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© Annals of Palliative Medicine. All rights reserved. Ann Palliat Med 2016;5(2):125-134apm.amegroups.com
Review Article
Manipulating the Hippo-Yap signal cascade in stem cells for heart
regeneration
Wen-Feng Cai1, Lei Wang1, Guan-Sheng Liu2, Pin Zhu3, Christian Paul1, Yigang Wang1
1Department of Pathology & Lab Medicine, 2Department of Pharmacology & Cell Biophysics, College of Medicine, University of Cincinnati,
Cincinnati, OH 45267-0575, USA; 3Department of Cardiac Surgery, Guangdong Cardiovascular Institute, Guangdong General Hospital,
Guangdong Academy of Medical Sciences, China
Contributions: (I) Conception and design: WF Cai, Y Wang; (II) Administrative support: Y Wang; (III) Provision of study materials or patients: L
Wang; (IV) Collection and assembly of data: WF Cai, L Wang, GS Liu; (V) Data analysis and interpretation: WF Cai, L Wang; (VI) Manuscript
writing: All authors; (VII) Final approval of manuscript: All authors.
Correspondence to: Dr. Yigang Wang. Department of Pathology & Laboratory Medicine, University of Cincinnati, College of Medicine, 231 Albert
Sabin Way, Cincinnati, OH, 45267-0575, USA. Email: wanyy@ucmail.uc.edu.
Abstract: The Hippo-Yap pathway was originally recognized as a crucial signal cascade controlling organ
size, and more recently identied as an important component involved in the regulation of cardiomyocyte
survival, proliferation, and regeneration. Negative stress responses can activate mammalian sterile 20-like
kinase 1 (Mst1) to suppress protective autophagy and promote cardiomyocyte apoptosis via phosphorylation
and inhibition of Bcl-xL. Moreover, decreased Yap activity and nuclear entry will decrease upon Mst1
activation, ultimately suppressing cardiomyocytes proliferation and regeneration. Based on these
observations, there are potential therapeutic opportunities in cardiac structural and functional regeneration
post myocardium infarction to be gained by manipulation of the Hippo-Yap signal cascade. This review will
summarize the main components of the Hippo-Yap pathway and their molecular biological functions. It
will then highlight the role of these signal modules in the acquisition of stem cell pluripotency, cardiogenic
differentiation, cardiomyocyte proliferation and maturation, and mitochondrial biogenesis in cardiac stem
cells. Finally, it will discuss the potential for future studies of Hippo-Yap pathway using induced pluripotent
stem cell (iPSC) technology.
Keywords: Hippo-Yap signal; stem cells; heart regeneration
Submitted Jan 06, 2016. Accepted for publication Jan 21, 2016.
doi: 10.21037/apm.2016.03.03
View this article at: http://dx.doi.org/10.21037/apm.2016.03.03
Introduction
Ischemic heart disease remains the leading cause of
death and disability in the U.S. Although the associated
acute coronary syndrome can be attenuated using
pharmacotherapy or angioplasty procedures, the heart
gradually deteriorates ultimately leading to the heart
failure as a consequence of lost cardiomyocytes and
pathological remodeling. Stem cell therapy is currently
a focus of intensive investigation in view of the fact that
it has the potential to regenerate or repair infarcted
tissue. Many techniques have adopted this promising
therapeutic strategy to treat ischemic heart diseases
through intravascular infusion, intramyocardial injection,
or bio-membrane/patch-based delivery of cells to the
myocardium. Randomized and controlled clinical trials
have also demonstrated that stem cell therapy can improve
the recovery of cardiac function in patients after acute
myocardial infarction. However, decreased cell survival
and engraftment efficiency are two major pitfalls that can
compromise efcacy. The vast majority of transplanted cells
are lost within days in situ due to the anoxic environment
inside heart tissue, and the formation of brosis hinders the
migration of transplanted cells to the site of damaged tissue.
Hippo complexes, also known as Salvador/Warts/Hippo
Cai et al. Stem cells Hippo-Yap signal for heart regeneration
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(SWH) pathway, were originally identified as a signal
cascade controlling Drosophila organ size through the
regulation of cell proliferation and apoptosis (1). Orthologs
of Hippo components have subsequently been recognized
in mammals, and this highly conserved signal axis appears to
be crucial in maintaining organ development in vertebrates.
In vivo experimental research has shown that temporal
activation of the principal Hippo effector Yap (Yes-associated
protein) led to massive hepatomegaly (2). A recent study
also demonstrated that Yap serves as a critical downstream
effector of the Hippo pathway controlling embryonic heart
size, in which constitutive activation of Yap significantly
enhanced cardiomyocyte number and increased heart size
while ablation of Yap resulted in myocardial hypoplasia and
mouse lethality during the embryonic stage (3).
Tissue regeneration is a repair and renewal process,
which is an essential and fundamental feature in
multicellular organisms in response to injury. It is Hippo’s
potency in coordinating cell proliferation, differentiation,
and tissue homeostasis that has encouraged further
research into regulation of this signaling pathway as a
potential therapeutic strategy in regenerative medicine.
Using Xenopus as a model organism, both Tead4 and Yap1
have been documented as Hippo pathway transcriptional
regulators required for general vertebrate epimorphic
regeneration, as well as for organ size control in appendage
regeneration. These Hippo transcription factors regulate
the growth and fate of tissue cells in Xenopus tadpole
tail, including spinal cord, notochord, muscles, and blood
vessels, in both time- and position-dependent manner (4).
This suggests a precisely programmed regulatory
mechanism that has contributed to the development of
appropriately sized three-dimensional organs in response
to injury. Unfortunately, heart muscles exert feeble
regenerative ability in response to a variety of damages,
which is one of the primary contributors to heart failure
post ischemic injury. As a result, cardiac Hippo signaling (5)
has become a focus of research in the field of cardiac
regenerative medicine. Pronounced cardiac regeneration
has been observed in myocardial infarcted regions when
the active form of Yap was constitutively expressed in heart
tissue, associated with a default brotic response (6). Using
Yap gain- and loss-of-function genome-wide analysis and
RNA sequencing techniques, the phosphoinositol-3-kinase-
Akt pathway was identified as a functional downstream
mechanism promoting mitogenic activity and stimulating
endogenous cardiomyocyte proliferation in response to the
heart tissue injury (7).
With the advent of human induced pluripotent
stem cell (iPSC) technology, it is now possible to treat
myocardial infarction with autologous iPSC-derived
cardiomyocytes. However, induction and acquirement
of mature cardiomyocytes from iPSCs and functional
integration of these committed differentiated cells into
injured myocardium often compromises the translational
therapeutic value of this technique in a clinical setting.
This article will discuss the role of the Hippo-Yap signaling
cascade in cardiogenic differentiation of stem cells and its
translational therapeutic value in myocardial infarction
treatment.
Core components of the Hippo-Yap signal
pathway
The Hippo signaling complex consists of a cluster
of cytoplasm-located protein kinases and two major
transcription factors associated with correspondent
regulators. Enrichment of these pathway components with
WW domains and their cognate proline-rich interacting
motifs provide an efficient signaling mechanism to sense
upstream input and start the downstream output (8). Briey,
Mst can be activated by a variety of stress signals including
mechanic stress, extracellular matrix stiffness, cytoskeletal
rearrangement, contact inhibition and anoxemia (9). This
signal activation can directly modulate mitochondrial
function to affect energy metabolism, or Mst can transduce
this activation to Lats1/2 (large tumor suppressor kinase)
to phosphorylate Yap, which will subsequently be degraded
by the ubiquitin proteasome pathway. Contrarily, inhibition
of Hippo signals will protect against Yap degradation and
promote its nuclear translocation (Figure 1).
Mammalian sterile 20-like kinase 1 (Mst1)/2
serves as a response kinase
The full-length of Mst1 is composed of N-terminal kinase
domain, a C-terminal Sav/Rassf/Hpo (SARAH) domain,
with an auto-inhibitory domain (AID) between them. Mst1
activity is regulated by the phosphorylation at threonine
residues (T120, T183, T387), tyrosine residue (Y433), and
serine residue (S438) (Figure 2). Increased phosphorylation
levels at T183 and Y433 will lead to Mst1 activation
(10,11), whereas Mst1 activity will be inhibited when it
is phosphorylated on T120, T387, and S438 (10,12,13).
Mst1 acts as a responsive kinase under stressful conditions
to phosphorylate Bcl-xL at Serine14 in the BH4 domain,
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thereby blockading the Bcl-xL/Bax covalent interactions
and subsequently triggering mitochondria-mediated
apoptotic death through the activation of Bax (14). Further
studies have demonstrated that Mst1 actually serves as a
switch between cardiac cell life and death through dual
regulation of autophagy and apoptosis (15). Indeed, during
cellular stress, the activated Mst1 can phosphorylate Beclin1
at threonine-108, leading to the separation of Beclin-1
from Atg14L-Vps34 complex. The dissociated Beclin1
subsequently competes with Bax to form complexes with
Bcl-2, which can suppress autophagy and result in the
accumulation of protein aggregates. Unbound Bax will
translocate to mitochondria triggering mitochondrial
perturbations, leading to the release of cytochrome c and
caspase activation resulting in apoptosis (15,16).
Regulation of Lats1/2 kinase activity in Hippo
complex
Mammalian Last1/2 kinase is classified as a subgroup of
the AGC protein kinase family, which is conserved among
eukaryotes. The structure of this serine/threonine kinase
features a conserved N-terminal regulatory domain (NTR)
and an insert between subdomains of the catalytic kinase
domain. It has been earlier reported that Lats locate to the
centrosome and negatively regulate cell cycle progression
through inhibition of cell cycle controller CDC2 kinase in
early mitosis (17). More recent studies have demonstrated
this kinase is also involved in the regulation of F-actin
binding, cell migration (18), and cell morphology (19).
Like other kinases, activation of Lats kinases result from
the phosphorylation of serine/threonine residues on the
activation segment motif (AS) and a C-terminally located
hydrophobic motif (HM) regulated by a group of upstream
protein kinases including Mst1/2, Cdk1, Aurora A,
CHK1/2, and PKA. Among them, Mst1/2-induced
phosphorylation on Thr1079 and Ser909 are essential
for maintaining kinase activity, and this activation can
be counteracted by PP2A-mediated dephosphorylation
within the AS and HM region (20,21). Notably, structural
and biochemical investigations strongly indicate that the
conserved NTR domain of Lats1/2 also contain a binding
motif that can interact with Mob, but it is unclear how
this regulatory protein affects Lats activity. One proposed
mechanism suggests that Mst1/2 kinases phosphorylate
Mob1 and thereby promote the formation of Mob1/Lats
complexes, and this covalent binding enables efficient
autophosphorylation within the AS domain of Lats (22).
Figure 1 Core components of the Hippo signaling pathway.
Hippo complexes are composed of a group of serine/threonine
kinases, which include MST1/2 and LATS1/2 in combination
with their activating adaptor proteins SAV1 and Mob1 respectively
(highlight in orange color). The pathway was activated upon stress
stimulation, and MST can phosphorylate on LATS proteins, which
in turn directly phosphorylates YAP on multiple sites leading to
ubiquitin-proteasome degradation. YAP and TAZ are the main
downstream effectors of the Hippo pathway. Dephosphorylated
YAP/TAZ goes into the nucleus and actively targets gene
expression with several transcription factors. Mst1, mammalian
sterile 20-like kinase 1.
Figure 2 Structure of mammalian sterile 20-like kinase. Mst is
composed by three different functional domains and the kinase
activity can be regulated through phosphorylation on serine or
threonine residues in different sites. AID, auto-inhibitory domain;
SARAH, Sav/Rassf/Hpo.
Nuclear
YAP/TAZ
YAP/TAZ PP
U
U
β-TRCP
YAP/TAZ
LATS1/2
Mob1
Mst1/2
SAV1
Cytoplasm
Cells interaction
Cytokines
Growth factors
P
P
P
TEADs/Smads...
Cell proliferation anti-apoptosis/
anti-differentiation stem/progenitor
cell self-renewal migration
T120 T183
N'
1300 331 394 431 487
C'Kinase AID SARAH
T387 Y433 S438
Cai et al. Stem cells Hippo-Yap signal for heart regeneration
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Yap, an effector for the Hippo complex
Yap is a transcriptional co-activator shuttling between the
nucleus and cytoplasm, and this spatial alteration is mainly
determined by the phosphorylation on residue Serine 127 or
Serine 379. In vitro experimental evidence shows that Yap is
subjected to cytoplasmic retention and ubiquitin-dependent
degradation upon Lats kinase-induced phosphorylation,
whereas phosphorylated Yap translocate into the nucleus.
The increased amount of Yap can be observed in the
nucleus when residue Serine 127 is changed into Alanine, a
mutation form that keeps this residue from phosphorylation.
Correspondingly, Yap-induced biological effects (such as
cell proliferation) are also enhanced to a greater extent
in S127A-mutated Yap when compared to wild type Yap.
Nuclear-translocated Yap cannot recognize and interact with
DNA binding domain per se. Instead, this Hippo effector
serves as co-activator along with TAZ to regulate the DNA-
binding activity of Tead, a crucial transcriptional factor
that triggers proliferative and prosurvival gene progression
programs. Tead contains a C-terminal protein binding
domain that interacts with the N-terminal of Yap. This is
supported by nuclear magnetic resonance (NMR) evidence
that the Yap-binding domain of Tead is characterized by
an immunoglobulin-like beta-sandwich fold with two extra
helix-turn-helix inserts, and this structural feature can
precisely recognize and covalently bind to the Tead-binding
domain of Yap (23). Correspondingly, Yap wraps around the
globular structure of Tead and forms extensive interactions
via three highly conserved interfaces (24). Alternatively,
Tead contains an N-terminal TEA domain, a DNA
binding module that can interact with canonical M-CAT
elements to regulate target gene expression. M-CAT
sequence motif (5’-TCATTCCT-3’) has been identified
in several gene promoters and is the decisive DNA region
controlling regulation of cell growth, differentiation, and
epithelial-mesenchymal transition. Notably, the enhanced
protein-protein interaction between Yap and Tead has
been identified as a molecular mechanism contributing
to oncogenesis and metastasis (especially hepatocellular
carcinoma and gastric cancer (25,26), and pharmacological
blockade of Yap-Tead complex formations may be an
important novel therapeutic strategy for inhibiting tumor
growth (27).
Hippo signals and stems cell pluripotency
Pluripotency, a hallmark characteristic of stem cells,
is defined by the dual features of self-renewal and
differentiation potential, giving rise to the spectrum of cell
types that comprise an organism and holding significant
promise in regenerative medicine therapeutics. Studies have
confirmed that iPSC techniques can enable somatic cells
to acquire pluripotency through genetic reprogramming,
providing an opportunity to diagnose and treat malignant
disorders by simply replacing dysfunctional cells with iPSC-
derived functional counterparts. However, barriers to
reprogramming become signicant in advanced developed
somatic cells, especially under the conditions of aging,
metabolism disturbance, and hypoxia. These restraints
include genes involved in transcription, chromatin
regulation, ubiquitination, dephosphorylation, vesicular
transport, and cell adhesion (28). Recent studies reveal the
Hippo pathway as an important regulator in reprogramming
process.
Comparison of transcriptional profiles between
pluripotent and somatic cells has revealed Lats as a
negative regulator of pluripotency. Silencing of the
endogenous LATS specifically enhances generation of
fully reprogrammed iPS cells without accelerating cell
proliferation, suggesting that Hippo pathway activation
may constitute a barrier to cellular reprogramming (29)
and blockade of Hippo signals may promote this process.
Correspondingly, constitutive overexpression of Yap5SA,
a Yap mutation form which is no longer suppressed by
the Hippo pathway, can maintain embryonic stem cell
morphology and pluripotency as evidenced by the stable
alkaline phosphatase staining and the sustained expressions
of Sox2 and Oct4 (30). Several studies have made strides to
elucidate the molecular mechanism of Hippo components
in maintaining stem cell pluripotency. Proteomics
approach has been utilized to identify that the promoter
of mesendodermal genes, a group of transcription factors
critically involved with self-renewal of undifferentiated
embryonic stem cells, is bound by OCT4, NANOG, and
SMAD2/3/4. At the pluripotent stage, YAP/TAZ and
TEAD act to recruit the NuRD (nucleosome remodeling
and deacetylase) complex to bind to a promoter, which
subsequently represses mesendodermal gene expression by
precluding the transcription factor-induced initiation. On
the contrary, YAP/TAZ and TEAD no longer bind to these
genes upon the mesendodermal differentiation (31,32). A
recent study also revealed that the Hippo pathway members
can enrich Sox2 to the inner cell mass (ICM) where it can
promote ICM fate in mouse blastocysts (33).
Considered a key component in maintaining pluripotency,
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the Hippo signal complex has been identified as a
therapeutic target that terminates the stemness of tumor
cells, and some small molecules [such as verteporfin
(VP)] have been designed to inhibit Yap-Tead interaction.
VP treatment can disrupt Yap-Tead signaling and
downregulate Oct4 expression in hepatoma cells and
human retinoblastoma cell lines (34). Interestingly, the Yap-
mediated hepatocyte reprogramming has been identified
as a mechanism contributing to human hepatocellular
carcinoma (HCC) pathogenesis, and the enriched Yap
targets have been detected in the aggressive human HCC
subtype, which features a proliferative signature and absence
of the CTNNB1 mutation. Importantly, targeting Yap with
small interfering RNA-lipid nanoparticles can signicantly
restore hepatocyte differentiation and result in pronounced
tumor regression in a genetically engineered mouse HCC
model (35). On the other hand, Hippo pathway modulation
can regulate stem cell proliferation and maintenance
and may be useful therapeutically for tissue repair and
regeneration following injury (36).
Hippo-Yap signal in heart development and
myocyte maturation
The importance of Yap in regulating heart development
has been explored in several studies using gain- and loss-of-
function approaches. In one experiment, slower heartbeat
and decreased number of cardiac Troponin-positive
cardiomyocytes were observed, which consequently resulted
in embryonic death in inducible Yap gene mutant embryos.
Although cardiac looping and chamber formation was not
affected, deletion of Yap diminished the proliferation of
cardiomyocytes, leading to a significant reduction in the
number of ventricular myocytes when compared with the
wild type littermates. Correspondingly, forced expression
of YapS112A, a Yap mutant form that is constitutively
active and localized to the nucleus, signicantly promoted
the proliferation of cardiomyocytes in the hearts of
transgenic embryos, and YapS112A transgenic mice
displayed an abnormally thickened myocardium and
expanded trabecular layer compared with that of Yap
transgenic mice (3). Compromised cellular phenotype was
similarly also observed in Mst deficient embryonic body
(Mst−/− EBs), in which beating cell clumps disappeared
and the expressions of cardiac progenitor markers (such as
Nkx2.5, Tbx5, Mesp1, Isl1 and Baf60c) were significantly
suppressed. Further studies revealed that Mst are involved
in cardiogenesis with a mechanism that regulates non-
canonical Wnt ligands. Expression and secretion of several
non-canonical Wnt ligands (such as Wnt2, Wnt2b, and
Wnt5a) were reduced in Mst−/− EBs, whereas canonical
Wnt ligand genes expression were not affected (37).
Numerous studies have provided evidence that Yap is a
nexus of multiple signaling pathways in governing cardiac
growth and survival. Actually, Yap builds up an interlink
among Hippo pathway, Wnt pathway, and the IGF pathway
to regulate β-catenin signaling and precisely control cardiac
development (3).
In the process of cardiac differentiation, cardiomyocyte
morphology maturation is characterized by enhanced
myofibril density and alignment, associated with visible
sarcomeres under bright-eld microscopy (38). Functional
maturation is indicated by increased ion channel expression
in the cell membrane, enhanced calcium storage capacity
in sarcoplasmic reticulum (SR), high density distribution
of adrenergic receptors, and robust contractility. These
differences can be revealed in comparisons between human
pluripotent cells, pluripotent cell-derived cardiomyocytes,
and adult cardiomyocytes (Figure 3) (39). There is no
evidence, so far, that Hippo-Yap signals directly regulate
the functional maturation of electrophysiology and
calcium handling during cardiomyocytes differentiation.
However, a most recent study demonstrated this signal
pathway is involved in the actin cytoskeletal remodeling
with protrusion formation, using the Salvador gene
knock out (Salv KO) mouse model and chromatin
immunoprecipitations (ChIP) sequencing. Mst1 activation
is dependent on the interaction with Salvador. Ablation
of Salvador will inhibit the kinase activity of whole Hippo
signals, thereby leading to the nuclear accumulation of non-
phosphorylated Yap. Actually, Yap-Chip sequencing and
mRNA expression profiling in Salv KO hearts revealed
that Yap is involved in gene transcription and regulation
of Sarcoglycan and Talin2, which can compose the
plasmalemmal complexes that link the actin cytoskeleton to
the extracellular matrix. Importantly, this was conrmed in
mouse ischemic hearts post left anterior descending artery
ligation. The greater extent of cytoskeleton rearrangement
was observed in Hippo kinase-compromised cardiomyocytes
than in wild type counterparts, which enabled the migration
of cardiomyocytes into infarct border-zone. Upregulation
of Sarcoglycan and Talin2 help Salv KO cardiomyocytes
extend sarcomere-filled protrusions into scar tissue in the
region of myocardial injury, as evidenced by the appearance
of costameres linking the ECM to the actin through the
integrin-vinculin-talin complex, which is essential cellular
Cai et al. Stem cells Hippo-Yap signal for heart regeneration
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event for heart regeneration (40).
Taken together, functional inhibition Hippo signals
can promote cardiac differentiation of stem cells, whereas
activation of these kinases impedes the process. In addition,
it would be interesting to investigate the involvement of
Hippo-Yap in stem cell electrophysiological mutation in
future studies.
Hippo-Yap signal and cardiomyocyte proliferation
Adult mammalian cardiomyocytes mostly withdraw
from the cell-cycle and therefore do not proliferate.
The mammalian heart is normally thought to grow by
enlargement without cardiomyocyte proliferation during
the postnatal period, thereby restraining the intrinsic
regenerative capability of the adult hearts. Interestingly,
cytokinesis and mitosis are detectable both in human and
murine cardiomyocytes throughout one’s life, and the
incidence of these myocytes proliferating events appear
more frequent at young age (7,41). Yap’s regulatory effect
on cardiomyocytes proliferation has been revealed for the
most part through gain- and loss-of-function experiments.
Yap deficiency impaired cardiomyocyte proliferation,
whereas overexpression promotes cell-cycle activity in
cardiomyocytes both in vitro and in infant hearts (42).
A recent study using genome-wide screen and high
throughput sequencing on Yap-ChIPs indicated that Yap
can facilitate Tead binding to the enhancer of Pi3kcb, which
encodes p110β as catalytic subunit of phosphoinositol-3-
kinase. Particularly, Pik3cb gain-of-function can promote
cardiomyocytes proliferation, while ablation of Pik3cb can
reduce Yap pro-mitogenic activity (7). Further studies have
revealed enhanced phosphorylation of AKT on Serine-473
upon Pi3kcb overexpression, which subsequently triggers
degradation of cell-cycle inhibitor P27 and results in the
cell proliferation (7). A post-transcriptional regulatory
mechanism for Hippo complexes has been recently
reported. A microRNA cluster miR302-367 can repress
Mst1, Lats, and Mob1b mRNA expressions through their
respective 3’-untraslatonal region, which subsequently
promotes the nuclear entry of Yap and cardiomyocyte
proliferation (43). Indeed, the expression of this microRNA
cluster is detected at peak levels at embryonic stage, but
disappears at postnatal and adult stages, indicating its
importance for cardiomyocytes proliferation during early
heart development. Interestingly, postnatal re-expression
of miR302-367, as well as systemic delivery of miR302
mimic can reactivate the cell-cycle and increase mitogenic
activity in adult cardiomyocytes, reducing scar formation
post experimental myocardial infarction (43). These
studies indicate that precise targeting on Hippo signals can
promote cardiomyocyte proliferation, producing protective
and benecial effects.
Hippo-Yap signal and cardiac stem cell migration
Precise regulation of stem cell movement is crucial for
organogenesis of the vertebrate heart during development,
and is critical for heart tissue repair after injury (especially
in the presence of a fibrotic barrier under hypoxic
conditions). Recently, a genetically modified zebrafish
system (44) was used to reveal the biological significance
of Hippo-Yap complexes in steering cardiac stem cell
migration. Temporal analysis of the Yap/Taz-Tead nuclear
Figure 3 Characteristics of hPSC, hPSC-cardiac progenitors and adult human cardiomyocytes. In comparison, adult cardiomyocyte are
larger, with multiple nuclei and visible sarcomeres, large numbers of mitochondria, large areas of sarcoplasmic reticulum (SR) and high
density distribution of SERCA2a. hPSC, human pluripotent stem cell; SERCA2a, SR Ca2+ ATPase.
L-type ca channel
T-type ca channel
SERCA2a
Mitochondria
Nuclear
Sarcoplasmic reticulum
Sarcomere
Adult human cardiomyocytehPC-cardiac progenitorshPSC
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signal and activity in zebrafish indicated that both are
intensied in cardiac precursors and cardiomyocytes, while
disruption of Yap/Taz activity lead to a failure of midline
migration for cardiac progenitors, resulting in significant
cardia bifida (44). It is still unknown which genes are
targeted by Yap/Taz to regulate cell movement in cardiac
stem cells, but the potential molecular mechanisms have
been partially demonstrated in cancer cell studies that
include upregulation of genes responsible for chemokine,
chemokine receptor, and cytoskeleton reorganization. As
an upstream kinase of Yap, Lats1 can steer cell migration
independent of the Yap-induced transcription regulatory
mechanism. The most recent study on breast cancer cells
demonstrated that p53 phosphorylation is reduced after
exposure to Lats1 siRNA, which subsequently affects
p53 activity with enhanced binding to p52, a member of
the NF-κB transcription factor family. This interactome
consequently promotes PTGS2 expression levels
resulting in increased cell movement and migration (45).
Interestingly, many canonical migration pathways, such as
epidermal growth factor receptor (EGFR) signaling (46)
and HMG-CoA reeducates (HMGCR) cascade (47), can
crosstalk with Hippo-Yap to produce synergistic effect on
cell migration, revealing the signicance of this pathway in
regulating cell mobility.
Hippo-Yap signal and mitochondrial biogenesis
in cardiac stem cell
Mitochondria occupy approximately one-third of cellular
volume in adult cardiomyocytes and play a central role in
energy metabolism, and ATP is predominantly generated
through oxidative phosphorylation in this cellular
powerhouse (48). Therefore, mitochondrial maturation,
both in morphology and function, has been recognized as
a hallmark event in differentiation of embryonic stem cells
and iPS cells. Mst1 is ascertained as an endogenous inhibitor
of autophagy (16), which has been recently recognized
as a cellular event during mitochondrial biogenesis in
myoblast myogenic differentiation (49), indicating that
Hippo signaling might be involved in the regulation of
mitochondrial maturation in cardiac stem cells maturation.
Indeed, the dynamic remodeling of mitochondrial network,
including autophagy-induced mitochondrial clearance
and optic atrophy 1 (OPA1)-mediated mitochondrial
repopulation, facilitates the switch of metabolic mechanism
from glycolysis to oxidative phosphorylation upon
myogenic differentiation. Upon activation, myoblasts
can fuse with other newly differentiated or preexisting
myoblasts to constitute myotubes, which is a metabolically
active cell type that depends predominantly on oxidative
phosphorylation. To meet and maintain this enhanced
energetic requirements, the expanded mitochondrial mass
and the formation of an intensive mitochondrial network
are demanded. Actually, dynamin-1-like protein (DNM1L)
is up-regulated but cleared soon after, during the early
phase of differentiation. The abrupt increased expression of
this GTPase can induce mitochondrial fission to facilitate
mitophagy, which will consequently break down the low
dense mitochondrial network and recycle the sparsely
distributed small size mitochondria. Post the elimination
and clearance of most preexisting mitochondria, the brisk
up-regulation of mitochondrial fusion protein, OPA1, will
lead to the replenishment of high density mitochondrial
network via peroxisome proliferator-activated receptor
gamma, coactivator 1 alpha (PPARGC1A)/PGC-1a-
mediated mitochondrial biogenesis. Notably, the autophagic
ux in early-phase of myogenic differentiation appeared to
be an essential stage for mitochondrial remodeling, since
the mitochondrial reconstitution phase won’t occur till
mitophagy-mediated clearance has been completed. Both
in vitro and in vivo evidences revealed that pharmacological
blockade, gene silencing or genetic ablation of essential
autophagy components, such as Atg5, BAF and Sqstm1,
can significantly inhibit autophagic flux and impair
mitochondrial remodeling in myoblasts, and subsequently
prevent myogenic differentiation (49). A recent study
indicated that Mst1, an autophagy suppressor, are required
for the proper cardiac lineage cell development. Although
Mst1 decient embryonic stem cells can differentiate into
mesoderm lineage, the further differentiation into cardiac
lineage cells is drastically inhibited, suggesting that Mst1
might be involved in the regulation of autophagic ux and
dynamic mitochondrial remodeling.
Conclusions and perspective
An accumulating amount of evidence implicates the
Hippo-Yap signal axis as an indispensable and paramount
mechanism in regulating cardiac development,
regeneration, and rejuvenation (50). At the level of
mitochondria, upregulating Hippo can modify cardiac
response to stress by modulating Mst1 activity, which
appears to suppress autophagy and can promote
cardiomyocyte apoptosis through phosphorylation and
inhibition of Bcl-xL (14,16). Importantly, inhibition of
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Mst1 activation using its dominant-negative mutation
can act as protection against cardiomyopathy by reducing
myocyte necrosis and apoptosis (51). The nuclear entry
of Yap can be mediated by Mst1 activation through
sequential phosphorylation of Hippo components, which
will regulate gene transcription by guiding Tead to the
corresponding transcription factor binding motif. It has
recently become apparent that this coordinated gene
transcription regulatory mechanism represents a nodal
point in regulation cardiac cell proliferation, differentiation,
and regeneration. Indeed, perturbation of Yap-Tead
interaction with a blocking peptide significantly inhibits
Yap overexpression-induced upregulation of cell-cycle-
related genes, such as Aurkb, cdc20, and Ccna2 (42). It is
important to note that these studies were performed mainly
in mouse models, and Hippo-Yap cascades are seldom
studied in specific types of cardiac stem cells, such as cell
lineages expressing c-Kit, Sca-1 and MDR-1. In addition,
the studies conducted in rodents differ significantly from
Hippo-Yap function studies done on humans. Therefore,
it remains necessary to investigate the role of Hippo-Yap
in cardiogenic differentiation, cardiomyocyte proliferation,
and cardiomyocyte regeneration in a clinical setting.
Fortunately, recent developments in iPSC technology
have provided researchers with a unique platform to assess
signaling pathways in specific types of human cells by
converting somatic cells into stem-like cells. As such, it
would be interesting to investigate the Hippo-Yap signaling
complexes in human iPSCs, especially in acquiring
pluripotency, cardiogenic differentiation, and functional
maturation of cardiomyocytes, which holds great promise
for future cardiac regenerative medical therapeutics.
Acknowledgements
Funding: This work was supported by grant from the
National Institutes of Health, USA (R01HL110740, and
R01HL107957 to Yigang Wang), the grant from National
Natural Science Foundation of China (grant No. 81370230
and No. 81570279); and the grant from Technology
Foundation for Selected Overseas Chinese Scholar,
Ministry of Human Resources and Social Security of China
(grant No. Z012013046).
Footnote
Conicts of Interest: The authors have no conicts of interest
to declare.
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Cite this article as: Cai WF, Wang L, Liu GS, Zhu P, Paul C,
Wang Y. Manipulating the Hippo-Yap signal cascade in stem
cells for heart regeneration. Ann Palliat Med 2016;5(2):125-
134. doi: 10.21037/apm.2016.03.03
