Roles and relationship of gasotransmitters hydrogen sulfide nitric oxide in myocardial infarction

ROLES AND RELATIONSHIP OF GASOTRANSMITTERS HYDROGEN SULFIDE AND NITRIC OXIDE IN MYOCARDIAL INFARCTION CHUAH SHIN CHET B.Sc. (Hons), National University of Singapore A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF PHARMACOLOGY NATIONAL UNIVERSITY OF SINGAPORE 2009  ACKNOWLEDGEMENTS First and foremost, I would like to express my heartfelt gratitude to my project supervisor, Associate Professor Zhu Yi- Zhun for the great opportunity to work on this interesting project and also for his invaluable advice, patient guida nce and encouragement throughout the course of this project. I would also like to thank Dr Wang Hong and Dr Wang Zhongjing for their helpful input and constructive suggestions which were instrumental to the development of the project. My sincere thanks also goes out to past and present members of Dr. Zhu’s lab for the friendship, support and bantering of ideas along the way. Specifically, I would like to thank Miss Wong Wan Hui, who has done an excellent job in maintaining an orderly lab environment, and also for providing assistance with the purchasing o f necessary materials. It was also a great pleasure to work alongside Ms Loh Kok Poh, my fellow post-graduate lab-mate, who has been very encouraging. I would like to extend my appreciation to members of the Life Sciences Institute Cardiovascular Biology Group, academic and non-academic staff of the Department of Pharmacology, N US for their kind help rendered a long the way. I would also like to express my gratitude to the National University of Singapore for granting me this Ph.D. research scholarship, hence allowing me to pursue my interest in research. Last but not least, I would like to express my heartfelt appreciation to my family and husband KG. Without their strong support and loving encouragements, this project would not ve reached fruition. i TABLE OF CONTENTS ACKNOWLEDGEMENTS i TABLE OF CONTENTS .ii SUMMARY viii LIST OF TAB LES .xi LIST OF FIGUR ES xii LIST OF ABBREVIATIONS .xvii LIST OF PUB LICATIONS xx CHAPTER I INTRODUCTION .1 1.1 GENERA L OVERVIEW .2 1.2 MYOCA RDIAL INFA RCTION (MI) .3 1.2.1 Pathophysiology and Management of Myocardial Infarct ion 1.2.2 Animal Models of Acute Myocardial Infarction (AMI) .4 1.2.3 Experimental Model of MI: Simu lation of Hypoxic Condit ions in vitro .5 1.2.3.1 H9c2 1.2.3.2 Rat Neonatal Cardio myocytes 1.3 GA SOTRA NSMITTERS .7 1.3.1 Hydrogen Sulfide 10 1.3.1.1 Overview o f H2 S .10 1.3.1.2 Biosynthesis of H2 S 10 1.3.1.3 Metabolism of H2 S 13 1.3.1.4 Roles of H2 S in the Card iovascular System 15 1.3.1.4.1 Effect of H2 S on Vascular Tone .15 1.3.1.4.2 Effect of H2 S on the Ischemic Heart 15 1.3.1.5 S-ally lcysteine (SAC) as a Novel H2 S Donor 17 1.3.1.5.1 Garlic as a Cardioprotective Agent .17 1.3.1.5.2 SAC as a Cardioprotective Agent 18 1.3.2 Nit ric Oxide .20 1.3.2.1 Overview of NO 20 1.3.2.2 Biosynthesis of NO .21 1.3.2.3 NOS Isoforms .22 1.3.2.4 NO Metabolism .23 1.3.2.5 Roles of NO in the Cardiovascular System 24 1.3.2.6 Sildenafil as a Novel Substrate for Endogenous NO Production 27 1.3.2.6.1 Overview of Sildenafil 27 1.3.2.6.2 Sildenafil as a Cardioprotective Agent 28 1.3.3 Crosstalk between H2 S AND NO .30 1.3.3.1 H2 S and NO: Co mmon Functions .31 1.3.3.2 H2 S and NO: Ev idence for Crosstalk 31 1.3.3.3 H2 S and NO: Nitrosothiol Format ion 33 1.4 HYPOXIA-INDUCIBLE FA CTOR-1α (HIF-1α) .34 1.4.1 Hypoxia 34 1.4.2 Discovery of HIF 35 1.4.3 Regulation of HIF .36 ii 1.4.4 HIF-1α and the Card iovascular System 39 1.4.5 Downstream Targets of HIF in the Heart 40 1.4.5.1 iNOS as a Do wnstream Target of HIF 41 1.5 PI3K/AKT SIGNA LING PATHWA Y 42 1.6 RESEA RCH INTEREST AND OBJECTIVES .44 CHAPTER II MATERIALS AND MET HODS 48 2.1 MATERIA LS .49 2.1.1 Drugs .49 2.1.2 Chemicals .50 2.2 METHODS 51 2.2.1 Animal 51 2.2.1.1 Animal Model of Acute Myocardial Infarct ion (AMI) 51 2.2.2 Cell Cu lture .52 2.2.2.1 H9C2 .52 2.2.2.2 Neonatal Rat Primary Cardio myocytes .52 2.2.2.3 Isolation of Primary Card io myocytes .53 2.2.2.4 Hypoxia Model .54 2.3 EXPERIM ENTA L PROTOCOL 55 2.3.1 Experimental Protocol 1: SA C exerts Card ioprotection in AMI via a H2 S-related pathway with Concomitant NO Production 56 2.3.2 Experimental Protocol 2: NO-med iated Card ioprotection by Sildenafil invo lves a CSE/ H2 S-related pathway during Myocardial Ischemia .58 2.3.3 Experimental Protocol 3: Relationship between H2 S and NO in a Rat Model o f AMI 60 2.3.4 Experimental Protocol 4: H2 S exerts Cardioprotection by enhancing HIF-1α activation and iNOS exp ression through the PI3K/Akt-dependent pathway .62 2.4 EXPERIM ENTA L M ETHODS .65 Animal 2.4.1 Measurement of Infarct Size .65 2.4.2 Measurement of Hypertrophy Index .65 2.4.3 Hemodynamic Measurements .66 2.4.3.1 Blood Pressure 66 2.4.3.2 Electrocardiograms and Heart Rate 66 2.4.4 Morphological Examination— Hemato xylin & Eosin staining .67 Cell Cu lture 2.4.5 Cell Count .68 2.4.6 MTT Assay .68 2.4.7 LDH Assay .69 2.4.8 Trypan Blue Exclusion Assay 70 Biochemical Assays 2.4.9 Measurement of CSE Activ ity in the Left Ventricle 71 2.4.10 Measurement of H2 S Concentration in the Plasma .72 2.4.11 Measurement of Nitrate/Nit rite (NOx) Concentration in Left Ventricle and Plas ma .73 2.4.12 Measurement of Nitrate/Nitrite Concentration in Cell Mediu m .74 2.4.13 Total RNA Isolation from Animal Tissue 74 2.4.14 Total RNA Isolation fro m Cell Samp le 75 2.4.15 RNA Quantitation .76 iii 2.4.16 Reverse transcription-Poly merase Chain Reaction (RT-PCR) .76 2.4.17 Collection of Nuclear and Cytoplasmic Ext racts 79 2.4.18 Electrophoretic Mobility Shift Assay (EMSA) 79 2.4.19 Protein Extraction fro m Animal Tissue 81 2.4.20 Protein Ext raction fro m Cells .82 2.4.21 Western Blot .82 2.5 STATISTICA L ANA LYSIS 84 CHAPTER III S-ALLYLCYS TEIN E MEDIAT ES CARDIOPROTECTION VIA A HYDROGEN SULPHIDE-RELATED PATHWAY WITH CONCOMITANT NITRIC OXIDE PRODUCTION .85 3.1 RESULTS………………………………… ……………………… …………86 3.1.1 Preliminary Study…………………………… .………………… .….…….86 3.1.2 Survival Rate after Myocardial Infarction (M I)… ………. ……… .………87 3.1.3 Exclusion Criteria……………… ………………………… .………… .…87 3.1.4 Infarct Size…………………………………………………… .……………88 3.1.5 Ventricu lar Hypertrophy………………………… …………… ……… .89 3.1.6 Hemodynamic Parameters………………………………………… .………90 3.1.6.1 Blood Pressure……………………………… .………………………… ……….90 3.1.6.2 Electrocard iograms (ECGs ) ……………… .……………………… ……… 91 3.1.6.3 Heart Rate……………………………………… .……………………….……….94 3.1.7 Morphological Examination………………………………………… .…….94 3.1.8 CSE Activ ity in the Left Ventricle…………………………………… ……95 3.1.9 Plas ma H2 S Concentration…………………………………………… ……97 3.1.10 CSE Protein Exp ression……………………………… .……………… ….98 3.1.11 Nitrate/Nitrite Levels in the Left Ventricle…………………… .……… 100 3.1.12 Plas ma NOx Concentration…………………………… .……………… 101 3.2 DISCUSSION………………………………………………………………… 103 3.2.1 H2 S as a Cardioprotective Agent………………………………………… .103 3.2.2 Garlic as a Cardioprotective Agent……………………………………… .104 3.2.3 S-allycysteine (SA C) as a Cardioprotective Agent……………………… .104 3.2.4 SAC Improved Surv ival and Infarct Size after AMI…………………… .105 3.2.5 Effect of SA C on BP, ECG and Heart Rate……………………………… 106 3.2.6 SAC Improved Morphology of Ischemic LV………………………….… .107 3.2.7 Participation of H2 S/ CSE Pathway in SA C-Mediated Cardioprotection… 108 3.2.8 Participation of NO in SA C-Mediated Cardioprotection………………… 110 3.2.9 Summary of Findings for Experiment Protocol 1……………………… .110 CHAPTER IV NO-MEDIATED CARDIOPROTECTION B Y S ILDENAFIL IN MYOCARDIAL ISCHEMIA INVOLVES A H2 S/CSE PATHWAY .……………………… .………………… .112 4.1 RESULTS……………………………… .………………………………………113 4.1.1 Survival Rate after Acute Myocardial Infarction … ………… .………113 4.1.2 Exclusion Criteria………………………………… .……………… …113 4.1.3 Infarct Size………………………… ……………………………114 4.1.4 Ventricular Hypertrophy……………………… .…………………………114 4.1.5 Hemodynamic Parameters………………………… ……………………115 4.1.5.1 Blood Pressure……… .……………………………… .…………………115 iv 4.1.5.2 Electrocard iograms …………… ……………… .…………………116 4.1.5.3 Heart Rate……………………………………… .………………119 4.1.6 NOx Content in the Left Ventricle……………… .……………119 4.1.7 Plas ma NOx Concentration………………………………… …………120 4.1.8 Protein Exp ressions of eNOS and nNOS……………… ………121 4.1.9 iNOS Gene Expression………………………………… .……123 4.1.10 iNOS Protein Expression……………………………… …124 4.1.11 Effect of Sildenafil on H2 S Production in the Left Ventricle……… .…124 4.1.12 Effect of Sildenafil on Plasma H2 S Concentration…… …… .…125 4.1.13 Effect of Sildenafil on CSE Protein Exp ression……… .………… …126 4.2 DISCUSSION…………………………… 128 4.2.1 Sildenafil as a Cardioprotective Agent………………… .………… 128 4.2.2 Sildenafil Imp roved Survival and Limits Infarct Development in AMI… 129 4.2.3 Effect of Sildenafil on Hemodynamic Parameters ……………… .130 4.2.4 Effect of Sildenafil on Nitrate/Nitrite Concentration in the Body……… 132 4.2.5 Effect of Sildenafil on the Exp ressions of NOS Isoforms…………… 133 4.2.6 H2 S Involvement in Sildenafil-mediated Cardioprotection……… 134 4.2.7 Summary of Findings for Experiment Protocol 2……………………… .135 CHAPTER V ROLES AND RELATIONS HIP OF HYDROGEN S ULFIDE AND NITRIC OXIDE IN A RAT MODEL OF ACUTE MYOCARDIAL INFARCTION.……………………… 137 5.1 RESULTS …… 138 5.1.1 Survival Rate after Acute Myocardial Infarction …… ………… .… 138 5.1.2 Exclusion Criteria……………………… .138 5.1.3 Infarct Size…………………………………………… .……… .139 5.1.4 Ventricular Hypertrophy……………………………… .… 139 5.1.5 Hemodynamic Parameters……………………………… .………… .140 5.1.5.1 Blood Pressure…………………………………… 140 5.1.5.2 Electrocard iograms …………………………………… .………… 141 5.1.5.3 Heart Rate……………… .…………… 144 5.1.6 CSE Gene Expression……………………… .…………144 5.1.7 CSE Protein Expression…………………………… .…….146 5.1.8 H2 S Production in the Left Ventricle………………………… .… .……147 5.1.9 Plas ma H2 S Concentration……………………………… …148 5.1.10 Gene Exp ressions of NOS Isoforms……………………………… .…149 5.1.11 Protein Expressions of NOS Isoforms…………………………… .…… .152 5.1.12 Nitrate/ Nitrite (NOx) Content in the Left Ventricle……………… .… .155 5.1.13 Plas ma Nitrate/Nitrite Concentration…………………… 156 5.1.14 HIF-1α Protein Expression………………………………………… .… 157 5.1.15 HIF-1α Gene Expression………………………………… 158 5.2 DISCUSSION……………………………………………………… .……….160 5.2.1 Interplay between H2 S and NO…………… ….160 5.2.2 Exogenous H2 S and NO A meliorates MI…………… .…162 5.2.3 Effect of H2 S and NO on BP, ECG and Heart Rate………… .164 5.2.4 Effect of H2 S and NO on the CSE/H2 S System……… 165 5.2.5 Effect of H2 S and NO on the NOS/NO System…………… 167 5.2.6 Effect of H2 S and NO on the HIF System………………………… 169 5.2.7 Summary of Findings for Experiment Protocol 3…………………… .170 v CHAPTER VI H2 S EXERTS CARDIOPROTECTION B Y EN HANCING HIF-1 α ACTIVATION AND i NOS EXPRESS ION WITH THE INVOLVEMENT OF PI3 K/AKT PATHWAY IN H9 C2 AND PRIMARY CARDIOMYOCYTES……………………… .…………… ……172 6.1 RESULTS………………………………… …………… .…… ………173 6.1.1 Optimization of Hypo xia Conditions………… .………… .…………173 6.1.1.1 Selection of Hypoxia Model………………… .………… ………173 6.1.1.2 Optimization of CoCl Concentration…………… .……174 6.1.1.3 Optimization of Hypo xia Durat ion………………… .……… .….176 6.1.1.4 Optimization of NaHS Concentration…………… .177 6.1.2 Assessment of Cell Viab ility …………………………………… … 178 6.1.2.1 MTT Assay…………………………………………………… .178 6.1.2.2 LDH Assay……………………………………… ……………179 6.1.2.3 Trypan Blue Exclusion Assay……………………… .………… 180 6.1.2.3.1 H9C2………………………………………… .……… 180 6.1.2.3.2 Card io myocytes……………………………………… 181 6.1.3 Involvement of HIF-1α/ iNOS Pathway in H2 S-Mediated Card ioprotection ……182 6.1.3.1 Protein Exp ression of HIF-1α in Total Cell Lysates……… .……182 6.1.3.2 Gene Expression of HIF-1α……… ……………. .……184 6.1.3.3 Protein Expression of HIF-1α in Nuclear and Cytoplasmic Fract ions .186 6.1.3.4 Electrophoretic Mobility Shift Assay (EMSA) for HIF-1α Binding 188 6.1.3.5 iNOS Gene Exp ression……………………………………… .190 6.1.3.6 iNOS Protein Exp ression……………………………………… 192 6.1.3.7 NO Production………………………………………… 193 6.1.4 Involvement of PI3K/Akt Pathway in H2 S-Mediated Card ioprotection……… 195 6.1.4.1 Protein Expression of Akt…………………… …………… .195 6.1.4.2 Protein Exp ression of p-Akt ………………………… 197 6.1.4.3 Akt Activation……………………………… ……… .199 6.1.4.4 Protein Expression of eNOS ………………… …… 201 6.1.4.5 Protein Expression of p-eNOS…………… .…… .203 6.1.4.6 eNOS Activation………………………………… .205 6.1.4.7 Nitrate/Nitrite Concentration …………….………… 207 6.1.5 Protein Expression of HIF-1α…………………………… 209 6.2 DISCUSSION…………………………………………………… …211 6.2.1 Overview of HIF-1……………………………………… .…… .….211 6.2.2 HIF-1 Involvement in NaHS-mediated Cardioprotection… … …212 6.2.3 Optimization of Hypo xia Model and Conditions……………… .213 6.2.4 Determination of NaHS Concentration………………………… .215 6.2.5 Cell Viabilities of H9C2 and Card io myocytes after Hypoxia…… 215 6.2.6 Effect of NaHS on HIF-1α Protein and Gene Exp ressions……… .218 6.2.7 Effect of NaHS on HIF-1α Transcriptional Activity …………… 219 6.2.8 Effect of NaHS on iNOS Exp ressions and NO Production……… 220 6.2.9 Involvement of PI3K/Akt Pathway in NaHS-mediated Cardioprotection 222 6.2.10 PI3K Lies Upstream of HIF-1α Signaling……………………… 224 6.2.11 Summary of Findings for Experiment Protocol 4……………… .225 vi CHAPTER VII S UMMARY OF CONTRIB UTIONS AND FUTUR E DIRECTIONS 227 7.1 SUMMARY OF CONTRIBUTIONS………………………………… 228 7.2 FUTURE DIRECTIONS………………………………………… 231 REFERENC ES ………………………………….…… ……………………………… 233 vii SUMMARY Hydrogen sulfide (H2 S) and nitric oxide (NO) are gasotransmitters endogenously synthesized in the body, sharing several common roles such as vasodilation. Additionally, both are implicated in the disease progression of myocardial infarction (MI), which will be examined in this study. Furthermore, several works have investigated their interaction in the vascular system, but due to the disparity in outcomes observed, their relationship is far from clear. Thus far, the interplay between H2 S and NO in the cardiovascular system has not been researched on. For this thesis, we aim to elucidate the roles and relationship of H2 S and NO in MI, and s hed light on the mechanisms involved. In the first study, S-allylcysteine (SAC) is proposed to be a novel H2 S donor as it exerted cardioprotection through a CSE (H2 S-synthesizing enzyme)/H2 S-related pathway. Pretreatment with SAC before MI induction lowered mortality and reduced infarct size. This was accompanied by an increase in left ventricular (LV) H2 S production and plasma H2 S concentration. Co-treatment with propargylglycine (PAG; CSE inhibitor) which blocked H2 S production and lowered plasma H2 S concentration was shown to abrogate the improvements in survival and infarct. Furthermore, SAC increased NO content in the LV and plasma, implicating NO involvement in SAC- mediated cardioprotection. In the next study, sildenafil brought about cardioprotection in MI via a NO-related pathway with the concomitant involvement of H2 S. Sildenafil improved survival and attenuated infarct size. This was via a NOS/NO pathway as protein and gene expressions of eNOS, nNOS and iNOS were drastically upregulated with an associated enhancement in LV and plasma NO levels. Interestingly, sildenafil also stimulated CSE activity by viii increasing H2 S production in the heart without affecting CSE’s protein expression, providing yet another evidence for the interaction between H2 S and NO. The third study examined this crosstalk on a common platform using both donors and inhibitors of H2 S and NO in in vivo MI mode ls. NaHS and molsidomine attenuated infarct enlargement and improved survival whilst inhibitors of CSE and NOS exacerbated these. Crosstalk is evidently present between H2 S and NO. Firstly, NaHS increased LV and plasma NO levels due to an upr egulation of eNOS and iNOS gene and protein expressions. Consistent stimulation of NOS/NO pathway by NaHS may involve HIF as NaHS upregulated HIF-1 protein expression drastically. This will be further examined in the next study. Secondly, blockade of H2 S production with PAG resulted in higher NO levels in both LV and plasma. This may be due to an increment in NOS activities as protein expressions were unaltered. Thirdly, NOS inhibitor L-NAME increased CSE protein expression, which was accompanied by an increase in LV H2 S production. Transcription factor HIF-1 plays a pivotal role in initiating the transcription of hypoxiasensitive genes to improve cellular adaptation to hypoxia. During hypoxia, NaHS enhanced HIF-1 protein expression and transcriptional activity in cardiac cells. Moreover, following NaHS treatment, HIF-1 activation upregulated downstream target iNOS and increased NO production. Additionally, numerous studies have implicated PI3K/Akt participation during hypoxia to mediate HIF-1α activation. Hence, its invo lvement was determined. NaHS-pretreated hypoxic cells had higher Akt and eNOS phosphorylations, which were abrogated when PI3K inhibitors were applied, indicating PI3K/Akt pathway involvement in this mode of cardioprotection. Furthermore, it was determined that this pathway lies upstream of HIF-1α. ix REFERENCES 94. JENNINGS R.B., REIMER K.A. The cell biology of acute myocardial ischemia. Annu Rev Med 1991, 42:225-246. 95. JI X., TAN B.K., ZHU Y.C., LINZ W., ZHU Y.Z. 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L-NAME Figure 4.12 CSE protein expression in MI-operated rats in saline, sildenafil and LNAME treated groups xiii Figure 5.1 Infarct sizes and hypertrophy indices of rats in saline, NaHS, molsidomine, PAG and L-NAME-treated groups Figure 5.2 Blood pressure (mmHg) of rats in saline, NaHS, molsidomine, PAG and L-NAME treated groups Figure 5.3 ECGs of saline, NaHS, molsidomine, PAG and L-NAME-treated rats... Survival rate of the animals in saline, sildenafil and L-NAME-treated groups following MI Table 4.2 Heart rates for saline, sildenafil and L-NAME treated rats Table 5.1 Survival rates of rats 48h after induction of myocardial infarction in saline, NaHS, molsidomine, PAG and L-NAME treated groups Table 5.2 Heart rates for saline, NaHS, molsidomine, PAG and L-NAME treated rats xi LIST OF FIGURES Figure... levels in the left ventricles of MI and sham-operated rats in saline, sildenafil and L-NAME treated groups Figure 4.5 NOx concentration in plasma samples of MI and sham-operated rats in saline, sildenafil and L-NAME treated groups Figure 4.6 eNOS protein expression in MI-operated rats in saline, sildenafil and LNAME treated groups Figure 4.7 nNOS protein expression in MI-operated rats in saline, sildenafil... cell signaling pathways and the underlying mechanisms involved such that a better understanding of the disease can be achieved to allow for the development of a novel, more superior, therapeutic intervention in the treatment of MI 1.2.2 Animal Models of Acute Myocardial Infarction (AMI) Several animal models of AMI have contributed to our understanding of the disease AMI can be initiated using several... Zhu Y.Z Hydrogen sulphide exerts cardioprotection by enhancing hypoxia-inducible factor 1 (HIF-1) activation and iNOS expression through regulation of PI3K/Akt signaling pathway during ischemia Oral presentation at The First International Conference of Hydrogen Sulfide in Biology and Medicine 2009, 26-28th June, Shanghai, China Recipient of the Young Investigator Award xx INTRODUCTION CHAPTER 1 INTRODUCTION... atheromatous plaques build up in coronary vessels supplying the myocardium, limiting oxygen and nutrients to result in myocardial damage Of all cardiovascular deaths in Europe and in the United States, CHD is the single largest killer, accounting for more than 1 in 5 deaths (Klocke et al., 2007) The number of CHD is escalating in both developed and developing countries and is claiming more lives than cancer... xv Figure 6.16 Binding o f HIF-1α protein from myocytes to HRE in iNOS gene promoter region Figure 6.17 iNOS gene expression in NC, HC, N300 and H300 groups of h9c2 Figure 6.18 iNOS gene expression in NC, HC, N100 and H100 groups of cardiomyocytes Figure 6.19 iNOS protein expression in NC, HC, N100 and H100 groups of cardiomyocytes Figure 6.20 NO production in various treatment groups of h9c2 Figure... protein kinase MI myocardial infarction mitoK ATP mitochondrial ATP-sensitive potassium channel MTT 3-(4,5-Dimethylthiazol- 2-yl)-2,5-dip henyltetrazolium bromide NAD nicotinamide adenine dinucleotide NADH reduced nicotinamide adenine dinucleotide NaHS sodium hydrosulfide NC normoxic control nNOS neuronal nitric oxide synthase NO nitric oxide NOS nitric oxide synthase NOx nitrate and nitrite O2 - superoxide... sulfhemoglobin Hemoglobin is also a common sink for both NO and CO in forming nitrosyl hemoglobin and scarlet carboxyhemoglobin respectively (Wang, 1998) As such, the binding of one gas will reduce the binding potential of the other gases to hemoglobin, and this will then alter the bioavailability of these gases to act on their targeted cells (Wang, 2002) 13 INTRODUCTION Figure 1.3 Metabolism of H2S in the body . ROLES AND RELATIONSHIP OF GASOTRANSMITTERS HYDROGEN SULFIDE AND NITRIC OXIDE IN MYOCARDIAL INFARCTION CHUAH SHIN CHET B.Sc. (Hons), National University of Singapore . AND RELATIONSHIP OF HYDROGEN SULFIDE AND NITRIC OXIDE IN A RAT MODEL OF ACUTE MYOCARDIAL INFARCTION. ……………………… 137 5.1 RESULTS …… 138 5.1.1 Survival Rate after Acute Myocardial Infarction … …………. platform using both donors and inhibitors of H 2 S and NO in in vivo MI models. NaHS and molsidomine attenuated infarct enlargement and improved survival whilst inhibitors of CSE and NOS exacerbated