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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online September 2, 2004 as doi:10.1096/fj.04-1619fje. |
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,1
* Department of Internal Medicine III, University of Cologne,
Center for Molecular Medicine, University of Cologne (CMMC), Germany
1Correspondence: Department of Internal Medicine III, University of Cologne, Joseph-Stelzmann-Str. 9, 50924 Cologne, Germany. E-mail: uta.hoppe{at}uni-koeln.de
SPECIFIC AIMS
The goal of the present study was to gain further insight into mechanisms promoting the differentiation of mammalian cardiac conduction tissue, with an aim to easily identify embryonic stem (ES) cell-derived pacemaker cells and possibly direct differentiation toward this cell type.
PRINCIPAL FINDINGS
1. Establishment of an ANP-EGFP-expressing ES cell line
D3 cells have been characterized as pluripotent ES cells that can develop cardiomyocytes with electrophysiological properties resembling sinus node and atrial and ventricular cells at the terminal differentiated stage (6+
9d). Our aim was to identify pacemaker-like cells by specific labeling and morphological criteria. Since it had been shown that ES cell-derived ventricular cardiomyocytes labeled by tissue-specific EGFP expression under the control of the Mlc2v promoter did not develop into cells exhibiting pacemaker properties, we speculated that selection of predominantly atrial cardiac precursor cells would include a sufficient number of pacemaker cells. Thus, we chose the human ANP promoter to stably express EGFP in ES cell-derived cells.
Generally, the ANP promoter was switched on at day 6+4, rarely on day 6+3 as indicated by the formation of cell clusters featuring bright EGFP fluorescence. All fluorescent areas within the embryoid bodies (EB) developed spontaneous contractions 24 h later. Two-dimensional planimetric calculation revealed that the EGFP-positive area within EBs amounted to 18.0 ± 3.3% (n=14) at the late stage of development (6+20 d). During all stages of cardiomyogenesis, EGFP fluorescence was exclusively detected in these beating areas, indicating the cardiac specificity of the human ANP promoter.
2. Cardiac-specific ANP-EGFP expression
To further confirm the cardiac nature of EGFP-expressing cells, immunostaining was performed on EBs and single ES cell-derived cells: 91% of EGFP-positive cells stained positively with an antibody against 
-actinin. Staining with an antibody specific for cardiac troponin I, a marker of late-stage cardiomyocytes in EBs, resulted in positive labeling of 83% of EGFP-positive cells isolated from 24- to 28-day-old EBs. Thus, immunocytochemistry corroborated the expression of cardiac-specific reporter genes in ANP-EGFP transfected ES cells.
3. ANP-EGFP cells differentiate into distinct cell populations
The hyperpolarization-activated inward current If is a characteristic ionic current of cardiac pacemaker cells; it is essential for spontaneous beating activity and for the modulation of pacing rate. Therefore, If current recordings and registrations of action potentials were performed to identify and further characterize ES cell-derived pacemaker cells of the terminal differentiation stage (6+9d). If was present in 74% of all EGFP-positive cells investigated with a mean current density of 20.4 ± 2.0 pA/pF at –150 mV (n=128). However, we observed distinct subpopulations of ANP-EGFP-positive cells. Cardiomyocytes enzymatically isolated from beating areas of embryoid body outgrowths exhibited a spindle-, round- or tri-/multiangular-shaped morphology after dissociation. While basic electrophysiological properties were reported to be independent of the cellular morphology in unlabeled cardiomyocytes, in this study ANP-EGFP-expressing cells with a spindle-like morphology all had a pacemaker-like phenotype (n=22) (Fig. 1
A). These spindle-shaped cardiocytes exhibited vigorous spontaneous beating with a rate of 173 ± 14 bpm and spontaneous sinus nodal action potentials (resting potential –45.5±2.2 mV; slow diastolic depolarization; overshoot 19 ± 3 mV, APD90 111.3±1.7 ms) (Fig. 1C
). Conversely, 58% of tri-/multiangular cells (n=77) (Fig. 1B
) were quiescent, the remaining beating at a significantly slower rate of 62 ± 3 bpm (P<0.001). All triangle-shaped cells displayed atrial-like action potentials with more negative resting potentials (–68.7±2.1 mV), more positive overshoots (37±1.8 mV), and shorter durations (APD90 48.0±2.1 ms) (Fig. 1D
). Consistent with these differences, all spindle-like cells revealed significantly larger If current densities (34.5±2.4 pA/pF at –150 mV), earlier first If current activation (–50 to –60 mV) and faster current activation kinetics (
395.3±30.7 ms at –150 mV) than tri-/multiangular cells (12.8±0.7 pA/pF at –150 mV; first activation at –80 to –90 mV; 681.1±30.3 ms at –150 mV; P<0.001) (Fig. 1E, F
). These distinct If current properties further support the pacemaker-like phenotype of spindle-shaped cells.
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By their morphology, 21.3 ± 3.1% of ANP-EGFP-expressing cells could not be classified to either sublineage. These cells exhibited a round-shaped morphology whereas their electrophysiological properties resembled those of spindle-shaped (33%) or triangular (67%) cardiocytes. It remains to be determined whether these cells were not well enough attached to the coverslip surface to show their typical morphology, exhibit an intermediate phenotype, or were not yet fully differentiated. No ventricle-like cells could be identified reflecting atrial-specific expression of the human ANP promoter in ES cells.
4. Endothelin-1 directs development of ANP-EGFP ES cells toward a pacemaker phenotype
So far no developmental factors promoting the differentiation of mammalian atrial-derived, presumably central conduction tissue have been reported. Thus, we tested the effect of endothelin-1 (ET-1) and ET-1 with ET receptor antagonists BQ123 or BQ788 on the differentiation of the two sublineages identified. Indeed, ET-1 significantly increased the relative percentage of spindle-shaped cells by 67% (from 18.3±1.7% to 30.3±2.1%; P<0.001) while decreasing the relative percentage of tri-/multiangular cells by 35% (from 60.3±4.2% to 39.2±3.5%, P=0.001). The concentration-response relation for ET-1 revealed an EC50 of 1.1 x 10–9 M. All spindle-like and tri-/multiangular cells exhibited action potential configurations and If current properties of pacemaker and atrial cells identical to untreated control cells with the respective morphologies. The effects of ET-1 could be prevented by BQ123 or BQ788. This indicated that ET-1 shifted differentiation of ANP-EGFP-positive, ES cell-derived cardiocytes toward pacemaker cells in an endothelin receptor-dependent manner without affecting electrophysiological properties.
The effect of ET-1 on the development of the cardiac conduction tissue was further confirmed by immunostaining and Western blot analyses. Exposure of EBs to ET-1 markedly increased the intensity of connexin 40 staining, a marker of the early differentiating conduction system in mice, compared with controls (Fig. 2
A, B). Expression of connexin 45, a marker of the mouse sinus node and conduction system, was increased by ET-1 (Fig. 2E, F
) whereas expression of connexin 43, a marker of working myocardium, remained unaffected (Fig. 2C, D
). Similar results were obtained by Western blot analysis. ET-1 increased the protein level of connexin 40 and connexin 45, while not affecting connexin 43. The ET-1-induced changes could be inhibited by BQ123 or BQ788.
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5. Neuregulin-1 shows no inductive effect on differentiation of pacemaker cells
We examined the effect of neuregulin-1 (NRG-1) on the development of our ANP-EGFP-expressing ES cells. However, no effect of NRG-1 on differentiation of ANP-EGFP-positive ES cells (spindle-shaped cells 15.1±1.6%; triangular cells 57.4±2.7%; P=NS vs. control) was observed. NRG-1 did not affect protein levels of connexins. Treatment with the combination of ET-1 and NRG-1 resulted in similar changes of cell differentiation (spindle-shaped cells 28.6±3.6%; triangular cells 37.5±6.8%; P=NS vs. ET-1) and protein amounts of connexins as ET-1 alone.
CONCLUSIONS AND SIGNIFICANCE
ES cells provide a useful model for the evaluation of early differentiation and development of various tissues. In the present study we established ANP-EGFP-expressing ES cell lines to further characterize the development of very early stages of the mammalian cardiac conduction tissue.
ANP-EGFP-positive cells revealed distinct morphological and electrophysiological subpopulations. The most common cell type was tri-/multiangular with atrial electrophysiological characteristics presumably further developing into working atrial myocardium. We could identify a second sublineage of cells with a spindle-like shape exhibiting a pacemaker-like phenotype. The differences seen in morphology, action potentials, and If properties indicate a very early diversification of electrophysiological and morphological parameters between pacemaker cells vs. atrial working myocardium.
ET-1 exposure resulted in a shift toward spindle-shaped cells with pacemaker-like electrophysiological characteristics and increased expression levels of connexin 40 and connexin 45, detected predominantly in the murine cardiac central conduction tissue and sinus node. In embryonic mice, NRG-1 converted ventriculocytes into cells of the ventricular conduction system, (Purkinje cells). Conversely, no effect of NRG-1 on the differentiation of ANP-EGFP-positive ES cells was observed in the present study (Fig. 3
). These results are consistent with an early diversity of paracrine signaling in the differentiation of the atrial-derived, presumably central vs. the ventricular (Purkinje-like) conduction system.
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Our observations give further insight into the differentiation of the cardiac conduction system. ANP-EGFP expression enabled the identification of ES cell-derived pacemaker cells only by their fluorescence and morphology. Since we could markedly enrich the percentage of pacemaker cells by ET-1, these results might be a valuable first step in the specific selection of pacemaker cells for the development of cell therapeutic strategies.
FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-1619fje
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