Vol.2, No.1, 24-31 (2010)
doi:10.4236/health.2010.21005
SciRes Copyright © 2010 http://www.scirp.org/journal/HEALTH/
Health
Openly accessible at
Applicability of the P19CL6 cells as a model of
cardiomyocytes – a transcriptome analysis
Iraj Khodadadi1, 2, Nick J. Plant2, Vassilis Mersinias3, Alfred E. Thumser2*
1Department of Biochemistry and Nutrition, Hamedan University of Medical Sciences, Hamedan, Iran
2Division of Biochemical Sciences, University of Surrey, Guildford, United Kingdom
3B.S.R.C. “Alexander Fleming”, Varkiza, Greece;
Received 15 November 2009; revised 4 December 2009; accepted 7 December 2009.
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
1. INTRODUCTION
There is a requirement for the development of realistic
cell culture models both for basic research and the de-
velopment of novel therapeutic agents. However, for
several tissues, including heart, no individual cell line has
been successfully validated for these purposes; such a
failure is usually the result of the loss of one or more
specific phenotypic features associated with the target
tissue in the cell line. One approach to mitigate these
issues has been the utilisation of chemically-stimulated
differentiation of stem cells, with the hope that these cell
lines will have a more realistic phenotype than cell lines
derived from fully differentiated tissue. Pluripotent em-
bryonic carcinoma cells have been reported as successful
in vitro models of cardiac differentiation; for example, the
P19 mouse embryonal carcinoma cell-line has been re-
ported to differentiate into an embryonic cardiac-muscle
phenotype in vitro [1] upon the addition of dimethyl
sulfoxide (DMSO) [2-5]. Differentiated P19 cells have
been reported to retain the ability to spontaneously con-
tract and shown to express transcripts in a temporal
manner during culture, suggestive of a cardiac-muscle
phenotype [5-7], and as such these cells have therefore
been extensively used to study cardiac cell physiology
[1,2,5,6,8], although with the caveat that these cells are
embryonic instead. However, in addition to these cardiac-
muscle-specific properties, P19 cells also display pluri-
potent properties and can be differentiated into cells dis-
playing either a skeletal muscle or neural phenotype [1,
3-5]. There has thus been some concern about the ho-
mogeneity of DMSO-differentiated P19 cultures, with a
heterogeneous cell population following differentiation
significantly reducing the utility of these cells as a car-
diac-muscle-specific model: There has thus been much
interest in identifying subclones of P19 cells that more
robustly differentiate into cardiomyocytes. The P19CL6
cell-line, a sub-clone of P19 embryonal cells, has been
reported to efficiently differentiate into beating cardio-
myocytes upon exposure to DMSO under adherent cul-
ture conditions [9] and has been widely used as an in vitro
model of cardiovascular cells [1,2,10-16].
It is clear that the P19CL6 cell line has potential as a
model system for the study of cardiomyocyte develop-
ment and differentiation, and indeed they are currently
used as such. However, full characterisation and valida-
tion is required before they can be used for this purpose
with full confidence. A review of the literature, focusing
on P19CL6 and P19 cell culture conditions, shows that
two separate methods are commonly used, namely ad-
herent and non-adherent culture conditions [1,5,9,10,17].

I. Khodadadi et al. / HEALTH 2 (2010) 24-31
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25
In addition, vitamins and hormones such as adrenaline
have been shown to act as potent inducers of P19 cell
differentiation into cardiomyocytes in addition to the
aforementioned DMSO [1,5,7,9]. In this study we have
characterized the P19CL6 cells in more detail under dif-
ferent culture conditions, focusing in particular on util-
ising microarray methodologies to compare the P19CL6
transcriptome against native cardiac-muscle and skele-
tal-muscle transcriptomes. These investigations represent
the first robust examination of both P19CL6 transcrip-
tome and cardiac phenotype, and demonstrate that the
P19CL6 cell-line displays only a limited cardiomyocyte
phenotype that is dependent on passage conditions. As
such we would advise caution in the use of this cell line as
a ‘complete’ in vitro model of cardiac-muscle cells.
2. MATERIALS AND METHODS
2.1. Materials
Cell culture media and reagents were obtained from In-
vitrogen Corporation (Paisley, U.K.) and Sigma-Aldrich
Company Ltd. (Poole, U.K.). Materials and kits for RNA
extraction, cDNA synthesis and RT-PCR were supplied
by Promega Corporation (Southampton, U.K.), Amer-
sham Biosciences (Chalfont St. Giles, U.K.) and Qiagen
Ltd. (Crawley, U.K.). Corning Life Sciences (Schi-
phol-Rijk, Netherlands) supplied the ProntoPlus mi-
croarray kit. Ambion Ltd. (Huntingdon, U.K.) supplied
mouse heart and embryonic total-RNA, whereas mouse
skeletal muscle and embryonic heart total-RNA were
purchased from Panomics Inc. (Redwood City, U.S.A.)
and Zyagen (San Diego, U.S.A.), respectively.
2.2. Cell culture
P19CL6 cells were purchased at passage 9 from Riken
Cell Bank (Ibaraki, Japan) in growing flask and cultured
in medium containing -MEM (minimal essential media)
supplemented with 10% FBS (foetal bovine serum) and
1% penicillin-streptomycin (10,000 Units/ml and 10
mg/ml, respectively) [9]. To differentiate P19CL6 cells
into cardiomyocytes, cells were plated in 6-well culture
plates (10 cm2) at a density of 2104 cells/cm2 in standard
medium containing 1% DMSO [9]. Cells were cultured
for 15 days at 37C and 5% CO2 with medium refreshed
every second day. For culturing P19CL6 cells under
non-adherent conditions, cells were stimulated to form
aggregates by incubation in bacterial petri dishes (1106
cells/dish; 78 cm2), containing a thin layer of 0.5% agar,
for 4 days with standard media containing 1% DMSO,
before transfer to regular cell culture flasks for the re-
mainder on the incubation period [5]. Cell aggregates
were collected by centrifugation and replated into culture
flasks for 15 days at 37C and 5% CO2 in the presence of
1% DMSO. The H9C2 (2-1) cell-line, a murine cell-line
that expresses a skeletal muscle phenotype, was obtained
from the European Collection of Cell Cultures (ECACC;
Salisbury, U.K.) and cultured under the same adherent
conditions as the P19CL6 cells.
2.3. Determination of mRNA Levels by
Reverse Transcriptase Polymerase
Chain Reaction (RT-PCR) and cDNA
Microarrays
Total RNA was isolated from cells with Trizol reagent, as
per manufacturer's instructions (Invitrogen Corporation,
Paisley, U.K.). Whole heart and skeletal muscle tissues
(upper leg muscle) were dissected from 10 week old male
CD1 wild-type mice (+/+), homogenised in Trizol reagent
using an Ultra-Turrax T8 homogeniser, and total RNA
isolated as per manufacturer’s instructions. Gene-specific
forward and reverse primers used in the one-step RT-PCR
or nested PCR reactions are shown in Table 1. In samples
that did not show detectable cDNA levels after an initial
RT-PCR amplification the PCR products were reampli-
fied by nested PCR [18]. PCR products were separated by
electrophoresis on 2% agarose gels containing ethidium
bromide, and the identity of PCR products verified by
sequencing.
Microarray experiments were designed as
dual-hybridisation optimal interwoven loops
(http://exgen.ma.umist.ac.uk) [19-21]. cDNA was synthe-
sised from purified total- RNA and labelled by a direct
labelling method in the presence of Oligo dT, nucleo-
tide mixture, Cy3-/Cy5- dCTP dyes, and SuperScript-II
Reverse Transcriptase, based on Human Genome Map-
ping Project protocols (http://www.hgmp.mrc.ac.uk/).
Purified Cy3- and Cy5- labelled cDNA samples (40 pmol
each) were mixed in pairs (total volume 40 µl), according
to the experimental plan, and hybridised to microarrays
by incubation in hybridization chambers for 20 hours at
50C, before post-hybridization washing and drying.
Microarray images were acquired using an Affymetrix
428 scanner (Affymetrix, Santa Clara, USA) and ana-
lysed with BlueFuse (BlueGnome Ltd., Cambridge,
U.K.), including quantification of pixel intensities of the
spots and excluding background intensity and artefact
areas on the arrays. The data was filtered by eliminating
low quality array spots, as determined by BlueFuse’s spot
uniformity and circularity measurements (spot uniformity
and circularity >0.5 in at least 50% of the arrays), and
normalised by intensity-dependent per spot and per chip
(LOWESS) normalisation with GeneSpring-7 data
analysis software (Agilent Technologies UK Ltd, Stock-
port, UK).
After Lowess normalisation and filtering of microarray
data linear modelling (LLAMA; Live Linear Analysis of
MicroArray) was performed (http://exgen.ma.umist.ac.uk)
to convert data from the loop experiment into a linear
model and generate differential gene expression estimates

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26
Openly accessible at
(a)
(b)
Figure 1. Determination of the effect of (a) passage number
and (b) adrenaline on the beating characteristic in P19CL6
cells cultured under adherent conditions. (a) P19CL6 cells
(passage 12) were cultured with 0-20 M adrenaline and
pulse rate was recorded on day 15 of incubation. Linear re-
gression analysis showed a correlation of pulse rate with
adrenaline concentration (y = 1.1x + 21.8, R2 = 0.541, P <
0.001) and one-way ANOVA confirmed a significant effect
of adrenaline on pulse rate in P19CL6 cells (P < 0.001). Cells
were cultured in three biological repeats and beating was
recorded in at least six localized areas in the well. a, b, and c
represent groups with a significant difference in average
pulse rate. (b) P19CL6 cells from different passage numbers
(12-16) were cultured in the presence of DMSO and pulse
rate (beats/min) was recorded on day 15 of incubation. Linear
regression analysis shows a significant effect of passage
number on pulse rate (y = -2.58x + 63.3, R2 = 0.486, P <
0.001) and two-way ANOVA confirmed significance differ-
ence between the different passage numbers of P19CL6 cells
(P < 0.01).
between contrast pairs, i.e. Cy3/Cy5 ratios [20,21,33]. The
P19CL6 samples consisted of three biological repeats, with
each sample analyzed on at least three microarray slides
(technical repeats), and these were reduced to a single
sample during the LLAMA analysis. The data reduction
generated by the LLAMA analysis was further evaluated
by PCA (principal component analysis) and PLS (partial
least squares regression) (Simca-P+, Umetrics, New Jersey,
U.S.A.). The PCA analysis generated correlations be-
tween the different samples. In order to simplify the in-
terpretation of the PLS analysis, the P19CL6 sample was
compared to skeletal muscle, adult and embryonic heart
samples, with the H9C2 (2-1) sample removed. The PLS
analysis was used to produce a variable importance (VIP)
list and all genes with a VIP > 1, i.e. having a significant
impact on the difference between the P19CL6 and other
samples, were further analysed with the DAVID software
suite (http://david.abcc.ncifcrf.gov/home.jsp) for func-
tional annotation clustering according to their biological
functions [24].
a, b,
c
a, b
c
b
a
3. RESULTS
a, b
In a series of separate cultures of P19CL6 cells it was
noted that the cultures did not consistently display a
“beating” phenotype, a characteristic that has previously
been used as evidence of the cardiomyocyte properties of
this P19 sub-clone [9].
c
a
b, c
3.1. P19CL6 Cells Exhibit a
Cardiac-Muscle-Like ‘Beating’
Phenotype under Specified Culture
Conditions
A characteristic of the P19CL6 cells is that they display
beating in localised nodes following differentiation in the
presence of DMSO [9]. The cardio-stimulatory chemical
adrenaline elicited a dose-dependent linear increase in the
observed pulse rate with statistically significant increases
observed between 0-20 M adrenaline (Figure 1a).
However, observation of P19CL6 cells over time in cul-
ture (passages 12-22) demonstrated that beating was
consistently only observed between passages 12-16, and
quantification of this beating showed a significant inverse
correlation of the pulse-rate with passage number (Figure
1b). Microscopic analysis of DMSO-differentiated P19-
CL6 cells demonstrated mono-nuclear cells with no evi-
dence of cell fusion; these characteristics were distinct
from the morphological characteristics of skeletal muscle
cells, as typified by the H9C2 (2-1) murine cell-line
(Figure 2), and added further weight to the assertion that
differentiated P19CL6 cells exhibit a cardiac-specific
muscle phenotype [5,10,34]. The microscopic analysis
and response to adrenaline provided an indication of a
cardiac-type phenotype, although the loss of beating with
passage number suggested that the phenotype may not be
robust with regards to culture duration.

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27
(A) (B)
Figure 2. Microscopic examination of P19CL6 and H9C2(2-1)
cells. Cells were cultured on coverslips under adherent condi-
tions in the presence of DMSO and collected on day 15 of
incubation. Cells were fixed using paraformaldehyde and
observed by light microscopy under 40× magnification. (a)
P19CL6 cells; (b) H9C2(2-1) cells.
3.2. The P19CL6 Cell-Line Expresses both
Cardiac- and Skeletal Muscle-Specific
Transcripts
To further examine the reported cardiac properties of the
P19CL6 cell-line, mRNA transcript levels of -MHC, -
MHC, MyoD and myogenin were determined by RT-
PCR followed by nested PCR. The expression of these
gene products are characteristic of cardiac- (-MHC,
-MHC) and skeletal muscle (MyoD, myogenin) tissues
[35-38]. Figure 3a shows that P19CL6 cells expressed
significant levels of -MHC, -MHC, MyoD and myo-
genin transcripts, inconsistent with a cardiac only phe-
notype: To examine if the unexpected expression of
skeletal muscle markers in P19CL6 cells was caused by
the culture conditions we also examined P19CL6 under
non-adherent culturing conditions; under these conditions
we once again observed both cardiac- and skeletal mus-
cle-specific markers (Figure 3b), suggestive of a mixed
cardiac/skeletal-muscle transcriptome in P19CL6 cells
regardless of culture conditons. The identity of the -
MHC, -MHC, MyoD and myogenin transcripts was
confirmed by sequencing of the nested PCR products
(data not shown). The marker transcript specificity was
confirmed using both the H9C2 (2-1) cell-line, which
expresses a skeletal muscle phenotype, and only ex-
pressed MyoD and myogenin transcripts and mouse car-
diac tissue, which only expressed the aforementioned
transcript markers of cardiac phenotype, -MHC and
-MHC (Figure 3c). These data were thus consistent
with P19CL6 cells exhibiting a mixed cardiac/skeletal
muscle phenotype, and therefore a global transcriptome
analysis was under-taken to examine this hypothesis,
comparing P19CL6 cells with the H9C2 (2-1) cell-line
and mouse cardiac and skeletal muscle tissue.
3.3. Characterisation of the P19CL6
Cell-Line by Microarray Analysis
Transcriptomes from three independent P19CL6 cultures
were compared by microarray analysis to the transcript-
GAPDH

-MHC -MHC MyoD
P19CL6
500 bp
Myogenin
400 bp
300 bp
200 bp
H9C2
(2-1)
P19CL6P19CL6 P19CL6P19CL6 H9C2
(2-1)
H9C2
(2-1)
H9C2
(2-1)
H9C2
(2-1)
a b
(a)
GAPDH
500 bp
300 bp
400 bp
-MHC -MHCMyoD Myogenin
(b)
Mouse Heart
GAPDH -MHC -MHC MyoD Myogenin
600
500
400
(c)
Figure 3 Expression of -MHC, -MHC, MyoD and myo-
genin, as detected by nested-PCR, in (a) P19CL6 and
H9C2(2-1) cells, cultured under adherent conditions, (b)
P19CL6 cells cultured under non-adherent conditions, upon
exposure to 1% DMSO, and (c) mouse heart tissue. Cells
were cultured under adherent or non-adherent conditions for
15 days in the presence of 1% DMSO, as described under
Methods. Heart tissue was dissected from 10 weeks old CD1
wild type (+/+) male mice. Total RNA was isolated and
mRNA levels determined by RT-PCR followed nested-PCR.
Theoretical sizes of PCR products are 275 bp or 413 bp
(GAPDH), 302 bp (-MHC), 410 bp (-MHC), 392 bp
(MyoD) and 337 bp (myogenin), as indicated by the arrows.
For Figure 3c the theoretical sizes of PCR products were:
556 bp (GAPDH); 491 bp (-MHC); 587 bp (-MHC); 513
bp (MyoD); and 608 bp (Myogenin), as indicated by arrows.
Data shown of one experiment that is representative of three
separate biological experiments.

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Openly accessible at
tomes of mouse cardiac and skeletal muscle tissue, H9C2
(2-1) cells, and a reference sample, which was a mixture
of cDNA from all the samples in equal proportions. An
interwoven loop design and data reduction by linear
modelling [21-23] was utilized to compare all samples to
the reference sample (Figure 4). The transcriptomes of
all samples were initially analysed by PCA and showed a
low correlation between the P19CL6 cells and other
samples (Table 2, Figure 5). Only the embryonic heart
sample did not differ significantly from the P19CL6 cells
(P>0.05), although even this comparison showed a very
low correlation of 0.007 (Table 2). Subsequent PLS
analysis, excluding the H9C2 (2-1) samples in order to
simplify interpretation, showed that principal component
1 (PC1) accounted for 52% of the total variance and
clearly separated the P19CL6 sample from the heart and
muscle tissue (Figure 5). The loading factors for PC1
showing a variable importance (VIP) of >1.0 represent
those transcripts driving the separation of P19CL6 cells
from the other samples; these were put into a biological
context by ascertaining Gene Ontology (GO) identifiers
that were significantly over-represented in the identified
transcript level changes, using the DAVID bioinformatics
suite [24]. Such over-representation is often indicative of
a significant biological effect in the pathway(s) associated
with the GO identifiers. Several annotation clusters with
an enrichment score of greater than 1.0, i.e. showing
significant enrichment, were identified (502 separate
genes) and the five main Biological Processes that were
identified are shown in Table 3. A more detailed analysis
of the genes identified within the “Regulation of cellular
processes” cluster showed that 57% (total number 146
separate gene products) of the identified mRNA levels
were up-regulated in P19CL6 cells compared to embry-
onic heart tissue, whereas the remainder were down-
regulated (data not shown). The latter tissue was the
primary focus for comparison as P19-derived cardio-
myocytes are embryonic in nature and have previously
been used as a model system for the embryonic heart
[1,6,8]. However, in the original P19CL6 paper this
cell-line was proposed to be a good model system for
adult heart [9].
4. DISCUSSION
The P19CL6 cell-line, a derivative of P19 embryonal
carcinoma cells, is widely used as an in vitro model of
cardiovascular cells [1,2,10,16], and has been shown to
differentiate into a beating phenotype that is reminiscent
of cardiomyocytes upon exposure to DMSO [9]. Data
presented here confirm that differentiated P19CL6 cells
do exhibit some markers of a cardiac phenotype in the
P19CL6 cells: a beating phenotype that is positively
responsive to adrenaline; expression of transcript markers
of cardiac phenotype (-MHC, -MHC); microscopic
3
1
2 3
1
2
Figure 4: Khodadadi et al 2009
2
4 1
3
7
6
3
1
2
5
4
4
7
6
3
1
2
5
4
4
A
C
B
ab
c
Figure 4. Representation of dual-hybridisation optimal in-
terwoven loop design for microarray experiments. Loop de-
signs showing three, four, and seven samples (nodes) that
were compared in three different experiments. The samples
analysed in each experiment were (a) P19CL6 cells, mouse
embryonic heart and mouse adult heart tissue (b) P19CL6
cells, H9C2 (2-1) cells, mouse adult heart and mouse skeletal
muscle tissue (c) P19CL6 cells (two different biological
samples), H9C2 (2-1) cells, mouse embryonic heart tissue,
mouse adult heart tissue, mouse skeletal muscle tissue and a
reference sample, the latter containing cDNA from all the
samples. The samples at the start of the arrows were labelled
with Cy3 and the target samples with Cy5.
-30
-20
-10
0
10
20
30
-30 -20 -100102030
Principal component 2
Principal component 1
-30
-20
-10
0
10
20
30
-30 -20 -100102030
Principal component 2
Principal component 1
P19CL6
Embryonic heart
tissue
Skeletal
muscle tissue
Adult heat
tissue
Figure 5. Analysis of transcriptomic expression data (microar-
rays) by PLS analysis. Gene expression data from microarray
analysis was reduced by linear regression, using the LLAMA
algorithm [19-21]. The data was further analysed by PCA and
subsequently PLS to demonstrate discrimination between the
samples (R2X for principal components 1 and 2 is 0.52 and 0.33,
respectively) and generate a list of variable importance that
could be used in the DAVID analysis.
analysis showing mono-nuclear cells with no evidence of
cell fusion [1,5,8,25]. However, closer examination of
these features raises some concerns, and is suggestive of
an unstable, mixed, cardiac/skeletal muscle phenotype.
We demonstrated that the pulse-rate for beating was
negatively correlated with the passage number of the cells,
with no beating observed after passage 16, suggesting

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Table 1. Sequences of oligonucleotide primers used for PCR.
Oligo name Sequences %GCTmC Application Product
size
GAPDH-F
ATT CAA CGG
CAC AGT CAA
GGC
5259.8
GAPDH-R
CAC ATT GGG
GGT AGG AAC
AC
5559.4
RT-PCR 556 bp
GAPDH-F
AGC TTG TCA
TCA ACG GGA
AGC
52 59.8
GAPDH-R
CTA AGC AGT
TGG TGG TGC
AG
55 59.4
nested PCR275 bp
GAPDH-F
GAC TCC ACT
CAC GGC AAA
TTC
5259.8
GAPDH-R
AGT CTT CTG
GGT GGC AGT
GAT
5259.8
nested PCR413 bp
-MHC-F
ATG GAG CAG
ACC ATC AAG
GAC
5259.8
-MHC-R
TTG TGT ATT
GGC CAC AGC
GAG
5259.8
RT-PCR 491 bp
-MHC-F
AAG AGT GAG
CGG CGC ATC
AA
5559.4
-MHC-R
CTG CTG GAG
AGG TTA TTC
CTC
5259.8
nested PCR302 bp
-MHC-F
CTT CAA CCA
CCA CAT GTT
CGT G
5060.3
-MHC-R
TCA CCC CTG
GAG ACT TTG
TCT
5259.8
RT-PCR 587 bp
-MHC-F
GAG GGC ATT
GAG TGG ACA
TTC
5259.8
-MHC-R
CTT TCT TTG
CCT TGC CTT
TGC C
5060.3
nested PCR412 bp
MyoD-F
AGT GAA TGA
GGC CTT CGA
GAC
5259.8
MyoD-R
CTG GGT TCC
CTG TTC TGT
GT
5559.4
RT-PCR 513 bp
MyoD-F
TAC CCA AGG
TGG AGA TCC
TG
5559.4
MyoD-R
GTG GTG CAT
CTG CCA AAA
GC
5559.4
nested PCR392 bp
MyoG-F
AGC TGT ATG
AGA CAT CCC
CCT
5259.8
MyoG-R
ACG ATG GAC
GTA AGG GAG
TG
5559.4
RT-PCR 608 bp
MyoG-F
ACC AGG AGC
CCC ACT TCT
AT
5559.4
MyoG-R
GCG CAG GAT
CTC CAC TTT
AG
5559.4
nested PCR337 bp
F and R indicate forward and reverse primers, respectively. Tm: an-
nealing temperature (C); bp: base pair.
Table 2. Correlation matrix of mRNA expression levels in
P19CL6 and H9C2 (2-1) cells, and skeletal muscle, adult heart
and embryonic heart tissue samples, as analyzed by microarray
analysis.
Samples H9C2 (2-1)Skeletal
muscle Adult heartEmbryonic heart
P19CL6 -0.058* -0.154** -0.210** 0.007
H9C2 (2-1) -- 0.031 0.175** -0.023
Skeletal muscle-- -- 0.299** -0.024
Adult heart -- -- -- 0.373**
Microarray data was reduced by linear analysis using LLAMA and the
data analyzed by PCA, with the data fitted to 5 components (explaining
100% of the cumulative variance). *P<0.05; **P <0.001.
that the P19CL6 cells are subject to phenotypic drift with
time in culture, and hence may not represent a stable
cardiomyocyte phenotype. In addition, while P19CL6
cells expressed the aforementioned transcript markers of
cardiac phenotype (-MHC, -MHC), they also ex-
pressed transcript markers for a skeletal muscle pheno-
type (MyoD and myogenin), again suggestive of a mixed
cardiac/skeletal muscle phenotype. Whereas examination
of single markers can give an indication of the potential
phenotype for a cell-line they are not necessarily indica-
tive of a fully functioning biological system. For example,
liver cell-lines such as HepG2 have been validated for use
in drug screening as hosts for both reporter genes and
marker transcripts [26,27]; however, in-depth analysis
demonstrates that this validation is simplified, with
HepG2 cells unable to support some genome-based
transcriptional activation and for the transcriptome to be
markedly affected by culture conditions [28,29]. There-
fore whereas low complexity measurements may be
suitable for validation of cell-lines for use in individual
assays, approaches such as transcriptome/proteome
analysis are more appropriate to fully characterize cell-
lines [30]; anchoring this data to known phenotypic
markers then allows an accurate assessment of the ap-
propriateness of any cell-line to the in vivo system they
are supposed to be modelling [31]. Transcriptome analy-
sis of P19CL6 cells demonstrated significant differences
in the transcript profile between these cells and other
samples, including importantly both embryonic and adult
cardiac cells. Gene Ontology over-representation analysis
suggests that these transcripts are linked to biological
pathways associated with cellular metabolism, an inter-
esting observation since it is generally accepted that car-
diac muscle cells have specific metabolic processes that
differentiate embryonic and adult cardiomyocytes, and
also skeletal and cardiac muscle cells.
In conclusion, we have both undertaken physiological
and transcriptome analysis of P19CL6 cells, assessing
their suitability as models of cardiomyocytes for in vitro
experimentation. Our data suggests that whereas the

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SciRes Copyright © 2010 http://www.scirp.org/journal/HEALTH/
30
Openly accessible at
P19CL6 cell-line has some phenotypic similarities to
cardiomyocytes (e.g. the ability to pulse) there exist sig-
nificant differences between these cells and the in vivo
situation. Our observations clearly demonstrate that the
P19CL6 cell line does not maintain a robust cardia-
myocyte phenotype as shown by the loss of cell beating
with time in culture. In addition, transcriptome analysis
clearly shows that even freshly differentiated cells do not
exhibit a clear cardiac or muscle transcript profile, further
questioning the utility of P19CL6 as a model system for
the study of cardiomyocyte physiology.
5. ACKNOWLEDGMENTS
Technical advice and guidance on cDNA microarray work from Dr
Giselda Bucca and Prof. Colin Smith, and Dr George Kass with mi-
croscopy, in the Faculty of Health and Medical Sciences, University of
Surrey are gratefully acknowledged. Mr Ben Routley and Dr Mark
Muldoon (The School of Mathematics, University of Manchester)
provided valuable assistance in the use and interpretation of Live Linear
Analysis of MicroArray (LLAMA) for analysis of microarray data. Mr
Peter Kentish helped with the animal tissue studies. Financial support
for this study was provided by the Iranian Ministry of Health and
Medical Sciences and the Hamedan University of Medical Sciences.
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ABBREVIATIONS
ANOVA: Analysis of variance;
DMSO: Dimethyl sulphoxide;
RT-PCR: Reverse transcriptase polymerase chain reaction;
PCA: Principle components analysis;
PLS: Partial least squares regression;
LLAMA: Live Linear Analysis of MicroArray.