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Breaking Down Cell-Cycle Barriers in the Adult Heart
Kelly M. Regula, Lorrie A. Kirshenbaum
O
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ver the past several years, there has been considerable interest in the ability of the adult myocardium to
re-enter the cell cycle. From a historical perspective,
the adult heart has generally been viewed as a nonproliferative organ with a limited and meager capacity for de novo
myocyte regeneration and/or self-renewal after injury.1,2 After birth, cardiac myocytes are believed to irreversibly exit
from the cell cycle. As a result, growth of the postnatal heart
occurs by hypertrophic rather than hyperplasic processes. The
loss of functional cardiac myocytes through apoptotic/necrotic processes during ischemia or traumatic injury in the
absence of de novo cell regeneration has been postulated to be
an underlying cause of ventricular remodeling and diminished ventricular pump function. However, the recent discovery of cardiac progenitors within the myocardium itself,3
coupled with reports documenting the presence of adult
myocytes synthesizing DNA in the nondiseased heart, has
challenged the current dogma and has led to a re-evaluation of
the true proliferative capacity of adult myocardium.4
Although it is generally accepted that adult ventricular
myocytes do possess some capacity for DNA synthesis, there
remains considerable debate as to whether differentiated
postmitotic ventricular myocytes readily traverse the cell
cycle, the frequency of synthetic events, and whether DNA
synthesis coincides with a concomitant increase in cell
number. Notwithstanding the acknowledged ability of adult
myocytes to synthesize DNA, these limited events alone do
not appear to be adequate to functionally restore diminished
ventricular performance in patients with heart failure postmyocardial infarction. Given that myocyte number can directly influence cardiac pump function, the ultimate therapeutic goal in reducing morbidity and mortality in patients
with heart failure would be either to preserve the number of
existing myocytes by suppressing cell death process or to
increase the number of functionally active myocytes by
regenerative processes. Recently, numerous exciting and
tantalizing strategies have been developed in efforts to
achieve this therapeutic endpoint. These include the phenotypic conversion of nonmuscle cells to the muscle lineage
with myogenic factors,5 the exogenous grafting of skeletal
and cardiac myoblasts directly into the diseased myocardium,6,7 the generation of de novo myocardium with pluripotent
and bone marrow-derived progenitors,8 and the genetic manipulation of key cellular factors to reactivate DNA synthesis
and cell-cycle progression.9,10
Whereas bone marrow-derived or embryonic-derived stem
cells have attracted considerable attention and hold promise
for the possibility of generating new cardiac muscle, several
issues surrounding their feasibility have been raised. In
particular, considerable debate still remains as to whether the
improved ventricular function after the implantation of pluripotent cells into the myocardium is a direct result of the
generation of new ventricular myocytes alone or from the
differentiation of cells into other cell lineages.11 Nevertheless,
overcoming these initial barriers hold promise for repopulating the diseased myocardium with cells capable of differentiating into functionally active ventricular myocytes. Alternative approaches for repopulating the adult myocardium with
functionally active ventricular myocytes include strategies
that manipulate cell-cycle progression.
Transition through the mammalian cell cycle is a tightly
regulated event involving the sequential and coordinated
activation of checkpoint proteins to ensure genomic stability
of the host cell DNA. Cell cycle is controlled by a complex
system of proteins that coordinate the biochemical events
within the cell necessary for cell division.12 These proteins
include cyclins, cyclin-dependent protein kinases (cdk), cdkactivating kinases (Cak), Cdk inhibitors, and members of the
retinoblastoma (Rb) family, which function at different stages
of the cell cycle.
Cyclins are the regulatory subunit for the activity of Cdks.
Cdks in complex with cyclins phosphorylate key checkpoint
proteins to drive otherwise quiescent cells into S phase. The
G1 cyclins include cyclin D1, D2, and D3 and cyclin E. These
are expressed consecutively throughout G1 phase and are
required for entry into S phase. D-type cyclins with Cdk 4 and
Cdk 6 play a critical role in the transition of a cell from Go to
G1 by facilitating the phosphorylation of Rb, p107, and p130.
Other cyclins, including cyclins A and B, are expressed
during S phase and G2/M. The assembly, activation, and
disassembly of cyclin B/cdc2 complexes modulate entry and
exit from mitosis.
The retinoblastoma gene product, Rb, was first identified
as a putative negative regulator of cell growth by the
observation that functional loss or inactivation of Rb correlated with the development of a variety of human malignancies.13 The biochemical mechanisms by which Rb suppresses
growth have largely been attributed to its ability to negatively
regulate the cell cycle by sequestering the transcription factor
E2F-1.14 The ability of Rb to interact with E2F-1 represents
a crucial step in the regulation of cellular E2F-1 activity.
The opinions expressed in this editorial are not necessarily those of the
editors or of the American Heart Association.
From The Institute of Cardiovascular Sciences, St. Boniface General
Hospital Research Centre and the Department of Physiology, Faculty of
Medicine, University of Manitoba, Winnipeg, Manitoba, Canada.
Correspondence to Dr Lorrie A. Kirshenbaum, Institute of Cardiovascular Sciences, St. Boniface General Hospital Research Centre, Room
3016, 351 Taché Avenue, Winnipeg, Manitoba R2H 2A6, Canada.
E-mail [email protected]
(Circ Res. 2004;94:1524-1526.)
© 2004 American Heart Association, Inc.
Circulation Research is available at http://www.circresaha.org
DOI: 10.1161/01.RES.0000134761.05562.a6
1524
Regula and Kirshenbaum
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E2F-1 is required for the activation of genes required for
DNA synthesis.12
Certain DNA tumor viruses encode transforming proteins
such as adenovirus E1A, human papilloma virus E6, and SV
40 large T antigen (SV40TAg). These proteins physically
interact with Rb and p107 and promote G1 exit of nonproliferating cells presumably by displacing E2F proteins.15
Expression of E1A proteins was found to reactivate cell-cycle
progression and DNA synthesis in postnatal ventricular myocytes.9 Similarly, expression of E2F-1 proteins in cardiac
muscle was sufficient to promote DNA synthesis in adult
ventricular myocytes in vitro and in vivo,10,16 supporting the
role of Rb and related family members as key regulators of
cell-cycle control in the heart. However, although these
exciting early studies hinted toward the possibility of manipulating cell-cycle constituents as a means to reactivate DNA
synthesis in the adult myocardium, one impediment to either
E1A or E2F-1 expression was an increased incidence of
apoptosis. This was attributed to the activation of the tumor
suppressor p53, a surveillance protein that protects against
aberrant or inappropriate DNA synthesis by initiating the cell
death program. Importantly, the cell death triggered by either
E1A or E2F-1 could be abrogated by Bcl-2 and related
adenovirus E1B proteins.9 Although E1A and the SV40TAg
share similarities in their ability to effectively interact with
Rb and p107, differences in the affinity for other cellular
factors, such as p300 as in the case of E1A and p53 in the case
of SV40TAg, may functionally account for biological differences of these oncoproteins. This point is perhaps best
highlighted by the fact that in contrast to E1A, SV40TAg can
promote G1 exit and augment growth of atrial and ventricular
myocytes without provoking apoptosis.17,18 This suggests that
alternative cellular binding partners (E1A versus SV 40 TAg)
may underlie differing abilities of either of the 2 proteins to
promote complete cell-cycle progression in the absence of
cell death.
As a step toward identifying putative cellular targets of the
SV40TAg that mechanistically underlie the ability of the
SV40TAg to override growth inhibitory signals by p53 to
promote G1 exit, studies by Loren J. Field’s laboratory
(Indiana University School of Medicine, Indianapolis, Ind.)
have identified a novel SV40TAg interacting protein in
cardiac muscle with a molecular weight of 193 kDa.19 The
exact function of p193 is undetermined; however, early
structure function studies revealed that full-length p193
protein is proapoptotic and provokes cell death when expressed in cells. Interestingly, deletion of the carboxyl terminus of p193 was found sufficient to support cell-cycle
re-entry and protect against p53-dependent and independent
apoptotic signals.19 In the same study, the authors demonstrated that inactivation of both p53 and p193 pathways in
cardiac-derived embryonic stem cells was required for E1Ainduced cell-cycle progression without cell death. These
studies position p193 as a key SV40TAg cellular target and
cell-cycle checkpoint protein that together with p53 coordinates signals that impinge on cell-cycle and cell death
pathways mediated by viral proteins. It further illustrates the
exciting possibility that functional inactivation of p193 may
Reactivation of DNA Synthesis in Adult Heart
1525
be required to overcome growth-arresting signals without
provoking cell death in the adult myocardium.
In this issue of Circulation Research, Nakajima et al20
extend these studies by testing the impact of dominantinterfering mutants of p193 and p53, respectively, on DNA
synthesis in the context of the normal and infarcted adult
myocardium. The authors demonstrate that cardiac restricted
expression of either transgene alone or in combination had no
apparent effect on DNA synthesis in the normal noninjured
myocardium. In contrast, however, a notable increase in
myocyte DNA synthesis was observed in the border zone of
infarcted hearts expressing either transgene. Interestingly,
despite the apparent increase in DNA synthesis by mutant
transgenes, cell-cycle reactivation was only observed in the
interventricular septa of transgenic mice expressing the p193
mutant. Moreover, a reduction in ventricular remodeling and
septal hypertrophy was observed, but only in mice expressing
mice the dominant-negative p193. No apparent difference in
the apoptotic index or caspase 3 activity was observed in the
septa of either p193 or p53 transgenic hearts after injury.
Detailed microscopic analysis revealed that minimal cardiac
fiber diameter was markedly reduced in the mutant p193
hearts in comparison to either mutant p53 or Lac Z control
hearts.
The studies presented are intriguing and indicate that
complete or partial inactivation of p193 and p53 activities
may be required to reactivate DNA synthesis in adult ventricular myocytes after myocardial injury. Perhaps most
compelling is the observed increase in DNA synthesis in the
interventircular septa of mutant p193 mice compared with
mutant p53 hearts or LacZ controls. These differential effects
suggest that the septum must be subjected to different
physiologic signals (loads) after injury compared with the
infarcted border zone. The findings also highlight the possibility for regional heterogeneity among cells within the
myocardium itself (septum versus ventricular free wall),
which may be differentially coupled to different subsets of
cell-cycle checkpoint proteins and regulated in a stimulusdependent manner. Alternatively, the preferential increase
DNA synthesis in the septum of the mutant p193 mice
compared with p53 mutant mice despite the abilities of both
to reactive DNA synthesis in the border zone may reflect
partial or incomplete unmasking of overlapping pathways
that regulate cell cycle.
Further, the data presented suggest that myocardial injury
likely activates conflicting but overlapping signals in attempts to limit or salvage damaged myocardium (Figure).
One pathway involved reinitiating cell cycle and repair,
whereas the other involved initiating cell death. The fact that
blockade of p53 function was sufficient to reactivate DNA
synthesis in the infarcted border zone without apoptosis
supports this contention. This raises the interesting possibility
that targeted inactivation of p193 and p53 cell-cycle checkpoints after myocardial infarction may permit G1 exit and
reactivation of the cell-cycle program without provoking cell
death.
The data presented provide new important insight into the
molecular switches that govern cell-cycle control and cell
death pathways in the adult myocardium, and support a
1526
Circulation Research
June 25, 2004
Model for the role of p193 and p53 for cell-cycle control and
apoptosis in adult cardiac muscle. Myocardial injury initiates
signals that are integrated by the cell for cell-cycle re-entry or
apoptosis. A, In the presence of functionally active p193 and
p53 proteins, the apoptotic program is preferentially activated.
B, Dual inactivation of both p193 and p53 checkpoint proteins
are required to reactivate the cell-cycle program in the adult
heart in absence of apoptosis.
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paradigm shift toward the regenerative capacity of the adult
myocardium. The question that arises is whether p193 or p53
regulates equivalent pathways in other models of cardiac
injury or cardiomyopathies, or whether inducible expression
or conditional ablation of p193 would have alternative effects
on DNA synthesis and mitosis in the nonischemic or infarcted
adult heart. Also, it is not clear whether expression of the
p193 mutant alone would be sufficient to prevent progression
to heart failure, given that only 1 time point was examined.
Nevertheless, the data presented are intriguing and support
the exciting possibility of manipulating cellular factors involved in cell-cycle control for reactivating DNA synthesis in
the adult heart. The data further suggest that genetic manipulation of p193 or related cell-cycle regulators may have
therapeutic advantage in modulating ventricular remodeling
and improve ventricular performance by enhancing regenerative cardiac growth.
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KEY WORDS: regeneration 䡲 cell cycle
proteins 䡲 ventricular myocytes
䡲
apoptosis
䡲
tumor-suppressor
Breaking Down Cell-Cycle Barriers in the Adult Heart
Kelly M. Regula and Lorrie A. Kirshenbaum
Downloaded from http://circres.ahajournals.org/ by guest on June 14, 2017
Circ Res. 2004;94:1524-1526
doi: 10.1161/01.RES.0000134761.05562.a6
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