Download 2. Ischemic heart disease and limited cardiac regeneration

Probing cardiac regeneration: immune
modulation by microRNAs.
John A.L. Meeuwsen supervised by Eva van Rooij.
Hubrecht Laboratory, Royal Netherlands Academy of Arts and Sciences, Utrecht, The
Netherlands.
Abstract | In this review we describe the basic biology of the heart, the involvement of the
immune system after myocardial infarction, and how microRNAs can influence the
inflammatory response to enhance cardiac regeneration. Cardiomyocytes are the key
players of cardiac contraction and their proliferation is abundant during embryonic
development. However, shortly after birth this regenerative potential occurs only at very
low levels. Upon myocardial infarction (MI) and subsequent reperfusion, a considerable
injury occurs to the heart and millions of viable cardiomyocytes are lost. Therefore, cardiac
function is diminished and renewal of cardiomyocytes is strongly required. The
inflammatory response plays a key role in protection and injury of cardiac cells post MI.
Different important pathways in cardiac inflammation are discussed to investigate the
possibility of reducing cardiac damage and enhancing cardiac repair and regeneration.
MicroRNAs are important regulators of mRNA abundance, involving a broad range of
biological processes, including inflammation. Therefore, the role of microRNAs involving
the cardiac inflammatory response post MI is reviewed. MicroRNAs appear to be critically
involved in cardiac regeneration by modulation of the immune system and therefore
deserve extensive attention in future research, with the ultimate goal to resolve a major
health problem of cardiovascular disease.
1. Basic biology of the heart
To obtain insights in the organization of the
heart, we will describe the different cell types in
the heart, which are required for proper cardiac
contraction. In addition, we will discuss how
proliferation of cardiomyocytes is regulated via
several proteins and pathways during
development.
1.1 Different cell types of the heart
The heart consists of four chambers, and
multiple cells, which together form a blood
pump, essential for life. The heart is mainly
composed from fibroblasts (more than 50 % of
the cells) and cardiomyocytes, which together
form the cardiac muscle of the atria and
ventricles. Endothelial cells cover the inside of
the heart and blood vessels. The conduction
system is formed by pacemaker cells and
Correspondence to: J.A.L. Meeuwsen
Email: [email protected]
Purkinje fibers. A group of pacemaker cells on
the right atrium, called the sinoatrial node,
generates impulses which are conducted by the
atrioventricular node from the atria to the
ventricles, ultimately resulting in heart
contraction (Xin et al., 2013). All these different
cell types present in the heart, are important for
normal
heart
function.
However,
cardiomyocytes are the most important cells in
the heart, because they are essential for the
contractile function of the heart.
1.2 Proliferation of cardiomyocytes
during development
The formation of the heart during development
requires proliferation of cardiac myocytes.
Insights into the mechanisms of cardiomyocyte
proliferation may catalyze the development of
new approaches to expand cardiomyocyte
numbers, which is required for replacement of
diseased or dead cardiomyocytes after cardiac
injury (Xin et al., 2013). Therefore, we will
discuss the essential players and pathways of
cardiomyocytes proliferation during fetal stage.
In addition to cyclins and related proteins,
several pathways are known to induce
cardiomyocyte proliferation. We will discuss
the Insulin-like growth factor (IGF)-1 pathway,
the Wnt pathway and the Hippo pathway
(figure 1). These pathways are important in
cardiomyocyte proliferation by inducing
transcription via Yes associated protein (YAP)
and β-catenin (Ahuja et al., 2007; Xin et al.,
2013). Lastly, we will briefly describe the role
of FGF1 in myocyte proliferation and survival.
transcription factors, which are important
players in the different phases of the cell cycle
(Ahuja et al., 2007), leading to the proliferation
of cardiomyocytes.
1.2.2 IGF pathway
The IGF1 pathway has been shown to play an
important
role
in
proliferation
of
cardiomyocytes (Ahuja et al., 2007). IGF1
binds to the IGF1 receptor (IGF1R) which
mediates
its
mitogenic
activity
via
phosphatidylinositol 3-kinase (PI3K) and Akt
(also known as Protein Kinase B). Upon
activation by IGF1R, PI3K phosphorylates Akt.
Subsequently, Akt phosphorylates glycogen
synthase kinase 3 β (GSK3β), thereby
preventing degradation of β-catenin by GSK3β.
β-catenin induces transcription of several genes
important for cardiomyocyte proliferation.
1.2.1 Cyclins and cyclin dependent kinases
During cardiac development several cyclins and
cyclin-dependent kinases (CDK), and CDKinhibitors are necessary for regulation of
cycling of cardiomyocytes in the heart. Cyclins
D1, D2 and D3, A, B1 and E, as well as several
kinases (Cdc2, CDK2, CDK4 and CDK6) are
abundantly expressed during cardiogenesis.
Inhibitors of these CDKs -and eventually of
proliferation- in the heart, include p16, p17,
p18, p21, p27 and p57. Together these Cyclins,
CDKs and their inhibitors tightly regulate
cardiomyocyte proliferation. Upon activation of
these CDKs by cyclins, Retinoblastoma (Rb)
family members Rb, p107, and p130 are
phosphorylated, resulting in release of E2F
Overexpression of IGF1 in neonatal mice shows
an increase of the heart mass, and a 50%
increased number of cardiomyocytes at day 210
(Reiss et al., 1996), while suppression of IGF1
levels hardly affected the number of
cardiomyocytes or its function (Lembo et al.,
1996). However, genetic deletion of IGF1 mice
was fatal in more than 95% of the mice, due to
underdeveloped muscle tissue (Powell-Braxton
et al., 1993). These data underline the
importance of activation of the IGF1 pathway in
cardiomyocyte proliferation.
- 2-
(TCF-LEF) and β-catenin. Three really
important inhibitors of this pathway are MST1,
MST2, and the scaffold protein Salvador (SAV,
mammalian homologue is WW45) (Xin et al.,
2013; Lee et al., 2008).
1.2.3 Wnt pathway
The Wnt pathway is involved in many
regulatory mechanisms in different stages of
cardiac development. β-catenin is important in
the IGF pathway, but also involved in the Wnt
pathway. Binding of the Wnt protein to the
Frizzled receptor activates disheveled (Dvl),
which represses the complex that induces βcatenin degradation. This complex consists of
axis inhibition protein (AXIN), adenomatosis
polyposis coli (APC) and GSK3β. Thus,
activation of the Wnt pathway rescues βcatenin, which plays an important role in
cardiomyocyte proliferation.
Activation of YAP has a role in regulation of
proliferation of cardiac cells. Genetic loss of
function resulted in perinatal lethality and
cardiac knockout of Salv, which inhibits YAP,
induced significant heart enlargement, not as a
result hypertrophy, but due to increased cell
proliferation in the developing mouse heart
(Heallen et al., 2011). Furthermore, a link
between the Hippo pathway with the Wnt/βcatenin signaling pathway was discovered,
since it appeared that nuclear unphosphorylated
YAP formed a complex with β-catenin.
Moreover, reducing levels of β-catenin
diminishes the enlargement of the heart upon
cardiac knockout of SAV (Heallen et al., 2011).
Thus, inactivation of the Hippo pathway
induces cardiac proliferation, and may act via
regulation of the Wnt/β-catenin pathway.
The importance of the Wnt/β-catenin pathway
was illustrated by the fact that ablation of βcatenin was lethal in mice (Tzahor, 2007).
Conversely, overexpression of β-catenin in the
heart resulted in proliferation of second heart
field progenitors (Tzahor, 2007). However, in
other periods of heart development, activation
of the Wnt/β-catenin pathway decreases
proliferation and differentiation (Ueno et al.,
2007). The exact mechanisms of Wnt/β-catenin
signaling and their effects on cardiac cell
proliferation remain unclear, which may be due
to the complexity of the different stages in
cardiogenesis and the other Wnt pathways, as
well as other factors involving β-catenin
regulation (Tzahor, 2007). Nonetheless, it is
clear that the Wnt/β-catenin pathway plays an
important role in cardiac cell proliferation and
later, also in differentiation.
1.2.5 FGF induces proper cardiomyocyte
proliferation
Besides IGF1, other growth factors, such as
FGF, influence the cardiac cell proliferation.
FGF is required for embryonic heart
development in chicken, since blockage of the
FGF receptor inhibited myocyte proliferation
and/or survival (Mima et al., 1995). In addition,
another paper showed that epicardial and
endocardial FGF expression is essential for
proper heart proliferation, since its absence
results in dilated cardiomyopathy (Lavine et al.,
2005).
1.2.4 Hippo pathway
The Hippo pathway is a key regulatory pathway
in proliferation and size of different organs,
including the heart. The pathway derives its
name from one of the important kinases in the
pathway, hpo (in mammals: STE20-like protein
kinase (MST) 1 and MST2). Activation of this
pathway via a complex consisting of large
tumor suppressor 1 (LATS 1) and LATS2 and
Mps one binder (MOB) inactivates the
transcription factor Yes-associated protein
(YAP) by phosphorylation, thereby restraining
cardiac proliferation. Active YAP however will
translocate to the nucleus and form a complex
with TEA domain family members (TEAD), T
cell factor - lymphoid enhancer-binding factor
Taken together, several mechanisms are known
and described for proliferation and survival of
cardiomyocytes. However, shortly after birth
most cardiomyocytes enter the cell cycle once
more, replicating DNA, but finishing without
cytokinesis, leaving the majority of the
contractile heart cells binucleated throughout
life (Ahuja et al., 2007). Hence, many adult
cardiomyocytes are terminally differentiated
and have lost the ability to proliferate (Maillet
et al., 2013). Therefore, loss of cardiomyocytes
by cardiac injury is a serious problem.
- 3-
2.1 Cardiomyocyte regeneration is
limited in human
2. Ischemic heart disease and
limited cardiac regeneration
For decades, researchers believed the heart was
a post-mitotic organ, and cardiomyocytes were
unable to proliferate during life. Indeed, the
majority of cardiomyocytes in the adult heart
are terminally differentiated (Bergman et al.,
2009). However, in the last decade, multiple
independent lines of evidence support a limited
level of cardiomyocyte proliferation in the
postnatal heart.
Myocardial infarction (MI) is defined as:
“myocardial cell death due to prolonged
ischemia” (Thygesen et al., 2012). The most
prevalent cause of myocardial ischemia is
coronary artery disease (CAD) with
superimposition of atherosclerotic plaque
rupture (Frangogiannis, 2006). Other causes of
MI include blood vessel spasm, endothelial
dysfunction, arrhythmias and stent occlusion
(Thygesen et al., 2012). Myocardial infarction
results in loss of millions of cardiac myocytes
reducing the contractile function of the heart.
The regenerative potential of the heart is shown
to exist in human, but appears too low for
sufficient re-establishment of cardiac function.
Therefore, the loss of cardiomyocytes can
eventually lead to heart failure, which is a major
cause of death worldwide (Go et al., 2013).
Heart failure contributes to one of each nine
deaths, and about 50 percent of the people who
are diagnosed with heart failure, die within five
years (Go et al., 2013). The prevalence of heart
failure in the USA is 5.8 billion, worldwide over
23 billion, and still these numbers are growing
(Bui et al., 2013).
2.1.1 Cardiac regeneration in human
The annually turnover of cardiomyocytes was
estimated 0.5 to 1.0 % (Bergmann et al., 2009).
The researchers used integrated C14 – a byproduct of nuclear bomb testing in the cold war
– to establish the age of cardiomyocytes in
humans (Bergmann et al., 2009). The turnover
decreases with age, and fewer than 50% of the
cardiomyocytes are replenished during life
(Bergmann et al., 2009). In another study, the
estimated annually turnover was 22% (Kajstura
et al., 2010). These researchers used postmortem hearts from patients with cancer, which
were treated with the thymidine analogue
iododeoxyuridine (IdU). These findings
suggests that the heart is fully replaced in 4 to 8
years (Kajstura et al., 2010). It is difficult to
reconcile these studies. Several arguments may
explain differences between studies on the
estimated turnover rate of cardiomyocytes. For
instance, Kajstura et al., (2010) report a 3-fold
higher proliferation in hearts from IdU treated
patients compared to hearts from controls.
Furthermore, several issues make it difficult to
determine the number of regenerating
cardiomyocytes concisely. These include: 1) the
relative low division rate of cardiomyocytes
compared to non-myocytes; 2) the lack of a
highly specific marker for cardiomyocytes; 3)
the presence of polyploidy in cardiomyocytes
(which allows DNA-synthesis without cell
division); and 4) differences in mathematical
modelling
to
calculate
turnover
of
cardiomyocytes. Nonetheless, these studies
demonstrate the existence of cardiac
regeneration in humans, although additional
research is required to elucidate the apparent
contradictions.
Upon MI, ischemic cardiomyocyte death occurs
as a result of a series of events initiated by
ischemia. Ischemia induces a rapid fall down of
the energy in the cell, resulting in a strong
decrease of the intracellular pH and large
changes in ion transport. Subsequently, there is
a lack of electronic activity and proper
contraction in the affected area. Once, the vessel
obstruction is removed by percutaneous
coronary intervention or thrombolysis, blood
flow through the infarcted area is restored.
Reperfusion of the ischemic area generates a
rapid recovery of the intracellular pH. The
recovery of the pH together with the changes in
ion transport induce intracellular Calcium
(Ca2+) overload. Besides that, pH recovery
causes enormous Reactive Oxygen Species
(ROS) generation. Intracellular Ca2+ overload
and ROS activate inflammatory cascades and
disrupt DNA and proteins, ultimately leading to
apoptosis and necrosis of the cells (Sanada et
al., 2011).
- 4-
2.1.2 Regenerative capacity in neonatal
mice
recruitment of leukocytes combined with
release of danger associated molecular patterns
(DAMPs) and alarmins characterize the initial
immune response (Arslan et al., 2011;
Frangogiannis, 2014). We will discuss four
major items of the immune system involved in
post MI remodeling: I) Cytokines, II) Toll Like
Receptor (TLR) pathway, III) Janus Kinase /
Signal Transducer and Activator of
Transcription (JAK/STAT) pathway and IV)
macrophages. Tight regulation of the different
immune key players is crucial to balance the
beneficial and detrimental effects of the
immune system after MI.
In neonatal mice, the regenerative capacity
appears strikingly high compared to adult
humans. Upon surgical resection of the apex in
one day old mice (P1), a regenerative response
was initiated and cardiac anatomy function was
restored (Porrello et al., 2011). In contrast,
regeneration was absent in mice undergoing
surgery at P7, instead, fibrotic scar tissue was
formed. To further extend this model to
myocardial infarction, Haubner and colleagues
induced myocardial infarction (MI) in mice
from P1.5 (Haubner et al., 2012). LAD induced
cardiac cell death, as was measured at P3 and
P4. Interestingly, at 7 days post-operation,
complete regeneration of the heart was observed
(Haubner et al., 2012). These studies
demonstrate that mammalian hearts have
regenerative potential shortly after birth, which
is diminished within 7 days. The difference in
regeneration after injury at P1, and fibrosis after
injury at P7, gives researchers opportunities to
investigate the mechanisms leading to either
fibrosis or regeneration upon cardiac injury.
There is no data available about the regenerative
capacity of neonatal human cardiomyocytes.
3.1 Cytokines
Cytokines are polypeptide or glycoprotein
factors, responsible for a broad range of
physiological responses which include
activation of pro- and anti-inflammatory
pathways. After cardiac injury, cytokines are
released by immune cells and all cell types in
the myocardium. Both, autocrine and paracrine
signaling is common in cytokine signaling,
which leads to a variety of inverse and
pleiotropic biological mechanisms (Prabhu,
2004; Arslan et al., 2011). A large number of
cytokines exist, of which the important
cytokines in the heart include the proinflammatory TNF-α, interleukin (IL)-1, IL-6
and IL-18 and the anti-inflammatory Stromal
Derived Factor (SDF)-1α (also known as
CXCL12), Granulocyte Colony Stimulating
Factor (G-CSF), Leukemia Inhibitory Factor
and IL-2 (Prabhu, 2004).
Thus, cardiac regeneration is present in neonatal
mice and sufficient to renew the injured heart
completely upon apical dissection and MI.
However, in the adult heart cardiac regeneration
occurs only at low levels which appears to be
insufficient to restore cardiac function. Along
this line, it is really interesting to research the
differences between young and adult mammals,
to obtain new insights in the mechanisms of
cardiac regeneration. It has been suggested that
the immune system in neonatal mice is one of
the major differences between neonatal and
adult mice, which influences the cardiac
regenerative capacity (Aurora et al., 2014).
Therefore, in the next chapter we will focus on
the inflammatory pathways involved in cardiac
repair and regeneration following MI.
3.1.1 Pro-inflammatory cytokines
The pro-inflammatory cytokines TNF-α, IL-1,
IL-6 and IL-18 play a central role in cardiac
injury. Their influence on cardiac function is
dependent of their expression level, duration of
presence and surrounding inflammatory players
(Coggins and Rosenzweig, 2012), although, it
seems that detrimental effects are primarily
described. It has been shown that TNF-α
triggers apoptosis after MI, and in addition that
TNF-α in combination with NF-κB is
responsible for cardiac damage due to Ca2+
overload (Zhang et al., 2005). However, many
research has been conducted to the role of TNF
3. Inflammatory pathways
involved in post MI remodeling
Upon infarction, a complex and comprehensive
inflammatory response occurs. Influx and
- 5-
and NF-κB upon MI, and conclusions remain
contradictive (Coggins and Rosenzweig, 2012).
studies with promising effects (Srinivas et al.,
2009).
Clinical
trials
with
G-CSF
administration in human with cardiac problems
had different outcomes. Some reported no
significant beneficial outcome, others reported
increased left ventricular ejection fraction
(LVEF), but at least no significant adverse
effects has been published (Srinivas et al.,
2009). In most of the clinical trials, mobilization
of CD34+ stem cells from the bone morrow was
achieved. Therefore, absence of beneficial
cardiac outcome in the studies may be due to
timing, dose and lack of homing of the CD34+
stem cells (Srinivas et al., 2009).
IL-18, another pro-inflammatory cytokine, is
converted to its active form by caspase-1, a proapoptotic protein. IL-18 levels are elevated in
animal
models
and
patients
with
ischemia/reperfusion (I/R) and infarction
(Coggins and Rosenzweig, 2012). Moreover,
IL-18
was
indicative
of
promoting
inflammation, and degrading the extracellular
matrix via MMP induction (Reddy et al., 2010).
These results indicate an important role for IL18 during cardiac remodeling.
Pro-inflammatory cytokines are also released
upon activation of inflammasomes. The
inflammasome is a complex of different
proteins, required for the initiation of the
immune response after cardiac injury
(Kawaguchi et al., 2014). The inflammasome is
formed after I/R injury and upon its activation,
different pro-inflammatory cytokines like IL-1β
are secreted, which leads to inflammation and
subsequently
to
infarct
development,
myocardial fibrosis and dysfunction. Upon
genetic deletion of the major protein of the
inflammasome, apoptosis-associated speck-like
protein containing a caspase (ASC),
inflammation and cardiac injury were
significantly diminished (Kawaguchi et al.,
2014).
Administration of another anti-inflammatory
cytokine, IL-2, after I/R in the isolated rat heart
was shown to reduce infarct size (Cao et al.,
2004). Furthermore, injection of recombinant
human IL-2 two days after MI reduced scar
formation, improved left ventricular fractional
shortening, and enhanced angiogenesis in mice
(Bouchentouf et al., 2011).
A class of chemotactic cytokines is also known
as chemokines (chemotactic cytokines), which
function is attracting cells to the site of highest
expression of these chemokines. For example,
SDF-1α is a chemokine. Overexpression of
SDF-1α in mice was shown to enhance
migration and homing of cardiac stem cells via
its receptor, CXCR4, and Phosphatidylinositol4,5-bisphosphate 3-kinase (PI3K) (Wang et al.,
2012). Moreover, overexpression of SDF-1α
reduced infarct size significantly compared to
sham operated mice (Wang et al., 2012).
Taken together, these pro-inflammatory
cytokines indicate both beneficial and adverse
effects on cardiac remodeling. Although, it
seems that we can distinguish two inflammatory
phases: acute inflammation in particular is
likely essential for cardiac repair, but chronic
inflammation profoundly shows detrimental
effects on cardiac remodeling after MI (Coggins
and Rosenzweig, 2012).
Overall, it seems that overexpression of antiinflammatory cytokines after MI impairs
cardiac injury, and are not detrimental. For
example, G-CSF administration in patients with
heart disease is tested in different clinical trials
(Srinivas et al., 2009; Achilli et al., 2014), but
no groundbreaking results have been reported
yet. Further research is required to elucidate
possible therapeutic mechanisms.
3.1.2 Anti-inflammatory cytokines
The anti-inflammatory cytokines include
granulocyte colony stimulating factor (G-CSF),
IL-2 and Stromal Derived Factor (SDF)-1α
(also known as CXCL12) (Linde et al., 2007).
Upon binding of G-CSF, JAK/STAT is
activated, which protects against reverse
remodeling (Harada et al., 2005). In addition,
G-CSF has been researched in many animal
3.2 The JAK/STAT pathway
Stress signals from plasma membrane are
transduced by the Janus Kinase / signal
transducer and activator of transcription
(JAK/STAT) pathway to the nucleus (figure 2).
- 6-
In the heart, several ligands as ILs, interferons
and hematopoietic growth factors can bind to
the receptor, glycoprotein (gp) 130, and will
induce dimerization. Hence, JAK proteins are
phosphorylated and activated and will
phosphorylate the receptor, creating docking
sites for STAT proteins. Next, STAT proteins
become phosphorylated and will dimerize, and
upon dissociation from the receptor, STAT is
transported to the nucleus, regulating several
target genes. The proteins encoded by this genes
are involved in processes like cell growth,
differentiation, angiogenesis and extracellular
matrix composition (Snyder et al., 2008;
Boengler et al., 2008a). Besides expression of
these target genes, as a negative feedback loop,
suppressor of cytokine signaling 1 (SOCS1) and
SOCS3 are expressed. SOCS 1 and 3 inhibit
JAK by binding to JAK or its kinase active
domains (Yasukawa et al., 2012). In addition,
SRC homology 2 (SH2)-domain-containing
PTP2 (SHP2) can inhibit JAK and STAT, as
well as protein inhibitor of activated STAT
(PIAS) can inhibit STAT3 dimers, thereby
inactivating the pathway, and constraining
cytokine signaling (Boengler et al., 2008a).
reduced STAT1 phosphorylation in vitro.
Cardioprotective effects of tempol in vivo were
overruled by IFN5 induced phosphorylation of
STAT1 (McCormick et al., 2006). Thus, the
adverse effects of STAT1 on cardiomyocytes
seems to be related to anti-oxidants, although
the anti-oxidant theory of STAT1 may not
explain the complete mechanism behind
cardiomyocyte death.
3.2.1 STAT1 and STAT3 display opposite
effects
Upon I/R injury, STAT1 and STAT3 appear to
be essential for heart protection from ischemic
injury. Cardiomyocyte specific genetic deletion
of STAT3 increased apoptosis and infarct size
in mice after one hour ischemia and 24 hours
reperfusion (Hilfiker-Kleiner et al., 2004), but
not after 30 min ischemia and two hours
reperfusion (Boengler et al., 2008b). These
results indicate that STAT3 contributes to
cardiac protection after longer I/R injury
duration. In accordance to these results,
constitutive overexpression of STAT3 after I/R
injury resulted in reduced infarct size via the
ROS scavengers metallothionein 1 and 2
(Oshima et al., 2005).
3.2.2 Genetic deletion of SOCS3 inhibits
infarct size after MI
SOCS1 and SOCS3 are potent inhibitors of
JAK/STAT3 signaling via binding of JAK. In
this way, SOCS proteins tightly regulate the
duration and intensity of JAK/STAT3 signaling
in the heart. Cardiac specific SOCS3 knockout
mice exhibited reduced infarct size after acute
MI. In addition, apoptosis and fibrosis was
decreased in the infarcted myocardium (Oba et
al., 2012). These cardioprotective effects of
genetic SOCS3 deletion underscore the
protective mechanism of JAK/STAT3 signaling
after MI. Thus, activation of the JAK/STAT3
signaling pathway is beneficial for cardiac
STAT1 also seems to be involved in I/R injury.
Upon administration of the anti-oxidant of
green tea, Epigallocatechin-3-gallate, STAT1
phosphorylation
was
decreased
and
cardiomyocyte death was protected (Townsend
et al., 2004). Tempol, a free radical scavenger
- 7-
outcome
after
MI,
reducing
apoptosis,
adverse inflammation and
oxidative stress, and
enhancing angiogenesis
(Oba et al., 2012;
Yasuwaka et al., 2012).
3.3 The TLR pathway
Toll like receptors (TLRs)
are expressed in the heart
and have important roles
in the innate immune
response after infarction
(Arslan et al., 2011)
(figure 3). Predominantly
expressed TLRs in the
heart are TLR2, TLR3 and
TLR4 (Coggins
and
Rosenzweig, 2012). TLRs
are activated by danger
associated molecular patterns (DAMPs), such
as high mobility group B1 (Frangogiannis,
2014). Upon TLR activation, myeloiddifferentiation primary response gene 88
(MyD88) is recruited, which in turn can recruit
different proteins, including transforming
growth factor-beta-activated kinase (TAK1;
also known as mitogen-activated protein kinase
kinase kinase 7), IRAK1/IRAK4 and TNF
receptor associated factor (TRAF) 6 (Coggins
and Rosenzweig, 2012; Arslan et al., 2011).
TAK1 induces apoptosis via activation Jun Nterminal Kinases and the MAP kinase pathway.
Alternatively, TAK1, but also IRAK1, can
induce NF-κB translocation to the nucleus. This
process needs activation of IκB Kinase (IKK),
which phosphorylates the inhibitor of NF-κB
(IκB), leading to its ubiquitylation and
degradation, hence NF-κB is allowed to enter
the nucleus (Mann et al., 2011). NF-κB is a very
important player integrated in many different
inflammatory processes initiated after I/R injury
and therefore, as reviewed by Jones et al.
(2003), responsible for a variety of
physiological and pathophysiological states.
However, genetic blockade of NF-κB resulted
in reduced infarct size after I/R, indicating that
NF-κB is important for cellular death after
infarction (Jones et al., 2003).
3.4 Macrophages
As described so far, the immune system is an
important determinant in repair and survival of
processes after MI. In addition, tight regulation
of the immune response is crucial for
regeneration. This has been revealed by several
studies, including regeneration the hind limb of
the Xenopus (King et al., 2012) and
remyelination of the nerve system in mice
(Ruckh et al., 2012).
Recently, the influence of macrophages and
monocytes was investigated in neonatal mice
(P1) and compared to two week old mice (P14)
(Aurora et al., 2014). The researchers showed
that the inflammatory response differs
significantly between both groups, regarding
the kinetics and abundance of monocytes and
macrophages. Upon depletion of macrophages
in neonatal mice, regeneration and neoangiogenesis was decreased, and instead
fibrotic scar tissue was formed. The authors
propose, based on gene expression experiments,
that the difference in polarization of
macrophages is responsible for the cardiac
regeneration
(Aurora
et
al.,
2014).
Macrophages achieve these cardioprotective
and regenerative effects by debris clearance,
activation of stem/progenitor cells, immune
modulation and angiogenesis (Aurora and
- 8-
Olson,
2014).
Concise
research
towards
the
differences in inflammatory
response and macrophage
polarization between neonatal
and adult mammals may
provide new tools to minimize
detrimental effects of the
inflammatory response while
supporting
cardiac
regeneration after MI.
Now we have discussed
different important players
during inflammation after
infarction. The role of
cytokines, the JAK/STAT
pathway, the TLR-pathway
and macrophages has been
described. It has been
demonstrated that the injured
heart
requires
enhanced
survival and regeneration of
damaged cardiomyocytes, to
retain its contractile function,
and that the immune system is
of great importance in these
processes. A boost of the
acute inflammatory response
induced by cytokines can
activate
a
beneficial
inflammatory response and
induces the mobilization and homing of stem
cells. In addition, activation of the JAK/STAT3
pathway improves cardiomyocyte survival after
MI. Moreover, inhibition of the TLR pathwayinduced inflammatory response mainly shows
attenuation of cardiac remodeling. Furthermore,
proper activation of macrophages would
probably enhance cardiac function after MI.
Together, these mechanisms are interesting
targets to increase cardiac repair and
regeneration post MI. It is important to realize
that the acute inflammatory response
profoundly exhibits favorable cardiac outcome,
but chronic inflammation exacerbates the
pathological remodeling of the heart.
4. MicroRNA function in the
cardiac inflammatory response
MicroRNAs (miRNAs) are pivotal players in
many biological processes through regulating
mRNA abundance. Also in the heart, miRNAs
are critically involved in different physiological
and pathological mechanisms (Bostjancic et al.,
2010; Tijsen et al., 2012; Wang and Martin,
2014). Different miRNAs have been implicated
in the cardiac inflammatory response. In the
next chapter we will briefly describe de
biogenesis and mechanisms of action of
miRNAs. Hereafter, we discuss the role of
miRNAs in the inflammatory response after MI.
4.1 Introduction of microRNAs
MiRNAs are ~22 nucleotides long RNAs and
play an important role in regulation of gene
expression (Bartel et al., 2009). The biogenesis
- 9-
of miRNAs starts in the nucleus of a cell during
transcription of a miRNA gene by RNA
polymerase II/III (figure 4). A primary miRNA
is formed and cleaved by Drosha-DGCR8 (also
known as Pasha) in the nucleus. Transport of the
resulting precursor miRNA to the cytoplasm is
facilitated by Exportin-5. The precursor
miRNA is then cleaved by Dicer in complex
with the double stranded RNA-binding protein
(TRBP) and a mature microRNA duplex is
formed. Hence, the mature miRNA (without the
passenger strand) together with Argonaut 2
proteins can form an RNA Induced Silencing
Complex (RISC). RISC next will bind the
miRNA target mRNA through Watson-Crick
base pairing which results in mRNA target
cleavage, translational repression or mRNA
deadenylation (Bartel et al., 2009). The
passenger strand of the mature miRNA is
degraded. Besides the described canonical
pathway of miRNA processing, other pathways
have been described as well (Winter et al.,
2009). MiRNAs can act via several mechanisms
to target a certain biological process (Small and
Olson, 2011). These mechanisms include the
binding of one miRNA to several players of one
biological process. On the other hand, other
mechanisms are described where different
miRNAs target one player of a certain pathway.
Thus, miRNAs are important regulators of
mRNA abundance and can act independently or
cooperate with other miRNAs.
cardioprotective agent, to rat cardiomyocytes
resulted in increased expression of miR-21.
Furthermore, administration of sodium sulfide
in mice caused a strong decrease in
inflammasome formation and infarct size
(Toldo et al., 2014). Intriguingly, these
cardioprotective effects were lost upon genetic
deletion or pharmacological inhibition of miR21. These data suggest an important role in
inflammation upon MI of sodium sulfide, which
is mediated by miR-21. However, before
therapeutic approaches can be explored, more
research is required, because miR-21 is
involved in an opposite manner in other
mechanisms after cardiac injury. For instance,
inhibition of miR-21 resulted in prevention of
cardiac fibrosis and attenuation of cardiac
dysfunction (Thum et al., 2008), although this
statement was challenged by other researchers,
using a miR-21 knockout model, and observed
no prevention of cardiac remodeling (Patrick et
al., 2010). Nonetheless, miR-21 is important in
cardiac injury and reduces the inflammatory
response after MI.
4.2.2 MicroRNA-150 inhibits mobilization
and migration of precursor/stem cells via
CXCR4
The anti-inflammatory cytokine, SDF1-α, was
shown to be involved in migration and homing
of cardiac stem cells via its receptor CXCR4
(Wang et al., 2012). Different studies have
identified miR-150 as an important player in
this process (Tano et al., 2011; Rolland-Turner
et al., 2013). Mobilization and migration of
CXCR4pos bone marrow mono nuclear cells
(BM MNCs) was increased after infarction in
mice and miR-150 levels were decreased (Tano
et al., 2011). In vivo experiments on migration
and binding of BM MNCs reveal that CXCR4
is targeted by miR-150. The researchers
developed a model where they transplanted BM
MNCs lacking miR-150 in irradiated wild type
mice. It appeared that these mice had increased
numbers of MNCs in peripheral blood, after MI
(Tano et al., 2011). These results support that
miR-150 has a regulatory function in MNC
mobilization and migration via its target
CXCR4, upon MI.
4.2 MicroRNAs and cytokines
As described before, cytokines can be proinflammatory or anti-inflammatory and effects
on cardiac injury post MI are varying from
beneficial to detrimental, depending on dose
and time range of the secreted cytokines
(Prabhu, 2004). In addition, inflammasomes are
necessary for initiation of the inflammatory
response after cardiac injury, and mediate
secretion of several cytokines. We will discuss
the role of several miRNAs involved in
regulation of these cytokines.
4.2.1 MicroRNA-21 is involved in reducing
inflammasome formation and activation
The role of miR-21 in cardiac inflammation was
investigated by Toldo et al., (2014).
Administration of sodium sulfide, a
- 10 -
These findings were further supported by a
recent study investigating the role of adenosine
in migration of endothelial progenitor cells
(EPCs) (Rolland-Turner et al., 2013).
Adenosine increased CXCR4 expression and
decreased miR-150 in vitro under ischemic
conditions. The increase of CXCR4 by
adenosine was abolished after addition of premiR-150. In vivo treatment with adenosine after
MI stimulated EPC recruitment and
angiogenesis (Rolland-Turner et al., 2013).
Another miRNA involved in JAK/STAT
signaling is miR-155. It was shown that
hypertrophy in different mouse models with
cardiac pressure overload involved miR-155
mediated inhibition of SOCS1, the suppressor
of STAT. MiR-155 effects on hypertrophy were
mediated by macrophages, and therefore this
subject will be discussed in paragraph 4.5.
4.4 MicroRNAs and the TLR pathway
Activation of one of the pathways downstream
of TLR leads to the release of NF-κB to the
nucleus, thereby inducing transcription of proinflammatory genes (Arslan et al., 2011).
Several key players in this TLR pathway are
targeted by miR-146a, i.e. TRAF6 and IRAK-1
(Taganov et al., 2006). To elucidate the function
of miR-146a in TLR signaling in the heart after
MI, researchers induced miR-146a expression
by a lentiviral system, seven days prior to I/R
(Wang et al., 2013). Lentiviral expression of
miR-146 in the mouse heart led to significantly
reduced infarct size and prevented decreases in
ejection fraction compared to control.
Moreover, miR-146a suppressed expression of
IRAK1 and TRAF6 in the myocardium and
prevented NF-κB activation (Wang et al.,
2013). Recently, another paper presented
cardioprotective effects of miR-146a (Ibrahim
et al., 2014). Cardiosphere derived cell (CDC)
exosomes
were
administered
to
immunodeficient mice after MI, and it was
shown that scar mass was decreased, viable
mass was increased and heart function was
improved compared to mice treated with
exosomes derived from normal human dermal
fibroblasts (NHDF) (Ibrahim et al., 2014).
Intriguingly, a major difference was seen in the
miRNA content of CDC exosomes compared to
NHDF exosomes, miR-146a in particular was
markedly increased in CDC exosomes (Ibrahim
et al., 2014). Knockdown of miR-146a in CDC
exosomes impaired the beneficial effects after
cardiac injury and single miR-146a
administration partly mimicked the improved
cardiac outcome after MI (Ibrahim et al., 2014).
In addition, investigation of miR-146a, secreted
by endothelial cells in exosomes, revealed that
neonatal rat cardiomyocytes were able to take
up these exosomes containing miR-146a
(Halkein et al., 2013). These studies underscore
Taken together, miR-21 inhibits inflammasome
formation and activation and miR-150 targets
CXCR4
and
therefore
influences
precursor/stem cell mobilization and migration,
which are important steps in cardiac repair and
regeneration post MI. MiR-150 was also
identified in humans, since the miR-150 levels
were increased in patients with MI (Zidar et al.,
2011). Therefore, therapeutical potential of
miR-150 should be investigated to enhance
cardiac repair and regeneration in patients with
MI.
4.3 MicroRNAs and the JAK/STAT
pathway
Targets downstream of the JAK/STAT
pathways play an important role in cell survival,
differentiation, angiogenesis and extracellular
matrix composition (Snyder et al., 2008;
Boengler et al., 2008a). Several miRNAs are
described to be involved in regulation of the
JAK/STAT pathway.
To investigate the role of STAT3 in heart
failure, researchers created STAT3 knockout
mice, and it appeared that they spontaneously
developed heart failure. Besides that, cardiac
miR-199 levels were elevated. MiR-199 targets
different components of the ubiquitinproteasome system, which may explain the
pathophysiological symptoms in the hearts of
STAT3 knockout mice (Haghikia et al., 2011).
In addition, a similar pattern was found in
humans. Failing hearts exhibited low STAT3
levels, increased miR-199 levels and decreased
expression of ubiquitin conjugating enzymes
(Haghikia et al., 2011). These findings indicate
an important role of miR-199 in development of
cardiac remodeling, which is induced by the
key-player of JAK/STAT signaling, STAT3.
- 11 -
the importance of miR-146a in regulating the
TLR pathway after MI. Manipulation of miR146a abundance appears to be cardioprotective,
an effect that may be achieved in
cardiomyocytes. Moreover, these results
indicate that cardiac stem and progenitor cells
achieve their cardioprotective and proliferative
effects, at least partly, by involvement of
miRNAs.
was due to constraining chronic JAK/STAT
signaling (Heymans et al., 2013). However, the
macrophage-specific statement was challenged
by Seok et al., (2014), because they identified
miR-155, expressed by cardiomyocytes, as an
inducer of hypertrophy.
Taken together, it appears that miRNAs have a
key role in regulation of different inflammatory
mechanisms after MI. MiRNAs are involved in
cytokine signaling and for instance miR-150 is
critically involved in mobilization and
migration of stem/progenitor cells post MI.
Besides that, inhibition of STAT3 in the
JAK/STAT pathway shows an important role
for miR-199. Manipulation of the TLR pathway
by cardiosphere derived exosomes was also
regulated by a miRNA. Lastly, macrophages
induce expression of miR-155. Inhibition of this
miRNA appeared to improve cardiac function
after MI. Thus, enhancing cardiac regeneration
by manipulation of the immune system provides
an interesting area for novel research. Along
this line, miRNAs, key regulators of the
inflammatory response, deserve considerable
attention in the field of cardiac regenerative
research.
4.5 MicroRNAs and macrophages
As described before, macrophages are
important immune players, necessary for
cardiac regeneration after MI (Aurora et al.,
2014). In addition, it has been shown that
different miRNAs secreted by macrophages are
important in several pro- and anti-inflammatory
responses post MI.
It was shown that miR-155 knockout mice were
immunodeficient, and that miR-155 is required
for normal function of immune cells, including
macrophages (Rodriguez et al., 2007). The
function of miR-155 in heart failure was
investigated by by Heymans et al. (2013). The
researchers used two models for cardiac
pressure
overload,
Angiotensin
II
administration
and
transverse
aortic
constriction (TAC). Upon pressure overload,
genetic deletion or pharmacological inhibition
of miR-155 reduced cardiac inflammation,
hypertrophy and dysfunction (Heymans et al.,
2013). In absence of miR-155 the abundance of
macrophages seemed to skew towards type 2
macrophages, which might be cardioprotective.
These results indicate that miR-155 is required
for mobilization and infiltration of macrophages
in the pressure overloaded heart and may induce
an adverse inflammatory response. These
results were at least partly achieved by binding
of miR-155 to its target, SOCS1, which
suppresses STAT3. Thus, upon miR-155
inhibition, JAK/STAT3 signaling was
restrained. This effect was not mediated by
cardiomyocytes,
because
cardiomyocyte
specific genetic deletion of miR-155 had no
effect on cardiac hypertrophy or dysfunction.
Chronic activation of JAK/STAT3 promotes
inflammation and adverse cardiac outcome.
Therefore, it was proposed that the beneficial
effect of miR-155 inhibition in macrophages
5. Looking to the future
After myocardial infarction and reperfusion
millions of cardiomyocytes are lost, which
diminishes cardiac function, and eventually
causes death, thereby forming a major health
problem worldwide. Current therapies for heart
failure temporally restore heart function, but are
not adequate to replenish the lost cells and
completely restore cardiac pump function.
Hence, chronic heart failure often occurs after
several years after MI. Even heart
transplantation patients survive approximately
10 years. Therefore, new therapies are strongly
required to cure patients with cardiac injury.
In the last few years, several promising new
therapies were tested in clinical trials, with a
focus on stem/progenitor cells including the
CAUDUCEUS trial using cardiosphere derived
cells (Malliaras et al., 2014) and the STEMAMI trial, which involved G-CSF treatment
(Achilli et al., 2014). However, after several
years of clinical trials, no significant
- 12 -
improvement in cardiac function has been
observed.
different people may respond in diverse ways to
the drugs.
Although at a low level, it has been shown that
the human heart has potential for regeneration.
In addition, a crucial role of the immune system
during cardiac repair and regeneration has been
highlighted. Therefore, ways to enhance these
mechanisms could maybe potentiate cardiac
repair post MI. MiRNAs involved in regulating
of the inflammatory response upon MI are very
interesting targets for future research. Post MI
enhancement of regeneration by miRNAs
involving the immune system has already been
evidenced in the study of Ibrahim et al., (2014).
Exosomes of CDCs, containing miR-146a,
significantly increased the viable mass and
cardiac function in mice after MI.
In conclusion, heart disease is a major problem
worldwide. Loss of viable cardiomyocytes after
MI is neither prevented nor restored by current
therapies. Regeneration of the heart in humans
has been shown to exist in human, albeit at a
low level. Therefore, enhancement of the
regenerative response by modulation of the
inflammatory response seems promising.
MiRNAs have key roles in regulating the
inflammatory response after cardiac injury and
further research will elucidate the therapeutical
potential of these micromanagers of protein
output.
A potential intriguing way to manipulate the
cardiac inflammatory response to enhance heart
regeneration would be by modulating in vivo
levels of miRNAs (Olson, 2014). The miRNA
levels can be regulated by miRNA mimics to
increase, or anti-miRNAs to decrease the
abundance of active miRNAs. However, a
problem is the broad expression patterns of
miRNAs, and systemic delivery of miRNA
therapeutics could also influence miRNAs in
undesired organs or mechanisms, possibly
leading to adverse side effects. Therefore, more
localized delivery of miRNA mimics of antimiRNAs would be preferred. Recently, a paper
was published about the site directed and
sustained delivery of IGF by a hydrogel in the
heart upon MI (Koudstaal et al., 2014). To
address the issue of systemic delivery and
possible detrimental side effects of miRtherapeutics, a hydrogel could deliver the
solution, and provide site directed and sustained
drug delivery.
Xin M, Olson EN, Bassel-Duby R. Mending broken
hearts: cardiac development as a basis for adult heart
regeneration and repair. Nat Rev Mol Cell Biol. 2013.
14:529-41.
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