University of Hull

Investigation of the Impact of Metforminon Metabolism and Functionin Cardiac Ischaemia and Reperfusion

School of Biological, Biomedical and Environmental Sciences

Sophie OakleyBSc Biomedical ScienceMay 2014

201005529Abstract

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.

AcknowledgementsMany 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.Contents

Page

Abstract2

Acknowledgements2

Contents3

Abbreviations5

Introduction7

Cardiac Ischaemia8

Reduction of ATP9

Reperfusion10

Reactive Oxygen Species 12

Calcium Overload12

Opening of the Mitochondrial Permeability Transition Pore12

Fatty Acid Metabolism13

AMPK mediated fatty acid metabolism16

PPAR17

Cardioprotection18

Metformin19

Objectives19

Method21

Perfusion Buffer21

Langendorff Perfusion22

Physiological Measurements25

LVDP26

Myocardial Oxygen Consumption27

Efficiency of the Heart27

Perfusion Protocol27

Control27

Metformin28

Protein Expression29

Sample Expression29

SDS Page30

Visualisation32

Data Presentation and Statistical Analysis32

Results33

Cardiac Function in Normoxia, Ischaemia and Reperfusion33

PPAR expression41

Discussion43

Normoxia43

Contractile Function43

PPAR44

Oxygen Consumption45

Cardiac Efficiency45

Ischaemia46

ReperfusionContractile Function4848

PPAR49

MVO249

Cardiac Efficiency50

Limitations51

Future51

Conclusion52

Appendices54

References59

Abbreviations

Akt/PKBAMPK ATP BPMBSA BwtCa2+cAMPCKCLCHDCOCO2CPTCVDDP ETCFAFADHFAO FAT FFAHIF-HRIL-6iNOSIRIRS-1KH LVDP

MIMPTPMVO2NADHP38-MAPK PAGEPCIPFK2 PGC-1 PIPP2PPARRISKROSRPPSDSSERCASODSPSTEMITGF-TNFSerine/threonine kinase or Protein kinase B5'adenosine monophosphate-activated protein kinaseAdenosine triphosphate Beats per minuteBovine Serum Albumin Body WeightCalciumCyclic adenosine monophosphateCreatine kinaseCardiolipinCoronary heart DiseaseCardiac outputCarbon dioxideCarnitine palmitoyltransferaseCardiovascular diseaseDiastolic pressureElectron transport chainFatty acidFlavin adenine dinucleotideFatty acid oxidationFatty acid translocaseFree fatty acidHypoxia-inducible factorHeart rateInterleukin 6Induce nitric oxide synthaseIschaemia reperfusionInsulin receptor subtrateKrebs Hensleit (Buffer)Left Ventricular Developed Pressure

Myocardial InfarctionMitochondrial permeability transition poreMyocardial oxygen consumptionNicotinamide adenine dinucleotidep38 mitogen-activated protein kinasesPolyacrylamide gel electrophoresisPercutaneous coronary interventionPhosphofructokinase 2Peroxisome proliferator-activated receptor gamma coactivator 1-alphaInoganic phosphates Protein phosphatase 2Peroxisome proliferators activated recptor Reperfusion injury signalling kinaseReactive oxygen speciesRate Pressure ProductSodium dodecyl sulphateSarco/endoplasmic reticulum Ca2+-ATPaseSuperoxide dismutaseSystolic pressureSt-elevation myocardial infarctionTumor growth factorTumor necrosis factor

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.

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 IschaemiaThe 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)Table 1: Summarising the time-scale of acute events following a large myocardial infarction.Adapted from (Leonard, 2011b).TimeEvent

1-2 minLevels of ATP drop leading to cessation of contraction. Energy supplies are depleted and increased levels of inorganic phosphates (Pi).

10 minHalf 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 minIrreversible cell damage begins and vascular permeability increases causing further edema.

Reduction of ATPUnder 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), FADH2 (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 bc1) 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).

ReperfusionTime 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 ActionConsequenceLiterature

Highly oxygenated blood flows into the myocardiumExcess 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 pHUtilisation of H+/Na+ exchangers leads to excess Na+ in the cell.(Anderson et al., 2010)

Excess Na+ in myocytesNa+ / Ca2+ exchangers lead to calcium overload as Na+ is removed.(Hill & Turer 2010).

Calcium overloadActivation of different enzymes and pathways lead to ROS production,the opening of the MPTP and hypercontracture.(Garcia-Doradoet al., 2012).

High pressure and flow of bloodEndothelial cell damage triggering an inflammatory response. (Gong et al., 2012).

Inflammatory responseActivation of chemokines and cytokines leads to recruitment of leukocytes and T-cells leading to tissue damage.(Andreoli et al., 2011).

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 (O2-), H2O2, 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). Calcium OverloadUpon 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+/ HCO3- exchange proteins and increases the Na+ concentration in the myocyte. To reduce these levels Na2+/Ca2+ 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 PoreThe 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.

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 MetabolismThe 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 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.SubstrateConsequence

PhaseGlucoseFatty Acid

Normoxia20-40% of energy generated60-80% of energy generatedMetabolism is balanced

IschaemiaMajority of energy generated through glycolysisVery little in depleted oxygen conditionsFA, lactate and H+ accumulate

ReperfusionSome energy generatedAccelerated energy generation from high FA levelsRepression 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).

Table 4: Examination of the efficacy of glucose and palmitate metabolism .Adapted from (Aksentijevic, 2008)PalmitateGlucose

Energy Efficiency6.6 ATP per CO25.3 ATP per CO2

Oxygen Efficiency4.6 ATP per O2 5.3 ATP per O2

Overall ATP 10632

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 FADH2 and impairs several key enzymes and pH. Adapted from (Fillmore & Lopaschuk, 2011).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 metabolismAMPK 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 metabolismbut 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 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).

PPARPPAR 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).

CardioprotectionCurrent 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).

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 Ca2+ influx and disruption of both mitochondrial networks and Ca2+ buffering capacity (Criollo et al., 2013).

MetforminMetformin 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.

ObjectivesMetformin 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 reperfusion2. to analyse the impact of Metformin on cardiac metabolism3. to assess the effect of Metformin on the expression of PPAR

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 BufferThe 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.CompoundMolarity (mM)

NaCl118

NaHCO325

KCl4.5

KH2PO41.2

MgSO4 7H2O1.2

CaCl2 H2O1.25

Glucose5

Palmitate3

Lactate1

Pyruvate0.1

Glutamine0.55

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.

Langendorff PerfusionAnimal 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% O2 / 5% CO2 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.

20100552922

Figure 7: The Langendorff perfusion apparatus.

Figure 8: The cannulated heart.

201005529 24

Physiological MeasurementsHeart 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).

LVDPLeft 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.

LVDP = Systolic Pressure Dyastolic Pressure

Equation 1: Calculation for LVDP

RPP = LVDP x HR

Equation 2: Calculation for RPP

Myocardial Oxygen ConsumptionThe Myocardial Oxygen Consumption (MVO2) was calculated by measuring the oxygen content of the perfusate and buffer and the heart at each stage of the experiment. The pO2 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).

MVO2 = (((pOs (buffer) pO2 (effluent)) / 760mmHg) x solubility of water in O2) x flow rate) / weight of heart

Equation 3: Calculation for MVO2. Solubility of water in O2 (0.199mmol/ml).Flow Rate (14ml/min)

Efficiency of the HeartEfficiency of the heart is calculated from the work done by the heart per O2 consumed. Using RPP and MVO2 this value was calculated using Equation 4.RPP /MVO2= Efficiency

Equation 4: Calculation for Efficiency

Perfusion ProtocolControlAfter 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.

MetforminAfter 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.

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).

Protein ExpressionPPAR expression was analysed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS PAGE) and western blotting.

Sample PreparationFrozen 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 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.

Component3% Stacking GelVolume (ml)12% Running GelVolume (ml)

H2O7.43.35

10% (w/v) SDS0.10.1

1.5M Tris (pH 8.8)6.82.5

Acyrlamide/ bis (30% w/v)1.254

Ammonium persulfate (10% w/v)1.30.05

TEMED0.020.01

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 14.1. Molecular Marker2. 10g of liver tissue3. 25g of liver tissue4. 50g of liver tissue5. 75g of liver tissue6. 100g of liver tissue7. 125g of liver tissue8. 150g of liver tissue9. 125g of liver tissue10. 100g of liver tissue11. 75g of liver tissue12. 50g of liver tissue13. 25g of liver tissue14. 10g of liver tissue

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.Table 7: Summary of antibody, dilutant and blocking buffer compositions.

TypeDilutionDilutantBlocking Buffer

Primary PPAR AntibodyPolyclonal Rabbit1:10000.25% TBS-Tween with 1% milk powder0.25% TBS Tween with 5% milk powder

Secondary PPARAntibodyDonkey Anti-rabbit(HRP conjugated)1:40000.25% TBS-Tween with 1% milk powder0.25% TBS Tween with 5% milk powder

VisualisationVisualisation 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 AnalysisResults are presented as the means SEM where appropriate with any statistical significance identified using an unpaired student's T test for statistical analysis.20100552932Results

Cardiac Function in Normoxia and ReperfusionThe 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.MVO2 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 MVO2 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 protocolin either the presence or absence of Metformin.

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 RPPin the presence and absence of Metformin.

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 protocolin either the presence or absence of Metformin.

20100552936Table 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. Table 10: Myocardial function in the presence and absence of Metformin during reperfusionWhere 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 201005529 38During 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

Figure 19: Cessation of function (T1), Onset of Contracture (T2) and Time till max contracturein 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.

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

PPAR expressionPPAR 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.

1. 10g of Heart tissue2. Molecular Marker3. 25g of Heart tissue4. 50g of Heart tissue5. 50g of Heart tissue6. 25g of Heart tissue7. 50g of Fat8. 50g of Liver

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.

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 210. Metformin Reperfusion 311. Liver12. Fat

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.DiscussionThe 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.

NormoxiaContractile FunctionDuring 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).

PPARPPAR 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 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 ConsumptionDuring 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 Metformin model.

Cardiac EfficiencyCardiac 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 (MVO2) 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.

IschaemiaDuring 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.

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 Ca2+ and AMPK, several studies (Dyck et al., 2009; Budas et al,. 2007) have provided not only a link between AMPK and Ca2+, particularly with ageing but also as a consequence of AMPK reduction they found depletion in Ca2+ 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.

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).

ReperfusionContractile 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 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.

PPARDuring 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 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?

MVO2During reperfusion MVO2 was unchanged compared to the control mode but with Metformin promoting glucose metabolism it would be expected that the MVO2 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 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.

LimitationsAlthough 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 studies.FutureMany 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.

20100552944Appendix A: Physiological function values for control model.Notes: Original results table from the control hearts. Appendix B: Physiological data for Metformin models.Notes: Original results table from Metformin treated hearts.

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

Notes: Original results table from control hearts during ischaemia.

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

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

LiverCN1CN2MN1MN2CIR1CIR2CIR3MIR1MIR2MIR3

Sample 1.2437433220062117523352104546150161910760711852212245173618039641658569

Sample 2.39900225939804616023488407943067882958184286881231942764991725445608937771280

Actin19118737937015833868924436681914785952896436881602243798221408951849404

Ratio 102.601274262.639051981.8955723.086233751.910573421.200387982.100961661.005641550.842621430.8968127

Ratio 20.020869682.768344165.535675915.283306796.315734833.763822733.200241853.623263112.04748232.0814140820.4234878

Av.0.020869682.684809214.087363953.58943944.700984292.837198072.200314912.862112381.526561931.4620177510.6601502

Av.24.7284911824.1452118442.6332084564.549576641

SDV0.1181362572.0482224212.3954902442.2836021141.3104451531.414110231.0764296760.7366926610.87595868613.80744436

Appendix E: PPAR optical density values.

Notes: Original results table from the original PPAR data

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