Thermal Remodelling of the Ectothermic Heart A thesis submitted to the University of Manchester for the degree of Doctor of Philosophy in the Faculty of Biology, Medicine and Health Doctoral Academy. 2016 Adam Nicholas Keen Thermal remodelling of the ectothermic heart Page | 2 Thermal remodelling of the ectothermic heart Contents ABSTRACT ............................................................................................................................... 7 Declaration ............................................................................................................................... 9 Copyright Statement .............................................................................................................. 11 About the Author .................................................................................................................... 13 Acknowledgements ................................................................................................................ 15 Organisation of Thesis ........................................................................................................... 17 List of Abbreviations ............................................................................................................... 21 1. INTRODUCTION ................................................................................................................. 23 1. 1. THE FUNCTION OF THE HEART .................................................................................... 23 1. 1. 1. Pressure-Volume Relationships During the Cardiac Cycle ...................................... 23 1. 1. 2. Cardiac Muscle Excitation and Contraction .............................................................. 26 1. 1. 3. Contractile Proteins ................................................................................................... 28 1. 1. 4. Length-Tension Relationships .................................................................................. 29 1. 1. 5. Length-Dependent Myofilament Ca2+ Sensitivity ...................................................... 31 1. 1. 6. Passive tension ......................................................................................................... 32 1. 1. 6. 1. Cardiac wall thickness ...................................................................................... 32 1. 1. 6. 2. The cardiac extracellular matrix ........................................................................ 32 1. 1. 6. 3. Intracellular structural proteins .......................................................................... 34 1. 2. CARDIAC REMODELLING .............................................................................................. 34 1. 2. 1. Pressure and Volume Overload Cardiac Remodelling ............................................. 34 1. 2. 1. 1. Cardiac hypertrophy .......................................................................................... 35 1. 2. 1. 2. Signalling pathways in hypertrophic growth ...................................................... 37 1. 2. 1. 3. Remodelling of cellular energetics .................................................................... 38 1. 2. 1. 4. Remodelling of connective tissue ..................................................................... 39 1. 2. 1. 5. Intracellular structural remodelling .................................................................... 40 1. 3. CARDIAC REMODELLING IN ECTOTHERMS.................................................................. 40 1. 3. 1. The Effects of Temperature on Ectotherms ............................................................... 40 1. 4. CARDIAC REMODELLING IN FISH ................................................................................. 41 1. 4. 1. Anatomy and Physiology of the Fish Heart ............................................................... 41 1. 4. 1. 1. The sinus venosus ............................................................................................ 42 1. 4. 1. 2. The atrium ......................................................................................................... 42 Page | 3 Thermal remodelling of the ectothermic heart 1. 4. 1. 3. The ventricle ...................................................................................................... 43 1. 4. 1. 4. The outflow tract (OFT) ...................................................................................... 43 1. 4. 2. Cardiac Function in Fish with Acute Temperature Change....................................... 44 1. 4. 2. 1. Effects on myofilaments .................................................................................... 45 1. 4. 2. 2. Effects on ion channel flux and the action potential .......................................... 45 1. 4. 2. 3. Acute effects on the passive properties of the heart ......................................... 47 1. 4. 3. Cold Acclimation and Remodelling of Fish Heart ...................................................... 47 1. 4. 3. 1. Remodelling of calcium handling....................................................................... 48 1. 4. 3. 2. Remodelling of myofibrils .................................................................................. 49 1. 4. 3. 3. Cardiac morphology .......................................................................................... 50 1. 4. 3. 4. The extracellular matrix ..................................................................................... 52 1. 4. 3. 5. Length-dependent changes in force generation ................................................ 52 1. 4. 3. 6. Remodelling of cellular energetics .................................................................... 52 1. 5. CARDIAC REMODELLING IN TURTLES .......................................................................... 53 1. 5. 1. Anatomy and Physiology of the Turtle Heart ............................................................. 53 1. 5. 1. 1. The atria ............................................................................................................ 53 1. 5. 1. 2. The ventricle ...................................................................................................... 53 1. 5. 2. Cardiac Function in Turtles with Acute Temperature Change .................................. 55 1. 5. 3. Cold Acclimation and Remodelling of the Turtle Heart ............................................. 56 1. 5. 3. 1. Remodelling contractile force ............................................................................ 56 1. 5. 3. 2. Remodelling of the passive properties of the heart ........................................... 57 1. 5. 3. 3. Remodelling of cellular energetics .................................................................... 57 1. 6. SUMMARY ...................................................................................................................... 58 1. 7. AIMS .............................................................................................................................. 58 2. GENERAL METHODS ......................................................................................................... 61 2. 1. IN VIVO CARDIAC FUNCTION ....................................................................................... 61 2. 2. WHOLE HEART .............................................................................................................. 62 2. 2. 1. Isolation of the Fish Heart ..................................................................................... 62 2. 2. 2. Ex Vivo Pressure-Volume Curves ......................................................................... 62 2. 3. TISSUE TECHNIQUES .................................................................................................... 65 2. 3. 1. Tissue Histology .................................................................................................... 65 2. 3. 1. 1. Haematoxylin and Eosin (H & E) ................................................................... 65 2. 3. 1. 2. Picro-Sirus Red ............................................................................................. 66 2. 3. 1. 3. Miller’s Elastic Stain ....................................................................................... 68 2. 3. 1. 4. Oil Red O Stain .............................................................................................. 69 Page | 4 Thermal remodelling of the ectothermic heart 2. 3. 1. 5. Periodic Acid Schiff (PAS) Stain ................................................................... 69 2. 3. 2. Fourier Transform Infrared (FTIR) Spectroscopy ................................................. 70 2. 3. 2. 1. Background to FTIR .......................................................................................... 70 2. 3. 2. 2. Recording a Spectrum .................................................................................. 72 2. 3. 2. 3. FTIR Imaging Spectroscopy ......................................................................... 73 2. 3. 3. Atomic Force Microscopy (AFM) ........................................................................... 76 2. 3. 4. In Situ Zymography ............................................................................................... 77 2. 4. 1. SDS-PAGE Gelatin Zymography .......................................................................... 78 2. 4. 2. Real-Time Quantitative PCR (RT-qPCR).............................................................