1. Developmental Biology and Stem Cells
Download icon

Calcium handling precedes cardiac differentiation to initiate the first heartbeat

  1. Richard CV Tyser
  2. Antonio MA Miranda
  3. Chiann-mun Chen
  4. Sean M Davidson
  5. Shankar Srinivas Is a corresponding author
  6. Paul R Riley Is a corresponding author
  1. University of Oxford, United Kingdom
  2. University College London and Medical School, United Kingdom
  3. Wellcome Trust, United Kingdom
Research Article
Cited
1
Views
3,868
Comments
0
Cite as: eLife 2016;5:e17113 doi: 10.7554/eLife.17113

Abstract

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 Ca2+-oscillations (SACOs) in the forming cardiac crescent (stage E7.75) prior to overt beating. 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.

https://doi.org/10.7554/eLife.17113.001

eLife digest

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.

https://doi.org/10.7554/eLife.17113.002

Introduction

The heart is the first organ to form and function during mammalian embryonic development. In the mouse, mesoderm originating from the primitive streak forms a bilateral pool of progenitor cells that at E7.5 give rise to the cardiac crescent (CC). 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) (reviewed in Buckingham et al., 2005). The first cardiac contractions have been described during the transition from CC to LHT. Studies of heart development in model organisms have historically focused on the origin and spatial-temporal allocation of cardiac progenitors and cardiovascular lineage determination (Buckingham et al., 2005; Saga et al., 1996; Cai et al., 2003; Meilhac et al., 2004; Wu et al., 2006; Moretti et al., 2006; Evans et al., 2010; Devine et al., 2014). Whilst insight into the identification and regulation of cardiac cell types is important for improved understanding of congenital heart disease (Bruneau, 2008), an anatomical and cellular bias has overlooked a role for the onset of cardiac function. Early descriptions of initial cardiac contractions (Navaratnam et al., 1986), including suggested pacemaker activity on either side of the embryonic midline (Goss, 1952), as well as optical mapping of spontaneous action potentials performed in both chicken (7 somite stage) and rat (3-somite stage) (Fujii et al., 1981; Hirota et al., 1985), have been informative, but lack resolution. Subsequent studies in mouse (3-somite stage) could only infer early electrical activity based on irregular fluctuations in basal Ca2+ (Nishii and Shibata, 2006). Given the forming heart contracts from an early stage, this raises the important question of when and how contractile activity of cardiomyocytes is first initiated during development and to what extent this influences the progression of differentiation and subsequent cardiac morphogenesis. This is especially important as the forces exerted by cardiac contractions have been shown in several models to be required for proper heart development (Granados-Riveron and Brook, 2012), and to modulate gene expression (Miyasaka et al., 2011) at later developmental stages.

In mature cardiomyocytes, coordinated electrical excitation is coupled to physical contraction in a process termed excitation contraction coupling (ECC) (Bers, 2002). ECC relies on changes in the intracellular concentration of the second messenger Ca2+ via release from the sarcoplasmic reticulum (SR) in a process termed Ca2+ induced Ca2+ release (CICR) (Fabiato and Fabiato, 1979). Increases in the concentration of intracellular Ca2+ result in cardiomyocyte contraction due to Ca2+ binding to troponin and myofilament activation. ECC involves a number of specific proteins including L-type Ca2+ channels (LTCC, sarcolemmal Ca2+ influx), ryanodine receptors (RyR2, SR Ca2+ release), the sarco (endo) plasmic reticulum Ca2+ ATPase (SERCA, SR Ca2+ uptake) and the Na+/Ca2+ exchanger (NCX, Sarcolemmal Ca2+ efflux). 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 (Conway et al., 2002; Seki et al., 2003) and whilst they express a variety of ion channels and exchangers present in the adult heart (Seisenberger et al., 2000; Cribbs et al., 2001; Linask et al., 2001), the expression and activity of these proteins is distinct from that in mature cardiomyocytes (Liang et al., 2010). Using isolated cells as well as genetically manipulated animals, two contrasting mechanisms have been proposed for how Ca2+ transients are generated in the developing heart from approximately E8.5 onwards. Early studies suggested that myocyte contraction is triggered by sarcolemmal Ca2+ influx through voltage activated Ca2+ channels with little or no contribution from the SR (Nakanishi et al., 1988; Takeshima et al., 1998). In contrast, more recently it has been shown that at ~E8.5–9, Ca2+ transients originate from the SR, via RyR together with InsP3 channels, to trigger electrical activity as well as contraction (Viatchenko-Karpinski et al., 1999; Méry et al., 2005; Sasse et al., 2007; Rapila et al., 2008). Whilst these studies characterised SR function at ~E8.5–9, they did not investigate how Ca2+ transients are regulated at the earliest stages of cardiac crescent development when contraction is initiated, and relied on experiments performed using isolated cells cultured for between 12 to 70 hr (Sasse et al., 2007; Rapila et al., 2008). Thus there is a lack of cellular resolution in vivo and no current mechanistic insight into the onset of Ca2+ handling and its impact on differentiation and cardiogenesis.

We report here, for the first time, high-resolution live imaging of Ca2+ 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.

Results

Staging of early cardiac development and sarcomeric assembly

It is commonly stated that initiation of contraction begins with the formation of the LHT (Bruneau, 2008), and whilst cardiac contractions have been reported just prior to the ‘linear heart tube’ stage (Navaratnam et al., 1986; Nishii and Shibata, 2006; Linask et al., 2001; Kumai et al., 2000; Porter and Rivkees, 2001), a precise study on the initiation of cardiac function has not been conducted. A difficulty with these reports is the use of ‘embryonic day’ or ‘somite number’ to stage the developing heart. Somite number is variable in its correlation to the overall embryonic stage (Kaufman and Navaratnam, 1981), can depend on genetic background (Méry et al., 2005; Porter and Rivkees, 2001) and importantly, is not a sufficiently fine-grained proxy for the developmental stages of the heart. This can lead to ambiguities, as a ‘3-somite’ embryo may range from the cardiac crescent to early LHT stages. We, therefore, created a staging system specific to the early heart, from early crescent to LHT (Supplementary file 1a), similar to studies at later stages when a more precise morphological characterization is necessary (Biben and Harvey, 1997). On this basis, we defined four stages (0, 1, 2 and 3) of cardiac crescent development prior to the LHT stage, based on clear morphological differences. Stage 0 hearts represented the first discernible crescent structure situated beneath the developing head folds, being the widest (360–390 µm along the medio-lateral axis) and thinnest (70–80 µm along the rostro-caudal axis) of the crescent stages (Supplementary file 1a). Whilst stage 1 was morphologically similar to stage 0, the cardiac crescent had become narrower (300–370 µm) and thicker (75–95 µm). By stage 2, folding of the cardiac crescent is evident based on the formation of a trough at the embryonic midline and two lobes on either side. As the embryo transitions to stage 3 this trough becomes less obvious with a rostral-caudal elongation of the heart as the LHT begins to form. Transition from stage 3 to the LHT was defined by the complete fusion of the two lobes and loss of the central trough (Figure 1; Supplementary file 1a).

Figure 1 with 1 supplement see all
Sarcomeric assembly occurs in the forming cardiac crescent during heart development.

Maximum intensity projections of alternating myomesin (Myom) and sarcomeric alpha-actinin (α-Actinin) immunostaining from cardiac crescent formation to the linear heart tube stage (LHT; AE; A, 11 stacks; B, 36 stacks; C, 35 stacks; D, 31 stacks; E, 36 stacks). Analysis by qRT-PCR revealed a significant increase in the expression of Myom1, Actn2, Tnnt2 (encoding Myomesin, sarcomeric alpha-actinin and cardiac troponin t), in isolated cardiac crescents between stage 0 and stage 1 (F). cc, cardiac crescent (lateral plate mesoderm); hf, head folds (neural ectoderm). Scale bars: AE, 100 μm, A’E’, 10 μm. Statistics: ANOVA and Tukey test for multiple comparisons (*p<0.05; **p<0.01; ***p<0.001).

https://doi.org/10.7554/eLife.17113.003

To molecularly characterize these stages, we performed immunostaining for three proteins of the cardiac contractile machinery: sarcomeric α-actinin (α-Act), 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 (Figure 1; Figure 1—figure supplement 1). At stage 0, cTnT was the most evident contractile protein within the early cardiac crescent, albeit without sarcomeric banding (Figure 1—figure supplement 1A). Both α-Actinin and Myomesin appeared in small clusters of cells at stage 0 (Figure 1A). Sarcomeric banding of these proteins, indicative of contractile capability, only manifested later in discrete regions within stage 1 and 2 crescents (Figure 1B,C; Figure 1—figure supplement 1B,C). This was surprising given that the crescent stages of early heart development are thought to correspond to non-differentiated cardiac mesoderm (reviewed in Harvey, 2002), without prior reports of contractile machinery or functionality. By stage 3, sarcomere assembly and myofibrilar banding became more uniform, coincident with coalescence of the paired crescent primordia to the embryonic midline. This increased through to the linear heat tube stage, consistent with progressive cardiomyocyte maturation (Figure 1D,E; Figure 1—figure supplement 1D,E). To further characterize stage development in relation to sarcomere assembly, qRT-PCR was performed on isolated cardiac crescents, to assess the corresponding gene expression of Myom1 (encoding Myom), Actn2 (encoding α-actinin) and Tnnt2 (encoding cTnT) (Figure 1F). Myom1, Actn2 and Tnnt2 expression significantly increased between stages 0 and 1 and continued to increase until formation of the LHT (Supplementary file 1b).

The onset of Ca2+handling in the cardiac crescent

Coincident with sarcomere formation at stage 1 (Figure 1B; Figure 1—figure supplement 1B), we observed the onset of beating in discrete foci in the lateral regions of cardiac crescents of cultured mouse embryos, by differential interference contrast (DIC) imaging (Video 1), significantly earlier than previously described (Navaratnam et al., 1986; Nishii and Shibata, 2006). These foci generally contracted at the same rate, indicative of either synchronization or a shared intrinsic beat rate for nascent cardiomyocytes (Figure 2—figure supplement 1). The earliest cardiac contractions had a frequency of approximately 30 beats per minute (BPM), which increased significantly by stage 3 (after approximately 5 hr) to around 60 BPM (Figure 2A). Contractile activity requires cytosolic Ca2+ 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 (Figure 2B), even through regions of non-contractile tissue (Video 2). As the cardiac crescent starts to fuse to form the LHT, the transients switch from a lateral to a more caudal-rostral propagation (data not shown). At stage 0 spontaneous asynchronous Ca2+ oscillations (SACOs) were observed within the forming crescent, in the absence of contractile activity and sarcomeric banding of cTnT (Figure 2C; Video 3). SACOs were observed in all stage 0 cardiac crescents imaged (n = 35), propagated within individual cells (Figure 2D’’) and displayed a range of Ca2+ dynamics with variable frequencies and durations (Figure 2D’, Video 4). Compared to Ca2+ transients at later stages (Figure 2A) 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 (Figure 2—figure supplement 1C). During a ~20 s recording period we observed only 10.3 ± 0.7 individual SACOs per embryo (n = 35) occurring in different sites. Consecutive SACOs in the same site were rarely observed within the 20 s imaging window and, therefore, we conclude that SACOs in individual cells occur at a frequency < 3 bpm. The appearance of Ca2+ transients in the embryonic heart prior to beating (Figure 2A,B) is consistent with the idea that Ca2+ signalling within early cardiac progenitors may be important to promote sufficient differentiation for subsequent contractile function (Mesaeli et al., 1999; Li et al., 2002).

Video 1
Representative movie of beating regions in the cardiac crescent at Stage 1.

DIC imaging of a stage 1 embryo highlighting beating regions in the lateral regions (dotted circle) of the developing cardiac crescent. Acquisition was performed at 10 frames per second (fps) with a 20x objective and movie played at 8 fps. Scale bar: 100 µm.

https://doi.org/10.7554/eLife.17113.005
Figure 2 with 3 supplements see all
Initiation of contraction begins within the forming cardiac crescent and is preceded by spontaneous asynchronous Ca2+ oscillations during heart development.

Quantitative analysis from the onset of cardiac contraction at stage 1 of crescent formation to formation of the LHT (see Figure 1 and Supplementary file 1a for morphological staging. Stage 1, n = 12; stage 2, n = 8; stage 3, n = 10; LHT, n = 7), revealed a significant increase in heart rate from stages 2 to 3 (A). Ca2+ signal following Cal520 loading of stage 1 embryos revealed lateral propagations of transients across the crescent that correlated with the onset of beating at stages significantly earlier than previously described (B). Ca2+ signal following Cal-520 loading of stage 0 embryos revealed spontaneous asynchronous Ca2+ oscillations in individual cells prior to beating, highlighted by white arrows (C), temporal maximum intensity projection of Cal-520 fluorescence (MaxFluo) over a period of 30 s. Higher resolution imaging of stage 0 single cell Ca2+ oscillations represented as a temporal maximum intensity projection over a period of 100 s (D) revealed variation in SACO transient size and frequency (D’) and could be observed slowly propagating throughout cells (D’’). Scale bars: B, C, 100 μm, D, 20 μm. Statistics: ANOVA and Tukey test for multiple comparisons (*p<0.05; **p<0.01; ***p<0.001).

https://doi.org/10.7554/eLife.17113.006
Video 2
Representative movie of a Ca2+ transient at Stage 1.

Confocal time-lapse of a stage 1 embryo loaded with Cal-520. Cal-520 emission (rainbow) was captured simultaneously with DIC imaging (gray). Embryo the same as that shown in Figure 2D. Acquisition was performed at 10 fps with a 40x water immersion objective. Background fluorescence was removed by subtracting the signal at a resting phase. Movie played at 8 fps to better show the propagation of the Ca2+ transient. Scale bar: 100 µm.

https://doi.org/10.7554/eLife.17113.010
Video 3
Representative movie of SACOs at Stage 0.

Confocal time-lapse of a stage 0 embryo loaded with Cal-520. Cal-520 emission (rainbow) was captured simultaneously with DIC imaging (gray). Acquisition was performed at 10 fps with a 20x objective. Background fluorescence was removed by subtracting the signal at a resting phase. Scale bar: 100 µm.

https://doi.org/10.7554/eLife.17113.011
Video 4
Representative high-resolution movie of SACOs at Stage 0.

Confocal time-lapse of a stage 0 embryo loaded with Cal-520. Cal-520 emission (rainbow) was captured simultaneously with DIC imaging (grey). Acquisition was performed at 10 fps with a 20x objective. Movie playback is at 5x original speed. Background fluorescence was removed by subtracting the signal at a resting phase. Scale bar: 10 µm.

https://doi.org/10.7554/eLife.17113.012

Ca2+ signalling and contraction in embryonic stem cell-derived cardiomyocytes

We next used an embryonic stem cell (ESC)-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 (reviewed in Kattman and Keller, 2007; Willems et al., 2009; Van Vliet et al., 2012), this has not been rigorously mapped onto embryonic stages of heart development. We, therefore, performed a principal component analysis (PCA) and hierarchical clustering of 12 cardiac related gene expression profiles of whole embryos across embryonic stages E7.5 to E8.5, compared with ESC-derived embryoid bodies (EBs) from days 0 to 7 inclusive and day 14 of differentiation. Whole embryos were used to reflect the myriad of cell types present in the ESC-cardiomyocyte differentiation assay.

Hierarchical gene clusters were evident with the onset of beating at E7.5 and day 4 and 5 of EB formation, coincident with cardiac progenitor gene expression (Figure 2—figure supplement 2A). Expression profiles by days 6 and 7 correlated with E8.0 (stage 0 to stage 2) when beating was well established in both ESC-derived EBs and the embryonic heart, whereas day 14 was equivalent to the more mature E8.5 (Figure 2—figure supplement 2A). Key cardiac genes Myh6, Tnnt2 and Mef2c revealed comparable trends of increased expression over time of differentiation in both EBs and embryos (Figure 2—figure supplement 2). The cardiac specification program, characterized by expression of Mesp1, was evident by day 4 in EBs (Figure 2—figure supplement 2I) following Brachyury expression, an indicator of mesoderm formation, and loss of pluripotency markers such as Pou5f1 (encoding Oct-4; Figure 2—figure supplement 2J–K). Mesp1 up-regulation in EBs was consistent with expression at E7.5 in the embryo (Figure 2—figure supplement 2E) and preceded beating at day 6 and 7. These later stages revealed an up-regulation of Ca2+-handling genes such as Slc8a1, Cacna1c and Ryr2 (Figure 2—figure supplement 2L–N).

Sarcomere assembly in ESC-derived cardiomyocytes accompanied the onset of beating at day 7 (Figure 2—figure supplement 3A,B) and, interestingly, the rate of beating was comparable at the outset (day 7 of differentiation) with that observed in the developing heart at stage 1 (Figure 2—figure supplement 3C), consistent with an intrinsic rate for early cardiomyocytes contractions. Furthermore, Ca2+ transients were observed in day 7 EBs as both large propagating waves (Figure 2—figure supplement 3D,F; Video 5) and within small regions of cells prior to beating, similar to that observed in the stage 0 embryonic heart (Figure 2—figure supplement 3E,G; Video 6).

Video 5
Representative movie of a propagating Ca2+ transient at day eight of ESC cardiomyocyte differentiation.

Confocal time-lapse of a day eight EB loaded with Cal-520. Cal-520 emission (rainbow) was captured simultaneously with DIC imaging (grey). Acquisition was performed at 10 fps with a 20x Objective. Background fluorescence was removed by subtracting the signal at a resting phase. Scale bar: 100 µm.

https://doi.org/10.7554/eLife.17113.013
Video 6
Representative movie of SACOs at day six of ESC cardiomyocyte differentiation.

Confocal time-lapse of a day six EB loaded with Cal-520. Cal-520 emission (rainbow) was captured simultaneously with DIC imaging (grey). Acquisition was performed at 10 fps with a 20x objective. Background fluorescence was removed by subtracting the signal at a resting phase. Scale bar: 10 µm.

https://doi.org/10.7554/eLife.17113.014

Ion channels are expressed during the earliest stages of heart development

ECC components, and specifically 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. We, therefore, assessed ECC gene expression on embryonic hearts at the different stages defined herein; focusing specifically on Slc8a1 (encoding NCX1), Cacna1c and Cacna1d (encoding the LTCC subunits, CaV1.2 and CaV1.3 respectively; Figure 3A), Atp2a2 (encoding SERCA2), Iptr2 (encoding InsP3 type 2 channels) and Ryr2 (encoding RyR2) that collectively are key components of SR Ca2+ regulation (Figure 3—figure supplement 1A–D; Supplementary file 1b). Between E7.75 (as defined by the presence of clear head-folds but not a cardiac crescent) and stage 0, Slc8a1 significantly increased 21 fold (p-value<0.001), whilst Cacna1c revealed a modest but non-significant increase of 2.5 fold (p-value>0.05) and Cacna1d a significant 4-fold increase (p-value <0.01). From stage 1 onwards Slc8a1, Cacna1c and Cacna1d expression continued to significantly increase until stage 3 (Figure 3A,B; Figure 3—figure supplement 1A, Slc8a1, 234-fold; Cacna1c, 38-fold; Cacna1d, 41-fold). Between stage 3 and the LHT, expression of Slc8a1 and Cacna1c was maintained whilst Cacna1d significantly decreased (Supplementary file 1b; p-value<0.05). Previous studies have suggested a non-ECC dependent role for the SR during development, implicating both InsP3 and RyR channels (Méry et al., 2005; Sasse et al., 2007; Rapila et al., 2008). While Ryr2 significantly increased 30 fold between E7.75 and stage 0 (p-value<0.001), expression of Atp2a2 only increased 1.7 fold (p-value<0.05) and Iptr2 did not significantly change (p-value>0.05; Figure 3—figure supplement 1C,D).

Figure 3 with 2 supplements see all
The ECC components NCX1 and LTCC are expressed within the early embryonic heart and ESC-derived cardiomyocytes.

Analyses by qRT-PCR revealed a significant increase in the expression of Slc8a1 (encoding NCX1) in the heart from E7.75 to stage 0 (A, n = 5 per stage) and from day 2 of differentiation of ESC-derived cardiomyocytes (B, n = 5 per stage). In contrast expression of Cacna1c (encoding the LTCC subunit CaV1.2), increased at a later stages from stage 0 to stage 1 and from day 4 of ESC-derived cardiomyocyte differentiation (C, n = 5 per stage). Confocal imaging section of a stage 0 embryo following immunostaining for cTnT (red) and NCX1 (green), indicated membrane localization of NCX1 within the forming crescent (D; white arrows in lower panel), whereas CaV1.2 (green) was absent from cTnT+ (red) regions at the same stage (E). A maximum intensity projection of day 7 ESC-derived cardiomyocytes revealed complete overlap of staining for cTnT (red) and NCX1 (green; F, 33 stacks), whereas CaV1.2 (green) overlapped in part with cTnT (red) but there were also extensive cTnT+/CaV1.2- regions (dotted box) emphasizing the later requirement for LTCC (G, 22 stacks). Confocal imaging section of a stage 2 embryo following immunostaining for both NCX (H) and CaV1.2 (I) revealed the expression of both proteins at later stages of heart development. cc, cardiac crescent; hf, head folds. Scale bars: D, F 50 μm, H, I 100 μm. All error bars are mean ± S.E.M; Statistics: one-way ANOVA and Tukey test for multiple comparisons (*p<0.05; **p<0.01; ***p<0.001).

https://doi.org/10.7554/eLife.17113.015

We performed a similar analysis on EB derived cardiomyocytes. Slc8a1 expression increased significantly in EBs prior to Cacna1c (day 2 versus day 4; Figure 3B,C) and to a much greater extent with the onset of beating (110 fold versus 38 fold; Figure 3B,C), suggesting NCX1 might play a more immediate role in the onset of beating. This was supported by the lack of any further increase in Slc8a1 expression from E9.5 through to birth (P0) and adulthood (Figure 3—figure supplement 2), whereas Cacna1c expression fluctuated across later developmental stages and significantly increased post-natally (Figure 3—figure supplement 1A,B) with increased maturation.

Since the sarcolemmal channel genes revealed the most significant increases over the developmental timecourse, we proceeded to investigate spatiotemporal protein expression of NCX1 and CaV1.2 in embryos (Figure 3D,E), and ESC-derived cardiomyocytes (Figure 3F,G). Whilst NCX1 was clearly detectable within the cardiac crescent at stage 0 (Figure 3D), CaV1.2 was absent (Figure 3E). Differences in the expression of NCX1 and LTCC prior to beating were not maintained at later stages, after established contraction (stage 2 onwards), when both channels were expressed (Figure 3H,I). In EBs at day 7 of culture, both NCX1 and CaV1.2 were expressed, but whilst NCX1 was consistently co-expressed with cTnT (Figure 3F), there were cTnT+ foci that were negative for CaV1.2 (Figure 3G). Collectively, this expression data suggests that NCX1 precedes Cav1.2 within the developing cardiac crescent at stage 0, being present prior to and at the onset of both SACOs and beating, whereas Cav1.2 became upregulated later from stage 1 onwards. Of note, while NCX1 and CaV1.2 signal was detected in other regions of the embryo, notably in the head folds, in contrast to the heart it was not membrane localised in these regions, indicative of non-functional protein. Furthermore qRT-PCR data for isolated head folds did not reveal any significant increases in Slc8a1 or Cacna1c expression, from E7.75 through to LHT stages (p-value>0.05; Figure 3—figure supplement 2E,F; Supplementary file 1c) even though the head folds were maturing, as shown by morphological changes and expression of the neural ectoderm marker Sox1 (E7.75 versus LHT, p-value<0.001; Supplementary file 1c)

Ca2+ transients within the forming cardiac crescent are dependent upon NCX1

To functionally assess the roles of the sarcolemmal and SR channels in establishing and maintaining heartbeat, we employed pharmacological blockade on embryos maintained ex-vivo and ESC-derived cardiomyocyte cultures. We inhibited NCX1 using the specific inhibitors CB-DMB (Secondo et al., 2009) or KB-R7943 (Kimura et al., 1999), the LTCC using nifedipine (McDonald et al., 1994) and Ryanodine together with 2-APB to simultaneously block RyR and InsP3, similar to that described previously (Sasse et al., 2007; Rapila et al., 2008). Treated embryos were imaged for contractile activity by DIC imaging and Ca2+ transients were recorded in parallel with confocal fluorescence imaging of Cal-520. Acute treatment (for a maximum of 30 min) with NCX inhibitors (CB-DMB and KB-R7943) or LTCC inhibitor (Nifedipine), affected the embryos in a stage dependent-manner (Figure 4). Both NCX1 and the LTCC were required for Ca2+ transients during stages 1 and 2 (Figure 4A), and their inhibition led to an initial confinement of Ca2+ transients to one side of the crescent (within approximately 5 min of treatment, which persisted through to 15 min) followed shortly afterwards by complete loss of Ca2+ signal (Figure 4C,D). At stage 3 and later, only the LTCC was required for Ca2+ transient generation (Figure 4B). Whilst NCX1 can function in both forward (Ca2+ 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 (Hoyt et al., 1998; Iwamoto, 2004), recapitulated the results with CB-DMB and acted as a control for off-target effects of the latter. These data suggests that reverse mode NCX function may contribute to Ca2+ transient generation at the earliest stages of cardiac contraction, potentially via inward Ca2+ flux. The inhibitor experiments were repeated on day 7 and day 14 EBs. The relative effects of CB-DMB, KB-R7943 and nifedipine were consistent with those observed in stages 1 and 2 of the embryonic heart (Figure 4E–G). Treatment with Ryanodine + 2-APB only affected more mature embryos that had already undergone both cardiac looping and embryonic turning (Figure 4—figure supplement 1A) and did not prevent Ca2+ transients within cardiac crescents at stage 3 (Figure 4—figure supplement 1B). This suggest that a functional SR is only required once cardiac looping has been fully initiated, more than 18 hr later than the first observable SACOs within the cardiac crescent.

Figure 4 with 1 supplement see all
Both NCX1 and LTCC are required for Ca2+ transients associated with beating cardiomyocytes during cardiac crescent development.

Inhibition of Ca2+ transients upon treatment of stage 1-LHT embryos with either NCX1 inhibitors CB-DMB, KB-R7943 or the LTCC inhibitor nifedipine, relative to DMSO control after 5, 15 and 30 min of drug application (A, B). Inhibition of NCX1 with either CB-DMB (20 μM) or KB-R7943 (30 μM) affected only stage 1 and 2 embryos (A), whereas inhibition of LTCC with nifedipine (10 μM) effected both stages 1 and 2 and the later stage 3/LHT (B; stage1/2: DMSO, n = 5; CB-DMB, n = 9; KB-R7943, n = 6; nifedipine, n = 6; stage3/LHT: DMSO, n = 9; CB-DMB, n = 19; KB-R7943, n = 8; nifedipine, n = 8). Time series of Ca2+ transients on stage1-2 embryos at different time points of either CB-DMB or nifedipine treatment, revealed a confinement to the right side of the embryo prior to complete block (C, D). ESC-derived cardiomyocytes at different days of differentiation were treated with the same channel blockers: inhibition of NCX1 with CB-DMB (10 μM) significantly reduced contractions only in day 7 cardiomyocytes, whereas KB-R7943 (30 μM) affected cardiomyocytes at both day 7 and 14 (E). Inhibition of LTCC with nifedipine (10 μM) significantly reduced contractions in both day 7 and 14 cardiomyocytes and to a much greater extent than KB-R7943 at the later stage (F; day 7: DMSO, n = 38; CB-DMB, n = 36; KB-R7943, n = 7; nifedipine, n = 10; day 14: DMSO, n = 25; CB-DMB, n = 36; KB-R7943, n = 15; nifedipine, n = 15). Time series of Ca2+ transients on day 7 ESC-derived cardiomyocytes at different time points of either CB-DMB or nifedipine treatment, revealed a confinement prior to complete block (F, G), equivalent to that observed in the treated embryos (C, D). Treatment of stage 0 embryos prior to the onset of beating with CB-DMB (20 μM) and KB-R7943 (30 μM) resulted in inhibition of slow asynchronous Ca2+ transients after 15 min application relative to baseline (H, I; DMSO, n = 10; CB-DMB, n = 8; KB-R7943, n = 9) whereas treatment with nifedipine (10 μM) had no discernible effect on the slow transients (H, J; nifedipine n = 8), supporting the earlier role for NCX1 in initiating Ca2+ handling and beating. All scale bars 100 μm. Statistics: Freeman-Halton extension of Fisher exact probability test for embryos for embryos; Chi-square test with Bonferroni correction for ESCs (*p<0.05; **p<0.01; ***p<0.001).

https://doi.org/10.7554/eLife.17113.018

Taking into account the earlier upregulation of NCX1 relative to Cav1.2 at stage 0 (Figure 3A), we tested the possibility that NCX1 might be pivotal in initiating the SACOs observed in the early cardiac crescent (Figure 2C). Treatment of stage 0 embryos with CB-DMB and KB-R7943 resulted in a 90% decrease in the number of cells with SACOs after drug application when compared to baseline. This was significantly greater than following application of nifedipine to inhibit LTCC, or the DMSO control (CB-DMB, 90% inhibition + 6.6, mean + SEM, n = 10 embryos, p-value <0.01; KB-R7943, 91% inhibition + 7.0, n = 9 embryos, p-value <0.001; nifedipine, 39% inhibition + 11.4; n = 8 embryos, p-value=0.44; DMSO, 29% inhibition + 7.6, n = 10; Figure 4H–J; Supplementary file 1d). These data suggest that NCX1 is required for establishing the earliest pre-contractile SACOs.

Ca2+ channel blockade inhibits ESC differentiation into cardiomyocytes

Chronic treatment of Eomes-GFP ESC cultures (ESCs containing a knock-in of a GFP reporter into the endogenous Eomes locus (Arnold et al., 2009); marking nascent mesoderm (Ciruna and Rossant, 1999), with CB-DMB or Nifedipine, from days 0 to 14 of differentiation, resulted in a reduction in the percentage of beating EBs at day 14, down to 22% and 52% (Percentage beating EBs; DMSO, 76.53% (n = 196); CB-DMB, 22.37%, p-value<0.001 (n = 152); Nifedipine, 52.11%, p-value<0.001 [n = 71]) in the presence of CB-DMB and nifedipine respectively, compared to controls (Figure 5A), whilst not effecting cell number (Figure 5—figure supplement 1A–C). The reduction in beating EBs was supported by a down-regulation of key cardiac genes Myh6 (mean+ SEM: 0.57 ± 0.058; p-value<0.05; n = 6; Figure 5B) and Tnnt2 (0.66 ± 0.095; p-value<0.05; n = 6; Figure 5B) specifically following treatment with CB-DMB relative to control DMSO treatment. The decrease in cardiac gene expression associated with CB-DMB treatment could be observed from day 4 of Eomes-GFP ESC differentiation, at the initiation of cardiomyocyte differentiation (Figure 5—figure supplement 1D–N). In an Nkx2.5-EGFP ESC line (ESCs containing a vector expressing eGFP under the control of a murine Nkx2.5 promoter and regulatory region; marking cardiac progenitor cells [Wu et al., 2006]) this was associated with loss of GFP+ cardiac progenitors by day 14 (Figure 5—figure supplement 2A–E) and the significant down-regulation of key cardiac markers following treatment with CB-DMB relative to control DMSO treatment (Figure 5—figure supplement 2F). Importantly not all cardiac genes were effected by chronic exposure to the channel inhibitors including Slc8a1, Cacna1c and Camk2d downstream of Ca2+ signalling (Figure 5—figure supplement 3A), suggesting that the inhibitors did not simply have a global negative effect on gene expression or cardiomyocyte survival.

Figure 5 with 3 supplements see all
Influx of Ca2+ and CaMKII signalling are required for early and late cardiac gene expression and crescent formation.

Following chronic exposure of embryoid bodies for 14 days to CB-DMB (1 μM) there was a significant decrease in the incidence of beating from 80% to 22% as compared to a reduction to 52% following nifedipine treatment (10 μM) (A; DMSO, n = 196; CB-DMB, n = 152; nifedipine, n = 71). Prolonged exposure resulted in a significant decreases in mature cardiomyocyte genes, Mef2c, Myh6, Myh7 and Tnnt2 (B) gene expression following treatment with CB-DMB (n = 6) but not with nifedipine (n = 6). EBs cultured for 14 days in different concentrations of extracellular Ca2+ (1.8 mM is the normal culture medium concentration) revealed significantly decreased incidence of beating following culture with reduced Ca2+ when assessed in media containing 1.8 mM Ca2+ (C; 1.8 mM, n = 74; 1.0 mM, n = 61; 0.1 mM n = 80). Cultured ESC derived-cardiomyocytes exposed to NCX1 inhibitors effected downstream Ca2+ signalling via alterations in the levels of phosphorylated CaMKII (pCaMKII; D). pCamKII to total CamKII ratio was decreased in the presence of 1 μM CB-DMB as compared to 10 μM nifedipine and DMSO (D’; n = 3). E7.5 embryos were dissected and cultured for 12 hr in media containing either DMSO, nifedipine (10 μM) or CB-DMB (3 μM) and stained for cTnT and Nkx2.5 (E; maximum intensity projections, 30 stacks each). Embryos developed normally in culture, as indicated by head fold formation, coalescence of the cardiac crescent and addition of somites (not shown). Embryos cultured in CB-DMB were delayed in terms of cardiac crescent formation and show a weaker cTnT signal compared to either DMSO alone or nifedipine-treated (E; number of affected embryos: DMSO – 1/7; CB-DMB – 7/8; Nifedipine – 1/6). Cultured E7.5 embryos in the presence of either CB-DMB or nifedipine for 12 hr, revealed that CB-DMB significantly down-regulated the expression of both early Nkx2.5 and Mef2c and late Tnnt2 (F, G) cardiac genes, coincident with impaired cardiac crescent formation, whereas nifedipine-treatment did not appear to have any effect on cardiac gene expression (H). All error bars are mean ± S.E.M. Statistics: B, D, G and H: one-way ANOVA and Tukey test for multiple comparisons; A, C: Chi-square test with a Bonferroni correction for multiple comparisons (*p<0.05; **p<0.01; ***p<0.001). CC, Cardiac crescent; HF, Head folds; EM, Embryonic midline. All scale bars 50 μm.

https://doi.org/10.7554/eLife.17113.020

Given the proposed role for inward Ca2+ 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 (Figure 5C). At the end of the differentiation protocol (14 days) we accessed the percentage of beating EBs after returning to media with normal levels of Ca2+ for 2 hr. Culture in 0.1 mM Ca2+ resulted in an inhibitory effect on EB beating to the same extent (percentage beating EBs; 1.8 mM, 78.38%, n = 74; 0.1 mM, 28.75%, n = 80; p-value<0.001) as with CB-DMB treatment (Figure 5C) and culture in 1.0 mM resulted in an equivalent inhibitory effect to that following treatment with nifedipine (Figure 5C; 1.0 mM, p-value<0.01; n = 61). This suggested that influx of external Ca2+ is required for cardiomyocyte maturation/beating and supports a potential role for NCX1 working in reverse mode to bring Ca2+ into early cardiac progenitors.

We next investigated whether Ca2+ handling prior to, and concurrent with, the earliest contractile function impacted on known Ca2+-signalling pathways to activate foetal gene expression, as has been reported during adult pathological hypertrophy (Molkentin et al., 1998). Calmodulin-dependent kinase II (CaMKII) is a key component of Ca2+ and calcineurin signalling which directly impacts on downstream hypertrophic gene expression by induction of Mef2c (Wu et al., 2006; Molkentin et al., 1998; Passier et al., 2000; Zhang, 2007). Following CB-DMB-induced NCX1 inhibition at day 7 of EB differentiation, activated phospho-CaMKII (pCaMKII) levels were significantly reduced compared to either DMSO control or nifedipine treatment (mean ± SEM: DMSO, 1.18 ± 0.13; CB-DMB, 0.69 ± 0.15; nifedipine, 1.13 ± 0.06. DMSO vs. CB-DMB, p-value<0.01; CB-DMB vs. nifedipine, p-value<0.05; n = 3; Figure 5D,D'). NCX1 inhibition at day 4, a stage prior to cardiac progenitor specification, had no effect on pCaMKII levels (Figure 5—figure supplement 3B,B'). NCX1 inhibition reduced Ca2+ influx and decreased activation of CaMKII accompanied by significantly reduced expression of Mef2c and Myh7 relative to DMSO control (mean ± SEM: Mef2c, 0.71 ± 0.13; Myh7, 0.55 ± 0.18; p-value<0.05; n = 6) which, whilst previously associated with pathological cardiac hypertrophy, contribute here towards (physiological) cardiomyocyte differentiation (Figure 5B). In contrast, nifedipine treatment resulted in decreased Myh7, whereas Mef2c remained unaffected at early stages (Figure 5B).

NCX1 is essential for cardiomyocyte differentiation and cardiac crescent formation

To determine the effect of NCX1 blockade on the subsequent development of the heart, we cultured embryos isolated at E7.25 (the onset of head fold formation and pre-cardiac crescent stage) in the presence of DMSO, CB-DMB or Nifedipine for 12 hr (Figure 5E). Whilst the embryos remained viable during the culture period, initiation of heart development was impaired in those treated with CB-DMB compared to DMSO controls (Figure 5E). This was in contrast to treatment with Nifedipine alone, in which the crescent developed normally and progressed to an equivalent stage as DMSO treated embryos (Figure 5E). More specifically embryos cultured in the presence of CB-DMB had reduced expression of cTnT compared to embryos cultured in other conditions. However, most cells were still positive for Nkx2.5, suggesting delayed or impaired differentiation likely accounted for the failure of crescent formation under conditions of NCX1 blockade. The relative effects of NCX1 and LTCC inhibition on developmental progression and crescent formation were supported by corresponding gene expression data from cultured embryos (Figure 5F,G). While Tnnt2 was down-regulated in both CB-DMB and Nifedipine treated embryos, Nkx2.5 was only down-regulated in CB-DMB treated embryos (Figure 5F,G). Equally Slc8a1, was down-regulated exclusively in the presence of CB-DMB (Figure 5—figure supplement 3C), whereas Cacna1c was unaltered in the presence of either inhibitor (Figure 5—figure supplement 3C,D). These data collectively support a role for NCX in cardiomyocyte differentiation and crescent formation.

Discussion

Previous studies have attempted to investigate how cardiac function develops within the early embryo. Whilst these studies are informative they rely on the dissociation and culture of embryonic cardiomyocytes to facilitate physiological measurements resulting in the loss of critical spatial and temporal information regarding Ca2+-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 (Sasse et al., 2007; Rapila et al., 2008), our data did show that the SR becomes functional at later stages during cardiac looping (~E8.5). This may represent a period in which SR Ca2+ filling is required before a threshold for Ca2+ release from the SR can occur, as has been proposed in the adult setting (Stokke et al., 2011). Overall our data reveals that during the time frame from the earliest cardiac morphogenesis (crescent maturation) to looping of the heart (approximately 18 hr) there is a rapid transition in the mechanism by which the intracellular concentration of Ca2+ is elevated, based on distinct channel and exchanger function.

The observation of spontaneous asynchronous calcium oscillations (SACOs) 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 Ca2+-dependent signalling (via the CAMKII pathway) 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 (Video 2). Since blockade of NCX1 prior to the formation of the cardiac crescent, and chronic treatment in ESCs, leads to impaired cardiac differentiation it is also possible that SACOs may be present in mesoderm cells earlier than reported here, which we were not able to image due to the limitations of the current experimental set-up.

In the embryonic heart, Ca2+ 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 (non-functional) SR in the developing mouse heart (Conway et al., 2002). In zebrafish embryos it has recently been demonstrated that Ca2+ handling and not contraction per se is essential for regulating cardiomyocyte development (Andersen et al., 2015). Previous studies characterised functional expression of NCX from E8.5-E9.5 (post-LHT formation) in mouse (Linask et al., 2001; Reppel et al., 2007) coincident with cardiac looping and significantly later than the onset of the first heat beat described herein. NCX1 knock-out mice have been generated by multiple groups, with conflicting results in regards to the phenotype, extent of mutant heart development and the stage at which embryonic lethality occurs (Wakimoto et al., 2000; Koushik et al., 2001; Reuter et al., 2002; Cho et al., 2003). This suggests that loss of NCX1 may be compensated for at the level of early cardiomyocyte specification, differentiation and contraction. In mammals there are three different Na+-Ca2+ exchangers (NCX1, NCX2 and NCX3), and it has been previously demonstrated that these three exchangers share similar physiological properties (Linck et al., 1998; Lytton, 2007). Furthermore, NCX1 has several splicing variants with exon 1 being mutually exclusive to exon 2 (Lytton, 2007; Quednau et al., 1997). It is, therefore, possible that either a different NCX or an alternative splice variant will compensate for loss of the NCX1 variant targeted in the previous studies. Indeed, in all of the previous NCX1 generic loss-of-function studies, mutant mice were created by targeting exon 2, supporting the possibility that an alternately spliced variant may be able to compensate. Furthermore, precedent for genetic ablation being compensated for over the course of development exists, whereby patterning defects were muted over time (Bloomekatz et al., 2012). In contrast, our use of pharmacological channel blockers CB-DMB and KB-R7943, resulted in an acute stage-specific-loss of NCX1 function which attributed an essential role for NCX1 in generating SACOs, and potentially acting as a trigger to establish the onset of beating in the cardiac crescent and subsequent cardiomyocyte differentiation and morphogenesis. These findings are consistent with the situation in tremblor (tre) zebrafish which have a mutation in NCX1 leading to absence of normal calcium transients and cardiac fibrillation (Langenbacher et al., 2005) as well as earlier findings in rodents demonstrating a requirement for elevated cytoplasmic calcium to drive cardiac myofibrillogenesis in developing cardiomyocytes (Webb and Miller, 2003).

Parallel analyses on ESC-derived cardiomyocytes revealed that lowering the concentration of Ca2+ in the media had a similar effect on reducing the number of beating EBs as treatment with CB-DMB. This both reinforced the relative importance of NCX1 and also provided the first indication that the exchanger may be working in reverse mode, facilitating inward Ca2+ as necessary for cardiomyocyte differentiation. Furthermore, our use of the inhibitor KB-R7943, previously reported to specifically inhibit reverse mode NCX1 activity (Brustovetsky et al., 2011), replicated observations made with CB-DMB, such that at stage 1 and 2 both LTCC and NCX1 are required for sufficient Ca2+ influx. Whilst inhibition of NCX1 clearly blocked SACOs in stage 0 cardiac crescents, the mechanism by which NCX1 alone could lead to periodic oscillations in Ca2+ is still unclear, and may require oscillations in other ions, such as Na+, and/or contribution of other sarcolemmal proteins, such as the Plasma membrane Ca2+ ATPase (PMCA), 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 (Sasse et al., 2007; Rapila et al., 2008) at later stages than were the focus in this study. To further understand the mechanism of NCX1 in SACO generation will require in vivo electrophysiological analysis to more accurately determine whether the inhibitory effect of NCX1 blockade with CB-DMB and KB-R7943 is due to effects on reverse mode NCX1 function (inhibition of translemmal Ca2+ influx) 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.

As cardiomyocytes matured, transition to LTCC became the predominant mechanism for inward Ca2+ 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 (Ebert et al., 2005). Increased expression of NCX1 has also been linked with pathological hypertrophy and heart failure (reviewed in Sipido et al., 2002), whereby elevated NCX1 is thought to compensate for defective ECC and depressed function of SERCA but also produce arrhythmogenic-delayed after-depolarisations (Gómez et al., 1997; Schultz et al., 2004; Venetucci et al., 2007). Transgenic mice over-expressing NCX1 within the myocardium exhibited a proportional decrease in contractile function and increased incidence of heart failure, suggesting a decompensatory mechanism with regards to Ca2+ handling (Roos et al., 2007).

Delta isoforms of CaMKII predominate in the heart and are involved in multiple signalling cascades to regulate gene expression, as well as cardiomyocyte physiology including Ca2+ and Na+ homeostasis (Wagner et al., 2006; Aiba et al., 2010). In this study, CaMKII activation was impaired following NCX1 inhibition in embryonic stem cells, and, moreover, canonical hypertrophy genes, including Mef2c and Myh7 were down-regulated in response to impaired NCX function during development. Whilst changes in gene expression did differ between embryos and ESCs in response to treatment with different drugs, this likely reflects inherent differences between the in vitro versus in vivo models, as well as the timing and length of drug exposure. ESCs were exposed at a relatively earlier stage of cardiac lineage specification and for longer periods, which was not feasible in the live embryo cultures, resulting in a stronger down-regulation of the genes investigated. Overall though these findings suggest analogous roles for NCX1 in early heart development and adult hypertrophy, with regards Ca2+-handling and foetal cardiac gene induction to promote physiological or pathological myocyte growth, respectively.

There is recent precedent for a role for Ca2+ in the establishment of other embryonic lineages; most notably Ca2+ signals are involved in the earliest steps of neurogenesis, including neural induction and the differentiation of neural progenitors into neurons (Leclerc et al., 2012). Here we show that pharmacological inhibition of NCX1 and dysregulation of Ca2+ handling from the outset had an adverse effect on early cardiomyocyte differentiation and led to impaired cardiogenesis in the embryo (Figure 5). Thus, an early induction of Ca2+-handling preceding beating within cardiac muscle is pivotal for subsequent terminal differentiation and normal heart development.

Materials and methods

Mouse strains, husbandry and embryo collection

All animal experiments were carried out according to UK Home Office project license PPL 30/3155 and 30/2887 compliant with the UK animals (Scientific Procedures) 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 (Charles River, England). 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 (Sigma-Aldrich, England).

Embryo staging

Progressive crescent stages were defined based on morphological criteria: the length (medio-lateral axis) and the maximum height (rostral-caudal axis) 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 (Supplementary file 1a).

Live imaging – DIC, Ca2+, embryos and cells

Live imaging of embryos, including Ca2+ imaging was performed as previously described, with some adaptations (Chen et al., 2014). Briefly, freshly dissected embryos were imaged in a mix of 50% phenol red-free CMRL (PAN-Biotech, Germany) supplemented with 10 mM L/glutamine (Sigma-Aldrich) and 50% Knockout Serum Replacement (Life Technologies, England). Initial characterisation of cardiac contractions was performed using Differential Interference Contrast (DIC) imaging on a Spinning Disk Confocal microscope at 37°C and an atmosphere of 5% CO2 + 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 (MatTek Corporation, Framingham, MA) 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 (Sigma-aldrich) was used at a final concentration of 10 μM, CB-DMB (Sigma-aldrich) at 20 μM, KB-R7943 (Sigma-aldrich) at 30 μM, Ryanodine (Tocris Bioscience, England) at 100 μM and 2-APB at 200 μM, all diluted in DMSO (Sigma-aldrich). 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.

Embryo culture

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% CO2+ 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.

ES cell culture and cardiomyocyte differentiation

Cardiomyocyte differentiation from ESCs was carried out as previously described using an Nkx2.5-GFP (Moretti et al., 2006) and Eomes-GFP (Arnold et al., 2009) cell line. Briefly, ESCs were maintained in an undifferentiated state by culturing with KO-DMEM (Gibco, England) supplemented with glutamax (2 mM; Gibco), embryomax FBS (15%; Millipore), nonessential amino acids (0.1 mM; Invitrogen, England), penicillin (60U/mL; Gibco), streptomycin (60 μg/mL; Gibco), β-mercaptoethanol (0.1 mM; Sigma-aldrich) and Leukaemia inhibitory factor (1000 U/ml; Millipore, England). Cardiomyocyte differentiation was induced using the hanging drop culture method (Kattman and Keller, 2007). Approximately 500 ESCs were plated in 20 μl drops of differentiation media (4.5 g glucose/DMEM, embryomax FBS (20%), nonessential amino acids (0.1 mM), penicillin (60 U/ml), streptomycin (60 μg/ml), β-mercaptoethanol [0.1 mM]) on the lids of petri dishes and cultured as hanging drops throughout the first four days of differentiation, allowing embryoid bodies (EBs) to form. At day 4, the EBs were transferred onto 0.1% gelatin coated plates for a further 10 days of culture before being collected at day 14 of differentiation. 1 μM CB-DMB and 10 μM Nifedipine were added to the differentiation media at day 0. For control experiments, DMSO was added at the same concentration as drug containing media. Once the EBs had been plated at day 4, drug-containing media was changed every two days up until day 14. To assess the percentage of EBs which were beating, drug containing media was removed 2 hr prior to assessment and replaced with fresh drug-free culture media. Culturing of ESCs in different Ca2+ concentrations was achieved using Ca2+ free DMEM (Gibco) with Ca2+ being added back to the desired concentration (0.1 mM, 1.0 mM, 1.8 mM). 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.

Immunostaining

Dissected 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 (5% donkey serum, 1%BSA in PBT-0.1%) overnight (o/n) at 4°C. Primary antibodies (Supplementary file 1e) were then added to the solution and incubated o/n at 4°C. The embryos were washed 3x in PBT-0.1% and incubated o/n 4°C in PBT-0.1% with the secondary antibodies (Supplementary file 1e), then subsequently washed 3x PBT-0.1% for 15 min and mounted in Vectashield mounting media with DAPI for at least 24 hr at 4°C. After fixation ESC samples were rinsed with PBS before being permeabilised with PBT-0.1% for 10 min, followed by blocking with 10% goat serum, 1% BSA in PBT-0.1% for 1 hr. Incubation with primary antibodies, diluted in blocking buffer, was carried out o/n at 4°C. After incubation with primary antibodies samples were washed for 3x for 10 min with PBT-0.1% and then incubated with secondary antibodies (Supplementary file 1e) diluted in blocking buffer for 1 hr at room temperature. After incubation with secondary antibodies samples were washed 5x for 5 min in PBS. Samples were mounted using Vectashield mounting media with DAPI for at least 24 hr prior to imaging with either a 40x oil (1.36 NA) or water (1.2 NA) objective. Images were captured at a 512 × 512 pixel dimension and tiled 2x2 with a Z-step of 1.5 µm. For embryos each staining was repeated for at least three litters (6–10 embryos per litter). Both experiments involving embryos and ESCs were performed on at least three independent days and with two different secondary antibodies.

RNA-extraction and qRT-PCR

RNA extraction of whole embryos, embryonic hearts, head folds and ESC samples was performed using an RNeasy Micro Kit (Qiagen, England) 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 (Invitrogen, England), according to the manufacturer’s instructions, additional DNase I (Promega, England) 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 (Supplementary file 1f), together with endogenous controls (treated embryos and differentiated ES cells, HPRT, GAPDH, 18 s; in vivo timecourse, GAPDH & HPRT; ES cell differentiation, GAPDH & 18 s (Murphy and Polak, 2002) were measured in triplicate for each sample by quantitative real-time PCR using SYBR Green (Applied Biosystems, England). Each reaction contained: 8 ng cDNA, 0.5 μl of each primer, 6.5 μl water and 12.5 μl 2 x SYBR Green, made up with H2O to a final volume of 22 μl. Primers (Sigma-aldrich) were either designed using Primer-BLAST (National Center for Biotechnology Information, National Institutes of Health) or obtained from PrimerBank (http://pga.mgh.harvard.edu/primerbank; Primer 3) or previous publications (primer sequences in Supplementary file 1f). Primers were designed to span exon-intron boundaries, have annealing temperatures around 60°C and generate amplicons between 50–200 bp. The reaction mixture and samples were loaded into either a MicroAmp Optical 96-Well Reaction Plates or MicroAmp Fast Optical 96-Well Reaction Plates and sealed with Optical Adhesive Films (Life Technologies). Quantification was performed on a ViiA 7 Detection System (Applied Biosystems) using a PCR programme of 95°C 15 min followed by 40 cycles of (95°C 15 s melting phase and 60°C 1 min annealing and extension). Amplification of a single amplicon was confirmed by obtaining dissociation curve (melt curve) profiles as well as using gel electrophoresis to separate the reaction product. Cycle threshold (Ct) values were generated using either Viia7 software (Applied Biosystems). Relative gene expression levels were obtained using the ΔΔCt method, in which expression of each gene of interest was normalised to endogenous controls (Schmittgen and Livak, 2008) (Murphy and Polak, 2002), and presented as fold change over a reference sample. For time course data fold-change was calculated in relation to the earliest stages (E7.75 in vivo, D0 ESC models), 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.

Western blot analysis

Protein was extracted on ice using direct lysis of cells with NP-40 extraction buffer (150 mM NaCl, 1.0% NP-40, 50 mM Tris (pH8.0)) and 1x protease and phosphatase inhibitor cocktail (Roche, England). 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 (Bio-rad, England). 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 (GE Healthcare, England).

Statistics

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 (Ward, 1963).

Image analysis

A variant of absolute image filter was used to visualize and plot measurements of cardiac contractions in the developing cardiac crescent as described elsewhere (Chen et al., 2014). Briefly, pixel displacement, indicative of contractions, was visualized and represented by increased grey levels within the crescent. Change in pixel intensity was assessed in a selected region, to reveal the contraction dynamics. Background Ca2+ 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.

References

  1. 1
  2. 2
  3. 3
  4. 4
  5. 5
  6. 6
  7. 7
  8. 8
  9. 9
  10. 10
  11. 11
  12. 12
  13. 13
  14. 14
  15. 15
  16. 16
  17. 17
  18. 18
  19. 19
  20. 20
  21. 21
  22. 22
  23. 23
  24. 24
  25. 25
  26. 26
  27. 27
  28. 28
  29. 29
    Early differentiation of the heart in mouse embryos
    1. MH Kaufman
    2. V Navaratnam
    (1981)
    Journal of Anatomy 133:235–246.
  30. 30
  31. 31
  32. 32
    Loss of connexin45 causes a cushion defect in early cardiogenesis
    1. M Kumai
    2. K Nishii
    3. K Nakamura
    4. N Takeda
    5. M Suzuki
    6. Y Shibata
    (2000)
    Development 127:3501–3512.
  33. 33
  34. 34
  35. 35
  36. 36
  37. 37
  38. 38
    Functional comparison of the three isoforms of the Na+/Ca2+ exchanger (NCX1, NCX2, NCX3)
    1. B Linck
    2. Z Qiu
    3. Z He
    4. Q Tong
    5. DW Hilgemann
    6. KD Philipson
    (1998)
    The American Journal of Physiology 274:C415–C423.
  39. 39
  40. 40
    Regulation and modulation of calcium channels in cardiac, skeletal, and smooth muscle cells
    1. TF McDonald
    2. S Pelzer
    3. W Trautwein
    4. DJ Pelzer
    (1994)
    Physiological Reviews 74:365–507.
  41. 41
  42. 42
  43. 43
  44. 44
  45. 45
  46. 46
  47. 47
  48. 48
  49. 49
    Differentiation of the myocardial rudiment of mouse embryos: an ultrastructural study including freeze-fracture replication
    1. V Navaratnam
    2. MH Kaufman
    3. JN Skepper
    4. S Barton
    5. KM Guttridge
    (1986)
    Journal of Anatomy 146:65–85.
  50. 50
  51. 51
  52. 52
    Ontogeny of humoral heart rate regulation in the embryonic mouse
    1. GA Porter
    2. SA Rivkees
    (2001)
    American Journal of Physiology. Regulatory, Integrative and Comparative Physiology 281:R401–R407.
  53. 53
    Tissue specificity and alternative splicing of the Na+/Ca2+ exchanger isoforms NCX1, NCX2, and NCX3 in rat
    1. BD Quednau
    2. DA Nicoll
    3. KD Philipson
    (1997)
    The American Journal of Physiology 272:C1250–C1261.
  54. 54
  55. 55
  56. 56
  57. 57
  58. 58
    MesP1: a novel basic helix-loop-helix protein expressed in the nascent mesodermal cells during mouse gastrulation
    1. Y Saga
    2. N Hata
    3. S Kobayashi
    4. T Magnuson
    5. MF Seldin
    6. MM Taketo
    (1996)
    Development 122:2769–2778.
  59. 59
  60. 60
  61. 61
  62. 62
  63. 63
  64. 64
  65. 65
  66. 66
  67. 67
  68. 68
  69. 69
  70. 70
  71. 71
  72. 72
  73. 73
  74. 74
  75. 75
  76. 76
  77. 77

Decision letter

  1. Margaret Buckingham
    Reviewing Editor; Institut Pasteur, France

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.

[Editors’ note: a previous version of this study was rejected after peer review, but the authors submitted for reconsideration. The previous decision letter after peer review is shown below.]

Thank you for choosing to send your work entitled "Calcium handling precedes cardiac differentiation to initiate the first heart beat" for consideration at 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.

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.

We consider that the issues raised by the reviewers cannot be addressed in the short time frame required by 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.

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 Figure 4 as neither Slc8a1 or Cacna1c are solely restricted to the heart and the gene expression was performed on whole embryos.

More information is needed to evaluate the gene expression studies. At minimum, primer details, amplicon details, replicates (both biological and technical) 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 Figure 5A-F but at 1mM in those in Figure 6A-D. This is a massive difference. The authors must provide full rationale for these choices in light of the issue raised under point 2.

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.

5) In the last paragraph of the subsection “NCX1 is essential for the initiation of Ca2 transients in the developing cardiac crescent”. the effects on GFP from Nkx2-5GFP should be quantified, preferably by FACS.

6. 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 (only 4 genes analysed, two of these encoding the channels, and only at one time point). Effects could be secondary.

7) In the subsection “Inward 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?

8) 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:

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 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.

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, 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.

Most 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.

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 (please see for instance: Excitation-Contraction Coupling of the Mouse Embryonic Cardiomyocyte" Rapila R et al. 2008 and the other papers by the Tavi group or "Intracellular Ca2 Oscillations, a Potential Pacemaking Mechanism in Early Embryonic Heart Cells" Sasse P et al. 2007).

Furthermore, it is well known that intact Ca2 cycling is important for cardiac gene expression and heart development. The authors should clearly cite the previous work and specifically describe the novelty and mechanism of their study.

The main conclusion of the blocker experiments is that NCX reverse mode is important for 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 (although controversial if this really happens, or if the effect is only a less effective forward mode). 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 (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 (e.g. "Direction-independent block of bi-directional Na /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.

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.

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 (which is easy, because of the nice in vivo data).

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 (especially stage 1 2) would help. Is cross striation completely absent in stage 0?

The finding of Ca2 transients at stage 0 is fascinating. Maybe better call them "spontaneous asynchronous Ca2 oscillations" also to highlight the difference semantically to the (action potential-based?) 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.

Figure 3: Normalizing gene expression data to D0 or E7.5 (where most genes are not expressed) make less sense, adds noise and masks information. Please consider normalization to the time point of the highest value.

Unfortunately, Cacna1d (Cav1.3), which is the dominant isoform of LTCC in the early embryo (see "Subtype switching of L-Type Ca 2 channel from Cav1.3 to Cav1.2 in embryonic murine ventricle". Takemura H et al. 2005) 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 (see also "Functional Embryonic Cardiomyocytes after Disruption of the L-type α1C (Cav1.2) 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 (Figure 6O) 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?

Was the data in Figure 5A, B, E, F obtained from Ca imaging or from video microscopy? If here video microscopy was used, also data from Ca imaging should be added to see the acute (>1-2 minutes) effects of the three blockers on Ca2 transients and remaining Ca2 oscillations (before all Ca2 has left the cell and the impaired Ca2 homeostasis is blocking pacemaking).

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.]

Thank you for submitting your article "Calcium handling precedes cardiac differentiation to initiate the first heartbeat" for consideration by 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.

The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.

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 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.

Firstly, in the experiments with inhibitors, the stage 0 experiments are critical. The authors use nifedipine to block voltage dependent 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.

The SACO shown in Figure 2D and Video 3 is one of the most important pieces of data and in fact is the major novelty. Please provide more data here including n-numbers, statistics and report reproducibility of these findings. Are SACO observed in every stage 0 embryo? What is the Ca2 cycling frequency and amplitude and how often are single uncoupled (Figure 2D´) and multiple coupled cells (Figure 2D´´) observed?

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 (example traces, videos) 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 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 novel Figure 3—figure supplement 1E on RyR/APD treatment is confusing as it does not follow the authors (very good) stage classification (stage 0, 1, 2, 3, LHT) but has a novel classification (turned/pre-turned embryo). Please label data as stage 0, stage 1 and LHT, at least. Please add statistics, error bars and original Ca2 imaging traces. A DMSO control for stage 3 is missing.

Figure 4 A, B, E, F: the error bars are missing. In fact, it is not even fully clear from the methods what% inhibition means. Please discriminate between complete block of beating (movement in DIC images), reduction of frequency, complete block of Ca2 transients and reduction of Ca2 transient amplitude. Also please provide time courses and report the delay of drug action (should be easy as Ca2 imaging was performed at baseline and at 5, 15 and 30 minutes post drug treatment).

In general, in the text it is important to clarify what is meant by inhibition and partial inhibition.

Although the pharmacological block using CB-DMB shown in Figure 4I-L lets the authors conclude that 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 (under special circumstances such as elevated intracellular Na or depolarized membrane potential) 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.

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 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 (NCX block-based Ca2 overload can also inhibit gene expression), 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.

A 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 (normalised only to housekeeping genes) and then the meaningful differences in time course/relative levels discussed.

[Editors' note: further revisions were requested prior to acceptance, as described below.]

Thank you for resubmitting your work entitled "Calcium handling precedes cardiac differentiation to initiate the first heartbeat" for further consideration at eLife.

Before publication, the following remaining concerns of one reviewer will need to be addressed:

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 Ca2 -handling in the cardiac crescent” ("variable frequencies and durations"). From the authors comment in the rebuttal and from Video 4 (very nice) it is well understandable that precise numbers are difficult to give but at lease state something like "Compared to Ca2 transients at later stages (Figure 2A) the SACO were rare. During a ~20 s recording period we observed only 10.3 -x individual SACO per embryo (n=24) occurring in different sites. Consecutive SACO in a same site were rarely observed within the 20 s imaging windows and therefore we conclude that SACO in individual cells occur at a frequency < 3 bpm."

2) Please describe the meaning of Figure 2—figure supplement 1C with something like "During a SACO, Ca2 rises slowly and reaches the peak between 0.5 and 10 s. Cells with slow Ca2 influx also showed similar slow Ca2 efflux (Figure 2—figure supplement 1C)."

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).

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 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).

https://doi.org/10.7554/eLife.17113.025

Author response

[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.

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 vitro analyses of isolated cells (Sasse et al. 2007; Rapila et al., 2008). 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.

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;

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 in vitro studies;

We exclude an essential role for the SR and reveal that sarcolemmal Ca2

flux 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.

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 Ca2 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.

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.

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 Ca2 -handling and contractile function feed back onto cardiomyocyte differentiation/maturation and early cardiac morphogenesis functioning.

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.

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 (see revised Figure 1A-E; subsection “Staging of early cardiac development and sarcomeric assembly”, second paragraph). In addition, we have performed qRT-PCR on the genes encoding cardiac troponin T, myomesin and sarcomeric α-actinin, which all revealed significant up-regulation in isolated cardiac crescents between stage 0 and stage 1 (See revised Figure 1F; and the aforementioned paragraph).

The gene expression studies should be complemented by whole mount in situ hybridization. This is especially important for Figure 4 as neither Slc8a1 or Cacna1c are solely restricted to the heart and the gene expression was performed on whole embryos.

We have expanded our data to now include gene expression from both isolated cardiac crescents and head folds to address the issue of Slc8a1 or Cacna1c being expressed both in heart and extra-cardiac lineages. By isolating tissues from within the embryo and performing qRT-PCR we are able to more accurately quantify gene expression, as well as assess multiple genes per time point, which is why we have retained a qRT-PCR based-approach as opposed to performing whole mount in situhybridisation. Whilst Slc8a1 and Cacna1c both increased in the heart samples there were no changes in expression within the head folds (see Figure 3—figure supplement 1A-C; subsection “Ion channels are expressed during the earliest stages of heart development”) confirming our original data. In addition, our immunostaining analyses for NCX and LTCC provides spatial localisation on protein expression, negating the need to perform whole mount in situ hybridisation.

More information is needed to evaluate the gene expression studies. At minimum, primer details, amplicon details, replicates (both biological and technical) 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 (see revised Supplementary file 1F; subsection “RNA-extraction and qPCR”). Supplementary file 1F includes a list of primers used as well as sequence information, amplicon size, primer source, PCR parameters and further details are now also included in the revised Methods section. All samples were run in triplicate to account for technical variability and figure legends now state the number of biological replicates. Reference genes were initially validated by qPCR and this was the rationale for not using HPRT as a housekeeping gene when assessing gene expression during ESC differentiation, given the HPRT Ct values decreased during differentiation, as previously reported (Murphy and Polak, 2002). We have now used multiple housekeeping genes for each experiment, to provide us with more comprehensive reference expression levels, as now stated in the revised methods. For time course data fold-change was calculated in relation to the earliest stages (E7.75 in vivo, D0 ESC models), whilst for drug treated experiments fold change was calculated relative to control (DMSO) samples; this is now included in the revised methods as requested.

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.

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.

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 -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.

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.

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 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.

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 Figure 5A-F but at 1mM in those in Figure 6A-D. This is a massive difference. The authors must provide full rationale for these choices in light of the issue raised under point 2.

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 Ca2 measurements. Unfortunately, we are unable to definitively measure the concentration of drug to which the tissue was actually exposed.

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.

We thank the reviewer for pointing this out and have included new experiments in revised Figure 4 and reworded the accompanying text (subsection “Ca2 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.

5) In the last paragraph of the subsection “NCX1 is essential for the initiation of Ca2 transients in the developing cardiac crescent”. the effects on GFP from Nkx2-5GFP should be quantified, preferably by FACS.

We have now quantified Nkx2.5-GFP by assessing the percentage of GFP area across multiple images taken from different experiments (see Figure 5—figure supplement 3, subsection “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.

6. 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 (only 4 genes analysed, two of these encoding the channels, and only at one time point). Effects could be secondary.

These experiments include four key cardiac genes, but also additional analysis of Slc8a1, 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 (see Figure 5—figure supplement 1A-C).

7) In the subsection “Inward 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?

Percentage of beating embryoid body analysis was performed in media containing 1.8 mM 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 .

8) 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 (see Figure 5E; subsection " NCX1 is essential for cardiomyocyte differentiation and cardiac crescent formation”). By performing this more in-depth analysis, we are confident in concluding that inhibition of NCX by CB-CMB leads to a decrease in mature cardiomyocytes which is not observed with inhibition of LTCCs by nifedipine treatment.

Reviewer #3:

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 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.

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, 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.

We are grateful for the positive comment regarding 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.

Most 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.

Pace-making appears not to apply to stages earlier than the looping heart tube, consistent with our focus on the initiation of Ca2 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.

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 (please see for instance: Excitation-Contraction Coupling of the Mouse Embryonic Cardiomyocyte" Rapila R et al. 2008 and the other papers by the Tavi group or "Intracellular Ca2 Oscillations, a Potential Pacemaking Mechanism in Early Embryonic Heart Cells" Sasse P et al. 2007).

Furthermore, it is well known that intact Ca2 cycling is important for cardiac gene expression and heart development. The authors should clearly cite the previous work and specifically describe the novelty and mechanism of their study.

We have now significantly expanded the Introduction to cite previous studies that have looked at early cardiac development and contractile function and more clearly highlighted the novelty of our data. The manuscripts mentioned in the comment above have looked at cells from later stages than us (linear heart tube stage or later) 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 is not required for the earliest manifestation of SACOs which precedes pacemaking activity.

The main conclusion of the blocker experiments is that NCX reverse mode is important for 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 (although controversial if this really happens, or if the effect is only a less effective forward mode). 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.

We are focusing on the earliest onset of 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.

NCX has been shown to act in both forward and reverse mode in embryonic cardiomyocytes (Reppel et al. 2007, Annals of the New York Academy of Sciences) and the directionality of NCX1 activity is dependent on the concentration gradient of intra- and extra-cellular Ca2 and Na in addition to the membrane potential (Reppel, Reuter, et al. 2007). 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 (Sasse et al. 2007; Rapila et al. 2008), 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.

We have qualified our interpretation of NCX1 acting in reverse mode in the revised text:

“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 influx at this early stage.”

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 (e.g. "Direction-independent block of bi-directional Na /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.

We agree that the concept of NCX working in reverse mode is controversial despite the fact that it has been reported that KB-R9743 preferentially blocks reverse mode NCX1 activity (Hoyt et al. 1998; Iwamoto 2004). 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 (which is easy, because of the nice in vivo data).

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 (especially stage 1 2) 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 Figure 1A-E and Figure 1—figure supplement 1A-E, inserts are provided highlighting the formation of sarcomeres from stage 1 onwards. From this more detailed analysis we have been unable to detect cross striation within the stage 0 cardiac crescent, as is now addressed in the subsection “Staging of early cardiac development and sarcomeric assembly”.

The finding of Ca2 transients at stage 0 is fascinating. Maybe better call them "spontaneous asynchronous Ca2 oscillations" also to highlight the difference semantically to the (action potential-based?) 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.

We thank the reviewer for their positive comments and have taken on-board the suggested terminology of "spontaneous asynchronous 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 Figure 2D now includes characterisation of SACOs within a 90 sec video and highlights the variation in frequency and oscillation dynamics. We believe there is evidence that transients are coupled between some individual cells and we are planning future studies to measure membrane potential within these cells. However, this requires extensive further experimentation which is beyond the scope of this submission, but will be important in understanding the progression of SACOs to synchronised/paced transients.

Figure 3: Normalizing gene expression data to D0 or E7.5 (where most genes are not expressed) make less sense, adds noise and masks information. Please consider normalization to the time point of the highest value.

After 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 (see "Subtype switching of L-Type Ca 2 channel from Cav1.3 to Cav1.2 in embryonic murine ventricle". Takemura H et al. 2005) 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 (see also "Functional Embryonic Cardiomyocytes after Disruption of the L-type α1C (Cav1.2) 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.

We thank the reviewer for this suggestion and have now investigated the expression of Cav1.3 (Figure 3—figure supplement 1A; subsection “Ion channels are expressed during the earliest stages of heart development”). However, by performing pharmacological inhibitor studies using Nifedipine, we have effectively inhibited all LTCC isoforms and not just Cav1.2 (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 Figure 4A-K and we have revised the text to limit our conclusions, for relative roles of NCX versus LTCC, to the earliest manifestation of Ca2 handling.

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 (Figure 6O) 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 (DMSO, n=6; CB-DMB, n=8; Nifedipine, n=6) 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 Figure 5E) and clearly show the inhibitory effect of pharmacological NCX blockade on cardiomyocyte differentiation, which also correlated with a decrease in cardiac specific gene expression (revised Figure 5F). Due to the clear and consistent decrease in cTnT staining after NCX inhibition, we have not quantified the immunofluoresence, particularly as quantification of immunostaining is of limited accuracy. However, we present quantitative data on changes in gene expression in the CB-DMB treated embryos through qRT-PCR. Whilst being able to study a developmental timecourse of cardiac crescent development with and without pharmacological inhibition would be ideal, it is not currently feasible due to issues associated with staging embryos, variations in extent of development between embryos and the technical limitations with the live-imaging.

Was the data in Figure 5A, B, E, F obtained from Ca imaging or from video microscopy? If here video microscopy was used, also data from Ca imaging should be added to see the acute (>1-2 minutes) effects of the three blockers on Ca2 transients and remaining Ca2 oscillations (before all Ca2 has left the cell and the impaired Ca2 homeostasis is blocking pacemaking).

Ca2 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 (Figure 4A-H (stage1/2: DMSO, n=4; CB-DMB, n=7; KB-R7943, n=6; nifedipine, n=6; stage3/LHT: DMSO, n=5; CB-DMB, n=14; KB-R7943, n=7; nifedipine, n=8) at specific time- points (5mins, 15mins and 30mins).

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.

We apologize for this confusion. While CB-DMB and KB-R7943 were used for the acute, short term embryo culture experiments, only CB-DMB and Nifedipine were used in the long term embryo culture studies. Due to the difficulties associated with obtaining large numbers of embryos at the same development stage (dissection, handling and maintaining constant culture conditions) 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.]

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 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.

We 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.

Firstly, in the experiments with inhibitors, the stage 0 experiments are critical. The authors use nifedipine to block voltage dependent 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.

We have now performed follow-up experiments using KB-R7943 at stage 0, and have included this data in the revised Figure 4H. Whilst we are aware of the controversy surrounding the mode of KB-R7943 action (addressed in the fourth paragraph of the Discussion), we have found that two independent NCX inhibitors block the SACOs observed at Stage 0, allowing us to conclude that NCX is required for their generation.

The SACO shown in Figure 2D and Video 3 is one of the most important pieces of data and in fact is the major novelty. Please provide more data here including n-numbers, statistics and report reproducibility of these findings. Are SACO observed in every stage 0 embryo? What is the Ca2 cycling frequency and amplitude and how often are single uncoupled (Figure 2D´) and multiple coupled cells (Figure 2D´´) observed?

We have now provided more data to describe the stage 0 SACOs including the number of stage 0 embryos assessed (n=35) as well as more data on the Ca2 transient dynamics including the time to peak Ca2 signal (Ca2 influx) and the time to ½ maximum Ca2 signal (Ca2 efflux) (Figure 2—figure supplement 1C). In general, as long as the endoderm was properly removed to allow penetration of the dye and better visualization of the cardiac mesoderm, SACOs were observed in every stage 0 embryo, revised text in subsection “The onset of Ca2 -handling in the cardiac crescent”. We believe that further studies on SACO dynamics, especially in terms of uncoupled and coupled cells, is beyond the scope of this current manuscript but is something we are actively addressing by developing methodology to accurately quantify Ca2 transient amplitude and synchronisation in live embryos, given this requires longer imaging periods than we are currently able to perform.

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 (example traces, videos) 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 signals? How is the variance of cells with SACO per embryo? ANOVA comparisons are required to take these variations in control embryos into account.

Following on from the new experiments conducted with KB-R7943 at stage 0, we have reanalysed our data and now report SACO inhibition after drug treatment as the percentage inhibition of SACOs compared to baseline (subsection “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 (Figure 4H-J, Supplementary file 1D). Regarding the statistics, we have chosen to perform Fisher Exact Probability Test (FEPT) and treat this data as categorical, i.e. presence or absence of SACOs, since the variance for CB-DMB and KB-7943 is virtually non-existent (almost all embryos had zero SACOs after treatment) and hence statistical tests which assume a continuous data model are not suitable and cannot be applied to data.

The novel Figure 3—figure supplement 1E on RyR/APD treatment is confusing as it does not follow the authors (very good) stage classification (stage 0, 1, 2, 3, LHT) but has a novel classification (turned/pre-turned embryo). Please label data as stage 0, stage 1 and LHT, at least. Please add statistics, error bars and original Ca2 imaging traces. A DMSO control for stage 3 is missing.

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, (indicating the Ca2 at the earliest stages is not SR dependent). 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 Figure 4—figure supplement 1. The data lacks error bars because in this instance it is binary (either it was inhibited or not) hence also why we applied a Freeman-Halton extension of the Fisher exact probability test.

Figure 4 A, B, E, F: the error bars are missing. In fact, it is not even fully clear from the methods what% inhibition means. Please discriminate between complete block of beating (movement in DIC images), reduction of frequency, complete block of Ca2 transients and reduction of Ca2 transient amplitude. Also please provide time courses and report the delay of drug action (should be easy as Ca2 imaging was performed at baseline and at 5, 15 and 30 minutes post drug treatment).

In general, in the text it is important to clarify what is meant by inhibition and partial inhibition.

We have now included an explicit definition of what is meant by% inhibition in the Methods section (subsection “Live imaging – DIC, Ca2 561, Embryos and Cells”) 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 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 Figure 4 to show the time-course of inhibition requested above. We are unable to determine Ca2 transient amplitude given the method for imaging Ca2 transients in intact tissue does not allow for ratiometric measurements.

Although the pharmacological block using CB-DMB shown in Figure 4I-L lets the authors conclude that 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 (under special circumstances such as elevated intracellular Na or depolarized membrane potential) 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.

We agree with the reviewer that the precise mechanism for periodic oscillations of Ca2 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.

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 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 (NCX block-based Ca2 overload can also inhibit gene expression), 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.

We have now reworded the text to be less dogmatic regarding 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 (e.g. by electroporation, or virus), 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.

A 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 (normalised only to housekeeping genes) 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:

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 Ca2 -handling in the cardiac crescent” ("variable frequencies and durations"). From the authors comment in the rebuttal and from Video 4 (very nice) it is well understandable that precise numbers are difficult to give but at lease state something like "Compared to Ca2 transients at later stages (Figure 2A) the SACO were rare. During a ~20 s recording period we observed only 10.3 -x individual SACO per embryo (n=24) occurring in different sites. Consecutive SACO in a same site were rarely observed within the 20 s imaging windows and therefore we conclude that SACO in individual cells occur at a frequency < 3 bpm."

The suggested sentences above are now included with correct values on page 6:“Compared to Ca2 transients at later stages (Figure 2A) 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(Figure 2—figure supplement 1C). During a ~20 s recording period we observed only 10.3 ± 0.7 individual SACOs per embryo (n=35) occurring in different sites. Consecutive SACOs in the same site were rarely observed within the 20 second imaging window and therefore we conclude that SACOs in individual cells occur at a frequency < 3 bpm.”

2) Please describe the meaning of Figure 2—figure supplement 1C with something like "During a SACO, Ca2 rises slowly and reaches the peak between 0.5 and 10 s. Cells with slow Ca2 influx also showed similar slow Ca2 efflux (Figure 2—figure supplement 1C)."

We have incorporated an explanation similar to that suggested above about Figure 2—figure supplement 1C on page 6:

“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(Figure 2—figure supplement 1C).”

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:

“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 pacemaking mechanism (Sasse et al. 2007 and Rapilla et al. 2008), at later stages than were the focus in this study.”

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 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).

We agree with the reviewer that we have not tested SR inhibition within the stage 0 embryo and have, therefore, included the following statement: 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).

https://doi.org/10.7554/eLife.17113.026

Article and author information

Author details

  1. Richard CV Tyser

    1. Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, United Kingdom
    2. The Hatter Cardiovascular Institute, University College London and Medical School, London, United Kingdom
    Contribution
    RCVT, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article
    Contributed equally with
    Antonio MA Miranda
    Competing interests
    The authors declare that no competing interests exist.
  2. Antonio MA Miranda

    Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, United Kingdom
    Contribution
    AMAM, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article
    Contributed equally with
    Richard CV Tyser
    Competing interests
    The authors declare that no competing interests exist.
  3. Chiann-mun Chen

    Wellcome Trust, London, United Kingdom
    Contribution
    C-mC, Acquisition of data, Analysis and interpretation of data, Contributed unpublished essential data or reagents
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon 0000-0002-7007-3649
  4. Sean M Davidson

    The Hatter Cardiovascular Institute, University College London and Medical School, London, United Kingdom
    Contribution
    SMD, Conception and design, Analysis and interpretation of data, Drafting or revising the article
    Competing interests
    The authors declare that no competing interests exist.
  5. Shankar Srinivas

    Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, United Kingdom
    Contribution
    SS, Conception and design, Analysis and interpretation of data, Drafting or revising the article
    Contributed equally with
    Paul R Riley
    For correspondence
    shankar.srinivas@dpag.ox.ac.uk
    Competing interests
    The authors declare that no competing interests exist.
  6. Paul R Riley

    Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, United Kingdom
    Contribution
    PRR, Conception and design, Analysis and interpretation of data, Drafting or revising the article
    Contributed equally with
    Shankar Srinivas
    For correspondence
    paul.riley@dpag.ox.ac.uk
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon 0000-0002-9862-7332

Funding

British Heart Foundation (4-year DPhil programme FS/12/69/3008)

  • Richard CV Tyser
  • Antonio MA Miranda

British Heart Foundation (BHF Oxbridge Regenerative Medicine Centre RM/13/3/30159)

  • Shankar Srinivas
  • Paul R Riley

Biotechnology and Biological Sciences Research Council (REI BB/F011512/1)

  • Shankar Srinivas

The Wellcome Trust (WTSIA 103788/Z/14/Z)

  • Shankar Srinivas

The Wellcome Trust (105031/C/14/Z)

  • Shankar Srinivas

British Heart Foundation (CH/11/1/28798)

  • Paul R Riley

British Heart Foundation (RG/13/9/303269)

  • Paul R Riley

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

This work was generously supported by the British Heart Foundation (RCVT, AMAM, SMD; CH/11/1/28798 and RG/13/9/303269 to PRR) including core support from the BHF Oxbridge Regenerative Medicine Centre (RM/13/3/30159), the BBSRC (REI BB/F011512/1 to SS) and The Wellcome Trust (C-mC; WTSIA 103788/Z/14/Z and 105031/C/14/Z to SS). We thank Liz Robertson for provision of the Eomes-GFP ESC line and Sean Wu for the Nkx2.5-eGFP ESC line.

Ethics

Animal experimentation: All animal experiments were carried out according to UK Home Office project license PPL 30/3155 Compliant with the UK Animals (Scientific Procedures) Act 1986.

Reviewing Editor

  1. Margaret Buckingham, Reviewing Editor, Institut Pasteur, France

Publication history

  1. Received: April 20, 2016
  2. Accepted: September 13, 2016
  3. Version of Record published: October 11, 2016 (version 1)

Copyright

© 2016, Tyser et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

Metrics

  • 3,868
    Page views
  • 469
    Downloads
  • 1
    Citations

Article citation count generated by polling the highest count across the following sources: Crossref, PubMed Central, Scopus.

Comments

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Download citations (links to download the citations from this article in formats compatible with various reference manager tools)

Open citations (links to open the citations from this article in various online reference manager services)

Further reading

    1. Computational and Systems Biology
    2. Developmental Biology and Stem Cells
    Qixuan Wang et al.
    Research Article Updated
    1. Cell Biology
    2. Genes and Chromosomes
    Wahid A Mulla et al.
    Research Article Updated