THE ROLE OF FOLLISTATIN-LIKE 3 (FSTL3) IN CARDIAC HYPERTROPHY AND REMODELLING Kalyani Panse A thesis submitted for the degree of Doctor of Philosophy and the Diploma of Imperial College Harefield Heart Science Centre National Heart & Lung Institute Imperial College London May 2011 1 ABSTRACT Follistatins are extracellular inhibitors of TGF-β family ligands like activin A, myostatin and bone morphogenetic proteins. Follistatin-like 3 (Fstl3) is a potent inhibitor of activin signalling and antagonises the cardioprotective role of activin A in the heart. The expression of Fstl3 is elevated in patients with heart failure and upregulated by hypertrophic stimuli in cardiomyocytes. However its role in cardiac remodelling is largely unknown. The aim of this thesis was to analyse the function of Fstl3 in the myocardium in response to hypertrophic stimuli using both in vivo and in vitro approaches. To explore the role of Fstl3 in cardiac hypertrophy, cardiac-specific Fstl3 knock-out mice (Fstl3 KO) were subjected to pressure overload induced by trans-aortic constriction. They showed attenuated cardiac hypertrophy and improved cardiac function compared to wild type littermate control mice. Knock-out of Fstl3 specifically from cardiomyocytes was sufficient to reduce the total expression of Fstl3 in the heart following pressure overload, implying that cardiomyocytes are the major source of Fstl3 in the heart after TAC. Fstl3 KO mice also showed reduced expression of typical hypertrophic markers including ANP, BNP, α-skeletal actin and β-MHC. Similarly, treatment of neonatal rat cardiomyocytes with Fstl3 resulted in hypertrophy as measured by increased cell size and protein synthesis. This was also accompanied by activation of p38 and JNK signalling pathways. Microarray analysis of ventricular samples from these mice indicated reduced expression of genes involved in protein binding and extracellular matrix. Verification by quantitative real-time RT-PCR revealed reduced expression of collagens and TGF- β1 in the myocardium, indicating reduced fibrosis. This was supported by histological analysis showing reduced interstitial fibrosis. As an in vitro model of pressure overload, cardiomyocytes were subjected to mechanical stretch, which elevated the expression of Fstl3. In order to explore the role of cardiomyocyte-derived Fstl3 in modulating fibroblast function, cardiac fibroblasts were treated with the conditioned medium from stretched cardiomyocytes and collagen synthesis was measured. Fstl3 was found to be necessary for conditioned medium to induce an increase in collagen synthesis in fibroblasts. Fstl3 was also shown to induce low levels of cell death in cardiac fibroblasts. In order to identify stretched 2 cardiomyocyte derived factors necessary for Fstl3 action on fibroblast collagen synthesis, a yeast two hybrid analysis was undertaken. Results indicated that Fstl3 may act, at least in part, through binding to proteins of the extracellular matrix as well as pro-fibrotic factors, including connective tissue growth factor (CTGF). While CTGF did not affect fibroblast collagen synthesis in the presence of Fstl3, it inhibited Fstl3 induced cell death, indicating a protective role. In summary, data presented in this thesis demonstrates that Fstl3 is upregulated after cardiac injury and functions by activating the pro-hypertrophic and pro-fibrotic responses in the myocardium, making it an attractive therapeutic target for intervention of cardiac pathologies. 3 ACKNOWLEDGEMENTS I would like to thank my supervisor Dr. Enrique Lara-Pezzi for all the help and support he has offered me during the course of my PhD. I feel lucky to have him as a supervisor and friend who could offer me his expert guidance as well as an honest opinion. Without Enrique, I would not have been able to express my views in the thesis and he helped me immensely at every stage of writing it. I cannot thank him enough! I am also very grateful to Prof. Nadia Rosenthal for giving me this opportunity and motivating me at every step. At times when things came to a standstill, speaking to her always encouraged me to explore different aspects and made me believe that the world was brighter than it looked! I am greatly indebted to Dr. Paul Barton for always believing in me and showing me the ‘big picture’ to put things in perspective. Thank you so much Paul for always being there for me and offering me your support when I needed it the most! I would also like to thank Imperial College for awarding me the ORS funding to undertake this research project. I am deeply indebted to Dr. Kenneth Walsh and his laboratory for helping me carry out some experiments at the Boston University Whittaker Cardiovascular Institute. Thanks Ken for giving me this opportunity and for your scientific input. Special thanks to Dr. Kazuto Nakamura for teaching me the in vivo techniques and to Dr. Shimano for taking the project forward and carrying out some in vivo procedures for me. Without their help, this project would not have been completed! Special thanks go to the EMBO short-term fellowship for funding my research at Boston University. I am lucky to have had a great set of colleagues and friends to support me throughout this challenging project. Special thanks go to Dr. Leanne Felkin for her expert knowledge and training towards RNA work and qRT-PCR as well as yeast two-hybrid analysis. I would also like to thank Dr. Maria Paola Santini for her advice at various stages and especially during the writing up period. I am deeply indebted to Dr. Padmini Sarathchandra for teaching me various immunohistochemistry procedures and Dr. Renee Germack for carrying out the radioactivity experiments. I would also like to thank Dr. Nigel Brand and Dr. Sagair Hussain for all their help and advice. Special thanks go to Anne Helness, Jonas Lexow, Borggia Seemampillai, Bhawana Poudel, Dr. Elham Zarrinpashneh, Tommaso Poggioli, Elisa Belian, Dr. Athina Meli, Dr. Francesca 4 Colazzo and Napachanok ‘Poom’ for making this experience more enjoyable! Thanks for the fun times we shared together and for being patient listeners to all my cribbing! I would also like to thank all members of the Heart Science Centre and especially the BSU staff for their support throughout the course of my PhD. I would like to express my deepest gratitude towards my family – Dr. Anastasios Papageorgiou for encouraging me and making sure the thesis came out perfectly. I couldn’t have done it without your love and support! I would also like to thank my parents Mr. Dilip Panse and Mrs. Shubhangi Panse for trusting in me to undertake this degree and for all their encouragement. Thanks also to my brother Kushal Panse for always being there for me. This thesis is dedicated to all of you! 5 DECLARATION The work presented in this thesis was conducted by the author and any contributions from other members are appropriately referenced. Specifically for the data presented in Chapter 3, experimental protocols including RNA extraction from human cardiac tissues and quantitative real-time RT-PCR were carried out by Dr. Leanne Felkin. Microarray analysis was performed by Dr. Enrique Lara- Pezzi and immunohistochemical studies on human cardiac tissues were carried out by Dr. Padmini Sarathchandra. In vivo procedures of myocardial infarction and ischemia- reperfusion on mice as well as analysis of Fstl3 and Activin βA mRNA expression were carried out by Dr. Yuichi Oshima and Kyriakos Papanicolaou. All in vivo procedures in this project were carried out at Whittaker Cardiovascular Institute, Boston University by the author, except for the following: 1. Trans-aortic constriction on 10 mice by Dr. Shimano 2. ELISA for activin A from mouse serum samples by Dr. Shimano Additional contributions from other members are referenced in Chapter 2 in the respective methods sections. 6 TABLE OF CONTENTS ACKNOWLEDGEMENTS ....................................................................................................................... 4 DECLARATION ........................................................................................................................................ 6 TABLE OF CONTENTS ........................................................................................................................... 7 LIST OF TABLES .................................................................................................................................... 11 LIST OF ABBREVIATIONS ................................................................................................................ 112 CHAPTER 1: INTRODUCTION ........................................................................................................... 13 1.1 EPIDEMIOLOGY OF CARDIOVASCULAR DISEASES .......................................................................... 13 1.2 CARDIAC HYPERTROPHY ................................................................................................................ 13 Figure 1.1: Classification of left ventricle geometry in pathological cardiac hypertrophy. ............. 15 1.3 INITIATION OF PATHOLOGICAL CARDIAC HYPERTROPHY ............................................................. 16 1.3.1 Mechanosensors in the heart .................................................................................................. 16 1.3.2 Neurohumoral stimuli ............................................................................................................. 20 Figure 1.2: G protein mediated intracellular signalling pathways in cardiac hypertrophy.