Tead1 is required for perinatal cardiomyocyte proliferation
Tead1 is required for perinatal cardiomyocyte proliferation
Ruya Liu 0 1 2
Rajaganapathi Jagannathan 1 2
Feng Li 0 1 2
Jeongkyung Lee 0 1 2
Nikhil Balasubramanyam 1 2
Byung S. KimID 1 2
Ping Yang 0 1 2
Vijay K. YechoorID 0 1 2
Mousumi Moulik (MM 1 2
0 Division of Diabetes, Endocrinology and Metabolism, Department of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania, United States of America, 2 Division of Diabetes, Endocrinology and Metabolism, Department of Medicine, Baylor College of Medicine , Houston , Texas, United States of America, 3 Division of Cardiology, Department of Pediatrics, University of Pittsburgh, Pittsburgh, Pennsylvania, United States of America, 4 Division of Cardiology, Department of Pediatrics, UTHealth McGovern Medical School , Houston, Texas , United States of America
1 Editor: Enzo Porrello, Murdoch Childrens Research Institute , AUSTRALIA
2 the National Institutes of Health (NIH) K08- HL091176 (M.M.), Veterans Affairs VA-ORD- BLR&D I01BX002678 and NIH R01 DK097160 (V. College of Medicine Advanced Core Labs: Mouse Metabolism and Phenotyping Core funded by NIH (UM1HG006348 , 1R01DK114356) , Pathology and Histology Core funded by P30 Cancer Center
Adult heart size is determined predominantly by the cardiomyocyte number and size. The cardiomyocyte number is determined primarily in the embryonic and perinatal period, as adult cardiomyocyte proliferation is restricted in comparison to that seen during the perinatal period. Recent evidence has implicated the mammalian Hippo kinase pathway as being critical in cardiomyocyte proliferation. Though the transcription factor, Tead1, is the canonical downstream transcriptional factor of the hippo kinase pathway in cardiomyocytes, the specific role of Tead1 in cardiomyocyte proliferation in the perinatal period has not been determined. Here, we report the generation of a cardiomyocyte specific perinatal deletion of Tead1, using Myh6Cre deletor mice (Tead1-cKO). Perinatal Tead1 deletion was lethal by postnatal day 9 in Tead1-cKO mice due to dilated cardiomyopathy. Tead1-deficient cardiomyocytes have significantly decreased proliferation during the immediate postnatal period, when proliferation rate is normally high. Deletion of Tead1 in HL-1 cardiac cell line confirmed that cell-autonomous Tead1 function is required for normal cardiomyocyte proliferation. This was secondary to significant decrease in levels of many proteins, in vivo, that normally promote cell cycle in cardiomyocytes. Taken together this demonstrates the non-redundant critical requirement for Tead1 in regulating cell cycle proteins and proliferation in cardiomyocytes in the perinatal heart.
Data Availability Statement: All relevant data are
within the paper and its Supporting Information
Mammalian adult heart size is achieved by a combination of proliferation (hyperplasia) and an
increase in cardiomyocyte size (hypertrophy). Cardiomyocytes proliferate at a high rate in the
perinatal period, setting a range for the eventual cardiomyocyte cell number in the adult heart.
Shortly after birth, proliferation declines drastically, physiological hypertrophy, in turn,
constitutes the major mechanism of further heart growth [
]. Similar to other highly specialized post
mitotic cells, cardiomyocyte proliferation is restricted in the adult heart, thus limiting
Support Grant (NCI-CA125123), and Gene Vector
Core. The funders had no role in study design, data
collection and analysis, decision to publish, or
preparation of the manuscript.
regeneration after injury. Therefore, gaining more insights into and understanding the
molecular mechanisms underlying cardiomyocyte proliferation is critical towards developing
cardiomyocyte replacement as potential therapeutic approach for cardiac diseases.
The highly conserved Hippo-Tead signaling pathway, which regulates cell proliferation and
apoptosis, has emerged as one of the very important regulators of organ size control [
inhibitory Hippo signaling pathway is activated by high cell density and other extracellular
cues to Mst kinases 1/2 (mammalian STE20-like protein kinase)-Sav1 (Salvador homolog 1)
complex, which gets activated and subsequently phosphorylates and activates Lats kinases 1/2
(large tumor suppressor kinase)-Mob1?/1? (Mob kinase activator 1). Lats1/2, in turn,
phosphorylates transcriptional co-activators Yap and Taz, which are then sequestered in the
cytoplasm via association with 14-3-3 family members, and then degraded in a
proteasomedependent manner. In the absence of this inhibitory phosphorylation by the Hippo kinase
pathway, Yap/Taz translocate to the nucleus and bind to transcription factors, including
Tead1, to induce genes promoting cell cycle and survival. Inactivation of Hippo pathway?
either by silencing upstream kinases such as Mst1/2, Lats2 or its binding partner Salvador [
or by activation of downstream kinase effectors Yap [
]?resulted in an increased heart size
and cardiomyocyte number at both embryonic and postnatal stages with evidence of
regenerative myocardium post-injury [
]. While all these studies demonstrate the importance of the
mammalian hippo kinase components including Yap, the downstream transcriptional effector
has not been conclusively demonstrated. Mechanistically, Yap protein, as a co-activator,
possesses a transcriptional activation domain, but lacks a DNA binding domain. Hence, it requires
other DNA-binding transcription factors to regulate transcription, of which the Tead family
serve as the major transcriptional effectors [
], with one study demonstrating that Tead1-Yap
interaction was required for the proliferative effects of Yap1 in cardiomyocytes [
Tead proteins (Tead1-4) are ubiquitously expressed in all organs in a spatial and temporal
]. Global deletion of Tead1, in mice, caused in utero lethality, at embryonic day 11.5,
due to myocardial hypoplasia, but without overt disturbances in cardiac patterning, indicating
its non-redundant role in early embryonic cardiomyocyte proliferation and cardiac
]; while deleting both Tead1 and Tead2 led to severe morphological defects at
embryonic day 8.5 with a failure of heart tube formation [
]. However, whether Tead1 is required
for normal cardiomyocyte proliferation at a later stage, such as in the perinatal period, is not
known. We have recently generated mice carrying the Tead1 floxed allele to inducibly delete
Tead1 and study its function later developmental and postnatal stages. We demonstrated that
Tead1 had a critical function in adult cardiomyocytes and that its loss of function leads to
rapid-onset severe dilated cardiomyopathy [
]. We now asked the question if Tead1 is
required for cardiomyocyte proliferation in the perinatal period, when cardiomyocyte
proliferation is very high, by deleting Tead1, perinatally, in cardiomyocytes using Myh6-Cre.
Our results demonstrated that cell-autonomous Tead1 function in cardiomyocytes was
critical and its loss of function led to lethality by postnatal day 9, accompanied by significantly
reduced cardiomyocyte proliferation, as well as systolic dysfunction and dilated
cardiomyopathy. This was secondary to significant decrease in the levels of many cell cycle related proteins
that promote cell cycle and proliferation. These results demonstrate that Tead1 is required and
has a critical role in promoting cardiomyocyte proliferation in the perinatal period.
Materials and methods
All animal procedures were performed at the Baylor College of Medicine, approved by the
Institutional Animal Care and Use Committee (IACUC) of Baylor College of Medicine conforming
2 / 16
to the Guide for the Care and Use of Laboratory Animals published by the U.S. National
Institutes of Health. The conditional Tead1F/F allele was constructed as previously described [
Cardiomyocyte-specific deletion of Tead1 (Tead1-cKO) was achieved by crossing Tead1F/F mice
with Myh6-Cre mice (011038, The Jackson Laboratory). Mice were maintained under standard
12-h light-dark cycles with ad lib access to food and water, unless specified otherwise. For tissue
collection, mice were euthanized after isoflurane anesthesia, per approved protocols by the
IACUC. To analyze the survival rate of Tead1-cKO pups, time to death (day)?when pups are
found dead in the cage?was monitored and used as the endpoints. Animal health and behavior
were monitored daily by trained researchers or veterinarians, no signs of severe suffering or
distress were observed. Kaplan?Meier survival curves were generated from n of 15 for each group.
Under 1.0% isoflurane anesthesia, echocardiographic examination was conducted on the
mouse pups using Vevo2011. End systolic and diastolic dimensions were obtained from
Mmode ultrasound, ejection fraction and fractional shortening were calculated accordingly.
Echocardiograms were performed blinded to genotype.
Antibodies for immunoblotting and immunofluorescence
The antibodies used for immunoblotting are listed below: Tead1 [EPR3967(2)] (ab133533,
Abcam; 1:2,000), Hsp90 (4874S, Cell Signaling; 1:2,000), Cell Cycle Regulation Antibody
Sampler Kit (9932, Cell Signaling; 1:1,000 for all) & Sampler Kit II (9870, Cell Signaling; 1:1,000 for
all). The antibodies for immunofluorescence are: cardiac Troponin T [1C11] (ab8295, Abcam;
1:500), Tead1 [EPR3967(2)] (ab133533, Abcam; 1:100), phospho-Histone H3 (Ser10) (9701,
Cell Signaling; 1:100), Ki67 (ab16667, Abcam, 1:200).
For Masson?s Trichrome staining, dissected hearts were immediately fixed in 10% formalin
overnight at room temperature, followed by paraffin embedding. Coronal or cross sectioned
tissues (5 ?m) were deparaffinized in Histo-Clear (National Diagnostics), rehydrated and fixed
in Bouin?s Solution at 56?C for 1 hr. Following washes in deionized water, sections were
sequentially stained with Weigerts? Iron Hematoxylin, Beibrich Scarlet-Acid Fuchsin solution,
phosphotungstic/phosphomolybdic acid and Aniline blue (25088, Masson?s Trichrome stain
kit from Polysciences). Sections were dehydrated via ethanol series, cleared in Xylene and
mounted in resin-based medium. Images were analyzed using a Zeiss Axioplan-2 imaging
For wheat germ agglutinin (WGA) staining of tissue sections, following antigen retrieval in
boiling 10mmol/L sodium citrate (pH 6.0) for 30min, sections were blocked with 10% goat
serum, followed by incubation of the sections with Rhodamine-WGA (RL-1022, Vector Lab)
at 5g?/ml and mounted with ProLong (ThermoFisher). Terminal deoxynucleotidyl transferase
(TdT) dUTP Nick-End Labeling (TUNEL) assay was carried out following the manufacturer?s
protocol using In Situ Cell Death Detection Kit (11684817910, Roche). For cytology staining,
cells were washed with PBS and fixed in 10% formalin for 15 min at room temperature,
incubated in 0.5% Triton X-100/PBS and blocked in 2% BSA/PBS. Cells were then incubated with
primary antibodies as indicated, subsequently with Alexa Fluor conjugated anti-mouse IgG
(Invitrogen, 1:200). Images were taken with a DeltaVision (Deconvolution) image restoration
microscope or a Zeiss Axioplan-2 imaging system.
Mouse cardiomyocytes culture
Mouse primary cardiomyocytes were isolated from the hearts of 0?3-day-old neonatal mice as
described previously [
]. Ex vivo Tead1 knockout in neonatal cardiomyocytes was achieved
3 / 16
as previously described [
]. Mouse HL-1 cardiac muscle cell line (SCC065, Sigma-Aldrich)
was maintained according to manufacturer?s protocol. HL-1 stable knockout cell lines were
generated as previously described [
]. Briefly, Cas9 (blasticidin resistant) and Tead1-targeted
sgRNA (sgRNA sequence: TAGCCAGATACATCAAACTC; puromycin resistant) lentiviruses
were transduced to generate the Tead1 knockout cells, followed by blasticidin 1?g/ml and
puromycin 1?g/ml selection for 3 weeks. Similarly, Cas9-control cells were generated by
transduction of Cas9-lentivirus only and followed by blasticidin selection. Knockout efficiency was
validated by further experimentation. LentiCas9-Blast was a gift from Feng Zhang (Addgene
plasmid # 52962).
RNA extraction and real-time PCR
Total RNAs were extracted and cleaned up using RNeasy Mini Kit (QIAGEN) according to
manufacturer?s protocol. Complementary DNA were synthesized using q-Script cDNA
Supermix kit (Quanta Biosci.). Realtime PCR was performed using Perfecta SYBR Green Supermix
(Quanta Biosci.) in a Roche 480 Light Cycler machine. Relative mRNA expression levels were
determined by normalization of target genes to 36B4 as internal control. Primer sequences are
available upon request.
Western blot analysis
Tissue protein samples were prepared in ice-cold lysis buffer (100 mmol/L TrisHCl pH7.4, 10
mmol/L NaCl, 1 mmol/L EDTA, 1 mmol/L EGTA, 10% glycerol, 0.5% deoxycholate, 1%
Triton X-100, 0.1% SDS, 20mmol/L Na4P2O7, 2 mmol/L Na3VO4, 1 mmol/L NaF, 1 mmol/L
PMSF) supplemented with 1X protease inhibitor and phosphatase inhibitor (Roche). Equal
amount of protein samples was loaded and fractionated by SDS-PAGE, then transferred to
0.45 ?m pore-size nitrocellulose membranes (Millipore). Membranes were blocked in 5%
non-fat milk-TBS (w/v), then incubated with indicated primary antibodies and
HRP-conjugated secondary antibodies, enhanced chemiluminescence substrate (Thermo Sci.) was applied
for detection. Band densities were quantified by ImageJ software.
The data are presented as means ? s.e.m. Significance tests were determined by the two-tailed,
unpaired Student?s t test, or two-way ANOVA followed by Sidak?s multiple comparisons test.
p< 0.05 was considered statistically significant. GraphPad Prism 7.04 software was used for
Cardiomyocyte-specific deletion of Tead1 leads to early neonatal death
Global deletion of Tead1 leads to fetal demise at embryonic day e11.5 [
], precluding the
investigation of its function in later cardiac developmental stages. We have previously
generated mice with conditional Tead1 floxed alleles (Tead1F/F) and have demonstrated that
Tead1 is essential in the regulation of adult cardiomyocyte function, using an inducible
knockout mouse model [
], wherein Tead1 was deleted only in adult cardiomyocytes in 5
week old mice. We also found that Tead1 expression peaked during late embryonic and
early postnatal stages, which correspond with periods of high proliferation in
]. Therefore, to determine if Tead1 plays a non-redundant role in the perinatal
heart, when proliferation of cardiomyocytes is high, we generated mice with a
cardiomyocyte-specific deletion of Tead1, using myosin heavy chain 6 (Myh6)-Cre deletor mice, when
4 / 16
cardiac patterning and chamber formation are completed [
] (hereafter referred as
Tead1cKO; Fig 1A). Tead1-cKO pups were born at Mendelian frequency, overtly
indistinguishable at birth from littermates (Fig 1B). However, after postnatal day 5, there was increased
mortality in the Tead1-cKO pups, and none survived beyond postnatal day 9, as shown in
the survival curve in Fig 1C (p<0.001). Thus, deletion of Tead1 in perinatal cardiomyocytes
led to 100% mortality with early neonatal death.
Immunostaining of the heart ventricular sections from 1 day old pups showed nuclear
expression of Tead1 co-localizing with Troponin T staining in control mouse cardiomyocytes
and an efficient Tead1 deletion in Tead1-cKO cardiomyocytes (Figure A in S1 File). WGA and
DAPI co-staining suggested disorganized myofibers in Tead1-cKO compared to flox control
hearts (Fig 1D). These results demonstrated Tead1 function in the cardiomyocytes is
nonredundant in the maintenance of postnatal viability.
Tead1 loss-of-function results in neonatal dilated cardiomyopathy
To evaluate the systolic function of Tead1 deficient hearts, M-mode echocardiography on
left ventricles was performed in 1-day-old pups (Fig 2A). Ejection fraction and fractional
shortening were significantly decreased by 20.4% (p = 0.01) and 28.0% (p <0.01),
respectively, in Tead1-cKO pups compared with flox littermates (Fig 2B and 2C). The
left-ventricular anterior wall thickness (LVAWs; 0.39 ? 0.02 mm compared with 0.48 ? 0.02 mm,
p = 0.02) was lower (Fig 2D), while internal diameter (LVIDs; 1.37 ? 0.04 mm compared
with 1.04 ? 0.12 mm, p = 0.0220) at systole, was significantly higher, as was the
left-ventricular internal diameter at diastole (LVIDd), which showed a trend towards being increased
(1.76 ? 0.03 mm compared with 1.61 ? 0.10 mm, p = 0.15) (Fig 2E and 2F). The calculated
relative wall thickness (2 X posterior wall thickness / left ventricular diastolic diameter) was
unchanged (0.41 ? 0.04 mm:mm compared with 0.46 ? 0.06 mm:mm, p = 0.52) (Fig 2G). In
all, the echocardiographic analyses indicated that Tead1-cKO pups showed significant left
ventricular systolic dysfunction and displayed features of chamber enlargement, consistent
with dilated cardiomyopathy (DCM).
As shown in Fig 3A?3C, heart weight of 1-day-old Tead1-cKO pups was significantly lower
compared to flox littermates (5.92 ? 0.169 g compared with 6.54 ? 0.20 g, p = 0.03), though the
body weight was the same (1.28 ? 0.03 g compared with 1.28 ? 0.03 g, p = 0.95). However, the
heart weight normalized to body weight also being significantly lower in Tead1-cKO
(5.120 ? 0.153 mg/g compared with 4.62 ? 0.07 mg/g, p = 0.005). As the pups grew older, heart
weight and body weight of the floxed controls rose significantly, while this rise was
significantly less in the Tead1-cKO pups, such that at postnatal day 7, the Tead1-cKO pups heart
(18.546 ? 0.67 g compared with 22.480 ? 1.28 g, p = 0.01) and body weight (2.37 ? 0.15 g
compared with 3.85 ? 0.23 g, p < 0.001) was significantly less than their floxed control pups. Since
the rise in body weight was far lower in the Tead1-cKO pups their normalized heart weight to
body weight became significantly higher than the control flox littermates (8.075 ? 0.501 mg/g
compared with 5.848 ? 0.100 mg/g, p = 0.0005), a finding consistent with their ongoing heart
A significant enlargement of the Tead1-cKO hearts was observed by postnatal day 5, as
shown in Fig 3D. Trichrome staining of the heart on day 1 old Tead1-cKO pups did not
reveal any structural abnormalities in chamber development consistent with
echocardiographic studies (Fig 3E). Along with chamber enlargement, a significant increase in
intercellular fibrotic reaction was evident on Trichrome staining in Tead1-cKO pups, as early as
postnatal day 1 (Fig 3F).
5 / 16
Fig 1. Cardiomyocyte-specific deletion of Tead1 leads to early neonatal death by day 9 after birth. (A) Generation of Tead1
cardiomyocytespecific knockout mice (Tead1-cKO) using knockout-first strategy: promoter-driven selection cassette. (B) Representative photograph of a
Tead1-cKO breeding litter, demonstrating Tead1-cKO were born in Mendelian manner and not overtly distinguishable from littermates. (C)
Kaplan-Meier survival curves of Tead1F/F littermates and Tead1-cKO mice. n = 15 mice per group. p<0.001 by log-rank (Mantel-Cox) test
(D) Immunostaining in day 1 heart section verifying Tead1 deletion. Scale bar, 50?m.
6 / 16
Fig 2. Tead1 is required for postnatal cardiac function. Echocardiography on postnatal day 1 neonatal pups. (A) Representative image
of the echocardiography. (B) ejection fraction, (C) fractional shortening (FS), (D) left ventricular anterior wall at systole (LVAWs), (E-F)
left ventricle internal diameter at systole and diastole (LVIDs and LVIDd), and (G) relative wall thickness (RWT, 2xLVPWd/LVIDd) of
the TeadF/F and Tead1-cKO pups (n = 4?5). p < 0.01, p < 0.05; Student?s t-test.
Molecular markers of fetal program following Tead1 ablation in cardiomyocytes
To characterize the mechanisms underlying the regulatory role of Tead1 in cardiomyocytes in
the perinatal period, we assessed changes in gene expression in Tead1 knockout hearts. RNA
isolated from the ventricles from 1-day-old Tead1-cKO and control flox littermate pups was
assayed for molecular markers associated with heart failure and the cardiac fetal program (Fig
4A). Many of the markers often associated with heart failure were differentially changed with
Tead1 deletion. Changes in ?-skeletal actin (Acta1?6 fold increase), atrial natriuretic peptide
(Nppa? 1.9 fold increase), brain natriuretic peptide (Nppb? 2.6 fold increase), cardiac
troponinT (cTnT? 14% decrease), consistent with expected molecular signature of heart failure.
Heart failure-associated fetal gene program is also characterized by upregulation of ?-MyHC
(Myh7), the dominant isoform of myosin heavy chain in the fetal cardiomyocytes, and a down
regulation of ?-MyHC (Myh6), the dominant isoform in the adult cardiomyocytes [
However, in the Tead1-cKO pups, despite a DCM phenotype, Myh7 was not upregulated, and was
7 / 16
Fig 3. Tead1 loss-of-function results in heart failure associated with myocardium hypoplasia. (A) Heart weight (B) Body weight and (C)
heart weight to body weight ratios of Tead1-cKO mice at postnatal days 1 and 7 (n = 10?13). p < 0.001, p < 0.01; Student?s t-test. (D)
TeadF/F and Tead1-cKO postnatal days 5 hearts. Scale bar, 1mm. (E-F) Representative coronal section Trichrome staining of day-1 TeadF/F
and Tead1-cKO mice hearts. Scale bar, 1mm (E), 20?m (F).
in-fact downregulated significantly by 30%, compared to control flox littermates. To confirm
that these changes were primarily due to the cell-autonomous loss-of-function of Tead1, and
not secondary (non-cell autonomous) changes from heart failure, we used our, previously
described, ex vivo knockout model [
], wherein we compared gene expression changes with
Tead1 deletion ex vivo, in Tead1F/F neonatal cardiomyocytes treated with Ad-Cre, as
compared to that with control Ad-lacZ treatment. Ex vivo Tead1 deletion in these neonatal
cardiomyocytes mirrored the in vivo Tead1 deletion findings of up-regulation of ?-skeletal actin
(Acta1) and a down-regulation of both ?-, ?-MyHC (Myh6, Myh7) (Fig 4B), suggesting Tead1
regulation of these fetal program genes [
] in cardiomyocyte is unique and cell-autonomous
and that tead1 is required for ?-MyHC upregulation in heart failure.
8 / 16
Fig 4. Tead1 deletion in perinatal cardiomyocytes leads to elevated markers of heart failure. Data are presented as
mean ? s.e.m. p < 0.001, p < 0.01, p < 0.05; data were analyzed by Student?s t-test. Comparisons between (A)
Tead1-cKO pups and TeadF/F pups (n = 7), (B) TeadF/F neonatal cardiomyocytes transduced with adeno-virus Cre (Tead1
deletion) or lacZ (control).
Tead1 is required for cardiomyocyte proliferation
We next sought to test if cardiomyocyte proliferation was impaired in Tead1-cKO pups.
Detection for cell proliferation in heart sections from 1-day-old pups, by immunostaining for
Ki67 (marker for G1/S/G2/M phases of the cell cycle) (Fig 5A), indicated that there were
significantly less (82% reduction) number of Tead1-deficient cardiomyocytes entering cell cycle
(1.4 ? 1.14% Ki67 pos CMs in Tead1-cKO compared with 7.7 ? 1.65% in floxed controls,
p = 0.03) (Fig 5C). Similarly, decreased (45% reduction) number of phospho-Histone H3 at
serine 10 (pHH3 ?marker for M phase) positive cardiomyocytes in heart sections from
1-dayold pups (0.5 ? 0.03% pHH3 positive CMs in Tead1-cKO compared with 1.2 ? 0.11% in floxed
controls, p = 0.0012) indicated that there was a decrease in number of cells entering mitosis in
the Tead1-cKO hearts (Fig 5B and 5D). We also assessed for apoptosis by the terminal
deoxynucleotidyl transferase (TdT) dUTP nick-end labeling (TUNEL) assay and we did not detect a
difference in apoptosis in either cKO compared to floxed control hearts (Figure B in S1 File).
These results demonstrated that Tead1 is required for cardiomyocytes to enter cell cycle
proliferation during the perinatal period.
To confirm that Tead1 regulated cell proliferation in a cell-autonomous manner, we
established stable mouse HL-1 cardiac cell line with Tead1 deletion (Figure C (i) in S1 File) using
9 / 16
Fig 5. Tead1 is required for cardiomyocyte proliferation. (A, B) Cardiomyocyte proliferation (Ki67) and mitosis (phospho-Histone 3Ser10
positive immunostaining) in postnatal day 1 TeadF/F and Tead1-cKO hearts. (C, D) Quantitative analysis represents counting of multiple
sections from three independent samples per group (approximately 1.2?1.5 X 105 cardiomyocytes were counted). p < 0.01; Student?s t-test.
Scale bar, 20 ?m.
10 / 16
CRISPR/Cas9 system and characterized cell proliferation. Tead1-KO and control cells were
synchronized at G0 phase by serum depletion and cell numbers were determined every 48
hours. Growth curves demonstrated a significant decrease in Tead1-KO cells compared to
control cells, by 37% on day 4 and day 6 (Figure C (ii) in S1 File). This result confirmed that
Tead1-regulation of cell cycle is cell-autonomous.
Loss of Tead1 leads to decreased levels of critical cell cycle proteins
The expression of essential cell cycle proteins was assessed in Tead1-deleted myocardium as
compared to control flox littermates, by Western blotting. There was a significant decrease in
the levels of G1/S phase regulating proteins, Cdk4, Cdk6, hyperphospho-RbSer807/811 levels,
which suggested that Tead1 is required for cardiomyocytes to progress through G1-S phase.
Similarly, the levels of phospho-Wee1Ser642 and cyclin B1, which regulate cell cycle progression
from S to G2 and G2/M phases, were decreased in Tead1-cKO hearts (Fig 6A and 6B).
Taken together, these data demonstrate that Tead1 is critical for the proliferation of CM
perinatally via regulation of the levels of many critical cell cycle markers in all phases of cell
cycle and its perinatal loss leads to heart failure and DCM.
In the present study, we examined the role of Tead1 in proliferating cardiomyocytes by
generating perinatal cardiomyocyte-specific Tead1 knockout mice using Myh6-Cre- mediated
deletion of Tead1 floxed alleles. None of the Tead1-cKO mice survived beyond postnatal day 9 and
died of severe heart failure. Decreased expression of cell cycle promoting genes accompanied
by decreased cardiomyocyte proliferation was observed, demonstrating the non-redundant
role of Tead1 in proliferating cardiomyocytes in the perinatal period. In addition, absence of
upregulation of ?MHC (Myh7), a fetal gene program marker of heart failure, in the failing
hearts of the Tead1-cKO mice demonstrates requirement of Tead1 for heart failure-associated
fetal program increase in ?MHC.
Canonical Hippo-Tead signaling consists of a kinase cascade, modulated by both extra- and
intra-cellular cues to regulate multiple cellular functions, including, cell proliferation. Previous
studies on Hippo signaling in the heart have demonstrated that upstream components of the
mammalian Hippo pathway, including Mst1/2, Sav, Lats1/2 all negatively regulate
cardiomyocyte proliferation [
], while Yap was shown to be a positive regulator of cardiomyocyte
proliferation . While Tead1 has been shown to indirectly mediate the effects of upstream
Hippo components, there has not been any definitive study to demonstrate the role of Tead1
in cardiomyocyte proliferation in the perinatal period.
Heart specific inactivation of Yap1, using early cardiac developmental marker-driven Cre
lines?such as Nkx2.5, Myh7 and Tnnt2 ?are embryonic lethal due to decreased CM
]. While using the Myh6-Cre, expressed at embryonic day 10.5 and upregulated
during fetal and postnatal cardiac development, to inactivate Yap, death occurred only when the
mice were 11 to 20-week-old, with dilated cardiomyopathy. In the same study, Yap was also
shown to be critical for neonatal cardiac regeneration . While, Taz knockout mice, using
Myh6-Cre, were viable without evident cardiac impairment, deletion of both cardiac Yap and
Taz led to death by post-natal day 1, significantly earlier than when either of the two genes was
deleted alone, indicating redundancy between the functions of Yap and Taz, as well as possible
gene dosage dependent effects [
]. Since, both Yap and Taz act as co-activators of Tead1, early
neonatal death due to Yap/Taz combined deletion is consistent with our observation of early
neonatal death secondary to heart failure with decreased cardiomyocyte proliferation after
Tead1 deletion in cardiomyocytes, using the same Myh6-Cre line. It is possible that the slightly
11 / 16
Fig 6. Loss of Tead1 leads to decreased expression of cell cycle proteins. (A) Western blot analysis of cell cycle related proteins in postnatal
day 7 TeadF/F (n = 7) and Tead1-cKO (n = 6) hearts. Loading control, Hsp90. (B) Densitometry analyses of relative protein expression,
normalized by Hsp90. Data are presented as mean ? s.e.m. p < 0.001, p < 0.01, p < 0.05; data were analyzed by Student?s t-test.
earlier death seen in cardiomyocyte specific Yap/Taz double knockout mice could be due to
the loss of function of critical Tead1-dependant [
], and Tead1-independent [
Though it has been suggested that Tead1 mediates the effects of upstream Hippo
components in the heart, this has largely been based only on supportive evidence. The only direct
evidence for a role of Tead1 in CM proliferation was seen with the global Tead1 KO, which die
embryonically, at e11.5 [
], from cardiac hypoplasia due to a decrease in CM proliferation.
While this demonstrated the role of Tead1 in embryonic CM proliferation, there has not been
any study to demonstrate a role for Tead1 in subsequent CM proliferation in the perinatal
period. In this study we used the Myh6-Cre deletor line to address this. The expression of
Myh6-Cre in the heart has been reported to be as early as e8.5 [
], predominantly in atria,
with ventricular expression being lower until just before birth, when it rises significantly.
Hence Myh6 promoter-driven Cre mediated recombination in ventricular cardiomyocytes
12 / 16
could have incomplete and variable efficacy prior to birth. At e14.5, the expression of Tead1 in
cardiomyocytes was variable in our Tead1-cKO mice with some CMs with Tead1 deletion and
others with persistent presence (data not shown), unlike that seen at P1 pups (Fig 1 and
Figure A in S1 File), wherein most CMs had deletion of Tead1. Hence, while we can conclude
that Tead1 is required for CM proliferation in the perinatal period, the Myh6-Cre deletor
model cannot address whether Tead1 is required in the embryonic periods.
Our data demonstrated significantly decreased levels of cardiomyocytes that were entering
cell cycle, in vivo, along with a significant decrease in many proteins that promote progression
through the various stages of the cell cycle, in Tead1 ablated myocardium, consistent with that
observed in vitro, in Tead1-deficient HL1 cells. Based on these findings, we infer that Tead1 is
required for cardiomyocyte cell cycle progression and that lack of cell-autonomous Tead1
function leads to cell cycle withdrawal. These data are also consistent with previous studies in
other cell types. Tead1 germline mutant embryos presented decreased percentage of
myocardial cells in S phase [
] as was also seen in breast cancer cells, wherein knockdown of all Tead
family members led to similar cell cycle changes [
]. Interestingly, Tead1 overexpression in
cardiomyocytes, in vivo, was not sufficient to induce significant hyperplasia of the
]. The lack of hyperplasia with Tead1 overexpression, in vivo, taken together with our
current findings indicate that Tead1 is required, but not sufficient for cardiomyocyte
We had previously demonstrated that in adult cardiomyocytes Tead1 is a direct
transcriptional regulator of Ppp1r1a (Inhibitor-1) and that loss-of-function of Tead1 leads to increased
PP1 activity, decreased (PLN) phospholamban phosphorylation and consequent decrease in
Serca2a activity, along with heart failure [
]. In the current study, we observed a similar
decrease in Inhibitor-1 and decreased phosphorylation of phospholamban (data not shown),
suggesting that this is a conserved mechanism even in neonatal CMs and may contribute to
the observed early lethality observed in the Tead1 cKO mice.
Heart adaptation to pathophysiologic insults, such as myocardium ischemia,
pressure/volume overload, often includes an induction of a ?fetal gene program? [
]. In rodent
myocardium the predominant isoform of myosin heavy chain (MHC) is the ?-MHC and post-natally
this transitions to the ? isoform so that in the adult rodent myocardium ?-MHC is the
predominant isoform. An isoform switch from ?-MHC to ?-MHC, due to an increased
expression of ?-MHC with a concomitant repression of ?-MHC, is a prominent feature in the
stressed rodent myocardium. However, in the Tead1-depleted cardiomyocytes, ex vivo (ex
vivo Tead1 deletion in neonatal cardiomyocytes) and in vivo (Tead1-cKO) models, both
?MHC and ?-MHC are significantly repressed despite the evident heart failure phenotype in
vivo. This is evidence that Tead1 is a transcriptional regulator of both ?- and ?-MHC [
and this demonstrates that cell-autonomous function of Tead1 is required for the ?normal?
induction of the fetal gene program associated upregulation of ?-MHC in the stressed rodent
myocardium. In stressed myocardium, there is an induction of ?-skeletal actin (Acta1), as was
seen in Tead1 deletion models, both in vivo and ex vivo, indicating that this regulation of
Acta1 by Tead1 is cell-autonomous and repressive. Repression of certain genes by Tead1 has
been previously reported, especially in concert with repressors, such as Vgll4  in the heart,
and other cell types [
]. Tead1 has been reported to upregulate Acta1 during skeletal muscle
] but a repressive regulation in cardiomyocyte has not been previously
noted and will need to be addressed in future work.
In conclusion, our study demonstrates the critical non-redundant role of Tead1 in
proliferating cardiomyocytes, in the perinatal period. Understanding the regulatory mechanisms
underlying Tead1 function in cardiomyocyte proliferation could allow identification of new
13 / 16
targets for promoting cardiomyocyte proliferation for regenerative therapies for heart diseases
in the future.
S1 File. Supporting figures and legends.
Figure A. Cardiomyocyte Tead1 knockout validation on heart sections from 1.5-day-old
pups. Representative immunostaining showing Tead1 (green), cardiac Troponin T (red),
DAPI (blue). Scale bar, 50 ?m.
Figure B. Tead1 ablation did not impact apoptosis. (A) Representative figures of the TUNEL
staining in control and Tead1-cKO heart sections. Scale bar, 100 ?m. (B) Quantitative analysis
represents counting from three independent samples per group (approximately 0.7?1.7 X
105cells were counted). Data were analyzed by Mann-Whitney t-test.
Figure C. Tead1 regulates cardiomyocyte growth and cell cycle in a cell-autonomous
manner. (i) Western blot validation for Tead1 knockout in stable HL-1 cells. (ii) Cell counts in
HL1-Cas9 and HL1-Tead1 knockout (HL1-Tead1KO) cells. Equal number of cells were plated
and synchronized by overnight serum starvation, and cell numbers were counted at the
indicated time points after reintroduction of complete growth medium (n = 4). p< 0.01;
p< 0.001; data were analyzed by two-way ANOVA followed by Sidak?smultiple comparisons
This work was also supported by Baylor College of Medicine Advanced Core Labs: Mouse
Metabolism and Phenotyping Core, Pathology and Histology Core, and Gene Vector Core.
Conceptualization: Ruya Liu, Vijay K. Yechoor, Mousumi Moulik.
Data curation: Ruya Liu.
Formal analysis: Ruya Liu, Rajaganapathi Jagannathan, Feng Li, Vijay K. Yechoor, Mousumi
Funding acquisition: Vijay K. Yechoor, Mousumi Moulik.
Investigation: Ruya Liu, Rajaganapathi Jagannathan, Feng Li, Jeongkyung Lee, Nikhil
Balasubramanyam, Byung S. Kim, Ping Yang, Vijay K. Yechoor, Mousumi Moulik.
Methodology: Ruya Liu, Rajaganapathi Jagannathan, Feng Li, Jeongkyung Lee, Nikhil
Balasubramanyam, Byung S. Kim, Vijay K. Yechoor, Mousumi Moulik.
Project administration: Vijay K. Yechoor, Mousumi Moulik.
Resources: Vijay K. Yechoor.
Supervision: Vijay K. Yechoor, Mousumi Moulik.
Validation: Ruya Liu.
Visualization: Vijay K. Yechoor, Mousumi Moulik.
Writing ? original draft: Ruya Liu.
Writing ? review & editing: Vijay K. Yechoor, Mousumi Moulik.
14 / 16
15 / 16
1. von Gise A , Lin Z , Schlegelmilch K , Honor LB , Pan GM , Buck JN , et al. YAP1, the nuclear target of Hippo signaling, stimulates heart growth through cardiomyocyte proliferation but not hypertrophy . Proceedings of the National Academy of Sciences of the United States of America . 2012 ; 109 ( 7 ): 2394 - 9 . Epub 2012/02/07. https://doi.org/10.1073/pnas.1116136109 PMID: 22308401; PubMed Central PMCID : PMC3289361 .
2. Halder G , Johnson RL . Hippo signaling: growth control and beyond . Development . 2011 ; 138 ( 1 ): 9 - 22 . Epub 2010/12/09. https://doi.org/10.1242/dev.045500 PMID: 21138973; PubMed Central PMCID : PMCPMC2998162 .
3. Heallen T , Zhang M , Wang J , Bonilla-Claudio M , Klysik E , Johnson RL , et al. Hippo pathway inhibits Wnt signaling to restrain cardiomyocyte proliferation and heart size . Science . 2011 ; 332 ( 6028 ): 458 - 61 . Epub 2011/04/23. https://doi.org/10.1126/science.1199010 PMID: 21512031; PubMed Central PMCID : PMC3133743 .
4. Xin M , Kim Y , Sutherland LB , Qi X , McAnally J , Schwartz RJ , et al. Regulation of insulin-like growth factor signaling by Yap governs cardiomyocyte proliferation and embryonic heart size . Science signaling . 2011 ; 4 ( 196 ): ra70 . Epub 2011 /10/27. https://doi.org/10.1126/scisignal.2002278 PMID: 22028467; PubMed Central PMCID : PMC3440872 .
5. Xin M , Kim Y , Sutherland LB , Murakami M , Qi X , McAnally J , et al. Hippo pathway effector Yap promotes cardiac regeneration . Proceedings of the National Academy of Sciences of the United States of America . 2013 ; 110 ( 34 ): 13839 - 44 . Epub 2013/08/07. https://doi.org/10.1073/pnas.1313192110 PMID: 23918388; PubMed Central PMCID : PMCPMC3752208 .
6. Lin Z , von Gise A , Zhou P , Gu F , Ma Q , Jiang J , et al. Cardiac-specific YAP activation improves cardiac function and survival in an experimental murine MI model . Circ Res . 2014 ; 115 ( 3 ): 354 - 63 . Epub 2014/ 05/17. https://doi.org/10.1161/CIRCRESAHA.115.303632 PMID: 24833660; PubMed Central PMCID : PMCPMC4104149 .
7. Morikawa Y , Zhang M , Heallen T , Leach J , Tao G , Xiao Y , et al. Actin cytoskeletal remodeling with protrusion formation is essential for heart regeneration in Hippo-deficient mice . Science signaling . 2015 ; 8 ( 375 ): ra41 . Epub 2015 /05/07. https://doi.org/10.1126/scisignal.2005781 PMID: 25943351; PubMed Central PMCID : PMCPMC4442128 .
8. Vassilev A , Kaneko KJ , Shu H , Zhao Y , DePamphilis ML . TEAD/TEF transcription factors utilize the activation domain of YAP65, a Src/Yes-associated protein localized in the cytoplasm . Genes Dev . 2001 ; 15 ( 10 ): 1229 - 41 . Epub 2001/05/19. https://doi.org/10.1101/gad.888601 PMID: 11358867; PubMed Central PMCID : PMCPMC313800 .
9. Chen Z , Friedrich GA , Soriano P . Transcriptional enhancer factor 1 disruption by a retroviral gene trap leads to heart defects and embryonic lethality in mice . Genes Dev . 1994 ; 8 ( 19 ): 2293 - 301 . Epub 1994/ 10/01. PMID: 7958896 .
10. Sawada A , Kiyonari H , Ukita K , Nishioka N , Imuta Y , Sasaki H . Redundant roles of Tead1 and Tead2 in notochord development and the regulation of cell proliferation and survival . Mol Cell Biol . 2008 ; 28 ( 10 ): 3177 - 89 . Epub 2008/03/12. https://doi.org/10.1128/ MCB . 01759 -07 PMID: 18332127; PubMed Central PMCID : PMCPMC2423158 .
11. Liu R , Lee J , Kim BS , Wang Q , Buxton SK , Balasubramanyam N , et al. Tead1 is required for maintaining adult cardiomyocyte function, and its loss results in lethal dilated cardiomyopathy . JCI Insight . 2017 ; 2 ( 17 ). Epub 2017 /09/08. https://doi.org/10.1172/jci.insight.93343 PMID: 28878117; PubMed Central PMCID : PMCPMC5621883 .
12. Ehler E , Moore-Morris T , Lange S. Isolation and culture of neonatal mouse cardiomyocytes . J Vis Exp . 2013 ; (79) . https://doi.org/10.3791/50154 PMID: 24056408; PubMed Central PMCID : PMCPMC3857885 .
13. Sanjana NE , Shalem O , Zhang F. Improved vectors and genome-wide libraries for CRISPR screening . Nat Methods . 2014 ; 11 ( 8 ): 783 - 4 . Epub 2014/07/31. https://doi.org/10.1038/nmeth.3047 PMID: 25075903; PubMed Central PMCID : PMCPMC4486245 .
14. Agah R , Frenkel PA , French BA , Michael LH , Overbeek PA , Schneider MD . Gene recombination in postmitotic cells. Targeted expression of Cre recombinase provokes cardiac-restricted, site-specific rearrangement in adult ventricular muscle in vivo . J Clin Invest . 1997 ; 100 ( 1 ): 169 - 79 . Epub 1997/07/01. https://doi.org/10.1172/JCI119509 PMID: 9202069; PubMed Central PMCID : PMCPMC508177 .
15. Rajabi M , Kassiotis C , Razeghi P , Taegtmeyer H . Return to the fetal gene program protects the stressed heart: a strong hypothesis . Heart Fail Rev . 2007 ; 12 ( 3-4 ): 331 - 43 . Epub 2007/05/23. https:// doi.org/10.1007/s10741-007-9034-1 PMID: 17516164 .
16. Del Re DP , Matsuda T , Zhai P , Maejima Y , Jain MR , Liu T , et al. Mst1 promotes cardiac myocyte apoptosis through phosphorylation and inhibition of Bcl-xL . Mol Cell . 2014 ; 54 ( 4 ): 639 - 50 . Epub 2014/05/ 13. https://doi.org/10.1016/j.molcel. 2014 . 04 .007 PMID: 24813943; PubMed Central PMCID : PMCPMC4074544 .
17. Heallen T , Morikawa Y , Leach J , Tao G , Willerson JT , Johnson RL , et al. Hippo signaling impedes adult heart regeneration . Development . 2013 ; 140 ( 23 ): 4683 - 90 . Epub 2013/11/21. https://doi.org/10.1242/ dev.102798 PMID: 24255096; PubMed Central PMCID : PMC3833428 .
Wackerhage H , Del Re DP , Judson RN , Sudol M , Sadoshima J . The Hippo signal transduction network in skeletal and cardiac muscle . Science signaling . 2014 ; 7 ( 337 ): re4 . Epub 2014 /08/07. https://doi.org/ 10.1126/scisignal.2005096 PMID: 25097035 .
19. Santucci M , Vignudelli T , Ferrari S , Mor M , Scalvini L , Bolognesi ML , et al. The Hippo Pathway and YAP/TAZ-TEAD Protein-Protein Interaction as Targets for Regenerative Medicine and Cancer Treatment . Journal of medicinal chemistry . 2015 ; 58 ( 12 ): 4857 - 73 . Epub 2015/02/27. https://doi.org/10. 1021/jm501615v PMID: 25719868 .
20. Ota M , Sasaki H. Mammalian Tead proteins regulate cell proliferation and contact inhibition as transcriptional mediators of Hippo signaling . Development . 2008 ; 135 ( 24 ): 4059 - 69 . doi: dev.027151 [pii]; https://doi.org/10.1242/dev.027151 PMID: 19004856
21. Gupta MP . Factors controlling cardiac myosin-isoform shift during hypertrophy and heart failure . J Mol Cell Cardiol . 2007 ; 43 ( 4 ): 388 - 403 . Epub 2007/08/28. https://doi.org/10.1016/j.yjmcc. 2007 . 07 .045 PMID: 17720186; PubMed Central PMCID : PMCPMC2701247 .
22. Zanconato F , Forcato M , Battilana G , Azzolin L , Quaranta E , Bodega B , et al. Genome-wide association between YAP/TAZ/TEAD and AP-1 at enhancers drives oncogenic growth . Nat Cell Biol . 2015 ; 17 ( 9 ): 1218 - 27 . Epub 2015/08/11. https://doi.org/10.1038/ncb3216 PMID: 26258633 .
23. Tsika RW , Ma L , Kehat I , Schramm C , Simmer G , Morgan B , et al. TEAD-1 overexpression in the mouse heart promotes an age-dependent heart dysfunction . The Journal of biological chemistry . 2010 ; 285 ( 18 ): 13721 - 35 . Epub 2010/03/03. https://doi.org/10.1074/jbc.M109.063057 PMID: 20194497; PubMed Central PMCID : PMC2859535 .
24. Taegtmeyer H , Sen S , Vela D . Return to the fetal gene program: a suggested metabolic link to gene expression in the heart . Ann N Y Acad Sci . 2010 ; 1188 : 191 - 8 . Epub 2010/03/06. https://doi.org/10. 1111/j.1749- 6632 . 2009 . 05100 . x PMID : 20201903 ; PubMed Central PMCID : PMCPMC3625436 .
25. Iwaki H , Sasaki S , Matsushita A , Ohba K , Matsunaga H , Misawa H , et al. Essential role of TEA domain transcription factors in the negative regulation of the MYH 7 gene by thyroid hormone and its receptors . PLoS One . 2014 ; 9 ( 4 ): e88610 . Epub 2014 /05/02. https://doi.org/10.1371/journal.pone.0088610 PMID: 24781449; PubMed Central PMCID : PMCPMC4004540 .
26. Lin Z , Guo H , Cao Y , Zohrabian S , Zhou P , Ma Q , et al. Acetylation of VGLL4 Regulates Hippo-YAP Signaling and Postnatal Cardiac Growth . Developmental cell . 2016 ; 39 ( 4 ): 466 - 79 . Epub 2016/10/11. https://doi.org/10.1016/j.devcel. 2016 . 09 .005 PMID: 27720608; PubMed Central PMCID : PMC5121000 .
27. Pan D. The hippo signaling pathway in development and cancer . Developmental cell . 2010 ; 19 ( 4 ): 491 - 505 . Epub 2010/10/19. https://doi.org/10.1016/j.devcel. 2010 . 09 .011 PMID: 20951342; PubMed Central PMCID : PMCPMC3124840 .
28. Joshi S , Davidson G , Le Gras S , Watanabe S , Braun T , Mengus G , et al. TEAD transcription factors are required for normal primary myoblast differentiation in vitro and muscle regeneration in vivo . PLoS Genet . 2017 ; 13 ( 2 ): e1006600 . Epub 2017 /02/09. https://doi.org/10.1371/journal.pgen.1006600 PMID: 28178271; PubMed Central PMCID : PMCPMC5323021 .