CARDIOVASCULAR JOURNAL OF AFRICA • Volume 25, No 3, May/June 2014
118
AFRICA
Cardiac preconditioning with sphingosine-1-phosphate
requires activation of signal transducer and activator of
transcription-3
Roisin F Kelly-Laubscher, Jonathan C King, Damian Hacking, Sarin Somers, Samantha Hastie,
Tessa Stewart, Aqeela Imamdin, Gerald Maarman, Sarah Pedretti, Sandrine Lecour
Abstract
Aims:
Sphingosine-1-phosphate (S1P) is a cardioprotec-
tive agent. Signal transducer and activator of transcription 3
(STAT-3) is a key mediator of many cardioprotective agents.
We aimed to explore whether STAT-3 is a key mediator in
S1P-induced preconditioning.
Methods:
Langendorff-perfused hearts from Wistar rats
and wild-type or cardiomyocyte-specific STAT-3 knockout
mice were pre-treated with S1P (10 nmol/l), with or without
the STAT-3 pathway inhibitor AG490, before an ischaemia–
reperfusion insult. Triphenyltetrazolium chloride and Evans
blue staining were used for the determination of infarct size.
Western blot analysis was carried out on the S1P pre-treated
hearts for detection of cytosolic, nuclear and mitochondrial
phosphorylated and total STAT-3 proteins.
Results:
Pre-treatment with S1P decreased the infarct size
in isolated rat (5
±
3% vs control 26
±
8%,
p
<
0.01) and
wild-type mouse hearts (13
±
1% vs control 33
±
3%,
p
<
0.05). This protective effect was abolished in the rat hearts
pre-treated with AG490 (30
±
10%,
p
=
ns vs control) and in
the hearts from STAT-3 knockout mice (35
±
4% vs control 30
±
3%,
p
=
ns). Levels of phosphorylated STAT-3 were signifi-
cantly increased in both the nuclear (
p
<
0.05 vs control)
and mitochondrial (
p
<
0.05 vs control) fractions in the S1P
pre-treated hearts, but remained unchanged in the cytosolic
fraction (
p
=
ns vs control).
Conclusion:
These novel results demonstrate that pharma-
cological preconditioning with S1P in the isolated heart is
mediated by activation of mitochondrial and nuclear STAT-3,
therefore suggesting that S1P may be a novel therapeutic
target to modulate mitochondrial and nuclear function in
cardiovascular disease in order to protect the heart against
ischaemia–reperfusion.
Keywords:
STAT-3, cardioprotection, preconditioning, sphingo-
sine-1-phosphate, myocardial infarction
Submitted 9/12/13, accepted 31/3/14
Cardiovasc J Afr
2014;
25
: 118–123
DOI: 10.5830/CVJA-2014-016
Signal transducer and activator of transcription 3 (STAT-3) is
a downstream mediator of many cardioprotective agents, most
notably, of ischaemic pre- and postconditioning,
1-5
i.e. protection
brought about by repeated bouts of brief ischaemia before
(preconditioning) and after (postconditioning) a prolonged
period of ischaemia. Many pharmacological conditioning agents
such as adenosine,
6
opioids,
7
erythropoietin,
8
ethanolamine,
9
melatonin,
10
leptin,
11
and tumour necrosis factor alpha (TNF
α
)
4,12
also protect via the activation of STAT-3.
These findings have led to the description of a novel
pathway involved in both mechanical and pharmacological
preconditioning: ‘survivor activating factor enhancement’
(SAFE).
5,13
This study focused on the role of the SAFE pathway,
more specifically STAT-3, in S1P-induced preconditioning.
Sphingolipids and their metabolites are important signalling
molecules in the heart. There is growing evidence that major
components of the sphingolipid pathway, such as ceramide,
sphingosine and sphingosine-1-phosphate (S1P) can protect
the heart against an ischaemia–reperfusion insult, but the exact
mechanism remains unclear.
14-18
Signalling molecules such as
protein kinase C
ε
,
15
the pro-survival protein kinase-B/Akt
18
and extracellular signal-regulated kinase 1/2, which are major
components of the ‘reperfusion injury salvage kinase’ pathway
(RISK),
19,20
are implicated in S1P-induced preconditioning.
Recent data have demonstrated that S1P upregulates STAT-
3 phosphorylation in other organ systems both
in vitro
21
and
in vivo
.
22
It was also demonstrated that STAT-3 mediates
S1P-induced protection against doxorubicin-induced toxicity in
isolated ventricular cardiomyocytes.
23
Similarly, pharmacological
postconditioning with S1P protects isolated mouse hearts against
a global ischaemia–reperfusion insult via STAT-3 activation in
the mitochondrion and nucleus,
24
therefore suggesting a link
between S1P and STAT-3, and hence activation of the SAFE
pathway by S1P. However whether the same mechanism of
protection is involved in S1P-induced preconditioning remains
unknown.
In this study, we used cardiomyocyte-specific STAT-3
knockout mice and a STAT-3 pathway inhibitor to investigate the
role of STAT-3 in the cardioprotective effect of pharmacological
preconditioning with S1P against both global and regional
ischaemia–reperfusion injury.
Hatter Institute for Cardiovascular Research in Africa,
Chris Barnard Building, Medical School Campus,
University of Cape Town, Cape Town, South Africa
Roisin F Kelly-Laubscher, PhD,
Jonathan C King, MMed
Damian Hacking
Sarin Somers
Samantha Hastie
Tessa Stewart
Aqeela Imamdin
Gerald Maarman
Sarah Pedretti
Sandrine Lecour, PhD
