REVIEW Circ J 2009; 73: 1184 – 1192

GSK-3β, a Therapeutic Target for Cardiomyocyte Protection

Tetsuji Miura, MD; Takayuki Miki, MD

Glycogen synthase kinase-3β(GSK-3β) is a multifunctional Ser/Thr kinase that plays important roles in necrosis
and apoptosis of cardiomyocytes. A major mechanism of cell necrosis is the opening of the mitochondrial per-
meability transition pore (mPTP), which consists of multiple protein subunits, including adenine nucleotide
translocase (ANT). The threshold for mPTP opening is elevated by phosphorylation of GSK-3β at Ser9, which
reduces activity of this kinase. How inactivation of GSK-3βsuppresses mPTP opening has not been fully under-
stood, but evidence to date suggests that preservation of hexokinase-II in the mPTP complex, inhibition of
cyclophilin-D-ANT binding, inhibition of p53 and inhibition of ANT into the mitochondria are contributory.
GSK-3βphosphorylation is a step to which multiple protective signaling pathways converge, and thus GSK-3β
phosphorylation is crucial in cardioprotection of a variety of interventions against ischemia/reperfusion injury.
Apoptosis of cardiomyocytes by pressure overload or ischemia/reperfusion is also suppressed by inactivation of
GSK-3β, in which reduced phosphorylation of p53, heat shock factor-1 and myeloid cell leukemia sequence-1 and
inhibition of Bax translocation might be involved. Considering predominant roles of GSK-3β in cardiomyocyte
death, manipulation of this protein kinase is a promising strategy for myocardial protection in coronary artery
disease and heart failure.   (Circ J 2009; 73: 1184 – 1192)
Key Words: Apoptosis; Heart failure; Myocardial infarction; Preconditioning; Signal transduction

G
lycogen synthase kinase-3β (GSK-3β) is a constitu-
tively active 47-kDa Ser/Thr protein kinase that
was purified more than 2 decades ago as a kinase
that reduces glycogen synthase activity. However, GSK-3β
is now known as a multifunctional kinase having more than
40 substrates, playing roles not only in glycogen metabolism
but also cell proliferation, growth and death.1–3 To properly
execute its functions, GSK-3βhas multiple regulatory mech-
anisms including phosphorylation at Ser and Tyr residues,
complex formation with scaffold proteins, priming of sub-
strates and intracellular translocation. Recently, pathophysi-
ological roles of GSK-3β have also received attention
because it is involved in common and serious diseases such
as Alzheimer’s disease, bipolar mood disorder, cancer and
ischemia/reperfusion injury.1,3 In the cardiovascular system,
GSK-3β has major roles in glucose metabolism,4 cardio-
myocyte hypertrophy5 and cell death. The roles of GSK-3β
in hypertrophy have recently been reviewed by Sugden et
al5, so therefore we have focused on the roles of this protein
kinase in myocyte death, particularly that after ischemia/
reperfusion. We first briefly overview mechanisms of
ischemia/reperfusion injury and then discuss the contribu-
tion of GSK-3β to cardiomyocyte death and manipulation
of GSK-3βfor myocardial protection.
(Received April 22, 2009; revised manuscript received May 13, 2009;
accepted May 14, 2009; released online June 9, 2009)
Division of Cardiology, Second Department of Internal Medicine,
Sapporo Medical University School of Medicine, Sapporo, Japan
Mailing address:  Tetsuji Miura, MD, Division of Cardiology, Second
Department of Internal Medicine, Sapporo Medical University School
of Medicine, South-1 West-16, Chuo-ku, Sapporo 060-8543, Japan.
E-mail: miura@sapmed.ac.jp
All rights are reserved to the Japanese Circulation Society. For permis-
sions, please e-mail: cj@j-circ.or.jp
Mechanisms of Cardiomyocyte Necrosis
During Ischemia/Reperfusion
When myocardial blood perfusion is interrupted, for
example by occluding the coronary artery, ischemic car-
diomyocytes suffer from several critical intracellular events
that can lead to cell necrosis and/or prime the cells to reper-
fusion-induced necrosis. First, intracellular level of high-
energy phosphate is reduced due to oxygen deficiency.
Although ischemic cardiomyocytes cease contraction within
a few minutes after the onset of ischemia, adenosine triphos-
phate (ATP) consumption continues during ischemia mainly
by mitochondrial ATPase for maintenance of mitochondrial
membrane potential.6,7 Anaerobic glycolysis supplies ATP
during the early period of ischemia, but it is ultimately halted
by inhibition of glyceraldehyde phosphate dehydrogenase
due to accumulated H+ and NADH.7,8 Second, intracellular
Na+ accumulates due to Na+ influx via Na+–H+ exchangers
and Na+ channels and due to reduced Na+ efflux via the
Na+–K+ pump.9–11 This Na+ overload predisposes ischemic
cardiomyocytes to Ca2+ influx by the Na+–Ca2+ exchanger.
Third, Ca2+ influx via the Na+–Ca2+ exchanger, reduced Ca2+
uptake into the sarcoplasmic reticulum and reduced Ca2+
efflux via the sarcolemmal Ca2+ pump induce Ca2+ overload
in ischemic cardiomyocytes.9–12 However, elevation of intra-
cellular Ca2+ is modest during ischemia because acidosis
inhibits the Na+–Ca2+ exchanger and cytosolic Ca2+ is taken
up by the mitochondria as long as its membrane potential is
maintained by use of ATP.9–11,13
Severely ischemic cardiomyocytes ultimately ‘starve to
death’, although sarcolemmal damage by detergent actions
of accumulated long-chain Acyl-CoA and by Ca2+-activated
proteases and phospholipases might also be involved in
ischemia-induced necrosis.7 Nevertheless, reperfusion is nec-
essary to salvage ischemic cardiomyocytes from necrosis.
However, reperfusion per se can trigger lethal mechanisms.
Reperfusion washes out H+ in the extracellular space, which

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GSK-3βin Cardioprotection
retracts inhibition of the reverse mode operation of the
Na+–Ca2+ exchanger, resulting in massive Ca2+ influx into
cardiomyocytes.9–11 The abrupt increase in intracellular Ca2+
level induces activation of calpain, a Ca2+-activated protease,
which compromises Na+–K+ pump function10 and Ca2+
overload of the mitochondria.6,7,9,11 At the same time as Ca2+
overload, cardiomyocytes abruptly develop cell edema after
reperfusion as a result of osmotic loading during ischemia.
ATP production restituted by re-supply of oxygen in Ca2+-
overloaded and swelling myocytes with fragile sarcolemma
induces hypercontraction and sarcolemmal rupture. Involve-
ment of this mechanical factor in cardiomyocyte necrosis is
supported by findings that suppression of myocardial con-
traction during the early period of reperfusion by the use of
butanedione monoxime significantly reduced infarct size.14,15
However, a major mechanism of lethal reperfusion injury is
likely to be opening of the mitochondrial permeability tran-
sition pore (mPTP) at the time of reperfusion, which will be
discussed in detail below.
Apoptosis of Cardiomyocytes After
Ischemia/Reperfusion
Most, if not all, of dead cardiomyocytes after ischemia/
reperfusion show morphology of necrosis under examina-
tions with macro- and micro-histochemistry and electron
microscopy. However, contribution of apoptosis to myocyte
loss after ischemia/reperfusion is rather inconclusive.16 A
major problem in this issue is limitations of methods used for
detecting apoptosis. Terminal deoxynucleoidyl tranferease-
mediated dUTP in-situ nick end labeling (TUNEL) and
detection of non-random fragmentation of DNA (DNA lad-
dering) are somewhat non-specific, and TUNEL-positive
cardiomyocytes after ischemia/reperfusion in situ actually
showed necrotic morphology under electron microscopy.17
However, there are some observations suggesting that
Circulation Journal Vol.73, July 2009
1185
Figure 1.   Models of mitochondrial permeability tran-
sition pore (PTP). (A) A classic model. OMM, outer
mitochondrial membrane; IMM, inner mitochondrial
membrane; IMS, intermembrane space; HK, hexokinase;
PBR, peripheral benzodiazepine receptor; CK, creatine
kinase; CypD, cyclophilin-D; VDAC, voltage-dependent
anion channel; ANT, adenine nucleotide translocator.
The pore consists of VDAC and ANT. In response to
cellular stress, CypD in the matrix binds to ANT, which
sensitizes the pore to opening stimuli such as Ca2+.
(B) A new model. Components of the pore itself (PTP)
are not unidentified but are known to be regulated by
CypD, ANT and other elements depicted in the figure.
From Juhaszova et al28 with permission.
apoptosis is involved in cardiomyocyte death after ischemia/
reperfusion. Ischemia/reperfusion induces activation of
caspase-3, -8, -9 and translocation of Bax and Bcl-2 in the
myocardium.18,19 Myocardial infarct size (ie, size of necrotic
tissue mass) was reduced to 37–78% of controls by caspase
inhibitors20,21 or genetic deletion of Fas,22 and this protec-
tion was associated with reduction in TUNEL-positive cells
in reperfused regions. Furthermore, recent studies have
shown that apoptotic process is converted to necrotic process
when essential co-factors for apoptosis are lacking.23–25
ATP is one of such co-factors, and switching from apoptosis
to necrosis by inhibition of mitochondrial ATP generation
has been shown in non-cardiac cells.23,24 Taken together,
these findings are consistent with the notion that apoptotic
cell death triggered by intrinsic mechanisms and/or by
Fas-Fas ligand system is ultimately converted to necrotic
cell death in the myocardium during ischemia/reperfusion.
However, this possibility remains to be critically tested.
mPTP and Reperfusion Injury
The mPTP is a non-selective large conductance channel in
the mitochondrial inner membrane, which is physiologically
closed. Opening of mPTPs is involved in cell death induced
by a variety of causes (for example, ischemia/reperfusion,
alcohol, endotoxin, anti-cancer agents).26 Although the
molecular structure of the mPTP has not yet been clarified,
several proteins have been suggested to be subunits of this
multi-protein complex.26–29 In a classic model of mPTP, the
pore is formed by adenine nucleotide translocase (ANT) in
the inner membrane and voltage-dependent anion channel
(VDAC) (Figure 1A). In this model, binding of cyclophilin-
D (CypD), a matrix protein, to ANT increases sensitivity of
ANT to Ca2+, a trigger of mPTP opening. A crucial role of
CypD in mPTP opening has been indicated by 3 indepen-
dent studies demonstrating that deletion of a gene coding
1186 MIURA T et al.

Figure 2.   Interaction of phospho-GSK-3βwith mitochondrial permeability transition pore subunit proteins in rat hearts.
Isolated perfused rat hearts were subjected to 25-min ischemia and reperfusion with or without IPC and EPO infusion
before ischemia. Tissue samples were taken for IP and IB at 5 min after reperfusion. (A) IPC + EPO increased level of
ANT in phospho-GSK-3β immunoprecipitates by 50%, whereas phospho-GSK-3β-VDAC interaction was not detected.
(B) Level of CypD in ANT immunoprecipitates was reduced by IPC + EPO to 40% of the control level, though ANT-
VDAC binding was unchanged. ANT, adenine nucleotide translocase; CypD, cyclophilin-D; EPO, erythropoietin; GSK,
glycogen synthase kinase; IB, immunoblotting; IP, immunoprecipitation; VDAC, voltage-dependent anion channel.
*P<0.05 vs Control. From Nishihara et al35 with permission.

mitochondrial CypD (Ppif) markedly increased resistance
of cells to mPTP opening and necrosis.30–32 However, recent
studies using ANT- and VADAC-deficient mice indicate
that ANT and VDAC are not indispensable for mPTP
opening.33,34 Based on these findings, a new model of mPTP
was recently proposed by Sollott’s group (Figure 1B).28 In
this new model, ANT and VDAC are regulatory factors of
an unknown channel protein.
In addition to Ca2+, reactive oxygen species (ROS), accu-
mulated inorganic phosphate and depletion of ATP promote
opening of mPTPs.35,36 Although the relative importance of
each factor is not clear, all of these mPTP opening stimuli
are induced in cardiomyocytes subjected to long-sustained
ischemia and reperfusion. Studies using radio-labeled 2-
deoxyglucose as a tracer of opened mPTPs indicated that
mPTPs open within 5–10 min after reperfusion but not
during ischemia.37 This finding is consistent with the notion
that ROS production and Ca2+ influx upon reperfusion are
triggers of mPTP opening in the reperfused myocardium.
Because molecules smaller than 1.5 kDa pass through
mPTPs, irreversible mPTP opening abolishes mitochondrial
membrane potential, disabling the mitochondria to produce
ATP. Without ATP generation, reperfused cardiomyocytes
cannot recover Na+ and Ca2+ homeostasis and thus cannot
maintain cell viability. Results of experiments using phar-
macological inhibitors of mPTP and CypD knockout mice
generally support the notion that opening of mPTPs is
responsible for cardiomyocyte necrosis after ischemia/reper-
fusion.32–34,38–40
In contrast to its role in cell necrosis, the role of mPTP in
apoptosis is still controversial. Earlier studies using non-
cardiac cells have shown that mPTP opening induces Bax
translocation to the mitochondria, leading to apoptosis.41,42
Furthermore, inhibition of mPTP by pharmacological inhib-
itors (cyclosporine A, bongkrekic acid, NIM811) suppressed
apoptosis induced by H2O2 in vitro43–45 and by ischemia/
reperfusion in vivo.46 However, susceptibility of CypD
deleted cells to apoptosis induced by etoposide, saturosporin,
TNF-α plus cyclohexamide was not different from that of
normal control cells.32 There is no clear explanation for
the contradictory observations, but it may reflect complex
cross-talks of redundant signaling pathways of apoptosis
and necrosis.24
GSK-3β and mPTP
Johaszova et al first reported that GSK-3β activity is a
determinant of the threshold for mPTP opening in car-
diomyocytes.47 They showed that the threshold for ROS-
induced opening of mPTPs is elevated by inhibitory phos-
phorylation of GSK-3β, pharmacological inhibitors of
GSK-3β and knockdown of GSK-3β protein expression
using siRNA. Recently, Gomez et al examined the role of
GSK-3βin mPTP regulation by using the amount of loading
Ca2+ required to induce irreversible Ca2+ release in isolated
mitochondria as an index of the threshold for mPTP
opening.48 Postconditioning significantly elevated the thresh-
old of mPTP opening in cardiac mitochondria from wild-
type mice, but such a protective effect was not detected in
mitochondria from transgenic mice expressing GSK-S9A.
These findings indicate that GSK-3β activity promotes
mPTP opening in response to ROS and/or Ca2+ overloading.
How inactivation of GSK-3β (or its phosphorylation at
Ser9) increases the threshold for mPTP opening remains
unclear. However, several mechanisms have been suggested
to date. First, hexokinase II (HK-II) might be preserved
in the mPTP complex by inactivation of GSK-3β.28,49,50
Although experiments were conducted by using non-cardiac
cells, Pastorino et al showed that activation of GSK-3β
induces release of HK-II from the mitochondria via phos-
phorylation of VDAC, which enhanced susceptibility of the
cells to necrosis.49 A role of HK-II as a stabilizer of mPTP
was also indicated by finding that the inhibitory effect of
leukemia inhibitory factor on mPTP opening was sig-
nificantly attenuated by dissociation of HK-II from the
mitochondria by the use of glucose-6-phosphate or HK-II

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GSK-3βin Cardioprotection
dissociating peptide.50
Second, our recent study suggests that binding of
phospho-GSK-3β to ANT suppresses interaction of ANT
with CypD, a trigger of mPTP opening.51 In isolated rat
hearts, reperfusion after sustained ischemia induced trans-
location of cytosolic GSK-3βto the mitochondria, where it
formed a complex with ANT and VDAC. Phosphorlylation
of GSK-3βby ischemic preconditioning and erythropoietin
(EPO) receptor activation was dependent on protein kinase
C (PKC) and phosphatidiylinositol 3-phosphate kinase
(PI3K), and phospho-GSK-3β was bound to ANT but not
to VDAC at reperfusion (Figure 2A). Interestingly, this
phopsho-GSK-3β-ANT interaction was associated with
reduction of CypD-ANT binding by 60% compared with
the untreated control group (Figure 2B). Because neither
ANT nor CypD is a putative substrate of GSK-3β3, modifi-
cation of CypD-ANT interaction by phospho-GSK-3β is
presumably an indirect one.
Third, p53-mediated regulation of mPTP might be sup-
pressed by inhibition of GSK-3β activity. Phosphorylation
of p53 by GSK-3βenhances its functional activity and trans-
location to the nucleus and mitochondria.52 Venkatapuram
et al showed that an inhibitor of p53, pifithrin-α, sensitized
the myocardium to isoflurane-induced protection, which is
presumably phospho-GSK-3β-mediated.53 This beneficial
effect of pifithrin-αwas abolished by an activator of mPTP,
atractyloside, indicating that a target of the p53 inhibitor
was mPTP.
As another possible mechanism of protection by GSK-3β
inactivation, Das et al recently showed that inhibition of
GSK-3β suppressed ATP hydrolysis by reducing ATP
transport from the cytosol to the mitochondria.54 This effect
was associated with reduced phosphorylation of VDAC,
suggesting modification of ATP transport through VDAC.
Suppression of ATP hydrolysis during ischemia would
prevent both ATP depletion and accumulation of inorganic
phosphate, 2 factors promoting mPTP opening. All of the
mechanisms discussed here are not mutually exclusive and
might contribute in concert to mPTP inhibition (Figure 3).
GSK-3β and Tolerance of Cardiomyocytes
to Necrosis
Involvement of GSK-3β phosphorylation in myocardial
Circulation Journal Vol.73, July 2009
1187
Figure 3.   Current hypothesis of GSK-3β-
mediated regulation of cell death and survival
in the heart. ANT, adenine nucleotide trans-
locase; ATP, adenosine triphosphate; CypD,
cyclophilin-D; EPO-R, erythropoietin receptor;
ERK, extracellular signal-regulated kinase;
GPCRs, G-protein-coupled receptors; HK-II,
hexokinase-II; HSF-1, heat shock factor-1;
IGF-R, IGF-1 receptor; I-R, insulin receptor;
mATPase, mitochondrial ATPase; MCL-1,
myeloid cell leukemia sequence-1; mPTP,
mitochondrial permeability transition pore;
OMP, outer membrane permeabilization; PI3K,
phosphatidylinositol 3-phosphate kinase; Pi,
inorganic phosphate; PKC, protein kinase C;
PP1, protein phosphatase-1; PP2A, protein
phosphatase-2A; VDAC, voltage-dependent
anion channel.
protection has been suggested in a wide variety of inter-
ventions against infarction. Elevation (or preservation) of
phospho-Ser9-GSK-3βlevel on reperfusion and anti-infarct
tolerance have been observed in rat, rabbit or mouse hearts
treated with ischemic preconditioning,51,55–57 ischemic post-
conditioning,48,58 bradykinin,59 opioids,60,61 EPO,57,62 an ade-
nosine A1/A2 receptor agonist,63 an A3 receptor agonist,64
PKC-ε activator,65 PKC-δ inhibitor,66 isoflurane,67 lipopoly-
saccharide,68 sildenafil,69 xenon70 or resveratrol.71 GSK-3β
phosphorylation by these apparently unrelated interven-
tions is explained by the fact that GSK-3β is a substrate
of multiple pro-survival protein kinases, including Akt,
PKC-ε, extracellular signal-regulated kinase (ERK) and
protein kinase G, and GSK-3βphosphorylation is therefore
a step to which multiple protective signaling pathways
converge. Direct inhibition of GSK-3β by the use of struc-
turally different pharmacological inhibitors (SB-216763,
lithium chloride) administered before either ischemia or
reperfusion limits infarct size.55,57,60,72,73 It is interesting to
note that inhibitors of GSK-3βincrease the level of phospho-
GSK-3β, which is explained by GSK-3β-mediated positive
regulation of protein phosphatase-1 (Figure 3).74 Neverthe-
less, these findings support the notion that inactivation of
GSK-3β by phosphorylation at Ser9 is a common mecha-
nism of protection of cardiomyocytes against necrosis in
many cardioprotective interventions.
We hypothesized that phospho-Ser9-GSK-3βat the time
of reperfusion determines the extent of reperfusion-induced
myocardial necrosis, because it suppresses mPTP opening.
To test this possibility, different levels of GSK-3β phos-
phorylation were induced in rat hearts in situ by IPC, admin-
istration of EPO, the combination of these two, or inhibitors
of PKC or PI3K together with IPC-EPO combination before
20 min ischemia/2 h reperfusion. Levels of phospho-GSK-
3β at 5 min after reperfusion were tightly correlated with
infarct sizes (% of ischemic area) 2 h after reperfusion
(r=0.809, P<0.05), indicating that approximately 60% of
infarct size variation is explained by variation in the level
of GSK-3β phosphorylation.57 This relationship between
phospho-GSK-3β and infarct size is unlikely to be non-
specific, because there was no correlation between infarct
size with level of phospho-Ser473-Akt or phospho-Tyr705-
STAT3 on reperfusion.
However, that role of phospho-GSK-3βin cardiomyocyte
1188
protection might not be equivalent across animal species. To
our surprise, protective effects of ischemic preconditioning
and postconditioning were not lost in GSK-3α/β knock-in
mice in which Ser21 of GSK-3α and Ser9 of GSK-3β were
changed to Ala.75 Furthermore, pharmacological inhibitors
of GSK-3βfailed to limit infarct size in these knock-in mice
and also in wild-type mice. In contrast, another study using
transgenic mice showed apparently opposite results. Gomez
et al reported that the infarct size-limiting effect of post-
conditioning was lost in mice cardiospecifically expressing
GSK-3β-S9A and that SB216763 could mimic postcondi-
tioning in wild-type mice.48 The reasons for this apparent
discrepancy in results of the 2 mouse studies remains
unclear, although differences in intracellular distribution of
mutant GSK-3β, function of extra-cardiac GSK-3β and
function of GSK-3αare possibilities. In addition, Skyschally
et al recently reported that the infarct size-limiting effect of
ischemic postconditioning was maintained in pigs in which
phosphorylation of Akt, ERK and GSK-3β was inhibited
by inhibitors of PI3K and ERK.76 Collectively, the negative
results in mouse and pig studies suggest that role of phospho-
GSK-3β as a determinant of myocyte tolerance to reperfu-
sion-induced necrosis may be species dependent.
Role of GSK-3β in Apoptosis
of Cardiomyocytes
It is known that GSK-3β has 2 opposite roles in apop-
totic death of non-cardiac cells depending on the trigger of
apoptosis. GSK-3β activity restrains pro-apoptotic signal-
ing from death receptors (ie, TNF-R1, Fas, DR4, DR5).77–80
Inhibition of GSK-3βin the cells with activated death recep-
tors enhanced activation of caspases-8 and -3, Bid cleavage
and apoptosis, indicating that GSK-3βsuppresses pro-apop-
totic signals upstream of caspase activation.78–80 Thus, inac-
tivation of GSK-3β under the condition of stimulation of
death receptors promotes apoptosis.
In contrast, apoptosis induced by intrinsic mechanisms
is facilitated by active GSK-3β. Pro-apoptotic functions of
GSK-3β have been shown in apoptosis by withdrawal of
growth factors,81–83 DNA damage,52,84 mitochondrial toxins,85
ischemia,86,87 oxidant stress88 and other conditions that trigger
apoptosis via the mitochondrial pathway. Phosphorylation
of 4 GSK-3β substrates (p53, heat shock factor-1 [HSF-1],
myeloid cell leukemia sequence-1 [MCL-1], Bax) is involved
in the pro-apoptotic functions. Phosphorylation of p53 in the
nucleus leads to p53-mediated transcription of pro-apoptotic
genes52,89 phosphorylation of HSF-1 inhibits its function as
a survival-promoting transcription factor,90,91 phosphory-
lation of MCL-1 induces ubiquitinylation and subsequent
degradation of this anti-apoptotic Bcl-2 protein,92,93 and phos-
phorylation of Bax promotes its localization in the mito-
chondria and apoptosis.81 Contribution of the 4 GSK-3βsub-
strates to apoptosis appears different depending on cell types,
experimental conditions and triggers of apoptosis.
Although the role of GSK-3βin apoptosis of cardiomyo-
cytes has not been fully clarified, evidence to date supports
its significant contribution to apoptosis induced by ischemia/
reperfusion,69,94–96 hypoxia/re-oxygenation,97β-adrenoceptor
activation98 and pressure overload.99 Apoptosis by these
insults was suppressed by overexpression of the adreno-
medullin gene95 or kallikrein gene96 or treatment with
statins,97 all of which induced phosphorylation of GSK-3β.
Furthermore, this protection from apoptosis was inhibited
by dominant-negative Akt or active GSK-3β mutant and
MIURA T et al.
mimicked by pharmacological inhibitors of GSK-3β69,94–98
.
Transgenic mice that cardio-specifically express dominant-
negative GSK-3β showed significantly fewer apoptotic
cardiomyocytes after pressure overloading by aortic con-
striction.99 These findings indicate that GSK-3β activity
determines the fate of cardiomyocytes exposed to apoptosis
inducers. However, the intracellular localization of phospho-
GSK-3βresponsible for the anti-apoptotic function and the
mechanism downstream of GSK-3β phosphorylation in
cardiomyocytes remain unclear.
Recently, we tested the hypothesis that translocation of
phosphorylated GSK-3β to the mitochondria is important
for inhibition of oxidant stress-induced apoptosis of car-
diomyocytes.100 Apoptosis of H9c2 cells was induced by
hydrogen peroxide or hypoxia/reoxygenation. EPO and
insulin-like growth factor-1 significantly suppressed apop-
tosis of H9c2 cells, which was associated with activation of
Akt and phosphorylation of GSK-3β at Ser9. Furthermore,
H9c2 cells transfected with S9A were insensitive to EPO-
induced protection, and siRNA knockdown of GSK-3βcould
mimic the effect of the EPO receptor activation on H2O2-
induced apoptosis. However, translocation of GSK-3βto the
mitochondria after EPO receptor activation was not detected
by tracing EGFP-tagged GSK-3βor by co-immunostaining
of GSK-3βand mitochondria. Instead, these methods showed
that GSK-3β was phosphorylated within the mitochondria
after EPO receptor activation in a PI3K-dependent manner.
No significant changes were detected for p53, MCL-1 or
Bcl-2 after H2O2 challenge or EPO treatments in our proto-
cols, but translocation of Bax to mitochondria in response
to oxidant stress was suppressed. These results suggest that
phosphorylation of GSK-3β pre-existing in the mitochon-
dria by Akt affords protection from oxidant stress-induced
apoptosis, possibly by suppressing Bax translocation, in car-
diomyocytes. Phosphorylation of GSK-3βin the mitochon-
dria100 is possibly achieved by translocation of Akt to the
mitochondria.50,62
Do We Need a Novel Therapy for Cardiomyocyte
Protection in the PCI Era?
Introduction of reperfusion therapy and its continuous
refinement have improved myocardial salvage in patients
with acute myocardial infarction (AMI) and their progno-
sis. Our recent analysis of data on infarct size and ischemic
zone size in the literature indicates that current reperfusion
therapy salvages more than 50% of the ischemic myocar-
dium in approximately half of the patients with AMI.101
However, there is wide variation of the effect of reperfu-
sion therapy, and the infarct size is larger than 75% of the
ischemic zone despite successful coronary reperfusion in
25% of AMI patients.101 Patients with infarct size larger than
20% of the left ventricle at the acute phase of AMI were
also approximately 25% of the total patients. Interestingly,
the variation of infarct size cannot be explained by symptom-
to-treatment time alone. Collectively, it is clear that a novel
and potent therapy is necessary for at least approximately
25% of AMI patients in order to improve their clinical out-
comes.
Recently, 2 randomized clinical studies, AMISTAD II102
and J-WIND103 showed that administration of adenosine
and that of human atrial natriuretic peptide, respectively,
significantly reduced infarct size in AMI patients. However,
the protection afforded by these pharmacological agents was
not associated with improvement of prognosis or suppres-
Circulation Journal Vol.73, July 2009
GSK-3βin Cardioprotection
sion of ventricular remodeling after AMI. Possible reasons   
for the apparent discrepancy between infarct size limitation
and lack of clinical benefit were extensively discussed in   
our recent review.101 Nevertheless, it is notable that ‘signifi-   
cant’ limitation of infarct size might not always reduce mor-
tality after AMI or its surrogate markers such as ventricular
dimension.   
Myocardial necrosis is a major problem not only in coro-
nary artery disease but also in heart failure. Necrosis of car-
diomyocytes, in addition to their apoptosis, has been observed   
in animal models of heart failure.104 and in end-stage heart
failure of patients with dilated cardiomyopathy and aortic   
stenosis.105,106 Recent studies have shown that plasma tropo-
nin concentration increases during acute severe heart failure   
and during exacerbation of chronic heart failure, and their
prognosis correlates with the concentration of troponin.107–112   
However, no current therapy directly targets myocardial
necrosis in failing hearts.
 
GSK-3β Inhibitors for Clinical Application
Lithium chloride is the only GSK-3βinhibitor in clinical  
use for bipolar mood disorders at the present time.2,3 Find-
ings in experiments discussed in the present article indicate
that GSK-3β inhibitors are promising agents for use in  
adjunctive therapy with PCI for AMI patients. Side effects
of the agent for this use could be minimal, because a single  
dose administration of a GSK-3β inhibitor would be suffi-
cient to inhibit mPTP opening at the time of reperfusion.
However, chronic use of a GSK-3β inhibitor for heart fail-  
ure, for example, could be problematic because its effects
on ventricular hypertrophy and tumor growth might result
 
in adverse outcomes.2,3 Actually such serious side effects
have not been reported for chronic use of lithium chloride.
However, more specific and potent inhibitors of GSK-3β  
could have different side effects.
Although pre-clinical studies except for one using  
mice55,57,60,72,73,75 have shown that inhibitors of GSK-3β
could limit infarct size in animal models of AMI, efficacy
of the GSK-3βinhibitors has not yet been assessed in human
cardiac tissues. However, protection of the human heart was  
reportedly achieved by an inhibitor of mPTP, cyclosporine  
A,110 ischemic preconditioning,113–115 and postconditioning.116
If the roles of GSK-3βin mPTP opening, ischemic precon-
ditioning and postconditioning are similar between human,  
rat and rabbit hearts, we could expect that GSK-3β inhibi-
tors afford significant protection in human hearts as well.  
However, a recent preliminary study by Yang et al suggests
that the extent of myocardial protection by inhibition of
reperfusion injury is much less in the primate than in the rat
 
and rabbit because ischemic injury is more predominant as a
mechanism of myocardial necrosis in this species.117 Never-
theless, roles of GSK-3β in the human myocardium and
efficient strategies to directly or indirectly inhibit GSK-3β  
for protection of cardiomyocytes warrant further investiga-
tion.  
Acknowledgements
 
The scientific work of the authors is supported by Grants-in-Aid for Sci-
entific Research from the Japanese Society for the Promotion of Science.  
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