CARDIOVASCULAR JOURNAL OF AFRICA • Volume 25, No 3, May/June 2014
AFRICA
121
To investigate the role of STAT-3 in S1P-induced
preconditioning, we administered the Jak/STAT-3 inhibitor,
AG490 (Fig. 3). PerfusionofAG490abolished thecardioprotective
effect of S1P (30
±
10% vs ischaemic control,
p
=
ns,
n
=
6).
There was no significant difference in the size of the area at risk
between the four groups (data not shown).
After 30 minutes of regional ischaemia and 120 minutes of
reperfusion, the LVDP, heart rate and coronary flow were not
significantly different between the four groups (Table 1). No
significant differences in heart rate were found within any group
at the different time points measured.
As expected, all groups showed a significant decrease (
p
<
0.05) in LVDP by the end of the reperfusion period compared
to pre-ischemic values. Interestingly, only groups treated with
AG490 demonstrated a significant decrease in LVDP five
minutes into reperfusion compared to baseline values (
p
<
0.05).
All groups except the control group demonstrated a significantly
decreased coronary flow rate by the end of reperfusion compared
to baseline values (
p
<
0.05).
S1P induced an increase in phosphorylated STAT-3
in the nucleus and mitochondrion
Western blot analysis of tissue extracted from isolated rat hearts
revealed an increase in nuclear (control 1 vs S1P; 3.42
±
0.83
arbitrary units,
p
<
0.05) and mitochondrial (control 1 vs S1P;
1.52
±
0.10 arbitrary units,
p
<
0.05) phosphorylated/total STAT-
3 after S1P pre-treatment (Fig. 4B, C). S1P pre-treatment did not
significantly alter the cytoplasmic phosphorylation of STAT-3
(control 1 vs S1P; 1.00
±
0.27 arbitrary units,
p
=
ns). There was
no significant change in total STAT-3 in the cytosolic, nuclear or
mitochondrial fractions.
Discussion
Our present study demonstrates that pre-treatment with S1P
protected against ischaemia–reperfusion injury via the activation
of STAT-3. This was evidenced by several of our findings. Firstly,
S1P-induced preconditioning was inhibited in STAT-3 knockout
mice. Secondly, S1P-induced preconditioning was inhibited
by the STAT-3 inhibitor, AG490. Thirdly, S1P upregulated the
phosphorylation of both nuclear and mitochondrial STAT-3.
S1P can activate the JAK/ STAT-3 pathway
S1P is now recognised as a cardioprotective agent both
in vivo
and
ex vivo.
17,18,29,30
S1P can induce cardioprotection as a pre- or
postconditioning stimulus.
14,17,18,31
Furthermore, S1P mediates
the cardioprotective effects of other preconditioning agents, e.g.
TNF
α
,
4
and ethanolamine.
9
In fact, TNF
α
and STAT-3 are both
Table 1. Haemodynamic parameters of isolated
rat hearts exposed to regional ischaemia and
S1P-induced preconditioning
Parameters
Pre-
ischaemia
Ischaemia
(5 min)
Reperfusion
(5 min)
Reperfusion
(120 min)
LVDP (mmHg)
IC
86
±
7
54
±
10
69
±
8
46
±
8*
S1P
83
±
5
35
±
12
71
±
7
45
±
7*
S1P + AG
99
±
3
65
±
15
81
±
3
67
±
3*
AG
92
±
5
57
±
17
75
±
4
66
±
4*
Heart rate (bpm)
IC
287
±
18 263
±
43 270
±
14 293
±
11
S1P
280
±
20 250
±
55 288
±
42 268
±
28
S1P + AG
273
±
17 290
±
60 297
±
18 283
±
21
AG
293
±
18 270
±
64 240
±
15 257
±
24
Coronary flow (ml/min)
IC
10.8
±
1.4
8
±
5
11.2
±
1.7 7.8
±
1.9
S1P
9.7
±
0.9
4
±
1
9.8
±
0.8 5.9
±
0.8*
S1P + AG
9.8
±
1.0
5
±
1
8.4
±
0.6 5.8
±
0.7*
AG
8.1
±
0.3
5
±
1
8.2
±
0.2 5.0
±
0.2*
Parameters measured prior to ischaemia (pre-ischaemia), at five minutes
into ischaemia and at five minutes and 120 minutes after reperfusion,
respectively. IC
=
ischaemic control, S1P
=
sphingosine-1-phosphate,
AG
=
AG490, LVDP
=
left ventricular developed pressure, (
n
=
6 per
group.*
p
<
0.05 reperfusion at 120 minutes vs pre-ischaemia).
4
3
2
1
0
Control
S1P
P-STAT-3/Total STAT-3
Cytoplasm
P-STAT-3 (ser)
Total STAT-3
Beta actin
Control
S1P
4
3
2
1
0
Control
S1P
P-STAT-3/Total STAT-3
*
Nucleus
P-STAT-3 (ser)
Total STAT-3
Beta actin
Control
S1P
4
3
2
1
0
Control
S1P
P-STAT-3/Total STAT-3
*
Mitochondria
P-STAT-3 (ser)
Total STAT-3
VDAC
Control
S1P
Fig. 4.
S1P pre-treatment increased phosphorylation of nuclear and mitochondrial STAT-3. Representative Western blots demon-
strating levels of phosphorylated-STAT-3/total STAT-3 in (A) the cytoplasm, (B) the nucleus, and (C) the mitochondria after
seven minutes of S1P pre-treatment in isolated rat hearts (
n
=
4 per group, *
p
<
0.05 vs control).
A
B
C
