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Identification and characterization of embryonic stem cell-derived pacemaker and atrial cardiomyocytes

E. Kolossov^*,1, Z. Lu^,1, I. Drobinskaya, N. Gassanov, Y. Duan, H. Sauer, O. Manzke, W. Bloch, H. Bohlen^*, J. Hescheler and B. K. Fleischmann^||,2

^* Axiogenesis AG, Cologne;
Institutes of Neurophysiology and
Internal Medicine I, University of Cologne, Cologne;
Department of Molecular and Cellular Sport Medicine, German Sports University, Cologne; and
^|| Institute of Physiology I, University of Bonn, Bonn, Germany

²Correspondence: Institute of Physiology I, Argelanderstr. 2a, University of Bonn, Bonn D-53115, Germany. E-mail: bernd.fleischmann{at}uni-bonn.de

SPECIFIC AIMS

Little is known about the characteristics of individual cardiac cell types during early embryonic development because of the very small size of the heart tube, a lack of morphological differences, and the expression of specific markers. The aim of this study was the identification and functional characterization of cardiac subtypes during early stages of development. For this purpose, we used the embryonic stem (ES) cell in vitro differentiation system.

PRINCIPAL FINDINGS

1. -MHC promoter-driven EGFP expression pattern in EBs
To identify early cardiomyocytes, transgenic ES cells were generated using the -myosin heavy chain promoter, driving the expression of the enhanced green fluorescent protein (EGFP). EGFP expression was not detected at the ES cell stage or in EBs until day 7 of development. EGFP-positive areas were observed on the first or second day after plating (day 8/day 9); these started to beat spontaneously 1 day later. The most remarkable feature was the large diversity of EGFP fluorescence intensities among cells, corroborated by flow cytometry, where only a small percentage of EGFP-positive cells were detected 2 days after plating. Only 1–4% (n=10) of the whole EB population was EGFP-positive and differences in fluorescence intensity ranged from 10² to 10⁴ counts. Immunostaining with cardiac--actinin on whole EBs proved that EGFP-positive cells were detected exclusively in -actinin-labeled areas. Specificity of the -MHC-driven EGFP expression in ES cell-derived cardiomyocytes was corroborated by staining isolated cells with -MHC antibody (Fig. 1 A, B). EGFP and -MHC staining were detected in 95% of cells counted (n=150), whereas no concomitant EGFP fluorescence was detected in 5% of -MHC-positive cells.

View larger version (14K): [in this window] [in a new window]   Figure 1. Specificity of EGFP expression and correlation between EGFP fluorescence intensity and shape. A, B) Isolated early stage ES cell-derived cardiomyocytes display intense EGFP expression (left) after fixation and positive -MHC immunostaining (right). Note the typical round (upper panels) and triangular morphology (lower panels). The triangle-like shaped cell displays cross striation. C) Fluorescence pictures show the typical correlation between shape and EGFP intensity in isolated ES cell-derived early stage living cells. The histogram demonstrates that early stage ES cell-derived cardiomyocytes with round morphology are preferentially characterized by higher EGFP fluorescence intensities than those with triangular morphology. A, B: bar = 10 µm; C: bar = 20 µm.

2. Morphologically distinct cells display different EGFP fluorescence intensity
To better understand the EGFP labeling of ES cell-derived cardiomyocytes, fluorescence intensity and morphological phenotype were correlated using single-cell imaging. Two morphologies, triangular and round, coincided with EGFP-positive cells (Fig. 1A, B ). EGFP fluorescence intensity differed significantly as triangle-shaped cells displayed relatively low levels of EGFP fluorescence (794±71 counts, n=71) when compared with the round shaped cells (2959±208 counts, n=40) (Fig. 1C ). Confocal sections through the cells confirmed that round-shaped cells displayed higher fluorescence intensities throughout the cell than did triangular cardiomyocytes.

3. Pacemaker-like cells display higher EGFP fluorescence intensities than atrial-like cells
To identify the cardiac subtype and correlate it with its EGFP fluorescence intensity, patch clamp experiments were performed on spontaneously beating isolated ES cell-derived cardiomyocytes. Pacemaker-, atrial-, and ventricular-like action potentials (APs) could be discerned in early ES cell-derived cardiomyocytes starting from day 9. AP shape differed between cardiac subtypes with the atrial cells displaying typical triangle-like APs, the pacemaker cells a more pronounced "shoulder," and the ventricular cells a prominent plateau phase (Fig. 2 A). At this stage, the atrial-like cells were found to display the shortest (63±6 ms, n=12) APD₉₀, whereas pacemaker-like (109±9 ms, n=14) and early embryonic ventricular (307±51 ms, n=8) cardiomyocytes in particular were characterized by significantly longer APD₉₀ (Fig. 2B ). Our experiments clearly showed that fluorescence intensities were significantly higher in the pacemaker-like (6053.7±1158.2 counts, n=14) compared with the atrial-like (2204.8±705.4 counts, n=10) cardiomyocytes (Fig. 2C ), whereas all ventricular-like cells were EGFP-negative. The functional characteristics were corroborated by performing electrophysiological experiments on atria- and ventricle-derived cells isolated from early embryonic (E10.5) mouse hearts, where almost identical APs as in ES cell-derived cardiomyocytes were detected (Fig. 2B ). The link between high level of -MHC promoter-driven EGFP expression and pacemaker-like cells was confirmed by using multielectrode recordings on EBs. These experiments (n=5) showed that bright EGFP-positive areas are the "primary pacemaker" areas, whereas EGFP-negative areas are not initiating the population field potentials. Thus, quantitative reporter gene expression allows the identification of pacemaker-like cells after in vitro differentiation.

View larger version (15K): [in this window] [in a new window]   Figure 2. Early stage ES cell-derived cardiomyocytes are functionally committed; pacemaker-like cells display highest EGFP fluorescence intensities. A) Overlay of representative APs recorded from early stage ES cell-derived cardiomyocytes showed distinct differences among the three cardiac subtypes. For the overlay, the initiation of the fast upstroke was used as a reference point (data do not reflect the real membrane potential). B) Statistical analysis of the APD90 demonstrates significant differences among the three cardiac subtypes. The similarity among ES cell- and murine-derived pacemaker-/atrial-like cardiomyocytes is shown. Note the difference in APD90 between the ventricular cells harvested from EBs and from mouse. C) Fluorescence intensity measurements of individual cells demonstrate that pacemaker-like cells display higher levels. The phenotype of the cells included in the analysis was identified in current-clamp recordings. The average fluorescence intensity differed significantly between the two groups.

4. Functional characteristics of early pacemaker- and atrial-like cardiomyocytes
The in vitro differentiation of ES cell-derived cardiomyocytes permitted for the first time an analysis of the functional characteristics of pacemaker- and atrial-like early embryonic cardiomyocytes. Because of the spontaneous electrical activity of the cells, we have focused our analysis on the hyperpolarization-activated nonselective cation (I_f) and the inward rectifier K⁺ current (I_K1). The early stage pacemaker- and atrial-like cells displayed spontaneous electrical activity and concomitant diastolic depolarization. As expected, both cell types were found to functionally express I_f at similar densities at the early embryonic stage. Conversely, at later stages the spontaneous electrical activity declined along with down-regulation of I_f in atrial-like cardiomyocytes. We also analyzed I_K1, known to be a critical determinant for stable resting membrane potential in adult cardiomyocytes, particularly ventricular cells. When comparing the expression pattern of I_K1, we noticed that it was preferentially detected in atrial-like cells and at significantly higher densities. This indicated that fundamental differences in the expression pattern of ion currents exist between pacemaker- and atrial-like cardiomyocytes at this early embryonic stage.

CONCLUSIONS AND SIGNIFICANCE

In the present work we have addressed the question of whether cardiac subpopulations can be targeted and characterized. We show that quantitative reporter gene expression under control of the -MHC promoter allows the discrimination of early pacemaker- from early atrial-like cardiomyocytes.

To understand the functional characteristics of early embryonic atria-derived cells, we have used the -MHC promoter, known to be expressed in atria of the early mouse embryo. Our data are fully in line with the pattern of -MHC expression during early mouse development. In the midgestation stage (from E9,5 to E14,5), -MHC expression is restricted mainly to the atrial- and ß-MHC to the ventricular portion of the developing heart (Fig. 3 ). While the early heart tube shows a gradient transition of MHC- to ß-MHC isoforms from the sinoatrial toward the ventricular part, respectively, we could detect EGFP expression restricted to the atrial- and pacemaker-like cells. The EGFP-positive population displayed a large variation in the fluorescence intensity of individual cells. This was not merely due to the cellular geometry, as confocal sections corroborated our findings with flow cytometry and microscopy. The round EGFP-positive cardiomyocytes displayed preferentially high fluorescence, whereas triangle-shaped cells showed significantly lower EGFP fluorescence intensities. The combination of EGFP intensity measurements and single-cell patch clamp analysis demonstrated that EGFP fluorescence intensity levels are a good indicator for pacemaker- vs. atrial-like cardiomyocytes. Specific EGFP expression allowed us to functionally characterize (to our knowledge for the first time) early embryonic pacemaker- and atrial-like cells. The different subtypes could be clearly distinguished based on their AP shape and duration. In contrast to differentiated cardiomyocytes, clear differences became evident: all cells were spontaneously beating, which was likely related to the functional expression of I_f. The physiological relevance of our findings was strengthened by experiments on murine embryonic cardiomyocytes where almost identical characteristics were detected. I_f was found to be expressed at similar densities in both early embryonic pacemaker- and atrial-like cells, which displayed prominent diastolic depolarization. During further development, we noticed that both I_f expression and the diastolic depolarization declined in atrial-like cells which provided indirect evidence for its physiological role for pacemaking.

View larger version (12K): [in this window] [in a new window]   Figure 3. Schematic diagram of MHC/ßMHC gradients in the early (E8.5) embryonic heart and MHC promoter driven EGFP expression in transgenic ES cell-derived cardiac subtypes. The early embryonic heart tube consists of the inflow (IFT) and the outflow (OFT) tract, where sinusnode (SA), atria (A), and ventricles arise from (left). Within the early embryonic heart, opposite gradients for MHC and ßMHC are reported (center). The MHC-EGFP transgenic ES cells differentiate into precardiac mesodermal (PCM), and then into atrial (a)- and pacemaker-like (sa) cells (EGFP-positive) and ventricular-like (v) cells (EGFP negative, right). The pacemaker-like cardiomyocytes display the highest EGFP content.

To our knowledge, our findings show for the first time that quantitation of -MHC promoter-driven EGFP expression allows highly reliable identification of pacemaker- and atrial-like cells, thus circumventing the problem of the lack of specific markers of pacemaker cells. The use of double reporter gene constructs (i.e., - and ß-MHC, Fig. 3 ) may provide a more definitive means of distinguishing among all cardiac subtypes. In addition to furnishing insight into the functional characteristics of cardiac subtypes, this approach may also provide a better understanding of the mechanisms underlying the differentiation of the various cardiac subtypes.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-1451fje;

¹ These authors contributed equally to this work.

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This Article Full Text (PDF) All Versions of this Article: 19/6/577 03-1451fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Kolossov, E. Articles by Fleischmann, B. K. Search for Related Content PubMed PubMed Citation Articles by Kolossov, E. Articles by Fleischmann, B. K.