M.M. Mocanu et al
Special report
199
than 80 mmHg or heart rate less than 300 beats min71. A
further two hearts were excluded due to failure to reperfuse.
Fast ventricular tachycardia or ventricular ®brillation
occurred in several hearts during reperfusion (incidence 11 ±
42% among the dierent experimental groups) but converted
to sinus rhythm within 2 min. No hearts were excluded as a
result of tachyarrhythmias. Thus we report infarct data from
45 successfully completed experiments.
Table 1 summarizes myocardial contractile function (as
rate-pressure product) and coronary ¯ow at the end of
ischaemia, during early reperfusion and at the end of
reperfusion. During prolonged Langendor perfusion with-
out coronary occlusion, coronary ¯ow rate and contractile
function decline steadily during the experimental time
course. Coronary occlusion resulted in immediate decline
in coronary ¯ow rate which increased upon reperfusion
and thereafter declined steadily during the reperfusion
period. At the end of the 120 min reperfusion period there
were no signi®cant variations between groups with regard
to coronary ¯ow rate or contractile performance which is
consistent with previous experience with this model
(Mocanu et al., 1999). Although contractile function and
coronary ¯ow were not primarily endpoints assessed in
this study, a tendency towards better recovery of both
¯ow and contractility was noticeable in the caspase-9
inhibitor group.
The ischaemic risk zone volume (i.e. the volume of
myocardium at risk of infarction) was similar in all the
experimental groups at around 0.5 cm3 (Figure 2a). In control
hearts, the percentage of infarction within the risk zone was
38.5+2.6%, a value consistent with previous reports using this
experimental model of ischaemia-reperfusion (Mocanu et al.,
1999). Within control hearts 38.5+2.6% of the risk zone was
infarcted. The percentage of infarction was markedly reduced
in all the caspase inhibitor-treated groups compared with the
control group (Figure 2b). It is of interest to note that
treatment with the non-selective caspase inhibitor (Z-
VAD×fmk) did not confer greater protection against infarction
than any of the selective inhibitors when these were
administered alone (Z-VAD×fmk 24.6+3.4%; Z-LEHD×fmk
19.3+2.4%; Z-IETD×fmk 23.0+5.4%; Ac-DEVD×cmk
27.8+3.3%, dierences not signi®cant).
activation via death ligands or mitochondrial damage is a
crucial event in apoptosis and since apoptosis appears to be
accelerated during the process of reperfusion, we were
interested to study the eect upon reperfusion injury of
caspase inhibitors given at the onset of reperfusion. Our
primary hypothesis was that apoptosis contributes substan-
tially to myocardial infarction and that its role is most
relevant during reperfusion. Although some studies provide
evidence that apoptosis occurs during experimental myocar-
dial ischaemia (Anversa et al., 1998), work from our own
and other laboratories suggests that subsequent reperfusion
increases the extent of cardiomyocyte and vascular
endothelial cell apoptosis quite substantially (Gottlieb et al.,
1994; Fliss & Gattinger, 1996; Scarabelli et al., 1999).
Previous studies have shown that application of caspase
inhibitors prior to ischaemia confers protection against
ischaemia-reperfusion injury in myocardium. For example,
Z-VAD×fmk administered before, and continuously during
and after an ischaemic insult resulted in better recovery and
a smaller infarct size in rat heart in vivo (Yaoita et al.,
1998). Holly et al. (1999) have reported that caspase
inhibition with the non-selective inhibitor Y-VAD×cmk prior
to coronary artery occlusion in the rabbit heart in vivo led to
limitation of infarct size. Since these compounds inhibit
caspases irreversibly it is likely that caspases were also
inhibited during reperfusion. In contrast, in the present
study the caspase inhibitors were given speci®cally at early
reperfusion. We demonstrate that under these circumstances
they also reduce the extent of infarction signi®cantly,
providing evidence that the key signaling pathways
controlling apoptosis may mediate reperfusion injury.
It is not clear to what extent apoptosis and necrosis
individually contribute to tissue infarction. Relatively recent
information suggests that necrosis and apoptosis are governed
by similar mechanisms, and contrary to earlier belief they may
share common molecular pathways (Shimizu et al., 1996;
Gottlieb & Engler, 1999). Moreover, if the high energy
phosphate reserves are exhausted, cells undergoing apoptosis
can switch to secondary necrosis (Leist & Nicotera, 1997;
Daemen et al., 1999). Thus, the precise mechanism(s) by which
caspase inhibitors lead to limitation of infarction is not certain.
In spite of the fact that the inhibitors we used are reported to
exert speci®c and selective anti-caspase eects at the concentra-
tion used, their speci®city should be accepted with some caution.
Indeed, the fact that all the selective inhibitors were eective to
approximately the same degree as the non-selective inhibitor was
surprising to us. Therefore, we cannot exclude the possibility
that caspase inhibitors may exert non-speci®c actions and might
inhibit other proteases, such as calpains for example, which have
been previously implicated in ischaemia-reperfusion injury
(Iwamoto et al., 1999). There is evidence that calpains, which
are structurally related to caspases, are involved not only in
necrotic processes but also in apoptosis (McGinnis et al., 1999).
Conversely, there is accumulating evidence that apoptosis and
necrosis are linked phenomena sharing common pathways, and
in the pathology of ischemic reperfused myocardium itis dicult
to distinguish between these two cell death pathways (Shimizu et
al., 1996; Gottlieb & Engler, 1999). Indeed, recent evidence
suggests that in addition to their well-established role in
apoptosis, caspases may also mediate necrotic injury (Edelstein
et al., 1999).
Discussion Infarct size is a critical determinant of acute and
long-term prognosis in patients following coronary throm-
bosis. At present, prompt reperfusion is regarded as the
primary means of salvaging ischaemic myocardium but,
paradoxically, reperfusion may be associated with further
exacerbation of the infarct process (`lethal reperfusion
injury') (Yellon & Baxter, 2000). The identi®cation of drugs
which in¯uence the rate and extent of tissue death during
both ischaemia and reperfusion is a major therapeutic goal.
In particular, agents which could be administered as
adjuncts to thrombolysis to attenuate lethal reperfusion
injury, and would thereby provide further bene®t than
reperfusion therapy alone, might signi®cantly in¯uence
clinical outcome from myocardial infarction. In the present
study, we report that a range of caspase inhibitors when
administered as adjuncts to reperfusion in an experimental
model of myocardial infarction, provide substantial protec-
tion. There are several lines of evidence that myocardial cell
death during ischaemia-reperfusion can take place via
apoptosis and necrosis but the individual contributions of
these two phenomena and at what point they contribute to
tissue death is unclear (Bromme & Holtz, 1996; Buja &
Entman, 1998; Gottlieb & Engler, 1999). Since caspase
In conclusion, we present the ®rst evidence that caspase
inhibitors administered as adjuncts to reperfusion limit infarct
size. Although the precise mechanism underlying the protec-
tion remains to be clari®ed, these observations indicate that
inhibition of caspases may be
a promising route for
British Journal of Pharmacology, vol 130 (2)
Schamschurin
