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Cardiovascular Research 2003 58(2):410-422; doi:10.1016/S0008-6363(03)00247-5
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Copyright © 2003, European Society of Cardiology

P19 embryonal carcinoma cells: a suitable model system for cardiac electrophysiological differentiation at the molecular and functional level

Marcel A.G. van der Heydena,*, Marjan J.A. van Kempena, Yukiomi Tsujia,b, Martin B. Rooka, Habo J. Jongsmaa and Tobias Opthofa

aDepartment of Medical Physiology, University Medical Center Utrecht, P.O. Box 85060, 3508 AB Utrecht, The Netherlands
bDepartment of Circulation, RIEM, University of Nagoya, Nagoya, Japan

m.a.g.vanderheyden{at}med.uu.nl

* Corresponding author. Tel.: +31-30-253-8418; fax: +31-30-253-9036.

Received 19 September 2002; accepted 17 January 2003


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 Acknowledgments
 References
 
Objective: Murine P19 embryonal carcinoma (EC) cells can differentiate into spontaneously beating cardiomyocytes in vitro and have revealed important insight into the early molecular processes of cardiomyocyte differentiation. We assessed the suitability of the P19 cell model for studying cardiac ion channel regulation at the molecular and functional level. Methods: P19 cells were induced to differentiate towards cardiomyocytes. mRNAs for cardiac markers and ion channels were determined by RT-PCR at six timepoints during the differentiation process. Action potentials and individual ion currents were measured by whole cell patch clamp. Results: Ion channel mRNA expression of several channels is temporally regulated during differentiation, while others show little or no regulation. L-type calcium and transient outward channels are expressed from very early on, while sodium and delayed and inward rectifier channels are upregulated at somewhat later stages during differentiation, which mirrors the in vivo murine cardiomyocyte differentiation during embryogenesis. Spontaneous cardiomyocyte action potentials exhibit a low upstroke velocity, which often can be enhanced by hyperpolarizing the cells, hence activating thusfar dormant ion channels to contribute to the action potential upstroke. Action potential duration decreases considerably during the differentiation of spontaneously beating cells. In late stages, non-beating myocytes can be found which only generate action potentials upon electrical stimulation. Their shape is comparable to neonatal/juvenile ventricular mouse myocytes in culture. Finally, we show that P19-derived cardiomyocytes display a very complete set of functional ion channels. Conclusion: P19 cells represent a powerful model to study the regulation of myocardial electrophysiological differentiation at the molecular and functional level.

KEYWORDS Cell culture; Developmental biology; Gene expression; Ion channels; Membrane currents


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 Acknowledgments
 References
 
Cardiac myocytes generate action potentials (APs) to elicit cellular contraction. APs are produced by coordinated action of voltage operated ion channels. In adult ventricular myocytes, diastolic membrane potential is kept constant by the inward rectifier potassium current IK1. The AP upstroke in these cells results from transient activation of inward sodium and calcium currents, directly followed by the activation of the repolarizing transient outward potassium current ITO. Subsequent activation of delayed rectifier channels, and eventually the inward rectifier repolarize the cell again to the resting diastolic membrane potential [1].

The core of an ion channel is formed by a main ({alpha})-subunit which comprises the actual pore through the plasma membrane. Additional subunits of the ion channel contribute to the further tuning of its gating and ion selectivity [1,2]. Numerous studies have focused on ion channel characteristics at protein level in isolated neonatal and adult cardiomyocytes of various species. Study of the molecular regulation of ion channel expression in cardiomyocytes has gained less emphasis so far [2] because of the lack of versatile cardiac cell lines. P19 embryonal carcinoma (EC) stem cells are derived from an induced murine teratocarcinoma [3] and they can differentiate into cardiomyocytes in vitro. During this process, cells progress through a sequence of phases that is to a large extent identical to in vivo cardiomyocyte differentiation during embryonic maturation [4].

P19 EC cells have been used successfully to unravel genetic pathways involved in cardiomyocyte differentiation (for reviews, see Refs. [5,6]). Due to its susceptibility to incorporate and express ectopic genes, easy cell culture conditions and differentiation in large quantities, the P19 model system provides some important advantages over mouse ES (mES) cells [7]. Thusfar however, electrophysiological characterization of P19 derived cardiomyocytes has received little attention [8–11]. A study on the onset and maturation of electrophysiological activity of P19 derived cardiomyocytes is a prerequisite for future studies on the regulation of electrical differentiation.

In the present study we investigate the electrophysiological phenotype of cardiomyocytes evolving during differentiation of P19 EC cells. We have focused on ion channel expression at mRNA level in combination with the functional properties. We demonstrate the sequence of functional ion channel expression during cardiomyocyte differentiation, resulting in early nodal-like APs to late ventricular-like APs. Finally, we demonstrate the presence of a complete set of functional ion channels in P19 derived cardiomyocytes.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 Acknowledgments
 References
 
2.1 P19 cell culture and differentiation
P19 cells are amenable for subcloning resulting in more specialized clones [12]. We aimed at isolating clones displaying cardiac differentiation without dimethylsulfoxide (DMSO) to circumvent any direct influence of DMSO on ion channel expression. Therefore, P19S18 cells [13] were transfected with the neomycin resistance gene to facilitate subsequent subcloning. Following G418 (neomycin analog, Sigma) selection for 3 weeks (400 µg/ml), 13 individual clones were isolated of which three were tested positive for differentiation into beating muscle without DMSO. P19-ND (no DMSO) gave the highest efficiency of cardiac differentiation and was used for subsequent experiments.

P19S18 and P19-ND cells were maintained in DMEM–F12 (DF) (1:1) (Gibco, Breda, The Netherlands) containing 10% fetal calf serum (Gibco), 2 mM L-glutamine, 50 U/ml penicillin and 50 µg/ml streptomycin. Three serum batches were tested for the highest efficiency for differentiation of P19 cells. The resulting serum batch (40A0196K) was exclusively used for all differentiation assays.

Differentiation protocol: day 0, spontaneous aggregation of P19 cells was accomplished by transferring the cells into 10-cm bacterial dishes containing a thin layer of 0.5% agar/DF medium to prevent adhesion, in a concentration of 1.106 cells/ml in a volume of 9 ml culture medium in the presence of 1% DMSO (P19S18) or without DMSO (P19-ND). After 1 day, 7 ml of fresh medium was added and on day 2, 7 ml medium was removed and replaced by fresh medium. Day 4, aggregates (embryoid bodies) were transferred to 6-cm tissue culture dishes and allowed to adhere. Medium was refreshed every second day. Beating cardiac myocytes were first observed at day 10, and clusters of beating myocytes could be isolated from day 14 onwards. Beating cell clusters were excised, collected in Hanks saline solution without Ca2+ and Mg2+, and dissociated in the same buffer supplemented with 0.13% trypsin and 20 µg/ml DNAse (Roche, Mannheim, Germany) at 37°C. After 25 min, dissociated cells were collected by centrifugation, suspended in DF medium containing differentiation serum (40A0196K), and replated on 0.1% gelatin coated coverslips or 3.5 cm dishes for immunofluorescence and electrophysiology, respectively.

2.2 Immunofluorescence
To establish cardiac differentiation, cells were labeled essentially as described before [14]. The following antibodies were used: {alpha}-actinin (clone EA-53, Sigma), MLC2v (clone F109.3E1, Alexis, San Diego, CA, USA), Troponin-T (clone JLT-12, Sigma), Connexin43 (Clone 2, Transduction Labs., Lexington, KY, USA) as primary and Texas Red or fluorescin isothiocyanate (FITC) conjugated donkey anti-mouse (Jackson Immunoresearch, West Grove, PA, USA) as secondary antibody.

2.3 Semi-quantitative reverse transcription polymerase chain reaction (RT-PCR)
RNA was isolated using Trizol (Gibco) and reverse transcribed using M-MLV-RT (Gibco). Primers, annealing temperature, product size and number of PCR cycles are depicted in Table 1, including references for primers described previously. Primers for β-tubulin, {alpha}1c, SCN5A and KvLQT1 were designed using VectorNTI software (InforMax, North Bethesda, MD, USA). Products were analyzed in 1.5% agarose, ethidium bromide stained gels. β-Tubulin was used as an internal standard.


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  		Table 1 PCR primer pairs

 
2.4 Electrophysiological recordings
The standard whole-cell patch-clamp method was used for recording membrane potential and currents. Patch pipette resistances were 3–5 M{Omega}; after filling with the pipette solution: 120 mM potassium gluconate, 10 mM KCl, 5 mM 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid (HEPES), 5 mM EGTA, 2 mM MgCl2, 4 mM Na2ATP and 0.6 mM CaCl2, (pH 7.2, pCa 8.0). During recordings cultures were superfused with a salt solution consisting of: 143 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl2, 0.5 mM MgCl2, 0.25 mM NaH2PO4, 5 mM HEPES and 5.6 mM glucose, pH adjusted to 7.4 with NaOH. The bath temperature was kept constant at 35–36 °C. Voltage and current signals were recorded using a custom-built amplifier (lowpass filtered at 5 kHz) and stored on a MacIntosh computer using custom made acquisition software (Scope) and analyzed offline with custom made software (MacDaq), kindly provided by Drs. Jan Zegers and Antoni van Ginneken (Academic Medical Centre, University of Amsterdam, Amsterdam, The Netherlands).


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 Acknowledgments
 References
 
3.1 Cardiac differentiation of P19 cells
To analyze the cardiac differentiation of the P19-ND clone, cells were allowed to form embryoid bodies for 4 days, and subsequently plated to tissue culture dishes (Fig. 1A). Ten days after the start of aggregation, contracting cells were observed in the cultures. When observed with a phase contrast microscope, undifferentiated cells are relatively small with a high nucleus to cytoplasm ratio (Fig. 1B), a common characteristic of embryonal stem cells. Dissociated and replated beating myocytes displayed a denser cytoplasmic appearance combined with a bright halo around the cells (Fig. 1C), in contrast to non-myocytes. This characteristic appearance remained in later stages (not shown). To confirm cardiac differentiation, beating cell clusters were dissociated between days 15 and 17, cultured on coverslips and subsequently stained for cardiac muscle markers on days 17–24. Cells expressed the muscle marker {alpha}-actinin, regularly seen in a striated pattern due to its subcellular localization in Z-bands (Fig. 1D). A broader (I and A bands) striated pattern was often observed with f-actin staining (not shown). Cells expressed cardiac markers MLC2v (Fig. 1F), striated in A-bands, and Troponin-T (Fig. 1G). Finally, a proportion of the {alpha}-actinin positive cells also expressed {alpha}MHC (<10%) (not shown) and the gap junction protein Connexin43 (25–30%) (Fig. 1E).


Figure 1
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  		Fig. 1 Cardiac differentiation of P19-ND. (A) P19 differentiation and experimental timeframe. Suspended P19 cells were allowed to aggregate at day 0. Resulting embryoid bodies were plated at day 4. First beating cells appear at day 10 in aggregate outgrowths. Samples for RT-PCR (see Figs. 2 and 3Go) were taken at the indicated time points (arrows). Dissociation of beating cell clusters was performed on day 14 or 18. Immunofluorescence (IF) (see Fig. 1) was performed on dissociated cell cultures between days 16–24. Electrophysiology (EP) (see Figs. 4–8GoGoGoGo) was performed on dissociated cells in two time periods, days 16–18 and days 21–25. (B) Phase contrast image of undifferentiated P19-ND cells. (C) Phase contrast image of a dissociated day 18 beating cardiomyocyte. (D) {alpha}-Actinin IF staining in dissociated myocyte on day 22 displays striated staining. (E) Connexin43 IF staining in dissociated myocyte on day 22 (co-staining of panel D) displays gap-junctional plaques between the two cells, and intracellular staining. (F) MLC2v IF staining in dissociated myocyte on day 17 displays fibrillar and striated staining. (G) Troponin-T IF staining in dissociated myocytes on day 24 displays mostly fibrillar staining and some striated staining at the thick edges of the cell. Bar, 20 µm.

 
To determine and compare the time frame of cardiac marker expression in P19S18 and P19-ND cells, total RNA was isolated at the indicated timepoints from whole cultures (days 0, 4, 8 and 12) and from excised clusters of beating myocytes (days 16 and 21) and further processed for semi-quantitative RT-PCR (Fig. 2). MLC2v mRNA expression could be detected in undifferentiated P19S18 cells. Increased expression levels could be observed at day 8 in P19S18 and P19-ND cells. Cardiac actin is expressed in the undifferentiated state of both cell types and became upregulated during the cardiac differentiation. Expression of other forms of muscle actin (smooth and skeletal muscle actin) do not match with cardiac actin in either cell type. Thus, P19S18 and P19-ND cells can be differentiated into cardiac myocytes.


Figure 2
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  		Fig. 2 Cardiac differentiation of P19S18 and P19-ND cells as revealed by semi-quantitative RT-PCR. Undifferentiated cells (0 days) were allowed to aggregate for 4 days (4) and subsequently plated. On days 8 and 12 samples were made from whole cultures (8, 12), on days 16 and 21, beating clusters were excised and used to extract RNA (16*, 21*). RNA was isolated, DNAse treated and converted to cDNA. Samples were amplified with primers for ventricular myosin light chain 2 (MLC2v), cardiac, skeletal and smooth muscle actin and β-tubulin as input control. No amplification product was found without prior reverse transcription reaction or in water controls (not shown).

 
3.2 Ion channel mRNA expression during cardiac differentiation of P19 cells
To determine the time course of ion channel expression during differentiation of P19S18 and P19-ND cells, we analyzed the mRNA expression levels of the {alpha}-subunits of the main cardiac ion channels. Again, RNA samples were obtained during the course of cardiac differentiation, followed by semi-quantitative RT-PCR for seven different ion channels (Fig. 3). In general, for most ion channels similar expression profiles were obtained from the two cell types. Many ion channel mRNAs were already present in undifferentiated cells (Fig. 3, 0 days), despite the absence of electrical activity (data not shown). mRNAs for inward L-type Ca2+ ({alpha}1c) and delayed rectifier IKr (MERG B variant) channels became upregulated between days 4 and 8 in both cells. ITO channel mRNA (Kv4.3) increased between days 8 and 12 in P19S18 and between days 0 and 4 in P19-ND. Message of the cardiac sodium channel (SCN5A) was increased between days 8 and 12 in both cell types while the mRNA for delayed rectifier IKs (KvLQT1) became slightly upregulated about 4 days later. mRNA for the inward rectifier IK1 (Kir2.1) became upregulated between days 4 and 8 in both cell types while downregulation was observed on day 21 when compared to beating clusters of day 16. HCN1/4 (If) mRNA expression was transiently increased in P19-ND cells between days 8 and 16. Regulation of {alpha}1c and Kv4.3 were very alike in P19-ND cells, which could indicate common upstream signaling pathways leading to their transcriptional activation. No consistent regulation was found of MERG A in either cell type or HCN1/4 in P19S18 cells.


Figure 3
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  		Fig. 3 Semi-quantitative RT-PCR of cardiac ion channel expression in differentiating P19S18 and P19-ND cells. Cell differentiation and sample preparation is identical as described in Fig. 2. Samples were amplified with primers for pacemaker channels (HCN1 and 4), sodium channel (SCN5A), L-type calcium channel ({alpha}1c), transient outward channel (Kv4.3), delayed rectifier IKr (MERG A and MERG B variants) and IKs (KvLQT1) and inward rectifier (Kir2.1). β-Tubulin serves as input control. No amplification product was found without prior reverse transcription reaction or in water controls (not shown).

 
3.3 P19 derived cardiomyocytes display shortening of the action potentials during differentiation
Following cell dissociation, action potentials were recorded from individual cells or small cell groups (up to five cells) between days 16 and 25 of differentiation in order to characterize electrical differentiation of P19-ND derived cardiomyocytes. Mean cell capacitance, measured in single cells, was 73.5±7.7 pF (n = 35 cells; ±S.E.M.). Fig. 4A shows spontaneous action potentials with diastolic depolarization in a day 16 cardiomyocyte. The AP morphology was more or less uniform in day 16–18 cells, and of an early embryonal or nodal-like type (Fig. 4B), while in day 21–25 cells, action potential configurations were more diverse and were nodal-like, atrial-like or ventricular-like in nature (Fig. 4C, D and E). APD50 shortened from 108 ms to 56 ms during differentiation (Table 2), although beating frequency, maximal diastolic potential and upstroke velocity remained similar in these spontaneously active cells (Table 2). Within the population of day 21–25 cells, a small subpopulation did not contract, although these were morphologically indistinguishable from spontaneous beating cells. Such cells were electrically quiescent and had comparatively negative resting membrane potentials suggesting an increased expression of inward rectifier channels. Moreover, they could be stimulated to generate murine ventricular-like action potentials (Fig. 4F) with increased upstroke velocities and substantially decreased APD50 (Table 2) when compared to spontaneously beating cells, suggesting the activation of sodium channels due to their negative membrane potential.


Figure 4
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  		Fig. 4 Action potential shapes in P19-ND derived cardiomyocytes. (A–E) Spontaneous action potentials on day 16, day 17, day 22, and day 23 after the initiation of differentiation, respectively. Action potentials are of an nodal-like (A, B, C), early ventricular-like (D) and atrial-like (E) type. (F) Triggered, ventricular-like, action potential in a quiescent cardiomyocyte 25 days after the initiation of differentiation. Note the difference in maximal diastolic membrane potential in spontaneous vs. quiescent cardiomyocytes. Action potentials of panels B–F are plotted on identical time scales.

 

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  		Table 2 P19 derived cardiomyocyte action potential characteristics

 
3.4 Functional ion channel expression in P19-ND derived cardiomyocytes
Already at day 0, mRNAs for the {alpha}-subunits of six ion channels were present in undifferentiated P19-ND cells (Fig. 3). However, virtually no corresponding currents could be measured in whole cell patch clamp experiments at this stage (50 cells, data not shown).

In day 16 cardiomyocytes of P19-ND cells, mRNA for all {alpha}-subunits of the cardiac ion channels investigated could be detected. In isolated spontaneously beating cells, several currents could also be measured from day 16 onwards, although the amplitude of the currents differed considerably between individual cells. Fig. 5A shows the inward L-type calcium current in a day 24 cell induced by applying depolarizing voltage steps form a holding potential of –40 mV, which could be blocked by 600 µM Cd2+ (not shown). IV curves constructed from these measurements (Fig. 5B, left panel) reveal the typical threshold for activation of the L-type calcium current at a voltage between –40 and –30 mV and a peak amplitude at 0 mV. It has been demonstrated before that P19 derived cardiomyocytes are able to express fast inward sodium currents as well [9]. To establish whether our P19-ND cells also expressed fast inward currents when depolarized from more negative membrane potentials, we applied depolarizing test potentials from a holding potential of –80 mV. In about 50% of the spontaneous beating cells, we were able to detect an additional inward current that activates at more negative potentials than L-type calcium current (Fig. 5A, B, right panels). The threshold for activation of the current measured after depolarization from –80 mV was between –70 and –60 mV and peak amplitude occurred between –30 and –40 mV which indicates the presence of either sodium or T-type calcium currents, or both. Regardless of the exact identity of the fast inward current, our data indicate that its contribution to the AP formation is negligible in these spontaneous cells, since the channels are largely inactivated due to the low maximal diastolic membrane potentials.


Figure 5
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  		Fig. 5 Examples of representative recordings, indicate the presence of L-type calcium and fast inward currents on day 24. IV curves of L-type calcium channels (average of 14 recordings), and fast inward channels, presumable sodium and/or T-type calcium channels, (average of seven recordings) of day 21–25 cells. In these experiments, L-type calcium currents were recorded first, followed by fast inward current recordings. Voltage protocol for L-type calcium channels (–40 mV holding potential) and fast inward channels (–80 mV holding potential) are depicted on top.

 
To test the hypothesis that fast inward currents do not contribute to the action potential upstroke due to low diastolic membrane potential, a constant hyperpolarizing current was injected to increase the diastolic membrane potential. Fig. 6 depicts such an experiment. After recording spontaneous action potentials, 325 pA hyperpolarizing current was applied leading to a membrane potential of –75 mV and the disappearance of spontaneous activity. Next, an action potential was elicited by stimulation with a 15 ms/150 pA current pulse. Upstroke velocity was increased while APD50 was not significantly changed in the hyperpolarized/triggered AP compared to the spontaneous APs. In four out of seven cells, the upstroke velocity was increased more than three times (Table 3).


Figure 6
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  		Fig. 6 Representative example of spontaneous (left panel) and subsequently reconstituted (right panel) action potentials in P19-ND derived cardiomyocyte (day 21). To hyperpolarize the cell to quiescence, 325 pA repolarizing current was delivered constantly, followed by a 15 ms long rectangular pulse of 150 pA to elicit an action potential as displayed in the current protocol in the top right panel.

 

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  		Table 3 P19 derived cardiomyocyte reconstituted action potential characteristics

 
Inward currents, activated upon hyperpolarization, were measured to assess the presence of IK1 and If. As depicted in Fig. 7A, most spontaneously beating cells revealed only slight linear inward currents. If-currents could be measured in a few spontaneous active cells (three out of 22) (Fig. 7B). IK1 currents could only be detected in quiescent myocytes (three out of eight) (Fig. 7C).


Figure 7
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  		Fig. 7 Hyperpolarization-induced inward currents in P19-ND derived cardiomyocytes. Three hundred millisecond hyperpolarizing voltage steps between –120 and –40 mV from a holding potential of –40 mV were applied. (A) The majority of cells displayed a slight linear inward current, example from day 23 cell. (B) If currents were rare [three out of 22 measured cells (13.6%)], example from day 22 cell. (C) IK1 currents found only in three out of eight quiescent cells (37.5%), example from day 25 cell.

 
To measure outward potassium currents, 200 ms depolarizing voltage steps between –30 and 70 mV from a holding potential of –40 mV were applied, while 0.6 mM CdCl2 was added to the bathing solution to block L-type calcium currents. In most cells, rapidly activating potassium currents were present. The rate of inactivation and steady state amplitude however, was rather variable. Nevertheless, the majority of cells expressed a current component with activation and inactivation rates that resemble an ITO-like current (Fig. 8A). Fig. 8B displays an example of a delayed rectifier potassium current with no inactivation and a clear tail current. Most cells displayed outward currents which were a mixture of the extremes displayed in Fig. 8A and B. Average peak and steady state current densities of such currents are given in Fig. 8C and D.


Figure 8
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  		Fig. 8 Several types of outward currents are present in P19-ND derived cardiomyocytes. L-type calcium channels were blocked by the addition of 0.6 mM CdCl2. Depolarizing voltage steps between –30 and 70 mV were applied from a holding potential of –40 mV as depicted. (A, B) Rapidly activating currents with different inactivating kinetics, examples from day 24 and day 23 cells, respectively. (C, D) IV curves of peak (closed circles) and steady state (open circles) current densities in ITO-like currents (C), example from day 18 cell, and delayed rectifier tail currents (D), example from day 24 cell.

 

   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
Acknowledgments
 References
 
4.1 mRNA expression of cardiac markers and ion channels during P19 cell differentiation
We have described the electrophysiological differentiation of P19 EC cells at the molecular and functional level. First, we assessed mRNA expression of a number of differentiation markers and of ion channels in undifferentiated cells. MLC2v mRNA was present in undifferentiated P19S18 cells, although no MLC2v protein could be detected by immunofluorescence microscopy (data not shown). Similarly, despite the presence of ion channel mRNAs in undifferentiated cells, no corresponding ion currents could be measured, as noted before with respect to the sodium channel [9]. In most RT-PCR profiles displaying mRNA expression in undifferentiated cells, expression levels decreased during the initial aggregation phase (day 4), but subsequently increased in later phases. In P19-ND cells a gradual increase can be seen in smooth and skeletal muscle actin between day 0 and day 12. Selection of beating clusters on day 16 and day 21 led to an increase in cardiac actin, while the other two actin forms were not enhanced. This could be explained by either co-differentiation of whole cell culture into other directions than cardiac muscle, or by expression of other actin forms in cardiac differentiating cells, as observed also during in vivo cardiac differentiation [15,16].

From day 16 onwards, well defined expression of cardiac protein markers and ion channels was observed.

4.2 Temporal appearance of membrane currents and action potential generation
It has been suggested that early action potentials in mES cells mainly are driven by L-type calcium and ITO currents [17,18]. Our results provide molecular as well as functional evidence for this in P19 cardiomyocytes as well. The message for the {alpha}-subunits of channels underlying these two currents increased almost simultaneously. In P19-ND cells this increase was even obvious before the onset of contraction. The message for delayed rectifiers MERG B and KvLQT1 became upregulated later. Furthermore, the low upstroke velocity of the spontaneously beating cells at days 16–18 points to the absence of a contribution of the sodium current to the action potential. Nevertheless, at this early stage of differentiation, the presence of sodium channels cannot be excluded, since upregulation of SCN5A mRNA shortly followed after that of {alpha}1c and Kv4.3 (Fig. 3). However, due to the relatively low maximal diastolic membrane potential, sodium channels, or other fast inward channels with similar activation kinetics, would remain inactivated.

We noticed a significant decrease in APD50, when comparing day 16–18 and day 21–25 spontaneous action potentials. This may be explained by the enhanced expression of MERG and KvLQT1 channels leading to increased delayed rectifier current, while no significant effect was observed on the maximal diastolic membrane potential.

Voltage clamp experiments confirmed that L-type calcium currents and ITO-like currents were present in all beating myocytes from at least as early as 16 days and onwards. In addition, delayed rectifier currents could be measured in most cells. Based on the data presented in this study, it is not possible to unequivocally attribute these delayed rectifier currents to Kv1.5, MERG or KvLQT-1 {alpha}-subunits. Although KvLQT-1 mRNA is already strongly expressed in day 16 cells, typical slowly activating KvLQT-1 currents were never observed. In addition, the accessory β-subunit MinK became detectable and upregulated during differentiation from day 8 onwards (data not shown). It remains a possibility that no or minor amounts of KvLQT and/or MinK protein is produced, or that the kinetics of the IKs channel are very different in mouse embryonal-like cells, such as our P19-derived cardiomyocytes, compared to isolated adult cells.

When depolarizing from a holding potential of –80 mV, we were able to detect a fast inward current. From its IV characteristics, this current could be attributed to either sodium or T-type calcium channels or both. In an earlier study, evidence for the presence of sodium currents was shown in P19 cells [9] and here we show upregulation of the sodium channel mRNA. However, besides sodium channels, differentiating mouse ES cells [19] and the developing embryonic mouse heart [20] express T-type calcium channels and currents also. Specific follow up studies are required to unequivocally attribute this current to its underlying ion channel(s).

It is of interest that in voltage clamp experiments, appreciable fast inward currents could be measured in spontaneously beating cells with diastolic membrane potentials that render SCN5A sodium or T-type calcium channels inactivated. Furthermore, in day 21–25 cultures a small population of quiescent cardiomyocytes was present in which the more negative membrane potential correlated with the presence of IK1-current. These myocytes generated short action potentials with relatively high upstroke velocities, thus resembling fully differentiated murine ventricular myocytes. This strongly suggests that during differentiation most cells host a more or less complete set of depolarizing and repolarizing ion channels—apart from IK1, which only appears in cells that enter the final stage of differentiation into working myocardial cells. Another intriguing sub-population of spontaneously beating cells is the one that displayed If-currents. These cells might represent precursors of typical nodal cells.

On day 10 the first beating cells could be observed in our cultures. In subsequent days, both the total number as well as the size of the beating clusters increased. This suggests that the onset of cardiac differentiation is not synchronous. This could in part explain the variability in action potential format and current densities. However, clear distinctions could be made between different time frames days 16–18 vs. days 21–25 with respect to APD50 and the presence of quiescent ventricular-like cardiomyocytes. This indicates the occurrence of cardiomyocyte maturation during the differentiation process.

4.3 Comparison of P19 cardiomyocyte electrophysiology with mES and embryonal/fetal cardiomyocytes
Our results are in some aspects different from action potential data as described for differentiating mES cells [17]. Due to the fact that mouse APs consist of a rapid first phase of repolarization in which already 80–90% of the final maximal diastolic membrane potential is reached, followed by a second relatively long final phase of repolarization, we were only able to determine reliable APD50 values, in contrast to Maltsev et al. who managed to determine and describe APD90 values in mES [17]. This complicates a direct comparison of the APDs. Maltsev et al. [17] demonstrated that spontaneous early action potentials of mES derived cardiomyocytes were uniform with an upstroke velocity between 1 and 10 V/s and an APD90 between 70 and 100 ms. Our day 16–18 cells have a similar upstroke but a longer APD since our APD50 is already exceeding the mES APD90 at this stage. In later stages, Maltsev et al. [17] found two types of spontaneous action potentials in mES cells, with an upstroke velocity of 10–50 V/s and an APD90 of 100–200 ms or 2–10 V/s with an APD90 of 70 ms, respectively. We classified our day 21–25 spontaneous action potentials in P19-ND cells as one class with a similar upstroke as day 16–18 cells (10.0 vs. 13.5 V/s), while APD50 decreased significantly (108 vs. 56 ms). In late mES cardiomyocytes, a stable resting membrane potential was found at a more negative membrane potential (–74.5 mV). Triggering of these mES cells revealed upstrokes of 227 and 235 V/s for different action potential formats, respectively, [17], and APD90 values of 124 and 148 ms. In our quiescent late cells, upstroke velocities were also increased, although to a lesser degree (62.5 V/s) which may be attributed to the difference in mean resting membrane potential between mES and P19 cells (–74.5 mV in mES vs. –59.0 mV in our quiescent P19-ND cells). As a result there may be a more pronounced role for fast inward sodium and/or T-type calcium channels in upstroke velocities in mES cells than in quiescent P19-ND-derived cardiomyocytes. Furthermore, the APD50 in our quiescent cells was shortened dramatically (16 ms). Therefore, the main difference between the APs of mES and P19 cells is the strong decrease in APD during differentiation in P19 cells in contrast to mES. Initial upstroke velocities are quite similar in early mES and day 16–18 P19-ND cells but they seem to differ significantly at later stages.

Action potential recordings from mouse embryonic ventricular myocytes demonstrate a shortening of APD during development [21,22]. In our quiescent P19-ND cells, the APD50 is comparable with that of 3 day old neonatal ventricular cells (18 ms). However, the upstroke velocities in the neonatal cells are three-times higher and this aspect of the AP is therefore more comparable to the late mES derived cardiomyocytes as described by Maltsev et al. [17].

Developmental expression of ion current on isolated embryonic cardiomyocytes from timed-pregnant females, demonstrate that L-type calcium currents play a dominant role in early murine cardiomyocytes, while inward sodium currents were increased dramatically just before birth [23]. In the same study, inward rectifier currents were only seen in late stages of murine development. These in vivo data resemble to a certain extent our observation in differentiating P19-ND, where L-type calcium currents are present in virtual all cardiomyocytes, while fast inward channels (such as sodium channels) were less frequently expressed. Moreover, the inward rectifier, responsible for the ability of sodium and/or T-type calcium channel activation due to increasing the resting membrane potential, were observed in some late stage cardiomyocytes only.

In conclusion, P19 cells represent a valuable model system to study cardiac electrophysiological differentiation. It is similar, but not identical, to the mouse ES model system, and mirrors in vivo differentiation to a certain extent. However, these comparisons demonstrate that the resulting electrophysiological phenotype is of a late embryonal phenotype. Molecular analysis of ion channels can be performed on mRNA level, and studying the underlying gene regulation is now in reach, although caution has be taken in extrapolation of mRNA levels to ion currents. In our study, mRNA analyses have been performed on whole cell populations, which thereby excludes the possibility to determine ion channel mRNA expression in individual cells. Future application of single cell based RT-PCR techniques would enable such studies. As shown in this paper, differentiated P19-ND derived cardiomyocyte populations are electrophysiological heterogeneous. One of the future challenges will be to understand and manipulate differentiation into a predefined electrophysiological homogeneous phenotype.

Time for primary review 28 days.


   Acknowledgments
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 Acknowledgments
References
 
We thank Dr. Antoni van Ginneken for careful reading of the manuscript. This study was supported by the Netherlands Science Organization, section Medical Sciences (NWO-MW) grant 902-16-193 (M.vdH., M.vK.), and Japan Heart Foundation & Bayer Yakuhin Research Grant Abroad (Y.T.).


   References
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 Acknowledgments
 References
 

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