The granin protein family in cardiac disease Helge R. Røsjø, MD1,2,3 1 Division of Medicine, Akershus University Hospital, Lørenskog, Norway 2 Institute of Experimental Medical Research, Oslo University Hospital, Ullevål, Oslo, Norway 3 Center for Heart Failure Research and K.G. Jebsen Cardiac Research Centre, Institute of Clinical Medicine, University of Oslo, Oslo, Norway © Helge R. Røsjø, 2012 Series of dissertations submitted to the Faculty of Medicine, University of Oslo No. 1333 ISBN 978-82-8264-359-7 All rights reserved. No part of this publication may be reproduced or transmitted, in any form or by any means, without permission. Cover: Inger Sandved Anfinsen. Printed in Norway: AIT Oslo AS. Produced in co-operation with Unipub. The thesis is produced by Unipub merely in connection with the thesis defence. Kindly direct all inquiries regarding the thesis to the copyright holder or the unit which grants the doctorate. Contents Supported by ......................................................................................................................... 4 Acknowledgments ................................................................................................................ 5 Abbreviations ........................................................................................................................ 8 List of papers in thesis: ....................................................................................................... 10 Introduction ........................................................................................................................ 11 Cardiovascular disease ................................................................................................ 11 The syndrome of heart failure ..................................................................................... 15 Basic pathology of heart failure .................................................................................. 17 Biomarkers in cardiovascular disease ......................................................................... 19 Production of granin proteins ...................................................................................... 26 Conceptual model for granin proteins in cardiac disease ............................................ 34 Aim of thesis ....................................................................................................................... 37 Methodological considerations ........................................................................................... 38 Clinical biomarker studies ........................................................................................... 38 Experimental HF model .............................................................................................. 39 Echocardiography ........................................................................................................ 42 Cardiomyocyte cell culture experiments ..................................................................... 43 Ischemia-reperfusion model of the isolated perfused rat heart ................................... 44 Real-time reverse transcription polymerase chain reaction (RT-qPCR) ..................... 45 1-D gel electrophoresis and immunoblotting .............................................................. 47 Enzyme-Linked ImmunoSorbent Assay ..................................................................... 48 Radioimmunoassay ..................................................................................................... 49 Immunohistochemistry ................................................................................................ 49 2 Summery of results ............................................................................................................. 50 Paper #I: ...................................................................................................................... 50 Paper #II: ..................................................................................................................... 51 Paper #III: .................................................................................................................... 52 Paper #IV: ................................................................................................................... 53 Discussion ........................................................................................................................... 54 Production of granin proteins in HF ............................................................................ 54 Functional aspects of granin proteins in cardiovascular disease ................................. 56 Granin proteins as cardiovascular biomarkers ............................................................ 58 Conclusion .......................................................................................................................... 61 References .......................................................................................................................... 62 3 Supported by South-Eastern Norway Regional Health Authority Akershus University Hospital The University of Oslo The Center for Heart Failure Research The Norwegian Research Council The Norwegian National Health Association The Anders Jahre´s Fund for Promotion of Science The Raagholt Trust The Blix Trust The Sigrid Wolmar Trust The K.G. Jebsen Cardiac Research Centre 4 Acknowledgments There are things in life which are not planned, but still turn out to be exciting and truly rewarding. This is the case with me and academic medicine. I was introduced to research at the Norwegian Defense Research Establishment, but it was first after I moved back to Akershus University Hospital (Ahus) that I started to consider going into research. Luckily for me, just prior to my return, Torbjørn Omland had moved to Ahus to serve as Professor in Cardiology and was looking for a research fellow to study the role of chromogranin A in cardiac ischemia and heart failure. The project, funded by the South-Eastern Norway Regional Health Authority, was a joint effort with Professor Geir Christensen, Institute for Experimental Medical Research (IEMR), Ullevål. The opportunity to serve under two of Norway's best cardiac researchers, and to learn both basic and clinical research, was tempting and I decided to try the life of the research fellow. Today, I am truly grateful to Torbjørn and Geir for providing me with this opportunity, and although there were some early challenges, in general, I feel that the project has been a great success. All of this work would not have been possible without the help of many talented colleagues at Ahus and IEMR. I would especially like to thank Mai Britt Dahl, who has been instrumental in our effort to establish basic cardiac research at Ahus. I would also like to acknowledge the work of Cathrine Husberg, IEMR, for helping us establishing the molecular biology required for this thesis; Ivar Sjaastad, IEMR, for providing encouragement during the difficult first months and expert small animal echocardiography; Anna Frengen, Ahus, for supporting us from the start and providing lab facilities at the EpiGen Institute; and Professor Ole M. Sejersted for graciously letting me be a part of the well-established research facility of IEMR. There are several other colleagues at IEMF and Ahus that have also made significant contributions to the work herein, not least my co-authors, and I am very grateful for the interest you have all shown in our joint projects. I would also like to thank the Section for Comparative Medicine at Oslo University Hospital, Ullevål for excellent animal care and Vigdis Bakkelund and Anne Grethe Hokstad, Ahus, for blood sampling in papers #3 and #4. The great spirit among the young researchers in the Center for Heart Failure Research, which is headed by Professor Christensen, has also inspired me throughout my years as a research fellow. 5 Outside of the Oslo region, I would especially like to thank Dr. Mats Stridsberg, University of Uppsala, Sweden for the expert knowledge on granin proteins and the continuous support of my projects. The contribution by Professor Allan Flyvbjerg, Århus, Denmark to chromogranin A measurements in papers #1 and #2 is also highly appreciated. Furthermore, I had the pleasure of collaborating with Dr. Anna Maria Jansson, Professor Kenneth Caidahl, and the PRACSIS research group to study the role of chromogranin A in patients with acute coronary syndrome. I hope we can continue this collaboration also in the future. Furthermore, it was very inspiring to work with the renowned researchers of the GISSI-Heart Failure group, including Professor Roberto Latini and Serge Masson, to examine chromogranin A as a biomarker in stable heart failure. I would be honored to work with such a leading cardiovascular research group again. Although many have contributed, this work would not have been possible without the dedicated work of Professor Torbjørn Omland and Professor Geir Christensen. I am privileged to have worked with, and to have learned from, two such inspiring researchers and leaders. I have especially enjoyed the complementary strengths that you represent in cardiovascular research, but also as mentors and leaders of two strong research groups. I am humble of the confidence you have trusted in me during my period as a research fellow, and I am grateful for all the formal and informal knowledge you have shared with me since I started working with you in