THE REGIONALITY OF CARDIAC ΒETA-2- ADRENOCEPTOR SIGNALLING PETER THOMAS WRIGHT NATIONAL HEART AND LUNG INSTITUTE, IMPERIAL COLLEGE LONDON A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DECEMBER 2013 1 ABSTRACT The dysfunction of the apical myocardium is observed following chronic exposure to catecholamines, or after acutely stressful scenarios, in the syndrome of Takotsubo cardiomyopathy. The physiological functions of the β2AR were assessed via measurement of cell shortening; β2AR-cAMP signalling was assessed by FRET microscopy, comparing cells from the apical and basal myocardium. The effects of pre-stimulation of the β2AR with high levels of endogenous catecholamines were investigated to simulate pathological scenarios. The structure of the cellular membranes of cells from different myocardial regions was assessed and manipulated to investigate their role in β2AR signalling. An improved animal model of the regional pathology in Takotsubo was investigated using ovariectomized female rats. Apical cardiomyocytes are sensitized to β2AR stimulation, displaying larger increases in inotropy and lusitropy in comparison to basal cells. This was not due to differences in cAMP levels induced by β2AR within the cell cytosol. Differences were apparent in the amount of cAMP reaching the RII-PKA domains (the main arbiters of cellular inotropy and lusitropy). β2AR-cAMP signalling was discovered to be more persistent in the apical cardiomyocytes. Basal cardiomyocytes were found to have a larger number of caveolae within their membranes, caveolae have been demonstrated to modulate β2AR. Following the disruption of caveolar domains via chelation of cholesterol, apical and basal responses to β2AR stimulation were equalized; inhibition of phosphodiesterase 4 had the same effect. Catecholamine pre-stimulation reduced β2AR responses; adrenaline pre-stimulation exerts a more Gi-dependent desensitization than noradrenaline. Female rats are shown to be relatively protected from the effects of adrenergic overstimulation and ovariectomized female rats suffer acutely high mortality. Cells from different regions of the myocardium may control their receptor signalling in different ways to produce desirable physiological activity. In settings of pathology this phenomena may leave certain regions vulnerable to damage. 2 PUBLICATIONS Wright PT, Nikolaev VO, O'Hara T, Diakonov I, Bhargava A, Tokar S, Schobesberger S, Shevchuk, A, Sikkel MB, Wilkinson R, Trayanova NA, Lyon AR, Harding SE, Gorelik J. Caveolin-3 regulates compartmentation of cardiomyocyte beta2-adrenergic receptor-mediated cAMP signaling. Journal of Molecular and Cellular Cardiology, Accepted (in press) Gorelik J, Wright PT, Lyon AR, Harding SE, 2013, Spatial control of the βAR system in heart failure: the transverse tubule and beyond. Cardiovasc Res, Vol: 98, 0008-6363, Pages: 216-224 Tranter MH, Wright PT, Sikkel MB, Lyon AR, 2013, Takotsubo cardiomyopathy: the pathophysiology, Heart Failure Clinics, Vol: 9, 1551-7136, Pages: 187-196 Lab MJ, Bhargava A, Wright PT, Gorelik J. 2012, The Scanning Ion Conductance Microscope (SICM) for Cellular Physiology, American Journal of Physiology-Heart and Circulatory Physiology Paur H, Wright PT, Sikkel MB, Tranter MH, Mansfield C, O'Gara P, Stuckey DJ, Nikolaev VO, Diakonov I, Pannell L, Gong H, Sun H, Peters NS, Petrou M, Zheng Z, Gorelik J, Lyon AR, Harding SE. 2012, High levels of circulating epinephrine trigger apical cardiodepression in a β2-adrenergic receptor/Gi- dependent manner: a new model of Takotsubo cardiomyopathy., Circulation Journal, Vol: 6, Pages: 697-706 Miragoli M, Moshkov A, Novak P, Shevchuk A, Nikolaev VO, El-Hamamsy I, Potter CMF, Wright PT, Sheik Abdul Kadir SH, Lyon AR, Mitchell JA, Chester AH, Klenerman D, Lab MJ, Korchev YE, Harding SE, Gorelik J. 2011, Scanning ion conductance microscopy: a convergent high-resolution technology for multi-parametric analysis of living cardiovascular cells, Journal of the Royal Society Interface, Vol: 8, Pages: 913-925 International Conferences Oral presentations Wright PT, Moshkov A, Lyon AR, Harding SE, Gorelik J. 2011, Functional imaging of the effects of catecholamine shock on B1/B2-Adrenergic signaling in ventricular cardiomyocytes by SICM/FRET, ISHR European Subsection (Haifa, June 2011) Wright PT, Diakonov I, Nikolaev VO, Lyon AR, Harding SE, Gorelik J. 2012, Abstract 15647: Myocardial Gradients in Caveolar Number Modulate Cardiomyocyte Contractile Response to Specific {beta}2AR Stimulation, Scientific Sessions of the American Heart Association, Pages:A15647-A15647 (Los Angeles, November 2012) (Recipient of International Travel Award) UK Conferences Oral Presentations Wright PT ‘Beta-2 adrenoceptor signalling in cardiomyocyte regions - location, location, location’ (Highly Commended Oral Presentation by a basic scientist). NHLI Postgraduate Research Day June 2013 3 Posters Wright PT, Diakonov I, Zaccolo M, Lyon AR, Harding SE and Gorelik J. Differences in cardiomyocyte cAMP compartmentation may underlie regional variation in the contractile response upon β2AR stimulation. ‘Targeting Cyclic AMP Signalling to Combat Cardiovascular Diseases’ meeting of the Biochemical Society (London December 2013) (Best Poster Prize) Wright PT, Nikolaev VO, Tokar S, Bhargava A, Diakonov I, Sikkel MB, Lyon AR, Harding SE, Gorelik J, 2012, Abstract 15670: Caveolin3 Depletion Modifies {beta}2AR-cAMP Responses at Both Cellular and Sub-Cellular Levels in Cardiomyocytes, Scientific Sessions of the American Heart Association, Pages:A15670-A15670 (Los Angeles, 2012) Wright PT, Pannell LMK, Nikolaev VO, Lyon AR, Harding SE, Gorelik J, Imaging regional differences in beta-2 adrenergic receptor function at the cellular level. Joint BAS/BSCR Spring Meeting Manchester, UK (28th-29th of May 2012) Wright PT, Diakonov I, Tokar S, Lyon AR, Sikkel M, Harding SE, Gorelik J, 2012 Beta-2 adrenergic receptor signaling in adult rat ventricular myocytes at 4, 8 and 16 weeks after myocardial infarction (Judges Choice in Excitation-Contraction Coupling Section) European Society of Cardiology: Frontiers in Cardiovascular Biology London, UK (30th of March-1st of April 2012) Wright PT, Pannell LMK, Lyon AR, Harding SE, Gorelik J. The negatively inotropic effect of adrenaline stress is not completely linked to the reduction of cAMP release mediated by the, Gs/Gi switch. European Society of Cardiology: Frontiers in Cardiovascular Biology London, UK (30th of March-1st of April 2012) DECLARATION All work presented in this thesis is my own unless otherwise indicated or credited within the text. ‘The copyright of this thesis rests with the author and is made available under a Creative Commons Attribution Non-Commercial No Derivatives licence. Researchers are free to copy, distribute or transmit the thesis on the condition that they attribute it, that they do not use it for commercial purposes and that they do not alter, transform or build upon it. For any reuse or redistribution, researchers must make clear to others the licence terms of this work’ 4 ACKNOWLEDGEMENTS First and foremost I must thank Dr. Julia Gorelik for her supervision and guidance during my time as both an MRes student and a PhD student in her laboratory. Her tenacity in the pursuit of new knowledge and desire to push forward the development of new technologies provided me with a great source of motivation, as well as a belief in the power of academic science. I thank Professor Sian Harding for her guidance on both scientific matters and pastoral ones, concerning my interest in the communication of science to society at large. She also deserves thanks for her clear/critical analysis of my findings and providing me with wider perspectives on the path of science in general. I thank Dr. Alexander Lyon for his enthusiasm, encouragement and for giving my work a clinically relevant aspect, without which my work would have much less meaning. I would like to thank Dr. Ivan Diakonov, Dr. Sergiy Tokar and Dr. Alexey Moshkov, members of the Gorelik group, for their assistance with microscopic and biochemical studies. I would also like to thank them for their creative input into my work and suggestions for technical improvements. All members of the Gorelik group and collaborators deserve thanks for their creative input during the course of this work and their friendship throughout this time. Thanks are given in no particular order to Sophie Schobesberger, Francisca Schultz, Oladipupo Adeyemi, Dr. Jose Sanchez Mardones- Alonso, Dr. Alexey Gluhkov and Dr. Michele Miragoli. I should also thank Professor Max Lab for his insights and impertinent questions, which as the great Jacob Bronkowski pointed out so often lead to pertinent answers. I thank Dr. Viacheslav Nikolaev and the members of his research group (Ruwan Perrira, Julia Sprenger, Konrad Gotz and Karina Zimmerman) for their support with matters related to FRET microscopy, their provision of the Epac2 and pmEpac2 sensors, and their contribution of biochemical studies which appear in this work. I thank Professor Manuela Zaccolo for her gift of the RII_Epac sensor. I thank Laura Pannell and Ami Mehta who performed studies under my supervision as part of their medical training. Their hard work and attention to detail allowed me to move into other areas of study and made my first supervisory responsibilities much easier. I thank Dr. Helen Paur for passing on her work with the in vivo model of Takotsubo cardiomyopathy. I also thank Matthew Tranter with whom I have shared this work and who has become a great friend. I thank