Cardiovascular Research

Cardiovascular Research 2003 58(2):292-302; doi:10.1016/S0008-6363(02)00771-X
© 2003 by European Society of Cardiology

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Copyright © 2003, European Society of Cardiology

Twenty one years of P19 cells: what an embryonal carcinoma cell line taught us about cardiomyocyte differentiation

Marcel A.G. van der Heyden^a,* and Libert H.K. Defize^b

^aDepartment of Medical Physiology, University Medical Center Utrecht, PO Box 85060, 3508 AB Utrecht, The Netherlands
^bHubrecht Laboratory, Netherlands Institute for Developmental Biology, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands

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 25 October 2002

	Abstract

Top Abstract 1 Introduction 2 P19 cell origin... 3 In vitro differentiation... 4 P19 cells in... 5 P19 derived cardiomyocyte... 6 Developmental stage of... 7 Conclusions and future... Acknowledgments References

 
Many different stem cell types have been shown to differentiate into cardiac muscle cells in vitro but P19 embryonal carcinoma (EC) cells were one of the first examples described and have been the most extensively characterized. P19 EC cells, isolated from an experimental embryo-derived teratocarcinoma in mice, are multipotent and can differentiate into cell types of all three germ layers. Because of their capacity to form cardiomyocytes however, they have been used to dissect the role of cardiac-specific transcription factors and upstream signalling pathways in cardiac cell differentiation. Furthermore, they have shed light on unique aspects of cardiac cell physiology during heart cell differentiation, including regulation of the proteins underlying the electrical and contractile systems. Here, we review studies on different subclones of P19 cells, and what they have taught us about cardiac differentiation and physiology.

KEYWORDS Cell culture/isolation; Developmental biology; Gene expression; Signal transduction; Stem cells

	1 Introduction

Top Abstract 1 Introduction 2 P19 cell origin... 3 In vitro differentiation... 4 P19 cells in... 5 P19 derived cardiomyocyte... 6 Developmental stage of... 7 Conclusions and future... Acknowledgments References

 
Modern biological research makes good use of an ancient enemy: cancer cells. The classic example of human HeLa cells illustrates that the use of these aggressive cancer cells that caused the death of a young woman, led to invaluable insights that gave us the opportunity to fight these same aggressive and life threatening cells successfully [1]. By analogy, developmental biology has benefited from the existence of a special class of tumor cell: embryonal carcinoma (EC). These cells have the ability to change phenotype from malignant into non-malignant via the process of cellular differentiation. EC cells are derived from teratocarcinomas. These malignant tumors arise spontaneously in the testes of mice and humans from defective germ cells or can be induced artificially by transplantation of early murine embryos to extrauterine sites. They may contain many kinds of differentiated tissues, even complete teeth, hair and limbs [2–4]. In contrast to benign teratomas, malignant teratocarcinomas also contain populations of undifferentiated stem cells. Once isolated into culture, they grow indefinitely as undifferentiated cell lines [5–7]. Examples of such cells that have been useful in developmental biology are the human Tera-2 EC cells [8,9], and mouse F9 EC cells [10–12] and P19 EC cells [13,14].

In this review, we will focus on the role P19 EC cells have played in our understanding of cardiomyocyte differentiation and the physiology of fully differentiated heart muscle cells. Excellent reviews already exist on neural and skeletal muscle differentiation of P19 EC cells [13–15].

	2 P19 cell origin and subclones

Top Abstract 1 Introduction 2 P19 cell origin... 3 In vitro differentiation... 4 P19 cells in... 5 P19 derived cardiomyocyte... 6 Developmental stage of... 7 Conclusions and future... Acknowledgments References

 
Teratocarcinomas can develop in some mouse strains from early embryos transferred from the uterus into ectopic sites [3,4]. The experiments in which P19 EC cell lines were derived were designed to establish cell lines from female embryos which were heterozygous for X-linked alleles [16]. For this purpose, 7.5-day-old embryos derived from crossing a C3H/He female with males carrying an X-chromosome derived from a feral mouse bearing a number of variant alleles, were transplanted into the testis of an acceptor C3H/He mouse. The undifferentiated P19 EC cells derived from the primary tumor had a euploid male karyotype (40:XY) and grew rapidly in culture without feeder cells. The stem cell nature of the P19 EC cells was subsequently verified by injection into blastocysts of a different strain. P19 EC derived cells were found in tissues of all three germ layers in the resulting chimaeric mice, even when only a single P19 EC cell was injected [17]. This confirmed the multipotent character of the undifferentiated cells.

Early work from the McBurney group [17–19] who derived the line, showed that P19 EC cells are very suitable for isolating clonal sub-lines, with or without prior mutagenesis. Thus, P19 S18 cells, P19 D3 cells which were unable to form muscle and P19 RAC65 cells which are not responsive to retinoic acid and hence do not differentiate into neuronal cell types, were isolated [17–19]. More recently, P19Cl6 was cloned following long term culture under conditions promoting mesoderm differentiation [20]. This line differentiates easily to cardiac muscle. Furthermore, many clonal cell lines have been made by overexpressing genes that drive cellular differentiation into specific lineages (see below). Several cell lines have also been derived from differentiated P19 cell cultures; these include and epithelioid ectodermal-like cell EPI-7, a mesoderm-like cell MES-1, a visceral endoderm-like cell END-2 [21,22] and bipotential mesodermal progenitor cells MR16 and MR322 [23].

	3 In vitro differentiation of P19 cells

Top Abstract 1 Introduction 2 P19 cell origin... 3 In vitro differentiation... 4 P19 cells in... 5 P19 derived cardiomyocyte... 6 Developmental stage of... 7 Conclusions and future... Acknowledgments References

 
Several years before P19 EC cells were established in culture, it was observed that efficient differentiation of EC cells depends on the prior formation of non-adhering aggregates or embryoid bodies, which resemble the inner cell mass of an embryo [5,6]. In embryoid bodies, initial differentiation occurs when the outer cells of the aggregate differentiate into endoderm-like cells that surround an undifferentiated core. In addition, it was observed that hormones, like retinoic acid, induced differentiation of some EC cells, like F9, in monolayer culture, without an aggregation phase [24]. A combination of embryoid body formation and drug application was then used to induce P19 EC cells to differentiate into derivatives of all three germ layers. It was shown that relatively high concentrations of retinoic acid induced the formation of neuronal and glial tissues [25], while 0.5–1% DMSO led to the formation of a wide variety of endodermal and mesodermal tissues of which mononucleated cardiac and multinucleated skeletal muscle attracted the most attention [18,26]. DMSO induced differentiation is not unique for P19 EC cells. For example, DMSO is found as an inducer of differentiation in Friend erythroleukemia (MEL) cells [27], neuroblastoma cells [28], lung cancer cells [29] and recently even in mouse ES cells [30]. Nevertheless, DMSO appears a rather cell line specific inducer, though DMSO elicited signalling pathways may be more general. Application of DMSO in P19 and several other cell types, led to a transient increase in intracellular calcium, released from intracellular stores, that could trigger downstream signalling events crucial for cardiac muscle differentiation [31].

Aggregation is required for DMSO-induced differentiation of P19 EC cells into cardiac myocytes [26]. During the first 4 days, TROMA-1 (an endoderm marker) positive cells formed at the periphery of the aggregates, while prolonged incubation for several days resulted in extensive morphological changes of these cells, which then resembled visceral endoderm cells [32]. Only a minority of the endodermal cells express visceral endoderm markers (e.g. alpha-fetoprotein) and display the true morphological characteristics of visceral endoderm cells, i.e microvilli and pinocytotic vesicles. The molecular events that occur during aggregation and that are necessary for cardiac differentiation are not yet fully understood. Remarkably, many EC cells, such as P19 and F9, but also embryonic stem cells express high levels of the gap-junction protein connexin43 [33–35]. When native P19 EC cells were aggregated with P19 D3 cells, incapable of DMSO-induced muscle formation, the D3 cells remained undifferentiated as expected, while the P19 EC cells differentiated much less efficiently to muscle when compared to homogeneous P19 aggregates. No change in gap-junction formation in D3 cells was seen and it was concluded that DMSO-induced differentiation seemed dependent on cellular interactions, with or without direct exchange of cellular substances, between neighbouring cells [36]. Following aggregation, the percentage of phosphorylated and membrane associated connexin43 increases in F9 and P19 cells [35] (MvdH unpublished observations). Whether this results in enhanced cellular communication and is involved in cardiac differentiation remains to be elucidated for P19 cells. However, in F9 EC cells, aggregation leads to enhanced communication, but this seems not to be crucial for parietal endoderm marker expression and hence differentiation [35]. In addition, cellular aggregation could lead to enhanced cell to cell signaling mediated by adhesion molecules. In P19 cells overexpressing the transcription factor MyoD, it was observed that aggregation led to differentiation into skeletal muscle, without changes in MyoD mRNA or protein levels, suggesting that aggregation resulted in an increased accessibility of MyoD to muscle-specific genes in chromatin [37]. Upstream signalling pathways however were not resolved.

P19Cl6 cells differentiate efficiently to cardiomyocytes upon DMSO-treatment in the absence of prior embryoid body formation [20]. However, the protocol for this differentiation under adherent conditions requires very high cell densities in which cells are packed in multiple layers. Local cell densities are probably not unlike those in embryoid bodies.

Besides DMSO, other factors have been described to induce cardiac differentiation in P19 cells. A co-culture of P19 EC cells with the P19 derived visceral endoderm-like cell line (END-2) induced aggregation and cardiac differentiation, a process which is inhibited by Activin A [38,39]. Butyrate, 6-thioguanine and retinoic acid were shown to induce aggregation-dependent cardiac differentiation in a dose-dependent fashion [40]. The inducing capacity of retinoic acid suggested that transcription factors of the steroid–thyroid–retinoic superfamily could be involved in cardiac differentiation of P19 cells. Experiments showing the ability of 30 nM 3,5,3'-triiodo-l-thyronine (T3) or thyroid hormone to induce cardiomyocytes [41], which is accompanied by the occupancy of specific thyroid receptor response elements in T3-treated P19 cells [42], are in line with this suggestion. Recently, another intriguing hormone was demonstrated to induce cardiomyocyte differentiation in P19 EC cells [43]. This hormone, oxytocin, was long thought to be primarily involved in uterine contraction upon parturition, milk ejection during lactation and regulating ovulation. However, oxytocin turned out to be involved in many more processes including body fluid and cardiovascular homeostasis, cell growth and differentiation (reviewed in Ref. [44]). In aggregated P19 cells, oxytocin induces beating muscle formation 4 days earlier than DMSO. Furthermore, an oxytocin antagonist not only blocks oxytocin induced cardiac myocyte differentiation, but also DMSO-induced differentiation, suggesting that DMSO acts via the oxytocin pathway. Interestingly, the oxytocin receptor has been shown to become upregulated in butyrate induced differentiation of a salivary adenocarcinoma clone [45]. Finally, oxytocin expression is responsive to retinoic acid and thyroid hormone (T3) [46,47] and therefore might be downstream of retinoic acid and T3-induced P19 cardiac differentiation as well.

	4 P19 cells in deciphering cardiac differentiation pathways

Top Abstract 1 Introduction 2 P19 cell origin... 3 In vitro differentiation... 4 P19 cells in... 5 P19 derived cardiomyocyte... 6 Developmental stage of... 7 Conclusions and future... Acknowledgments References

 
4.1 Transcription factors
The ability to generate clonal cell lines efficiently in combination with high susceptibility for ectopic gene incorporation and expression, make P19 cells an attractive system to study genetic pathways of cardiac differentiation.

The cardiac specific transcription factor GATA-4 was exclusively expressed in the cardiac lineages of differentiating P19 cells, and near maximal expression was found after 3 days of DMSO treatment [48]. This expression preceded the upregulation of putative GATA-4 target genes such as the cardiac muscle markers BNP, cTpC and MHC. To investigate the requirement for GATA-4 in cardiac differentiation, antisense GATA-4 RNA was stably expressed in P19 EC cells [48]. This had no effect on undifferentiated growth or RA-induced neuronal differentiation. However, DMSO-induced P19 aggregates spread poorly after aggregation, cultures contained many undifferentiated cells and apoptosis was observed by day 5 of differentiation [48,49]. Most importantly, neither beating muscle nor cardiac marker expression was found, in contrast to control cells. Endoderm and early mesoderm were formed normally as indicated by the specific markers cytokeratin55, brachyury T and goosecoid, respectively, indicating no requirement for GATA-4 in very early differentiation. After analysis of early cardiac marker expression (MEF2c, Msx-1, Nkx2.5, MHox, Msx-2 and MLP) it was concluded that antisense GATA-4 P19 clones were unable to differentiate beyond the precardiac, or cardioblast stage [49]. GATA-4 overexpression (P19[GATA-4]) on the other hand, increased cardiac differentiation even without DMSO treatment, although it was still dependent on aggregation. The combination of GATA-4 and DMSO potentiated differentiation in time and degree [49].

As GATA-4, the transcription factors Nkx2.5 and MEF2c, which are upregulated after GATA-4 [49], also induced beating muscle in stably transfected P19 cells independently of DMSO when simply aggregated [50]. Upregulation of MEF2c and GATA-4 was observed in differentiating P19[Nkx2.5] cell cultures and vice versa; Nkx2.5 and GATA-4 were enhanced in differentiating P19[MEF2c] cells [50]. Like GATA-4, Nkx2.5 is essential for cardiac differentiation of P19 cells as demonstrated by the introduction of a dominant negative version of Nkx2.5 (P19[Nkx/EnR]) [51]. Cells remain arrested in the precardiac stage as upregulation of GATA-4 and MEF2c was not observed, while mesoderm markers such as brachyury T and Wnt5b remained unaffected. Specificity for the cardiac pathway was demonstrated by the observation that skeletal muscle differentiation occurred normally in P19[Nkx/EnR] cells.

GATA-4, Nkx2.5 and MEF2c thus most likely form a self amplifying genetic system leading to cardiac commitment and differentiation.

The T-box transcription factor Tbx5 associates with Nkx2.5, and mutations in Tbx5 led to limb and heart malformations in the Holt–Oram syndrome [52,53]. Tbx5 and Nkx2.5 complexes induced transcription of the cardiac specific natriuretic peptide precursor type a (Nppa) gene synergistically [54]. P19Cl6 was used to investigate the functional consequence of Tbx5 activity in cardiac differentiation. It was shown that Tbx5 overexpression resulted in enhanced cardiac differentiation, while cell lines expressing a mutated form of Tbx5 were unable to differentiate into beating cardiomyocytes [54].

To find genes involved in the very early phases of cardiac differentiation, P19Cl6 cells were used to perform a differential display analysis. This resulted in the cloning of Midori (myocyte induction/differentiator originator) [55]. Midori is a nuclearly localized protein and appears to be a muscle specific transcription factor. Antisense Midori sequences inhibited DMSO-induced cardiac differentiation of P19Cl6, while overexpression of functional Midori resulted in increased endogenous Midori levels and in more efficient cardiac differentiation [55]. Whether Midori is an upstream inducer of the GATA-4, Nkx2.5, MEF2c network, or is just an extra player, remains to be determined.

4.2 Signalling pathways
In recent years, efforts have been made to understand the regulation of the expression of the cardiac specific transcription factors described above. P19 EC cells have been used in some of these analyses, and were instrumental in elucidating several of the signal transduction pathways leading to cardiac differentiation.

Involvement of bone morphogenetic proteins (BMPs) in cardiac differentiation was strongly suggested by the observation that BMP soaked beads elicit ectopic expression of Nkx2.5 and GATA-4 in chick embryos [56]. Application of noggin, a natural inhibitor of BMPs, antagonized cardiac differentiation. BMP-2 and BMP-4 were detected in undifferentiated P19Cl6 cells, and remained expressed during their DMSO-induced differentiation [57]. This suggested that DMSO is not involved in the induction of BMP expression. Overexpression of noggin in P19Cl6 cells (P19Cl6[noggin]) prevented DMSO-induced cardiac differentiation as determined by absence of spontaneously beating cells, cardiac transcription factor and contractile protein gene expression [57]. This could be rescued by overexpression of BMP-2 or simply by addition of BMP protein to the culture medium. Therefore, BMP signalling is essential for P19 cardiac differentiation.

BMP signalling occurs via at least two pathways. First, the MAPKKK pathway. The MAPKK-kinase TAK1 is expressed in P19Cl6 cells and therefore could act in the signalling pathway. Overexpression of dominant negative TAK1 led to an inhibition of cardiac muscle formation in P19Cl6 while overexpression of wildtype TAK1 or constitutively active TAK1 rescued cardiac differentiation in P19Cl6[noggin] cells, although it still required the presence of DMSO [57]. Constitutively active raf-1, another MAPKKK member, was unable to rescue cardiac differentiation in P19Cl6[noggin] cells, emphasizing the specificity of TAK1. The direct downstream effector of TAK1 remains to be elucidated. Data on the role of MAPK pathways in cardiac differentiation of P19 cells, provided strong evidence that p38 MAPK is absolutely required for differentiation, while ERK1/2 only partly contributes to the differentiation pathway [58,59]. One way in which p38 might exert its function is by activating the transcription factors MEF2c [60,61] and ATF-2 [62], the latter of which is essential in P19 cardiomyocyte differentiation [63]. Another known target for p38 MAPK is the heat shock protein 25 (HSP25). During cardiac differentiation, HSP25 expression in strongly increased from day 6 onward. Its expression is essential for the differentiation process as shown by antisense overexpression, although the mechanism by which it regulates cardiac differentiation is thus far unknown [58]. Moreover, the critical time period for p38 activity in cardiac differentiation does not overlap with HSP25 expression [58].

The other major BMP signalling pathway operates via the activation of Smad1, Smad5 and Smad8 and consecutive association to the co-activator Smad4 [64]. Overexpression of Smad1 and Smad4 was sufficient to rescue cardiac differentiation in DMSO-induced P19Cl6[noggin] cells, and further application of exogenous BMPs increased the differentiation efficiency [63]. To support further the involvement of the Smad pathway, the inhibitory Smad6 was overexpressed. Smad6 blocks BMP signalling by preventing interaction and phosphorylation of the receptor Smads 1, 5 and 8 by the activated receptors [64]. The resulting P19Cl6[Smad6] cells were unable to differentiate into cardiac muscle upon DMSO induction [63].

Both BMP pathways act in parallel as demonstrated in experiments where dnTAK1 inhibits Smad1/4 rescue and Smad6 inhibits caTAK1 rescue of P19Cl6[noggin] cells, respectively. In addition, both pathways probably culminate in activation of the transcription factor ATF-2 that is a target for Smad and TAK1 pathways in TGF-β signalling [65]. In fact, ATF-2 was upregulated at the mRNA level during cardiomyocyte differentiation of P19Cl6 cells, and an increase in the activated form of ATF-2 protein was found. Finally, overexpression of dominant negative forms of ATF-2, lacking the regulating SAPK phosphorylation sites, inhibited the differentiation into cardiac myocytes, while wild type ATF-2 potentiates the already observed rescue of Smad1/4 and TAK1 [63].

In another study, it was noted that during cardiac differentiation, AP1 activity was increased. AP1 complexes were formed by c-Jun, JunD and Fra-2, while involvement of ATF-2 was not found [59]. In this study a dominant negative c-Jun (c-JunbZIP) inhibited cardiac differentiation, probably in a non-autonomous way. Although not detected in the AP1 complex, ATF-2 was able to enhance cardiac differentiation when overexpressed, in accordance to the data of Monzen et al. [63]. Overexpression of c-Jun too, led to cardiac differentiation [59].

Independent experiments in P19 cells also indicated the requirement for BMP-4 signalling [66]. P19[noggin] cells failed to differentiate into cardiac myocytes upon aggregation and DMSO induction. When co-aggregates were made of P19[noggin] and P19[Nkx2.5] cells, no cardiac myocytes were formed, while P19[Nkx2.5] aggregated alone or with control P19 cells do form beating muscle, even in the absence of DMSO. Apparently, aggregation of P19[Nkx2.5] cells resulted in effective BMP signalling required for proper cardiac differentiation. To verify this hypothesis, various concentrations of BMP-4 were added to P19[Nkx2.5] cells in monolayer, this indeed resulted in a concentration dependent cardiac myocyte differentiation. Differentiation was not observed in control P19 cells. Using the aggregation independent P19Cl6, simultaneous overexpression of Nkx2.5 and GATA-4 in P19Cl6[noggin] cells induced cardiac differentiation while differentiation was not observed when the genes were expressed alone [63].

In a recent study by Pandur et al. [67], P19 cells were used to demonstrate the capacity of Wnt11 conditioned medium, collected from NIH-3T3 cells expressing and secreting murine Wnt11, to induce cardiomyocyte differentiation. Wnt11 signals via the non-canonical Wnt signalling pathway, that subsequently activates protein kinase C and Jun N-terminal kinase.

In summary, many signalling pathways involved in cardiac differentiation have been uncovered using P19 cells (Fig. 1). Many questions however remain. To what extent aggregation is involved in all signalling pathways described and what the cellular effectors are, still has to be elucidated. The transcription factor Midori still has to be placed correctly in the pathways. Potential co-operation between the AP1, ATF-2 and GATA-4/MEF2c/Nkx2.5 transcription factors is conceivable. The role of the oxytocin/oxytocin receptor system and its downstream signalling events needs pursuing. In all of these aspects, P19 cells can play a useful role. Ultimately though, it has to be established which of these pathways regulates cardiac differentiation in vivo.

View larger version (26K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 1 Signal transduction cascades involved in cardiac differentiation of P19 embryonal carcinoma cells. See text for details.

 

	5 P19 derived cardiomyocyte cell physiology

Top Abstract 1 Introduction 2 P19 cell origin... 3 In vitro differentiation... 4 P19 cells in... 5 P19 derived cardiomyocyte... 6 Developmental stage of... 7 Conclusions and future... Acknowledgments References

 
During the differentiation of P19 cells to beating cardiomyocytes, many cardiac specific proteins become expressed such as contractile proteins, ion channels and receptors. Recently, the first microarray-based analysis of global changes in gene expression during cardiomyocyte differentiation of P19Cl6 cells was reported [68], showing the upregulation of many expected and unexpected mRNAs, accompanied by a vast number of EST sequences. Anisimov et al. [69] performed a sequential analysis of gene expression (SAGE), which also revealed a number of expected and unexpected changes in gene expression during P19 cardiomyocyte differentiation, that could be important for both differentiation and cell physiology.

During cardiomyocyte differentiation, the lipid composition of the plasma membrane changes, which could be important for tuning the electrical excitability of the cell. Table 1 provides an overview of the reported presence of more or less cardiac specific molecules.

View this table: [in this window] [in a new window]   Table 1 Occurrence of (cardiac-specific) molecules in P19 derived cardiomyocytes

 
5.1 Contraction apparatus
The rhythmic beating of P19 derived cardiac myocytes demonstrates the existence of a fully operational contraction apparatus. Though fully functional, electron microscopy showed the immature nature of the contractile apparatus of the mononucleated cardiac cells [26]. mRNA for cardiac actin, either endogenous or transcribed from stably transfected human genes, was detected when the cells start to beat (day 6). Subsequently, mRNA levels became elevated, followed by a decrease [70,71]. The decrease could be caused by the fact that only about 10% of the cells differentiate into cardiac actin expressing cardiomyocytes with a low proliferation rate [72], which may become overgrown by non cardiac actin expressing cells proliferating faster. On the other hand, the decrease could have reflected a genuine developmental regulation of the genes. The expression profiles of skeletal actin and MLC1v were very similar to that of cardiac actin, while MHC steadily increased from day 6 to 9 and then slightly decreased [71]. Interestingly, the mononucleate cardiac myocytes co-express MLC1v, MLC2a, -MHC and β-MHC [71]. Together, these observations indicate that the cardiomyocytes resemble embryonic cardiomyocytes. Besides cardiac actin and the myosin light and heavy chains, expression of other contractile apparatus associated proteins (cardiac troponin C, desmin) has been shown (Table 1).

5.2 Plasmamembrane composition
An essential property of cardiac myocytes is the generation of action potentials. This involves the action of a number of transmembrane ion channels. Plasmamembrane lipid composition influences the functional properties of ion channels [73–75] and therefore regulation of membrane constitution could be expected to occur during differentiation. Indeed, following induction of cardiac differentiation in P19 cells, the plasma membrane composition is enriched in phosphatidylethanolamine (PE) while a decrease was seen for phosphatidylcholine (PC) [72,76] and thereby the membrane composition becomes very similar to that of native cardiac myocytes (reviewed in [74]). No change was observed in the relative fatty acid content for each phospholipid [72]. The rise in PE results from de novo synthesis and concomitant with the altered membrane composition, also the underlying enzyme function is regulated during the differentiation process. Thus it was found that in P19 cells the activity of PE synthesizing and modifying enzymes ethanolaminephosphotransferase and lysophosphatidylethanolamine-acyltransferase was enhanced [72,76], in accordance with increased biosynthesis. Cholinephosphotransferase and membrane associated phosphocholine cytidylyltransferase activities were increased as well, which together with the decrease in PC levels is indicative for an enhanced PC turnover.

A substantial decrease in the level of diacyl-glycerol has been suggested to be responsible for the translocation of protein kinase C (PKC) from the particulate (membrane) fraction to the cytosolic compartment during cardiac differentiation of P19 cells following DMSO induction [77].

The observation that an increase in PE was observed before the onset of cardiac marker expression, prompted an investigation into the importance of PE change for the differentiation process itself. From these experiments it was concluded that blocking PE synthesis in P19 cells did not affect the differentiation capacity. Moreover, RA induced neural differentiation, also led to an increase in PE levels [76]. Therefore, the change in plasma membrane composition might be a more general aspect of stem cell differentiation, and meet the specific needs of differentiated cells, such as for cardiomyocyte and neuron physiology.

5.3 Electrophysiological properties
Studies on the electrical make-up of the P19 cardiac myocytes are limited. Undifferentiated P19 cells express functional Na⁺/H⁺ exchanger, but its potential regulation during cardiac differentiation has not been studied [78]. Of the inward currents, expression of functional voltage dependent sodium channels has been reported [68,79,80]. While some undifferentiated cells display low inward sodium currents, cardiac differentiation increased TTX sensitive sodium currents in amplitude and frequency in the cell populations. Functional expression of calcium channels was found in differentiated P19 cells but not in undifferentiated cells [68,80,81]. Although sodium channels were present, they did not contribute to the action potential as demonstrated for example by TTX application to beating cardiomyocytes which had no effect on action potential characteristics [80,81]. Finally, the existence of potassium currents was reported [68,80]. Chronotropic effects were found for adrenaline, isoprenaline, clenbuterol, phenylephrine and forskolin indicating the presence of regulatory actions of - and β-adrenoceptors and adenylyl cyclase dependent regulation in beating frequency [26,81]. Differentiation of P19 cells into cardiomyocytes increased basal adenylyl cyclase activity [82]. Furthermore, adenylyl cyclase activity could not be activated in undifferentiated cells by β-adrenoceptor stimulation, while in differentiated cells, -adrenoceptor stimulation led to a decrease in adenylyl cyclase activity, and beta stimulation slightly increased frequency [82]. Remarkably, carbachol caused a slight positive chronotropic effect [81], in contrast to a negative effect in mouse ES derived cardiomyocytes [83,84]. Therefore, in P19, no muscarinic cholinoceptor response was detected in contrast to embryonic and neonatal heart cells [81]. Finally, expression of a nicotinic receptor response was observed [81].

Our recent data reveal evidence that besides L-type calcium and sodium channels, P19 derived cardiomyocytes express functional I_TO, I_Ks, I_Kr, I_K1 and I_f currents and the corresponding ion channel -subunits (SCN5a, 1c, Kv4.3, KvLQT1, mERG, Kir2.1, HCN1 and HCN4) [80]. Furthermore, we find a shortening of the action potential duration during differentiation from early to late cardiomyocytes, and the presence of quiescent, but inducible, cardiac myocytes with ventricular like action potentials upon stimulation [80].

	6 Developmental stage of P19 derived cardiomyocytes

Top Abstract 1 Introduction 2 P19 cell origin... 3 In vitro differentiation... 4 P19 cells in... 5 P19 derived cardiomyocyte... 6 Developmental stage of... 7 Conclusions and future... Acknowledgments References

 
During heart development, cardiomyocytes alter their transcriptional activity, morphology and electrical properties, resulting in stage dependent characteristics [85,86].

In P19 derived cardiomyocytes, the contractile apparatus consists of a mixture of atrial and ventricular light and heavy chains, and in addition to cardiac actin, the skeletal form of actin is expressed, another characteristic of embryonic cardiomyocytes [71]. The contractile proteins are organized in sarcomeres, however not of the robust type found in adult, working myocytes.

P19 derived cardiomyocytes beat spontaneously, they express calcium channels and only limited amounts of functional sodium channels [79–81]. The maximal diastolic potential is rather low (–40 to –60 mV). The action potential shape and characteristics resemble primary isolated embryonic cardiomyocytes, in that they have a relatively long action potential duration with a slow upstroke velocity.

Based on the studies performed thus far on expression of contractile markers, the functional properties of the ion channels and the resulting action potentials, P19 derived cardiomyocytes appear to have an embryonic phenotype.

	7 Conclusions and future directions

Top Abstract 1 Introduction 2 P19 cell origin... 3 In vitro differentiation... 4 P19 cells in... 5 P19 derived cardiomyocyte... 6 Developmental stage of... 7 Conclusions and future... Acknowledgments References

 
To date, P19 EC cells have given us invaluable information on processes involved in cardiac differentiation. The realization that these tumor cells can be turned into a model system to study cardiomyocyte differentiation led to detailed insights into the functional role of cardiac specific transcription factors, signalling pathways and cardiac commitment, regulation of contractile protein expression and functional ion channel expression. No doubt within the next few years, much more information will be obtained using this model system in which bulk biochemical approaches as well as single cell studies can be carried out.

Besides P19 cells, also mouse ES cells can be differentiated efficiently towards cardiomyocytes. Compared to P19 cells, mouse ES cells have a normal karyotype, differentiate DMSO independent to cardiomyocytes, and once genetically manipulated, mouse ES cells can be used easily to generate transgenic animals. This opens the opportunity to combine in vitro and in vivo experiments using the same cells. However, undifferentiated mouse ES cells depend on feeder cells and LIF addition in culture, which makes them less attractive for bulk biochemical approaches or well defined culture conditions. Nevertheless, both model systems have their specific strengths and limitations and the choice for either cell system depends heavily on the topic of investigation.

P19 cells can easily be manipulated to express markers under the control of a heart specific promoter. Such modified cells could be used as an in vitro biological screening system to test the activity of numerous molecular agents and secreted proteins on heart cell differentiation.

As mentioned, the P19 derived cardiac myocytes are of an embryonic nature. This opens the possibility of extending research into the direction of resolving the later steps in heart development and maturation. Like for early differentiation, this process could also depend on specific transcription factors and upstream signalling pathways.

Cocultures between P19 derived (embryonic) cardiomyocytes and isolated adult cardiomyocytes could give insight into processes taking place after cardiomyocyte cell transplantation, envisioned as potential therapy for damaged regions in the human heart following for instance myocardial infarction. The role of direct interaction, electrical coupling and exchange of secreted proteins could be studied in detail using such in vitro cell systems.

The electrophysiological analysis of P19 derived cardiomyocytes is still in its early days. Using this system, which allows a combination of biochemical and single cell approaches, a functional relationship could be made between the presence of (cardiac) specific transcription factors and signalling proteins and the generation of the cardiac action potential resulting from its underlying ion channels.

The premature death of a 7.5-day-old mouse embryo created an immortal cell line that gave us the opportunity to understand the making of one of the icons of biology: the beating heart.

Time for primary review 26 days.

	Acknowledgments

Top Abstract 1 Introduction 2 P19 cell origin... 3 In vitro differentiation... 4 P19 cells in... 5 P19 derived cardiomyocyte... 6 Developmental stage of... 7 Conclusions and future... Acknowledgments References

 
This study is financed in part by NWO-MW grant #902-16-193 (MvdH). We thank Christine Mummery for fruitful discussions and advice.

	References

Top Abstract 1 Introduction 2 P19 cell origin... 3 In vitro differentiation... 4 P19 cells in... 5 P19 derived cardiomyocyte... 6 Developmental stage of... 7 Conclusions and future... Acknowledgments References

 

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