https://theses.gla.ac.uk/ Theses Digitisation: https://www.gla.ac.uk/myglasgow/research/enlighten/theses/digitisation/ This is a digitised version of the original print thesis. Copyright and moral rights for this work are retained by the author A copy can be downloaded for personal non-commercial research or study, without prior permission or charge This work cannot be reproduced or quoted extensively from without first obtaining permission in writing from the author The content must not be changed in any way or sold commercially in any format or medium without the formal permission of the author When referring to this work, full bibliographic details including the author, title, awarding institution and date of the thesis must be given Enlighten: Theses https://theses.gla.ac.uk/ [email protected] IDENTIFICATION OF LEFT VENTRICULAR MASS QTL IN THE STROKE-PRONE SPONTANEOUSLY HYPERTENSIVE RAT This being a thesis submitted for the degree of Doctor of Philosophy in the Faculty of Medicine, University of Glasgow ©Kirsten Gil day B.Sc. Hons (University of Glasgow), M.Sc. (University of Glasgow) University of Glasgow Division of Cardiovascular & Medical Sciences BEDF Glasgow Cardiovascular Research Centre 126 University Place Glasgow G12 8TA O ct 2006 ProQuest Number: 10390645 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a com plete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest ProQuest 10390645 Published by ProQuest LLO (2017). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States C ode Microform Edition © ProQuest LLO. ProQuest LLO. 789 East Eisenhower Parkway P.Q. Box 1346 Ann Arbor, Ml 48106- 1346 [GLASGOW 1 hlNlVERSlTY ll.lU R AKW J DECLARATION I declare that this thesis has been written entirely by myself and has not been submitted previously for a higher degree. The work recorded has been carried out by me under the supervision of Professor Anna Dominiczak and Dr Delyth Graham, unless stated otherwise in acknowledgements. KIRSTEN GILDAY ACKNOWLEDGEMENTS I would like to express my sincere appreciation to my supervisors Dr Delyth Graham and in particular Professor Anna F Dominiczak, for their constant support, guidance and help throughout the course of my study for this thesis. I would also like to thank Dr Barrie Condon and the technical staff at 7T MRI unit, Garscube. I am also very grateful to Drs Martin McBride and Wai Kwong Lee for their teaching in molecular techniques and advice throughout and Dr John McClure for statistical direction and help in analysing large volumes of data. Finally and most importantly, this thesis would not have been possible without the constant support and encouragement from my family, in particular my husband, without whom I would not have been able to accomplish a higher education. Ill CONTENTS PAGE DECLARATION ii ACKNOWLEDGEMENTS iii CONTENTS iv LIST OF TABLES vii LIST OF FIGURES ix ABBREVIATIONS xii SUMMARY XV 1 CHAPTER 1 INTRODUCTION 1.1 COMPLEX CARDIOVASCULAR TRAITS 1 1.1.1 BLOOD PRESSURE; REGULATION AND MAINTENANCE 3 1.1.2 HUMAN ESSENTIAL HYPERTENTION 6 1.1.3 LEFT VENTRICULAR HYPERTROPHY (LVH) 11 1.2 EVIDENCE FOR GENETIC EFFECTS 14 1.2.1 MENDELIAN FORMS OF HYPERTENSION 14 1.2.2 MENDELIAN FORMS OF CARDIAC HYPERTROPHY 18 1.2.3 HUMAN STUDIES OF HYPERTENSION 20 1.2.4 HUMAN STUDIES OF LVH 22 1.3 EXPERIMENTAL ANIMAL MODELS 25 1.3.1 ANIMAL MODELS OF CARDIOVASCULAR DISEASE 25 1.3.2 MOUSE MODELS FOR CARDIOVASCULAR DISEASE 25 1.3.3 RAT MODELS OF HYPERTENSIVE CARDIOVASCULAR 26 DISEASE 1.3.4 THE STROKE-PRONE SPONTANEOUSLY HYPERTENSIVE RAT 27 1.3.5 STUDIES IN ANIMAL MODELS 29 1.3.6 GENDER DIFFERENCES 33 1.4 GENETIC MARKERS/MAPS 35 1.4.1 GENOME WIDE QTL ANALYSIS 35 1.4.2 COMPARATIVE GENOME ANALYSIS 39 1.4.3 CANDIDATE GENE ANALYSIS 39 1.4.4 CONGENIC STRAIN AND SUBSTRAIN PRODUCTION 40 IV 1.5 GENE EXPRESSION PROFILING 41 1.5.1 MICROARRAY ANALYSIS AND QUANTITATIVE RT-PCR 41 1.6 HIGH FIDELITY PHENOTYPING 45 1.6.1 HAEMODYNAMIC PROFILING 45 1.6.2 CARDIAC IMAGING TECHNIQUES 46 1.7 AIMS OF THE STUDY 48 2 CH APTER 2 METHODS 2.1 GENERAL LABORATORY PRACTICE 51 2.2 ANIMAL STRAINS 51 2.2.1 Fi and Fz PRODUCTION 52 2.2.2 CONGENIC STRAINS 52 2.3 PHENOTYPIC ANALYSIS 55 2.3.1 TAIL CUFF PLETHYSMOGRAPHY 55 2.3.2 RADIO-TELEMETRY 55 2.3.3 BODY AND CARDIAC MEASUREMENTS 56 2.3.4 ECHOCARDIOGRAPHY 56 2.3.5 MAGNETIC RESONANCE IMAGING (MRI) 57 2.4 GENETIC ANALYSIS 59 2.4.1 DNA EXTRACTION 59 2.4.2 POLYMERASE CHAIN REACTION (PCR) 61 2.4.3 GEL ELECTROPHORESIS USING AGAROSE GELS 62 2.4.4 GEL ELECTROPHORESIS USING POLYACRYLAMIDE GELS 62 AND AUTORADIOGRAPHY 2.4.5 FLUORESCENT PCR AND ABI DNA SEQUENCER ANALYSIS 64 2.5 QTL ANALYSIS 64 2.5.1 JOINMAP VERSION 3.0 65 2.5.2 MAPMANAGER QTX VERSION B20 66 2.5.3 WINDOWS QTL CARTOGRAPHER VERSION 2.5 67 2.6 QUANTATITIVE RT-PCR 68 2.6.1 RNA EXTRACTION 68 2.6.2 DNAse TREATMENT OF RNA 69 2.6.3 QUALITY ASSESSMENT OF RNA 69 2.6.4 TWO STEP RT-PCR (TAQMAN) 69 2.7 SEQUENCING OF CANDIDATE GENES 71 2.7.1 CLEANING PCR PRODUCT 71 2.7.2 SEQUENCING PCR PRODUCT 72 2.8 MICROARRAY 73 2.8.1 TISSUE PREPARATION 73 2.8.2 TARGET HYBRIDISATION 74 2.8.3 FLUIDIC STATION AND PROBE ARRAY SETUP 75 2.8.4 DATA ANALYSIS 77 3 CHAPTER 3 IDENTIFICATION OF LEFT VENTRICULAR MASS QTL IN SHRSP 3.1 INTRODUCTION 79 3.2 METHODS 82 3.2.1 EXPERIMENTAL ANIMALS AND GENETIC CROSSES 82 3.2.2 GENOTYPING, IMPROVED GENETIC LINKAGE MAP AND QTL 83 ANALYSIS 3.3 RESULTS 99 3.4 DISCUSSION 98 4 CHAPTER 4 COMPARATIVE MAPPING AND IDENTIFICATION OF CANDIDATE GENES 4.1 INTRODUCTION 104 4.2 METHODS 106 4.2.1 COMPARATIVE MAPPING 106 4.2.2 PHARMACOLOGICAL INTERVENTION 106 4.2.3 RNA EXTRACTION AND cDNA SYNTHESIS 106 4.2.4 REAL-TIME PCR QUANTIFICATION 107 4.2.5 SEQUENCING CANDIDATE GENES 107 4.2.6 SEQUENCE ANALYSIS 110 4.2.7 SNP GENOTYPING 110 4.3 RESULTS 110 4.4 DISCUSSION 126 5 CHAPTER 5 MRI AS A METHOD FOR IN VIVO LV QUANTIFICATION AND GEOMETRY 5.1 INTRODUCTION 133 5.2 METHODS 135 5.2.1 EXPERIMENTAL ANIMAL STRAINS 135 5.2.2 ECHOCARDIOGRAPHIC LEFT VENTRICLE DETERMINATION 135 5.2.3 ANIMAL PREPARATION FOR MRI IMAGING 138 5.2.4 MRI ASSESSED LV DETERMINATION AND CALCULATIONS 140 5.2.5 POSTMORTEMEVALVATION 140 5.3 RESULTS 142 5.4 DISCUSSION 147 6 CHAPTER 6 PRODUCTION OF CONGENIC STRAINS 6.1 INTRODUCTION 155 6.2 METHODS 157 6.2.1 CONGENIC STRAIN PRODUCTION 157 VI 6.2.2 TAIL-TIPPING AND DNA EXTRACTION 159 6.2.3 PCR AND GENOTYPING 159 6.2.4 DESIGN OF MICROSOFT ACCESS DATABASE 159 6.2.5 PHENOTYPE MEASUREMENTS 160 6.3 RESULTS 160 6.4 DISCUSSION 164 7 CHAPTER 7 GENOME WIDE EXPRESSION ANALYSIS 7.1 INTRODUCTION 173 7.2 METHODS 175 7.2.1 EXPERIMENTAL ANIMALS, DIETARY MANIPULATION AND 175 PHENOTYPIC MEASUREMENTS 175 7.2.2 RNA EXTRACTION AND MICRO ARRAY EXPERIMENT 175 7.2.3 MICROARRAY DATA ANALYSIS 176 7.3 RESULTS 176 7.4 DISCUSSION 211 8 CHAPTER 8 GENERAL DISCUSSION 215 REFERENCES 219 APPENDICES I-V 252 Vll LIST OF TABLES Page Table 1.1: Key regulatory hormones and neurotransmitters involved in the regulation and maintenance of blood pressure Table 1.2: The JNC-7 guidelines for blood pressure classification Table 1.3: Mendelian forms of hypertension, mutations and molecular mechanism involved in blood pressure modulation 16 Table 1.4: Disease genes involved in Familial Hypertrophic 20 Cardiomyopathy (FHC) Table 1.5: Genetically inbred hypertensive rat strains in common use for 28 cardiovascular research Table 3.1 Polymorphic microsatellite marker oligonucleotide sequences 82 Table 4.1 PCR and sequencing primers for Dbp 109 Table 4.2 PCR and sequencing primers for Corin 110 Table 4.3 Pairwise analysis of candidate genes 114 Table 5.1 MRI summary data for animals 146 Table 6.1 Background heterogeneity in backcross strains generated 164 Table 7.1 Differentially expressed genes mapping to chromosome 14 184 Table 7.2 Networks identified from RP analysis for each sample group 185 comparison Table 7.3- List of 119 genes identified from highest significant network 190 Table 7.4: Two-way ANOVA strain-salt interaction 210 Vlll LIST OF FIGURES Page Figure 1.1: Illustration of the polygenic, multifactorial nature of a complex trait 2 Figure 1.2: Diagram outlining the sequence of events during a heartbeat 4 Figure 1.3: The rate at which a viscous fluid flows through a cylindrical shaped ^ pipe Figure 1.4: Diagrammatic outline of the RAS regulatory cascade in blood pressure ^ regulation Figure 1.5: Organ damage resulting from elevated blood pressure 10 Figure 1.6: Outline of sarcomere organisation 12 Figure 1.7: Left ventricular geometric patterns, consequences of different ^ ^ combinations of pressure and/or volume overload Figure 1.8: Choices of analysis for quantitative trait locus mapping 39 Figure 1.9: Cartoon representing the basic principals behind the production of congenic strains Figure 2.1: Diagram depicting congenic strain production 54 Figure 2.2A: Representative echocardiogram 59 Figure 2.2B: Correlation of echocardiograph estimated LVM and post mortem evaluation Figure 2.3A: Short axis view image of 1mm slice through the heart in diastole 61 Figure 2.3B: Short axis view image of 1mm slice through the heart in systole 61 Figure 2.4: An example of output from GeneMapper v3.7 66 Figure 2.5 : Diagram of the GeneChip® Probe Array 77 Figure 3.1: A representative agarose gel of 47 F2 animals genotyped with marker D14Arbl7 Figure 3.2A: