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CARDIOVASCULAR JOURNAL OF AFRICA • Volume 31, No 4, July/August 2020
AFRICA
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in a rat model of pressure overload hypertrophy.
38
It has also been
reported that the negative inotropic and chronotropic effects of
chloroquine on isolated perfused rabbit hearts were due to a
reduction in mitochondrial calcium binding and accumulation.
39
These negative effects of chloroquine on mitochondrial function
are in contrast with those observed in the present study and may
be due to differences in dosage and experimental conditions.
Mitophagy in ischaemia/reperfusion
Mitophagy, responsible for the degradation and recycling of
damaged mitochondria, is a dynamic cellular process from the
formation of autophagosome, autophagosome–lysosome fusion
to final degradation of mitochondria and has been shown to
be essential for cardioprotection under both physiological and
pathophysiological conditions (for review see reference 40). It
is also generally accepted that the PINK1/Parkin mitophagy
pathway is a major mitochondrial quality-control mechanism
and critical for maintenance of function at baseline in adult
hearts.
41
As far as we are aware, the temporal relationship between
ischaemia/reperfusion-induced changes in mitochondrial
ox-phos function and mitophagy has received little attention. It
is well established that mitochondrial depolarisation induced by
various stimuli is a common trigger for mitophagy. In the present
study, markers of mitophagy were evaluated in mitochondria
isolated from perfused hearts after stabilisation, exposure to
ischaemia alone as well as after reperfusion following ischaemia.
Mitochondrial PINK1, Parkin, p62/SQSTM1 and TOM70
expression were used in the present study as indicators of
mitophagy in view of the fact that Parkin-mediated mitophagy
required loss of mitochondrial membrane potential.
42,43
According
to Gottlieb and co-workers,
12
p62/SOSTM1 expression can be
regarded as a useful marker of autophagy: it is a polyubiquitin-
binding protein that is degraded by autophagy and its protein
levels are inversely related to autophagic activity.
44,45
Therefore
accumulation of p62/SQSTM1 would be indicative of impaired
autophagosome clearance in the case of a snapshot approach.
There are pitfalls however in determining autophagic activity
based on snapshot measurements only during an experimental
protocol,
12
since static levels give an incomplete indication of
autophagy without assessment of flux. The interruption of
autophagy by chloroquine-induced inhibition of the acidification
of lysosomes that fuse with autophagosomes
15,16
will rescue p62/
SQSTM1. Therefore, increased accumulation of this marker
in the presence of chloroquine will be indicative of increased
autophagic flux.
Evaluationof thetemporalrelationshipbetweenmitochondrial
ox-phos and mitophagy was initially done using snapshot
measurements made at different times during the ischaemia/
reperfusion protocol. In view of the fact that depolarisation of
the mitochondrial membrane leads to activation of mitophagy as
well as the gross ultrastructural changes visible after exposure of
the perfused heart to 25 minutes of global ischaemia,
33
changes in
the PINK1/Parkin pathway were expected. However, apart from
a reduction in TOM70 expression during ischaemia/reperfusion,
no significant changes in mitochondrial PINK1, Parkin and p62/
SQSTM1 expression were observed throughout the perfusion
protocol, suggesting that removal of damaged mitochondria by
the mitophagic process probably occurs at a later stage.
Based on these assumptions, it was concluded that the
changes in mitochondrial oxidative phosphorylation caused by
25 minutes of ischaemia are not yet associated with measureable
changes in the PINK1/Parkin mitophagy pathway. In view of the
role of TOM70 in the import of PINK1 into mitochondria,
46
it is
possible that changes in TOM70 precede the mitophagic process.
In contrast with our findings, increases in mitophagy after
exposure to ischaemia/reperfusion were reported in many studies
using a longer period of ischaemia. For example, increased
expression of PINK1 and Parkin was reported in mouse (30
minutes of ischaemia/two hours of reperfusion)
47
and rat hearts
(30 minutes of regional ischaemia/two hours of reperfusion).
48
Short episodes of ischaemia/reperfusion (three minutes of
ischaemia/three minutes of reperfusion) have also been shown
to translocate Parkin from the cytosol to the mitochondria.
49
This suggests that the perfusion protocol may have an important
effect on the mitophagy process. It should be kept in mind that
these studies were done in the absence of chloroquine.
Pre-treatment with chloroquine had a profound effect
on the parameters studied: not only did the drug influence
the expression of a number of markers of mitophagy when
compared to their corresponding untreated counterparts,
but it also affected the pattern of the response to ischaemia
and reperfusion. Apart from a significant increase in PINK1
levels after ischaemia, the most significant changes induced
by chloroquine occurred during the reperfusion period: the
reduction in PINK1, Parkin and p62/SQSTM1 levels observed
at this stage suggested a
downregulation of mitophagic flux
during reperfusion. In contrast, the significant upregulation of
PINK1 levels during ischaemia may be indicative of increased
mitophagy occurring at this stage. Therefore, based on flux
measurements after chloroquine treatment, it appears that the
changes in mitochondrial oxidative phosphorylation function
induced by exposure of hearts to 25 minutes of global ischaemia
coincides with changes in mitophagic flux (in contrast with using
snapshot evaluation of events).
The possibility of chloroquine wash-out during reperfusion
accounting for the reduction in expression of PINK1, Parkin,
p62/SQSTMI and Rab 9 is unlikely, since one would expect
values to be similar to those obtained in the absence of
treatment. In addition, our results are in agreement with those
obtained by Ma and co-workers,
13
namely that the increase in
p62/SQSTM1 in chloroquine-treated hearts subjected to 30
minute of ischaemia/90 minutes of reperfusion was not affected
by chloroquine treatment, indicating impaired mitophagic flux
under these conditions.
In addition to the conventional markers of mitophagy,
the effects of ischaemia/reperfusion as well as chloroquine
pre-treatment were also evaluated by determining their effects
on mitochondrial fission, as indicated by expression and
phosphorylation of the DRP1. DRP1, the master mediator of
fission, is located in the cytosol and when activated, translocates
to the mitochondrial outer membrane where it interacts with
other proteins such as human fission factor (Fis 1), and
mitochondrial fission factor (MFF) (for a review see references
8 and 9). Once at the outer mitochondrial membrane, DRP1
mediates mitochondrial fragmentation and loss of membrane
potential, and facilitates release of cytochrome C.
50
Our results show that while the expression of total
mitochondrial DRP1 was not changed by the ischaemia/