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.

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.

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 InsP_3. 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.

1. 1. Aiba T
   2. Hesketh GG
   3. Liu T
   4. Carlisle R
   5. Villa-Abrille MC
   6. O'Rourke B
   7. Akar FG
   8. Tomaselli GF

   (2010) Na+ channel regulation by Ca2+/calmodulin and Ca2+/calmodulin-dependent protein kinase II in guinea-pig ventricular myocytes

   Cardiovascular Research 85:454–463.

   https://doi.org/10.1093/cvr/cvp324

   • Google Scholar
2. 1. Andersen ND
   2. Ramachandran KV
   3. Bao MM
   4. Kirby ML
   5. Pitt GS
   6. Hutson MR

   (2015) Calcium signaling regulates ventricular hypertrophy during development independent of contraction or blood flow

   Journal of Molecular and Cellular Cardiology 80:1–9.

   https://doi.org/10.1016/j.yjmcc.2014.12.016

   • Google Scholar
3. 1. Arnold SJ
   2. Sugnaseelan J
   3. Groszer M
   4. Srinivas S
   5. Robertson EJ

   (2009) Generation and analysis of a mouse line harboring GFP in the Eomes/Tbr2 locus

   Genesis 47:775–781.

   https://doi.org/10.1002/dvg.20562

   • Google Scholar
4. 1. Bers DM

   (2002) Cardiac excitation-contraction coupling

   Nature 415:198–205.

   https://doi.org/10.1038/415198a

   • Google Scholar
5. 1. Biben C
   2. Harvey RP

   (1997) Homeodomain factor Nkx2-5 controls left/right asymmetric expression of bHLH gene eHand during murine heart development

   Genes & Development 11:1357–1369.

   https://doi.org/10.1101/gad.11.11.1357

   • Google Scholar
6. 1. Bloomekatz J
   2. Grego-Bessa J
   3. Migeotte I
   4. Anderson KV

   (2012) Pten regulates collective cell migration during specification of the anterior-posterior axis of the mouse embryo

   Developmental Biology 364:192–201.

   https://doi.org/10.1016/j.ydbio.2012.02.005

   • Google Scholar
7. 1. Bruneau BG

   (2008) The developmental genetics of congenital heart disease

   Nature 451:943–948.

   https://doi.org/10.1038/nature06801

   • Google Scholar
8. 1. Brustovetsky T
   2. Brittain MK
   3. Sheets PL
   4. Cummins TR
   5. Pinelis V
   6. Brustovetsky N

   (2011) KB-R7943, an inhibitor of the reverse Na+ /Ca2+ exchanger, blocks N-methyl-D-aspartate receptor and inhibits mitochondrial complex I

   British Journal of Pharmacology 162:255–270.

   https://doi.org/10.1111/j.1476-5381.2010.01054.x

   • Google Scholar
9. 1. Buckingham M
   2. Meilhac S
   3. Zaffran S

   (2005) Building the mammalian heart from two sources of myocardial cells

   Nature Reviews Genetics 6:826–837.

   https://doi.org/10.1038/nrg1710

   • Google Scholar
10. 1. Cai CL
    2. Liang X
    3. Shi Y
    4. Chu PH
    5. Pfaff SL
    6. Chen J
    7. Evans S

    (2003) Isl1 identifies a cardiac progenitor population that proliferates prior to differentiation and contributes a majority of cells to the heart

    Developmental Cell 5:877–889.

    https://doi.org/10.1016/S1534-5807(03)00363-0

    • Google Scholar
11. 1. Chen CM
    2. Miranda AM
    3. Bub G
    4. Srinivas S

    (2014) Detecting cardiac contractile activity in the early mouse embryo using multiple modalities

    Frontiers in Physiology 5:508.

    https://doi.org/10.3389/fphys.2014.00508

    • Google Scholar
12. 1. Cho CH
    2. Lee SY
    3. Shin HS
    4. Philipson KD
    5. Lee CO

    (2003) Partial rescue of the Na+-Ca2+ exchanger (NCX1) knock-out mouse by transgenic expression of NCX1

    Experimental & Molecular Medicine 35:125–135.

    https://doi.org/10.1038/emm.2003.18

    • Google Scholar
13. 1. Ciruna BG
    2. Rossant J

    (1999) Expression of the T-box gene Eomesodermin during early mouse development

    Mechanisms of Development 81:199–203.

    https://doi.org/10.1016/S0925-4773(98)00243-3

    • Google Scholar
14. 1. Conway SJ
    2. Kruzynska-Frejtag A
    3. Wang J
    4. Rogers R
    5. Kneer PL
    6. Chen H
    7. Creazzo T
    8. Menick DR
    9. Koushik SV

    (2002) Role of sodium-calcium exchanger (Ncx1) in embryonic heart development: a transgenic rescue?

    Annals of the New York Academy of Sciences 976:268–281.

    https://doi.org/10.1111/j.1749-6632.2002.tb04749.x

    • Google Scholar
15. 1. Cribbs LL
    2. Martin BL
    3. Schroder EA
    4. Keller BB
    5. Delisle BP
    6. Satin J

    (2001) Identification of the t-type calcium channel (Ca(v)3.1d) in developing mouse heart

    Circulation Research 88:403–407.

    https://doi.org/10.1161/01.RES.88.4.403

    • Google Scholar
16. 1. Devine WP
    2. Wythe JD
    3. George M
    4. Koshiba-Takeuchi K
    5. Bruneau BG

    (2014) Early patterning and specification of cardiac progenitors in gastrulating mesoderm

    eLife 3:e03848.

    https://doi.org/10.7554/eLife.03848

    • Google Scholar
17. 1. Ebert AM
    2. Hume GL
    3. Warren KS
    4. Cook NP
    5. Burns CG
    6. Mohideen MA
    7. Siegal G
    8. Yelon D
    9. Fishman MC
    10. Garrity DM

    (2005) Calcium extrusion is critical for cardiac morphogenesis and rhythm in embryonic zebrafish hearts

    PNAS 102:17705–17710.

    https://doi.org/10.1073/pnas.0502683102

    • Google Scholar
18. 1. Evans SM
    2. Yelon D
    3. Conlon FL
    4. Kirby ML

    (2010) Myocardial lineage development

    Circulation Research 107:1428–1444.

    https://doi.org/10.1161/CIRCRESAHA.110.227405

    • Google Scholar
19. 1. Fabiato A
    2. Fabiato F

    (1979) Use of chlorotetracycline fluorescence to demonstrate Ca2+-induced release of Ca2+ from the sarcoplasmic reticulum of skinned cardiac cells

    Nature 281:146–148.

    https://doi.org/10.1038/281146a0

    • Google Scholar
20. 1. Fujii S
    2. Hirota A
    3. Kamino K

    (1981) Optical indications of pace-maker potential and rhythm generation in early embryonic chick heart

    Journal of Physiology 312:253–263.

    https://doi.org/10.1113/jphysiol.1981.sp013627

    • Google Scholar
21. 1. Goss CM

    (1952) Development of the median coordinated ventricle from the lateral hearts in rat embryos with three to six somites

    The Anatomical Record 112:761–796.

    https://doi.org/10.1002/ar.1091120405

    • Google Scholar
22. 1. Granados-Riveron JT
    2. Brook JD

    (2012) The impact of mechanical forces in heart morphogenesis

    Circulation: Cardiovascular Genetics 5:132–142.

    https://doi.org/10.1161/CIRCGENETICS.111.961086

    • Google Scholar
23. 1. Gómez AM
    2. Valdivia HH
    3. Cheng H
    4. Lederer MR
    5. Santana LF
    6. Cannell MB
    7. McCune SA
    8. Altschuld RA
    9. Lederer WJ

    (1997) Defective excitation-contraction coupling in experimental cardiac hypertrophy and heart failure

    Science 276:800–806.

    https://doi.org/10.1126/science.276.5313.800

    • Google Scholar
24. 1. Harvey RP

    (2002) Patterning the vertebrate heart

    Nature Reviews Genetics 3:544–556.

    https://doi.org/10.1038/nrg843

    • Google Scholar
25. 1. Hirota A
    2. Kamino K
    3. Komuro H
    4. Sakai T
    5. Yada T

    (1985) Early events in development of electrical activity and contraction in embryonic rat heart assessed by optical recording

    Journal of Physiology 369:209–227.

    https://doi.org/10.1113/jphysiol.1985.sp015897

    • Google Scholar
26. 1. Hoyt KR
    2. Arden SR
    3. Aizenman E
    4. Reynolds IJ

    (1998) Reverse Na+/Ca2+ exchange contributes to glutamate-induced intracellular Ca2+ concentration increases in cultured rat forebrain neurons

    Molecular Pharmacology 53:742–749.

    https://doi.org/10.1124/mol.53.4.742

    • Google Scholar
27. 1. Iwamoto T

    (2004) Forefront of Na+/Ca2+ exchanger studies: molecular pharmacology of Na+/Ca2+ exchange inhibitors

    Journal of Pharmacological Sciences 96:27–32.

    https://doi.org/10.1254/jphs.FMJ04002X6

    • Google Scholar
28. 1. Kattman SJ
    2. Adler ED
    3. Keller GM

    (2007) Specification of multipotential cardiovascular progenitor cells during embryonic stem cell differentiation and embryonic development

    Trends in Cardiovascular Medicine 17:240–246.

    https://doi.org/10.1016/j.tcm.2007.08.004

    • Google Scholar
29. 1. Kaufman MH
    2. Navaratnam V

    (1981)

    Early differentiation of the heart in mouse embryos

    Journal of Anatomy 133:235–246.

    • Google Scholar
30. 1. Kimura J
    2. Watano T
    3. Kawahara M
    4. Sakai E
    5. Yatabe J

    (1999) Direction-independent block of bi-directional Na+/Ca2+ exchange current by KB-R7943 in guinea-pig cardiac myocytes

    British Journal of Pharmacology 128:969–974.

    https://doi.org/10.1038/sj.bjp.0702869

    • Google Scholar
31. 1. Koushik SV
    2. Wang J
    3. Rogers R
    4. Moskophidis D
    5. Lambert NA
    6. Creazzo TL
    7. Conway SJ

    (2001) Targeted inactivation of the sodium-calcium exchanger (Ncx1) results in the lack of a heartbeat and abnormal myofibrillar organization

    The FASEB Journal 15:1209–1211.

    https://doi.org/10.1096/fj.00-0696fje

    • Google Scholar
32. 1. Kumai M
    2. Nishii K
    3. Nakamura K
    4. Takeda N
    5. Suzuki M
    6. Shibata Y

    (2000)

    Loss of connexin45 causes a cushion defect in early cardiogenesis

    Development 127:3501–3512.

    • Google Scholar
33. 1. Langenbacher AD
    2. Dong Y
    3. Shu X
    4. Choi J
    5. Nicoll DA
    6. Goldhaber JI
    7. Philipson KD
    8. Chen JN

    (2005) Mutation in sodium-calcium exchanger 1 (NCX1) causes cardiac fibrillation in zebrafish

    PNAS 102:17699–17704.

    https://doi.org/10.1073/pnas.0502679102

    • Google Scholar
34. 1. Leclerc C
    2. Néant I
    3. Moreau M

    (2012) The calcium: an early signal that initiates the formation of the nervous system during embryogenesis

    Frontiers in Molecular Neuroscience 5:3.

    https://doi.org/10.3389/fnmol.2012.00064

    • Google Scholar
35. 1. Li J
    2. Pucéat M
    3. Perez-Terzic C
    4. Mery A
    5. Nakamura K
    6. Michalak M
    7. Krause KH
    8. Jaconi ME

    (2002) Calreticulin reveals a critical Ca(2+) checkpoint in cardiac myofibrillogenesis

    Journal of Cell Biology 158:103–113.

    https://doi.org/10.1083/jcb.200204092

    • Google Scholar
36. 1. Liang H
    2. Halbach M
    3. Hannes T
    4. Fleischmann BK
    5. Tang M
    6. Schunkert H
    7. Hescheler J
    8. Reppel M

    (2010) Electrophysiological basis of the first heart beats

    Cellular Physiology and Biochemistry 25:561–570.

    https://doi.org/10.1159/000315075

    • Google Scholar
37. 1. Linask KK
    2. Han MD
    3. Artman M
    4. Ludwig CA

    (2001) Sodium-calcium exchanger (NCX-1) and calcium modulation: NCX protein expression patterns and regulation of early heart development

    Developmental Dynamics 221:249–264.

    https://doi.org/10.1002/dvdy.1131

    • Google Scholar
38. 1. Linck B
    2. Qiu Z
    3. He Z
    4. Tong Q
    5. Hilgemann DW
    6. Philipson KD

    (1998)

    Functional comparison of the three isoforms of the Na+/Ca2+ exchanger (NCX1, NCX2, NCX3)

    The American Journal of Physiology 274:C415–C423.

    • Google Scholar
39. 1. Lytton J

    (2007) Na+/Ca2+ exchangers: three mammalian gene families control Ca2+ transport

    The Biochemical Journal 406:365–382.

    https://doi.org/10.1042/BJ20070619

    • Google Scholar
40. 1. McDonald TF
    2. Pelzer S
    3. Trautwein W
    4. Pelzer DJ

    (1994)

    Regulation and modulation of calcium channels in cardiac, skeletal, and smooth muscle cells

    Physiological Reviews 74:365–507.

    • Google Scholar
41. 1. Meilhac SM
    2. Esner M
    3. Kelly RG
    4. Nicolas JF
    5. Buckingham ME

    (2004) The clonal origin of myocardial cells in different regions of the embryonic mouse heart

    Developmental Cell 6:685–698.

    https://doi.org/10.1016/S1534-5807(04)00133-9

    • Google Scholar
42. 1. Mesaeli N
    2. Nakamura K
    3. Zvaritch E
    4. Dickie P
    5. Dziak E
    6. Krause KH
    7. Opas M
    8. MacLennan DH
    9. Michalak M

    (1999) Calreticulin is essential for cardiac development

    Journal of Cell Biology 144:857–868.

    https://doi.org/10.1083/jcb.144.5.857

    • Google Scholar
43. 1. Miyasaka KY
    2. Kida YS
    3. Banjo T
    4. Ueki Y
    5. Nagayama K
    6. Matsumoto T
    7. Sato M
    8. Ogura T

    (2011) Heartbeat regulates cardiogenesis by suppressing retinoic acid signaling via expression of miR-143

    Mechanisms of Development 128:18–28.

    https://doi.org/10.1016/j.mod.2010.09.002

    • Google Scholar
44. 1. Molkentin JD
    2. Lu JR
    3. Antos CL
    4. Markham B
    5. Richardson J
    6. Robbins J
    7. Grant SR
    8. Olson EN

    (1998) A calcineurin-dependent transcriptional pathway for cardiac hypertrophy

    Cell 93:215–228.

    https://doi.org/10.1016/S0092-8674(00)81573-1

    • Google Scholar
45. 1. Moretti A
    2. Caron L
    3. Nakano A
    4. Lam JT
    5. Bernshausen A
    6. Chen Y
    7. Qyang Y
    8. Bu L
    9. Sasaki M
    10. Martin-Puig S
    11. Sun Y
    12. Evans SM
    13. Laugwitz KL
    14. Chien KR

    (2006) Multipotent embryonic isl1+ progenitor cells lead to cardiac, smooth muscle, and endothelial cell diversification

    Cell 127:1151–1165.

    https://doi.org/10.1016/j.cell.2006.10.029

    • Google Scholar
46. 1. Murphy CL
    2. Polak JM

    (2002) Differentiating embryonic stem cells: GAPDH, but neither HPRT nor beta-tubulin is suitable as an internal standard for measuring RNA levels

    Tissue Engineering 8:551–559.

    https://doi.org/10.1089/107632702760240472

    • Google Scholar
47. 1. Méry A
    2. Aimond F
    3. Ménard C
    4. Mikoshiba K
    5. Michalak M
    6. Pucéat M

    (2005) Initiation of embryonic cardiac pacemaker activity by inositol 1,4,5-trisphosphate-dependent calcium signaling

    Molecular Biology of the Cell 16:2414–2423.

    https://doi.org/10.1091/mbc.E04-10-0883

    • Google Scholar
48. 1. Nakanishi T
    2. Seguchi M
    3. Takao A

    (1988) Development of the myocardial contractile system

    Experientia 44:936–944.

    https://doi.org/10.1007/BF01939887

    • Google Scholar
49. 1. Navaratnam V
    2. Kaufman MH
    3. Skepper JN
    4. Barton S
    5. Guttridge KM

    (1986)

    Differentiation of the myocardial rudiment of mouse embryos: an ultrastructural study including freeze-fracture replication

    Journal of Anatomy 146:65–85.

    • Google Scholar
50. 1. Nishii K
    2. Shibata Y

    (2006) Mode and determination of the initial contraction stage in the mouse embryo heart

    Anatomy and Embryology 211:95–100.

    https://doi.org/10.1007/s00429-005-0065-x

    • Google Scholar
51. 1. Passier R
    2. Zeng H
    3. Frey N
    4. Naya FJ
    5. Nicol RL
    6. McKinsey TA
    7. Overbeek P
    8. Richardson JA
    9. Grant SR
    10. Olson EN

    (2000) CaM kinase signaling induces cardiac hypertrophy and activates the MEF2 transcription factor in vivo

    Journal of Clinical Investigation 105:1395–1406.

    https://doi.org/10.1172/JCI8551

    • Google Scholar
52. 1. Porter GA
    2. Rivkees SA

    (2001)

    Ontogeny of humoral heart rate regulation in the embryonic mouse

    American Journal of Physiology. Regulatory, Integrative and Comparative Physiology 281:R401–R407.

    • Google Scholar
53. 1. Quednau BD
    2. Nicoll DA
    3. Philipson KD

    (1997)

    Tissue specificity and alternative splicing of the Na+/Ca2+ exchanger isoforms NCX1, NCX2, and NCX3 in rat

    The American Journal of Physiology 272:C1250–C1261.

    • Google Scholar
54. 1. Rapila R
    2. Korhonen T
    3. Tavi P

    (2008) Excitation-contraction coupling of the mouse embryonic cardiomyocyte

    Journal of General Physiology 132:397–405.

    https://doi.org/10.1085/jgp.200809960

    • Google Scholar
55. 1. Reppel M
    2. Sasse P
    3. Malan D
    4. Nguemo F
    5. Reuter H
    6. Bloch W
    7. Hescheler J
    8. Fleischmann BK

    (2007) Functional expression of the Na+/Ca2+ exchanger in the embryonic mouse heart

    Journal of Molecular and Cellular Cardiology 42:121–132.

    https://doi.org/10.1016/j.yjmcc.2006.10.008

    • Google Scholar
56. 1. Reuter H
    2. Henderson SA
    3. Han T
    4. Ross RS
    5. Goldhaber JI
    6. Philipson KD

    (2002) The Na+-Ca2+ exchanger is essential for the action of cardiac glycosides

    Circulation Research 90:305–308.

    https://doi.org/10.1161/hh0302.104562

    • Google Scholar
57. 1. Roos KP
    2. Jordan MC
    3. Fishbein MC
    4. Ritter MR
    5. Friedlander M
    6. Chang HC
    7. Rahgozar P
    8. Han T
    9. Garcia AJ
    10. Maclellan WR
    11. Ross RS
    12. Philipson KD

    (2007) Hypertrophy and heart failure in mice overexpressing the cardiac sodium-calcium exchanger

    Journal of Cardiac Failure 13:318–329.

    https://doi.org/10.1016/j.cardfail.2007.01.004

    • Google Scholar
58. 1. Saga Y
    2. Hata N
    3. Kobayashi S
    4. Magnuson T
    5. Seldin MF
    6. Taketo MM

    (1996)

    MesP1: a novel basic helix-loop-helix protein expressed in the nascent mesodermal cells during mouse gastrulation

    Development 122:2769–2778.

    • Google Scholar
59. 1. Sasse P
    2. Zhang J
    3. Cleemann L
    4. Morad M
    5. Hescheler J
    6. Fleischmann BK

    (2007) Intracellular Ca2+ oscillations, a potential pacemaking mechanism in early embryonic heart cells

    Journal of General Physiology 130:133–144.

    https://doi.org/10.1085/jgp.200609575

    • Google Scholar
60. 1. Schmittgen TD
    2. Livak KJ

    (2008) Analyzing real-time PCR data by the comparative C(T) method

    Nature Protocols 3:1101–1108.

    https://doi.org/10.1038/nprot.2008.73

    • Google Scholar
61. 1. Schultz JJ
    2. Glascock BJ
    3. Witt SA
    4. Nieman ML
    5. Nattamai KJ
    6. Liu LH
    7. Lorenz JN
    8. Shull GE
    9. Kimball TR
    10. Periasamy M

    (2004) Accelerated onset of heart failure in mice during pressure overload with chronically decreased SERCA2 calcium pump activity

    AJP: Heart and Circulatory Physiology 286:H1146–H1153.

    https://doi.org/10.1152/ajpheart.00720.2003

    • Google Scholar
62. 1. Secondo A
    2. Pannaccione A
    3. Molinaro P
    4. Ambrosino P
    5. Lippiello P
    6. Esposito A
    7. Cantile M
    8. Khatri PR
    9. Melisi D
    10. Di Renzo G
    11. Annunziato L

    (2009) Molecular pharmacology of the amiloride analog 3-amino-6-chloro-5-[(4-chloro-benzyl)amino]-n-[[(2,4-dimethylbenzyl)-amino]iminomethyl]-pyrazinecarboxamide (CB-DMB) as a pan inhibitor of the Na+-Ca2+ exchanger isoforms NCX1, NCX2, and NCX3 in stably transfected cells

    Journal of Pharmacology and Experimental Therapeutics 331:212–221.

    https://doi.org/10.1124/jpet.109.152132

    • Google Scholar
63. 1. Seisenberger C
    2. Specht V
    3. Welling A
    4. Platzer J
    5. Pfeifer A
    6. Kühbandner S
    7. Striessnig J
    8. Klugbauer N
    9. Feil R
    10. Hofmann F

    (2000) Functional embryonic cardiomyocytes after disruption of the L-type alpha1C (Cav1.2) calcium channel gene in the mouse

    Journal of Biological Chemistry 275:39193–39199.

    https://doi.org/10.1074/jbc.M006467200

    • Google Scholar
64. 1. Seki S
    2. Nagashima M
    3. Yamada Y
    4. Tsutsuura M
    5. Kobayashi T
    6. Namiki A
    7. Tohse N

    (2003) Fetal and postnatal development of Ca2+ transients and Ca2+ sparks in rat cardiomyocytes

    Cardiovascular Research 58:535–548.

    https://doi.org/10.1016/S0008-6363(03)00255-4

    • Google Scholar
65. 1. Sipido KR
    2. Volders PG
    3. Vos MA
    4. Verdonck F

    (2002) Altered Na/Ca exchange activity in cardiac hypertrophy and heart failure: a new target for therapy?

    Cardiovascular Research 53:782–805.

    https://doi.org/10.1016/S0008-6363(01)00470-9

    • Google Scholar
66. 1. Stokke MK
    2. Briston SJ
    3. Jølle GF
    4. Manzoor I
    5. Louch WE
    6. Øyehaug L
    7. Christensen G
    8. Eisner DA
    9. Trafford AW
    10. Sejersted OM
    11. Sjaastad I

    (2011) Ca(2+) wave probability is determined by the balance between SERCA2-dependent Ca(2+) reuptake and threshold SR Ca(2+) content

    Cardiovascular Research 90:503–512.

    https://doi.org/10.1093/cvr/cvr013

    • Google Scholar
67. 1. Takeshima H
    2. Komazaki S
    3. Hirose K
    4. Nishi M
    5. Noda T
    6. Iino M

    (1998) Embryonic lethality and abnormal cardiac myocytes in mice lacking ryanodine receptor type 2

    The EMBO Journal 17:3309–3316.

    https://doi.org/10.1093/emboj/17.12.3309

    • Google Scholar
68. 1. Van Vliet P
    2. Wu SM
    3. Zaffran S
    4. Pucéat M

    (2012) Early cardiac development: a view from stem cells to embryos

    Cardiovascular Research 96:352–362.

    https://doi.org/10.1093/cvr/cvs270

    • Google Scholar
69. 1. Venetucci LA
    2. Trafford AW
    3. O'Neill SC
    4. Eisner DA

    (2007) Na/Ca exchange: regulator of intracellular calcium and source of arrhythmias in the heart

    Annals of the New York Academy of Sciences 1099:315–325.

    https://doi.org/10.1196/annals.1387.033

    • Google Scholar
70. 1. Viatchenko-Karpinski S
    2. Fleischmann BK
    3. Liu Q
    4. Sauer H
    5. Gryshchenko O
    6. Ji GJ
    7. Hescheler J

    (1999) Intracellular Ca2+ oscillations drive spontaneous contractions in cardiomyocytes during early development

    PNAS 96:8259–8264.

    https://doi.org/10.1073/pnas.96.14.8259

    • Google Scholar
71. 1. Wagner S
    2. Dybkova N
    3. Rasenack EC
    4. Jacobshagen C
    5. Fabritz L
    6. Kirchhof P
    7. Maier SK
    8. Zhang T
    9. Hasenfuss G
    10. Brown JH
    11. Bers DM
    12. Maier LS

    (2006) Ca2+/calmodulin-dependent protein kinase II regulates cardiac Na+ channels

    Journal of Clinical Investigation 116:3127–3138.

    https://doi.org/10.1172/JCI26620

    • Google Scholar
72. 1. Wakimoto K
    2. Kobayashi K
    3. Kuro-O M
    4. Yao A
    5. Iwamoto T
    6. Yanaka N
    7. Kita S
    8. Nishida A
    9. Azuma S
    10. Toyoda Y
    11. Omori K
    12. Imahie H
    13. Oka T
    14. Kudoh S
    15. Kohmoto O
    16. Yazaki Y
    17. Shigekawa M
    18. Imai Y
    19. Nabeshima Y
    20. Komuro I

    (2000) Targeted disruption of Na+/Ca2+ exchanger gene leads to cardiomyocyte apoptosis and defects in heartbeat

    Journal of Biological Chemistry 275:36991–36998.

    https://doi.org/10.1074/jbc.M004035200

    • Google Scholar
73. 1. Ward JH

    (1963) Hierarchical grouping to optimize an objective function

    Journal of the American Statistical Association 58:236–244.

    https://doi.org/10.1080/01621459.1963.10500845

    • Google Scholar
74. 1. Webb SE
    2. Miller AL

    (2003) Calcium signalling during embryonic development

    Nature Reviews Molecular Cell Biology 4:539–551.

    https://doi.org/10.1038/nrm1149

    • Google Scholar
75. 1. Willems E
    2. Bushway PJ
    3. Mercola M

    (2009) Natural and synthetic regulators of embryonic stem cell cardiogenesis

    Pediatric Cardiology 30:635–642.

    https://doi.org/10.1007/s00246-009-9409-2

    • Google Scholar
76. 1. Wu SM
    2. Fujiwara Y
    3. Cibulsky SM
    4. Clapham DE
    5. Lien CL
    6. Schultheiss TM
    7. Orkin SH

    (2006) Developmental origin of a bipotential myocardial and smooth muscle cell precursor in the mammalian heart

    Cell 127:1137–1150.

    https://doi.org/10.1016/j.cell.2006.10.028

    • Google Scholar
77. 1. Zhang T
    2. Kohlhaas M
    3. Backs J
    4. Mishra S
    5. Phillips W
    6. Dybkova N
    7. Chang S
    8. Ling H
    9. Bers DM
    10. Maier LS
    11. Olson EN
    12. Brown JH

    (2007) CaMKIIdelta isoforms differentially affect calcium handling but similarly regulate HDAC/MEF2 transcriptional responses

    Journal of Biological Chemistry 282:35078–35087.

    https://doi.org/10.1074/jbc.M707083200

    • Google Scholar

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.

   "This ORCID iD identifies the author of this article:" 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.

   "This ORCID iD identifies the author of this article:" 0000-0002-9862-7332

• 11,360

  views

• 1,022

  downloads

• 84

  citations

Views, downloads and citations are aggregated across all versions of this paper published by eLife.