Download Protective effects of sphingosine-1

Cardiovascular Research (2009) 83, 285–293
doi:10.1093/cvr/cvp137
Protective effects of sphingosine-1-phosphate
receptor agonist treatment after myocardial
ischaemia–reperfusion
Ulrich Hofmann*, Natalie Burkard, Carolin Vogt, Annemarie Thoma, Stefan Frantz, Georg Ertl,
Oliver Ritter, and Andreas Bonz
Department of Internal Medicine I, University of Würzburg, Medizinische Klinik und Poliklinik I, Josef-Schneider-Str. 2,
D-97080 Würzburg, Germany
Received 18 July 2008; revised 21 April 2009; accepted 27 April 2009; online publish-ahead-of-print 5 May 2009
Time for primary review: 24 days
KEYWORDS
Sphingosine-1-phosphate;
FTY720;
Myocardial ischaemia–
reperfusion injury
Aims Several experimental studies have demonstrated protection against cardiac ischaemia–reperfusion
injury achieved by pre-treatment with exogenous sphingosine-1-phosphate (S1P). We tested the hypothesis
that pharmacological S1P receptor agonists improve recovery of function when applied with reperfusion.
Methods and results Isolated rat cardiomyocytes were stimulated with exogenous S1P, the selective S1P1
receptor agonist SEW2871, or the S1P1/3 receptor agonist FTY720. Western blot analysis was performed
to analyse downstream signalling pathways. Ischaemia–reperfusion studies were conducted in rat cardiomyocytes, isolated Langendorff-perfused rat hearts, and in human myocardial muscle strip preparations
to evaluate the effect of S1P receptor agonists on cell death and recovery of mechanical function. All S1P
receptor agonists were able to activate Akt. This was associated with transactivation of the epidermal
growth factor receptor. In isolated cardiomyocytes, selective stimulation of the S1P1 receptor by
SEW2871 induced protection against cell death when administered either before or after ischaemia–reperfusion. In isolated rat hearts, treatment with FTY720 during reperfusion attenuated the rise in left ventricular end-diastolic pressure (LVEDP) and improved the recovery of left ventricular developed pressure without
limiting infarct size. However, selective S1P1 receptor stimulation did not improve functional recovery but
rather increased LVEDP. Additional experiments employing a human myocardial ischaemia–reperfusion
model also demonstrated improved functional recovery induced by FTY720 treatment during reperfusion.
Conclusion Pharmacological S1P receptor agonists have distinct effects on ischaemia–reperfusion injury.
Their efficacy when applied during reperfusion makes them potential candidates for pharmaceutical postconditioning therapy after cardiac ischaemia.
1. Introduction
It has been demonstrated recently that cardioprotective
pathways that are important for preconditioning can also
be induced effectively by ischaemic postconditioning or
pharmaceutical postconditioning treatment during the
reperfusion period.1,2 With these findings in mind, there is
now increasing interest to develop pharmacological reperfusion strategies to reduce infarct size and improve clinical
outcome after acute myocardial infarction and subsequent
reperfusion.
The lysophospholipid sphingosine-1-phosphate (S1P) is a
high-nanomolar serum constituent. S1P is released during
platelet activation,3 by immune cells during inflammatory
activation4 and by endothelial cells.5 S1P receptors are
widely expressed on different cell types.6 S1P1 receptor
* Corresponding author. Tel: þ49 931 201 1; fax: þ49 931 201 36280.
E-mail address: [email protected]
stimulation induces peripheral lymphocytopenia and was
demonstrated to be a new promising therapeutic principle
in several models of inflammation and autoimmunity.7
S1P and the sphingosine kinase activator GM-1 confer protection against ischaemia–reperfusion injury in mouse
hearts when given before ischaemia.8 There is further
experimental evidence that sphingosine kinase activation
contributes to the beneficial effect of ischaemic preconditioning.9 Human10 as well as rodent11 myocytes express
the S1P receptors S1P1 and S1P3. Both receptor subtypes
have recently been associated with cardioprotective signalling.12–14 In different cell types, experimentally applied S1P
(via S1P receptors) activated downstream protein kinases,
especially Akt and ERK.15 Akt and ERK are pro-survival
kinases, which were recently identified as intracellular
mediators of ischaemic or pharmacological postconditioning. Activation of Akt and ERK is a common mechanism
downstream of diverse cardioprotective interventions.16
Published on behalf of the European Society of Cardiology. All rights reserved. & The Author 2009.
For permissions please email: [email protected].
286
Several synthetic S1P receptor modulators are now available. FTY720 is a substrate of sphingosine kinase 2. Its phosphorylated metabolite (FTY720-P) acts as a high-affinity
agonists at four of the five S1P receptors, namely S1P1,
S1P3, S1P4, and S1P5 (Kd , 10 nM) but not S1P2 (Kd .
10 mM).17 FTY720 was demonstrated to reduce both
hepatic and renal ischaemia–reperfusion injury in vivo.18,19
SEW2871 also effectively attenuates renal ischaemia
reperfusion injury.20 The compound is a selective S1P1
receptor agonist, which recapitulates S1P effects on S1P1
receptors and does not activate S1P2, S1P3, S1P4, or S1P5
receptors at micromolar concentrations.21
In the present study, we tested the hypothesis that
pharmacological S1P receptor agonists can improve functional recovery when applied with reperfusion after myocardial ischaemia.
U. Hofmann et al.
2.4 Isolated rat heart model
Our setup for Langendorff experiments was previously described in
detail.24 After equilibration, global normothermic hypoxia was
induced by perfusion stop. Constant temperature was maintained
throughout the whole experiment by immersing the heart in the
perfusion medium (Krebs–Henseleit buffer) at 378C. Following
30 min of global ischaemia, hearts were reperfused for 90 min.
Stimulation protocols are described in the Supplementary material
online, Methods.
2.5 Infarct size measurement
At the end of the reperfusion period, hearts were cut into four to six
parallel transverse slices, which were stained with triphenyl tetrazolium chloride (TTC) for 30 min. After fixation in 4% formaldehyde,
slices were photographed by a digital camera. Total area and area of
necrosis (non-red ¼ TTC-negative) was quantified by planimetry
(Image J software).
2. Methods
2.6 TUNEL staining
The investigation conforms to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health
(NIH Publication No. 85-23, revised 1996) and with the principles
outlined in the Declaration of Helsinki. Local government approved
of the animal experiments. Use of human specimen was reviewed by
the Ethics Board of the medical faculty at the University of
Würzburg.
For TUNEL staining, cryo-sections were prepared from the
paraformaldehyde-fixed apical part of each heart. Sections were
then stained with a commercially available TUNEL kit as indicated
by the manufacturer (Roche diagnostics). Nuclei were counterstained with DAPI. The percentage of TUNEL-positive nuclei was calculated as sum of all double-positive nuclei divided by the sum of all
nuclei per high-power field. At least 1500 nuclei were analysed for
each heart. For identification of cardiomyocytes, counterstaining
by Texas Red-labelled Phalloidin (Invitrogen) was performed after
TUNEL staining.
2.1 Cardiomyocyte stimulation procedures
Cardiac myocytes were isolated and cultured from neonatal rat
hearts based on the method reported previously.22 Before stimulation, cardiomyocytes were cultured serum-free for at least 8 h
to avoid unspecific receptor stimulation by sphingolipids, which
can be found in serum preparations.
One millimolar stock solutions of SEW2871, S1P, and FTY720phosphate were prepared in acidified dimethylsulfoxide (containing
5% 1 N HCl). Stock solutions were made of AG 1478 (3 mM), PP2
(3.3 mM), GM 6001 (2.6 mM), Wortmannin (2.5 mM), and HB-EGF
(50 mg/mL) by dissolving the compounds in dimethylsulfoxide.
SEW2871 was supplied by Cayman Chemicals, Ann Arbor, MI, USA.
FTY720-P was a generous gift from Novartis, Basel, and AG1478,
PP2, and GM 6001 were from Calbiochem. All other compounds
were from Sigma, Deisenhofen.
2.2 Assessment of cell injury after simulated
ischaemia–reperfusion
For ischaemia–reperfusion studies, cardiomyocytes were set on 1%
foetal calf serum 24 h prior to simulated ischaemia–reperfusion.
The protocol has been described in detail previously by another
group.23 In brief, cardiomyocytes cultured on 24-well plates (5 105 cells/well) were made hypoxic for 6 h in a humidified incubator
containing an atmosphere of 5% CO2 and 95% N2 (378C). Cells were
then reoxygenated for 2 h. Supernatant and trypsinized cells from
individual wells were collected and centrifuged. The cell pellets
were washed and stained with Annexin V-FITC and propidium
iodide according to the manufacturer (BD Bioscience Annexin-FITC
FACS staining kit). Cells were analysed on a BD FACSCalibur flow cytometer. Data were analysed by cellquest software.
2.3 Western blot analysis
Protein phosphorylation was analysed by standard western blotting
procedures followed by probing with phospho-specific antibodies.
More details are provided in the Supplementary material online,
Methods.
2.7 Human ischaemia–reperfusion model
The experimental setup (Scientific instruments Heidelberg, Germany)
and the fibre mounting procedure were previously described in
detail.25 The ischaemia–reperfusion and stimulation protocols are
described in the Supplementary material online, Methods.
2.8 Statistical analysis
Data are reported as mean + SE. Statistical analysis was performed
using WinStat software (Benecke & Schwippert, Staufen, Germany)
working on Excel spreadsheets. Multiple comparisons were done by
ANOVA analysis for repeated measurements. Comparison of variables between two groups was done by using the unpaired Student’s
t-test. P-values ,0.05 were considered significant.
3. Results
3.1 S1P receptor agonists induce Akt activation
Different cardioprotective signalling pathways converge to
PI3-kinase-mediated Akt activation. We investigated
whether S1P receptor agonists stimulate Akt activity in cardiomyocytes. Akt activation was assessed by screening for
(Ser473)-phosphorylated Akt in S1P, SEW2871, or
FTY720-P-stimulated rat cardiomyocytes by western blotting. The physiological S1P receptor ligand S1P, as well as
the S1P1/3 receptor agonist FTY720-P and the selective
S1P1 receptor agonist SEW2871, significantly induced Akt
phosphorylation in cardiomyocytes (S1P 100 nM: 5.7 + 1.3,
FTY720-P 100 nM: 4.9 + 1.2, SEW 10 mM: 5.2 + 1.1-fold
control, P , 0.05 vs. control, Figure 1B). After 15 min of
incubation, Akt phosphorylation had reached a maximum
(data not shown). Further western blot analyses of cardiomyocytes were therefore performed after 15 min of
stimulation.
Cardio-protective effects of sphingosine-1-phosphate receptor agonists
287
Figure 1 Phosphorylation status of Akt (p-Akt) and EGFR (p-EGFR) was determined by western blot analysis. Band intensities were normalized for GAPDH and
results were expressed as x-fold control. Cardiomyocytes were co-incubated with inhibitors for Src kinase (PP2 3.3 mM), EGFR tyrosin kinase (AG: AG 1478 3 mM),
metalloproteinases (GM: GM 6001 2.6 mM), and PI3-kinase (Wort: Wortmannin 2.5 mM). (A) Representative western blots. (B) p-Akt, *P , 0.05 vs. control, n ¼ 7.
(C ) p-EGFR, *P , 0.05 vs. control, n ¼ 6. (D–G) p-Akt: *P , 0.05 vs. S1P 100 nM, n ¼ 6; #P , 0.05 vs. SEW 10 mM, n ¼ 7; and ‡P , 0.05 vs. FTY720-P 100 nM, n ¼ 8.
(H ) Schema of the suggested S1P receptor downstream events.
3.2 S1P receptor agonists transactivate the EGF
receptor
The stimulating effect of S1P, FTY720-P, and SEW2871 on Akt
phosphorylation was sensitive to PI3K inhibition by Wortmannin. This suggests that the pathway downstream of the S1P
receptor leading to Akt phosphorylation engages PI3-kinases
(S1P þ Wort: 1.0 + 0.4, FTY720-P þ Wort: 1.4 + 0.3, SEW þ
Wort: 0.4 + 0-fold control, P , 0.05 vs. S1P, FTY720, or
SEW2871, Figure 1D). As previously demonstrated, S1P
receptors are able to transactivate the EGF receptor
(EGFR). We therefore assessed EGFR tyrosine kinase phosphorylation in response to S1P receptor agonists.26 Again,
both S1P and S1P receptor agonist induced phosphorylation
of the EGFR, indicating that this receptor is transactivated
by S1P receptors (S1P 100 nM: 2.9 + 0.7, FTY720-P 100 nM:
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U. Hofmann et al.
Figure 1 Continued.
2.7 + 0.7, SEW 10 mM: 2.5 + 0.8-fold control, P , 0.05 vs.
control ¼ 1, Figure 1C). To further elucidate whether Akt activation via the S1P receptor engages the EGFR, we incubated
cardiomyocytes with an EGFR tyrosine kinase inhibitor
(AG1478). AG1478 significantly attenuated S1P, SEW2871, and
FTY720-P-induced Akt phosphorylation (S1P þ AG: 0.5 + 0.3,
FTY720-P þ AG: 1.0 + 0.5, SEW þ AG: 0.7 + 0.2-fold control,
P , 0.05 vs. S1P, FTY720-P, or SEW2871, Figure 1E). Myocytes
incubated with the endogenous EGFR ligand HB-EGF were
used as a positive control. As supposed, HB-EGF induced both
EGFR and Akt phosphorylation (Figure 1B and C).
Different G-protein-coupled receptors are capable of
transactivating EGFR by metalloproteinase-mediated shedding of HB-EGF. We therefore analysed EGFR and Akt phosphorylation status after co-incubation with GM 6001, a
broad-spectrum metalloproteinase inhibitor that has been
shown to inhibit HB-EGF shedding.26 The metalloproteinase
inhibitor significantly attenuated SEW2871-induced Akt
phosphorylation. This indicates that the S1P1 receptor activates Akt by a mechanism involving metalloproteinasemediated HB-EGF shedding and EGFR activation (S1P þ GM:
2.3 + 1.1, FTY720-P þ GM: 1.3 + 0.1, P ¼ n.s.; SEW þ GM:
0.6 + 0.3-fold, P , 0.05 vs. SEW2871, Figure 1F).
To further test the hypothesis that the Src kinase is also
involved in EGFR-mediated Akt activation, we co-incubated
myocytes with the selective Src kinase inhibitor PP2. The
experiments revealed that S1P, FTY720-P, and SEW2871induced Akt phosphorylation was sensitive to Src kinase inhibition (S1P þ PP2: 0.5 + 0.1, FTY720-P þ PP2: 1.0 + 0.3,
SEW þ PP2: 0.5 + 0.1-fold, P , 0.05 vs. S1P, FTY720-P, and
SEW2871, Figure 1G).
In summary, the results show that EGFR transactivation by
S1P1 receptor engages HB-EGF cleavage. Both S1P1 and S1P3
receptors induced EGFR transactivation and downstream Akt
phosphorylation additionally involves Src kinase activity
(Figure 1H).
3.3 SEW2871 inhibits ischaemia–
reperfusion-induced cell death in cardiomyocytes
In order to assess the functional relevance of S1P
receptor-mediated Akt activation for protection from
Cardio-protective effects of sphingosine-1-phosphate receptor agonists
289
Figure 2 Representative density blots of Annexin V-FITC/propidium iodide-stained cardiomyocytes (A). Necrotic cells are Annexin V/propidium iodide-positive
(upper right quadrant, cumulative results depicted in (C )] and apoptotic/early necrotic cells are Annexin V-positive [lower right quadrant, cumulative results in
(B)]. In (B and C ), data are presented as the percentage of all gated cells (R1). Ten micromolar SEW2871 both added before simulated ischaemia (SEW 10 I, n ¼
14) and with reperfusion (SEW 10 R, n ¼ 14) protected against cell death. The preconditioning effect of SEW2871 was sensitive to PI3K inhibition by Wortmannin
(SEW 10 I þ Wort I, n ¼ 3). *P , 0.05 vs. control experiments.
ischaemia–reperfusion-induced cell death, we studied
the effect of the compounds on necrosis/apoptosis in
reperfused rat cardiomyocytes. SEW2871, when administered at the onset of reperfusion, protected myocytes
against necrosis (Annexin-V/propidium iodide doublepositive cells: 3.4 + 0.4%, P , 0.05 vs. control 4.9 +
0.4%, Figure 2). Furthermore, administration of SEW2871
significantly protected against apoptotic/early necrotic
cell death (Annexin V-positive cells: 39.7 + 4.4% when
given before ischaemia and 39.2 + 3% when given with
reperfusion, P , 0.05 vs. without treatment 49.8 +
3.5%). The preconditioning effect of SEW2871 was
reversed
by
Wortmannin
indicating
that
PI3-kinase-mediated Akt activation is required for protection. FTY720-P did not protect against ischaemia–
reperfusion-induced cell death.
3.4 FTY720 improves recovery of function in rat
hearts
After demonstrating that S1P1 receptor stimulation protects
cardiomyocytes against cell injury induced by ischaemia–
reperfusion, we thought to investigate the functional
effect of S1P receptor agonists on recovery of function.
Hence, isolated rat hearts were treated with SEW2871 or
FTY720 at the onset of reperfusion. Mean left ventricular
developed pressure (LVDP) was 129.9 + 4.6 mmHg at baseline before ischaemia. After 30 min of ischaemia and
90 min of reperfusion, LVDP was 33.3 + 7.6 mmHg in
control hearts, 29.5 + 11.3 mmHg in 50 nM FTY720 (non-
significant vs. control), and 56.5 + 5.4 mmHg (P , 0.05
vs. control) in 500 nM FTY720-treated hearts (Figure 3A).
Accordingly, left-ventricular end-diastolic pressure (LVEDP)
after 90 min of reperfusion was 50.7 + 4.3 mmHg in
untreated control hearts, but significantly lower in 500 nM
FTY720-treated hearts (35.3 + 3.6 mmHg, P , 0.05 vs.
control) (Figure 3B). Fifty nanomolar FTY720 did not attenuate the rise in LVEDP. Neither 50 nor 500 nM FTY720 showed a
significant effect on coronary flow (Figure 3C) or heart rate
(data not shown) during reperfusion.
In contrast to FTY720, the selective S1P1 receptor agonist
SEW2871 was neither able to improve the recovery of LVDP
nor to reduce LVEDP. After 90 min of reperfusion, LVDP was
37.9 + 7.2 mmHg in the control and 35.4 + 7.6 mmHg in
the 500 nM SEW2871 group (Figure 3D). LVEDP was even
increased in SEW2871-treated hearts (n.s., Figure 3E).
Western blot analysis of myocardial lysates revealed a significant activation of Akt in the 500 nM FTY720 group compared with control hearts after 30 (Figure 4B) and 90 min
(Figure 4C) of reperfusion (p-Akt 0.9 + 0.1 vs. control 0.5 +
0.1, P , 0.05). Fifty nanomolar FTY720 showed no effect.
Reperfusion with FTY720 for only 5 min was not sufficient to
activate Akt (Figure 4A). SEW2871 only transiently activated
Akt as revealed after 30 min (Figure 4B). FTY720 treatment
had no effect on eNOS phosphorylation (data not shown).
Neither FTY720 nor SEW2871 was able to activate ERK.
To study whether the observed improved recovery of function can be attributed to attenuated cell death, infarct size
was determined in reperfused Langendorff hearts. As quantified by planimetry after TTC staining, neither FTY720 nor
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U. Hofmann et al.
Figure 3 Effect of FTY720 (A–C ) or SEW2871 (D–F ) on the recovery of mechanical function in rat hearts subjected to 30 min of no-flow ischaemia and 90 min of
reperfusion. Isolated hearts were perfused in a Langendorff apparatus with KHS buffer. FTY720 or SEW2871 was infused continuously starting at reperfusion. Data
are presented as mean + SEM for KHS (n ¼ 7), FTY 50 nM (n ¼ 5), FTY 500 nM (n ¼ 7), control (n ¼ 7), and SEW (n ¼ 7). *P , 0.05 vs. control (KHS).
SEW2871 was able to significantly reduce infarct size.
However, the treatment of rat hearts by either FTY720 or
SEW2871 during reperfusion significantly reduced the frequency of TUNEL-positive cells (FTY 50 nM: 2.5 + 0.5, FTY
500 nM: 1.4 + 0.3, SEW2871 500 nM 0.5 + 0.2, P , 0.05 vs.
control: 5.3 + 0.82, Figure 5). We could clearly identify
the majority of TUNEL-positive cells as cardiomyocytes.
3.5 FTY720 improves recovery of function in
human myocardium
As there might be differences in subtype-specific receptor
expression between rodent and human myocardium,10 we
decided to do some further experiments in human myocardium to confirm the relevance of our findings in isolated
rat hearts. The recovery of mechanical function of human
right atrial muscle strips was studied after 90 min of simulated ischaemia and 120 min of reperfusion. Preconditioning
by 5 min of no-flow ischaemia, followed by 5 min of normoxic reperfusion, before index ischaemia significantly
improved the recovery of developed force (4.3 + 1.6 vs.
13.0 + 3.6% baseline isometric force, P , 0.05) after
120 min of reperfusion (Figure 6A). Comparable to the
effect of ischaemic preconditioning, administration of
1 mM FTY720 during reperfusion significantly improved the
recovery of developed pressure (19.8 + 5.2 vs. control:
4.3 + 1.6%, P , 0.05, Figure 6A).
In the human atrial preparations, FTY720 significantly
increased ERK 1/2 phosphorylation but did not influence
Akt phosphorylation status (ERK1: 1.0 + 0.0 vs. control
0.7 + 0.1, P , 0.05; ERK2: 0.7 + 0.0 vs. control 0.5 + 0.1,
P , 0.05, Figure 6B).
Cardio-protective effects of sphingosine-1-phosphate receptor agonists
291
Figure 4 Western blot analysis of Akt phosphorylation status 5 (A), 30 (B), and 90 min (C ) after start or reperfusion. At 30 and 90 min (E: *P , 0.05 vs. control,
n ¼ 7), 500 nM FTY720 significantly increased Akt phosphorylation, whereas treatment for 5 min did not alter p-Akt levels (D). Akt phosphorylation was only transiently increased by SEW2871 after 30 min (B).
4. Discussion
The effects of S1P receptor agonists on contractile function
and cell viability in myocardial tissue might result from the
activation of S1P receptors on different cell types. Therefore, we started our investigations on the therapeutic
potential of S1P receptor agonists with experiments conducted in isolated cardiomyocytes aiming to establish their
effect on intracellular signalling in cardiomyocytes. Here,
we could show that the endogenous S1P1 and S1P3 receptor
ligand (S1P), the selective synthetic S1P1 receptor agonist
(SEW2871), and combined S1P1 and S1P3 receptor stimulation by the compound FTY720-P induce Akt phosphorylation in cardiomyocytes.
Different previous studies reported a cardioprotective
effect of S1P when applied before or during ischaemia.8,13,27
The beneficial effects in mouse myocardium were attributed
to either an S1P113 or S1P3 receptor12-mediated pathway
involving Akt activation. Recent experiments in S1P2 and
S1P3 receptor knockout mice further suggested a possible
role for S1P2 in conferring protection by activating Akt.14
Akt downstream pathways were implicated in cardioprotective signalling induced by ischaemic preconditioning,28
ischaemic postconditioningn29 or pharmacological postconditioning.30 Both kinases, Akt and ERK, were shown to
reduce mitochondrial membrane transition pore opening
and caspase activation during reperfusion, events contributing to ischaemic cell death.29 Akt activation in cardiomyocytes
further
protects
cardiomyocytes
from
hypoxia-induced dysfunction as indicated by reduced contracture and normalized calcium handling.31
Cardioprotection mediated by G-protein-coupled receptors also involves transactivation of EGFRs.32 As our findings
clearly indicated that both S1P1 and S1P3 receptors are
capable of activating Akt in cardiomyocytes, we studied
the possible involvement of receptor transactivation in activating Akt. We could demonstrate that Akt activation via S1P
receptors is indeed mediated by EGFR transactivation. Akt
phosphorylation induced by S1P or FTY720-P, which are
both agonists at S1P1 as well as S1P3 receptors, was sensitive to Src inhibition indicating that Src mediates EGFR
transactivation. However, Akt activation induced by the
selective S1P1 receptor agonist was additionally sensitive
to metalloproteinase inhibition. This suggests that S1P1
receptor-mediated transactivation of the EGFR engages an
additional mechanism involving HB-EGF shedding from the
plasma membrane by metalloproteinases.
After demonstrating that S1P receptor agonists can
induce cardioprotective Akt signalling, we examined both
S1P receptor modulators regarding their therapeutic potential as postconditioning mimetics. In isolated cardiomyocytes, the selective S1P1 receptor agonist SEW2871
protected against cell death, when applied either before
or immediately after ischaemia. The S1P1 and S1P3 receptor agonist FTY720 was ineffective in attenuating cell death
of isolated cardiomyocytes, however. On the other hand,
FTY720 significantly reduced LVEDP and preserved LVDP,
whereas SEW2871 even increased LVEDP in rat hearts. As
there are inconsistent data in the literature concerning
the cell-type-specific expression pattern of S1P1 and S1P3
receptors in human and rodent myocardium,10,11,33,34 we
performed additional experiments in human myocardium
to confirm the relevance of our findings in rat myocardium.
Here, we demonstrated an analogous effect of FTY720 on
functional recovery in the higher dose group. Availability
of the active, phosphorylated form of FTY720 depends on
sphingosine kinase activity. It is therefore difficult to
292
Figure 5 In ischaemia-reperfused rat hearts, the percentage of TUNELpositive nuclei was determined as the sum of double-positive-stained nuclei
(TUNEL þ DAPI) divided by the sum of all nuclei. Both FTY720 (A) and
SEW2871 (B) significantly reduced the number of TUNEL-positive cells (*P ,
0.05 vs. individual control group, n ¼ 7 hearts/group).
extrapolate the in vivo dose required for comparable
effects from ex vivo experiments.
The observed induction of Akt phosphorylation in Langendorff hearts by FTY720 probably accounts for the significant
reduction of TUNEL-positive cells. However, the frequency
of TUNEL-positive cells—some of them might also be noncardiomyocytes—in reperfused myocardium was quite low
and we could not demonstrate a reduction of infarct size
in FTY720-treated hearts. Furthermore, the compound
FTY720 has also been demonstrated to induce pro-apoptotic
signalling.35 Both might contribute to the observed discrepancy between Akt activation and lack of effect on ischaemia–reperfusion-induced cell death.
Improved contractile performance in human myocardium
was not associated with Akt but with ERK activation. ERK
has also been recognized as a mediator of cardioprotective
signalling.16,36 This further argues against an indispensable
role of an Akt-mediated pro-survival pathway for the
observed effect on functional recovery. However, we
cannot entirely exclude the possibility that Akt is transiently
activated during early reperfusion in human myocardium.
Reduction of LVEDP can be interpreted as improved
calcium handling leading to diminished contracture and
improved LVDP during reperfusion. Nakajima et al.34 demonstrated that S1P1 receptor stimulation leads to calcium
U. Hofmann et al.
Figure 6 Human atrial myocardial preparations were subjected to 90 min of
simulated ischaemia and 120 min of normoxic reperfusion. Ischaemic preconditioning (IP) was performed by 5 min of ischaemia followed by 5 min of
reperfusion before index ischaemia. (A) Comparable to the effect of IP,
FTY720 dose-dependently improved the recovery of isometrically developed
force as indicated in per cent of baseline before ischaemia. *P , 0.05 vs.
control, n ¼ 6. (B) One micromolar FTY720 (grey bars) induced ERK1/2 phosphorylation but not Akt phosporylation, *P , 0.05 vs. control (white bars).
overload and contractile impairment in isolated cardiomyocytes. Keeping these results in mind, we would like to
suggest that the beneficial effect of FTY720 is at least in
part mediated by functional antagonistic properties at
S1P1 receptors.37 Inhibition of S1P1 receptor stimulation
by FTY720 might attenuate calcium overload and thus
improve mechanical function. Our finding that the S1P1
receptor agonist SEW2871 increased LVEDP during reperfusion further supports the suggestion that FTY720 has a beneficial antagonistic effect on S1P1 receptors. Unfortunately,
the lack of selective S1P1 receptor antagonists precludes for
the moment further investigations on this possible
mechanism.
In summary, we were able to demonstrate that S1P receptor stimulation in cardiomyocytes induces Akt phosphorylation by transactivation of the EGFR. Selective S1P1
receptor stimulation by SEW2871 limits cell death even
when applied during reperfusion. Reperfusion treatment by
the S1P1/S1P3 receptor modulator FTY720 effectively
improves functional recovery during reperfusion. To the
Cardio-protective effects of sphingosine-1-phosphate receptor agonists
best of our knowledge, this is the first report demonstrating
the feasibility of pharmacologic S1P receptor modulation for
pharmacological treatment during reperfusion after myocardial ischaemia.
Supplementary material
Supplementary material is available at Cardiovascular
Research online.
Acknowledgements
The compounds FTY720 and FTY720-P were kind gifts of Volker
Brinkman, Novartis Pharma, Basel, Switzerland. We thank Katharina
Meder and Helga Wagner for excellent technical assistance and
Tatjana Williams for critical reviewing of the manuscript.
Conflict of interest: none declared.
Funding
This work was supported by the Deutsche Forschungsgemeinschaft (SFB 688, TP A10 to G.E. and S.F.; Ri 1085/4-1
to O.R.), by the interdisciplinary center for clinical research
(IZKF) Würzburg (Z-2/26 to U.H.; E-33, E-37, E-40 to O.R.)
and by the Deutsche Stiftung für Herzforschung (F24/04 to
O.R.).
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