Applicability of the P19CL6 cells as a model of cardiomyocytes – a transcriptome analysis

Iraj Khodadadi, Nick J. Plant, Vassilis Mersinias, Alfred E. Thumser

.

DOI: 10.4236/health.2010.21005                

Abstract

The P19CL6 cell-line, a clone of the P19 mouse embryonal carcinoma cell-line, has been exten-sively used as a model for cardiomyocytes as these cells can be differentiated into a cardio-myocyte phenotype upon incubation with di-methyl sulfoxide. Uniquely, these cells can be observed to “beat” when monitored by mi-croscopy. We started investigating the response of P19CL6 cells to fatty acids, but highly vari-able results lead us to investigate the phenotype of the P19CL6 cells in more depth. In this study we demonstrated that the P19CL6 cells are re-sponsive to adrenaline, but loose the “beating” phenotype after 16 passages in culture. Analysis of specific mRNA transcripts indicated that the P19CL6 cells express both cardiac- and skeletal muscle-specific genes, while global analysis of microarray data showed clear differences be-tween the P19CL6 cells and cardiac tissue of embryonic or adult origin. In conclusion, our observations suggest caution in the use of the P19CL6 cells as a model of cardiomyocytes unless rigorous validation for the intended analysis has been undertaken.

Keywords

Gene expression; Cardiomyocyte; P19CL6 Cell-line

Conflicts of Interest

The authors declare no conflicts of interest.

References

[1]	Van der Heyden, M.A. and Defize, L.H. (2003) Twenty one years of P19 cells: what an embryonal carcinoma cell line taught us about cardiomyocyte differentiation. Car-diovascular Research, 58, 292-302.
[2]	Anisimov, S.V., Tarasov, K.V., Riordon, D., Wobus, A.M., and Boheler, K.R. (2002) SAGE identification of differ-entiation responsive genes in P19 embryonic cells induced to form cardiomyocytes in vitro. Mechanisms of Devel-opment, 117, 25-74.
[3]	McBurney, M.W., Jones-Villeneuve, E.M., Edwards, M.K. and Anderson, P.J. (1982) Control of muscle and neuronal differentiation in a cultured embryonal carcinoma cell line. Nature, 299, 165-167
[4]	McBurney, M.W. (1993) P19 embryonal carcinoma cells. International Journal of Developmental Biology, 37, 135-140.
[5]	Skerjanc, I.S. (1999) Cardiac and skeletal muscle devel-opment in P19 embryonal carcinoma cells. Trends Car-diovascular Medicine, 9, 139-143.
[6]	Skerjanc, I.S., Petropoulos, H., Ridgeway, A.G. and Wil-ton, S. (1998) Myocyte enhancer factor 2C and Nkx2-5 up-regulate each other's expression and initiate cardio-myogenesis in P19 cells. Journal of Biological Chemistry, 273, 34904-34910.
[7]	Wobus, A.M., Kleppisch, T., Maltsev, V. and Hescheler, J. (1994) Cardiomyocyte like cells differentiated in vitro from embryonic carcinoma cells P19 are characterized by functional expression of adrenoceptors and Ca2+ channels. In Vitro Cellular Development and Biology, 30A, 425-434.
[8]	Rudnicki, M.A., Jackowski, G., Saggin, L. and McBurney, M.W. (1990) Actin and myosin expression during development of cardiac muscle from cultured embryonal carcinoma cells. Developmental Biology, 138, 348-358.
[9]	Habara-Ohkubo, A. (1996) Differentiation of beating cardiac muscle cells from a derivative of P19 embryonal carcinoma cells. Cell Structure and Function, 21, 101-110.
[10]	Van der Heyden, M.A., van Kempen, M.J., Tsuji, Y., Rook, M.B., Jongsma, H.J. and Opthof, T. (2003) P19 embryonal carcinoma cells: a suitable model system for cardiac electrophysiological differentiation at the molecular and functional level. Cardiovascular Research, 58, 410-422.
[11]	Eaton, S., Chatziandreou, I., Krywawych, S., Pen, S., Clayton, P.T. and Hussain, K. (2003) Short-chain 3-hydroxyacyl-CoA dehydrogenase deficiency associated with hyperinsulinism: a novel glucose-fatty acid cycle? Biochemical Society Transactions, 31, 1137-1139.
[12]	Monzen, K., Hiroi, Y., Kudoh, S., Akazawa, H., Oka, T., Takimoto, E., Hayashi, D., Hosoda, T., Kawabata, M., Miyazono, K., Ishii, S., Yazaki, Y., Nagai, R. and Komuro, I. (2001) Smads, TAK1, and their common target ATF-2 play a critical role in cardiomyocyte differentiation. Journal of Cell Biology, 153, 687-698.
[13]	Paquin, J., Danalache, B.A., Jankowski, M., McCann, S.M. and Gutkowska, J. (2002) Oxytocin induces differ-entiation of P19 embryonic stem cells to cardiomyocytes. Proceedings of the National Academy of Sciences USA, 99, 9550-9555.
[14]	Peng, C.F., Wei, Y., Levsky, J.M., McDonald, T.V., Childs, G. and Kitsis, R.N. (2002) Microarray analysis of global changes in gene expression during cardiac myocyte dif-ferentiation. Physioogical Genomics, 9, 145-155.
[15]	Ridgeway, A.G., Wilton, S. and Skerjanc, I.S. (2000). Myocyte enhancer factor 2C and myogenin up-regulate each other's expression and induce the development of skeletal muscle in P19 cells. Journal of Biological Chemistry, 275, 41-46.
[16]	Young, D.A., Gavrilov, S., Pennington, C.J., Nuttall, R.K., Edwards, D.R., Kitsis, R.N. and Clark, I.M. (2004) Ex-pression of metalloproteinases and inhibitors in the dif-ferentiation of P19CL6 cells into cardiac myocytes. Bio-chemical and Biophysical Research Communications, 322, 759-765.
[17]	Morley, P. and Whitfield, J.F. (1993) The differentiation inducer, dimethyl sulfoxide, transiently increases the in-tracellular calcium ion concentration in various cell types. Journal of Cellular Physiology, 156, 219-225.
[18]	Newton, C.A. and Graham, A. (1997) PCR. BIOS Scien-tific Publishers Ltd, Oxford.
[19]	Goldsmith, Z.G. and Dhanasekaran, N. (2004) The mi-crorevolution: applications and impacts of microarray technology on molecular biology and medicine. Interna-tional Journal of Molecular Medicine, 13, 483-495.
[20]	Vinciotti, V., Khanin, R., D'Alimonte, D., Liu, X., Cattini, N., Hotchkiss, G., Bucca, G., de Jesus, O., Rasaiyaah, J., Smith, C.P., Kellam, P. and Wit, E. (2005) An experi-mental evaluation of a loop versus a reference design for two-channel microarrays. Bioinformatics, 21, 492-501.
[21]	Wit, E. and McClure, J. (2004) Statistics for microarrays: design, analysis, and inference. John Wiley & Sons Ltd.. Chichester, United Kingdom.
[22]	Dobbin, K. and Simon, R. (2002) Comparison of mi-croarray designs for class comparison and class discovery. Bioinformatics, 18, 1438-1445.
[23]	Simon, R., Radmacher, M.D. and Dobbin, K. (2002) Design of studies using DNA microarrays. Genetic Epidemiology, 23, 21-36.
[24]	Dennis, G., Sherman, B.T., Hosack, D.A., Yang, J., Gao, W., Lane, H.C. and Lempicki, R.A. (2003) DAVID: Da-tabase for Annotation, Visualization, and Integrated Dis-covery. Genome Biology, 4, 3.
[25]	Rudnicki, M.A., Sawtell, N.M., Reuhl, K.R., Berg, R., Craig, J.C., Jardine, K., Lessard, J.L. and McBurney, M.W. (1990) Smooth muscle actin expression during P19 em-bryonal carcinoma differentiation in cell culture. Journal of Cellular Physiology, 142, 89-98.
[26]	El-Sankary, W., Gibson, G.G., Ayrton, A. and Plant, N.J. (2001) Use of a reporter gene assay to predict and rank the potency and efficacy of CYP3A4 inducers. Drug Me-tabolism & Disposition, 29, 1-6.
[27]	Morgan, K.T., Ni, H., Brown, H.R., Yoon, L., Qualls, C.W., Crosby, L.M., Reynolds, R., Gaskill, B., Anderson, S.P., Kepler, T.B., Brainard, T., Liv, N., Easton, M., Merrill, C., Creech, D., Sprenger, D., Conner, G., Johnson, P.R., Fox, T., Sartor, M., Richard,E., Kuruvilla, S., Casey, W. and Benavides, G. (2002) Application of cDNA mi croarray technology to in vitro toxicology and the selec-tion of genes for a real-time RT-PCR based screen for oxidative stress in Hep G2 cells. Toxicologic Pathology, 30, 4
[28]	Morgan, K.T., Casey, W., Easton, M., Creech, D., Ni, H., Yoon, L., Anderson, S., Qualls, C.W., Crosby, L.M., MacPherson, A., Bloomfield, P. and Elston, T.C. (2003) Frequent sampling reveals dynamic responses by the transcriptome to routine media replacement in HepG2 cells. Toxicologic Pathology, 31, 448-461.
[29]	Phillips, A., Hood, S.R., Gibson, G.G. and Plant, N.J. (2005) Impact of transcription factor profile and chromatin conformation on human hepatocyte CYP3A gene expression. Drug Metabolism and Disposition, 33, 233-242.
[30]	Plant, N. (2004) Strategies for using in vitro screens in drug metabolism. Drug Discovery Today, 9, 328-336.
[31]	uo, W., Fan, W., Xie, H., Jing, L., Ricicki, E., Vouros, P., Zhao, L.P. and Zarbl, H. (2005) Phenotypic anchoring of global gene expression profiles induced by N-hydroxy-4 acetylaminobiphenyl and benzo[a]pyrene diol epoxide reveals correlations between expression profiles and mechanism of toxicity. Chemical Research in Toxicology, 18, 619-629.
[32]	[Laderas, T. and McWeeney, S. (2007) Consensus framework for exploring microarray data using multiple clustering methods. OMICS, 11, 116-128
[33]	Smyth, G.K. (2005) Linear Models and Empirical Bayes Methods for Assessing Differential Expression in Mi-croarray Experiments. Statistical Applications in Genetics and Molecular Biology, 3, 1-26.
[34]	Kimes, B.W. and Brandt, B.L. (1976) Properties of a clonal muscle cell line from rat heart. Experimental Cell Research, 98, 367-381.
[35]	Buckingham, M. (2001) Skeletal muscle formation in vertebrates. Current Opinion in Genetics & Development, 11, 440-448.
[36]	Gulick, J., Subramaniam, A., Neumann, J. and Robbins, J. (1991) Isolation and characterization of the mouse cardiac myosin heavy chain genes. Journal of Biological Chem-istry, 266, 9180-9185.
[37]	Metzger, J.M., Lin, W.I., Johnston, R.A., Westfall, M.V. and Samuelson, L.C. (1995) Myosin heavy chain expression in contracting myocytes isolated during embryonic stem cell cardiogenesis. Circulation Research, 76, 710-719.
[38]	Sabourin, L.A. and Rudnicki, M.A. (2000) The molecular regulation of myogenesis. Clinical genetics, 57, 16-25.