CARDIOVASCULAR JOURNAL OF AFRICA • Volume 25, No 3, May/June 2014
122
AFRICA
members of the cardioprotective SAFE pathway and S1P may act
via TNF
α
to activate STAT-3.
24
Using a cardiomyocyte-specific STAT-3 knockout mouse
model and the STAT-3 inhibitor, we demonstrated the requirement
of STAT-3 for S1P-induced preconditioning in a whole-organ
model. Although STAT-3 in other cell types of the heart has
also been implicated in ischaemic preconditioning, the current
results suggest that cardiomyocyte STAT-3 is required for
S1P-induced cardioprotection. This is supported by experiments
looking at ischaemic preconditioning, which showed that part
of the protective response mediated by endothelial STAT-3 was
caused by upregulation of cardiomyocyte-specific STAT-3.
32
Less
evidence is available on the preconditioning role of STAT-3 in
other cardiac cell types.
Cellular localisation of STAT-3 activation
S1P pre-treatment significantly increased nuclear levels of
phosphorylated STAT-3. Phosphorylation of STAT-3 is suggested
to increase translocation of STAT-3 from the cytoplasm to the
nucleus where it acts as a transcription factor. However, if STAT-
3 did translocate from the cytoplasm to the nucleus, one would
expect a concomitant increase in total STAT-3 in the nucleus and
possibly a decrease in total cytoplasmic STAT-3.
Our results do not show an increase in total nuclear STAT-3
or a decrease in cytosolic STAT-3. This may suggest either that
an increase in STAT-3 export from the nucleus to the cytoplasm
compensates for the movement of the phosphorylated form of
STAT-3 into the nucleus, and/or that phosphorylation occurs for
STAT-3 already present in the nucleus.
STAT-3 is best known as a transcription factor, however, the
results of transcription are unlikely to produce the protective
effects seen in these short-term experiments. This may suggest
that phosphorylated STAT-3 also plays a non-transcriptional role
in the nucleus, e.g. DNA repair in response to oxidative stress,
33
or
interaction with other signalling molecules within the nucleus.
34
S1P pre-treatment also significantly increased mitochondrial
levels of phosphorylated STAT-3. Recently, it has been suggested
that rather than the cytosolic or nuclear pool of STAT-3
accounting for the protective effects of pre- and postconditioning,
the mitochondrial pool of STAT-3 may also be important.
35
The
mechanism by which the mitochondrial STAT-3 acts remains
unknown. However evidence from other studies suggests that it
may affect cellular respiration and opening of the mitochondrial
permeability transition pore.
23,35,36
The dual site activation of STAT-3 is in agreement with
the findings of Somers
et al
.,
24
who found that S1P-induced
postconditioning caused an increase in STAT-3 activation in
the nucleus and mitochondrion. Despite these similar findings,
Somers
et al
.
24
observed a concurrent decrease in cytosolic STAT-
3 activation, which was not seen in the present study. The main
difference in the protocols used in these two studies was the time
at which S1P was administered. In S1P-induced preconditioning,
S1P was administered before ischaemia to a healthy heart under
physiological conditions. In S1P-induced postconditioning, the
stimulus was provided in a pathological (post-ischaemic) state.
The reduction of infarct size seen in the current study was
similar to that seen when S1P was given as a postconditioning
agent. This may suggest that the levels of STAT-3 activation in
the cytosolic fraction do not affect S1P-mediated protection but
it is possible that they could affect long-term recovery from
cardiovascular disease, such as remodelling.
However, it should be noted that the changes in activation of
STAT-3 seen in this study focused on phosphorylation levels seven
minutes after S1P treatment, which may not be representative
of the changes over time. Furthermore, the current study only
looked at phosphorylation of the serine residue of STAT-3.
Future studies should explore the changes in phosphorylation
of STAT-3 on both the serine and tyrosine residues over time in
response to S1P-induced pre- and postconditioning to confirm
different patterns of activation.
In humans with myocardial infarction, other cardiovascular
risk factors are normally present, such as hypertension and
diabetes. These may affect the ability of some pharmacological
agents to protect the heart.
37
The experiments described in this
article were carried out on healthy animals. Therefore, it is
imperative that S1P-induced preconditioning be confirmed in
animal models that include these co-morbidities.
Conclusion
Our data strongly suggest that the cardioprotective effects of
S1P-induced preconditioning may be mediated by dual activation
of STAT-3 in the nucleus and mitochondria. Our data provide
a unique therapeutic opportunity to target survival against
ischaemia–reperfusion injuries, especially since S1P and its
sphingolipid pathway form part of the high-density lipoproteins
(HDL). Addition of S1P to already existing synthetic HDL may
be considered a therapeutic option in the prevention of cardiac
damage associated with ischaemia–reperfusion.
This work was supported in part by the National Research Foundation of
South Africa, the Inter-University Cape Heart Group of the South African
Medical Research Council and the Servier Heart Failure Project. Dr RF
Kelly-Laubscher was supported by the Claude Leon Foundation and Prof S
Lecour was partly supported by the Medical Research Council Career Award.
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