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CARDIOVASCULAR JOURNAL OF AFRICA • Volume 32, No 3, May/June 2021
142
AFRICA
Unless stated otherwise, drugs and chemicals were obtained
from Sigma-Aldrich (SA). Streptozotocin (STZ) was used to
induce a moderate form of diabetes mellitus, as previously
described.
14
Rats were fasted of food (but not water) for six
hours to improve the uptake of STZ before being injected
intraperitoneally (i.p.) with STZ (50 mg/kg). The STZ was freshly
dissolved in 0.1 M citrate buffer (pH 4.5) before administration.
Blood glucose was measured from tail vein blood samples
obtained at similar times of the day using a glucometer
(Accu-Chek, Roche, SA).
14
Rats with a random blood glucose
concentration ≥ 15 mmol/l were considered diabetic.
Magnesium was administered as MgSO
4
(270 mg/kg, i.p.)
dissolved in normal saline.
15,16
The i.p. route was chosen for Mg
2+
to achieve more reliable uptake compared to oral administration
in water or food where the uptake may vary in diabetes due to
polydipsia and polyphagia.
The rats were randomly divided into four treatment groups,
and each rat was identified by a unique label on the tail. The
control group was injected i.p. with a single dose of citrate buffer
on the first day, and with saline i.p. once daily for 28 consecutive
days. The STZ group was injected i.p. with a single dose of STZ
50 mg/kg on the first day, and with saline i.p. once daily for 28
days. The STZ + Mg
2+
group was injected i.p. with a single dose
of STZ 50 mg/kg on the first day, and with MgSO
4
270 mg/kg
i.p. once daily for 28 days. The Mg
2+
group was injected i.p. with
a single dose of citrate buffer on the first day, and with MgSO
4
270 mg/kg i.p. once daily for 28 days.
Rat hearts were surgically removed under anaesthesia to
euthanise the rats, as previously described.
16
Briefly, rats were
anticoagulated with heparin (500 IU/kg, i.p.) and anaesthetised
with sodium pentobarbital (70 mg/kg, i.p., Vetserv, SA). Upon
loss of the pedal withdrawal reflexes, the hearts were excised via
a thoracotomy incision and placed in cold (4°C), filtered (7-
μ
m
pore Whatman filter paper, Sigma-Aldrich, SA), modified Krebs-
Henseleit (KH) solution containing (in mmol/l): 118.5 NaCl, 4.7
KCl, 25 NaHCO
3
, 1.2 MgSO
4
, 1.8 CaCl
2
, 1.2 KH
2
PO
4
and 11
glucose (pH 7.4). CaCl
2
was added after the optimisation of pH
to prevent precipitation of calcium with phosphate. Some hearts
were used for cardiac perfusion studies, whereas the others were
either histologically analysed or snap-frozen in liquid nitrogen and
stored at –80°C for Western blot analysis.
For perfusion studies, the hearts were retrogradely perfused
with K-H solution through an aortic cannula on a constant-
pressure (74 mmHg) Langendorff apparatus. To ensure optimal
cardiac tissue viability, the time lapse between excision of the
heart and commencement of perfusion was limited to three
minutes. The K-H solution was gassed with carbogen (95% O
2
and 5% CO
2
) and was maintained at 37°C. The coronary flow
rate was measured by collecting coronary effluent over time and
was normalised to heart weight. Blood samples used for Mg
2+
assays were collected at the time of removal of the heart and
centrifuged at 15 000
g
(Beckman microfuge, USA) to obtain
plasma, which was frozen until further analysis.
Electrocardiographic (ECG) and haemodynamic parameters
were measured using the PowerLab data-acquisition system
and LabChart Pro 7 software (ADInstruments, Australia), as
previously described.
16
ECG was recorded using apex-to-base
electrodes via a transducer (ML136) and was analysed using
the LabChart Pro ECG module (ADInstruments, Australia).
The QT interval, corrected for heart rate (QTc) was calculated
using Bazett’s formula. Left ventricular (LV) pressure was
measured using a water-filled, intraventricular balloon connected
to a pressure transducer (MLT1199) and amplifier (ML221,
ADInstruments, Australia).
The hearts were stabilised for 20 minutes and the LV
end-diastolic pressure (LVEDP) was set at 5–10 mmHg. The
LabChart 7 Pro blood pressure module (ADInstruments,
Australia) was used to analyse haemodynamic data and to derive
the maximal rate of pressure increase (+dP/dt
max
), the maximal
rate of pressure decline (–dP/dt
max
), contractility index and the
time constant of ventricular relaxation (
tau
). The LV developed
pressure (LVDP) was calculated as the difference between LV
peak systolic pressure and LVEDP.
Transverse sections of cardiac ventricular tissue were
stained with either haematoxylin and eosin (H&E) or Masson’s
trichrome, as previously described.
16
Histological images were
taken using a charge-coupled device camera (Zeiss AxioCam,
Germany) attached to an optical microscope (Zeiss AxioSkop,
Germany). The cardiomyocyte width on H&E images was
analysed using ImageJ software (NIH, USA). The average width
of five cells on each of four sections of the heart was calculated
for each heart. The degree of interstitial and perivascular fibrosis
on Masson’s trichrome images was semi-quantitatively scored, as
done previously,
16
based on a scoring system described by Buwa
et al
.
17
as follows: none (–), mild (+), moderate (++), and severe
(+++).
Frozen LV tissues were homogenised on ice by sonication
in a modified radioimmunoprecipitation assay buffer (50 mM
Tris-HCl, 150 mM NaCl, 1% Triton X-100, 0.5% sodium
deoxycholate, 0.1% sodium dodecyl sulphate, pH 7.4) containing
a protease/phosphatase inhibitor cocktail (Thermo Scientific,
USA). Protein concentrations were quantified (Pierce protein
assay kit, Thermo Scientific, USA) and protein samples (40
µg) were loaded and electrophoresed on 12% sodium dodecyl
sulphate-polyacrylamide gels (Mini-Protean Tetra Cell, BioRad,
SA) and transferred to isopropanol-soaked polyvinylidene
fluoride membranes (Trans-Blot Turbo, Bio-Rad, SA).
The membranes were blocked with 5% bovine serum albumin
(BSA) in 0.1% Tween20 phosphate-buffered saline (PBS-T) for
one hour at room temperature, and incubated with anti-ATP5A
mouse antibody (1:5000, #136178, Santa Cruz Biotechnology,
USA) in 5% BSA in PBS-T overnight at 4°C. The primary
antibody was excluded in the negative control in order to rule out
non-specific binding of the secondary antibody. The membranes
were washed with PBST and incubated with horseradish
peroxidase-conjugated secondary antibody (1:10000, #170-6516,
Bio-Rad, SA) in 5% BSA in PBS-T for two hours at room
temperature.
The membranes were then washed with PBS-T, incubated
with enhanced chemiluminescence substrate (Bio-Rad, SA)
and exposed to X-ray film in the dark room. The membranes
were stripped, blocked and re-probed with anti-
β
-actin rabbit
antibody (1:10000, #16039, Abcam, USA) and goat anti-rabbit
secondary antibody (1:10 000, #6721, Abcam, USA). The bands
on films were analysed using ImageJ software (NIH, USA) and
were normalised to those of the housekeeping protein
β
-actin.
The Mg
2+
concentration was measured in the plasma samples
prepared at exsanguination, 18–24 hours after the final dose
of MgSO
4
had been administered. Ionised Mg
2+
concentration
was measured using automated spectrophotometric and