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Am J Physiol Heart Circ Physiol 284: H2114-H2123, 2003. First published February 6, 2003; doi:10.1152/ajpheart.01106.2001
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Vol. 284, Issue 6, H2114-H2123, June 2003

Development of electrical activity in cardiac myocyte aggregates derived from mouse embryonic stem cells

K. Banach1, M. D. Halbach1, P. Hu1, J. Hescheler1, and U. Egert2

1 Institut für Neurophysiologie, Universität zu Köln, 50931 Köln; and 2 Institut für Neurobiologie und Biophysik, Institut für Biologie III, Albert-Ludwigs Universtität Freiburg, 79104 Freiburg, Germany


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Embryonic stem cells differentiate into cardiac myocytes, repeating in vitro the structural and molecular changes associated with cardiac development. Currently, it is not clear whether the electrophysiological properties of the multicellular cardiac structure follow cardiac maturation as well. In long-term recordings of extracellular field potentials with microelectrode arrays consisting of 60 substrate-integrated electrodes, we examined the electrophysiological properties during the ongoing differentiation process. The beating frequency of the growing preparations increased from 1 to 5 Hz concomitant to a decrease of the action potential duration and action potential rise time. A developmental increase of the conduction velocity could be attributed to an increased expression of connexin43 gap junction channels. Whereas isoprenalin elicited a positive chronotropic response from the first day of spontaneous beating onward, a concentration-dependent negative chronotropic effect of carbachol only developed after ~4 days. The in vitro development of the three-dimensional cardiac preparation thus closely follows the development described for the mouse embryonic heart, making it an ideal model to monitor the differentiation of electrical activity in embryonic cardiomyocytes.

cardiac development; microelectrode array; excitation spread; connexin43; mouse embryonic stem cells


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

EMBRYONIC STEM (ES) cells differentiate in vitro into various cell types including cardiac myocytes (7). Previous studies on this system have shown that ES cell-derived cardiac myocytes (ESCs) express cardiac-specific proteins including alpha -actinin and alpha -myosin heavy chain in a time-dependent manner. The cell morphology gradually changes from a small and round shape to larger rod-shaped cells with recognizable myofibrillar assemblies (13, 15, 28, 37). Furthermore, electrophysiological measurements on isolated ESCs demonstrate a time-dependent expression of ion channels and signal transduction pathways (1, 12, 24, 25). The differentiation of the ESCs in culture therefore seems to recapitulate steps of the in vivo embryonic differentiation of cardiac muscle. Action potential (AP) recordings on isolated ESCs suggest that these cells differentiate into specific cardiac cells such as atrial, ventricular, and pacemaker cells (15, 25). Although their distribution within the beating aggregate of ESCs has not been completely analyzed, in vivo stainings and tissue-specific expression of green fluorescent protein indicate that different cellular phenotypes are arranged in aggregates rather than randomly distributed (27, 28). However, it is currently not clear how the developmental and tissue-specific differentiation relates to the electrophysiological properties of the intact three-dimensional preparation of ESCs. In the present study, we analyzed the developmental changes of spontaneous electrical activity with repeated multielectrode recordings using substrate-integrated microelectrode arrays (MEAs). This technique allows not only the long-term culture of the preparation but also the analysis of the spatial electrophysiological differences within the preparation. In the present study, we analyzed different field potential (FP) parameters to characterize the development of the spontaneous electrical activity of multicellular aggregates of ESCs and their electrophysiological properties. Furthermore, the use of an ES cell line deficient in the expression of connexin43 (Cx43-/-), the major cardiac gap junction protein, enabled us to distinguish developmental changes of the active, voltage-dependent membrane properties from changing passive components like the intercellular resistance.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Culture of ES cells. ES cells of the cell lines D3 and R1 [Cx43-/- (31, 34)] were propagated in culture and differentiated within three-dimensional embryo-like structures called embryoid bodies (EBs) as described previously (16, 25). Briefly, ES cells were propagated on feeder cell layers with leukemia-inhibiting factor (LIF) added to the culture medium. Differentiation was initiated by the "hanging drop" method (400 cells/20-µl drop) and withdrawal of LIF. After 2 days, the drops were washed off the lid of the culture dish, and EBs were maintained in suspension [DMEM plus supplements (46)] for another 5 days. After these 7 days, EBs were plated onto MEAs, which served as the FP recording device and culture dish at the same time. The EBs attached to the bottom of the MEA within 1 day after being plated (day 7+1) and subsequently spread to form a three-dimensional cell layer containing various cell types including cardiac myocytes (16, 46).

MEA recording. We used substrate-integrated, planar MEAs (Multi Channel Systems; Reutlingen, Germany) for long-term recordings of the spontaneous electrical activity from cultures of cardiac myocytes and EBs (8, 16). EBs were positioned in the middle of a sterilized MEA consisting of 60 titanium nitride-coated gold electrodes (diameter = 30 µm; interelectrode distance 200 µm in a square grid). For recording, a separate sterile Ag/AgCl electrode was temporarily inserted into the dish as the ground electrode. The MEA was connected to the amplifier and data-acquisition system (Multi Channel Systems), which included a heating device to maintain a constant temperature of 37°C. Data were recorded simultaneously from up to 60 channels (sampling frequency up to 40 kHz) under sterile conditions. The data were analyzed off-line with a customized toolbox programmed for MATLAB (Mathworks; Natick, MA) to detect FPs. Figure 1A shows an example of voltage traces recorded from a small EB at day 7+4 in culture. Electrical activity is reflected in the negative deflections of the voltage traces. In the magnification of the FP recorded from electrode 56 (Fig. 1A, left), the FP parameters analyzed are depicted: the interspike intervals (ISI), the size of the largest negative peak (FPMin), the last positive peak in a cycle (FPMax), the duration of the FP (FPdur; defined as the interval between FPMin and FPMax), and the FP descent phase [FPrise; defined as the time from the onset of the FP (baseline) to FPMin].


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  		Fig. 1.   A: field potentials (FPs) recorded from an embryonic body (EB) with a microelectrode array (MEA). The 8 × 8 arrangement of the traces reflects the arrangement of the electrodes. The parameters analyzed are indicated for the magnified FP. The interspike interval (ISI; C) was recorded from continuous FP recordings (B) on single MEA electrodes and is a reliable measure of the beating frequency (ISI = 859.5 ± 39.9 ms, mean ± SE). FPMin, size of the largest negative peak; FPMax, last positive peak in a cycle; FPdur, duration of the FP; FPrise, FP descent phase.

These parameter were chosen according to previous analysis and interpretations of FPs by Regeher et al. (36), Spach et al. (39), and Yamamoto et al. (47), who directly correlated FP parameters to simultaneously recorded APs. After these interpretations, the decay of the extracellular waveform (here, FPrise) is proportional to the duration of the upstroke of the intracellular potential (39). Furthermore, according to the data published by Yamamoto et al. (47), the slow return of the FP to the baseline coincides with the repolarization of the APs. We found, however, that the interval from FPMin to FPMax was a more reliable, relative correlate of the AP duration (FPdur). Even though this analysis does not allow us a quantitative analysis of the currents contributing to the APs, changes in the parameters can be interpreted as changes in the contribution of different ion channels to the FP. The ISI represents the beating frequency of the preparation and could be measured for extended periods. Figure 1B shows the FP recorded from one MEA electrode over the period of 2 min. The ISI measured from this electrode is shown in Fig. 1C and revealed an ISI of 859.5 ± 39.9 ms (mean ± SE). To control the validity of this measurement, usually the ISI at different electrodes within one preparation was analyzed and compared.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Development of spontaneous pacemaker activity in ES cells. ES cells differentiate in vitro into cardiac myocytes, thereby expressing cardiac-specific proteins in a time-dependent manner. By plating EBs on the MEA, we examined when electrical activity can be recorded from spontaneously beating cardiac myocytes and whether developmental changes of the electrophysiological properties can be quantified during the developmental process. One or two days after being plated (i.e., days 7+1 and 7+2), cardiac cells were identified within beating aggregates of EBs. The development of visible beating coincided with the occurrence of negative deflections in the FPs recorded with the MEA. Figure 2A illustrates FPs recorded from the same EB preparation on subsequent days. Counting the number of electrodes with electrical activity gives us a relative measure for the size of the beating area and most likely reflects the growth of the cluster of electrically active cardiac cells (Fig. 2B; means ± SE, n = 21 EBs). The number of active electrodes increased almost sevenfold during the first 10 days of culture. Concurrently, the amplitude of FPMin increased. Although the degree of this change varied between electrodes within the beating area, the overall increase of FPMin was reproducible. The increase of the beating area as well as the increase of FPMin most likely indicates the developmental increase in the number of electrically active cardiac cells.


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  		Fig. 2.   A: FPs from one EB preparation recorded on days 7+1, 7+5, and 7+8 of culture. Traces from positions without detectable activity were omitted (lines indicate 0-µV level only). The number of electrodes and FPMin increased during development. B: the number of electrodes with electrical activity increased sevenfold during the first 10 days of culture (means ± SE, n = 21). C: in the same period, the ISI (means ± SE, n = 19) decreased.

In addition, there was a clear developmental change of the spontaneous beating frequency of the ESC clusters. The ISI decreased with the age of the culture to ~0.2 s around day 7 + 10 (Fig. 2C). The reproducibility of these findings in different EB preparations indicates that this change of the ISI was not random but reflects a stable differentiation process.

Developmental change of the shape of the FP. During cardiac development of embryonic mice, the electrophysiological properties of the cells, like the AP upstroke velocity and AP duration, undergo significant changes (5, 41). To examine whether the developmental changes of the AP are reflected in changes of the FP during developmental differentiation within a beating aggregate of ESCs, we examined the waveform of FPs recorded from the same preparation on successive days. On the first day of spontaneous activity, FPs were small in amplitude and FPrise was long; a representative example is shown in Fig. 3A. FPs throughout the aggregate exhibited a high degree of similarity at this state of development; however, in later stages, a temporal as well as developmental change could be observed. The average of all FPs within single preparations reveals an overall decrease of FPrise up to day 7+9 of development (Fig. 3B; means ± SE, n = 13 EBs). However, during this period, the FPs within a single preparation grew more heterogeneous. Examining FPrise along the path of excitation spread revealed that in ESCs FPrise was long at the origin of excitation and shortened significantly with increasing distance from the pacemaker region (Fig. 3C).


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  		Fig. 3.   FPs (A) recorded simultaneously on day 7+2 of development on electrodes 35, 34, 33, and 42. The succession of FPMin indicates the delay of excitation spread. B: FPrise (means ± SE, n = 10 EBs) plotted versus time in culture. C: normalized FPrise as a function of the distance from the origin of excitation (means ± SE, n = 5).

Taking the data of Yamamoto et al. (47) as a basis that the repolarization of the AP coincides with the return of the negative potential to the baseline, we were able to characterize the developmental changes of AP duration in ESCs. To assess the overall development of FPdur on successive days of development, we averaged the FPdur over all active electrodes for each preparation and found a developmental decrease that stabilized around day 7+10 (Fig. 4A; n = 19 EBs). To demonstrate that FPdur also follows experimentally induced changes of the AP duration, we exposed ESCs to increasing concentrations of 4-aminopyridine (4-AP), a blocker of the transient outward K+ current (Ito) known to prolong AP duration in mouse ventricular cells (32, 43) and ESCs (12). A concentration-dependent increase of FPdur could be observed (Fig. 4C), concomitantly with a decrease of the ISI (Fig. 4B).


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  		Fig. 4.   A: FPdur plotted as a function of time in culture (means ± SE, n = 19 EBs). FPdur estimated from FPs from the experiment illustrated in C are plotted versus ISI (B). Numbers indicate the concentrations of 4-aminopyridine (4-AP; in mmol/l) in the bath. Each cluster represents FPs recorded during a time period of 30 s. Note that the ISI shortened, although the action potential lengthened, with increasing concentrations. C: representative voltage traces of FPs recorded at the 4-AP concentrations indicated.

Developmental change of the propagation velocity. The MEA system enables us to determine the origin and direction of excitation spread from the temporal succession of FPMin (16) on the surrounding electrodes. In Fig. 5A, the delay of excitation spread recorded from a single preparation at days 7+2 and 7+5 of culture is plotted against the respective distance of the electrodes from the origin of excitation. At the onset of beating (day 7+2), a delay of almost 30 ms for a 200-µm distance can be observed, corresponding to a conduction velocity of 6 × 10-3 m/s. Examination of the same preparation at day 7+5 in culture reveals an increase of the beating area and an increase of the velocity of excitation spread. The path of excitation spread is again marked on the sketch of the MEA (open circles, Fig. 5A). Plotting the interelectrode delay against the distance from the origin of excitation, now located at the bottom left end of the MEA, reveals that the delay is markedly decreased, reflecting a conduction velocity of ~15 × 10-3 m/s. Calculating absolute propagation velocities requires detailed information about the exact path of excitation propagation within the cell syncytium of the EB. Because information about these parameters is not readily available in this preparation, the values can only be taken as approximations. The increase of the conduction velocity could be based on a developmental increase in the current density of fast-activating voltage-dependent channels, increased expression of gap junction channels (2, 40), increased tissue organization, or an increase in cellular size. To rule out that the increase is solely due to the structural features of the preparation, we studied the developmental change of the conduction velocity in preparations of ESCs lacking the expression of Cx43, the major cardiac gap junction protein whose expression is upregulated in the cardiac tissue at embryonic day 9.5 postcoitum (E9.5) (2). Cardiomyocyte aggregates derived from Cx43-/- cells exhibited the same time-dependent change of ISI, FPrise, and FPdur as observed in wild-type cells. In contrast, differences in the developmental change of the conduction velocity could be observed. At the onset of beating at day 7+1/7+2 (open symbols, Fig. 5B), both cell lines exhibited similar low conduction velocities (6 × 10-3 m/s), although the developmental increase of the conduction velocity in cultures older than 7+7 days (open symbols, Fig. 5B) was significantly higher in the wild-type than in the Cx43-/- cell line. Whereas conduction velocities of ~15 × 10-3 m/s were reached in the Cx43-/- preparation, wild-type preparations reached an average of 45 × 10-3 m/s. This observation suggests that the developmental expression of Cx43 underlies the structural maturation of the tissue and is responsible for the developmental increase in conduction velocity.


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  		Fig. 5.   The delay of excitation spread decreased during time in culture. A: path of excitation spread in 7+2 (open circle )- and 7+5 ()-day-old EB preparations. In both cases, excitation started at the lowermost electrode. Plotting the delay of each electrode versus its distance from the origin of excitation reveals a significant decrease in the delay of excitation spread during time in culture. B: delay of excitation spread in 7+2 (open circle )- and 7+7 ()-day-old embroyic stem cell-derived cardiac myocytes (ESCs) derived from wild-type and connexin43-deficient (Cx43-/-) preparations. (Four different EB preparations from each cell line at both developmental stages were included in the analysis.)

Development of hormonal sensitivity. The responsiveness of the early murine embryonic heart to beta -adrenergic stimulation is controversial in the literature. Whereas some reports describe that the positive chronotropic and inotropic responses to beta -adrenergic stimulation lag behind the expression of beta -adrenergic receptors (3, 4), the newer studies of Liu et al. (22) describe positive chronotropic effects on whole hearts as well as on single cardiac myocytes of 9-day-old mouse embryos. The negative chronotropic effect in the heart is mediated by carbachol (CCh) via the activation of muscarinic K+ current (IK,ACh). In chicken as well as rat hearts, a negative chronotropic response to CCh develops only later in development. To test whether the susceptibility of the pacemaker to adrenergic and cholinergic stimulation in ESCs repeated this change during the differentiation process in culture, we perfused the cells at different stages of development with either the beta -receptor activator isoprenalin (Fig. 6) or CCh (Fig. 7). Already at the first day of spontaneous electrical activity (in this preparation, day 7+2), we observed a positive chronotropic effect of beta -receptor stimulation (n = 3; Fig. 6A). Application of CCh induced a concentration-dependent negative chronotropic effect only around day 7+5 (Fig. 7A) of development (n = 3 for preparations older than 7+5 days and n = 4 for preparations younger than 7+5 days). A transient and a steady-state component can be distinguished in the frequency response. The sensitivity of the culture toward CCh increased during subsequent days of development (Fig. 7B). In the same culture, identical concentrations of CCh (10 and 50 µM) now induced a transient block of pacemaker activity before beating resumed at a decreased steady-state frequency (Fig. 7B). The antagonizing effect of CCh on beta -adrenergic stimulation was likewise only detected in older cultures. Application of isoprenalin to a preparation at day 7+8 in culture induced a decrease of the ISI from 500 to 200 ms (Fig. 6B). Additional transient perfusion with CCh resulted in a reversible decrease of the beating frequency. The experiments demonstrate that ESCs not only change their spontaneous electrical activity in culture but also that their sensitivity to hormonal stimuli changes during the differentiation process.


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  		Fig. 6.   ISI plotted against the time of recording. A: isoprenalin (Iso) induced a decrease of ISI in a preparation that started beating at day 7+2 of culture. B: at day 7+8, carbochol (CCh; 1 µM) antagonized reversibly the positive chronotropic effect induced by Iso (100 nM).



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  		Fig. 7.   A: at day 7+5, the negative chronotropic effect of CCh is reflected in a small increase of the ISI. Only 1 day later (B; day 7+6), the same preparation responded to the identical concentration of CCh with a transient block of the spontaneous activity and a more pronounced increase in the steady-state ISI.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Previous characterizations of the developmental steps associated with segregation of electrically and functionally different cell types in the developing heart relied on either whole organ preparations (19, 20) or the isolation of single cells from the embryonic heart (32, 43, 44). With both techniques, electrophysiological properties at the stage of organ isolation can be described, but continuous measurement of the differentiation process is limited. Furthermore, culture conditions are known to initiate remodeling of isolated cells (9, 14). Mouse as well as human ES cells, on the other hand, are known to recapitulate molecular stages of the developmental process of cardiac myocytes (21, 28, 37, 45), and electrophysiological and intrinsic properties of the APs could be described in single-cell cardiac myocyte preparations (1, 12, 23).

We report here for the first time how the sequence of change of the electrophysiological properties and its interplay with the structural change of the multicellular cardiac preparation result in the spontaneous electrical activity recorded in a multicellular in vitro model of cardiac development. With the differentiating EBs plated on a MEA, we were able to continuously monitor the spatial and temporal structure and the dynamics of electrical activity of a developing multicellular structure of cardiac myocytes. Besides the analysis of the beating frequency and the velocity of excitation spread by means of the FP, we analyzed developmental changes of FP parameters that are proportionally related to the duration of the AP upstroke (39) and AP duration (47). With the repeated, comparative analysis of these parameters within the same specimen of wild-type and genetically manipulated preparations, the importance of individual components for the developmental or pathophysiological change of spontaneous electrophysiological properties can be revealed.

Comparison of FPs recorded from ESCs and isolated cardiac myocytes. In differentiating EBs, cells of all three germ layers can be detected (7), although the distribution of different cell types inside an EB needs to be analyzed in detail. Oyamada et al. (33) reported that no dye coupling takes place between cardiac and noncardiac cells in the EB, indicating that cardiac cells are electrically isolated from the surrounding tissue. Because electrical coupling could influence the shape of the FPs recorded at electrodes not in direct contact with electrically active cells, we compared FPs recorded from ESCs and, presumably more homogeneous, monolayers of cardiac myocytes isolated from the embryonic mouse heart (K. Banach, unpublished data). Although the structure of ESCs is more complex, the recordings revealed no qualitative or quantitative differences of the waveforms. The FP types recorded in cardiac myocyte cultures were also found in EB recordings, suggesting that the same mechanisms generate these fields and that the same currents likely form their basis.

Developmental change of the FP in ESCs. The average number of electrodes detecting electrical activity in recordings from differentiating ESCs increased sevenfold during the first 10 days of culture. An increase in the surface of the beating aggregate due to the increase in cell size or migration of cells from the EB as described for this preparation (46) cannot be ruled out; however, previous studies report the withdrawal of ESCs from the cell cycle only at day 16 (day 7+9) (26) and increasing cardiomyocyte numbers until day 7+14 of differentiation (30). Therefore, at least during the first 10 days after the cells were plated, the mitotic activity seems to play a significant role in the increase of the beating aggregate. During the increase of the beating aggregate, the beating frequency increased from initially 1 to 5 Hz. The time course of the functional development of the cardiomyocyte structure within the EB compares well with that of the developing embryonic mouse heart (35), where electrical as well as contractile activity start at 8.5 days postcoitum, when myocardium is still added to the heart tube (10, 29, 30).

The electrophysiological basis of the increased beating frequency could depend on multiple factors. In isolated ESCs, intracellular Ca2+ oscillations as well as ATP-dependent K+ channels, Ito, and the pacemaker current (If) have been described to play an important role in the development of pacemaker activity (1, 12). The developmental increase in frequency therefore could be based on either a switch between the major mechanisms of pacemaker activity or an increased current density of the responsible current component as described for If in ESCs (1).

Besides an increase of the beating frequency, we observed within the area of differentiating cardiac myocytes 1) an increase of FPrise, 2) a decrease of FPdur, and 3) an increase of the propagation velocity. These parameters relate to the following changes of the intrinsic electrophysiological properties described for isolated ESCs and cardiac cells isolated from embryonic mice.

First, all FPs recorded at the time of onset of visible beating, i.e., days 7+1 or 7+2, exhibited a homogenous shape with a long FPrise and prolonged FPdur (Figs. 3A and 4, A and B). This result corresponds to the slow rise times described for APs recorded from the embryonic chicken (11, 19, 20) and from isolated ESCs up to day 7+3 in culture (24). The potentials were similar to those recorded from the origin of excitation in EBs at later stages of development (Fig. 3C). These data indicate that all cells are functionally homogeneous and pacemaker-like at the onset of beating and that no fast voltage-activated depolarizing currents like Na+ current are involved in the generation of the AP, consistent with the findings in isolated ESCs (24). As a caveat, it must be noted that FPMin also depends on the absolute current flowing at the location of an electrode. This is influenced by the increasing number of electrically coupled cells and their intercellular resistance within the range of the MEA electrode, which, in turn, is suggested by the growth of the EB described above. It is important to note that pacemaker-like FPs with long descent times found at the origin of excitation spread within the multicellular aggregate (Fig. 3C) did not necessarily produce the smallest FPMin values within a culture, as would be expected for small, weakly coupled populations. This supports the interpretation that the decrease of FPrise reflects the development of fast depolarizing currents.

Second, the developmental decrease of the AP duration points out the ongoing differentiation process. In the mouse heart, a developmental decrease of the AP duration was attributed to complex changes of the expression level of different ion channels responsible for repolarization. The current density of Ito from the neonatal to adult heart, as well as the current density of the rapid and slow components of the delayed rectifier K+ current, increases during embryonic development (5, 44) and during the developmental differentiation of ESCs (12). Up to now, we did not identify the current component responsible for the accelerated repolarization, but a developmentally increased expression of outward rectifying currents could be demonstrated in single ESCs isolated from beating aggregates (12). Additionally, the marked increase of FPdur after application of 4-AP indicates the expression of Ito in our multicellular preparations and supports a shortening of the AP due to the differentiation of these K+ currents.

Finally, in the ESCs derived from wild-type as well as Cx43-/- ES cells, a developmental increase in the conduction velocity could be observed, whereas the increase in the wild-type preparations exceeded that of the Cx43-/- cells by a factor of 3. Besides the increased density of fast voltage-activated channels, the propagation velocity inside a cardiac preparation depends on the intercellular resistance as well as the size and structural arrangement of the cells in the electrical syncytium (40). An increased density of the voltage-dependent Na+ current was previously described during embryonic development (4) and in differentiating ESCs (25) and is in good agreement with the increased FPrise observed in our FP measurements. However, because cardiac myocytes isolated from Cx43-/- mice exhibit similar densities of the voltage-dependent Na+ current to those of wild-type cells (18), the increase in conduction velocity cannot completely be attributed to the increased AP rise time. The cellular structure of ESCs, which is similar to that of cardiac myocytes, develops from a small and round cell to a larger, rod-shaped cell with recognizable myofibrillar assemblies (13, 15, 45, 46). These changes could also be observed in our culture, although no differences between the wild-type and Cx43-/- preparations could be observed. The earliest gap junction channel protein expressed in the embryonic heart is Cx45. Cx45, which is also expressed in ES cells (33, 45), forms a low-conductance channel that is believed to be the reason for the slow peristaltic contractions of the embryonic mouse heart (2). Expression of the cardiac gap junction proteins Cx40 and Cx43 follows at E9.5 of the embryonic mouse heart development (2). The expression of all three connexins is described for ES cells. Increased expression of Cx40 was found to coincide with the occurrence of cardiac myocytes (33), and Cx43 expression could be demonstrated within the ESCs by immunohistochemistry (33, 45). The structural reorganization of the cells in conjunction with an increased density of fast voltage-dependent ion channels (13, 25) might thus account for the developmental decrease in the conduction velocity of Cx43-/- and wild-type ESCs, although the further reduction in the wild-type cells might be attributed to the developmentally increased expression of Cx43.

Development of hormonal sensitivity. Besides the expression of voltage-dependent ion channels, electrical activity of cardiomyocytes depends crucially on their sensitivity to modulation by the vegetative or autonomous nervous system. We could demonstrate that adrenergic and cholinergic sensitivities are regulated differentially during the differentiation of ESCs in vitro. Whereas the positive chronotropic effect of isoprenalin is present from the onset of spontaneous beating activity (day 7+2), the negative chronotropic effect of CCh developed only around day 7+4 of in vitro differentiation. Our data are thus in agreement with results obtained from the intact embryonic mouse heart, in which a positive inotropic effect of isoprenalin was observed on whole hearts and isolated ventricles of 9.5 days postculture, 1 day after the initiation of spontaneous beating (22). Only later in development does the negative chronotropic effect of CCh on embryonic heart start. Davies et al. (4) described the expression of IK,ACh only after 17 days postculture in mouse atrial cells. In the rat and chicken, the negative chronotropic effect of CCh is not established from the beginning of contractile activity (6, 42). However, in previous experiments on the sensitivity of ESCs for CCh and isoprenalin on isolated ESCs in the whole cell patch-clamp configuration, Maltsev et al. (23) described that L-type Ca2+ current was insensitive to isoprenalin, forskolin, and cAMP until day 7+9 in culture. Furthermore, stimulation of If by isoprenalin could only be found in late-stage cells. In contrast, the inhibitory effect of CCh on If was already developed in early cells (1, 17). This contrast to our findings might be caused by the isolation procedure and the dedifferentiation of isolated cells in culture, which lack the influence of the surrounding noncardiac tissue in the EB. Another possibility is a spatially, in terms of their receptor expression, heterogeneous differentiation of the cells in the multicellular aggregate that cannot be distinguished after the isolation procedure.

In conclusion, in this study, we demonstrated that the developmental succession of the electrophysiological properties of multicellular preparations of ESCs matches the sequence of electrophysiological changes described for the embryonic heart. The combination of the ES cell and the MEA technique not only allowed the characterization of the beating frequency and the propagation velocity during development, but also enabled the analysis of concurrent changes of intrinsic AP properties, such as AP rise time and AP duration. Additionally, the study on the differentiation of wild-type and Cx43-/- ESCs revealed the electrophysiological and structural interplay during the development of pacemaker activity and excitation spread.


    ACKNOWLEDGEMENTS

We thank Dr. J. Rossant for providing the Cx43-/- cell line.


    FOOTNOTES

This research was supported by Bundesministerium für Bildung und Forschung Grants FKZ 0310967, 0310965, and 0310964D. K. Banach was funded by the Lise Meitner-Habilitationsstipendium of Nordrhein Westfalen.

Address for reprint requests and other correspondence: K. Banach, Dept. of Physiology, Loyola Univ. Chicago, 2160 S. First Ave., Maywood, IL 60153 (E-mail: kbanac1{at}lumc.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

First published February 6, 2003;10.1152/ajpheart.01106.2001

Received 19 December 2001; accepted in final form 3 February 2003.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Heart Circ Physiol 284(6):H2114-H2123
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