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CARDIOVASCULAR JOURNAL OF AFRICA • Volume 31, No 4, July/August 2020
AFRICA
175
and phosphorylated DRP1 was observed, in contrast with an
increase in Rab9 levels.
Using the snapshot approach, the perfusion protocol had
very little effect on the expression of PINK1, Parkin and p62/
SQSTM1; the only significant change being a reduction in the
expression of TOM70 at reperfusion, compared to stabilisation.
The alternative pathway showed more significant changes:
exposure to ischaemia reduced the expression of Rab9, while
reperfusion upregulated its levels as well as the pDRP1 and p/t
DRP1 ratio (Fig. 5B).
Effects of chloroquine pre-treatment on the mitophagic
process in rat hearts exposed to ischaemia and reperfusion
are shown in Figs 5A and B. Although chloroquine had no
effect on the levels of PINK1, Parkin and p62/SQSTM1, as
well as TOM70 after 40 minutes of stabilisation, compared
with untreated controls, it markedly increased PINK1 levels
after ischaemia, while reducing the expression of PINK1, p62/
SQSTM1 and particularly Parkin during reperfusion, with no
effect on TOM70.
Chloroquine had no effect on the levels of mitochondrial
total and pDRP1 but caused a reduction in the p/tDRP1 ratio
after stabilisation, while not having an effect after ischaemia/
reperfusion, when compared to its untreated counterparts. A
marked inhibitory effect during reperfusion was also seen in the
expression of Rab9.
Discussion
The aims of this study were: (1) to assess the temporal
relationship between ischaemia/reperfusion-induced changes in
mitochondrial function and mitophagy (steady state and flux),
(2) to evaluate mitophagy by comparing snapshot measurements
at specific times during the perfusion protocol with mitophagic
flux, obtained by pre-treatment of the experimental animals with
chloroquine, as suggested by Gottlieb
et al.
,
12
and (3) to evaluate
the appropriateness of chloroquine use in this regard.
Of paramount importance in studies aimed at evaluation
of autophagic flux is the presence of the drug at all times
throughout the protocol. Chloroquine has been administered
one to four hours before experimentation,
24-27
a rather long
period, which could lead to loss of drug effects. Another
approach could be to add chloroquine directly to the perfusate
of the isolated rat heart.
28
In view of the results obtained by Ma,
Zhang and co-workers,
24,25
we decided to use a time period of
one hour between administration of chloroquine and onset of
ischaemia. The marked effects observed during reperfusion after
ischaemia (Fig. 4) led us to believe that chloroquine still exerted
its effects in the isolated heart after a total perfusion period of
75 to 80 minutes.
Chloroquine (9-aminoquinoline) is an old drug, known
for its anti-malarial, anti-rheumatic and immunomodulatory
effects. Although cardiac side effects of chloroquine have rarely
been reported, it could be severe and irreversible (for review see
reference 18). In addition, chloroquine has been shown to protect
against ischaemia/reperfusion damage in the heart
29,30
and liver
31
via inhibition of phospholipase A, preventing phospholipid
breakdown. It also is a known inhibitor of autophagy: it
disrupts autophagy by inhibiting the acidification of lysosomes
that fuse with autophagosomes,
15,16
which forms the basis of
its use for the study of autophagic flux. These multiple effects
of chloroquine could indeed affect the response of the heart
to ischaemia/reperfusion injury, mitochondrial function and
thus the mitophagic process, apart from its direct effects on the
autophagosomal and lysosomal interaction.
Interestingly, in the present study, hearts from rats pre-treated
with chloroquine exhibited a slight but significant reduction
in aortic and cardiac output (as measured 60 minutes after
injection). However the inhibitory effects of chloroquine on
myocardial function observed in the present study were rather
small but significant (Fig. 2) and unlikely to affect the outcome
of the results.
Unfortunately functional recovery during reperfusion could
not be assessed in our working heart model, since hearts were
freeze-clamped after 10 minutes of reperfusion only, when
working heart measurements could not yet be done
.
However,
other studies from our laboratory
32
showed that pre-treatment
(one hour) with chloroquine had no effect on the ischaemia-
induced infarction after 35 minutes of regional ischaemia/120
minutes of reperfusion. It also was without effect on functional
recovery during reperfusion after 20 minutes of global ischaemia,
suggesting that the changes in mitochondrial function and
mitophagy observed in the present study were not caused by the
effects of chloroquine on function.
Mitochondrial function after ischaemia/reperfusion
Subsarcolemmal mitochondria were used for the purpose of
this study. As expected, exposure of the hearts to ischaemia/
reperfusion had marked effects on the parameters of oxidative
phosphorylation, regardless of the substrate used. Chloroquine
pre-treatment increased mitochondrial QO
2
states 3 and 4, the
ox-phos rate and RCI of mitochondria isolated after 30 minutes
of global ischaemia in particular, while having relatively little
effect on mitochondrial behaviour during reperfusion (Figs 3, 4).
The effects of ischaemia/reperfusion on mitochondrial
function of the isolated rat heart model are well established:
exposure to a relatively short period of ischaemia is characterised
by metabolic, ultrastructural and functional changes.
33
Inactivation of mitochondrial respiratory complexes during
ischaemia is known to be time dependent, progressive and
heterogeneous: a reduction in mitochondrial state 3 is known
to occur in ischaemic hearts from rats and rabbits (see for
example, references 34–36). As was also observed in the present
study, reperfusion after an ischaemic incident is associated
with improvement in subsarcolemmal mitochondrial ox-phos
rate.
33,35
Interestingly, mitochondrial oxygen uptake (state 4) after
reperfusion appeared to be higher with palmitoyl-L-carnitine/
malate as substrates, and may indicate a degree of uncoupling in
the presence of fatty acids in the incubation medium.
Chloroquine pre-treatment resulted in an upregulation in
state 3 respiration after exposure of the hearts to 25–30 minutes
of global ischaemia (Figs 3, 4). This may be due to inhibition of
phospholipase A, but this remains to be determined. In contrast,
in vivo
treatment with anti-malarials (chloroquine, primaquine
and quinine) adversely affected oxidative energy metabolism in
rat liver mitochondria, namely a marked depression in states 3
and 4 respiration rates, while these drugs also had uncoupling
effects on sites II and III phosphorylation.
37
High-dose chloroquine has been shown to be metabolically
cardiotoxic by inducing lysosomal andmitochondrial dysfunction