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CARDIOVASCULAR JOURNAL OF AFRICA • Volume 31, No 4, July/August 2020
AFRICA
171
above, the hearts were plunged into ice-cold KE medium (0.18
M KCl/0.01 M EDTA, pH adjusted to 7.4 with Tris base) and
homogenised with a Polytron PT 10 homogenizer (4
×
4 seconds,
4°C, setting 2). The mitochondria were isolated by differential
centrifugation, as described by Sordahl
et al
.
20
The mitochondrial
pellet was divided into two: one half was dispersed in KE
medium for immediate measurement of mitochondrial function,
while the other half was dissolved in lysis buffer (see below) and
stored at –80°C for subsequent Western blot analysis. Protein
determination for mitochondrial functional studies was done
with the technique of Lowry and co-workers.
21
Immediately
after
preparation,
subsarcolemmal
mitochondrial oxidative phosphorylation (oxphos) was
measured polarographically at 27°C using an oxygraph
(Hansatech Instruments, Bannan UK) and Clark electrode. The
mitochondrial incubation medium contained (in mM): sucrose
0.25, Tris-HCl 10, pH 7.4, K
2
HPO
4
8.5 and glutamate 5/malate
2 or palmitoyl-L-carnitine 0.45/malate 2 as substrates (pH 7.4).
ADP (250–350 nmoles) was added to initiate state 3 respiration.
Parameters investigated were the ADP/O ratio, state 3
respiration (mitochondrial respiration in the presence of ADP)
and state 4 respiration (mitochondrial respiration in the absence
of ADP). Mitochondrial respiratory rates (states 3 and 4)
were expressed as nAtoms oxygen uptake/mg protein/min. The
respiratory control index (RCI) was calculated according to the
following formula: state 3/state 4 respiration.
The amount of ADP added to the incubation system was
obtained spectrophotometrically (molar extinction coefficient
of ADP: 15.4 at 259 nm). The oxidative phosphorylation rates
(nmoles ATP produced/mg protein/min) were calculated as
follows: QO
2
(state 3)
×
ADP/O ratio.
To evaluate the ability of the isolated mitochondria to
withstand oxidative stress, mitochondrial preparations were
also exposed to 20 minutes of anoxia in the oxygraph chamber,
followed by six minutes of re-oxygenation, as described by Essop
and co-workers.
22
Recovery of respiratory function (state 3)
after these interventions was calculated as a percentage of the
respiratory rate (state 3) before exposure of the mitochondria to
anoxia. Mitochondrial preparations from five to six hearts were
studied in each group.
For Western blot technique, an aliquot of the mitochondrial
pellet was homogenised in 200 μl of lysis buffer containing
(in mM): Tris-HCl 20, p-nitrophenyl phosphate 20, EGTA
1, EDTA 1, NaCl 150, tetra-sodium-pyrophosphate 2.5,
β
-glycerophosphate 1, sodium orthovanadate 1, phenylmethyl
sulphonyl fluoride (PMSF) 1, aprotinin 10 μg/ml and leupeptin
10 μg/ml, Triton-X100 1%, pH 7.4 using a Bullet Blender
®
(Next
Advance Inc, USA) at 4°C for five minutes with a scoop of 0.15
mm zirconium oxide beads equivalent to the pellet size. Samples
were then microfuged at 15 000 rpm for 10 minutes to obtain the
supernatant, the protein content of which was determined using
the Bradford technique.
23
The lysates were adjusted accordingly by dilution with lysis
buffer to an equal protein concentration, followed by Laemmli
sample buffer and boiled for five minutes. Depending on the
protein of interest, 30 to 60 μg were loaded and separated by
sodium dodecyl sulphate polyacrylamide gel electrophoresis
(SDS-PAGE) using the standard Bio-Rad Criterion system.
The running buffer contained (in mM): Tris 25, glycine 192 and
sodium dodecyl sulphate (SDS) 1%.
After separation, the proteins were transferred to a PVDF
membrane (Immobilon
TM
P, Millipore) using wet electrotransfer.
The transfer buffer contained (in mM): Tris-HCl 25, glycine
192, and methanol (20% v/v). Non-specific binding sites on the
membranes were blocked with 5% fat-free milk in TBST (Tris-
buffered saline + 0.1% Tween 20) for one to two hours at room
temperature. After washing with TBST (five by five minutes),
membranes were incubated overnight at 4°C with the primary
antibodies.
The primary antibodies were diluted in TBST or in 5%
fat-free milk in TBST solution. After overnight incubation,
membranes were washed with TBST (five by five minutes)
and then incubated for one hour at room temperature, with
a diluted horseradish peroxidase-labelled secondary antibody
(Cell Signaling Technology
®
). The secondary antibody was
either diluted in TBST or in 5% fat-free milk/TBST solution.
The following primary antibodies were used: TOM 70 (Santa
Cruz, Dallas Tex, USA), PINK1, p62/SQSTM1, Rab9, total (t)
and phospho (p) DRP1 (Cell Signaling, Danvers, MA, USA),
Parkin (Abcam, Cambridge UK). After thorough washing
with TBST, membranes were covered with ECL (enhanced
chemiluminescence) detection reagents (Bio-Rad Clarity) and
quantified using a ChemiDoc-XRS imager (Bio-Rad).
Twenty-six-well Criterion
TM
4–20% pre-cast gradient gels
and stain-free technology were used throughout the study. With
stain-free technology, the transferred proteins on the PVDF
membrane can be visualised in the ChemiDoc to confirm equal
loading. Furthermore, the intensity of bands detected by the
ECL reaction are normalised to the total proteins that were
transferred in each lane, negating the use of a loading control
.
Four samples/group were included on the same gel plus a sample
prepared from a heart of an unperfused age-matched control
animal to act as standard for normalisation of all data.
Statistical analysis
Statistical analysis was performed using GraphPad Prism version
5 software (GraphPad Software, Inc). Comparisons between the
groups were performed using one-way ANOVA. If two groups
were compared, the unpaired Student’s
t
-test was used. A
p
-value
≤
0.05 was deemed as statistically significant.
Results
Myocardial function
Baseline function of perfused working hearts from untreated
and chloroquine-treated rats was monitored over a period of 40
minutes without any interventions. No differences in heart rate,
coronary flow and peak systolic pressure were observed between
the two groups. However, chloroquine pre-treatment caused
a slight but statistically significant reduction in aortic output,
cardiac output and work total performed (Fig 2). Since hearts
were freeze-clamped after 10 minutes of reperfusion, which was
too short for measurement of working heart function, functional
recovery during reperfusion could not be evaluated.
Mitochondrial function
Figs 3 and 4 show the effect of ischaemia/reperfusion on
mitochondrial function with glutamate/malate and palmitoyl