The developing chick embryonic heart forms in a rostrocaudal manner from the bilateral mesoderm in the heart fields between stages 4–8 (16–29 hr; staging according to Hamburger and Hamilton, 1951; see also Rawles, 1943). The developing heart forms a contractile tube by approximately 33 hr of development. Therefore, the development of the chick cardiac cell phenotype, heart morphology, and cardiac function must be tightly coordinated in the relatively short time period of approximately 17 hr in the chick (i.e., between 16–33 hr after fertilization). In the mouse, the development of beating cardiomyocytes is compressed into an even shorter time frame between 7.5 to 8.0 days of gestation: In the 3-somite mouse embryo in the anterior regions, feebly beating cardiomyocytes in the mouse are already present, while more caudad cells are only being committed to the cardiac lineage at this time.
Differentiation of the cardiomyocyte during heart development must synchronize myofibrillogenesis and sarcomere assembly with mechanisms regulating ion fluxes to drive cell contractions. The cardiac Na+-Ca++ exchanger serves as the main calcium extrusion mechanism in heart muscle and is important in maintaining intracellular calcium homeostasis. Based on the KB-R7943 induced inhibition of NCX activity and ouabain block of early cardiac differentiation, it appears that regulation of [Ca++]i is important in two separate phases of heart development: (1) in early cardiac cell differentiation whereby an apparent low cytosolic calcium steady state is maintained leading from precardiac to early cardiac stages, and (2) later, after cardiac cell differentiation is complete to control calcium influx and efflux to support the contraction-relaxation cycle.
The NCX-1 isoform is widely distributed in adult tissues and cells, including cardiac, skeletal, and smooth muscle cells, neurons, astrocytes, kidney, lung, and spleen cells (Blaustein and Lederer, 1999). In the chick embryo, at early stages we show that NCX-1 is expressed not only in the heart regions, but also earlier in the Hensen's Node. The NCX-2 and NCX-3 isoforms so far have been found only in brain and skeletal muscle in the adult in mammals and have not been examined in embryos. Current literature in this field suggests that the three NCX isoforms have similar functions. This indicates that possibly in NCX-1 transgenic knockout animals, other isoforms may be able to compensate for each other in those tissues where multiple isoforms are present or in those tissues where other isoforms may be upregulated.
Cytosolic calcium ions play a key role in intracellular signaling in virtually all types of animal cells. Therefore, it follows that abnormal calcium regulation may have profound pathophysiological consequences. That these types of ion exchangers, as a sodium/calcium-potassium exchanger Nckx30C, have a role in embryonic cell signaling was indicated recently in Drosophila (Haug-Collet et al., 1999). Additionally, it was reported recently that calcium gradients play a role in dorsal-ventral patterning during early Drosophila development (Creton et al., 2000). In this report, we suggest that calcium signaling based upon NCX-1 localization may be part of left/right patterning, as well. NCX-1 asymmetry in the Hensen's Node may result in asymmetric calcium fluxes in the Node. This may relate also to an importance for the asymmetric distribution of gap junctions in the Hensen's Node in the chick embryo (Levin and Mercola, 1998, 1999). At stage 5, Cx 43 is expressed in the streak and shows asymmetry, being present on the right side of Hensen's Node, the same side where NCX-1 protein is expressed. The asymmetric expression in chick and mouse embryos leads to the possibility of differential activation and regulation of numerous intracellular Ca++-mediated signaling cascades via activation of key transducing molecules as calmodulin and protein kinase C in a left-right manner.
Calcium-dependent signaling is mediated by a number of specific receptors, including calmodulin, calcineurin, troponin C, and protein kinase C. Intracellular Ca++ concentration must be tightly regulated for normal cellular processes. Which calcium-dependent signal transduction pathway(s) in the early embryo is (are) involved in cardiac differentiation has not been defined. However, two calcium-dependent signaling molecules, protein kinase C (PKC) and calcineurin, have been implicated in signal transduction pathways that affect myogenesis (Li et al., 1992; Molkentin, et al., 1998).
In addition to Ca++ influx pathways, cells must also have mechanisms to remove free Ca++ from the cytosol. Two important mechanisms for reducing cytosolic free Ca++ are Na+ Ca++ exchange across the sarcolemmal membrane and the Ca++ pumps in the endoplasmic reticulum (or sarcoplasmic reticulum) for sequestering Ca++ in intracellular stores. By the initial beating stages, the sarcoplasmic reticulum is only beginning to develop as a modification of pre-existing smooth endoplasmic reticulum.
NCX-1 Coordination With Myofibrillogenesis to Regulate Cardiac Contractility in the Chick Embryo
During early heart development, the early cardiac mesodermal cells must assemble the molecular machinery that will drive Ca++ sequestration and release cycles, as well as myofibril assembly, for cardiac cell contraction. Myofibrils begin to be assembled, in stage 7–8 chick embryos (approximately 24–29 hr) on the apical sides of the cardiac epithelium (Han et al., 1992; Linask et al., 1997; Imanaka-Yoshida et al., 1998). It is not until the 5–6 somite stage that some sarcoplasmic reticulum-like segments are observed close to the cardiac cell plasma membrane and a complete meshwork of sarcoplasmic reticulum surrounding myofibrils is formed by 9–12 days in the chick (Komazaki and Hiruma, 1997). Furthermore, it was shown that the two types of Ca channels, dihydropyridine receptor and the ryanodine receptor, will not form a functional coupling until 48 hr of development. On this basis, it is suggested that the initial chick heartbeats before 48 hr (beginning at approximately 33 hr) will be mainly dependent on an influx of Ca++ from the medium through the plasma membrane. Our results on NCX-1 expression in chick and mouse embryos and KB-R7943 inhibition of NCX in the chick at initial beating stages indicate the first cardiac contractions in the embryo occur as a result of Na+-Ca++ exchanger activity. This is substantiated by its enhanced expression in the outer curvature of the heart, the region that is the first to beat, as well as in the ventricular region of the heart in general.
Collectively, these experiments indicate that an early calcium-mediated signaling pathway is being perturbed. At stage 4 in the heart-forming areas, apparently normal signaling had taken place in the anterior bilateral regions, and cells in these small regions within the heart fields continued to differentiate as assayed by stable cNkx2.5 gene expression or by MF-20 immunolocalization for sarcomeric myosin heavy chain expression. Control embryonic hearts incubated in normal medium without inhibitors added (Fig. 8B) or normal medium plus vehicle (Fig. 9D) developed normally.
The fact that some stage-5 embryos formed looping hearts upon KB-R7943 exposure indicates that a relatively early signaling pathway is being perturbed, possibly activated as early as stages 3+/4. This early signal may be initiated at the midline Hensen's node region where NCX is first detected in the chick embryo. It has been shown by DiI labeling that cells that will form the myocardium and endocardium of the future heart are localized in the most rostral part of the primitive streak at the earliest stages of gastrulation (Garcia-Martinez and Schoenwolf, 1993). Alternatively, the signal may relate to NCX-1 expression at the mesoderm-endoderm interface in the bilateral regions. This would coincide with the polarized expression of the sodium pump in the mesoderm cells within this same area. Once the early Ca++-mediated signaling event has been completed and differentiation ensues, KB-R7943 (ouabain or ionomycin) exposure no longer has an influence on the ongoing differentiation program. In later embryos exposed to the inhibitor at stages 8–11 and monitored to stages when the embryonic heart begins to beat, KB-R7943 was found to have an inhibitory effect on heartbeat.
KB-R7943 is known to inhibit both the outward NCX current (reverse mode) and the inward NCX current (forward-mode) (Kimura et al., 1999). The major function of NCX-1 in the cardiac cells is probably to extrude calcium. The activity of NCX-1 is dependent on membrane potential and the gradients of Na+ and Ca++. NCX can reverse its action to bring calcium into the cell at membrane potentials that are more positive than the reversal potential for NCX. With ouabain alone, the Na+ may rise, but possibly only slightly. Rather than causing reversal of the exchanger and calcium influx, calcium efflux via the exchanger may be reduced. This results in an intracellular elevation of free calcium. KB-R7943 alone would have a similar effect. Upon inclusion of ouabain with KB-R7943, it may not have much additional effect. Without detailed knowledge of the parameters involved at the early stages, including membrane potentials, intracellular Na+ and Ca++ concentrations, Na/K-ATPase activity, or sodium pump current, and NCX-1 activity during these stages, the mechanisms underlying our results remain speculative.
At stage 12, after the cardiomyocytes have differentiated and formed a functioning, tubular structure, NCX-1 apparently regulates calcium fluxes to drive cardiac contractions to initiate a coordinated embryonic heartbeat. During the revision of our manuscript, this result was recently substantiated from two independent studies on NCX-1-deficient mice in which hearts form, but do not beat (Wakimoto et al., 2000; Koushik et al., 2001). In the stage 11–12 chick embryo within 8 hr of KB-R7943 exposure, heart rate is significantly reduced and, by 22 hr, is totally arrested while control hearts are still beating. At these relatively early stages, a sarcoplasmic reticulum is not yet present. This indicates the embryonic cardiac cell plasma membrane Na+-Ca++ exchanger has a major and direct role in regulating cardiomyocytes' intracellular Ca++ levels driving the embryonic heartbeats. Interestingly, one study also indicates that cardiac myogenesis is abnormal (Koushik et al., 2001). This would also be similar to our results on a block of cardiac differentiation as assayed by sarcomeric myosin heavy chain localization, except occurring to a milder degree in the NCX-1 null mutant.
Where our results differ from the NCX-1 null embryos generated by gene targeting in which case hearts develop, is that in the chick embryo by modulating [Ca]i levels pharmacologically during stage 4, cardiac cell differentiation and hence heart development can be blocked. This result would suggest that compensatory Ca++ regulatory pathways exist in the chronic mouse model of NCX-1 deficiency to allow normal early calcium signaling to take place. In our acute perturbation experiments using the chick embryo, compensatory pathways are not able to be established to overcome the arrest in the experimental time period. However, if chick embryos are allowed to recover in normal medium lacking inhibitors, cells can recover and continue to differentiate (see Linask and Gui, 1995). That a requirement for NCX-1 exists in regulating the embryonic heartbeat, as well as myogenesis, is substantiated by both experimental approaches.
Previous studies have indicated that in the late fetal and newborn rabbit the Na+/Ca++ exchange current (INaCa) can directly support contraction of ventricular myocytes (Artman et al., 1995; Boerth et al., 1994; Haddock et al., 1997). The immature mammalian heart, i.e., the late fetal or newborn rabbit or rat heart, has been shown to be more dependent upon transsarcolemmal Ca++ fluxes for regulating contraction and relaxation, than upon the sarcoplasmic reticulum, as seen in the adult. This appears to be true also for the fetal/neonatal chick heart (Vetter and Will, 1986). Our results suggest that the cardiac sarcolemmal Na+-Ca++ exchanger has a major role in regulating intracellular Ca++ from the earliest stages of cardiomyocyte differentiation leading to the first heartbeats. It then continues to support cardiac contractility throughout development, as well as in the early newborn vertebrate heart (Artman et al., 1995; Boerth et al., 1994; Koban et al., 1998).