FInal Draft Sophie V5

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University of Hull

Investigation of the Impact of Metformin
on Metabolism and Function
in Cardiac Ischaemia and Reperfusion

School of Biological, Biomedical and Environmental Sciences

Sophie Oakley
BSc Biomedical Science
May 2014

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Abstract

Many studies have previously demonstrated the cardioprotective effect Metformin has when the
heart undergoes ischaemia reperfusion. This study set out to investigate the impact Metformin had
on metabolism and function in cardiac ischaemia and reperfusion. Using male Sprague-Dawley
rats, hearts were isolated and perfused in a modifed isovolumic retrograde Langendorff mode.
These were subjected to either 30 mins of Krebs-Henseleit (KH) buffer or 20 mins of KH buffer and
10 mins of KH plus 1mMol of Metformin. This was followed by 25 mins of ischaemia then 30
mins of reperfusion. This investigation showed that Metformin improved oxygen consumption
during normoxia, delayed the onset of contracture but accelerated time till max contracture during
ischaemia. Metformin also improved time to recovery and PPAR expression in reperfusion. In
conclusion Metformin altered both metabolism and function of the heart in this limited study and
with the data gathered and the current literature we have speculated the mechanisms involved.
Broader investigations into AMPK, substrate utilisation and the role of PPAR in cardioprotection
may provide further evidence for the theories speculated in this study.

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. Her enthusiasm and patience
provided me with the confidence and reassurance to put my all into this work.

Thank you Kath Bulmer, your invaluable assistance and vast practical knowledge which led to the
success of both the perfusions and the analytical processing. Rob Atkinson and Faisal Nuhu for
your help and patience whilst working within the lab.

To my lab partners Laura Morris, Naomi Whitwman and Nina Peters for all the laughs and fun we
had during this project and to Tracy Kew and Kathleen Nelson for your invaluable help.

Finally my sincerest thanks go to my mum and Lars, without your strength and moral support I
could not have completed this.
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Contents

Page
Abstract 2
Acknowledgements 2
Contents 3
Abbreviations 5
Introduction 7
Cardiac Ischaemia 8
Reduction of ATP 9
Reperfusion 10
Reactive Oxygen Species 12
Calcium Overload 12
Opening of the Mitochondrial Permeability Transition Pore 12
Fatty Acid Metabolism 13
AMPK mediated fatty acid metabolism 16
PPAR 17
Cardioprotection 18
Metformin 19
Objectives 19
Method 21
Perfusion Buffer 21
Langendorff Perfusion 22
Physiological Measurements 25
LVDP 26
Myocardial Oxygen Consumption 27
Efficiency of the Heart 27
Perfusion Protocol 27
Control 27
Metformin 28
Protein Expression 29
Sample Expression 29
SDS Page 30
Visualisation 32
Data Presentation and Statistical Analysis 32
Results 33
Cardiac Function in Normoxia, Ischaemia and Reperfusion 33
PPAR expression 41

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Discussion 43
Normoxia 43
Contractile Function 43
PPAR 44
Oxygen Consumption 45
Cardiac Efficiency 45
Ischaemia 46
Reperfusion

Contractile Function
48
48
PPAR 49
MVO
2
49
Cardiac Efficiency 50
Limitations 51
Future 51
Conclusion 52
Appendices 54
References 59

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Abbreviations

Akt/PKB
AMPK
ATP
BPM
BSA
Bwt
Ca
2+

cAMP
CK
CL
CHD
CO
CO
2

CPT
CVD
DP
ETC
FA
FADH
FAO
FAT
FFA
HIF-
HR
IL-6
iNOS
IR
IRS-1
KH
LVDP

Serine/threonine kinase or Protein kinase B
5'adenosine monophosphate-activated protein kinase
Adenosine triphosphate
Beats per minute
Bovine Serum Albumin
Body Weight
Calcium
Cyclic adenosine monophosphate
Creatine kinase
Cardiolipin
Coronary heart Disease
Cardiac output
Carbon dioxide
Carnitine palmitoyltransferase
Cardiovascular disease
Diastolic pressure
Electron transport chain
Fatty acid
Flavin adenine dinucleotide
Fatty acid oxidation
Fatty acid translocase
Free fatty acid
Hypoxia-inducible factor
Heart rate
Interleukin 6
Induce nitric oxide synthase
Ischaemia reperfusion
Insulin receptor subtrate
Krebs Hensleit (Buffer)
Left Ventricular Developed Pressure

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MI
MPTP
MVO
2
NADH
P38-MAPK
PAGE
PCI
PFK2
PGC-1
PI
PP2
PPAR
RISK
ROS
RPP
SDS
SERCA
SOD
SP
STEMI
TGF-
TNF

Myocardial Infarction
Mitochondrial permeability transition pore
Myocardial oxygen consumption
Nicotinamide adenine dinucleotide
p38 mitogen-activated protein kinases
Polyacrylamide gel electrophoresis
Percutaneous coronary intervention
Phosphofructokinase 2
Peroxisome proliferator-activated receptor gamma coactivator 1-alpha
Inoganic phosphates
Protein phosphatase 2
Peroxisome proliferators activated recptor
Reperfusion injury signalling kinase
Reactive oxygen species
Rate Pressure Product
Sodium dodecyl sulphate
Sarco/endoplasmic reticulum Ca
2+
-ATPase
Superoxide dismutase
Systolic pressure
St-elevation myocardial infarction
Tumor growth factor
Tumor necrosis factor

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Introduction

Cardiovascular Disease (CVD) was the leading cause of death in 2008, where 17.3 million people
died of CVD and of these 7.3 million deaths were due to coronary heart disease (CHD) WHO
(2013). These numbers are estimated to increase significantly by 2040 if no major changes are
made in treatment. This is in line with the impact of an increasing ageing population (Bibbins-
Domingo et al., 2011). The severity of the damage caused by ischaemia from CHD, reversible or
permanent, is determined by the degree and length of the disproportionate oxygen supply and
demand. Survival of a first myocardial infarction (MI) has increased to 90% but despite this
subsequent infarcts are associated with more considerable mortality (Butany et al., 2012). Overall
30% of all patients with a large MI will die before reaching hospital and the majority of these will
have died in under an hour reflecting just how quickly irreversible damage can occur (Morrow-
Frost 2013). With more people surviving their first MIs and larger subsequent infarcts causing
considerable mortality, it is of the utmost importance that not only short-term cardioprotection is
applied but also long term protection.

Ischaemia can come in many forms and varying degrees of severity and ultimately leads to death of
the myocardium and accounts for the morbidity and mortality of coronary heart disease. Metabolic
changes during normoxia, ischaemia and reperfusion are varied, however those in ischaemia and
reperfusion contribute to the damage seen as a consequence to inadequate blood supply and the long
term changes to the myocardium.

Reperfusion injury in the myocardium was first observed in the 1960s using a canine model and
was shown to exacerbate the necrosis of the myocardium. This led to the investigation of
cardioprotective mechanisms and the elucidation of the pathogenesis behind ischaemia (Turer &
Hill, 2010). Oscar Langendorff developed the retrograde technique of perfusion of the isolated
mammalian heart in 1895 which although modified, is still used today to explore heart physiology
and the pathogenesis and implications of various conditions on the heart (Bell et al. 2011). By
understanding the changes involved physiologically in the heart at each stage: normoxia, ischaemia
and reperfusion, we can use the Langendorff retrograde perfusion technique to examine the
functional and metabolic changes at these points individually and interpret them as both individual
environments and collectively as a chain of events.

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The Langendorff technique used to reperfuse the isolated heart remains the most widely used tool
for exploring the pathways, physiology and signalling surrounding the heart. This technique offers
a simple, cost effective and easily reproducible way of collecting data over long periods of time
whilst specifically looking at the heart without interference from other organ systems (Bell et al.,
2011). Although isolated heart perfusions may be run for several hours, it should be noted that a 5-
10% reduction in heart rate and contractility per hour has been reported (Sutherland & Hearse,
2000) but the Langendorff technique still remains the best method for the aims of the experiment.

Cardiac Ischaemia
The general description of an MI is the necrosis of the myocardium as a consequence to insufficient
blood supply. Following ischaemia, metabolic and functional changes occur within the hypoxic
tissue that activate apoptosis or necrotic pathways after prolonged periods in this state.
Consequently contraction of the myocardium ceases, phosphocreatine and ATP levels fall and
activation of Hypoxia Induction Factor (HIF-) pathways leads to the change into anaerobic
respiration (Semenza, 2011). Without intervention such as oxygen therapy, thrombolysis or
angioplasty the myocardium will be permanently damaged and the patient may die (Eckle &
Eltzschig, 2011). Figure 1 summarises the short-term effects of myocardial hypoxia where Table 1
demonstrates a time line of acute events with a large myocardial infarction.

Figure 1: Acute consequences of myocardial ischaemia and their mechanisms.
Adapted from (Leonard, 2011b)
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Table 1: Summarising the time-scale of acute events following a large myocardial infarction.
Adapted from (Leonard, 2011b).
Time Event
1-2 min Levels of ATP drop leading to cessation of contraction. Energy supplies are
depleted and increased levels of inorganic phosphates (Pi).
10 min Half of ATP levels are depleted, intracellular swelling begins, membrane
potential becomes altered and the heart becomes increasingly susceptible to
arrhythmias through changes in ion balance.
20-24 min Irreversible cell damage begins and vascular permeability increases causing
further edema.

Reduction of ATP
Under normal conditions oxidative phosphorylation provides 90% of the ATP used in the heart.
However in ischaemia these conditions inhibit HIF Hydroxylases allowing the formation of HIF-1
and leading to the stimulation of anaerobic glycolysis and use of endogenous substrates. Anaerobic
glycolysis is essential for the formation of ATP through the modification of pyruvate to lactate in
depleted oxygen conditions. Without adequate blood supply there is an accumulation of lactate,
NADH (nicotinamide adenine dinucleotide), FADH
2
(Flavin adenine dinucleotide) and H
+

,consequently impairing the enzymes involved in fatty acid oxidation and decreasing the pH.
Reduction of ATP alters ionic homoeostasis by impairing ATPases, specifically Na
+
K
+
ATPase.
Inhibition of these enzymes involved in ion transport leads to osmotic loading, calcium overload
and uncoupling of the respiratory chain. Changes in pH also alter myofilament sensitivity to
calcium and cause the cessation of contraction (Jaswal et al., 2011). Alterations in ion homeostasis
that lead to mitochondrial injury may impair complexes III (cytochrome bc
1
) and IV (cytochrome C
oxidase) limiting the recovery of oxidative phosphorylation during ischaemia. Mitochondrial
membrane dysfunction allows proton leakage into the mitochondrial matrix and restoration of the
electrochemical gradient is required to provide the proton motive force needed for oxidative
phosphorylation. This is accomplished by reversing ATP synthase, hydrolysing ATP to pump
protons and paradoxically further diminishing ATP levels (Sack, 2006).

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Reperfusion
Time dependent damage to the myocardium through ischaemia and the mechanisms discussed
previously emphasises the importance of restoring blood supply quickly in order to reduce the size
of ischaemic/necrotic tissue and at risk area. However reperfusion comes with its own
consequences and exacerbates the damage caused by the hypoxic myocyte but is necessary to limit
the overall injury. Revascularisation leads to the production of ROS, depletion of high energy
phosphates, structural damage and alterations, contractile dysfunction and calcium overload which
may lead to apoptosis or necrosis if not mediated (Seymour, 2013). Table 2 and Figure 2
summarise the key effects and consequences of reperfusion on the heart (Fillmore & Lopaschuk
2011).

Table 2: A summary of the effects of reperfusion on the ischaemic heart when reperfused..
Reperfusion Action Consequence Literature
Highly oxygenated blood
flows into the myocardium
Excess oxygen and impaired oxidative
phosphorylation in the ETC during respiration
leads to the formation of oxygen radicals.
(Chen and Zweier
2014)
H
+
washed out of myocytes
normalises pH
Utilisation of H
+
/Na
+
exchangers leads to
excess Na
+
in the cell.
(Anderson et al.,
2010)
Excess Na
+
in myocytes
Na
+
/ Ca
2+
exchangers lead to calcium overload
as Na
+
is removed.
(Hill & Turer
2010).
Calcium overload
Activation of different enzymes and pathways
lead to ROS production,the opening of the
MPTP and hypercontracture.
(Garcia-Dorado
et al., 2012).
High pressure and flow of
blood
Endothelial cell damage triggering an
inflammatory response.
(Gong et al.,
2012).
Inflammatory response
Activation of chemokines and cytokines leads
to recruitment of leukocytes and T-cells leading
to tissue damage.
(Andreoli et al.,
2011).

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Figure 2: A summary of the cellular changes in both ischaemia and reperfusion and their
consequences. Adapted from (Seymour, 2013).

Reactive Oxygen Species
Reactive oxygen species (ROS) such as superoxide (O
2
-), H
2
O
2
, OH and peroxynitrate can inflict
irreparable cellular injury to both nuclear, mitochondrial and cytosolic components of the cell if not
neutralised. Proteins, nucleic acids and lipids react and are modified by these species leading to
in/activation of various enzymes and dysfunction of several pathways. Under normal
circumstances many reactions produce ROS but scavenger systems such as superoxide dismutase
(SOD) and catalase convert them to more stable products. In reperfusion injury the flood of rich
oxygenated blood back into the ischaemic heart overloads these systems as oxidative
phosphorylation restarts in the dysfunctional mitochondria. Cardiolipin (CL) is a component of the
mitochondrial membrane that prevents deleterious effects and affects 5 main processes of
mitochondrial function including ATP production, ROS formation, cytochrome C anchoring,
regulation of mitofusion and the import of proteins (Han and He 2014). However CLs are
particularly vulnerable to oxidation because of their composition of peroxidozable fatty acids (De
Benedictis et al. 2013) which results in the impairment of complex 1 as well as release of
cytochrome C and consequential formation of the apoptosome through the opening of the
mitochondrial permeability transition pore (MPTP) (Bhakuni et al., 2009).
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Calcium Overload
Upon reperfusing the heart, pH rapidly returns to normal levels as H
+
ions are flushed from
myocyte. This process however requires the use of Na
+
/

H
+
and Na
+
/

HCO
3
-

exchange proteins and
increases the Na
+
concentration in the myocyte. To reduce these levels Na
2+
/Ca
2+
exchangers are
activated to remove the sodium and consequentially lead to calcium overload (Hill & Turer 2010).
With the restoration of pH and oxygen into the cell oxidative phosphorylation is restarted, however
in the presence of such high levels of calcium this leads to hypercontracture through rapid cycling
of calcium in the sarcoplasmic reticulum. Repeated contraction and the structural damage caused
by this dysfunctional calcium cycling and consequential activation of the contractile mechanisms
have been shown to lead to sarcolemmal membrane rupture. Calpains activated by high levels of
calcium interfere with the structural integrity and strength of the cytoskeleton and the sarcolemma
membrane through proteolysis, therefore increasing the likelihood of this event (Garcia-Dorado et
al., 2012).

Calcium mediated activation of various other enzymes including proteases, protein kinases,
endonucleases, nucleases and capsases results in the dysfunction of mitochondria, DNA damage,
structural break down and ROS production. Dislocation of cytochrome C from the mitochondrial
membrane with calcium competing for binding sites on the mitochondrial membrane then enables
the opening of the MPTP and induction of apoptosis pathways (Asp et al., 2013; Jou & Peng, 2010).

Opening of the Mitochondrial Permeability Transition Pore
The mitochondrial permeability transition pore is a channel of the mitochondrial membrane that is
non selective. It has been indicated as a major contributor to reperfusion injury as drugs inhibiting
the opening have shown large reductions in the size of the area of necrotic tissue caused by MIs
(Frolich et al., 2013). Swelling of the mitochondria occurs as a consequence of the high
concentration of proteins in the inter membrane space however calcium is released along with
compounds such as cytochrome C and ROS into the cytosol which induces apoptosis (Halestrap,
2010). Figure 3 summarises this process.
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Figure 3: The Caspase cascade triggered as a consequence of MPTP opening.
Adapted from (Anderson et al., 2004).

Clinically, despite the consequences of reperfusion injury, the aim is not only to limit the infarct
size but also to return as much functionality to the heart whilst providing pain management. In the
hospital setting oxygen therapy, thrombolysis and angioplasty are the main ways of reperfusing the
heart (Dejean et al., 2011).

Fatty Acid Metabolism
The myocardium requires 6kg of ATP to be cycled a day in order to meet its metabolic demand.
With limited stores of phosphocreatine to convert in the absence of glucose, the heart must be able
to utilise different substrates such as fatty acids (FA), lactate, pyruvate, aminoacids and ketones in
order to produce adequate ATP to function. Figure 4 highlights the pathways involved in FA and
glucose metabolism. Fatty acid -oxidation under normal circumstances generates 60-80% of ATP
during normoxia. However during ischaemia lack of oxygen promotes glycolysis, reducing fatty
acid oxidation significantly. Build up of fatty acids in ischaemia in the absence of oxygen can lead
to lipotoxic environments where instead of being converted to acyl-CoA FAs interferes with
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insulin receptor substrate 1 (IRS-1) pathways leading to a reduction in glucose intake (Dickerson et
al. 2010).

At reperfusion, FA levels rise further due to elevated plasma FA concentrations during ischaemia.
This consequently inhibits signalling pathways for insulin receptors and pyruvate dehydrogenase
leading to reduction of glucose uptake and efficacy of the heart. This is summarised in Table 3
(Jaswal et al., 2011). High FA levels not only inhibit glucose metabolism but act as a detergent,
disrupting the membranes of the cell causing further damage to the myocardium (Arouri and
Mouritsen, 2013). Accumulation of palmitate and serine leads to the formation of ceramides which
in larger amounts lead to induction of protein phosphatase 2 (PP2A) apoptopic pathways (Gudz &
Novgorodov, 2009). Large amounts of ceramides can induce Nitric Oxide Synthase (iNOS) and
consequentially the formation of nitric oxide, a reactive oxygen species which reacts with
superoxides to produce peroxynitrite and induce apoptosis (Cai et al., 2010).

Table 3: Summarising the changes in metabolism throughout normoxia, ischaemia and reperfusion
and the consequences associated with each.
Substrate
Consequence
Phase Glucose Fatty Acid
Normoxia 20-40% of energy generated 60-80% of energy generated Metabolism is balanced
Ischaemia
Majority of energy generated
through glycolysis
Very little in depleted
oxygen conditions
FA, lactate and H+
accumulate
Reperfusion Some energy generated
Accelerated energy
generation from high FA
levels
Repression of glucose
metabolism and
reduction of cardiac
efficiency

Reduction of cardiac efficiency through increased FA metabolism is demonstrated in Table 4
showing that although fatty acid oxidation (FAO) through palmitate is less efficient than glucose per
molecule of oxygen, it provides more ATP per carbon atom (Fillmore & Lopaschuk, 2011). Both in
ischaemia and heart failure the myocardium has shown to change to the more oxygen efficient
glucose metabolism in reaction to hypoxia, expression of particular enzymes in FAO are decreased
as the myocardium becomes more dysfunctional (Ardehali et al., 2012).
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Table 4: Examination of the efficacy of glucose and palmitate metabolism .Adapted from
(Aksentijevic, 2008)
Palmitate Glucose
Energy Efficiency 6.6 ATP per CO
2
5.3 ATP per CO
2

Oxygen Efficiency 4.6 ATP per O
2
5.3 ATP per O
2

Overall ATP 106 32

Figure 4: Pathways of fatty acid and glucose oxidation in the cell. In the absence of oxygen there
is a build up of H+, lactate, NaDH and FADH
2
and impairs several key enzymes and pH.
Adapted from (Fillmore & Lopaschuk, 2011).
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Imbalance of either FA or glucose leads to problems in ionic homeostasis in the myocardium.
Excess lactic acid and proton production from prolonged glycolysis exacerbates acidosis and
increases the risk of calcium overload at reperfusion. Consequentially ATP has to be redirected to
dealing with these products causing contractile function and efficiency to be reduced until sufficient
energy levels are maintained upon reperfusion (Dyck et al., 2002). The degree of recovery upon
reperfusion is determined by the type of metabolism used throughout. As previously mentioned
prior, high levels of circulating FA and consequential oxidation lead to repression of glucose
oxidation and reduced cardiac efficiency. Studies promoting increased glucose metabolism in
reperfusion have shown promising results towards cardioprotection (Clanachan et al., 2012;
Arrhenius et al., 2004) however the activation of AMPK itself still promotes FAO and raises the
question of whether sufficient glucose oxidation can be obtained to counter this effect.

AMPK mediated fatty acid metabolism
AMPK is an enzyme regulating the pathways involved with the production and use of energy.
Phosphorylation of AMPK causes activation leading to the inhibition of energy consuming
pathways and the stimulation of the generation of energy instead as demonstrated in Figure 5
(Hardie et al., 2012).

Figure 5: Summarises some of the outcomes of activation of AMPK on not only metabolism
but also other cellular functions. Adapted from (Hardie et al., 2012)

Although FA levels are elevated in ischaemia the primary substrate used is glucose. On reperfusion
these FA are rapidly oxidated. However, the Randals cycle dramatically inhibits pyruvate
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dehydrogenase and as consequence reduces glucose oxidation (Boisvenue et al., 2011). FA
metabolism is mediated through increased uptake of substrate by membrane transport proteins.
First, fatty acid translocase (FAT)/CD36 is responsible for 50% of FA transport into the cell. AMPK
promotes its recruitment from intracellular storage to the cell membrane (Bonen et al., 2010; Dyck
and Lopaschuk, 2006). Secondly, AMPK inhibits carnatine palmityltransferase-1 which regulates
fatty acid entry (Abrahani et al., 2005; Birnbaum et al., 2004). Thirdly, AMPK promotes the
transport of lipoprotein lipase which facilitates FA oxidation by extracting fatty acids from
triglycerides (Atherton et al., 2013; Beauloye et al., 2000).

PPAR
PPAR is one of a family of hormone receptors. It is expressed particularly in tissues which are
metabolically very active. PPAR in particular plays a key role in FA metabolism not only through
peroxisomes but also the expression of enzymes involved in majority of the FAO (Ardehali et al.,
2012). Genetic knock-out studies have shown that in the absence of PPAR there is reduced FAO,
increased utilisation of glucose, decreased expression of both FA transport proteins and enzymes.
Contractile failure at high workload was more commonly seen in the knock-out mice, highlighting
the importance of FAO in more stressful situations (Altarejos et al., 2002). Despite this, the
addition of insulin and the consequent up-regulation, Glut-1 allowed these mice to overcome the
ATP requirements solely through increased transport and glucose metabolism (Balschi,J et al.,
2005) and it is these principles that are the basis for several cardioprotective drugs.

PPAR has not only been implicated in FAO studies of cardioprotection and vice versa but also in
fibrosis, inflammation and endothelial function. Studies involving PPAR agonists resulted in a
variety of associated benefits. Reduced fibrosis from the decrease in endothelin 1 mediated Tumor
Growth Factor- (TGF-) expression (Fan et al., 2012). Increased levels of nitrous oxide and
decreased vascular tone have improved endothelial function (Cianflone et al., 2006) and decreased
expression of inflammatory cytokines such as interleukin-6 (IL-6) and tumor necrosis factor
(TNF) all of which can contribute to attenuation of injury of the myocardium (Paintlia et al. 2013).
Additionally other studies showed that PPAR activation in mice significantly reduced the high
levels of free fatty acids at reperfusion and potentially attenuating potential lipotoxic
cardiomyopathy (Chen et al., 2006).

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Cardioprotection
Current clinical therapies for cardiac ischaemia are limited due to the mostly unpredictable nature of
MIs. Percutaneous Coronary Intervention (PCI) is the first intervention used in an ST-elevation MI
(STEMI) and then thrombolytic therapy is started (Boura et al., 2003). Both of these techniques
work on a mechanical based approach of removing the obstruction to return blood flow but do little
to prevent damage from reperfusion injury (Beatt et al., 2006). New pharmacological approaches
focus more on reperfusion injury and the cellular and metabolic mechanisms of injury, aiming to
inhibit or promote pathways associated with cardioprotection. Inhibition of the opening of the
MPTP and apoptosis cascades are one such target whereas metabolic changes in substrate use and
the promotion of survival pathways and better calcium handling are other targets (Boengler et al.,
2009). AMPK is one such pathway linked to cardioprotection and particularly emphasised in one
mechanism of action of the cadrioprotective agent Metformin. Figure 6 demonstrates the
relationship between AMPK and several metabolic processes in both ischemia and reperfusion.

Figure 6: Schematic of the suggested effects of AMPK on metabolic control in the cell during
ischaemia and reperfusion. Adapted from (Balschi and Tian, 2006).

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Several studies have demonstrated a correlation between increased AMPK activated glucose uptake
through upregulated GLUT4 activity and cardioprotection, particularly the inhibition of FAO and
attenuating the consequential production of ceramides which increase mitochondrial calcium
(Clanachan et al., 2011). High glucose levels inhibit carnitine palmitoyltransferase activity through
action of manoyl-coA and thus lower FAO (Bartha et al., 2013). This decrease in FAO and
consequential ceramide production could provide an answer to AMPKs role in delaying calcium
overload, as ceramides not only increase plasma calcium levels but also induce apoptosis and
necrosis through Ca
2+
influx and disruption of both mitochondrial networks and Ca
2+
buffering
capacity (Criollo et al., 2013).

Metformin
Metformin is clinically used in diabetics to help increase insulin sensitivity, lower blood glucose
and increase uptake of glucose through suggested stimulation of the adenosine monophosphate-
activated protein kinase (AMPK) and RISK pathways. Peroxisome proliferator-activated receptor
gamma co-activator 1- (PGC-1) is one such survival factor which Metformin increases the
expression of in order to manipulate energy metabolism and mitochondrial homeostasis. By
attenuating respiratory complex 1 in the mitochondria, ATP levels initially decrease leading to the
activation of AMPK (Algire et al.,2012). Despite this, not all of the morbidity and mortality
associated with ischaemia is immediate. Mitochondrial alterations as mentioned previously, as well
as changes in metabolism, have been implicated in decreased efficacy and functionality of the
reperfused heart and may present later as a much bigger problem associated with heart failure (De
Boer et al., 2013). Experimental investigations in animal models perfused using the Langendorff
technique and inducing ischaemia-reperfusion (IR) showed marked decrease in infarct size
compared to the control. This suggested that the mechanisms of Metformin were not purely based
on substrate utilisation (Hall et al., 2013). However Metformin will be used in this investigation
into the metabolic changes in the ischaemic and reperfused heart and compared inorder to explore
how both cardioprotection occurs and how metabolic and functional processes are altered as a
consequence.

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Objectives
Metformin is traditionally used to treat high blood glucose in diabetics. Increasingly literature has
demonstrated the cardioprotective effects of Metformin through a variety of different mechanisms
and pathways. Thus it is hypothesised that Metformin should show some cardioprotective action.
By understanding the changes at each stage: normoxia, ischaemia and reperfusion, we can use the
Langendorff retrograde perfusion technique to examine the functional and metabolic changes at
these points individually and interpret them as both individual environments and collectively as a
chain of events in both the presence and absence of Metformin.

This study aims to examine not only the metabolic changes but also the functional changes when
hearts are treated with Metformin. More specifically the objectives of this investigation are:

1. to determine the impact of Metformin on cardiac function during normoxia, ischaemia and reperfusion
2. to analyse the impact of Metformin on cardiac metabolism
3. to assess the effect of Metformin on the expression of PPAR

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Method

All procedures using Male Sprague-Dawley rats of 308-380g supplied by Charles River (Kent)
conformed to the recent amendments of the Animals (scientific procedure act) Home Office (2012).

Perfusion Buffer
The Krebs-Henseleit (K-H) buffer pH 7.4 contained the following components as shown in Table 5
made up in 18M ultra pure water and filtered through a 0.45micron filter.

Table 5: Components of the K-H buffer.
Compound Molarity (mM)
NaCl 118
NaHCO
3
25
KCl 4.5
KH
2
PO
4
1.2
MgSO
4
7H
2
O 1.2
CaCl
2
H
2
O 1.25
Glucose 5
Palmitate 3
Lactate 1
Pyruvate 0.1
Glutamine 0.55

During the Metformin protocol 1mMol of Metformin was added to the K-H buffer. No dose
responsive curve was used. This value was chosen based on previous studies.

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Langendorff Perfusion
Animal were anaesthetised with sodium thiopentane (100mg/kg) before the heart was quickly
excised, rinsed and placed into a small petri dish of ice cold K-H buffer containing Heparin. The
heart and dish were then weighed before the heart was cannulated via the aorta within 5mins on the
perfusion apparatus Figure7 and 8 (Sample et al., 2006).

Hearts were perfused in a modified isovolumic retrograde Langendorff mode equilibrated with
95% O
2
/ 5% CO
2
K-H buffer (pH 7.4) via a special contractile oxygenator which was jacketed to
maintain the temperature at 37C and at 14ml/min (Sample et al., 2006). The apex of the left
ventricle was pierced, passing through the mitral valve to prevent fluid build up as a consequence of
Thebesian circulation Liao et al., (2012). Copper pacing wires were inserted and connected to the
PowerLab stimulator output, pacing the heart with a 1 msec pacing pulse at 400-1500mV at 5Hz
ensuring (HR) would be 300 BPM.

Contractile function was monitored using a cling film balloon inserted into the left ventricle via the
mitral valve and attached via a fluid filled line. This was then connected to the SensoNor pressure
transducer, bridge amplifier and Powerlab (4/35) and its volume then adjusted to a left ventricular
diastolic pressure of 8mmHg. with the 2.0ml micrometer syringe (Gilmont Instruments, Barrington
USA). With the pacing wires connected to the system the heart was then placed into perfusion
chamber and contractile function recorded. At the end of each protocol, hearts were removed and
freeze clamped with Wollendberger tongs before being stored in liquid nitrogen at -196C until
further analysis.

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Figure 7: The Langendorff perfusion apparatus.

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Figure 8: The cannulated heart.

201005529 25

Physiological Measurements
Heart rate (HR), Systolic Pressure (SP) and Diastolic Pressures (DP) were recorded throughout the
perfusion SensoNor pressure transducer, bridge amplifier and Ad instruments Powerlab (4/35). HR
was a constant 300 bpm and systolic and dystolic pressure means were acquired from Labchart
traces over the period of normoxia as seen in Figure 9.

Figure 9: Recorded trace of cardiac function. Diastolic Pressure (DP), Systolic Pressure (SP),
Left Ventricular Pressure (LVDP).

LVDP
Left Ventricular Developed Pressure (LVDP) was calculated by using Equation 1. LVDP was then
used to calculate Rate Pressure Product (RPP). A measure of cardiac work load or stress was the
calculated by Equation 2.
201005529 26

Equation 1: Calculation for LVDP

Equation 2: Calculation for RPP

Myocardial Oxygen Consumption
The Myocardial Oxygen Consumption (MVO
2
) was calculated by measuring the oxygen content of
the perfusate and buffer and the heart at each stage of the experiment. The pO
2
was measured using
an AB Radiomoles blood gas analyser. This was then used to calculate the oxygen consumption
(moles/g wet weight/min) via Equation 3 (Morgan and Neely, 1974; Batteby et al., 1967).

Equation 3: Calculation for MVO
2.
Solubility of water in O
2
(0.199mmol/ml).
Flow Rate (14ml/min)

Efficiency of the Heart
Efficiency of the heart is calculated from the work done by the heart per O
2
consumed. Using RPP
and MVO
2
this value was calculated using Equation 4.

Equation 4: Calculation for Efficiency

LVDP = Systolic Pressure Dyastolic Pressure
RPP = LVDP x HR

MVO
2
= (((pO
s
(buffer) pO
2
(effluent)) / 760mmHg) x solubility of water in O
2
) x flow rate)
/ weight of heart
RPP /MVO
2
= Efficiency
201005529 27
Perfusion Protocol
Control
After a 10 min equilibration period normoxia protocol was started. Perfusion was left to run for 30
mins under the K-H buffer, then ischaemia was induced by turning off the K-H buffer and allowing
the perfusion chamber to fill up. After 20 mins of ischaemia the K-H buffer was turned back on,
balloon was deflated and the perfusion tank allowed to drain. 5 mins later the balloon was re-
inflated and reperfusion started and perfused for a further 30 mins before hearts were freeze
clamped and stored at -196C. This is summarised in Figure 10.

Figure 10: Control Perfusion Protocol in the absence of Metformin.

Metformin
After a 10 min equilibration period normoxia protocol was started. Perfusion was left to run for 20
mins under K-H Buffer. The buffer was then switched to K-H + 1mM Metformin buffer and run for
a further 10 mins. Ischaemia was induced at 30 mins by turning off the K-H + 1mM Metformin
buffer and allowing the perfusion chamber to fill up. After 20 mins of ischaemia the K-B buffer
was turned back on, balloon was deflated and the perfusion tank allowed to drain. 5 mins later the
balloon was re-inflated and reperfusion started and perfused for a further 30 mins before hearts
were freeze clamped and stored at -196C. This is summarised in Figure 11.

201005529 28

Figure 11: Perfusion protocol in the presence of 1mM Metformin.

From traces during ischaemia the values of time till cessation of function (T1), onset of contracture
(T2), time till max contracture (T3) and extent of contracture (E1) are shown in Figure 12.

Figure 12: 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).

201005529 29
Protein Expression
PPAR expression was analysed by sodium dodecyl sulphate polyacrylamide gel electrophoresis
(SDS PAGE) and western blotting.

Sample Preparation
Frozen cardiac tissue was ground in a pestle and mortar in liquid nitrogen to prevent thawing.
Approximately 0.150g of tissue sample was mixed with 1ml of extraction buffer containing 50mM
Tris (pH 7.4), 1% w/v SDS and protein inhibitor cocktail. Samples were homogenised 3x for 5 secs
at 4C with an Ultra-Turax homogeniser. Samples were then centrifuged at 13000rpm for 5 mins at
4C and the supernatent removed and kept on ice. Protein concentration for loading was determined
by the BioRad spectrophotometric assay. A dilution series of 0 to 1mg/ml Bovine Serum Albumin
(BSA) was used as a standard by adding 60l BSA to 3ml of BioRad dye and incubating at room
temperature for 5 mins. The absorbance of each was then read at 595nm resulting in the standard
shown in Figure 13.

Figure 13: Protein standard curve

10l of sample supernatant was diluted with 190l of ultra pure water and vortexed for a 1 in 200
dilution to give an amount of protein with the linear range of the standard curve. 60l of diluted
sample was added to 3ml of BioRad dye. This was added to the curvette and the solution mixed
201005529 30
before being incubated for 5 mins at room temperature and then absorbance read at 595nm from the
spectrophotometer.

Protein samples were diluted to a 1:2 dilution by the addition of Laemmli buffer containing Tris pH
6.8 125mM, SDS 2% (w/v), glycerol 10% (v/v), bromophenol blue 0.001% (w/v) and 2-
mercaptoethanol 5% (v/v)(Laemmli,1970) to achieve a final protein concentration of 5g/l. This
solution was then boiled for 5 mins and split up into smaller aliquots and excess samples were
frozen for storage.

SDS Page
Proteins were separated using a BioRad Mini Protean II and sodium dodecyl sulphate
polyacylamide gel electrophoresis (SDS-Page). A 12% (v/v) running gel, a 3% (v/v)
polyacrylamide stacking gel consisting of the components listed in Table 6 and a 10% running
buffer (Tris 25mM, SDS 0.1% (v/v), glycine 192mM) pH 8.3 were used.

Table 6: Composition of the stacking and running gels.

Component
3% Stacking Gel
Volume (ml)
12% Running Gel
Volume (ml)
H
2
O 7.4 3.35
10% (w/v) SDS 0.1 0.1
1.5M Tris (pH 8.8) 6.8 2.5
Acyrlamide/ bis (30% w/v) 1.25 4
Ammonium persulfate (10% w/v) 1.3 0.05
TEMED 0.02 0.01

201005529 31
The initial gels were used to practise technique and optimise protein loading. These were stained
with Brilliant Blue polyacrylamide gel staining to show protein separation as shown in Figure 14.

Figure 14: Brilliant Blue staining of practice gel.

10l of prepared sample was loaded into the gel with 5l of molecular marker and liver and fat
samples for positive and negative controls in the final gels before being run for an hour. Gels were
then washed of SDS with transfer buffer (tris 25mM, glycine 192mM) pH 8.3 for 30 mins to
remove any SDS before the proteins were transferred to a nitrocellulose membrane for 1.5 hours at
4C using a mini-transblot cell (BioRad laboratories,UK).

After transferring the proteins, the membrane was washed in TBS Tween for 5x 5mins. Following
this the membrane was blocked in 5% milk blocking buffer for an hour to reduce unspecific
binding. After another wash, 5x 5mins in TBS Tween PPAR Santacruz rabbit primary antibodies
were incubated over night at 4C. The membrane was then once again washed 5x 5min in TBS
Tween before the secondary antibody Donkey anti-rabbit was applied and incubated for 1 hour at
room temperature (Akki and Seymour, 2009). The composition of the blocking buffers, dilutants
and antibodies can be found in Table 7.
1. Molecular Marker
2. 10g of liver tissue
201005529 32
Table 7: Summary of antibody, dilutant and blocking buffer compositions.

Type Dilution Dilutant Blocking Buffer
Primary PPAR
Antibody
Polyclonal
Rabbit
1:1000
0.25% TBS-Tween with
1% milk powder
0.25% TBS Tween
with 5% milk powder
Secondary
PPAR
Antibody
Donkey
Anti-rabbit
(HRP conjugated)
1:4000
0.25% TBS-Tween with
1% milk powder
0.25% TBS Tween
with 5% milk powder

Visualisation
Visualisation was achieved using an enhanced chemiluminescence technique (ECL) kit with
scanning densitometry and ImageJ software. Liver tissue and adipose tissue were used as control
groups. Membranes were subjected to Actin probing to determine the protein expression compared
to protein loading in order quantify expression.

Data Presentation and Statistical Analysis
Results are presented as the means SEM where appropriate with any statistical significance
identified using an unpaired student's T test for statistical analysis.
201005529 33
Results

Cardiac Function in Normoxia and Reperfusion
The effect of Metformin on LVDP showed no statistical significance in contractility during
normoxia compared to the control. Similarly in reperfusion Metformin showed no significant
change in contractility during this time. As expected decreased contractility from normoxia to
reperfusion was seen in both models. This is shown in Figure 15, derived from Table 8-11.

Figure 15: LVDP at different stages of the protocol in either the absence or presence of Metformin.
201005529 34
MVO
2
during normoxia was increased in the Metformin model, however in reperfusion the control
group was unchanged between the control and Metformin treated animals. As expected MVO
2
was
decreased in both models in reperfusion compared to normoxia. This is shown in Figure 16, derived
from Tables 8-11.

Figure 16: Oxygen consumption at different stages of the protocol
in either the presence or absence of Metformin.

201005529 35
RPP in normoxia showed minor reductions with no statistical significance between the Metformin
and control models. Similarly only minor decreases in myocardial energy demand were seen but
not deemed significant. Despite this, comparing normoxia to reperfusion the control model
remained higher than Metformin whilst both were reduced in reperfusion. This is shown in Figure
17, derived from Table 8-11.

Figure 17: Myocardial energy demand as demonstrated by the RPP
in the presence and absence of Metformin.

201005529 36
Cardiac efficiency in normoxia showed a decrease in the Metformin model compared to the control.
This was further shown to greater extent in reperfusion where Metformin was once again decreased
compared to the control. However comparing both models in normoxia and reperfusion, reperfusion
saw Metformin treated and control hearts increase in cardiac efficiency. This is shown in Figure 18,
derived from Table 9-11.

Figure 18: Cardiac Efficiency at different stages of the protocol
in either the presence or absence of Metformin.

201005529 37
Table 8: Myocardial Function in the presence and absence of Metformin during normoxia. Where HR is heart rate.

Table 9: Myocardial Function in the presence and absence of Metformin during normoxia
.
Model
Control (n=3) 71.129.0
0.28.6 1198.2683.2 -267.91699.4 300
Metformin (n=3)
83.933.0
15.75.8 1645840.5 -1469.3789.4
300
Systolic Pressure(mmHg) Diastolic Pressure (mmHg) Max dP/dT (mmHg/s) Min dP/dT(mmHg) HR (bpm)
Model
Control (n=3) 70.820.3 0.90.1 21240.36077.2 23903.27756.9
Metformin (n=3) 68.230.2 1.00.1 20472.09074.9 20995.37516.9
LVDP (mmHg) MVO2(mol/g wet heart weight/min) RPP( mmHg/min) CE(mmHg/mol/g)

201005529 38
Table 10: Myocardial function in the presence and absence of Metformin during reperfusion
Where RPP is rate pressure product and CE is cardiac efficiency.

Table 11: Myocardial function in the presence and absence of Metformin during reperfusion.Where RPP is rate pressure product and CE is cardiac
efficiency
Model
Control (n=3)
60.336.7 0.70.2 19599.012946.3 32461.329402.3
Metformin (n=2)
61.919.6 0.70.2 18565.55865.5 25861.92514.8
LVDP (mmHg) MVO2(mol/g wet heart weight/min) RPP (mmHg/min) CE (mmHg/mol/g)
Model Systolic Pressure (mmHg) Diastolic Pressure(mmHg) Max dP/dT (mmHg/s) Min dP/dT(mmHg) HR (bpm)
Control (n=3) 62.746.6 1387.61159.6 -1474.21317.1 300
Metformin (n=2) 67.336.9 5.417.4 1259.8311.7 -1384.21222.09 300
2.4 11.8

201005529 39
During ischaemia cessation of function, onset of contracture, time till max contracture and extent of
contracture were recorded in the presence and absence of Metformin. Table 10 contains the data
collected and Figure 19 also shows the contrast between the two protocols. At cessation of function
(T1) no significant differences were seen between the control and Metformin models. However at
the onset of contracture (T2) Metformin models showed a delay whereas time till max contracture
(T3) was reduced in the Metformin model. The extent of contracture differences in the Metformin
and control models were insignificant.

Table 10: Data values for ischaemia. T1 = Cessation of function, T2 = Onset of Contracture,
T3 = Time till max contracture, E1 = Extent of contracture

Model
Control (n=2) 8:181.4 05:320.11 57.939.7
Metformin (n=2) 1:510.01 09:400.11 04:020.07 65.336.4
T1 (mins) T2 (mins) T3 (mins) E1 (mmHg)
02:040.01

201005529 40

Figure 19: Cessation of function (T1), Onset of Contracture (T2) and Time till max contracture
in the absence and presence of Metformin.

During reperfusion time to recovery and extent of recovery were recorded. Table 11 shows the data
collected and observed in the presence and absence of Metformin during reperfusion. Although
Metformin improved time to recovery the control model was substantially higher for the extent of
recovery.

201005529 41
Table 11: Time to recovery and extent of recovery in the presence and absence of Metformin.

PPAR expression
PPAR protein expression was determined by Western Blotting as described in the methods section.
Samples volume was optimised initially by running the gel displayed in Figure 20.

PPAR

Figure 20: PPAR expression optimization by loading different amounts of sample upon the gel.

Figure 21 shows both the actin and PPAR gels, whereas Figure 22 demonstrates how the
normalised values show that in normoxia PPAR expression was not significantly different between
the Metformin and control models although PPAR expression was increased significantly at
reperfusion.

Control (n=2)
Metformin (n=1)
Time to Recovery (mins) Extent of Recovery (mmHg)
08:410.19 37.525.5
6:150 50
1. 10g of Heart tissue
2. Molecular Marker
7. 50g of Fat
8. 50g of Liver

201005529 42

PPAR

ACTIN

Figure 21: PPAR expression normalised by actin, visualised by Western Blotting.

Figure 22: PPAR expression quantified using the relative optical density of PPAR gel bands
against the actin gel bands where absorbance is compared to expression.
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
11. Liver
12. Fat

201005529 43
Discussion
The ability of Metformin to lower blood glucose is one of the prime reasons it is used in the
treatment of type 2 diabetes. Its ability to reduce both the risk factors and damage of CVD itself
make it a good candidate for clinical cardioprotection. Instead of increasing levels of insulin
Metformin reduces it by improving insulin sensitivity and reducing glucose release from the liver.
Originally the cardioprotective effects were thought only to occur through the regulation of glucose.
However UKPDS showed that overweight patients had a 39% less risk of developing myocardial
infarction compared to those on dietary therapy alone, independent of blood glucose levels (Bethel
et al., 2008; UKPDS, 2008). Other studies showed that Metformin did not alter glucose levels in
non-diabetic rats but still improved both cardiac function and reduced infarct size after an ischaemic
attack (Bestermann et al., 2008).

This study showed that the addition of Metformin had an impact on several aspects of cardiac
function, recovery and protein expression. The 4 key findings in this investigation were;

1. Metformin improved oxygen consumption in normoxia.
2. Metformin delayed the onset of contracture but accelerated time till max contracture during
ischaemia.
3. Metformin improved time to recovery but not extent of recovery during reperfusion.
4. Metformin increased the expression of PPAR in reperfusion only.

These results suggested Metformin had some aspects of cardioprotection.

201005529 44
Normoxia
Contractile Function
During Normoxia no changes were seen between the control and Metformin treated models for
LVDP. Although this lack of change in LVDP was consistent with some studies administering
Metformin 24 hours before the ischaemic attack (Btker et al., 2008) it was not consistent with
more acute administrations of Metformin. One such study by Barreto-Torres et al. showed an
increase in LVDP in the Metformin model during Normoxia when Metformin was administered 10
mins before the onset of ischaemia. This study used a 2mMol concentration of Metformin whereas
we used only 1mMol. This might suggest that the 1mMol concentration was not sufficient to
promote these changes. Levels of AMPK phosphorylation were increased in the study of Barreto-
Torres et al. as well as showing that attenuation of PPAR reduced these changes considerably as
demonstrated by the PPAR inhibitor GW6471 suggesting that the mechanism of action was
mediated through PPAR and AMPK activation (Barreto-Torres et al., 2012).

RPP similarly was unchanged during normoxia between the control and Metformin models. This
was consistent with a study using 2mM of Metformin throughout the perfusion (Allard et al., 2008)
however was inconsistent with two other studies. As previously mentioned, Barreto-Torres et al.
used 2mMol of Metformin at the same time as our study but instead showed an increase in RPP
which again was attenuated when PPAR was inhibited, further implicating the action of PPAR
and AMPK in these mechanisms. Though less comparable to the present study, another
investigation saw an increase in RPP when Metformin was administered two weeks in advance in
line with increased levels of AMPK activity (Hauton, 2011).

The differences seen in our investigation in both LVDP and RPP compared to other similar studies
could thus be explained by the lower concentration of Metformin used. Other studies showed that
to promote increases in glucose metabolism a minimum of 2mMol of Metformin was required and a
maximum of 5mMol of Metformin to prevent reduced energy states of the heart (Balschi et al.,
2007).

PPAR
PPAR expression was slightly lower in the Metformin treated hearts during normoxia compared to
the control, so the connection previously shown between both LVDP and RPP increases and PPAR
expression could explain why the results of this investigation were not consistent with other

201005529 45
studies. Although Metformin was unable to directly affect PPAR expression in acute studies,
probably because of the small time scales involved, this suggested that Metformin's action is likely
through the activation of AMPK and PPAR instead rather than expression (Barreto-Torres et al.,
2012).

Though no changes in LVDP and RPP were seen during normoxia in the Metformin model, this
may have been because of low concentrations of Metformin or low expression of PPAR. Further
studies with dose alterations, AMPK and PPAR would need to be carried out to give weight to
these speculations.

Oxygen Consumption
During normoxia, Metformin treated hearts showed increased oxygen consumption compared to the
control model. Activation of AMPK through the use of Metformin would be one explanation for
these changes. Conversely, high concentrations of Metformin (10mMol) have shown to
significantly reduce oxygen consumption through the inhibition of the mitochondrial respiratory
chain complex 1 but still activate AMPK (Foretz et al., 2011; Bantandier et al., 2004).

Current literature indicates that the cardioprotection Metformin provides is mediated by AKT and
AMPK pathways (Choi and Sung, 2012). AKT pathways ultimately increase both the transport and
metabolism of glucose (Chambers et al,. 2011; Dai et al,. 2011). However one study showed that
Metformin increased AKT phosphorylation threefold when infused at reperfusion but there was no
increase when Metformin was introduced pre-ischaemia (Bhamra et al,. 2008). Conflicting studies
showed in both pre-ischaemic and post-ischaemic delivery of Metformin, AMPK phosphorylation
was increased or was unaltered. Inhibitors of AMPK did not alter the metabolic actions of
Metformin, whereas inhibitors of P38 MAPK or protein C did, suggesting that Metformin's effects
on the cell can occur independently of AMPK and instead via other associated pathways such as
Liver kinase B1(Lkb1) (Allard et al,. 2008). Despite this AMPK deletions lead to twofold increases
in glycogen content and threefold reduction in glucose uptake (Athea et al., 2007). AMPK
activation acts to maintain cellular energy levels and reserves by promoting the production of ATP
and inhibiting the pathways using it through mitochondrial biogenesis and improved oxidative
metabolism. This has shown not only to occur chronically through transcriptional changes but also
acutely (Auwerx et al., 2009) suggesting the activation of AMPK may well explain the increase of
oxygen consumption through mechanisms of increased ATP generation and consumption in the

201005529 46
Metformin model.

Cardiac Efficiency
Cardiac efficiency, determined by oxygen consumption and contraction of the heart was reduced in
the Metformin model during normoxia. This is consistent with the previous findings where
although contractile function (LVDP and RPP) was the same as the control model, oxygen
consumption (MVO
2
) was increased. This would suggest that Metformin is decreasing the
efficiency in the heart. By inhibiting respiratory complex 1 ATP is reduced and AMPK activated
(Algire et al.,2012). Increased oxygen consumption through increased AMPK or AKT activation
would theoretically increase the ATP generated but with little changes in contractile function, thus
decrease efficiency of the heart overall.

Ischaemia
During ischaemia cessation of function was accelerated in the presence of Metformin. It could be
speculated that in line with the decreased cardiac efficiency seen in normoxia, Metformin's
increased generation of ATP then quick cessation of function in ischaemia may suggest mechanisms
which lead to the consumption of ATP. Increased glycolysis particularly as a consequence of
AMPK activation would lead to a decreased cessation of function and the build up of inorganic
phosphates which lead to the reduced calcium sensitivity of myofilaments in the myocardium (Buja,
2005).

Given the delay of contracture and accelerated time of max contracture in the Metformin model
demonstrated in our investigation and the well-studied affect of Metformin on AMPK activation,
one such cause for this affect could be related to ion homoeostasis within the cardiac myocyte.
AMPK activation is said to be rapidly increased in ischaemia as several studies have shown
increased levels of AMPK in response to stresses such as ischaemia, hypoxia, ROS and exercise in
both acute and chronic environments (Burgess et al., 2012; Alexander and Walker, 2011). To
compensate for the depleted ATP one action of AMPK is to restore energy in the cell through fatty
acid or glucose oxidation and the inhibition of biosynthetic pathways (Hardie et al., 2012). Not
only this but AMPK is thought to have influence over several ion channels and transporters which
may contribute to dysfunctional calcium handling and arrhythmias within the heart (Harada et al.,
2012). This is summarised in Figure 23.

201005529 47

Figure 23: Summary of the affects AMPK activation may have on ion handling and consequent
arrhythmias that may arise. Adapted from (Harada et al., 2012).

AMPK knock-out (KO) mice demonstrated significantly reduced activation of ATP-dependent
potassium channels shown to confer with cardioprotection in previous studies (Harada et al., 2012).
Another study demonstrated AMPK (KO) mice had increased sensitivity to contracture and
accelerated contracture (Athea et al., 2007). Despite further elucidation being needed concerning
the relationship between Ca
2+
and AMPK, several studies (Dyck et al., 2009; Budas et al,. 2007)
have provided not only a link between AMPK and Ca
2+
, particularly with ageing but also as a
consequence of AMPK reduction they found depletion in Ca
2+
uptake pumps, contractile function
and the sarcoplasmic reticulum Ca2+-ATPase-2A (SERCA2A). One such study even demonstrated
that treatment with Metformin reduced these contractile problems in the attenuated AMPK mice,
thus suggesting this may be the reasoning behind our results (Du et al., 2010). However, further
research and evidence would be required to support these theories. Thus the activation of AMPK
through Metformin may explain the changes in contracture seen in this investigation.

201005529 48
Metformin increased the extent of contracture compared to the control. One theory to explain this
would be the significant levels of glycolysis seen in ischaemia in other studies. This would lead to
increased levels of both protons and calcium,.Ion imbalance causes Ca2+ to be imported into the
cell and subsequently increases calcium overload and mitochondrial dysfunction (Garcia-Dorado et
al., 2012). This effect would be exacerbated within the Metformin model due to increased AMPK
activation, promoting further glycolysis and an increase in detrimental byproducts (Dyck et
al.,2002).

Reperfusion
Contractile function was not significantly different between control and Metformin models during
reperfusion. LVDP showed very little change compared to the control model. This again was
inconsistent with the findings of Barreto-Torres et al., where Metformin treated hearts showed a
76% improvement in LVDP against the control model during reperfusion. RPP in reperfusion was
again unchanged compared to the control model. These results were inconsistent with other studies
investigating the cardioprotective properties of Metformin where a consequential increased in
glucose uptake and fatty acid oxidation were associated with improved cardiac function (Barreto-
Torres et al., 2012; Hauton, 2011; Allard et al., 2008). This may again suggest the difference in
Metformin concentration and account for the lack of changes seen. However compared to both
normoxia and LVDP, RPP were decreased in reperfusion as expected and consistent with ischaemia
reperfusion injury. Various mechanisms could explain this reduction including calcium overload,
opening of the MPTP and ROS and endothelial dysfunction, all of which can lead to apoptosis and
necrosis of the myocardium. However, as suggested previously, altered sensitivity of calcium in
myofilaments and the dysfunction of ion homeostasis may also lead to myocardial stunning and
consequent reduction in contractility (Alhaj et al., 2012).

Time to recovery during reperfusion was accelerated in the Metformin model compared to the
control. This result was consistent with a study in AMPK KO mice where contractile recovery of
the myocardium was delayed (Athea et al., 2007). This might suggest that AMPK signalling may
explain the accelerated recovery seen in our investigation. Although no other comparable studies
could be found, a similar drug exhibiting the same AMPK activating mechanism as Metformin
showed similar responses (Chang et al., 2012). Even though recovery was improved by Metformin
the extent of recovery was not. This was not consistent with other studies which demonstrated that

201005529 49
Metformin in fact improved the extent of recovery after ischaemia-reperfusion (Barreto-Torres et
al., 2012; Btker et al., 2008; Houston et al., 2002). Considering there was only one Metformin
reperfusion sample it can be assumed that the sample size for both time of recovery and extent of
recovery was too small and thus may speculate that it should have been an improvement on the
control value.

It has been suggested that the inhibition of the mitochondrial respiratory complex 1 through the
action of Metformin and subsequent eNOS promotion reduces the production of ROS caused by
calcium overload (Jou and Peng, 2010). This is suggested to be mediated through SIRT1 and
PPAR expression (Arunachalam et al., 2013) and would be congruent with the increased
expression of PPAR at reperfusion seen in our investigation.

PPAR
During reperfusion PPAR expression was significantly increased in the Metformin treated hearts
compared to the control model. Although during normoxia it was previously suggested that the
Metformin concentration may have been too low or too short to increase expression by reperfusion,
time enough may have passed to activate the necessary gene translation and protein synthesis
though other studies showed no change in PPAR expression in Metformin treated hearts (Barreto-
Torres et al., 2012).

Increased AMPK activity is one such explanation for the increase in expression seen in reperfusion.
AMPK itself has been shown to regulate PGC-1 which is a transcriptional co-activator that
interacts and modulates PPAR and PPAR to regulate the genes of mitochondrial biogenesis and
the uptake of fatty acids through transcription of proteins including CD36 (Dirkx et al., 2011). Thus
PPAR plays a crucial role in the regulation of metabolism in the heart.

PPAR has been shown to inhibit the opening of the MPTP during reperfusion in Metformin
treated animals. However, immunoprecipitation studies demonstrated this was not a direct
interaction with ANT, Cyp-D or VDAC components of the MPTP, instead suggesting PPARs
influence was through other indirect routes such as P13k/AKt and NO pathways (Dong et al.,
2010).

Several studies have demonstrated the relationship of PPAR signalling and cardioprotection
through inhibition of NO production. P13K attenuated the cardioprotection from PPAR in one

201005529 50
study, where PPAR activation was suggested to maintain the activity of NO through P13K/ AKT
signalling (Bulhak et al., 2008). PPAR and NO synthase inhibitors in combination prevented all
cardioprotective effects mediated by activated PPAR suggesting this may be another role of
PPAR outside of fatty acid metabolism (Barreto-Torres et al., 2012; Boysen et al., 2004). Another
study explored PPAR signalling in relation to attenuating apoptosis by reducing NF- transport
into the nucleus, inhibiting the release of inflammatory cytokines and caspase 3 activation whilst
preserving cardiac contractility (Chen et al., 2006). Cardioprotective action through the increased
expression of PPAR by Metformin could explain the increased recovery. Despite increased
expression RPP and LVDP were unchanged in reperfusion, was expression too late?

MVO
2

During reperfusion MVO
2
was unchanged compared to the control mode but with Metformin
promoting glucose metabolism it would be expected that the MVO
2
would be increased through
greater access to substrate. Glucose provides a substantial source of ATP when the myocardium
undergoes ischaemia as both fatty acid and glucose oxidation can no longer meet the metabolic
demand and instead glycolysis takes over. Not only does this reduce the production of toxic fatty
acid metabolites but also glucose metabolism is more oxygen efficient and glycolysis is anaerobic
which is advantageous in this oxygen depleted environment. Activation of AMPK would therefore
be congruent with the idea of the maintenance of cellular energy (Fu et al., 2013; Clanachan et al.,
2011). Glucose metabolism is first mediated by translocation of GLUT1and GLUT4 transporters
from intracellular storage to the cell membrane via exocytosis. At the same time GLUT4
endocytosis is reduced, preventing the recycling of transporters (Atherton et al., 2013; Holman and
Yang, 2005). In the hypoxic environment of the ischaemic myocardium, oxidative metabolism is
decreased and glycolysis is increased to produce ATP in the absence of oxygen. AMPK mediates
this by promoting the enzymatic activity of phosphofructokinase-2 (PFK-2) to produce fructose 2,6-
biphosphate which in turn activates PFK-1 (Beauloye et al,. 2000). Regulation of glycolysis is
primarily done by PFK-1. In the presence of ATP it is inhibited preventing excess glycolysis when
cell energy levels are high (Cheung et al., 2011). Ergo AMPK mediates both the transport of
glucose and the initiation of glycolysis during ischaemia and early reperfusion so the expected
result would be the increase in oxygen consumption.

Compared to normoxia, oxygen consumption was reduced in reperfusion. One explanation would
be the induction of HIF-1 during the hypoxia in ischaemia. HiF1 stimulates glycolysis and inhibits
mitochondrial function and consequently oxygen consumption through the activation of pyruvate

201005529 51
dehydrogenase kinase. High levels of AMPK activity remaining after ischaemia and into
reperfusion could also promote glycolysis to remain elevated and instead cause greater uncoupling
and consequential proton generation during reperfusion preventing high enough levels of ATP to
inhibit glycolysis (Dyck et al., 2002). Given that both the control and Metformin oxygen
consumption is the same, it is likely this reduction can be explained by reperfusion injury and the
consequential metabolic dysfunction.

Cardiac Efficiency
During reperfusion, Metformin showed a reduction in cardiac efficiency compared to the control,
suggesting that although aerobic respiration was the same as the control there was an increased
amount of glycolysis and ATP in the Metformin model. Increased by-products of glycolysis such as
lactic acid and proton production exacerbate acidosis and increase the risk of calcium overload at
and through reperfusion. Consequentially ATP has to be redirected to dealing with these products
causing contractile function and efficiency to be reduced until aerobic respiration fulfil energy
requirements. Despite this both Metformin and the control group showed increased cardiac
efficiency in reperfusion compared to the normoxia value this would indicate either more of the
oxygen consumed and consequent ATP production was being used for contraction or the contractile
dysfunction from ischaemia reperfusion injury matched the reduction in oxygen consumption.

Limitations
Although our study explored other mechanisms of actions beyond the investigation to prove these
theories beyond speculation, additional analysis would be required. Such examples would be the
activation of AMPK, NOS and PPGC-1 as well as a dose-response curve of Metformin. However
the majority of the studies and evidence presented in this investigation was executed in vitro and in
animal models, not an accurate representation of the human body's environment. Use of animal
models itself is limited by trying to gather enough information and samples to create a reliable and
accurate set of data, whilst being sparse with resources for ethical reasons and learning the
techniques involved at the same time. We shared animals between four of us to try and reduce the
amount of animals used but analysed the data separately.

The learning process brought inevitable mistakes and loss of hearts that may have been otherwise
viable and in contrast there were hearts that should have been viable but did not function
adequately. This was further complicated by the fact some aspects of our data had no comparable

201005529 52
studies.
Future
Many of the pathways and mechanisms of cardioprotection afforded by Metformin give rise to
potential new pharmaceutical targets. Although some of these pathways and relationships require
further elucidation to be obtained through future research. From this many new methods of
cardioprotection may be found not only for revascularization but also for surgery, transplantation
and the treatment of other aspects of cardiovascular disease. Ideally in the pursuit of this
knowledge we will be able to reduce the need to sacrifice so many animals and potentially use PET
or MRI to replace this particular method of data collection.

With better inhibitors of AMPK and further studies looking not only into glucose metabolism but
also into fatty acid metabolism, many of the speculations in this study may be further investigated
and explored.

Conclusion

Our investigation into the metabolic and functional changes in Metformin has shown not only the
positive impacts on cardiac function but also the promotion of metabolic changes and expression of
PPAR within the myocardium after ischaemic injury. In this acute setting and brief investigation
we have looked at limited aspects of Metformin's impact on the heart and literature that might
explain why. However future, .broader investigations are required to further elucidate the
speculations we have made here.

201005529 53

201005529 54
Appendix A: Physiological function values for control model.
Notes: Original results table from the control hearts.
Control Heart Weight (g)
Normoxia 1 103.2 9.7 1971.6 -1659.0 300.0 93.5 596.2 204.7 1.5 1.0 28056.9 29490.4
Normoxia 2 62.3 -2.0 946.0 1626.3 300.0 64.3 560.3 279.0 1.5 0.7 19275.0 27172.2
Normoxia 3 47.5 -7.2 677.0 -771.0 300.0 54.6 570.0 213.8 1.2 1.1 16389.0 15046.9
Ischaemia 1 95.0 -12.4 -1940.0 3607.5 300.0 107.5 581.3 252.0 1.7 0.7 32235.0 44766.6
Ischaemia 2 55.7 12.3 1072.0 -854.0 300.0 43.4 369.0 246.0 1.8 0.3 13020.0 51943.6
Reperfusion 1 126.6 18.8 2618.0 -2976.0 300.0 107.8 504.0 213.8 2.2 0.5 32349.0 65903.6
Reperfusion 2 21.8 0.2 315.0 -515.5 300.0 21.6 510.8 281.3 1.4 0.6 6465.0 10669.9
Reperfusion 3 67.0 0.4 1229.7 -931.0 300.0 66.6 569.3 182.3 1.5 1.0 19983.0 20810.3
72.4 2.5 861.2 -309.1 300.0 69.9 532.6 234.1 1.6 0.7 20971.6 33225.5
SD 33.8 10.4 1346.2 2034.0 0.0 31.0 73.7 36.0 0.3 0.5 9297.0 19255.3
Err 12.0 3.7 476.0 719.1 0.0 11.0 26.0 12.7 0.1 0.5 3287.0 6807.8
Sys (mmHg) Dys (mmHg) Max dP/dT (mmHg) Min dP/dT (mmHg) HR (bpm) LVDP (mmHg) O2 Buffer (mmHg) O2 Effluent (mmHg) MVO2 (mol/g wet heart weight/min) RPP (mmHg) CE (mmHg/mol/g)
Av.

201005529 55
Appendix B: Physiological data for Metformin models.
Notes: Original results table from Metformin treated hearts.

Metformin Heart Weight
Normoxia 1 49.1 10.0 693.3 -560.0 300.0 39.0 585.0 225.0 1.5 0.9 11712.0 12854.6
Normoxia 2 114.5 15.1 2225.4 -1980.0 300.0 99.4 597.0 174.0 1.4 1.1 29832.0 27673.6
Normoxia 3 88.1 21.9 2016.4 -1867.8 300.0 66.2 579.0 321.0 1.1 0.9 19872.0 22457.7
Ischaemia 1 82.3 6.5 1564.3 -1253.5 300.0 5.0 552.0 231.0 1.5 0.8 1500.0 1935.5
Ischaemia 2 56.1 -3.5 953.6 -684.0 300.0 59.6 540.0 197.3 1.9 0.7 17883.0 27155.4
Reperfusion 1 41.1 -7.0 615.1 -520.0 300.0 48.1 506.3 210.0 1.8 0.6 14418.0 24083.7
Reperfusion 2 93.4 17.7 1904.5 -2248.3 300.0 75.7 525.0 213.8 1.4 0.8 22713.0 27640.2
75.0 8.7 1424.7 -1301.9 300.0 56.2 554.9 224.6 1.5 0.8 16847.1 20542.9
SD 26.8 10.8 665.0 732.8 0.0 29.8 33.5 46.5 0.3 0.2 8954.0 9716.8
Sys (mmHg) Dys (mmHg) Max (dP/dT (mmHg) Min dP/dT (mmHg) HR (bpm) LVDP (mmHg) O2 Buffer (mmHg) O2 Effluent (mmHg) MVO2 (mol/g wet heart weight/min) RPP (mmHg) CE (mmHg/mol/g)
Av.

201005529 56

Appendix C: Physiological data gathered in ischaemia in the control model.

Notes: Original results table from control hearts during ischaemia.

Control
Ischaemia 1 01:24 06:00 09:30 78.0
Ischaemia 2 02:10 09:30 05:00 5.0
Reperfusion 1 02:30 09:20 03:35 96.5
Reperfusion 2 02:15 08:25 04:05 52.0
Average 02:04 08:18 05:32 57.9
0.0197672852 0.0672318228 0.1126378043 39.7
0.0098836426 0.0336159114 0.0563189021 19.8
Cessation of Function (mins) Onset of Contracture (mins) Time till max contracture (mins) Extent of Contracture (mmHg)
Standard Dev
StandardError

201005529 57

Appendix D. Physiological data gathered in ischaemia in the Metformin model.

Notes: Original results table from Metformin treated hearts during ischaemia.

Metformin
IM1 01:50 06:40 06:00 81.0
IM2 01:35 11:15 04:35 89.0
RM1 02:10 12:35 02:15 11.0
RM2 01:52 08:12 03:20 80.0
Average 01:51 09:40 04:02 65.3
0.0099570677 0.1132554936 0.0673587247 36.4
0.0388020833 0.2015625 0.0842013889 32.6
Cessation of Function (min) Onset of Contracture (min) Time till max contracture (min) Extent of Contracture (mmHg)
Standard Dev
StandardError

201005529 58
Appendix E: PPAR optical density values.

Notes: Original results table from the original PPAR data

Liver CN1 CN2 MN1 MN2 CIR1 CIR2 CIR3 MIR1 MIR2 MIR3
Sample
1.

2437433 2200621 1752335 2104546 1501619 1076071 1852212 2451736 1803964 1658569
Sample
2. 399002 2593980 4616023 4884079 4306788 2958184 2868812 3194276 4991725 4456089 37771280
Actin 19118737 937015 833868 924436 681914 785952 896436 881602 2437982 2140895 1849404
Ratio 1 0 2.60127426 2.63905198 1.895572 3.08623375 1.91057342 1.20038798 2.10096166 1.00564155 0.84262143 0.8968127
Ratio 2 0.02086968 2.76834416 5.53567591 5.28330679 6.31573483 3.76382273 3.20024185 3.62326311 2.0474823 2.08141408 20.4234878
Av. 0.02086968 2.68480921 4.08736395 3.5894394 4.70098429 2.83719807 2.20031491 2.86211238 1.52656193 1.46201775 10.6601502
Av.2

4.728491182

4.145211844

2.633208456

4.549576641

SDV

0.118136257 2.048222421 2.395490244 2.283602114 1.310445153 1.41411023 1.076429676 0.736692661 0.875958686 13.80744436

201005529 59

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