SIGNALLING THROUGH RHO GTPASES IN CARDIOMYOCYTES ANDREW KEITH MARSHALL BSc (Hons) A thesis submitted to Imperial College London for the degree of Doctor of Philosophy National Heart & Lung Institute, Faculty of Medicine Imperial College of Science, Technology and Medicine Dovehouse Street, London SW3 6LY February 2011 Page | 1 ABSTRACT Endothelin-1 (ET-1) promotes changes in gene/protein expression in cardiomyocytes leading to hypertrophy. This results from activation of intracellular signalling pathways including small G proteins that activate protein kinases. Thus, ET-1 activates RhoA that stimulates ROCK and PKN, and Ras that promotes activation of extracellular signal-regulated kinases 1/2 (ERK1/2). Microarrays were used to dissect the roles of ERK1/2 vs RhoA in the cardiomyocyte transcriptomic response to ET-1 using PD184352 and C3 endotoxin from C. botulinum (C3T) for selective inhibition of the ERK1/2 cascade and RhoA, respectively. Microarray data were analysed using GeneSpring and data were validated by qPCR. ERK1/2 signalling positively regulated ~65% of the early gene expression response to ET-1 with a small (~2%) negative effect, whereas RhoA signalling positively regulated ~11% of the early gene expression response to ET-1 with a greater (~14%) negative contribution. Of RNAs non-responsive to ET-1, 66 or 448 were regulated by PD184352 or C3T, respectively, indicating that RhoA had a more significant effect on baseline RNA expression. mRNAs upregulated by ET-1 encoded several receptor ligands (e.g. Ereg, Areg) and transcription factors (e.g. Abra/STARS, Srf) that potentially propagate the response. Published studies suggest that PKN1 (activated by RhoA) is important in cardiomyocyte gene expression. Adenoviruses were generated to overexpress FLAG-tagged PKN1 in cardiomyocytes for protein kinase studies. Unexpectedly, PKN1 was not activated by ET-1, but was activated by oxidative stress, insulin, or hyperosmotic shock, stimuli that do not activate RhoA. Thus, PKN1 is not necessarily an effector of RhoA in cardiomyocytes. In conclusion, ERK1/2 dominates over RhoA in the early transcriptomic response to ET-1. RhoA plays a major role in maintaining baseline RNA expression but, as with upregulation of Abra/Srf by ET-1, RhoA may regulate changes in RNA expression over longer times. However, the effects of RhoA on cardiomyocyte gene expression are unlikely to be mediated through PKN1. Page | 2 ACKNOWLEDGEMENTS The research described here would not have been possible without the consistent help, advice and patience provided by my supervisors, Professor Angela Clerk and Professor Peter Sugden, for which I am very grateful. The constant encouragement and the wisdom of years of experience in the laboratory provided by Dr Stephen Fuller have also been invaluable. Similarly, Dr Timothy Cullingford‟s experience and advice on large microarray studies was crucial when I undertook my own such experiment. Furthermore, I would like to acknowledge and thank all of my colleagues at the NHLI who have provided advice when needed and an excellent working environment over the years. This research was fully and generously funded by the British Heart Foundation. Page | 3 STATEMENT OF CONTRIBUTION The research which forms the basis of my thesis was carried out during my time as a full-time post- graduate research student in the Department of Cardiac Medicine, National Heart and Lung Institute, Imperial College London. The research was conducted in part in the laboratories of the Flowers Building (South Kensington campus) and in the Guy Scadding Building (Royal Brompton Campus). The RBD-GST fusion protein was expressed and purified in association with Professor Peter Sugden. Dr Stephen Fuller prepared and purified the initial FLAG-pShuttle vector which was used to begin the process of generating my FLAG-PKN1 overexpressing adenoviral vector. Furthermore, his advice throughout the preparation of the adenoviruses was crucial. On a weekly basis, the cardiomyocyte cultures were in the large part prepared by Professor Angela Clerk and Miss Elli Tham. Cardiomyocyte proteins were partially-purified by Mono-Q ion-exchange chromatography by Professor Angela Clerk. Fragmentation of the antisense cRNA and hybridisation to the Affymetrix rat genome arrays was performed at the CSC Microarray Centre, Hammersmith Campus, Imperial College London. All other experimental work and data analyses were performed by me unless otherwise stated. Page | 4 PUBLICATIONS, ABSTRACTS AND PRESENTATIONS PUBLICATIONS Marshall AK, Barrett OP, Cullingford TE, Shanmugasundram A, Sugden PH, Clerk A. ERK1/2 signaling dominates over RhoA signaling in regulating early changes in RNA expression induced by endothelin-1 in neonatal rat cardiomyocytes. PLoS One. 2010 Apr 2;5 (4):e10027. Markou T, Marshall AK, Cullingford TE, Tham El L, Sugden PH, Clerk A. Regulation of the cardiomyocyte transcriptome vs translatome by endothelin-1 and insulin: translational regulation of 5' terminal oligopyrimidine tract (TOP) mRNAs by insulin. BMC Genomics. 2010 May 29;11:343. Yndestad A, Marshall AK, Hodgkinson JD, Tham El L, Sugden PH, Clerk A. Modulation of interleukin signalling and gene expression in cardiac myocytes by endothelin-1. Int J Biochem Cell Biol. 2010 Feb;42 (2):263-72. Pikkarainen S, Kennedy RA, Marshall AK, Tham El L, Lay K, Kriz TA, Handa BS, Clerk A, Sugden PH. Regulation of expression of the rat orthologue of mouse double minute 2 (MDM2) by H2O2- induced oxidative stress in neonatal rat cardiac myocytes. J Biol Chem. 2009 Oct 2;284 (40):27195- 210. Cullingford TE, Butler MJ, Marshall AK, Tham El L., Sugden PH, Clerk A. Differential regulation of Krüppel-like factor family transcription factor expression in neonatal rat cardiac myocytes: effects of endothelin-1, oxidative stress and cytokines. Biochim Biophys Acta. 2008 Jun;1783 (6):1229-36. Page | 5 ABSTRACTS AND PRESENTATIONS 33rd FEBS Congress & 11th IUBMB Conference 2008, Athens, Greece. Poster presentation: RhoA/B/C regulation of early phase gene expression in cardiomyocytes exposed to hypertrophic stimulus endothelin-1. European Society for Cardiology ‘Heart Failure 2009’/ International Society for Heart Research European Section. 2009, Nice, France. Oral and poster presentations: Regulation of gene expression in cardiomyocytes by endothelin-1 (ET-1) via Rho signalling. British Society for Cardiovascular Research Conference on ‘Myocardial Energetics and Redox in Health and Disease’, 2009, Oxford, UK. Regulation of mRNA expression by H2O2 in cardiomyocytes. Page | 6 Table of contents TABLE OF CONTENTS ABSTRACT ...........................................................................................................................................2 ACKNOWLEDGEMENTS .................................................................................................................3 STATEMENT OF CONTRIBUTION ................................................................................................4 PUBLICATIONS, ABSTRACTS AND PRESENTATIONS ............................................................5 PUBLICATIONS ............................................................................................................................................................. 5 ABSTRACTS AND PRESENTATIONS ....................................................................................................................... 6 TABLE OF CONTENTS .....................................................................................................................7 TABLE OF FIGURES ........................................................................................................................12 ABBREVIATIONS .............................................................................................................................15 CHAPTER 1: INTRODUCTION ......................................................................................................18 1.1. Cardiac growth and development ................................................................................................................... 18 1.2. Cardiac hypertrophy in experimental systems .............................................................................................. 20 1.3. Stimuli and biochemical features of hypertrophy ......................................................................................... 21 1.4. Transmembrane receptors associated with cardiomyocyte hypertrophy.................................................... 22 1.4.1. G protein-coupled receptors in the heart .................................................................................................... 22 1.4.2. Gαq/11 signalling in the heart ......................................................................................................................... 24 1.4.3. Receptor protein tyrosine kinases ................................................................................................................ 26 1.5. Downstream signalling from transmembrane receptors............................................................................... 28 1.5.1. Protein kinase C and cardiac hypertrophy ................................................................................................. 28 1.5.2. Small G proteins ............................................................................................................................................ 32 1.5.2.1. Ras family small G proteins ......................................................................................................................