Pkc£ And Cardioprotection: An Exploration Of Putative Mechanisms

Table of Contents

Declaration

Acknowledgements

Summary

Table of Contents

Glossary of molecular Biology Terms

Abbreviations

List of Figures and Tables

Chapter 1: Introduction

A. BACKGROUND

1.1 Heart Disease: global and local burden

B. DEFINITIONS

1.2 Ischemia

1.2.1 Ischemia and Infarction

1.2.2 Ischemic Damage

iI

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1.2.3 Reperfusion Injury 22

1.2.4 IschemiaJReperfusion vs Hypoxia/reoxygenation 23

1.3 Preconditioning Stimuli

1.3.1 Ischemic preconditioning

1.3.2 Pharmacological preconditioning

1.3.3 Remote preconditioning

1.3.4 Postcondltloning

C PKC: PHYSIOLOGY AND BIOCHEMISTRY

1.4 PKC family of proteins

1.5 PKC and the heart

1.6 PKCs and IschemiaJreperfusion

1.7 PKC and ischemic preconditioning

1.8 PKCs and cardloprotectlon

D MITOCHONDRIA: BACKGROUND AND OVERVIEW

E MITOCHONDRIAL ENERGY PRODUCTION

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1.8.1 Tricarboxylic acid cycle (TCA cycle) 42

1.8.2 Cytosolic Reducing Equivalents 43

1.8.3 Fatty acid J3-oxidatlon 45

1.8.4 Oxidative Phosphorylation 48

1.9 Perturbed mitochondrial energy production 55

1.10 Mitochondria control cell death 59

1.11 Hypothesis 69

Chapter 2: Materials and Methods 71

2.1 Animals 72

2.1.1 Ethics 72

2.1.2 PKCs overexpressing transgenic mouse model 72

2.1.3 Genotyping of PKCs transgenic mouse 74

2.1.3.1 DNA Extraction 74

2.1.3.2 Polymerase Chai n Reaction 75

2.1.3.3 Agarose Gel Electrophoresis 77

2.2 IschaemlaJReperfusion experiments: Infarct size

2.2.1 Isolated mouse heart perfusion model 78

2.2.2 Cardiac Functional assessment 79

2.2.3 Infarct size determination 80

2.3 Mitochondrial Respiration Studies

2.3.1 Mitochondrial isolation 81

2.3.2 Lowry protein determination 82

2.3.3 Determination of Mitochondrial respiration coupling 83

2.3.4 Determination of basal functional ANT content 84

2.3.5 Determination of basal mitochondrial proton leak 85

2.3.6 Determination of total inorganic phosphate 85

2.3.7 Rate of mitochondrial ATP synthesis 85

2.4 FACS analysis 86

2.5 Western Blot Analysis 87

2.5.1 Polyacrylamide Gel electrophoresis 88

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2.6

2.7

2.8

Adaptation for ANT1

Cytochrome c

UCP2

VDAC1

Anoxialreoxygenation experiments

2.6.1 Mitochondrial endpoints

2.6.2 Determination of functional ANT content in response

to anoxialreoxygenatlon

Hypobaric Hypoxia

2.7.1 Hypobaric hypoxic chamber

2.7.2 Real Time quantitative RT -PCR

2.7.3 Functional assessment

2.7.4 Mitochondrial respiration

2.7.5 Electron microscopy

2.7.6 Rate of ATP synthesis

Statistics

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Chapter 3: Characterization of the protective phenotype of the

PKCE cardiac-specific overexpresslng mouse

3.1 Introduction 99

3.2 Methods 101

3.2.1 Characterization of the protective phenotype 101

3.2.2 Mitochondrial functional phenotype 102

3.3 Statistics 104

3.4 Results 105

3.4.1 Characterization of the protective phenotype 105

3.4.2 Mitochondrial functional phenotype 108

3.4.3 Acute oxidative stress 113

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3.5 Discussion

3.6 Conclusion

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Chapter 4: A cardioprotective role for constitutively active PKCE

In response to hypobaric hypoxia

4.1 Introduction 130

4.2 Materials and methods

4.2.1 Transgenic mouse model 134

4.2.2 Mitochondrial Isolation 135

4.2.3 Characterization of mitochondrial function 135

4.2.4 Electron microscopy of left ventricular tissue 135

4.2.5 Heart perfusion studies 136

4.2.6 Assessment 0" hypertrophy 136

4.2.7 Quantitative RT -PCR 137

4.3 Statistics 137

4.4 Results 138

4.5 Discussion 150

Chapter 5 : Conclusion

5.1 Acute Oxygen lack 164

5.2 Chronic Oxygen lack 165

5.3 Proposed molecular mechanism for PKC E 169

5.4 Future Directions 171

5.5 Possible Clinical Applications 172

5.6 Limitations of the study 174

5.7 Publications arising from this work 176

References 178