are depolarizing the cell but then cells should be synchronized (no SACO). Maybe Na levels are cycling or the Na/K-ATPase is involved? This could be clarified by testing whether SACO at stage 0 are blocked by RyR/2-APB or by SERCA blockers (Thapsigargin/CPA). These mechanistic considerations should be taken into account in the authors' Discussion.Although the pharmacological block using CB-DMB shown in 2 entry may involve other factors beyond NCX1, however, we have now shown, using two different inhibitors, that NCX is required for the generation of SACOs within the cardiac crescent at stage 0. We have taken into account the limitations on mechanistic insight as suggested and have addressed them within the revised Discussion, as well as detailing further experiments which would be of interest, but are currently beyond the scope of this manuscript.We agree with the reviewer that the precise mechanism for periodic oscillations of Ca2 cycling mechanism and introduce notes of caution in their Discussion. The authors are encouraged to attempt electrophysiological analysis since without this it remains unclear if the inhibitory effect on beating and cardiac crescent formation of NCX by KB-R7945 or CB-DMB is due to effects on SACO, translemmal NCX reverse mode Ca2 entry (mechanism unclear) or NCX forward mode Ca2 exit , imbalance of Ca2 homeostasis or off-target effects. These analyses as well as straight forward siRNA experiments, proposed in the first reviews prior to resubmission, should be feasible with the elegant embryo culture technology set up by the authors.On the mechanistic front, as it stands the paper does not permit definitive conclusions; the authors must be less dogmatic about the SACO/Ca2 cycling mechanism as well as introducing notes of caution within the revised Discussion. The siRNA experiments suggested by the reviewer, though elegant, are not straightforward. They involve optimisation of a delivery method , followed by prolonged culture to ensure knock-down of transcript and clearance of protein through turnover, only after which can the experiments be attempted. It is not clear that embryos subjected to such interventions will retain normal electrophysiological behaviour and what level of knock-down is required to ablate function. Therefore, whilst such experiments would be of interest and something we plan to pursue in the future we believe they are beyond the scope of this manuscript given the aims of the current study: to understand the effects of Ca2 handling and early contractile function on downstream cardiomyocyte differentiation and morphogenesis, during which we have provided strong evidence implicating a role for NCX1.We have now reworded the text to be less dogmatic regarding the SACO/CaA second aspect which requires further revision is the gene expression data. This should be normalised relative to housekeeping control genes. Normalising to E7.75 when most genes are not expressed and there is mainly noise is not informative, preventing meaningful comparison between genes. For instance, in the current presentation the time course of NCX and Cav1.3 seems to be superimposable. The data should be represented as dCT  and then the meaningful differences in time course/relative levels discussed.We have now repeated all the analysis of qRT-PCR data and expressed everything as deltaCT as well as normalising only to housekeeping genes as suggested above by the reviewer(s).[Editors’ note: the author responses to the re-review follow.]Before publication, the following remaining concerns of one reviewer will need to be addressed:2 -handling in the cardiac crescent” ("variable frequencies and durations"). From the authors comment in the rebuttal and from 2 transients at later stages (1) Please provide information on the frequency, duration and regularity of SACO at stage 0 as requested and as stated in the subsection “The onset of Car stages  the SACO2 transients at later stages  Please describe the meaning of 2 efflux ."We have incorporated an explanation similar to that suggested above about “SACOs were significantly slower, with fluorescence reaching peak intensity between 0.79 and 11.9 s and decreasing with a similar slow rate of efflux.”3) Because of the very slow nature of SACO, also oscillations in energy production and ATP levels can be involved (in fact the most likely explanation of SR is not involved). Please add this to the Discussion (fourth paragraph).We thank the reviewer for suggesting another potential mechanism of SACO generation and have now included this in the discussion on page 15:2 transients, we have not tested the involvement of SR function at stage 0 and, therefore, cannot fully exclude that periodic Ca2 releases from the SR are involved in the generation of SACOs. The latter has been reported in cultured E8.5-9 embryonic cardiomyocytes as a pacemaking mechanism , at later stages than were the focus in this study.”“Due to the slow nature of SACOs, oscillations in energy production along with adenosine triphosphate levels could also be involved in SACO generation, especially with reduced SR function. Whilst SR inhibition did not prevent stage 3 Ca2 releases from the SR are involved in the generation of SACO as reported before as a pacemaking mechanism in early embryonic cardiomyocytes (cite adequate references).4) The authors state that Ryanodine 2-APB only affected embryos that had already undergone cardiac looping but the earliest stage tested was stage 3. Although requested, the effect of Ryanodine 2-APB on SACO was not analyzed. Thus it seems to be fair to include in the revised Discussion on the SACO mechanism (fourth paragraph) something like: "Because we have not tested the involvement of SR function at stage 0, we cannot fully exclude that periodic Ca“we have not tested the involvement of SR function at stage 0 and, therefore, cannot fully exclude that periodic Ca2 releases from the SR are involved in the generation of SACOs”.However, we respectfully disagree with inclusion of the “reported before” statement, since the two studies which have previously looked at the role of SR in pace making have studied cardiomyocytes cultured for 12 – 70 hours and collected at a significantly later time point (E8.5/E9) as stated in the Introduction. We, therefore, feel that referring to these studies directly against our early stage analyses is inappropriate; albeit our results recapitulate theirs when looking at the more mature hearts (i.e. looping at E8.5).We agree with the reviewer that we have not tested SR inhibition within the stage 0 embryo and have, therefore, included the following statement:


Decreased energy production and increased oxidative stress are considered to be major contributors to aging and aging-associated pathologies. The role of mitochondrial calcium homeostasis has also been highlighted as an important factor affecting different pathological conditions. Here, we present evidence that loss of a small mitochondrial protein Fus1 that maintains mitochondrial homeostasis results in premature aging, aging-associated pathologies, and decreased survival. We showed that Fus1KO mice develop multiple early aging signs including lordokyphosis, lack of vigor, inability to accumulate fat, reduced ability to tolerate stress, and premature death. Other prominent pathological changes included low sperm counts, compromised ability of adult stem cells to repopulate tissues, and chronic inflammation. At the molecular level, we demonstrated that mitochondria of Fus1 KO cells have low reserve respiratory capacity (the ability to produce extra energy during sudden energy demanding situations), and show significantly altered dynamics of cellular calcium response.Our recent studies on early hearing and memory loss in Fus1 KO mice combined with the new data presented here suggest that calcium and energy homeostasis controlled by Fus1 may be at the core of its aging-regulating activities. Thus, Fus1 protein and Fus1-dependent pathways and processes may represent new tools and targets for anti-aging strategies. Ample evidence suggests that defects in mitochondrial activities occur during aging -4. Howev2+, NO, NAD, etc. c) c while mitochondrial Ca2+ ([Ca2+]m) dynamics was analyzed by co-staining of cells with MitoTracker Green  and Rhod-2  . Fura-2,d-2 Fig. .2+]c response and a 50% higher amplitude of initial [Ca2+]m elevation as compared to WT cells  elevations and low-amplitude oscillations (Osc), were observed  and increase (SI), steady-state (SS) level, and oscillations (Osc)  Fig. . Since tern Fig. . Howeverern Fig.  while Fuges Fig. .+/Ca2+ exchanger (mNCX) that exports Ca2+ from mitochondria in exchange for Na+ [2+]m response to LPS in Fus1 KO MEFs was due to elevated activity of mNCX, we pre-treated MEFs with CGP37157, a chemical inhibitor of mNCX. Indeed, pharmacological suppression of mNCX in LPS-treated Fus1 KO MEFs resulted in suppression of SI-type of [Ca2+]m changes shifting the LPS-induced response towards SS levels of [Ca2+]m  to identify a set of human genes that strongly correlate with the Fus1 gene expression. The first 500 probes that overall recognize 386 genes showed Fus1 co-expression coefficient with p-values between 2.6e-61 and 3.2e-29. Thus, we analyzed this 386 gene set with GSEA  software (http://software.broadinstitute.org/gsea/msigdb/annotate.jsp) to identify Fus1-associated molecular processes and diseases. Inquiry to identify the “hallmark gene sets” came up with the “oxidative phosphorylation” process as number one on the list with the p ≥ 4.57e-54 unequivocally confirming the association of Fus1 expression with the main mitochondrial functions - respiration and energy production. Next on the list were “DNA repair”, “adipogenesis”, “unfolded protein response”, “UV radiation response” as well as a groups of proteins involved in “glycolysis and gluco-neogenesis”  software  and found that Fus1 levels decline in aging muscle tissue. These data support the clinical significance of Fus1 and its link with aging that may be related to a compromised mitochondrial activity   Fig.  or AO ac+ myeloid cells in the peripheral blood of Fus1 KO mice  mechanism, mitochondria maintain generation of Ca2+ oscillations via emptying an ER Ca2+ store that maintains Ca2+ elevations and prevents autoinhibition of Ca2+ channels c levels. In the absence of Fus1, mitochondrial efficiency in handling Ca2+ levels is impaired, thus affecting overall Ca2+ signaling [2+]m levels are increased to a higher amplitude than in WT cells, but drops to a steady-state level with a faster rate. Thus, we propose that Fus1 plays a gatekeeper role for mtCU by restraining the initial accumulation of Ca2+ in mitochondria during Ca2+ load and preventing mitochondria from Ca2+ uptake at low cytosolic Ca2+ concentration , prevents autoinhibition of MCU by Ca2+ thereby providing mitochondrial Ca2+ uptake with a positive feedback loop c accumulation while mito-chondria experience deficiency in Ca2+ uptake  opening depends on ROS and mitochondrial Ca2+ elevations m), fresh epithelial cells were grown overnight on 8-chamber coverglass slides . The following day, cells were simultaneously treated in situ with the AM ester of Ca2+ sensor dye rhod-2   and mitochondria-specific fluorescent marker MitoTracker Green  (Molecular Probes) at 37°C for 30 min , w, w2+ res2+ sensor Fura-2. Cells were grown in a similar manner as described for [Ca2+]m analysis. The next day, cells were treated with 1 μM fura 2-AM (Molecular Probes) for 30 min at 37°C [2+ imaging was performed using a Nikon TE2000-E wide-field fluorescence microscope equipped with a xenon lamp for fluorescence excitation. Fura 2-loaded cells were excited using 340 and 380 excitation filters and a polychroic mirror. Fluorescence was captured by a CoolSNAP HQ2 camera (Roper Scientific). Ratio measurements (340/380) were recorded at 5-s intervals over a 20-min time period. The [Ca2+]c concentration for cells treated with Ca2+ agonists was estimated by 340/380 ratio (F) normalized to the basal level of corresponding 340/380 ratio in the same cells (Fo).For measurement of [Ca2+]c, we utilized ratiometric Ca at 37°C .]. Ca2+


As the major Ca2+ repositories inside the cell, the endoplasmic reticulum (ER) and the sarcoplasmic reticulum (SR) of muscle cells are central in maintaining and amplifying the intracellular Ca2+ signal. The morphology of these organelles, along with the distribution of key calcium-binding proteins (CaBPs), regulatory proteins, pumps, and receptors fundamentally impact the local and global differences in Ca2+ release kinetics. In this review, we will discuss the structural and morphological differences between the ER and SR and how they influence localized Ca2+ release, related diseases, and the need for targeted genetically encoded calcium indicators (GECIs) to study these events.The flow of intracellular calcium (Ca The efficiency and speed of Ca2+ signaling is powered by the ability of cells to maintain the near 20,000-fold gradient between intracellular and extracellular Ca2+ concentrations  by refilling the ER/SR with Ca2+ upon signal termination  remains high after stimulus removal. This sustained level of cytosolic Ca2+ contributes directly to muscle stiffness. Administration of the drugs dantrolene and verapamil was shown to restore cytosolic Ca2+ levels back to basal  of the target organelle. It must have good folding and bright fluorescence at 37 °C for mammalian cell application. It should possess a large signal enhancement upon Ca2+ binding with a high signal to noise ratio (SNR). It should display rapid binding and release kinetics for measuring fast calcium fluctuations. The binding stoichiometry of the GECI–Ca2+ complex should be 1:1 for simple, quantitative interpretation of the fluorescence signal. Additionally, the probe should be unaffected by the pH of the environment being monitored [To decipher the irregularities in Caignaling . MagFuraectively ,134,135.ganelles . The abiganelles . Using s imaging ,138. To onitored ,138,139.onitored ,138.2+ dynamics from the ER/SR and cytosol [2+. D1ER and D4cpv-CASQ1 are cameleons that utilize CFP and Citrine, a type of YFP, and CFP and circularly permutated Venus, respectively. D1ER and D4cpv-CASQ1 are targeted to the ER/SR and have been used to study Ca2+ release from these organelles [Kds of 0.8 and 60 µM with an in situ Kd of 220 µM determined in HeLa cells. D1ERCmR2 can be used in tandem with fura-2 to measure ER and cytosolic Ca2+ concentrations [Ƭrt) of 83 ms. YC-Nano140 has a Ƭrt of 303 ms [GECIs fall into two major categories: single fluorophore and Förster resonance energy transfer (FRET) pair. Single fluorophore sensors are non-ratiometric and experience a change in fluorescence intensity when calcium binds. Most of the single fluorophore sensors are large chimeras of a fluorescent protein (FP) connected to a native CaBP, such as CaM or troponin C (TnC). Chelation of calcium by the CaBP induces a conformational change that rearranges the chromophore environment, inducing fluorescence change. For single fluorophore sensors, calcium binding produces changes either in absorbance or the quantum yield that lead to the change in fluorescence intensity. Single fluorophore sensors are commonly referred to as intensiometric because the fluorescence intensity change at only one wavelength is measured . FRET pa cytosol  but primganelles ,143. Recganelles . The newtrations . As for f 303 ms .2+ are high with Kd values in the nanomolar range [2+Kd of 235 nM with a dissociation time constant of ~200 ms [2+ induced fluorescence change mechanism was discovered from crystal structure analysis of GCaMP2. The Ca2+ induced conformational change in CaM creates new contacts between CaM and cpGFP. CaM residues change the chromophore environment of cpGFP, preventing solvent access to the chromophore aiding fluorescence increase [2+ transients in different environments such as mouse cardiac cells in vivo (GCaMP2) [Caenorhabditis elegans and Drosophila melanogaster (GCaMP3) [Caenorhabditis elegans, Drosophila melanogaster, and zebra fish (GCaMP5s) [2+ nanosparks that initiate EC coupling in the junctional zone dyad space in rat cardiomyocytes. By targeting the sensor to the junctional zone using resident proteins junctin and triadin, Ca2+ nanosparks that are 50 times smaller than standard sparks were seen [2+ that limit their use to cellular environments with minimal Ca2+ concentrations like the cytosol, and only use cpGFP. None of the initial GCaMPs have Ca2+ affinities low enough to target to organelles like the ER/SR [2+ indicators for optical imaging (GECOs). Green (G-GECO), red (R-GECO), and ratiometric blue-green (GEM-GECO) variants were created. Although the color palette was expanded, the new GECO variants still have high Ca2+ sensitivity [2+Kds of 24 and 12 µM, respectively. LAR-GECO1 was used to monitor thapsigargin inhibition of ER refilling in HeLa, HEK293, and U2-OS cells co-transfected with CatchER. LAR-GECO had larger decreases in intensity over all three cell lines [2+Kd of 368 µM and a large dynamic range. This new variant termed 2+Ca-measuring organelle-Entrapped Protein IndicAtor 1 in the ER (CEPIA1er) was able to monitor ER Ca2+ dynamics in HeLa cells with thapsigargin and histamine treatment [2+ affinities of 565, 672, and 558 µM, respectively [2+ binding and release kinetics. For more localized measurements of rapid Ca2+ transients, in processes like muscle contraction, it is crucial to utilize a Ca2+ biosensor with reported, fast kinetics [Because of the large size of FRET pairs and limited signal intensity, probes called GCaMP were developed using a single circularly permuted enhanced green fluorescent protein (cpEGFP) fused to calmodulin and M13. The use of cpEGFP increased the signal intensity of the probes for use in cells but their affinities for Caar range ,146. The ~200 ms . The Ca2(GCaMP2) , chemose(GCaMP3) , and in GCaMP5s) . As mentere seen . All of he ER/SR ,147,149.sitivity . Robert ll lines . In 2014reatment . Variantectively . Table 1kinetics .2+Ca sensor for detecting high concentration in the ER) was designed [2+ binding and a Kd between 120–180 µM. Its unmatched Ca2+ off rate (koff) of 700 s−1 . FLIM provides better spatial resolution on smaller timescales extending its quantitative ability beyond standard fluorescence microscopy [In the past, we demonstrated our knowledge and skill for designing novel CaBPs using non-native CaBPs as the scaffold ,155, anddesigned  (Figure  700 s−1 b, which ll types . In respll types  (Figure e sensor . CatchER2+ bound , a propecroscopy .2+ release from the SR lumen. A better understanding of local Ca2+ signaling and the essential components that comprise the underlying machinery will guide the development of innovative methods to treat the numerous diseases that develop as a result of impaired ER/SR Ca2+ signaling.The intricacies of the intracellular calcium signaling network are impacted by the structural organization of the ER/SR. Events such as muscle contraction are greatly influenced by the local organization of the RyR, CASQ, JP45, and other regulatory proteins that initiate Ca


Increased use of nanomaterials poses potential human health risk. It is therefore critical to understand the nature and origin of the toxicity imposed by nanomaterials (nanotoxicity). In this article we review the toxicity of the transition metal oxides in the 4th period that are widely used in industry and biotechnology. Nanoparticle toxicity is compellingly related to oxidative stress and alteration of calcium homeostasis, gene expression, pro-inflammatory responses, and cellular signaling events. The precise physicochemical properties that dictate the toxicity of nanoparticles have yet to be defined, but may include element-specific surface catalytic activity , nanoparticle uptake, or nanoparticle dissolution. These  Nanotechnology involves the study of the control of matter on atomic and molecular scales. Nanomaterials have at least one dimension in the range of 1–100 nm in) plays major regulatory roles in cellular metabolism, signal transduction, and gene expression. For instance, calcium can influence cell cycle by activating protein kinase C (PKC), Ca2+/calmodulin-dependent protein kinases (CaMK), and mitogen-activated protein kinase (MAPK). Accordingly, [Ca2+]in is tightly regulated, and alterations of [Ca2+]in are associated with cellular dysfunction, metabolic and energetic imbalance, disease states, and cell death. A host of environmental toxicants elevate [Ca2+]in directly or indirectly by promoting Ca2+ influx, releasing Ca2+ from intracellular stores, inhibiting Ca2+ sequestration, or blocking Ca2+ efflux from the cell [2+]in, that could be partially attenuated by the antioxidant N-acetylcysteine (NAC), indicating an effect of oxidative stress on calcium homeostasis [2+]in and cell viability suggests a role for calcium in cell death. The moderation of this increase by nifedipine suggests that a portion of this increase reflects the influx of extracellular calcium. Membrane disruption  may also play a role in this influx. The involvement of Ca2+ release from intracellular stores has yet to be evaluated.The intracellular calcium concentration (in of Chinese hamster ovary (CHO) cells expressing M3 muscarinic receptors from 40 to 130 nm without compromising calcium homeostatic mechanisms and the CHO cells had no store operated calcium entry response in the presence of 10 µg/ml ZnO. Hence, ZnO particles had minimal effects on IP3- or thapsigargin-mediated release of intracellular calcium from the endoplasmic reticulum, but strongly inhibited store operated calcium entry. This effect was seen a decrease in Ca2+ entry upon introduction of calcium to the extracellular medium following thapsigargin-induced depletion of calcium from the endoplasmic reticulum (EC50’s ≈ 2 µg/ml). Thus, ZnO nanoparticles interfere the M3 signaling pathway (at least) via disruption of store operated calcium entry.In another study, the effects of ZnO nanoparticles on store operated calcium entry was studied in Chinese hamster ovary (CHO) cells stably transfected with a M3 muscarinic receptor. M3 receptors activate Gαβ (PLCβ) . PLCβ hy2+] and OS are a likely contributing factor. While [Ca2+]in and OS affect the activity of each other, they both induce cell death by distinct pathways. Finally, though calcium-dependent kinase activation pathways leading to cell cycle regulation or cell death have been well documented, studies in this area with transition metal oxides are lacking.Nanoparticle-induced cell death by various means is summarized in Inflammation is clinically defined as the presence of redness, swelling and pain. Histologically, it is defined as the presence of edema fluid and the infiltration of tissues by phagocytic cells. Chronic inflammation can lead to diseases such as atherosclerosis, pulmonary diseases, and cancer. Oxidative stress is closely related to inflammatory response by activating the nuclear factor-kappaB (NF-κB) signaling pathway that controls the transcription of pro-inflammatory genes such as IL-1β, IL-8, and tumor necrosis factor-α (TNF-α)  at both mRNA and protein levels in vitro ,84,85,86Recent studies have increased our understanding of the nanotoxicity of metal oxide particles, particularly with respect to oxidative stress-induced cascade pathways that lead to inflammatory responses. Several important challenges remain. First, inter-laboratory discrepancies contribute to uncertainties in risk assessment. Second, as new nanomaterials continue to emerge, a systematic approach to identifying the physicochemical properties of nanomaterials that determine toxicity is required. This is problematic using nanomaterials of industrial origin since they 1) vary in multiple physical characteristics, 2) are synthesized using different methods, and 3) vary widely in purity. Third, to attribute toxicity to a particular factor, one needs to consider the extent to which the test design is relevant to the cellular environment. It is likely that toxicity is a non-linear function of multi-variables, which highlights the necessity of developing a theoretical computing model for predictive nanotoxicity.