Published online before print May 10, 2001, doi: 10.1161/hh1001.090878
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© 2001 American Heart Association, Inc.
Article |
Coexpression of Mutant p53 and p193 Renders Embryonic Stem Cell–Derived Cardiomyocytes Responsive to the Growth-Promoting Activities of Adenoviral E1A
From the Wells Center for Pediatric Research and Krannert Institute of Cardiology, Indiana University School of Medicine, Indianapolis, Ind. Present address of S.-C.T. is Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan, Republic of China.
Correspondence to Dr Loren J. Field, Herman B Wells Center for Pediatric Research, James Whitcomb Riley Hospital for Children, 702 Barnhill Dr, Room 2600, Indianapolis, IN 46202-5225. E-mail ljfield{at}iupui.edu
Abstract
Abstract—Expression of adenoviral E1A in cardiomyocytes results in the activation of DNA synthesis followed by apoptosis. In contrast, expression of simian virus 40 large T antigen induces sustained cardiomyocyte proliferation. Previous studies have shown that T antigen binds to 2 proapoptotic proteins in cardiomyocytes, namely the p53 tumor suppressor and p193 (a new member of the BH3-only proapoptosis subfamily). Structure-function analyses identified a p193 C-terminal truncation mutant that encodes prosurvival activity. This mutant was used to test the role of p193 in E1A-induced cardiomyocyte apoptosis. E1A induced apoptosis in cardiomyocytes derived from differentiating embryonic stem cells. Expression of the prosurvival p193 mutant alone or a mutant p53 alone did not block E1A-induced apoptosis. In contrast, combinatorial expression of mutant p193 and mutant p53 blocked E1A-induced apoptosis, resulting in a proliferative response indistinguishable from that seen with T antigen. These results confirm the hypothesis that there are 2 proapoptotic pathways, encoded by p53 and p193, respectively, which restrict cardiomyocyte cell cycle activity in differentiating embryonic stem cell cultures. Furthermore, these results explain in molecular terms the phenotypic differences of E1A versus T-antigen gene transfer in cardiomyocytes.
Key Words: cardiomyocyte proliferation • cardiac myocyte apoptosis • heart regeneration
The capacity of adult mammalian cardiomyocytes to initiate DNA synthesis and to subsequently divide appears to be quite limited.1 2 Consequently, the ability of the heart to undergo regenerative growth in disease states is also limited, a fact underscored by the high morbidity and mortality rates for individuals with severe cardiovascular disease. It is generally believed that increasing the number of functional cardiomyocytes in a diseased heart would have a positive impact on contractile activity provided that the new cells participate in a functional syncytium. This belief has fostered the development of a number of novel approaches to increase cardiomyocyte number.3 4 One approach has focused on the identification of key cardiomyocyte cell cycle–regulatory proteins, with the hope that exploitation of these proteins (or their respective regulatory pathways) could promote regenerative myocardial growth.3 Toward that end, a number of gene transfer approaches have been used to target expression of the simian virus 40 (SV40) large T antigen (T-Ag) or the adenoviral E1A oncoproteins to cardiomyocytes in an effort to induce proliferation. Targeted expression of T-Ag to atrial or ventricular myocardium in transgenic mice can induce a proliferative response,5 6 7 which in most instances results in the formation of tumors comprising differentiated, proliferating cardiomyocytes. In contrast, the ability of E1A to induce cardiomyocyte proliferation appears to be more restricted. Although E1A expression in cultured fetal or neonatal cardiomyocytes can reactivate DNA synthesis, this is followed rapidly by apoptosis.8 9 10 11 12
It is well established that DNA tumor virus oncoproteins subjugate cell cycle activity by binding to and altering the activity of endogenous cell cycle regulators.13 In light of this paradigm, identification of the cardiomyocyte T-Ag and E1A binding partners could provide insight into cell cycle regulation in the myocardium. In the case of T-Ag, 3 prominent cardiomyocyte binding proteins have been identified. They are the p53 tumor suppressor,14 15 p107 (a member of the retinoblastoma protein gene family),14 16 and p193 (a novel member of the BH3-only family of proapoptosis proteins).14 17 The same set of T-Ag binding proteins was detected in multiple independently derived tumor cell lines, as well as in primary cardiomyocyte cultures from the transgenic tumors. In the case of E1A, retinoblastoma protein family members (pRB, p107, and p130) and p300 (a transcriptional coactivator with intrinsic histone acetylase activity) were identified as cardiomyocyte binding proteins.10 Thus, T-Ag possesses several antiapoptotic activities that are lacking in E1A (namely the ability to bind to 2 proapoptotic proteins, p53 and p193). Given the cell death observed in response to E1A expression, we would anticipate that expression of E1A in conjunction with blockade of both the p53 and the p193 pathways would induce cardiomyocyte proliferation, rather than apoptosis, as was reported previously where one or both of these proapoptotic pathways were intact.8 9 10
In this report, a preliminary structure-function study was initiated to further characterize the proapoptotic activity encoded by p193. Surprisingly, a mutant molecule encoding p193 amino acid residues 1 to 1152 possessed growth-promoting activity in an NIH-3T3 assay similar to that observed with a p193 antisense expression construct. Additional analyses revealed that expression of this mutant rendered NIH-3T3 cells resistant to methyl methanesulfonate (MMS)–induced apoptosis. The availability of a prosurvival p193 variant and a mutant p53 (which is able to block p53-induced apoptosis) enabled us to directly test the hypothesis that DNA tumor viral oncoprotein activation of the cardiomyocyte cell cycle requires blockade of both the p193 and the p53 pathways. Expression of E1A in embryonic stem (ES) cell–derived cardiomyocytes led to widespread apoptosis. E1A induced similar levels of apoptosis in cardiomyocytes expressing mutant p53 or mutant p193. In contrast, coexpression of mutant p53 and mutant p193 blocked E1A-induced apoptosis, resulting in a proliferative response indistinguishable from that seen with SV40 T-Ag gene transfer. These results provide an explanation in molecular terms for the different phenotypes observed in cardiomyocytes expressing T-Ag versus E1A.
Materials and Methods
Recombinant Clones
cDNAs encoding
full-length17 and
C-terminal–deleted p193 were subcloned into the pRC/cytomegalovirus
(CMV) expression vector (Invitrogen). The mouse 
-cardiac myosin
heavy chain (MHC) promoter18
was used to generate expression constructs for the ES cell–derived
cardiomyocyte colony growth assay. For generation of a
mutant p53 expression cassette, a p53 genomic clone from CB7 cells was
used.19 20 For
generation of an E1A expression cassette, a wild-type E1A genomic clone
was used.
NIH-3T3 Colony Growth Assay
NIH-3T3 cells (American Type Culture Collection) were
transfected using a calcium phosphate
approach,21 selected in G418
(300 µg/mL) for 15 days, and stained with gentian violet. Stable cell
lines were obtained by prolonged G418 selection. Transfection
efficiencies were calculated by monitoring expression of a
cotransfected CMV-ßGAL reporter gene at 48 hours after
transfection.
Assessment of Apoptosis
NIH-3T3 cells were grown at a concentration of
3x106 cells per dish and treated with MMS.
In other experiments, cells from microdissected beating clusters from
the ES cell–derived cardiomyocyte colony growth assay were
analyzed. Cells were processed using a DNA ladder kit (Roche).
For terminal
deoxynucleotidyltransferase–mediated
dUTP nick-end labeling (TUNEL), dispersed cell preparations were plated
onto fibronectin-coated chamber slides and processed using an in situ
cell death detection kit (Roche).
ES Cell Culture Conditions and Cardiomyocyte
Colony Growth Assay
Undifferentiated R1 ES cells were transfected via
electroporation with a construct carrying both an
-MHC–aminoglycoside phosphotransferase
(MHC-neor) and a phosphoglycerate kinase
(pGK)-hygromycin–resistant transgene in a common pBM20 vector
backbone
(Boehringer-Mannheim).22
In some studies, the cells were cotransfected with multiple expression
vectors. Transfected cells were selected by incubation in growth medium
containing hygromycin B (200 µg/mL) for 7 days and dissociated, and
4x106 cells were plated per 100-mm
bacterial dish and cultured in growth medium lacking leukemia
inhibitory factor (LIF). After 4 days of culture, the resulting
embryoid bodies were plated onto cell culture dishes. When spontaneous
contractile activity was noticed, growth medium supplemented with G418
(200 µg/mL) was added to eliminate the noncardiomyocytes.
Transfection efficiencies were calculated by monitoring expression of a
cotransfected CMV-ßGAL reporter gene at 48 hours after transfection.
To visualize cardiomyogenic induction, the plates were stained with
periodic acid–Schiff (PAS) reagent.
Assessment of Gene Expression
For Western blots, protein samples were displayed on
polyacrylamide gels; transferred to nitrocellulose; and reacted
with anti-p53 PC35 (Oncogene), anti-E1A M73 (Santa Cruz Biotechnology),
anti–T-Ag PAB-416 (Oncogene), or anti–sarcomeric myosin MF20
(Developmental Studies Hybridoma Bank, University of Iowa, Iowa City,
Iowa) as
described.17 23 24
For Northern blots, total RNA was purified and processed as
described.17 21 25
An expanded Materials and Methods section can be found in the online data supplement available at http://www.circresaha.org.
Results
Identification of a p193 Mutant Encoding
Prosurvival Activity
In a preliminary effort to establish structure-function
relationships, a series of expression vectors encoding p193 molecules
with nested C-terminal truncations was generated and tested in an NIH
colony growth assay. Transfection with a control expression vector
lacking a cDNA insert indicates the cumulative sum of cell growth and
death under the conditions used. In agreement with previous
results,17 transfection with
an expression vector encoding full-length p193 failed to give rise to
colonies because of its proapoptotic activity, and transfection
with an expression vector encoding a full-length p193 in the antisense
orientation resulted in enhanced growth (as evidenced by a marked
increase in both colony size and number) compared with the
control construct
(Figure 1A
). Surprisingly, transfection with an expression
vector encoding p193 amino acid residues 1 through 1343 (1343stp)
resulted in slightly enhanced growth in a subset of the resultant
colonies. This effect was even more pronounced with an expression
vector encoding p193 amino acid residues 1 through 1152 (1152stp);
transfection with this construct resulted in the formation of a large
number of colonies with, on average, markedly increased size as
compared with the other constructs tested. Progressively greater
C-terminal truncations (912stp, 309stp, and 243stp) resulted in a loss
of the growth-promoting phenotype seen with 1152stp. The level
of growth enhancement obtained with 1152stp was similar to that
obtained with the p193 antisense construct
(Figure 1).17
Importantly, similar transfection efficiencies were obtained with all
of the p193 constructs tested, as determined by cotransfection with a
reporter gene encoding ß-galactosidase activity
(Figure 1 legend).
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Because p193 encodes a BH3-only proapoptosis
activity, the marked growth enhancement observed with the 1152stp
construct was rather surprising. Stable cell lines carrying either the
control or the 1152stp expression vectors were generated to determine
whether 1152stp encoded prosurvival activity. The cells were incubated
with increasing concentrations of MMS, an agent that promotes DNA
damage and that has previously been shown to induce apoptosis
via both p53-dependent and p53-independent
pathways.26 27 28 29 30 31
DNA prepared from the control cells incubated in MMS at high
concentration exhibited pronounced internucleosomal cleavage
(Figure 2), indicative of a strong apoptotic
response. In contrast, DNA prepared from the 1152stp cells incubated
with MMS showed no evidence of internucleosomal cleavage
(Figure 2), suggesting that apoptosis was either
delayed or blocked in cells expressing 1152stp. These data indicate
that C-terminal truncation of p193 at amino acid residue 1152 bestows a
prosurvival activity on the molecule.
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Coexpression of Mutant p53, p193, and E1A
Markedly Increases the Yield of ES Cell–Derived
Cardiomyocytes
To directly determine the importance of the p53 and
p193 proapoptotic pathways in cardiomyocyte cell
cycle regulation, expression constructs encoding a mutant p53
(designated CB7; see Reference 1919 ), the prosurvival p193 mutant
(1152stp), and E1A under the regulation of the mouse -cardiac MHC
promoter were generated and used in an ES cell–derived
cardiomyocyte colony growth assay. The
cardiomyocyte colony growth assay was based on the previous
observation that essentially pure cardiomyocyte cultures
suitable for long-term studies can easily be generated from
differentiating ES cells that carry an
MHC-neor/pGK-hygror
transgene.22
Undifferentiated ES cells were transfected with an
MHC-neor/pGK-hygror
transgene alone or in combination with the MHC-CB7, MHC-1152stp, and/or
MHC-E1A transgenes. The presence of
pGK-hygror sequences allowed for the
selection of colonies of transfected, undifferentiated ES cells. The
resulting colonies were pooled and used to generate embryoid bodies for
in vitro differentiation. The presence of
MHC-neor sequences allowed for enrichment of
cardiomyocytes after in vitro differentiation; once
cardiomyogenic induction was observed (as evidenced by the presence of
spontaneous contractile activity), G418 was added to the cultures to
eliminate nonmyocytes. After 60 days of culture, the dishes
were stained with PAS reagent to visualize the
cardiomyocytes (PAS stains glycogen-containing cells
intense violet).
The results are shown in
Figure 3. The control dish depicts
cardiomyocytes obtained by transfection with the
MHC-neor/pGK-hygror
transgene alone and is indicative of the baseline rate of ES
cell–derived cardiomyocyte growth under the conditions
used. Expression of CB7 or 1152stp did not have a marked impact on the
yield of cardiomyocytes. In contrast, expression of E1A
dramatically decreased cardiomyocyte yield,
consistent with the proapoptotic activity of this
molecule in cardiomyocytes as described by others.
Coexpression of E1A and CB7 or E1A and 1152stp did not result in
enhanced cardiomyocyte yield as compared with expression of
CB7 or 1152stp alone, although the yield was increased as compared with
cultures transfected with E1A only. In contrast, coexpression of E1A,
CB7, and 1152stp markedly increased the yield of
cardiomyocytes, with levels similar to those obtained from
transfection with T-Ag.
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Coexpression of CB7 and 1152stp Inhibits
E1A-Induced Apoptosis in ES Cell–Derived
Cardiomyocytes
The results presented above are
consistent with the hypothesis that coexpression of CB7 and
1152stp blocks cardiomyocyte apoptosis in response
to E1A expression. Several analyses were performed to further
test this hypothesis. DNA was prepared from parallel cultures of ES
cell–derived cardiomyocytes after 15 days of
differentiation. Gel electrophoresis revealed the presence of marked
internucleosomal DNA fragmentation from cultures expressing E1A alone,
E1A and CB7, or E1A and 1152stp
(Figure 4). In contrast, no evidence for internucleosomal DNA
fragmentation was observed in cultures coexpressing E1A, CB7, and
1152stp, consistent with the absence of apoptosis in
these cells. DNAs prepared from the other cultures in this series were
included as additional controls
(Figure 4).
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TUNEL analysis was also used to monitor
apoptosis. Dispersed cell preparations were generated after 15
days of differentiation, and the cardiomyocytes were
replated at low density on chamber slides. After an additional 24 hours
of culture, the samples were processed for TUNEL analysis.
TUNEL-positive cardiomyocytes were readily detected in
cultures expressing E1A alone, but not in cultures coexpressing E1A,
CB7, and 1152stp
(Figure 5A). The frequency of TUNEL positivity was scored in
all of the cultures
(Figure 5B). Frequencies of TUNEL positivity ranging from
40% to 50% were observed in cultures expressing E1A alone, E1A and
CB7, or E1A and 1152stp, whereas the frequency observed in cultures
cotransfected with E1A, CB7, and 1152stp was comparable with the
control cultures. These data collectively indicate that coexpression of
CB7 and 1152stp blocks E1A-induced apoptosis in ES
cell–derived cardiomyocytes.
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ES Cell–Derived Cardiomyocytes Expressing E1A
and CB7 or E1A and 1152stp Are Not Viable
Transgene expression was monitored using protein and
RNA prepared from cultures after 60 days of differentiation
(Figure 6). Seventy-five micrograms of protein or 10 µg of
RNA from each sample was analyzed. High levels of CB7 protein
and 1152stp mRNA were observed in all cultures transfected with the
MHC-CB7 and MHC-1152stp constructs, respectively. In contrast, E1A
protein was detected only in the cultures cotransfected with MHC-E1A,
MHC-CB7, and MHC-1152stp. As expected, T-Ag protein was only detected
in cultures transfected with the MHC–T-Ag construct. The absence of
E1A protein in colonies arising from ES cells transfected with the
MHC-E1A alone, or from ES cells cotransfected with MHC-E1A and MHC-CB7
or MHC-E1A and MHC-1152stp, is consistent with the high levels
of DNA degradation and cardiomyocyte TUNEL positivity
observed in these cultures. Importantly, these expression
analyses confirm that cardiomyocytes expressing E1A
are only viable if both CB7 and 1152stp are expressed.
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To quantify the differential effect of transgene expression
on cardiomyocyte yield, MHC expression was monitored. One
percent of the total protein from each culture dish was subjected to
Western blot analysis using an anti-MHC antibody (MF20; see
Figure 6
). Consistent with the qualitative
observations from PAS-stained cultures, the cardiomyocyte
yield was markedly enhanced in cultures coexpressing E1A, CB7, and
1152stp, with total yields similar to that seen with cultures
expressing T-Ag. Importantly, all of the ES cell–derived cultures
contained differentiated, spontaneously contracting
cardiomyocytes. The relatively low MHC signal in the
control and singly transfected cultures is due to the low protein input
on the Western blot; because of the marked enhancement of
cardiomyocyte yield in the cotransfected and T-Ag cultures,
low protein input was necessary to capture signals in the linear range
of the film. Finally, the ES cell–derived cardiomyocyte
colony growth assay was highly reproducible, and a similar effect of
transgene expression on cardiomyocyte growth has been
obtained in 5 independent experiments.
Discussion
The results presented here indicate that E1A-induced cell cycle activation in ES cell–derived cardiomyocytes provokes an apoptotic response similar to that observed after adenovirus-mediated E1A gene transfer in fetal or neonatal cardiomyocyte cultures. Furthermore, coexpression of mutant p53 and mutant p193 blocked E1A-induced apoptosis in an ES cell–derived cardiomyocyte colony growth assay, resulting in a proliferative response indistinguishable from that seen with SV40 T-Ag gene transfer. These results provide an explanation in molecular terms for some of the phenotypic differences observed in response to T-Ag versus E1A expression in cardiomyocytes. Studies with transgenic mice have indicated that targeted expression of T-Ag promotes sustained proliferation of differentiated cardiomyocytes.5 6 7 Biochemical analyses of cardiomyocytes derived from the transgenic animals identified 2 proapoptotic T-Ag binding proteins, p53 and p193,14 17 leading to the hypothesis that there are 2 prominent proapoptotic activities, encoded by p53 and p193, respectively, that function to block cardiomyocyte proliferation induced by targeted expression of DNA tumor virus oncoproteins.17
In contrast, targeted expression of E1A using either the
atrial natriuretic factor (ANF) or -cardiac actin
promoters failed to produce viable transgenic mice (K.B.S.P. and
L.J.F., unpublished results, 1991). This observation is
consistent with the profound cardiomyocyte
apoptosis observed in response to adenoviral delivery of E1A to
fetal
cardiomyocytes8 9 10 11 12
or in response to E1A expression in ES cell–derived
cardiomyocytes as described in this report. The observation
that the combinatorial activities encoded by mutant p53 and mutant p193
block E1A-induced apoptosis in ES cell–derived
cardiomyocytes and thereby renders the cells proliferative
supports the hypothesis that these 2 proapoptotic pathways must
be compromised for successful cell cycle progression to occur in
response to DNA tumor virus oncoprotein expression. This scenario is
further supported by the previous observations that E1A expression
induced apoptosis in fetal or neonatal
cardiomyocytes in which one or both of these
proapoptotic pathways were
intact.8 9 10
However, because the molecular activities of 1152stp are not fully
characterized, we cannot at present rule out the possibility that
expression of mutant p193 has a direct cell cycle effect in ES
cell–derived cardiomyocytes, in addition to the clearly
documented antiapoptotic effects. Indeed, although an overt
cell cycle effect was not evident in the singly transfected ES
cell–derived cardiomyocyte cultures (see
Figure 3), expression of 1152stp appeared to have a cell
cycle effect in NIH-3T3 cells (see the colony growth assay in
Figure 1). It is also noteworthy that our studies used a
genomic E1A clone, which can encode the 9S, 12S, and 13S splice
variants. Most early cardiomyocyte E1A studies used cDNAs
encoding the 12S splice variant only. Western analyses clearly
indicate that the products of both the 12S and 13S transcripts are
expressed in our study
(Figure 6). Finally, preliminary studies revealed that
expression of Bcl-2 and E2F-1 are not markedly increased in response to
coexpression of E1A, CB7, and/or 1152stp in the ES cell–derived
cardiomyocytes (see the online data supplement available at
http://www.circresaha.org), which suggests that the altered
survival and proliferation observed in this model do not simply result
from altered expression of these molecules.
Members of the Bcl family of apoptosis regulators share homology to Bcl-2, the prototypical family member, at one or more motifs known as Bcl-2 Homology domains (BH1, BH2, BH3, and/or BH4). Family members with the greatest homology to Bcl-2 tend to promote cell survival, whereas those more distantly related tend to promote apoptosis.32 The proapoptosis activity of the BH3-only proteins resides largely in their ability to heterodimerize with, and thereby alter the activity of, prosurvival Bcl-2 family members. Deletion of the BH3 domain blocks the ability of BH-3–only proteins to form heterodimers and in most instances concomitantly abolishes their apoptotic activity.33 34 It was therefore not surprising that deletion of the C-terminal BH3 domain abolished the proapoptotic activity of p193.17 In contrast, the observation that 1152stp encodes prosurvival activity was quite surprising.
There are several instances wherein modification of a prosurvival Bcl family member produced a proapoptotic variant. For example, alternative splicing of Bcl-XL produces a proapoptotic variant, Bcl-xS, containing BH3 and BH4 domains.35 Similarly, alternative splicing of Mcl-1 and Bok-L (prosurvival proteins that contain BH1, BH2, and BH3 domains) disrupts the BH1 and BH2 domains in these molecules, resulting in the formation of proapoptotic BH3-only splice variants.36 37 38 In contrast, we are unaware of previous examples in which modification of a proapoptotic BH3-only protein resulted in a prosurvival phenotype. At present the molecular basis for the prosurvival and growth-enhancing phenotypes encoded by 1152stp is not clear. It is unlikely that these activities result from simple titration of the normal p193 binding partners, as marked and consistent growth enhancement was only observed with the 1152stp construct. Rather, disequilibrium resulting from the titration of only a subset of the proteins that bind to full-length p193 might underlie the activities encoded by 1152stp. Unfortunately, the primary amino acid sequence and subcellular localization of p193 do not suggest an obvious mechanistic explanation for the activity encoded by 1152stp.
Expression of p193-antisense constructs in an NIH-3T3 colony
growth assay resulted in decreased levels of the endogenous
p193 transcript with a concomitant increase in cell
growth17 (see also
Figure 1). The phenotype of cells expressing 1152stp
closely mimicked that of cells with antisense-mediated p193 loss of
function, raising the possibility that 1152stp may directly antagonize
the proapoptotic activity of endogenous p193. This
view is supported by the observation that 1152stp expression bestows a
potent prosurvival phenotype in 2 independent model systems
(resistance to MMS-induced apoptosis in NIH-3T3 cells and, in
combination with coexpression of mutant p53, resistance to E1A-induced
apoptosis in ES cell–derived cardiomyocytes). The
enhanced proliferation and prosurvival phenotypes resulting
from 1152stp expression are both consistent with the notion
that this molecule encodes dominant negative p193 activity. Despite
these circumstantial arguments, additional experimentation is required
before 1153stp can be designated as a dominant negative
p193.
The ES cell–derived cardiomyocyte colony growth
assay relied on cotransfection of an
MHC-neor/pGK-hygror
transgene in combination with the genes of interest. On
differentiation, imposition of G418 selection resulted in cultures that
were markedly enriched for cardiomyocytes. This approach is
quite analogous to and enjoys the benefits of standard fibroblast
colony growth assays and also provides a relatively rapid system with
which to examine the effects of a given gene on
cardiomyocyte proliferation and/or survival. Moreover, the
cultures are not subject to temporal limitations resulting from
proliferation of noncardiomyocytes. However, because
multiple genes were introduced via cotransfection, the approach as
described does suffer from the possibility of partial gene transfer.
For example, it is likely that the presence of
CB7-positive/E1A-negative and 1152stp-positive/E1A-negative
cardiomyocytes
(Figures 3 and 6) is attributable to the differentiation of
progenitor cells that were not transfected with the E1A expression
construct. In support of this, polymerase chain reaction
analysis of DNA prepared from these cultures indicated that the
E1A transgene was absent (see the online data supplement).
Similarly, the slight increase in TUNEL positivity in cultures
cotransfected with E1A, CB7, and 1152stp as compared with control
cultures is also likely attributable to partial gene transfer
(Figure 5). Importantly, coexpression of E1A, CB7, and
1152stp had a similar effect on cardiomyocyte proliferation
when using either ANF-promoted transgenes or a combination of ANF- and
MHC-promoted transgenes (data not shown), indicating that
promoter squelching is not a problem with this assay.
The observation that E1A induces proliferation only in ES cell–derived cardiomyocytes that also express mutant p53 and mutant p193 supports the notion that there are 2 proapoptotic pathways that can negatively regulate cell cycle activity in ES cell–derived cardiomyocytes. This finding may have important implications for therapeutic myocardial regeneration. For example, if no additional cell cycle checkpoints are activated on terminal differentiation, the results presented here would predict that abrogation of p53 and p193 activity should render adult cardiomyocytes responsive to cell cycle activation. In light of this, it is of interest to note that several groups have shown that E1A and E2F-1 have very similar activities when expressed in fetal cardiomyocytes in vitro.8 9 10 11 12 39 Furthermore, E2F-1 expression in adult cardiomyocytes results in initiation of DNA synthesis followed by apoptosis.40 It will thus be of great interest to determine whether coexpression of E2F-1 (or E1A), CB7, and 1152stp is sufficient to promote proliferation in adult cardiomyocytes. The p53 and p193 may also impact on pathophysiologic apoptosis in the heart. Forced expression of p53 is sufficient to induce cardiomyocyte apoptosis, and although p53 has been implicated in some forms of myocardial apoptosis, its role as a causative agent in this process remains controversial. Combinatorial blockade of p53 and p193 may have a greater protective effect during pathophysiologic apoptosis, similar to that seen after E1A gene transfer.
Acknowledgments
This work was supported by grants from the National Heart, Lung, and Blood Institute. We thank Dr Betty Moran (Fels Institute for Cancer Research and Molecular Biology, Philadelphia, Pa) for sequences encoding E1A, Alan Bernstein (Canadian Institutes of Health Research, Ottawa, Ontario, Canada) for the CB7 genomic clone, and Andres Nagy (Samuel Luenfeld Research Institute, Toronto, Ontario, Canada) for R1 ES cells.
Footnotes
Original received February 2, 2001; revision received April 2, 2001; accepted April 2, 2001.
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