Download Cardiomyocyte proliferation in cardiac development and regeneration

Am J Physiol Heart Circ Physiol 309: H1237–H1250, 2015.
First published September 4, 2015; doi:10.1152/ajpheart.00559.2015.
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Review
Cardiac Regeneration and Repair: Mechanisms and Therapy
Cardiomyocyte proliferation in cardiac development and regeneration: a guide
to methodologies and interpretations
Marina Leone,1* Ajit Magadum,2* and Felix B. Engel1
1
Experimental Renal and Cardiovascular Research, Institute of Pathology, Department of Nephropathology, FriedrichAlexander-Universität Erlangen-Nürnberg (FAU), Erlangen, Germany; and 2Department of Cardiology, Icahn School of
Medicine at Mount Sinai Hospital, New York, New York
cardiac regeneration; cardiomyocyte proliferation; newt; zebrafish
THE MAMMALIAN HEART FORMS from the first and second heart
field. The heart field precursors migrate and fuse at the midline
of the embryo to form the primary linear heart tube (81). The
heart tube then elongates, which occurs primarily via addition
of precursor-derived cardiomyocytes to both poles. Subsequent
“ballooning” of the chambers and growth of the heart are
achieved by proliferation of contracting cardiomyocytes (109).
After birth, mammalian cardiomyocytes exit the cell cycle and
stop proliferating (Fig. 1) (107, 115).
Cardiovascular diseases are among the leading causes of
death worldwide, presenting a major socioeconomic burden
whose incidence is expected to increase further (39). Upon
injury the adult mammalian heart does not regenerate, neither
by activating stem cells nor by induction of cardiomyocyte
proliferation (129). Instead, the heart compensates for the loss
* M. Leone and A. Magadum contributed equally to this work.
Address for reprint requests and other correspondence: F. B. Engel, Experimental
Renal and Cardiovascular Research, Inst. of Pathology, Dept. of Nephropathology,
Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU), Schwabachanlage 12,
91054 Erlangen, Germany (e-mail: [email protected]).
http://www.ajpheart.org
of cardiomyocytes by cardiomyocyte hypertrophy (increase in
cell size). In contrast to reversible physiological hypertrophy
during pregnancy or endurance training, this compensatory
hypertrophy results in concentric or dilative remodeling (Fig.
1). It is accompanied by inflammation, fibrosis, increased wall
stiffness, cardiomyocyte slippage, and continuous apoptotic
loss of cardiomyocytes, resulting in reduced heart function (47,
57, 76).
Currently, there are no effective therapies available to reverse cardiac damage and remodeling, and, for a large group of
patients, further deterioration cannot be efficiently prevented.
Consequently, there is an urgent need for the development of
novel therapies. In contrast to adult mammals, newborn mice,
newts, and zebrafish regenerate their hearts after experimentally induced injury by the induction of cardiomyocyte proliferation (14, 38, 63). Thus, extensive efforts have been invested
to determine whether it is possible to induce adult mammalian
cardiomyocyte proliferation. A large body of evidence has
accumulated in recent years suggesting that in the future it
might be possible to prevent functional deterioration after
0363-6135/15 Copyright © 2015 the American Physiological Society
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Leone M, Magadum A, Engel FB. Cardiomyocyte proliferation in cardiac
development and regeneration: a guide to methodologies and interpretations. Am J
Physiol Heart Circ Physiol 309: H1237–H1250, 2015. First published September 4,
2015; doi:10.1152/ajpheart.00559.2015.—The newt and the zebrafish have the
ability to regenerate many of their tissues and organs including the heart. Thus, a
major goal in experimental medicine is to elucidate the molecular mechanisms
underlying the regenerative capacity of these species. A wide variety of experiments have demonstrated that naturally occurring heart regeneration relies on
cardiomyocyte proliferation. Thus, major efforts have been invested to induce
proliferation of mammalian cardiomyocytes in order to improve cardiac function
after injury or to protect the heart from further functional deterioration. In this
review, we describe and analyze methods currently used to evaluate cardiomyocyte
proliferation. In addition, we summarize the literature on naturally occurring heart
regeneration. Our analysis highlights that newt and zebrafish heart regeneration
relies on factors that are also utilized in cardiomyocyte proliferation during
mammalian fetal development. Most of these factors have, however, failed to
induce adult mammalian cardiomyocyte proliferation. Finally, our analysis of
mammalian neonatal heart regeneration indicates experiments that could resolve
conflicting results in the literature, such as binucleation assays and clonal analysis.
Collectively, cardiac regeneration based on cardiomyocyte proliferation is a promising approach for improving adult human cardiac function after injury, but it is
important to elucidate the mechanisms arresting mammalian cardiomyocyte proliferation after birth and to utilize better assays to determine formation of new
muscle mass.
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Fetal heart
Neonatal heart
Adult heart
Polyploidization
M
C
Cell cycle
M
Injury
C
F
A
I
L
U
R
E
Cell cycle
M
Injury
Regeneration
H
E
A
R
T
G2
S
Hypertrophy
G0
Cell cycle
Birth
G1/0
G2
Pregnancy
Sport
G2
S
G1
Polyploidization
G1
G1
C
S
Binucleation
Endomitosis
Polynucleation
injury and even to reverse cardiac damage by inducing cardiomyocyte proliferation.
Assays to Determine Cardiomyocyte Proliferation
Cardiomyocyte cell cycle activity does not necessarily
equate with proliferation as it can also reflect pathological
hypertrophy, polyploidization, or polynucleation (115, 129).
Therefore, it is important to be careful with the interpretation
of data from cell cycle assays (Table 1). Misinterpretation of
cell cycle assays might provide an explanation for the controversies in the field of cardiac regeneration, as to the extent of
cardiomyocyte proliferation. Therefore, we will discuss here
their advantages and disadvantages.
Nucleotide analog incorporation. A commonly used approach in determining the extent of cardiomyocyte proliferation is the use of a nucleotide analog-incorporation assay
(BrdU, EdU, tritiated thymidine). Nucleotide analogs will be
incorporated during DNA synthesis, and thus cells will be
labeled during S phase. A major advantage of this assay is that
it permits the ability to perform long-term labeling, as well as
pulse-chase experiments. Long-term labeling is especially
helpful for the detection of cell cycle entry when the time
course of cell cycle activation is unclear and, furthermore,
helps to determine the total number of cycling cardiomyocytes.
However, DNA synthesis occurs not only during semiconservative DNA replication during S phase but also during DNA
repair. Moreover, semiconservative DNA replication is merely
an indicator of S-phase cell cycle progression (115, 129). Thus,
a disadvantage of this assay is that it does not predict whether
a cell will divide or undergo G2/M arrest, polyploidization, or
polynucleation. Pulse-chase experiments permit the identification of colony formation and thus, by deduction, cell division,
as the label will be reduced by 50% per each cell division. Yet,
to our knowledge, this approach has so far not been utilized in
the field of cardiac regeneration.
Colorimetric assays. Several colorimetric proliferation assays are available. These assays, such as MTT, Alamar Blue, or
ViaLight assays, are based on a color change due to the
cleavage of tetrazolium salts, reduction of resazurin, or the
detection of cellular ATP. An increase in the color change is
interpreted as an increase in cell number. However, these
assays work on the assumption that mitochondrial activity or
the amount of cellular ATP per cell is constant. In this regard,
these assays have limitations when applied to cardiomyocytes,
as mitochondrial activity is increased during hypertrophy or
elevated contractility (100). Moreover, as these assays are not
cell-type specific, it cannot be excluded that an increase in the
colorimetric signal results from an increase in proliferation of
Table 1. Assays used to determine cardiomyocyte proliferation and their specificity
Bi-/
DNA Synthesis DNA Repair Polyploidization Mitosis Endomitosis Polynucleation Cytokinesis Proliferation Hypertrophy Cardiac specific
Nucleotide incorporation
Ki67
Histone H3 phosphorylation
Aurora B/Anillin
Sarcomere disassembly
FACS
Colorimetric assays
Cell count
Clonal analysis
X
X
X
X
X
?
X
X
X
X
X
X
X
?
X
?
X
X
X
X
X
?
X
X
?
X
X
X
FACS, fluorescence-activated cell sorting.
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X
X
X
X
X
X
X
X
X
X
X
X
?
X
X
X
X
X
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Fig. 1. Cell cycle activities in fetal, neonatal, and adult mammalian cardiomyocytes. In development the ballooning of the chamber and the further growth of
the heart are accomplished by fetal cardiomyocyte proliferation. After birth, cardiomyocytes gradually stop proliferating and exit the cell cycle (G0/G1 phase).
Initially this arrest is reversible, as newborn cardiomyocytes can re-enter the cell cycle and proliferate upon injury. Yet during postnatal development
cardiomyocytes undergo one more incomplete cycle, resulting in binucleated or polyploid neonatal cardiomyocytes. In the case of cell cycle re-entry of adult
cardiomyocytes, such as during pathological hypertrophy in contrast to physiological hypertrophy (e.g., sports and pregnancy), cells undergo polyploidization,
endomitosis, and polynucleation but not cell division. This is a common feature of heart failure.
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Cell division
G2 phase
mitosis. In contrast, during endomitosis the sarcomeric apparatus stays intact.
Ki67. Ki67 has been shown to be required for cell proliferation, but its function is poorly understood. Ki67 is expressed
in all cell cycle phases of proliferating cells but is not expressed in non-cycling cells. Thus, Ki67 has been considered a
reliable marker for evaluating cellular proliferation (18, 30).
Furthermore, based on its intranuclear staining pattern it is
even possible to determine in what cell cycle phase a cell
resides, a feature that has not been utilized yet in cardiomyocytes. However, there is evidence that Ki67 is not a reliable
marker for determining if a cardiomyocyte can proliferate. For
instance, Meckert and coworkers (78) showed that Ki67positive cardiomyocytes undergo endomitosis. Moreover, it
has been shown that cell cycle genes as well as Ki67 are
re-expressed in hypertrophy (28, 129). Thus, as with nucleotide
analog incorporation and H3P labeling for mitosis, Ki67 does
not predict whether a cycling cell will divide, undergo polyploidization, or will binucleate.
Aurora B and Anillin. In 2005 we introduced Aurora B as a
marker of cytokinesis in cardiomyocytes (33). Aurora B is
expressed in G2 phase, phosphorylates histone H3 at Ser-10,
Binucleation
H3P/Anillin
H3P
Anillin
Aurora B
DAPI
Prophase
Anaphase
Cytokinesis
Fig. 2. Schematic representation of H3P, Anillin, and Aurora B localization in dividing vs.
binucleating cardiomyocytes. In G2 phase, histone H3 becomes phosphorylated (H3P) while
Anillin (diffuse) and Aurora B (dotty) accumulate in the nucleus. During prophase, Aurora B
associates with centromeric heterochromatin,
while Anillin is translocated to the cytoplasm.
At this stage, these markers are not suitable to
distinguish proliferating from binucleating cardiomyocytes. During anaphase, Anillin accumulates at the cell cortex, and Aurora B in the
central spindle. Yet, as in binucleating cells,
cleavage furrow ingression is impaired; one can
find anaphase-like cells that are H3P-negative
(telophase-like nuclei) in contrast to proliferating cardiomyocytes. In cytokinesis of dividing
cardiomyocytes, Anillin localizes around the
midbody, and Aurora B concentrates at its
flanking regions. In binucleating cardiomyocytes, Aurora B behaves similarly; however,
Anillin fails to concentrate around the midbody.
Costaining of Aurora B and Anillin highlights
the asymmetric furrow ingression, a characteristic of binucleating cardiomyocytes.
Cycling product
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nonmyocytes, which are present, albeit to a lesser degree, in
primary cardiomyocyte cultures.
Histone H3 phosphorylation. Histones undergo a wide variety of posttranslational modifications, some of which are
specific to mitosis (118). The most common histone modification used to identify mitotic cells is the phosphorylation of
histone H3 at serine 10 (Ser-10) (H3P). Phosphorylation at
Ser-10 begins in G2 phase and dephosphorylation is completed
“immediately prior to detectable chromosome decondensation
in telophase” (Fig. 2) (24, 44). Thus, the often-used term
“mitotic index” for the percentage of H3P-positive cells is
misleading, as part of the H3P-positive cells are in G2 phase.
In addition, as with detection of S-phase activity, mitosis does
not predict cell division as it can lead to polyploidization via
endomitosis (78) or binucleation due to failure of cytokinesis
(34). Furthermore, unlike nucleotide analog incorporation assays, H3P staining cannot be combined with a cardiac-specific
nuclear marker (e.g., Nkx2.5), as prometaphase is coupled with
nuclear envelope breakdown. Therefore, to determine if an
H3P-positive cell is a cardiomyocyte, H3P staining is generally
coupled with staining of the sarcomeric apparatus, which loses
in cardiomyocytes its organized structure upon entry into
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we have demonstrated that this complex disassembly-reassembly
process occurs not only in neonatal and adult cardiomyocytes after
induction of cell division but also during binucleation (33, 34).
Thus, sarcomere disassembly, often misleadingly called “dedifferentiation,” is not an unambiguous marker of cardiomyocyte
proliferation. However, it can be utilized to exclude proliferation as sometimes H3P-positive cardiomyocytes are reported
with an intact sarcomeric apparatus, indicative of endomitosis
(32, 115).
Cell count experiments. Cell count experiments are used as
a direct measurement of cardiomyocyte proliferation. Yet,
often investigators measure only the total cell number, not
distinguishing between cardiomyocytes and nonmyocytes. In
contrast, we have for example shown that the total number of
neonatal cardiomyocyte increases over time after FGF1/p38
inhibitor stimulation based on total cell number corrected for
the percentage of cardiomyocytes as analyzed by FACS (33).
In addition, in some studies the cell number after stimulation is
compared with control stimulation. A higher cell number can
also be explained by a cell-protective effect, as it is known that
neonatal as well as adult cardiomyocytes undergo apoptosis in
culture. In addition, cell numbers are often determined by
isolating cardiomyocytes from tissue or trypsination from culture dishes. Here, it should be considered that alterations in
extracellular matrix composition, cell-to-cell adhesion, and cell
size can significantly affect the cell count due to inefficient
enzymatic digestion or altered efficiency in cell harvest by
centrifugation.
Clonal analysis. Clonal analysis from single cells unambiguously determines if a stimulus induces proliferation. In vitro,
clonal analysis has not yet been reported for cardiomyocytes.
However, we have introduced the monitoring of cardiomyocytes over time, demonstrating an increase in cardiomyocyte
density after FGF1/p38 inhibitor stimulation (33). Recently,
Lin and coworkers (65) performed clonal analyses in vivo. The
authors utilized the Brainbow technology (68) to induce, in a
small number of randomly distributed cardiomyocytes, expression of a fluorescence protein (to obtain individual labeled
cardiomyocytes). In addition, they utilized a cardiomyocytespecific doxycycline-inducible system to induce Yes-associated protein expression in cardiomyocytes (65). If induction of
proliferation were successful, one would expect the formation
of clones from the individual labeled cardiomyocytes (Fig. 3).
It should be noted, however, that the Brainbow system for
clonal analysis requires that labeled individual cells with the
same color not be adjacent to one another. Given that noninduced/control hearts in the study by Lin and coworkers occasionally exhibited clones of two adjacent cardiomyocytes of
the same color, this assay is thus useful only if a significant
number of clones of at least four labeled cardiomyocytes are
observed. A more direct proof of cardiomyocyte division has
been introduced by Ali and coworkers (4), who have utilized
the “mosaic analysis with double markers” mouse model (132),
which can distinguish between parental and daughter cells. In
this system parental cells are GFP- and RFP-double positive.
After cell division daughter cells will be RFP or GFP positive,
while binucleated cells will be GFP- and RFP-double positive
(Fig. 3). As this system is dependent on Cre recombination,
proliferation can be analyzed specifically in cardiomyocytes.
While this system has been used to determine naturally occurring cardiomyocyte proliferation (4), it has not yet been uti-
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and associates with centromeric heterochromatin early in mitosis (Fig. 2). In contrast to H3P as a marker, Aurora B can be
used to analyze cytokinesis as it transfers first from the kinetochore during metaphase to the central spindle during anaphase, ultimately contributing to midbody formation during
telophase (24, 121). Unfortunately, we realized later that Aurora B alone is also not a reliable marker for cell division, as its
staining pattern in dividing cells is indistinguishable from the
pattern in binucleating cells (34). However, Aurora B staining
combined with a sarcomere staining allowed us to visualize
cytokinesis failure during binucleation based on asymmetric
constriction of the cleavage furrow. Subsequently, we introduced Anillin as an additional marker to evaluate whether a
cardiomyocyte will binucleate or divide. Like Ki67, Anillin is
absent in G0 phase. In proliferating cells it accumulates in the
nucleus during G1, S, and G2 phase, then localizes to the cell
cortex at the onset of mitosis, and finally locates around the
midbody in cytokinesis (36, 111). Aurora B and Anillin
costaining identifies binucleating cells based on asymmetric
constriction of the cleavage furrow and failure of Anillin to
focus at the cortex in anaphase and to concentrate around the
midbody during cytokinesis (34). Collectively, the sole presence of Aurora B or Anillin is not proof of cell division.
However, the cellular localization of Anillin will indicate if a
cell will divide or binucleate.
Fluorescence-activated cell sorting. A method that has
rarely been utilized to characterize cell cycle progression in
cardiomyocytes is fluorescence-activated cell sorting (FACS).
Measuring cell proliferation via FACS is based on labeling
isolated cardiomyocytes with a cardiomyocyte-specific marker
and a fluorescent DNA dye, which permits determination of the
DNA content based on fluorescence intensity. Using this approach, one can determine what fraction of the cardiomyocytes
is for example in S phase. Time series experiments help to
determine whether DNA synthesis results in binucleation (increase in the G2/M phase peak), polyploidization/polynucleation (DNA content ⱖ 4n), or cell division (no change in the
G1, S, and G2/M distribution) (34). In addition, one can
perform a BrdU pulse-chase experiment (diluting the BrdU
label due to cell division) or a BrdU-Hoechst assay to prove
that cell division occurs (29). The principle of a label being
diluted because of cell division (such as BrdU or membrane
labeling) even enables investigators to determine how often a
cell has divided, as each cell division results in a 50% decrease
of the label (71).
Sarcomere disassembly/dedifferentiation. Previously, it has
been suggested that newt and zebrafish cardiomyocytes dedifferentiate before undergoing proliferation based on the observation that their sarcomeric apparatus disassembles (52).
Yet Ahuja and coworkers (3) have shown in detail that fetal
cardiomyocytes disassemble their sarcomeric apparatus in a
highly ordered fashion during mitosis and reassemble it during
cell division. This suggests that sarcomere disassembly is not a
sign of dedifferentiation but is simply a necessary process to
allow cardiomyocyte proliferation. That adult mammalian cardiomyocytes are also able to restructure or disassemble their
sarcomeric apparatus has been long known. For example, adult
cardiomyocytes with disassembled/disorganized/restructured
sarcomeric apparatus has been observed in long-term in vitro
cultures (92, 110), hypocontractile hibernating myocardium
(72, 106), and Tako-Tsubo cardiomyopathy (83). Importantly,
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A
Myh6CreERT2; MADM-11 GT/TG
Cardiomyocyte proliferation
Myh6CreERT2
Tamoxifen
No
cytokinesis
Mitosis
G2-X
Cytokinesis
Myh6-MerCreMer; ROSA26 fs.rt TA/+; TRE-YAP; CAG-fsBrainbow
Proliferation
Cardiomyocyte proliferation
Tamoxifen
Doxycycline
rtTA
aYAP
rtTA
Myh6-MCM
aYAP
rtTA
aYAP
rtTA
aYAP
rtTA
aYAP
rtTA
aYAP
rtTA
aYAP
Centromere
rtTA
Promoter
LoxP site
N-GFP
C-GFP
N-RFP
C-RFP
Stop
aYAP
RFP
GFP
Cre Recombinase
Doxycycline-dependent
reverse tetracycline
activator gene
Doxycycline-dependent
reverse tetracycline
activator protein
“Yes-associated
protein” gene
Fig. 3. Scheme of 2 available cardiac-specific mouse models for clonal analysis. In the literature, 2 in vivo systems have been described to visualize clonal growth
of cardiomyocytes. A: the mosaic analysis with double markers (MADM) mouse model is based on split genes encoding for NH2- and COOH-terminal parts of
the fluorescent proteins red fluorescent protein (RFP) and green fluorescent protein (GFP). The system is designed such that, upon Cre recombination during
mitosis, the split genes are recombined to allow the expression of RFP in 1 daughter cell and GFP in the other (G2-X). If cell division/cytokinesis is not
completed, cells will be binucleated, expressing both RFP and GFP and appearing yellow. This model has been used to determine homeostasis in the heart of
the Myh6CreERT2 mouse. B: the 2nd model is based on the Brainbow mouse model containing several genes encoding different fluorescent proteins under the
control of a general promoter. Cre recombination results in the random expression of individual fluorescent proteins. Thus, the use of a cardiac-specific promoter
(Myh6) driving Cre will result in the labeling of individual cardiomyocytes. Activation of proliferation in such an individual labeled cardiomyocyte will then
result in single color-labeled clones. Yet this system works well only if few cardiomyocytes are labeled, several different fluorescent proteins are utilized, and
cardiomyocytes are induced to undergo several cell division. However, there is always an uncertainty, especially in the case of small-sized clones, whether a
single color-labeled clone arises from proliferation or by the same recombination event in neighboring cells. This system was utilized as a dual-color system (RFP
and GFP) to determine the effect of Yes-associated protein (YAP) on cardiomyocyte proliferation. YAP was expressed under the control of a doxycyclinedependent reverse tetracycline activator protein.
lized to test the efficiency of factors to induce cardiomyocyte
proliferation.
Cardiomyocyte specificity. A major issue, especially in vivo,
is to determine whether the signal of a cell cycle marker indeed
belongs to a cardiomyocyte. This is especially critical as often
there is an increase of fibroblast proliferation and infiltration of
inflammatory cells upon injury. As has been previously shown
and discussed, cardiomyocyte-specific cytoplasmic markers
are prone to misinterpretations; also the presentation of Zstacks cannot solve this issue (32, 108). Thus, the detection of
Ki67, BrdU, or cell cycle regulators such as cyclins should be
combined with staining for a cardiomyocyte-specific nuclear
marker such as GATA4 or MEF2. Besides a nuclear marker,
cardiomyocyte-specific cytoplasmic membrane markers such
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B
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as caveolin 3 can be utilized to identify unambiguously a
cycling cardiomyocyte (32).
The analysis of commonly used methods to determine cardiomyocyte proliferation shows that while many of these
techniques can detect cell cycle activity, distinguishing between proliferation, polynucleation, or polyploidization is
more difficult and in some cases impossible. Not acknowledging this issue often results in misinterpretation of results and is
likely the reason for the contradictions, and even controversies,
in the field of cardiac regeneration.
Naturally Occurring Cardiac Regeneration
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Pondering regeneration is as old as civilization. First written
reports by Empedocles and Aristotle describing the observation
that certain animals can regenerate their appendages can be
found as early as 400 BC. In modern times the first known
publication regarding regeneration was by René Antoine Ferchault de Réaumur in 1712, where he described limb regeneration in crustaceans (crayfish, crabs, and lobsters) (41, 99). In
1740, Abraham Trembley perturbed, analyzed, and dissected
the process of regeneration in hydra, which is considered to be
the birth of “experimental biology” (60). In contrast to common belief, regeneration is a widely distributed event with
regenerative species in almost every phylum (101).
Urodele heart regeneration. Urodeles (salamanders and
newts) are considered to be the champions of regeneration.
They can regenerate a variety of structures such as limbs, tails,
jaws, eyes, and the heart (16, 114). Urodele heart regeneration
was mentioned in the literature as early as 1974 (9, 88). After
Oberpriller and Oberpriller (87) reported the appearance of
mitotic adult newt cardiomyocytes in Diemictylus viridescens
16 days after apical resection, they studied the response of the
adult newt ventricle to injury in Notophthalmus viridescens
(88). The authors resected around 12.5% of the ventricle. At
the site of injury a blood clot occluded the ventricle and
macrophages accumulated at 10 days within and around it. To
determine if the injury induced cardiomyocyte proliferation the
authors injected tritiated thymidine 3 h before tissue collection
to determine the frequency of cycling cardiomyocytes. The
maximal thymidine-incorporation index (9 –10%) and mitotic
index (0.9%) were observed at 20 days postinjury. Becker and
coworkers (9) provided additional support for the capacity of
urodeles to regenerate their hearts. They demonstrated that
Triturus viridescens at the adult aquatic stage survives the
resection of 30 –50% for at least 2 wk with a mortality rate of
10% (several hundred resections were performed). Normal
circulatory dynamics were re-established after 5 h based on
microscopic analysis of skin capillaries in the tail fin using
transmitted light. These data suggested that, upon injury, the
adult newt heart might be able to regenerate its heart by
cardiomyocyte proliferation. However, the authors observed
the formation of scar tissue at 30 days postinjury, indicating a
limited regenerative response (88).
To demonstrate that the newt heart regenerates from cardiomyocyte proliferation several assays have been performed.
Mitotic cardiomyocytes in newts were initially identified by
electron microscopy based on condensed mitotic chromatin,
“fibrils, which appeared to be myofibrils approximately the
length of a sarcomere” in the periphery of the cell, a basal
lamina, and glycogen granules (87). In addition, these mitotic
cells are characterized by the absence of thin filaments and Z
bands (87). These features have also been observed in dividing
fetal mammalian cardiomyocytes, indicating that the adult
newt heart might indeed regenerate its heart by cardiomyocyte
proliferation. However, this “transient disassembly of the sarcomeric apparatus” occurs also when cells fail to undergo cell
division after mitosis resulting in binucleated cardiomyocytes
(34). Thus, it was important to determine whether the number
of binucleated cardiomyocytes increases upon injury. Oberpriller and coworkers (89) reported that the vast majority
(⬎98%) of adult newt cardiomyocytes are mononucleated.
Importantly, they have demonstrated that the majority of newt
cardiomyocytes (⬃85%) that underwent DNA synthesis 45
days after injury (apex resection and reintroduction after mincing) were still mononucleated and diploid. These data strongly
indicated that the vast majority of adult newt cardiomyocytes
that enter the cell cycle complete cell division.
To prove that adult newt cardiomyocytes are indeed able to
complete cell division and to proliferate, Matz and coworkers
(77) performed life cell imaging experiments (day 8 –19 in
culture). These experiments demonstrated that 80% of mononucleated cardiomyocytes produced mononucleated daughter
cells, while the remaining failed to complete cytokinesis,
resulting in binucleated cells. In addition, 32% of binucleated
cardiomyocytes produced binucleated daughter cells. Bettencourt-Dias and coworkers (12) confirmed the proliferative
competence of adult newt cardiomyocytes in vitro. They observed that approximately one-third of the cells completed cell
division and part of those entered successive cell divisions.
These data suggest that the newt heart contains a subpopulation
of cardiomyocytes that retain a higher proliferative potential,
while the remaining newt cardiomyocytes behave similarly to
their mammalian counterparts.
To determine whether new myocardium is formed during
newt regeneration, Piatkowski and coworkers (91) introduced
“scanning electron microscopy analysis in combination with
stereological estimation”. Injury induced by “repeated orthogonal squeezing with fine forceps” was followed by cardiac
regeneration associated with a significant increase in the volume fraction of trabeculae. In contrast to initial studies, this
and other studies have demonstrated, by investigating later
time points, that cardiac newt regeneration results in a morphologically fully regenerated heart with no signs of scarring
(91, 124).
To provide evidence that newts can functionally regenerate
their hearts, a standardized ventricular resection model was
established, whereby “a more lateral region of the ventricle, as
opposed to the apex,” (5–10%), was resected (124). Echocardiography was utilized for the analysis of heart function and
revealed that heart function was reduced at day 7 postinjury
from around 38-34% fractional shortening. Full function was
reported at 23 days and 60 days postinjury (⬃40% fractional
shortening) (124). However, it remains unclear whether the
functional improvement is indeed due to newly formed heart
tissue. Considering that a rather small amount of muscle tissue
was resected, the rather small drop in function might also be
due to the initial formation of the blood clot and scar at day 7,
impairing wall motion. In the future it would be important to
repeat such a study in a model with more severe damage to the
heart. This is especially important as recent studies in neonatal
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ocytes or by stem cells (or some other cardiomyocyte progenitor cell) differentiating into cardiomyocytes. In addition, in
contrast to newt cardiomyocytes (12, 77), no protocol exists to
induce proliferation of isolated adult zebrafish cardiomyocytes
(64, 102) in vitro.
Thus, the source of newly formed cardiomyocytes remained
controversial for several years until Jopling and coworkers (52)
as well as Kikuchi and coworkers (54) introduced the tamoxifen (4-OHT)-inducible Cre/lox system to the zebrafish model
to conduct lineage-tracing experiments. These lineage-tracing
approaches demonstrated that stem/progenitor cells are not
significantly involved in zebrafish cardiac regeneration but
indicated that regenerated heart muscle in zebrafish is derived
from proliferation of differentiated cardiomyocytes. Jopling
and coworkers (52) observed a global proliferative response of
cardiomyocytes to mechanical injury, which was supported by
the finding that an inhibitor of the cell cycle kinase Plk1
blocked cardiac regeneration. In contrast, Kikuchi and coworkers (54) identified a cardiomyocyte subpopulation (GATA4positive) throughout the subepicardial ventricular layer that
contributed to heart regeneration. The importance of this subpopulation for cardiac regeneration has been proven by inducible, tissue-specific overexpression of a dominant-negative
Gata4 cassette (g4DN), which blocked regeneration and caused
severe scarring. The data indicate that proliferation of trabecular myocytes was not affected while cortical cardiomyocyte
proliferation was markedly reduced (43).
Earlier studies were significantly limited by the lack of
longitudinal studies (observing the process of regeneration of
an individual heart over time) and functional studies. As it is
Table 2. Summary of cardiac regeneration in model systems
Cardiomyocyte Proliferation
Heart Regeneration/Cardiac Tissue Regrowth after:
in vitro
Species
Neonatal
Adult
in vivo after
Injury
Urodeles
X
X
X
Resection
Cryoinjury
Genetic
Ablation
LAD
Mechanical
Injury
X
X
X
X
Functional
Improvement
X
X
X
X
X
X
Zebrafish
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
Mammals (fetal/neonatal)
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
LAD, left anterior descending coronary artery ligation.
AJP-Heart Circ Physiol • doi:10.1152/ajpheart.00559.2015 • www.ajpheart.org
Reference
88
9
87
89
12, 77
91
124
97
21
25
119
52
54
43
50
127
40
61, 115
19
26
74, 75
93
94
6
5
51
56
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mice suggest that the regenerative response of the heart depends on the size of injury (19, 26).
Collectively, the available data demonstrate that adult newt
cardiomyocytes can proliferate in vitro and indicate that newts
can repair their hearts after several different kinds of injury by
cardiomyocyte proliferation (Table 2). To further characterize
newt heart regeneration, echocardiographic studies should be
performed comparing the functional recovery in injury models
of varying degrees of damage. Finally, it remains unclear how
adult proliferating newt cardiomyocytes differ from nonproliferating newt or mammalian cardiomyocytes.
Zebrafish heart regeneration. Zebrafish are known to regenerate a variety of organs, and, in contrast to urodeles, zebrafish
are amenable to molecular analysis and genetic manipulation.
In 2002, Poss and coworkers (97) tested whether zebrafish can
regenerate their hearts. Similar to the experiments with urodeles, Poss and coworkers amputated around 20% of the apex. On
the basis of histological data, nucleotide analog (BrdU)-incorporation assays, and an analysis of H3P for mitosis, the authors
concluded that the zebrafish can regenerate its heart by cardiomyocyte proliferation after mechanical injury. Subsequently,
similar data were obtained for other injury models including
cryoinjury (21) and genetic ablation (25, 119). That zebrafish
heart regeneration is based on cardiomyocyte proliferation was
supported by the observation that zebrafish carrying a mutation
in a cell cycle gene failed to regenerate, forming a scar (97).
However, this mutation was not cell-type specific and might
have affected heart regeneration by inhibiting proliferation of
another cell type. Thus, it remained unclear if regeneration
occurred by proliferation of remaining endogenous cardiomy-
Review
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CARDIOMYOCYTE PROLIFERATION
paired wall motion by increased pumping capacity of the
remainder of the ventricular wall. However, it remains unclear
if lost cardiac tissue is fully replaced by cardiomyocyte proliferation or whether hypertrophy and/or binucleation contribute to this process. In the future, it will be important to
demonstrate that adult zebrafish cardiomyocytes can proliferate
in vitro and to elucidate how adult zebrafish cardiomyocytes
differ from postnatal mammalian cardiomyocytes.
Newborn mouse heart regeneration. Fetal and newborn
cardiomyocytes have the ability to proliferate (115). Consequently, the question was raised if this ability is enough to
allow the fetal, embryonic, or newborn heart to regenerate after
injury. In 1975 the first evidence for fetal heart regeneration
after mechanical injury in rabbits was reported (74, 75). In
2008, Drenckhahn and coworkers (27) introduced a mouse
model that demonstrated that the fetal heart can compensate for
the genetic inhibition of proliferation in around 50% of fetal
cardiomyocytes. Proliferation was inhibited through inactivation of the X-linked gene encoding holocytochrome c synthase
(Hccs). While deletion of Hccs in knockout males resulted in
midgestational lethality, heterozygous knockout female mice
were not affected, even though around 50% of the cardiomyocytes were Hccs deficient because of the random nature of
X-chromosome inactivation. The gradual increase of complex
III activity of the mitochondrial respiratory chain, the decrease
in Hccs-deficient cardiomyocytes, and a detailed cell cycle
analysis showed that the reduced proliferation rate of Hccsnegative cells is compensated for by an increased proliferation
rate of the healthy cardiomyocytes. However, hearts of Hccsdeficient female mice at postnatal day 1 were still hypoplastic.
Importantly, despite an increased proliferation rate adult Hccsdeficient females exhibit a range of cardiac phenotypes (28).
These data suggest that the fetal heart has only a limited
capacity for cardiac regeneration.
While fetal cardiomyocytes contribute significantly to heart
growth by proliferation, cardiomyocytes of newborn mice have
a limited proliferation capacity, and cell cycle activity generally results in binucleation instead of cell division (61). Despite
this fact, Porrello and coworkers (93) observed that neonatal
mice at postnatal day 1, but not day 7, exhibit a transient
cardiac regeneration capacity. In addition, Naqvi and coworkers (82) have recently reported a brief but intense proliferative
burst of predominantly binuclear cardiomyocytes at postnatal
day 15 in mice. Moreover, they report that mice at postnatal
day 15 were able to partially regenerate upon experimental
myocardial infarction. Apex resection (15%) at postnatal day 1
was restored within 21 days following a similar process as
described for zebrafish (see above) (93). Cardiomyocyte proliferation was assessed by BrdU incorporation assays, phosphorylation of histone H3, aurora B kinase expression, and
sarcomere disassembly, indicating an up to sevenfold increase
in cell cycle activity. As described above these markers do not
allow one to distinguish between proliferation and binucleation
(34). However, the observation that ventricular weight and
surface area in the absence of hypertrophy were recovered
within 21 days supports the conclusion that the rate of cardiomyocyte proliferation is increased upon resection. In addition,
cardiac function of resected animals was comparable to shamoperated hearts. Yet it remains unclear whether heart function
was affected and whether the normal differentiation process
resulting in binucleation was inhibited by the resection or not.
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difficult to identify the amputation plane and to identify newly
formed cardiomyocytes, it remained unclear if zebrafish can
fully restore lost myocardium and whether the observed morphological regeneration is indeed due to cardiomyocyte proliferation. For example, the morphologically repair of a ventricle
might be due to cardiomyocyte migration, which has been
shown to be essential for zebrafish cardiac regeneration (50).
To address this issue, the group of Ken Poss (22) recently
introduced so-called “zebraflash” transgenic lines for in vivo
bioluminescence imaging. Expression of firefly luciferase under the cardiomyocyte-specific promoter cmcl2 allows for
direct monitoring of the recovery of heart size. However, this
transgenic line cannot distinguish whether recovery is due to
either proliferation or hypertrophy. Moreover, these transgenic
lines cannot be used to determine functional recovery.
To demonstrate that the zebrafish can fully regenerate heart
function after injury, physiological parameters have been analyzed, resulting in contradictory results. Analyses of cardiac
conduction postresection in regenerating and scarring hearts
initially suggested that zebrafish might not fully recover function. J-point depression was statistically significant 10 days
postinjury and gradually normalized at 60 days postresection,
independent of scarring or regeneration. In contrast, QTc
intervals remained prolonged in both scenarios, indicating
delayed electric repolarization (127). However, Kikuchi and
coworkers (54) reported normalization of conduction 30 days
postresection, which was later confirmed in the cryoinjury
model (21). While these studies provided a more physiological
insight into cardiac regeneration in zebrafish, recovery of
cardiac conduction is a weak indicator of improved cardiac
function.
In 2011, Wang and coworkers (119) introduced “swimming
endurance” as a read-out to evaluate functional recovery after
injury. Zebrafish were placed in a swim tunnel respirometer
(MiniSwim-170; Loligo Systems, Tjele, Denmark) and allowed to acclimatize for 20 min at a low flow velocity of 3
cm/s. Swimming speed was then slowly increased to a velocity
of ⱖ20 cm/s, and the endurance of zebrafish was monitored.
The authors utilized this system to test the effect of inducible
genetic ablation of a high percentage of cardiomyocytes
through overexpression of cytotoxic diphtheria toxin A chain.
They demonstrated that inducible genetic ablation of ⬎60% of
cardiomyocytes in contrast to apex resection results in reduced
performance in a swim tunnel. Recovery of this phenotype,
considering an initial loss of ⬍60% together with recovery of
the density of cardiomyocyte nuclei, provided strong evidence
of regeneration from proliferation, even though binucleation
had not been excluded (119).
Recently, Gonzalez-Rosa and coworkers (40) introduced the
use of 2D echocardiography to directly determine the pump
function of the zebrafish heart. In a longitudinal study analyzing individual zebrafish over time the authors observed that
global systolic function but not ventricular wall motion was
fully recovered following cardiac cryoinjury. This was in
agreement with an increased density of cardiomyocyte nuclei
in the injured ventricular wall that might affect normal contraction due to ultrastructural changes.
In conclusion, the available data indicate that zebrafish can,
to a large extent, regenerate their hearts by means of cardiomyocyte proliferation (Table 2). In addition, it appears that in
case of incomplete regeneration zebrafish compensate for im-
Review
CARDIOMYOCYTE PROLIFERATION
What Can We Learn from Naturally Occurring Cardiac
Regeneration for Human Cardiac Therapy?
It is assumed that studying animal models that can naturally
regenerate their hearts will result in novel therapies to treat
human cardiac disease. In regard to cardiomyocyte prolifera-
tion it is important to understand the molecular mechanisms
controlling proliferation and to determine whether the identified molecular mechanisms can be applied to human cardiomyocytes. Critical questions are:
Are adult cardiomyocytes from model organisms comparable to adult mammalian/human cardiomyocytes?
Are positive regulators enough to induce adult mammalian/
human cardiomyocyte proliferation?
Urodele and zebrafish heart regeneration. Urodeles have
been used in classical experiments to study cardiogenesis as its
development is slow and its embryos are large and develop
externally (84). However, due to the lack of molecular tools the
use of urodeles is limited with regard to the elucidation of
molecular mechanisms regulating heart development and thus
also heart regeneration. Consequently, so far only microRNA
(miR)-128 (123) and the process of RNA editing (125) have
been implicated in newt heart regeneration. To harness the
advantages of newt heart regeneration the group of Thomas
Braun (15, 69, 70) has made significant progress in pioneering
work to elucidate the transcriptome and proteome of newt heart
development and regeneration.
Even though the zebrafish heart has only two chambers, one
ventricle and one atrium, the myocardial wall is similar in
structure to that of mammals, being composed of three layers:
myocardium, epicardium, and endocardium. In addition, as in
mammals, the zebrafish myocardium can be divided into compact and trabecular layers (48). That the zebrafish is a useful
model in which to study the human heart and human disease
has been demonstrated in a large number of embryonic studies
and forward genetic screens (7). However, in contrast to
mammalian cardiomyocytes, zebrafish cardiomyocytes are
small and mononucleated and retain proliferative potential
throughout life (96, 122). Electrophysiological studies revealed
that the adult zebrafish myocardium is very similar to that of
humans based on acetylcholine-activated K⫹, Na⫹, L-type
Ca2⫹, and IKr channels. However, these studies also revealed
that adult zebrafish cardiomyocytes display a robust T-type
Ca2⫹ current, which, in mammals, is observed only in development or disease. These data further suggest that adult zebrafish cardiomyocytes are less mature than adult mammalian
cardiomyocytes (85).
Whether the underlying mechanisms of adult zebrafish regeneration can be utilized to induce mammalian cardiomyocyte
proliferation remains uncertain. To date only a few regulatory
mechanisms underlying zebrafish cardiomyocyte proliferation
have been identified. Ventricular injury induces raldh2 expression, which is known to result in retinoic acid (RA)-induced
fetal cardiomyocyte proliferation. In addition, induced transgenic inhibition of RA signaling blocked cardiac regeneration
(53). Besides RA signaling, it has been suggested that IGF2
signaling (49) and Notch signaling (131) are required for
zebrafish cardiomyocyte proliferation and cardiac regeneration. In addition, it has been shown that the cardiomyocytespecific miR-133 is a negative regulator of zebrafish cardiomyocyte proliferation (126). All of these pathways/regulators
have previously been shown to be involved in cardiomyocyte
proliferation during mammalian development (62, 67, 105,
112, 116). However, none of these factors has been shown to
have a significant effect on adult mammalian cardiomyocyte
proliferation. While they might be necessary, to date they have
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In a subsequent study Porrello and coworkers (94) investigated
whether the neonatal heart also regenerates after an ischemic
insult induced by permanent ligation of the left anterior descending (LAD) coronary artery, a more disease-related injury.
A major advantage of this study is the marked decrease in
cardiac function 4 days after ligation, which was normalized at
day 21. Moreover, the scar that formed within 7 days was
resolved at day 21. In addition, in contrast to the Hccs-deficient
female mice, LAD-ligated mice showed no obvious pathology
at 9 mo of age, indicating full regeneration. Utilizing the same
assays as in their previous studies the authors concluded that
regeneration is due to induction of cardiomyocyte proliferation. In addition, Aurora and coworkers (6) have shown that the
regenerative response in the LAD model depends on macrophages.
Recently, it has been suggested that, similar to the study of
Drenckhahn and coworkers (28), the regenerative capacity of
newborn mouse hearts is limited. Andersen and coworkers (5)
published the first study that challenged the apex resection
model of regeneration. The authors observed neither a recovery
in heart size and weight nor an increased proliferation rate of
cardiomyocytes, even though they used the same mouse strain
and a similar injury protocol as Porrello and coworkers. However, loss and recovery of heart tissue were assessed differently
(heart to body weight vs. ventricular weight and surface).
Instead of regeneration, the authors observed fibrosis and
cardiac hypertrophy. Interestingly, Jesty and coworkers (51)
had already shown that cryoinjury in neonatal mice does not
result in scar-free regeneration within 3 mo. In addition, signs
of fibrosis and hypertrophy were reported in a diphtheria toxin
receptor-based injury model studying the dependence of neonatal regeneration on macrophages (59). Recently, Konfino and
coworkers (56) challenged the regeneration upon LAD ligation. While they observed complete regeneration after apical
resection, they reported scar formation after LAD ligation in
neonatal mice with a lower cardiomyocyte proliferation rate
than in the apex resection model.
Collectively, these studies demonstrate that, under certain
circumstances, neonatal hearts can recover from injury through
cardiomyocyte proliferation (Table 2). It is possible that the
observed differences are due to varying injury protocols. Recently, two reports have indicated that the regenerative response depends on the severity of the injury. Darehzereshki
and coworkers (26) demonstrated that, in contrast to superficial
cryoinjuries, transmural cryoinjuries resulted, even after 120
days, in severe scarring and marked reduced function. In
addition, Bryant and coworkers (19) reported that the regenerative capacity depends on the size of the resected piece of the
apex. In the future, it will be important to determine how robust
the cardiac regenerative capacity of newborn mice is and to
identify the reasons for the failure of inducing regeneration
upon different injury models in several laboratories. Finally, as
for zebrafish, it will be important to analyze the effect of the
injuries on binucleation.
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Review
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CARDIOMYOCYTE PROLIFERATION
or required for neonatal heart regeneration with the potential to
induce adult mammalian cardiomyocyte proliferation or at
least to improve cardiac function (95, 104). For example,
Mahmoud and coworkers (73) have shown that the transcription factor Meis1 is downregulated upon myocardial infarction
at postnatal day 1, yet only by around 25%. siRNA-mediated
knockdown in rat neonatal cardiomyocytes (age is unclear)
increased rates of histone H3 phosphorylation. Cardiomyocytespecific (␣MHC) Meis1 knockout mice exhibited increased
cell cycle activity (BrdU, H3P, and Aurora B) at low levels
(BrdU index of 0.3%) 14 days after birth and contained an
increased number of total and mononuclear cardiomyocytes.
Analyses of adult Meis1 knockout hearts suggested that cardiomyocyte proliferation markedly decreased with age. Considering that the use of ␣MHC-Cre results in Meis1 deletion
before birth, these data suggest that deletion of Meis1 might
prevent or delay cardiomyocyte differentiation. Yet the authors
confirmed induction of cell cycle activity in adult cardiomyocytes upon Meis1 deletion utilizing ␣MHC-MerCreMer mice,
which allowed Meis1 deletion in cardiomyocytes upon tamoxifen administration. In fact, the cell number data indicate that
Meis1 deletion induced the production of 2 million cardiomyocytes in less than 5 wk by an H3P as well as Aurora B fold
increase of less than fourfold. This is an increase of around
40% of the total cardiomyocyte. The heart-to-body weight ratio
increased by 20%. In conclusion, these data support the idea
that inhibitors control the cell cycle arrest in cardiomyocytes
and that this arrest is reversible. This is in accordance with the
observation that Meis1 deletion results in upregulation of
positive cell cycle regulators and downregulation of other
negative regulators (73). Still, it is important that these data are
confirmed independently, ideally by clonal assays.
Porrello and coworkers (94) reported that the miR-15 family
regulates neonatal heart regeneration. They reported that neonatal hearts of miR-195-overexpressing transgenic mice failed
to regenerate after LAD ligation because of reduced cardiomyocyte proliferation. Subsequently, they tested whether inhibition of the miR-15 family would enable adult cardiomyocyte
proliferation. To address this question they injected locked
nucleic acid-modified anti-miRs postnatally at days 1, 7, and
14 and induced cardiac injury at day 21 by using an LAD
ligation/reperfusion model. Even though the authors observed
an increase from 1 to around 5 H3P-positive cardiomyocytes
per section, the treatment had only a minor effect on cardiac
function.
Critical Open Questions Regarding Human Heart
Regeneration
Is the cell cycle arrest in adult mammalian/human cardiomyocytes actively maintained and reversible? Decades ago it
was demonstrated that the expression of a large variety of
positive cell cycle factors is downregulated in cardiomyocytes
during development, while negative regulators, such as cyclindependent kinase inhibitors (CDKI), are transiently upregulated (113, 115, 129). After birth, the majority of cardiomyocytes become binucleated or polyploid (Fig. 1) (17, 45, 107),
the contractile apparatus changes dramatically into a highly
ordered structure (66), and cardiomyocytes increase in size
almost 20-fold (13, 37). These changes might explain why the
proliferative potential and thus the efficiency to induce cardi-
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not been shown to be sufficient to induce proliferation in adult
mammalian cardiomyocytes.
In contrast to the above-mentioned factors, Fang and coworkers (35) have reported a Stat3-dependent program as
injury-specific regulator of zebrafish cardiomyocyte proliferation. They identified several Jak1/Stat3 pathway members by
screening translating RNAs in zebrafish cardiomyocytes during
heart regeneration. Cardiomyocyte-specific Stat3 inhibition
blocked proliferation during regeneration but not development.
Retro-orbital introduction of the secreted protein Rln3 partially
rescued Stat3-inhibited cardiomyocyte proliferation (35). In
addition, Aguirre and coworkers (2) have recently suggested
that a microRNA program (miR-99/100, Let-7a/c, fntb,
smarca5) responsible for zebrafish heart regeneration can be
utilized to regenerate the mammalian heart. Manipulation of
these factors indicated that they regulate zebrafish cardiomyocyte proliferation after cardiac injury. The authors subsequently utilized these factors to induce adult mammalian cardiomyocyte proliferation. They provided in vitro-only data
regarding re-expression of proliferating cell nuclear antigen
(PCNA), a DNA clamp that acts as a processivity factor for
DNA polymerase-␦ in eukaryotic cells and is essential for
DNA replication. In vivo, they determined expression of
GATA4, PCNA, Aurora B kinase, and Anillin as well as
histone H3 phosphorylation, and BrdU incorporation. While
these data indicate proliferation, they don’t prove cardiomyocyte proliferation (see above). Yet cardiac function (% fractional shortening and ejection fraction) was also significantly
improved, and ventricular wall thickness was recovered to
around 50% (2). Whether this is due to adequate levels of
cardiomyocyte proliferation, activation of a compensatory hypertrophic response, or a combination of both remains unclear.
Newborn mouse heart regeneration. The mouse heart is
anatomically highly similar to the human heart (120). However, the difference in size results in different physiological
demands and thus in differences to human cardiac physiology.
In contrast to the human heart, the mouse heart can beat up to
800 times per minute and exhibits significantly different calcium handling. On a molecular level, the human and mouse
hearts differ, for example, in the phosphorylation and isoform
expression of a variety of contractile proteins (e.g., myosin
heavy chain, titin, troponin) (79, 86). Whether these differences affect cardiomyocyte proliferation is unclear. Yet the
human heart might have a greater regeneration potential than
those of mice and rats, as ⬎60% of adult human cardiomyocytes remain mononucleated (but mainly polyploid) (80),
while ⬎90% of adult mouse and rat cardiomyocyte are
bi-/polynucleated (61, 107). In addition, it has been suggested that low proliferation rates of human cardiomyocytes
can be observed until the second decade of life (80). Yet a
recent paper challenges this view and provides data demonstrating that the final number of human cardiomyocytes is
reached at 1 mo of birth and remains constant thereafter
with a low turnover rate of around 1% (11).
Newborn heart regeneration as well as development studies
will help to identify mechanisms required for cardiomyocyte
proliferation. Moreover, they might help to elucidate the mechanism underlying the cell cycle arrest. However, it remains
unclear whether this knowledge will lead to reversal of cell
cycle arrest in adult cardiomyocytes, allowing heart regeneration. Yet several genes have been identified as associated with
Review
CARDIOMYOCYTE PROLIFERATION
H1247
to be confirmed independently. A major issue here is that often
the limitations of the different proliferation assays are not
considered. In addition, there are no studies that follow individual cardiomyocytes to determine if they divide, if the
resulting daughter cells are viable and functional, and whether
they can continue to proliferate. Moreover, available clonal
assays that can unambiguously demonstrate cardiomyocyte
proliferation in vivo are rarely utilized. Importantly, the induction efficiency of mammalian cardiomyocytes decreases dramatically with age, which might be at least in part due to
epigenetic mechanisms (103) and centrosome disassembly
(130).
An important goal in achieving mammalian heart regeneration is to understand naturally occurring heart regeneration.
While newt and zebrafish regeneration is well established, it
appears that cardiomyocytes of these species do not undergo a
similar cell cycle arrest as mammalian cardiomyocytes after
birth (130). Thus, it might not be possible to learn from newt
and zebrafish regeneration how to reverse the cell cycle arrest
in adult mammalian cardiomyocytes. The same might be true
for neonatal heart regeneration. In addition, it remains unclear
to what extent neonatal mice can regenerate their hearts (19,
26, 28).
Furthermore, one has to consider carefully the mammalian
model system in which one studies the effect of candidate
factors in improving cardiac function. For example, pigs are
commonly used in large-animal studies for translational cardiac studies as they share more similar function and structure
to humans than do rodents. However, porcine cardiomyocytes
contain 4 –16 nuclei (1, 42). Thus, it is unclear whether
large-animal models are suitable for studies of cardiac regeneration based on cardiomyocyte proliferation. However, data
are accumulating, at least in the adult mouse heart, for the
existence of a subpopulation of cardiomyocytes with neonatal
characteristics (55). These cells might be responsive to the
mechanisms utilized during naturally occurring heart regeneration. Thus, in future study it will be important to determine in
which species such subpopulation of cardiomyocytes exist.
Finally, it should be considered that the heart does not
consist only of cardiomyocytes. Cardiomyocytes contribute
only around 40 – 60% of the cells in mammalian hearts (8, 23,
46, 90, 117). Thus it appears important to better understand
how the current regenerative approaches contribute to the
formation of new complex cardiac tissue, how the dynamic of
the process is, and whether dividing cardiomyocytes actively
contribute to it.
Conclusions
ACKNOWLEDGMENTS
A large amount of data has been accumulated that demonstrates that, in newt, zebrafish, and newborn mice, a variety of
injuries induce heart regeneration by cardiomyocyte proliferation. In addition, it has been shown that it is possible to
efficiently induce neonatal mammalian cardiomyocyte proliferation. In contrast, with regard to adult cardiomyocytes, little
evidence exists supporting true proliferation (i.e., successful
completion of cytokinesis resulting in two cells). Moreover,
much of the data suggestive of adult cardiomyocyte proliferation is mainly based on immunofluorescence analysis, which,
depending on the scrutiny and method of analysis, can lead to
misinterpretations. Ultimately, much of the data underlying
claims of regeneration by cardiomycoyte proliferation has yet
We thank the rest of the Engel lab for critical reading and suggestions.
GRANTS
This work was supported by grants from the Emerging Fields Initiative
(EFI) for Cell Cycle in Disease and Regeneration (CYDER) from the
Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU to F. B. Engel) and
the Deutsche Forschungsgemeinschaft (IRTG 1566, PROMISE; EN 453/9-1 to
F. B. Engel).
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the author(s).
AUTHOR CONTRIBUTIONS
Author contributions: M.L. and F.B.E. analyzed data; M.L. and F.B.E.
prepared figures; M.L., A.M., and F.B.E. drafted manuscript; M.L., A.M., and
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omyocyte proliferation are markedly decreased in adult mammalian cardiomyocytes. Yet the mechanism that induces
CDKI-dependent cell cycle arrest of mammalian cardiomyocytes after birth, as well as its purpose, remains elusive. Sdek
and coworkers (103) have shown that the regulation of cell
cycle genes in cardiomyocytes is mediated by histone methylation. Possible inducers of this epigenetic regulation might be
alterations of the extracellular matrix around birth, as well as
elevated levels of reactive oxygen species inducing DNA
damage (98, 128, 129). Yet there is also evidence that the cell
cycle arrest might be initiated even before birth. For example,
fetal cardiomyocytes cannot be maintained in a proliferative
state in vitro. They exit the cell cycle in vitro in a similar
temporal pattern as it occurs in vivo, suggesting that an
intrinsic process is activated in cardiomyocytes before birth,
limiting the proliferative capacity of cardiomyocytes to a
distinct number of cell divisions (20). Moreover, it has recently
been shown that mammalian cardiomyocytes, in contrast to
newt and zebrafish cardiomyocytes, start to disassemble their
centrosomes in rats as early as embryonic day 15, a process
that is completed shortly after birth (130). As recent data have
demonstrated that the centrosome is a critical signaling hub for
the cell cycle regulatory machinery, these data indicate the loss
of centrosomes after birth as a possible reason why the hearts
of mammals are unable to regenerate after injury. Yet the
observations that inhibition of p38 MAP kinase enhances
cardiomyocyte proliferation in the absence of a functional
centrosome (31, 130) and that newborn mice can regenerate
their heart after apex resection through cardiomyocyte proliferation (93), as well as several other studies (reviewed in Refs.
95, 104), provide some optimism for heart regeneration via
proliferation of endogenous cardiomyocytes.
Does a subpopulation of adult mammalian/human cardiomyocytes with a regenerative potential exist? In recent years
evidence has accumulated suggesting that cardiomyocyte proliferation turnover occurs in both mice and humans throughout
their lifetimes (10, 108). Yet it is unclear if all cardiomyocytes
contribute to this turnover or whether a subpopulation with a
higher proliferative capacity exists. The existence of such a
subpopulation of mononucleated cardiomyocytes was suggested as early as in 2007 (58) and has recently been substantiated by a fate mapping study identifying a “rare population of
hypoxic cardiomyocytes that display characteristics of proliferative neonatal cardiomyocytes, such as smaller size, mononucleation and lower oxidative DNA damage” (55).
Review
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CARDIOMYOCYTE PROLIFERATION
F.B.E. edited and revised manuscript; M.L., A.M., and F.B.E. approved final
version of manuscript.
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