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Characterising myocardial remodelling in hypertensive heart disease
Structural and functional changes in the Spontaneously Hypertensive Rat
Adèle Joanna Pope
A thesis submitted in partial fulfilment of the requirements for the degree of Doctorate in
Philosophy, The University of Auckland, 2011.
ii
Abstract
The organisation of myocardium into a laminar structure has been shown to be both mechanically and electrically important for cardiac function. Myocytes of varying orientation across the ventricular wall are grouped into layers, separated by cleavage planes. The role of extracellular matrix, in particular collagen, in this laminar organisation has not been fully delineated due to an inability to truly appreciate its 3D relationships.
This thesis utilised an unique laser confocal scanning microscopy system to produce high resolution 3D images of normal adult rat myocardium to elucidate the arrangement of collagen with respect to myocytes. Perimysium was shown to have an ordered arrangement that played a direct role in laminar organisation. There were three distinct forms of perimysial structures seen in the midwall: an extensive meshwork on laminar surfaces, convoluted fibres connecting adjacent layers, and longitudinal cords. The subepicardium had a different structure that lacks distinct layers and the only perimysial collagen present was as longitudinal cords.
Changes in perimysial collagen organisation were then studied using a rat model of hypertensive heart disease. Spontaneously Hypertensive Rats were studied at 3 months (hypertensive), 12 months (associated hypertrophy), 18 months (compensated failure) and 24 months or when determined to be in decompensated heart failure. Age-matched Wistar Kyoto rats were used as controls. Clinically used assessments, namely blood pressure measurements, echocardiography measurement of active left ventricular (LV) function and neurohormone BNP measurements were carried out on each rat to demonstrate disease progression. Hearts were then removed and passive LV function was determined using pressure-volume loop measurements on a Langendorff apparatus. Finally, hearts were then imaged as above.
High resolution imaging of the diseased hearts showed the expected increased fibrosis.
Moreover, there were distinct changes in perimysial and endomysial collagen that disrupted
and in some places eradicated the myocardial laminar organisation. These changes
persisted into LV decompensation along with LV hypertrophy and passive compliance, which
is contrary to the current understanding of HHD. The mechanical and electrical
consequences of this remodelling are likely to contribute to the transition from compensated
to decompensated heart failure.
iii
Acknowledgements
I would like to express my special thanks to my parents Antoinette and Dr Kevin Pope for their ongoing support and to my sisters, Sandra and Hazel, for their love no matter where in the world we are. I also would like to acknowledge the support I have received from my best friends Dr Amanda Lorier and Dr Kushlin Higgie who have been there for me throughout the entire process and Dr Janet Rhodes for encouraging me during the last stages.
I would like to thank my supervisors at the Physiology Department, Faculty of Medicine and Health Sciences: Associate Professor Ian LeGrice for introducing me to the wonderful world of cardiac structure and Associate Professor Bruce Smaill for providing a guiding hand throughout my PhD project.
I greatly appreciate the technical assistance during my research from Dr Gregory Sands, Dane Gerneke, Linley Nisbet and Helen Walsh.
I would also like to mention Dr Denis Loiselle, Dr Marie-Louise Ward and Professor Alistair Gunn for their support and encouragement while I have been in the Physiology Department.
I want to acknowledge the support that I received from Dr Gillan Whalley, Unitech Institute and Associate Professor Rob Doughty, University of Auckland who made it possible for me to include echocardiography measurements.
I also would like to acknowledge the support that I received from Professor Mark Richards, University of Otago who assisted with my plasma BNP measurements.
At different stages of my PhD project I have also received valuable input from Associate
Professor Lindsay Brown, University of Queensland, Professor Andrew McCulloch,
University of California San Diego, and Professor Thomas Borg, previously of University of
South Carolina. Thank you.
iv He whakataukī tenei:
Whāia e koe ki te iti kahurangi Ki te tūohu koe, me maunga teitei
This is the philiosophy that I live by:
Seek out the dreams you hold most dearly
If you must bow your head, let it be to a tallest mountain
v
Table of Contents
Abstract ... ii
Acknowledgements ... iii
Table of Contents ... v
List of Figures ... viii
List of Tables ... xiii
Terms and Abbreviations with Definitions ...xiv
Chapter 1. Introduction ... 1
1.1. Motivation ... 1
1.2. Anatomy and function of the heart ... 2
1.3. Cardiac structure and function at the cell level ... 5
1.4. Left ventricular pressure-volume loop analysis ... 8
1.5. Muscular architecture of the heart ... 11
1.6. Role and regulation of collagen in myocardium ... 16
1.7. Objectives of this research project ... 24
Chapter 2. Transmural arrangement of 3D ECM in normal myocardium ... 25
2.1. Introduction ... 25
2.2. Methodology ... 27
2.2.1. Animals and tissue preparation ... 27
2.2.2. Image acquisition ... 27
2.2.3. Image analysis ... 29
2.2.4. Statistics ... 31
2.3. Results ... 32
2.4. Discussion ... 39
2.4.1. 3D perimysial collagen organisation. ... 39
2.4.2. Myocardial architecture and mechanical function ... 42
2.4.3. Limitations ... 44
2.5. Conclusion ... 44
Chapter 3. Myocardial remodelling and mechanical dysfunction in HHD: Introduction and motivation ... 45
3.1. Introduction ... 45
3.2. Myocardial remodelling in HHD ... 46
3.3. The spontaneously hypertensive rat as a model of HHD ... 53
vi
3.4. Measurements of cardiac dysfunction in humans ... 56
3.4.1. Echocardiography ... 57
3.4.2. Brain natriuretic peptide ... 58
Chapter 4. Myocardial remodelling and mechanical dysfunction in HHD: Methodology and Procedures ... 60
4.1. Research Context ... 60
4.1.1. Research location ... 60
4.1.2. Animal colonies ... 60
4.2. Procedures and Instrumentation ... 61
4.2.1. Tail cuff sphygmomanometry ... 61
4.2.2. Echocardiography ... 61
4.2.3. Extraction of organs ... 63
4.2.4. Plasma collection and BNP assay ... 64
4.2.5. Semi-automated pressure-volume pump and recording system ... 65
4.2.6. Tissue preparation for LV pressure-volume testing ... 70
4.2.7. LV pressure-volume experimental protocol ... 71
4.2.8. Non-filtered LV pressure-volume data ... 72
4.2.9. Histological staining and tissue processing ... 75
4.2.10. Confocal imaging and extended image volume handling ... 75
4.2.11. Immunohistological staining and confocal imaging ... 76
4.3. Data Analysis ... 76
4.3.1. General measurements and in vivo physiological parameters ... 76
4.3.2. Pressure-volume loops... 77
4.3.3. Quantiative image analysis ... 77
4.3.4. Statistical analysis ... 79
4.4. Summary ... 79
Chapter 5. Myocardial remodelling and mechanical dysfunction in HHD: Results and discussion . 80 5.1. Haemodynamic and morphologic characteristics... 80
5.2. Echocardiograph assessment of LV geometry and function ... 84
5.3. Neuropeptide levels ... 89
5.4. Passive LV pressure-volume relations... 91
5.5. Myocardial remodelling with ageing and HHD ... 93
5.5.1. Initial findings ... 93
5.5.2. Transmural variation in myofibre orientation ... 93
vii
5.5.3. Myocyte dimensions ... 94
5.5.4. Collagen content ... 96
5.5.5. Laminar organisation of myocytes ... 96
5.6. Discussion ... 105
5.6.1. Clinical assessment of disease progression ... 105
5.6.2. Passive LV function ... 111
5.6.3. Mechanical properties of passive cardiac tissue ... 114
5.6.4. Myocyte arrangement and laminar organisation in HHD ... 117
5.6.5. Validity of Analysis ... 118
5.6.6. Study Limitations... 119
5.7. Conclusion ... 120
Chapter 6. Myolaminae in normal and diseased myocardium: Final discussion and future directions ... 121
6.1. Summary of principal findings ... 121
6.1.1. Objective 1 ... 121
6.1.2. Objective 2 ... 122
6.1.3. Objective 3 ... 124
6.2. Future Directions ... 127
6.2.1. Further questions about myocardial remodelling in HHD ... 127
6.2.2. Other cardiac pathologies ... 129
6.2.3. Anti-remodelling agents... 130
References ... i Appendix...I
viii
List of Figures
Figure 1-1. The anatomy of the heart cut in the coronal plane ... 2
Figure 1-2. Wiggers Diagram: The events of the cardiac cycle ... 3
Figure 1-3. The anatomical organisation of the heart muscle ... 6
Figure 1-4. The passive and active force-length relations of cardiac muscle ... 7
Figure 1-5. Alterations of the LV pressure-volume loop ... 10
Figure 1-6. The layered structure of myocardium ... 12
Figure 1-7. The collagen hierarchy in the heart. ... 13
Figure 1-8. Systolic thickening in the subendocardium ... 15
Figure 2-1. Schematic of a tissue block that is imaged to create an extended image volume ... 29
Figure 2-2. Transmural variation of fibre angles ... 30
Figure 2-3. Collagen linearity in different transmural locations ... 31
Figure 2-4. 3D volume renderings extracted from images of WKY (A) and Wistar (B) rat LVs ... 32
Figure 2-5. Transmural variation of myocyte orientation (A) and collagen fraction (B) in WKY and Wistar LV blocks ... 33
Figure 2-6. A curved transmural cutting plane was defined normal to the local fiber orientation at every point ... 34
Figure 2-7. Comparison of subepicardial (A), midwall (B), and subendocardial (C) blocks extracted from the Wistar image volume ... 36
ix
Figure 2-8. High resolution volume of normal adult rat heart midwall... 37
Figure 2-9. Perimysial collagen organisation revealed by high-resolution subvolumes ... 38
Figure 2-10. A curved transmural plane ... 42
Figure 3-1. Scanning electron microscopy images of ECM collagen skeletons from human adult hearts ... 52
Figure 4-1. Examples of echocardiography views ... 62
Figure 4-2. Excised hearts from representative 24 month old WKY and SHR in failure ... 64
Figure 4-3. Schematic of the biaxial pump ... 66
Figure 4-4. Pressure-volume experimental equipment. ... 66
Figure 4-5. Balloon and tubing ... 67
Figure 4-6. User interface window of the software controlling the pressure-volume experiments. ... 68
Figure 4-7. Pump displacement protocol 3. ... 69
Figure 4-8. Representative calibration plot for pump displacement ... 70
Figure 4-9. The process of positioning a balloon in a suspended LV ... 72
Figure 4-10. Typical raw data obtained from one pressure-volume experiment ... 73
Figure 4-11. Effect of filtering on data ... 74
Figure 5-1. Systolic blood pressure in WKY and SHR rats ... 81
x
Figure 5-2. Heart rate measurements derived from blood pressure trace recordings in WKY and SHR
rats ... 81
Figure 5-3. Heart mass determined by wet weight measurement normalised to average TL ... 82
Figure 5-4. Lungs wet weight measurement normalised to average TL ... 82
Figure 5-5. RV mass determined by wet weight measurement normalised to average TL ... 83
Figure 5-6. Liver wet weight to dry weight ratios ... 83
Figure 5-7. Echocardiograph assessment of the internal LV end diastolic diameter from WKY and SHR hearts ... 84
Figure 5-8. Echocardiograph assessment of the internal LV end systolic diameter from WKY and SHR hearts ... 85
Figure 5-9. Echocardiograph assessment of LV posterior wall thickness in diastole from WKY and SHR hearts ... 85
Figure 5-10. Echocardiograph assessment of interventricular septum wall thickness in diastole from WKY and SHR hearts ... 86
Figure 5-11. Echocardiograph assessment of LV relative wall thickness in diastole from WKY and SHR hearts ... 86
Figure 5-12. Echocardiograph derived LV wall mass from WKY and SHR hearts ... 87
Figure 5-13. Comparison of echocardiograph derived LV wall mass with heart wet weight measurements from WKY and SHR hearts ... 87
Figure 5-14. Echocardiograph determination of LV fractional shortening from WKY and SHR hearts 88 Figure 5-15. Echocardiograph determination of mitral E wave DT from WKY and SHR hearts ... 88
Figure 5-16. Plasma brain natriuretic peptide concentration from WKY and SHR hearts ... 89
xi
Figure 5-17. Passive LV filling relationships for WKY rats and SHR ... 91
Figure 5-18. LV compliance as a function of filling pressure. ... 91
Figure 5-19. LV midwall blocks from WKY ratsand SHR... 93
Figure 5-20. Transmural variation of myocyte fibre orientation in all WKY and SHR ... 94
Figure 5-21. Myocyte minimum diameter in the midwall region of hearts from WKY and SHR from 3 to 24 months of age ... 95
Figure 5-22. Variation of LV myocyte cross-sectional area with age for SHR and WKY rats. ... 95
Figure 5-23. Variation of LV collagen fraction with age for SHR and WKY rats. ... 97
Figure 5-24. Average layer width at midwall of LV free wall from WKY and SHR ... 98
Figure 5-25. Average myocytes per layer in midwall regions for WKY and SHR ... 98
Figure 5-26. High resolution images of LV midwalls from a 12 month WKY rat and SHR ... 100
Figure 5-27. High-resolution comparison of 12-month old WKY and SHR LV midwall structure. ... 101
Figure 5-28. Quantification of type I collagen in LV midwall from hearts from 24 months old SHR and WKY ... 103
Figure 5-29. Presence of Type 1 Collagen in hearts from 24 months old WKY and SHR ... 104
Figure A-1. Illustration of a midwall image shown to impartial viewer to qualitatively judge structural characteristics using a scoring criteria ... I
Figure A-2. Qualitative assessment of the degree of organisation of myocardium into layers. ... II
Figure A-3. Qualitative assessment of myocardial laminae being separated by open spaces.. ... III
xii
Figure A-4. Qualitative assessment of amount of endomysial collage visible.. ... III
Figure A-5. Qualitative assessment of extent of myocyte replacement evident. ... IV
xiii
List of Tables
Table 1. Collagen fraction at 5 transmural locations (subepicardium 7% to subendocardium 93%) across 7 normal adult rat LV walls. ... 34
Table 2. Normalised variance of collagen colinearity in 3 regions across 7 hearts. ... 37
Table 3. MANOVA results including all the independent parameters measured in this study ... 90
Table 4. Parameters characterising mean inflation curves to a 30 mmHg maximum for all WKY and SHR age groups. ... 92
Table 5. Global collagen fractions sampled at sub-epicardium (7%), midwall (50%) and
sub-endocardium (93%) for WKY and SHR groups aged at 3, 12, 18 and 24 months ... 96
Table 6. Quantification of segmented WKY and SHR images ... 102
xiv
Terms and Abbreviations with Definitions
1D: One-dimension/al
2D: Two-dimensions/al
3D: Three-dimensions/al
ACEI: Angiontensin converting enzyme inhibitor
AngII: Angiotensin II
BNP: Brain natriuretic peptide
CPF: Coiled perimysial fibre
Diastole: The phase of the cardiac cycle when the left ventricle relaxes and then fills with blood
DT: Deceleration time
DT-MRI: Diffusion tensor magnetic resonance imaging
ECG: An electrocardiogram is the recording of spread of electrical activation in the heart
ECM: Extracellular matrix
EF: Ejection fraction
Endocardium: The inner surface of the heart
Epicardium: The outer surface of the heart
FS: Fractional shortening
Hypertrophy: Enlargement of myocardium by increasing myocyte size and sometimes by
addition of myocytes
HHD: Hypertensive heart disease
Interstitium: The space around myocytes in myocardium excluding blood vessels
xv
LV: Left ventricle/ar
MMP: Matrix metallo-proteinases
Midwall: The centre region of the heart wall
Myocardium: The muscular wall of the heart
PSR: Picrosirius red dye
RAAS: Renin-Angiotensin-Aldosterone System
RV: Right ventricle/ar
SBP: Systolic blood pressure is the maximum measured arterial blood pressure
Subendocardium: Inner one third of the heart wall
Subepicardium: Outer one third of the heart wall
Systole: The phase of the cardiac cycle that involves contraction and ejection of blood from the ventricles
TIMP: Tissue inhibitor of metallo-proteinases
TDI: Tissue Doppler imaging
TL: Tibial length
