Cardiovascular Journal of Africa: Vol 27 No 6 (November/December 2016)

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CARDIOVASCULAR JOURNAL OF AFRICA • Volume 27, No 6, November/December 2016 382 AFRICA
Surprisingly, FTY720 exerted a considerably different effect
on infarct size (Fig. 9), in contrast to the effects seen on
functional recovery after regional ischaemia. Administration of
1 µM FTY720 as a pre-treatment showed a very strong tendency
to increase IFS (control: 39.89 ± 3.93% vs PreFTY: 51.73 ±
2.36%, n = 6; p = 0.066), while administration during reperfusion
limited the development of infarction (control: 39.89 ± 3.93% vs
PostFTY: 23.96 ± 3.99%, n = 6–7; p < 0.05).
In contrast to this dependence on the time point of
administration relative to sustained ischaemia, 2.5 µM FTY720
consistently reduced IFS (control: 39.89 ± 3.93% vs PreFTY:
29.97 ± 1.03% and PostFTY720: 30.45 ± 2.16%, n = 6; p < 0.05).
The area at risk, relative to the total area, did not differ among
any of the groups.
We are therefore left with these two disparate observations:
on one hand, 1 µM FTY720 had no effect on post-ischaemic
functional recovery and 2.5 µM reduced functional recovery,
while on the other hand FTY720 caused a significant reduction
in IFS, with the exception of the increase observed when 1 µM
FTY720 was administered prior to ischaemia.
Discussion
Recently there has been great interest in the sphingosine
analogue, FTY720, mostly because of its immunomodulatory
functions, but also because of its potential to stimulate similar
pathways to sphingosine-1-phosphate. In this context, several
researchers have investigated its potential to confer protection
against myocardial ischaemia/reperfusion injury, however, with
divergent results.
In this study we investigated the effects of two different
concentrations of FTY720, administered prior to ischaemia
or during initial reperfusion in two models of ischaemia with
function and infarct size as endpoints. We found that 1 µM
FTY720 exerted no effect on functional recovery regardless of
the time of administration, although pre-treatment augmented
infarction, while reperfusion treatment reduced IFS. Increasing
the dose to 2.5 µM proved severely detrimental to functional
recovery, although it was associated with an unexpected
reduction in IFS.
FTY720 is a sphingosine analogue, therefore it can elicit
similar effects to sphingosine and it can also be phosphorylated by
intracellular SK2 to generate a phosphorylated form (P-FTY720)
similar to S1P, which can exit the cell of origin and then bind on
any one of the S1P receptors (except receptor 2).53 This leads
to a conundrum when administering and experimenting with
FTY720, which has also been reported by others,30,37 namely, are
the effects observed due to FTY720 simulating sphingosine or
P-FTY720 mimicking S1P?
Although we were unable to distinguish between these
two forms of the drug in our experimental setup, there is an
observation and an argument, which both point to a possible
significant involvement of P-FTY720. First, a significant increase
in coronary flow was observed during the administration of
FTY720 at both dosages tested. This vasodilatory effect has
been linked to the activation of S1P receptor 1 and/or 3 in
the endothelium, which then recruits a PI3-kinase pathway,
as well as a Ca2+-mediated mechanism to activate endothelial
nitric oxide synthase (eNOS) to produce nitric oxide (NO),
which ultimately facilitates vascular relaxation.54-56 Although
the IC50 values for P-FTY720 binding to both S1P1 and S1P3
are extremely low (0.21 ± 0.17 nM for S1P1 and 5.0 ± 2.7 nM
for S1P353), the observed vasodilation in our experimental
hearts nonetheless implies the presence of P-FTY720 in the
system. This possibility is further confirmed by the fact that
the enzyme responsible for phosphorylating FTY270, SK2, is
the predominant sphingosine kinase in the heart57,58 and is also
activated by FTY720.57 It is therefore probable that the relatively
high dose of FTY720 employed in this study would further
stimulate FTY720 phosphorylation.
In this regard, Tölle and colleagues55 found that SK2 in
human umbilical vein endothelial cells (HUVECs) and whole
aortae converted FTY720 to P-FTY720 at a notable rate: 70%
of 1 µM FTY720 was phosphorylated after two minutes, and
90% after 10 minutes. Taken together, it is highly likely that a
large portion of the FTY720 administered in our study had been
phosphorylated.
The S1P receptors are all G-coupled protein receptors, which,
when activated, can induce the activation of several pathways
associated with cardioprotection, such as protein kinase C
(PKC), phosphatidylinositol-3-kinase (PI3-kinase) and protein
kinase B (PKB/Akt).20-24,59 It is therefore no surprise that several
researchers have investigated the ability of S1P to limit infarct
size.21-24 Even unphosphorylated sphingosine has been associated
with cardioprotection if administered concentrations are low
enough (~0.4 µM22).
These observations have led to the question whether FTY720
can also be used to confer protection. In 2009, Hofmann and
colleagues addressed this in an ex vivo rat heart preparation and
found that although FTY720 increased post-ischaemic function,
it failed to limit IFS,32 a result repeated in an in vivo model
of ischaemia.29 In 2011, however, Egom et al.31 illustrated the
ability of FTY720 to increase cell viability in isolated neonatal
rat cardiomyocytes exposed to either simulated ischaemia or
hypoxia. These observations were subsequently confirmed by
Vessey and co-workers,30 who reported that 600 nM FTY720,
administered as a post-conditioning intervention, exerted an
infarct-sparing effect in an isolated mouse heart model exposed
to global ischaemia.
Wang and colleagues60 administered FTY720 for a period
of one or three weeks in a mouse model and found that this
relatively chronic treatment regime also reduced IFS. Therefore
our results agree with the latter set of findings that FTY720
is able to reduce infarct size. These results can probably be
explained by the expected activation of S1P receptor-mediated
pro-survival pathways and/or suppressed inflammation in the
heart, as has been shown by others (although we did not assess
the mechanism of protection).
The surprising exception was pre-treatment with 1 µM, which,
at best, did not influence IFS, while not having an effect on post-
ischaemic functional recovery. To our knowledge, this is the first
study to have experimented with the acute administration of
FTY720 prior to ischaemia, although both Hofmann et al. 29 and
Wang et al.60 administered the drug to their respective animal
models for a substantial period of time (from one day once off
to three weeks’ chronic treatment) prior to ischaemia. We did
not investigate the mechanism by which FTY720 pre-treatment
influences injury. We however propose that it can be explained, at
least in part, by the concurrent activation of protein phosphatase
2A (PP2A). The concentration of FTY720 used in the present