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SA JOURNAL OF DIABETES & VASCULAR DISEASE
RESEARCH ARTICLE
VOLUME 17 NUMBER 2 • NOVEMBER 2020
49
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-μm
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 constantpressure
(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 potentiometric
analyses (Beckman AU Chemistry Analyzer, PathCare, SA).
14
Statistical analysis
Data are expressed as mean and standard error of the mean
(SEM) or as box plots and the mean, and
n
indicates the number