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Keywords:

  • NaCa exchanger;
  • NCX-1;
  • calcium;
  • heart development;
  • asymmetry;
  • cardiac contractility;
  • KB-R7943 inhibitor

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

Ouabain-induced inhibition of early heart development indicated that Na/K-ATPase plays an important role in maintaining normal ionic balances during differentiation of cardiomyocytes (Linask and Gui [1995] Dev Dyn 203:93–105). Inhibition of the sodium pump is generally accepted to affect the activity of the Na+-Ca++ exchanger (NCX) to increase intracellular [Ca++]. These previous findings suggested that Ca++ signaling may be an important modulator during differentiation of cardiomyocytes. In order to identify a connection between heart development and NCX-mediated Ca++ regulation, we determined the embryonic spatiotemporal protein expression pattern of NCX-1 during early developmental stages. In both chick and mouse embryos, NCX-1 (the cardiac NCX isoform) is asymmetrically expressed during gastrulation; in the right side of the Hensen's node in the chick, in the right lateral mesoderm in the mouse. At slightly later stages, NCX-1 is expressed in the heart fields at comparable stages of heart development, in the chick at stage 7 and in the mouse at embryonic day (ED) 7.5. By ED 8 in the mouse, the exchanger protein displays a rostrocaudal difference in cardiac expression and an outer curvature-inner curvature ventricular difference. By ED 9.5, cardiac expression has increased from that seen at ED8 and NCX-1 is distributed throughout the myocardium consistent with the possibility that it is important in regulating initial cardiac contractile function. Only a low level of expression is detected in inflow and outflow regions. To substantiate a role for the involvement of calcium-mediated signaling, using pharmacologic approaches, ionomycin (a Ca++ ionophore) was shown to perturb cardiac cell differentiation in a manner similar to ouabain as assayed by cNkx2.5 and sarcomeric myosin heavy chain expression. In addition, we show that an inhibitor of NCX, KB-R7943, can similarly and adversely affect early cardiac development at stage 4/5 and arrests cardiac cell contractility in 12-somite embryos. Thus, based upon NCX-1 protein expression patterns in the embryo, experimental Ca++ modulation, and inhibition of NCX activity by KB-R7943, these results suggest an early and central role for calcium-mediated signaling in cardiac cell differentiation and NCX's regulation of the initial heartbeats in the embryo. © 2001 Wiley-Liss, Inc.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

The major functional characteristics of cardiac cells, as with all cells whose function is based on membrane polarization/depolarization cycles, are their contractile and electrical properties. A connection between ionic regulation and differentiation in these cells may also exist. The time course of the development of electrical properties in the cardiomyocyte during early heart development remains inadequately defined. The development of cardiac electrophysiology is significant for normal heart development, as well as for understanding the possible later development of pathologies in the adult myocardium. For example, disruption of the normal order of expression of ion currents in skeletal muscle can significantly compromise the later development of skeletal muscle cells (Dallman et al., 1998; Linsdell and Moody, 1995).

Our interest in the development of early ionic properties as a factor in cardiac development arises from previously reported observations on cell–cell adhesion mediated events involving N-cadherin that coordinate chick cardiac cell differentiation and cardiac compartment formation (Imanaka-Yoshida et al., 1998; Linask et al., 1997). The development of a polarized epithelial cardiac compartment from an unpolarized precursor mesoderm cell population follows the induction of apical, N-cadherin-mediated, cell–cell contacts. Epithelialization of the cardiac cells results in a resorting of proteins into different membrane domains. This includes the redistribution of Na,K-ATPase, the sodium pump, a marker for the polarized phenotype. From an initial circumferential plasma membrane localization in the precursor mesenchymal cell, the sodium pump becomes localized to the basolateral walls of the splanchnic, epithelial cardiomyocyte population (Linask, 1992).

Previously, we used ouabain, a highly specific Na, K-ATPase inhibitor that has been widely used to inhibit sodium pump activity (Lingrel et al., 1991), to experimentally assess the importance of sodium pump inhibition on the formation of the cardiac compartment and the pericardial cavity (Linask and Gui, 1995). Ouabain exposure of whole embryos demonstrated that pericardial coelom development and cardiomyocyte differentiation were blocked principally between stages 4 to 7 in a rostrocaudad gradient. After stage 7+/8 ouabain no longer affected these cardiac developmental processes (Linask and Gui, 1995). Ouabain inhibition of cardiac cell differentiation suggested that intracellular ionic concentrations may be closely tied to early cardiac signaling and that this may involve also the sodium-calcium exchanger (NCX).

To date, the primary focus of efforts in early heart development and cardiac cell differentiation has been on the identification of regulatory genes and growth factors that control development. Our experiments indicate that maintenance of specific intracellular ionic levels including calcium are critical during the early stages of cardiac cell differentiation. Additionally, as shown here, NCX-1 activity in its regulation of calcium is important in driving the first heartbeats of the embryo. The potential contribution of calcium signaling remains largely unexplored during early stages of heart development.

In mature myocardiocytes, the sarcolemmal Na+Ca++ exchanger rapidly transports Ca++ during excitation-contraction coupling and is the dominant myocardial Ca++ efflux mechanism (for a comprehensive review of NCX, see Blaustein and Lederer, 1999). The Na+Ca++ exchanger uses the transmembrane Na+ gradient to catalyze countertransport of Ca++ against its electrochemical gradient. Inhibiting sodium pump activity alters the driving force for the Na+Ca++ exchanger, such that cellular calcium stores increase. The highest levels of exchange activity have been observed in cardiac myocytes where the NCX-1 variant is expressed (Quednau et al., 1997). Na+ - Ca++ exchange appears to be the dominant Ca++ transport pathway for contraction and relaxation in late fetal and early newborn cardiac ventricular myocytes (Artman et al., 1995; Boerth et al., 1994; Haddock et al., 1997). However, the role of the exchanger in early development remained incompletely defined.

This study determined that NCX-1 protein is expressed at the earliest stages of heart development and up through beating heart stages. Secondly, we show that modulation of calcium by means other than ouabain produced similar results to block early cardiac cell differentiation and, as a result, heart development. Thirdly, we show that pharmacologic inhibition of NCX activity in early (stage 4/5) embryos using the inhibitor KB-R7943 can affect normal cardiac cell differentiation. Later, in 12-somite embryos it arrests cardiac contractility. Thus, we suggest that calcium signaling and maintenance of specific intracellular Ca++ concentrations are important in early heart development leading to differentiation of the cardiomyocytes. The sodium-calcium exchanger NCX-1 apparently has a role in this regulation and subsequently regulates calcium fluxes in the myocardium to drive the first embryonic heartbeats.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

Our previously published results indicated that ouabain, an inhibitor of the sodium pump, arrested cardiac cell differentiation at a step associated with cardiac cell epithelialization (Linask and Gui, 1995). The sole function of ouabain has been shown experimentally to be an inhibition of Na-K-ATPase (sodium pump) activity. As a result the intracellular sodium [Na+]i increases, followed by an increase in [Ca++]i, due to the influence of the Na/K-ATPase on the activity of the Na+-Ca++ exchanger. As there is no information on the expression pattern of the Na+-Ca++ exchanger in early chick embryos, we undertook a qualitative RT-PCR analyses of NCX-1 expression during various stages of chick development and an in situ hybridization study of its possible localization in the chick heart-forming regions. The NCX-1 isoform has been shown to be the primary isoform present in the myocardium. Because there is not always a close correlation between message and protein expression (Gygi et al., 1999), we also analyzed for NCX-1 protein expression in both the chick and mouse embryos.

Spatiotemporal Localization of the NaCa Exchanger (NCX-1) During Chick and Mouse Heart Development: Protein and mRNA Expression

Immunohistochemical and in situ analyses for NCX-1 in the chick embryo.

After cleavage of a signal peptide, the mature NCX-1 protein is formed that has 11 α-helical transmembrane segments (Nicoll et al., 1999). RT-PCR amplification of a 493-bp fragment was carried out using primers designed from the partial chick mRNA sequence for NCX-1 (see Experimental Procedures). This is a region that is approximately 81% homologous with the mouse sequence and is in the 5′ portion of Exon 2, which encodes for ∼90% of the NCX protein. RT-PCR for NCX-1 expression was carried out on total RNA from early chick embryos (stages 4–7) and on total RNA isolated from the chick heart at later stages (stages 9 and 15). The NCX-1 isoform message was present at all stages analyzed (not shown).

To be certain that the above RT-PCR data reflected NCX-1's presence in the heart fields at early stages, immunohistochemistry (stages 5/7) and in situ hybridization (stage 12) analyses were carried out in chick embryos (shown in Figs. 1 and 2). At stage 4/5, NCX-1 protein in whole mounts was detectable only in the Hensen's Node region (arrow, Fig. 1A). By stage 7, NCX-1 protein is present within the heart-forming regions (HFR, Fig. 1B; control embryo is shown in Fig. 1C). At stages 12–15, positive signal for NCX-1 message is present throughout the looping heart, which is now rhythmically beating (Fig. 2A): whereas control hearts (using sense probes) show no detectable signal (Fig. 2B). In sections through the stage-12 embryonic chick heart, expression is present throughout the heart tube, but the predominant expression is in the outer loop of the myocardial wall (arrows, Fig. 2C). A section through the heart of a control embryo at stage 12 using sense probes in Figure 2D shows an absence of alkaline phosphatase signal.

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Figure 1. NCX-1 protein localization in the early chick embryo. A: Asymmetric localization of NCX-1 protein in Hensen's Node at stage 5 in this ventral view of the embryo. More signal is observed in the right side of the node (see arrow) than in the left. At this stage no signal was detectable in the heart-forming regions. HP, head process. B: At stage 7, NCX-1 is expressed in the heart-forming regions (HFR). Here the right side is shown. Localization in the left side is the same. This signal is also apparent in the developing neural tube (NT) at this stage. C: Control stage 7 embryo. Photographic exposure time for control was same as for B. Magnification bar in C (for all panels) = 50 μm.

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Figure 2. In situ hybridization for NCX-1 mRNA expression in stage 12 chick embryos was carried out using digoxigenin-labeled sense and antisense riboprobes. A: At stage 12, positive signal for NCX-1 message using antisense probe is present throughout the looping chick heart as a darkened blue color (arrow). Magnification bar (A,B) = 80 μm. B: No detectable specific signal in the heart in a similar stage embryo treated with sense riboprobes. C: Stage 12 embryos sectioned through the heart show relatively high levels of message primarily in the outer curvature of the ventricular region of the looping heart (arrows). With longer development of the alkaline phosphatase reaction, some signal becomes detectable in the developing brain that is not evident in this sectioned embryo. D: A section through a control embryonic heart hybridized with sense riboprobe as in B; no alkaline phosphatase signal is detected in the heart (D). Magnification bars (C,D) = 50 μm.

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Differential Spatiotemporal Localization of the NaCa Exchanger (NCX-1) Protein During Mouse Heart Development

A spatiotemporal in situ hybridization study of the ion exchanger at the messenger RNA level has been reported in the mouse (Koushik et al., 1999). It was indicated that this message is cardiac specific at day 7.75–8 of gestation. This would place detection of the message, however, later than we detected the protein immunohistochemically (Figs. 3 and 4). Also, we observed that NCX-1 protein expression is not restricted to the heart at the earliest stages of detection, but already localizes to the mesoderm asymmetrically after gastrulation and before the cardiac compartment has formed.

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Figure 3. NCX-1 protein expression in Day 7 mouse embryos. In the early head-fold, presomite embryo, as one sections transversely from the region of the ectoplacental cone towards the distal tip of the embryo, NCX-1 is present primarily in the right mesoderm layer. Sparse localization is detectable also in the neural ectoderm on both sides (arrows in A). B: Lateral mesoderm at higher magnification. NCX-1 signal (arrows) is primarily localized to the mesoderm cell membranes, especially at the interface between the mesoderm and ectoderm, and has a punctate localization pattern throughout the ectoderm. ECT, ectoderm; MES, mesoderm. Magnification bar (A) = 25 μm; (B) = 25 μm.

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Figure 4. Day 7.5 mouse. In sections cut through heart-forming regions, a punctate patterning of the Na+Ca++ exchanger protein is detectable primarily in cells at the mesoderm-endoderm interface in the region near the anterior intestinal portal (small white arrows point to NCX-1 localization). The exchanger is detectable in the endoderm (END) and at the basal aspect of the epithelial cardiac mesoderm (MES; A). In sections more caudad (B), but still within the heart forming areas, NCX-1 is present primarily associated with the endoderm cells, underlying the cardiac mesoderm, and on the basal aspect of the cardiac mesoderm cells (small white arrows in B). Only sparse localization is present in the ectoderm. Note bright signal on outer wall of foregut is nonspecific (see control section in Fig. 5F). Magnification bar (A,B) = 25 μm.

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Day 7–day 7.5.

In the early head-fold, presomite mouse embryo, as one sections transversely from the region of the ectoplacental cone towards the distal tip of the embryo, NCX-1 is present in the neural ectoderm extending to the lateral ectoderm; it localizes in the lateral mesoderm on the right side of the embryo (arrows in Fig. 3A and B). This localization is primarily punctate and generally distributed throughout the ectoderm, along cell membrane boundaries, especially at the interface of the ectoderm and mesoderm (Fig. 3B).

At day 7.5 of gestation in the mouse the Na+Ca++ exchanger is detectable in transverse sections through the anterior heart-forming regions in a punctate pattern in cells at the mesoderm-endoderm interface. It localizes primarily in the endoderm and at the basal aspect of the epithelial cardiac mesoderm (Fig. 4A, white arrows). Spatially, this coincides with the basolateral expression of Na/K-ATPase reported earlier in the chick (Linask, 1992). More posteriorly, but still within the heart-forming areas, the Na+-Ca++ exchanger continues to be expressed in the endoderm, underlying the cardiac mesoderm (Fig. 4B). This region of expression, i.e., the bilateral mesoderm-endoderm interface, is an area where inductive signaling is taking place at this time to specify the cardiac compartment (Jacobson and Sater, 1988; Lough and Sugi, 2000).

Day 8 (approximately 7-somite stage).

As above, mouse embryos were sectioned transversely from anterior regions through the developing head and brain regions posteriorly through the developing inflow of the heart. In sections cut through anterior regions of the heart (Fig. 5A and B), a sparse, punctate pattern of Na+-Ca++ exchanger expression is detectable in the outflow tract (OFT) region of the heart (Fig. 5A and B). To the right of the OFT in the primitive ventricular (V) region, NCX-1 localization is detectable within the myocardium in a differential manner (Figs. 5B). The left side of the heart tube shows more signal in the cells than the right (RV) side (Fig. 5C). More posteriorly, focal regions showing higher immunoreactivity are seen in cells in the outer and inner curvature of the heart (arrows; Fig. 5D). The outer curvature coincides with the region of the myocardium that first begins to contract. As shown here, the nonuniform pattern of expression continues in the ventricular regions, but becomes more sparse in its localization posteriorly. In the sinus venosus, the most posterior region of the heart, little signal is detectable (Fig. 5E). Thus, the inflow (sinus venosus) and outflow regions (OFT) of the heart show only a sparse punctate NCX-1 localization pattern at this stage in contrast to ventricular regions. The bright immunostaining in the foregut (FG) is nonspecific (compare Fig. 5A and F) and thought to be due to mucopolysaccharide in the foregut as also seen in negative control embryos treated with Cy-3 conjugated secondary antibody only (Fig. 5F). Similar nonspecific localization in the mouse foregut at a similar stage was reported in other immunolocalization studies on matrix molecules in the mouse (see Tsuda et al., 1998). Throughout these stages, the endocardium is primarily negative for NCX-1 specific signal, except for some immunostaining beginning to appear in the endocardium of the ventricular region.

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Figure 5. NCX-1 protein expression in the heart of the day 8 mouse. Differential localization patterns of NCX-1 in the embryonic heart are apparent on this day of gestation. Progressively rostrocaudal sections are shown in A–E. A: Lower magnification of the mouse embryo sectioned at the level of the outflow tract (OFT). Note bright immunofluorescence in foregut is nonspecific, as this is also detectable in control embryos treated without primary antibody (see F). Only sparse, punctate pattern of NCX-1 expression is seen in the outflow region. B: Cut slightly posterior where outflow tract (OFT) is still apparent on left and the ventricular (V) region on the right is primarily shown with a relatively high expression of NCX-1 (white signal). Some cells have a higher level of expression than others. C: Slightly more posteriorly, this section through the ventricular region shows the right ventricular region (RV) has only a sparse level of expression in comparison to that seen on the left side (top right). D: Section further caudad in the common ventricular region. More label is seen in the outer and inner curvature of the heart than in other regions of the myocardium. Arrows point to more brightly fluorescing regions in the curvatures. As one progresses more caudad, increasingly lower levels of NCX-1 localization are seen, although a nonuniform expression pattern is still evident. E: Section through the sinus venosus (SV) or inflow region of the heart. As seen also in the anterior outflow region (compare Fig. 6A–B), the inflow has only a sparse, punctate pattern of expression (arrow). The ventricular region (V) that is still apparent continues to show more expression of NCX-1 in the plasma membranes. F: Section through the heart region of a negative control, day 8 embryo that was not treated with primary antibody. Nonspecific fluorescence is present only in the foregut. OFT, outflow tract; V, ventricular region, SV, sinus venosus. Magnification bar = 50 μm (A–E); 200 μm (F).

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Cardiac localization on day 9.5.

At day 9.5 of gestation in most anterior to posterior regions of the developing heart, NCX-1 is expressed more uniformly than in day-8 embryos. Most cells of the myocardium (M), primarily within the ventricular (V) regions, express NCX-1. At this time the heart is beating regularly. In Figure 6, the boxed areas indicate the areas that are shown at a higher magnification. Figure 6A–D (anterior regions through the heart) shows a high intensity of NCX-1 expression on the ventral aspect of the heart tube (arrows in Fig. 6B) and also in association with the dorsal mesocardium (DM). As seen at higher magnification in Figure 6C and D, the endocardium, as well as myocardium, express NCX-1 at this stage of development, which was not apparent on day 8 of gestation. More caudad in Figure 6E, the outflow, atrial and ventricular regions all show a relatively high level of expression throughout the myocardium and endocardium of the heart. Figure 6F shows a higher magnification of the boxed-in region in Figure 6E. The inflow (SV) region of the heart shows a low level of NCX-1 localization (Fig. 6G), as was also seen earlier in day 8 embryos. In the ventricular region, both myocardium and endocardial cells express NCX-1 (Fig. 6H). Control embryos at this stage showed only the nonspecific foregut immunolabeling as shown in Figure 5F.

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Figure 6. NCX-1 protein expression in the mouse embryonic heart on Day 9.5 of gestation. Progressively rostrocaudad sections through the developing heart (A,B,E,G) indicate an increased level of NCX-1 expression than was apparent on day 8. C,D, F, and H are higher magnification images of boxed-in regions indicated in B, E, and G. A: At the level of the right atrium (At), increased expression is apparent and notably on the ventral side. B: Increased levels of expression are apparent on the ventral side of the atria (arrows). Boxed-in regions are shown in C and D, respectively. Localization of NCX-1 is detectable in the dorsal mesocardium. C,D: Both myocardial and endocardial cells express NCX-1. E: Outflow, atrial, and ventricular regions all show a relatively high level of NCX-1 expression. The endocardium shows expression that was not apparent on day 8 (see Fig. 5). F: Higher magnification of a comparable region to that outlined in E, but located slightly more posteriorly. G: More posteriorly within region where sinus venosus is now apparent, as well as posterior part of the ventricle. Note a reduced level of expression of NCX-1 is apparent in the sinus venosus region. H: Outlined region of ventricle in G is shown slightly rotated to the left in this micrograph. In addition to the myocardial expression, note NCX-1 continues to be expressed by the endocardial cells at this level. OFT, outflow tract; At, common atrial region; V, ventricular region, SV, sinus venosus; Myo, myocardium; FG, foregut; ENDO, endocardium. Magnification bar =100 μm (A,B,E,G); 50 μm (C,D,F,H).

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Figure 7. Abnormal heart and neural tube development occurred in chick embryos pharmacologically perturbed using the Ca++ ionophore, ionomycin. Heart development along the anterior/posterior axis within the heart field was assessed by detection of MF-20 antibody made against sarcomeric myosin heavy chain and used here as a marker of cardiac differentiation. A: Embryos exposed to 1.0 μM ionomycin at stage 4+ showed cardiabifida with MF-20 expression primarily in two small tubes in the most anterior regions. The whole anterior part of the embryo appeared shortened with little extension of the developing brain region. AIP designates the anterior intestinal portal. B: Exposure at stage 5/5+ shows an increased area of expression along the A/P axis across the heart field and fusion of the heart tubes has taken place rostrad in this embryo. D: The embryo shown here was sectioned at the level of the black arrow (section shown in C). The myocardial wall is expressing MF-20 (small arrow in C). The neural region (N) is apparent with notochord beneath it, but the neural plate has not completely folded up to form a tube, as occurs in control chick embryos. E: After 22-hr incubation, control untreated embryos formed a normal looping and beating heart as shown in this panel. The brighter signal in the mid part of the heart reflects level of plane of focus. Magnification bar = 200 μM (A,B,D,E); 50 μM (C).

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Treatment of Whole Chick Embryos With Ionomycin to Elevate [Ca]i

Ionomycin is a polyether antibiotic with properties characteristic of a divalent cation ionophore. Cation displacement studies have shown a similar specificity of ionomycin for divalent cations and a greater affinity for Ca++ over Mg++ than with the calcium ionophore A23187 (Kaufmann et al., 1980). Ionomycin has been widely used ever since its initial discovery to elevate intracellular calcium levels.

Since ouabain is known to elevate calcium levels indirectly by modulating Na+Ca++ exchanger activity, we treated embryos with 0.01, 0.1, or 1.0 μM ionomycin 24 hr, to more directly increase intracellular calcium levels and to determine whether ouabain inihibition of cardiac cell differentiation is due to increased calcium levels. The latter we found to be the case during cardiac differentiation in a stage- and dose-dependent fashion along the rostrocaudal axis within the heart fields. In chick embryos exposed to 1.0 μM ionomycin at stage 4/5 and incubated for 24 hr, the most anterior regions were unaffected and continued to differentiate. As with ouabain exposure, it is suggested that these anterior areas are composed of cells that were already past the calcium concentration-sensitive signaling event. These cells, hence, were not perturbed, and continued to differentiate. However, in more posterior heart-forming regions, cardiac cell differentiation was blocked (Fig. 7).

In general, ionomycin-treated stage 4 embryos developed abnormally with the ionomycin-induced increase in intracellular calcium. Effects were similar to what we previously reported for ouabain (Linask and Gui, 1995) and MF-20 localization of sarcomeric myosin heavy chain in the heart region was inhibited in a stage- and dose-dependent manner (Table 1). Heart and neural tube development were severely affected. The effects on heart development along the anterior/posterior (A/P) axis is shown here using MF-20 localization as a marker of heart differentiation. As shown in Figure 7A and B, two embryos exposed to ionomycin at stages 4 and 5 show varying degrees of cardiabifida with MF-20 expression detectable in anterior regions. Exposure at stage 5 resulted in an increase in the area of MF-20 localization along the A/P axis across the heart field, and fusion of the heart tubes occurred over a longer distance (Fig. 7B). Reduced or no levels of MF-20 localization were apparent in more posterior regions. Contractions of cardiac tissue were often limited to the most anterior regions. Similarly, brain development was inhibited with ionomycin treatment. As seen earlier with ouabain inhibition (Linask and Gui, 1995), the effects of ionomycin on heart development were reversible after return of the embryo after 8 hr to ionomycin-free medium; cells recovered from the block to differentiation, and developed into tubular, beating hearts similar to control, untreated hearts shown at the end of the incubation period (Fig. 7E). Bright intensity of localization signal in E reflects only the plane of focus. Embryos exposed at stage 7/7+ to ionomycin formed apparently normal hearts (similar to heart in Fig. 9E). The ionomycin experiments shown here and the previously reported ouabain experiments taken together suggest high calcium levels affect an early upstream signaling event blocking further cardiac cell differentiation as assessed by cNkx2.5 expression (see Fig. 8A) and sarcomeric myosin heavy chain expression (Fig. 9A).

Table 1. Embryos Treated With Ionomycin (1 μM)
StageLooping heartTubular heartAnterior cardiac tissue only
  • a

    Number of embryos showing the specified phenotype.

40012a
4 (control)510
5 or 6415
5 (control)620
7+400
7+ (control)300
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Figure 8. Ouabain inhibition of cNkx2.5 expression in bilateral heart regions and arrest of heart development. Experimental and control embryos are shown after the 22-hr incubation period. A: Localization of cNkx2.5 gene expression is detectable only in short anterior heart tubes extending approximately 300 μm in length. Due to the rostrocaudal manner of heart development, the extent of caudad cardiac differentiation blocked by ouabain is dependent upon embryonic stage at which exposure to the inhibitor occurs. This is shown schematically in the diagram below A. In ouabain-treated chick embryos at stages 4/5, the green filled-in region depicts the approximate anterior heart region that is unaffected by ouabain and continues to differentiate during the 22-hr incubation period. The green outlined posterior region of the heart field is detectably affected by the drug and cells are blocked from further differentiation. Magnification bar for A = 150 μm. B: A control stage 4 embryo incubated for the same time period of 22 hr in normal medium without ouabain. This embryo is shown at lower magnification than in A. Development of the 1-mm beating heart is normal. The dark blue alkaline phosphatase signal within heart depicts cNkx2.5 expression. Magnification bar in B = 250 μm.

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Figure 9. Abnormal heart development occurred in chick embryos exposed for 22–24 hr to ouabain, the NCX inhibitor KB-R7943 (KB-R), or KB-R7943 plus ouabain. The extent of cardiac differentiation along the anterior/posterior axis of the heart field was assessed here by using MF-20 antibody localization for sarcomeric myosin heavy chain. Positive signal is seen as white. A: In this ouabain-exposed embryo (here analyzed by laser confocal microscopy), two small bilateral regions of cardiac cells are differentiating in the anterior part of the embryo after an incubation period. This would be similar to the region expressing cNkx2.5 shown in 8A, except differentiating tissue remains further apart. The anterior intestinal portal (AIP) is identified. Due to increased gain during confocal analysis, the embryo appears gray. B: Embryo exposed to the NCX inhibitor KB-R7943 at stage 5 displaying two short, anterior bilateral regions where cardiac cells are differentiating. In this particular embryo, the regions are closer together than in A. C: A stage 4 embryo preincubated in KB-R9743 and then transferred into KB-R9743 plus ouabain developed two short cardiac tubes that have differentiated, but have not quite fused. Heart development has not progressed at all to the level seen in control. D: A normal looping, tubular heart is seen in control embryos exposed to DMSO vehicle only without the inhibitor. Due to heart looping, only a part of the heart tube is in the plane of focus, resulting in the high intensity of the immunolocalization signal seen within the midregion of this normal heart. Magnification bar in A–D = 150 μm.

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Since the major action of ouabain is to inhibit the sodium pump, which, in effect, activates the Na+-Ca++ exchanger to increase intracellular Ca++ levels, we next proceeded to determine what effect inhibiting the exchanger activity would have on early cardiac cell differentiation and initial heartbeats.

Treatment of Whole Chick Embryos With Ouabain and KB-R7943, an Inhibitor of NCX-Activity, Affects Heart Development Early at Stages 4/5 and Arrests Initial Heartbeats in Chick Embryos at Stage 12

In contrast to Na/K-ATPase, which has selective natural inhibitors such as ouabain and other cardiac glycosides, there has been no similar inhibitor of the Na+-Ca++ exchanger. Only a few drugs have been found to inhibit NCX. KB-R7943 (initially called no. 7943) is a synthetic drug that has been shown to inhibit the cardiac exchange current with higher affinity than another earlier inhibitor, DCB. KB-R7943 is known to inhibit both the outward NCX current (reverse mode) and the inward NCX current (forward-mode) (Kimura et al., 1999).

Stages 4–5 (early cardiac stage).

Some stage-4 embryos were treated first with (experimental embryos) and without (control embryos) the addition of ouabain and were assayed by in situ hybridization for the marker gene cNkx2.5 at the end of a 22-hr incubation. Figure 8A shows the heart-forming areas of an ouabain-exposed embryo after incubation in the sodium pump inhibitor for 22 hr. After the incubation period, cNkx2.5 as detected by alkaline phosphatase, is expressed only in two small fusing cardiac tubes (∼ 300 μm in length) in the anterior region of the embryo. The positive region of cNkx2.5 localization coincides with the anterior regions of the heart fields of ouabain-treated embryos that had epithelialized before exposure to ouabain (Linask and Gui, 1995) and were shown to express sarcomeric myosin heavy chain by MF-20 antibody localization (see also Fig. 9A). Stage-4 control embryos that were not exposed to ouabain, but were incubated for the same 22-hr period, developed normal, beating, looping hearts. All of the control, untreated, embryonic hearts expressed cNkx2.5 throughout the heart, as would be expected (Fig. 8B). The untreated, beating, control heart tube that has formed is approximately 1 mm in length. Thus, exposing the embryo to the sodium pump inhibitor ouabain at stage 4 blocked cardiac differentiation in approximately the posterior two-thirds of the heart fields. This is shown diagrammatically below Figure 8A where only the region filled in green at stage 4 continues to differentiate. The green outlined region represents areas that are blocked from further differentiation. Ouabain was shown also by RT-PCR to decrease Nkx2.5 gene expression and ventricular myosin heavy chain 1 gene expression in precardiac mesoderm explants removed at stage 5/6 (Searcy and Yutzey, 1998). The in vivo expression pattern substantiates the RT-PCR data.

In Figure 9A, sarcomeric myosin heavy chain protein expression is shown in a ouabain-treated embryo at the end of the 22-hr incubation period. This is very similar to the result shown in Figure 8A, but the parts of the cardiac tubes that have formed are not close enough to begin fusing. KB-R7943 exposed embryos showed similar results of blocking further cardiac differentiation in the more posterior regions at the end of a 22-hr incubation period (Fig. 9B). Additionally, experimental sets of embryos were first pretreated with KB-R7943 (5 μM) for 1 hr and then transferred into KB-R7943 (10 μM) with ouabain (10 μM; shown in Fig. 9C). This was done to determine whether there is any type of suppression of the ouabain effect by KB-R7943 as has been reported (Watano et al., 1999). Little, if any, protective effect was seen (Fig. 9C).

As shown here and in our previous studies, modulating intracellular calcium levels in embryos results in only two small bilateral anterior areas of cardiac tissue differentiating (see Figs. 8A and 9A). Therefore, in all three experimental conditions that cause a change in intracellular calcium, i.e., by ouabain, ionomycin, or NCX inhibition using KB-R7943, cardiac cell differentiation is arrested in the as yet undifferentiated posterior parts of the heart fields. Anterior areas apparently already committed to differentiation before exposure, continue to differentiate. Inclusion of KB-R7943 with ouabain, however, appeared to have only a slight protective effect on ouabain's effects on heart development. The embryonic hearts usually were observed to form longer, tubular structures undergoing varying degrees of fusion (Fig. 9C). Of the two doses of inhibitor that were used, 5 μM and10 μM KB-R7943, the higher dose was found to be more effective in inhibiting early heart development in embryos up to the two-three somite stage (Table 2). Heart of a control, untreated embryo, showed normal development (Fig. 9D).

Table 2. Embryos Treated With KB-R7943: Stage Dependent Effects
StageLooping heartCardiabifidaAnterior cardiac tissue onlyNo cardiac tissue
  • a

    Number of embryos showing the specified phenotype.

45a4103
4 (control)10300
5 or 65531
5 (control)6200
88000
8 (control)3000
Stage 12 (beating heart stage).

Embryos (11–12 somites) with beating, looping hearts were treated with 30 μM KB-R7943. After an 8-hr exposure, the activity of the embryonic hearts was videotaped and revealed a decrease in heart rate of experimentally treated embryos (see Table 3) with the average beat rate being 70 bpm (beats per minute; standard deviation SD ± 6). Control hearts were beating at 97 bpm (SD ± 4). In another experiment set up with slightly younger embryos (10 somites), the inhibitor-treated embryos averaged a heart rate around 31 bpm (SD ± 9), while controls were beating at 95 bpm (SD ± 2). The control embryos in the first group displayed strong, normal contractile function with sinoatrial and ventricular contractions following each other in a rhythmic manner. The KB-R7943 embryos displayed weak, predominantly sino-atrial, contractions of much shorter duration, and at a significantly reduced rate, indicating that NCX-1 activity is necessary for regulating calcium fluxes during normal contractility at these early heartbeat stages. After an additional 14-hr incubation, hearts of the KB-R7943-exposed embryos were no longer beating at all; control embryonic hearts were still beating, although had considerably slowed down to 39 bpm under these culture conditions.

Table 3. KB-R7943 Inhibition of Heart Ratea
 Stage 11Stage 11 ControlStage 12Stage 12 Control
  • a

    Number of embryos shown in parentheses.

Beats/min31 (6)95 (6)70 (7)97 (8)
Standard deviation±9±2±6±4

DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

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.

KB-R7943 Inhibition

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).

EXPERIMENTAL PROCEDURES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

Embryos

Chick.

White Leghorn chick embryos (Gallus gallus; Truslow Farm, Chestertown, MD) were used for the experimental studies and for in situ hybridization. Staging is according to Hamburger and Hamilton (1951).

Mice.

C57Bl/6J (Jackson Labs, Bar Harbor) and Swiss Webster (Taconic, Germantown, NY). Both strains showed similar immunohistochemical localization patterns for NCX-1 protein.

Whole Chick Embryo Cultures

Detailed methodology has been reported previously (Linask and Gui, 1995; Linask and Lash, 1988). Briefly, whole embryos including intact area opaca are removed from yolk and placed on Nuclepore filters with ventral side facing up. They are then transferred into wells of 3-well slides and placed on holes in filter paper rings over nutrient medium. Experimental cultures included addition of either 10-5M ouabain (Na/K-ATPase inhibitor; Sigma, St. Louis, MO) or 1 μM ionomycin (Sigma). Embryos are incubated at 37°C for 24 hr and then fixed in Histochoice (Amresco, Soho, IO) or 4% p-paraformaldehyde/PBS, respectively, for either immunohistochemistry or in situ hybridization.

RNA Isolation and RT-PCR

Isolation.

Cultured chick embryos for different stages were removed and 1 ml of RNA STAT60™ (Tel-Test, Inc) was added to each embryo (about 100 mg of tissue). After chloroform treatment and isopropanol precipitation, RNAs were resuspended in DEPC-dH20 by heating at 65°C for 10 min.

RT-PCR.

First strand cDNA was made using Superscript II reverse transcriptase (Gibco BRL) followed by digestion with RNAse H. PCR was carried out by mixing 2 μl cDNA with 5 μl 10× buffer, 1 μl 10 mM dNTPs, 2 μl primer for each (100 ng/μl), 5 μl BSA (2.5 g/μl), 0.5 μl Taq polymerase (5 U/μl) and 32.5 μl d H20. PCR reactions were carried out for 35 cycles. PCR products (10 μl) were run on a 1.4% agarose gel in 1× TAE. GAPDH (330 bp) was used as a control. The primers were prepared corresponding to the nucleotide sequences of the chick NCX-1 (accession no. AJ012579 for partial chick sequence). The amplified region corresponds to the coding region beginning just before the transmembrane 2 (TM2) domain and extends into the intracellular loop, just past the inhibitor peptide site (XIP).

The forward reaction (20 mer) is primed by: 5′ –GACTGTTTCCAACCTCACAC-3′ and reverse reaction (19 mer): 5′ –AGCAACCTTTCCGTCCATC-3′.

This results in a 493-bp amplified product of the NCX-1 fragment. The mouse and chick sequences show 81% homology (chick to human sequences is 81%; mouse to human is 89%).

The RT-PCR product for chick cNCX-1 was subsequently subcloned into a pGEM-T Easy vector (Promega, Madison, WI) and sense and antisense riboprobes were synthesized and labeled with digoxigenin (Boehringer Mannheim, Indianaopolis, IN). Both sense and anti-sense riboprobes were sequenced to confirm faithfulness of PCR amplification by the Molecular Resource Facility at the New Jersey Medical School of UMDNJ, Newark. In situ hybridization was carried out on various stages of chick development. Detailed protocol for in situ hybridization has been reported elsewhere (Linask et al., 1997).

Ouabain and Ionomycin Incubation and cNkx2.5 In Situ Hybridization

Whole chick embryos at stage 4/5 were incubated in the presence of 10-5 M ouabain or 1 μM ionomycin for 22–24 hr. At end of incubation embryos were rinsed quickly in PBS and fixed in 4% paraformaldehyde for in situ hybridization. Digoxigenin labeled riboprobes for cNkx2.5 were used for in situ hybridization on pharmacologically treated and control chick embryos. An alkaline phosphatase secondary antibody detection system was used.

KB-R7943 Inhibition

Whole chick embryos at stages 3,4,5, and 12 were incubated in the presence of varying concentrations (5–30 μM) of the NCX inhibitor KB-R7943(2-[2-[4-nitrobenzyloxyl]phenyl]ethyl)isothiourea methanesulphonate, New Drug Research Laboratories, Kaneba Ltd., Osaka, Japan) (Kimura et al., 1999; Watano et al., 1996). Some embryos were pretreated for 1 hr with K-BR7943 (5 μM) and then transferred into 10 μM K-BR7943 plus 10 μM ouabain (39 embryos; see Fig. 9). At the end of incubation, digitized images of the embryos were captured and the younger stages were fixed in Histochoice (Amresco, OH) and immunostained for MF-20 to determine effects on cardiac cell differentiation and cardiac organogenesis. Using videomicroscopy, the 12-somite embryos (18 embryos) incubated in K-BR7943 inhibitor after 8- and 24-hr incubation periods were videotaped for 2-min periods to record effects on heart contractility at these two time points and to obtain heart rate. Control embryos were incubated in normal 2:2:1 medium with equivalent amounts of DMSO added as used for dilution of KB-R7943. Embryos were set up in filter paper ring cultures as previously described.

Immunohistochemistry and Microscopy

Immunohistochemistry was carried out on mouse and chick embryos using a high resolution technique whereby the embryos are fixed, pre-embedded immunostained with a NCX-1 mouse monoclonal antibody, embedded in araldite, and sectioned at 2 μm through the anterior embryo through the heart region (for detailed immunohistochemistry methodology using chick and mouse embryos, seeTsuda et al., 1998). A mouse monoclonal IgM antibody made against purified canine Na+Ca2+ exchanger (Affinity BioReagents, Inc., Golden, CO) was used for immunolocalization. This antibody recognizes an epitope in the conserved region of amino acids 371–525, which is on the intracellular side of the plasma membrane. MF-20 antibody for sarcomeric myosin heavy chain was obtained from Developmental Studies Hybridoma Bank (Iowa University, IO). Fluorescence microscopy was carried out using a Nikon Optiphot II microscope equipped with epifluorescence attachments. Digitized images were captured directly using a Princeton Micromax cooled CCD camera interfaced with MetaMorph Image Processing Analysis software (Universal Imaging, West Chester, PA).

Videomicroscopy.

Videomicroscopy was done using a Hamamatsu CCD camera attached to a Nikon SMZ-10 stereo microscope. The camera is interfaced with a Panasonic Video Cassette Recorder and a SONY TV monitor. A temperature controlled air blower (Nikon Incubator NP2) was used to maintain the embryos at 38°C on the stage of the microscope, which is kept in an enclosed chamber to maintain constant temperature.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

This work was partially supported by an Established Investigator Grant from the American Heart Association (K.K.L.) and also by the UMDNJ Foundation (K.K.L.). Many thanks to Drs. Peter Haddock, William A. Coetzee, and Tomoe Y. Nakamura, in the Department of Pediatrics at New York University Medical Center, for helpful discussions on the Na+ Ca++ exchanger and for the very generous sharing of reagents.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES