The Link between
Connexin 43 and
Fibrosis Regulation
In relation to Heart Diseases
Mark Schreuder
3/27/2012
Abstract
Cell-to-cell coupling, excitability of the
cardiomyocytes and the intercellular tissue
architecture are important determinant factors for
the proper electrical conduction through the
myocardium. However, cardiac remodeling is
often hallmarked by alterations in these factors.
Reduced connexin 43 (Cx43) expression and
enhanced fibrosis formation, which affect cell-tocell coupling and ECM architecture, are frequently
reported as a consequence of cardiac remodeling.
Recent studies provided evidence for a direct link
between reduced expression of Cx43 and enhanced
cardiac fibrosis. At present it is unknown how
Cx43 expression is able to regulate fibrosis
formation. This thesis will first summarize the
important regulators of Cx43 expression and
fibrosis. Hereafter, a new model is proposed for the
link between Cx43 remodeling and fibrosis
formation during cardiac stress and remodeling.
Introduction
Cardiopathologies like myocardial infarction
(MI), heart failure and pressure-overloaded
hearts are often hallmarked by cardiac
remodeling. This involves alterations in
cardiac function, structure, cell survival and
collagen synthesis and breakdown. These
alterations make the heart susceptible for
conduction defects that may lead to ventricular
arrhythmias. Arrhythmias and sudden cardiac
death remain one of the major unresolved
problems
in
the
clinic.
Annually,
approximately 250.000 to 300.000 individuals
in the United States die from the consequences
of sudden cardiac failure making it the most
common sudden death (Mirza 2012; Myerburg
1992; Stevenson 1997).
The electrical conduction through the
myocardium is mainly determined by the
balance between cell-to-cell coupling,
excitability of the cardiomyocytes and tissue
architecture of the heart (Bowers 2010; Jansen
2010). During cardiac remodeling these
factors can be altered, namely cell-to-cell
coupling and the architecture. (Karaahmet
2010; Kostin 2003). Cell-to-cell coupling is
mediated by specialized gap junctions
consisting of connexin proteins, mainly
connexin 43 (Cx43). Cardiac remodeling is
often accompanied by Cx43 downregulation,
resulting in reduced cell-to-cell coupling
(Severs 2004). As a consequence, the
electrical conduction through the heart may
become impaired which could make the heart
susceptible for life-threatening ventricular
arrhythmias (de Vuyst 2011; Severs 2008). An
alteration commonly present in the tissue
architecture of the heart is the development of
fibrosis. This occurs when cardiac fibroblasts
are stimulated, producing more collagens and
other extracellular matrix components.
Fibrosis formation may result in increased
myocardial stiffness and diastolic impairment
(de Jong 2011a). Moreover, cardiac fibrosis
may cause alterations in the electrical
conduction of the heart, making the heart
vulnerable to cardiac arrhythmias (de Jong
2011a).
Recent studies have provided evidence for a
link between Cx43 expression and fibrosis
formation (de Jong 2011b; Jansen 2012).
Instead of working independently from one
another, it seems that Cx43 expression can
influence fibrosis formation (de Jong 2011b;
Jansen 2012). However, at present it is unclear
how Cx43 expression may regulate fibrosis
formation.
This thesis will focus on the link between
reduced Cx43 expression levels and increased
cardiac fibrosis. First, the different regulators
of Cx43 and collagen/fibrosis expression will
be discussed after which a model is proposed
for the link between Cx43 and fibrosis.
Connexins
In the human heart, electrical impulses arising
spontaneously from the sinus node control the
heartbeat. From the sinus node the impulses are
rapidly propagated through the conduction
system towards the ventricles innervating the
cardiomyocytes resulting in cardiac contraction.
The rapid conduction of these electrical
impulses is mediated by special transmembrane
channels located in the intercalated discs,
specialized structures at the cell poles of the
cardiomyocytes. These channels are the socalled gap junctions and directly connect the
cytoplasms of the adjacent cells, providing
electrical coupling between cardiomyocytes
(Figure 1). Gap junctions are formed by two
hemichannels or connexons, composed of 6
connexin proteins in which each apposing cell
delivers a hemichannel (Teunissen 2004; Vuyst
2011).
Connexins, named after its molecular mass, are
part of a large family consisting of 20 gene
members in the mouse and 21 gene members in
the human genome (Jansen 2010; Söhl 2004).
Each connexin protein has a different expression
pattern and distribution. The main isoforms
expressed in the human heart are Cx43,
connexin 40 (Cx40) and connexin 45 (Cx45)
(Coppen 2003; Jansen 2010; Vozzi 1999).
Cx43 is the predominant connexin expressed in
the adult mammalian heart. It is extensively
expressed in the working myocardium, the atrial
and ventricular myocytes (Fontes 2012; Severs
2004). Cx43 expression is also found in the
conduction system, although only in limited
amounts in the distal parts (Fontes 2012; Jansen
2010). The sinus node mainly expresses Cx45
and small amounts of Cx40 but not Cx43 (Davis
1995; Jansen 2010). Furthermore, Cx40 and
Cx45 expression is found in other components
of the conduction system, including the
atrioventricular (AV) node, the bundle of His
and the bundle branches (Jansen 2010; Severs
2004). Cx40 and Cx45 are also detectable at low
levels in the atria and ventricles, with a higher
expression towards the atria (Davis 1995;
Severs 2004; Vozzi 1999). The expression
pattern of the individual connexin proteins
seems to vary greatly, suggesting different
Figure 1. Gap junction formation in
cardiomyocytes (de Vuyst 2011). Connexin
proteins are synthesized in the endoplasmic
reticulum (ER) and 6 connexins form a
hemichannel/connexon. Each cell provides a
hemichannel at the intercalated disc,
completing the formation of the gap junction
channel.
conduction properties and regulatory
mechanisms in the cardiac cells.
Hereafter the regulation of Cx43 will be
discussed. Alterations in Cx43 expression
are frequently found during cardiac
remodeling and seem to play a crucial role in
the induction of life-threatening ventricular
arrhythmias.
Nkx2.5
One of the earliest genes expressed in the
embryonic development of the heart is the
Nkx2.5 gene, regulating atrial septation and
formation of the left ventricle (Fodor 2005), but
continues to be expressed throughout the adult
life (Komuro 1993). Nkx2.5 is a homeobox
protein highly conserved in evolution (Kasahara
2003). This homeobox protein receives special
interest for its apparent restricted tissue
specificity (Turbay 1996).
To date, several mutations in the Nkx2.5 gene
have been found in humans (Benson 1999;
Dupays 2005; Schott 1998). These mutations
are associated with certain classes of congenital
heart diseases resulting in conduction defects
and ventricular dysfunction. Of these mutations,
four are single missense mutations in the
homeodomain resulting in a reduced DNA
binding ability but preserved homodimerization
capacity (Kasahara 2000). A similar non-DNA
binding Nkx2.5 mutant was found in Xenopus.
When mutant Nkx2.5 mRNA was injected in
Xenopus eight-cell stage embryos it would lead
to the expression of the mutant Nkx2.5 from the
earlier stages of cardiac development. The
expression of this mutant resulted in smaller
hearts or in some cases even no heart formation
(Grow 1998).
To further examine the function of Nkx2.5 in
the mammalian embryonic heart, transgenic
mice were generated expressing Nkx2.5(I183P),
the non-DNA binding mutant of Nkx2.5. In
contrast to the Xenopus, these mice were born
with an apparently normal heart development.
However, accumulation of the Nkx2.5(I183P)
protein resulted in progressive cardiac
conduction defects and heart failure (Kasahara
2001). The conduction defects could be
observed at 2 weeks of age shown by P-R
prolongations in the electrocardiogram (ECG).
These defects rapidly progressed into complete
AV-blocks at 4 weeks of age.
Moreover, mRNA and protein levels of Cx43 as
well as Cx40 were normal at birth but were
rapidly reduced after birth in Nkx2.5(I183P)
transgenic mice compared with wild-type (WT)
mice. Already at 1 week of age, a reduction in
ventricular Cx43 expression could be detected
which continued to decline until expression was
barely visible at 3 weeks of age (Kasahara
2001).
These results suggest an activating role of
Nkx2.5 in the regulation of Cx43. However,
expression of endogenous Nkx2.5 in
transgenic Nkx2.5(I183P) hearts was
elevated by 2-4-fold compared with WT
mice. In addition, Nkx2.5 downregulation
did not occur in Nkx2.5(I183P) mice after
birth (Kasahara 2001), while expression of
Nkx2.5 is normally gradually downregulated
directly after birth (Kasahara 2003).
Therefore, the phenotype of Nkx2.5(I183P)
mice may be influenced by the elevated
expression of endogenous Nkx2.5. In this
case, endogenous Nkx2.5 may be responsible
for the reduced Cx40 and Cx43 levels which
would suggest a repressive function of
Nkx2.5 rather than an activating function.
In a different study, transgenic mice were
generated expressing WT Nkx2.5 and a
carboxyl-terminus deletion mutant ΔC (a
putative transcriptional active mutant)
(Kasahara 2003). Mice from both groups
died within 4 months of age due to heart
failure and conduction abnormalities.
Isolated cardiomyocytes from WT and ΔC
mutant mice revealed altered sarcomere
structures and dramatically reduced Cx43
protein expression. Furthermore, adenoviral
mediated overexpression of Nkx2.5 in
cultured adult ventricular cardiomyocytes
showed that Cx43 mRNA expression was
downregulated at 16 hours after infection
and was further downregulated at 48 hours
(Kasahara 2003). Similar results were found
in another study, which showed a Cx43
downregulation at 24 hours (Teunissen
2003). These results indicate that Cx43 is
sensitive to Nkx2.5 upregulations and
therefore it may function as a transcriptional
repressor on the Cx43 gene.
Since different results are found in the
regulation of Cx43 by Nkx2.5 for the
embryonic and postnatal stadium, Nkx2.5
may have different functions in these stages.
Nkx2.5 may act as an activator in the
embryonic stage, while functioning as a
repressor in the postnatal period. The fact
that Nkx2.5 may have a distinct role in the
regulation of Cx43 may suggest that other
regulatory proteins are involved, influencing the
action of Nkx2.5.
family. Tbx2 and Tbx3 may stimulate the
repressive function of Nkx2.5 while Tbx5
may serve the opposite, activating Nkx2.5
function.
T-box family
The T-box (Tbx) family consists of a large
group of transcription factors known to be
important in the embryonic development of the
heart (Hoogaars 2007). These transcriptions
factors are named after their T-domain, a 180
amino acid residue DNA binding domain.
Besides its role in cardiac development, several
T-box factors are also implicated in connexin
regulation.
Transfection of Tbx2 in an osteoblast-like cell
line resulted in decreased levels of Cx43
protein, while transfection of an antisense Tbx2
revealed an increase in Cx43 expression (Borke
2003; Chen 2004), indicating that Tbx2 is a
negative regulator of Cx43 expression. A Tbx3
knockout and mutant study showed also a
repressive role of Tbx3 on Cx43 expression
since Tbx3 knockout and mutant mice expressed
Cx43 in the AV node while Cx43 is normally
not expressed in this region (Bakker 2008).
Another study demonstrated a role for the
homeobox proteins Msx1 and Msx2, which
functionally interacted with Tbx2 and Tbx3 to
repress Cx43 expression in a rat heart-derived
cell line (Boogerd 2008).
Several studies reported an opposite role for
Tbx5 as it activated several of the genes that
were downregulated by Tbx2 and Tbx3
(Boogerd 2008; Bruneau 2001; Greulich 2011).
One of these target genes of Tbx5 was Cx40
(Bruneau 2001; Moskowitz 2004). However, no
reports about Tbx5-dependent Cx43 expression
are currently known.
Interestingly, several studies showed that the Tbox factors Tbx2, Tbx3 and Tbx5 were able to
bind to Nkx2.5 (Boogerd 2009; Habets 2002;
Hiroi 2001). T-box proteins are expressed in
many different tissues regulating different
processes. Therefore, binding with tissue
specific proteins like the cardiac-specific
Nkx2.5 may be important to regulate tissue
specific processes. A possible mechanism in
determining the action of Nkx2.5 may be the
interaction with different members of the T-box
Adrenoreceptors
Adrenoreceptors
and
muscarinic
acetylcholine receptors play important roles
in the human heart regarding cardiac
physiology and pathophysiology. Heart
frequency, contractility and conduction
velocity are controlled by the autonomic
nervous system, consisting of the sympathic
and parasympathic nervous systems. These
systems are controlled by adrenoreceptors
and muscarinic acetylcholine receptors,
respectively (Brodde 1999). Adrenoreceptors
are G-protein coupled receptors and consist
of two different classes, the α-receptors and
β-receptors, each with several subtypes. In
the human heart, the β1 receptor is the main
expressed isoform while β2 and α1adrenoreceptors are expressed in a lesser
extent (Bristow 1988; Salameh 2011). There
are also other subtypes of receptors found in
the human heart (i.e. β3 and α2-receptors) but
these are expressed at very low levels
(Brodde 1999).
Previous studies have shown that chronic αadrenergic stimulation resulted in gap
junctional changes. Phenylephrine, an αadrenergic stimulant, activated protein kinase
C (PKC), which seemed to be responsible for
the reduced connexin coupling seen in
ventricular cardiomyocytes (De Mello 1997).
This PKC-dependent uncoupling was
thought to be mediated by specific PKC
isoforms. Activation of PKCε seemed to
reduce gap junctional conductance and Cx43
expression (Bao 2004; Imanaga 2004; Lin
2006), while PKCα was implicated in
increasing gap junctional coupling (Salameh
2005).
24-hour stimulation with phenylephrine in
adult rat cardiomyocytes in vitro and in vivo
resulted in increased Cx43 mRNA and
protein levels. The increased Cx43
expression was accompanied with enhanced
intercellular coupling, suggesting that
increased Cx43 expression levels resulted in
functional gap junctions (Salameh 2006).
Further research showed that p38 mitogenactivated protein kinase (MAPK), c-Jun Nterminal kinase (JNK) and extracellular signalregulated kinase (ERK) were phosphorylated
after phenylephrine stimulation, indicating the
activation of these factors. The increased
expression of Cx43 could be blocked by a PKC
inhibitor (Bisindolylmaleimide I), a mitogenactivated protein kinase kinase (MEK)-inhibitor
(PD98059) and partially by a MAPK inhibitor
(SB203580) (Salameh 2008). These results
indicate that α-adrenergic stimulation enhances
intercellular conduction by increasing Cx43
expression via MAPK-dependent pathways.
Interestingly, in this study c-fos was found to be
translocated to the nucleus after phosphorylation
of ERK and JNK. C-fos is able to form a
complex with Jun-family proteins to ultimately
form the transcription factor activator protein 1
(AP-1) (Herrera 1990; Lee 2012). It is known
that the promoter of Cx43 contains several
putative transcription binding sites, including
two AP-1 binding sites, a proximal AP-1 site
and a distal AP-1 site (Echetebu 1999;
Geimonen 1996; Salameh 2011). Of these two
AP-1 binding sites, the proximal site seems to
be the most important in the regulation of Cx43
expression. Deletion of the distal AP-1 site in
transfection assays with Jun and Fos family
members did not abrogate Cx43 expression,
while a 2-basepair mutation in the proximal AP1 site significantly reduced Cx43 expression
(Mitchell 2005). Together, these studies indicate
that AP-1 is activated after α-adrenergic
stimulation via ERK and JNK kinase pathways
and that AP-1 binding to the proximal AP-1
binding site of the Cx43 promoter may be an
important regulator of Cx43 expression.
Research on chronic β-adrenergic stimulation
revealed that rat cardiomyocytes had elevated
levels of Cx43 mRNA and protein in vitro and
in vivo. Cultured rat cardiomyocytes stimulated
24 hours with cyclic adenosine monophosphate
(cAMP), a downstream product of β-adrenergic
receptors revealed an upregulated Cx43
expression (Darrow 1996). This was supported
by findings in adult rat cardiomyocytes in vivo
after 24-hour infusion of a β-adrenergic
agonist. These cardiomyocytes revealed an
increase in mRNA and protein Cx43 levels,
which could be abolished by protein kinase
A (PKA) inhibition (Salameh 2006),
indicating that Cx43 upregulation during
chronic β-adrenergic stimulation was
regulated by PKA activation. Furthermore,
upregulation of Cx43 resulted in an
increased intercellular electrical coupling,
suggesting that the increased connexin
synthesis also leaded to an enhanced
functional electrical coupling between
cardiomyocytes.
Surprisingly, further studies revealed that
Cx43 upregulation in chronic β-adrenergic
stimulated cardiomyocytes was accompanied
by phosphorylation of p38 MAPK, ERK1/2
and JNK and translocation of transcription
factors AP-1, cAMP response elementbinding protein (CREB) and nuclear factor
of activated T-cells (NFAT) to the nucleus,
which are all coupled to the α-adrenergic
pathway. P38 MAPK and JNK activation
could be inhibited by the β-adrenergic
antagonist propranolol and by PKA
inhibition (Salameh 2009; Zheng 2000),
suggesting that p38 MAPK and JNK were
phosphorylated by PKA during β-adrenergic
stimulation. In addition, another study
showed increased ERK activation in βadrenergic stimulated cardiomyocytes, which
could be inhibited by cAMP and PKA
inhibitors (Zou 1999). Together these studies
indicate that both α-adrenergic and βadrenergic receptors can act on the same
pathways to induce similar effects like
upregulation of Cx43 expression.
As mentioned above chronic α- or βadrenergic stimulation during cardiac
hypertrophy enhances Cx43 expression and
intercellular electrical coupling. However, in
dilated cardiomyopathy and heart failure
with an ejection fraction below 40%, Cx43
was downregulated (Kostin 2003; Kostin
2004). This may indicate that in the earlystage of cardiac hypertrophy the heart tries to
compensate
by
upregulating
Cx43
expression. On the other hand, the
downregulation of Cx43 during dilated
cardiomyopathy and heart failure may reflect
the end-stage of heart diseases.
Although patients with early-stage hypertrophic
hearts often have enhanced Cx43 expression,
these hearts instead of having an improved
cardiac function, become more prone to reentrant arrhythmias. The newly synthesized
Cx43 proteins are not only incorporated in the
specialized intercalated discs but distribution of
Cx43 is seen over the whole cell membrane
(Emdad 2001; Kostin 2004; Salameh 2009;
Uzzaman 2000). This lateralization of Cx43
may give rise to transverse conduction through
the myocardium and may be sufficient to induce
re-entrant arrhythmias.
Renin-angiotensin system
The renin-angiotensin system (RAS) is an
important hormone system regulating many
physiological processes including blood
pressure and fluid balances. Juxtaglomerular
cells in the kidneys produce and secrete renin
into the circulation which then converts
angiotensinogen from the liver into angiotensin
I. This protein will be further converted into
angiotensin II (ANG II) by angiotensinconverting enzyme (ACE), a critical step in this
system, since ANG II is the major effector
protein of RAS (Fyhrquist 2008).
The enhanced activation of RAS is thought to be
one of the important mechanisms increasing the
risk for arrhythmias in various cardiopathological conditions. Enhanced levels of
ANG II have been associated with the increased
risk of arrhythmias (Kasi 2007) and might
explain the successful use of ACE inhibitors and
ANG II receptor blockers in the clinic
(Domanski 1999; Gavras 2000). Furthermore,
increased cardiac ANG II levels accompanied
by a downregulation of Cx43 expression have
been observed in patients with heart failure
(Imanaga 2010), which may suggest a role for
ANG II in the regulation of Cx43.
In order to investigate the role of ANG II in the
initiation of cardiac arrhythmias, a mouse model
was generated overexpressing cardiac-specific
ACE. These mice, called ACE 8/8 revealed a
100-fold increased expression of ACE (Xiao
2004). Due to this ACE overexpression, cardiac
ANG II levels were 4.3-fold higher
compared with WT mice. The mice
developed
cardiac
arrhythmias
that
correlated with the increased incidence of
sudden deaths. Furthermore, ACE 8/8 mice
revealed evidence of AV blocks and slow
atrial and ventricular conduction (Kasi
2007). Further analysis of these mice
revealed the downregulation of ventricular
Cx43 mRNA levels and atrial and ventricular
Cx43 protein levels.
Several studies reported the downregulation
of Cx43 due to increased levels of ANG II.
However, others described an upregulation
of Cx43 protein in response to ANG II. 24hour ANG II stimulation of rat neonatal
ventricular myocytes resulted in a 2-fold
upregulation of Cx43 protein expression
(Dodge 1998). Inhibition of ANG II by
losartan, an ANG II receptor antagonist,
blocked this response. Further support came
from studies in 9-week old spontaneously
hypertensive rats (SHR) which developed
left ventricular hypertrophy. Examination of
the hearts revealed an increased Cx43
expression that was characterized by Cx43
lateralization (Zhao 2008). Treatment with
losartan reduced left ventricular hypertrophy
as well as gap junction remodeling since
Cx43 expression was reduced and the
distribution became more confined to the cell
poles (Kansui 2004; Zhao 2008). Looking
more into the mechanism of ANG II
mediated Cx43 expression, nuclear factorkappa beta (NF-κB) was found to be
upregulated upon ANG II stimulation while
ANG II inhibition by losartan reduced NFκB levels (Zhao 2008). Interestingly, NF-κB
is a downstream factor of the p38-MAPK
pathway, which has been reported as an
important pathway in the regulation of Cx43
expression by adrenoreceptors, as mentioned
before. These results suggest that activation
of RAS in hypertrophy enhances Cx43
expression by ANG II which acts on the p38MAPK pathway activating NF-κB. NF-κB
may then translocate to the nucleus and
directly or indirectly increase Cx43
expression. However, further studies are
needed in order to unravel the exact role of NFκB in the regulation of Cx43.
The role of RAS in connexin remodeling is still
far from understood. An explanation for the
discrepancy of Cx43 expression response after
ANG II stimulation is that Cx43 remodeling
may be dependent on the stage of hypertrophy.
Herein, upward and downward remodeling of
Cx43 by ANG II may be modulated by an acute
and a chronic effect of cardiac ANG II (Imanaga
2010). In the early stages of hypertrophy,
connexin remodeling may be mediated by the
acute effects of ANG II resulting in the
upregulation of Cx43 in cardiomyocytes.
However, in later stages connexin remodeling
may be mediated by the chronic effects of ANG
II, now resulting in a downregulation of Cx43.
Although the acute and chronic effects of ANG
II may result in a different connexin remodeling
pattern, both upward and downward remodeling
of Cx43 could result in enhanced cardiac
arrhythmia susceptibility.
TGF- β
The type β transforming growth factor
superfamily (TGF-β) is an important family of
proteins regulating various cellular processes
such as tissue development and repair. To date,
35 members of this TGF-β superfamily have
been found in vertebrates (Ramos-Mondragón
2008). Besides its role in physiological
processes, TGF-β signaling is involved in
various human pathologies, including cardiac
arrhythmias and heart failure (RamosMondragón 2008).
One member of the TGF-β superfamily is TGFβ1 and is, together with TGF-β3, the main TGFβ expressed in cardiomyocytes (Long 1996).
The cardiac expression level of TGF-β1 is
upregulated after myocardial stress and during
several cardiomyopathies, including idiopathic
hypertrophic cardiomyopathy (Li 1997) and
dilated cardiomyopathy (Pauschinger 1999). In
fact, experimental models and human studies
showed that TGF-β1 was particularly
overexpressed during the transition from stable
hypertrophy to heart failure (Boluyt 1994;
Ramos-Mondragón 2008).
TGF-β signaling has been reported to affect
the electrophysiological properties of the
heart (Ramos-Mondragón 2008). Cultured
cardiomyocytes were stimulated with TGFβ1 for 48 hours. This stimulation resulted in
a downregulation of Cx43 accompanied with
a punctate and disorganized Cx43 expression
(Waghabi 2009), indicating that TGF-β
signaling affected Cx43 expression. This
result was reinforced by treatment with SB431542, a specific TGF-β1 inhibitor, which
caused an increased expression of Cx43.
However, another study reported the
upregulation of Cx43 in cultured neonatal rat
ventricular cardiomyocytes after 1-hour
stimulation with TGF-β1 or vascular
endothelial growth factor (VEGF), a
downstream factor of TGF-β (Pimentel
2002). This upregulation of Cx43 could be
blocked by a specific anti-VEGF antibody,
suggesting that stimulation of the TGF-β
pathway leaded to an upregulation of Cx43
rather than a downregulation.
The discrepancy in the regulation of Cx43
following TGF-β1 stimulation may be
explained
by
differences
in
the
concentrations used in the studies.
Stimulation with 2 ng/ml of TGF-β1 resulted
in the downregulation of Cx43 expression
(Waghabi 2009), whereas a 10 ng/ml
stimulation showed an upregulation of Cx43
expression (Pimentel 2002). On the other
hand, this difference may be explained by
the acute and chronic effects of TGF-β
stimulation. Acute stimulation of TGF-β may
lead to the upregulation of Cx43, as
represented by the 1-hour TGF-β1
stimulation (Pimentel 2002). In contrast,
chronic stimulation may result in the
downregulation of Cx43 expression, as
shown by the 48-hour stimulation with TGFβ1 (Waghabi 2009). However, further studies
are needed to fully elucidate the role of TGFβ pathways in the regulation of Cx43
expression and to determine whether
concentration differences or acute and
chronic stimulation with TGF-β1 differently
alter Cx43 expression.
miRNA
MicroRNA’s (miRNA’s) are endogenous small
non-coding RNA’s consisting of approximately
20 to 23 nucleotides. They are transcribed as
long transcripts and several modifications
ultimately result in mature miRNA molecules.
miRNA’s negatively regulate gene expression
via the inhibition of translation and mRNA
degradation (Feng 2011). This is achieved by
binding to almost complementary sequences in
the 3’-untranslated regions of target mRNA’s
(Yang 2007). Each miRNA can target multiple
mRNA molecules and each mRNA molecule
can be targeted by multiple miRNA’s, making a
complex
network of posttranscriptional
regulators. MiRNA’s have been shown to be
important players in the regulation of many
physiological but also pathophysiological
processes (Xu 2012). Among the miRNA’s,
several are expressed in a tissue-dependent
matter, including miR-1 which is specifically
expressed in cardiac and skeletal muscle cells.
In fact, miR-1 is the most abundant miRNA in
the human heart, representing 24% of the total
miRNA in cardiomyocytes (Feng 2011).
Various changes in miR-1 expression have been
detected in different heart diseases. Decreased
miR-1 levels were reported in patients with
aortic stenosis, dilated cardiomyopathy and
atrial fibrillation (Girmatsion 2009; Ikeda 2007).
In addition, miR-1 downregulation was reported
in murine models for cardiac hypertrophy and
heart failure (Sayed 2007). On the other hand, a
study reported that cardiac miR-1 levels were
elevated 2.8-fold in coronary artery disease
(CAD) patients compared with healthy controls
(Yang 2007). This result was confirmed in adult
rat hearts subjected to MI, which showed similar
elevated miR-1 levels. Furthermore, injection of
miR-1 in the infarcted myocardium promoted
the incidence of cardiac arrhythmia, while a
specific miR-1 inhibitor (AMO-1) repressed
arrhythmias (Yang 2007).
To investigate how miR-1 overexpression
resulted in enhanced arrhythmia susceptibility,
Cx43 levels were analyzed. When rats were
subjected to MI, cardiomyocytes showed a
lower expression of Cx43. MiR-1 transfection
resulted in a further reduction of Cx43
expression, whereas pretreatment with AMO-1
rescued Cx43 expression (Yang 2007).
These results were supported by another
study which reported upregulated levels of
miR-1 accompanied by reduced Cx43
expression levels in a viral myocarditis
model (Xu 2012). Interestingly, Cx43
mRNA contained two independent binding
sites for miR-1 in the 3’-untranslated region
of which both were needed to inhibit Cx43
translation effectively (Anderson 2006; Yang
2007).
In a recent study, administration of the βblocker propranolol did reduce the incidence
of cardiac arrhythmias in a rat model of MI
(Lu 2009). Looking more into the
mechanism, the study reported that
propranolol administration dose-dependently
inhibited the upregulation of miR-1 after MI
and thereby rescued Cx43 expression. These
results suggested a role for the β-adrenergic
system in upregulating miR-1 expression
after MI. To support this idea, stimulation of
cultured ventricular myocytes with forskolin,
a cAMP activator, greatly enhanced miR-1
expression (Lu 2009). The PKA inhibitor
H89 abolished this upregulated miR-1
expression, showing that the β-adrenergiccAMP-PKA pathway was involved in the
regulation of miR-1 and possibly played a
role in the upregulation of miR-1 expression
after MI.
This pathway is commonly
involved in cardiopathologies, therefore it
would be interesting to see whether βadrenergic stimulation also stimulates miR-1
expression in heart diseases like hypertrophy
and heart failure.
Besides miR-1, miR-206 may also play a
role in the regulation of Cx43. It has been
shown that Cx43 contained two independent
binding sites for miR-206 (Anderson 2006).
Furthermore this study showed that miR-206
was able to inhibit Cx43 protein expression
in skeletal myoblasts, while Cx43 mRNA
levels remained unchanged. Anti-miR-206
treatment rescued Cx43 expression almost
completely. Cardiac-specific overexpression
of E2F6, a member of the E2F family, in
transgenic
mice
induced
dilated
cardiomyopathy
and
heart
failure
(Westendorp 2012). These mice revealed a
markedly reduction in Cx43 protein expression
and a 10-fold upregulation of miR-206
compared with WT mice. Interestingly, the
MAPK-pathway was significantly activated in
these transgenic mice. Since ERK is able to
influence Cx43 as well as miR-206 expression,
(Westendorp 2012) this suggests that the
activation of ERK downregulates Cx43
expression and upregulates miR-206 expression.
MiR-206 may in turn inhibit Cx43 translation,
resulting in the reduction of Cx43 protein.
PPARγ
Diseases like diabetes and obesity affect cardiac
energy metabolism and increase the lipid
accumulation in cardiomyocytes. This has been
frequently associated with mechanical and
electrical alterations in the heart and sudden
cardiac death (Morrow 2011). The peroxisome
proliferator–activated receptor-γ (PPARγ) is a
transcription
factor
regulating
lipid
accumulation and glucose metabolism and is
normally expressed at low levels in the heart.
However, PPARγ was frequently upregulated in
the human heart in patients with the metabolic
syndrome and cardiac hypertrophy (Krishnan
2009; Marfella 2009). In order to investigate the
role of metabolic abnormalities on cardiac
arrhythmias, transgenic mice with cardiacspecific overexpression of PPARγ were studied.
These mice showed abnormal high levels of
accumulated lipids in cardiomyocytes and
developed dilated cardiomyopathy with
spontaneous
ventricular
arrhythmias,
resulting in an increased incidence of sudden
cardiac deaths (Morrow 2011). Furthermore,
overexpression of PPARγ in these mice
resulted in a 70% reduction of Cx43 mRNA
levels and an 85% reduction of Cx43 protein
levels. Therefore, this study suggested a role
for PPARγ in Cx43 downregulation in
patients with the metabolic syndrome or
cardiac hypertrophy.
Altogether, several factors are involved in
the regulation of Cx43 (Figure 2). Nkx2.5 is
one of the transcription factors influencing
Cx43 expression, which may be modulated
by T-box factors. During cardiac remodeling,
RAS and adrenoreceptors are activated
which stimulate the translocation of CREB,
NFAT, NF-κB and AP-1 to the nucleus via a
MAPK/ERK
pathway.
The
βadrenoreceptors are also involved in the
activation of miR-1, which together with
miR-206 targets Cx43 translation. Finally,
TGF-β and PPARγ may also be able to
modulate Cx43 expression, although the
mechanisms are not known.
Figure 2. Schematic figure representing the regulation of Cx43 expression in cardiomyocytes.
Due to cardiac stress, several pathways and factors are activated including Nkx2.5 and the T-box
factors Tbx2, Tbx3 and Tbx5 (Tbx2/3/5). Nkx2.5 translocates to the nucleus while the T-box factors
modulate Nkx2.5 function, either to upregulate or downregulate Cx43. Furthermore, several receptors
are activated like the ANG II-receptor (AT-R) and the α- and β-adrenoreceptors (α-AR and β-AR,
respectively). These receptors activate p38-MAPK and ERK. ERK allows the translocation of the
transcription factors CREB, NFAT and NF-κB to the nucleus and activates JNK, which together with
C-Fos forms the transcription factor AP-1. MiR-1 and miR-206 are negative regulators of Cx43
translation whereby miR-1 is activated by β-AR. Finally, TGF-β and PPARγ play a role in the
regulation of Cx43 although the mechanisms are not known.
Fibrosis
The human heart consists mainly of
cardiomyocytes, the contractile cells giving the
heart its pumping function. However, besides
cardiomyocytes, the heart consists of an
extracellular matrix (ECM) network which
provides mechanical support to the working
cardiomyocytes and influences signaling
pathways regulating cell behavior (Lukashev
1998). ECM is formed by several different
components. It includes the interstitial fluid and
several proteins including proteoglycans and
fibrillar collagen that are produced and secreted
by cardiac fibroblasts (de Jong 2011a). Collagen
is the main ECM component and the most
abundant protein expressed in humans
comprising 30% of the total human proteins
(Kavitha 2008). It has an important role in
ensuring that the heart maintains its original
shape and provides elastic strength to the
cardiomyocytes.
The regulation of collagen is a highly dynamic
process involving the newly synthesis and
secretion of collagen fibers and the breakdown
of old collagen fibers. To date, about 27
different types of collagen are known, mostly
consisting of collagen types I, II and III (Deyl
2003). In the human heart, collagen type I is the
most abundant type of collagen comprising 85%
of the total collagen fibers (de Jong 2011a).
Collagen type III comprises 11% of the ECM.
In the heart, the mRNA molecules of these
collagens are only found in cardiac fibroblasts,
indicating that only this type of cells are able to
synthesize collagen proteins (Eghbali 1988).
Collagen is synthesized by the ER as preprocollagen and can be turned into mature
collagen fibers by several posttranscriptional
modifications (Figure 3). Collagen molecules
are composed of long protein chains with a
specific triplet repeat. Due to this triplet motif,
collagen chains can bind to each other forming a
triple helix structure. This structure is known as
procollagen and is secreted into the ECM.
Procollagen contains two prodomains, one at the
C-terminus and one at the N-terminus. In the
ECM, these prodomains are cleaved off by
specialized proteinases to form mature
collagen molecules, which can be integrated
in the ECM to form long collagen fibers
(Kavitha 2008). Furthermore, the different
types of collagen fibers interact with each
other as well as with other proteins resulting
in a highly complex and organized ECM.
Just like the newly synthesis of collagen
fibers, collagen breakdown is an important
process for the homeostasis of the cardiac
ECM. This is regulated by different
proteases, mainly the matrix metalloproteinases (MMP) (Rodriguez-Feo 2005).
The MMP’s are a large family of zincdependent enzymes that are synthesized by
cardiac fibroblasts and leukocytes (Rizas
2009). MMP’s cleave their substrate in two
parts which can then be further fragmented
by a different subset of MMPs. Of the MMP
family, MMP-1, MMP-8 and MMP-13 are
highly specific for the turnover of collagen
types I and II, therefore they are also known
as collagenases (de Jong 2011a).
One of the characteristics of cardiac
remodeling is an altered ECM homeostasis
and collagen turnover. Collagen deposition
increases, resulting in cardiac fibrosis. This
process may stimulate the progression to
heart failure and cardiac arrhythmias (Edgley
2012;
Seferovic
2006)
since
the
accumulation of collagen may reduce
contractility, increase stiffness and cause
conduction abnormalities in the heart. The
formation of fibrosis can be triggered by
different processes like pressure and volume
overload, inflammation and MI. Due to these
triggers, cardiac fibroblasts start proliferating
and differentiate into myofibroblasts
(Okayama 2012). Myofibroblasts are
important cells in physiological wound
healing, producing cytokines, growth factors
and ECM components more effectively
compared
with
normal
fibroblasts.
Nowadays myofibroblasts appear also to be
responsible for the development of fibrosis
in heart diseases (Okayama 2012; Petrov
2002).
Figure 3. Schematic figure representing the development of collagen fibers (Lodish 2000).
Collagen proteins are synthesized by the ER as pre-pro-collagens. Several posttranscriptional
modifications in the ER and Golgi turn pre-pro-collagen into pro-collagen. Pro-collagen is
then secreted and turned into mature collagen proteins. These will be integrated in the
collagen fibers, creating a complex network of collagen fibers in the ECM.
Renin-angiotensin system
Besides its role in cardiac hypertrophy and
conduction abnormalities, RAS may play an
important role in the development of fibrosis in
heart diseases. The main effector molecule of
RAS, ANG II, can act on cardiomyocytes as
well as on cardiac fibroblasts. Whereas ANG II
induces hypertrophy in cardiomyocytes, it
induces hyperplasia and ECM synthesis in
cardiac fibroblasts (Zou 1998).
In multiple studies, adult rat cardiac fibroblasts
were stimulated with either ANG II or
aldosterone, another RAS effector molecule. 24hour stimulation revealed increased collagen
mRNA and protein expression (Brilla 1994;
Zhou 1996). This stimulation could be
abolished by a specific ANG II or
aldosterone receptor antagonist, suggesting a
direct role of RAS on fibrosis formation.
Further evidence was provided by in vivo
studies. Transgenic mice with cardiac
specific overexpression of ANG II showed a
10-fold higher expression of ANG II
compared with WT mice. Under basal
conditions, these mice revealed enhanced
collagen depositions in the heart. However,
they do not show any evidence for
hypertrophy, therefore the effects on the
interstitial fibrosis may be attributed to the ANG
II overexpression rather than a consequence of
cardiac hypertrophy (Xu 2007). When subjected
to MI or long-term hypertension, these
transgenic mice developed severe interstitial
fibrosis compared with WT mice (Xu 2007; Xu
2010).
In another study an aged mouse model (52
weeks) was chronically treated (36 weeks) with
two types of RAS inhibitors, an aldosterone
antagonist and an ANG II antagonist. This aged
model was characterized by enhanced interstitial
and replacement fibrosis resulting in agedinduced arrhythmias (Stein 2010). In this model,
chronic RAS inhibition revealed a significantly
reduced interstitial and replacement fibrosis
(Stein 2010). Furthermore, the incidence of
cardiac arrhythmias was reduced in treated mice
directly correlating with the reduction in cardiac
fibrosis.
Besides stimulating the ECM production, there
is evidence for a role of RAS in the regulation
of ECM breakdown. Stimulation of cultured
adult rat cardiac fibroblasts with ANG II
revealed a reduced collagenase activity (Brilla
1994; Zhou 1996), while other studies showed
the downregulation of MMP-1, one of the
collagenases, after ANG II stimulation (Lijnen
2006; Lijnen 2008). Furthermore, TIMP-1 and
TIMP-2, inhibitors of MMP’s, were found to be
upregulated after ANG II stimulation (Lijnen
2006; Lijnen 2008). These results may suggest
that RAS inhibits collagen breakdown by
downregulating
MMP
expression
and
upregulating MMP inhibitors. The reduced
collagen degradation would favor collagen
synthesis and may therefore contribute to the
formation of fibrosis.
TGF- β
TGF-β1 is thought to be one of the crucial
factors in the process of cardiac hypertrophy
and the progression to heart failure by initiating
fibrosis formation (Border 1994; Dean 2005).
In the last decades, studies have shown that
myocardial TGF-β is markedly and consistently
upregulated in various cardiopathologies in both
animal models and humans (Bujak 2007;
Debaczewski 2011). One study showed that
higher levels of TGF-β1 were positively
correlated with an increased risk for heart
failure in humans (Glazer 2012). Another
clinical study showed that fibrosis was a
crucial process in the transition to heart
failure and was associated with increased
levels of TGF-β1 (Hein 2003). TGF-β1 was
mainly upregulated in the transition phase
from stable hypertrophy to heart failure,
around the same time as the upregulation of
collagen (Boluyt 1994). Therefore, TGF-β1
may be, together with collagen, one of the
few markers discriminating between stable
and unstable hypertrophy.
TGF-β1 is known as a potent cytokine
regulating collagen production in fibroblasts.
In vitro stimulation with TGF-β1 as well as
adenovirus-mediated overexpression of
TGF-β1 in cardiac fibroblasts revealed the
enhanced expression of collagen type I and
III (Eghbali 1991; Ignotz 1986; Villareal
1996). Inhibiting TGF-β1 stimulation by
specific
inhibitors
abolished
this
upregulation. TGF-β1 may also be an
important factor in the transition from
fibroblasts to myofibroblasts as it induces the
expression of α-smooth muscle actin (αSMA), a marker for myofibroblasts (Jester
1999; Tomasek 2002). This transition is a
crucial process in connective tissue
remodeling during normal and pathological
wound healing (Tomasek 2002) since
myofibroblasts produce collagen and other
ECM components more effectively than
normal fibroblasts (Petrov 2002). Moreover,
TGF-β1 seems to induce its own expression
in myofibroblasts, initiating an autocrine
loop which further stimulates fibrosis
formation (Rosenkranz 2004). Blocking the
TGF-β1 pathway reduced the amount of
fibrosis as well as the transition to
myofibroblasts (Okayama 2012). Therefore,
myofibroblasts are thought to be responsible
for various fibrotic pathologies initiated by
TGF-β1 stimulation (Wynn 2008).
Also in vivo studies revealed an important
role for TGF-β1 in the process of cardiac
fibrosis. Transgenic mice overexpressing
TGF-β1 developed cardiac hypertrophy
accompanied with enhanced interstitial
fibrosis (Rosenkranz 2002). Blocking TGF-β1 in
a MI model resulted in a markedly reduction of
interstitial fibrosis and a reduced collagen
expression in cardiac fibroblasts (Ellmers 2008;
Kuwahara 2002; Sakata 2008).
TGF-β signaling is mediated by TGF-β
receptors. These are serine/threonine receptors
that dimerise and exist in several isoforms.
When the receptor is activated, a signaling
cascade is initiated involving Smad proteins.
These Smad proteins are phosphorylated and
associate with Co-Smad to translocate to the
nucleus where they act as transcription factors.
In the nucleus, the interaction with other
transcription factors, activators and repressors
determine gene expression (Rosenkranz 2004).
In a recent study, the effects of TGF-β signaling
were investigated in a mouse model of heart
failure with progressive myocardial fibrosis.
This study revealed that increased levels of
TGF-β resulted in enhanced cardiac fibrosis and
nuclear translocation of Smad 2 and Smad 3. In
addition, treatment with a TGF-β receptor
antagonist markedly decreased fibrosis as well
as the translocation of these transcription factors
(Sakata 2008). These results provide evidence
that increased TGF-β signaling activates Smad
proteins including Smad 2 and Smad 3, which
translocate to the nucleus and may directly or
indirectly influence myocardial fibrosis
formation.
Interestingly, recent studies have provided
evidence that TGF-β and ANG II are able to act
together in an integrated signaling pathway
promoting cardiac remodeling and fibrosis
formation (Leask 2010). Cultured cardiac
fibroblasts and myofibroblasts treated with
ANG II revealed an upregulation of TGF-β
mRNA levels while losartan abolished this
effect (Campbell 1997; Lee 1995). ANG II also
increased collagen type I expression in cardiac
fibroblasts whereas a TGF-β inhibitor attenuated
this effect (Gao 2009), indicating that ANG II
upregulated collagen type I via TGF-β.
Furthermore, blocking ANG II with an ANG II
receptor antagonist reduced the levels of TGF-β
and fibrosis formation in a rat model for cardiac
fibrosis (Tomita 1998). Together, these studies
provided evidence for the collaboration of ANG
II and TGF-β in the process of cardiac
remodeling rather than acting independently
of each other. In fact, TGF-β may be a
downstream factor of ANG II.
Besides its role in the synthesis of ECM
components, TGF-β1 may also have a role in
the regulation of ECM breakdown factors
such as MMP’s. However, the role of TGFβ1 in the regulation of MMP expression is not
well understood since some studies report an
upregulation of MMPs, whereas others
report downregulated levels of MMPs
(Ogawa 2004; Risinger 2010; Yan 2007).
Endothelin
Endothelins (ET) are important proteins in
the regulation of the vascular tone and blood
pressure but are also proposed to have
mitogenic properties. The endothelin family
consists of three isoforms, namely ET-1, ET2 and ET-3. Of these isoforms, ET-1 is the
isoform mostly expressed in humans.
Endothelins are mainly secreted by
endothelial cells, however, it is shown that
cardiac fibroblasts also express endothelin
and endothelin receptors (Leask 2010).
In vitro studies revealed a role for ET-1 in
the process of ECM remodeling. Stimulation
of adult rat and human cardiac fibroblasts
with ET-1 promoted the synthesis of
collagen types I and III via its receptors ETA
and ETB (Guarda 1993; Hafizi 2004).
Furthermore, ET-1 stimulation caused a 5.8fold reduction in collagenase activity
(Guarda 1993).
Mice with a specific deficiency of ET-1 in
the vascular endothelium were subjected to
cardiac hypertrophy and fibrosis by ANG II
administration. These mice had reduced
cardiac fibrosis and collagen expression
levels (Adiarto 2012). In another study,
chronic hypertensive rats were characterized
by enhanced cardiac fibrosis, which could be
attenuated by treatment with bosentan, an
endothelin receptor antagonist (Karam
1996).
Treatment with a non-specific endothelin
receptor antagonist markedly decreased
infarct size in patients with MI, which was
thought to be mediated by the regulation of
cardiac myofibroblasts (Katwa 2003). Further
analysis showed that these myofibroblasts had
upregulated levels of collagen mediated by ET-1
and its receptors (Katwa 2003).
Interestingly, endothelins and TGF-β are often
upregulated in the same conditions and diseases
(Clozel 2005). Stimulation with TGF-β strongly
increased ET-1 expression levels in endothelial
cells (Rodriguez-Pascual 2003) but also in
cardiac fibroblasts (Clozel 2005). Furthermore,
two DNA elements in the human promoter of
the ET-1 gene were identified as being regulated
by TGF-β (Rodriguez-Pascual 2003). At last, a
different study demonstrated that bosentan is
able to prevent the profibrotic effects of TGF-β,
including the upregulation of collagen and
fibronectin (Katwa 2003). Together, these
studies indicate a direct relationship between
TGF-β and endothelin and suggest ET-1 as a
possible downstream factor of TGF-β.
Connective tissue growth factor
The connective tissue growth factor (CTGF) is a
matricellular protein and member of the CCN
family. It modifies the signaling responses in
many biological processes, including cell
adhesion, proliferation, angiogenesis and
migration (Leask 2008). CTGF is a surrogate
marker for activated fibroblasts during wound
healing and fibrosis and is a factor consistently
activated in cardiac remodeling (Leask 2010).
CTGF has often been associated with TGF-β as
a
co-factor
or
downstream
protein.
Subcutaneous injection of TGF-β in mice
resulted in the formation of granulation tissue
which disappeared after several days, whereas
an injection with CTGF only resulted in slight
granulation (Mori 1999). However, injection of
both cytokines leaded to long-term fibrotic
tissue, suggesting that both growth factors
cooperated to form fibrotic tissue. In this case,
TGF-β may have initiated the fibrosis formation
while CTGF was needed for the maintenance of
fibrosis (Mori 1999). So CTGF may be a
modulating factor, maximizing the response of
pro-fibrotic cytokines like TGF-β. This was also
demonstrated in a study in embryonic mouse
fibroblasts. Compared with WT fibroblasts,
CTGF deficient fibroblasts revealed a reduced
expression of pro-fibrotic genes, including
collagen type I and α-SMA after TGF-β
stimulation (Kennedy 2007). Furthermore,
anti-CTGF treatment with oligonucleotides
significantly reduced collagen type I
expression in a liver fibrosis mouse model
(Uchio 2004). Interestingly, also the TGF-β
expression level was reduced by this
treatment, suggesting that CTGF could
induce TGF-β expression and was thereby
able to form an autocrine loop. A TGF-β
autocrine loop in cardiac fibroblasts has
already been suggested (Lee 1995) and now
CTGF seems to be involved in this loop.
Various studies have provided evidence for
CTGF acting as a downstream factor of
TGF-β. Moreover, studies have suggested
that ET-1 expression was induced by TGF-β
(Clozel 2005; Rodriguez-Pascual 2003). In
turn, TGF-β expression has been associated
with ANG II activation (Leask 2010), while
ET-1 was able to induce CTGF expression
(Fonseca 2011). Altogether, these results
suggest that the pro-fibrotic factors work
together in a common pathway existing of
ANG II  TGF-β  ET-1  CTGF.
Adrenoreceptors
In various large clinical trials, β-blocker
treatment showed a favorable effect on
morbidity and mortality in patients with
heart failure (Kubon 2011). Although most
studies focused on the hypertrophic effects,
several linked the β-adrenergic system with
the regulation of cardiac fibrosis.
In a recent study, β1- and β2-adrenoreceptor
knockout and WT mice were subjected to
aortic banding to induce ventricular
remodeling. WT mice responded with
cardiac hypertrophy, upregulation of several
inflammatory
cytokines
and,
more
importantly, upregulation of fibrogenic
growth factors and severe cardiac fibrosis
(Kiriazis 2008). In contrast, the hypertrophic
response was only partially observed in the
β1- and β2-adrenoreceptor knockout mice.
Furthermore, knockout mice did not show
any upregulation of inflammatory cytokines
and fibrogenic growth factors and interstitial
fibrosis was virtually absent.
Transgenic
mice
overexpressing
β1 adrenoreceptor revealed prominent interstitial
fibrosis throughout the left ventricle with a 4.8fold increase in collagen content (Engelhardt
2002; Seeland 2007). Blocking the β-adrenergic
pathway abolished this effect. Furthermore,
chronic isoproterenol administration to Wistar
rats, a non-selective beta-adrenergic agonist,
markedly enhanced cardiac fibrosis (Brouri
2004). Subsequent treatment with a β1adrenoreceptor antagonist or pretreatment with a
β2-agonist,
to
induce
β2-adrenoreceptor
desentitization, significantly reduced ventricular
interstitial fibrosis. The effects of these β1- and
β2-treatments were comparable, showing that
both adrenoreceptors were equally involved in
the process of fibrosis.
Further evidence was provided by a study on
male rats subjected to aortic stenosis, which
presented with higher expression levels of
fibronectin, collagen types I and III and
increased fibroblast proliferation rate compared
with WT mice (Grimm 2001). Carvidilol
administration, a β-blocker, dose-dependently
reduced this proliferation rate accompanied by
the downregulation of fibronectin and collagen.
In a different study, β1-adrenoreceptor
overexpressing mice developed compensated
ventricular hypertrophy (5 months) that
progressed to ventricular dilatation and
dysfunction (12 months) correlated with
progressive interstitial fibrosis. Expression
analysis revealed that compared with WT mice,
collagen types I and III mRNA and protein
expression were increased 4-fold in the
compensated state and 17-fold in the dilated
state (Seeland 2007). Whereas the structure of
the collagen network was still organized in mice
with compensated hypertrophy, mice with
dilated hypertrophy revealed a more progressive
and heterogeneous distribution of collagen
fibers compared with WT mice.
There is currently not much known about the
involvement of the β-adrenergic system in the
regulation of collagen breakdown. However,
increased expression levels of proMMP-2,
MMP-2, TIMP-2, the membrane bound MT1MMP and the collagenases MMP-1 and MMP-
13 were observed in compensated
hypertrophic mice (Seeland 2007). However,
in dilated hypertrophic mice, MMP-1 and
MMP-13 expression levels were reduced
compared with compensated hypertrophic
mice whereas proMMP-2, MMP-2, TIMP-2
and MT1-MMP expression levels were
further increased. While reduced expression
of MMP-1 and MMP-13 suggests an
involvement in the formation of interstitial
fibrosis, the activation of MMP-2 seems
contradictory. However, MMP-2 degrades
gelatins and fibronectin thus promoting the
cleavage of fibronectins attached to β1integrins at places that connect cardiac cells
with the ECM (Seeland 2007). This cleavage
could impair β1-integrin signaling in cardiac
cells. Integrin signaling is important in
maintaining the myocardial structure and
providing
supportive
strength
to
cardiomyocytes. Furthermore, it is involved
in cell survival and proliferation (Dedhar
1999). Interestingly, in the transgenic mice
overexpressing
β1-adrenoreceptor,
β1 integrin signaling was impaired suggesting a
role for MMP-2 (Seeland 2007). Impaired
β1-integrin signaling may lead to cardiac cell
death and replacement fibrosis, facilitating
the transition from compensated to
decompensated hypertrophy.
TIMP-2 is known as an inhibitor of MMP-2.
However, TIMP-2 is also able to form a
complex
with
pro-MMP-2
thereby
facilitating the binding of pro-MMP-2 to
MT1-MMP, which activates both MMP’s
(Hernandez-Barrantes 2000). The exact role
of these proteins is currently not known but
they may be involved in the α2β1-integrin
signaling (Zigrino 2001). MMP-1 and MMP13 are known for their specific degradation
of collagen types I and III (de Jong 2011a).
The fact that these MMP’s were upregulated
during compensated hypertrophy but
downregulated during dilated hypertrophy
may suggest a role for these enzymes in the
transition
from
compensated
to
decompensated
hypertrophy.
Fibrosis
formation has already been associated with
this transition (Edgley 2012). Thus, the
downregulation of these MMP’s may lead to
a reduced collagen breakdown which together
with an increased collagen synthesis may result
in the formation of interstitial fibrosis, thereby
triggering the transition of hypertrophy.
miRNA
There is increasing evidence supporting that
miRNA’s play important roles in the process of
cardiac
remodeling
including
cardiac
hypertrophy and fibrosis. One of the most
extensively studied miRNA in the regulation of
cardiac fibrosis is miR-21. In healthy hearts,
expression of miR-21 is minimal. However,
miR-21 was markedly upregulated in animal
models and in humans with cardiac hypertrophy
and heart failure (Bauersachs 2010; Matkovich
2009). MiR-21 is predominantly expressed in
cardiac fibroblasts, whereas only low expression
levels are found in cardiomyocytes (Thum
2008).
Transfection of cardiac fibroblasts with miR-21
resulted in the upregulation of collagen and αSMA, suggesting the involvement of miR-21 in
the differentiation from cardiac fibroblasts to
cardiac myofibroblasts (Liang 2012). In various
models of heart failure, miR-21 overexpression
was accompanied by an increased activation of
ERK-MAPK (Thum 2008). Furthermore,
silencing of miR-21 increased the amount of
apoptotic
cardiac
fibroblasts,
whereas
overexpression reduced this amount. The ERKMAPK pathway is known to be involved in
fibroblast activation and cell survival (Pagès
1993; Raffetto 2006). This suggests that miR-21
is an important factor in the activation and
survival of cardiac fibroblasts during stress
conditions. In line with these results, miR-21
was found to target Spry1, a potent negative
regulator of the ERK-MAPK pathway (Thum
2008). Overexpression of miR-21 in cardiac
fibroblasts resulted in a strong repression of
Spry1 protein expression, while Spry1 mRNA
expression remained unaffected. Furthermore,
the 3’-untranslated region of Spry1 contained
several miRNA binding sites. Of these
miRNA’s, only miR-21 was upregulated during
heart failure (Thum 2008).
Another miRNA specific for cardiac fibroblasts
is miR-29. MiR-29 expression is often
downregulated during cardiac remodeling
(Van Rooij 2008; Vettori 2012). This
downregulation has been associated with
increased expression of ECM components,
including collagen type I and III (Vettori
2012). By using computational predictions,
elastin, fibrillin-1 and several collagen types
were found as possible targets of miR-29.
Mice hearts subjected to MI revealed a
downregulation of miR-29 accompanied by
upregulated levels of fibrillin 1, elastin and
several types of collagen (Van Rooij 2008).
One of the potential regulators of miR-29 is
TGF-β. In TGF-β stimulated cardiac
fibroblasts, miR-29 expression was reduced
(van Rooij 2008), suggesting that the
downregulated levels of miR-29 may have
been TGF-β dependent. Therefore, these
results may give a possible mechanism for
TGF-β to induce collagen expression in
cardiac fibroblasts.
MiR-133 is another miRNA often associated
with cardiac hypertrophy and heart failure. It
has been consistently downregulated in
models for pathological hypertrophy
(Duisters 2009) and may be a protective
factor for cardiac fibrosis. Recently it was
found that miR-133 was able to target
collagen type I since the gene activity of
collagen type I was suppressed by miR-133
(Castoldi 2012). In mice subjected to
transverse aortic constriction (TAC), miR133 levels were consistently downregulated
accompanied
by
fibrosis
formation
(Matkovich 2009). However, in transgenic
mice overexpressing miR-133, myocardial
fibrosis was reduced without affecting
cardiac hypertrophy.
Interestingly, a miR-133 binding site was
found in the 3’-untranslated region of CTGF
(Duisters 2009). Knockdown of miR-133 in
cultured cardiomyocytes and cardiac
fibroblasts resulted in the enhanced
expression
of
CTGF,
whereas
overexpression of miR-133 reduced CTGF
levels (Duisters 2009). This indicates that the
upregulation of CTGF during cardiac
remodeling may be caused by reduced levels
of miR-133.
Finally, miR-206 may play a role in the
regulation of fibrosis. In a recent study, adult
female mice subjected to coronary artery
ligation were treated with High Mobility Group
Box-1 protein (HMGB1), a factor involved in
inflammation and tissue repair (Limana 2011).
Three days after treatment, miR-206 expression
was 4-5-fold higher compared with controls,
accompanied by a decreased TIMP-3
expression, another MMP inhibitor. By using
prediction analysis, TIMP-3 was identified as a
possible target of miR-206. This was confirmed
in cultured cardiac fibroblasts (Limana 2011).
Overexpression of miR-206 resulted in the
downregulation of TIMP-3 whereas anti-miR206 treatment counteracted this downregulation.
Furthermore, a luciferase assay revealed that
miR-206 was able to target TIMP-3 directly at a
binding site in the 3’-untranslated region. These
results suggest that miR-206 may modulate
fibrosis formation by directly targeting TIMP-3.
Altogether, several factors are involved in the
enhanced cardiac fibrosis during cardiac
remodeling (Figure 4). Cardiac stress may
activate a pathway, in which ANG II, TGF-β,
ET-1 and CTGF ultimately result in the
activation and proliferation of cardiac
fibroblasts, the transition to myofibroblasts and
collagen synthesis. Furthermore, the βadrenergic system and several miRNA’s play a
role in the regulation of fibrosis. At last, besides
stimulating collagen synthesis, ANG II, TGF-β,
miR-206 and the β-adrenergic system are
involved in ECM degradation. They are able to
influence the expression of MMP’s and TIMP’s,
resulting in the inhibition of collagen
breakdown.
Figure 4. Schematic diagram illustrating the regulation of collagen and cardiac fibrosis in
cardiac fibroblasts.
Certain triggers and cardiac stress, like cardiopathologies, activate ANG II. ANG II induces a
pathway in which TGF-β, ET-1 and CTGF are involved. Together with activated βadrenoreceptors, this results in the stimulation of collagen synthesis in cardiac fibroblasts.
Furthermore, TGF-β downregulates miR-29, which normally inhibits fibrosis formation. MiR-21
is upregulated, thereby preventing the inhibition of ERK/MAPK, whereas downregulated levels of
miR-133 stimulate CTGF and collagen synthesis. At last, ANG II, TGF-β, miR-206 and βadrenoreceptors are involved in the regulation of MMP’s and TIMP’s and ECM degradation.
Linking Cx43 and fibrosis
Recent studies provided evidence that the
regulation of Cx43 expression and fibrosis are
related in various pathophysiological conditions,
instead of having an independent regulation. In
the heart, areas with enhanced fibrosis
formation were often accompanied by reduced
levels of Cx43 (Kostin 2003; van Veen 2005).
Furthermore, in aged mice heterozygous for
Cx43, the amount of cardiac fibrosis was
significantly increased compared with agerelated WT mice (Jansen 2008), indicating that
reduced levels of Cx43 may have induced
collagen synthesis. In addition, chronic
administration of a RAS inhibitor in senescent
mouse hearts largely prevented fibrosis
formation (Stein 2010). RAS inhibition also
prevented Cx43 remodeling whereas in nontreated aged mice Cx43 expression was
heterogeneous and reduced.
At present, it is unclear how Cx43 expression
and fibrosis formation are exactly related. In
arrhythmogenic right ventricular dysplasia/
cardiomyopathy (ARVD/C), an inherited
cardiomyopathy, Cx43 expression was reduced
in the early stages of the disease (Saffitz 2009).
In contrast, cardiac fibrosis was formed in a
later stadium when Cx43 levels were already
lowered. This gives rise to the idea that reduced
levels of Cx43 precede the formation of cardiac
fibrosis and that Cx43 expression may play a
role in the regulation of fibrosis.
To investigate whether reduced Cx43
expression levels may be involved in the
formation of cardiac fibrosis, the effects of
normal and reduced Cx43 levels were
investigated in an aging and TAC mouse model
(Jansen 2012). Both models are known to
induce the development of fibrosis (Jansen
2008; Müller 2008). In the physiological aging
model, WT mice and Cx43 heterozygous mice
were aged 18 to 20 months, while in the
pathophysiological model TAC was induced at
3 months and mice were sacrificed after 4
months. Both models revealed that Cx43
heterozygous mice developed more severe
cardiac fibrosis under stress conditions.
Furthermore, Cx43 heterozygous mice were
more susceptible to ventricular arrhythmias
compared with WT mice. Interestingly, mice
developing ventricular arrhythmias had more
severe fibrosis than mice without
arrhythmias, indicating that the severity of
fibrosis may be associated with the
susceptibility for arrhythmias.
One of the possible mechanisms for the link
between Cx43 expression and fibrosis may
be the increased fibroblast activation and
proliferation. Mice subjected to TAC
revealed an increase in expression levels of
P1NP and P3NP, the propeptides for
collagen I and III, respectively (Jansen
2012). Also COL1A2, encoding for the proα2 chain of collagen I increased after TAC.
Cx43 heterozygous mice had higher
expression levels of these genes compared
with WT mice, suggesting that the reduction
of Cx43 may have activated cardiac
fibroblasts leading to increased collagen
synthesis.
In carcinogenesis, connexins are thought to
have tumor suppressive effects by inhibiting
cell proliferation. Impaired connexin
expression and communication are often
observed in various tumors and restoration of
this impairment contributes to the reversion
of the transformed tumor cells (Ionta 2009).
Various studies described that reduced levels
of Cx43 in tumor cells were inversely
correlated to tumor proliferation (Avanzo
2007; Huang 2002; Ionta 2009; Roger 2004;
Song 2009). It was recently demonstrated
that cardiac fibroblasts exhibit the same
effects (Zhang 2008). Increasing Cx43
expression by adenoviral infection strongly
reduced fibroblast proliferation accompanied
by enhanced intercellular communication. In
contrast, Cx43 heterozygous fibroblasts
revealed an increased cell proliferation and
reduced cell-to-cell coupling. These studies
suggest an important role for Cx43 in
fibroblast activation and proliferation.
However, it is not known whether this effect
is caused directly by the reduced expression
of Cx43 or by the subsequent reduced
intercellular
communication
between
fibroblasts or between cardiomyocytes and
fibroblasts.
While in the above studies the reduced
expression levels of Cx43 were mainly induced
by genetic ablation, in humans connexin
remodeling is initiated by cardiopathologies. In
stress conditions such as during cardiac
hypertrophy and heart failure, several pathways
are (chronically) activated thereby inducing
connexin remodeling and fibrosis. Especially
TGF-β, ANG II and the α- and βadrenoreceptors are activated during cardiac
remodeling. It is possible that these factors
downregulate Cx43 in cardiomyocytes, resulting
in a reduced communication between
cardiomyocytes and cardiac fibroblasts. The
reduced coupling may then activate the
fibroblasts to proliferate, transform into
myofibroblasts and produce collagens and other
ECM components leading to cardiac fibrosis.
On the other hand, ANG II and TGF-β may also
have a direct effect on cardiac fibroblasts. They
may be able to downregulate Cx43 in these
fibroblasts which can directly or via the reduced
inter-fibroblast coupling activate the cardiac
fibroblasts.
Although α- and β-adrenergic stimulation is
mainly associated with cardiac hypertrophy, it is
also implicated in the regulation of Cx43. In
contrast to ANG II and TGF-β, the α- and βadrenergic stimulation appears to upregulate
Cx43 expression. It seems contradictory that
several systems chronically activated in
cardiopathologies
downregulate
Cx43
expression, whereas other factors upregulate
Cx43. However, the adrenoreceptors may be
involved in the heterogeneous expression of
Cx43 as frequently seen in heart failure patients.
The balance between upregulating and
downregulating systems may be an important
mediator and could vary between different areas
of the heart. This would result in higher Cx43
expression levels in one area compared with
other areas, leading to heterogeneous Cx43
expression. The heterogeneous Cx43 expression
has major implications since it causes variations
in conduction velocity within the myocardium
and may thereby trigger ventricular arrhythmias.
On the other hand, β-adrenoreceptors are able to
induce the expression of miR-1 via PKA. MiR-1
is a negative regulator of Cx43 expression,
suggesting that β-adrenergic stimulation may
also lead to Cx43 downregulation.
Besides the role for ANG II, TGF-β and the
adrenoreceptors in Cx43 remodeling, these
factors may also play an important role in the
formation of cardiac fibrosis. Studies have
shown that ANG II, TGF-β and the
adrenoreceptors are, together with ET-1 and
CTGF, potent stimulants for fibroblasts
activation,
proliferation,
myofibroblast
transition, collagen synthesis and fibrosis.
The fact that the same factors are
independently responsible for connexin
remodeling and fibrosis formation may
provide a link between the reduced Cx43
expression and enhanced fibrosis. It may be
possible that the activation of these factors
during cardiac remodeling lead to the
activation of independent pathways resulting
in Cx43 downregulation in cardiomyocytes.
On the other hand, activation of these factors
may also induce other independent pathways
resulting in fibrosis formation by cardiac
fibroblasts. Together this will lead to the
reduced Cx43 expression and enhanced
fibrosis as often seen during cardiac
remodeling.
To conclude, recent studies provide evidence
for a direct link between Cx43 expression
and cardiac fibrosis formation. However, at
present the mechanisms are still unknown.
This thesis proposes a new model for the link
between the reduced Cx43 expression and
enhanced fibrosis often observed in cardiac
diseases (Figure 5). In this model, cardiac
fibroblasts play a crucial role. During cardiac
stress, several factors affect Cx43
expression. Especially ANG II, TGF-β and
the adrenoreceptors are potent Cx43
regulators. The reduced Cx43 expression
results in a decreased cell-to-cell coupling.
The
reduced
cardiomyocyte-fibroblast
coupling may result in cardiac fibroblasts
activation and proliferation. These activated
fibroblasts
will
transform
into
myofibroblasts and produces collagens more
effectively, resulting in cardiac fibrosis. On
the other hand, ANG II, TGF-β and the βadrenergic system are able to regulate both
the
Cx43
and
collagen
expression
independently. Therefore, it is possible that the
activation of these factors during cardiac
remodeling induce independent pathways
resulting
in
connexin
remodeling
in
cardiomyocytes while other pathways stimulate
fibrosis formation in cardiac fibroblasts.
Figure 5. Schematic figure representing the link between reduced Cx43 expression and enhanced
fibrosis. Stimulation by ANG II and activation of the α- and β-adrenoreceptors (α-AR and β-AR,
respectively) in cardiomyocytes results in the activation of p38-MAPK and ERK. Activated ERK
stimulates the translocation of the transcription factors AP-1, CREB, NF-κB and NFAT to the nucleus
affecting Cx43 expression, whereas TGF-β affects Cx43 expression by an unknown mechanism. Also
miR-1 and miR-206 affect Cx43 expression by inhibiting Cx43 translation. Together, these factors
downregulate Cx43 expression, resulting in a reduced cell-to-cell coupling that may lead to ventricular
arrhythmias. Due to the reduced intercellular communication, fibroblasts become activated. Furthermore,
the
ANG
IITGF-βET-1CTGF
pathway,
the
β-adrenoreceptors,
the
miR21Spry1ERK/MAPK pathway and several miRNA’s contribute to the activation of cardiac
fibroblasts resulting in the proliferation, transformation into myofibroblasts and enhanced collagen
synthesis. As a consequence, this may lead to an enhanced cardiac fibrosis formation, which in turn may
be involved in the initiation of ventricular arrhythmias.
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