Modulation of Connective Tissue Growth Factor and Activin Receptor 2B Function in Cardiac Hypertrophy and Fibrosis

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.

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 jatkuessaan 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 vajaatoiminnan 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

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 helpful and supportive in my studies. And last, but not least, let me thank Professor Jaana Rysä, who instructed me about the responsibilities working with experimental animals, who showed me how to work with rats and who always motivated me with great humour. Luckily she visits our department frequently and never misses a chance to ask me about how this thesis book is progressing. I wish to offer thanks to Hanna-Marja Voipio, the lead veterinarian of the Animal Center and also to Sakari Laaksonen, the practical vet. Along with the staff members of the Animal Center, they always helped me to find the appropriate solutions to my animal related problems. And needless to stay, I had many. Végezetül szeretném megköszönni drága szüleimnek es Zsuzsa tesómnak, hogy mindig segítettek, bíztattak, es bíztak benne, hogy a meglehetősen hosszúra nyúlt tanulmányi idő ellenére vegül is sikerül elnyernem a tudományos fokozatot. Nagy szerepe volt ebben szerelmemnek, Évának, aki azóta a feleségem, és akivel gyakran éjszakába nyúlóan videotelefon kapcsolat közben, egymást bíztatva, és egymás virtuális közelségéből erőt merítve dolgoztunk, ki-ki a saját munkáján. Once again to all those mentioned above, and also to those who I was not able to mention by name due to the lack of space in KEKS, Pathology Department and electron microscopy lab: a huge thank you to all of you!

Oulu, September 2019 Zoltán Szabó

8 Abbreviations

ACE angiotensin converting enzyme ACVR2B activin receptor type 2 B ALK activin receptor-like kinase Ang II angiotensin II ANOVA analysis of variance ANP atrial natriuretic peptide ARB angiotensin receptor blocker AS aortic valve stenosis AT1 angiotensin receptor type 1 AT2 angiotensin receptor type 2 ATx angiotensin II receptor subtype BMP bone morphogenetic protein BNP B-type natriuretic peptide Ca calcium CNP C-type natriuretic peptide CNS central nervous system CTGF connective tissue growth factor GDF growth differentiation factor ECM extracellular matrix EF ejection fraction ERK extracellularly regulated kinase ESPVR end-systolic pressure-volume relation ET-1 endothelin-1 ETx endothelin receptor subtype FBS fetal bovine serum FGF fibroblast growth factor FS fractional shortening HF heart failure HFpEF heart failure with preserved ejection fraction HFrEF heart failure with reduced ejection fraction HR heart rate HSPG heparin sulphated proteoglycan IL interleukin IRI ischemia-reperfusion injury i.p. intraperitoneally

I/R ischemia-reperfusion IVS;s or d interventricular septum thickness in systole or diastole JNK c-Jun N-terminal kinase KO knock-out LAD left anterior descending (coronary artery) LRP lipoprotein receptor-related protein LV left ventricular LVEF left ventricular ejection fraction LVID;s or d left ventricular internal diameter in systole or diastole LVH left ventricular hypertrophy LVPW;s or d left ventricular posterior wall thickness in systole or diastole mAb monoclonal antibody MAPK mitogen activated protein kinase MI myocardial infarction MMP matrix metalloproteinase NF-κB nuclear factor-κ B NPR natriuretic peptide receptor PAI-1 plasminogen activator inhibitor 1 PBS phosphate buffered saline PDGF platelet-derived growth factor Pi phosphate RAS renin-angiotensin-system RT-PCR reverse transcriptase polymerase chain reaction s.c. subcutaneously SD standard deviation α-SMA α-smooth muscle actin TAC transverse aortic constriction TAK1 TGF-β–activated kinase 1 TGF-β transforming growth factor-β TIMP tissue inhibitor of metalloproteinase TNF tumor necrosis factor TSP thrombospondin VCI vena cava inferior VEGF vascular endothelial growth factor

10 Original publications

This thesis is based on the following publications, which are referred throughout the text by their Roman numerals:

I Szabó Z, Magga J, Alakoski T, Ulvila J, Piuhola J, Vainio L, Kivirikko KI, Vuolteenaho O, Ruskoaho H, Lipson KE, Signore P, Kerkelä R. Connective tissue growth factor inhibition attenuates left ventricular remodeling and dysfunction in pressure overload-induced heart failure. Hypertension. 2014;63(6):1235-40. II Vainio LE, Szabó Z, Lin R, Ulvila J, Yrjölä R, Alakoski T, Piuhola J, Koch WJ, Ruskoaho H, Fouse SD, Seeley TW, Gao E, Signore P, Lipson KE, Magga J, Kerkelä R. 2019 Connective Tissue Growth Factor Inhibition Enhances Cardiac Repair and Limits Fibrosis After Myocardial Infarction. JACC Basic Transl Sci. 2019 Feb 25;4(1):83- 94. III Szabo Z, Vainio LE, Lin R, Swan J, Hulmi J, Serpi R, Pasternack A, Ritvos O, Kerkelä R, Magga J. Systemic blockade of ACVR2B ligands reverses muscle wasting in chronic heart failure model without compromising cardiac function. Manuscript.

12 Contents

Abstract Tiivistelmä Acknowledgements 7 Abbreviations 9 Original publications 11 Contents 13 1 Introduction 15 2 Review of literature 17 2.1 Remodeling in hypertensive heart diseases ...... 17 2.1.1 Principles of cardiac anatomy ...... 17 2.1.2 Cardiac hypertrophy ...... 18 2.1.3 Cardiac fibrosis ...... 21 2.1.4 Identifying and origin of cardiac fibroblasts ...... 21 2.1.5 Collagens ...... 23 2.1.6 Myocardial inflammation ...... 28 2.2 Modulators of myocardial fibrosis ...... 30 2.2.1 Angiotensin II ...... 30 2.2.2 Endothelin ...... 31 2.2.3 Aldosterone ...... 32 2.2.4 Natriuretic peptides ...... 33 2.2.5 Transforming growth factor beta superfamily ...... 34 2.2.6 Activin receptor signaling ...... 37 2.2.7 Interleukin 6 ...... 41 2.2.8 Connective tissue growth factor ...... 42 2.3 Cardioprotective and antifibrotic pharmacotherapies ...... 44 2.3.1 Pharmacological strategies to inhibit cardiac fibrosis ...... 46 2.4 Cardiomyocyte fibroblast interaction ...... 47 3 Aims of the present study 51 4 Materials and methods 53 4.1 Transverse aortic constriction surgery...... 53 4.1.1 Anatomical background ...... 53 4.1.2 Surgical procedure ...... 53 4.1.3 Postoperative care ...... 54 4.1.4 Results of the TAC surgery ...... 54

4.2 Acute myocardial infarction (MI) and ischemia-reperfusion (IR) models ...... 55 4.2.1 MI surgery ...... 55 4.2.2 IR surgery ...... 55 4.2.3 Results of the MI and IR surgeries ...... 56 4.3 Echocardiography ...... 56 4.3.1 Introduction ...... 56 4.3.2 Preparation ...... 57 4.3.3 B-mode image ...... 57 4.3.4 M-mode image ...... 58 4.3.5 Pulse - wave Doppler image ...... 58 4.3.6 Strain analysis ...... 59 4.4 Pressure – Volume loop analysis ...... 60 4.4.1 Surgery methods for PV catheter insertion, approaches ...... 60 4.5 Application of neutralizing monoclonal antibody for CTGF ...... 61 4.6 Application of a trap for the ACVR2B ligands ...... 61 4.7 Histology ...... 62 4.8 TUNEL assay ...... 62 4.9 MMP2 activity assay ...... 63 4.10 Quantitative PCR analysis ...... 63 4.11 RNA sequencing analysis ...... 63 4.12 Western blot analysis ...... 64 4.13 Statistical Analysis ...... 64 5 Summary of results and discussion 65 5.1 CTGF in pressure-overload (TAC) mouse model ...... 65 5.2 CTGF in cardiac ischemia and MI models ...... 68 5.3 Activins and GDFs in cardiac ischemia ...... 74 5.3.1 Ligands of the activin receptor type 2B are regulated differently after MI ...... 74 5.3.2 Systemic blockade of ACVR2B ligands does not alleviate post-MI remodeling ...... 75 5.3.3 Systemic blockade of ACVR2B ligands reduces skeletal muscle atrophy in MI-induced cardiac cachexia ...... 76 6 Conclusions 79 References 81 Original publications 117

14 1 Introduction

Cardiovascular diseases are the leading cause of death in the industrialized world, with heart failure (HF) being the fastest growing subclass during the last 10 years. Severe (New York Heart Association class III-IV) HF is associated with 40% mortality within the first year, and the 5-year survival rate of all patients suffering from HF is approximately 50% (Levy et al., 2002). One follow-up study revealed that the overall 10-year mortality is dire, 99% (Chun et al., 2012). By definition, HF is present when structural or physiological abnormalities lead to failure in carrying respiration gases according to the demands of the peripheral metabolizing tissues (McMurray et al., 2012). Whereas hypoxic damage within the life-supporting organs, such as kidneys, lungs, central nervous system (CNS) determines the severity of the disease, the majority of the HF patients die from cardiovascular causes such as sudden cardiac death or progressive HF (Mosterd & Hoes, 2007). Clinically, HF is diagnosed by its symptoms (e.g. dyspnoe, fatigue, oedema in the lower extremities, continuous coughing, gasping, fluid retention, ascites) and signs of anatomical or functional abnormalities examined by evaluation methods at resting (Dickstein et al., 2008). Cardiac function is usually measured by transthoracic echocardiography. The key parameter in the analysis of systolic function is the left ventricular (LV) ejection fraction (EF). The EF% is calculated from the volume of blood ejected from the left ventricle relative to the end-diastolic (maximal) left ventricular volume. The EF is highly dependent on preload, afterload conditions, heart rate and valvular function as well as hormonal / neurohumoral influence, but usually reduced after temporal or permanent ischemic condition. On the other hand, HF can occur when the EF is still normal, this condition being termed HF with preserved ejection fraction (HFpEF). This sort of diastolic dysfunction occurs when abnormal LV stiffness prevents sufficient relaxation. Although the calculated EF is within the normal range in this case, the decreased diastolic LV volume and reduced stroke volume result in a decrease in the absolute pumped blood volume that is not adequate to ensure a proper oxygen supply. The common cause for the increase in ventricular stiffness is the increased volume of fibrotic tissue in the interstitium, especially in the perivascular regions of the left ventricle. The cardiomyocytes in HF patients also show signs of degeneration due to the hypoxia associated with myocardial infarction (MI) and ischemia-reperfusion (IR) injuries, or due to the increased mechanical stress (i.e. in valve diseases or in severe

hypertension). The increased intracellular Ca2+ concentration and the increased production in mitochondria of reactive oxygen species due to the impaired function of the electron transport chain following the hypoxic condition, initiate the apoptotic signals in cardiomyocytes. HF is also associated with increased inflammation in the myocardium, at least partly due to the release of antigens from the dead cells that act as a chemoattractant for the cells of the immune system. An increasing number of strategies to prevent or cure heart failure have been investigated in laboratories, but translating these putative therapies into the clinical practice has been mainly unsuccessful (Perricone & Vander Heide, 2014). One reason is that it is very challenging to extrapolate the results of a highly controlled animal model to those obtained in heterogeneous human patient populations (Vander Heide & Steenbergen, 2013). Furthermore, even the key molecular mechanisms behind the cardiac diseases are poorly understood. By understanding these mechanisms, novel targets for heart failure therapy may be discovered.

16 2 Review of literature

2.1 Remodeling in hypertensive heart diseases

2.1.1 Principles of cardiac anatomy

The cardiac wall is composed of three major layers i.e. from outside to inside: epicardium, myocardium, and endocardium. The heart is also covered with extensive serous membranes. The function of these membranes is mechanical protection and decreasing the friction of the “inner” (visceral), and “outer” (parietal) layer by secreting lubricant into the cavity. The parietal layer, named the pericardium, is firmly attached to the sternum by the sterno-pericardial ligaments and extends into the diaphragm. The visceral layer, named the epicardium, lies on the outer myocardial surface. Between the two layers, there is a space filled with fat tissue. The pericardiac adipose tissue has important functions, protecting coronary arteries from tension or torsion, providing an energy source for the myocardium, and having a role in the cardiac nervous system (Yamada & Sata, 2015). The myocardium is the functional layer that generates force to maintain the blood circulation by producing a pressure gradient. In macroscopic terms, it seems to be composed by fibers. The unit of the myocardium is the cardiomyocyte cell; these cells are connected to each other by gap junctions and desmosomes. The gap junctions provide electrical links between the cells, which makes the whole myocardium like one syntitium. The correct structural alignment of cardiac muscle fibers is necessary for proper contraction of the cardiac muscle (Opie, 2003). It has been shown that the direction of cardiac fibers varies transmurally from approximately −70° in the epicardium to approximately +80° in the endocardium (Wong & Kuhl, 2014). The cardiomyocytes responsible for the impulse generation and conduction system are morphologically distinct from the contractile ventricular and atrial cells. In addition to cardiomyocytes, endothelial cells can also transmit the electrical signal in the myocardium (Travers, Kamal, Robbins, Yutzey, & Blaxall, 2016). All surfaces in the body that are in direct contact with the blood must be covered by endothelium which is the innermost layer of the endocardium. The endothelial cells sit on the basement membrane that is formed by a loose connective tissue (lamina reticularis) and secrete a microenvironment around themselves

(lamina basalis). There is also a third layer embedded into this connective tissue; this is an aponeurosis-like fibrous structure conferring strength to the endocardium. This strong layer represents the skeleton for the valves. The circulation of blood is intended to meet the body’s high demand of oxygen; the blood is transported via a well-developed vascular network, with a very low numbers of anastomosis from the level of arterioles.

2.1.2 Cardiac hypertrophy

Remodeling of the myocardium involves a multitude of structural changes that facilitate the adaptation of the heart to meet the increased demand. The direct causes are mechanical stress and/or neurohumoral activation (D L Mann, Barger, & Burkhoff, 2012). These changes have beneficial properties in the short term for maintaining the anatomical integrity and physiological LV function. On the other hand, the reorganization (remodeling) of histological elements (cells and surrounding extracellular matrix) leads to pathological processes, including cardiomyocyte death by apoptosis and necrosis, autophagy, extracellular matrix (ECM) degradation by matrix metalloproteinases (MMPs), and the accumulation of fibrotic tissue (D L Mann et al., 2012). The restructuring of the cardiac wall may stem from benign causes, such as increased physical activity or pregnancy, or may be due to pathological conditions such as systemic hypertension, MI, valvular disease, genetic causes, irregular heart rhythms, anemia, thyroid conditions, sepsis or toxins. The increased physical activity and pregnancy result in physiological LV hypertrophy that is reversible and may enhance cardiac function, while pathological cardiac hypertrophy is most often progressive and may result in heart failure. Pathological LV hypertrophy is characterized by specific molecular changes that occur in the cell. The main characteristics of LV hypertrophy at the cellular level is the increase in the cross-sectional area of individual cardiomyocytes, which is associated with increased protein synthesis, and an increase in the expression of certain fetal cardiac genes such as atrial natriuretic peptide (ANP), brain derived natriuretic peptide (BNP), c-myc and c-fos (Rajabi, Kassiotis, Razeghi, & Taegtmeyer, 2007). Cardiomyocytes accumulate more contractile protein elements: actin, myosin and supporting organelles such as mitochondria, sarcoplasmic reticulum and ribosomes. The signaling mechanisms regulating the development of cardiac hypertrophy and heart failure have been investigated for the past 25 years in great detail. Data from experimental heart failure models have revealed a number

18 of signaling molecules which participate in cardiac hypertrophy and the adverse LV remodeling. The most abundant data exists for the role of calcineurin, glycogen synthase kinase 3 beta (GSK-3), histone deacetylases, extracellular signal- regulated kinase (ERK) and β-adrenoceptor signaling (Frey & Olson, 2003). However, despite promising findings in experimental rodent models, none of the so-far identified targets have led to the development of novel therapies. In addition to LV hypertrophy, the pathological, irreversible remodeling consists of a massive deposition of ECM elements such as different types of collagen fibers (mainly type I and type III), produced by cardiac fibroblasts, but with a relatively lower number of supplying vessels and a reduced density of capillaries (Cokkinos & Pantos, 2011). The incompressible, non-elastic, solid ECM elements decrease the compliance and increase the stiffness of the cardiac wall. The modified elasticity of the heart has an influence on its pumping function. In the case of HFpEF, the ejection fraction (EF), or stroke volume, drops until the oxygen demand of the body can no longer be met by the actual capacity of the heart, and heart failure occurs. Anatomically, cardiac hypertrophy takes two distinct forms concerning the relation of the LV wall thickness and LV diameter. In the case of concentric hypertrophy, the ventricular diameter / LV wall thickness ratio shifts downwards i.e. thickening of the myocardium without any significant change or even with a decrease in the ventricular diameter. Concentric hypertrophy is usually caused by increased resistance in pressure-overload conditions (Lips, deWindt, van Kraaij, & Doevendans, 2003). The other form is called eccentric hypertrophy, when the ventricular diameter / cardiac wall thickness ratio shifts upwards where there is more ventricular dilation and less myocardial thickening compared to concentric hypertrophy (Hill & Olson, 2008) (Figure 1).

Fig. 1. Representative images of the different types of cardiac hypertrophy visualized by transthoracic echocardiographic sonograms from short axis B-mode. Images shown are from a mouse with (A) concentric cardiac hypertrophy, (B) normal heart and (C) eccentric cardiac hypertrophy. All images were captured by using VEVO 2100 ultrasound device in diastole. LVID;d: left ventricular internal diameter in diastole,

LVPW;d: left ventricular posterior wall thickness in diastole. LVIDA< LVIDB< LVIDC, and

LVPWA >LVPWB >LVPWC. All images are shown in the scale shown in panel C. Eccentric LV hypertrophy occurs after MI and volume-overload conditions such as aortic valve regurgitation. While concentric hypertrophy attenuates cardiac wall stress, eccentric hypertrophy can normalize the stroke volume (Lips et al., 2003). Interestingly, the structural changes observed in LV hypertrophy are not necessary for survival, since the attenuation of these phenomena does not result in cardiac decompensation (Drazner, 2011; Hill & Olson, 2008). It is actually rather the opposite, the presence of LV hypertrophy is an important risk factor and a strong predictor of cardiovascular morbidity and mortality (Schillaci et al., 2000; R S Vasan & Levy, 1996; Verdecchia et al., 2001). The healing process after MI is intended to repair the damaged area of the cardiac wall. The necrosis of myocytes in the infarcted region first initiates an inflammatory phase of cardiac repair (up to 72 hours); this is characterized by the increased infiltration of leukocytes and the clearance of the infarct site of the debris from dead cells and extracellular matrix (Frangogiannis, 2014). The proliferative phase of infarct repair occurs at 3-7 days after injury, and is associated with an inhibition of pro-inflammatory signals and enhanced formation of fibrous tissue and angiogenesis (Dobaczewski, Gonzalez- Quesada, & Frangogiannis, 2010; Prabhu & Frangogiannis, 2016). The fibroblasts in the infarct area differentiate into contractile myofibroblasts that have a capacity to secrete large amounts of ECM proteins. The infarct repair is completed by a maturation phase that can last up to two months and is characterized by cross-

20 linking of ECM proteins and the formation of a mature scar. The subsequent result from the compensational responses of the noninfarcted myocardium that resets the counterbalanced force / wall stress ratios is eccentric hypertrophy (Gonzalez, Ravassa, Beaumont, Lopez, & Diez, 2011; Lorell & Carabello, 2000).

2.1.3 Cardiac fibrosis

The accumulation of ECM and increased angiogenesis, associated with the development of LVH and HF, implies that the non-muscle cells residing in the myocardium likely play an important role in the pathogenesis of both cardiac hypertrophy and heart failure. In fact, cells other than cardiomyocytes account for approximately 70% of the total cell number in the heart. Methodological challenges and the presence of large amounts of ECM in the diseased myocardium led to the proposal that fibroblasts would be the most abundant cell type in the heart. However, Pinto at al. showed that the endothelial cells are in fact the most abundant cell type in the healthy heart and there is only a relatively small number of cardiac fibroblasts (Pinto et al., 2016). This fibroblast population has a major role producing ECM proteins, which form a structural scaffold for cardiomyocytes as well as distributing mechanical forces in the myocardium, and participating in electrical conduction (Travers et al., 2016). In addition, cardiac fibroblasts produce a variety of growth factors that are involved in the interplay between cardiac fibroblasts and cardiomyocytes. However, activation of fibroblasts in pathological cardiac conditions results in excessive deposition of ECM proteins, which leads to structural changes in the myocardium and may result in LV dysfunction. The accumulation of collagen and other ECM proteins also disrupts the normal electrical cell-to-cell coupling and impulse conduction, predisposing to arrhythmias and sudden cardiac death (T. P. Nguyen, Qu, & Weiss, 2014).

2.1.4 Identifying and origin of cardiac fibroblasts

The classical method to characterize a cardiac fibroblast is based on its morphology, but these cells are difficult to identify at the molecular level. Thus, there is a clear demand for selective molecular fibroblast markers. For instance, vimentin antibody labels fibroblasts with high sensitivity but also labels endothelial cells (Ausma et al., 1995; Goldsmith et al., 2004; Kjorell, Thornell, Lehto, Virtanen, & Whalen, 1987). Fibroblast Specific Protein 1 (FSP1) is a promising marker, although it is also expressed by metastatic cancer cells (Zeisberg et al., 2007). On the other hand,

FSP1 is expressed only by a subset of cardiac fibroblasts, and FSP1-positive cells are rarely encountered in the normal heart (Zeisberg et al., 2007). In the healthy heart, valvular, but not interstitial, fibroblasts express -smooth muscle actin (SMA), in the myocardium (Snider et al., 2009). During the process of cardiac fibrosis, most cardiac fibroblasts become SMA-positive. Those cells are termed as myofibroblasts, and they are regarded to be the activated form of the fibroblast (Weber, 1997). SMA is also expressed in vascular smooth muscle cells and pericytes, often residing adjacent to the fibroblasts hampering the identification of the cell type. Discoidin domain receptor 2 (DDR2) has also been used to identify cardiac fibroblasts (Camelliti, Borg, & Kohl, 2005; Goldsmith et al., 2004), but DDR2 is also expressed by lymphocytes. Periostin is a matricellular protein expressed specifically by fibroblasts that developmentally originate from epicardial-derived progenitor cells (Weber, 1997). Unfortunately it is a secreted ECM protein and this complicates the exploitation of periostin as a fibroblast marker. Recently it has become evident that there are multiple developmental origins of cardiac fibroblasts, and there are no specific markers which can identify all of these cells. The so-called fate-mapping is an innovative, but complex method. The principle behind this technique is that the cell acquires a specific “imprinted tag” which remains unchanged. One can reveal this tag by molecular biological techniques, cellular and tissue engineering, and recombinant, transfection or transduction methods (Zeisberg & Kalluri, 2010). Fate-mapping studies have revealed fibroblasts originating from endothelial cells, epicardial cells, bone marrow, stroma cells and pericytes in the heart (Tallquist & Molkentin, 2017). Ali et al. concluded that the cellular expansion driven by pathological remodeling in the pressure-overload condition induced by thoracic aortic constriction (TAC) was not restricted to any single developmental subset of fibroblasts (Ali et al., 2014). The simultaneous activation of heterogeneous fibroblast populations points to the presence of some common signaling mechanism. However, further studies will be needed to determine how these different cell lines contribute to the remodeling process in the heart in other pathological conditions, such as after MI.

22 2.1.5 Collagens

Synthesis, maturation, fibril formation

Collagens are the most abundant ECM element in mammalian tissues. To date, 28 different collagens have been identified (Zvackova, Matalova, & Lesot, 2017). One class is the fibril-forming collagens: type I, II, III, V, XI, XXIV, XXVI, (Kadler, 1995), the other class is associated with the fibrillar collagens (Fibril Associated Collagens with Interrupted Triple helices (FACIT)): type IX, XII, XIV, XIX, XXI (van der Rest, Dublet, & Champliaud, 1990). These two classes of collagens confer great tensile resistance for the tissues. On the other hand, there are several cell surface collagen binding receptors (integrins, discoidin domain receptors (DDRs), glycoprotein VI (GPVI), leukocyte-associated Ig-like receptors (LAIR-1), GPR56, and osteoclast-associated receptor (OSCAR) that transmit the signals from the ECM to the adjacent cells (Coelho & McCulloch, 2016). In this sense, the collagen matrix forms an extracellular three-dimensional web that is able to gather and transmit mechanical information from the surrounding tissue. The vast majority of the ECM in the myocardium consists of fibrillar collagens type I and III. The type I collagen forms elongated, linear structural fibers, whereas the type III collagen forms reticular fibers (Fleischmajer, Gay, Perlish, & Cesarini, 1980; Montes et al., 1980). The collagen is mainly synthetized by activated fibroblasts. The pre-pro-peptide is synthesized on the ribosomes on the rough endoplasmic reticulum (RER) and contains a signal peptide at its N-terminal. The first step in the maturation process is the cleavage of signal peptide in the cavity of RER. The second step is hydroxylation of proline and lysine and glycosylation exclusively on lysine side-chains. Each propeptide chain has the repeating structure Gly-Pro-X or Gly-X-hPro, where Gly is the glycine amino acid, hPro is the hydroxyproline and X can be various other amino acids, for example, lysine or hydroxylysine. The post-translational modification of Pro requires the prolyl- hydroxylase enzyme with an ascorbate co-factor (Stassen, Cardinale, & Udenfriend, 1973). This means that in the absence of ascorbic acid, new collagen synthesis is not possible. Interestingly, only humans, primates and guinea pigs are unable to synthesize vitamin C, because they do not possess a functional gulonolactone oxidase gene (Nishikimi, Fukuyama, Minoshima, Shimizu, & Yagi, 1994; Nishikimi, Kawai, & Yagi, 1992; Nishikimi, Koshizaka, Ozawa, & Yagi, 1988; Nishikimi & Udenfriend, 1976; Ohta & Nishikimi, 1999). If there is lysine amino acid in the chain, usually its side chain can also be hydroxylated by lysyl-

hydroxylase (Kellokumpu, Sormunen, Heikkinen, & Myllyla, 1994). The hydroxylated and glycosylated collagen pre-propeptide, named as procollagen, is transferred into the Golgi apparatus where its oligosaccharide chains are attached and packed into secretory vesicles. The procollagen molecule is composed of three major parts: globular N-, and C-telopeptides, and the 300 nm long fibrillar part in the middle that forms a right-handed triple helix structure which is enforced by hydrogen bonds (Figure 2). Further maturation of procollagen results in the removal of the telopeptides by C-, and N-telopeptidases releasing the middle part which is named as the tropocollagen molecule (Leung, Fessler, Greenberg, & Fessler, 1979). The collagen fiber formation goes in a side-to-side direction by adding new molecule to the bundle and also with foot – head connections elongating the new fiber (Birk, Nurminskaya, & Zycband, 1995; Graham, Holmes, Watson, & Kadler, 2000), (Figure 2).

Fig. 2. Collagen maturation. The extracellularly secreted procollagen molecule is cleaved by telopeptidases producing tropocollagen. The 300 nm long tropocollagen is organized in a head-to-foot direction with a 40 nm gap between the sections. The collagen fibril grows by adding new layers.

The collagen bundle is stabilized by cross-links. These links are provided by the lysyl oxidase (LOX) enzyme (Eyre, Paz, & Gallop, 1984), (Figure 3). LOX

24 expression is induced in the heart post-MI and knocking-down the enzyme is able to attenuate adverse LV remodeling (Lu et al., 2012). Chemical inhibition of the LOX or antagonizing the function of the enzyme by polyclonal antibody has also shown beneficial effects following MI in mice (Gonzalez-Santamaria et al., 2016). In heart failure patients, the grade of cross-links correlates with the severity of LV dilation and the degree of stiffness as well as with the cardiac function (Lopez, Querejeta, Gonzalez, Larman, & Diez, 2012).

Fig. 3. Action of the lysyl oxidase enzyme. Step 1 involves oxidizing the lysyl residues into carbonyl groups (allysine). Step 2 is a passive procedure when two carbonyl groups form a covalent bond via an aldol condensation.

There are several members of modulator / regulator proteins that are essential for the formation of collagen bundles. Small leucine-rich repeat proteoglycans (SLRPs) like lumican, decorin or fibromodulin have an effect on fibrillarization in vitro (Hedbom & Heinegard, 1989; Oldberg, Antonsson, Lindblom, & Heinegard, 1989; Rada, Cornuet, & Hassell, 1993; Vogel, Paulsson, & Heinegard, 1984).

Collagen degradation

The matrix metalloproteinases (MMPs) are calcium-dependent, zinc ion-containing endopeptidases that are responsible for the degradation of the extracellular matrix. The MMP family has 28 members (Peng, Chen, Drachenberg, Raufman, & Xie, 2019), numbered from 1 to 28. MMPs share common structural similarities, with each of them being composed of an inhibitory pro-peptide domain, a zinc- containing catalytic domain, a linker peptide, and a hemopexin domain (Q. Chen et al., 2013). They are classiﬁed as collagenases, membrane type MMPs, gelatinases, stromelysins, matrilysins, and another, non-classified group (Jabłońska-Trypuć, Matejczyk, & Rosochacki, 2016). They have shown to play a role in ECM remodeling by collagen breakdown, in combatting bacterial or viral invasion, in promoting inflammation by augmenting the migration of leukocytes,

and also in metastasis formation by preparing room for malign cells to gain access into the tissues (Marchant et al., 2014). The collagenase MMP subfamily is composed of MMPs 1, 8, 13, 14, 16, 18 (Coelho & McCulloch 2016). They can cleave collagen in a specific glycine-isoleucine/leucine site, creating a more relaxed tertiary structure. This allows other MMPs (MMP2, 8, gelatinases) for further processes. Originally one criterion for an enzyme to be considered as an MMP was that it had to exert proteolytic activity on at least one ECM protein (Q. Chen et al., 2013; Woessner Jr, 1991). Later, non-ECM protein substrates have been identified such as interleukin-1β, that is cleaved into its active forms by several MMPs, including MMP2 and MMP9 (Ito et al., 1996; Schonbeck, Mach, & Libby, 1998). At present, multiple cytokines, chemokines and growth factors are known to be substrates for MMPs. Tissue inhibitors of metalloproteinases (TIMPs) act as endogenous inhibitors of metalloproteinases and play important roles in the regulation of ECM turnover, tissue remodeling and cellular behavior. One direction of cancer research has focused on the inhibition of MMPs as novel therapeutic approaches (Deryugina & Quigley, 2006). For instance, batimastat and its derivative marimastat have demonstrated beneficial effects in cancer models in vivo (Lohi, Wilson, Roby, & Parks, 2001; Winer, Adams, & Mignatti, 2018). However, clinical trials with MMP inhibitors have failed either due to the lack of efficacy or to the appearance of severe side effects (Winer et al., 2018).

MMP2 (Gelatinase A) and MMP9 (Gelatinase B)

MMP2 is a 72kDa, Zn-containing endopeptidase that predominantly cleaves nonfibrillar proteins like collagen IV, V, VII and X, gelatin and fibronectin (Spinale, 2007; Turner & Porter, 2012). In addition, troponin I (TnI) has been identified as a possible intracellular target of MMP2. MMP2 cleaves TnI in vitro and the TnI loss can be blocked with MMP inhibitors after I/R injury (W Wang et al., 2002). In humans, the MMP2 gene is located on chromosome 16. Some classical regulatory elements, such as TATA box and AP-1 sites, are missing from its promoter and hence MMP2’s transcription is constitutive in many cell types. Its activity is regulated at the post-transcriptional level by growth factors, cytokines, oxidative and mechanical stress (Porter & Turner, 2009). Growth factors can have diverse effects, for instance TGF- induces MMP2 in fibroblasts but inhibits MMP1 (Overall, Wrana, & Sodek, 1991). With respect to the activation of pro-MMP2 zymogen, a trimolecular complex composed of the pro-MMP2, MT1-MMP

26 (MMP14) and TIMP2 is needed (Kandasamy, Chow, Ali, & Schulz, 2010). Cytokines, growth factors, ROS and collagen enhance the MMP2 activity, whereas angiotensin II (Ang II) and hypoxia decrease MMP2 activity in cultured cardiac fibroblasts (Porter & Turner, 2009). Plasma levels of MMP2 are elevated in patients with acute MI (Tang et al., 2009). In experimental models, there are increases in myocardial MMP2 activity after MI or in response to Ang II stimulation as well as in pressure overload conditions (Coker et al., 2017; Herzog, Gu, Kohmoto, Burkhoff, & Hochman, 1998; Spinale, 2007). In MI, it appears that the major source of MMP2 is the cardiac fibroblasts and not the infiltrating inflammatory cells as originally postulated (Mukherjee et al., 2006). Targeted deletion of MMP2 improved post-MI survival by hindering macrophage infiltration and reducing the rate of LV rupture (Matsumura et al., 2005). Enhanced MMP2 activity has recently been linked to increased rupture following MI (Gong, Ma, Li, Shi, & Nie, 2018). In addition, pharmacological inhibition of MMP2 e.g. by doxycycline, has been shown to improve the recovery from acute ischemia in both experimental models and human patients (Cerisano et al., 2014). The MMP9 has the same substrate profile as the MMP2 (nonfibrillar collagens, gelatine, fibronectin). MMP9 expression is also increased following MI (Heymans et al., 1999; Spinale, 2007). In contrast to MMP2, MMP9 is predominantly secreted by inflammatory cells, but its release is also upregulated in cardiac fibroblasts. Studies in MMP9 KO mice have shown that MMP9 deficiency reduces myocardial inflammation, collagen deposition, rupture, LV dilatation and improves angiogenesis and survival after MI (Ducharme et al., 2000; Heymans et al., 1999; Lindsey et al., 2005). However, prompt pharmacological inhibition of MMP9 after MI was able to delay the resolution from inflammation and this exacerbated cardiac dysfunction (Iyer et al., 2016). It remains to be elucidated whether these contrasting results are due to activation of compensatory mechanisms in MMP9 KO mice, attributable to non-specific effects of the pharmaceutical agent, or for some other reason.

PAI-1

PAI-1 is a serine protease inhibitor (serpin). It functions as the principal inhibitor of tissue plasminogen activator (tPA) and urokinase (uPA), the activators of plasminogen and hence it is involved in fibrinolysis. In addition, PAI-1 controls the MMP proteolytic activities and thus maintains tissue homeostasis. The expression

of the PAI-1 gene is tightly regulated by a wide variety of cytokines and growth factors including TGF-β, interleukin-1β, epidermal growth factor (EGF), insulin, lipopolysaccharide, and lipoproteins. Numerous transcription factors are involved in controlling the expression of PAI-1 in a cell type – dependent manner and in the context of both physiological and pathological conditions (R. M. Liu & Gaston Pravia, 2010; Nagamine, 2008; Samarakoon & Higgins, 2008). An increase in circulating PAI-1 levels has been detected in young survivors of acute myocardial infarction, and elevated plasma PAI-1 levels have been determined as a risk factor for recurrent myocardial infarction (Hamsten et al., 1987; Hamsten, Wiman, de Faire, & Blombäck, 2010). PAI-1 has been shown to serve a profibrotic role in the lung, liver, kidney and skin that has been attributed to the inhibition of the plasmin- MMP system (Flevaris & Vaughan, 2017; A K Ghosh & Vaughan, 2012). However, seemingly contradictory findings have been observed in the context of cardiac fibrosis as aging PAI-1 deficient mice have been shown to exhibit enhanced cardiac fibrosis (Asish K. Ghosh et al., 2010; Z. Xu, Castellino, & Ploplis, 2010). There is a rare frameshift mutation in the SERPINE-1 gene present in some human population (W P Fay, Parker, Condrey, & Shapiro, 1997; William P. Fay, Shapiro, Shih, Schleef, & Ginsburg, 2010). A recent study revealed that these individuals with the mutation- induced reduction of circulating PAI-1 levels exhibited myocardial fibrosis and had elevated levels of circulating TGF- (Flevaris et al., 2017). The investigators also found that a deficiency of PAI-1 augmented Ang II – induced cardiac hypertrophy and cardiac fibrosis in mice. Transcriptomic analysis further showed that PAI-1 deficiency enhanced expression of TGF-β signaling elements and transcriptional targets (Flevaris et al., 2017). These data thus indicate an upstream regulatory role for PAI-1 in the TGF-β-dependent cardiac fibrosis.

2.1.6 Myocardial inflammation

The inflammatory process in the myocardium occurs following many diseases e.g. after injury of myocardial wall. It can be acute, subacute or chronic causing structural and functional deficits (Marchant et al., 2012). In the case of an ischemia – reperfusion (IR) injury (IRI), the reperfusion is essential for the tissue survival, but paradoxically, the intensive oxygen burst during reperfusion may cause further damage to cardiomyocytes. While the mechanism for this is not clear, the excess production of reactive oxygen species (ROS) during reperfusion is regarded as a major contributor to the reperfusion injury. Reactive oxygen species are produced by mitochondria (Di Lisa & Bernardi, 2006),

28 cytochrome p450 (Granville et al., 2004; M Sato, Yokoyama, Fujita, Okumura, & Ishikawa, 2011), endothelial nitric oxide synthase (eNOS) (Vasquez-Vivar et al., 1998; Xia, Tsai, Berka, & Zweier, 1998), xanthine oxidase (Brown et al., 1988; B. E. Lee, Toledo, Anaya-Prado, Roach, & Toledo-Pereyra, 2009), and neutrophil granulocytes (Jordan, Zhao, & Vinten-Johansen, 1999; F. M. Williams, 1996). Various approaches to target the formation of ROS in the treatment of reperfusion injury have displayed efficacy in preclinical models, but the clinical benefit of those therapies has been very limited (D. N. Granger & Kvietys, 2015). The immune response is initiated by different antigens released from the injured, dead cells and cell debris following MI (Mustonen, Ruskoaho, & Rysä, 2012). This is accompanied by the activation of the complement system and increased expression of cytokines such as IL-1β, IL-6, TNF-α and adhesion molecules. Cytokines attract leukocytes into the infarcted area. In parallel to these reactions, suppressor molecules (TGF-β, IL-10) are also released (Sutton & Sharpe, 2000). In addition, the infiltrating monocytes also begin to differentiate into macrophages. Macrophages play an important role in clearing the tissue debris, producing proangiogenic factors, and secreting a variety of chemokines, growth factors, inflammatory cytokines, and MMPs that regulate the extent of the inflammation and degrade the ECM (Ben-Mordechai et al., 2015; Ma, Mouton, & Lindsey, 2018). An impairment of macrophage phagocytic capacity has actually been shown to prolong inflammation and impede post-MI infarct repair (Wan et al., 2013). Post-MI inflammation is also associated with increased cytokine expression as well as that of ANP and BNP in the remote myocardium (Nian, 2004). Hemodynamic stress also leads to inflammation via the release of cytokines. Elevated levels of TNF-α, and other pro-inflammatory molecules (IL-2, ET-1) have been detected in HF patients and in hearts in murine models (Celis, Torre-Martinez, & Torre-Amione, 2008; Moilanen et al., 2012). In the case of chronic renal insufficiency, the inflammatory arsenal is activated and there is evidence that it is a predictor of increased cardiovascular mortality (Gross & Ritz, 2008). It seems most likely that the chronic kidney dysfunction maintains oxidative stress and inflammation by accumulating uremic toxins. The activation of local RAS has also been demonstrated, and increased levels of C - reactive protein (CRP), TNF-α and IL-6 have been detected (Schiffrin, Lipman, & Mann, 2007).

2.2 Modulators of myocardial fibrosis

2.2.1 Angiotensin II

Angiotensin II (Ang II) is an octapeptide (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe), and functions as the primary effector molecule of the renin – angiotensin – aldosterone system (RAAS) that regulates the volume of the extracellular fluid and osmotic homeostasis. The precursor molecule of Ang II, angiotensinogen (2-globulin fraction in the blood) is produced in the liver and released constitutively. Human angiotensinogen consists of 453 amino acids, and other species have angiotensinogens of varying sizes. The first 12 amino acids (Asp-Arg-Val-Tyr-Ile- His-Pro-Phe-His-Leu-Val-Ile-...) are essential for the biological activity of Ang II. Angiotensin I is formed by the activity of renin. Renin is a protease that cuts the peptide bond between the leucine (Leu) and valine (Val) of angiotensinogen. Renin is secreted by the juxtaglomerular cells of the afferent arterioles in the kidneys, and its expression is regulated by a) blood pressure, b) osmotic pressure of the distal tubule through the macula densa, and c) sympathetic nervous system by β1 adrenergic receptors. Angiotensin I has no known biological effects, it is simply the precursor for Ang II and the other angiotensin peptides. The transformation proceeds in the lung endothelial cells which have a surface-linked enzyme, the angiotensin-converting enzyme (ACE). ACE produces a potent vasoconstrictor, Ang II, that acts on the smooth muscle of precapillary arterioles and increases the total peripheral resistance (TPR). The TPR has a major influence on the blood pressure (BP): BP = CO x TPR (where CO is the cardiac output). Ang II also acts as an important regulator of glomerular filtration rate (GFR) in the kidney (Willenheimer, 1999). The biological effects of Ang II are mediated by the

Ang II receptors (ATRs). AT1R activation initiates vasoconstriction, increases blood pressure, cardiac hypertrophy through intracellular Ca2+ increase following phospholipase C activation and cardiac fibrosis due to fibroblast activation. Ang II also influences the central nervous system by stimulating thirst (McKinley et al., 2004), promoting the secretion of vasopressin (Matsukawa & Miyamoto, 2011), and by increasing the activity of the sympathetic nervous system (Ramchandra, Yao,

& May, 2013). Binding of Ang II on AT1R activates Ras and downstream mitogen- activated protein kinase (MAPK) pathways, inducing the expression of growth promoting proto-oncogenes (de Gasparo, Catt, Inagami, Wright, & Unger, 2000).

When Ang II acts on AT2R, it counteracts many of the effects of AT1R activation

30 e.g. inhibiting apoptosis, arterial stiffness, cardiac hypertrophy and fibrosis (Horiuchi, Akishita, & Dzau, 1999; Kaschina, Namsolleck, & Unger, 2017). In addition to Ang II, there are other peptides with biological activity in the angiotensin peptide family (Figure 4.) Angiotensin 1-7 peptides were described in 2003 (Averill, 2003). ACE2 catalyzes the conversion of angiotensin I to angiotensin 1-9 from which ACE catalyzes angiotensin 1-7 that acts on Mas receptors. Their actions as well as the activation of AT4R have been shown exert cardioprotective effects attenuating hypertension, myocardial fibrosis, and cardiac hypertrophy (Santiago et al., 2010). There is also some evidence that Ang II modulates cardiac contractility. Petroff et al. have shown positive inotropic effects, that results from enhanced L-type Ca2+ currents (Petroff, Aiello, Palomeque, Salas, & Mattiazzi, 2000). In addition to systemic RAAS, there is evidence for the existence of local renin-angiotensin systems (RAS) in the different tissues such as heart, kidney and other organs (Paul, Poyan Mehr, & Kreutz, 2006).

Fig. 4. The angiotensin activation cascade. ACE: angiotensin convertase enzyme, ACE2: type 2 angiotensin convertase enzyme, AT1R: angiotensin type 1 receptor, AT2R: angiotensin type 2 receptor, AT4R: angiotensin type 4 receptor, APA: aminopeptidase A enzyme

2.2.2 Endothelin

Ang II have been shown to stimulate endothelin-1 (ET-1) secretion in vascular smooth muscle cells in vitro (Herizi, Jover, Bouriquet, & Mimran, 1998), but the most abundant source of ET-1 is the vascular endothelial cells (Yanagisawa, Kurihara, Kimura, Goto, & Masaki, 1988). Endothelins are potent vasoconstrictors

and their structure is very similar to the snake venom, safarotoxin. There are three isoforms of endothelin (ET-1, 2, 3) with different binding characteristics (Roubert, Gillard, Plas, Chabrier, & Braquet, 1991). The endothelin action is mediated by endothelin receptors which are G-protein coupled, seven transmembrane domain, rhodopsin-like receptors. Three isoforms are known (ETA, ETB, ETC) with the B- type having two isoforms B1 and B2 (Hynynen & Khalil, 2006). Several actions occur through the activation of these receptors, such as phospholipase C activation, phosphatidylinositol (1, 4, 5)-trisphosphate release leading to an increase in the intracellular Ca2+ concentration (Neylon, 1999), and they also stimulate sarcolemmal Ca2+ channels, increasing the Ca2+ influx from the extracellular space (Smith, Payne, Sedeek, Granger, & Khalil, 2003). Endothelin receptor activation can also stimulate phospholipase D leading to the generation of diacylglycerol, Src- family tyrosine kinases, phospholipase A2 with the release of arachidonic acid, the Na+/H+ exchanger, ERK, c-Jun-NH2-terminal kinase (JNK), and p38 MAPK (Daou & Srivastava, 2004; Kedzierski & Yanagisawa, 2001). Homozygous knockout of any component of the ET system (ET-1, ET-2, ET-3, ETA, ETB) has a lethal phenotype, complicating functional studies of the endothelin system (Ahn et al., 2004). Kisanuki et al. generated mice with endothelial cell specific endothelin-1 knockout, and showed that ET-1 plays an important role in maintaining the basal vascular tone and blood pressure (Kisanuki et al., 2010). In addition, pharmacological blockade with the ETA/ETB receptor inhibitor, bosentan, has been shown to reduce blood pressure in different experimental models (Doucet, Gonzalez, & Michel, 1996; Li, Larivière, & Schiffrin, 1994; Verma, Bhanot, &

McNeill, 2017). Recently, inhibition of ETA by macitentan was shown to reduce right ventricular blood pressure in pulmonary artery banding -induced experimental pulmonary hypertension in rabbits (Ramos et al., 2018). There is also data from some cardiac fibrosis models, such as after Ang II infusion and in a model of diabetes, showing that ET-1 can regulate cardiac fibrosis (Adiarto et al., 2012; Widyantoro et al., 2010).

2.2.3 Aldosterone

Aldosterone is the main mineralocorticoid hormone produced by the zona glomerulosa of the adrenal cortex (Jaisser & Farman, 2016). Chemically, it is a steroid hormone regulating sodium, potassium and water homeostasis and blood pressure. An increased aldosterone concentration causes sodium retention, hypervolemia and expansion of the extravascular space (Lijnen & Petrov, 2000).

32 The local increase in aldosterone concentration within the heart evokes many deleterious effects, e.g. stimulating myocyte growth, cardiac hypertrophy and fibrosis (Mentz et al., 2013; Okoshi et al., 2004). Aldosterone also has other detrimental effects on the cardiovascular system: myocyte apoptosis, vascular injury, endothelial dysfunction, oxidative stress, ventricular arrhythmia, and sudden death (Messaoudi, Azibani, Delcayre, & Jaisser, 2012; Nolly et al., 2014). Early studies showed that if the effects of aldosterone could be antagonized, then this reduced mortality in patients with systolic heart failure of any cause as well as MI (B Pitt et al., 1999; Bertram Pitt et al., 2003).

2.2.4 Natriuretic peptides

The natriuretic peptides (NPs) are a hormonal family involved in the maintenance of sodium and fluid homeostasis of the body. Three NPs have been identified: atrial (ANP), brain type (or b-type, BNP), and c-type (CNP) (Daniels & Maisel, 2007). ANP is released by the cardiac atria in response to increased atrial pressure and fluid overload (Lang et al., 1985). BNP, on the other hand, is produced in the left ventricle in response to the increased filling pressure occurring in a pressure overload condition. Both ANP and BNP promote natriuresis and diuresis and antagonize cardiac hypertrophy and fibrosis (Kerkelä, Ulvila, & Magga, 2015). The NPs exert their function through cell surface receptors. Thus far, three NP receptors (NPRs) have been identified: guanylyl cyclase-coupled (NPRA and NPRB), and natriuretic peptide clearance receptor (NPRC) (Potter, Abbey-Hosch, & Dickey, 2006). The assay of plasma ANP and BNP levels can be used as diagnostic and prognostic markers in heart failure patients (London, 2006; Ruskoaho, 2003). The NPs have prevented cardiomyocyte hypertrophy in vitro (Horio et al., 2000). In animal models, a significant correlation has been detected between the blood pressure and the ANP precursor gene activity, but not that of the BNP in spontaneously hypertensive rats (Ye & West, 2003). Destruction of the ANP gene led to hypertension and hypertrophy in homozygous knockouts, and salt-sensitive hypertension in heterozygotes (John et al., 1995). The overexpression of ANP appears to be protective during hypertrophic stimuli (Klinger et al., 1993). The activation NPRA and NPRB receptors are responsible for the protective effect via an increase in the cGMP level and induction of protein kinase G (PKG) (Mangiafico, Costello-Boerrigter, Andersen, Cataliotti, & Burnett, 2013). Targeted disruption of natriuretic peptides receptor type A (NPRA) also induces hypertension and cardiac hypertrophy (Oliver et al., 1997). As well as the

mechanical stretching, there are other factors that can induce ANP and BNP production like endothelin-1 (ET1), angiotensin II (AngII), isoproterenol and phenylephrine (Kerkelä et al., 2015). Several signaling pathways, such as MAPKs, have been shown to regulate NP expression in cardiomyocytes (Clerk et al., 2007).

2.2.5 Transforming growth factor beta superfamily

Transforming growth factors (TGFs) are a group of biologically active polypeptides that induce anchored cells to undergo modifications that result in their independent growth (J E de Larco & Todaro, 1978). Two subclasses have been identified based on the relationship with epidermal growth factor (EGF), TGF-α and TGF-β (Roberts, Frolik, Anzano, & Sporn, 1983). TGF-α has affinity for the EGF receptor, which means that it can compete with EGF (Joseph E. De Larco, Preston, & Todaro, 1981; Marquardt & Todaro, 1982; Ozanne, Fulton, & Kaplan, 1980; Todaro, Fryling, & De Larco, 1980), while TGF-β does not have any ability to bind directly to the EGF receptor (Anzano, Roberts, Meyers, et al., 1982; Anzano, Roberts, Smith, Lamb, & Sporn, 1982).

Transforming growth factor beta receptors

The receptor for the members of the TGF- superfamily has a conserved intracellular domain while the extracellular component differs and shows ligand specificity (Frangogiannis, 2018). The intracellular domain possesses a serine / threonine kinase, and also tyrosine kinase activity therefore it is classified as dual specificity kinase (Hata & Chen, 2016). Two main types of TGF- receptors exist, type I and II. During the activation process, two type I and two type II receptors become assembled into a heteromeric complex upon binding their ligand. The ligand stabilizes the tetramer and the constitutive active type II can activate the type I receptor (ALK) by phosphorylation of a glycine-serine rich domain (GS-box) (Massagué, 2012; Moustakas & Heldin, 2009; Weiss & Attisano, 2013; Wrana et al., 1992; P. Xu, Liu, & Derynck, 2012). There are seven type I and five type II receptors in the human TGF- superfamily (Hata & Chen, 2016) and type I receptors are further divided into subgroups: those that phosphorylate smad2/3 and those that phosphorylate smad1/5 (Y G Chen et al., 1998; Lo, Chen, Shi, Pavletich, & Massagué, 1998). The smad2/3 and smad1/5 activation by phosphorylation and their downstream regulatory events is known as a canonical pathway. Furthermore, the active TGF- receptor complex can activate another, so-called noncanonical

34 pathway, the first element of which is the TGF--activated kinase I (TAK1). Koitabashi et al. produced myocyte specific TGF- type 2 receptor (TR2) knock- down mice. These mice displayed a reduced activation of smad and TAK1 and there was blockade of all maladaptive responses in the chronic pressure-overload condition. Eliminating the TR2 enhanced the cardioprotective bone morphogenic protein 7 (BMP7) level (Koitabashi et al., 2011). In addition, the fibroblast-specific deletion of TR1/2 or smad3 (not smad2) significantly reduced fibrosis after the TAC operation (Khalil et al., 2017).

The “small” form of mothers against decapentaplegic, alias smad proteins

Members of the TGF-β family share a common downstream signaling pathway regulating gene expression by activation of smad2/3 transcription factor. Active, phosphorylated smad2/3 or smad1/5 form a heterotrimeric complex with smad4 and target many genes causing either their activation or repression (Massague, Seoane, & Wotton, 2005). Kong et al. produced fibroblast and cardiomyocyte specific smad3 KO mouse lines. They found that mice with fibroblast-specific loss of Smad3 displayed less adverse remodeling after MI but exhibited a higher incidence of rupture. There was more extensive myofibroblast proliferation but less collagen deposition in the scar and peri-infarcted area (Kong et al., 2018). Smad3 is involved in the regulation of fibroblast functions by activating the expression of integrin-mediated nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-2 (NOX-2). The smad3 KO fibroblasts exhibit reduced expression of - SMA after TGF-1 stimulation and show downregulated late (24-72 hours after reperfusion) CTGF expression (Dobaczewski, Bujak, et al., 2010; Kong et al., 2018). In cardiomyocyte-specific Smad3 KO mice, there was less apoptosis of cardiomyocytes, accompanied by a reduction in the level of myocardial NOX-2 and lowered expression of MMP2 (Kong et al., 2018). On the other hand, the ALK can activate other kinases, such as TGF- activated kinase 1 (TAK1), Erk-JNK, p38-MAPK, PI3K/AKT and other GTPases. This smad independent pathway is also called the noncanonical signaling pathway (Y. E. Zhang, 2009). The transforming growth factor beta (TGF-) superfamily is comprised of several subgroups including TGF-s, growth differentiation factors (GDFs), bone morphogenetic proteins (BMPs), and the activin-inhibin family.

Transforming growth factor beta (TGF-) subfamily

The TGF-β is a disulfide linked 2x25kDa homodimer (TGF-β1 and TGF-β2), or heterodimer (TGF-β1.2 called TGF-3) (Cheifetz, Andres, & Massague, 1988), ubiquitous ECM modulator profibrotic cytokine molecule (Sporn, Roberts, Wakefield, & Assoian, 1986). It has a role in the development of fibrosis in the case of almost all organs (Lenz, Costabel, & Maier, 1996; Manoury et al., 2005; Psathakis et al., 2006; Rottoli et al., 2005). TGF-β increases the level of reactive oxygen species (ROS) by compromising mitochondrial function and increasing NADPH oxidase (NOX) activity, especially NOX-4. TGF-β also suppresses the antioxidant glutathione (GSH) impairing the redox balance and evoking oxidative stress (R. M. Liu & Desai, 2015). It is well known that TGF-β is a potent inducer of CTGF (M. Ahmed, 2004; M. M. Chen, Lam, Abraham, Schreiner, & Joly, 2000; Chuva De Sousa Lopes et al., 2004). M. Ikeuchi et al. concluded that the knock-down of TGF- signaling by administration of a soluble TGF- type II receptor (TIIR) produced by plasmid- transfected cells, exacerbated early LV contractile dysfunction and attenuated LV hypertrophy and interstitial fibrosis in mice with myocardial infarction (Ikeuchi et al., 2004).

Targeting TGF-β1 signaling in the myocardium

TGF- activated kinase 1 (TAK1) is the main effector of the noncanonical TGF- signaling pathway. TAK1 activation followed by enhanced CTGF production due to TGF- stimulation in cardiomyocytes. TAK1 activates p38 MAPK, induces cardiomyopathy and depresses the response of myofibrils to calcium (Liao et al., 2002; Vahebi et al., 2007). TAK1 is also stimulated by IL-1 and TNF- (Adhikari, Xu, & Chen, 2007). TNF receptor associated factor 6 (TRAF6) is also involved in the TGF- mediated TAK1 activation (Sorrentino et al., 2008). Compromised diastolic function is usually encountered in patients with hypertensive heart disease or hypertrophic cardiomyopathy. The prognosis of HFpEF is usually better than HFrEF, but morbidity is substantial in both conditions (Ramachandran S. Vasan, Benjamin, & Levy, 1995). TGF- blockade by a monoclonal antibody in the TAC model (Wistar rats), not only inhibited fibroblast activation and myocardial fibrosis but also prevented diastolic dysfunction, while not affecting mean arterial pressure, myocyte hypertrophy, and systolic function (Kuwahara et al., 2002). In another study, the systemic blockade by TGF-β

36 neutralizing antibody reduced the amount of fibrosis, but failed to suppress chamber dilation and diastolic dysfunction, while TGF-β type 2 receptor myocyte specific knock-down protects against maladaptive responses (Koitabashi et al., 2011). Although the smad2 KO is embryonically lethal (Nomura & Li, 1998), smad3 KO mice are viable and have been examined for alterations in the fibrotic response. Smad3 takes part in the induction of TGF-β–responsive genes (Acta2, Col1a1, Col3a1, Fn, and Ctgf) and the smad3 mutant mice are protected against renal fibrosis in a unilateral ureteral obstruction model (Misako Sato, Muragaki, Saika, Roberts, & Ooshima, 2003), epithelial-mesenchymal transition in a lens injury of the eye (Saika et al., 2004) and angiotensin II– induced fibrosis of the blood vessels (Wansheng Wang et al., 2006).

2.2.6 Activin receptor signaling

Activin / Inhibin subfamily

Activin and inhibin form protein complexes that present hormonal activity. They were identified in 1986 and shown to play a role in activation and inhibition effect on follicle stimulating hormone (FSH) biosynthesis and secretion (Ling et al., 1986; Vale et al., 1986). Since then many other functions have been found: including role in cell proliferation, differentiation, apoptosis, metabolism, homeostasis, immune response, wound repair and endocrine function (Ye Guang Chen et al., 2006; Sulyok, Wankell, Alzheimer, & Werner, 2004). The complexes are dimers, linked by a single disulfide bound and the subunits are composed of two polypeptide chain  and . The  subunit has two isoforms: A and B (Table 1.) (Burger, 1988; Ying, 1987).

Table 1. Subunit composition of the most common activins and inhibins

Class Complex Subunits 1 2 Activin Activin A A A Activin AB A B Activin B B B Inhibin Inhibin A  A Inhibin B  B

In addition to its vast actions in human body, activin A also plays a role in the heart in cardiogenesis and in cardiomyocyte differentiation and physiology. Furthermore, activin A levels are elevated in heart failure and activin A may contribute to heart failure pathology (Bloise et al., 2018). Adenovirus-mediated overexpression of activing A has been shown to result in systolic and diastolic cardiac dysfunction in mice (Roh et al., 2019). Activins bind to activin receptor type 2B (ACVR2B) in mice, like many of the TGF-β family members: myostatin (MSTN), bone morphogenetic proteins (BMPs) 9, 10, 11 and GDF-11 (Figure 5.). Overexpression of inactive ACVR2B in gene modified mice results hypermuscularity like in case of MSTN KO mice (S. J. Lee & McPherron, 2001). After receptor-ligand interaction, the ACVR2B heterodimerises with activin type I receptor ALK4 or ALK5. The active receptor phosphorylates and activates smad2/3. The active smads translocate to the nucleus and with the cofactor smad4 serve as a gene transcription activator (Tsuchida et al., 2009). Expression of follistatin-like 3 (FSTL3) is induced by all of the major ligands through ACVR2-induced smad2/3 signaling (Bartholin et al., 2002; Tsuchida et al., 2009). Roh et al. recently showed that systemic ACVR2 activity, determined by analysis for circulating FSTL3 levels, increases with aging and correlates with an age-related increase in activins (Roh et al., 2019). Further, blockade of ACVR2 signaling with a monoclonal antibody that blocks both ACVR2A and ACVR2B restored cardiac function in different animal models of aging, pressure overload or sarcomere mutation (Roh et al., 2019). On the other hand smad2/3 inhibits Akt signaling and mammalian target of rapamycin (mTOR) pathways (Morissette, Cook, Buranasombati, Rosenberg, & Rosenzweig, 2009; R Sartori et al., 2009; Trendelenburg et al., 2009; Welle, 2009). mTOR activation promotes skeletal muscle hypertrophy but blocking mTOR leads just 40% decrease in muscle mass (R Sartori et al., 2009; Welle, 2009). Akt inhibition within the myostatin signaling pathway leads to enhanced nuclear translocation of forkhead box O1 (FOXO) that will increase the transcription of atrogin-1. Atrogin-1 is a ubiquitin ligase that labels its targets for proteosomal degradation.

38 Fig. 5. Signal transduction scheme of TGF-ß family members acting on Activin receptor type II

Growth differentiating factor (GDF) subfamily

Several GDFs have been identified in the heart (GDF8, GDF11, GDF15) and efforts have been made to reveal their function (Kempf et al., 2011; Loffredo et al., 2013; Morissette et al., 2006; Poggioli et al., 2016; Rodgers et al., 2009). GDF8 and GDF11 have a highly homologous structure and they share a common receptor complex and signal transduction pathways (Heldin & Moustakas, 2016). GDF15 is produced and secreted into the ECM in conditions of oxidative stress, inflammation and hypoxia (Wollert, Kempf, & Wallentin, 2017). GDF15, an antihypertrophic factor in the heart (Kempf et al., 2011; Wollert et al., 2017; J. Xu et al., 2006) also activates smad2/smad3 similar to GDF8 and GDF11 but binds to the glial cell- derived neurotrophic factor (GDNF) -like receptor (GFRAL), a structure that has

been found in the brain (Emmerson et al., 2017; Hsu et al., 2017). GDF15, through GFRAL, activates the Erk, Akt and PLC pathways in neurons leading to a reduction in energy production and weight loss (Emmerson et al., 2017; Hsu et al., 2017; Mullican et al., 2017; Saarma & Goldman, 2017).

Myostatin

The myostatin (MSTN), is also known as growth differentiation factor 8 (GDF8). The mature, 12.4 kDa peptide’s best-known function is to regulate muscle mass. MSTN KO mice created in 1997 by McPherron et al. have muscular hypertrophy and hyperplasia (McPherron, Lawler, & Lee, 1997). MSTN overexpression induces cachexia (T A Zimmers et al., 2002). The presence and enhanced expression of myostatin within the heart supposes its involvement in physiological and pathological processes (Sharma et al., 1999). The fractional shortening (FS) of the left ventricle on transthoracic echocardiograms is better, the phosphorylation of the SERCA activator phospholamban is higher in MSTN KO aging mice comparing to same age WT, and MSTN KO mice show less interstitial fibrosis (Morissette, Stricker, et al., 2009). Based on these results, MSTN inhibition has beneficial effects on the heart. Inhibiting the Akt signaling, MSTN attenuates the phenylephrine-induced hypertrophy of rat neonatal cardiomyocytes while phenylephrine increases cardiac growth substantially in MSTN KO mice (Morissette et al., 2006). MSTN overexpressing mice show smaller heart and LV diameter than the MSTN KO, but they have no difference in EF (Artaza et al., 2007). MSTN may play role in the pathophysiology of heart failure. In response to chronic pressure-overload induced by aorto-caval shunt in rat (Shyu, Lu, Wang, Sun, & Chang, 2006), and after MI in rat (Sharma et al., 1999), myocardial MSTN gene and protein expression is increased.

Growth differentiation factor 11 (GDF11)

The GDF11 is a member of TGF- family and show extensive homology with myostatin (Mosher et al., 2007; Parsons, Millay, Sargent, McNally, & Molkentin, 2006). The structural similarity suggests that they have a similar action on cardiac and skeletal muscle (Xing et al., 2007). In some studies, elevated circulating level of GDF11 decreased the cardiac and skeletal muscle mass (Hammers et al., 2017; Teresa A. Zimmers et al., 2017). Daily i.p. injections of GDF11 are beneficial

40 effects on age-induced cardiac hypertrophy (Loffredo et al., 2013), functions of skeletal muscle (Sinha et al., 2014), and brain vascularization (Katsimpardi et al., 2014). However, the function of GDF11 as a potential therapeutic target for aging- dependent muscle defects has been challenged and there is even some indication that GDF11 could inhibit muscle regeneration (Egerman et al., 2015; Harper et al., 2016). Harper et al. concluded that GDF11 administration could reduce pressure- overload induced pathological hypertrophy and associated fibrosis, but at a high dose, it can cause severe cachexia and premature death (Harper et al., 2018).

Bone morphogenetic protein (BMP) subfamily

BMPs are important embryonic morphogens that drive the development of lungs, kidneys and are also involved in ectopic bone formation from which their name derives. Recently, more than 20 BMPs have been identified (J. Wu, Jackson-Weaver, & Xu, 2018). BMP2, BMP4 and BMP10 are expressed in the myocardium and coronaries, BMP6 and BMP9 are secreted into the circulation by the liver (Andriopoulos et al., 2009; Wei, Salmon, Upton, Morrell, & Li, 2014). The different BMPs are regulated in a different way in cardiac symptoms (Chang et al., 2008; Merino et al., 2016; X. Wu et al., 2014). For instance, the level of BMP4 is elevated in pressure overload-induced hypertrophy whereas the BMP7 level is reduced (Merino et al., 2016; X. Wu et al., 2014). BMPs have also been shown to regulate skeletal muscle mass (Roberta Sartori et al., 2013).

2.2.7 Interleukin 6

Interleukin 6 (IL-6) is a proinflammatory, pleiotropic cytokine. The human IL-6 is a single glycoprotein chain composed by 212 amino acid residues, and approximately 26 kDa with 2 glycosylation sites (Hirano et al., 1986). The coding gene of human IL-6 is located on the chromosome 7 (Sutherland et al., 1988). The IL-6 receptor is a heterodimer, composed by α and β subunits where the β has catalytic activity (Taga et al., 1989). IL-6 blood concentration is elevated in patients with congestive heart failure (Tsutamoto et al., 1998). IL-6 is thought to play role in the reduced contractility, in the development of hypertrophy and HF following ischemia. In case of MI, IL-6 is released from the peri-infarction region (Gwechenberger et al., 1999). Cardiomyocytes release IL-6 under hypoxia (Yamauchi-Takihara et al., 1995), as a result of the activation of the adenosine A3-receptor (Wagner, Kubota, Sanders,

McTiernan, & Feldman, 1999). IL-6 signaling can be protective or pathogenic as it protects the heart tissue in acute damage, whereas long term IL-6 signaling may lead to chronic inflammation and fibrosis in cardiovascular disease (Fontes, Rose, & Čiháková, 2015).

2.2.8 Connective tissue growth factor

CCN family members

The acronym CCN is derived from the first three members of the family discovered, namely CYR61 (cysteine-rich angiogenic protein 61 or CCN1), CTGF (connective tissue growth factor or CCN2), and NOV (nephroblastoma overexpressed or CCN3). Together with three Wnt-induced secreted proteins (Wnt1 inducible signaling pathway protein (WISP) 1-3, or CCN4-6), they comprise the CCN family of matricellular proteins (Bork, 1993). These proteins have been named CCN1-6 by international consensus (Brigstock et al., 2003). Members of the CCN protein family have four conserved cysteine residue-rich domains. These domains are, from N- to C-termini: insulin-like growth factor binding protein (IGFBP) domain, von Willebrand type C repeats (vWC) domain, thrombospondin type 1 repeat (TSR) domain, C-terminal domain (CT) with a cysteine knot motif (Figure 6.). In CCN5 this last one is missing (Holbourn, Acharya, & Perbal, 2008).

Fig. 6. Schematic presentation of modular structure of CTGF with its interacting sites

CTGF’s actions

The CCN proteins mediate their functions through binding to various cell surface molecules in a context-dependent manner, however there is no specific receptor which bind them with high affinity (Bornstein & Sage, 2002). The IGFBP domain can bind insulin-like growth factors (IGF); the vWC domain interacts with

42 members of the TGF-β superfamily, the TSP domain interacts with VEGF, lipoprotein receptor-related proteins (LRP), and TrkA, and the CT domain can bind to fibronectin, perlecan, fibulin-1, mucins and heparan sulfate proteoglycans (HSPGs) (Jun & Lau, 2011). The complex, mosaic structure of CTGF can facilitate opposing actions. According to Lipson et al, there are five different mechanisms for CTGF actions (Lipson, Wong, Teng, & Spong, 2012). Firstly. CTGF can facilitate or even inhibit response interacting with cytokines such as TGF- and BMPs (Abreu, Ketpura, Reversade, & De Robertis, 2002). For instance CTGF can induce angiogenesis by linking to TGF- and on the other hand can inhibit angiogenesis interacting with VEGF (Inoki et al., 2002). This VEGF dependent inhibition can be alleviated by MMP protease action (A. Leask & Abraham, 2006). Secondly, CTGF can influence the local concentration and therefore the effect of heparin-binding growth factors by interaction of sulfate proteoglycans (HSPGs) (Lipson et al., 2012). Thirdly CTGF can block, mask or opposingly, produce new binding sites in the ECM altering with cell-ECM interactions, adhesion and mobility (Babic, Chen, & Lau, 2015; Y. Chen, 2004; Nishida et al., 2003). Fourth possibility is that the CTGF binds directly to a cell surface receptor stimulating classical signal transduction pathways (Babic et al., 2015; Segarini et al., 2001; Wahab, Weston, & Mason, 2005). Finally the CTGF may be taken up into the cell and modulate activity of signal transduction pathways (Wahab, Brinkman, & Mason, 2015). CTGF is essential during development. CTGF null mice show impaired chondrocyte proliferation and extracellular matrix composition and die shortly after birth due to respiratory failure caused by skeletal defect (Ivkovic et al., 2003). Expression of CTGF is induced in cultured cardiomyocytes and fibroblasts in response to a variety of pro-hypertrophic and pro-fibrotic stimuli (M. Ahmed, 2004; Frangogiannis, 2012). Cardiac expression of CTGF is upregulated in various models of cardiac fibrosis, including those induced by myocardial infarction, Ang II, and hypertrophy (Andrew Leask, 2015). Accumulating evidence shows that binding of CCN2 (CTGF) to TGF-β potentiates the effects of TGF-β (Frangogiannis, 2012; Grotendorst, 1997). TGF-β stimulus induces maturation of fibroblasts and transformation of fibroblasts to myofibroblasts. Myofibroblasts actively synthetize and secrete CTGF and CTGF has been shown to increase TGF- β (H. Yang et al., 2010) and VEGF production (F. Y. Liu et al., 2007). Blocking the function of CTGF by a mouse monoclonal antibody (mAb) can cut these positive feedback loops and the organs could restore the normal structure and function (Lipson et al., 2012).

In cardiac models, mice overexpressing CTGF show increased fibrosis in response to pressure overload (Yoon et al., 2010). Data from a cardiomyopathy model caused by lamin A/C gene mutations shows that extracellularly secreted CTGF protein mediates fibrosis and alters cardiac function (Chatzifrangkeskou et al., 2016). Blocking the function of CTGF significantly also improved LV function and slowed the progression of LV expansion in a mouse cardiomyopathy model induced by overexpression of protein kinase C(Koshman et al., 2015). CTGF inhibition significantly decreased the expression of many genes linked to tissue remodeling, while the total collagen deposition was not attenuated (Koshman et al., 2015). Interestingly, though, neither deletion nor overexpression of CTGF in cardiomyocytes had effect on LV remodeling and cardiac function during aging or stress response (Accornero et al., 2015). On the other hand, Dorn at al. in 2018 showed that deletion of CTGF specifically in activated cardiac fibroblasts protected from Ang II –induced cardiac fibrosis (Dorn, Petrosino, Wright, & Accornero, 2018). Data from clinical studies indicates that CTGF may serve as a diagnostic marker as circulating level of CTGF are associated with clinical grading and severity of HF (Behnes et al., 2014).

2.3 Cardioprotective and antifibrotic pharmacotherapies

Angiotensin converting enzyme inhibitors (ACEI) and angiotensin receptor blockers (ARB) reduce mortality and morbidity in cases of HF (Garg & Yusuf, 1995; C. B. Granger et al., 2003), MI (Cleland, Erhardt, Murray, Hall, & Ball, 1997; Kober et al., 1995; Pfeffer et al., 2003) and reduce the consequences in hypertensive conditions (Dahlof et al., 2002). ACE inhibitors are effective not just in case of hypertonia and HF. As shown in experimental models, they also reduce re- infarction, and infarct size and prevent reperfusion-induced arrhythmias (Linz, Scholkens, & Han, 1986), dilate the coronaries and improve contractile and metabolic function in ischemia reperfusion injury (Hartman, 1995; Kitakaze et al.,

1995; Linz, Wiemer, & Scholkens, 1996; Y. H. Liu et al., 1996). AT1-receptor KO mice have been shown to exert less adverse LV remodeling and increased survival after MI (Hamawaki et al., 1998). Combination of ARB with neprilysin inhibitor sacubitril, which prolongs the action of ANP, may provide an additional anti-fibrotic strategy to HFpEF (Zile et al., 2019).

44 Calcium channel blockers (CCBs) are extensively used in cases of angina, hypertension or other cardiovascular conditions (Elkayam, Weber, McKay, & Rahimtoola, 1985; Packer et al., 1996; Yasue, Ogawa, & Okumura, 1989). For instance, the CCB nifedipine upregulates endothelial superoxide dismutase by stimulating VEGF, and reduces oxidative stress (Ghiadoni et al., 2003; Taddei et al., 2001). CCBs also reduce the onset of HF and the need of coronary angioplasty in patients with stable angina (Poole-Wilson et al., 2004). The CCBs could enhance the cardioprotective effect of ARBs and the combination of these drugs may be preferable (Okuda et al., 2005). Beta-blockers were long considered to be the first-line agents in the cardioprotective therapy according to the international guidelines (Committee, 2003; Jones & Hall, 2004). They are used in hypertension, angina, atrial fibrillation, diabetes and stable coronary artery disease (Messerli, Bangalore, Yao, & Steinberg, 2009). Surprisingly, for more than three decades, no studies have shown any benefit of beta-blockers in the monotherapy of hypertension as compared with placebo (Bangalore, Messerli, Kostis, & Pepine, 2007). A recent meta-analysis revealed that beta-blockers do not confer any benefit for the end points of all-cause mortality and MI compared with placebo (Wiysonge, 2017). However they reduce the risk of stroke by 16-22%, but the protective effect is suboptimal, less than in the case of other agents (Wiysonge, 2017). Diuretics have been used for the last five decades in the therapy of hypertension. In combination with beta-blockers, they were the most commonly used drug until the early 1980’s, when ACEIs and CCB were introduced (Cutler & Davis, 2008), but even today, the appropriate use of diuretics is a safe and effective way to treat hypertension (Kaplan, 2009). Preclinical studies have provided evidence that blockade of mineralocorticoid receptors can attenuate the process of atherosclerosis (Deuchar et al., 2011; Raz- Pasteur, Gamliel-Lazarovich, Gantman, Coleman, & Keidar, 2014), modulate endothelial function in animal models (Sartorio et al., 2007; Schafer et al., 2010), and in diverse patient groups (Fujimura et al., 2012; Studen, Sebestjen, Pfeifer, & Prezelj, 2011), e.g. they can limit myocardial IR injury and prevent IR-induced arrhythmias (Lammers et al., 2012; Swedberg et al., 2012), and beneficially modulate cardiac remodeling (van den Berg et al., 2014). Spironolactone is a nonselective aldosterone receptor antagonist; it can decrease fibroblast collagen I mRNA expression (Bunda, Liu, Wang, Liu, & Hinek, 2007), and is beneficial in the development of cardiac hypertrophy and collagen deposition in spontaneously hypertensive rats (SHRs) (Zhao, Gu, Li, Ge, & Li, 2015). Although the efficacy of

spironolactone is clearly supported by clinical trials and treatment experiences, the use of spironolactone is limited by its side effects. Spironolactone has a tendency to bind none selectively to estrogen and androgen receptors causing gynecomastia and erectile dysfunction as well as menstrual abnormalities. Eplerenone is a more selective mineralocorticoid antagonist producing less adverse effects (Epstein & Calhoun, 2011). From 2018, the international guidelines have proposed that anti-hypertensive therapy is initiated with a dual therapy: combination of ACE inhibitor (ACEi) or angiotensin receptor blocker (ARB) and a calcium channel blocker (CCB) or diuretic. In the second line, there is a triple combination: ACEi (or ARB) with CCB and diuretic. In the third line, in the case of resistant hypertension, spironolactone or some other agent, such as a diuretic or beta-blocker should be added to the triple combination (B. Williams et al., 2018).

2.3.1 Pharmacological strategies to inhibit cardiac fibrosis

The continuous loss of compliance in the myocardium by the accumulation of rigid collagen structures accompanied by a decrease of contractile elements is thought to be the fundament of heart failure (Davis & Molkentin, 2014; Kong, Christia, & Frangogiannis, 2014; Andrew Leask, 2015; Moore-Morris, Guimarães-Camboa, Yutzey, Pucéat, & Evans, 2015; Porter & Turner, 2009). Among the currently used therapies, ACE inhibitors, ARBs and spironolactone have shown efficacy in reducing cardiac fibrosis (Brilla, Funck, & Rupp, 2000; Kong et al., 2014; Maron et al., 2018; Shibasaki et al., 2006). Statins inhibit 3-hydroxy-3-methylglutaryl-coenzyme A reductase and are used to reduce the level of cholesterol and to prevent the intra-arterial plaque formation (Abulhul et al., 2012; Goldstein & Brown, 2015). Preclinical studies have revealed that statins also inhibit scarring, fibrosis and cardiac remodeling (Bauersachs, Galuppo, Fraccarollo, Christ, & Ertl, 2001; Patel et al., 2001). It appears that statins can modulate TGF- signaling i.e. simvastatin was shown to attenuate TGF- - induced α -SMA expression (Shiroshita-Takeshita et al., 2007). It would appear that antagonizing the TGF- signaling is a logical target to inhibit cardiac fibrosis and that has appeared as a useful strategy in preclinical studies (Kagitani et al., 2004; Koitabashi et al., 2011; Mirkovic et al., 2002; Tank et al., 2014). The usefulness of long term inhibition of TGF- receptors, however, is hampered by cardiac toxicities (Herbertz et al., 2015; Park, Nguyen, Pezhouman, & Ardehali, 2019). A novel TGF-  inhibitor, GW788388, which inhibits ALK5 signaling, has displayed potent anti-

46 fibrotic effects in the heart (Derangeon et al., 2017; Tan, Zhang, Connelly, Gilbert, & Kelly, 2010) and has a better safety profile (Petersen et al., 2008). Pirfenidone and tranilast are clinically approved drugs that have been shown to inhibit TGF- signaling and to inhibit the development of cardiac fibrosis in experimental models (Martin et al., 2005; Mirkovic et al., 2002; D. T. Nguyen, Ding, Wilson, Marcus, & Olgin, 2010). While the use of pirfenidone and tranilast may cause hepatic toxicity and liver failure (Fang, Murphy, & Dart, 2017), pirfenidone has been studied in a clinical trial to evaluate its antifibrotic effects in patients with HFpEF. Among the other novel strategies to inhibit cardiac fibrosis, studies in clinical and preclinical field have targeted MMP action (Spinale, 2007), mast cell function (Kim et al., 2006; S. Oyamada, Bianchi, Takai, Chu, & Sellke, 2011), monocyte chemo-attractant protein 1 (MCP-1) (Hayashidani et al., 2003), chemokine interferon-gamma-inducible protein (IP-10/CXCL10) (Bujak et al., 2009), endothelin pathway (Rodríguez-Pascual, Busnadiego, & González-Santamaría, 2014), Ang II pathway (De Mello & Specht, 2006; Shibasaki et al., 2005) and Wnt- signaling (Daskalopoulos, Hermans, Janssen, & Matthijs Blankesteijn, 2013; He et al., 2010). miRNA-based strategies are another promising approach to inhibit cardiac fibrosis (Small, Frost, & Olson, 2010; Thum, 2012). miR29 was the first identified microRNA identified to target ECM proteins such as collagen in the heart (van Rooij et al., 2008). The researchers found that miR-29 was downregulated in the peri-infarct region and furthermore, down-regulation of miR-29 induced the expression of collagens, whereas overexpression of miR-29 in fibroblasts reduced collagen expression (van Rooij et al., 2008). In addition, inhibition of miR-21 by antagomir in vivo has been shown to decrease cardiac fibrosis in the pressure- overload model (Thum et al., 2008). It also appears that the miRNAs do not only act intracellularly, but some miRNAs, such as miR-21, also have some paracrine potential (Bang et al., 2014).

2.4 Cardiomyocyte fibroblast interaction

Cardiac fibroblasts may regulate cardiomyocyte phenotype through paracrine hormonal regulation, and vice versa, cardiomyocyte modulate the function of adjacent cardiac fibroblasts. Cardiac fibroblasts show extensive communication with the cardiomyocytes not only during development but also in the adult heart. The embryonic cardiac fibroblasts reside in the developing myocardium and express many components of extracellular matrix, such as fibronectin, collagens, periostin, hyaluronic acid, proteoglycans link protein 1 (Kakkar & Lee, 2010).

Interestingly, co-culturing of adult mouse cardiac fibroblasts together with cardiomyocytes resulted in a hypertrophic response in cardiomyocytes, whereas co- culturing of embryonic fibroblasts with cardiomyocytes enhanced cardiomyocyte proliferation (Ieda et al., 2009). Thus, these data indicate that the function of cardiac fibroblasts shifts during development. Cardiac fibroblasts produce several active molecules that are implicated in cardiomyocyte growth and development (Fig. 7). One of the first observations into the interplay between cardiomyocytes and fibroblasts emerged from a neonatal rat cardiomyocyte model showing that Ang II induced paracrine release of TGF-1 and ET-1 from cardiac fibroblasts which evoked a hypertrophic response in cardiomyocytes (Gray, Long, Kalinyak, Li, & Karliner, 1998). Fibroblast –derived fibroblast growth factors (FGF) have also been shown to play a role in cardiomyocyte growth. FGF2 and FGF4 induce transcription factors and ventricular specific markers in the chick embryo (Lopez- Sanchez, Climent, Schoenwolf, Alvarez, & Garcia-Martinez, 2002), and other FGFs may influence cardiac morphogenesis via induction of Wnt/-catenin signaling (Cohen et al., 2007). In addition, humoral factors secreted by cardiac fibroblasts, including cardiotrophin-1, ET-1, IL-6, periostin, and leukemia inhibitory factor, have been shown to induce hypertrophic responses in cultured cardiomyocytes (Kakkar & Lee, 2010). A number of factors have been similarly implicated in mediating the function of fibroblasts, such TGF-, FGF2, IL-6 family members and IL-33 (A Leask, 2010; Andrew Leask, 2015). In addition, recently evidence has appeared that non-coding RNAs could also play a role in fibroblast- cardiomyocyte signaling (Bang et al., 2014). The role of cardiac fibroblasts in the cardiac regeneration after injuries is unclear. There is evidence for the concept that fibroblast activity impairs the regeneration (Palatinus, Rhett, & Gourdie, 2010). For instance, the majority of mammalian tissues can heal without extensive fibrosis. The main difference between cardiac tissue and other peripheral tissue is the persistence of activated cardiac fibroblasts during and after the regeneration, whereas fibroblasts undergo an apoptotic cell death in the peripheral tissues (Desmouliere, Chaponnier, & Gabbiani, 2005; Sun, Kiani, Postlethwaite, & Weber, 2002). Non-mammalian vertebrates such as zebrafish and urodele amphibians repair wounds with only minor fibrosis. The robust cardiac regeneration potential occurs either by progenitor cells or de-differentiation and replication of terminally differentiated resident cells (Ausoni & Sartore, 2009; Poss, Wilson, & Keating, 2002).

48 Fig. 7. Illustration of secreted humoral factors participating in cardiac fibroblast- myocyte paracrine communication (modified from Kakkar and Lee, 2010).

In addition to the paracrine regulation and interaction transmitted by the extracellular matrix components, there is evidence for direct cell-to-cell interaction via gap junctions. Cardiac fibroblasts express connexin-43, and they start to express connexin-45 in close proximity with cardiomyocytes (Camelliti, Green, LeGrice, & Kohl, 2004; M. Oyamada et al., 1994; Y. Zhang et al., 2008). At the moment, the communication pathways between the cardiac fibroblasts and cardiomyocytes are only partially understood, but it seems to be complex, involving direct and indirect regulatory mechanisms that have important roles in cardiac regeneration and repair.

50 3 Aims of the present study

The level of CTGF is elevated in several cardiac disease (hypertrophy, myocardial infarction, fibrosis) indicating that CTGF may play a substantial role in cardiovascular pathologies. The low level of CTGF proved to be cardioprotective in fibroblast-specific knock-out fibrosis models. Our first hypothesis was that a general blockade of the action of CTGF would decrease cardiac fibrosis and improve cardiac function in TAC and MI mouse models. One major problem encountered in the myocardial infarction in addition to the loss of functional myocardium is remodeling and the metabolic alteration of the remote myocardium that further deteriorates cardiac function. The action of the TGF-β family members, such as activin, myostatin, GDF11, and BMPs, on activin receptor type 2B, is involved in the regulation of metabolism, growth and survival of striated muscle. Our second hypothesis was that the systemic blockade of ACVR2B ligands would be able to prevent the loss of cardiac and skeletal muscle and improve cardiac function in MI model.

The specific aims were: 1. to determine the potential of antagonizing the function of CTGF in protecting the heart from experimental hemodynamic overload –induced left ventricular hypertrophy, fibrosis and heart failure 2. to determine the potential of antagonizing the function of CTGF in protecting the myocardium from the long-term deleterious effects of myocardial infarction 3. to investigate the potential of blocking activin receptor 2B signaling to modify/target cardiac hypertrophy and fibrosis and skeletal muscle wasting in ischemic heart failure

52 4 Materials and methods

4.1 Transverse aortic constriction surgery

4.1.1 Anatomical background

The largest blood vessel, the aorta, after leaving the heart, is subdivided into several parts. The very first part - an onion-like bulb is situated between the auricles, is the root. Within the root of the aorta, the three leaflets of the semilunar type valve are located, as well as the openings of the coronary arteries from the space behind the two anterior pockets of the valve. Following the root, the next section is the ascending aorta, which turns into the descending aorta at the level of the 2nd rib, immediately behind the manubrium sterni. This connecting part is the aortic arch. The aortic arch has three primary branches; in the direction of blood flow these are: brachiocephalic trunk (arteria anonyma), left common carotid artery, and left subclavian artery. There is a fatty connective tissue situated around these major vessels to provide mechanical support and protection. The target region for TAC surgery is the part of the aortic arch, which is located between the first two primary branches.

4.1.2 Surgical procedure

C57Bl/6 mice were anesthetized with ketamine (100 mg/kg, Ketaminol) - xylazine (15 mg/kg, Rompun) cocktail i.p., intubated (BD VenflonTM Pro IV. Canule), and continuously ventilated (Minivent Type 845, 150 strokes/min, Tidal volume (μl) = mouse body weight x 10). After the midline incision of the skin, midsagittal sternotomy was applied until the third rib from the jugular notch, and the chest was kept open by a chest extractor. The two lobes of thymus were separated from each other, and between them the pericardium that covers the whole ascending aorta, was opened. The aortic arch was exposed by removal of the surrounding supporting tissue with 3.5mm edge-length fine forceps. After careful cleaning of the aortic arch, 7-0 surgical silk (Ethicon K-804) was hooked around the aorta. Before the bow was tightened firmly, a dull 27G L-shape needle was inserted within the hook together with the aorta. Two stitches were made, and the aorta was completely closed after which the needle was carefully removed. The total closure of the aorta is tolerable for up to one minute, but the time can be somewhat prolonged if pure oxygen is

used for inhalation. There were no signs of hypoxia (hyperpnoea, gasping) observed during surgery. The thoracic cage was firmly closed by one stitch, and the skin was closed by a running suture with 6-0 surgical silk. As a result of the procedure, the size of the lumen of the ligated aorta was constricted to the size of 27G needle, the pressure was elevated before the ligation site, and thus pressure overload conditions developed in the heart.

4.1.3 Postoperative care

Ventilation and external heating were maintained until the spontaneous breathing and protecting reflexes (thermoregulation, vestibular, and whisker reflex) recovered (approx. 15 min). We provided postoperative care, e.g. pain alleviation was applied three days following operation (buprenorphine, Vetergesic, 0.05 mg/kg s.c. 2x/day and carprofen (Rimadyl) 5 mg/kg s.c., 1x/day). The total postsurgical mortality was approximately 5-6%.

4.1.4 Results of the TAC surgery

As the result of aortic constriction, the heart has to work against an increased pressure due to the resistance of the smaller lumen, and the narrowing of the path of blood flow increases blood velocity which converts originally laminar flow into becoming turbulent. This turbulent flow is visible on color Doppler sonograms, although if the blood velocity is extremely increased, then it will be beyond the measurable range of the Vevo 2100 ultrasound device. On the one hand, the low blood pressure distal from the ligation site transmits a signal through the baroreceptors, stimulating the basal vasoconstrictor tone of the sympathetic nervous system; on the other hand, it has an effect on the juxtaglomerular cells of the renal glomeruli and triggers an increased secretion of renin, activating the renin-angiotensin system, augmenting the structural changes occurring in the myocardium. Due to the pressure-overload condition, the heart undergoes a massive myocardial hypertrophy. On weeks 2-4, the thickness of the myocardium continuously increases as does the level of the interstitial fibrosis. The accumulation of non-contractile, rigid, type I collagen fibers impairs both systolic and diastolic function. After week 4, the ventricles begin to dilate and eventually dilated cardiomyopathy occurs.

54 4.2 Acute myocardial infarction (MI) and ischemia-reperfusion (IR) models

4.2.1 MI surgery

C57BL/6 mice were anesthetized by 1.5-2% isoflurane without intubation. Fur was removed from the surgical window. Skin incision was applied on the left side from the xiphoid process in the direction of the left shoulder. Immediately after the cut of the skin, a 4-0 surgical monofilament was positioned and secured appropriately into the skin for later, rapid closure. The greater and lesser pectoralis muscles were separated and the region between them was exposed. This gap is located at the level of 5th intercostal space, where the apex cordis is facing the thorax wall and it serves as the surgical entrance site. A small artery clamp was used to penetrate the intercostal space and with the help of the equipment, the hole can be kept open while the heart is popped out by gently applying pressure with the operator’s supporting finger. A previously prepared 6-0 silk in small needle holder was used to ligate the left anterior descending coronary artery (LAD, ramus interventricularis anterior in humans). Due to the pressure change of the externalized heart, the artery starts to lose its color and definition after being exposed, thus requiring rapid ligation of the artery. After tightening of the suture, the heart was pushed back into the thorax, the pneumothorax (PTX) was removed by finger pressure, and the previously prepared skin suture is closed. The duration of the surgery was 2 minutes per mouse, and the time while the chest was opened and breathing stopped took 20 seconds. The immediate survival rate of the surgery was greater than 90%. The researchers must consider that with this experimental model, there is extensive variability in the area supplied by the coronary arteries and therefore the size and the severity of the infarction will differ.

4.2.2 IR surgery

The IR surgery is technically similar to the MI. The difference is in the method of LAD ligation. In this case, a sliding bow that could be released was used for the suturing of the LAD. The end of silk suture was left out of the body where it could be easily untied and be noninvasively accessible after the closure of the skin. In the

reperfusion procedure, the mice were re-anesthetized in an isoflurane chamber, and 30 minutes after the ligation, the suture around the LAD was released.

4.2.3 Results of the MI and IR surgeries

In hypoxic conditions, the myocardium suffers damage and loses its proper function. There is a deterioration of the contractile parameters (IR), whereas in MI the ischemic region stops moving (MI). The changes, which are detectable 24 hours after surgery, were evaluated by transthoracic echocardiography. The infarct size was measured at 24 hours after the IR injury, according to the protocol described earlier (Gao et al., 2010). After the 24-hour reperfusion time, the mice were anesthetized, with a high concentration of isoflurane (terminal anesthesia), the chest was opened, and the heart was exposed. After identification of the original surgical suture, the bow was retied through the original ligation site while the aorta was clamped. 2% Evans blue dye solution was injected into the aortic bulb which circulated via the coronaries through the entire heart. The stain cannot reach the region supplied by the ligated vessel therefore the area not supplied by the circulation (ischemic) remains pale whereas the dark-blue dye stains the perfused myocardium. The heart was dissected sliced into 5 thick pieces by sharp scalpel, each of the same thickness. Then the slices were incubated in 1% triphenyltetrazolium chloride (TTC) (Sigma) and imaged with a Canon EOS 450D digital camera with 100 mm macro lens. The blue stained “area not at risk” (ANAR), red viable area and pale infarct area were measured with Nikon NIS-Elements BR 2.30 software.

4.3 Echocardiography

4.3.1 Introduction

Transthoracic echocardiography is a powerful noninvasive tool for evaluating the cardiac morphology and function in vivo, providing immediate, online image without causing any tissue damage. The focused ultrasound waves penetrate into the body and are reflected from histological, acoustically dense tissues and thus provide a linear, 2-dimensional image. The higher the frequency, the better the axial resolution, but the scattering of beam also increases with frequency. This means that waves with a higher frequency have a lower penetration and thus it is possible

56 to image and visualize a thinner layer. For transthoracic echocardiography, the 30- 40 MHz probe is used in mice whereas it is 16-21MHz used in rats due to the size differences. In the current study, a Vevo 2100 high frequency high resolution ultrasound system was used with MS-550D (40 MHz, axial resolution: 40 μm, lateral resolution: 90 μm, Fujifilm-Visualsonics, Toronto, Canada) linear array transducer.

4.3.2 Preparation

The animal must be immobilized during the procedure. Isoflurane gas anesthesia is generally used to immobilize the subject and thus 4% isoflurane (at 1 bar pressure) was used for the induction and 2% for maintaining the anesthesia during the echocardiography. A heated evaluation table was used because the body temperature has an influence on the cardiac parameters, and the narcosis disturbs the animal’s thermoregulation. The evaluation was carried out with a free hand method, without taping and restraining thus allowing the heart to remain in its physiological position, while the animal was lying on its left side. The physiological position of the heart provides good quality images even in a diseased, morphologically modified heart.

4.3.3 B-mode image

The B-mode (“brightness mode”) is the conventional, two-dimensional image of an “acoustical section”. The image is composed of black and white pixels, where the depth of color is related to the amplitude of the reflected ultrasound wave. The sonogram is a row of consecutive steady images, where the frame rate is 400 bpm. On this “high velocity video”, the greater abnormalities of morphology (cardiac hypertrophy) as well as impaired motion (cardiac infarction) are detectable. Two main approaches are possible, the parasternal long axis view (PSLA) that is equivalent to the longitudinal section, and the short axis view (SAX) that represents the cross-section. In order to guarantee comparability, there are landmarks that help the sonographer to pinpoint the proper planes and angles. In PSLA, the landmarks are the apex cordis and the root of the aorta, in SAX, the papillary muscles.

4.3.4 M-mode image

The M-mode (“motion” mode), is a two-dimensional image, where the second dimension is time. The morphological change of the tissue is captured along a specified line and the time dependent motion is imaged. The following key parameters were acquired from the M-mode recordings: the left ventricular internal diameter in systole and diastole (LVID;s, LVID;d), left ventricular posterior wall thickness in systole and diastole (LVPW;s, LVPW;d), interventricular septum thickness in systole and diastole (IVS;s, IVS;d). The heart rate (HR) was also recorded. These parameters were used to calculate key parameters of LV structure and systolic function by using the following formulas:

– Left ventricular volume (LV Vol) = (7.0 / (2,4 + LVID)) x LVID3 – Fractional shortening (FS) = 100 x ((LVID;d-LVID;s) / LVID;d) – Ejection fraction (EF) = 100 x ((LV Vol;d-LV Vol;s) / LV Vol;d) – Stroke volume (SV) = LV Vol;d – LV Vol;s – Cardiac output (CO) = SV x HR – Left ventricular mass (LV Mass) = 1,053 x ((LVID;d + LVPW;d + IVS;d)3– LVID;d) 3

4.3.5 Pulse - wave Doppler image

The pulse – wave Doppler method is useful for recording fast motion. Our approach was the apical 4 chamber view, when the transducer active window is facing toward the apex cordis, and the plane of the acoustic section passes through both atrioventricular valves. From this viewpoint, the transmitral flow velocity that represents the speed of the blood flowing through the bicuspid (left atrioventricular) valve, and the mitral annular velocity that is the axial movement of the annulus fibrosus of mitral valve during the whole cardiac circle, were determined.

Transmitral flow velocity

The Doppler window was positioned behind the mitral valve within the left ventricle. The target velocity range was 400-1500 mm/s. The following parameters represent the diastolic functions: early and atrial filling (E and A peaks), deceleration and deceleration time, isovolumetric relaxation time (IVRT),

58 isovolumetric contraction time (IVCT), aortic ejection time (AET), non-filling time (NFT = IVCT+IVRT+AET). These data were further used to obtain:

– Mitral valve E to A ratio = E/A – Myocardial performance index (Tei-index) (MPI) = (NFT – AET) / AET

Mitral annular velocity

In this Doppler analysis, the target velocity range was smaller (5-40 mm/s), and the Doppler window was positioned on the septal side of mitral annulus fibrosus. Although all the events of the cardiac circle are represented by the tissue Doppler graph, we only measured the early (E’) and atrial (A’) peaks that are the same events as the E and A - peaks assessed in transmitral flow. Tissue Doppler calculations were the following: E’/A’, A’/ E’. Therefore, it is possible to combine measurements from the two forms of Doppler data, such as E / E’ that describe the mechanical characteristics of the wall of the left ventricle.

4.3.6 Strain analysis

Strain echocardiography allows for evaluation of dynamic parameters of the heart. These parameters are: radial, longitudinal and circular displacement; radial, longitudinal and circular velocity; radial and longitudinal strain; and radial and longitudinal strain rate. The connections between the dynamical cardiac parameters are shown in the figure (Fig. 8).

Fig. 8. Values of strain analysis and their definitions

The starting point of the strain analysis was a B-mode cine loop image set. The Vevo Strain (Visualsonics) software is capable to follow the dislocation of a pixel from frame to frame. In the traditional ultrasound strain imaging, these are the “speckles”, the particles, and it is their movements which are followed. The program selects 6 segments, and in each segment multiple speckles are situated. The strain analysis allows for evaluating the movements of these speckles in the different directions, as well as comparing them to each other. The velocity pattern is also recorded. In a healthy heart, there is synchronicity in the motion of the different segments. They exhibit the top of the peak of each feature at the same time. In case of a diseased heart, the different speckles within the different segment will achieve their top position in a different time and thus there is asynchronicity (time-to-peak analysis). This time-shift was clearly evident for instance in mouse hearts subjected to experimental myocardial infarction.

4.4 Pressure – Volume loop analysis

The pressure-volume relation method is used to evaluate the cardiac physiology by inserting an electrode directly into the left ventricle. This probe contains a pressure sensor and electrically conductive rings. These rings form an electrical field within the heart and its morphology determines the relative conductivity of the blood. The sensors detect the conductivity and thus the volume of the chamber can be calculated. Conductance catheter (1.2F, “F” is “French”, the value for the outer diameter of catheters, 1F = 0.33 mm, Scisense) was used. Implanting the probe demands a surgical procedure that causes severe damage in the vascular system as well as in the endothelium, therefore, the procedure is terminal.

4.4.1 Surgery methods for PV catheter insertion, approaches

There are two ways to gain access to the left ventricle. We used the technique where the catheter was inserted into the left ventricle through the right common carotid artery. Cervical surgery provided an opportunity for the straightforward cannulation of the external jugular vein. The second approach is the abdominal route. In this case, the whole heart was exposed by opening the chest, and therefore intubation and assisted respiration were needed. In order to prevent blood loss, electrocautery was used for cutting the tissues instead of scissors. The probe was inserted into the LV through the apex

60 cordis. The muscular pressure around the catheter created a good enough seal to prevent bleeding. The widely open thoracic cage allowed access to the inferior vena cava (VCI) to permit occlusion. For VCI occlusion, intratracheal intubation was performed and the same anesthesia method was used as in the TAC surgery. Data acquisition and analysis were performed with LabScribe2 (IWorx) software.

4.5 Application of neutralizing monoclonal antibody for CTGF

FG-3149 (provided by Fibrogen Inc, San Francisco, CA) is a chimeric, mouse IgG2a class antibody against CTGF. It is similar to FG-3019 (pamrevlumab), a human monoclonal antibody that binds to CTGF in the vWC domain and has been used in clinical trials. TAC-operated C57BL/6 mice were treated 3x/week with FG- 3149 (CTGF mAb, 10mg/kg dose, ip). Other groups were treated in a similar way with biologically inactive IgG or PBS as control groups. In the MI study, the mice were treated with control IgG or CTGF mAb 3x/week in the 7-day study, and 2x/week in the 7-week study.

4.6 Application of a trap for the ACVR2B ligands

Soluble activin receptor type 2 B linked to conservative C-fragment (Fc) of human IgG1 antibody (sACVR2B-Fc) was used as described (Magga et al., 2019). The subcutaneously administered ligand trap eliminated the circulating members of TGF- subfamily such as activin B, activin A, GDF11, myostatin. The procedure resulted in a substantial reduction in skeletal muscle and body weight growth with a loss of subcutaneous fat. ACVR2B-Fc (5 mg/kg s.c.) was administered immediately after the ligation of the LAD and continued twice per week for 4 weeks (4wk study) or started at 4 weeks after the LAD ligation and continued twice per week for 6 weeks (4+6wk study). PBS or human IgG1-Fc control was used as a vehicle control. ACTB-Fc (5 mg/kg s.c.), which specifically blocks activin B ligand, was administered immediately after the ligation of LAD and continued twice per week for 4 weeks (4wk study).

4.7 Histology

Five μm thick sections were cut from the conventionally paraffin-embedded blocks of formalin-fixed LV tissue. Sections were deparaffinized in xylene and rehydrated in a graded ethanol series. Masson’s trichrome, Picrosirius red and wheat germ agglutinin (WGA)-Alexa Fluor 488-conjugate (Molecular Probes) stains were used to visualize cardiomyocytes, cell borders, cell nuclei and fibrous tissue. The cross- sectional myocyte cell size was quantified (Nikon NIS-Elements) based on average of 50 cardiomyocytes in each section obtained from five representative fields (with a 20× objective) from the left ventricles. The length of the infarction was measured from Masson’s trichrome stained sections. Infarct size was quantified with a 1x objective by measuring the length of the infarct in relation to the whole LV circumference. The myocardial collagen content was quantified by staining the sections with Picrosirius Red (Direct Red 80, Sigma). The sections were analyzed under the microscope with linear polarized light (Olympus BX51) and images were captured with a color digital camera (with 10x objective, Olympus, DP71). Red and green birefringence of collagen fibers was quantified with Image J software from the left ventricle, and separately from the infarcted area, peri-infarct zone and remote areas from the hearts subjected to MI. The status of capillaries in myocardium was assessed by staining the sections with Pecam-1 (CD31, Santa Cruz Biotechnology). The size and number of capillaries were analyzed from five representative fields with a 40x objective. A primary antibody for slow myosin heavy chain MHCI (Chemicon/Merck) was used to detect the slow-twitch myocytes in quadriceps skeletal muscle. The sections were double-stained with WGA and MHCI and counterstained with DAPI.

4.8 TUNEL assay

In the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL), we used a kit from Merck Millipore (Billerica, MA, USA) according to the manufacturer’s instructions. Images were viewed with a Nikon Eclipse 80i microscope (Nikon Inc., Tokyo, Japan) and NIS elements software was used to record immunofluorescence images.

62 4.9 MMP2 activity assay

Cardiac tissue was homogenized in lysis buffer (20 mM Tris, 150 mM NaCl, 1% Triton X-100, pH 7.4) supplemented with protease inhibitors (Sigma) and briefly dispersed with an Ultra-Turrax homogenizer (Ika). LV tissue homogenates were centrifuged at 12500 rpm for 20 min at 4°C. The supernatant was collected, and protein concentration determined with the Bradford protein assay (Bio-Rad). MMP2 activity was measured with a mouse MMP2 activity assay kit (QuickZyme) according to the manufacturer’s instructions with 120 μg of the total protein being used per assay.

4.10 Quantitative PCR analysis

Total RNA was extracted from the LV cardiac tissue samples (hypertrophied heart) and from the non-ischemic area of the IR and MI hearts with Trizol reagent (Life technologies). cDNA was synthetized from 500 ng of RNA with Transcriptor First- Strand cDNA Synthesis Kit (Roche). Expression levels were detected by real-time quantitative polymerase chain reaction (qPCR) analysis with the use of TaqMan or SYBR Green chemistry on an ABI Prism 7700 Sequence Detector System (Applied Biosystems). The results were normalized to 18S ribosomal RNA.

4.11 RNA sequencing analysis

RNA sequencing analysis was performed by using single-end sequencing chemistry with a 75bp read length (Illumina NextSeq). After de-multiplexing the sequences, FASTQ generation was performed (Basespace, Illumina), the sequences were aligned and annotated using RefSeq Gene 2013.04.01 build. Finally, the gene expression levels were quantitated using reads per kilobase of transcript, per million mapped reads (RPKM). Only genes with a raw read count of >20 in at least 1 sample were used for further analysis. Genes with >1.5-fold difference in expression level with p < 0.05 (t-test) were considered as being altered. Gene ontology (GO) analysis was performed using GO Consortium software. Pathway Studio MammalPlus 12.0.1.9 (Elsevier) was used to identify common upstream regulators.

4.12 Western blot analysis

Frozen LV tissue samples were homogenized in ice-cold, 20 mM Tris (pH=7,5), 10 mM NaCl, 1 mM EDTA, 1 mM EGTA containing lysis buffer. The buffer was made with 1 mM β-glycerophosphate, 2 mM dithiothreitol (DTT), 1 mM Na3VO4, 10 μg/mL leupeptin, 10 μg/mL aprotinin, 2 μg/mL pepstatin, 2 mM benzamidine, 1 mM PMSF 20 mM NaF and 1% Triton X-100. Samples were centrifuged at 12500 rpm for 20 min at 4 °C and the supernatant was collected. The protein concentration was determined with the Bradford method. Protein extracts were matched for protein concentration and stored denatured in SDS loading buffer at -70 °C. Equal volumes (30 µg) of protein samples were loaded onto 10% gel in SDS-PAGE and transferred to nitrocellulose membranes. Appropriate antibodies were used to recognize the different protein bands. Protein levels were detected by using fluorescent secondary antibodies with Odyssey Fc imaging system (LI-COR Biosciences, Lincoln, NE, USA). Image Studio software (LI-COR) was used for the quantification.

4.13 Statistical Analysis

Statistical analysis was performed with IBM SPSS Statistics software. For the comparison of multiple groups, one-way ANOVA was used, followed by Tukey post-hoc tests for equal variances, or the Games-Howell post-hoc test for unequal variances. The Kruskall-Wallis test was performed when data did not have a normal distribution. Normality of variables was tested with Kolmogorov-Smirnov and Shapiro-Wilk tests. When two groups were compared, Student’s t-test or Mann Whitney U-test was performed. Data are shown as mean ± SD. Differences were considered statistically significant at the level of P<0.05.

64 5 Summary of results and discussion

5.1 CTGF in pressure-overload (TAC) mouse model

Chronic hemodynamic pressure overload results in an accumulation of ECM fibers in the LV tissue. With the increase of cardiac wall stress, there is increased expression and secretion of numerous circulating and paracrine factors from resident cardiac cells. These factors, i.e. released in either a paracrine or autocrine fashion, may induce a hypertrophic response in the heart (F. Yang, Chung, Huang, & Lan, 2009). The formation of excess fibrous tissue in various organs and tissues is associated with increased expression of CTGF. Prior studies indicate that CTGF may directly induce proliferation of fibroblasts and it has been suggested that inhibiting CTGF may be useful as an anti-fibrotic therapy (C. C. Chen & Lau, 2009; Mori et al., 1999; Panek et al., 2009). In order to analyze the induction of CTGF in a mouse model, we subjected mice to thoracic aortic constriction for four and eight weeks. qPCR analysis after 4 week and 8 weeks of TAC showed 2,1 and 14-fold increase in CTGF expression in the left ventricle comparing to sham (Fig. 9A). We investigated the role of CTGF in cardiac pressure overload –induced fibrosis by subjecting mice to eight weeks of TAC and treating the animals with CTGF mAb or control IgG. Transthoracic echocardiography at the end of the study revealed that CTGF mAb had significantly decreased the TAC –induced LV dilatation (LVID;d) and resulted in better preserved LV systolic function as evidenced by increased ejection fraction and radial strain. (Fig. 9B-D). These findings indicate that the CTGF mAb treated mice had been protected from TAC-induced adverse LV remodeling and LV dilatation. Invasive hemodynamic analysis by intracardiac pressure – volume catheter after 8 weeks of TAC caused a reduction in LV end diastolic volume in CTGF mAb treated mice (Fig 9E). The slightly steeper slope in the end-systolic pressure- volume relation (ESPVR) after vena cava inferior (VCI) occlusion was indicative of a better inotropic performance in CTGF mAb treated mice (Fig. 9F). However, no difference was observed in end-diastolic pressure-volume relation (EDPVR) between the TAC groups indicating that there had been a similar passive compliance of the LV in both treatment groups.

Fig. 9. (A) Mice were subjected to TAC for 4 and 8 weeks and cardiac samples were analyzed for expression of CTGF. (B-E) Mice were subjected to TAC for 8 weeks and treated with vehicle, control IgG or CTGF mAb. CTGF mAb treatment decreased the TAC –induced LV dilation (B), and attenuated the decrease in LV systolic function (C-D). Pressure-volume loops after 8 weeks of TAC show less dilation (E) as well as sharper ESPVR by VCI occlusion (F) in CTGF mAb treated mice. Data are presented as mean ± SD; *p<0.05; **p<0.01; ***p<0.001 vs sham, #p<0.05 vs TAC IgG. (Modified from I. Fig. 1) qPCR analysis of LV samples showed that antagonizing the function of CTGF with a mAb reduced the TAC-induced increase in the expression of fibroblast-derived collagen 1 (Fig. 10A). CTGF mAb treatment also resulted in a modest decrease in TAC-induced expression of collagen 3 and SMA (Fig. 10A). Interestingly, CTGF mAb significantly decreased the TAC –induced increase in the expression of CTGF as well as TAC –induced expression of IL-6 (Fig. 10B). The quantification of fibrous tissue in the LV using picrosirius red staining under polarized light detected a non-significant decrease in actual fibrosis in the CTGF mAb treated hearts versus control IgG treated mice following TAC (Fig. 10 C-D). Interestingly, CTGF mAb reduced TAC-induced MMP2 activity analyzed at four weeks after TAC (Fig. 10E) and expression of MMP2 analyzed at eight weeks after TAC (Fig. 10F).

66 Fig. 10. Mice were subjected to TAC for 8 weeks and treated with vehicle, control IgG or CTGF mAb. (A-B) Analysis for the expression of collagen 1 (Col1a1), collagen 3 (Col3a1), -smooth muscle actin (-SMA), CTGF and interleukin-6 (IL-6) in the left ventricles. (C-D) Representative images of picrosirius red stained LV sections and quantification of the fibrotic area. (E-F) Analysis for MMP2 activity at 4 weeks after TAC and MMP2 mRNA levels at 8 weeks after TAC. Data are presented as mean ± SD; *p<0.05; **p<0.01; ***p<0.001 vs sham, #P<0.05, ##P<0.01 vs TAC IgG. (Modified from I. Fig. 3)

To understand the role of CTGF in the development of cardiac fibrosis in more detail, we then infused the mice with Ang II. To our surprise, neutralizing CTGF mAb had no effect on the Ang II –induced cardiac fibrosis, expression of fibrosis- related genes or on cardiomyocyte hypertrophy and expression on hypertrophic genes (I. Fig. S6). Thus, in contrast to the protection seen against the hemodynamic overload evoked by TAC, the profibrotic and hypertrophic responses to Ang II were not alleviated by antagonizing the function of CTGF. Cardiac fibrosis is activated by multiple stimuli, including direct mechanical stretch and by circulating and local hormones such as ET-1, TGF-, platelet-derived

growth factor, and Ang II (A Leask, 2010). CTGF appears to modulate the signaling by these factors to enhance the activation of resident fibroblasts, which then secrete various factors that act as hypertrophic stimuli to adjacent cardiomyocytes. Cardiomyocytes undergoing hypertrophy, on the other hand, secrete factors that further activate the adjacent fibroblasts (Pathak, Sarkar, Vellaichamy, & Sen, 2001). There are only a limited number of studies dissecting the function of CTGF, and the majority of those reports are based on transgenic, CTGF overexpressing mice. In the current study, we investigated whether antagonizing the function of CTGF would exert protective effects on TAC-induced LV remodeling and dysfunction. We found that antagonizing CTGF had beneficial effects on LV remodeling in TAC model, but not against chronic Ang II infusion. This finding suggests that there are different mechanisms driving cardiac hypertrophy and fibrosis followed by TAC and chronic Ang II infusion. Ang II is a central mediator of fibrosis in the heart and blocking the actions of Ang II has shown efficacy in reducing cardiac fibrosis in various experimental models as well as in patients with myocardial disease (Shibasaki et al., 2005). Ang II signals through both AT1 and AT2 receptors. The activation of the AT1 receptor induced TGF- dependent early (15min) and TGF-  independent late (24h) smad2/3 phosphorylation (F. Yang et al., 2009). Interestingly, the profibrotic effect of Ang II appears to be independent of its arterial pressor effect (Tokuda et al., 2004). In fact, Ang II -induced activation of cardiac fibroblasts has been shown to be a nodal step for Ang II -induced cardiomyocyte hypertrophy (Odenbach et al., 2011) and ARBs represent the most efficient class of drugs to reduce the LV mass in patients with hypertension (Fagard, Celis, Thijs, & Wouters, 2009). While the reason for the lack of effect of CTGF mAb in Ang II – induced hypertrophy and fibrosis is not clear, it may originate from TGF- independent effects of Ang II on cardiac fibroblasts and cardiomyocytes where CTGF has either no role or makes only a minor contribution.

5.2 CTGF in cardiac ischemia and MI models

The cardiac repair procedure after an ischemic period is composed of three consecutive phases. These periods can overlap with each other, and the duration depends on several factors that are rather poorly elucidated. The first state is inflammatory, followed by a proliferative stage with the last phase representing maturation (W. Chen & Frangogiannis, 2013). The inflammatory phase in the mouse heart after ischemia-reperfusion injury lasts up to 72 hours, and even longer in the case of permanent infarction. This is followed by the proliferative phase

68 (days 3-7), in which fibroblast proliferation, and differentiation into myofibroblasts predominate. Following the infarct scar formation, activation of fibroblasts occurs in the peri-infarct and remote myocardium, which contributes to the adverse remodelling of the left ventricle. The elevation of CTGF expression during wound healing has been detected in different tissues (Gibson et al., 2014; Igarashi, Okochi, Bradham, & Grotendorst, 1993; Shi-Wen, Leask, & Abraham, 2008). The role of CTGF in infarct repair was examined by initiating CTGF mAb treatment during the proliferative phase (starting at day 3 after MI) with the mice being treated until day 7. CTGF mAb treatment following MI resulted in better survival (Fig. 11A) and better preserved LV systolic function as compared to mice treated with control IgG (Fig. 11B). No significant difference was detected in LV dilatation or LV posterior wall thickness (Fig. 11B). The CTGF mAb treatment also had beneficial effects on the infarct expansion index, which is derivative of LV septum and scar thickness and LV chamber size and LV tissue area (Iexp = LV septum/scar thickness x chamber area/LV muscle “ring” area) (Fig. 11C).

Fig. 11. Mice were subjected to experimental myocardial infarction, and 3 days after surgery treated with IgG vehicle or CTGF mAb for 4 days. The figure shows (A) survival of animals during the experiment, (B) echocardiography analysis for LV ejection fraction (EF), end-diastolic dimension (LVID;d), and posterior wall thickness (LVPW;d) at the end of the study, and (C) analysis for ratio of septum versus infarct scar thickness and infarct expansion index. Data are presented as mean ± SD; *p<0.05; **p<0.01; ***p<0.001. (Modified from II. Fig. 2)

In the current study, it was somewhat surprising that even though CTGF mAb treated mice had a better survival, the mice that survived also exhibited better LV systolic function. In this context, it is important to note that at three days after MI, the mice were randomized to treatment (with either control IgG or CTGF mAb) based on the analysis of infarct size and ejection fraction by echocardiography. The improved survival and LV systolic function in CTGF mAb treated mice thus does not stem from a smaller initial infarct size in the CTGF mAb treated mice. Immunostaining for the leukocyte marker, CD45, showed no difference in the degree of inflammation in the infarcted area between MI groups, nor were there any differences in the expression of inflammation-related genes in the remote left

70 ventricle (II. Fig. S1). Furthermore, no difference was observed in the amount of fibrosis in LV in the remote myocardium (II. Fig. 2D). The role of CTGF in post-MI LV remodeling was investigated by subjecting the mice to MI by permanent LAD ligation. One week later we randomized the mice based on the echocardiographic parameters for treatment with control IgG or CTGF mAb. Intraperitoneal injections were given twice a week for six weeks. Post- mortem analysis of mice revealed a significant reduction in MI –induced heart weight to body weight ratio in CTGF mAb treated mice (Fig. 12A). This was also supported by the echocardiography analysis indicating lower MI -induced LV mass in CTGF mAb treated mice (Fig. 12A). Echocardiography analysis also revealed that CTGF mAb had attenuated the MI –induced left atrial dilatation (Fig. 12A). Left atrial dilatation is common in HF and reflects the increased burden to left atria due to increased pressure or volume load and is a powerful predictor of cardiovascular mortality (Abhayaratna et al., 2006; Zile et al., 2011) On the other hand, no difference was observed in LV systolic function between the MI groups (Fig. 12A). The collagen content that is marker of the grade of fibrosis was decreased to the level of the sham operated animals in MI mice treated with CTGF mAb (Fig. 12A). The role of CTGF in the regulation of cardiac gene expression was assessed by comparing LV samples from control mice and MI mice treated with CTGF mAb or control IgG for six weeks by RNA sequencing analysis. We found that more than 1000 genes had been significantly regulated in response to MI, and the CTGF mAb treatment significantly altered the expression of 72 genes (II. Table S3 and Dataset 1). Gene ontology enrichment analysis indicated that 24 of the 72 genes regulated by CTGF mAb were related to the fibrotic phenotype or inflammation. Further analysis indicated that a number of fibrosis and inflammation related genes regulated by CTGF mAb were downstream target genes TGF-1, TNF- and IL- 1 pathways (Fig. 12B). In addition, CTGF mAb treatment resulted in an upregulation of genes that have been shown to play a role in cardiac development and repair, such as Cited4, Nkx-2-5 and GATA4 (II. Fig. 3F) (Akazawa & Komuro, 2005; Boström et al., 2010; Pikkarainen, Tokola, Kerkelä, & Ruskoaho, 2004).

Fig. 12. Mice were subjected to MI and one week later randomized for treatment with control IgG or CTGF mAb for six weeks. (A) Analysis for heart weight to body weight (HW/BW) ratio, LV mass, left atrial end-diastolic area (LAA;d), ejection fraction (EF) and analysis of interstitial fibrosis from picrosirius red-stained LV sections under polarized light. Data are presented as mean ± SD; *p<0.05; **p<0.01; ***p<0.001. (B) Hierarchical clustering of RNAseq data for transcripts that were altered by MI and at least partially normalized by CTGF mAb. The data indicates that many of these genes are regulated by transforming growth factor-b1 (TGF-ß1), tumor necrosis factor (TNF), or interleukin (IL)-1ß. (Modified from II. Fig. 3)

72 In order to investigate the role of CTGF in acute cardiac ischemic injury, we subjected mice to an ischemia-reperfusion injury, and treated the mice with CTGF mAb. The treatment with CTGF mAb was initiated at 24h prior to ischemia, or only at reperfusion. We found that antagonizing the function of CTGF by a monoclonal antibody did not increase infarct size or compromise the recovery of LV function following ischemia, as might be expected if CTGF should be cardioprotective (II. Fig. 4). However, the administration of the CTGF mAb was sufficient to induce JNK and STAT3 signalling in the ischemic hearts (II. Fig. 4). Previous data obtained from transgenic mice with cardiomyocyte-specific overexpression of CTGF suggesting that CTGF could protect the myocardium from an acute I/R injury (M. S. Ahmed et al., 2011). Kaasboll et al also reported that recombinant, human CTGF was able to mimic the cardioprotective phenotype when Langendorff-perfused mouse hearts were exposed to CTGF before or after the onset of ischemia (Kaasboll et al., 2016). While the role of CTGF in I/R injury has not been studied in a knockout model, previous studies have suggested that augmenting the levels of CTGF would exert protective effects following acute cardiac I/R injury. However, our data shows that antagonizing the function of CTGF was not able to prevent the injury. These contrasting data may stem from the mosaic domain structure of CTGF and augmenting the protein levels allows CTGF to modulate different biological functions of the cellular environment in the myocardium. Myocardial infarction is the most common cause of HF with a dismal prognosis. In the case of HF, contractility is compromised by increased fibrosis, which results in decreased ventricular compliance. The accumulation of fibrillar ECM elements also disrupts normal electro-mechanical coupling and leads to conduction abnormalities, cardiac arrhythmias and even sudden cardiac death. The production of CTGF is elevated in fibrotic conditions in every organ as well as in the failing heart (Lipson et al., 2012). Clinical trials investigating the efficacy of CTGF monoclonal antibody for treatment of Duchenne muscular dystrophy, pulmonary fibrosis and pancreatic cancer are ongoing. In the current study, we showed that antagonizing the function of CTGF with a mAb conferred protection from adverse LV remodelling in the hearts of the mice subjected to MI. Interestingly, we also found that CTGF mAb treatment was also beneficial in the proliferative phase of cardiac repair. There is recent data shows that genetic targeting of fibroblast smad3, a downstream target of TGF-β, is detrimental during cardiac repair (Kong et al., 2018). It is thus possible that CTGF mAb does not abrogate TGF-β signalling in the infarcted heart, but rather mitigates the signalling

and thus attenuates the excessive ECM production. In addition, CTGF mAb may have an effect on post-MI cardiac inflammation, a condition that peaks prior to the time points that were analysed in the current study.

5.3 Activins and GDFs in cardiac ischemia

Signaling through activin receptors, especially ACVR2B, regulates skeletal muscle mass. ACVR2B ligands have also been postulated to participate in the pathology associated with myocardial infarction (MI). We determined the effect of systemic blockade of ACVR2B ligands on cardiac function in experimental MI as well as in MI-induced ischemic heart failure. During cardiac remodeling, humoral factors including myostatin and activins are released and adversely affect the skeletal muscle (Douglas L Mann, Zipes, Libby, Bonow, & Braunwald, 2014). Cardiac cachexia is associated with chronic HF and it predicts decreased survival and serves as a prognostic factor for HF (Rossignol et al., 2015). Thus, we also examined the effect of systemic blockade of ACVR2B ligands on skeletal muscle hypertrophy and its potential to revert muscle wasting in ischemic heart failure.

5.3.1 Ligands of the activin receptor type 2B are regulated differently after MI

We aimed to determine the activation profile of the different ligands following MI. The expression levels of activin A and activin B and also the TGF- were upregulated after the infarction and remained higher throughout the first two weeks. In contrast, the myostatin level was downregulated during the proliferative phase after MI (Fig. 13).

74 Fig. 13. Analysis for myostatin, activin A, activin B and TGF-ß mRNA expression in the left ventricles after myocardial infarction. Data are presented as mean ± SD; *p<0.05; **p<0.01 vs sham. (Modified from III. Fig. 1)

5.3.2 Systemic blockade of ACVR2B ligands does not alleviate post- MI remodeling

Our further goal was to evaluate whether systemic blockade of ACVR2B ligands, would be cardioprotective and could reduce post-MI remodeling and improve cardiac function. In our previous study (Magga et al., 2019), the presence of soluble ACVR2B receptor reduced cardiac remodeling after an ischemic injury and restored cardiac functions. Eliminating the ACVR2B ligands by ACVR2B-Fc was initiated immediately after the surgery and continued for 4 weeks (inflammatory and reparative phases). We used either soluble nonfunctioning ACVR2B receptor to block a wider range of ligands (activin A, activin B, myostatin and GDF11), or an inhibitor which specifically inhibits the activin B (ActB-Fc). Echocardiography at the 4 week time-point showed that ACVR2B-Fc or ActB- Fc did not affect LV dimensions or systolic functions (III. Fig 2A). ACVR2B-Fc or ActB-Fc did not affect either the amount of fibrosis or the degree of hypertrophy (III. Fig. 2B-C). Administration of the inhibitors did not modify the expression of hypertrophic, fibrotic or angiogenetic genes (III. Fig. S1B-C). ACVR2B-Fc or ActB-Fc did not cause any compensatory upregulation of ACVR2B ligands or type II receptors (III. Fig. S1E).

Fig. 14. Mice were subjected to myocardial infarction (MI) and treated with vehicle or ACVR2B-Fc. Shown are analyses for cardiac hypertrophy (A) and fibrosis (B) at 4 weeks after MI. Data are presented as mean ±SD; *p<0.05; ***p<0.001 vs sham. (Modified from III. Fig. 3)

In the next step, we aimed to study whether ACVR2B-Fc, which is known to be a potent hypertrophic agent in the skeletal muscle, could improve cardiac function in ischemic heart failure. ACVR2B-Fc treatment was started 4 weeks after MI, and continued for 6 weeks (4+6wk study). Similar to the 4wk study post-MI, ACVR2B- Fc did not affect systolic function nor LV dimensions as compared to vehicle- treated MI animals. ACVR2B-Fc did not increase cardiac hypertrophy during 6- weeks of treatment (Fig 14A). Administration of ACVR2B-Fc only slightly reduced cardiac fibrosis in late-remodeling stage post-MI (Fig. 14B). In sham mice treated with ACVR2B-Fc, we detected that ACVR2B-Fc seemed to downregulate the expression of ANP (III. Fig. S2A) and interfere with cardiac diastolic function. This was accompanied with slightly elevated Lox expression, however, collagen deposition was not increased by ACVR2B-Fc in sham mice (III. Fig S2B).

5.3.3 Systemic blockade of ACVR2B ligands reduces skeletal muscle atrophy in MI-induced cardiac cachexia

We then aimed to evaluate whether ACVR2B-Fc as a potent anabolic agent, could reduce skeletal muscle loss in cardiac cachexia. ACVR2B-Fc treatment post-MI in the 4wk study increased the body weight by causing hypertrophy of skeletal muscle. (Fig. 15A).

76 Fig. 15. Mice were subjected to myocardial infarction (MI) and treated with vehicle or ACVR2B-Fc. Shown are analysis of body weight and cardiomyocyte cross-sectional area after 4 weeks of treatment (A). Analysis for body weight (B), cardiomyocyte cross- sectional area (C-D) and expression of phosphorylated S6 (pS6), phosphorylated extracellular signal-regulated kinase (pERK) and peroxisome proliferator activated receptor gamma coactivator 1 alpha (PGC1) in the left ventricles (E) after 6-week treatment started at 4 weeks after MI. Data are presented as mean ±SD; #p<0.05; ##p<0.01; ###p<0.001 vs MI+vehicle. (Modified from III. Fig. 4)

ACVR2B-Fc treatment exerted effects on other organs such as spleen (splenomegaly) and adipose tissue, causing reduced fat mass in MI mice. There was also a clear difference in the body weight between the treated and sham animals during the 6wk treatment (4+6wk study) that was visible already from the first week (Fig. 15B). We quantified the cell size of quadriceps muscle, and found that ACVR2B-Fc had increased the skeletal muscle mass (Fig. 15C). There was a shift in the muscle cell population to the direction of larger myocyte size (Fig. 15D). Although ACVR2B-Fc decreased the expression of MHCβ (III. Fig 4), there was no change in the proportion of MHCI slow-twitch myocytes in skeletal muscle (III. Fig 4). The qPCR analysis showed that the ACVR2B-Fc downregulated PGC1α (III. Fig. S4A); this was confirmed also at the protein level (Fig 15E). In addition, ACVR2B-Fc downregulated Pfkm but upregulated Cited4 in quadriceps skeletal muscle (III. Fig S4A-B). ACVR2B-Fc slightly downregulated atrogenes: Atrogin1

and Murf1 (III. S4B). Western blot evaluation detected an increase in phosphorylation of S6 (hypertrophy indicator) while the ERK phosphorylation was not affected (Fig. 15E). These changes in the genetic regulation point to alterations in the cell-homeostasis in addition to hypertrophy. There was no change in the expression level of TGF-β, activin A and B, nor in the expression level of several receptors (ACVR2A and B, BMP receptor type 2 (BMPR2) (III. Fig S4C). The ACVR2B-Fc treatment increased the expression levels of GDF11 in MI mice; the treatment also elevated GDF11 and myostatin levels in sham mice (III. Fig S4C). It was speculated that there might be a compensatory increase in the amounts of antihypertrophic factors after ACVR2B ligand blockade. Since BMPs can also regulate muscle mass (Sartori et al, 2013), we decided to determine the BMP expression levels after the ACVR2B-Fc treatment in MI mice. We did not detect any significant changes in the expression of BMPs although MI induced upregulation of BMP13 in skeletal muscle (III. Fig S4D). A similar effect also occurred in the heart (III. Fig S3E), where MI reduced the expression of BMP7 while increasing the expression of BMP13. Collectively, our results with regard to ACVR2B signaling suggest that although it increased skeletal muscle mass in ischemic HF, pharmacological blockade of ACVR2B ligands did not compromise cardiac function nor exacerbate cardiac remodeling after an ischemic injury.

78 6 Conclusions

The presence of cardiac hypertrophy and fibrosis predispose to heart failure, cardiac arrhythmias and sudden cardiac death. Several studies have shown that the connective tissue growth factor has a central role in the regulation of fibrotic processes, wound healing, ECM formation, even in migration processes in different tissues. The modular structure of the CTGF provide different actions in the presence of different ECM elements (HSPGs, collagen), growth factors (TGF-β, VEGF) or cell surface receptors (integrins) (Lipson et al., 2012). We investigated the effect of the systemic blockade of CTGF with a mouse monoclonal antibody on the stressed, diseased heart. Cardiac function was investigated by transthoracic echocardiography, intracardiac pressure-volume electrode and molecular biological methods were exploited to determine hypertrophic, fibrotic, inflammatory and functional responses in the heart. In the first study, the role of CTGF was investigated in cardiac pressure overload induced by TAC or Ang II. We found that blockade of CTGF with mAb resulted in better preserved cardiac systolic function in TAC mouse model and attenuated the LV dilation. However, CTGF mAb could not protect from Ang II – induced cardiac hypertrophy and fibrosis. In the second study, we investigated if antagonizing the function of CTGF could modulate infarct repair and development of HF after MI, and if CTGF mAb would be able to alleviate an acute cardiac IR injury. We found that CTGF mAb treatment during infarct repair improved survival; it resulted in better preserved LV systolic function and decreased the infarct expansion index. CTGF mAb treatment during post-MI LV remodelling decreased cardiomyocyte size and LV mass, and decreased the amount of cardiac fibrosis. CTGF mAb treatment had no effect on acute cardiac IR injury. Therapies for HF patients with angiotensin-converting enzyme inhibitors, angiotensin II receptor antagonists and mineralocorticoid receptor antagonists have been shown to attenuate the development of cardiac fibrosis. In addition, targeting TGF- signalling might be an effective approach in fibrosis therapy. However, TGF- targeted therapies have been associated with undesirable side effects. Antagonizing the function of CTGF with a mAb (FG-3019, pamrevlumab) has not been associated with serious side effects in clinical trials. Targeting CTGF may thus provide a novel therapeutic approach for preventing cardiac fibrosis and the subsequent development of heart failure. While CTGF mAb treatment mitigated the adverse cardiac effects induced by pressure overload or MI, it did not alleviate Ang II –induced cardiac hypertrophy or fibrosis. It would therefore be interesting

to evaluate if combining CTGF mAb with either ACEi or ARB would enhance the efficacy of RAS targeted therapy in the treatment of cardiac fibrosis and heart failure. Activins, similar to the BMPs and GDFs, are ligands of the TGF- superfamily. They act on activin receptors that are similar to the TGF- receptor; all ligands in the TGF- superfamily share their signalling through seven type I and five type II receptors which form a heterotetrametric complex. There are both canonical (smad2/3 or smad 1/5/8 activation by phosphorylation) and non-canonical (PI3K, Akt, mTOR signalling) pathways through which activins and GDFs regulate cell survival, muscle growth and muscle atrophy. Apoptosis of myocytes is one of the pathologies occurring in the heart after myocardial infarction. The loss of functioning cardiomyocytes further deteriorates the cardiac function. Our research question was: could we restore the function of the myocardium by blocking the activin signalling in a sustained myocardial ischemia model. Although we found that the inhibition of activin receptor activity by competitive, injectable and soluble but non-functioning activin receptor, caused skeletal muscle hypertrophy and therefore might be beneficial to alleviate cardiac cachexia, the treatment exerted no effect on cardiac function nor on the scar formation and healing in MI mouse model. Cachexia occurs when there is more than a 5% body weight loss during one year or the body mass index (BMI) is less than 20 kg/m2 skin surface. Characteristic for cachexia is decreased muscle strength, fatigue, anorexia and an increase in inflammatory markers (Evans et al., 2008). Cardiac cachexia deteriorates the prognosis of HF, and paradoxically obese persons have a greater chance for survival (Horwich, Fonarow, & Clark, 2018), however obesity itself is a key cardiovascular risk factor (Aune et al., 2016). This phenomenon is called the obesity survival paradox. Interestingly, blockade of ACVR2B induces skeletal muscle hypertrophy but also reduces adipocyte size and fat mass, suggesting that it could possibly target cachexia without causing harmful effects from excess adipose tissue and obesity. Cachexia treatment currently includes neurohormonal blockade, anaemia treatment with iron supplementation, appetite stimulants and nutritional support, anabolic hormones and physical exercise (Ebner, Elsner, Springer, & Von Haehling, 2014). Non-steroid anabolic treatment may offer considerable advantages in the therapy of cardiac cachexia.

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116 Original publications

Reprinted with permission from Wolters Kluwer (I) and Elsevier (II).

Original publications are not included in the electronic version of the dissertation.

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118 ACTA UNIVERSITATIS OULUENSIS SERIES D MEDICA

1511. Korhonen, Tommi (2019) Bone flap survival and resorption after autologous cranioplasty 1512. Sliz, Eeva (2019) Genetics and molecular epidemiology of metabolic syndrome- related traits : focus on metabolic profiling of lipid-lowering therapies and fatty liver, and the role of genetic factors in inflammatory load 1513. Ollila, Meri-Maija (2019) The role of polycystic ovary syndrome (PCOS) and overweight/obesity in women’s metabolic and cardiovascular risk factors and related morbidities 1514. Kotiaho, Antti (2019) Radiation dose determination using MOSFET and RPL dosimeters in x-ray imaging 1515. Hänninen, Sandra Lynn (2019) Transcriptional control of muscle cell excitation- contraction coupling : the role of activity and mitochondrial function 1516. Väyrynen, Otto (2019) Factors affecting aggressive oral tongue cancer invasion and development of in vitro models for chemoradiotherapy assay 1517. Euro, Ulla (2019) Risk factors for sciatica 1518. Lotvonen, Sinikka (2019) Palvelutaloon muuttaneiden ikääntyneiden fyysinen toimintakyky, sen muutos ja toimintakykyyn yhteydessä olevat tekijät ensimmäi- sen asumisvuoden aikana 1519. Tuomikoski, Anna-Maria (2019) Sairaanhoitajien opiskelijaohjausosaaminen ja ohjaajakoulutuksen vaikutus osaamiseen 1520. Raza, Ghulam Shere (2019) The role of dietary fibers in metabolic diseases 1521. Tiinanen, Suvi (2019) Methods for assessment of autonomic nervous system activity from cardiorespiratory signals 1522. Skarp, Sini (2019) Whole exome sequencing in identifying genetic factors in musculoskeletal diseases 1523. Mällinen, Jari (2019) Studies on acute appendicitis with a special reference to appendicoliths and periappendicular abscesses 1524. Paavola, Timo (2019) Associations of low HDL-cholesterol level and premature coronary heart disease with functionality and phospholipid composition of HDL and with plasma oxLDL antibody levels 1525. Karki, Saujanya (2019) Oral health status, oral health-related quality of life and associated factors among Nepalese schoolchildren

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