From the Departments of Molecular Biology and Internal Medicine,
University of Texas Southwestern Medical Center, Dallas, Texas 75390 and Duke University Medical School,
Durham, North Carolina 27710
Received for publication, December 23, 2002, and in revised form, January 27, 2003
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Studies of cardiac muscle gene expression and
signaling have been hampered by the lack of immortalized cardiomyocyte
cell lines capable of proliferation and irreversible withdrawal from the cell cycle. With the goal of creating such cell lines, we generated
transgenic mice using cardiac-specific cis-regulatory elements from the mouse Nkx2.5 gene to drive the expression
of a simian virus 40 large T-antigen (TAg) gene flanked by sites for
recombination by Cre recombinase. These transgenic mice developed tumors within the ventricular myocardium. Cells isolated from these
tumors expressed cardiac markers and proliferated rapidly during serial
passage in culture, without apparent senescence. However, they were
unable to exit the cell cycle and failed to exhibit morphological
features of terminal differentiation. Introduction of Cre recombinase
to these cardiac cell lines by adenoviral delivery resulted in the
elimination of TAg expression, accompanied by rapid cessation of cell
division, and increase in cell size without an apparent induction of
cellular differentiation. Incubation of cells lacking TAg in
serum-deficient media with various pharmacological agents
(norepinephrine, phenylephrine, or bone morphogenetic
protein-2/4) or constitutively active
calcium/calmodulin-dependent protein kinase I and/or calcineurin
led to the formation of sarcomeres and up-regulation of cardiac genes
involved in excitation-contraction coupling. The combination of TAg
expression under the control of an early cardiac promoter and
Cre-mediated recombination allowed us to derive an immortal cell line
from the ventricular myocardium that could be controllably withdrawn
from the cell cycle. The conditional expression of TAg in this manner
permits propagation and regulated growth termination of cell types that
are otherwise unable to be maintained in cell culture and may
have applications for cardiac repair technologies.
Studies of cardiac development have been hampered by
the lack of immortalized cell lines capable of proliferation and
differentiation. The major obstacle to deriving such cell lines is the
phenomenon of permanent withdrawal of mammalian cardiac muscle cells
from the cell cycle shortly after birth (1, 2). Although a small fraction of adult mammalian cardiomyocytes can re-enter the cell cycle
and replicate DNA upon physiological or pathological stimulation in vivo, there is no significant contribution to cardiac
repair by hyperplasia of cardiac cells following damage
(i.e. myocardial infarction) (3). Neonatal or embryonic
cardiac muscle cells will progress through limited rounds of cell
division in cell culture, but they ultimately withdraw permanently from
the cell cycle, and cultured adult cardiomyocytes will not divide and
eventually die.
There have been several attempts to establish cardiac muscle cell
lines. Cardiomyocytes have been derived from embryonic stem cells (4),
P19 cells (5), and hematopoietic stem cells (6). During the course of
differentiation these cells differentiated into different cell types
and therefore did not represent a homogeneous cell population. To
overcome this problem the cardiomyocytes derived from embryonic stem
cells were subjected to enrichment by using a selectable marker driven
by a cardiac-specific promoter (7, 8). Nonetheless, all of these
approaches failed to provide a sustainable homogeneous cardiac cell line.
Other cell lines, QCE-6 and H9c2, derived from precardiac mesoderm of
quail (9) or embryonic rat myocardium (10), respectively, provided
useful models for studying early cardiac fate specification and cardiac
ion channel function. However, upon induction of differentiation, the
QCE-6 cell line transformed into a mixed population of cells and failed
to differentiate into mature cardiomyocytes. The H9c2 cell line was
shown to express a number of muscle-specific channels but displayed few
muscle structural proteins.
Many investigators have used ectopic expression of oncogenes to
transform embryonic or adult cardiac muscle cells into immortalized cell lines. Rat embryonic ventricular cardiomyocytes infected with
recombinant retrovirus expressing v-myc and
v-H-ras resulted in cells exhibiting some myocyte
characteristics; however, these cells do not form sarcomeres or
contract (11).
Promising results have come from studies utilizing simian virus 40 (SV40) T-antigen (TAg)1 as a
transforming factor in murine and human primary cells (12). Cell lines
derived by expression of TAg in cardiac, skeletal, and smooth muscle
showed rapid proliferation and, in the case of conditional expression,
retained some degree of differentiation (13-20). In particular, the
AT-1 cell line derived from atrial tumors of transgenic mice expressing
SV40 TAg driven by the atrial natriuretic factor promoter was the first
cardiac cell line that was able to maintain contractility in
vitro after being passaged several times (21, 22). The main
drawback of this line is that it must be propagated as ectopic grafts
in syngeneic mice and cannot be passaged in vitro or
frozen. In contrast, the HL-1 cell line derived from the original AT-1
line can be passaged in conventional cell culture and frozen, although
it must be maintained in a proprietary media of unknown composition
supplemented with growth factors and hormones such as insulin, retinoic
acid, and norepinephrine (23). Neither AT-1 nor HL-1 cells become
quiescent by serum deprivation. These drawbacks significantly limit
their use in signal transduction and biochemical studies.
The Nkx2.5 gene encodes a homeodomain transcription factor
that is among the earliest known markers of cardiogenesis in the vertebrate embryo (24). Recently, a cardiac cell line that expresses TAg under the control of the proximal cardiac enhancer of the Nkx2.5 gene was derived (13). This cardiac cell line
exhibited a cardiac embryonic like phenotype. However, it was devoid of striations or contractile properties and unable to exit the cell cycle.
In the present study, we developed immortalized cardiac cell lines from
hearts of transgenic mice carrying cardiac-specific regulatory
sequences from the mouse Nkx2.5 gene to drive the expression of the TAg coding region flanked by loxP sites. We implemented the
Cre-lox system as an efficient method to inactivate genes permanently
(25, 26), allowing for Cre recombinase-mediated deletion of the TAg
gene. We show that these cell lines proliferate rapidly until they are
infected with an adenovirus encoding Cre recombinase, at which time
they cease expressing TAg and exit the cell cycle. Cessation of cell
division in these cells is accompanied by an alteration of cell
morphology, assembly of an organized stress fiber network, and changes
in gene expression characteristic of differentiated myocytes. This
strategy for generating reversibly transformed cell lines may be widely
applicable for the generation of cell lines from a variety of tissues
that are otherwise unable to be maintained in proliferative or
differentiated states in culture.
Generation of Transgenic Mice--
DNA constructs, Nk-TAg (Fig.
1A) and NkL-TAg (Fig. 4A), were used to generate
transgenic mice expressing SV40 large TAg under the control of the
mouse Nkx2.5 gene heart-specific enhancer ( Cell Culture--
Cells were isolated from subendocardial
tumor-like structures in the hearts of transgenic mice as described
previously (28) with some modifications. Briefly, dissected tissues
were minced and dissociated using an enzyme mix containing 0.2%
collagen type II (Worthington) and 0.6 mg/ml pancreatin (Sigma).
Cardiac cells derived from Nk-TAg and NkL-TAg mice were maintained in
Dulbecco's modified Eagle's/F-12 media supplemented with 100 IU/ml
penicillin, 100 µg/ml streptomycin, 2 mM
L-glutamine, and 10% fetal bovine serum (FBS) on plates
coated with 12.5 µg/ml fibronectin and 0.1% gelatin.
Cloning cylinders were used to harvest cell clones. Cell growth curves
were generated by plating 1.5 × 105 or 2 × 105 cells (as indicated) in growth medium onto 100-mm
plates and counting total cell number every 24 h for 5 days.
Differentiation medium contains Dulbecco's modified Eagle's
medium/F-12 supplemented with 100 IU/ml penicillin, 100 µg/ml
streptomycin, 2 mM L-glutamine, 5%
heat-inactivated horse serum, 10 µg/ml insulin, 5.5 µg/ml
transferrin, and 6.7 ng/ml sodium selenite.
Histochemical Analysis--
Heart tissue was fixed in 10%
phosphate-buffered formalin and stained with hematoxylin-eosin. Nk-TAg
and NkL-TAg cells were cultured on coverslips and fixed for 10 min with
either Measurements of Contractility and Calcium Transients in Response
to Electrical Stimulation--
Excitation-contraction coupling
function was assessed as described previously (29, 30) by measuring
cell contractility and calcium transients. Briefly, cells were grown in
35-mm tissue culture dishes for 2-3 days until 70-80% confluent and
loaded with 2 µM fura 2-AM (Molecular Probes) in minimum
Eagle's medium containing 0.5 mM probenecid for 15 min at
37 °C. A platinum electrode ring was placed in the tissue culture
dish. Myocyte contractions and calcium transients were elicited by
field stimulation at 1.5 Hz (Ion Optix) with current pulses of 4-ms
duration and voltages of 40 V. The polarity of the stimulating
electrodes was alternated at every pulse to prevent accumulation of
electrochemical byproducts. Myocyte contractions were imaged and
scanned at a rate of 240 Hz (Ion Optix). Calcium transients were
observed by exciting the fura 2-AM-loaded cells with alternating
wavelengths of 340 and 380 nm and recording the emission intensity at
510 nm. Contraction and calcium transient data for each myocyte were
recorded from a minimum of 12 consecutive stimuli.
Drug Treatment--
Drugs were added into cell culture media as
indicated at the following concentrations: 100 µM
phenylephrine (PE), 10 µM norepinephrine (NE), 10 ng/ml
recombinant human transforming growth factor- Viral Infection--
Cells were infected with recombinant
adenoviruses (Ad) at a multiplicity of infection of 100 for 3-12 h.
The medium was replaced with growth medium, and NkL-TAg cells were
cultured for the indicated times before analyses. Recombinant
adenoviruses were obtained from the following sources: Ad-Cre
recombinase (Ad-Cre) was provided by Dr. Frank Graham (McMaster
University) (25); GATA4 (Ad-GATA4), Nkx2.5 (Ad-Nkx2.5), MEK6 (Ad-MEK6),
and green fluorescent protein (Ad-GFP) were generated using the
"Easy-Track" system as described (31); antisense HDAC4 and
HDAC5 (Ad-HDAC4 or -5), expressing coding regions of HDAC genes in
reverse orientation, were provided by Dr. Chun Zhang; MEF2C (Ad-MEF2C)
was provided by Dr. Rebekka Nicol (HDAC 4/5 and MEF2C viruses were
generated using the pAC-CMV vector); constitutively active calcineurin
(Ad-CnA), IGF-I receptor (Ad-IGFI), constitutively active CaMKI
(Ad-CaMKI), and DNA Synthesis Assay--
NkL-TAg cells were infected with Ad-Cre
virus as described above and cultured in growth medium for 2 more days
followed by incubation with BrdUrd for 2 h. DNA synthesis was
determined based on BrdUrd incorporation using a BrdUrd assay kit
(Roche Molecular Biochemicals) according to the manufacturer's instructions.
Western Blot Analysis--
Cells were lysed in 10 mM
Tris-HCl (pH 6.8), 100 mM NaCl, 1% SDS, 1 mM
EDTA, 1% EGTA buffer. Total protein (50 µg) from lysate was resolved
on a 10% SDS-PAGE gel and transferred to a polyvinylidene difluoride
membrane. Membranes were incubated with primary antibodies recognizing
GATA4 (Santa Cruz Biotechnology) and TAg (Santa Cruz Biotechnology) at
1:1000 dilution followed by incubation with secondary antibodies
against rabbit IgG or mouse IgG (1:7500) conjugated to horseradish
peroxidase (Santa Cruz Biotechnology). Signal was detected using ECL
reagent (Amersham Biosciences).
RT-PCR and Northern Blot Analysis--
RNA was isolated from
cells using Trizol Reagent (Invitrogen) and used in Northern blot
analysis with a probe generated from the coding region of Nkx2.5 or
TAg. RT-PCR was performed using the Superscript II kit
(Invitrogen). Primers used for amplification are listed in the
Supplemental Material Table S1.
Microarray Hybridizations--
Microarray analyses for NkL-TAg
cells versus NIH3T3 cells were performed using the InCyte
Genomics Mouse cDNA microchip. Microarray analyses for NkL-TAg
cells infected with Ad-Cre or Ad-lacZ were performed at the University
of Texas Southwestern Core Facility using Affymetrix mouse oligo
microchip (MU74A). NkL-TAg cells were infected either with adenovirus
expressing Cre recombinase (Ad-Cre) or Generation of Nkx2.5-TAg Transgenic Mice with Cardiac
Tumors--
The Nkx2.5 cis-regulatory sequences
consisting of the early cardiac-specific enhancer region ( Isolation of Immortalized Cardiac Cells--
A heart harvested
from a 3.5-week-old (F0) transgenic Nkx2.5-TAg mouse
displaying mild cyanosis exhibited protruding masses in the left
ventricle. These tumors were excised from the myocardium and
dissociated into single cells. The cells were seeded at low density
onto fibronectin/gelatin-coated plates and grown for 10 days. During
this period several well defined cell colonies emerged consisting of
small spindle-shaped cells growing on top of each other. They showed no
contractile activity. Cell colonies were cloned using cloning cylinders
and independently subcultured in 24-well plates. Twenty one individual
colonies were isolated, although only 18 survived subsequent passages.
These cell lines were named Nk-TAg lines.
Following dissociation of the colonies, the plated cells grew as a
monolayer. Different growth rates were observed for individual clones
(Fig. 2A). Upon reaching
confluence the cells continued to proliferate, showing no evidence of
contact inhibition. When cells were plated at 1.5 × 105 cells per 10-cm plate and grown in medium containing
20% FBS, we observed a doubling time of less than 24 h.
Decreasing the serum content to 15% slightly reduced the doubling time
indicating that Nk-TAg cells respond to factors in serum (data not
shown).
Gene Expression Profile of Nk-TAg Cells--
All of the Nk-TAg
cell lines expressed TAg, but expression levels of TAg varied in the
different cell lines (Fig. 2B). A correlation was seen
between the expression level of TAg and growth rate of cells. However,
there was no relation seen between expression levels of TAg and
cardiac-enriched gene expression. The majority of Nk-TAg cell lines
expressed transcripts encoding proteins characteristic of
cardiomyocytes, such as the Nkx2.5, GATA4, and MEF2C transcription factors (Fig. 2B and see Table
I for a list of other genes). However,
many structural proteins that are essential for contraction of cardiac
myocytes, including titin and sodium, calcium-exchanger (Ncx-1), were
not detected even by RT-PCR (Table I).
Immunohistochemistry analysis of Nk-TAg cells showed a low level of
expression of Generation of Conditional TAg-transformed Cardiomyocyte Cell
Line--
Based on the finding that mitomycin C enhanced
Two loxP sites were added to flank the TAg-encoding region in the
Nk-TAg DNA construct (Fig.
4A), and this construct,
NkL-TAg, was used to generate transgenic mice. Dissection of
the hearts of NkL-TAg transgenic mice at 3.5 weeks of age
revealed one mouse with gross cardiomegaly due to multiple ventricular
myocardial tumors similar to those found in Nk-TAg mice (data not
shown, see Fig. 1). Histological analysis of the transgenic heart
revealed a phenotype similar to that observed in the hearts of Nk-TAg
transgenic animals (data not shown).
Cells were dissociated from the tumor regions of the NkL-TAg transgenic
hearts and plated onto fibronectin/gelatin-coated dishes at a low
density. Following 10 days in growth medium containing 10% FBS, the
NkL-TAg cells showed different characteristics than the
Nk-TAg cells. In contrast to the Nk-TAg cells, NkL-TAg cells did not
form well defined colonies and did not grow on top of each other.
Remarkably, some of the plated cells maintained contractile activity
during the initial passages, although this characteristic gradually
disappeared after a few cell passages. Immunohistochemistry confirmed
TAg expression in the NkL-TAg cells and also showed that some cells
expressed NkL-TAg Cells Exit the Cell Cycle following Cre-recombinase
Expression--
NkL-TAg cells grew without apparent senescence even
after 50 serial cell passages. Immunocytochemistry using TAg antibody showed that all NkL-TAg cells expressed TAg protein regardless of the
passage number. Infection of NkL-TAg cells with recombinant adenovirus
expressing Cre recombinase (Ad-Cre) effectively removed the TAg gene
from the cellular genome as confirmed by PCR (data not shown) and
immunocytochemistry using TAg antibody (Fig. 4B). Deletion
of the TAg gene was followed by a dramatic decrease in BrdUrd
incorporation (Fig. 4C) and eventual cessation of cell growth of the NkL-TAg cells (Fig. 4D). In contrast,
infection of NkL-TAg cells with a recombinant adenovirus expressing
Characterization of NkL-TAg Cells before and after Removal of
Tag--
Although NkL-TAg cells showed a gene expression pattern
similar to Nk-TAg cells (Table I), additional cardiac-specific genes were expressed in NkL-TAg cells including myoglobin,
Deletion of the TAg and subsequent withdrawal from the cell
cycle resulted in a dramatic increase in cell surface area (up to
10-fold and dependent on cell density). Immunocytochemistry showed that
NkL-TAg cells depleted of TAg exhibited pronounced stress fibers that
were immunoreactive for smooth muscle actin (Fig. 5, C and
D). After removal of TAg, many of the NkL-TAg cells become
binucleated. However, depletion of TAg had no apparent effect on the
level of expression of selected cardiac-specific genes as tested by
RT-PCR and/or immunocytochemistry (data not shown).
Induction of Differentiation of NkL-TAg Cells by Various
Stimuli--
We surveyed the effect of different drugs, hormones, and
overexpression of signaling proteins in attempts to induce further differentiation of NkL-TAg cells. Differentiation was assessed using
immunocytochemistry with an antibody against sarcomeric NkL-TAg Cells Exhibit Calcium Transients in Response to Electrical
Stimulation--
Calcium current and cell contractility were analyzed
to determine whether NkL-TAg cells were excitable. NkL-TAg cells
expressing TAg showed no response to electrical stimulation. However,
when NkL-TAg cells depleted of TAg and cultured for 7 days in
differentiation medium containing PE were subjected to electrical
stimulation, calcium transients were readily detected in ~30% of the
cells examined (Fig. 7A). This
implies that the sarcoplasmic reticulum of NkL-TAg cells releases
calcium in response to electrical stimulation. Despite this fact, cells
failed to contract. In comparison to freshly isolated adult cardiac
myocytes (Fig. 7B), NkL-TAg cells showed a diminished
response. In addition, calcium transients in NkL-TAg cells were
elicited in response to every other stimulus at the 50-Hz stimulation
frequency, whereas freshly isolated cardiac myocytes responded to each
electrical stimulus. The inability of NkL-TAg cells to respond to every
stimulus may be attributable to delayed restoration of excitability
because intracellular calcium in these cells may return to the basal
level more slowly than in normal adult myocytes.
Microarray Analysis of Nk-TAg and NkL-TAg Cell Lines--
Gene
expression profiles were examined using microarray analysis comparing
RNA transcripts of NkL-TAg cells with NIH/3T3 fibroblasts. Scatter plot
analysis of the microarray results revealed three distinct groups of
genes. The first and largest group contains genes that are equally
represented in both of the cell lines, consisting primarily of
housekeeping genes. The second group contains genes that are
predominantly or exclusively expressed in NIH/3T3 cells and consists of
non-muscle genes. The third group of genes is predominantly or
exclusively expressed in NkL-TAg cells and consists mainly of
cardiac-specific genes, supporting the premise that the NkL-TAg cell
line is a cardiomyocyte cell line (see Supplemental Material, Table
S2). Notably, many of the genes revealed by microarray analysis, such
as those encoding calponin, smooth muscle actin, skeletal actin, and
atrial natriuretic factor, are characteristic of embryonic
cardiomyocytes. Furthermore, analysis of the microarray data revealed
several uncharacterized ESTs expressed in NkL-TAg cells. These ESTs
were shown to be selectively expressed in heart by Northern blot and
in situ hybridization (data not shown).
Microarray analysis was also performed on RNA transcripts isolated from
NkL-TAg cells (grown in differentiation media with PE) expressing or
depleted of TAg. Infection of NkL-TAg cells with Ad-LacZ showed an
up-regulation of 298 genes; 197 of these genes were also up-regulated
in NkL-TAg cells infected with Ad-Cre, and 232 genes were
down-regulated, including 74 genes that were down-regulated in
Ad-Cre-infected cells (Fig.
8A). Further studies were not
done on these genes, viewing them as a cellular response to adenovirus
infection. Deletion of the TAg gene by Cre-recombinase led to
significant alterations of gene expression in NkL-TAg cells; 313 genes
were down-regulated (>2-fold), and 214 genes were up-regulated (>2-fold) (Fig. 8, B and C). Specifically, the
majority of genes down-regulated in TAg-deleted NkL-TAg cells were
involved in cell cycle regulation, including those encoding cyclin A2,
cyclin E2, cyclin B1, cyclin B2, cdc45, cdc46, cdc6, cdc7, Mcm2, cdc25,
and cdk2. In contrast, many of the transcripts that were up-regulated upon deletion of TAg were cardiac-associated genes including those encoding slow/cardiac troponin C; ZASP, a cardiac-specific Z-band protein; skeletal muscle actin; brain natriuretic peptide; fatty acid
transport protein 4; myotonic dystrophy protein kinase; and sarcoglycan, a dystrophin-associated glycoprotein. Expression of some
of these genes was confirmed with immunohistochemistry on NkL-TAg cells
in the absence or presence of Ad-Cre (data not shown).
The results of this study demonstrate that cardiomyocyte cell
lines capable of proliferation and irreversible withdrawal from the
cell cycle can be generated using a conditional cardiac-specific TAg
transgene. Through the use of Cre recombinase delivered via adenoviral
infection, such cell lines can be propagated in culture and rapidly
switched from a proliferative to a terminally quiescent state. This
strategy should have applications for the creation of diverse types of
cell lines, as well as for tissue repair and regeneration.
We used Nkx2.5 regulatory sequences, which are active from
the initial stages of cardiogenesis, to direct the expression of TAg.
The rationale for this strategy was to derive an early cardiac precursor cell line, which upon appropriate stimulus can be induced to
differentiate. Although it is clear that NkL-TAg cells exhibit a
cardiac phenotype, we cannot be certain whether these cells are
arrested at a specific early stage in the cardiac developmental pathway
or whether they progress partially toward a more mature stage of
cardiac development. Our gene expression data as well as the data of
Brunskill et al. (13) support the notion that cells derived
from hearts transformed by the Nkx2.5 promoter-driven TAg tend to express genes present in both embryonic and adult cardiomyocytes.
It is interesting that only a fraction of the cardiomyocytes from
the Nk-TAg transgenic mice appear to be transformed, thus forming
subendocardial tumors and allowing the remainder of the heart to
develop and function normally. We believe the most likely explanation
for this heterogeneity is mosaicism of transgene expression in founder
transgenic mice, because it would seem that embryonic lethality would
result from mice with fully transformed cardiac cells lacking
contractile machinery. Consistent with this notion, we have not
observed transmission of the transgene to the F1 generation. Other
explanations for observed mosaicism include the following: 1)
additional sources of cardiac progenitors from outside the early
heart-forming region in which the Nkx2.5 transgene is expressed; 2) a
"second hit" in cells that form the TAg-transformed tumors; or 3)
an expression gradient of the Nkx2.5 transgene from the endocardium
into the ventricular walls.
Our observation that NkL-TAg cells permanently withdraw from the cell
cycle upon removal of TAg disputes the second hit theory and
implies that expression of TAg is sufficient and necessary for
propagating these cardiac cells. It also indicates that NkL-TAg cells
even after multiple rounds of proliferation retain the cell cycle
arrest mechanism that is characteristic of cardiomyocytes. These
findings as well as increased expression of cell cycle inhibitor genes
(data not shown) indicate that these cells could be used as a reagent
to study cell cycle events in cardiomyocytes.
Expression of TAg is compatible with the expression of many early
cardiac lineage markers but is incompatible with growth arrest and
formation of mature sarcomeres. The use of a "floxed" TAg transgene
circumvents these effects and allows for a rapid and reversible switch
in the proliferative properties of cardiac muscle cells. To our
knowledge, this type of conditional Cre-lox approach has not been
utilized previously to expand and control the differentiation of
specific populations of progenitor cells in vitro. It may be
useful in the future to use similar Cre-lox methods to expand and study
many progenitor or mature cell lines depending on the promoter or
transforming agent applied.
The "floxed" TAg approach proved to be highly efficient and
efficacious for the control of proliferation of the derived NkL-TAg cell lines. However, withdrawal of NkL-TAg cells from the cell cycle
was not sufficient to induce differentiation as assessed by sarcomere
formation. It required cultivation of TAg-depleted NkL-TAg cells in low
serum media with addition of several factors or overexpression of genes
to induce formation of sarcomeres. This was accompanied by dramatic
changes in gene expression as revealed by the microarray data.
Subpopulation of these cells exhibited Ca2+ current upon
electrical stimulation. Although TAg-depleted NkL-TAg cells do not
contract spontaneously or upon electrical stimulation, further studies
may focus on this process, perhaps utilizing different culture strategies.
In summary, we have created cardiomyocyte cell lines using the
Nkx2.5 promoter/enhancer and TAg. These cell lines provide interesting opportunities for the study of cardiomyocytes particularly because the NkL-TAg cell line can be expanded in culture and induced to
a degree of differentiation with expression of Cre recombinase. In
general, the Cre-lox method we have utilized may be useful for the
development of different cell lines capable of proliferation followed
by differentiation. NkL-TAg cells may serve as a useful tool for
studies of cardiac gene regulation and cellular signaling, as well as
for novel gene discovery.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
9435 through
7353 bp) and its proximal promoter (
265 through +262 bp) (27).
NkL-TAg DNA was constructed by inserting 34-bp loxP sequences on either
side of the TAg of Nk-TAg DNA. The orientation of the loxP recognition
sequences was confirmed by sequencing. Nk-TAg and NkL-TAg DNA fragments
were excised from the pBluescript plasmid using XhoI and
XbaI and were gel-purified prior to microinjection of the
DNA into pronuclei of fertilized B6C3F1 oocytes. Genotyping of
F0 mice was performed by Southern blot and PCR analysis
using genomic DNA. The probe used for Southern blot analysis was a
1209-bp BamHI fragment excised from the Nk-TAg construct.
PCR genotyping was performed using primers for TAg, generating a 500-bp
band. Primer sequences are as follows: TAg forward,
5'-CGCCAGTATCAACAGCCTGTTTGGC-3'; TAg reverse,
5'CATAT- CGTCACGTCGAAAAAGGCGC-3'.
20 °C methanol or 4% paraformaldehyde for
immunohistochemistry. Blocking was performed by incubating fixed cells
with 1.5% bovine serum albumin and 10% normal goat serum in
phosphate-buffered saline for 20 min. Primary antibodies were incubated
for 30-60 min in 1.5% bovine serum albumin in phosphate-buffered
saline as follows: monoclonal anti-myosin (smooth) (1:100, Sigma),
polyclonal anti-myosin (skeletal) (1:100, Sigma), monoclonal
anti-
-smooth muscle actin (1:100, Sigma), monoclonal anti-skeletal
myosin (slow) (1:100, Sigma), polyclonal anti-connexin-43 (1:100,
Sigma), monoclonal anti-
-sarcomeric actin clone 5C5 (1:100, Sigma),
monoclonal anti-desmin (1:100, Sigma), monoclonal anti-calponin (1:100,
Sigma), monoclonal anti-
-actinin (sarcomeric) clone EA-53 (1:200,
Sigma), and monoclonal anti-SV40 T-antigen (1:100, Santa Cruz
Biotechnology). Secondary antibodies conjugated to either fluorescein
isothiocyanate or Texas Red (1:200, Vector Laboratories) were diluted
in phosphate-buffered saline and incubated for 30 min at room
temperature. In some cases, nuclei were co-stained with
4,6-diamidino-2-phenylindole (10 µg/ml) for 1 min. Coverslips were
mounted with Vectashield (Vector Laboratories), and fluorescence or
confocal images were captured using Leica DMRXE or Zeiss 3.95 microscopes, respectively.
1 (TGF-
1) (R & D
Systems), 1 µM dynorphin-
(Peninsula Laboratories), 1 µM trans- or cis-retinoic acid
(Sigma), 15 ng/ml bone morphogenetic protein (BMP)-2/4 (Genetics
Institute, Cambridge, MA), 1 µM angiotensin II (R & D
Systems), 20 nM endothelin-1 (R & D Systems), 100 ng/ml insulin-like growth factor I (IGF-I) (Roche Molecular Biochemicals), 5-azacytidine (Sigma), and 10 µg/ml mitomycin C (Sigma).
-galactosidase (Ad-LacZ) were generated by Dr.
Robert Gerard (University of Texas Southwestern) and were constructed
using an in vitro Cre-lox recombination system (32, 33).
-galactosidase (Ad-LacZ) at
100 multiplicity of infection per cell for 3 h. Cells were
incubated for 4 days in differentiation media (described above)
containing 100 µM PE followed by RNA isolation using
Trizol reagent (Invitrogen). Data were analyzed using GeneSpring and
Affymetrix Suite software.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
9435 to
7353 bp) fused to the endogenous promoter (
265 to +262 bp) was
linked to the coding region for SV-40 large T-antigen (Fig.
1A) and used to generate transgenic mice. Founder transgenic mice appeared normal at birth and
showed no abnormalities during the neonatal period. However, one female
(Fo) transgenic mouse died spontaneously at 5 weeks of age.
An autopsy showed that the heart was grossly enlarged with multiple
sessile masses protruding into the left ventricular chamber from the
interventricular septum and anterior surface of the ventricular free
wall (Fig. 1B). Histological analysis confirmed that the
masses were localized subendocardially and consisted of small poorly
differentiated, spindle shaped cells with small eosin-rich cytoplasm
without apparent striation (Fig. 1C). Many loci of
myocardial hyperplasia were noted, none of which involved the
endocardium. The architecture of the remaining myocardium was preserved
although many cardiomyocytes had excessively large hematoxylin-rich
nuclei as a possible sign of polyploidy. It was not possible to
determine the cause of death of the animal; however, it is plausible
that either outflow obstruction or ventricular arrhythmia led to sudden
death.
View larger version (61K):
[in a new window]
Fig. 1.
Nk-TAg transgene and cardiac tumors in
Nk-TAg transgenic mouse. A, diagram of the
transgenic construct Nk-TAg containing the Nkx2.5
cis-regulatory sequences consisting of the early
cardiac-specific enhancer region ( 9435 to
7353 bp), fused to the
endogenous promoter (
265 to +262 bp), and linked to the SV-40 large
T-antigen coding region. B, cross-section of a heart of a
3.5-week-old mouse expressing the Nk-TAg transgene showing
multiple tumors protruding into the left ventricular chamber
(arrowheads). C, hematoxylin-eosin staining
of a histological section of a Nk-TAg transgenic heart. The cardiac
tumor (indicated by arrow) is localized
subendocardially and consists of poorly differentiated eosin-rich
spindle-shaped cells.
View larger version (33K):
[in a new window]
Fig. 2.
Characterization of Nk-TAg cardiac cell
lines. A, two independent Nk-TAg cell lines
(lines 5 and 20) were plated at a low density
(1.5 × 105 cells/100-mm plate) and were counted for 5 subsequent days. Cells reached confluence on day 4 but failed to
undergo growth arrest. B, expression of Nkx2.5 by
Northern blot analysis and T-antigen and GATA4 by Western blot analysis
in isolated Nk-TAg cell lines. All of the established cell lines
expressed Nkx2.5, GATA4, and TAg genes as detected by RT-PCR (data not
shown), although the level of expression varied for each cell
line.
Genes expressed in Nk-TAg cellsa
-actinin, a prototypical Z-line protein in
cardiomyocytes (data not shown). Growing the cells on plates coated
with laminin or type II collagen at normal (10%) or low serum content
(2 or 5%) did not increase expression of
-actinin (data not shown).
However, addition of mitomycin C to the growth medium abated
proliferation of the Nk-TAg cells and enhanced expression of
-actinin in the cytoplasm as unassembled Z-lines (Fig.
3). This finding suggested that
inhibition of DNA synthesis in Nk-TAg cells promoted a cardiogenic
phenotype. Addition of hypertrophic agents including endothelin-1, PE,
and angiotensin II did not further induce the assembly of sarcomeres in
Nk-TAg cells (data not shown).
View larger version (58K):
[in a new window]
Fig. 3.
Mitomycin C induces expression of
-actinin in Nk-TAg cells. Nk-TAg cells treated
with mitomycin C were stained with 4,6-diamidino-2-phenylindole
(A) and with anti-
-actinin antibody (B and
C). Images were taken at ×40 magnification (A
and B) and ×100 magnification (C).
-actinin
expression in Nk-TAg cells, we postulated that the lack of growth control is counterproductive to establishing a cardiomyocyte cell line.
Therefore, in an effort to induce cellular differentiation, we sought
to generate cardiac cell lines capable of terminating cell division. We
chose the Cre-lox system as an efficient method to permanently remove
and thereby inactivate TAg.
View larger version (35K):
[in a new window]
Fig. 4.
Analysis of NkL-TAg transgenic mouse cell
lines. A, diagram of the NkL-TAg transgenic
construct containing the Nkx2.5 cis-regulatory
sequences consisting of the early cardiac-specific enhancer region
( 9435 to
7353 bp), fused to the endogenous promoter (
265 to +262
bp), and linked to the SV-40 large T-antigen coding region flanked by
loxP sites (red diamonds). B,
immunocytochemistry using TAg antibody of NkL-TAg cells
(control) and NkL-TAg cells infected with Ad-Cre. An
equivalent number of cells was placed on plates. All the cells stained
for TAg in the absence of Cre recombinase (left panel),
whereas less than 1% of cells stained for TAg after addition of Cre
recombinase (right panel); C, BrdUrd
incorporation was measured in NkL-TAg cells (control) and
NkL-TAg cells infected with Ad-Cre. Equivalent numbers of cells were
counted at ×20 magnification. D, NkL-TAg
(control) or NkL-TAg cells infected with Ad-LacZ or Ad-Cre
were plated at 2 × 105 cells per 100-mm plate. Cells
were counted for 5 subsequent days. NkL-TAg cells stop proliferating in
response to excision of the TAg gene.
-actinin in a non-striated pattern (data not shown).
-galactosidase (Ad-LacZ) initially caused a decrease in the cell
growth rate, but ultimately resulted in no change in cell proliferation
rate when compared with non-infected cells (Fig. 4D).
-myosin heavy
chain, FHL2/DRAL, MCIP1, MEF2D, and atrial natriuretic factor. Immunocytochemistry revealed expression of desmin and cadherin (Fig.
5, A and B) as well
as connexin-43 (data not shown) that was localized to the cell
junctions.
View larger version (101K):
[in a new window]
Fig. 5.
Immunocytochemistry of NkL-TAg cells.
A, NkL-TAg cells were immunostained with antibodies
recognizing desmin or cadherin. B, NkL-TAg cells
non-infected (control) or infected with Ad-Cre were
immunostained with antibody recognizing -smooth muscle actin.
Magnification at ×40.
-actinin.
Prior to depletion of TAg, addition of caffeine, potassium chloride,
5-azacytidine, low oxygen, PE, NE, IGF-I, cis- or
trans-retinoic acids, dynorphin, TGF-
1, ionomycin, basic
fibroblast growth factor, or recombinant adenovirus containing various
transcripts (CMV-LacZ, CMV-Calsarcin1, CMV-GATA4, CMV-Nkx2.5, CMV-IGFI,
CMV-HDAC5, CMV-CaMKI, CMV-MEK6, and CMV-calcineurin A) had no effect on
inducing sarcomere formation as assessed by
-actinin
immunocytochemistry. However, expression of
-actinin was increased
in NkL-TAg cells upon removal of TAg and switching the medium to
contain low serum (5% horse serum), insulin, transferrin, and selenium
(Fig. 6). Moreover, addition of PE,
BMP2/4, or NE, to the media led to further induction of sarcomere
formation in some cells (Fig. 6). A similar induction was seen when
NkL-TAg cells depleted of TAg were grown in differentiation media and
infected with recombinant adenovirus expressing constitutively active
CaMKI or constitutively active calcineurin A (Fig. 6), known effectors
of cardiomyocyte hypertrophy (34, 35).
View larger version (116K):
[in a new window]
Fig. 6.
Effect of different treatments on the
morphology of NkL-TAg cells before and after deletion of TAg.
Uninfected NkL-TAg cells (control) or NkL-TAg cells infected
with Ad-Cre were cultured in differentiation media containing PE, NE,
BMP2/4, Ad-CaMKI, or Ad-calcineurin. Immunocytochemistry was performed
using anti- -actinin (sarcomeric) antibody. Magnification at
×40.
View larger version (29K):
[in a new window]
Fig. 7.
Measurement of calcium transients in NkL-TAg
cell lines. A, NkL-TAg cells infected with Ad-Cre were
subjected to an electrical stimulation at 1.5 Hz (Ion Optix) with
current pulses of 4-ms duration and voltages of 40 V. Calcium
transients were observed by exciting the fura 2-AM-loaded cells with
alternating wavelengths of 340 and 380 nm and recording the emission
intensity at 510 nm. Calcium transient data for each myocyte were
recorded from a minimum of 12 consecutive stimuli. B,
similar measurements were performed on mouse adult cardiomyocytes.
Pulses are indicated with arrowheads.
View larger version (17K):
[in a new window]
Fig. 8.
Microarray analysis of gene
expression profiles of the NkL-TAg cell line. Scatter plot of
microarray analysis using RNA isolated from uninfected NkL-TAg cells
(control) and NkL-TAg cells infected with Ad-lacZ
(A); uninfected NkL-TAg cells (control) and
NkL-TAg cells infected with Ad-Cre (B). C,
NkL-TAg cells infected with Ad-LacZ and NkL-TAg cells infected with
Ad-Cre. Each dot represents a gene on the microarray slide.
Black dots denote genes that are expressed in both samples.
Green dots represent genes exclusively expressed in NkL-TAg
cells infected with Ad-lacZ (A), NkL-TAg cells infected with
Ad-Cre (B), and NkL-TAg cells infected with Ad-Cre
(C). Blue dots represent genes exclusively
expressed in A, and NkL-TAg cells (control) in B,
and NkL-TAg cells infected with Ad-lacZ in C. Diagonal
lines on the graphs show 2-, 5-, and 10-fold differences.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
![]() |
ACKNOWLEDGEMENTS |
---|
We thank John McAnally for generating transgenic mice; Dr. Ralph Shohet and Teresa Gallardo for assistance with microarray data analysis; Dr. Robert Gerard for providing adenoviruses; Dr. Robert Hammer for providing DNA encoding SV-40 T-antigen; Dr. James Richardson for assistance with histological analyses; and Alisha Tizenor for assistance with images.
![]() |
FOOTNOTES |
---|
* This work was supported by grants from the National Institutes of Health, Bridging Project for the Alliance for Cellular Signaling (NIGMS, National Institutes of Health), the Donald W. Reynolds Foundation, and the Texas Advanced Technology Program.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The on-line version of this article (available at
http://www.jbc.org) contains Tables S1 and S2.
§ To whom correspondence should be addressed: University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75390-9148. Tel.: 214-648-1187; Fax: 214-648-1196; E-mail: Eric.Olson@utsouthwestern.edu.
Published, JBC Papers in Press, February 17, 2003, DOI 10.1074/jbc.M213102200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
TAg, simian virus 40 large T-antigen;
BrdUrd, 5-bromo-2'-deoxyuridine;
Ad, adenoviruses;
Ad-Cre, recombinant adenovirus expressing Cre recombinase;
Ad-lacZ, recombinant adenovirus expressing -galactosidase;
BMP, bone
morphogenetic protein;
FBS, fetal bovine serum;
PE, phenylephrine;
NE, norepinephrine;
TGF-
1, transforming growth factor
1;
IGF-I, insulin-like growth factor I;
RT, reverse transcriptase;
CaMKI, calcium/calmodulin-dependent protein kinase I;
HDAC, histone
deacetylases.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Soonpaa, M. H., and Field, L. J. (1997) Am. J. Physiol. 272, H220-H226[Medline] [Order article via Infotrieve] |
2. | MacLellan, W. R., and Schneider, M. D. (2000) Annu. Rev. Physiol. 62, 289-319[CrossRef][Medline] [Order article via Infotrieve] |
3. | Rumyantsev, P. P. (1977) in International Review of Cytology (Bourne, G. H. , and Danielli, J. F., eds) , pp. 187-273, Academic Press, New York |
4. | Doetschman, T. C., Eistetter, H., Katz, M., Schmidt, W., and Kemler, R. (1985) J. Embryol. Exp. Morphol. 87, 27-45[Medline] [Order article via Infotrieve] |
5. | Edwards, M. K., Harris, J. F., and McBurney, M. W. (1983) Mol. Cell. Biol. 3, 2280-2286[Medline] [Order article via Infotrieve] |
6. |
Makino, S.,
Fukuda, K.,
Miyoshi, S.,
Konishi, F.,
Kodama, H.,
Pan, J.,
Sano, M.,
Takahashi, T.,
Hori, S.,
Abe, H.,
Hata, J.,
Umezawa, A.,
and Ogawa, S.
(1999)
J. Clin. Invest.
103,
697-705 |
7. |
Muller, M.,
Fleischmann, B. K.,
Selbert, S.,
Ji, G. J.,
Endl, E.,
Middeler, G.,
Muller, O. J.,
Schlenke, P.,
Frese, S.,
Wobus, A. M.,
Hescheler, J.,
Katus, H. A.,
and Franz, W. M.
(2000)
FASEB J.
14,
2540-2548 |
8. |
Klug, M. G.,
Soonpaa, M. H.,
Koh, G. Y.,
and Field, L. J.
(1996)
J. Clin. Invest.
98,
216-224 |
9. |
Eisenberg, C. A.,
and Bader, D. M.
(1996)
Circ. Res.
78,
205-216 |
10. | Kimes, B. W., and Brandt, B. L. (1976) Exp. Cell Res. 98, 367-381[Medline] [Order article via Infotrieve] |
11. | Engelmann, G. L., Birchenall-Roberts, M. C., Ruscetti, F. W., and Samarel, A. M. (1993) J. Mol. Cell. Cardiol. 25, 197-213[CrossRef][Medline] [Order article via Infotrieve] |
12. | Manfredi, J. J., and Prives, C. (1994) Biochim. Biophys. Acta 1198, 65-83[CrossRef][Medline] [Order article via Infotrieve] |
13. | Brunskill, E. W., Witte, D. P., Yutzey, K. E., and Potter, S. S. (2001) Dev. Biol. 235, 507-520[CrossRef][Medline] [Order article via Infotrieve] |
14. |
Jahn, L.,
Sadoshima, J.,
Greene, A.,
Parker, C.,
Morgan, K. G.,
and Izumo, S.
(1996)
J. Cell Sci.
109,
397-407 |
15. | Jat, P. S., Noble, M. D., Ataliotis, P., Tanaka, Y., Yannoutsos, N., Larsen, L., and Kioussis, D. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 5096-5100[Abstract] |
16. | Gu, W., Schneider, J. W., Condorelli, G., Kaushal, S., Mahdavi, V., and Nadal-Ginard, B. (1993) Cell 72, 309-324[Medline] [Order article via Infotrieve] |
17. | Miller, C., Rulfs, J., Jaspers, S. R., Buckholt, M., and Miller, T. B., Jr. (1994) Mol. Cell. Biochem. 136, 29-34[Medline] [Order article via Infotrieve] |
18. | Morgan, J. E., Beauchamp, J. R., Pagel, C. N., Peckham, M., Ataliotis, P., Jat, P. S., Noble, M. D., Farmer, K., and Partridge, T. A. (1994) Dev. Biol. 162, 486-498[CrossRef][Medline] [Order article via Infotrieve] |
19. | Mouly, V., Edom, F., Decary, S., Vicart, P., Barbert, J. P., and Butler-Browne, G. S. (1996) Exp. Cell Res. 225, 268-276[CrossRef][Medline] [Order article via Infotrieve] |
20. | Tedesco, D., Caruso, M., Fischer-Fantuzzi, L., and Vesco, C. (1995) J. Virol. 69, 6947-6957[Abstract] |
21. | Kline, R. P., Sorota, S., Dresdner, K. P., Steinhelper, M. E., Lanson, N. A., Jr., Wit, A. L., Claycomb, W. C., and Field, L. J. (1993) J. Cardiovasc. Electrophysiol. 4, 642-660[Medline] [Order article via Infotrieve] |
22. | Steinhelper, M. E., Lanson, N. A., Jr., Dresdner, K. P., Delcarpio, J. B., Wit, A. L., Claycomb, W. C., and Field, L. J. (1990) Am. J. Physiol. 259, H1826-H1834[Medline] [Order article via Infotrieve] |
23. |
Claycomb, W. C.,
Lanson, N. A.,
Stallworth, B. S.,
Egeland, D. B.,
Delcarpio, J. B.,
and Izzo, N. J.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
2979-2984 |
24. |
Lints, T. J.,
Parsons, L. M.,
Hartley, L.,
Lyons, I.,
and Harvey, R. P.
(1993)
Development
119,
419-431 |
25. | Anton, M., and Graham, F. L. (1995) J. Virol. 69, 4600-4606[Abstract] |
26. | Kawamoto, S., Niwa, H., Tashiro, F., Sano, S., Kondoh, G., Takeda, J., Tabayashi, K., and Miyazaki, J. (2000) FEBS Lett. 470, 263-268[CrossRef][Medline] [Order article via Infotrieve] |
27. |
Lien, C. L.,
Wu, C.,
Mercer, B.,
Webb, R.,
Richardson, J. R.,
and Olson, E. N.
(1999)
Development
126,
75-84 |
28. |
Paradis, P.,
MacLellan, W. R.,
Belaguli, N. S.,
Schwartz, R. J.,
and Schneider, M. D.
(1996)
J. Biol. Chem.
271,
10827-10833 |
29. | Cyran, S. E., Ditty, S. E., Baylen, B. G., Cheung, J., and LaNoue, K. F. (1992) J. Mol. Cell. Cardiol. 24, 1167-1177[Medline] [Order article via Infotrieve] |
30. | De Young, M. B., and Scarpa, A. (1989) Am. J. Physiol. 257, C750-C758[Medline] [Order article via Infotrieve] |
31. |
He, T. C.,
Zhou, S.,
Da Costa, L. T., Yu, J.,
Kinzler, K. W.,
and Vogelstein, B.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
2509-2514 |
32. | Aoki, K., Barker, C., Danthinne, X., Imperiale, M. J., and Nabel, G. J. (1999) Mol. Med. 5, 224-231[Medline] [Order article via Infotrieve] |
33. | Ng, P., Parks, R. J., Cummings, D. T., Evelegh, C. M., Sankar, U., and Graham, F. L. (1999) Hum. Gene Ther. 10, 2667-2672[CrossRef][Medline] [Order article via Infotrieve] |
34. |
Passier, R.,
Zeng, H.,
Frey, N.,
Naya, F. J.,
Nicol, R. L.,
McKinsey, T. A.,
Overbeek, P.,
Richardson, J. A.,
Grant, S. R.,
and Olson, E. N.
(2000)
J. Clin. Invest.
105,
1395-1406 |
35. | Molkentin, J. D., Lu, J. R., Antos, C. L., Markham, B., Richardson, J., Robbins, J., Grant, S. R., and Olson, E. N. (1998) Cell 93, 215-228[Medline] [Order article via Infotrieve] |