This study investigated the potential cardioprotective effects of metformin during ischemia-reperfusion in rat hearts. Hearts were isolated and perfused with or without metformin. Metformin accelerated the cessation of cardiac function during ischemia but delayed the onset of contracture and increased the time until maximum contracture, suggesting it reduces contractile injury. Metformin increased expression of PPARα during reperfusion only, indicating its mechanisms of action occur during ischemia-reperfusion. While metformin had no effect on left ventricular developed pressure, the small sample size limits conclusions about its impact on cardiac function.

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Investigation of the Impact of Metformin on Metabolism and Function in Cardiac Ischaemia
and Reperfusion
Sophie Oakley, Department of Biological Science, University of Hull, Hull
INTRODUCTION
Ischaemia can come in many forms and varying degrees of severity but
ultimately leads to death of the myocardium and accounts for the morbidity
and mortality of coronary heart disease (1)
.
Damage caused by ischaemia is determined by the degree and length of
the disproportionate oxygen supply and demand. However the act of
reperfusing the heart comes with it's own consequences and exacerbates
the injury caused by ischaemia (2)
.
Cardioprotective agents such as Metformin aim to reduce the damage
caused my ischaemia-reperfusion injury. Thus leading to the hypothesis
that Metformin should show evidence of cardioprotection (3)
.
AIM
To investigate the potential cardioportive effects of Metformin on metabolism
and function during ischaemia reperfusion.
METHODS
Using male Sprague-Dawley rats, hearts were isolated and perfused in a
modifed isovolumic retrograde Langendorff mode using the apparatus in
Figure 1 (4)
.
Hearts were perfused with Krebs-Henseleit buffer containing (NaCl 118mM;
NaHCO3
25mM; KCl 4.5mM; KH2
PO4
1.2mM; MgSO4
◦ 7H2
O 25mM; CaCl2
◦
H2
O 1.25mM; Glucose 5mM; Palmitate 3mM; Lactate 1mM; Pyruvate 0.1mM;
Glutamine 0.55mM. 1MM of Metformin was added in the Metformin model
noted in Figure 2..(4)
PPARα expression was analysed by sodium dodecyl sulphate polyacrylamide
gel electrophoresis (SDS PAGE) and western blotting. Actin was used to
normalise the optical density values. (5)
Results are presented as the means ±SEM where appropriate with any
statistical significance identified using an unpaired student's T test for
statistical analysis.
Figure 1: The Langendorff perfusion apparatus.
Figure 2:Control Perfusion Protocol in the absence and
presence of Metformin.
RESULTS
Figure 7 : Cessation of function (T1), Onset of Contracture (T2) and Time till max contracture
in the absence and presence of Metformin. N=2 in both groups.
Figure 3: Recorded trace of cardiac function. Diastolic Pressure (DP),
Systolic Pressure (SP), Left Ventricular Developed Pressure (LVDP)
Figure 5: LVDP at different stages of the protocol in either the absence or
presence of Metformin. N=3 in each group.
CONCLUSIONS
Metformin had no affect on left ventricular developed pressure
(LVDP) (Table 4) and(Figure 4). This was inconsistent with other
studies(6)
suggesting that either the sample size (n=3) was too
small or the concentration of Metformin (1mM) was not sufficient
to elicite a change.
Metformin accelerated the cessation of function during ischaemia
indicating Metformin might affect cardiac function within the
heart.
Metformin delayed the onset of contracture within ischaemia and
increased the time till max contracture (Table 2 and Figure 5)
suggesting that Metformin reduced contractile injury.
Metformin increased expression of PPARα in reperfusion only
(Figures 8 & 9) indicating that these changes are not instant and
may be consequence to mechanisms of action during ischaemia
-reperfusion.
Myocardial Contractile Function
Table 1. Left Ventricular Developed (LVDP) Pressure in the presence and
absence of Metformin during normoxia and reperfusion.
Cardiac Function During Ischaemia
PPARα expression
Figure 6: Recorded trace of cardiac function during ischaemia.
Time till cessation of function (T1), onset of contracture (T2), time
till max contracture (T3) and extent of contracture (E1).
REFERENCES
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Morrow,D, Murphy,S,Nahrendorf,M, Niessen,M, Piek,J, Pittet,M, M, Robbins,C,
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Waring,M Waterman,P, Wei,Y and Weissleder,R. (2012) Myocardial infarction accelerates
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Seymour, AM. (2013, October 16
th
). Cardioprotection 2.[PowerPoint slides]. Presented at
Clinical Chemistry lecture at Hull University.
Boengler,K, Heusch,G & Schulz,R. (2010) Inhibition of mitochondrial permability transition
pore opening: the holy grail or cardioprotection. Basic Research in Cardiology, 105,
(2),p151-154.
Cleland,J, Sample,J and Seymour,AM. (2006) Metabolic remodelling in the aging heart.
Journal of Molecular and Cellular Cardiology, 40, (1), p56-63.
Smith,K. (2006)The Impact of Erythropoietin on Uraemic Cardiomyopathy. PhD Thesis, Hull
University
.Barreto-Torres,G, Jaradov,S and Parodi-Rullan,R. (2012) The Role of PPAR α in
Metformin-induced attenuation of mitochondrial dysfunction in acute cardiac
ischemia/reperfuion in rats. International Journal of Molecular Sciences, 13, (6), p7694-
7709.
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2.
3.
4.
5.
6.
ACKNOWLEDGEMENTS
Many thanks and much gratitude goes to my supervisor Dr
Anne-Marie Seymour. Without her support and guidance this
project would not have been possible. Thank you Kath Bulmer,
your invaluable assistance and vast practical knowledge which
led to the success of both the perfusions and the analytical
processing.
Model
LVDP (mmHg)
Normoxia Reperfusion
Control (n=3) 60.3±36.7 69.4±38.91
Metformin (n=3) 61.9±19.55 64.8±13.5
T1 (mins) T2 (mins) T3 (mins) E1 (mmHg)
Control
(n=2) 02:04±0.01 8:18±1.4 05:32±0.11 57.9±39.7
Metformin
(n=2) 1:51±0.01 09:40±0.11 04:02±0.07 65.3±36.4
Table 2. Cardiac function in the presence and absence of Metformin
during ischaemia.
PPARα
Actin
Figure 8: : PPARα expression normalised by actin,
visualised by Western Blotting
1. Control Normoxia 1
2. Control Normoxia 2
3. Metformin Normoxia 1
4. Metformin Normoxia 2
5. Control Reperfusion 1
6. Control Reperfusion 2
7. Control Reperfustion 3
8. Metformin Reperfusion 1
9. Metformin Reperfusion 2
10. Metformin Reperfusion 3
11. Liver
12. Fat
Figure 9: PPARα expression quantified using the relative optical density of PPARα
gel bands against the actin gel bands where absorbance is compared to expression.