Yangchen Dhondup Toll-like receptor 9 signalling in heart failure

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Doctoral thesis at Oslo, 2018

Yangchen Dhondup

Toll-like receptor 9 signalling in

heart failure

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© Yangchen Dhondup, 2018

Series of dissertations submitted to the Faculty of Medicine, University of Oslo

ISBN 978-82-8377-156-5

reproduced or transmitted, in any form or by any means, without permission.

Cover: Hanne Baadsgaard Utigard.

Print production: Reprosentralen, University of Oslo.

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To Passang and Lhakpa

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Acknowledgements

Science is teamwork. The work that resulted in this thesis was carried out at the Research Institute for Internal Medicine (IMF) at Oslo University Hospital, Rikshospitalet and at the Institute for Experimental Medical Research (IEMR) at Oslo University Hospital, Ullevaal in Oslo.

I would like to thank my supervisor, Leif Erik, for giving me the opportunity to be a part of the

“Heart failure group”. Thank you for your continuous engagement and availability during my PhD studies.

Next, I would like to give a special thanks to my supervisor, Arne. You taught me the value of positivity, curiosity as well as humbleness and critical thinking in science. I’m truly grateful for your engagement in my projects, and for always being open to my ideas and suggestions as well as for your feedback when writhing my thesis. Thank you for your friendship.

Also, I would like to thank Pål for always keeping an eye on my PhD projects, making sure our end goals were reached at all time. Your combination of kindness, righteousness, down-to-earth attitude and supreme knowledge is what makes you a unique role model and an inspiration to all leaders. I would also thank my senior supervisor Lars for providing me with human tissue samples and for your engagement in my project.

During my first year I learned that in science hard work does not necessarily equal results worth publishing. Laboratory work was challenging and for someone who had just graduated from medical school, my confidence and motivation quickly reached bottom. I realized that this was a whole new academic field of learning for me, and I gained a tremendous respect for science, the psychological aspect of it and for the work behind every scientific paper. I learned that

guidelines for clinical everyday use truly are guidelines, as opposed to universal truths that can be applied for each individual, and that there are so many variables that may influence the

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resulted in an interesting side-project when I developed a glove to use in laboratory work with mice. I was thrilled of having established a novel double KO mouse model, the SERCA2a-TLR9 KO. I hope this will encourage scientists to do follow-up studies on TLR9 in the future. My third year was mostly enticed by writing papers and keeping my motivation up.

As mentioned above, the work behind this thesis could never have been carried out without collaborators. I would like to thank Helge for his positivity, compassion and for always

reminding me how fun medicine is! I will miss our meetings discussing histological slides, and I wish you the best of luck in your retirement years. Moreover, I would like to thank my research group: pharmacist, Ingrid, for her patients with me in the lab, Katrine and Azita for assisting me with genotyping, as well as the rest of the group: Alexandra, Maria, Marina, Øystein, Jonas, Mieke, Linn, Trine, Aurelia and lastly Thor, for helping me with the statistics. Thanks to all the excellent co-authors, especially: Christen, Erik, Shakil, Håvard, Jan Magnus, Lily and Solveig.

Thank you, Ivar, for your availability, and for performing echocardiography on the mice. Thank you, Geir, for keeping an overall perspective on the project and for your contribution to the papers.

I’m truly grateful for have had the opportunity to work with such inspirational people, and I hope that my acquired scientific knowledge will be of use in the future. I would like to thank my family and my dear Magnus for your unconditional love and support. Also, thanks to all my friends for your support. I learned a lot during my three years at the institute, but mostly I

learned about myself and it gave me perspective of what truly is important in life. Finally, thanks to the patients and to all supporters of The Norwegian Health Association.

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List of papers

Paper 1

Low circulating levels of mitochondrial and high levels of nuclear DNA predict mortality in chronic heart failure

Yangchen Dhondup, Thor Ueland, Christen Peder Dahl, Erik Tandberg Askevold, Øystein Sandanger, Arnt Fiane, Ingrid Kristine Ohm, Ivar Sjaastad, Alexandra Vanessa Finsen, Anne Wæhre, Lars Gullestad, Pål Aukrust, Arne Yndestad*, Leif ErikVinge*

J Card Fail. 2016;22(10):823-8.

Paper 2

Sustained TLR9 activation promotes systemic and cardiac inflammation, and aggravates diastolic heart failure in SERCA2a KO mice

Yangchen Dhondup, Ivar Sjaastad, Helge Scott, Øystein Sandanger, Lili Zhang, Solveig Bjærum Haugstad, Jan Magnus Aronsen, Trine Ranheim, Sigve Dhondup Holmen, Katrine Alfsnes, Muhammad Shakil Ahmed, Håvard Attramadal, LarsGullestad, Pål Aukrust, Geir Christensen, Arne Yndestad, Leif Erik Vinge.

PLoS One. 2015;10(10):e0139715.

Paper 3

Toll-like receptor 9 promotes survival in SERCA2a KO heart failure mice

Yangchen Dhondup, Ivar Sjaastad, Øystein Sandanger, Jan Magnus Aronsen,Muhammad Shakil Ahmed, Håvard Attramadal, Alexandra Vanessa Finsen, Lili Zhang, Trine Ranheim, Katrine Alfsnes, Pål Aukrust, Geir Christensen,Arne Yndestad*, Leif Erik Vinge*

Mediators of inflamm. 2017;2017:9450439.

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Selected abbreviations

Absent in melanoma Coronary artery disease

Cyclic guanosine monophosphate C-type Lectin receptors

Cardiomyocyte

Cytosin phosphate Guanine Cardiovascular disease

Danger associated molecular pattern Dendritic cells

Dilated cardiomyopathy Ejection fraction

Extracellular matrix Heart failure

Heart failure with midrange ejection fraction Heart failure with preserved ejection fraction Heart failure with reduced ejection fraction Interferon beta

Inhibitory protein of kappa B Interleukin

Knock out

Lipopolysaccharide Leucine rich repeats Left ventricle MerCreMer

Myocyte chemoattractant peptide 1 Myocardial infarction

Macrophage inflammatory protein 1 Matrix metalloproteinase

Messenger RNA Mitochondrial DAMP Mitochondrial DNA

Myeloid differentiation factor 88 Nuclear factor kappa B

Nuclear DNA

Nucleotide binding oligomerization domain New York Heart Association

Pathogen associated molecular pattern Protein kinase G

Pattern recognition receptor Retinoic acid inducible gene I

Sarco/endoplasmic reticulum calcium ATP-ase AIM

CAD cGMP CLRs CM CpG CVD DAMP DC DCM EF ECM HF HFmrEF HFpEF HFrEF IFNß IκB IL KO LPS LRR LV MCM MCP-1 MI MIP-1 MMP mRNA MTD mtDNA MyD88 NF-κB nDNA NOD NYHA PAMP PKG PRR RIGI SERCA

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1. Introduction

Cardiovascular diseases (CVDs) are leading causes of death and disability in the world (1,2). A great majority of these deaths are caused by arterial atherosclerosis (3,4) resulting in stroke and ischemic heart disease, e.g. myocardial infarction (MI). Heart failure (HF) is a severe condition caused by the heart’s inability to maintain a blood flow that meets the body’s requirement.

Though MI is the most common cause of HF (5), other frequent causes may be hypertension, valvular diseases or cardiomyopathies. Moreover, other causes may be: congenital heart disease, pulmonary hypertention, heart arrhythmias (e.g. atrial fibrillation), myocarditis, pericarditis and cardiotoxic substances (e.g. alcohol) (5), as well as chronic diseases such as diabetes, HIV, hyperthyroidism, hypothyroidism, hemochromatosis and amyloidosis (6). HF involves a substantial risk of morbidity and mortality, and it is the most common condition for hospital admissions in people aged >65 years, making it a major socioeconomic burden. Although there have been great improvements in the management of this disease over the past decade, the mortality and morbidity is still high, and the disease prevalence is continuing to rise, due to an aging population, earlier diagnosis and increased awareness.

Since the 1990s numerous clinical and experimental studies have demonstrated that low-grade inflammation may play a role in the progression of HF. Cardiac stress or injury involves the release of intracellular cell debris, which initiates recruitment of inflammatory cells in an attempt to clean and heal the affected area. Though cardiac inflammation conveys protective means during initial tissue damage or infection, it may also be detrimental if deregulated or prolonged

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incomplete and increased knowledge on activation of the inflammatory pathways during HF is needed until therapy targeting inflammation can be introduced in the management of HF.

1.1 Heart failure

1.1.1. Definition and epidemiology

HF is a clinical syndrome defined by the European Society of Cardiology as an “abnormality of cardiac structure or function, leading to failure of the heart to deliver oxygen at a rate

commensurate with the requirements of the metabolizing tissues” (9). HF is recognized by the following typical symptoms: dyspnoea, fatigue, reduced exercise tolerance, orthopnoea, nocturnal cough and signs; elevated jugular venous pressure, ankle oedema, tachycardia and pulmonary crackles (5,9).

HF is a major public health issue with a prevalence of over 23 million worldwide (10) and accounts for approximately 2% of the adult population in developed countries (5,9,10) with the prevalence rising to ≥10% among people of 70 years of age or older (9). The prevalence of HF is continuing to increase due to earlier diagnosis and awareness, as well as improvements in

therapy and management of other forms of CVD (10). Moreover, HF disorder involves a 5-year mortality of 45–60% (5,11).

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1.1.2 Systolic and diastolic HF

The most common clinical parameter used to describe HF is based on measurement of left ventricular (LV) ejection fraction (EF). Mathematically, EF is described as the stroke volume divided by the end-diastolic volume ((EDV-ESV)/EDV) (9). HF with reduced ejection fraction (HFrEF) is defined as ejection fraction (EF) <40% (12), also termed systolic HF, and it is the best characterized type of HF in terms of pathophysiology and treatment (9).

Over the years, an increased awareness has been devoted to HF with preserved ejection fraction (HFpEF) (8), defined as EF≥50% (12), also termed diastolic HF. Recently, a new term for HF patients with EF 40-49% (12) was described as HF with midrange EF (HFmrEF). These patients are believed to have a mild systolic dysfunction, but with characteristics of diastolic dysfunction (12). Patients with diastolic HF accounts for at least 50% of HF cases, with only slightly lower mortality compared with patients with systolic HF (10,13). Whereas evidence-based HF

treatment has greatly improved the prognosis of systolic HF patients over the past three decades, the prognosis of diastolic HF patients has remained unchanged (14). This is supported by several clinical trials, which demonstrates positive effects on systolic HF patients by using standard HF therapy and only neutral effects on diastolic HF patients (15). However, at the current time there is an on-going debate as to whether treatment with the aldosterone antagonist, spironolactone, (TOPCAT study) could improve clinical outcomes in these patients (16) The main reason for these inadequate effects is most likely the different underlying pathological mechanisms in diastolic HF and the higher prevalence of metabolic disorders (15,17,18), as well as non-cardiac

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1.1.2.1 Diastolic HF

Diastolic dysfunction is characterized by left ventricular (LV) stiffness. The pathogenesis is complex and probably involves several mechanisms, ultimately leading to one common macroscopic phenotype featured with increased LV-filling pressure. As the main theme of this thesis is inflammation the focus in this subchapter will be on this.

Some of the main driving mechanisms behind the phenotype are alterations in the extracellular matrix (ECM) and/or in the cardiomyocyte (CM). Studies have suggested that TGF-β induced trans-differentiation of fibroblasts into myofibroblasts promotes increased myocardial collagen.

This, in addition to the level of inflammatory cells has been correlated with diastolic HF (21).

Moreover, the Health ABC study reported a strong association between inflammatory cytokines, such as interleukin (IL)-6 and tumour necrosis factor (TNF), and diastolic HF (22).The

preceding inflammation with the changes in ECM ultimately leads to fibrosis. Moreover, metabolic disorders, oxidative stress, reduced nitric oxide bioavailability and down-regulated NO-mediated cyclic guanosine monophosphate (CAMPS) and protein kinase G (PKG)

signalling, have been linked to LV dysfunction. These factors may all contribute to the changes in titin, a sarcomere protein, and are believed to enhance CM and LV stiffness. The consequence of a stiffened heart is increased filling pressures to preserve normal LV end-diastolic volumes (23). Other important driving mechanisms behind diastolic HF are impaired LV active

relaxation. Interruptions in the Ca²⁺-handling of cardiac cells may lead to impaired relaxation of LV during the diastolic phase of the cardiac cycle. The change in CM Ca²⁺ homeostasis is a hallmark of HF pathogenesis, and is thought to underlie both mechanical and

electrophysiological dysfunction in HF (24).

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1.1.3 Sarco/endoplasmic reticulum Ca

²⁺

-ATPase (SERCA) and regulation of Ca

²⁺

homeostasis in HF

The Sarcoplasmic reticulum (SR) constitutes the main compartment for Ca²⁺ storage in cardiac cells (25). Excitation-contraction coupling includes Ca²⁺ influx through sarcolemma L-type channels. This involves Ca²⁺ induced Ca²⁺ release from the SR through ryanodine receptor channels with subsequent binding of Ca²⁺ to myosin, triggering contraction. Ca²⁺ re-enters into the SR via the SERCA pump and cellular efflux is conducted through the Na⁺-Ca²⁺ exchanger (Figure 1)(25). In human HF, excitation-contraction coupling is impaired (24) with less

SERCA2a protein expression (25) and altered phosphorylation of phospholamban, the regulator of SERCA activity (26). Along with increased Ca²⁺ leakage through ryanodine receptor

channels, this causes high cytosolic and low SR Ca²⁺ concentrations resulting in increased diastolic Ca²⁺ content (27). In mammals, there are three genes encoding several SERCA protein isoforms. SERCA1a and SERCA1b are expressed in adult and neonatal fast-twitch skeletal muscles, whereas SERCA2a is selectively expressed in heart and slow-twitch skeletal muscles.

SERCA2b is expressed nearly ubiquitously, thus considered the housekeeping isoform and SERCA3 is expressed in a limited number of non-muscle cells (28). Brody´s and Darier´s disease are two human genetic diseases associated with mutations in the SERCA pump (28).

Though these conditions are relatively rare, gene modified SERCA2a KO mice are used experimentally to study diastolic dysfunction (29,30). As opposed to other murine HF models, the SERCA2a KO mice display prolonged relaxation deficit due to slowed rate of Ca²⁺uptake (31) This results in a diastolic dysfunction, with subsequent enlargement of the left atrium,

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Modified from Bers DM: Nature. 2002;415:198-205

Action potential

SERCA2a

Figure 1. Excitation-contraction coupling

An action potential induces Ca²⁺influx through sarcolemma L-type channels. Ca²⁺induced Ca²⁺release from the SR through ryanodine receptor channels with subsequent binding of Ca²⁺ to myosin, triggers contraction. Ca²⁺ re-enters into the SR via the SERCA pump and cellular efflux is conducted through the sodium Na⁺-Ca²⁺exchanger.

1

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1.1.4 Myocardial remodelling

Myocardial remodelling may be caused by tissue loss, pressure overload (aortic stenosis) and/or hypertension), inflammatory heart muscle disease (myocarditis), idiopathic dilated

cardiomyopathy (DCM) or volume overload (valvular regurgitation) (32). Moreover, remodelling involves an increase in heart size, a more spherical shape and altered cardiac function in response to cardiac injury (32,33). At a molecular/cellular level, remodelling is characterized by cardiac myocyte growth, re-expression of foetal genes, changes in the

expression of proteins involved in excitation-contraction coupling, changes in myocyte energetic and metabolic state, as well as necrosis, apoptosis, oxidative stress and changes in the ECM (32).

When cardiac function is disrupted, the body elicits countermeasures to maintain hemodynamic homeostasis, e.g. fluid retention, release of neurohormones, increased sympathetic drive (34).

However, over time these mechanisms turn maladaptive and promote development of HF. To characterize the mechanisms that turn from adaptive into maladaptive responses is one of the major tasks in HF research.

Though the CM is an essential cell involved in the remodelling process, the interstitium, fibroblasts, coronary vasculature (32) and inflammatory cells (35) are likewise important contributors. Fibroblasts play a key role in ventricular remodelling (36), and are responsible for maintaining the balance between synthesis and degradation of the ECM, which consists of collagens, proteoglycans, glycoproteins, growth factors, cytokines and proteases. Such proteases termed matrix metalloproteinases (MMPs) degrade collagen-especially MMP-9 in humans.

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these changes become maladaptive if the inflammatory response is prolonged, leading to fibrosis and myocardial dilatation eventually causing systolic HF (36).

Several studies have suggested that the neurohormones that are involved in the renin-

angiotensin-aldosterone system (RAAS) and the adrenergic system, contribute to inflammation (37,38). Animal experiments on angiotensin II (ATII) have shown inflammatory cytokine induced inflammation in endothelium (39). Also, AT II (40) and aldosterone (41) induced intracellular ROS production with subsequent inflammation has been demonstrated. Moreover, as monocytes and lymphocytes express β-adrenergic receptors, experimental studies have shown that increased catecholamine may induce inflammatory responses (42). This may suggest that neurohormones, induced by RAAS-and the β-adrenergic activation, perhaps represents

components that interact with inflammatory cytokines rather than them being separate systems (43).

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1.2 Inflammation in CVD/HF

In 1990 Levine and colleagues reported elevated levels of circulating TNF in patients with chronic HF (44), and this finding lead to a new research area: inflammation in HF. Since then, numerous clinical studies have implied activation of inflammatory pathways both locally in the heart and in blood as a potentially important pathological event in the initiation and progression of the syndrome (45,46,47,48).

1.2.1 Inflammation and cytokines

Inflammation is a generic response, and therefore considered a mechanism of innate immunity, which is vital for host defence to eliminate the initial cause of cell injury, clear out necrotic cells and induce tissue repair (49). In response to infections, cascades of signals lead to the

recruitment of inflammatory cells to the affected area such as neutrophils and macrophages.

These cells phagocytize infectious agents and produce inflammatory mediators such as cytokines and chemokines which are pharmacologically active low weight proteins that are secreted from a variety of different cell types, and may promote either autocrine and/or paracrine effects. This leads to activation of lymphocytes and elicit adaptive responses. However, in the absence of pathogens the inflammatory response is also essential for tissue and wound repair, e.g. ischemia- reperfusion (I/R) injury or chemical damages. This type of inflammation is termed “sterile inflammation” indicating the absence of microorganisms (50). Though the initial phase of sterile inflammation is beneficial, e.g. post- MI, too much and prolonged inflammation is detrimental, causing harmful remodelling and eventually HF with severe hemodynamic stress.

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1.2.2 Inflammation in clinical HF

Several clinical studies have shown that some inflammatory markers play a significant role and can provide prognostic information in HF patients (51,52,53). Reports show that increased cytokines such as interleukin (IL)-1 (54), IL-6 (55) TNF and IL-1β concentrations are associated with poorer prognosis, which may suggests that they reflect important pathogenic pathways during HF (56,57,58). Moreover, a paper in 2014 demonstrated that inflammatory markers such as IL-6, TNF and C-reactive protein (CRP) were associated with HF risk and could predict the development of diastolic HF (HFpEF) (59).

In chronic HF the most important and most studied cytokine is TNF (55) and it has been recognised as a key cytokine involved in the remodelling process (7, 60,61,62,63). However, despite the knowledge that TNF and other cytokines are strongly associated with HF, several clinical trials have been unsuccessful to reach primary end points in the attempt to antagonize inflammatory mediators, e.g. TNF (58). Several studies have been conducted on the soluble TNF receptor, etanercept on HF patients. The RENEWAL trial resulted in chronic HF hospitalization and/or death (58,64) and the RECOVER and RENAISSANCE trial observed no increase in mortality, however none of them showed improvement in HF either (58). Another anti-TNF therapy against HF was the ATTACH trial, with the use of a human monoclonal antibody with an anti-TNF murine Fab, named infliximab. High-dose infliximab resulted in increased mortality and HF hospitalization and as a consequence of this study, high-dose infliximab became

contraindicated for HF patients (58). In addition to anti-inflammatory therapy, other approaches to counteract inflammation in HF have been attempted, broad-based immunomodulators, e.g.

intravenous immunoglobulin (IVIg) against a total imbalanced cytokine network, rather than only one cytokine (56,65). Also, methotrexate and immune modulation therapy and even

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autoimmune therapies have been suggested (58). At the current time, there are exciting on-going trials on inflammation as a target for CVD.The CIRT trial (66) is assessing whether low-dose methotrexate reduces MI, stroke or death in patients with type 2 diabetes or metabolic syndrome who have had heart attack or stable CAD. The CANTOS trial (67) is assessing whether

inhibition of IL-1β with canakinumab can reduce MI, stroke and death in post MI patients with increased CRP.

Of other cytokines associated with HF, is IL-6, in which increased circulating levels have been associated with CM hypertrophy, myocardial dysfunction and myocyte atrophy (55). Other cytokines, such as IL-8 and macrophage inflammatory protein-1 (MIP-1), have also strongly been associated with cardiac remodelling (7,48,65,68,69,70). Of the anti-inflammatory cytokines, IL-10 is considered the most important as it down regulates TNF, IL-6 and IL-1 synthesis (55).

Though we do not know the complete underlying mechanism, systemic inflammation is believed to play a central role in the progression on HF (43). This is particularly relevant as many HF patients have comorbid diseases, and may be increasingly important with a growing elderly population. Nevertheless, it seems that no single inflammatory cytokine provides sufficient discrimination to justify the transition to everyday clinical use as a prognosticator in HF.

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1.2.3 Pathogenic role of local and systemic inflammation in HF

Clinical data supports the hypothesis of persistent low-grade myocardial and systemic inflammation in HF. Still, we do not fully understand the implications of this inflammatory activation in patients with HF, i.e., whether it is beneficial, detrimental or simply that it does not affect the progression of the clinical disorder. However, although clinical evidence is still ambiguous, numerous experimental studies demonstrate a pathogenic role of several inflammatory cytokines in HF.

Primarily, HF involves inflammation induced by non-infectious pathological events such as hemodynamic overload and stress in several cell types, or tissue hypoxia and ischemia through the production of reactive oxygen species (ROS) and Nuclear factor kappa B (NF-κB)

transcription which all lead to cytokine production (43). NF-κB is the main regulator of transcription of inflammatory genes. As indicated initially in this chapter, the disease is characterized with local cardiac inflammation involving the myocardium itself with both CMs and non-CMs such as fibroblasts, smooth muscle cells, endothelial cells, e.g. TNF production from the heart (71,72). Such local inflammation may activate immune mediators and lead to the activation of proinflammatory cytokines (55) in an autocrine or paracrine manner (43). However, such release from the heart may activate extra-myocardial tissue cells that contribute to this inflammation, e.g. leukocytes, platelets and macrophages as well as peripheral organs, e.g. liver and lungs (43), thus inducing a low-grade systemic inflammation (44,48,71,72). As there is strong evidence of increased myocardial and circulating proinflammatory cytokines during HF, this has been suggested to be a consequence of increased NF-κB activity (55).

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As in human HF trials, TNF has been thoroughly studied in animal HF models. TNF binds to TNF receptors, and while type I (TNF-RI) activation has shown detrimental effects, type II (TNF-RII) activation has shown protective effects (73). Still, most animal studies seem to indicate beneficial effects by inhibiting TNF in general (74, 75). Reports on rats (76) and dogs (72) have shown that infusion with TNF reversibly impairs cardiac function, and the latter lead to systolic dysfunction (77). Moreover, several studies on transgenic mice that overexpress cardiac TNF have been conducted and demonstrate detrimental effects (78, 79,80,81).

Among other inflammatory cytokines that have been studied in animals is IL-6, which has been shown to induce negative inotropic effects (82,83,84) as well as hypertrophy, fibrosis, and diastolic dysfunction (85). IL-1β is a cytokine in the IL-1 family that has gained growing attention as previous clinical studies have shown a significant beneficial effect by inhibiting IL- 1β (55,86,87). Studies have shown that IL-1β deficient mice display cardiac dysfunction (63,87).

This suggests that IL-1β may aggravate hypertrophic remodelling (88).

As in clinical HF trials, inhibition of proinflammatory responses have been thoroughly investigated as a therapeutic strategy experimentally. IL-10 is a major anti-inflammatory cytokine, and IL-10 deficient mice have demonstrated increased cardiac hypertrophy, fibrosis and cardiac dysfunction in response to isoproterenol (89). However, IL-10 injections have even shown to significantly reduce cardiac hypertrophy, fibrosis and preserve cardiac function in different models of cardiac hypertrophy as well (89).

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1.2.4 The role of macrophages in the failing heart

More recently the research focus has changed from cytokines to the role of inflammatory cells per se. In healthy murine hearts, resident macrophages constitute about 6-8% of the non-CMs (90,91). Macrophages are specialized mononuclear phagocytes (92) that ultimately derive from the haematopoietic CD34+ stem cells in bone marrow (93). However, before entering the organ or tissue, the precursor cells of macrophages while circulating in the blood, are monocytes.

When they finally enter the injured myocardium, they differentiate into macrophages in response to different chemokines (92): macrophage colony stimulating factor (M-CSF)- which is

important for macrophage survival (94), TNF (95), platelet derived endothelial cell growth factor (PD-ECGF) and transforming growth factors α and β (TGFα and β), which indirectly contribute to fibrosis (96). Also IL-1 and insulin-like growth factor (IGF) are among the important

mediators (97).

Post MI remodelling involves three stages: inflammation, scar formation and scar remodelling with overlapping time frames (98). The removal of dead cell debris followed by wound healing is considered the primary role of the macrophage post-MI (98). The initial innate immune response is characterized by an early phase and a reparative phase, the latter occurs around day 3. The early phase involves mobilisation of neutrophils and monocytes to the necrotic tissue and the reparative phase involves macrophage phenotype transformation, followed by fibroblast activation and collagen synthesis, which is necessary for scar formation. Both of these phases are crucial for both mice and humans (99). Moreover, macrophages stimulate endothelial cell

induced angiogenesis, which is initiated by tissue hypoxia (98,99,100). Macrophages express MMPs, in which MMP9 may be the most important in post- MI remodelling (100). In addition to contributing to angiogenesis, MMPs degrade collagen during remodelling. Macrophages also express tissue inhibitor of metalloproteinases (TIMPs), which inhibit MMPs, thus the balance

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between MMPs and TIMPs determine net LV remodelling (101). The two macrophage

activation patterns are: 1) the proinflammatory M1 macrophage activation (classical) and 2) the anti-inflammatory M2 macrophage activation (alternative), and they display different markers.

Porcheray and colleagues demonstrated that macrophage activation occurs first through the M1 pathway, and then shifts to the M2 pathway, and this shift between pathways is reversible.

However, as both pathways are activated at varying time-point post-MI, both subgroups are most likely to be present simultaneously (102). The role of cardiac-resident macrophages in chronic post-MI remodelling is not yet fully understood (99). The balance between the two macrophage activation phases as well as the interaction between collagen synthesis and degradation are among the many factors that determine the net LV remodelling process. At this point, we do not completely understand this interaction and how the quantitative relationships of the mediating factors are regulated. Though inflammation is necessary for optimal wound healing in post-MI remodelling, scenarios where too much inflammation resulting in prolonged remodelling, seems to be detrimental.

As mentioned above, mechanical overload and oxidative stress may initiate inflammation.

However, studies show that certain components of the innate immune system are able to recognize specific molecular patterns in pathogens. These pattern recognition receptors (PRRs) are able to activate signalling pathways that lead to the production of cytokines and chemokines, which attract leukocytes to the affected area and ultimately combat harmful microbes (103). One subgroup of PRRs is the Toll-like receptors (TLRs), in which they do not only respond to

microbes but potentially also non-infectious agents with similar structures. Studies have shown

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1.3 The innate immune system

1.3.1 PRRs and PAMPS

As discussed above, an important step in the elucidation of new inflammatory target for therapy in HF, is to more precisely characterize the inflammatory pathways that are activated during these complex disorders. In the present thesis we focus on the role of Toll-like receptor-9 (TLR9), a component of the innate immune system.

The innate immune system consists of germ line-encoded PRRs, and recognizes evolutionary conserved structures termed pathogen associated molecular patterns (PAMPs). PAMPs are found in bacteria, viruses or fungi (104,105) and include microbe-specific carbohydrates, lipids and peptides or combinations of these components. Different PRRs recognize different agents, and they are expressed in macrophages, dendritic cells (DC), phagocytes and B-lymphocytes

(104,105) as well as in non-immune cells such as endothelial cells, fibroblasts (106,107,108) and CMs (109,110). Until now, five major groups of PRRs have been discovered, and they have been classified depending on their location within the cell: cytoplasmic PRRs include Retinoic acid inducible gene I (RIGI)-like receptors (RLRs), Nucleotide binding oligomerization domain (NOD)-like receptors (NLRs) and Absent in melanoma (AIM)-like receptors (ALRs)

(111,112,113). Transmembrane PRRs include C-type Lectin receptors (CLRs) and the TLRs.

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1.3.2 TLRs

Recent studies have shown that TLRs are involved in the inflammatory response during HF, and represent a bridge between infectious and non-infectious derived inflammation in stressed, injured and dead cells (114,115). The first TLR, “Toll”, was first identified in 1985 in a fruit fly (Drosophila melanogaster) (116,117). Over the years, ten human and twelve murine

homologues of the Drosophila TLR have been found, of which TLR 1-9 are the best characterized. TLRs are transmembrane signalling receptors localized either in the plasma membrane (TLR 1, 2, 4, 5 and 6) or within endolysosomes (TLR 3,7,8 and 9). The

transmembrane TLRs recognize microbial cell wall structures such as lipopeptides (TLR2), glycolipids (TLR4) or flagellin (TLR5) while endolysosomal TLRs recognize nucleic acids as RNA (TLR3, TLR7, TLR8) or DNA (TLR9) (108, 118,119). They have mostly been studied in immune cells, however TLR2, -3, -4, -5 and -9 have been identified in CMs as well

(43,120,121).

TLRs are type 1 integral membrane glycoproteins composed of a horseshoe shaped N-terminal ectodomain consisting of leucine rich repeats (LRR) which are responsible for ligand binding.

The trans membrane section is composed of a single membrane spanning helix terminating in a C-terminal cytoplasmic domain (122), and binding is either direct or indirectly dependent on the presence of a co-receptor. The cytoplasmic domain is termed the Toll/interleukin-1 receptor (TIR) domain, homologous to IL-1/IL-18 receptors. Upon activation, all TLRs form either homo-or heterodimers in M-shaped complexes, and the two dimerized TLRs frequently share a

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signalling may either be 1) myeloid differentiation factor 88 (MyD88) adaptor protein- dependent or 2) TIR-domain-containing adaptor-inducing interferon beta (IFNß)-dependent (TRIF) (123) (Figure 2). Each TLR recognizes specific PAMPs or danger associated molecular patterns (DAMPs). The latter is released in the absence of pathogens and induces “sterile inflammation” (124).

Modified from Kawai T: Immunity. 2011: 637–50

Figure 2. Overview of TLR signalling pathways

Signalling pathways in inflammatory cell types such as macrophages (MP), inflammatory monocytes (iMO), plasmacytoid dendritic cells (pDCs), conventional dendritic cells (cDCs) and lamina propria DCs (LPDCs).

Heterodimers of TLR1-2 and TLR2-6, as well as TLR4 and TLR5 are expressed on the cell surface. PAMP induced recruitment of adaptor proteins such as MyD88, TIRAP, TRIF, and TRAM leads to NF-κB induced production of inflammatory cytokines. In steady state, TLR3, TLR7, and TLR9 are primarily localized in the endoplasmic reticulum (ER). Upon activation, they traffic to the endosomes mediated by the chaperon protein, UNC93B1. TLR7 and TLR9 may initiate two signalling pathways depending on cell type; In pDCs, TLR7 and TLR9 signalling may lead to MyD88-dependent 1) NF-κB mediated signalling from the endosome or 2) IRF7 mediated signalling from the lysosome-related organelle (LRO) after the receptors are transported from the endosome. These two pathways lead to

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1.3.3 DAMPs-Mediators in CVD

DAMPS are endogenous molecules, largely released upon pathological stimuli, e.g. cellular stress or damage. This results in sterile inflammation (125,126) through activation of PRRs.

Different classes of DAMPs have been suggested: (i) cell derived, e.g. crystalline uric acid (127), heat shock proteins (128), high mobility group box 1 (HMGB1) (129), and nucleic acids (130) (ii) derived from breakdown of ECM, e.g. hyaluronic acid (131) and fibronectin (132,133) or (iii) plasma derived, e.g. oxidized LDL and palmitate (134). Increased circulating levels of DAMPs have been described in several clinical studies, e.g. patients with massive lung embolism (135), patients with cancer (136,137) or autoimmune diseases, e.g. rheumatoid arthritis (138,139,140). Most importantly, experimental studies using mice deficient in different PRRs strongly suggest pathogenic effects of DAMPs during sterile inflammation. Recently, Zhang and colleagues found elevated plasma levels of mitochondrial DNA (mtDNA) in trauma patients (141), and reports suggest that mitochondrial DAMPs consisting of N-formyl peptides and mtDNA are able to induce inflammation, and more specifically activate TLR9 (142).

1.3.4 TLR9 can be activated by mtDNA

Hemmi and colleagues first described TLR9 in 2000 as a TLR recognizing bacterial DNA (142).

TLR9 was primarily studied in B-lymphocytes and pDCs. However, recent reports suggest that TLR9 is also expressed in numerous other cellular entities like cardiac fibroblasts, monocytes, neutrophil granulocytes, respiratory epithelial cells, endothelial cells, vascular smooth muscle

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conformational changes as well as cleavage of the ligand binding LRR-domain, resulting in a functional receptor (147,150). Researchers have proposed that it is the cleavage of the C-

terminal fragment, which mediates ligand recognition (151,152) however the details behind this are not yet fully understood yet.

TLR9 is activated by specific nucleotide sequences named unmethylated cytosine-phosphate- guanine (CpG)-DNA-repeats. These are abundant in bacterial DNA and mtDNA, however rather limited in nuclear mammalian DNA. The similarities of mtDNA to bacterial DNA are thought to be a consequence of a distant common evolutionary step. According to the endosymbiotic

theory, a prokaryotic cell fused with a pre-eukaryotic cell, thus establishing the mitochondrion, necessary for multicellular life. When mtDNA is endocytosed into the endolysosomes, it

activates the residing TLR9 leading to a cascade of downstream signalling. Still, we do not know which part of the CpG structure is responsible for TLR9 activation. Studies have shown that oxidation of mtDNA may be of importance to the binding process. Others propose that the binding is independent of the CpG-motif base composition and that mtDNA phosphodiester 2’- deoxyribose backbone is responsible for activation (153). Furthermore, there have been reports suggesting that the preceding stages of endosomal compartmentalization of TLR9 is essential for the receptor to discriminate between endogenous and pathogenic DNA, and subsequent ligand binding to TLR9 (147,153,154).

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1.3.5 TLR9 in the heart

Since the report by Hemmi and colleagues (142), only a handful of studies have investigated TLR9’s role in the heart and even fewer studies have examined its role in HF. At this stage most studies have been conducted on animal models, although recently there have been a few studies on mtDNA in humans.

In 2010, Zhang and colleagues observed increased circulating mitochondrial DAMPs (MTDs), consisting of formal peptides and mtDNA, in trauma patients with muscle injuries. Moreover, the researchers found that MTDs from human liver, myositis and fracture haematoma attracted polymorph nuclear neutrophils (PMN). This was also demonstrated in rat muscle and liver. As several studies demonstrate that CpG can activate TLR9 in PMNs (155,156,157), Zhang and colleagues wondered if mtDNA induces similar responses at clinical plasma levels. Thus, in vitro, they incubated PMN with CpG or mtDNA, and observed co-existent low dose N-formal- Met-Leo-Phi (fly), a synthetic peptide that simulates bacteria, lead to IL-8 release. This finding demonstrated clinically significant activation of PMN secretion by mtDNA/TLR9 and that activated TLR9 could elicit organ injury in a sepsis-like manner (139). Moreover, they introduced these DAMPs as representatives of a link between trauma and systemic inflammation, i.e. systemic inflammatory response syndrome (SIRS). In 2012, our group compared circulating mtDNA in patients with ST-segment elevation MI (STEMI) to patients with stable angina, after being treated with percutaneous coronary intervention (PCI)(158). We found that 3 hours post PCI mtDNA levels were significantly increased in the STEMI group,

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group, mtDNA levels after 3 hours correlated with maximum troponin T levels. This study demonstrated for the first time that focal myocardial necrosis due to MI could lead to the release of mtDNA, and that the plasma levels correlated with the degree of myocardial damage.

As mentioned initially in this chapter, most studies on TLR9 in the heart have been conducted on various animal models. Experiments on mice have shown that 4 hours of pre-treatment with CpG followed by pressure overload HF induced by transverse aortic constriction (TAC) 12 hours later, can attenuate cardiac hypertrophy and function (159). The study by Velten and colleagues demonstrated that priming with CpG could attenuate the inflammatory response by modulating cardiac gene expression as well as cellular growth and proliferation. The latter was seen as reduced CCL2 and CCL4 and reduced macrophage activation and infiltration (159).

Moreover, CpG pre-treatment attenuated collagen deposition in TAC induced HF. The net result was attenuated hypertrophy, remodelling and LV function. Another study observed that priming with CpG 1 hour prior to myocardial ischemia followed by reperfusion, could attenuated

apoptosis and reduced infarct size determined by Triphenyltetrazolium chloride (TTC) staining through PI2K/Act signalling pathway (160). A recent study from our group, investigated mice subjected to 30 minutes of ischemia, immediately followed by injections with CpG and 24 hours later hearts were re-perfused (146). Though TLR9 activation displayed evidence of reduced cardiac monocyte and granulocyte infiltration, paradoxically there were increased circulating immune cells. Moreover, TLR9 activation increased several cardiac inflammatory genes.

However, CpG induced systemic TLR9 activation upon ischemia/reperfusion (I/R) did not influence infarct size despite several alterations in inflammatory parameters (146).

Though TLR9 activation evidently have shown salutary effects, some studies challenge this view

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by demonstrating adverse effects. One study by Knuefermann and colleagues demonstrated that CpG stimulation wild type (WT) mice with intact TLR9 resulted in a clear inflammatory

response by several cytokines, e.g. TNF, IL-6 and IL-1β (161). This was not seen in the TLR9 knock out (KO) mice. Moreover, isolated CMs from the CpG induced WT mice displayed reduced contractility (161). Boehm and colleagues supported this finding by demonstrating septic HF and increased mortality in WT mice injected with CpG i.p., followed by

pharmaceutical inhibition 30 minutes after. By comparison, these effects were not present in TLR9 KO mice (162). Finally, in 2012 Oka and colleagues published a scientific work,

supporting the harmful effects that had been reported in previous research. They demonstrated that mtDNA that escapes from autophagy, can activate cardiac TLR9 within lysosomes resulting in inflammation and cardiac dysfunction. The group studied in vivo CM-specific

deoxyribonucleic (DNase)2a inhibition, i.e. DNase2a KO mice, that were subjected to TAC which lead to increased intracellular mtDNA induced TLR9 signalling, since mtDNA was not degraded. This involved early increase in cardiac infiltration of inflammatory cells and increased messenger RNA (mRNA) expressions of several cytokines. Ten days after TAC, the DNase2a KO mice developed severe HF and demonstrated increased mortality (163). Moreover, both TLR9 depletion and pharmaceutical inhibition of TLR9 demonstrated attenuated cardiac function. Even WT mice with intact DNase2a and TLR9 presented with less inflammation and improved cardiac function.

The above-mentioned studies emphasize the complexity of studying TLR9 activation in hearts, as there are many considerations to make when interpreting the ambiguous results. The research on TLR9 in the heart is far from settled. As long as HF remains a challenge worldwide and more

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2. Aims of the thesis

We hypothesized that mtDNA is released during chronic HF and may impact cardiac function by activating TLR9. This hypothesis was investigated using differential experimental approaches combined with analyses in clinical material from patients with HF. Our specific aims were to:

1. Analyse circulating levels of mtDNA and nuclear DNA (nDNA) in HF patients and to investigate their correlations to clinical and biochemical parameters.

2. Determine the pathophysiological consequence of sustained systemic TLR9 stimulation in experimental chronic HF.

3. Explore the pathophysiological consequence of attenuated TLR9-signalling in experimental chronic HF.

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3. Summary of results

Paper 1

Low circulating levels of mitochondrial and high levels of nuclear DNA predict mortality in chronic heart failure

Aim: We aimed to investigate circulating levels of mtDNA and nDNA from 84 chronic HF patients with New York Heart Association (NYHA) functional class I-IV.

Our main findings:

• High circulating levels of nDNA are associated with increased mortality.

• High circulating levels of mtDNA are associated with increased survival.

• Patients with HF have increased circulating mtDNA and nDNA compared to controls.

Conclusion: Plasma levels of mtDNA and nDNA are elevated in human HF. High levels of nDNA are associated with mortality, whereas elevated levels of mtDNA are associated with increased survival. This study suggests a rationale for exploring TLR9, a putative mtDNA receptor, as a new target in treatment of human HF.

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Paper 2

Sustained TLR9 activation promotes systemic and cardiac inflammation, and aggravates diastolic heart failure in SERCA2a KO mice

Aim: We aimed to investigate the impact of sustained, systemic TLR9 activation on cardiac and systemic inflammation in SERCA2a KO HF mice and the consequences on HF progression and phenotype.

• Sustained TLR9 stimulation increases cardiac monocyte/macrophage infiltration and cytokine mRNA expression, as well as systemic lymphocyte infiltrations in lungs and liver in SERCA2a KO mice.

• Sustained TLR9 stimulation aggravates HF and promotes premature death in SERCA2a KO mice.

Conclusion: Sustained activation of TLR9 causes cardiac and systemic inflammation, and deterioration of SERCA2a depletion-mediated HF.

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Paper 3

Toll-like receptor 9 promotes survival in SERCA2a KO heart failure mice

Aim: We aimed to investigate the consequences of endogenous TLR9 signalling in SERCA2a KO HF mice.

• The absence of TLR9 promotes a significant premature death in SERA2a KO HF mice despite no echocardiography, biochemical or histological evidence of altered HF phenotype.

Conclusion: In mice with SERCA2a depletion-mediated diastolic HF, the absence of TLR9 reduces life expectancy compared to mice with cardiac TLR9 present. Despite thorough investigation to what may have caused the premature death, we were unsuccessful to pinpoint what was causing the difference in HF phenotype between mice with and without TLR9. Thus, further studies on alternative explanations need to be conducted.

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4. Methods

4.1 Establishment of SERCA2a KO model

In paper 2 and 3 in this thesis, we used the conditional SERCA2a KO model based on the Cre- lox P method first described in the early 1980s (164,165). Christensen and colleagues (30,166) established the lox-P-flanked SERCA2a model in which the gene resides between two loxP sites (170). By crossing this model with a mouse with the Cre-lox P gene, this resulted in the

MerCreMer (MCM) SERCA2a^flox/flox. MCM is a fusion protein, consisting of the Cre enzyme and two oestrogen-binding domains sensitive to tamoxifen (anti-oestrogen). MCM activation by tamoxifen, leads to Cre-recombinase mediated excision of the SERCA2a gene (173). Cre is controlled by a CM-specific mediated promotor; α-myosin heavy chain (α-MHC) (Figure 3).

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Reprinted and modified with permission from Professor Ole M. Sejersted: ”Lessons from the SERCA knock-out mouse” (Sejersted O.M. M.D. PhD. Lessons from the SERCA knock-out mouse. Lecture obtained as power point presentation on 4^th December 2015,IEMR.)

Figure 3. SERCA2 gene modification in CMs of adult mice

A) SERCA2 prior to gene excision is named SERCA2 ^flox/floxwith loxP sites on both sides of the target gene. After gene excision, i.e. SERCA2 KO, the gene is inactivated, i.e. not able to re-distribute Ca²⁺ into the sarcoplasmic reticulum during diastole. This leads to increased Ca²⁺ concentration in the cytosol and thus a relaxation deficit.

B) MCM is a fusion protein, consisting of the Cre enzyme and two oestrogen-binding domains, sensitive to tamoxifen. MCM activation leads to Cre redistribution into nucleus and SERCA2a gene excision. Cre is controlled by a CM-specific mediated promotor; α-myosin heavy chain (α-MHC).

FF

KO SERCA2a^flox/flox

Exon Exon

LoxP1 LoxP2

LoxP1/2 SERCA2a KO

3A

Inducible SERCA2a excision

(Andersson KB 1998-2003) SERCA2a gene modification

(Christensen G 1996-1998)

M M M

3B

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4.2 Establishment of SERCA2a-TLR9 KO model

In paper 3, we employed a three-generation breeding strategy by crossing the αMHC-MCM- SERCA2a ^flox/flox model with the single TLR9 KO, giving rise to four comparable mouse lines consisting of two HF models (SERCA2a KO and SERCA2a-TLR9 KO) and two control models:

WT and TLR9 KO (Figure 4). The breeding strategy was based on expected number of offspring per female mouse. By crossing the conditional KO and the TLR9 KO, there were three sets of genes that had to be merged into one animal. These genes were Cre, the floxed SERCA gene and the deleted TLR9. The breeding strategy is illustrated in Figure 4A in which the SERCA KO was crossed with the TLR9 KO, establishing a 50% chance of an offspring with all three genes (heterozygous). In the second generation, displayed in Figure 4B, we crossed two heterozygous animals, which resulted in the desired homozygous floxed SERCA, the TLR9 KO and Cre.

Normal gene

^MCM

^SERCA2a ^TLR9

4A G1

Normal gene

^MCM

^SERCA2a ^TLR9

G2

4B

Normal gene

^MCM

^SERCA2a ^TLR9

G3 4C

Figure 4. Establishment of the SERCA2a-TLR9KO model

A) In Generation one (G1), a MCM-SERCA2a ^flox/flox was crossed with the single TLR9 KO, giving rice to mice (G2)

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4.3 Ethics

In paper 1, all patient and control subjects that were recruited at Oslo University Hospital entered the study voluntarily after receiving appropriate study information and signing consent forms. The study protocol and all human tissue sampling were approved by the Regional

Committee for Medial and Health Research Ethics and conformed to the Declaration of Helsinki.

In paper 2 and 3, all animals were cared for according to the Norwegian Animal Welfare Act, which conforms to the National Institutes of Health guidelines (NIH publication no. 85– 23, revised 1996). Experiments were approved by the Norwegian National Animal Research

Committee (paper 2 FOTS ID 5319; paper 3 FOTS ID 6941) and conformed to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1985).

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5. Methodological considerations

5.1 Human study and control subjects

A prerequisite in clinical studies is to have suitable patient populations and comparable healthy control subjects. Patients with chronic HF in NYHA I-IV with stable LVEF ≤ 55 were recruited and consisted of a mixture of HFrEF, HFmrEF and HFpEF. Though, the majority of the

population consisted of HFrEF patients (n=71), and the rest consisted of HFmrEF patients (n=12) and one HFpEF patient. As the patient samples were assessed at our tertiary hospital;

Oslo University Hospital, Rikshospitalet, Oslo, Norway, most of them were categorized as NYHA II and III (Distribution: NYHA I n=5; NYHA II, n= 29; NYHA III n=40, NYHA IV n=10). This may lead to a systematic patient population bias, however by merging NYHA I/II and III/IV, we were able to minimize this effect. The patients were carefully clinically

characterized according to patient history, routine physical examinations, echocardiography and coronary angiography. Based on these measurements, the underlying cause of HF was classified as coronary artery disease (CAD), DCM (genetic) or other sub groups (hypertrophic

cardiomyopathies, aortic insufficiency, unknown aetiology). NYHA classification was based on the patient’s subjective report. To limit confounding factors and increase homogeneity, patients with acute coronary syndromes within the last 6 months, congenital heart disease, post-radiation affected hearts and right ventricular (RV) diseases as well as concomitant diseases, e.g.

malignancies, autoimmune disorders, or liver or kidney failure, were excluded.

Since age and gender affects immune responses (167,168), we found it crucial to include comparable healthy controls. Seventy-two age- and gender matched healthy blood donors with

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no prior medication requirements except contraceptives, allergy medication or medication for hypothyroidism, served as controls. In addition, controls were selected based on case history and clinical examination and a few selected blood samples within normal range limits (CRP,

proBNP, haemoglobin, leukocyte count, creatinine, cholesterol and metabolic tests).

Unfortunately, we did not obtain complete data of all patients and controls.

Venous blood samples were analysed for mtDNA and DNase1, however as they may be influenced by several factors it was necessary to standardize the collection criteria, processing and storage. To avoid contamination, samples were kept in pyrogen-free tubes with EDTA as anticoagulant (plasma) or no addition (serum). To further avoid induction of inflammatory responses, storage at room temperature was kept to a minimum by immediately placing samples on ice and centrifuging them within 15 minutes at 2000g for 20 minutes (plasma) or allowed to clot at room temperature for <1 hour before centrifugation at 1500g for 15 minutes (serum).

Similar to cytokine measurements from blood samples, DNA is affected by time and temperature as well as the frequencies of freeze and thaw cycles prior to analysis (169) and repeated freeze thawing will inevitably accelerate the activity of circulating DNase1. In paper 1, all blood samples had been frozen and thawed minimum 2 times prior to the experiments, whereas control samples had been thawed less than three times which is considered acceptable (169). However, all samples had been stored at -80°C until assayed. For most analyses in this thesis the choice of serum or plasma was dictated by the availability of samples.

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5.2 Mouse models of HF

The overall goal in medical research is to increase the knowledge of human diseases and to discover new treatment modalities. However, the majority of research projects with goals of reaching mechanistic insight are not immediately accessible using human patients, due to ethical considerations, and this warrants the use of experimental animal models. In our projects we have used the mouse as an experimental animal model.

There are several genetic murine models to study HF, among others: 1) overexpression of a specific gene 2) Gene KO mice. One example of gene overexpression models that may represent clinical HF is muscle lim protein KO mice in which one gene encoding muscle lim, an actin- based cytoskeletal protein that regulates myogenic differentiation, is interrupted (170). This may represent Lamin A/C mutations resulting in DCM in humans (171). However, some murine models may serve as models for cardiomyopathies, without mimicking human diseases. As opposed to the monogenetic causes of HF, these models presents with a single protein of importance in general human HF. One example may be mice with nuclear Ca²⁺/calmodulin kinase II overexpression in CMs (172) or mice with disrupted Ca²⁺ handling within CMs. 3) In gene KO models one may study conditional KO mice and in paper 2-3 in this thesis, we used SERCA2a KO mice (30). As opposed to most experimental models of HF, which primarily represents systolic HF, the complete KO of SERCA, with a short transition phase of

compensated function, eventually leads to diastolic dysfunction (30,166). As mentioned in the introduction of this thesis (chapter 1.1.3), SERCA2 mutations have been reported in humans (28). Moreover, several mutations in phospholamban, the protein involved in regulating SERCA, have been reported to lead to DCMs in humans (173,174). This may indicate a clinical relevance

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of studying such mutations in human HF. As recent studies have suggested that the pathogenesis of systolic and diastolic HF development is somewhat different (21), the relevance of using specific models that represent either one of these sub conditions becomes obvious. Moreover, as previously mentioned in this thesis, the SERCA2a KO is a conditional KO restricted to CMs allowing for cell specific studies and flexibility as for study initiation. It is however important to emphasize that overexpression of the Cre enzyme alone has been reported to express around 35% (175) of the genes, which makes it crucial to select appropriate control groups when

designing experimental studies using MCM SERCA2a KO mice (30,166). Due to the possibility of uncontrolled Cre induced gene regulations, we used MCM control mice for MCM SERCA2a KO mice. In traditional gene KO models, one single gene has been inactivated, making it possible to study the gene of interest in a given disease context.

As different as they appear, mice share a surprisingly similar biology to humans, which e.g., makes their immune systems comparable in several aspects (176). Mice are cost and space effective, and due to their short life span are they appealing as an animal model of chronic human diseases. Also, they reproduce quickly and can be genetically engineered. Thus, they can act as a bridge between in vitro and in vivo experiments to proof of concept data. Despite the advantages of utilizing mouse models (177), it is essential to keep in mind that mice are not furry humans with a tail, and interpretations from mouse models must be made with caution as the lack of the complexity of humans is inevitable (178). Among the disadvantages of using mice are that, unlike humans, mouse hearts have adapted to function at very high heart rates. Also,

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slow β-MHC is up regulated at the protein level in both species during HF. The SERCA protein, which is responsible for re-distributing Ca²⁺back into the SR during diastole filling, accounts for 90-92% of Ca²⁺re-distribution in rodents (179,180). In comparison, it only accounts for 76% of the re-distribution in humans, whereas NCX accounts for the majority of the rest in both groups (181). The use of KO mice is not un-problematic as one particular gene of interest may serve different roles in humans and in mice(182). Another challenge in KO mice is that since the gene most often is inactivated in all cells from birth, the body may early adapt to the physiological changes with unknown compensating factors that may impact cardiac function -and that are not present in human HF. In contrast, human HF is most often developed from the adult ages (apart from congenital HF) involving a gradual detrimental development. Other considerations are that it is important that littermates are crossed to reduce gene pool variations (183) and that

upbringing environments should be the same (temperature, food, etc.). All animals in our studies were stationed at the same animal facility.

It seems that is no “ideal” animal model of the human cardiovascular system, and obviously with all the limitations one should assess different animal models, both small and large, when

studying CVDs.

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5.3 Histological scoring of inflammation

Though HF is a source of systemic inflammation, secondary organ damage, e.g. lung and liver congestion, renal failure etc., as well as comorbidities, e.g. diabetes, hypertension, rheumatic diseases etc., may all contribute to systemic inflammation. As HF and systemic inflammation often co-exist, we investigated the impact of these two conditions combined in paper 2 using the SERCA2a KO.

Among our collaborators, a trained pathologist analysed haematoxylin eosin stained slides of hearts, livers and lungs, and established a scoring system based on pure histological

observations. He then applied this system to score organ inflammation while being blinded to genotype and intervention. As we could not find any predefined scoring system for hearts in the literature, we designed a novel system in which scoring of hearts was based on standard methods used to analyse biopsies from heart transplants to evaluate the degree of rejection. Initially, we tried to indicate the number of muscle fibres as cells per visual field with 200X magnification.

However, due to poor reproducibility we quickly had to discard this approach in favour of another approach: scoring muscle fibres relative to nuclei as nuclear-to-cytoplasm ratio. We decided to score gradual changes in the cytoplasm from 0, in which there was absence of nuclear variation, to 4 in which there were many light cells with large nuclei and with nucleoli present.

Very few heart samples (n =3) displayed vacuolization or necrosis. Red cytoplasm, often glass- like, and pyknosis (shrinkage due to condensation of chromatin) of nuclei is considered a sign of cell death, and were rarely seen. As such, the majority of evaluating cardiac cell stress and/or

Yangchen Dhondup Toll-like receptor 9 signalling in heart failure

Doctoral thesis at Oslo, 2018