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


CONCLUSIONS
P19 cells represent a powerful model to study the regulation of myocardial electrophysiological differentiation at the molecular and functional level.
van der Heyden
Marcel A G
MA
Department of Medical Physiology, University Medical Center Utrecht, P.O. Box 85060, 3508 AB, Utrecht, The Netherlands. m.a.g.vanderheyden@med.uu.nl
van Kempen
Marjan J A
MJ
Tsuji
Yukiomi
Y
Rook
Martin B
MB
Jongsma
Habo J
HJ
Opthof
Tobias
T
D000185:Actinin / Q000032:analysis; D000200:Action Potentials / Q000502:physiology; D000818:Animals; D020746:Calcium Channels, L-Type / Q000235:genetics; D018236:Carcinoma, Embryonal / Q000473:pathology*; D018929:Cell Culture Techniques / Q000379:methods; D002454:Cell Differentiation / Q000502:physiology; D018031:Connexin 43 / Q000032:analysis; D054278:Embryonal Carcinoma Stem Cells; D007473:Ion Channels / Q000502:physiology; D051656:KCNQ Potassium Channels; D051379:Mice; D023421:Models, Animal; D032383:Myocytes, Cardiac / Q000502:physiology*; D062554:NAV1.5 Voltage-Gated Sodium Channel; D014411:Neoplastic Stem Cells / Q000473:pathology*; D015221:Potassium Channels / Q000235:genetics; D024642:Potassium Channels, Voltage-Gated; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015222:Sodium Channels / Q000235:genetics; D020107:Troponin T / Q000032:analysis; D014404:Tubulin / Q000235:genetics; D014407:Tumor Cells, Cultured
D020746:Calcium Channels, L-Type; D018031:Connexin 43; D007473:Ion Channels; D051656:KCNQ Potassium Channels; C419385:L-type calcium channel alpha(1C); D062554:NAV1.5 Voltage-Gated Sodium Channel; D015221:Potassium Channels; D024642:Potassium Channels, Voltage-Gated; C568321:Scn5a protein, mouse; D015222:Sodium Channels; D020107:Troponin T; D014404:Tubulin; D000185:Actinin