1 Department of Biochemistry, University College London, London WC1E 6BT,
UK
2 Centre for Cell and Molecular Biology, The Institute of Cancer Research,
Chester Beatty Laboratories, 237 Fulham Road, London, UK
Author for correspondence (e-mail:
mbcd2{at}cam.ac.uk)
Accepted 4 June 2003
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Summary |
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Key words: Plasticity, Cardiomyocytes, Regeneration, Heart, Newt, Cell cycle
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Introduction |
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It is possible that the plasticity of the differentiated state in
cardiomyocytes is related to that in other newt tissues. An adult newt is also
able to regenerate its limbs and tail and ocular tissues such as the lens and
retina (Brockes, 1997;
Goss, 1969
). In these
contexts, the plasticity of the differentiated state is a key mechanism for
the generation of progenitor cells (Brockes
and Kumar, 2002
; Brockes et
al., 2001
). For example, after amputation of the limb or tail,
skeletal myofibres in the distal stump as well as implanted myotubes are able
to re-enter the cell cycle and to fragment into viable mononucleate cells
(Echeverri et al., 2001
;
Kumar et al., 2000
;
Lo et al., 1993
;
McGann et al., 2001
;
Velloso et al., 2000
). In the
case of the cultured newt myotube, DNA synthesis can be stimulated by
mammalian serum and depends on phosphorylation of the retinoblastoma protein
(Rb) (Tanaka et al., 1999
;
Tanaka et al., 1997
).
Here we address several questions regarding the cellular and molecular mechanisms that underlie the plasticity of the cardiomyocyte. First, is this ability to re-enter the cell cycle in response to injury a general property of the differentiated state in newt cardiomyocytes, as described for other newt differentiated cells, or are there defined sub-populations of cells that have this ability? Do newt cardiomyocytes progress through several cycles, or do they show the complex regulation of cell cycle progression of the mammalian cardiomyocyte? Finally, is cell cycle re-entry triggered by mammalian serum and is it dependent on Rb phosphorylation? To answer these questions we have exploited a culture system where S phase entry and cell cycle progression can be analysed in single cells.
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Materials and Methods |
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Cells were labelled with 3-bromo-2-deoxyuridine (BrdU; 10 µg/ml) or with [14C]thymidine (0.01 µCi/ml). For cumulative labelling with [14C]thymidine, fresh medium with label was added every 3 days until day 15, and then medium without label was added for 3 days prior to fixation and autoradiography with Ilford K5 emulsion. The concentration of [14C]thymidine chosen for these experiments did not affect cell cycle progression (results not shown). For pulse labelling, BrdU was added to the medium and cells were fixed 8, 9 or 18 hours afterwards depending on experimental convenience, as described in the figure legend.
Microinjection of cells
Injection of plasmid DNA or fluorescently labelled dextrans was performed
with a Narishige manipulator connected to a pneumatic picopump PV820 mounted
on a Zeiss Axiovert microscope, 5 days after plating. At 1 hour before
microinjection of the cells, the medium was changed to serum free AL15 with
2,3-butanedione monoxime (BDH, 4 mM) to inhibit myofibril contraction. DNA was
injected into the nucleus and dextrans (Texas Red or fluorescein-conjugated 70
kDa dextrans, Molecular Probes; used as an additional control for the effect
of microinjection) into the cytoplasm. After microinjection, the medium was
changed to AMEM with 10% FBS for 7 days prior to addition of BrdU for 18
hours. In plasmid pTL1-p16 the coding sequence for human
p16INK4 is expressed under control of the SV40 promoter in
the expression vector pTL1, as described previously for the retinoic acid
receptor (Ragsdale et al.,
1989). In plasmid pCAP the coding sequence for human placental
alkaline phosphatase is expressed under control of the SV40 promoter in the
expression vector pSG5 as described earlier
(Schilthuis et al., 1993
).
Lineage tracing with tracker dye
Cardiomyocytes were labelled in suspension with the PKH-26 fluorescent
tracker dye (Sigma) according to the manufacturer's instructions. The
PKH-labelled cells were mixed with unlabelled cells (1:24) and
4x104 cells were seeded onto a 35 mm dish scored with a grid.
After 4-5 days in culture the cell numbers were adjusted to 1 labelled
mononucleate cell per grid square by removing supernumeraries with a
microinjection pipette attached to a Narishige micromanipulator. The cells
were fixed and examined 24 days after plating.
Imaging
Live cardiomyocytes were viewed under phase contrast objectives and images
were captured with a monochrome CCD (SONY) camera and image contrast was
enhanced with Image Pro Plus software. For continuous time lapse analysis,
cells were maintained at room temperature in AL15 with 10% FBS overlaid with
mineral oil to prevent evaporation. For observation once or twice a day, cells
were plated in a dish scored with an oriented grid and kept in the incubator
in AMEM with 10% FBS. For sequential observations cells could readily be
recognised by their position in the grid. Images for immunofluorescence were
collected on a CV-12 (Photonic Sciences) cooled monochrome camera. Z-series
stacks were acquired under a Leica TCS SP confocal system and projections
generated using Leica TCS imaging software.
Antibodies and cell staining
Mouse monoclonal antibodies were used against BrdU (BU-20; Amersham),
sarcomeric myosin heavy chain (A4.1025; Dr Simon Hughes, Randall Institute,
King's College, London), p16 (DCS-50.2, used at 10 µg/ml; Dr Gordon Peters,
Imperial Cancer Research Fund, London), titin and troponin T (clones 9D10 and
CT3, Dr Elisabeth Ehler, ETH, Zurich), Rb [51ß7, used at 13 µg/ml; Dr
Sybille Mittnacht, ICR, London, available from Serotec (MCA2104), see Barrie
et al. (Barrie et al., 2003)
for more details on the antibody]. 51ß7 is specific for Rb as it stained
SF 295 Rb-positive cells (NCI) but not the matching SF 539 cells
(NCI; Rb deletion). The other antibody was polyclonal rabbit:
anti-phospho histone H3 (Upstate Biotechnology, New York, USA). For BrdU,
anti-phospho histone H3, p16 and sarcomeric proteins staining, cultured cells
were fixed, after rinsing with PBS, with 100% methanol at -20°C for 5
minutes. Cells were processed for BrdU and MyHC staining as previously
described (Tanaka et al.,
1999
). PKH-labelled cells were fixed with 4% paraformaldehyde (10
minutes), permeabilised with 0.5% saponin (BDH) in PBS for 30 minutes and
stained for MyHC. Cells labelled with dextrans were fixed in 4%
paraformaldehyde in PBS containing 0.2% Triton X-100 pH 7.4 for 10 minutes and
stained for BrdU and MyHC. Cells containing alkaline phosphatase were fixed
with acid alcohol (5% glacial acetic acid in ethanol) at -20°C for 5
minutes. Endogenous alkaline phosphatase activity was destroyed by incubation
in PBS at 65°C for 15 minutes, and cells were developed using ELF-97
(Molecular Probes). Cells for Rb staining were fixed in 4% PFA for 5 minutes
followed by permeabilisation with TBS-0.1% Tween 20 for 10 minutes.
Phosphatase treatment was performed by incubating 100 U/coverslip of lambda
phosphatase (NEB) at 30°C for 30 minutes. Controls were performed using
phosphatase inhibitors (NAF: 10 mM; B-glycerolphosphate: 10 mM). Controls for
all antibodies were performed by omitting primary antibody incubation or by
using mouse IgG as a primary. DNA was stained with Hoechst 33258 (1
µg/ml).
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Results |
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In an initial analysis of cell cycle progression, we determined if cardiomyocytes could incorporate BrdU and undergo mitosis as assessed by staining for phosphorylated histone H3. The cells entered S phase (Fig. 2A,B and D) with a peak at 10 days after plating (Fig. 2D) when 25.8% were strongly labelled with a pulse of BrdU. A peak of mitotic activity was also observed at 10 days (Fig. 2C,D), and mitotic cells were readily detected under phase contrast optics with their prominent chromosomes (Fig. 2E1,E2). They often remained flat and attached to neighbouring cells, and during mitosis most of the myofibrils seem to have disassembled and the remaining were seen in the cell periphery (Fig. 2E1-E4). Occasionally, myofibrils extended into the cleavage furrow at cytokinesis. The daughter cells often resumed beating after division.
|
The time course and extent of DNA synthesis
(Fig. 2D) were comparable to
those reported for adult newt ventricular cells after injury in situ
(Bader and Oberpriller, 1979).
In Bader and Oberpriller's experiment, the tip of the ventricles was cut,
minced and grafted back into the ventricle in order to increase the number of
myocytes near the wound surface. Animals were sacrificed 1 hour after being
injected intraperitoneally with tritiated thymidine. The percentage of
labelled cells in the minced graft (morphological criterion used to
distinguish cardiomyocytes from other cells) was determined. They reported a
peak of DNA synthesis at 16 days after injury where 24% of the cells in the
graft incorporated the label. These similarities suggest that this culture
system is appropriate to study the mechanisms regulating plasticity.
Progression and arrest in single cells; S phase and mitosis
In several mammalian species, cardiomyocytes become polyploid after birth
as a result of a G2 arrest or inability to finish cytokinesis
(Brodsky, 1991;
MacLellan and Schneider, 2000
;
Soonpaa et al., 1996
). In
order to assess the proliferative potential of newt cardiomyocytes, we
determined the proportion of these cells that enter S phase and the subsequent
progression of each cell through the cycle. We analysed the progression of
single cells by following them for 18 days by time lapse microscopy.
Cardiomyocytes were plated onto a dish scored with a numbered grid and
pictures of the grid squares were taken once or twice a day. Since cells did
not move between different squares, we could follow every cell division in
each square for 18 days. To avoid any effect of density-dependent inhibition
of mitosis, the squares selected for analysis had on average only 2-3 cells.
In order to identify which cells were entering the cycle and synthesising DNA,
cells were continuously labelled in three experiments with
[14C]thymidine for 15 days, followed by a further 3 days in
unlabelled medium. This incubation in unlabelled medium was chosen to allow
labelled cells to finish mitosis before the end of the experiment. The 3 day
period was chosen based on the average duration of (G2+M) phases in blastemal
cells in regenerating limbs, which is 43 hours
(Wallace and Maden, 1976
).
After analysis by autoradiography, we observed that 75% of the cells initially
chosen for analysis entered S phase, and that 76% of those subsequently
entered mitosis. These results show that the majority of adult newt
cardiomyocytes can be activated to enter S phase, and although a portion of
these cells appear to undergo a subsequent block, 60% have the ability to
progress into mitosis.
Progression and arrest in single cells; mitosis and cytokinesis
Approximately 29% of the initial cells (n=195) progressed through
one or more complete rounds of cell division (including karyokinesis and
cytokinesis) giving rise to beating mononucleate progeny
(Table 1,
Fig. 3A,B). Some cells gave
rise to clones that showed weaker and more disorganised staining for MyHC,
compared to cells that did not divide (Fig.
3A, final panel). A detailed description of the variety of
lineages produced by dividing clones is illustrated in
Fig. 3B. Although there was
great variation of the proliferative potential between different clones, there
was a tendency for sister cells to be similar in two important respects
(Fig. 3B). First, in 52
divisions that gave rise to mononucleate cells and occurred at least 5 days
before the end of the experiment, there was a significant propensity for
symmetric divisions. Second, there was a correspondence in cell cycle time
between sibling cells that was also significant. The detailed analysis of both
parameters is given in the supplementary information
(http://jcs.biologists.org/supplemental).
|
|
Approximately 31% of the initial cardiomyocytes gave rise to a binucleate cell in their first mitosis (Table 1, Fig. 3C). We observed a small number of candidate fusion events between non-sister cells (less than 7%) but most multinucleate cells (2 or more nuclei) clearly resulted from incomplete mitosis (Fig. 3C). This was apparently due to a problem in resolving the cleavage furrow, as mitosis often resulted in two partially separated cells with distant nuclei (Fig. 3C, at 12 days), which eventually became closer (Fig. 3C, 13 days). Interestingly, 19% of the multinucleate cells subsequently entered S phase and completed mitosis and cytokinesis, giving rise to a variety of outcomes (Fig. 3D); this shows that the formation of a multinucleate cell does not preclude further proliferation. In the population that divides more than once (Fig. 3B,D) most of the cells that go through one complete cycle also finish cycles in subsequent divisions.
The finding that more than half of the cardiomyocytes can undergo
karyokinesis and that half of these can successfully complete cell division
was confirmed using another technique. Cells were labelled with a fluorescent
tracker dye PKH-26 (Sigma), seeded onto a gridded surface and adjusted so
there was one labelled cell per square (see Materials and Methods). This
fluorescent tracker dye is coupled to long aliphatic tails that incorporate
into lipid regions of the cell membrane. PKH-26 has been characterised in a
wide variety of systems for in vitro and in vivo cell tracking applications
and has been used previously on newt cells
(Kumar et al., 2000). After 24
days in culture we observed that 35% of the initial cardiomyocytes
(myosin-positive cells) had divided at least once
(Fig. 4A) and that 38% of the
initial cells had become multinucleate
(Fig. 4B). The fact that more
cells in this experiment became multinucleate, as compared to those in the
time lapse experiment described above, could be because of the longer
incubation time or the unavoidable inclusion of putative fusion events
(Przybylski and Chlebowski,
1972
) or membrane overlapping events in this category.
|
Our results clearly indicate that the ability to enter S phase is a general
property of the differentiated state in adult cardiomyocytes. This is possibly
related to the similar property in other newt differentiated cells, such as
the cultured skeletal myotube, where at least 75% of the cells may enter S
phase (Tanaka et al., 1997).
This similarity suggests that the same mechanism may underlie entry into S
phase in these cells.
S phase re-entry is enhanced by mammalian serum and dependent on Rb
regulation
To determine whether DNA synthesis in newt cardiomyocytes was stimulated by
mammalian serum, we performed a dose response assay for FBS. We found that
newt cardiomyocytes respond to serum by S phase entry, a response that is
maximal at about 10% FBS (Fig.
5A).
|
In order to analyse the phosphorylation state of the retinoblastoma protein
in the newt cardiomyocyte, we used a monoclonal antibody that specifically
recognises an epitope that includes phosphoserine 608 in human Rb and is
conserved in the newt protein (Barrie et
al., 2003; Tanaka et al.,
1997
). This residue is hypophosphorylated in cells in G0/G1,
becomes phosphorylated prior to entry into S phase, and remains phosphorylated
throughout the cell cycle (Zarkowska et
al., 1997
). This antibody stained the nucleus of 59% of the
cardiomyocytes at 9 days after plating in medium containing 10% FBS
(Fig. 5B,C), and the staining
was significantly diminished by phosphatase digestion of the fixed cells (see
Materials and Methods). This result indicates that cell-cycle associated Rb
phosphorylation arises in these cells. The presence of Rb phosphorylation in
the overall population was confirmed using immunoprecipitation followed by
western blotting using a pan specific Rb antibody (data not shown).
In order to evaluate the functional role of Rb in S phase entry, we
injected adult cardiomyocytes with a plasmid encoding human
p16INK4, a CDK inhibitor that specifically inhibits CDK4/6
(Ruas and Peters, 1998). The
regulation of Rb activity, and possibly one other member of the pocket protein
family, is absolutely required for p16-mediated cell cycle arrest
(Bruce et al., 2000
;
Lukas et al., 1995
;
Medema et al., 1995
). After
exposure to a pulse of BrdU, the cells were stained with antibodies to BrdU,
human p16INK4 and MyHC. Expression of the p16INK4
protein produced an approximately 13-fold inhibition of S phase entry relative
to uninjected cells, whereas cells injected with a control plasmid were only
inhibited 1.3 fold (Table 2). We conclude that a serum-activated pathway leading to phosphorylation of Rb is
a strong candidate to mediate re-entry to the cell cycle by the adult
cardiomyocyte.
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Discussion |
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Although culture conditions may enhance dedifferentiation in cardiomyocytes
(Claycomb, 1991;
Eppenberger et al., 1988
) and
could in principle induce an artifactual response, several lines of evidence
suggest that the results described in this work reflect the properties of the
population of newt cardiomyocytes after injury and account for them at the
single cell level. First, the cells in long term culture show a time
dependence and extent of entry into S phase
(Fig. 2D) which is comparable
to that observed after injury to the newt ventricle
(Bader and Oberpriller, 1979
;
Bader and Oberpriller, 1978
).
Second, 19% of the cells go through more than one round of cell division in
culture which may account for the 2.5 fold increase in the number of
cardiomyocytes described in vivo, after injury
(Bader and Oberpriller, 1979
).
Finally, the blocks to cell cycle progression described here are in agreement
with the observations that 45 days after mincing the tip of the newt ventricle
and grafting it back to the heart, 6% of the cells in the graft are binucleate
and 7% are mononucleate with a polyploid nucleus
(Oberpriller et al., 1989
;
Oberpriller et al., 1995
). In
control animals, only 1% of the cardiomyocytes have a polyploid nucleus and
less than 1% are binucleate (Oberpriller
et al., 1989
).
An adult newt has an extraordinary regenerative ability, being able to
regenerate not only large sections of its heart, but also its jaws, lens,
retina, limbs and tail in response to tissue damage or removal. The
regenerative ability of adult urodeles is associated with high plasticity of
the differentiated state (Brockes and
Kumar, 2002). This is manifest in different ways depending on
tissue type. Iris pigmented epithelial cells transdifferentiate and
proliferate during lens regeneration
(Eguchi et al., 1974
).
Multinucleate newt myotubes and myofibres re-enter the cell cycle and undergo
conversion to mononucleate cells during limb and tail regeneration
(Echeverri et al., 2001
;
Kumar et al., 2000
;
Lo et al., 1993
;
Velloso et al., 2000
). We have
shown that the majority of newt cardiomyocytes can enter into S phase and the
differentiated state is compatible with complete cycles of division in 29% of
newt cardiomyocytes. In each of these cases of plasticity, there is entry into
the cell cycle and it is restricted to the zone adjacent to the wound
(Brockes and Kumar, 2002
;
Oberpriller et al., 1989
). Our
results suggest that the same pathway drives newt cardiomyocytes and skeletal
myotubes into S phase (Tanaka et al.,
1997
), since in both cell types this is enhanced by mammalian
serum and is dependent on Rb inactivation, as evidenced by the strong
inhibitory activity of p16INK4. Work of Sadoshima and Izumo
(Sadoshima et al., 1997
)
suggests that serum leads to Rb phosphorylation but not DNA synthesis in
cultured neonatal rat cardiomyocytes. To explain that difference it will be
necessary to investigate how factors in mammalian serum stimulate entry into S
phase in newt cardiomyocytes, namely whether Rb phosphorylation is regulated
differently.
A surprising result from our work is that newt cardiomyocytes have a
heterogeneous proliferative potential. Although all cells are exposed to
serum-containing medium, only a small subset seem to be responsible for the
increase in cell number observed upon regeneration. The behaviour of the
remaining cells resembles that observed for their mammalian counterparts at
several stages of development. The G2/M boundary and the ability to undergo
cytokinesis have long been recognised as critical checkpoints to the
proliferation of mammalian cardiomyocytes. Thus cardiomyocytes in several
mammalian species become polyploid and/or multinucleate after birth
(Brodsky, 1991;
MacLellan and Schneider, 2000
;
Poolman et al., 1998
;
Soonpaa et al., 1996
). In
cases where neonatal or adult mammalian mouse and rat cardiomyocytes traverse
S phase, mitotic figures are rarely seen and cytokinesis is not observed
(Claycomb and Bradshaw, 1983
;
MacLellan and Schneider, 2000
;
Soonpaa and Field, 1998
).
Finally, the presence of striated myofibrils in the equatorial region of the
cell has been noted as a possible factor in the formation of binucleate
cardiomyocytes in neonatal mammals (Li et
al., 1997
), and this was also observed here for the newt
cells.
Why do newt cardiomyocytes show this heterogeneity in proliferative
potential? The cells do not show any apparent distinction in terms of their
differentiated state as they have very homogeneous morphology, they all
express muscle markers and contract (our observations in culture) and they are
all quiescent in the adult newt
(Oberpriller et al., 1989).
However, a clue to this variable behaviour is the fact that these
cardiomyocyte clones show a similar pattern of cell division to the one
previously described for embryonic cardiomyocytes
(Burton et al., 1999
). We
found that sister-cell cardiomyocytes are significantly correlated, both in
terms of undergoing a subsequent cell cycle and also in respect of their cell
cycle time. It may be that this subgroup of cells has not undergone the
complete programme of terminal differentiation, and the absence of signals
keeps these cells quiescent in a non-injured animal. A molecular comparison of
the cells in this culture should help us to analyse the regulation of the
differentiated state and cell cycle progression in an adult cardiomyocyte.
The recent finding that zebrafish can regenerate the heart through
cardiomyocyte division (Poss et al.,
2002) shows that the potential for cardiomyocyte division is more
widespread then previously thought. The similarities between the regulation of
the cell cycle of newt and mammalian cardiomyocytes suggest that the large
difference in regenerative ability may reflect differences in regulation of
the same pathways. Consequently, one might expect that such differences could
be subject to genetic variability. It is noteworthy that cardiac repair has
recently been described after cryogenic infarction of the right ventricle in
the MRL strain of mice (Leferovich et al.,
2001
). This strain has an enhanced capacity to heal surgical
wounds, a complex trait that maps to at least seven genetic loci, and
significant re-entry to S phase was noted after injury to the heart
(Leferovich et al., 2001
).
Additionally, it is possible that mammalian cardiomyocytes may also display a
heterogeneous proliferative potential, as telomere shortening has been shown
in a small percentage of adult rat cardiomyocytes
(Anversa and Nadal-Ginard,
2002
; Kajstura et al.,
2000
). The results presented here raise the possibility that heart
regeneration through cardiomyocyte proliferation, while not normally a
significant occurrence, might become possible in mammals. It is worthwhile to
explore further the possibility that mammalian cardiomyocytes may also show a
heterogeneous proliferative potential and to investigate whether there may
exist populations more susceptible to stimulation to proliferate.
Additionally, the newt cardiomyocyte culture system offers an opportunity to
further analyse the molecular regulation of the differentiated state and cell
cycle progression in an adult cardiomyocyte by directly comparing cells with
different abilities to proliferate. These efforts might complement the current
approaches to heart regeneration that are based on implantation of cells
(Grounds et al., 2002
;
Kessler and Byrne, 1999
;
Orlic et al., 2001
;
Reinlib and Field, 2000
).
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Acknowledgments |
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![]() |
Footnotes |
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* Present address: Department of Genetics, University of Cambridge, Downing
Site, Cambridge CB2 3EH, UK
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