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CARDIOVASCULAR JOURNAL OF AFRICA • Volume 27, No 6, November/December 2016
378
AFRICA
Statistical analysis
Animals were randomly assigned to different experimental
groups on the day of experimentation. Where functional recovery
was assessed as the primary endpoint (i.e. after 20 minutes’ GI)
data were collected from a minimum of four to a maximum of
10 rats per group. Infarct size was compared between groups
containing a minimum of six rats per group. All statistical
analyses were done using Graphpad Prism (version 6.01). For all
comparisons (unless otherwise specified) a one-way analysis of
variance (ANOVA) was performed, followed by Dunnett’s test
to compare all relevant experimental groups to control alone. A
p
-value of less than 0.05 was considered statistically significant.
Results
The effect of FTY720 in a model of 20 minutes’ GI
Isolated, working rat hearts exposed to 20 minutes’ GI were
treated with either 1 or 2.5
µ
M FTY720, either prior to sustained
ischaemia or immediately at the onset of reperfusion (Fig. 1).
All functional data recorded in this study are shown in Table 1.
Baseline functional measurements of the hearts, prior to both
FTY720 administration and ischaemia, are shown in Table 2.
There were no significant differences in the basal capacity of any
of the groups compared to the control.
Administration of FTY720 at a dose of 1
µ
M had no effect
on functional recovery (Fig. 3), irrespective of whether it was
administered before ischaemia or during early reperfusion.
Similar to the effects associated with 1
µ
M, reperfusion
administration of 2.5
µ
M FTY720 did not exert any significant
effects on post-ischaemic function (Fig. 4).
Table 1. Functional parameters of isolated, perfused rat hearts recorded at baseline (i.e. prior to both the administration of FTY720, as well as exposure to sustained
ischaemia) and at the end of work-mode reperfusion (following sustained ischaemia). Reperfusion function was compared to baseline function using a paired
t
-test
Group
Coronary flow
†
(ml/min) Aortic output (ml/min) Cardiac output (ml/min)
Total work (mW)
Heart rate (beats/min) Systolic pressure (mmHg)
n
Baseline Post-ischaemic Baseline Post-ischaemic Baseline Post-ischaemic Baseline Post-ischaemic Baseline Post-ischaemic Baseline Post-ischaemic
20 minutes’ global ischaemia
Control
8.45
±
0.75 7.50
±
0.80 44.20
±
3.41 16.00
±
3.75* 60.55
±
4.10 28.35
±
4.29* 13.17
±
1.22 5.92
±
1.22
#
317.4
±
19.8 263.4
±
13.5 90.25
±
3.75 78.88
±
3.84
@
5–10
1
µ
M FTY720
PreFTY 9.13
±
1.13 6.31
±
0.43
@
47.13
±
4.11 13.50
±
3.62* 63.81
±
4.78 24.25
±
4.83* 14.41
±
0.56 5.94
±
1.09
$
278.6
±
18.4 255.0
±
15.9 96.25
±
4.05 82.25
±
3.39 5–8
PostFTY 6.70
±
0.47 12.20
±
0.93* 51.50
±
1.78 14.00
±
3.66* 66.39
±
2.45 23.72
±
5.45* 14.02
±
0.74 4.96
±
1.24* 267.0
±
10.2 189.0
±
37.1 94.89
±
3.01 72.11
±
14.01 9–10
2.5
µ
M FTY720
PreFTY 6.50
±
0.52 6.58
±
0.37 41.00
±
5.21 0.0
±
0.0
$
55.25
±
6.48 5.92
±
0.45
$
13.98
±
0.41 0.40
±
0.40* 266.8
±
23.5 24.3
±
24.3
#
101.7
±
4.78 16.00
±
16.00
#
4–6
PostFTY 5.50
±
0.49 9.57
±
0.98
#
44.29
±
2.88 8.21
±
1.83
$
58.93
±
4.81 24.00
±
4.73* 11.54
±
1.09 4.60
±
0.92* 282.1
±
15.2 259.4
±
9.6 88.71
±
5.47 77.14
±
3.23
@
6–7
35 minutes’ regional ischaemia
Control
9.00
±
0.52 9.27
±
0.46 48.33
±
3.56 14.33
±
6.82
#
62.83
±
4.34 29.83
±
7.09
#
13.46
±
1.13 5.60
±
1.49
#
292.7
±
10.3 277.5
±
8.0
@
96.00
±
1.88 81.33
±
2.72
#
6
1
µ
M FTY720
PreFTY 8.92
±
0.45 9.50
±
1.71 55.50
±
1.63 9.50
±
3.38* 69.08
±
3.98 19.75
±
4.52
$
15.46
±
0.63 3.98
±
0.55* 274.7
±
9.9 216.5
±
38.8 96.50
±
1.50 76.33
±
5.40
#
6
PostFTY 10.00
±
0.82 12.07
±
1.14 44.00
±
4.30 2.57
±
1.49
$
58.14
±
5.49 14.16
±
3.59
#
11.67
±
1.13 2.31
±
0.74
$
285.6
±
15.5 205.1
±
40.8
@
90.29
±
1.11 59.29
±
12.03
@
7
2.5
µ
M FTY720
PreFTY 10.25
±
0.54 9.42
±
0.27 47.00
±
3.53 0.33
±
0.33* 63.17
±
4.39 12.08
±
4.09
$
13.02
±
0.98 1.82
±
0.67
$
279.7
±
17.4 146.3
±
47.3 92.67
±
1.20 45.50
±
15.74
@
6
PostFTY 9.67
±
0.76 10.67
±
0.76 52.33
±
3.20 1.00
±
1.00* 69.42
±
3.94 9.42
±
3.25
$
14.62
±
0.87 1.55
±
0.58
$
269.8
±
13.2 156.0
±
50.2 94.83
±
1.08 48.33
±
15.66
@
6
†
As measured during retrograde perfusion; *
p
<
0.0001 versus baseline;
$
p
<
0.001;
#
p
<
0.01 versus baseline;
@
p
<
0.05 versus baseline.
Table 2. Baseline functional ability of isolated rat hearts prior to
exposure to both 20 minutes’ global ischaemia (GI),
as well as treatment with FTY720 (1 or 2.5
µ
M) either
before sustained ischaemia or during initial reperfusion
Group
Aortic output
(ml/min)
Cardiac output
(ml/min)
Total work
(mW)
Number
Control
44.20
±
3.41 60.55
±
4.10 13.17
±
1.22 5–10
1
µ
M FTY720
PreFTY
47.13
±
4.11 63.81
±
4.78 14.41
±
0.56
5–8
PostFTY
51.50
±
1.78 66.39
±
2.45 14.02
±
0.74 9–10
2.5
µ
M FTY720
PreFTY
41.00
±
5.21 55.25
±
6.48 13.98
±
0.41
4–6
PostFTY
44.29
±
2.88 58.93
±
4.81 11.54
±
1.09
6–7
Control
PreFTY PostFTY
Functional recovery
50
40
30
20
10
0
Control
PreFTY PostFTY
Functional recovery
60
50
40
30
20
10
0
Control
PreFTY PostFTY
Functional recovery
60
50
40
30
20
10
0
Fig. 3
The effect of FTY720 (1
μ
M) administered either immediately before sustained ischaemia (PreFTY), or for the first 15 minutes
of reperfusion (PostFTY), on functional recovery in isolated hearts exposed to 20 minutes’ GI, followed by 35 minutes’ reper-
fusion. Functional recovery was assessed as post-ischaemic (A) aortic output (AO); (B) cardiac output (CO); and (C) total
work expressed as a percentage of pre-ischaemic values,
n
=
5–10.
A
B
C