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Investigation Into the Cardiotoxic Effects of B- Adrenergic Receptor Agonists in Myocardial Ischaemia/Reperfusion Injury

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Investigation Into the Cardiotoxic Effects of B- Adrenergic Receptor Agonists in Myocardial Ischaemia/Reperfusion Injury Investigation into the cardiotoxic effects of b- adrenergic receptor agonists in myocardial ischaemia/reperfusion injury Nagra, A. S. Submitted version deposited in Coventry University’s Institutional Repository Original citation: Nagra, A. S. (2016) Investigation into the cardiotoxic effects of b- adrenergic receptor agonists in myocardial ischaemia/reperfusion injury. Unpublished PhD Thesis. Coventry: Coventry University Copyright © and Moral Rights are retained by the author. A copy can be downloaded for personal non-commercial research or study, without prior permission or charge. This item cannot be reproduced or quoted extensively from without first obtaining permission in writing from the copyright holder(s). The content must not be changed in any way or sold commercially in any format or medium without the formal permission of the copyright holders. Some materials have been removed from this thesis due to third party copyright. Pages where material has been removed are clearly marked in the electronic version. The unabridged version of the thesis can be viewed at the Lanchester Library, Coventry University. Investigation into the cardiotoxic effects of b- adrenergic receptor agonists in myocardial ischaemia/reperfusion injury By Aarondeep Singh Nagra January 2016 Supervisory team: Dr. Afthab Hussain, Professor Helen Maddock & Dr. Christopher Mee A thesis submitted in partial fulfilment of the University’s requirements for the Degree of Doctor of Philosophy Acknowledgements I would like to give thanks to my supervisory team Dr. Afthab Hussain, Professor Helen Maddock and Dr. Christopher Mee, whose guidance throughout has been invaluable allowing me to confidently go on to complete my thesis. I would also like to give special thanks to my colleagues; Mayel Gharanaei, Jawad Khan, Katherine Harvey and Maryam Babba for making our time in the lab together a period for us all to look back on and reminisce the good times. I am also grateful to Mark Bodycote and Bethan Grist for their continued technical support throughout. Thank you to all my family, Mandy, Sukh, Jay and in particular my mum, whose support and patience has allowed me to accomplish this success. 2 Abbreviations % Percentage 7TM 7 transmembrane region aARs a Adrenergic Receptor bAR b Adrenergic Receptor b1AR b1 Adrenergic Receptor b2AR b2 Adrenergic Receptor µM Micromole AC Adenylate Cyclase ACh Acetylcholine ACOS Asthma-Chronic Obstructive Pulmonary Disorder AIF Apoptosis inducing factor Akt Protein Kinase B ANT Adenine nucleotide translocator APAF-1 Apoptotic Protease Activating Factor-1 ASK1 Apoptosis signal regulated kinase 1 ATP Adenosine triphosphate CABG Coronary artery bypass graft CAD Coronary artery disease CaMKII Calmodulin dependent Protein Kinase II cAMP Cyclic Adenosine Monophosphate CF Coronary flow CHF Chronic Heart failure CICR Calcium induced calcium release CL Cytoplasmic loop 3 COPD Chronic Obstructive Pulmonary Disease CsA Cyclosporin A CVD Cardiovascular diseases Cyp D Cyclophilin D DISC Death-inducing-signalling-complex DMSO di-methyl sulfoxide ER Endoplasmic Reticulum Erk 1/2 Extracellular signal-regulated kinase 1 and 2 FADD Fas-associated via death domain Form Formoterol GAPDH Glyceraldehyde 3-phosphate dehydrogenase GDP Guanosine diphosphate GPCR G protein coupled receptors GSK3β Glycogen synthase kinase 3β GTP Guanosine triphosphate H+ Hydrogen ions H2O2 Hydrogen peroxide HF Heart Failure HR Heart Rate IHD Ischaemic Heart Disease IL-3, 4, 5 Inflammatory cytokines IMM Inner mitochondrial membrane IR Ischaemia reperfusion IsoP Isoproterenol JNK c-Jun N-terminal kinase 4 KHB Krebs Henseleit Buffer LABA Long acting b agonist LVDP Left ventricular diastolic pressure M1-5 Muscarinic Receptor MAPK Mitogen activated protein kinases MI Myocardial infarction mPTP Mitochondrial permeability transition pore MTT (3−[4,5−dimethylthia−zol−2−yl]−2,5−diphenyl tetrazolinum bromide) NADH Nicotinamide adenine dinucleotide phosphate NCX Na+/Ca2+ exchanger OMM Outer mitochondrial membrane PDK Phosphoinositide dependent kinase-1 PKA Protein Kinase A PI3K Phosphoinositide 3-kinase PIP2 Phosphatydilylinositol 3,4-trisphate PIP3 Phosphatidylinositol 3,4,5-trisphosphate PVDF Hybond-P Polyvinyl Difluoride RISK Reperfusion injury salvage kinase RyR Ryanodine Receptors ROS Reactive oxygen species SABA Short acting b agonist SalB Salbutamol SalM Salmeterol SMAC Second mitochondria-derived activator of caspases 5 SERCA Sarcoendoplasmic reticulum calcium ATPase SOD Superoxide dismutase SR Sarcoplasmic reticulum TBS Tris-buffered saline TMRM Tetramethylrhodamine methyl ester TNFa Tumor necrosis factor VDAC Voltage dependent anion channel WHO World Health Organization 6 Abstract The treatment of asthma still relies on primary therapy with bronchodilators; in particular b adrenergic receptor (bAR) agonists with a diverse range of short acting and long acting bARs available. An increase in the number of cardiovascular events with the use of bronchodilators have recently been reported including hypertrophy, heart failure, myocardial ischaemia and infarction. Several subtypes of bAR receptors exist including the b1 Adrenergic Receptor (b1AR) and b2 Adrenergic Receptor (b2AR), both located in the heart. The effects of selective b2AR agonists were investigated in the Langendorff model of myocardial ischaemia reperfusion injury, isolated perfused rat hearts underwent 35 minutes of ischaemia and 120 minutes of reperfusion. The selective b2AR long acting b agonists Formoterol and Salmeterol had no significant effect on infarct to risk ratio or time taken to depolarisation and hypercontracture in isolated cardiomyocytes. The non-selective b1AR agonist Isoproterenol has been show to induce myocardial ischaemia and infarction in rat hearts previously, here we demonstrated Isoproterenol (0.5µM) significantly decreased time taken to depolarisation and hypercontracture in isolated cardiomyocytes. The short acting b2AR agonist Salbutamol (0.01µ-1µM) significantly increased infarct to risk ratio in the Langendorff in addition to significantly decreasing time to hypercontracture in cardiomyocytes in the oxidative stress model highlighting a potential role of the mitochondrial permeability transition pore (mPTP). Activation of phosphorylated Akt and phosphorylated Erk1/2 via the PI3K/Akt signalling pathway and p44/p42 MAPK pathway were investigated by western blot analysis. Salbutamol significantly elevated expression of p-Akt in rat hearts exposed to reperfusion for 20 and 120 minutes whilst reducing expression of p-Erk. Recorded elevated cleaved caspase 7 3 expression in Salbutamol treated hearts can be associated as a marker of increased in cardiomyocyte cell death. The b1AR antagonist CGP 20712 was administered in the presence of Salbutamol with minimal reduction in infarct size in rat hearts recorded and no significant change in time taken to hypercontracture in isolated cardiomyocytes suggesting that Salbutamol mediated toxicity is via b2AR activation. Confirmation of this was verified with the b2AR antagonist ICI 118, 551. Significant decrease in infarct size was recorded in addition to a significant increase in time to hypercontracture in the oxidative stress model. Further to this, caspase 3 expression was significantly reduced in addition with p-Akt expression. With a potential role of the mitochondria and the mPTP contributing to Salbutamol induced myocardial injury, the Cyclophilin D inhibitor Cyclosporin A was administered in hearts and cardiomyocytes in the presence of Salbutamol. Infarct size was significantly reduced whilst time taken to hypercontracture significantly increased, suggesting that CsA treatment inhibits Salbutamol mediated injury via Cyclophilin D inhibition of the mPTP. To conclude, our results demonstrated that Salbutamol caused cardiotoxicity at tissue, cellular and protein level in conditions of ischaemia reperfusion injury. Further to this, inhibition of Cyclophilin D by CsA, or the use of the b2AR antagonist ICI 118, 551 inhibits Salbutamol induced toxicity. 8 CONTENTS FIGURE LIST ..................................................................................................................................................................... 15 1 INTRODUCTION ................................................................................................................................. 26 1.1 RESPIRATORY DISORDERS ............................................................................................................................. 26 1.2 CHRONIC OBSTRUCTIVE PULMONARY DISEASE ........................................................................................ 26 1.3 ASTHMA ............................................................................................................................................................ 27 1.4 BRONCHODILATORS ........................................................................................................................................ 28 1.4.1 Muscarinic Antagonists ........................................................................................................................... 29 1.4.2 b Adrenergic Receptor Agonists ........................................................................................................... 30 1.4.3 Isoproterenol ................................................................................................................................................ 31 1.4.4 Salbutamol ...................................................................................................................................................
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