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.

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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

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