Download Therapeutic opportunities for cell cycle re-entry

Cardiovascular Research 64 (2004) 395 – 401
www.elsevier.com/locate/cardiores
Review
Therapeutic opportunities for cell cycle re-entry and cardiac regeneration
Kelly M. Regula, Marek J. Rzeszutek, Delphine Baetz,
Charit Seneviratne, Lorrie A. Kirshenbaum*
The Institute of Cardiovascular Sciences, St. Boniface General Hospital Research Centre, and the Department of Physiology, Faculty of Medicine,
University of Manitoba, Rm. 3016, 351 Taché Avenue, Winnipeg, Manitoba, R2H 2A6, Canada
Received 17 June 2004; received in revised form 20 August 2004; accepted 3 September 2004
Time for primary review 20 days
Abstract
Over the last two decades, considerable effort has been made to better understand putative regulators and molecular switches that govern
the cell cycle in attempts to reactivate cell cycle progression of cardiac muscle. Rapid advancements on the field of stem cycle biology
including evidence of cardiac progenitors within the adult myocardium itself and reports of cardiomyocyte DNA synthesis, which each
suggest that the adult myocardium may in fact have the capacity for de novo myocyte regeneration. Augmenting cardiomyocyte number by
targeting specific cell cycle regulatory genes or by stimulating cardiac progenitor cells to differentiate into cardiac muscle may be of
therapeutic value in repopulating the adult myocardium with functionally active cells in patients with end-stage heart failure. Advancements
in the area of cardiomyocyte cell cycle control and regeneration and their therapeutic potential are discussed.
D 2004 European Society of Cardiology. Published by Elsevier B.V. All rights reserved.
Keywords: Cell cycle; DNA synthesis; Ventricular myocytes; Regeneration
1. Introduction
Heart disease represents a major cause of morbidity and
mortality and is reaching pandemic proportions worldwide.
In particular, the increased incidence of patients with heart
failure has largely been attributed to the widespread
adoption of western diet in developing and third world
countries. Risk factors including uncontrolled hypertension,
diabetes, smoking, sedentary life style, and viral infection
have been identified as key underlying factors that contribute to the demise of ventricular performance and heart
failure. Given the meager and limited ability of cardiac
muscle for repair and/or self-renewal after injury, an
inordinate loss of working ventricular muscle cells has been
suggested to be a predominant underlying cause of
contractile failure in patients with ischemic heart disease
or impaired coronary reserve.
* Corresponding author. Tel.: +1 204 235 3661; fax: +1 204 233 6723.
E-mail address: [email protected] (L.A. Kirshenbaum).
Following myocardial injury, a series of biochemical and
local neurohormonal events are initiated resulting in release
and activation of bioactive molecules such as angiotensin II,
endothelin, aldosterone, and inflammatory cytokines. These
act in a paracrine or autocrine manner to reprogram the
myocardium at the cellular and molecular levels [1]. Distinct
phenotypic changes at the cellular and subcellular levels
result in both structural and functional changes to the
myocardium resulting in heart hypertrophy. However, in a
number of individuals for reasons unknown, these adaptive
physiological processes eventually fail, resulting in a
diminished cardiac pump function and overt heart failure.
While the molecular events and signaling pathways that
underlie heart failure remain cryptic, there is general
consensus that heart failure results from one or more
mutually related events. In this context, defects in calcium
handling proteins of the sarcoplasmic reticulum [2,3],
increased oxidative stress, from a reduction in antioxidant
reserve [4,5], mitochondrial defects [6,7], genetic alterations
to key myofibrillar proteins [8] and extracellular matrix
proteins [9] have been identified to be crucial events in the
0008-6363/$ - see front matter D 2004 European Society of Cardiology. Published by Elsevier B.V. All rights reserved.
doi:10.1016/j.cardiores.2004.09.003
396
K.M. Regula et al. / Cardiovascular Research 64 (2004) 395–401
remodeling process that underlie ventricular dysfunction
and heart failure.
Recently, there has been considerable interest in the role
of apoptosis in heart failure. Undoubtedly, the acute loss of
working ventricular myocytes in the absence of de novo
myocyte regeneration could be seen as a major clinical
impediment and underlying event leading to heart failure.
There is now considerable evidence from studies in animals
as well as in humans that documents an increased apoptotic
index in the failing heart [10,11]. Whether the loss of cells
through an apoptotic process is adaptive, maladaptive, or
contributes to ventricular remodeling and failure is less clear
and remains an active area of inquiry. However, the
development of new therapeutic strategies to improve
cardiac cell number has been identified as a means to
improve cardiac function after injury. Although recent
studies have detected stem cells within the myocardium,
the impact of myocardial infarction outweighs the repair
capacity of these cells [12–14]. Pharmacological and
surgical revascularization is a cytoprotective treatment, but
so far, they cannot compensate for cell loss. Inasmuch as
myocyte number can directly influence cardiac pump
performance, the definitive therapeutic goal in treating
patients with heart failure would be to either preserve the
number of pre-existing myocytes or to increase the number
of functionally active force generating cells through de novo
regenerative processes. Many exciting approaches have
been postulated in efforts to achieve this therapeutic
objective. These include the inhibition of apoptosis
[15,16], the transdifferentiation of nonmuscle cells to
muscle cells [17], the exogenous grafting of skeletal and
cardiac myoblasts into infarcted myocardium [18,19], the
incorporation of pluripotent bone marrow progenitors [20]
into the myocardium, and the genetic manipulation of key
cell cycle regulator factors to effects of cell cycle
progression and DNA synthesis in the postmitotic adult
myocardium [21,22].
2. Regulators of cell cycle—the basics
Organogenesis and the renewal of differentiated cell
types depend upon a delicate balance between the proliferation of cells and cell death. While this is true of most cell
types throughout their life, cardiomyocytes, among few
other cell types, withdraw from the cell cycle during the
perinatal period of development. Thereafter, cardiomyocyte
hypertrophy prevails as the primary mechanism of myocardial growth to support increases in myocardial demand and/
or to compensate for cardiomyocyte cell loss during cardiac
disease states [23–25].
Cell proliferation is a tightly controlled process mediated
by internal and external signals. Growth factors stimulate
the replication process, but it is an intricate intrinsic control
mechanism that determines whether the cell is ready to
proceed through the ordered set of events, the cell cycle,
which culminate in the production of two daughter cells.
The cell cycle consists of four phases (Fig. 1): M (Mitosis),
G1 (Gap 1), S (Synthetic), G2 (Gap 2), and M phase or
mitosis, the period of the cell cycle during which nuclear
division occurs. In a carefully orchestrated manner, the
nuclear envelope breaks down, chromosomes condense and
align at the metaphase plate, and duplicated chromosomes
are segregated to opposite poles of the cell. Cytokinesis
marks the end of mitosis. The period between one mitosis
and the next is known as interphase and consists of the G1,
S, and G2 phases of the cell cycle. Immediately following
mitosis and in response to appropriate mitogenic signals, the
Fig. 1. The mammalian cell cycle. Cyclins/Cdk complexes control entry and exit through the 4 phases of the cell cycle. Growth factors through the action of
Ras, Raf, and ERK trigger the synthesis of D-type cyclins which complex with Cdk4 or Cdk6 to regulate cell cycle progression through Go. The
phosphorylation of pRb by G1 cyclins (D1, D2, D3, and E)/Cdk complexes promotes the release of E2F-1 from Rb. Through its association with p300, E2F-1
activates the transcription of genes important for the G1 transition to S phase.
K.M. Regula et al. / Cardiovascular Research 64 (2004) 395–401
cell enters G1. G1 is one of two important periods of cell
growth that occur prior to the next mitosis. During G1, the
cell produces enzymes necessary for nucleotide metabolism,
which will take place during the next phase of the cell cycle,
S phase. If the cell is not committed to entering S phase, it
can enter a state of quiescence known as Go. Depending on
the cell type and environmental factors, Go can vary in
length from days to years. Once exited from Go, cells
progress to S phase during which DNA replication occurs.
G2 is the second important period of cell growth and
precedes mitosis. G2 ensures that DNA replication is
complete before mitosis can occur and is the period during
which the chromosomes start to condense.
Cell cycle progression is controlled by a complex system
of proteins that coordinate the biochemical events within
the cell necessary for cell division. These proteins include
cyclins, cyclin-dependent protein kinases (Cdk), Cdkactivating kinase (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. Each cyclin contains a region called
the cyclin box which is involved in the binding of specific
Cdks [26]. In complex with cyclins, they phosphorylate
proteins at critical serine and threonine residues to drive the
cell cycle. The G1 cyclins include cyclin D1, D2 and D3,
and cyclin E. They are expressed consecutively throughout
G1 and are required for entry into S phase. Complexes of
the D-type cyclins with Cdk4 and Cdk6 play a critical role
in a cell’s transition from Go to G1. Growth factors induce
synthesis of D-type cyclins via the Ras/Raf/ERK signaling
pathway. In cycling cells, cyclin D is constitutively
produced but continuously turned over as a result of
phosphorylation of a conserved threonine by glycogen
synthase kinase-3g, followed by proteasome degradation
[27]. Cyclin E is expressed later in G1 and together with
Cdk2 promotes entry into S phase. Moreover, G1 cyclins
facilitate phosphorylation of members of the Retinoblastoma family of pocket proteins (pRb, p107, and p130),
promoting the activation of cellular transcription factors that
are required for cell proliferation. Other cyclins, cyclin A
and B, are expressed during S phase and G2/M. DNA
synthesis is contingent upon complexes of cyclin A and
Cdk2. Cyclin B synthesis begins in S phase, and the protein
accumulates through to G2 phase as it forms a complex
with Cdc2. The assembly, activation, and disassembly of
cyclin B/Cdc2 complexes modulate entry and exit from
mitosis.
397
loss or inactivation of pRb correlated with the development
of a variety of human malignancies. These include
retinoblastoma, a rare inherited childhood eye tumor [29]
as well as breast, bladder, and prostate carcinomas as well as
glioblastoma and leukemia [30,31]. The biochemical property of pRb, which enables it to suppress growth, has largely
been attributed to its ability to negatively regulate the cell
cycle by sequestering E2F transcription factors, particularly
E2F-1, via its large A/B pocket domain (amino acid residues
379–869). Hypophosphorylated pRb sequesters E2F1 in G0
and G1. The phosphorylation of pRb by G1 cyclins (D1,
D2, D3, and E)/Cdk complexes promotes the release of
E2F-1 from the inhibitory pocket. E2F-1 is then free to
modulate the transcription of genes important for the G1
transition to S phase, including DNA polymerase a, c-myc,
dihydrofolate reductase, and thymidine kinase. In S phase,
E2F-1 interacts with and becomes phosphorylated by cyclin
A/Cdk2 resulting in its inactivation [32], as shown in Fig. 1.
Subsequently during M phase, Rb becomes dephosphorylated, enabling it to reassociate with E2F during Go.
To date, seven homologues of E2F (E2F-1 to E2F-7)
have been identified. pRb interacts with E2F-1 to -3, while
E2F-4 and E2F-5 primarily associate with p107 and p130
[33,34]. E2F-6 does not interact with pocket proteins [35].
An association between E2F-7 and a pocket protein is
undetermined. In cells, E2F-1 to E2F-6 bind to DNA in
heterodimeric complex with a member of the DRFT1polypeptide family (DP1 and DP2) [36]. E2F-7 is unique in
that it binds to DNA independent of a DP partner [37]. The
basic structure of E2F proteins includes an N-terminal
DNA-binding domain, a central dimerization domain, and a
C-terminal transactivation domain. The C-terminal transactivation domain contains the docking site for the pocket
proteins. E2F-1, E2F-2, and E2F-3 also contain an Nterminal cyclin/Cdk binding site as well as a nuclear
localization sequence, whereas E2F-6 lacks the transactivation domain. E2F-7 is comprised only of two independent
DNA binding domains, setting it apart from the other E2Fs.
While the majority of E2F family members share significant
structural homology, they differ with respect to their abilities
to promote cell growth and cell death, respectively. E2F-1,
E2F-2, and E2F-3 can override G1-mediated growth arrest
and reactivate DNA synthesis in quiescent cells. In contrast,
E2F-4 and E2F-5 marginally stimulate S phase entry
[38,39]. The mechanism by which E2F factors influence
cell growth may be related to their ability to interact with
specific pocket protein members, basal transcription factors
such as TFIIH and TBP, as well as recruit the CBP/p300
histone acetylase [40,41].
3. The tumor suppressor proteins
The retinoblastoma gene product pRb is a tumor
suppressor protein and member of the bpocket proteinQ
family consisting of pRb, p107, and p130 (reviewed in Ref.
[28]). pRb was first identified as a putative negative
regulator of cell growth by the observation that functional
4. Regulation of Cdk activity during the cell cycle
The major transitions of the cell cycle are controlled by
the cyclin/Cdk complexes and as such, numerous mechanisms have evolved to regulate this important function
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K.M. Regula et al. / Cardiovascular Research 64 (2004) 395–401
(reviewed in Ref. [42]). Regulation begins with assembly of
the cyclin/Cdk complex. Cyclins display a periodic pattern
of expression. They accumulate during different phases of
interphase and then rapidly degrade before the next round of
the cell cycle. Once the Cdk has associated with its cyclin
partner, it is not catalytically active until it is phosphorylated
on a threonine around position 160 by Cdk-activating kinase
(CAK), an enzyme comprised of Cdk7 and cyclin H. The
cyclin/Cdk complexes are also subject to negative regulation. Proteins known as Cdk inhibitors (CKIs) bind to and
inhibit the activity of cyclin/Cdk complexes. CKIs are
categorized into two families: the Cip/Kip family and the
Ink4 family. Members of the Cip/Kip family which include
p21Cip1, p27Kip1, and p57Kip2 bind to and inhibit cyclin D-,
cyclin E-, and cyclin A-dependent kinases, whereas
members of the Ink4 family (p16INK4a, p15INK4b, p18INK4c,
and p19INK4d) bind directly to Cdk4 and Cdk6, and thus
inhibit only D-type cyclin/Cdk4/6 complexes. In addition,
Cdk2 and Cdc2 are regulated by inhibitory phosphorylation
of N-terminal tyrosine14 and tyrosine15 [43]. Members of
the Cdc25 family of protein phosphatases reverse this effect,
thus enabling Cdk activity.
5. Promoting cell cycle re-entry in cardiac myocytes
Cell cycle withdrawal is believed to be the primary
mechanism for the dramatic reduction in proliferative
capacity of cardiomyocytes. In the human heart, cardiomyocytes cease dividing between 3–6 months after birth,
whereas in the postnatal rat heart, this occurs between days
3 and 4, with evidence of Go/G1cell cycle arrest [44–47].
These and other studies have supported the longstanding
dogma that cardiomyocytes do not regenerate. Challenging
this dogma, however, are two recent reports of cardiomyocyte cell division in the failing and infarcted human heart
[48,49]. If adult myocytes do in fact undergo cell division, it
does not appear to be sufficient to balance the loss of
working myocytes that compromises heart function leading
to heart failure [50,51]. Therefore, among the therapeutic
strategies to reduce the morbidity and mortality of patients
with heart failure, reactivating cardiomyocyte proliferation
through the regulation of cell cycle regulators is particularly
appealing. Proliferating cells express high levels and activity
of cell cycle promoting factors such as cyclins D1, E, A, and
B, Cdk2, Cdk4/6, and Cdc2 as well as E2F family members
and low levels of the Cdk inhibitors p21Cip1and p27Kip1
[44,45,52]. In contrast, a marked down regulation of cell
cycle promoting factors and increased activity and/or
expression of cyclin/Cdk inhibitors is observed in the
postmitotic adult heart [44,45,53]. Immunodepletion of
p21Cip1 has been shown to increase Cdk2 activity in adult
cardiac cell lysate [45]. Strategies to induce cardiomyocyte
proliferation have therefore been directed towards the
genetic manipulation of cell cycle regulatory factors to
promote cell cycle progression.
In recent years, an increasing number of reports have
suggested that cardiomyocyte cell cycle reentry can be
achieved through the manipulation of specific cell cycle
regulatory proteins. For example, constitutive expression of
c-myc mRNA in the heart resulted in enhanced hyperplastic
growth during fetal development, presumably through the
activation of cyclin E/Cdk2 [54,55]. In another study,
transgenic hearts that expressed high levels of Cdk2 mRNA
showed significantly increased levels of Cdk4 and cyclins
A, D3, and E and augmented DNA synthesis in the adult
hearts but lacked significant differences in heart weight to
body weight ratios compared to wild-type mice [56].
Transgenic mice overexpressing cyclin D1 showed elevated
levels of Cdk2 and Cdk4 as well as increased DNA
synthesis and multinucleated cardiomyocytes in the mature
animal [57]. While the manipulation of these cellular factors
to promote cardiomyocyte cell division in the postmitotic
adult heart has been mostly unsuccessful, p27Kip1 knockout
mice have been shown to have larger hearts with more
cardiomyocytes compared to p27Kip1+/+ mice. Analysis of
myocytes demonstrated a greater proportion of cells in S
phase and fewer in Go/G1. The authors suggest that loss of
p27Kip1 prolongs the duration of cardiomyocyte division
after birth [58]. Interestingly, postnatal cardiomyocyte
hyperplasia has also been recently demonstrated in cyclin
A2 overexpressing mice [59]. Cyclin A2, which is silenced
in the postnatal heart, is an important mediator of S phase in
cells. Chaudhry et al. report cardiac enlargement and
evidence of mitosis during postnatal development of cyclin
A2 transgenic hearts that is absent in wild-type hearts.
Independently, these studies support the possibility of
modifying a cell’s internal cell cycle clock to extend its
proliferative potential [60].
The ability of Rb and related family members to
sequester E2F proteins also plays an important role in cell
cycle control. Viral proteins such as adenovirus E1A, human
papilloma virus E6, and SV40 large T antigen (SV40) that
bind to and inactivate Rb and p107, promote G1-exit of
nonproliferating cells. In postnatal ventricular myocytes,
E1A expression results in partial cell cycle reactivation
including DNA synthesis with cells arresting in G2/M [21].
Moreover, expression of E2F-1 in cardiac muscle alone is
sufficient to reactivate DNA synthesis in adult ventricular
muscles in vitro and in vivo [22,61], supporting the role of
Rb and related family members as key regulators of cell
cycle control. However, a compounding effect of reactivating DNA synthesis in cardiac muscle by E1A or E2F-1
proteins is an increased incidence of apoptosis that can be
abrogated by Bcl-2, adenovirus E1B proteins, or insulin-like
growth factor (IGF-1) [22,62]. In contrast, delivery of SV40
to cardiomyocytes has been shown to promote G1 exit and
augment growth of atrial and ventricular myocytes without
provoking apoptosis [63–65]. These differences may reflect
differential effects conferred by binding partners. Indeed,
SV40 has been found to have two unique binding partners,
the proapoptotic proteins p193 and p53. Interestingly,
K.M. Regula et al. / Cardiovascular Research 64 (2004) 395–401
inactivation of both p53 and p193 pathways block E1Ainduced apoptosis, allowing an unimpeded proliferative
response similar to that seen with SV40. Moreover, these
results suggest that p53 and p193 coordinate the signals
which control cell cycle and cell death pathways mediated
by viral proteins. Manipulation of one or more tumor
suppressor proteins in combination with increased expression of cyclin and CdK proteins may together be sufficient
to overcome the G2-M checkpoint without provoking
apoptosis.
399
Acknowledgements
This work was supported by grants to L.A.K. from the
Canadian Institutes for Health Research, an Interdisciplinary Heart Research Program grant from CHIR
(CHFNET). K.M.R. holds a studentship from the CIHR,
L.A.K. is a Canada Research Chair in Molecular
Cardiology.
References
6. Cell transplantation
An alternative approach for regenerating cardiac muscle
involves repopulating the damaged or diseased myocardium
with pluripotent stem cells. Stem cells are characterized by
their unique ability for prolonged self-renewal through cell
division and their ability to differentiate into one or more
mature cell types. Several recent studies have evaluated the
efficacy and feasibility of using pluripotent cells for as
transplantation therapy [66,67].
Embryonic stem cells (ESC) derived from the blastocyst
are pluripotent and capable of developing into different
cell types of the body. In contrast, however, adult stem
cells derived from bone marrow exhibit a more limited
ability to be phenotypically reprogrammed. Restrictions
placed on the use of embryonic stem cells for obvious
ethical considerations have limited their immediate experimental and clinical use, which has been the driving
impetus for developing alternative strategies using adult
stem cells for cell therapy. Advancements in the area of
stem cell biology raise the interesting possibility for de
novo generation or phenotypic conversion of endothelial
progenitor cells, bone marrow derived, or hematopoietic
stem cells into cells of the cardiac lineage [68–71].
Moreover, several reports now purport the exciting
prospects of using these cells to improve cardiac function
after injury [70]. Perhaps even more compelling is the
recent discovery of cardiac progenitor cells within the
adult myocardium itself [72,73]. These cells likely hold the
most promise as the bholy grailQ for regenerating adult
myocardium after injury.
7. Conclusions
Despite many unresolved issues, new innovative gene
therapy strategies to genetically reactivate DNA synthesis
and cell progression in the adult myocardium together with
cell transplantation therapies to repopulate damaged or
diseased myocardium with pluripotent progenitors each
suggest the exciting possibility for regenerating cardiac
muscle postmyocardial infarction and/or enhancing cardiac
performance in patients with heart failure or diminished
cardiac performance.
[1] Schwartz K, Chassagne C, Boheler KR. The molecular biology of
heart failure. J Am Coll Cardiol 1993;22:30A – 3A.
[2] Mercadier JJ, Lompre AM, Duc P, Boheler KR, Fraysse JB,
Wisnewsky C, et al. Altered sarcoplasmic reticulum Ca2(+)-ATPase
gene expression in the human ventricle during end-stage heart failure.
J Clin Invest 1990;85:305 – 9.
[3] Afzal N, Dhalla NS. Differential changes in left and right ventricular
SR calcium transport in congestive heart failure. Am J Physiol
1992;262:H868–74.
[4] Hill MF, Singal PK. Antioxidant and oxidative stress changes during
heart failure subsequent to myocardial infarction in rats. Am J Pathol
1996;148:291 – 300.
[5] Singal PK, Kirshenbaum LA. A relative deficit in antioxidant reserve
may contribute in cardiac failure. Can J Cardiol 1990;6:47 – 9.
[6] Liu J, Wang C, Murakami Y, Gong G, Ishibashi Y, Prody C, et al.
Mitochondrial ATPase and high-energy phosphates in 5 failing
hearts. Am J Physiol Heart Circ Physiol 2001;281:H1319–26.
[7] Sorescu D, Griendling KK. Reactive oxygen species, mitochondria,
and NAD(P)H oxidases in the development and progression of heart
failure. Congest Heart Fail 2002;8:132 – 40.
[8] Schwartz K, Carrier L, Lompre AM, Mercadier JJ, Boheler KR.
Contractile proteins and sarcoplasmic reticulum calcium-ATPase gene
expression in the hypertrophied and failing heart. Basic Res Cardiol
1992;87(Suppl 1):285 – 290.
[9] Brilla CG, Rupp H. Myocardial collagen matrix remodeling and
congestive heart failure. Cardiologia 1994;39:389 – 93.
[10] Narula J, Haider N, Virmani R, DiSalvo TG, Kolodgie FD, Hajjar RJ,
et al. Apoptosis in myocytes in end-stage heart failure. N Engl J Med
1996;335:1182 – 9.
[11] Wencker D, Chandra M, Nguyen K, Miao W, Garantziotis S, Factor
SM, et al. A mechanistic role for cardiac myocyte apoptosis in heart
failure. J Clin Invest 2003;111:1497 – 504.
[12] Anversa P, Kajstura J. Ventricular myocytes are not terminally
differentiated in the adult mammalian heart. Circ Res 1998;83:
1 – 14.
[13] Nadal-Ginard B, Kajstura J, Leri A, Anversa P. Myocyte death,
growth, and regeneration in cardiac hypertrophy and failure. Circ Res
2003;92:139 – 50.
[14] Orlic D. Stem cell repair in ischemic heart disease: an experimental
model. Int J Hematol 2002;76(Suppl 1):144 – 5.
[15] Regula KM, Ens K, Kirshenbaum LA. Inducible expression of BNIP3
provokes mitochondrial defects and hypoxia-mediated cell death of
ventricular myocytes. Circ Res 2002;91:226 – 31.
[16] Kirshenbaum LA, De Moissac D. The bcl-2 gene product prevents
programmed cell death of ventricular myocytes. Circulation 1997;96:
1580 – 5.
[17] Yeh ET, Zhang S, Wu HD, Korbling M, Willerson JT, Estrov Z.
Transdifferentiation of human peripheral blood CD34+-enriched cell
population into cardiomyocytes, endothelial cells, and smooth muscle
cells in vivo. Circulation 2003;108:2070 – 3.
[18] Murry CE, Wiseman RW, Schwartz SM, Hauschka SD. Skeletal
myoblast transplantation for repair of myocardial necrosis. J Clin
Invest 1996;98:2512 – 23.
400
K.M. Regula et al. / Cardiovascular Research 64 (2004) 395–401
[19] Dowell JD, Rubart M, Pasumarthi KB, Soonpaa MH, Field LJ.
Myocyte and myogenic stem cell transplantation in the heart.
Cardiovasc Res 2003;58:336 – 50.
[20] Orlic D, Kajstura J, Chimenti S, Bodine DM, Leri A, Anversa P.
Transplanted adult bone marrow cells repair myocardial infarcts in
mice. Ann N Y Acad Sci 2001;938:221 – 9.
[21] Kirshenbaum LA, Schneider MD. Adenovirus E1A represses cardiac
gene transcription and reactivates DNA synthesis in ventricular
myocytes, via alternative pocket protein- and p300-binding domains.
J Biol Chem 1995;270:7791 – 4.
[22] Kirshenbaum LA, Abdellatif M, Chakraborty S, Schneider MD.
Human E2F-1 reactivates cell cycle progression in ventricular
myocytes and represses cardiac gene transcription. Dev Biol
1996;179:402 – 11.
[23] Cluzeaut F, Maurer-Schultze B. Proliferation of cardiomyocytes and
interstitial cells in the cardiac muscle of the mouse during pre- and
postnatal development. Cell Tissue Kinet 1986;19:267 – 74.
[24] Zak R. Development and proliferative capacity of cardiac muscle
cells. Circ Res 1974;35(suppl II):17 – 26.
[25] Holtzer H, Schultheiss T, Dilullo C, Choi J, Costa M, Lu M, et al.
Autonomous expression of the differentiation programs of cells in the
cardiac and skeletal myogenic lineages. Ann N Y Acad Sci
1990;599:158 – 69.
[26] Kobayashi H, Stewart E, Poon R, Adamczewski JP, Gannon J, Hunt T.
Identification of the domains in cyclin A required for binding to, and
activation of, p34cdc2 and p32cdk2 protein kinase subunits. Mol Biol
Cell 1992;3:1279 – 94.
[27] Diehl JA, Cheng M, Roussel MF, Sherr CJ. Glycogen synthase
kinase-3beta regulates cyclin D1 proteolysis and subcellular localization. Genes Dev 1998;12:3499 – 511.
[28] Mulligan G, Jacks T. The retinoblastoma gene family: cousins with
overlapping interests. Trends Genet 1998;14:223 – 9.
[29] Benedict WF, Murphree AL, Banerjee A, Spina CA, Sparkes MC,
Sparkes RS. Patient with 13 chromosome deletion: evidence that
the retinoblastoma gene is a recessive cancer gene. Science
1983;219:973 – 5.
[30] Lee EY, To H, Shew JY, Bookstein R, Scully P, Lee WH. Inactivation
of the retinoblastoma susceptibility gene in human breast cancers.
Science 1988;241:218 – 21.
[31] Horowitz JM, Park SH, Bogenmann E, Cheng JC, Yandell DW, Kaye
FJ, et al. Frequent inactivation of the retinoblastoma anti-oncogene is
restricted to a subset of human tumor cells. Proc Natl Acad Sci U S A
1990;87:2775 – 9.
[32] Dynlacht BD, Flores O, Lees JA, Harlow E. Differential regulation
of E2F transactivation by cyclin/cdk2 complexes. Genes Dev
1994;8:1772 – 86.
[33] Classon M, Dyson N. p107 and p130: versatile proteins with
interesting pockets. Exp Cell Res 2001;264:135 – 47.
[34] Dyson N. pRB, p107 and the regulation of the E2F transcription
factor. J Cell Sci, Suppl 1994;18:81 – 7.
[35] Ogawa H, Ishiguro K, Gaubatz S, Livingston DM, Nakatani Y. A
complex with chromatin modifiers that occupies E2F- and Mycresponsive genes in G0 cells. Science 2002;296:1132 – 6.
[36] Stevens C, La Thangue NB. E2F and cell cycle control: a doubleedged sword. Arch Biochem Biophys 2003;412:157 – 69.
[37] Logan N, Delavaine L, Graham A, Reilly C, Wilson J, Brummelkamp
TR, et al. E2F-7: a distinctive E2F family member with an unusual
organization of DNA-binding domains. Oncogene 2004.
[38] Nevins JR, Leone G, DeGregori J, Jakoi L. Role of the Rb/
E2F pathway in cell growth control. J Cell Physiol 1997;173:
233 – 236.
[39] Lukas J, Petersen BO, Holm K, Bartek J, Helin K. Deregulated
expression of E2F family members induces S-phase entry and
overcomes p16INK4A-mediated growth suppression. Mol Cell Biol
1996;16:1047 – 57.
[40] Fry CJ, Pearson A, Malinowski E, Bartley SM, Greenblatt J, Farnham
PJ. Activation of the murine dihydrofolate reductase promoter by
[41]
[42]
[43]
[44]
[45]
[46]
[47]
[48]
[49]
[50]
[51]
[52]
[53]
[54]
[55]
[56]
[57]
[58]
[59]
[60]
[61]
E2F1. A requirement for CBP recruitment. J Biol Chem
1999;274:15883 – 91.
Trouche D, Kouzarides T. E2F1 and E1A(12S) have a homologous
activation domain regulated by RB and CBP. Proc Natl Acad Sci
U S A 1996;93:1439 – 42.
Obaya AJ, Sedivy JM. Regulation of cyclin-Cdk activity in
mammalian cells. Cell Mol Life Sci 2002;59:126 – 42.
Chow JP, Siu WY, Ho HT, Ma KH, Ho CC, Poon RY. Differential
contribution of inhibitory phosphorylation of CDC2 and CDK2 for
unperturbed cell cycle control and DNA integrity checkpoints. J Biol
Chem 2003;278:40815 – 28.
Brooks G, Poolman RA, McGill CJ, Li JM. Expression and activities
of cyclins and cyclin-dependent kinases in developing rat ventricular
myocytes. J Mol Cell Cardiol 1997;29:2261 – 71.
Poolman RA, Gilchrist R, Brooks G. Cell cycle profiles and
expressions of p21CIP1 and P27KIP1 during myocyte development.
Int J Cardiol 1998;67:133 – 42.
Brooks G, Poolman RA, Li JM. Arresting developments in the cardiac
myocyte cell cycle: role of cyclin-dependent kinase inhibitors.
Cardiovasc Res 1998;39:301 – 11.
Li JM, Poolman RA, Brooks G. Role of G1 phase cyclins and cyclindependent kinases during cardiomyocyte hypertrophic growth in rats.
Am J Physiol 1998;275:H814–22.
Kajstura J, Leri A, Finato N, Di Loreto C, Beltrami CA, Anversa P.
Myocyte proliferation in end-stage cardiac failure in humans. Proc
Natl Acad Sci U S A 1998;95:8801 – 5.
Beltrami AP, Urbanek K, Kajstura J, Yan SM, Finato N, Bussani R,
et al. Evidence that human cardiac myocytes divide after myocardial
infarction. N Engl J Med 2001;344:1750 – 7.
Colucci WS. Molecular and cellular mechanisms of myocardial
failure. Am J Cardiol 1997;80:15L – 25L.
Sabbah HN, Sharov VG, Goldstein S. Programmed cell death in the
progression of heart failure. Ann Med 1998;30(Suppl 1):33 – 8.
Li JM, Brooks G. Cell cycle regulatory molecules (cyclins, cyclindependent kinases and cyclin-dependent kinase inhibitors) and the
cardiovascular system; potential targets for therapy? Eur Heart J
1999;20:406 – 20.
Yoshizumi M, Lee WS, Hsieh CM, Tsai JC, Li J, Perella MA, et al.
Disappearance of cyclin A correlates with permanent withdrawal of
cardiomyocytes from the cell cycle in human and rat hearts. J Clin
Invest 1995;95:2275 – 80.
Jackson T, Allard MF, Sreenan CM, Doss LK, Bishop SP, Swain JL.
The c-myc proto-oncogene regulates cardiac development in transgenic mice. Mol Cell Biol 1990;10:3709 – 16.
Perez-Roger I, Kim SH, Griffiths B, Sewing A, Land H.
Cyclins D1 and D2 mediate myc-induced proliferation via
sequestration of p27(Kip1) and p21(Cip1). EMBO J 1999;18:
5310 – 5320.
Liao HS, Kang PM, Nagashima H, Yamasaki N, Usheva A, Ding B,
et al. Cardiac-specific overexpression of cyclin-dependent kinase 2
increases smaller mononuclear cardiomyocytes. Circ Res 2001;88:
443 – 450.
Soonpaa MH, Koh GY, Pajak L, Jing S, Wang H, Franklin MT,
et al. Cyclin D1 overexpression promotes cardiomyocyte DNA
synthesis and multinucleation in transgenic mice. J Clin Invest
1997;99:2644 – 54.
Poolman RA, Li JM, Durand B, Brooks G. Altered expression of cell
cycle proteins and prolonged duration of cardiac myocyte hyperplasia
in p27KIP1 knockout mice. Circ Res 1999;85:117 – 27.
Chaudhry HW, Dashoush NH, Tang H, Zhang L, Wang X, Wu EX,
et al. Cyclin A2 mediates cardiomyocyte mitosis in the postmitotic
myocardium. J Biol Chem 2004;279:35858 – 66.
Bicknell KA, Surry EL, Brooks G. Targeting the cell cycle machinery
for the treatment of cardiovascular disease. J Pharm Pharmacol
2003;55:571 – 91.
Agah R, Kirshenbaum LA, Abdellatif M, Truong LD, Chakraborty
S, Michael LH, et al. Adenoviral delivery of E2F-1 directs cell
K.M. Regula et al. / Cardiovascular Research 64 (2004) 395–401
[62]
[63]
[64]
[65]
[66]
cycle reentry and p53-independent apoptosis in postmitotic adult
myocardium in vivo. J Clin Invest 1997;100:2722 – 8.
von Harsdorf R, Hauck L, Mehrhof F, Wegenka U, Cardoso MC,
Dietz R. E2F-1 overexpression in cardiomyocytes induces downregulation of p21CIP1 and p27KIP1 and release of active cyclindependent kinases in the presence of insulin-like growth factor I. Circ
Res 1999;85:128 – 36.
Steinhelper ME, Lanson Jr NA, Dresdner KP, Delcarpio JB, Wit AL,
Claycomb WC, et al. Proliferation in vivo and in culture of
differentiated adult atrial cardiomyocytes from transgenic mice. Am
J Physiol 1990;259:H1826–34.
Katz EB, Steinhelper ME, Delcarpio JB, Daud AI, Claycomb WC,
Field LJ. Cardiomyocyte proliferation in mice expressing alphacardiac myosin heavy chain-SV40 T-antigen transgenes. Am J Physiol
1992;262:H1867–76.
Sen A, Dunnmon P, Henderson SA, Gerard RD, Chien KR.
Terminally differentiated neonatal rat myocardial cells proliferate
and maintain specific differentiated functions following expression of
SV40 large T antigen. J Biol Chem 1988;263:19132 – 6.
Torella D, Rota M, Nurzynska D, Musso E, Monsen A, Shiraishi
I, et al. Cardiac stem cell and myocyte aging, heart failure, and
insulin-like growth factor-1 overexpression. Circ Res 2004;94:
514 – 524.
401
[67] Lanza R, Moore MA, Wakayama T, Perry AC, Shieh JH, Hendrikx J,
et al. Regeneration of the infarcted heart with stem cells derived by
nuclear transplantation. Circ Res 2004;94:820 – 7.
[68] Makino S, Fukuda K, Miyoshi S, Konishi F, Kodama H, Pan J, et al.
Cardiomyocytes can be generated from marrow stromal cells in vitro.
J Clin Invest 1999;103:697 – 705.
[69] Jackson KA, Majka SM, Wang H, Pocius J, Hartley CJ, Majesky MW,
et al. Regeneration of ischemic cardiac muscle and vascular
endothelium by adult stem cells. J Clin Invest 2001;107:1395 – 402.
[70] Orlic D, Kajstura J, Chimenti S, Jakoniuk I, Anderson SM, Li B, et al.
Bone marrow cells regenerate infarcted myocardium. Nature
2001;410:701 – 5.
[71] Badorff C, Brandes RP, Popp R, Rupp S, Urbich C, Aicher A, et al.
Transdifferentiation of blood-derived human adult endothelial progenitor cells into functionally active cardiomyocytes. Circulation
2003;107:1024 – 32.
[72] Oh H, Bradfute SB, Gallardo TD, Nakamura T, Gaussin V, Mishina Y,
et al. Cardiac progenitor cells from adult myocardium: homing,
differentiation, and fusion after infarction. Proc Natl Acad Sci U S A
2003;100:12313 – 8.
[73] Beltrami AP, Barlucchi L, Torella D, Baker M, Limana F, Chimenti
S, et al. Adult cardiac stem cells are multipotent and support
myocardial regeneration. Cell 2003;114:763 – 76.