THE ROLES OF BIOLOGICALLY ACTIVE GASOTRANSMITTERS (NO AND H2S) IN MYOCARDIAL ISCHEMIA-REPERFUSION FU YILONG NATIONAL UNIVERSITY OF SINGAPORE 2008 THE ROLES OF BIOLOGICALLY ACTIVE GASOTRANSMITTERS (NO AND H2S) IN MYOCARDIAL ISCHEMIA-REPERFUSION FU YILONG (BSc, Beijing Medical University, Beijing, P.R China) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF PHARMACOLOGY NATIONAL UNIVERSITY OF SINGAPORE 2008 ACKNOWLEDGEMENT I would like to take this opportunity to acknowledge and thank all those who have helped me along the way For the both strands of this thesis - the scientific and the personal - I am grateful to a considerable number of people First and foremost, I would like to express my sincere respect and gratitude towards my supervisor, Associate Professor Zhu Yi Zhun I would like to thank him for giving me the opportunity to work on this interesting project, and also for his kind and patient guidance, critical comments, enlightening ideas and encouragements all the way I would also like to thank Prof Philip K Moore, for his co-supervision in my project I also owe my special thanks to the staff in Cardiovascular Group, Ms Wong Wan Hui, Ms Zhuo Yang, Mr Teo Eng Thiam, Daniel for their kind assistance and help in handling miscellaneous laboratory matters Appreciation also goes to Dr Wang Zhongjing, Dr Wang Hong, Ms Wong Wan Hui, Ms Chuah Shin Chet for their kind help in so many discussions and improving the outcome of this project I am also indebted to my collaborators Ms Chuah Shin Chet, Mr Tan Tong San , Ms Loh Kok Poh and Ms Zhang Huili for their industrious input in part of this work Thank all the former and current members in Cardiovascular Group for their company, encouragement, empathy, invaluable help and friendship I feel very lucky and happy having been with you in the last four years To the many scientists whose presentations I’ve listened to, whose papers I’ve read and whose company I have enjoyed while filling my head with so much new information i I say a heartfelt thank-you, especially those of you who were unaware that your contributions would end up here I wish to express my special appreciation to National University of Singapore for providing me this Ph.D research scholarship and so many opportunities in my academic pursuit and personal development Last but not the least, I extend my heartfelt gratitude to my family and my best friend Zhao Yan for their everlasting love and support throughout these years Without them, I would not have been here today FU Yilong Jan 25th, 2008 ii LIST OF ABBREVIATIONS AAR Area at Risk AMI Acute Myocardial Infarction ASA Acetylsalicylic acid (aspirin) BCA β-cyano-L-alanine BH4 Tetrahydrobiopterin BW Body Weight CABS Coronary Artery Bypass Surgery CBS cystathionine - β-synthase CHD Coronary Heart Disease CINODs Cyclooxygenase-Inhibiting Nitric Oxide Donors CO Carbon monoxide COX Cyclooxygenase CSE cystathionine γ-lyase CV Cardiovascular EDRF Endothelium-Derived Relaxing Factor eNOS endothelial Nitric Oxide Synthase ERK Extracellular signal-Regulated Kinase FAD Flavin Adenine Dinucleotide FMN Flavin Mononucleotide H2S Hydrogen Sulfide HW Heart Weight i.p Intraperitoneal I/R Ischemia-Reperfusion ICAM Intercellular Adhesion Molecule IL Interleukin iNOS inducible Nitric Oxide Synthase IS Ischemia iii LFA Lymphocyte Function-associated Antigen L-NAME NG-nitro-L-arginine methyl ester LPS Lipopolysaccharide LV Left Ventricular LVDevP Left Ventricular Developed Pressure LVW Left Ventricular Weight MI/R Myocardial Ischemia-Reperfusion MPO Myeloperoxidase N.S Not significant nNOS neuronal Nitric Oxide Synthase NO Nitric Oxide NOA NO-Aspirin NOP NO-Paracetamol NOS Nitric Oxide Synthase NOx Nitrites and Nitrates NSAID Nonsteroidal Anti-Inflammatory Drug ODFR Oxygen-Derived Free Radical ONOO- peroxynitrite PAG D, L propargylglycine PARA Paracetamol PGI2 Prostacyclin PLP pyridoxal-5’-phosphate ROS Reactive Oxygen Species RT-PCR Reverse Transcriptase – Polymerase Chain Reaction RyR2 ryanodine receptor SNP Sodium Nitroprusside SOD Superoxide Dismutase SR sarcoplasmic reticulum TNF Tumor Necrosis Factor TTC 2,3,5-Triphenyltetrazolium Chloride VEH Vehicle iv TABLE OF CONTENTS ACKNOWLEDGEMENT i LIST OF ABBREVIATIONS iii TABLE OF CONTENTS v LIST OF TABLES AND FIGURES ix SUMMARY…… xii LIST OF PUBLICATIONS xv CHAPTER INTRODUCTION 1.1 General Overview 1.2 Myocardial Ischemia-reperfusion Injury (MI/R) 1.2.1 Overview of MI/R 1.2.2 Regional rat MI/R model 1.3 Nitric Oxide (NO) 1.3.1 Overview of NO 1.3.2 Biosynthesis of NO 1.3.3 Metabolism of NO in the mammalian circulation 15 1.3.4 NO and inflammation 15 1.4 NO Donor 17 1.4.1 NO-Aspirin 18 1.4.1.1 Overview of NO-Aspirin 18 1.4.1.2 Effects of NO-Aspirin on inflammation 20 1.4.1.3 Cardiovascular effect of NO-Aspirin 21 1.4.2 NO-Paracetamol 22 1.4.2.1 Overview of NO-Paracetamol 22 1.4.2.2 Effects of NO-Paracetamol on inflammation 23 1.4.2.3 Cardiovascular effect of NO-Paracetamol 23 v 1.5 Hydrogen Sulfide (H2S) 25 1.5.1 A new member of gasotransmitter – H2S 25 1.5.2 Biosynthesis of H2S 26 1.5.3 Metabolism of H2S 27 1.5.4 Physiological functions of H2S 28 1.5.4.1 H2S and the cardiovascular system 28 1.5.4.2 H2S in inflammation 29 1.5.5 H2S releasing NSAIDs 32 1.6 Research Interests and Objectives 34 CHAPTER MATERIALS AND METHODS 40 2.1 Animals 41 2.2 Animal model of myocardial ischemia-reperfusion (MI/R) 41 2.3 Experimental protocols 43 2.3.1 Experimental protocol 43 2.3.2 Experimental protocol 45 2.3.3 Experimental protocol 48 2.4 Experimental methods 50 2.4.1 Systemic blood pressure and HR measurement 50 2.4.2 Measurement of LV haemodynamic parameters 50 2.4.3 Determination of infarct size 51 2.4.4 Measurement of plasma nitrate/nitrite 52 2.4.5 RNA extraction and reverse transcriptase – polymerase chain reaction (RT-PCR) amplification 53 2.4.6 Implantation of osmotic pumps 56 2.4.7 Assay of tissue H2S 57 2.4.8 Myocardial myeloperoxidase activity (MPO) 58 2.4.9 Myocardial cytokine assay 59 2.5 Statistical analysis 60 vi CHAPTER 3.1 RESULTS 61 Results of Experiment 1: Cardioprotective Effects of Nitric Oxide-Aspirin in Myocardial Ischemia-reperfused Rats 62 3.1.1 Mortality and infarct size 62 3.1.2 Systemic blood pressure and heart rate 64 3.1.3 Parameters of left ventricular function 66 3.1.4 Plasma nitrite/nitrate (NOx) concentration 69 3.1.5 Gene expression of NOS and COX 71 3.2 Results of Experiment 2: Role of Hydrogen Sulfide in the Rat Model of Myocardial Ischemia-Reperfusion 74 3.2.1 CSE mRNA expression in the ischemia left ventricle 74 3.2.2 H2S production in the ischemic left ventricle 76 3.2.3 Effects of PAG on H2S production 78 3.2.4 Effects of PAG and NaHS on infarct size 80 3.2.5 Effects of PAG and NaHS on left ventricular function 82 3.2.6 Effects of PAG and NaHS on MPO activity 87 3.2.7 Effects of PAG and NaHS on cytokine levels 89 3.3 Results of Experiment 3: NO-Paracetamol Reduces Pro-inflammatory Hydrogen Sulfide in Myocardial Ischemia Reperfused Rat 91 3.3.1 Effects of paracetamol and NO-Paracetamol on systemic BP and HR 91 3.3.2 Effects of paracetamol and NO-Paracetamol on infarct size 94 3.3.3 Effects of paracetamol and NO-Paracetamol on left ventricular function ……………………………………………………………………… 98 3.3.4 Effects of paracetamol and NO-Paracetamol on ischemic left ventricular H2S production 102 3.3.6 Effects of paracetamol and NO-Paracetamol on myocardial cytokine levels 108 CHAPTER DISCUSSION 112 4.1 Discussion of Experiment 113 4.1.1 Cardioprotection of pre-treatment NO-Aspirin 113 4.1.2 Cytotoxicity of iNOS-derived NO exacerbates LV dysfunction 115 vii 4.1.3 Pre-treatment of NO-Aspirin on COX mRNA expression 117 4.2 Discussion of Experiment 120 4.2.1 H2S production in the MI/R 120 4.2.2 Cardioprotective or detrimental effect of H2S in the reperfusion injury? ………………………………………………………………………122 4.2.3 H2S is a pro-inflammatory mediator in the reperfusion injury 122 4.3 Discussion of Experiment 125 CHAPTER GENERAL CONCLUSION AND FUTURE 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