Circulation Research

ORIGINAL RESEARCH

Modulation of Mammalian Cardiomyocyte

Cytokinesis by the Extracellular Matrix
Chi-Chung Wu, Sylvia Jeratsch, Johannes Graumann, Didier Y.R. Stainier

RATIONALE: After birth, cycling mammalian CMs (cardiomyocytes) progressively lose the ability to undergo cytokinesis and
hence they become binucleated, which leads to cell cycle exit and loss of regenerative capacity. During late embryonic and
early postnatal heart growth, CM development is accompanied by an expansion of the cardiac fibroblast (cFb) population
and compositional changes in the ECM (extracellular matrix). Whether and how these changes influence cardiomyocyte
cytokinesis is currently unknown.
OBJECTIVE: To elucidate the role of postnatal cFbs and the ECM in cardiomyocyte cytokinesis and identify ECM proteins that
promote cardiomyocyte cytokinesis.
METHODS AND RESULTS: Using primary rat cardiomyocyte cultures, we found that a proportion of postnatal, but not embryonic,
cycling cardiomyocytes fail to progress through cytokinesis and subsequently binucleate, consistent with published reports of
in vitro and in vivo observations. Direct coculture with postnatal cFbs increased cardiomyocyte binucleation, which could be
inhibited by RGD peptide treatment. In contrast, cFb-conditioned medium or transwell coculture did not significantly increase
cardiomyocyte binucleation, suggesting that cFbs inhibit cardiomyocyte cytokinesis through ECM modulation rather than
by secreting diffusible factors. Furthermore, we found that both embryonic and postnatal CMs binucleate at a significantly
higher rate when cultured on postnatal cFb-derived ECM compared with embryonic cFb-derived ECM. These cytokinetic
defects correlate with cardiomyocyte inefficiency in mitotic rounding, a process which is key to successful cytokinesis. To
identify ECM proteins that modulate cardiomyocyte cytokinesis, we compared the composition of embryonic and postnatal
Downloaded from http://ahajournals.org by on July 8, 2022

cFb-derived ECM by mass spectrometry followed by functional assessment. We found that 2 embryonically enriched ECM
proteins, SLIT2 and NPNT (nephronectin), promote cytokinesis of postnatal CMs in vitro and in vivo.
CONCLUSIONS: We identified the postnatal cardiac ECM as a nonpermissive environment for cardiomyocyte cytokinesis
and uncovered novel functions for the embryonic ECM proteins SLIT2 and NPNT (nephronectin) in promoting postnatal
cardiomyocyte cytokinesis.
GRAPHIC ABSTRACT: A graphic abstract is available for this article.

Key Words: cytokinesis ◼ coculture ◼ extracellular matrix ◼ mass spectrometry ◼ myocyte, cardiac

Editorial, see p 908 | In This Issue, see p 851 | Meet the First Author, see p 853

T
he proliferative and regenerative capacity of mam- mouse heart, the extent of cardiomyocyte proliferation
malian CMs (cardiomyocytes) declines with age. and functional recovery after myocardial infarction (MI)
Embryonic cardiomyocytes are highly proliferative1 negatively correlates with the degree of cardiomyocyte
and are able to regenerate after injury.2 Shortly after multinucleation.5 Similar observations have been made in
birth, cycling cardiomyocytes progressively lose the the regenerating zebrafish heart. Adult zebrafish cardio-
ability to complete cytokinesis and become binucle- myocytes are predominantly mononuclear and diploid,6
ated,3 leading to cell cycle exit and loss of regenerative and they can proliferate upon injury.7 However, experi-
capacity.4 Consistent with these findings, in the adult mentally induced cardiomyocyte polyploidization (both
 
Correspondence to: Chi-Chung Wu, PhD, Max Planck Institute for Heart and Lung Research, Department of Developmental Genetics, Ludwigstrasse 43, 61231, Bad
Nauheim, Germany, Email chi-chung.wu@mpi-bn.mpg.de; or Didier Y.R. Stainier, PhD, Max Planck Institute for Heart and Lung Research, Department of Developmental
Genetics, Ludwigstrasse 43, 61231, Bad Nauheim, Germany, Email didier.stainier@mpi-bn.mpg.de
The Data Supplement is available with this article at https://www.ahajournals.org/doi/suppl/10.1161/CIRCRESAHA.119.316303.
For Sources of Funding and Disclosures, see page 906.
© 2020 American Heart Association, Inc.
Circulation Research is available at www.ahajournals.org/journal/res

896   September 11, 2020 Circulation Research. 2020;127:896–907. DOI: 10.1161/CIRCRESAHA.119.316303

 Wu et al
Novelty and Significance
What Is Known?
• Mammalian cardiomyocytes (CMs) lose the ability to
complete cytokinesis and become binucleated shortly
after birth.
• The degree of multinucleation and polyploidization of
mammalian CMs negatively correlates with their prolif-
erative and regenerative capacity.
• cFbs (cardiac fibroblasts) and the cardiac ECM
(extracellular matrix) influence various aspects of CM
behavior.
What New Information Does This Article
Contribute?
• Cardiac fibroblasts induce CM binucleation through
modulation of the ECM.
• Postnatal cFb-derived ECM is a nonpermissive envi-
ronment for CM cytokinesis.
• Two embryonically enriched ECM proteins, SLIT2 and
NPNT (nephronectin), are able to function as soluble fac-
tors to promote CM cytokinesis both in vitro and in vivo.
Nonstandard Abbreviations and Acronyms
cFb cardiac fibroblast
Downloaded from http://ahajournals.org by on July 8, 2022
ECM extracellular matrix
MI myocardial infarction
NPNT nephronectin
multinucleation and increase in nuclear ploidy) in the
zebrafish heart impairs its proliferative and regenerative
capacity.8 These observations suggest that ploidy is a
critical factor in determining the degree of endogenous
cardiomyocyte proliferation and regeneration. However,
the regulatory mechanisms of cardiomyocyte cytoki-
nesis are poorly understood. Similar to their postnatal
counterpart, adult mammalian cardiomyocytes are also
prone to cytokinetic defects. Reactivation of cell cycle
activity in adult cardiomyocytes following MI9 or various
experimental or therapeutic approaches often results in
karyokinesis but not cytokinesis,10,11 which could impact
the potential of stimulating adult cardiomyogenesis via
cardiomyocyte proliferation as a therapeutic approach
for MI.10 A thorough understanding of cardiomyocyte cell
cycle control, in particular of the factors modulating car-
diomyocyte cytokinesis, is of great interest to the cardiac
development and regeneration fields.
In the past 2 decades, a number of factors and sig-
naling pathways have been identified as modulators
of the cardiomyocyte cell cycle including MEIS1,12 the
Hippo pathway,13–15 NRG1/ERBB2 signaling,16,17 thyroid
Circulation Research. 2020;127:896–907. DOI: 10.1161/CIRCRESAHA.119.316303
The ECM Regulates Cardiomyocyte Cytokinesis
Original Research
Mammalian CMs lose the ability to complete cyto-
kinesis and become binucleated shortly after birth,
which correlates with their cell cycle exit and the
loss of regenerative capacity. The regulatory mecha-
nisms of CM cytokinesis, in particular, the role of
cell nonautonomous factors, is poorly understood.
Using primary culture of postnatal CMs, we find that
cFbs promote CM binucleation through modulating
the ECM. We find that, unlike the embryonic cFb-
derived ECM, the postnatal cFb-derived ECM is a
nonpermissive environment for CM cytokinesis. Fur-
thermore, we uncover the novel function of 2 embry-
onically enriched ECM proteins, SLIT2 and NPNT,
in promoting CM cytokinesis both in vitro and in
vivo. These findings highlight the importance of the
nonmyocyte population and the ECM in modulating
CM cytokinesis, which may provide new therapeutic
targets to augment adult CM proliferation after myo-
cardial infarction.
hormone signaling,18 p38 MAPK,19,20 ECT2,21 TNNI3K,5
cell cycle genes,22 and hypoxia.23,24 However, little is
known about whether and how nonmyocyte cardiac cells
and the cardiac ECM (extracellular matrix) modulate car-
diomyocyte cytokinesis. Fibroblasts constitute ≈20% of
the nonmyocyte population in the mammalian heart25
and play important functions during heart development,
homeostasis, and disease.26 The number of cFbs (car-
diac fibroblasts) increases throughout development,27
and it continues to expand after birth with peak prolif-
eration at around postnatal day 4 (P4),28 coinciding with
the onset of cardiomyocyte binucleation.3 Similar to cFbs,
the ECM also plays important roles in the heart.29 The
ECM is a dynamic and complex protein network whose
composition30 and complexity31 changes as the heart
grows. Both cFbs and the ECM are known to interact
with cardiomyocytes and influence their behavior. Embry-
onic cFbs have been reported to promote cardiomyocyte
proliferation via paracrine signaling in contrast to the
hypertrophy-inducing adult cFbs.27 Several studies have
been conducted on ECM proteins in the heart including
PERIOSTIN, which has been shown to stimulate cardio-
myocyte proliferation and cardiac regeneration after MI
in neonates32 and adults.33 AGRIN, another ECM protein,
whose expression in the heart decreases from P1 to P7,
also promotes CM proliferation and regeneration after
MI.34 Despite their broad roles in CM behavior and car-
diac function, whether and how cFbs and the ECM influ-
ence CM cytokinesis remains unknown.
We addressed these questions using primary rat car-
diomyocyte cultures. We first show that postnatal cFbs
September 11, 2020   897
 Wu et al
induce cardiomyocyte binucleation through the ECM
rather than through diffusible factors. Using a cell-derived
Original Research
matrix approach, we further show that the postnatal ECM
is a nonpermissive environment for cardiomyocyte mitotic
rounding and cytokinesis, leading to cardiomyocyte binu-
cleation. By comparing the composition of embryonic
and postnatal cFb-derived ECM, we identify 2 ECM pro-
teins, SLIT2 and NPNT (nephronectin), which we show
are capable of promoting cardiomyocyte cytokinesis both
in vitro and in vivo. Our results reveal previously unap-
preciated roles for cFbs and the ECM in regulating mam-
malian cardiomyocyte cytokinesis.
METHODS
The data that support the findings of this study are available
from the corresponding authors upon reasonable request.
Other methods, sample sizes and the Major Resources Table
can be found in the Data Supplement.
Animal Procedures
Sprague Dawley rats (Rattus norvegicus) and C57/Bl6 mice
(Mus musculus) were housed at the Max Planck Institute for
Heart and Lung Research according to § 11 TierSchG and EU
Directive 2010/63/EU on the protection of animals used for
scientific purposes.
Rat Cell Isolation and Culture
Downloaded from http://ahajournals.org by on July 8, 2022
To isolate ventricular cardiomyocytes from rats, pooled
ventricles from E16 embryos or P3 pups of the same litter
were dissociated using the Neonatal heart dissociation kit
(Miltenyi Biotec). Hence, N (number of replicates) indicates
the number of independent cardiomyocyte isolations. After
dissociation, cardiomyocytes were purified using a 2-step
discontinuous Percoll gradient as previously described.35
Cardiomyocytes (1×105 cells/cm2 for E16 cardiomyocytes
and 1.2×105 cells/cm2 for P3 cardiomyocytes) were plated
on a cover glass coated with 1% gelatin and cultured in neo-
natal cardiomyocyte medium (DMEM/F12, GlutaMAX with
3 mmol/L Na-Pyruvate, 0.1 mmol/L Ascorbic acid, Insulin/
transferrin/Na-Selenite solution [1:200], 0.2% BSA and 1×
Pen/Strep) containing 5% horse serum and 0.02 mmol/L
ara-C at 37°C and 5% CO2 for 48 hours. To perform the
binucleation assay, unless otherwise specified, cardiomyo-
cytes were washed twice and cultured in neonatal cardiomyo-
cyte medium with 10% fetal bovine serum (FBS, HyClone)
for 24 hours. 0.02 mmol/L EdU was added to the medium
for 24 hours. Cells were washed twice to remove the EdU
and cultured for another 24 hours in neonatal cardiomyocyte
medium with 10% FBS until harvesting.
To isolate ventricular cFbs, ventricles were isolated, dis-
sociated, and cells were purified as described above. For P3
hearts, cFbs collected after the Percoll gradient were preplated
in uncoated Petri dishes in complete medium (DMEM/F12,
GlutaMAX, 1× Pen/Strep supplemented with 10% FBS) at
37°C for 1 hour. After removal of the unattached cells, cFbs
were cultured in complete medium. Fresh cFbs were isolated
for each experiment. For E16 hearts, cFbs obtained after the
898   September 11, 2020 Circulation Research. 2020;127:896–907. DOI: 10.1161/CIRCRESAHA.119.316303
The ECM Regulates Cardiomyocyte Cytokinesis
Percoll gradient were preplated for 30 minutes in complete
medium. After removal of the unattached cells, cFbs were
cultured in complete medium. To improve purity, embryonic
cFbs were passaged for 2× to 3× until no beating cells were
observed in the dish. cFbs were then expanded and frozen
down until use.
Cardiomyocyte cFb Cocultures
For the direct cocultures, isolated cardiomyocytes and cFbs
were resuspended in neonatal cardiomyocyte medium con-
taining 5% horse serum and 0.02 mmol/L ara-C and mixed
in 4:1 or 2:1 ratio (total cell number=1.2×105 cells/cm2), fol-
lowed by the binucleation assay. For the transwell cocultures,
isolated cardiomyocytes were seeded on a cover glass coated
with 1% gelatin in a 24 well plate. Forty thousand or 80 000
cFbs were plated on a Transwell permeable insert with a poly-
ester membrane of 0.4 μm pore size (Corning), followed by the
binucleation assay. For the cFb-conditioned medium experi-
ments, isolated P3 cFbs were cultured with complete medium
for 4 days. The collected medium was filtered (0.2 μm) and
stored at −20°C until use. For the binucleation assay, cardio-
myocytes were cultured in neonatal cardiomyocyte medium
with 10% FBS (ctrl) or 50% cFb-conditioned medium (1:1
mix with neonatal cardiomyocyte medium with 10% FBS) or
100% cFb-conditioned medium.
Preparation of cFb-Derived ECM
cFb-derived ECM was generated as previously described.36
E16 and P3 cFbs (6.25×104 cells/cm2) were plated in com-
plete medium on a cover glass coated with 0.1% gelatin cross-
linked with 1% glutaraldehyde. cFbs were cultured for 11 days
in complete medium with ascorbic acid (50 μg/mL), which was
exchanged every 48 hours. For decellularization, an extraction
buffer (DPBS with 0.5% (v/v) Triton X-100 and 20 mmol/L
NH4OH) was added and incubated for 5 minutes at 37°C. After
washing, DNA debris were removed by DNase I treatment (10
U/mL, Roche) for 45 minutes at 37°C. The resulting matrix was
washed with DPBS and stored at 4°C until use.
To generate a protein mixture of cFb-derived ECM, a solu-
bilization reagent (5 mol/L guanidine containing 10 mmol/L
dithiothreitol) was added to the matrix for 5 minutes on ice.
The ECM was scraped off the dish with a cell scraper and kept
in the solubilization reagent for 1 hour at 4°C. Afterward, the
solubilized matrix (supernatant) was collected by centrifuga-
tion (15 minutes, 12 000×g) and stored at 4°C until use. Cover
glass was coated with 10 μg/mL protein mix for at least 1
hour at 37°C.
RGD Peptide, ITGß1 Blocking Antibody, and
ECM Protein Treatments
The RGD peptide (Gly-Arg-Gly-Asp-Ser-Pro-Lys, Sigma) was
added to cardiomyocytes at 0.1 and 0.2 mmol/L. Water was
used as a control. Hamster anti-Rat ITGß1 antibody (#555002,
BD Pharmingen) or Hamster IgM control (#553957, BD
Pharmingen) was added to cardiomyocytes at 40 or 80 μg/
mL. The following recombinant ECM proteins were added to
cardiomyocytes at 0.5 or 1 μg/mL: mouse NPNT (#4298-NP-
050), mouse SLIT2 (#5444-SL-050), mouse NID2 (#6760-
ND-050), mouse SPON1 (#7950-SP-050), mouse TGM2
 Wu et al
(#5418-TG-010), human FN1 (#ACFP4305B; R&D Systems),
and human LAMA5 (#ab42326; Abcam).
Cardiomyocyte Cell Shape Measurements
Mitotic cardiomyocytes at metaphase or anaphase (PH3+ and
cTnI+) throughout the sample were randomly selected and
imaged. Cell boundaries of mitotic and neighboring nonmitotic
cardiomyocytes were manually outlined for aspect ratio mea-
surements using Fiji.
SLIT2 and NPNT Injections
About 1.5 μg of recombinant SLIT2 or NPNT protein (R&D
systems, see above) (PBS as a control) was injected daily
into wild-type C57/Bl6 pups from P1 to P4 while 50 mg/
kg EdU was injected once on P3. Ventricles were harvested
on P5, and cells were dissociated using 2 different fixation-
digestion protocols. Detailed cell dissociation protocols can be
found in the Data Supplement. Dissociated cells were spotted
on a glass slide and air-dried, which was followed by EdU and
alpha-actinin immunostaining. At least 100 EdU+ cardiomyo-
cytes were analyzed per sample, blindly. At least 3 crosses were
set up for each round of experiments. Litters were randomly
assigned to receive PBS, SLIT2, or NPNT; hence experiments
were not performed with littermate controls. No sex determina-
tion was performed and hence both sexes are assumed to have
been used in an unknown ratio.
RESULTS
Downloaded from http://ahajournals.org by on July 8, 2022
Establishing an EdU Pulse-Chase
Cardiomyocyte Binucleation Assay
During heart development, changes in the quantity and
properties (eg, transcriptome) of various cardiac cell
types, including CMs37 and Fbs,27,28 as well as in the
composition and complexity of the ECM,30,31 occur con-
comitantly. It is therefore difficult to decipher their indi-
vidual influence on cardiomyocyte cytokinesis in vivo. To
circumvent this complexity, we used primary rat cardio-
myocyte cultures as a model as they allow one to test
individual variables (eg, culturing cardiomyocytes of a
particular stage with different types of cFbs and ECM).
Detection of cardiomyocyte cytokinesis is conven-
tionally carried out by immunostaining or lineage tracing.
However, immunostaining for commonly used cytokinetic
markers like Aurora B-kinase has been shown to mark
both dividing and binucleating cardiomyocytes.38 While
lineage tracing-based systems including mosaic analy-
sis with double markers39 or live-cell imaging40 are more
reliable, they are not suitable for routine use because of
their low efficiency or throughput, respectively. Hence,
we first set out to establish a simple and robust cardio-
myocyte binucleation assay. Isolated rat cardiomyocytes
from various pre- and postnatal stages were cultured
with 10% FBS to stimulate cell cycle activity. To mark
the cycling population, EdU was added to the medium
for 24 hours and after washing out, cardiomyocytes were
Circulation Research. 2020;127:896–907. DOI: 10.1161/CIRCRESAHA.119.316303
The ECM Regulates Cardiomyocyte Cytokinesis
further cultured for another 24 hours (Figure 1A). The
nucleation status of EdU+ cardiomyocytes was then
Original Research
assessed by immunostaining for cardiac troponin I (car-
diomyocyte), N-cadherin (cardiomyocyte junctions), and
EdU at 24 and 48 hours after EdU addition (Figure 1B).
As the cell cycle length of postnatal mammalian cardio-
myocytes has been estimated to be around 35 hours,41
this regimen allows one to assess the degree of cyto-
kinesis completion in EdU+ cardiomyocytes within one
cell cycle.
Using this assay, we found that the percentage of
binuclear cells within the EdU+ cardiomyocyte population
increased significantly from 24 to 48 hours for P3 car-
diomyocytes, indicative of cytokinesis failure (Figure 1C),
and an observation consistent with previous reports.19,40
In contrast, no significant difference in cytokinesis was
observed using embryonic day 16 (E16) cardiomyocytes
(Figure 1C). Next, we repeated the above experiment with
P3 cardiomyocytes under serum-free condition to inhibit
cell cycle activity. Serum starvation did not induce signifi-
cant apoptosis in cardiomyocytes (Online Figure IA), and
we found no significant increase in the percentage of total
binuclear cardiomyocytes from 24 to 48 hours (Online Fig-
ure IB), suggesting that the observed increase in binuclear
P3 cardiomyocytes is cell cycle-dependent. Hereafter, the
degree of binucleation (ie, binucleation index) is calculated
as the relative increase in the percentage of binuclear cells
within the EdU+ cardiomyocyte population from 24 to 48
hours (Figure 1D). To further validate this approach, we
monitored the cell cycle behavior of E16 and P3 cardiomy-
ocytes by confocal time-lapse imaging with live tubulin and
DNA labeling (Online Movie I). We imaged >500 mitotic
events in total (195 for E16 and 372 for P3 cardiomyo-
cytes) and found that the majority of the mitotic P3 (58%)
and a small minority of the mitotic E16 (4.6%) cardiomyo-
cytes underwent binucleation (Figure 1E), consistent with
previous results (Figure 1C). Altogether, these data show
that the binucleation assay developed here is a reliable
readout of cardiomyocyte cytokinesis.
Postnatal Cardiac Fibroblasts Lead to an
Increase in Cardiomyocyte Binucleation
Through the Extracellular Matrix
To investigate the role of cFbs in cardiomyocyte cytokinesis,
we performed binucleation assays on isolated P3 cardio-
myocytes cocultured with increasing amounts of P3 cFbs
(Figure 2A). We found that the degree of binucleation posi-
tively correlated with cFb number (Figure 2B), suggesting
that postnatal cFbs promote cardiomyocyte binucleation.
Communication between cFbs and cardiomyocytes can
be mediated through diffusible factors, the ECM or direct
cell-cell contact.42 To investigate whether diffusible fac-
tors from cFbs could modulate cardiomyocyte binucleation,
we performed 2 types of indirect coculture experiments.
cFbs were cocultured with cardiomyocytes using Transwell
September 11, 2020   899
 Wu et al The ECM Regulates Cardiomyocyte Cytokinesis
Original Research

Figure 1. Postnatal rat cardiomyocytes binucleate in vitro.

A, Schematic of the CMs (cardiomyocytes) binucleation assay. Isolated CMs are cultured with 10% FBS and treated with EdU for 24 h. EdU
Downloaded from http://ahajournals.org by on July 8, 2022

is then washed out, and CMs are cultured for another 24 h to complete one cell cycle. B, Nucleation status of EdU+ CMs was assessed by
immunostaining for cTnI (cardiac troponin I), EdU, and N-cadherin (N-cad). C, Quantification of CM binucleation. The percentage of binuclear cells
within the EdU+ CM population increases significantly from 24 to 48 h in postnatal (P3) but not embryonic (E16) CM cultures. D, Binucleation
index, defined as the relative increase in the percentage of binuclear cells within the EdU+ CM population from 24 to 48 h, calculated from (C).
E, Cell cycle analysis of E16 and P3 CMs. Cardiomyocyte cell cycle was monitored by confocal time-lapse imaging with live tubulin and DNA
labeling. A majority of the mitotic P3 but not E16 CMs underwent binucleation. Endoreplication denotes failed karyokinesis. n=number of mitotic
CMs imaged. Values represent mean±SEM. (C, D) P values determined by unpaired t-test; (E) P values determined by χ2 test.

inserts with a pore size of 0.4 μm, which physically sepa-
rates the cells but allows diffusible factors secreted from
cFbs to reach cardiomyocytes. No significant difference in
the binucleation index was observed in cocultures of cardio-
myocytes with 40 000 or 80 000 cFbs compared with con-
trol (no extra cFbs; Figure 2C). Consistent with this result,
conditioned medium from P3 cFbs also did not increase
cardiomyocyte binucleation (Online Figure IIA). These data
suggest that diffusible factors are not mediating the effects
of postnatal cFbs on cardiomyocyte cytokinesis.
cFbs contribute in a major way to the production,
secretion, and maintenance of the cardiac ECM, and
ECM proteins have been shown to promote cardio-
myocyte proliferation27,34 and hypertrophic growth.43
We therefore hypothesized that postnatal cFbs pro-
mote cardiomyocyte binucleation by modulating the
microenvironment (ie, ECM protein expression and/or
assembly). In line with this hypothesis, we observed by
immunostaining increased expression and assembly into
fibrillar structures of FN (fibronectin) and COL 1 in 2:1
cardiomyocyte:cFb cocultures compared with control
(Online Figure IIB). To test whether ECM proteins could
promote cardiomyocyte binucleation, we decreased
cardiomyocyte-ECM interactions with the RGD peptide,
which mimics the RGD motif on a subset of ECM pro-
teins.44 Treating 2:1 cardiomyocyte:cFb cocultures with
the RGD peptide from 24 to 48 hours reduced the car-
diomyocyte binucleation index in a dose-dependent man-
ner (Figure 2D). Altogether, although we cannot rule out
the effect of cell-cell contacts, these data suggest that
cFbs increase cardiomyocyte binucleation through ECM
modulation rather than by secreting diffusible factors.
Postnatal Cardiac Extracellular Matrix
Hinders Cardiomyocyte Mitotic Rounding and
Cytokinesis
To further investigate the role of the ECM in cardio-
myocyte cytokinesis, we employed a cell-derived matrix
approach,36 which better mimics aspects of native tissue

900   September 11, 2020 Circulation Research. 2020;127:896–907. DOI: 10.1161/CIRCRESAHA.119.316303

 Wu et al
Downloaded from http://ahajournals.org by on July 8, 2022
microenvironments than conventional monolayer cul-
tures.45 In brief, isolated E16 and P3 cFbs were cultured
for 11 days during which ECM proteins were secreted
and deposited on the culture surface. After decellular-
ization, the native-like ECM was used for cardiomyocyte
cultures (Figure 3A; Online Figure IIIA). Binucleation
assays were performed on E16 and P3 cardiomyocytes
cultured on embryonic and postnatal ECM. We observed
that both E16 (compare columns 1 and 2) and P3 (com-
pare columns 3 and 4) cardiomyocytes binucleated at a
significantly higher rate on postnatal ECM (Figure 3B),
suggesting that, compared with the embryonic ECM, the
postnatal ECM is a nonpermissive environment for car-
diomyocyte cytokinesis (Figure 3B). In addition, E16 car-
diomyocytes consistently binucleated to a lesser extent
than P3 cardiomyocytes when cultured on the same type
of ECM (compare columns 1 and 3 for E16 ECM, 2 and
4 for P3 ECM; Figure 3B). Together, these data sug-
gest that both cell-autonomous (ie, intrinsic to the car-
diomyocytes) and cell-nonautonomous factors (ie, the
ECM) modulate cardiomyocyte cytokinesis. Integrins are
the major receptors that link the ECM to the cytoskel-
eton and they activate a number of intracellular signal-
ing pathways.46 We treated P3 cardiomyocytes cultured
Circulation Research. 2020;127:896–907. DOI: 10.1161/CIRCRESAHA.119.316303
The ECM Regulates Cardiomyocyte Cytokinesis
Original Research
Figure 2. Postnatal cardiac
fibroblasts increase cardiomyocyte
binucleation through the
extracellular matrix.
A, Direct cocultures of P3 cFbs (cardiac
fibroblasts) and cardiomyocytes (CMs).
Examples of CM (cTnI+) and cFb
(Vimentin+) cocultures shown on the
left. Percentage of cFbs in different
coculture conditions shown on the
right. B, Binucleation index in P3 CMs
cocultured with increasing amounts of
cFbs. C, Transwell cocultures of P3 cFbs
and CMs. Diffusible factors from cFbs do
not increase the CM binucleation index
compared with control. D, RGD peptide
treatment from 24 to 48 h decreases CM
binucleation (2:1 CM:cFb cocultures).
Values represent mean±SEM. P values
determined by 1-way ANOVA followed
by Tukey’s HSD test (P values of overall
ANOVA and post hoc tests indicated
between the parentheses and above the
lines, respectively).
on postnatal ECM with 40 or 80 μg/mL of Integrin ß1
(ITGß1) blocking antibody, which decreases cardiomyo-
cyte-ECM interactions47 and found that it reduced the
binucleation index compared with IgM control (Figure 3C,
Online Figure IIIB). These data further support our find-
ings that the ECM modulates cardiomyocyte cytokinesis.
Cells undergo a series of cell shape changes dur-
ing mitosis including mitotic rounding, and these
changes depend on the remodeling of cell-substrate
adhesion and are critical for successful cytokinesis.48
During early mitosis, postnatal cardiomyocytes retain
a flattened morphology with prominent sarcomeric
structures. From the metaphase stage onward, cardio-
myocytes partially lose contact with neighboring cells
and the substrate, and they start rounding up (Online
Figure IIIC). To investigate the effects of the ECM on
postnatal cardiomyocyte mitotic rounding, we quanti-
fied the aspect ratio of mitotic and neighboring non-
mitotic cardiomyocytes. While nonmitotic (PH3−)
cardiomyocytes exhibited a similar aspect ratio on both
embryonic and postnatal ECM, mitotic (PH3+) cardio-
myocytes reduced their aspect ratio (ie, rounded up) on
embryonic ECM but failed to do so on postnatal ECM
(Figure 3D). Moreover, treating P3 cardiomyocytes on
September 11, 2020   901
 Wu et al
Original Research
Downloaded from http://ahajournals.org by on July 8, 2022
postnatal ECM with 80 μg/mL of ITGß1 blocking anti-
body, which promotes cytokinesis (Online Figure IIIB),
rescued their mitotic rounding defects (Figure 3E).
These data suggest that the cardiomyocyte cytokinetic
defects on postnatal cFb-derived ECM can be at least
partly attributed to the perturbation of mitotic rounding.
NPNT and SLIT2 Promote Mitotic Rounding and
Cytokinesis of Postnatal Cardiomyocytes
Next, we generated protein mixtures of embryonic and
postnatal cFb-derived ECM through solubilization and
used them to coat the tissue culture surface before
seeding cardiomyocytes. This treatment eliminates the
original 3D organization/arrangement of the ECM and
allows one to test whether compositional differences
between two types of ECM alone are sufficient to affect
cardiomyocyte cytokinesis. Similar to our observations
902   September 11, 2020 Circulation Research. 2020;127:896–907. DOI: 10.1161/CIRCRESAHA.119.316303
The ECM Regulates Cardiomyocyte Cytokinesis
Figure 3. Postnatal cardiac
extracellular matrix increases
cardiomyocyte binucleation through
inefficient mitotic rounding.
A, Schematic of the production of cardiac
fibroblast (cFb)-derived ECM (extracellular
matrix). Native-like ECM was produced
by culturing cFbs for 11 d followed by
decellularization. B, Cardiomyocyte (CM)
binucleation index on embryonic and
postnatal ECM. Binucleation assay was
performed on CMs cultured on embryonic
or postnatal ECM. Both E16 and P3 CMs
binucleate at a significantly higher rate
on postnatal ECM. C, Integrin ß1 (ITGß1)
blocking antibody treatment from 24 to
48 h partially prevents CM binucleation.
D, CM mitotic rounding defects. Aspect
ratio of mitotic (PH3+, yellow dashed
line) and neighboring nonmitotic P3 CMs
was determined. Mitotic CMs reduce
their aspect ratio (ie, undergo mitotic
rounding) more efficiently on embryonic
compared with postnatal ECM. E, Integrin
ß1 blocking antibody treatment from 24
to 48 h increases CM mitotic rounding.
n=number of CMs analyzed. Values
represent mean±SEM. (B: E16 CMs, C) P
values determined by unpaired t-test; (B:
P3 CMs, E) P values determined by Mann-
Whitney U test; (D) P values determined by
Kruskal-Wallis test followed by Dunn test
(P values of overall ANOVA and post hoc
tests indicated between the parentheses
and above the lines, respectively).
with intact ECM (Figure 3B), P3 cardiomyocytes binu-
cleated at a significantly higher rate on postnatal-ECM
coated surfaces than on embryonic-ECM coated sur-
faces (Online Figure IVA).
To identify ECM proteins that modulate cardiomyocyte
cytokinesis, we compared the composition of the embry-
onic and postnatal cFb-derived ECM by mass spectrome-
try-based quantitative proteomics. We focused on proteins
enriched in the embryonic ECM as it promotes cardio-
myocyte mitotic rounding and cytokinesis. Out of the 270
proteins that were enriched in the embryonic ECM (Log2
fold change <1, false discovery rate <0.05; Online Figure
IVB), 44 are categorized as ECM or ECM-associated pro-
teins49 (Figure 4A). We then analyzed the gene expression
level for each of these proteins using a published data-
set50 and selected for further analysis 7 candidates whose
expression went down in the mouse heart within the first
postnatal week, that is, when cardiomyocyte binucleation
 Wu et al The ECM Regulates Cardiomyocyte Cytokinesis

begins (Figure 4A). Next, we assessed the ability of these role in cardiomyocyte cell cycle remains unaddressed. To
candidate proteins to promote cardiomyocyte cytokinesis test whether SLIT2 and NPNT are mitogenic, we assessed

in vitro. We performed binucleation assays using P3 car-
diomyocytes cultured on postnatal ECM and added recom-
binant proteins at 2 concentrations (0.5 and 1 μg/mL)
right after EdU removal (Figure 4B). Of the 7 candidate
proteins tested, SLIT2 and NPNT consistently promoted
cardiomyocyte cytokinesis (Figure 4B). While SLIT251 and
NPNT52 have been implicated in heart development, their
Downloaded from http://ahajournals.org by on July 8, 2022
Original Research
cardiomyocyte cell cycle activity by EdU incorporation upon
SLIT2 and NPNT treatment. No significant difference in cell
cycle activity was observed in starved P3 cardiomyocytes
treated with SLIT2 or NPNT compared with control (Fig-
ure 4C). As the embryonic ECM supports cardiomyocyte
mitotic rounding (Figure 3D) and subsequent cytokinesis
(Figure 3B), we tested whether SLIT2 and/or NPNT could

Figure 4. NPNT and SLIT2 promote mitotic rounding and cytokinesis of postnatal cardiomyocytes in vitro.
A, Workflow to identify ECM (extracellular matrix) proteins that modulate cardiomyocyte (CM) cytokinesis. The composition of the embryonic
and postnatal cardiac fibroblast (cFb)-derived ECM was compared by quantitative mass spectrometry-based proteomics. Seven embryonic ECM
proteins, whose gene expression went down in P7 hearts compared with P1, were selected for further analysis. B, Functional assessment of
embryonic ECM proteins. Binucleation assay was performed on P3 CMs cultured on postnatal ECM and recombinant ECM proteins were added
after EdU removal. NPNT and SLIT2 consistently promoted CM cytokinesis. C, NPNT and SLIT2 are not mitogenic. Treating starved P3 CMs with
NPNT or SLIT2 failed to promote cell cycle entry, as assessed by EdU incorporation. D, NPNT and SLIT2 promote mitotic rounding. Aspect ratio
of mitotic P3 CMs cultured on postnatal ECM. Treatment with NPNT or SLIT2 from 24 to 48 h promotes mitotic rounding. n=number of CMs
analyzed. Values represent mean±SEM. (B, C) P values determined by 1-way ANOVA followed by Tukey’s HSD test; (B) P values of all pairwise
comparisons shown in Online Table II; (D) P values determined by Kruskal-Wallis test followed by Dunn test (P values of overall ANOVA and post
hoc tests indicated between the parentheses and above the lines, respectively).

Circulation Research. 2020;127:896–907. DOI: 10.1161/CIRCRESAHA.119.316303 September 11, 2020   903

 Wu et al
rescue the mitotic rounding defects of P3 cardiomyocytes.
We treated P3 cardiomyocytes cultured on postnatal ECM
Original Research
with SLIT2 and NPNT for 24 hours and determined the
aspect ratio of mitotic cardiomyocytes. Compared with
controls (PBS and NID2, a candidate ECM protein that
failed to promote cardiomyocyte cytokinesis [Figure 4B]),
SLIT2 and NPNT promoted mitotic rounding (Figure 4D).
Next, we employed an approach similar to our binu-
cleation assay (Figure 1A) to test the role of SLIT2 and
NPNT in vivo. SLIT2 and NPNT recombinant proteins were
injected daily into wild-type mouse pups from P1 to P4. The
cycling cardiomyocyte population was marked by a single
injection of EdU on P3 (Figure 5A). Cell cycle entry and
nucleation status were subsequently determined on disso-
ciated P5 cardiomyocytes. Consistent with previous results
(Figure 4C), the percentage of EdU+ cardiomyocytes was
comparable between all groups (Figure 5A), suggesting
that cardiomyocyte cell cycle entry was not significantly
affected by SLIT2 or NPNT. On the other hand, compared
with PBS-injected controls, both SLIT2 and NPNT sig-
nificantly increased the percentage of mononuclear cells
within the EdU+ cardiomyocyte population (PBS: 22.3%,
SLIT2: 41% and NPNT: 40.3%, Figure 5B), suggesting a
higher degree of successful cytokinesis.
In summary, we have found that mammalian car-
diomyocyte cytokinesis is modulated by the postnatal
cardiac ECM, a nonpermissive environment for cardio-
myocyte cytokinesis. Moreover, through quantitative pro-
teomics analysis followed by candidate screening, we
Downloaded from http://ahajournals.org by on July 8, 2022
uncovered novel functions for the embryonic cardiac
ECM proteins SLIT2 and NPNT in promoting cardiomyo-
cyte mitotic rounding and cytokinesis.
DISCUSSION
Cell cycle control of mammalian cardiomyocytes has been
extensively investigated in the past decades. Recent stud-
ies in mouse5 and zebrafish8 have suggested polyploidy
as a critical factor limiting the endogenous proliferative
and regenerative potential of cardiomyocytes. How post-
natal cardiomyocyte cytokinesis is regulated has been a
904   September 11, 2020 Circulation Research. 2020;127:896–907. DOI: 10.1161/CIRCRESAHA.119.316303
The ECM Regulates Cardiomyocyte Cytokinesis
longstanding question in the field. While several cell-auton-
omous factors have been implicated in cardiomyocyte
cytokinesis, much less is known regarding the role of non-
myocyte cardiac cells and the cardiac ECM in this process.
Although originally considered as a bystander cell type
providing structural support to the heart, a growing body of
evidence has revealed a diverse role for cFbs during heart
development, homeostasis, and disease.26 Global genetic
inactivation of Tcf21, a transcription factor gene essential
for the formation of cFbs, leads to the loss of cFbs in the
embryonic mouse heart and hypoplastic ventricular cham-
bers.53 Notably, cFbs can influence cardiomyocyte behav-
ior in an age-dependent manner. In an in vitro coculture
system, embryonic cFbs reportedly induce cardiomyocyte
proliferation while adult cFbs induce cardiomyocyte hyper-
trophy.27 In 3D engineered cardiac tissue, adult cFbs sig-
nificantly worsen both electrical and mechanical functions
of the cocultured cardiomyocytes compared with embryonic
cFbs.54 The role of cFbs during early postnatal heart devel-
opment has also been the focus of recent studies. While
peak proliferation of cFbs in the postnatal heart coincides
with the onset of cardiomyocyte binucleation,28 evidence for
their role in cardiomyocyte cytokinesis regulation has been
lacking. Through direct coculture experiments, we found
that cFbs increase cardiomyocyte binucleation in a dose-
dependent manner (Figure 2B). Although cell density was
kept constant among the different conditions during cell
seeding, because of the high proliferative rate of cFbs, it
is possible that the 4:1 and 2:1 cardiomyocyte:cFb cocul-
tures have a higher cell density by the end of the experi-
ment (ie, 96 hours after seeding). And although we cannot
rule out a role for cell density in cardiomyocyte binucle-
ation, the effect of cFbs is mediated at least in part through
RGD-motif containing extracellular proteins as RGD pep-
tide treatment decreased the binucleation index in the 2:1
cardiomyocyte:cFb cocultures (Figure 2D). Consistent with
our in vitro data, genetic ablation of cFbs through overex-
pression of diphtheria toxin A in PDGFRα+ cells in P1 mice
promotes cardiomyocyte cell cycle activity as well as the
degree of successful cytokinesis at P5 (Michelle Tallquist,
personal communication, 2019). Altogether, these results
Figure 5. NPNT and SLIT2 promote
cytokinesis of postnatal cardiomyocytes
in vivo.
A, Injection of NPNT or SLIT2 recombinant
proteins from P1 to P4 did not significantly alter
the percentage of EdU+ CMs (cardiomyocytes).
B, NPNT and SLIT2 injections significantly
increased the percentage of mononuclear cells
within the EdU+ CM population. N=number of
hearts; n=number of CMs (A) or EdU+ CMs (B)
analyzed. Values represent mean±SEM. P values
determined by 1-way ANOVA followed by Tukey
HSD test (P values of overall ANOVA and post
hoc tests indicated between the parentheses and
above the lines, respectively).
 Wu et al
reveal a novel role for cFbs as modulators of cardiomyocyte
cytokinesis. Recently, multiple adult cFb populations with
distinctive transcriptional signatures have been reported
from a single-cell RNA sequencing study in mouse.55
Whether such cFb heterogeneity also exists in the early
postnatal heart and whether different cFb populations have
specific roles in controlling cardiomyocyte cytokinesis are
important questions to pursue in the future.
Embryonic to postnatal heart development is char-
acterized by concurrent changes intrinsic to cardiomyo-
cytes (eg, sarcomeric protein composition and structure,
metabolic shift from glycolysis to fatty acid oxidation),56
as well as in the microenvironment (eg, composition and
complexity of the ECM).30,31,34 It is therefore challenging
to determine their respective role in the cardiomyocyte
transition from cytokinesis to binucleation in vivo. We took
advantage of an in vitro system to address this question.
We found that the postnatal ECM is a nonpermissive envi-
ronment for cardiomyocyte cytokinesis irrespective of the
developmental stage of cardiomyocytes (Figure 3B). On
the other hand, P3 cardiomyocytes consistently exhibited
a higher degree of binucleation than E16 cardiomyocytes
when cultured on the same substrate, that is, gelatin (Fig-
ure 1D) or embryonic or postnatal ECM (Figure 3B). Of all
the conditions tested, the highest binucleation index was
observed when combining P3 cardiomyocytes with post-
natal ECM (Figure 3B). Altogether, these findings suggest
that both cardiomyocyte maturity and the ECM modulate
cardiomyocyte cytokinesis and that they may synergisti-
Downloaded from http://ahajournals.org by on July 8, 2022
cally induce cardiomyocyte binucleation. As ploidy was
not assessed in the current study, whether cFbs and/
or the ECM also influence cardiomyocyte karyokinesis
remains to be determined.
Although adult cardiomyocytes are able to undergo
self-renewal throughout life,57 their low proliferation rate
does not support efficient cardiac repair after MI. Thus,
identifying ways to induce adult cardiomyocyte cell cycle
activity has been a major goal in the cardiac repair field.
However, mammalian cardiomyocyte cell cycle activity
rarely leads to successful cytokinesis and the production
of new cells. In the adult heart, multi-isotope imaging mass
spectrometry revealed that only about 14% of the cycling
cardiomyocytes adjacent to the infarct zone after MI were
mononuclear and diploid, suggestive of a low cytokinesis
rate.9 Attempts to augment cardiomyocyte cell cycle activ-
ity by overexpressing cell cycle regulators including cyclin
D, A2, or the transcription factor c-Myc likewise result in
multinucleation rather than cytokinesis.58 Consequently,
identifying factors that can promote cardiomyocyte cytoki-
nesis, in addition to cell cycle re-entry, should be beneficial
for the development of efficient therapeutic strategies to
stimulate cardiac repair via cardiomyocyte proliferation.
However, commonly used readouts of cardiomyocyte pro-
liferation like Ki67,59 EdU,59,60 and Aurora B-kinase38 are
not suitable to unambiguously detect cytokinesis. In con-
trast, the binucleation assay introduced here (Figure 1A)
Circulation Research. 2020;127:896–907. DOI: 10.1161/CIRCRESAHA.119.316303
The ECM Regulates Cardiomyocyte Cytokinesis
provides a robust and reliable readout for cardiomyocyte
cytokinesis and is suitable for medium to high throughput
Original Research
screens of cardiomyocyte cytokinesis-promoting factors.
While FN, COL 3,27 and AGRIN34 have been implicated
in cardiomyocyte proliferation, ECM proteins that specifi-
cally modulate cardiomyocyte cytokinesis have not been
reported thus far. In this study, we found that SLIT2 and
NPNT promote mitotic rounding (Figure 4D) and cytokine-
sis (Figures 4B and 5B) but not cell cycle entry of postnatal
cardiomyocytes (Figures 4C and 5A). While both SLIT251
and NPNT52 have been implicated in heart development, it
is unclear how they interact with cardiomyocytes to regu-
late their cytokinesis. Since acute treatment (24 hours) with
SLIT2 or NPNT are sufficient to promote cardiomyocyte
mitotic rounding (Figure 4D) and cytokinesis (Figure 4B),
we postulate that they may act directly on cardiomyocytes
and activate intracellular signaling pathways critical for suc-
cessful cytokinesis. In line with this hypothesis, SLIT2 has
been shown to activate RhoA, a small GTPase essential
for cytokinesis,61 by activating Trio GEF62 and/or suppress-
ing the Myo9b RhoGAP activity.63 Analyzing the changes in
cytoskeletal organization and the activity of its regulators in
cardiomyocytes upon SLIT2 and NPNT treatment will shed
light on the molecular mechanisms underlying the effects
of SLIT2 and NPNT on cardiomyocyte cytokinesis.
ARTICLE INFORMATION
Received November 4, 2019; revision received May 28, 2020; accepted June
19, 2020.
Affiliations
From the Department of Developmental Genetics (C.-C.W., D.Y.R.S.), German
Centre for Cardiovascular Research (DZHK) Partner site Rhein Main (C.-C.W.,
S.J., J.G., D.Y.R.S.), and Biomolecular Mass Spectrometry (S.J., J.G.), Max Planck
Institute for Heart and Lung Research, Bad Nauheim, Germany.
Acknowledgments
We thank Arica Beisaw, Felix Gunawan, Paolo Panza, Rashmi Priya, and Michelle
Collins for critical reading of the manuscript, Mathilda Mommersteeg, Susann
Bruche, Eldad Tzahor, and Kfir-Baruch Umansky for collaboration, and all Stainier
lab members for helpful discussion and sharing of reagents.
Sources of Funding
This work was supported by a Croucher fellowship for postdoctoral research (C.-
C. Wu) and the Max Planck Society and Leducq foundation (D.Y.R. Stainier).
Disclosures
None.
Supplemental Materials
References64–75
REFERENCES
1. Sereti KI, Nguyen NB, Kamran P, Zhao P, Ranjbarvaziri S, Park S, Sabri
S, Engel JL, Sung K, Kulkarni RP, et al. Analysis of cardiomyocyte clonal
expansion during mouse heart development and injury. Nat Commun.
2018;9:754. doi: 10.1038/s41467-018-02891-z
2. Meilhac SM, Kelly RG, Rocancourt D, Eloy-Trinquet S, Nicolas JF,
Buckingham ME. A retrospective clonal analysis of the myocardium reveals
two phases of clonal growth in the developing mouse heart. Development.
2003;130:3877–3889. doi: 10.1242/dev.00580
September 11, 2020   905
 Wu et al
3. Soonpaa MH, Kim KK, Pajak L, Franklin M, Field LJ. Cardiomyocyte DNA
synthesis and binucleation during murine development. Am J Physiol.
Original Research
1996;271:H2183–H2189. doi: 10.1152/ajpheart.1996.271.5.H2183
4. Porrello ER, Mahmoud AI, Simpson E, Hill JA, Richardson JA, Olson EN,
Sadek HA. Transient regenerative potential of the neonatal mouse heart.
Science. 2011;331:1078–1080. doi: 10.1126/science.1200708
5. Patterson M, Barske L, Van Handel B, Rau CD, Gan P, Sharma A, Parikh S,
Denholtz M, Huang Y, Yamaguchi Y, et al. Frequency of mononuclear diploid
cardiomyocytes underlies natural variation in heart regeneration. Nat Genet.
2017;49:1346–1353. doi: 10.1038/ng.3929
6. Wills AA, Holdway JE, Major RJ, Poss KD. Regulated addition of new
myocardial and epicardial cells fosters homeostatic cardiac growth and
maintenance in adult zebrafish. Development. 2008;135:183–192. doi:
10.1242/dev.010363
7. Kikuchi K, Holdway JE, Werdich AA, Anderson RM, Fang Y, Egnaczyk GF,
Evans T, Macrae CA, Stainier DY, Poss KD. Primary contribution to zebrafish
heart regeneration by gata4(+) cardiomyocytes. Nature. 2010;464:601–
605. doi: 10.1038/nature08804
8. González-Rosa JM, Sharpe M, Field D, Soonpaa MH, Field LJ,
Burns CE, Burns CG. Myocardial polyploidization creates a barrier to
heart regeneration in zebrafish. Dev Cell. 2018;44:433–446.e7. doi:
10.1016/j.devcel.2018.01.021
9. Senyo SE, Steinhauser ML, Pizzimenti CL, Yang VK, Cai L, Wang M, Wu
TD, Guerquin-Kern JL, Lechene CP, Lee RT. Mammalian heart renewal
by pre-existing cardiomyocytes. Nature. 2013;493:433–436. doi:
10.1038/nature11682
10. Cahill TJ, Choudhury RP, Riley PR. Heart regeneration and repair after myo-
cardial infarction: translational opportunities for novel therapeutics. Nat Rev
Drug Discov. 2017;16:699–717. doi: 10.1038/nrd.2017.106
11. Zebrowski DC, Becker R, Engel FB. Towards regenerating the mamma-
lian heart: challenges in evaluating experimentally induced adult mam-
malian cardiomyocyte proliferation. Am J Physiol Heart Circ Physiol.
2016;310:H1045–H1054. doi: 10.1152/ajpheart.00697.2015
12. Mahmoud AI, Kocabas F, Muralidhar SA, Kimura W, Koura AS, Thet S,
Porrello ER, Sadek HA. Meis1 regulates postnatal cardiomyocyte cell cycle
arrest. Nature. 2013;497:249–253. doi: 10.1038/nature12054
13. Monroe TO, Hill MC, Morikawa Y, Leach JP, Heallen T, Cao S, Krijger PHL,
de Laat W, Wehrens XHT, Rodney GG, et al. YAP partially reprograms chro-
Downloaded from http://ahajournals.org by on July 8, 2022
matin accessibility to directly induce adult cardiogenesis in vivo. Dev Cell.
2019;48:765–779.e7. doi: 10.1016/j.devcel.2019.01.017
14. Heallen T, Zhang M, Wang J, Bonilla-Claudio M, Klysik E, Johnson RL,
Martin JF. Hippo pathway inhibits Wnt signaling to restrain cardiomyo-
cyte proliferation and heart size. Science. 2011;332:458–461. doi:
10.1126/science.1199010
15. Heallen T, Morikawa Y, Leach J, Tao G, Willerson JT, Johnson RL, Martin
JF. Hippo signaling impedes adult heart regeneration. Development.
2013;140:4683–4690. doi: 10.1242/dev.102798
16. Bersell K, Arab S, Haring B, Kühn B. Neuregulin1/ErbB4 signaling induces
cardiomyocyte proliferation and repair of heart injury. Cell. 2009;138:257–
270. doi: 10.1016/j.cell.2009.04.060
17. D’Uva G, Aharonov A, Lauriola M, Kain D, Yahalom-Ronen Y, Carvalho S,
Weisinger K, Bassat E, Rajchman D, Yifa O, et al. ERBB2 triggers mam-
malian heart regeneration by promoting cardiomyocyte dedifferentiation and
proliferation. Nat Cell Biol. 2015;17:627–638. doi: 10.1038/ncb3149
18. Hirose K, Payumo AY, Cutie S, Hoang A, Zhang H, Guyot R, Lunn D, Bigley
RB, Yu H, Wang J, et al. Evidence for hormonal control of heart regenerative
capacity during endothermy acquisition. Science. 2019;364:184–188. doi:
10.1126/science.aar2038
19. Engel FB, Schebesta M, Keating MT. Anillin localization defect in car-
diomyocyte binucleation. J Mol Cell Cardiol. 2006;41:601–612. doi:
10.1016/j.yjmcc.2006.06.012
20. Engel FB, Schebesta M, Duong MT, Lu G, Ren S, Madwed JB, Jiang H,
Wang Y, Keating MT. p38 MAP kinase inhibition enables proliferation of
adult mammalian cardiomyocytes. Genes Dev. 2005;19:1175–1187. doi:
10.1101/gad.1306705
21. Liu H, Zhang CH, Ammanamanchi N, Suresh S, Lewarchik C, Rao K, Uys
GM, Han L, Abrial M, Yimlamai D, et al. Control of cytokinesis by beta-adren-
ergic receptors indicates an approach for regulating cardiomyocyte endow-
ment. Sci Transl Med. 2019;11:eaaw6419.
22. Mohamed TMA, Ang YS, Radzinsky E, Zhou P, Huang Y, Elfenbein A, Foley
A, Magnitsky S, Srivastava D. Regulation of cell cycle to stimulate adult
cardiomyocyte proliferation and cardiac regeneration. Cell. 2018;173:104–
116.e12. doi: 10.1016/j.cell.2018.02.014
23. Puente BN, Kimura W, Muralidhar SA, Moon J, Amatruda JF, Phelps
KL, Grinsfelder D, Rothermel BA, Chen R, Garcia JA, et al. The
906   September 11, 2020 Circulation Research. 2020;127:896–907. DOI: 10.1161/CIRCRESAHA.119.316303
The ECM Regulates Cardiomyocyte Cytokinesis
oxygen-rich postnatal environment induces cardiomyocyte cell-cycle
arrest through DNA damage response. Cell. 2014;157:565–579. doi:
10.1016/j.cell.2014.03.032
24. Kimura W, Xiao F, Canseco DC, Muralidhar S, Thet S, Zhang HM,
Abderrahman Y, Chen R, Garcia JA, Shelton JM, et al. Hypoxia fate mapping
identifies cycling cardiomyocytes in the adult heart. Nature. 2015;523:226–
230. doi: 10.1038/nature14582
25. Pinto AR, Ilinykh A, Ivey MJ, Kuwabara JT, D’Antoni ML, Debuque
R, Chandran A, Wang L, Arora K, Rosenthal NA, et al. Revisit-
ing cardiac cellular composition. Circ Res. 2016;118:400–409. doi:
10.1161/CIRCRESAHA.115.307778
26. Furtado MB, Nim HT, Boyd SE, Rosenthal NA. View from the heart: car-
diac fibroblasts in development, scarring and regeneration. Development.
2016;143:387–397. doi: 10.1242/dev.120576
27. Ieda M, Tsuchihashi T, Ivey KN, Ross RS, Hong TT, Shaw RM, Srivastava
D. Cardiac fibroblasts regulate myocardial proliferation through beta1
integrin signaling. Dev Cell. 2009;16:233–244. doi: 10.1016/j.
devcel.2008.12.007
28. Ivey MJ, Kuwabara JT, Pai JT, Moore RE, Sun Z, Tallquist MD. Resident fibro-
blast expansion during cardiac growth and remodeling. J Mol Cell Cardiol.
2018;114:161–174. doi: 10.1016/j.yjmcc.2017.11.012
29. Rienks M, Papageorgiou AP, Frangogiannis NG, Heymans S. Myocar-
dial extracellular matrix: an ever-changing and diverse entity. Circ Res.
2014;114:872–888. doi: 10.1161/CIRCRESAHA.114.302533
30. Williams C, Quinn KP, Georgakoudi I, Black LD 3rd. Young developmen-
tal age cardiac extracellular matrix promotes the expansion of neo-
natal cardiomyocytes in vitro. Acta Biomater. 2014;10:194–204. doi:
10.1016/j.actbio.2013.08.037
31. Hanson KP, Jung JP, Tran QA, Hsu SP, Iida R, Ajeti V, Campagnola PJ,
Eliceiri KW, Squirrell JM, Lyons GE, et al. Spatial and temporal analysis
of extracellular matrix proteins in the developing murine heart: a blue-
print for regeneration. Tissue Eng Part A. 2013;19:1132–1143. doi:
10.1089/ten.TEA.2012.0316
32. Chen Z, Xie J, Hao H, Lin H, Wang L, Zhang Y, Chen L, Cao S, Huang X,
Liao W, et al. Ablation of periostin inhibits post-infarction myocardial regen-
eration in neonatal mice mediated by the phosphatidylinositol 3 kinase/
glycogen synthase kinase 3β/cyclin D1 signalling pathway. Cardiovasc Res.
2017;113:620–632. doi: 10.1093/cvr/cvx001
33. Kühn B, del Monte F, Hajjar RJ, Chang YS, Lebeche D, Arab S, Keating
MT. Periostin induces proliferation of differentiated cardiomyocytes and pro-
motes cardiac repair. Nat Med. 2007;13:962–969. doi: 10.1038/nm1619
34. Bassat E, Mutlak YE, Genzelinakh A, Shadrin IY, Baruch Umansky K,
Yifa O, Kain D, Rajchman D, Leach J, Riabov Bassat D, et al. The extra-
cellular matrix protein agrin promotes heart regeneration in mice. Nature.
2017;547:179–184. doi: 10.1038/nature22978
35. Golden HB, Gollapudi D, Gerilechaogetu F, Li J, Cristales RJ, Peng X,
Dostal DE. Isolation of cardiac myocytes and fibroblasts from neonatal
rat pups. Methods Mol Biol. 2012;843:205–214. doi: 10.1007/978-1-
61779-523-7_20
36. Franco-Barraza J, Beacham DA, Amatangelo MD, Cukierman E. Preparation
of extracellular matrices produced by cultured and primary fibroblasts. Curr
Protoc Cell Biol. 2016;71:10.9.1–10.9.34. doi: 10.1002/cpcb.2
37. DeLaughter DM, Bick AG, Wakimoto H, McKean D, Gorham JM, Kathiriya
IS, Hinson JT, Homsy J, Gray J, Pu W, et al. Single-cell resolution of temporal
gene expression during heart development. Dev Cell. 2016;39:480–490.
doi: 10.1016/j.devcel.2016.10.001
38. Hesse M, Doengi M, Becker A, Kimura K, Voeltz N, Stein V, Fleischmann
BK. Midbody positioning and distance between daughter nuclei enable
unequivocal identification of cardiomyocyte cell division in mice. Circ Res.
2018;123:1039–1052. doi: 10.1161/CIRCRESAHA.118.312792
39. Ali SR, Hippenmeyer S, Saadat LV, Luo L, Weissman IL, Ardehali R.
Existing cardiomyocytes generate cardiomyocytes at a low rate after
birth in mice. Proc Natl Acad Sci U S A. 2014;111:8850–8855. doi:
10.1073/pnas.1408233111
40. Leone M, Musa G, Engel FB. Cardiomyocyte binucleation is associated
with aberrant mitotic microtubule distribution, mislocalization of RhoA
and IQGAP3, as well as defective actomyosin ring anchorage and cleav-
age furrow ingression. Cardiovasc Res. 2018;114:1115–1131. doi:
10.1093/cvr/cvy056
41. Hashimoto H, Yuasa S, Tabata H, Tohyama S, Hayashiji N, Hattori F, Muraoka
N, Egashira T, Okata S, Yae K, et al. Time-lapse imaging of cell cycle
dynamics during development in living cardiomyocyte. J Mol Cell Cardiol.
2014;72:241–249. doi: 10.1016/j.yjmcc.2014.03.020
42. Kakkar R, Lee RT. Intramyocardial fibroblast myocyte communication. Circ
Res. 2010;106:47–57. doi: 10.1161/CIRCRESAHA.109.207456
 Wu et al
43. Laser M, Willey CD, Jiang W, Cooper G 4th, Menick DR, Zile MR, Kuppuswamy
D. Integrin activation and focal complex formation in cardiac hypertrophy. J
Biol Chem. 2000;275:35624–35630. doi: 10.1074/jbc.M006124200
44. Ruoslahti E. RGD and other recognition sequences for integrins. Annu Rev
Cell Dev Biol. 1996;12:697–715. doi: 10.1146/annurev.cellbio.12.1.697
45. Cukierman E, Pankov R, Stevens DR, Yamada KM. Taking cell-matrix
adhesions to the third dimension. Science. 2001;294:1708–1712. doi:
10.1126/science.1064829
46. Hynes RO. Integrins: bidirectional, allosteric signaling machines. Cell.
2002;110:673–687. doi: 10.1016/s0092-8674(02)00971-6
47. Milner R, Edwards G, Streuli C, Ffrench-Constant C. A role in migration
for the alpha V beta 1 integrin expressed on oligodendrocyte precursors. J
Neurosci. 1996;16:7240–7252.
48. Lancaster OM, Le Berre M, Dimitracopoulos A, Bonazzi D, Zlotek-Zlotkiewicz
E, Picone R, Duke T, Piel M, Baum B. Mitotic rounding alters cell geometry to
ensure efficient bipolar spindle formation. Dev Cell. 2013;25:270–283. doi:
10.1016/j.devcel.2013.03.014
49. Naba A, Clauser KR, Hoersch S, Liu H, Carr SA, Hynes RO. The matrisome:
in silico definition and in vivo characterization by proteomics of normal and
tumor extracellular matrices. Mol Cell Proteomics. 2012;11:M111.014647.
doi: 10.1074/mcp.M111.014647
50. O’Meara CC, Wamstad JA, Gladstone RA, Fomovsky GM, Butty VL,
Shrikumar A, Gannon JB, Boyer LA, Lee RT. Transcriptional reversion of
cardiac myocyte fate during mammalian cardiac regeneration. Circ Res.
2015;116:804–815. doi: 10.1161/CIRCRESAHA.116.304269
51. Zhao J, Mommersteeg MTM. Slit-Robo signalling in heart development. Car-
diovasc Res. 2018;114:794–804. doi: 10.1093/cvr/cvy061
52. Patra C, Diehl F, Ferrazzi F, van Amerongen MJ, Novoyatleva T, Schaefer L,
Mühlfeld C, Jungblut B, Engel FB. Nephronectin regulates atrioventricular
canal differentiation via Bmp4-Has2 signaling in zebrafish. Development.
2011;138:4499–4509. doi: 10.1242/dev.067454
53. Acharya A, Baek ST, Huang G, Eskiocak B, Goetsch S, Sung CY, Banfi S,
Sauer MF, Olsen GS, Duffield JS, et al. The bHLH transcription factor Tcf21
is required for lineage-specific EMT of cardiac fibroblast progenitors. Devel-
opment. 2012;139:2139–2149. doi: 10.1242/dev.079970
54. Li Y, Asfour H, Bursac N. Age-dependent functional crosstalk between car-
diac fibroblasts and cardiomyocytes in a 3D engineered cardiac tissue. Acta
Biomater. 2017;55:120–130. doi: 10.1016/j.actbio.2017.04.027
Downloaded from http://ahajournals.org by on July 8, 2022
55. Farbehi N, Patrick R, Dorison A, Xaymardan M, Janbandhu V, Wystub-Lis
K, Ho JW, Nordon RE, Harvey RP. Single-cell expression profiling reveals
dynamic flux of cardiac stromal, vascular and immune cells in health and
injury. eLife. 2019;8:e43882.
56. Tzahor E, Poss KD. Cardiac regeneration strategies: staying young at heart.
Science. 2017;356:1035–1039. doi: 10.1126/science.aam5894
57. Bergmann O, Bhardwaj RD, Bernard S, Zdunek S, Barnabé-Heider
F, Walsh S, Zupicich J, Alkass K, Buchholz BA, Druid H, et al. Evidence
for cardiomyocyte renewal in humans. Science. 2009;324:98–102. doi:
10.1126/science.1164680
58. Ahuja P, Sdek P, MacLellan WR. Cardiac myocyte cell cycle control in devel-
opment, disease, and regeneration. Physiol Rev. 2007;87:521–544. doi:
10.1152/physrev.00032.2006
59. Eulalio A, Mano M, Dal Ferro M, Zentilin L, Sinagra G, Zacchigna S, Giacca
M. Functional screening identifies miRNAs inducing cardiac regeneration.
Nature. 2012;492:376–381. doi: 10.1038/nature11739
Circulation Research. 2020;127:896–907. DOI: 10.1161/CIRCRESAHA.119.316303
The ECM Regulates Cardiomyocyte Cytokinesis
60. Magadum A, Ding Y, He L, Kim T, Vasudevarao MD, Long Q, Yang K,
Wickramasinghe N, Renikunta HV, Dubois N, et al. Live cell screening plat-
Original Research
form identifies PPARδ as a regulator of cardiomyocyte proliferation and
cardiac repair. Cell Res. 2017;27:1002–1019. doi: 10.1038/cr.2017.84
61. Piekny A, Werner M, Glotzer M. Cytokinesis: welcome to the Rho zone.
Trends Cell Biol. 2005;15:651–658. doi: 10.1016/j.tcb.2005.10.006
62. Backer S, Lokmane L, Landragin C, Deck M, Garel S, Bloch-Gallego E. Trio
gef mediates rhoa activation downstream of slit2 and coordinates telence-
phalic wiring. Development. 2018;145:dev153692.
63. Kong R, Yi F, Wen P, Liu J, Chen X, Ren J, Li X, Shang Y, Nie Y, Wu K, et al.
Myo9b is a key player in SLIT/ROBO-mediated lung tumor suppression. J
Clin Invest. 2015;125:4407–4420. doi: 10.1172/JCI81673
64. Lukinavičius G, Reymond L, D’Este E, Masharina A, Göttfert F, Ta H, Güther
A, Fournier M, Rizzo S, Waldmann H, et al. Fluorogenic probes for live-
cell imaging of the cytoskeleton. Nat Methods. 2014;11:731–733. doi:
10.1038/nmeth.2972
65. Lukinavičius G, Blaukopf C, Pershagen E, Schena A, Reymond L, Derivery E,
Gonzalez-Gaitan M, D’Este E, Hell SW, Wolfram Gerlich D, et al. SiR-Hoechst
is a far-red DNA stain for live-cell nanoscopy. Nat Commun. 2015;6:8497.
doi: 10.1038/ncomms9497
66. Graumann J, Hubner NC, Kim JB, Ko K, Moser M, Kumar C, Cox J,
Schöler H, Mann M. Stable isotope labeling by amino acids in cell cul-
ture (SILAC) and proteome quantitation of mouse embryonic stem cells
to a depth of 5,111 proteins. Mol Cell Proteomics. 2008;7:672–683. doi:
10.1074/mcp.M700460-MCP200
67. Billing AM, Ben Hamidane H, Graumann J. Quantitative proteomic
approaches in mouse: stable isotope incorporation by metabolic (SILAC)
or chemical labeling (reductive dimethylation) combined with high-res-
olution mass spectrometry. Curr Protoc Mouse Biol. 2015;5:1–20. doi:
10.1002/9780470942390.mo140156
68. Boersema PJ, Raijmakers R, Lemeer S, Mohammed S, Heck AJ. Multiplex
peptide stable isotope dimethyl labeling for quantitative proteomics. Nat
Protoc. 2009;4:484–494. doi: 10.1038/nprot.2009.21
69. Hsu JL, Huang SY, Chow NH, Chen SH. Stable-isotope dimethyl label-
ing for quantitative proteomics. Anal Chem. 2003;75:6843–6852. doi:
10.1021/ac0348625
70. Rappsilber J, Ishihama Y, Mann M. Stop and go extraction tips for matrix-
assisted laser desorption/ionization, nanoelectrospray, and LC/MS
sample pretreatment in proteomics. Anal Chem. 2003;75:663–670. doi:
10.1021/ac026117i
71. Kiweler M, Looso M, Graumann J. MARMoSET - extracting publication-
ready mass spectrometry metadata from raw files. Mol Cell Proteomics.
2019;18:1700–1702. doi: 10.1074/mcp.TIR119.001505
72. Cox J, Mann M. MaxQuant enables high peptide identification rates, indi-
vidualized p.p.b.-range mass accuracies and proteome-wide protein quanti-
fication. Nat Biotechnol. 2008;26:1367–1372. doi: 10.1038/nbt.1511
73. Cox J, Neuhauser N, Michalski A, Scheltema RA, Olsen JV, Mann M.
Andromeda: a peptide search engine integrated into the MaxQuant environ-
ment. J Proteome Res. 2011;10:1794–1805. doi: 10.1021/pr101065j
74. Ritchie ME, Phipson B, Wu D, Hu Y, Law CW, Shi W, Smyth GK. limma
powers differential expression analyses for RNA-sequencing and microar-
ray studies. Nucleic Acids Res. 2015;43:e47. doi: 10.1093/nar/gkv007
75. Xie Y, Allaire JJ, Grolemund G. R markdown: The definitive guide. Boca
Raton: Taylor & Francis, CRC Press; 2018.
September 11, 2020   907