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CALL FOR PAPERS Cardiac Regeneration and Repair: Mechanisms and Therapy
THE MAMMALIAN HEART FORMS from the first and second heart of cardiomyocytes by cardiomyocyte hypertrophy (increase in
field. The heart field precursors migrate and fuse at the midline cell size). In contrast to reversible physiological hypertrophy
of the embryo to form the primary linear heart tube (81). The during pregnancy or endurance training, this compensatory
heart tube then elongates, which occurs primarily via addition hypertrophy results in concentric or dilative remodeling (Fig.
of precursor-derived cardiomyocytes to both poles. Subsequent 1). It is accompanied by inflammation, fibrosis, increased wall
“ballooning” of the chambers and growth of the heart are stiffness, cardiomyocyte slippage, and continuous apoptotic
achieved by proliferation of contracting cardiomyocytes (109). loss of cardiomyocytes, resulting in reduced heart function (47,
After birth, mammalian cardiomyocytes exit the cell cycle and 57, 76).
stop proliferating (Fig. 1) (107, 115). Currently, there are no effective therapies available to re-
Cardiovascular diseases are among the leading causes of verse cardiac damage and remodeling, and, for a large group of
death worldwide, presenting a major socioeconomic burden patients, further deterioration cannot be efficiently prevented.
whose incidence is expected to increase further (39). Upon
Consequently, there is an urgent need for the development of
injury the adult mammalian heart does not regenerate, neither
novel therapies. In contrast to adult mammals, newborn mice,
by activating stem cells nor by induction of cardiomyocyte
proliferation (129). Instead, the heart compensates for the loss newts, and zebrafish regenerate their hearts after experimen-
tally induced injury by the induction of cardiomyocyte prolif-
eration (14, 38, 63). Thus, extensive efforts have been invested
* M. Leone and A. Magadum contributed equally to this work. to determine whether it is possible to induce adult mammalian
Address for reprint requests and other correspondence: F. B. Engel, Experimental
Renal and Cardiovascular Research, Inst. of Pathology, Dept. of Nephropathology,
cardiomyocyte proliferation. A large body of evidence has
Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU), Schwabachanlage 12, accumulated in recent years suggesting that in the future it
91054 Erlangen, Germany (e-mail: felix.engel@uk-erlangen.de). might be possible to prevent functional deterioration after
http://www.ajpheart.org 0363-6135/15 Copyright © 2015 the American Physiological Society H1237
Downloaded from journals.physiology.org/journal/ajpheart (192.167.074.006) on March 28, 2023.
Review
H1238 CARDIOMYOCYTE PROLIFERATION
Polyploidization Polyploidization
H
E
C Pregnancy A
M G2 Sport G2
S S R
G1
T
Hypertrophy
Cell cycle Birth Cell cycle G0 Cell cycle F
G1/0 M A
G2 Injury M I
Injury C
Regeneration G1 G1 L
C U
S R
Binucleation Endomitosis Polynucleation E
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.
injury and even to reverse cardiac damage by inducing cardi- However, DNA synthesis occurs not only during semiconser-
omyocyte proliferation. vative DNA replication during S phase but also during DNA
repair. Moreover, semiconservative DNA replication is merely
Assays to Determine Cardiomyocyte Proliferation
an indicator of S-phase cell cycle progression (115, 129). Thus,
Cardiomyocyte cell cycle activity does not necessarily a disadvantage of this assay is that it does not predict whether
equate with proliferation as it can also reflect pathological a cell will divide or undergo G2/M arrest, polyploidization, or
hypertrophy, polyploidization, or polynucleation (115, 129). polynucleation. Pulse-chase experiments permit the identifica-
Therefore, it is important to be careful with the interpretation tion of colony formation and thus, by deduction, cell division,
of data from cell cycle assays (Table 1). Misinterpretation of as the label will be reduced by 50% per each cell division. Yet,
cell cycle assays might provide an explanation for the contro- to our knowledge, this approach has so far not been utilized in
versies in the field of cardiac regeneration, as to the extent of the field of cardiac regeneration.
cardiomyocyte proliferation. Therefore, we will discuss here Colorimetric assays. Several colorimetric proliferation as-
their advantages and disadvantages. says are available. These assays, such as MTT, Alamar Blue, or
Nucleotide analog incorporation. A commonly used ap- ViaLight assays, are based on a color change due to the
proach in determining the extent of cardiomyocyte prolifera- cleavage of tetrazolium salts, reduction of resazurin, or the
tion is the use of a nucleotide analog-incorporation assay detection of cellular ATP. An increase in the color change is
(BrdU, EdU, tritiated thymidine). Nucleotide analogs will be interpreted as an increase in cell number. However, these
incorporated during DNA synthesis, and thus cells will be assays work on the assumption that mitochondrial activity or
labeled during S phase. A major advantage of this assay is that the amount of cellular ATP per cell is constant. In this regard,
it permits the ability to perform long-term labeling, as well as these assays have limitations when applied to cardiomyocytes,
pulse-chase experiments. Long-term labeling is especially as mitochondrial activity is increased during hypertrophy or
helpful for the detection of cell cycle entry when the time elevated contractility (100). Moreover, as these assays are not
course of cell cycle activation is unclear and, furthermore, cell-type specific, it cannot be excluded that an increase in the
helps to determine the total number of cycling cardiomyocytes. colorimetric signal results from an increase in proliferation of
Nucleotide incorporation X X X X X X X
Ki67 X ? X X ? ? X X X
Histone H3 phosphorylation X X X X X X
Aurora B/Anillin X ? X X X X ?
Sarcomere disassembly X X ? X X
FACS X X X X X X
Colorimetric assays X X
Cell count X X
Clonal analysis X X
FACS, fluorescence-activated cell sorting.
G2 phase
H3P/Anillin
H3P
Anillin
Fig. 2. Schematic representation of H3P, Anil-
Aurora B lin, and Aurora B localization in dividing vs.
DAPI binucleating cardiomyocytes. In G2 phase, his-
Prophase tone H3 becomes phosphorylated (H3P) while
Anillin (diffuse) and Aurora B (dotty) accumu-
late 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 car-
diomyocytes. During anaphase, Anillin accu-
mulates at the cell cortex, and Aurora B in the
Anaphase
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 proliferat-
ing cardiomyocytes. In cytokinesis of dividing
cardiomyocytes, Anillin localizes around the
midbody, and Aurora B concentrates at its
flanking regions. In binucleating cardiomyo-
Cytokinesis cytes, Aurora B behaves similarly; however,
Anillin fails to concentrate around the midbody.
Costaining of Aurora B and Anillin highlights
the asymmetric furrow ingression, a character-
istic of binucleating cardiomyocytes.
Cycling product
and associates with centromeric heterochromatin early in mi- we have demonstrated that this complex disassembly-reassembly
tosis (Fig. 2). In contrast to H3P as a marker, Aurora B can be process occurs not only in neonatal and adult cardiomyocytes after
used to analyze cytokinesis as it transfers first from the kinet- induction of cell division but also during binucleation (33, 34).
ochore during metaphase to the central spindle during ana- Thus, sarcomere disassembly, often misleadingly called “dedif-
phase, ultimately contributing to midbody formation during ferentiation,” is not an unambiguous marker of cardiomyocyte
telophase (24, 121). Unfortunately, we realized later that Au- proliferation. However, it can be utilized to exclude prolifera-
rora B alone is also not a reliable marker for cell division, as its tion as sometimes H3P-positive cardiomyocytes are reported
staining pattern in dividing cells is indistinguishable from the with an intact sarcomeric apparatus, indicative of endomitosis
pattern in binucleating cells (34). However, Aurora B staining (32, 115).
combined with a sarcomere staining allowed us to visualize Cell count experiments. Cell count experiments are used as
cytokinesis failure during binucleation based on asymmetric a direct measurement of cardiomyocyte proliferation. Yet,
constriction of the cleavage furrow. Subsequently, we intro- often investigators measure only the total cell number, not
duced Anillin as an additional marker to evaluate whether a distinguishing between cardiomyocytes and nonmyocytes. In
cardiomyocyte will binucleate or divide. Like Ki67, Anillin is contrast, we have for example shown that the total number of
absent in G0 phase. In proliferating cells it accumulates in the neonatal cardiomyocyte increases over time after FGF1/p38
nucleus during G1, S, and G2 phase, then localizes to the cell inhibitor stimulation based on total cell number corrected for
cortex at the onset of mitosis, and finally locates around the the percentage of cardiomyocytes as analyzed by FACS (33).
midbody in cytokinesis (36, 111). Aurora B and Anillin In addition, in some studies the cell number after stimulation is
costaining identifies binucleating cells based on asymmetric compared with control stimulation. A higher cell number can
constriction of the cleavage furrow and failure of Anillin to also be explained by a cell-protective effect, as it is known that
focus at the cortex in anaphase and to concentrate around the neonatal as well as adult cardiomyocytes undergo apoptosis in
midbody during cytokinesis (34). Collectively, the sole pres- culture. In addition, cell numbers are often determined by
ence of Aurora B or Anillin is not proof of cell division. isolating cardiomyocytes from tissue or trypsination from cul-
However, the cellular localization of Anillin will indicate if a ture dishes. Here, it should be considered that alterations in
cell will divide or binucleate. extracellular matrix composition, cell-to-cell adhesion, and cell
Fluorescence-activated cell sorting. A method that has size can significantly affect the cell count due to inefficient
rarely been utilized to characterize cell cycle progression in enzymatic digestion or altered efficiency in cell harvest by
cardiomyocytes is fluorescence-activated cell sorting (FACS). centrifugation.
Measuring cell proliferation via FACS is based on labeling Clonal analysis. Clonal analysis from single cells unambig-
isolated cardiomyocytes with a cardiomyocyte-specific marker uously determines if a stimulus induces proliferation. In vitro,
and a fluorescent DNA dye, which permits determination of the clonal analysis has not yet been reported for cardiomyocytes.
DNA content based on fluorescence intensity. Using this ap- However, we have introduced the monitoring of cardiomyo-
proach, one can determine what fraction of the cardiomyocytes cytes over time, demonstrating an increase in cardiomyocyte
is for example in S phase. Time series experiments help to density after FGF1/p38 inhibitor stimulation (33). Recently,
determine whether DNA synthesis results in binucleation (in- Lin and coworkers (65) performed clonal analyses in vivo. The
crease in the G2/M phase peak), polyploidization/polynucle- authors utilized the Brainbow technology (68) to induce, in a
ation (DNA content ⱖ 4n), or cell division (no change in the small number of randomly distributed cardiomyocytes, expres-
G1, S, and G2/M distribution) (34). In addition, one can sion of a fluorescence protein (to obtain individual labeled
perform a BrdU pulse-chase experiment (diluting the BrdU cardiomyocytes). In addition, they utilized a cardiomyocyte-
label due to cell division) or a BrdU-Hoechst assay to prove specific doxycycline-inducible system to induce Yes-associ-
that cell division occurs (29). The principle of a label being ated protein expression in cardiomyocytes (65). If induction of
diluted because of cell division (such as BrdU or membrane proliferation were successful, one would expect the formation
labeling) even enables investigators to determine how often a of clones from the individual labeled cardiomyocytes (Fig. 3).
cell has divided, as each cell division results in a 50% decrease It should be noted, however, that the Brainbow system for
of the label (71). clonal analysis requires that labeled individual cells with the
Sarcomere disassembly/dedifferentiation. Previously, it has same color not be adjacent to one another. Given that nonin-
been suggested that newt and zebrafish cardiomyocytes de- duced/control hearts in the study by Lin and coworkers occa-
differentiate before undergoing proliferation based on the ob- sionally exhibited clones of two adjacent cardiomyocytes of
servation that their sarcomeric apparatus disassembles (52). the same color, this assay is thus useful only if a significant
Yet Ahuja and coworkers (3) have shown in detail that fetal number of clones of at least four labeled cardiomyocytes are
cardiomyocytes disassemble their sarcomeric apparatus in a observed. A more direct proof of cardiomyocyte division has
highly ordered fashion during mitosis and reassemble it during been introduced by Ali and coworkers (4), who have utilized
cell division. This suggests that sarcomere disassembly is not a the “mosaic analysis with double markers” mouse model (132),
sign of dedifferentiation but is simply a necessary process to which can distinguish between parental and daughter cells. In
allow cardiomyocyte proliferation. That adult mammalian car- this system parental cells are GFP- and RFP-double positive.
diomyocytes are also able to restructure or disassemble their After cell division daughter cells will be RFP or GFP positive,
sarcomeric apparatus has been long known. For example, adult while binucleated cells will be GFP- and RFP-double positive
cardiomyocytes with disassembled/disorganized/restructured (Fig. 3). As this system is dependent on Cre recombination,
sarcomeric apparatus has been observed in long-term in vitro proliferation can be analyzed specifically in cardiomyocytes.
cultures (92, 110), hypocontractile hibernating myocardium While this system has been used to determine naturally occur-
(72, 106), and Tako-Tsubo cardiomyopathy (83). Importantly, ring cardiomyocyte proliferation (4), it has not yet been uti-
Myh6CreERT2
No
Tamoxifen
cytokinesis
Mitosis
G2-X
Cytokinesis
rtTA
Myh6-MCM aYAP
rtTA
aYAP
rtTA
aYAP
Centromere Doxycycline-dependent
rtTA Promoter rtTA reverse tetracycline
LoxP site activator gene
aYAP
rtTA
N-GFP Doxycycline-dependent
aYAP
C-GFP reverse tetracycline
N-RFP activator protein
rtTA C-RFP
aYAP Stop aYAP “Yes-associated
protein” gene
RFP
GFP
Cre Recombinase
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 doxycycline-
dependent reverse tetracycline activator protein.
lized to test the efficiency of factors to induce cardiomyocyte and discussed, cardiomyocyte-specific cytoplasmic markers
proliferation. are prone to misinterpretations; also the presentation of Z-
Cardiomyocyte specificity. A major issue, especially in vivo, stacks cannot solve this issue (32, 108). Thus, the detection of
is to determine whether the signal of a cell cycle marker indeed Ki67, BrdU, or cell cycle regulators such as cyclins should be
belongs to a cardiomyocyte. This is especially critical as often combined with staining for a cardiomyocyte-specific nuclear
there is an increase of fibroblast proliferation and infiltration of marker such as GATA4 or MEF2. Besides a nuclear marker,
inflammatory cells upon injury. As has been previously shown cardiomyocyte-specific cytoplasmic membrane markers such
as caveolin 3 can be utilized to identify unambiguously a cells are characterized by the absence of thin filaments and Z
cycling cardiomyocyte (32). bands (87). These features have also been observed in dividing
The analysis of commonly used methods to determine car- fetal mammalian cardiomyocytes, indicating that the adult
diomyocyte proliferation shows that while many of these newt heart might indeed regenerate its heart by cardiomyocyte
techniques can detect cell cycle activity, distinguishing be- proliferation. However, this “transient disassembly of the sar-
tween proliferation, polynucleation, or polyploidization is comeric apparatus” occurs also when cells fail to undergo cell
more difficult and in some cases impossible. Not acknowledg- division after mitosis resulting in binucleated cardiomyocytes
ing this issue often results in misinterpretation of results and is (34). Thus, it was important to determine whether the number
likely the reason for the contradictions, and even controversies, of binucleated cardiomyocytes increases upon injury. Ober-
in the field of cardiac regeneration. priller and coworkers (89) reported that the vast majority
(⬎98%) of adult newt cardiomyocytes are mononucleated.
Naturally Occurring Cardiac Regeneration Importantly, they have demonstrated that the majority of newt
cardiomyocytes (⬃85%) that underwent DNA synthesis 45
Pondering regeneration is as old as civilization. First written days after injury (apex resection and reintroduction after minc-
reports by Empedocles and Aristotle describing the observation ing) were still mononucleated and diploid. These data strongly
that certain animals can regenerate their appendages can be indicated that the vast majority of adult newt cardiomyocytes
found as early as 400 BC. In modern times the first known that enter the cell cycle complete cell division.
publication regarding regeneration was by René Antoine Fer- To prove that adult newt cardiomyocytes are indeed able to
chault de Réaumur in 1712, where he described limb regener- complete cell division and to proliferate, Matz and coworkers
ation in crustaceans (crayfish, crabs, and lobsters) (41, 99). In
(77) performed life cell imaging experiments (day 8 –19 in
1740, Abraham Trembley perturbed, analyzed, and dissected
culture). These experiments demonstrated that 80% of mono-
the process of regeneration in hydra, which is considered to be
nucleated cardiomyocytes produced mononucleated daughter
the birth of “experimental biology” (60). In contrast to com-
cells, while the remaining failed to complete cytokinesis,
mon belief, regeneration is a widely distributed event with
resulting in binucleated cells. In addition, 32% of binucleated
regenerative species in almost every phylum (101).
cardiomyocytes produced binucleated daughter cells. Betten-
Urodele heart regeneration. Urodeles (salamanders and
court-Dias and coworkers (12) confirmed the proliferative
newts) are considered to be the champions of regeneration.
They can regenerate a variety of structures such as limbs, tails, competence of adult newt cardiomyocytes in vitro. They ob-
jaws, eyes, and the heart (16, 114). Urodele heart regeneration served that approximately one-third of the cells completed cell
was mentioned in the literature as early as 1974 (9, 88). After division and part of those entered successive cell divisions.
Oberpriller and Oberpriller (87) reported the appearance of These data suggest that the newt heart contains a subpopulation
mitotic adult newt cardiomyocytes in Diemictylus viridescens of cardiomyocytes that retain a higher proliferative potential,
16 days after apical resection, they studied the response of the while the remaining newt cardiomyocytes behave similarly to
adult newt ventricle to injury in Notophthalmus viridescens their mammalian counterparts.
(88). The authors resected around 12.5% of the ventricle. At To determine whether new myocardium is formed during
the site of injury a blood clot occluded the ventricle and newt regeneration, Piatkowski and coworkers (91) introduced
macrophages accumulated at 10 days within and around it. To “scanning electron microscopy analysis in combination with
determine if the injury induced cardiomyocyte proliferation the stereological estimation”. Injury induced by “repeated orthog-
authors injected tritiated thymidine 3 h before tissue collection onal squeezing with fine forceps” was followed by cardiac
to determine the frequency of cycling cardiomyocytes. The regeneration associated with a significant increase in the vol-
maximal thymidine-incorporation index (9 –10%) and mitotic ume fraction of trabeculae. In contrast to initial studies, this
index (0.9%) were observed at 20 days postinjury. Becker and and other studies have demonstrated, by investigating later
coworkers (9) provided additional support for the capacity of time points, that cardiac newt regeneration results in a mor-
urodeles to regenerate their hearts. They demonstrated that phologically fully regenerated heart with no signs of scarring
Triturus viridescens at the adult aquatic stage survives the (91, 124).
resection of 30 –50% for at least 2 wk with a mortality rate of To provide evidence that newts can functionally regenerate
10% (several hundred resections were performed). Normal their hearts, a standardized ventricular resection model was
circulatory dynamics were re-established after 5 h based on established, whereby “a more lateral region of the ventricle, as
microscopic analysis of skin capillaries in the tail fin using opposed to the apex,” (5–10%), was resected (124). Echocar-
transmitted light. These data suggested that, upon injury, the diography was utilized for the analysis of heart function and
adult newt heart might be able to regenerate its heart by revealed that heart function was reduced at day 7 postinjury
cardiomyocyte proliferation. However, the authors observed from around 38-34% fractional shortening. Full function was
the formation of scar tissue at 30 days postinjury, indicating a reported at 23 days and 60 days postinjury (⬃40% fractional
limited regenerative response (88). shortening) (124). However, it remains unclear whether the
To demonstrate that the newt heart regenerates from cardi- functional improvement is indeed due to newly formed heart
omyocyte proliferation several assays have been performed. tissue. Considering that a rather small amount of muscle tissue
Mitotic cardiomyocytes in newts were initially identified by was resected, the rather small drop in function might also be
electron microscopy based on condensed mitotic chromatin, due to the initial formation of the blood clot and scar at day 7,
“fibrils, which appeared to be myofibrils approximately the impairing wall motion. In the future it would be important to
length of a sarcomere” in the periphery of the cell, a basal repeat such a study in a model with more severe damage to the
lamina, and glycogen granules (87). In addition, these mitotic heart. This is especially important as recent studies in neonatal
in vitro
in vivo after Genetic Mechanical Functional
Species Neonatal Adult Injury Resection Cryoinjury Ablation LAD Injury Improvement Reference
Urodeles X X 88
X X 9
X X 87
X X 89
X 12, 77
X X 91
X X X 124
Zebrafish X X 97
X X X 21
X 25
X X X X 119
X X 52
X X X 54
X X X 43
X X 50
X X 127
X X 40
Mammals (fetal/neonatal) X 61, 115
X X 19
X X X 26
X X 74, 75
X X X 93
X X X 94
X X X 6
X X 5
X 51
X X X X 56
LAD, left anterior descending coronary artery ligation.
difficult to identify the amputation plane and to identify newly paired wall motion by increased pumping capacity of the
formed cardiomyocytes, it remained unclear if zebrafish can remainder of the ventricular wall. However, it remains unclear
fully restore lost myocardium and whether the observed mor- if lost cardiac tissue is fully replaced by cardiomyocyte pro-
phological regeneration is indeed due to cardiomyocyte prolif- liferation or whether hypertrophy and/or binucleation contrib-
eration. For example, the morphologically repair of a ventricle ute to this process. In the future, it will be important to
might be due to cardiomyocyte migration, which has been demonstrate that adult zebrafish cardiomyocytes can proliferate
shown to be essential for zebrafish cardiac regeneration (50). in vitro and to elucidate how adult zebrafish cardiomyocytes
To address this issue, the group of Ken Poss (22) recently differ from postnatal mammalian cardiomyocytes.
introduced so-called “zebraflash” transgenic lines for in vivo Newborn mouse heart regeneration. Fetal and newborn
bioluminescence imaging. Expression of firefly luciferase un- cardiomyocytes have the ability to proliferate (115). Conse-
der the cardiomyocyte-specific promoter cmcl2 allows for quently, the question was raised if this ability is enough to
direct monitoring of the recovery of heart size. However, this allow the fetal, embryonic, or newborn heart to regenerate after
transgenic line cannot distinguish whether recovery is due to injury. In 1975 the first evidence for fetal heart regeneration
either proliferation or hypertrophy. Moreover, these transgenic after mechanical injury in rabbits was reported (74, 75). In
lines cannot be used to determine functional recovery. 2008, Drenckhahn and coworkers (27) introduced a mouse
To demonstrate that the zebrafish can fully regenerate heart model that demonstrated that the fetal heart can compensate for
function after injury, physiological parameters have been ana- the genetic inhibition of proliferation in around 50% of fetal
lyzed, resulting in contradictory results. Analyses of cardiac cardiomyocytes. Proliferation was inhibited through inactiva-
conduction postresection in regenerating and scarring hearts tion of the X-linked gene encoding holocytochrome c synthase
initially suggested that zebrafish might not fully recover func- (Hccs). While deletion of Hccs in knockout males resulted in
tion. J-point depression was statistically significant 10 days midgestational lethality, heterozygous knockout female mice
postinjury and gradually normalized at 60 days postresection, were not affected, even though around 50% of the cardiomy-
independent of scarring or regeneration. In contrast, QTc ocytes were Hccs deficient because of the random nature of
intervals remained prolonged in both scenarios, indicating X-chromosome inactivation. The gradual increase of complex
delayed electric repolarization (127). However, Kikuchi and III activity of the mitochondrial respiratory chain, the decrease
coworkers (54) reported normalization of conduction 30 days in Hccs-deficient cardiomyocytes, and a detailed cell cycle
postresection, which was later confirmed in the cryoinjury analysis showed that the reduced proliferation rate of Hccs-
model (21). While these studies provided a more physiological negative cells is compensated for by an increased proliferation
insight into cardiac regeneration in zebrafish, recovery of rate of the healthy cardiomyocytes. However, hearts of Hccs-
cardiac conduction is a weak indicator of improved cardiac deficient female mice at postnatal day 1 were still hypoplastic.
function. Importantly, despite an increased proliferation rate adult Hccs-
In 2011, Wang and coworkers (119) introduced “swimming deficient females exhibit a range of cardiac phenotypes (28).
endurance” as a read-out to evaluate functional recovery after These data suggest that the fetal heart has only a limited
injury. Zebrafish were placed in a swim tunnel respirometer capacity for cardiac regeneration.
(MiniSwim-170; Loligo Systems, Tjele, Denmark) and al- While fetal cardiomyocytes contribute significantly to heart
lowed to acclimatize for 20 min at a low flow velocity of 3 growth by proliferation, cardiomyocytes of newborn mice have
cm/s. Swimming speed was then slowly increased to a velocity a limited proliferation capacity, and cell cycle activity gener-
of ⱖ20 cm/s, and the endurance of zebrafish was monitored. ally results in binucleation instead of cell division (61). Despite
The authors utilized this system to test the effect of inducible this fact, Porrello and coworkers (93) observed that neonatal
genetic ablation of a high percentage of cardiomyocytes mice at postnatal day 1, but not day 7, exhibit a transient
through overexpression of cytotoxic diphtheria toxin A chain. cardiac regeneration capacity. In addition, Naqvi and cowork-
They demonstrated that inducible genetic ablation of ⬎60% of ers (82) have recently reported a brief but intense proliferative
cardiomyocytes in contrast to apex resection results in reduced burst of predominantly binuclear cardiomyocytes at postnatal
performance in a swim tunnel. Recovery of this phenotype, day 15 in mice. Moreover, they report that mice at postnatal
considering an initial loss of ⬍60% together with recovery of day 15 were able to partially regenerate upon experimental
the density of cardiomyocyte nuclei, provided strong evidence myocardial infarction. Apex resection (15%) at postnatal day 1
of regeneration from proliferation, even though binucleation was restored within 21 days following a similar process as
had not been excluded (119). described for zebrafish (see above) (93). Cardiomyocyte pro-
Recently, Gonzalez-Rosa and coworkers (40) introduced the liferation was assessed by BrdU incorporation assays, phos-
use of 2D echocardiography to directly determine the pump phorylation of histone H3, aurora B kinase expression, and
function of the zebrafish heart. In a longitudinal study analyz- sarcomere disassembly, indicating an up to sevenfold increase
ing individual zebrafish over time the authors observed that in cell cycle activity. As described above these markers do not
global systolic function but not ventricular wall motion was allow one to distinguish between proliferation and binucleation
fully recovered following cardiac cryoinjury. This was in (34). However, the observation that ventricular weight and
agreement with an increased density of cardiomyocyte nuclei surface area in the absence of hypertrophy were recovered
in the injured ventricular wall that might affect normal con- within 21 days supports the conclusion that the rate of cardi-
traction due to ultrastructural changes. omyocyte proliferation is increased upon resection. In addition,
In conclusion, the available data indicate that zebrafish can, cardiac function of resected animals was comparable to sham-
to a large extent, regenerate their hearts by means of cardio- operated hearts. Yet it remains unclear whether heart function
myocyte proliferation (Table 2). In addition, it appears that in was affected and whether the normal differentiation process
case of incomplete regeneration zebrafish compensate for im- resulting in binucleation was inhibited by the resection or not.
not been shown to be sufficient to induce proliferation in adult or required for neonatal heart regeneration with the potential to
mammalian cardiomyocytes. induce adult mammalian cardiomyocyte proliferation or at
In contrast to the above-mentioned factors, Fang and co- least to improve cardiac function (95, 104). For example,
workers (35) have reported a Stat3-dependent program as Mahmoud and coworkers (73) have shown that the transcrip-
injury-specific regulator of zebrafish cardiomyocyte prolifera- tion factor Meis1 is downregulated upon myocardial infarction
tion. They identified several Jak1/Stat3 pathway members by at postnatal day 1, yet only by around 25%. siRNA-mediated
screening translating RNAs in zebrafish cardiomyocytes during knockdown in rat neonatal cardiomyocytes (age is unclear)
heart regeneration. Cardiomyocyte-specific Stat3 inhibition increased rates of histone H3 phosphorylation. Cardiomyocyte-
blocked proliferation during regeneration but not development. specific (␣MHC) Meis1 knockout mice exhibited increased
Retro-orbital introduction of the secreted protein Rln3 partially cell cycle activity (BrdU, H3P, and Aurora B) at low levels
rescued Stat3-inhibited cardiomyocyte proliferation (35). In (BrdU index of 0.3%) 14 days after birth and contained an
addition, Aguirre and coworkers (2) have recently suggested increased number of total and mononuclear cardiomyocytes.
that a microRNA program (miR-99/100, Let-7a/c, fntb, Analyses of adult Meis1 knockout hearts suggested that cardi-
smarca5) responsible for zebrafish heart regeneration can be omyocyte proliferation markedly decreased with age. Consid-
utilized to regenerate the mammalian heart. Manipulation of ering that the use of ␣MHC-Cre results in Meis1 deletion
these factors indicated that they regulate zebrafish cardiomyo- before birth, these data suggest that deletion of Meis1 might
cyte proliferation after cardiac injury. The authors subse- prevent or delay cardiomyocyte differentiation. Yet the authors
quently utilized these factors to induce adult mammalian car- confirmed induction of cell cycle activity in adult cardiomyo-
diomyocyte proliferation. They provided in vitro-only data cytes upon Meis1 deletion utilizing ␣MHC-MerCreMer mice,
regarding re-expression of proliferating cell nuclear antigen which allowed Meis1 deletion in cardiomyocytes upon tamox-
(PCNA), a DNA clamp that acts as a processivity factor for ifen administration. In fact, the cell number data indicate that
DNA polymerase-␦ in eukaryotic cells and is essential for Meis1 deletion induced the production of 2 million cardiomy-
DNA replication. In vivo, they determined expression of ocytes in less than 5 wk by an H3P as well as Aurora B fold
GATA4, PCNA, Aurora B kinase, and Anillin as well as increase of less than fourfold. This is an increase of around
histone H3 phosphorylation, and BrdU incorporation. While 40% of the total cardiomyocyte. The heart-to-body weight ratio
these data indicate proliferation, they don’t prove cardiomyo- increased by 20%. In conclusion, these data support the idea
cyte proliferation (see above). Yet cardiac function (% frac- that inhibitors control the cell cycle arrest in cardiomyocytes
tional shortening and ejection fraction) was also significantly and that this arrest is reversible. This is in accordance with the
improved, and ventricular wall thickness was recovered to observation that Meis1 deletion results in upregulation of
around 50% (2). Whether this is due to adequate levels of positive cell cycle regulators and downregulation of other
cardiomyocyte proliferation, activation of a compensatory hy- negative regulators (73). Still, it is important that these data are
pertrophic response, or a combination of both remains unclear. confirmed independently, ideally by clonal assays.
Newborn mouse heart regeneration. The mouse heart is Porrello and coworkers (94) reported that the miR-15 family
anatomically highly similar to the human heart (120). How- regulates neonatal heart regeneration. They reported that neo-
ever, the difference in size results in different physiological natal hearts of miR-195-overexpressing transgenic mice failed
demands and thus in differences to human cardiac physiology. to regenerate after LAD ligation because of reduced cardiomy-
In contrast to the human heart, the mouse heart can beat up to ocyte proliferation. Subsequently, they tested whether inhibi-
800 times per minute and exhibits significantly different cal- tion of the miR-15 family would enable adult cardiomyocyte
cium handling. On a molecular level, the human and mouse proliferation. To address this question they injected locked
hearts differ, for example, in the phosphorylation and isoform nucleic acid-modified anti-miRs postnatally at days 1, 7, and
expression of a variety of contractile proteins (e.g., myosin 14 and induced cardiac injury at day 21 by using an LAD
heavy chain, titin, troponin) (79, 86). Whether these differ- ligation/reperfusion model. Even though the authors observed
ences affect cardiomyocyte proliferation is unclear. Yet the an increase from 1 to around 5 H3P-positive cardiomyocytes
human heart might have a greater regeneration potential than per section, the treatment had only a minor effect on cardiac
those of mice and rats, as ⬎60% of adult human cardiomy- function.
ocytes remain mononucleated (but mainly polyploid) (80),
while ⬎90% of adult mouse and rat cardiomyocyte are Critical Open Questions Regarding Human Heart
bi-/polynucleated (61, 107). In addition, it has been sug- Regeneration
gested that low proliferation rates of human cardiomyocytes
can be observed until the second decade of life (80). Yet a Is the cell cycle arrest in adult mammalian/human cardio-
recent paper challenges this view and provides data demon- myocytes actively maintained and reversible? Decades ago it
strating that the final number of human cardiomyocytes is was demonstrated that the expression of a large variety of
reached at 1 mo of birth and remains constant thereafter positive cell cycle factors is downregulated in cardiomyocytes
with a low turnover rate of around 1% (11). during development, while negative regulators, such as cyclin-
Newborn heart regeneration as well as development studies dependent kinase inhibitors (CDKI), are transiently upregu-
will help to identify mechanisms required for cardiomyocyte lated (113, 115, 129). After birth, the majority of cardiomyo-
proliferation. Moreover, they might help to elucidate the mech- cytes become binucleated or polyploid (Fig. 1) (17, 45, 107),
anism underlying the cell cycle arrest. However, it remains the contractile apparatus changes dramatically into a highly
unclear whether this knowledge will lead to reversal of cell ordered structure (66), and cardiomyocytes increase in size
cycle arrest in adult cardiomyocytes, allowing heart regenera- almost 20-fold (13, 37). These changes might explain why the
tion. Yet several genes have been identified as associated with proliferative potential and thus the efficiency to induce cardi-
A large amount of data has been accumulated that demon- We thank the rest of the Engel lab for critical reading and suggestions.
strates that, in newt, zebrafish, and newborn mice, a variety of GRANTS
injuries induce heart regeneration by cardiomyocyte prolifera-
This work was supported by grants from the Emerging Fields Initiative
tion. In addition, it has been shown that it is possible to (EFI) for Cell Cycle in Disease and Regeneration (CYDER) from the
efficiently induce neonatal mammalian cardiomyocyte prolif- Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU to F. B. Engel) and
eration. In contrast, with regard to adult cardiomyocytes, little the Deutsche Forschungsgemeinschaft (IRTG 1566, PROMISE; EN 453/9-1 to
evidence exists supporting true proliferation (i.e., successful F. B. Engel).
completion of cytokinesis resulting in two cells). Moreover, DISCLOSURES
much of the data suggestive of adult cardiomyocyte prolifera-
No conflicts of interest, financial or otherwise, are declared by the author(s).
tion is mainly based on immunofluorescence analysis, which,
depending on the scrutiny and method of analysis, can lead to AUTHOR CONTRIBUTIONS
misinterpretations. Ultimately, much of the data underlying Author contributions: M.L. and F.B.E. analyzed data; M.L. and F.B.E.
claims of regeneration by cardiomycoyte proliferation has yet prepared figures; M.L., A.M., and F.B.E. drafted manuscript; M.L., A.M., and
F.B.E. edited and revised manuscript; M.L., A.M., and F.B.E. approved final 23. Chottova Dvorakova M, Wiegand S, Pesta M, Slavikova J, Grau V,
version of manuscript. Reischig J, Kuncova J, Kummer W. Expression of neuropeptide Y and
its receptors Y1 and Y2 in the rat heart and its supplying autonomic and
spinal sensory ganglia in experimentally induced diabetes. Neuroscience
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