Cardiovascular Journal of Africa: Vol 31 No 4 (July/August 2020)

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CARDIOVASCULAR JOURNAL OF AFRICA • Volume 31, No 4, July/August 2020 178 AFRICA
reperfusion protocol, its phosphorylation was increased,
especially after reperfusion, causing a significant increase in
the pDRP1/tDRP1 ratio at this stage. Chloroquine had no
significant effect on mitochondrial fission during ischaemia/
reperfusion, as reflected by the pattern of unchanged DRP1
levels during the ischaemia/reperfusion protocol, apart from a
reduction in pDRP1/tDRP1 ratio after stabilisation.
Interestingly, in the presence of chloroquine, the very
significant reduction in the expression of the PINK1/Parkin/
p62 pathway occurring upon reperfusion, indicating reduced
mitophagic flux, did not coincide with changes in fission. In view
of the assumption that fission is a prerequisite for mitophagy, the
relationship between these events needs to be evaluated.
The link between the PINK1/Parkin pathway and fission has
been studied in COS cells.51 Workers have shown that Parkin
promotes fission independently from PINK1 and this effect
depends on pathways involved in DRP1 phosphorylation. Loss
of mitochondrial electron potential leads to recruitment of
DRP1 to mitochondria in the proximity of PINK1 and Parkin,
suggesting that mitochondrial division occurs at sites where the
PINK1/Parkin-dependent mitochondrial clearance programme
is initiated.
Although the translocation of DRP1 from the cytosol to
the mitochondria was not investigated in our study, previous
studies from our laboratory showed translocation occurring in
hearts exposed to ischaemia (unpublished data). Translocation
of DRP1 from the cytosol to the mitochondria is known to
occur in ex vivo hearts after exposure to 30 minutes of global
ischaemia/90 minutes of reperfusion, as well as in primary
cultured cardiomyocytes subjected to ischaemia/re-oxygenation.52
The effect of chloroquine on the alternative non-canonical
autophagy pathway was also evaluated by determining the
expression of an essential regulator of this process, Rab9, a
small GTP-binding protein, during exposure of the heart to
ischaemia/reperfusion. While Rab9 expression did not change
significantly during the ischaemia/reperfusion protocol in
untreated hearts, chloroquine almost completely abolished its
expression during reperfusion (Fig. 5). The results obtained
indicate that chloroquine also very significantly inhibited the
alternative autophagy pathway during reperfusion, suggesting
that this pathway should be considered when evaluating the
effects of this drug on autophagy/mitophagy.
Interestingly, chloroquine had its most significant effects
during reperfusion, as was seen in the expression of p62,
PINK1, Parkin and Rab9. The significance of the upregulated
state 3 ox-phos rate during reperfusion (Figs 3, 4) in the
observed changes in mitophagy needs to be further investigated.
Although the purpose of chloroquine treatment was to block
the autophagosome–lysosome interaction to allow evaluation
of flux, the data suggest that the effects of this treatment on
mitochondrial function per se may contribute to the effects on
mitophagy as seen specifically in reperfusion.
Conclusion
The results show that data obtained by snapshot measurements
of mitophagy differed markedly from flux measurements using
chloroquine pre-treatment. Reperfusion in particular is associated
with a significant inhibition of mitophagic flux. Whether this
indicates a salvage attempt to rescue damaged mitochondria, as
seen in the marked improvement in ultrastructural appearance
upon reperfusion, 33 remains to be determined.
The data obtained underscore the necessity of evaluation
of the process of mitophagy in the absence and presence of
lysosomal blockade, as suggested by Gottlieb and co-workers. 12
However the data also demonstrate that chloroquine per se
affected the response of the mitochondria to ischaemia, which,
in turn, may have affected the mitophagy process.
Another complicating factor in the interpretation of the
results is that the drug may have cardioprotective actions in a rat
heart model, attributed to its phospholipase A2 effects,29,30 which
may lead to an underestimation of the effects of ischaemia/
reperfusion on mitophagy. This possibility, however, was not
reflected by changes in infarct size and functional recovery after
global ischaemia. 32 Therefore although measurement of flux is a
prerequisite for evaluation of mitophagy, interpretation of data
should be done with care in view of the multiple other effects of
chloroquine on the heart.
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