Identification and functional characterization of anti-fibrotic natural compounds in vitro

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Aus dem Institut für Molekulare und Translationale Therapiestrategien (IMTTS) der Medizinischen Hochschule Hannover

Identification and functional characterization of anti-fibrotic natural compounds in vitro

Dissertation zur Erlangung des Doktorgrades der Medizin in der Medizinischen Hochschule Hannover

vorgelegt von

Lea Martha Grote-Levi

aus Göttingen

Hannover 2018

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Angenommen vom Senat: 03.02.2020

Präsident: Professor Dr. med. Michael P. Manns

Betreuer der Arbeit: Professor Dr. Dr. med. Thomas Thum

1. Referent: PD Dr. rer. nat. Nico Lachmann 2. Referentin: Prof.´in Dr. rer. nat. Ina Gruh

Tag der mündlichen Prüfung: 03.02.2020 Prüfungsausschuss:

Vorsitz: Prof. Dr. med. Alexander Kapp

1. Prüfer: PD Dr. med. Tomas Smith

2. Prüfer: PD Dr. med. Ingmar Mederacke

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Summary

Heart failure (HF) displays a deadly clinical syndrome worldwide. Patients suffering from HF can be divided into three subgroups, depending on their left ventricular ejection fraction (LVEF). Pathophysiologically, cardiac fibrosis, comprising excessive net accumulation of extracellular matrix, is one key factor within the orchestra of cardiac remodeling leading to HF.

Patients suffering from heart failure with preserved ejection fraction (HFpEF) exhibit more than 50% LVEF but suffer from cardiac dysfunction during the diastolic phase of the heart, which is mainly characterized by passive stiffness of the left ventricle (LV) caused by cardiac fibrosis. These patients actually face lack of therapeutic options, for example having no med- ication to modulate fibrotic outcome directly.

Within the first part of this thesis, induction of autophagy by Torin-1 and Quercetin within human cardiac fibroblasts (HCF) revealed beneficial effects on fibrotic response therefore de- creasing expression of fibrotic phenotype in vitro.

As main focus, the natural compounds Anisomycin, Geldanamycin, Bufalin and Gitoxigenin could be identified as potential anti-fibrotic structures in vitro, therefore acting as potential therapeutic options to address excessive cardiac fibrosis in patients with heart failure. Inves- tigating mechanistic correlation, Anisomycin, Geldanamycin and Bufalin induced cellular se- nescence whereas Gitoxigenin induced autophagy.

HCFs were cultivated and treated with different pre-selected compounds. Effects of compounds were investigated including proliferation capacity, apoptosis rate and cell cycle distribution as well as expression of fibrotic and autophagic markers on mRNA and protein level or secretion level of cytokines.

Next, profiling the miR transcriptome (miRnome) of HCFs treated with Anisomycin revealed three upregulated and therefore potentially anti-fibrotic acting miRNAs (miRNA-27a-5p, -29a- 5p and -181a-3p) and three downregulated miRNAs (miR-26a-5p, -129a-5p and -671-5p) displaying pro-fibrotic features respectively.

Within the second part, identification of circulating miRNAs acting as diagnostic biomarker to differentiate between patients suffering from heart failure with preserved ejection fraction versus reduced ejection fraction was aimed. Unfortunately, investigated miRNA (miR-451a, -4783-5p and -663b) failed to operate as diagnostic parameters.

In conclusion, this thesis presents potential therapeutic options targeting cardiac fibrosis in vitro and a trial to investigate non-coding RNAs as biomarkers in heart failure.

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Zusammenfassung

Herzinsuffizienz (HI) stellt ein weltweit verbreitetes, schwerwiegendes klinisches Syndrom dar. Patienten, die an HI erkranken, können abhängig von ihrer linksventrikulären Ejektions- fraktion (LVEF) in drei Untergruppen kategorisiert werden. Aus pathophysiologischer Sicht stellt kardiale Fibrose, die überschießende Zunahme extrazellulärer Matrix, einen Hauptfak- tor im Zusammenspiel des kardialen Remodeling-Prozesses dar, welcher zu HI führt. Patien- ten mit HI mit erhaltener Ejektionsfraktion (HFpEF) weisen mehr als 50% LVEF auf, leiden jedoch unter der kardialen Dysfunktion während der diastolischen Herzphase, welche haupt- sächlich durch passive Steifheit des linken Ventrikels durch kardiale Fibrose charakterisiert ist. Für diese Patientengruppe bestehen aktuell keine therapeutischen Optionen, beispiels- weise gibt es keine Medikamente zur direkten Modulierung des fibrotischen Prozesses.

Innerhalb des ersten Abschnitts dieser Dissertation konnte gezeigt werden, dass die Indukti- on von Autophagie durch Torin-1 und Quercetin in humanen kardialen Fibroblasten (HCF) förderliche Effekte auf deren fibrotische Reaktion in Form einer in vitro geminderten Expres- sion des fibrotischen Phenotyps offenbarte.

Außerdem konnten die Naturstoffe Anisomycin, Geldanamycin, Bufalin und Gitoxigenin als potentielle anti-fibrotische Strukturen in vitro identifiziert werden, um folglich als potentielle therapeutische Optionen zur Adressierung verstärkter kardialer Fibrose in Herzinsuffizienz- Patienten zu agieren. Im Hinblick auf mechanistische Korrelationen wurde aufgezeigt, dass einerseits Anisomycin, Geldanamycin und Bufalin zelluläre Seneszenz, Gitoxigenin anderer- seits Autophagie induziert.

HCFs wurden kultiviert und mit verschiedenen, vorselektierten Naturstoffen behandelt. Ne- ben den Auswirkungen der Naturstoffe auf das Proliferationsverhalten, auf Apoptose-Raten und die Zellzyklus-Verteilungen, wurden ebenfalls Expressionsanalysen von fibrotischen sowie Autophagie-anzeigenden Markern auf mRNA- und Protein-Ebene und Untersuchungen zum Sekretionsverhalten von Zytokinen mit aufgenommen.

Weiterhin ergab das Profiling des miR Transkriptom (miRnome) der mit Anisomycin behan- delten HCFs drei hochregulierte und daher potentiell anti-fibrotisch wirkende miRNAs (miRNA-27a-5p, -29a-5p und -181a-3p) sowie den Nachweis dreier herunterregulierter miR- NAs (miR-26a-5p, -129a-5p und -671-5p), welche möglicherweise pro-fibrotische Funktionen einnehmen.

Der zweite Abschnitt der Dissertation enthielt das Ziel, zirkulierende miRNAs als diagnostische Biomarker zu identifizieren, welche zwischen Patienten mit erhaltener sowie reduzierter linksventrikulärer Ejektionsfraktion diskriminieren können. Hier verfehlten die untersuchten miRNAs (miR-451a, -4783-5p und -663b) das Potential, als diagnostische Parameter zu agieren.

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Zusammenfassend zeigt diese Dissertation potentielle therapeutische Optionen der Modula- tion kardialer Fibrose in vitro sowie einen translationalen Ansatz, nicht-kodierende RNA als Biomarker in Herzinsuffizienz-Patienten zu identifizieren.

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Table of Contents VII

1 Introduction

1.1 Heart failure displays a major socioeconomic burden

Cardiovascular diseases (CVD) impose substantial medical burden on society, accounting for the highest death cause in the United States and encompassing a variety of different subgroups, such as coronary heart disease, sudden cardiac arrest or heart failure. (1)

The term Heart Failure (HF) as defined by the European Society of Cardiology describes “a clinical syndrome characterized by typical symptoms [e.g. breathlessness, ankle swelling and fatigue] that may be accompanied by signs [e.g. elevated jugular venous pressure, pulmo- nary crackles and peripheral oedema] caused by a structural and/or functional cardiac ab- normality, resulting in a reduced cardiac output and/or elevated intracardiac pressures at rest or during stress”. (2)

It is the final common stage of many heart diseases including myocardial disorders (for example due to ischaemia, inflammatory or metabolic influence), abnormal loading conditions (for example hypertension, valvular/myocardial structural disorders and volume overload) or arrhythmias. (2,3)

Pathophysiologically, those stressing conditions lead to pathological cardiac remodeling, comprising molecular, cellular and interstitial changes which manifest in alterations in size, shape and function of the heart. (4,5)

Three major subgroups of HF are defined based on left ventricular ejection fraction (LVEF) measurement: Patients suffering from heart failure with normal ejection fraction >50% LVEF (HFpEF), those exhibiting 40-49% LVEF and representing a ‘grey area’ called mid-ranged (HFmrEF), up to those patients clearly displaying reduced LVEF <40% (HFrEF). Patients suffering from HFpEF display cardiac dysfunction during the diastolic phase of the heart characterized by passive stiffness of the left ventricle (LV), prolonged isovolumic relaxation, and slow LV filling. Therefore, making a diagnosis of HFpEF remains challenging. (2,6,7) It is estimated that 2-3% of the worldwide population develops HF within their lives, rising up to over 10% among people over 70 years within developed countries. (3,8) Whereas incidence plateaus or even decreases, prevalence is growing, due to an aging population and improved treatment options. (3,9–11)

Up to now, HF is a deadly clinical syndrome with a 5-year survival rate about 35% even worse than most cancer species. (8,9)

Besides affecting a huge amount of people and CVD being one of every third death cause in the US, this disease also seizes a huge amount of money, being responsible for costs over US$ 39 billion annually within the US alone or 1-2% of health-care expenditures in developed countries. (1,3,9,12,13)

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Introduction 2

Although guideline-based treatment options, like β-Blocker and angiotensin-converting- enzyme inhibitors, reduce morbidity and mortality in patients suffering from HFrEF, in those patients suffering from HFpEF, displaying more than 50% of HF patients, no effective therapeutic option has been developed yet. (2,3)

1.2 Aim of this project

Until now, therapeutic modulation of fibrosis specifically within the context of heart failure has not been demonstrated in the clinics. This project aims to address and further understand development of cardiac fibrosis in order to investigate novel treatment options.

Aim of this project is to identify and further characterize anti-fibrotic natural compounds by treating human cardiac fibroblasts (HCFs) in vitro. Having an impact on the regulation of mi- croRNAs, compound-regulated microRNAs are to be identified that may be implicated in the anti-fibrotic effects of the compounds.

As one further part, a possible relationship between increased level of autophagy and fibrotic phenotype outcome of HCFs is addressed. Well-known autophagy inducers Torin 1, Spermi- dine and Quercetin, and diminished autophagy due to silencing APG 7, a protein that is essential for autophagy, are investigated for their fibrotic impact and anti-fibrotic natural compounds are explored for autophagic influence in vitro.

Third, investigations on circulating microRNAs as diagnostic biomarkers in the clinics are included comparing samples of patients suffering from heart failure with reduced versus preserved ejection fraction.

1.3 Proceeding and structure of thesis

This thesis is structured as followed: First, a short overview of actual knowledge about fibrosis in heart failure, relationship of autophagy and fibrotic phenotype and the impact of mi- croRNAs in the context of cardiac fibrosis are given.

Furthermore, selected candidates are introduced in respect of known attributes and used material and methods are explained.

Subsequently, results are presented comprising impact of autophagy-influencing compounds/silencing RNA on fibrotic phenotype and characterization of anti-proliferative natural compounds, followed by detailed analysis of identified anti-fibrotic natural compounds. More- over, a prospect of promising regulated microRNAs by identified anti-fibrotic natural compound Anisomycin is given. Translationally, microRNAs as diagnostic biomarkers, discriminating HFpEF versus HFrEF patients, within a miRNA profile analysis are highlighted.

Conclusively, results are discussed within the context of current research and an outlook of following investigations is given.

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2 State of the art

2.1 Fibrosis in heart failure

Cardiac stress, for example myocardial infarction, pressure overload due to hypertension/aortic stenosis or volume overload due to valve insufficiency, results in cardiac remodeling characterized by cardiomyocyte loss and/or hypertrophy, insulin resistance, electrophysi- ological changes and cardiac fibrosis. (6,14)

The term “cardiac fibrosis” describes excessive net accumulation of extracellular matrix proteins, mainly collagen, within the myocardium, which leads to stiffness of the heart, impair- ment of conductivity and of cardiomyocyte contraction, ultimately resulting in arrhythmias and declined ventricular function. (14,15) Although fibrosis represents an essential repair process in settings of tissue damage and wound healing, excessive fibrosis can result in scarring, organ dysfunction and, finally, organ failure. (14)

Various cell types are directly and indirectly involved in the fibrotic outcome including matrix protein producing cells, fibroblasts, or fibrogenic mediator secreting cells, like macrophages, mast cells or lymphocytes. (15)

Cardiac fibroblasts present up to 20% of non-myocyte cardiac cell population and balance extracellular matrix composition by regulating collagen turnover as well as outcome of cytokines, growth factors or matrix metalloproteinases (Figure 2.1). (15–18)

Figure 2.1: The versatile roles of cardiac fibroblasts. Being stimulated, they proliferate and transdif- ferentiate to myofibroblasts, balance extracellular matrix components, as well as secrete growth factors and cytokines. Taken from (18). Reprint with the permission of Wolters Kluwer Health, Inc.

Proliferation and transdifferentiation of fibroblasts to myofibroblasts, exhibiting combined characteristics of smooth muscle cells, formation of contractile stress fibres for migration, and

Cytokines and Growth Factors

ECM Secretion

MMPs

ECM Degradation Fibroblast

Proliferation

& Migration Myofibroblast

Chemokines

Recruitment of Inflammatory Cells

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State of the art 4

features of active fibroblasts, an extensive endoplasmatic reticulum for secretion, displays a key event of fibrosis. (6,15,16) Characterized by the expression of myofibroblast marker α- smooth muscle actin (α-SMA), myofibroblasts secrete, amongst others, different collagen isoforms I and III, as well as matricellular proteins, such as connective tissue growth factor (CTGF), or matrix metalloproteases (MMP), for example MMP-2. Latter may be predominantly expressed during late stage phase of cardiac remodeling resulting in cardiac dilata- tion.(15,16)

Collagen protein operates as scaffold and transmits contractile force of cardiomyocytes. Col- lagen I isoform covers almost 85% of total myocardial collagen and transfers tensile strength, collagen III isoform encloses 11% and promotes elasticity. TGF-β, a well-characterized fibrogenic factor, induces myofibroblast transdifferentiation and deposition of extracellular matrix via Smad3 signalling cascade. Three isoforms, TGF-β1, 2 and 3, are expressed in mammals, with isoform TGF-β1 being preponderantly expressed in the cardiovascular system. In the normal heart, TGF-β1 is presented as latent complex and becomes activated by a wide variety of molecular stressors, for example different proteases, raised by cardiac injury. (15) 2.2 Autophagy and proliferation are controlled by mTOR pathway

Autophagy is a highly-conserved recycling process which functions as significant protective survival mechanism. But in contrast, severe high levels of autophagy induce self-destruction, also seen in heart failure patients. (19,20)

Eukaryote cells exhibit at least two major recycling pathways: on the one hand degradation by the ubiquitin-proteasome system, on the other hand by organization of the autophagy- lysosome pathway. (19)

Lysosomal degradation of cellular debris, thus, can be subdivided into three major subforms:

chaperone-mediated autophagy, microautophagy and macroautophagy. (21) Hereinafter, the term ”autophagy” describes the macroautophagy pathway.

During autophagic degradation, complete cytoplasmic regions, including entire double- membraned organelles and soluble components, are sequestrated into autophagosomes, formed by phagophores. Followed by elongation and maturation, they fuse with lysosomes finally resulting into an autophagolysosome-system implying recycling of components by lysosomal hydrolases and generating free amino and fatty acids. (19)

Nucleation of autophagic vesicles requires activation of class III phosphoinositide 3-kinase Vps34, generating Phosphatidylinositol 3-phosphates and initiated by a protein-complex including beclin-1, p150, Bif1, beclin-1 interacting proteins UV irradiation resistance-associated tumour suppressor gene (UVRAG) and activating molecule in beclin 1-regulated autophagy (Ambra1). (19,22,23) Elongation of vesicles is arbitrated by two ubiquitin-like regimes: On the one hand, a covalent protein-protein conjugation of autophagy-related (APG) 5 and 12 pro-

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teins catalysed by APG7 and APG10 proteins, E1 respectively E2 ubiquitin-activating-like enzymes, links with APG16 protein resulting into an APG12-APG5-APG16 protein complex.

On the other hand, protein-lipid conjugation of microtubule-associated protein 1A/1B-light chain 3 (LC3) proceeds, from precursor proLC3 to unconjugated LC3-I isoform, catalysed by cysteine protease APG4, to phosphatidylethanolamine-conjugated LC3-II isoform, catalysed by APG7, E1-like, and APG3, E2-like, protein and influenced by APG5-APG12 protein complex. (19,24–26) Whereas APG12–APG5–APG16 protein complex is mostly associated with early autophagosome formation and liberated with ongoing maturation, LC3-II protein corre- lates with mature autophagosome formation. (19)

Nowadays, several studies confirm that, although a wide range of intracellular components are recycled, mechanistically, those components are specifically directed to the autophagy process. A protein called p62/sequestosome 1 (SQSTM1) selectively links poly- or mono- ubiquitinated debris with LC3 protein, therefore directly supplying depleting cargo to autophagic core system. (27)

As LC3 and p62 proteins are directly involved in the autophagy cascade, among other tech- niques, protein level detected by western blot can conveniently be used to monitor autophagy outcome. (24,28)

Since SQSTM1/p62 protein is directly integrated into autolysosomes and finally degraded, decreased SQSTM1/p62 protein levels indicate induction of autophagy whereas increased levels display inhibition of autophagy. Same applies for NBR-1, an autophagy protein struc- turally similar to SQSTM1/p62. (24,29)

Monitoring autophagy by LC3 protein levels remains more complex. Focusing on LC3-II isoform, increased protein level, and therefore increased number of autophagosomes, can either indicate induction of autophagy, known as on-rate autophagic flux, or arrest of lysosomal fusion/degradation, known as off-rate autophagosome accumulation. To differentiate between on- or off-rate, one well-accepted method is to additionally block LC3-II degradation, for example by using chloroquine. Chloroquine treatment results in pH neutralization of lysosomes and thus blocks lysosomal degradation. Raised LC3-II protein levels under treatment conditions including chloroquine treatment therefore indicate on-rate autophagic flux. (24,28) Autophagy is tightly regulated by different signalling pathways: Nutrient sensing by mechanistic target of rapamycin complex 1 (mTORC1) or Ras/cAMP dependent protein kinase A (PKA) signalling, the Insulin/growth factor pathway, energy sensing by 5′-AMP-activated protein Kinase (AMPK) signalling, stress response induced by endoplasmatic reticulum stress, hypoxia/oxidative stress or pathogen infection modify autophagy outcome. (27)

One well characterized negative regulator of autophagy is mTORC1. (24) mTORC1 displays one of two isoforms of mTOR complex, mTORC1 and mTORC2. The mTOR family presents atypical serine/threonine protein kinases, which emerge with different proteins to form com-

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State of the art 6

plexes, and was originally named after being a target of rapamycin, a macrolide produced by bacteria Streptomyces Hygroscopius, which exhibits extensive anti-proliferative characteristics. (30) The isoforms play crucial roles in regulating cell growth, proliferation, cell cycle, survival or apoptosis and motility. (31)

mTORC1 itself realizes its negative feed backed autophagy influence by either direct phosphorylation and thus repression of unc-51-like kinase1 (ULK1)/APG13/focal adhesion kinase family-interacting protein of 200kDa (FIP200) complex, therefore abolishing initiation of autophagy, or activation of death associated protein 1 (DAP1), a further autophagy inhibitor, or regulating WIPI2, a mTOR-dependent phosphoproteome, regulating early formation of autophagosomes. (30)

ULK1 acts as central leverage factor of autophagy induction. Its stability is dependent on a HSP90-CDC37 chaperone complex. Pharmacological inhibition of this complex leads to increased proteasome-mediated ULK1 turnover and reduces autophagy levels. (24)

As indicated by anti-proliferative outcome of eponym rapamycin, the mTOR pathway is not only involved in regulation of autophagy but also plays a crucial role in regulating cell proliferation or growth arrest, respectively (Figure 2.2).

mTORC2 mTORC1

Lysosomes

Raptor Rictor

S6K Akt SGK1

FoxO3a

Cyclin D1

SGK1 p21/p27

Foxo3a ULK1/ATG13

/FIP200 DAP1

WIPI2

Cytoplasm

Nucleus Autophagy

machinery

Proliferation PI3K PDK1

A

p18

Proliferation and autophagy inhibition

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Figure 2.2: Correlation between proliferation/growth arrest and autophagy modulated by mTORC pathway. (A) Induced mTORC1 promotes cell proliferation but inhibits autophagy pathway.

(B) Inactivated mTORC1 promotes via de-repressed mTORC2/Akt signalling pathway growth arrest and leads to autophagy induction. Modified from (32). Copyright © 2014 Mori et al, open access article permitting unrestricted use, distribution and reproduction.

Active mTORC1 supports cell growth and proliferation either by influencing S6 kinase 1, promoting cyclin D1 and inhibiting PI3K or directly suppressing FoxO3a expression. (24,30) Inactivated mTORC1 leads to activation of mTORC2 and induction of Akt pathway, followed by phosphorylation of FoxO3a and finally resulting in growth arrest. Thus, autophagic level and proliferation correlate inversely, being regulated by the mTOR pathway. (32)

Besides focusing only on proliferative manner, recent publications also promoted the hypoth- esis, that anti-autophagic phenotype may also be related to an inverse, thus induced, senes- cent outcome. (33)

In vivo, McMullen et al. already showed beneficial effects of mTOR-inhibitor rapamycin by regressing established cardiac hypertrophy and thus improving cardiac function, therefore exhibiting beneficial effect of upregulated autophagy levels but not discriminating between different involved cardiac cells. (34,35) Focussing specifically on cardiac fibroblast, Aránguiz- Urroz et al. demonstrated that increase in autophagy, triggered by b2-adrenergic stimulation, correlated with elevated degradation of collagen, therefore may giving first incidence of decreased fibrotic response. (35,36)

Nonetheless, Gupta and colleagues recently demonstrated that treatment of human atrial fibroblasts with TGF-β1 provoke both fibrogenisis and autophagy. (20)

These opposing results display the fragility of intracellular balance in regulating autophagy and the need of further investigations testing relationship between autophagic and fibrotic outcome of cardiac fibroblasts in the setup of heart failure.

mTORC2 mTORC1

Raptor Rictor

Akt SGK1

ULK1/ATG13 /FIP200 DAP1

WIPI2

Cytoplasm Autophagy

machinery

Growth arrest PI3K PDK1

B

Growth arrest and autophagy induction

mTORC2 Rictor

Rictor

SGK1

FoxO3a

p21/p27 Rictor/FoxO3a

Nucleus

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State of the art 8

2.3 The role of miRNAs in cardiac fibrosis

Approximately 1% of the human genome encodes for genes being transcribed to proteins, but the majority encodes for non-coding RNA including small RNAs like transfer-RNA, mi- croRNA, small nuclear RNA and small nucleolar RNA, long non-coding RNAs and pseudo genes. (37)

MicroRNAs (miRNA) are small non-coding RNAs of 21 to 25 nucleotides. They act as negative post-transcriptional modulators of gene expression in almost all biological processes and are involved in various cardiovascular disorders, including cardiac fibrosis, being explored by diverse in vitro and in vivo studies. (4,37,38)

MiRNA formation comprises several processing steps: Long primary transcripts (pri-miRNAs) are transcribed from genomic DNA and subsequently processed to precursor hairpin miR- NAs (pre-miRNAs) by Drosha and DiGeorge syndrome chromosomal region 8 (DGR8). After being exported to the cytoplasm, the endonuclease Dicer further cleaves pre-miRNA to approximately 22-nucleotide double-stranded miRNA. Divided into two strands, the so called guide strand or mature miRNA formats with Argonaute proteins within the RNA-induced silencing complex (RISC). With the aid of RISC, the mature miRNA acquires its modulating function by in-/complete binding to 3’-untranslated regions of messenger RNAs (mRNA), resulting in lowered levels of target genes because of mRNA degradation or inhibition of trans- lational machinery. The second strand, named passenger strand, has been thought to be degraded, but recent studies demonstrate this strand to either exist intracellularly or to be exported into exosomes, suggesting additional functions. (3,14)

Due to the fact, that miRNAs exhibit the possibility to bind to several mRNAs, they influence not only one single gene but can orchestrate a whole network of target genes. (37,39)

MiRNA-21 and miRNA-29 are prominent miRNAs regulating fibrotic phenotype of cardiac fibroblasts: miR-21 activates cardiac fibrosis by targeting sprouty homologue1 protein, MMP- 2 or TGFβ-Receptor 3. Expression levels are predominantly enriched in fibroblasts and increased levels are detected during cardiac remodeling in mice and patients with aortic stenosis. By contrast, reduced miR-29 expression levels are noticed under cardiac stress conditions, contributing to cardiac fibrosis development by de-repressing of fibrotic genes, therefore potentially acting as "anti-fibrotic" miRNA in vitro. (39)

Accordingly, miRNA-based therapies comprise promising innovative improvement addressing heart failure treatment, modulating miRNAs either by miRNA mimics or antimiR strate- gies. MiRNA mimics are synthetic double-stranded oligonucleotides, which resemble precursor miRNAs and are further processed within miRNA biogenesis pathway. Administrated by using adeno-associated viruses to encourage organ-specific uptake, miRNA mimics can re- store lowly expressed miRNAs. But overall, chemical modifications for optimizing outcome remain challenging due to further intracellular processing. AntimiRs, in contrast, are single-

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stranded antisense oligonucleotide molecules which directly block miRNAs function by binding to mature miRNAs, thus preventing binding to target mRNA. Chemical modifications pre- vent degradation and encourage cellular uptake, for example by adding cholesterol particles, 2'-O-methyl modifications, locked nucleic acid (LNA) modifications, non-nucleotide ZEN (N,N-diethyl-4-(4-nitronaphthalen-1-ylazo)-phenylamine) modifications, conjugation to N- acetylgalactosamine sugars or the use of miRNA sponges. (38) They have already displayed their therapeutic potential within phase two clinical trials. (40)

Additionally and due to the fact that miRNAs are on the one hand remarkably stable but are also released to extracellular space and found in body fluids, increasing interest addressing miRNAs as diagnostic or prognostic biomarkers has led to a substantial number of investiga- tive studies. (37–39)

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State of the art 10

2.4 Confirmation of preliminary work revealed six anti-proliferative natural compounds

Previously, BrdU-based screening of a library of natural compounds in HCFs, followed by validation within this project, led to the discovery of six anti-proliferative natural compounds:

Geldanamycin, Anisomycin, Bufalin, Gitoxigenin, 10-Hydroxycamptothecin and Piplartine (Figure 2.3).

Figure 2.3: Six natural compounds inhibit cell proliferation of HCFs. Cell proliferation analysis by BrdU-incorporation. (A) Anisomycin, (B) Geldanamycin, (C) Bufalin, (D) Gitoxigenin, (E) 10- Hydroycamptothecin and (F) Piplartine treatment significantly inhibit cell proliferation. Validation data of previous work (unpublished, Schimmel et. al., IMTTS) presented. n = 3 experiments. Unpaired t- test. *** = p < 0,001. **** = p < 0.0001

To investigate relationship between regulation of autophagy and fibrotic outcome, three already known autophagy-influencing compounds, named Torin 1, Spermidine and Quercetin, are included within this project. Previous data additionally identified Spermidine´s and Quer- cetin´s anti-proliferative influence on HCFs in vitro (Figure 2.4).

A B C

D E F

P ip la rtin e

HCFs: proliferation (relative to control)

D M S O c tr l.

0 ,1 µ M 1 µ M 1 0 µ M

0 .0 0 .5 1 .0

1 .5 ****

A n is o m y c in

D M S O c tr l.

0 ,1 µ M 1 µ M 1 0 µ M

0 .0 0 .5 1 .0 1 .5

***

G e ld a n a m y c in

D M S O c tr l.

0 ,1 µ M 1 µ M 1 0 µ M

0 .0 0 .5 1 .0 1 .5

****

B u fa lin

D M S O c tr l.

0 ,1 µ M 1 µ M 1 0 µ M

0 .0 0 .5 1 .0

1 .5 ****

1 0 -H y d ro x y c a m p to th e c in

D M S O c tr l.

0 ,1 µ M 1 µ M 1 0 µ M

0 .0 0 .5 1 .0

1 .5 ****

G ito x ig e n in

D M S O c tr l.

0 ,1 µ M 1 µ M 1 0 µ M

0 .0 0 .5 1 .0

1 .5 ****

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Figure 2.4: Two known autophagy-influencing compounds inhibit cell proliferation of HCFs. Cell proliferation analysis by BrdU-incorporation. (A) Quercetin and (B) Spermidine treatment significantly inhibit cell proliferation. Validation data of previous work (unpublished, Schimmel et. al., IMTTS) presented. n = 3 experiments. Unpaired t-test. **** = p < 0.0001

2.5 Description of natural compounds

Selected natural compound candidates exhibit various effects investigated both in vitro and in vivo.

2.5.1 Cardiotonic steroids: Bufalin and Gitoxigenin

Cardiotonic steroids (CTS), for example the well-known Digoxin, are actually used as therapeutic drugs in patients with symptomatic end-stage HF improving clinical symptoms but having no benefit on survival-rate. (2,41)

They act classically by inhibiting the ubiquitous expressed sodium-potassium adenosine tri- phosphatase (Na²⁺-K⁺-ATPase), which maintains sodium-potassium-gradient and therefore ensures cellular homeostasis. By blocking the Na²⁺-K⁺-ATPase, CTS acutely regulate calci- um concentration and therefore improve cardiac output, increase Na⁺-excretion as well as vasoconstriction. Within the last years it has become clear, that binding of CTS to Na²⁺-K⁺- ATPase also initiate an intracellular signalling cascade, leading to activation of tyrosine kinase Scr, transactivation of EGF receptor and phospholipase C and amongst others resulting in activation of mitogen-activated protein kinase (MAPK) as well as Akt. Within long term effects, CTS thus lead to remodelling changes, possibly resulting in hypertrophy or fibrotic effects. (41–43)

Recent studies also demonstrate that CTS exhibit prominent anti-cancer, as well as anti-viral activities, but being limited by cardiotoxicity. (44)

CTS are classified into two different subgroups, either cardenolides, being C23 steroids and having an butenolide ring, or bufadienolides, compromising C24 steroids with a hexadi- enolide ring. (45,46)

A B

Q u e rc e tin

D M S O c tr l.

1 0 µ M 2 0 µ M 3 0 µ M

0 .0 0 .5 1 .0

1 .5 ****

S p e rm id in e

H2O c tr l.

1 µ M 2 5 µ M 5 0 µ M

0 .0 0 .5 1 .0

1 .5 ****

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State of the art 12

Bufalin belongs to the subgroup of bufadienolides. Derived from the skin and parotid venom of different Bufonidae species, it is a component of traditional Chinese medicine Ch`an Su and widely used as alternative medicine in Asian countries. (45,47–49)

Well-known to have anti-cancer effects in various cancer cell lines via mediating Akt signalling pathway, high dose Bufalin induces apoptosis in malignant cells. (47,48,50)

Chang et al. have demonstrated, that the natural compound exhibits does-dependent anti- proliferative effects on rheumatic arthritis fibroblast-derived synoviocytes via the activation of MAPK and nuclear factor-kappa B (NF-κB). (49) Lower concentration of Bufalin lead to cell cycle arrest by downregulating cyclin A expression, upregulating p21 gene expression or influencing cyclin-dependent protein kinase (Cdk-2) and Cdk inhibitor (CKII) depending on the cell line. (48) Likewise, cells treated with low concentrations of Bufalin have been pro- tected against apoptosis due to raised NF-κB levels. (48) As Bufalin demonstrates equal af- finity to Na²⁺-K⁺-ATPase isoforms, it acts as potent vasopressor in rats, increasing natriuresis and reflectively kaliuresis, hence compiling raised diuresis, and increments heart rate.(42,51) Endogenous levels of Bufalin and Bufalin-like immune-reactive substances have been detected in human serum. (42,52)

Gitoxigenin or 16β-Hydroxydigitoxigenin, an aglycone/genin, belongs to the subgroup of cardenolides and can be isolated from Digitalis lantata or Digitalis purpurea. (53,54) Miluti- novic et al. have demonstrated that Gitoxigenin activates formation of promyelocytic leukae- mia protein nuclear bodies in HeLa cell line which induce apoptosis as well as functions as viral restriction factor. (44)

So far, Gitoxigenin effects have not been explored within the context of cardiac fibrosis.

2.5.2 Antibiotics: Anisomycin and Geldanamycin

Anisomycin is a member of pyrrolidine antibiotics produced by Streptomyces griseolus. Bind- ing to 60S ribosomal subunits and therefore blocking formation of peptide, Anisomycin acts as well-known protein synthesis inhibitor in various cell lines. It also interferes with some transcription factors related to protein synthesis, for example the eukaryotic initiation factor 4 family and ribosomal proteins. (55,56)

Activating MAPK signalling pathways, including p38 MAPK and especially c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK), as well as promoting the apoptotic mito- chondrial caspase pathway, Anisomycin is known to induce apoptosis (55,57,58). Further- more, Anisomycin accelerates fibrotic outcome in scar formation by resembling TGFβ1- effects and promoting inflammation. (56,59)

Contrary, via downregulation of TGFβ´s negative regulator Ski and SnoN, Anisomycin shows some promising anti-fibrotic patterns and moreover reduced myofibroblast-related α-SMA levels in human corneal limbal epithelial cell line. (60)

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Finn et al. have recorded Anisomycin to inhibit chaperone-mediated autophagy as well as macroautophagy. (61)

Geldanamycin is a benzoquinone ansamycin antibiotic and has been first isolated from Streptomyces hygrocopius. (62,63) Known to be a potent inhibitor of heat shock protein 90 (hsp90) by binding to the N-terminal ATP binding site, therefore inhibiting cancer-related raised chaperone activity of the protein, and finally stopping tumour progression, it has been considered as promising anti-cancer drug treatment. (63–66) Nevertheless, its anti-tumour effects not only include anti-proliferative mechanism, but also affects cell cycle inhibition and apoptosis induction. (62,64,65)

Furthermore, Tomcik et al. recently have showed that hsp90 inhibition affects TGFβ-pathway by increased degradation of TGFβ-receptor and therefore counteracting pro-fibrotic TGFβ- signalling. Geldanamycin treatment significantly decreased fibrotic outcome in cultured fibroblasts obtained from lesional skin biopsy samples from patients with systemic sclerosis in vitro as well as overexpressed, constitutively active TGFβ-receptor mouse model in vivo. (60) Finally, it has been reported that Geldanamycin treatment can activate chaperon-mediated autophagy. (61)

2.5.3 Piplartine

Piplartine, a member of phenylpropenes and also known as Piperlongumine, is an amide alkaloid component of different Piper species, being the main alkaloid of Piper longum.

(67,68) Fruits of piper longum are used as spice, but more interestingly, both, fruits and roots, are implicated in traditional Indian medicine - Ayurveda, Sidha and Unani. (69)

Piplartine exhibit a wide range of pharmacological effects, which have been intensively investigated by various groups. Its main focus, acting as promising in vitro as well in vivo anti- cancer drug in various cell lines, already resulted in a patent providing Piplartine as anti- cancer treatment component. Influencing selectively cancer cells, Piplartine´s cytotoxic and anti-tumour activities comprise cell cycle arrest in G1 or G2/M phase, induction of apoptosis as well as damaging DNA, induction of oxidative stress or addressing angiogenesis and me- tastasis. Besides those effects, the natural compound also inhibits platelet aggregation in vitro and exhibit anti-atherosclerotic as well as anti-diabetic effects in vivo. Furthermore, Piplartine effectively demonstrates anti-bacterial, anti-fungal and anti-parasite side effects.

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2.5.4 10-Hydroxycamptothecin

Camptothecin (CPT) and its analogues are well-known for exhibiting anti-tumorous response in various cancer cell lines. (70,71) The original substance of class CPT is a pentacyclic alkaloid and has been first isolated from the plant Camptotheca accuminata in 1966. (72,73) S-

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State of the art 14

phase specific cytotoxicity of this family of compounds arises from binding to DNA topoisomerase I, inhibiting religation of DNA and resulting in single-strand breaks, followed by colli- sions of the drug-enzyme-DNA-complexes with DNA replication fork and therefore resulting in double-stranded DNA-damage. (70,72,73)

Currently, Irinotecan, another member of CPT family, is clinically used as anti-cancer treatment. It converts in vivo into the active metabolite 7-ethyl-10-hydroxy-camptothecin. (72) 10-Hydroxycamptothecin demonstrate higher activity combined with reduced toxicity than other CPTs. (72) Beside described Topoisomerase I inhibition, 10-Hydroxy-campotothecin expose different other anti-malignant properties, for example the natural compound inhibits phosphorylation of histone H1 and H3 in murine hepatoma cells resulting in cell death or it inhibits cell growth and induces apoptosis of gastric cancer cells by activating p53, p21Waf1/Cip1 and p27Kip1 and suppressing B-cell lymphoma 2 and B-cell lymphoma XL. (73)

Recently it has been shown, that beside anti-cancer activity, 10-Hydroxy-camptothecin also induces apoptosis in epidural fibroblasts via increased NOXA expression. (70) It needs to be considered, that 10-Hydroxycamptothecin is water-insoluble, has a short half-life and poor biodistribution in vivo. (71)

2.5.5 mTOR inhibitor: Torin 1

Torin 1 belongs to the pyridinonequinoline class of kinase inhibitors. (74) It competitively inhibits ATP-binding site of both mTOR complexes, mTORC1 and mTORC2, therefore blocking selectively their kinase-dependent functions. (24,31,74)

As the mTOR pathway displays a crucial signalling pathway of cell growth, metabolism and proliferation and its dysregulation occurs in various malignant diseases, mTOR inhibitors exhibit promising anti-cancer effects. (31) Furthermore, Thoreen et al. have demonstrated Torin 1 to cause G1/S phase cell cycle arrest and impede proliferation in mouse embryogen- ic fibroblasts. (74)

Concentrating on cardiomyocytes, Sundararaj et al. have recently attributed Torin 1 to aug- ment phenylephrine-stimulated CTGF expression of those cardiomyocytes. (75)

Besides impairing proliferation in some cell lines, Torin 1 is a well-known autophagy inducer, too, serving as positive control in autophagy experiments, being stronger than

rapamycin. (24,31)

Another mTOR inhibitor, everolimus, has recently been presented to lead to suppression of human dermal fibroblast activation in vitro by restraining basal fibroblast growth factor- induced proliferation as well as repressing TGF-β1- mediated collagen 1 synthesis in vitro but failed to induce autophagy. (76)

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Taken together, it still remains unknown, how autophagy inducer Torin 1 affects fibrotic phenotype of human cardiac fibroblasts.

2.5.6 Caloric restriction mimetics: Spermidine and Quercetin

Both, Spermidine and Quercetin, are members of the caloric restriction mimetic family.

(77,78) Caloric restriction mimetics (CRM) are defined as agents revealing the same bio- chemical alterations as caloric restriction, and as caloric restriction is one of the most effective autophagy inducer, CRMs do so, too. (19,77,78)

This class provokes autophagy induction via three modes of action: either by causing the depletion of acetyl coenzyme A, by inhibition of acetyl transferase activity, mainly focusing on acetyl transferase E1A-binding protein p300 (EP300), or by activating deacetylases, mainly targeting sirtuine 1 (SIRT1). These events result in deacetylation of APG or foxhead box protein members, initiation of autophagosome formation as well as the pro-autophagic transcriptional response, leading to induction of autophagy. (78)

Spermidine induces autophagy by inhibiting histone acetyl transferases and therefore modulating the acetylation mode of over 100 proteins which are integrated into the central autophagy cascade. It therefore acts independent of mTOR-signalling. (78,79)

This polyamide is present in all organisms, with particular high concentrations in sperms, in citrus fruits and soybeans and it is also produced by the gut microflora. Spermidine is well known to increase life span in yeast, nematodes and flies, as well as health and life span of rodents, in an autophagy-dependent way. (77–79) Furthermore, Spermidine shows anti- cancer effects in mice developing colon cancer and, moreover, it reverses age-mediated memory deterioration in flies. Finally, treatment reduces arterial aging by reversing stiffness and ROS-mediated oxidative stress, as well as skin inflammation in mice. (78)

Gahl et al. have demonstrated that Spermidine inhibits proliferation of human skin fibroblasts obtained from patients with cystic fibrosis. (80)

Besides those effects, elevated Spermidine levels are observed in mice suffering from cardiac hypertrophy and heart failure induced by myocardial infarct or hypertension inducing in- terventions. (81)

Recently, Eisenberg et al. highlighted that Spermidine treatment extends lifespan of mice and displays cardio-protective effects in vivo by reducing cardiac hypertrophy and preserving diastolic function in an autophagy-dependent manner. (82)

Quercetin is a naturally occurring flavonoid and found in many plants, therefore also comprising fruits and vegetable. (83,84) It regulates autophagy by inducing the deacetylase SIRT1 and also exhibits anti-proliferative effects in various malignant cell lines in vitro. (78)

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State of the art 16

Besides, Quercetin treatment demonstrates a great variety of other pharmacological effects, comprising cardio-protective outcome, for example by reducing increased blood pressure and improving endothelial dysfunction as well as end-organ injury within the heart and kid- neys of rats suffering from hypertension. (83,85) Li et al. have furthermore demonstrated reduced fibrotic outcome in hearts of rats treated with Quercetin, previously being infused with isoproterenol. (85) Anti-inflammatory and anti-oxidative effects are likewise attributed to Quercetin treatment. (78,84)

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3 Material and methods

3.1 Material

3.1.1 Equipment

Table 3.1: Devices used for this work

Device Type Manufacturer

camera Digital Sight Ds-Qi1Mc Nikon

(Düsseldorf, Germany) camera software NIS-Elements BR Imaging

software 3.22.00

Nikon

(Düsseldorf, Germany)

Cell Counter Countess Invitrogen

(Karlsruhe, Germany)

Centrifuge Megafuge 1. OR Thermo Fisher Scientific

(Schwerte, Germany)

Centrifuge Multifuge X1R Heraeus

(Hanau, Germany) electrophoresis chamber SDS-

PAGE

Mini Protean® Tetra Cell Bio-Rad

(Munich, Germany) electrophoresis chamber West-

ern blot

Mini Trans-Blot Cell Bio-Rad

(Munich, Germany) FACS machine Guava easyCyte Single Sample

Flow Cytometer

Merck Millipore

(Schwalbach, Germany)

FlowJo software V10 MyCyte.org

(Ashland, USA)

fluorescence microscope ECLIPSE Ti-U Nikon

(Düsseldorf, Germany) Guava Easy Cyte Software guavaSoft 2.5 Merck Millipore

(Schwalbach, Germany)

heating block Thermomixer MHR13 HLC BioTech

(Bovenden, Germany)

ImageJ software ImageJ 1.50i Wayne Rasband

(NIH, USA)

multi-plate reader Synergy HT BIO-TEK

(Bad Friedrichshall, Germany)

photometer SmartPec Plus Bio-Rad

(Munich, Germany) Real-Time PCR device Thermalcycler C1000 Bio-Rad

(Munich, Germany)

Real-Time PCR device ViiA7 Applied Biosystems

(Foster City, CA, USA) Real-Time PCR software BioRad CFX Manager 3.1 Bio-Rad

(Munich, Germany) Real-Time PCR software QuantStudio™ Real-Time PCR

Software v.1.1

Applied Biosystems (Foster City, CA, USA)

X-ray film box RO13 Via Hartenstein

(Würzburg, Germany) X-ray film developer ECOMAX X-Ray Film Proces-

sor

Röntgen Bender

(Baden-Baden, Germany)

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Material and methods 18

3.1.2 Consumable material and chemicals Table 3.2: Material

Material Manufacturer

384-well plate Bio-Rad

(Munich, Germany)

blotting paper GE Healthcare

(Munich, Germany)

cell culture flask Sarstedt

(Nümbrecht, Germany)

cell culture plate TPP

(Trasadingen, Switzerland)

Falcon tubes Sarstedt

(Nümbrecht, Germany)

PVDF membrane Bio-Rad

(Munich, Germany)

1.5 ml safe-seal reaction tubes Eppendorf

(Hamburg, Germany)

1.5 ml easy-cap reaction tubes Sarstedt

(Nümbrecht, Germany) X-ray film development rea-

gents

Röntgen Bender

(Baden-Baden, Germany)

X-ray film Via Hartenstein

(Würzburg, Germany) Table 3.3: Chemicals

Material Manufacturer

ammonium persulphate (APS) Bio-Rad

(Munich, Germany)

chloroform Sigma-Aldrich

(Taufkirchen, Germany)

cell lysis buffer Cell Signaling Technology

(Danvers, USA)

dithiothreitol (DTT) New England Biolabs

(Frankfurt, Germany)

dimethyl sulfoxide (DMSO) Roth

ethanol Merck

(Darmstadt, Germany)

gelatine Roth

glycogen Invitrogen

H2O2 30% Merck

(Darmstadt, Germany)

isopropanol Roth

Lipofectamine 2000™ Invitrogen

Loading buffer (6x) Fermentas

(St. Leon-Rot, Germany)

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luminol Roth

methanol Roth

milk powder Roth

para-cumaric acid powder Roth

Rotiphorese® Gel 30 Roth

Roti®-Quant Roth

TEMED Roth

TRIS Roth

TritonX-100 Roth

TriFAST™ PEQLAB

(Erlangen, Germany)

Tween 20 Roth

(Karlsruhe, Germany) Table 3.4: Natural compounds for treatment of cells

Natural compound/compound Manufacturer

Anisomycin Green Pharma

Bufalin Green Pharma

Geldanamycin Green Pharma

Gitoxigenin Green Pharma

10-Hydroxycaomptothecin Green Pharma

Piplartine Green Pharma

Spermidine Sigma-Aldrich

Torin 1 Merck Millipore

Quercetin Sigma-Aldrich

3.1.3 Provided kit systems Table 3.5: Provided kit systems

Kit name Catalogue number Manufacturer

Absolute Blue qPCR Mix #AB-4138/B Thermo Fisher Scientific Cell proliferation ELISA BrdU

(colorimetric)

#11644807001 Roche Scientific

(Penzberg, Germany)

HotStar Taq Mastermix Kit #69504 Qiagen

(Hilden, Germany) iScript Select cDNA Synthesis

Kit

#170-8897 Bio-Rad

(Munich, Germany)

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Material and methods 20

iQ SYBR Green Supermix #172-5006CUST Bio-Rad

(Munich, Germany) TaqMan® MicroRNA Reverse

Transcription Kit

#4366597 Applied Biosystems

(Foster City, CA, USA) 3.1.4 Solutions and buffers

3.1.4.1 Cell culture media, components and used cells Table 3.6: Cell type used in this work

cell type Catalogue number Manufacturer

Human Cardiac Fibroblasts (HCFs)

C-12375 PromoCell

(Heidelberg, Germany) Mus musculus cardiac cell line

derived from AT-1 mouse atrial cardiomyocyte tumor lineage (HL-1)

Claycomb WC,

LSU Health Sciences Center (New Orleans, LA, USA) Human Embryonic Kidney Cells

(HEK-293)

Cooperation-Partner Prof.

Engelhardt

(TU Munich/University of Würz- burg, Germany)

Table 3.7: Cell culture medium and components

Medium/Component Catalogue number Manufacturer

Claycomb medium #51800C Sigma-Aldrich (Saint Louis, MI,

USA) Dulbecco´s Modified Eagle

Medium (DMEM)

#41965039 Life Technologies (Darmstadt, Germany)

Dulbecco´s Modified Eagle Medium (DMEM) phenol red free

Dulbecco´s Phosphate Buffered Saline (D-PBS)

Fetal Bovine Serum (FBS) #10270-106 Life Technologies (Darmstadt, Germany)

Fibroblast Basal Medium 3, phenol red-free

#C23235 PromoCell (Heidelberg, Ger-

many)

Fibroblast Growth Medium 3 Kit #C23130 PromoCell (Heidelberg, Ger- many)

L-Glutamine #56-85-9 Sigma-Aldrich (Saint Louis, MI,

USA)

Norepinephrine bitartrate salt #A0937-1G Sigma-Aldrich (Saint Louis, MI, USA)

Opti-MEM® #51985026 Life Technologies (Darmstadt,

Germany)

Penicillin-Streptomycin #15140-122 Life Technologies (Darmstadt, Germany)

Trypsin-EDTA 0.05% #25300-054 Life Technologies (Darmstadt,

Germany)

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Table 3.8: Fibroblast Growth Medium-3 (FGM-3) for HCFs

Component amount/concentration of

supplement/

500 ml FGM-3

FBM-3 450 ml

FBS 0.1 ml/ml

Basic Fibroblast Growth Factor (bFGF) (recombinant human)

1 ng/ml

Insulin (recombinant human) 5 µg/ml Penicillin/Streptomycin 5 ml

Table 3.9: Claycomb medium for HL-1

supplement/

500 ml Claycomb medium

Claycomb medium 500 ml

FBS 0.10 ml/ml

Norepinephrine 0.1 mM

L-glutamine 2 mM

Penicillin/Streptomycin 100 U/ml: 100 µg/mL

Table 3.10: DMEM medium for HEK-293

supplement/

500 ml DMEM

DMEM 450 mL

FBS 50 mL

Penicillin/Streptomycin 5 mL 3.1.4.2 SDS-Page and Western blot

Table 3.11: Gel recipe for collection gel (SDS-PAGE, 12%)

Component volume/gel

1.5 M Tris pH 8.8 / 0.4% (w/v) SDS

1.95 ml

Rotiphorese® Gel 30 3.0 ml

dH2O 2.5 ml

10% (w/v) APS 50 µl

TEMED 5 µl