27 / 70
CARDIOVASCULAR JOURNAL OF AFRICA • Volume 30, No 5, September/October 2019
AFRICA
269
Radiation Medicine Experimental Animal Centre. All study
protocols and use of rats were approved by the Institutional
Animal Care and Use Committee of Tianjin Medical University
(Tianjin, China).
Ca
2+
-free Tyrode solution contained (mM): NaCl 137, KCl
5.4, MgCl
2
1, NaH
2
PO
4
0.33, HEPES 10, and glucose 10 (pH
7.4 with NaOH). KB solution contained (mM): L-glutamic
acid 50, KCl 40, MgCl
2
3, KH
2
PO
4
20, taurine 20, KOH 70,
EGTA 0.5, HEPES 10, and glucose 10 (pH 7.4 with KOH).
The pipette solution contained (mM): CsCl 140, NaCl 10,
EGTA 5, HEPES 5, Na
2
ATP
5
(pH 7.3 with CsOH). The normal
extracellular solution contained (mM): choline-Cl 120, NaCl 25,
CsOH 4, CaCl
2
0.1, CoCl
2
2, MgCl
2
1, HEPES 10, and glucose
10 (pH 7.4 with CsOH). The simulated ischaemic extracellular
solution contained (mM): choline-Cl 120, NaCl 25, CsOH 4,
CaCl
2
0.1, CoCl
2
2, MgCl
2
1, HEPES 10, and natrium lacticum
20 (adjusted to pH 6.8 and filled with nitrogen for more
than five minutes before using). Atorvastatin calcium (USP
Corporation, Lot 344423-98-9) was dissolved in the ischaemic
extracellular solution to prepare the drug solution containing
5 μM atorvastatin (usually 3.02 mg of atorvastatin calcium was
dissolved in 500 ml of extracellular solution).
For isolation of the myocytes, single ventricular myocytes
were dissociated from hearts of Wistar rats using type II
collagenase (Gibco). Rats were weighted, heparinised (5 000 UI/
kg), anaesthetised with chloral hydrate (40 mg/kg), the chest was
opened and the hearts were removed, and then the rats were
euthanised. The heart was immersed in Ca
2+
-free Tyrode solution
(4°C) and immediately clipped.
The heart was cannulated through the aorta and mounted on
a Langendorff perfusion apparatus (100% O
2
, 37°C, perfusion
pressure 70 cm H
2
O). It was retrogradely perfused with Ca
2+
-free
Tyrode solution until the blood was washed out, followed by
perfusion with the same Ca
2+
-free Tyrode solution supplemented
with 0.6 mg/ml collagenase II and 0.5 mg/ml albumin bovine
serum (68 kD, Roche). As the drip rate reached 20 ml/min and
the colour of the heart changed to orange and transparent, the
perfusion was complete.
The heart was then removed into KB solution (37°C). The free
left ventricular wall was cut into approximately 8
×
2-mm sections
with a fine scissors and the endocardium and epicardium were
removed in the KB solution. The mid-myocardial section was
cut up and agitated with a dropper in order to obtain isolated
cells. The cell suspension was then filtered with a strainer (200
mesh). Before recording, the myocytes were placed in filtered KB
solution for more than two hours.
I
Na
was recorded at room temperature (25°C) using the
whole-cell configuration of the patch-clamp with Axopatch
700B amplifiers and pClamp 10.1 software (Axon Instruments,
USA). Pipettes were pulled from borosilicate capillary tubes
using a programmable horizontal micro-electrode puller (P-97,
Sutter Instruments, USA) and heat polished with a microforge
(MF-830, Narishige). Micropipette resistance was kept at 2–5
M
Ω
when filled with pipette solution and immersed in the
extracellular solution.
The cells were placed in normal extracellular solution
for rupture of the membrane, compensation for membrane
capacitance and series resistor (75%), and the currents were
recorded for baseline. Then the cell bath was perfused with the
simulated ischaemic solution (control group) or drug solution
(statin group) for three minutes (3 ml/min). At this time,
the extracellular solution was replaced completely and we
considered the time after one minute of perfusion as the zero
point for the start of ischaemia. The cells were then left standing
for one minute to avoid interference from mechanical vibration.
Thereafter
I
Na
was recorded every two minutes from three
minutes after the start of ischaemia to 21 minutes, in both the
statin and the control groups.
The holding potential was maintained at –90 mV and the
protocol for recording
I
Na
was composed of 50-ms pulses that
were imposed in 5-mV increments between –80 and +50 mV,
and pulse frequency was 2.5 Hz, which was matched with the
rat’s natural heart rate. In order to trace the inactivation curves,
a double-pulse protocol was set up: the first 50-ms conditioning
pulses were imposed in 5-mV increments between –80 and
+50 mV, each of which was followed by a test pulse to +10
mV. Finally, to describe the recovery curves after inactivation,
another double-pulse protocol was used: the first conditional
pulses were imposed at –40 mV for 50 ms, each of which was
followed by a fixed 80-ms test pulse from –90 to –40 mV, and the
interval between the two pulses was increased in 2-ms increments
from 2 to 76 ms.
Statistical analysis
In order to eliminate the effect of cell size on
I
Na
, the
I
Na
from
different myocytes should be standardised. As atorvastatin may
also affect the membrane capacitance, which may become a
confounding factor in the current density, we used the relative
current value as the normalised
I
Na
in order to evaluate the effects
of atorvastatin on the peak value of the
I
Na
.
The Boltzmann equation was used to fit the activation and
inactivation curves, and the recovery curve after inactivation was
fitted with an exponential equation. We observed the normalised
I
Na
, membrane potential at 50% maximal activation (
V
1/2,a
),
offsetting of the activation curve (
K
a
), membrane potential at
50% maximal inactivation (
V
1/2,i
), offsetting of the inactivation
curve (
K
i
)
and recovery constant (
τ
). The data were analysed
by means of variance analysis of repeated measurement data,
and the gating characteristics were analysed with the allogeneic
paired
t
-test;
p
<
0.05 indicated that the difference was statistically
significant.
Results
Effect of ischaemia on
I
Na
in the early stage after perfusion: Previous
experiments showed that ischaemia suppressed the amplitude of
I
Na
, but we observed the normalised
I
Na
was transiently increased in
the very early stage of ischaemia in the pre-experiment. In order to
verify the increased current was not associated with the mechanical
effect of perfusion, we compared the effect of ischaemic and
normal extracellular solutions on
I
Na
in the same way. We found
compared with normal extracellular solution, normalised
I
Na
was
transiently increased after perfusion with ischaemic extracellular
solution, while simulated ischaemia was for three minutes (0.92
±
0.04 vs 1.42
±
0.34 mA,
p
<
0.01; Fig. 1).
Effect of atorvastatin on
I
Na
in the early stage of ischaemia:
When entering the simulated ischaemic state, the whole-cell
currents of control and statin groups both changed over time
(Fig. 2). Because of the voltage-dependent characteristics, the