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 AFRICA 383
study was notably higher than those used by others. Egom et
al.28 used as little as 25 nM, Hofmann et al.32 used 500 nM, while
Vessey et al.30 used 600 nM. Our dose was based on work done
on FTY720 as an activator of PP2A in the setting of cancer and
cancerous cell lines.
In 2003, Matsuoka and colleagues38 reported that 8 µM
FTY720 could suppress the phosphorylation of PKB/Akt, Bad
and p70S6 kinase in T-cell leukaemia cells through the activation
of PP2A. Others have also reported on the ability of FTY720
to activate PP2A in the range of 2.5 to 10 µM.36,37,39 Recently it
was reported that FTY720 established this activation by binding
to SET (Suvar3-9, enhancer of zeste, trithorax), an endogenous
inhibitor of PP2A, thereby mediating the dissociation of
SET from PP2A.37,39 This mechanism is however dependent
on FTY720 not being phosphorylated, since P-FTY720 has
the potential to phosphorylate SET through a Jak2-mediated
pathway, thereby enhancing its PP2A inhibitory function.37
Others have however also shown that FTY720 can activate
PP2A through the activation of a Pak1-mediated signalling
pathway.61-63 It is therefore possible that the increase in infarct
size that we observed in the 1-µM pre-treatment group was due
to the activation of PP2A, which theoretically could suppress
the phosphorylation-mediated pro-survival pathways, also
co-activated by FTY720, which we have observed (unpublished
data) and others have reported.21,2230-32,63
In contrast to this, in the 2.5- µM pre-treatment group,
pro-survival activation probably dominated over PP2A
activation, thereby inducing protection. As will be discussed
in the following paragraphs, 2.5 µM FTY720 also induced a
profound reduction in post-ischaemic functional ability, thereby
reducing energy demand and also potentially contributing to a
reduction in IFS.
Contrary to the infarct-sparing effects associated with
FTY720 treatment in our study, we found that 1 µM exerted
no effect on functional recovery, while 2.5 µM significantly
suppressed post-ischaemic function in both the GI, as well as
RI models. This is in contrast to Hofmann et al.32 and Vessey
et al., 30 who both showed that FTY720 maintained functional
ability after ischaemia at doses of 500 nM, 600 nM and 1 µM.
It seems as if FTY720 has a dose-dependent effect, with an
increase in FTY720 concentration being detrimental to post-
ischaemic function. This was a surprising finding since, in the
context of cancer research, it has been reported that relatively
high doses of FTY720, administered chronically to mouse
models, did not exert any toxic effects.36,37 Neither of these studies
however investigated isolated heart performance or resistance to
ischaemic stress.
The concentrations of FTY720 used in our study were also
not as high as those of sphingosine, which have been shown
by others to be detrimental: Suzuki and colleagues64 reported
that 10 or 20 µM sphingosine induced apoptosis in several
cell lines, while Karliner18 specified 5 µM of sphingosine as
cardiotoxic. With regard to the phosphorylated form, Theilmeier
and co-workers59 reported that a dose as high as 10 µM of S1P
protected neonatal rat cardiomyocytes from apoptosis in a model
of glucose and growth factor withdrawal. It is therefore unlikely
that the FTY720 concentrations that we used were toxic. A
possible explanation for our seemingly controversial results may
be found in the perfusion model used, as well as the effects of
S1P receptor stimulation on heart function per se.
Our experimental model differed from those used by others.
Hofmann32 showed cardioprotection in retrogradely perfused
rat hearts and human myocardial muscle strip preparations,
while Vessey et al.30 used an isolated retrogradely perfused
mouse heart model, with FTY720 administered as a post-
conditioning intervention (four cycles of five seconds’ ischaemia
and reperfusion at the onset of reperfusion). We however
utilised an isolated working rat heart preparation, which has an
additional energy demand65 and free radical exposure66 associated
with it. This more challenging setting than normal retrograde
perfusion might explain the inability of FTY720 to protect
functional capacity in our model, and even contribute to its loss.
Although not investigated by us, it has been reported by
others that S1P receptor activation has the potential to suppress
both heart rate, as well as contractility, largely through effects on
intracellular calcium ion (Ca2+) dynamics. We propose that these
mechanisms, at least in part, explain the observed detrimental
effects of FTY720 on post-ischaemic function as follows. Heart
tissue expresses S1P receptors 1, 2 and 3.67 Of these, it is only 1
and 3 that can be activated by P-FTY720. 53 It has been reported
that activation of these receptors in the heart, especially receptor
3, induces a reduction in heart rate68-70 through the activation of
the inwardly rectifying atrial potassium ion channel (IKACh),
thereby allowing an increased inward flux of potassium ions into
the cell and hyperpolarising the sarcolemma.70,71
Activation of the S1P receptor 1 also exerts a negative
inotropic effect, through a reduction in the availability of
intracellular Ca2+ from the sarcoplasmic reticulum (SR) for the
initiation of contraction. Two mechanisms have been shown to
be involved in this inotropic effect: (1) similarly to the reduction
in heart rate, activation of IKACh leads to the hyperpolarisation
of the sarcolemma, leading to a subsequent reduction in action
potential duration, which in turn implies a reduced influx of
Ca2+ into the cardiomyocytes, thereby reducing the stimulus for
the Ca2+-induced release of Ca2+ from the SR; (2) linking with
the previous mechanism, S1P has been shown to reduce Ca2+
flux through the L-type Ca2+ channel, thereby also diminishing
the potency of Ca2+-induced Ca2+ release. This reduction in flow
through the L-type Ca2+ channel could be due to a Gi-mediated
reduction in cyclic AMP levels, associated with the stimulation
of the S1P receptors.67,72,73 Ironically, these same mechanisms that
reduce intracellular Ca2+ levels might also limit Ca2+ overload
during reperfusion, thereby contributing to the infarct-limiting
effects of FTY720.
In view of the effect of S1P activation on heart rate (as
discussed above), the divergent results that have been generated
regarding the effect of FTY720 on rhythmicity,28,29 and the
relevance of arrhythmia in the pathology of myocardial I/R
injury, it is a limitation of this study that we did not include the
incidence of arrhythmia in early reperfusion as an additional
endpoint.
We speculate that the FTY720-mediated reduction in
intracellular Ca2+ levels associated with a general reduction in
functional ability, in combination with the dual stressor of I/R
and work-mode perfusion could explain the severely detrimental
effects of FTY720 on post-ischaemic functional recovery. It
would therefore be interesting to assess the effects of acute
FTY720 administration at these doses in hearts either not
exposed to ischaemia and reperfusion, and/or not exposed to
work-mode perfusion.