Connexin43 phosphorylation and cytoprotection in the heart☆
Maya M. Jeyaramana,b, Wattamon Srisakuldeea,b, Barbara E. Nickelb, Elissavet Kardamia,b,c,⁎
aInstitute of Cardiovascular Sciences, St. Boniface Hospital Research Centre, University of Manitoba, Canada
bDepartment of Physiology, University of Manitoba, Canada
cDepartment of Human Anatomy and Cell Sciences, University of Manitoba, Canada
a b s t r a c ta r t i c l ei n f o
Received 6 May 2011
Received in revised form 17 June 2011
Accepted 27 June 2011
Available online 3 July 2011
The fundamental role played by connexins including connexin43 (Cx43) in forming intercellular
communication channels (gap junctions), ensuring electrical and metabolic coupling between cells, has
long been recognized and extensively investigated. There is also increasing recognition that Cx43, and other
connexins, have additional roles, such as the ability to regulate cell proliferation, migration, and
cytoprotection. Multiple phosphorylation sites, targets of different signaling pathways, are present at the
regulatory, C-terminal domain of Cx43, and contribute to constitutive as well as transient phosphorylation
Cx43 patterns, responding to ever-changing environmental stimuli and corresponding cellular needs. The
present paper will focus on Cx43 in the heart, and provide an overview of the emerging recognition of a
relationship between Cx43, its phosphorylation pattern, and development of resistance to injury. We will also
review our recent work regarding the role of an enhanced phosphorylation state of Cx43 in cardioprotection.
This article is part of a Special Issue entitled: The Communicating junctions, composition, structure and
© 2011 Elsevier B.V. All rights reserved.
Introduction: innate cellular ‘self-defense’ from injury . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Connexin43 and cytoprotection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Connexin43 phosphorylation and PKC-mediated cytoprotection. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.1.Ischemia-induced Cx43 dephosphorylation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.2.Chronic heart pathologies are linked to dephosphorylated Cx43 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.3.Cx43 is an interacting partner and a downstream target of PKCε . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.4.PKC-mediated cytoprotection is linked to 43–45 kDa phospho-Cx43 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.5.Increased Cx43 phosphorylation at S262 is a marker of the ‘conditioned’ heart . . . . . . . . . . . . . . . . . . . . . . . . . . .
Cx43 phosphorylation at S262 mediates the FGF-2-, and PKCε-induced cardiomyocyte resistance to ischemic injury . . . . . . . . . . . . .
Cx43 phosphorylation at S262 partially contributes to ischemic preconditioning of cardiomyocytes . . . . . . . . . . . . . . . . . . . . .
Conclusion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Introduction: innate cellular ‘self-defense’ from injury
Cells and tissues have innate, baseline, capabilities to resist injury
up to a certain threshold, as well as to enhance their resistance to
various forms of injury. The latter represents a ‘reserve’ of self-defense
that can be activated by a number of triggers. Subjecting cells and
organs to sub-lethal levels of stress, such as brief ischemic episodes or
heat shock, activates multiple signaling pathways that result in
enhanced resistance to a subsequent more severe insult. This
resistance is evident both acutely, resulting largely from post-
translational modifications such as phosphorylation, and in a more
sustained fashion, one day after the initiating event, accompanied by
changes in gene expression [1–3]. Ischemic preconditioning, consist-
ing of brief cycles of ischemia–reperfusion, is a highly potent inducer
of cytoprotection against subsequent severe ischemia, and this holds
Biochimica et Biophysica Acta 1818 (2012) 2009–2013
☆ This article is part of a Special Issue entitled: The Communicating junctions,
composition, structure and characteristics.
⁎ Corresponding author at: Institute of Cardiovascular Sciences, St. Boniface Research
Centre, 351 Taché Avenue, Winnipeg, Manitoba, Canada R2H 2A6. Tel.: +1 204 235
3519; fax: +1 204 233 6723.
E-mail address: email@example.com (E. Kardami).
0005-2736/$ – see front matter © 2011 Elsevier B.V. All rights reserved.
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true for numerous cell types including cardiomyocytes and neurons
[1,2,4]. Ischemic post-conditioning , which can be achieved after
the onset of severe ischemia, during the first minutes of reperfusion
(by brief cycles of reperfusion–ischemia before the final reperfusion
event) has also proven capable of calling upon endogenous protective
reserves . Multiple agents (adrenergic receptor agonists, KATP
channel openers, growth factors, cytokines, opioids) are recognized
as capable of conferring a degree of pharmacological ‘conditioning’,
simulating aspects of ischemic pre- and post-conditioning to a larger
describe any manipulation capable of enhancing cellular resistance to
Signal transduction pathways and key targets capable of trigger-
ing, mediating, and acting as effectors of the conditioning, cytopro-
tective response have been identified. In general, ischemic
preconditioning enhances release of agonists of G-protein coupled
receptor families (opioid, adenosine, bradykinin) leading to activation
of cell survival pathways (downstream activation of protein kinase C,
PKC, isoforms, in particular PKCε, ERK and PI3/Akt kinases) and
inhibition of cell death pathways [8–10]. These pathways converge
upon cellular effectors of the protective response which are found in
the plasma membrane as well as the mitochondria. Effectors of
cardioprotection include the sarcolemmal as well as mitochondrial
KATPchannels; and the mitochondrial permeability transition pore
. Several mitochondrial and/or mitochondria-associated proteins
have been implicated in the orchestration of the mitochondrial
response to conditioning and injury . Mitochondria-located Cx43
seems to play an important role in this context, in addition to its role
in forming gap junctions [13,14].
2. Connexin43 and cytoprotection
There is increasing evidence for an important role for connexin43
(Cx43) in the development of an injury-resistant phenotype in cells
including cardiomyocytes. A positive relationship exists between
Cx43 expression and the ability to develop cytoprotection. For
example, hearts from Cx43 (+/−) mice, as well as aging/failing
hearts, all of which have below normal Cx43 levels, are deficient in
mounting an ischemic preconditioning response [15,16]. Modest
overexpression of Cx43 in cardiomyocytes through transient gene
transfer raises resistance to simulated ischemia in vitro . A
positive relationship between Cx43 and neuroprotection has also
been reported .
The precise molecular mechanism or mechanisms by which Cx43
affects cytoprotection remain to be fully understood. Cx43 is found
at the plasma membrane forming gap junctions and hemi-channels,
but has also been localized at the inner mitochondrial membrane of
cardiac subsarcolemmal mitochondria [19,20], and brain synaptic
mitochondria , where it may also contribute hemi-channel action.
Thus it can potentially influence the cell in all of these sites.
The role of gap junction mediated intercellular communication
salutary effects of ischemic preconditioning against myocardial infarc-
[22,23]. GJIC may, on the other hand, allow propagation of deleterious
signals to neighboring cells, expanding the degree of injury as has been
proposed for brain damage, and also for cardiac reperfusion after
ischemia . Studies with isolated, non-gap junction-forming myo-
cytes, reported that cells from wild type hearts were still capable of
not, concluding that cytoprotection is dependent on Cx43 but not GJIC
Cx43 may be able to regulate a protective response by both GJIC-
dependent and independent mechanisms. Finally it would be reason-
able to expect that cells, including cardiomyocytes, possess both
connexin-dependent as well as connexin-independent pathways to
cytoprotection. Skeletal muscle cells, for example, lack connexins, but
are capable of a preconditioning response .
At the plasma membrane they have been implicated as effectors of
cytoprotection, by improving, for example, cell volume homeostasis
. Mitochondrial Cx43 hemi-channels can influence cell survival by
modulating mitochondrial integrity . Ischemic preconditioning
promotes translocation of Cx43 to mitochondria . Mitochondrial
Cx43 is required for mitochondrial reactive oxygen species (ROS)
generationwhichis needed for protective signaltransduction including
PKCε activation downstream of the diazoxide-sensitive mitochondrial
KATPchannel. Hearts from Cx43 deficient mice have a functional deficit
in ROS formation in response to diazoxide and this decreases their
ability to develop protection from injury . More recently, mito-
chondrial Cx43 was found to be required for the activation of
mitochondrial KATPchannel in cardiomyocytes .
3. Connexin43 phosphorylation and PKC-mediated cytoprotection
Cx43 is a phosphoprotein, and phosphorylation affects all of its
properties. Connexins possess multiple phosphorylation sites, both
serines and tyrosines, potential targets of different categories of
kinases and signal transduction pathways [28–30]. Cx43 undergoes
post-translational modifications throughout its life cycle; several
kinases such as protein kinase C (PKC), extracellular signal-regulated
kinase (ERK) , the tyrosine kinase src , casein kinase 1 ,
protein kinase A , protein kinase B (Akt)  and p34cdc2 have
been implicated in Cx43 phosphorylation, regulating, for instance, its
intracellular trafficking and assembly, electrical and chemical cou-
pling, protein–protein interactions, as well as turnover; for a
comprehensive review the reader is referred to . Phosphorylation
by different triggers may target different sites or combinations of sites
and have different consequences; for example the angiotensin-II and
endothelin-1-triggered increases in cardiomyocyte Cx43 phosphory-
lation are mediated by ERK and/or p38, and result in increased
intercellular communication . On the other hand, src-mediated
tyrosine phosphorylation, or PKC-mediated serine phosphorylations
at a number of sites are linked to metabolic and/or electrical
uncoupling of gap junctions . Phosphorylation can also affect the
ability of Cx43 to act as a suppressor of cell proliferation: mitotic
stimulation of either cardiomyocytes (by FGF-2) or HEK293 cells (by
serum) is inhibited by Cx43 overexpression, but allowed to proceed
under conditions simulating constitutive phosphorylation of Cx43 at
the PKC target site S262 [37,38]. There is emerging recognition that
the phosphorylation status of Cx43 is not static, but is intimately
linked to and reflective of cellular state, response to stress, and
resistance to injury. Diverse observations support this notion, as listed
in the following sections.
3.1. Ischemia-induced Cx43 dephosphorylation
Ischemia causes acute changes in Cx43, including its progressive
dephosphorylation in tissues such as the brain , heart  and
cardiomyocytes , through the action of protein phosphatase
(PP)-1, PP-2A, and PP-2B (calcineurin) [41,42]. In the heart, ischemia
causes Cx43 re-distribution along the lateral plasma membrane away
from the intercalated disk region . Lateralized Cx43 is dephos-
phorylated, whereas phosphorylated forms remain at the intercalated
disk region ; however dephosphorylated Cx43 is also present
at intercalated disks . These acute ischemic changes in Cx43
phosphorylation are followed by Cx43 loss due to degradation ,
coinciding with the development of arrhythmias. It is of interest that
functional recovery of isolated perfused hearts from ischemia and
reperfusion is closely linked to the ability of reperfusion to re-
establish Cx43 phosphorylation .
M.M. Jeyaraman et al. / Biochimica et Biophysica Acta 1818 (2012) 2009–2013
3.2. Chronic heart pathologies are linked to dephosphorylated Cx43
Chronic heart disease including hypertrophy and failure is
associated with arrhythmias as well as decreased total Cx43 but
increased dephosphorylated Cx43 . Prevention of the action of
PP-1 in failing hearts, which would be expected to increase baseline
Cx43 phosphorylation levels, results in significant restoration of
contractile function .
3.3. Cx43 is an interacting partner and a downstream target of PKCε
PKCε which is recognized as a central mediator of both baseline
and inducible resistance to injury  interacts with Cx43, and this
interaction is enhanced in FGF-2 stimulated cardiomyocytes .
PKCε is required for Cx43 phosphorylation at the PKC target sites S262
, as well as S368 , although other PKC isoforms may also be
capable of catalyzing these phosphorylations [29,49]. Both PKCε and
Cx43 are localized, and expected to interact, in plasma membrane
, and mitochondrial sites . Finally, both PKCε and Cx43
translocate to mitochondria in response to protective stimulation,
suggesting that the interaction, and its immediate consequences
(Cx43 phosphorylation) are a component of the mitochondrial role in
3.4. PKC-mediated cytoprotection is linked to 43–45 kDa phospho-Cx43
Modest cardiac overexpression of PKCε promotes a chronically
preconditioned phenotype,as wellas increasedCx43 phosphorylation
. Potent PKC-dependent cardioprotection against ischemia and
reperfusion injury (by ischemic preconditioning  or FGF-2
administration ) is associated with, and is likely dependent
upon, at least partially, the prevention of ischemia-induced Cx43
dephosphorylation and redistribution . Global ischemia of the
perfused heart for 30 min causes complete Cx43 dephosphorylation
which is not restored to any significant degree by simple reperfusion;
FGF-2-supplemented reperfusion, however, rapidly restores pre-
ischemic levels of phosphorylated 43–45 kDa Cx43 , a process
associated with potent activation of PKCε, and with significant
protection from ischemia–reperfusion associated cell death and
contractile dysfunction . Ischemia–reperfusion cell death and
injury are a consequence of the formation of mitochondrial perme-
ability transition pore; prevention of the deleterious effects of
ischemia–reperfusion by FGF-2, therefore, is very likely to target
cardiac mitochondria and prevent pore formation. One of the
potential avenues to do so would be by promoting/restoring
mitochondrial Cx43 phosphorylation.
Overall, preservation or restoration of the extensively phosphor-
ylated, ‘mature’ Cx43 in the heart would be expected to contribute to
the structural integrity of the intercalated disk and the cardiomyo-
cytes, and to avoid increases in hemi-channel permeability; hemi-
channel opening due to Cx43 dephosphorylation contributes to
ischemia–reperfusion induced cell death and injury .
3.5. Increased Cx43 phosphorylation at S262 is a marker of the
Ischemic preconditioning treatment, FGF-2, or diazoxide adminis-
as phospho-S368-Cx43 in non-ischemic hearts, correlating with PKCε-
S368-Cx43 remain unchanged even when the heart is subjected to
30 min of global ischemia, associated with complete prevention of the
appearance of the 41 kDa dephosphorylated Cx43 . It is of interest
encountered at the mitochondrial Cx43 level, where they would be
expected to influence Cx43 and mitochondrial responses; indeed
mitochondria from FGF-2-conditioned hearts are more resistant to
calcium-induced opening of the permeability transition pore .
It should be noted that phospho-S262-Cx43 and phospho-
S368-Cx43 likely belong to different pools of cellular Cx43, possibly
representing different aspects of Cx43 function and regulation by
phosphorylation [17,55]. Phospho-S368-Cx43 migrates around
41 kDa in normal hearts and cardiomyocytes, indicating that it
represented minimally phosphorylated Cx43, and suggesting that
phosphorylation at S368 may actually prevent phosphorylation at
other Cx43 sites. Phospho-S262-Cx43, migrating close to 45 kDa,
occurs in addition to phosphorylation at other sites (excluding S368),
migrating close to 45 kDa. One hypothetical scenario is that
phosphorylation at S368 occurs in newly synthesized, and/or non-
phosphorylated Cx43, and is creating a ‘closed’, inaccessible, confor-
mational state that, as well as promoting electrical and chemical
uncoupling , is also blocking access of the C-terminal to other
proteins including kinases as well as phosphatases. Phosphorylation
at S262, on the other hand, seems to occur on ‘mature’, already
phosphorylated Cx43, and may actually require previous phosphor-
ylations at other sites (except S368) that are present in hexamer-
forming Cx43 at gap junctions or hemi-channels. We propose that
phosphorylation at S262 renders all Cx43 phosphorylation sites
resistant or inaccessible to phosphatase action; and as a consequence,
Cx43 is also less vulnerable to the degradation which follows Cx43
4. Cx43 phosphorylation at S262 mediates the FGF-2-, and
PKCε-induced cardiomyocyte resistance to ischemic injury
Both FGF-2 and its downstream activated PKCε are strong inducers
of cardioprotection, and promote Cx43 phosphorylation at S262 in
vitro and in vivo. A series of in vitro studies in our laboratory has
shown that the ability of Cx43 to become phosphorylated at S262 is
essential for both baseline levels of resistance to injury, as well as for
the ability of either FGF-2, or overexpressed PKCε, to elicit enhanced
cytoprotection  against simulated ischemia (incubation in
‘ischemic medium’ and in a hypoxia chamber) in cardiomyocytes.
Typical data are shown in Fig. 1. Both FGF-2 pretreatment, and PKCε
overexpression, protect cardiomyocytes from cell death induced by
simulated ischemia in vitro, but this protection is completely lost in a
background of S262A-Cx43 overexpression which precludes phos-
phorylation at S262.
5. Cx43 phosphorylation at S262 partially contributes to ischemic
preconditioning of cardiomyocytes
An ischemic preconditioning protocol, consisting of 30 min
simulated ischemia, followed by 30 min of reperfusion (re-incubation
in non-ischemic, oxygenated buffer) of cardiomyocytes in vitro is
indeed strongly protective from cell death induced by subsequent
prolonged ischemia (Fig. 1). Expression of S262A-Cx43 results in
substantially higher incidence of cell death; ischemic preconditioning
was able to exert partial protection from the deleterious effects of
S262A-Cx43 expression. These findings indicate that the protective
effects of ischemic preconditioning only partially depend on Cx43
phosphorylation at S262, and they likely represent the combined
effect of both PKCε/phospho-S262-Cx43 dependent and independent
pathways. It would also appear that the signal transduction pathway
of FGF-2- or PKCε-induced cardiomyocyte protection is not identical
but has common elements (those dependent on PKCε/phospho-S262-
Cx43) with pathways activated by ischemic preconditioning.
Ischemic preconditioning cardioprotection requires the produc-
tion of short bursts of reactive oxygen species (ROS) to activate
downstream signals required for the net protective effect. There is a
recognition that mitochondria-derived ROS (which are stimulated by
diazoxide) make a major contribution to ischemic preconditioning
M.M. Jeyaraman et al. / Biochimica et Biophysica Acta 1818 (2012) 2009–2013
protection  and that diazoxide-dependent preconditioning re-
quires sufficient Cx43 expression, becoming inoperable in hearts with
reduced Cx43 content . On the other hand, hearts with reduced
Cx43 are still capable of a preconditioning response when ROS
production is elicited by a different, non-mitochondrial-exclusive
route . We speculate that the ischemic preconditioning and
mitochondria-dependent ROS production stimulate PKCε activity
and subsequent Cx43 phosphorylation at S262, and that this
component of the pathway is also triggered by FGF-2 stimulation, or
PKCε overexpression. Ischemic preconditioning-induced but non-
mitochondrial produced ROS exert protective effects that are
independent of Cx43 (and its phosphorylation at S262), and this
pathway may not be stimulated by FGF-2 stimulation or PKCε
overexpression, at least under the conditions tested.
Fig. 2 provides a ‘broad strokes’ type of diagram summarizing the
relationship between cardioprotection/cytoprotection, and Cx43
phosphorylation. Various cardioprotective agents and procedures
stimulate several signaling pathways linked to cytoprotection: these
include ERK, Akt, and PKC activating pathways, targeting various
cellular proteins including Cx43. We have used the term P*Cx43 to
denote particular, Cx43 ‘hyper’ phosphorylation events stimulated by
cardioprotective/cytoprotective agents and procedures. While PKC,
and particularly PKCε, is strongly linked to the induction of a P*Cx43
state, the other kinases (ERK, Akt), that promote cell survival during
the reperfusion phase may also contribute to Cx43 phosphorylation in
the scenario of cardioprotection. It will be important to obtain a full
profile of the Cx43 phosphorylation pattern or patterns associated
with an injury-resistant state; and to fully map the subcellular sites of
action of P*Cx43: in cardiomyocytes these include gap junction-
forming Cx43 at intercalated disks, hemi-channel-forming Cx43 at
lateral plasma membranes and mitochondria, as well as non-channel-
This work was supported by grants from the Heart and Stroke
Foundation of Canada, the Canadian Institutes for Health Research
(EK), and the St. Boniface General Hospital Research Foundation. MMJ
and WS were supported, respectively, by graduate studentship
awards from the Heart and Stroke Foundation of Canada, and the
Manitoba Institute for Health Research as well as the Institute of
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Fig. 1. The role of Cx43 phosphorylation at S262 in FGF-2, PKCε, and ischemic
preconditioning (IP)-induced cardiomyocyte protection from simulated ischemia-
induced cell death, as indicated. Neonatal cardiomyocytes were transduced with an
adenoviral vector expressing S262A-Cx43 (S262); control groups were transduced with
an empty adenoviral vector (V). Cultures were subsequently subjected to various
protective treatments, as indicated, followed by simulated ischemia for 6 h, and
determination of TUNEL staining index as described . Brackets indicate comparison
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