The control of formation and recovery of transverse tubules in the heart A thesis submitted to The University of Manchester for the degree of Doctor of Philosophy in the Faculty of Biology, Medicine and Health 2019 Jessica L. Caldwell School of Medical Sciences, Division of Cardiovascular Sciences Table of contents Table of contents .......................................................................................................... 1 List of figures ............................................................................................................... 9 List of tables ............................................................................................................... 11 List of abbreviations ................................................................................................... 12 Abstract ...................................................................................................................... 15 Declaration ................................................................................................................. 16 Copyright statement ................................................................................................... 17 Acknowledgments ...................................................................................................... 18 Contributions .............................................................................................................. 19 1. General Introduction ...................................................................................... 20 1.1. Introduction .............................................................................................. 20 1.2. Excitation contraction coupling ............................................................... 20 1.3. The transverse tubule network ................................................................. 23 1.3.1. Transverse tubules and EC coupling. ............................................... 24 1.3.2. Transverse tubules: Signalling pathways ......................................... 24 1.3.3. Transverse tubules: Cellular differences .......................................... 26 1.4. Transverse tubule alterations in disease................................................... 28 1.4.1. Heart failure ...................................................................................... 28 1.4.2. Transverse tubule remodelling in heart failure ................................. 28 1.4.3. Transverse tubule remodelling in atrial fibrillation .......................... 29 1 1.4.4. Transverse tubule remodelling: altered calcium handling. .............. 30 1.4.5. Reversal of transverse tubule remodelling ....................................... 32 1.5. Biogenesis of transverse tubules .............................................................. 34 1.5.1. Phosphatidylinositols (PI) ................................................................ 36 1.5.2. Amphiphysin II (BIN1) .................................................................... 37 1.5.3. Myotubularin (MTM1) ..................................................................... 41 1.5.4. Junctophilin (JPH2) .......................................................................... 43 1.5.5. Telethonin ......................................................................................... 45 1.5.6. Proteins of the z-line ......................................................................... 47 1.6. Aims ......................................................................................................... 48 2. Methods .......................................................................................................... 50 2.1. Sheep ........................................................................................................ 50 2.1.1. Induction of heart failure in sheep .................................................... 50 2.1.2. Recovery Sheep ................................................................................ 50 2.1.3. Isolation of sheep ventricular and atrial myocytes ........................... 51 2.2. Neonatal rats ............................................................................................ 51 2.2.1. Isolation of neonatal rat ventricular myocytes ................................. 51 2.3. Cell culture ............................................................................................... 53 2.3.1. Neonatal rat ventricular myocyte culture ......................................... 53 2.3.2. Induced pluripotent stem cell (iCells) culture .................................. 53 2.4. Generation of Plasmids and Transfection ................................................ 54 2 2.4.1. Plasmid DNA expansion and purification ........................................ 56 2.4.2. Generation of vectors with fluorescence tag .................................... 56 2.4.2.1. Digestion of vectors: ..................................................................... 56 2.4.2.2. Ligation of vectors: ....................................................................... 57 2.4.2.3. Sequencing of vectors: .................................................................. 59 2.4.2.4. Generated vector expansion .......................................................... 61 2.4.3. Transient Transfection ...................................................................... 61 2.5. T-tubule imaging...................................................................................... 62 2.5.1. Preparation of samples ..................................................................... 62 2.5.2. Confocal Microscopy ....................................................................... 62 2.5.3. Image analysis .................................................................................. 64 2.5.3.1. Distance maps ............................................................................... 64 2.5.3.2. Transverse tubule fractional area analysis .................................... 66 2.5.3.3. Transverse tubule orientation analysis .......................................... 66 2.5.3.4. Branching analysis ........................................................................ 67 2.5.3.5. Cell width ...................................................................................... 68 2.6. Measurements of intracellular calcium .................................................... 69 2.6.1. NRVM intracellular calcium ............................................................ 70 2.6.1.1. Measurements of intracellular calcium concentration .................. 70 2.6.1.2. Analysis of intracellular calcium: ................................................. 70 2.6.2. Sheep myocyte intracellular calcium................................................ 72 3 2.6.2.1. Measurements of intracellular calcium. ........................................ 72 2.6.2.2. Analysis of intracellular calcium: ................................................. 74 2.7. Protein assessment ................................................................................... 76 2.7.1. Protein extraction ............................................................................. 76 2.7.2. Protein quantification ....................................................................... 77 2.7.3. SDS-PAGE ....................................................................................... 77 2.7.3.1. Protein transfer .............................................................................. 78 2.7.3.2. Membrane blocking ...................................................................... 78 2.7.3.3. Detection of protein....................................................................... 79 2.7.3.4. Analysis of Western blots ............................................................. 79 2.7.4. Immunocytochemistry ...................................................................... 83 2.7.5. Co-localisation analysis .................................................................... 83 2.8. Statistics ................................................................................................... 84 3. Results: Disordered, yet functional, atrial t-tubules on recovery from heart failure. .................................................................................................................... 85 3.1. Introduction .............................................................................................. 85 3.1.1. Heart failure model ........................................................................... 86 3.1.2. Aims of the chapter ......................................................................... 88 3.2. Results ...................................................................................................... 89 3.2.1. Recovery of atrial t-tubules .............................................................. 89 3.2.1.1. Atrial t-tubule density ................................................................... 89 4 3.2.1.2. Atrial t-tubule structure and organisation ..................................... 92 3.2.1.3. Atrial t-tubule remodelling ............................................................ 95 3.2.2. Recovered atrial t-tubules were functional ....................................... 98 3.2.2.1. T-tubules were the main site for calcium release .......................... 98 3.2.2.2. T-tubule recovery restored synchronicity of calcium release. .... 101 3.2.2.3. Cellular distribution of calcium handling proteins ..................... 102 3.2.3.