D 1526 OULU 2019 D 1526 UNIVERSITY OF OULU P.O. Box 8000 FI-90014 UNIVERSITY OF OULU FINLAND ACTA UNIVERSITATIS OULUENSIS ACTA UNIVERSITATIS OULUENSIS ACTA DMEDICA Zoltan Szabo Zoltan Szabo University Lecturer Tuomo Glumoff MODULATION OF University Lecturer Santeri Palviainen CONNECTIVE TISSUE Senior research fellow Jari Juuti GROWTH FACTOR AND ACTIVIN RECEPTOR 2B Professor Olli Vuolteenaho FUNCTION IN CARDIAC University Lecturer Veli-Matti Ulvinen HYPERTROPHY AND FIBROSIS Planning Director Pertti Tikkanen Professor Jari Juga University Lecturer Anu Soikkeli Professor Olli Vuolteenaho UNIVERSITY OF OULU GRADUATE SCHOOL; UNIVERSITY OF OULU, FACULTY OF MEDICINE; Publications Editor Kirsti Nurkkala BIOCENTER OULU ISBN 978-952-62-2339-1 (Paperback) ISBN 978-952-62-2340-7 (PDF) ISSN 0355-3221 (Print) ISSN 1796-2234 (Online) ACTA UNIVERSITATIS OULUENSIS D Medica 1526 ZOLTAN SZABO MODULATION OF CONNECTIVE TISSUE GROWTH FACTOR AND ACTIVIN RECEPTOR 2B FUNCTION IN CARDIAC HYPERTROPHY AND FIBROSIS Academic dissertation to be presented with the assent of the Doctoral Training Committee of Health and Biosciences of the University of Oulu for public defence in the Leena Palotie auditorium (101A) of the Faculty of Medicine (Aapistie 5 A), on 27 September 2019, at 13 p.m. UNIVERSITY OF OULU, OULU 2019 Copyright © 2019 Acta Univ. Oul. D 1526, 2019 Supervised by Professor Risto Kerkelä Doctor Johanna Magga Reviewed by Professor Marjukka Myllärniemi Professor Eero Mervaala Opponent Professor Antti Saraste ISBN 978-952-62-2339-1 (Paperback) ISBN 978-952-62-2340-7 (PDF) ISSN 0355-3221 (Printed) ISSN 1796-2234 (Online) Cover Design Raimo Ahonen JUVENES PRINT TAMPERE 2019 Szabo, Zoltan, Modulation of connective tissue growth factor and activin receptor 2b function in cardiac hypertrophy and fibrosis. University of Oulu Graduate School; University of Oulu, Faculty of Medicine; Biocenter Oulu Acta Univ. Oul. D 1526, 2019 University of Oulu, P.O. Box 8000, FI-90014 University of Oulu, Finland Abstract The increase of cardiac hemodynamic load that requires increased mechanical performance drives adaptation of the heart to maintain cardiac function. Modification of protein synthesis in cardiomyocytes allows the cells to adapt to the increased load. Cardiomyocyte hypertrophy and activation of cardiac fibroblasts over the long term is maladaptive and leads to heart failure (HF). Members of the transforming growth factor-β (TGF-β) superfamily contribute to the remodeling process. TGF-β1 acts as a paracrine messenger between cardiomyocytes and cardiac fibroblasts. Connective tissue growth factor (CTGF) modulates TGF-β signaling and plays a role in the development of fibrosis. In the current study, we aimed to investigate whether blocking the actions of CTGF could alleviate ischemic injury and reduce cardiac remodeling. We determined whether blocking the action of these ligands would modulate cardiac hypertrophy and fibrosis. In the first study, we found that antagonizing the function of CTGF protected from transverse aortic constriction (TAC) -induced left ventricular remodeling. In the second study in myocardial infarction (MI) model, blocking the function of CTGF resulted in improved post-MI survival and this prevented to the decrease in left ventricular contractile function as compared to the situation in control mice. Treatment with CTGF mAb attenuated the development of dilated cardiomyopathy and limited the increase in cardiomyocyte size and deposition of interstitial fibrosis in a remote area. In the third study, targeting the TGF-β superfamily members myostatin and activins, by administration of a soluble decoy receptor of activin receptor 2B (ACVR2B-Fc) did not affect the extent of MI injury or cardiac remodeling in MI -induced ischemic HF. Understanding the complex and converging pathways regulating cardiac remodeling is a major challenge, but it may allow for opportunities to develop new therapies, new medicines and provide new hope for people with these life-threatening diseases. Keywords: cardiac fibrosis, connective tissue growth factor, growth factors, hemodynamic overload, hypertrophy, myocardial infarction Szabo, Zoltan, Sidekudoskasvutekijä ja aktiviinireseptori lääkehoidon kohteena sydämen hypertrofiassa ja fibroosissa. Oulun yliopiston tutkijakoulu; Oulun yliopisto, Lääketieteellinen tiedekunta; Biocenter Oulu Acta Univ. Oul. D 1526, 2019 Oulun yliopisto, PL 8000, 90014 Oulun yliopisto Tiivistelmä Sydämen lisääntynyt kuormitus vaatii lisääntynyttä supistusvoimaa, joka johtaa sydänlihaksen adaptaatioon pumppaustehon ylläpitämiseksi. Alkuvaiheessa sydämen liikakasvu on hyödyllis- tä, mutta pidempään jatkuessaan se johtaa lopulta pumppaustoiminnan heikkenemiseen ja sydä- men vajaatoimintaan. Useiden signalointimekanismien on osoitettu säätelevän sydänlihaksen adaptoitumista patologisille tiloille. Transformoiva kasvutekijä –β (TGF-β) proteiiniperhe säätelee sydämen adaptoitumista sekä vasemman kammion seinämän myötäävyyttä venytykselle. TGF-β1 indusoi supistuskykyisten myofibroblastien muodostumista sekä kollageenin tuotantoa. Runsas kollageenin tuotanto vah- vistaa sydämen seinämää ja on tarpeen sydäninfarktivaurion korjaamisessa, mutta pitkään jatku- essaan se heikentää sydämen toimintaa ja altistaa rytmihäiriöille, sydämen vajaatoiminnalle sekä sydänperäiselle äkkikuolemalle. Sidekudoskasvutekijä (CTGF) säätelee TGF-β1:n signalointia ja se osallistuu haavan paranemiseen sekä fibroosiin. Tutkimuksessa selvitettiin, voidaanko side- kudoskasvutekijän tai TGF-β -perheen proteiinien toimintaa estämällä lievittää sydämen vajaa- toiminnan kehittymistä. Koetuloksemme osoittavat, että CTGF:n toiminnan estäminen vasta-aineen (mAb) avulla vähentää hemodynaamisen liikakuormituksen indusoimaa vasemman kammion toiminnan heik- kenemistä, kammion laajenemista sekä fibroosia. CTGF mAb myös vähentää kuolleisuutta ja estää sydämen toiminnan heikkenemistä sydäninfarktin jälkeen sekä lievittää sydäninfarktin jäl- keistä dilatoivan kardiomyopatian kehittymistä. Aktiviinien ja myostatiinin toiminnan esto liu- koisen aktiviinireseptori 2B:n (ACVR2B-Fc) avulla sen sijaan ei vaikuta sydäninfarktivaurioon tai iskeemisen vajaatoiminnan kehittymiseen. ACVR2B-Fc kuitenkin lisää luurankolihaksen kasvua, estäen sydämen vajaatoimintaan liittyvää luurankolihaskatoa. Sydämen hypertrofian ja vajaatoiminnan syntymisen kannalta keskeisten signaalinvälitysreit- tien tunnistaminen ja niiden toiminnan ymmärtäminen auttaisi kehittämään tehokkaampia lääke- hoitoja sydänsairauksiin. Asiasanat: fibroosi, hemodynaaminen liikakuormitus, hypertrofia, kasvutekijät, sidekudoskasvutekijä, sydäninfarkti Acknowledgements This thesis work was conducted in the University of Oulu, Research Unit of Biomedicine during the years 2009-2019. First of all, I’m more than thankful to my principal supervisor, Professor Risto Kerkelä, who appreciated both my strong and weak sides and always treated me according to them to provide a possible relaxed environment. I wish to express my greatest gratitude to Dr. Johanna Magga, who became my co-supervisor. She taught me the importance of accuracy, endurance and modesty not only in the laboratory but also in real life. I want to express my deep gratitude to Professor Heikki Ruskoaho, who hired me as a PhD student. I would like to express my thanks to Loikkanen Ildikó and Markku Vaarala who helped me to come to Finland with a scholarship. I would like to express my thanks to Professor Jukka Hakkola, the current chair of the Research Unit of Biomedicine, and to Professor Emeritus Olavi Pelkonen, the former chair for providing space in this great research unit. Their management skills are crucial for the continuous, high level research work. I could always turn to them in case of need and they always listened to me carefully when I had questions, comments or suggestions. I wish to send many thanks to Sirpa Rutanen, an always patient, always kind, great assistance in animal work. I also acknowledge Kirsi Salo and Marja Arbelius, who helped a lot with teaching, investigating, organizing, staining, extracting and processing my samples, and also many thanks for his support to Esa Kerttula. The convivial atmosphere in the workspace comes from great colleagues. I spent excellent time in courses with Laura Vainio, who was my mate in the ultrasound laboratory, learning the echo methods from a really great teacher and person: Philippe Trochet. Many thanks to Professor Erhe Gao who visited our department a few times giving us humongous fruitful research work. Big thanks to Tarja Alakoski tried to lead me into the mystery of statistics on a statistical course, unfortunately with very low success, that was not her fault. I was lucky to collaborate with excellent researchers and students, from whom I managed to learn a lot. We spent great and fruitful times in the echo room with Professor Peppi Karppinen, Professor Lauri Eklund, Dr. Harri Elamaa, Dr. Mika Kaakinen and Dr. Joni Mäki. With Dr. Ann-Marie Auvinen I gathered useful knowledge about small animal electrocardiography, with Dr. Kati Richter, supervised by Professor Thomas Kietzmann, I became acquainted with the 3D 7 imaging in cancer biology. And I’m very thankful for the friendship and trust (in my echo results) to Dr. Teemu Kilpiö, Dr. Lea Rahtu-Korpela, Eveliina Halmetoja, Johanna Ulvila, Manar Elmadani, Saija Taponen, Ruizhu Lin, Dr. Jarkko Piuhola, Dr. Olli Tenhunen, and my Hungarian colleague, Dr. Ábel Perjés. I am also grateful to Kenneth Lipson and Pierre Signore and entire Fibrogen, Inc, who were always