Role of transforming growth factor beta1 (TGF-beta1) in the regulation of mitochondrial energy metabolism and cardiac function [Elektronische Ressource] / Robert Shahverdyan

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Aus dem Zentrum für Innere Medizin der Universität zu Köln Klinik III für Innere Medizin Direktor: Universitätsprofessor Dr. med. E. Erdmann Role of transforming growth factor-β (TGF-β ) in the regulation 1 1of mitochondrial energy metabolism and cardiac function Inaugural-Dissertation zur Erlangung der Doktorwürde der Hohen Medizinischen Fakultät der Universität zu Köln vorgelegt von Robert Shahverdyan aus Eriwan, Armenien Promoviert am 19. Mai 2010 Dekan: Universitätsprofessor Dr. med. J. Klosterkötter 1. Berichterstatter: Privatdozent Dr. med. S. H. Rosenkranz 2. Berichterstatterin: Frau Universitätsprofessor Dr. med. G. Pfitzer Erklärung: Ich erkläre hiermit, dass ich die vorliegende Dissertationsschrift ohne unzulässige Hilfe Dritter und ohne Benutzung anderer als der angegebenen Hilfsmittel angefertigt habe; die aus fremden Quellen direkt oder indirekt übernommenen Gedanken sind als solche kenntlich gemacht. Bei der Auswahl und Auswertung des Materials sowie bei der Herstellung des Manuskriptes habe ich Unterstützungsleistungen von folgenden Personen erhalten: Privatdozent Dr. med. S. Rosenkranz und Dr. med. M. Huntgeburth. Weitere Personen waren an der geistigen Herstellung der vorliegenden Arbeit nicht beteiligt. Insbesondere habe ich nicht die Hilfe einer Promotionsberaterin/eines Promotionsberaters in Anspruch genommen.
Publié le : vendredi 1 janvier 2010
Lecture(s) : 60
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Source : D-NB.INFO/1006979506/34
Nombre de pages : 67
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Aus dem Zentrum fr Innere Medizin der Universitt zu Kln
Klinik III fr Innere Medizin
Direktor: Universittsprofessor Dr. med. E. Erdmann
Role of transforming growth factor-β1 (TGF-β1) in the regulation
of mitochondrial energy metabolism and cardiac function
Inaugural-Dissertation
zur Erlangung der Doktorwrde
der Hohen Medizinischen Fakultt
der Universitt zu Kln
vorgelegt von
Robert Shahverdyan
aus Eriwan, Armenien

Promoviert am 19. Mai 2010

Dekan: Universittsprofessor Dr. med. J. Klosterktter
1. Berichterstatter: Privatdozent Dr. med. S. H. Rosenkranz
2. Berichterstatterin: Frau Universittsprofessor Dr. med. G. Pfitzer
Erklrung:
Ich erklre hiermit, dass ich die vorliegende Dissertationsschrift ohne
unzulssige Hilfe Dritter und ohne Benutzung anderer als der angegebenen
Hilfsmittel angefertigt habe; die aus fremden Quellen direkt oder indirekt
bernommenen Gedanken sind als solche kenntlich gemacht.
Bei der Auswahl und Auswertung des Materials sowie bei der Herstellung des
Manuskriptes habe ich Untersttzungsleistungen von folgenden Personen
erhalten:
Privatdozent Dr. med. S. Rosenkranz und Dr. med. M. Huntgeburth.
Weitere Personen waren an der geistigen Herstellung der vorliegenden Arbeit
nicht beteiligt. Insbesondere habe ich nicht die Hilfe einer
Promotionsberaterin/eines Promotionsberaters in Anspruch genommen. Dritte
haben von mir weder unmittelbar noch mittelbar geldwerte Leistungen fr diese
Arbeit erhalten, die im Zusammenhang mit dem Inhalt der vorgelegten
Dissertationsschrift stehen.
Die Dissertationsschrift wurde von mir bisher weder im Inland noch im Ausland
in gleicher oder hnlicher Form einer anderen Prfungsbehrde vorgelegt und
ist auch noch nicht verffentlicht.
Kln, den 23.September 2009

Die in dieser Arbeit angegebenen Experimente sind nach entsprechender

Anleitung durch Herrn Privatdozent Dr. med. S. Rosenkranz und Herrn Dr. med.

M. Huntgeburth von mir selbst ausgefhrt worden.

Die Dobutamin-Stress-Echokardiographie an den Versuchstieren

gemeinsam mit Privatdozent Dr. med. K. Tiemann durchgefhrt.

III

erduw

Acknowledgements
I hereby thank Prof. Dr. E. Erdmann for the opportunity to do my doctoral thesis
in the department of the Clinic III for Internal Medicine, University of Cologne.
I am especially grateful to Privatdozent Dr. S. Rosenkranz for his outstanding
supervision, advice and support in performing these studies. His enthusiasm
and his in-depth knowledge in research made a research passion for me.
I want to address special thank to Dr. M. Huntgeburth for assigning me to this
topic, for his scientific supervision and helpful and hortative support in all parts
of the work.
I gratefully acknowledge Prof. Dr. K. D. Schltter and Privatdozent Dr. K.
Tiemann for the fruitful collaborations.
I wish to extend special thanks to all lab members for providing an amicable and
stimulating working environment. It was a great pleasure for me to work with
.them I also show my appreciation to my wife for her reliable support, encouragement
and sound advice.
Thanks are also due to Dr. S.S. Thorgeirsson and Dr. N. Sanderson (National
Cancer Institute, NIH, Bethesda, MD/USA), for providing Alb/TGF-β1
(cys223,225ser) transgenic mice; Victor Kotelianski (Biogen Inc., Cambridge, MA)
for providing the TGF-β antagonist sTGFβR-Fc and Andrius Kazlauskas
(Harvard Medical School, Boston) for the granting of RasGAP antibody.
I am grateful to Kln-Fortune Funding for Medical Research, Medical Faculty of
the University of Cologne, Germany, for the financial support.

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Table of contents
1.Introduction....................................................................................................................1
1.1.Epidemiologyandpathogenesisofmyocardialhypertrophyandf......ail1ure
1.2.Transforminggrowthfactorβ1anditsroleforthepathogenesisof
myocardialhypertrophy..................................................................................................................2
1.3.Characterizationofuncouplingproteinsandregulationofenergy
metabolisminmitochondria.........................................................................................................6
1.4.TheimpactofGFTβ1,AngIIandadβrenergicsystemonUCPs....................11
1.5.Hypothesisandmaingoal................................................................................................12
2.MaterialsandMethods..............................................................................................15
2.1.Materials..................................................................................................................................15
2.1.1.Chemicalsandcompounds.................................................................................15
2.1.2.Antibodies.................................................................................................................17
2.1.3.Primers.......................................................................................................................18
2.1.4.TGFβ1transgenicmice.......................................................................................18
2.1.5.TreatmentofTGβF1transgenicmice...........................................................19
2.1.6.Humanhearttissue...............................................................................................19
2.2.Methods....................................................................................................................................20
2.2.1.Cardiomyocytecultures......................................................................................20
2.2.2.Measurementofcardiomyocytecontractility............................................21
2.2.3.InhibitionofUCPswithsiRNAandgenipinandmeasurementof
cardiomyocytecontractility...............................................................................................22
2.2.4.Preparationofwholeheartlysates................................................................23
2.2.5.Isolationofmitochondria...................................................................................23
2.2.6.Determinationofproteinconcentrations....................................................24
2.2.7.Westernblotting.....................................................................................................24
2.2.8.IsolationoftotalRNA...........................................................................................26
2.2.9.ReverseTranscriptasefirststrandcDNAsynthesis...............................27
2.2.10.TaqMan®RealTimequantitativeRTPCR..............................................27
2.2.11.Dobutaminestressechocardiography.......................................................28
2.2.12.Statistics...................................................................................................................29

VI

3.Results.............................................................................................................................30
3.1.RegulationoftheexpressionofUCPsinventricularcardiomyocytesunder
theinfluenceofTGβ1F..................................................................................................................30
3.2.RegulationoftheexpressionofUCnPsmoiuseheartandskeletalmuscle
undertheinfluenceofβTGF1....................................................................................................31
3.3.EffectofUCPinhibitiononcontractilefunctinionvitro.....................................34
3.4.TGFβ1doesnotregulatetheexpressionofothermylocprarodteiainssuch
asSERCA2,p38MAPKandANT...............................................................................................36
3.5.EffectofUCPinhibitiononcardiacfinunctionvivo..............................................38
3.6.TGFβ1andUCP3expressioninhumanheartfailure..........................................39
4.Discussion......................................................................................................................41
4.1.RegulationofmitochondrialUCPsthroughβTG1Fanditsroleinthe
regulationofenergymetabolism..............................................................................................42
4.2.InhibitionofUCPactivityinvivo...................................................................................44
4.3.InhibitionofUCPmediatedprotonleakbyaherbalsubstancenipineg.....45
4.4.UCPsinhumanheart..........................................................................................................46
4.5.Conclusions.............................................................................................................................46
5.Summary........................................................................................................................48
5.1.Englishsummary..................................................................................................................48
5.2.DeutscheZusammenfassung...........................................................................................49
6.References.....................................................................................................................51

IIV

Glossary
EAC PADAng II
TAN AP APS AT PAT BAT PCBM BSA 2+Ca CoA DCM EPCD DNA SED DTT LEC MEC ATED EF CET ADF FFA SBF g, ngµg, mg, HNE KPAT kb akD

angiotensin-converting enzyme
adenosine diphosphate
angiotensin II
adenine nucleotide translocator
activating protein
ammonium persulfate
angiotensin
adenosine triphosphate
brown adipose tissue
brain-specific mitochondrial carrier protein
bovine serum albumin
calcium
coenzyme A
dilatative cardiomyopathy
diethylpyrocarbonate
deoxyribonucleic acid
dobutamine stress echocardiography
dithiothreitol
electrochemiluminescence
extracellular matrix
ethylene diamine tetracetic acid
ejection fraction
electron-transport chain
flavine adenine dinucleotide
free fatty acids
first strand buffer
gram, milligram, microgram, nanogram
hexanitroethane
potassium channel
kilobase
kilodalton

IIIV

+K KO LV VML EFLV APKM MLV-M ADN HPADN PBS PKC SFPM FPVD AASR ASR RNA RI mRNA RPC-RT SSD A2CSER ANRsi RβT BS T EDEMT TRIS TG β-TGF UCP WT

potassium
knockout
left venticule
left ventricular mass
left ventricular ejection fraction
mitogen-activated protein kinase
moloney murine leukemia virus
nicotinamide adenine dinucleotide
nicotinamide adenine dinucleotide phosphate
phosphate buffered saline
proteinkinase C
phenylmethylsulphonyl fluoride
polyvinylidene difluoride
renin-angiotensin-aldosteron system
renin-angiotensin system
ribonucleic acid
resistive index
messenger ribonucleic acid
reverse transcription polymerase chain reaction
sodium dodecyl sulfate
sarco/endoplasmic reticulum Ca2+-ATPase
small interfering RNA
transforming growth factor-beta receptor
tris buffered saline
tetramethylethylenediamine
tris-(hydroxymethyl)-aminomethan
transgenic
transforming growth factor-beta
uncoupling protein
petywild

IX

Introduction

1. Introduction
1.1. Epidemiology and pathogenesis of myocardial
hypertrophy and failure
Cardiovascular diseases such as atherosclerosis, coronary heart disease
and heart failure are the most common causes of death in industrialized
countries (Rosamond et al. 2007). Cardiac hypertrophy can be the preliminary
phase in the clinical course of developing heart failure and is an independent
risk factor for cardiac morbidity and mortality (Kannel 2000). On the other hand,
the regression of hypertrophy leads to reduction of cardiovascular events
independently from other risk factors (Muiesan et al. 1995; Verdecchia et al.
1998). Multiple mechanisms, such as hormonal dysregulation, genetic
mutations of cardiac proteins as well as volume and/or pressure overload can
be identified as activators in the development of myocardial hypertrophy. The
above mentioned mechanisms are involved in morphological modification of
cardiomyocytes, cardiac non-muscle cells (fibroblasts, endothelial cells, etc.)
and extracellular matrix (ECM) as well as in certain modifications of cardiac
gene expression (Weber et al. 1993; Sugden and Clerk 1998). Nevertheless,
the pathophysiological mechanisms, which lead to myocardial hypertrophy and
particularly to the transition from adaptive hypertrophy to heart failure, remain
elusive. Extensive efforts in research have led to identification of some
important pathomechanisms both on the structural and molecular level. A
significant role belongs to the activation of neurohumoral systems, such as the
renin-angiotensin-system (RAS) and the β-adrenergic system (Sadoshima and
Izumo 1993; Rosenkranz et al. 1997; Bhm et al. 1999). Consequently, the
introduction of ACE-inhibitors, AT1-receptor-antagonists and β-blockers has led
to significant reduction of mortality in patients suffering from heart failure
(Heidenreich et al. 1997; Pitt et al. 2000; Pfeffer et al. 2003). A direct connection
between angiotensin II (Ang II) and transforming growth factor-β1 (TGF-β1) has

1

Introduction

been shown in vivo. Thus, Ang II upregulates the expression of TGF-β1 through
activation of AT1-receptors (Rosenkranz 2004).
It is established, that limited cardiac energy in form of ATP plays an
important role in the development of heart failure. In order to utilize the required
energy, the heart converts chemical energy stored in fatty acids and glucose in
mechanical energy of actin-myosin interactions of myofibrils. The inability to
produce an adequate amount of energy leads to cardiac dysfunction (Neubauer
2007). According to the current knowledge, the concentration of ATP in a
dysfunctional heart drops slowly and progressively up to 70-75% of original
values (Ingwall and Weiss 2004). Therefore, it can be postulated that heart
failure occurs as a result of an energy metabolism disorder (Òenergy starvationÓ
hypothesis).
Additionally, mitochondrial energy metabolism, which is regulated through
mitochondrial uncoupling proteins (UCP), is affected in the failing heart (Murray
et al. 2004). Thus far, a clear connection between cardiac TGF-β1 and UCPs
has not been characterized. However, in fetal brown adipocytes it has been
implicated that stimulation with TGF-β1 upregulates UCP mRNA (Teruel et al.
1995). Based on these finding we hypothesized that TGF-β1 affects energy
metabolism of the heart by regulating mitochondrial uncoupling proteins and
therefore plays an important role in the development of myocardial hypertrophy
and failure.
1.2. Transforming growth factor-β1 and its role for the
pathogenesis of myocardial hypertrophy
Transforming growth factor-β (TGF-β) is a pleiotropic cytokine, which
belongs to a family of more than 30 different structurally related cytokines.
There are three isoforms of TGF-β present in humans (TGF-β1, TGF-β2 and
TGF-β3), from which TGF-β1 is the prevalent one (Massague 1990).
TGF-β1 is a 25-kDa homodimeric protein, which is involved in a large
amount of cellular processes, such as stimulation and inhibition of cell growth or
2

Introduction

differentiation, morphological transformation of the cell and transcriptional
activation of genes encoding extracellular matrix proteins (Teruel et al. 1995). It
has a major role in the regulation of homeostasis between cell growth and
programmed cell death (apoptosis). Therefore, it represents an important factor
for the development of myocardial hypertrophy and fibrosis (Border and Noble
1994). There are two types of transmembrane serine/threonine kinase receptors
(TβR-I and TβR-II) that mediate signal relay by TGF-β1. Binding of TGF-β1 to its
receptors leads to phosphorylation of so-called Smads, the intracellular
mediators, which transduce TGF-β1 signals from the membrane to the nucleus,
bind to DNA and regulate the transcription of specific genes (Chang et al.
2002). There are three functional classes of eight identified Smad proteins
known to date: the receptor-regulated Smads (Smad1, 2, 3, 5 and 8), the co-
mediator Smad (Smad4) and inhibitory Smads (Smad6 and Smad7). There is
an interaction between the Smad molecules and other signaling pathways,
which affect the actions of TGF-β1 (Sun et al. 2005). One of the Smad-
independent pathways of TGF-β1-signaling is the stimulation of phosphatase 1
and 2A (Derynck and Zhang 2003). Figure 1 shows the detailed pathway of
TGF-β signaling known to date.

3

Introduction

Figure 1: Schematic diagram illustrating the signaling pathway of TGF-β.

Numerous studies in humans as well as in experimental models have
shown an increased expression of TGF-β1 during myocardial hypertrophy and
fibrosis. For instance, the expression of TGF-β1 mRNA was increased in left
ventricular myocardial tissue in patients with idiopathic hypertrophic or dilatative
cardiomyopathy (Li et al. 1998; Pauschinger et al. 1999). An upregulation of
TGF-β1 was also found in animal models after myocardial infarction, as well as
during chronic pressure load (Casscells et al. 1990; Takahashi et al. 1994; Hao
et al. 1999). TGF-β1 is particularly overexpressed in hypertrophic myocardium,
when compensated hypertrophy passes onto a non-compensated phase,
resulting in overt heart failure (Boluyt et al. 1994; Song et al. 1997). TGF-β1

4

Introduction

mediates cardiac hypertrophy through increased production of different
components of extracellular matrix (collagen, fibronectin), induction of
proliferation of myofibroblasts and heightened expression of fetal genes Ð all
characteristics of cardiac hypertrophy (Rosenkranz 2004). Some of the
preliminary works have substantiated that overexpression of TGF-β1 in
transgenic mice leads to cardiac hypertrophy, which is characterized both by
interstitial fibrosis and hypertrophic growth of cardiomyocytes (Rosenkranz et al.
2002). In parallel with that, it has been shown that fibrosis of the aging heart in
heterozygous (+/-) TGF-β1-deficient mice is reduced (Brooks and Conrad 2000).
Functional blockade of TGF-β1-mediated signal cascade in vivo prevented
myocardial fibrosis and cardiac dysfunction in pressure-loaded rat hearts
(Kuwahara et al. 2002). The pathogenesis of myocardial hypertrophy is
particularly imprinted with activation of neurohumoral systems such as the RAS
and the β-adrenergic system (Sadoshima and Izumo 1993; Rosenkranz et al.
1997; Bhm et al. 1999). ACE-Inhibitors, AT1-receptor-blockers and β-blockers
were shown to significantly reduce the mortality of patients with heart failure
(Heidenreich et al. 1997; Pitt et al. 2000; Pfeffer et al. 2003). A direct correlation
between Ang II and TGF-β1 has been shown in vivo, such that the expression of
TGF-β1 is upregulated by Ang II, an effect mediated through activation of AT1-
receptors (Rosenkranz 2004). Interestingly, induction of TGF-β1 by Ang II in
adult ventricular cardiomyocytes is mediated through NAD(P)H oxidase, which
leads to further activation of proteinkinase C (PKC), p38-mitogen-activated
protein kinase (p38-MAPK) and increased binding activity of nuclear activating-
protein-1 (AP-1) (Wenzel et al. 2001).
TGF-β1 is specifically expressed during the transition from stable
myocardial hypertrophy to heart failure (Song et al. 1997). The role of TGF-β1
for cardiac morphology and function was examined in vivo in wild type (WT)
(C57BL/6) and TGF-β1-transgenic (TGF-β1-TG) mice. After stimulation with the
selective β1-adrenoceptor agonist dobutamine in various concentrations, TGF-
β1-TG mice showed a clearly reduced contractile reserve in comparison with
WT (C57BL/6) mice during dobutamine stress echocardiography. Interestingly,
the contractility upon dobutamine stimulation was restored by chronic treatment
with the β-blocker metoprolol or a selective TGF-β1-antagonist (sTGFβR-Fc),
but not after administration of the AT1-receptor antagonist telmisartan
5

Introduction

(Rosenkranz 2004). These results were confirmed by studies on isolated
cardiomyocytes, during which the isoprenaline-induced increase of cell
shortening was less in cells isolated from TGF-β1-TG mice and was restored by
the use of metoprolol and sTGFβR-Fc, yet not of telmisartan (Rosenkranz
. 2004)It is important to note that a study on adipocytes showed a connection
between TGF-β1 and the regulation of UCPs (Teruel et al. 1995). This has led
us to the hypothesis that a currently unknown mechanism involving TGF-β1,
UCPs and the β-adrenergic system may be crucial for the pathogenesis of
myocardial hypertrophy and dysfunction.
1.3. Characterization of uncoupling proteins and regulation of
energy metabolism in mitochondria
In order to have proper cell function, external sources of energy in form of
nutrition, such as carbohydrates and fatty acids are needed. Cytosolic
intermediate metabolic products of free fatty acids (FFA) and glucose are
transported from the cytosolic compartment into the mitochondria, where
mitochondrial oxidative phosphorylation takes place. Catabolism of energy
sources (FFA and glucose) takes place through oxidation of nicotine adenine
dinucleotide (NADH) and flavin adenine dinucleotide (FADH2), which release
electrons in the respiratory chain (electron transport chain Ð ETC) in the
mitochondrial inner membrane (Krauss et al. 2005). Thereby, a defined number
of protons (H+) are pumped from the mitochondrial matrix into the
intermembrane space. As a result, a proton gradient (proton motive force, ∆P)
builds up in the mitochondrial inner membrane. This proton gradient repels the
protons with the help of ATP-synthase back into mitochondrial matrix and by
that makes the synthesis of ATP from ADP and non-organic phosphate (Pi)
possible. The synthesized ATP is available in the cell, enabling essential
cellular processes, such as function of ATP-dependent ion channels, protein
synthesis or muscle contraction. However, it has been shown that under certain
6

Introduction

conditions the proton gradient either is not used at all, or is used only partially
for the production of ATP. Thus, protons are being transmitted by UCPs back
into the matrix, which has been described as Òproton leakÓ (Krauss et al. 2005).
The proton leak, mediated via UCPs, uncouples the transaction of electron-
transport/proton-gradient on one hand and the generation of ATP-synthesis on
the other hand (Fig. 2) (Krauss et al. 2005).

Figure 2: Substrate oxidation and oxidative phosphorylation in mammalian cells
(modified from Krauss et al. 2005).
The cellular metabolism of substrates such as glucose and free fatty acids (arrows in the figure)
generates electrons (e-) in the form of the reduced hydrogen carriers - NADH and FADH2.
NADH and FADH2 donate electrons to the electron transport chain, which comprises protein
complexes that are located in the mitochondrial inner membrane. Electrons are ultimately
transported to molecular oxygen, which is reduced to water in the last step of the electron
transport chain. As electrons are transferred along the electron-transport chain, a fixed number
of protons (H+) are pumped from the mitochondrial matrix into the mitochondrial intermembrane
space, which establishes a proton gradient across the mitochondrial inner membrane. The
energy that is conserved in this proton gradient drives the synthesis of ATP from ADP and
inorganic phosphate (Pi) by ATP-synthase as protons are transported back from the
intermembrane space into the mitochondrial matrix. ATP is then made available to the cell for
various processes that require energy. Proton leak, which in part is mediated by the uncoupling
proteins, uncouples the processes of electron transport/proton-gradient generation on one hand
and ATP synthesis on the other. By dissipating the proton gradient, the energy that is derived
from the oxidized substrates is released as heat.
7

7

Introduction

UCPs are mitochondrial transporters, which are located on the
mitochondrial inner membrane and belong to the family of anion mitochondrial
carriers (Walker and Runswick 1993). They regulate the discharge of the proton
gradient, which is generated by the respiratory chain (Ledesma et al. 2002).
The members of that superfamily have similar structure and function. They
comprise a tripartite structure, which is one of the most typical traits of that
superfamily; with three repeats of approximately 100 amino acids, each of
which contains two hydrophobic stretches connected by a long hydrophilic loop
located on the matrix side of the protein, UCPs have a unique structure (Fig. 3)
(Ledesma et al. 2002).

Figure 3: The transmembrane arrangement of the UCP (modified from Ledesma et al.
).2002Six α-helical regions span the lipid bilayer with amino and carboxyl termini oriented to the
cytosolic side of the membrane and the long hydrophilic loops on the matrix side. Three portions
of the tripartite structure are separated with dotted lines.
UCPs are found in all mammalians and plants, as well as in fungi and
even protozoa (Jarmuszkiewicz et al. 1999; Rousset et al. 2004). Five UCP
homologues are present in mammals (UCP1-UCP5). UCP1-UCP3 are closely
8

Introduction

related to each other, whereas UCP4 and UCP5 (also known as brain-specific
mitochondrial carrier protein-1 - BMCP1) are more different (Fig. 4) (Ledesma et
. al. 2002)

Figure 4: Phylogenic tree of members of UCP family.
An unrooted phylogenetic tree depicting the evolutionary relationships among the members of
the UCP family. Six major classes into which the proteins cluster are illustrated. The tree was
constructed using the Neighbor-Joining method. The dotted lines indicate the more distant
relationship of the UCP4 and BMCP1 proteins to the other UCPs (Ledesma et al. 2002).

9

Introduction

Regulation of UCPs takes place at two levels: gene transcription and
protein activity in mitochondria.
UCP1 genes have been designated to mouse chromosome 8, rat
chromosome 19 and human chromosome 4. The UCP2 and UCP3 genes are
neighboring in each species. The human and mouse UCP2 genes are located
7-20 kilobases (kb) downstream of the UCP3 stop codon. The UCP2-UCP3
locus is situated on mouse chromosome 7, human chromosome 11, rat
chromosome 1 and porcine chromosome 9. The brain UCP homolog BMPC1 is
situated on the X chromosome in humans. Human UCP4 has been observed
close to generic marker SHGC-34952 (Ricquier and Bouillaud 2000).
Most of the UCPs (UCP1-UCP3) have molecular masses between 31 kDa
and 34 kDa, whereas the brain homologues (UCP4 and BMCP1) are larger
proteins with masses of 36-38 kDa (Ledesma et al. 2002).
UCP1 was found as the first protein member of this family. It mediates
proton leak in brown adipose tissue (BAT) with consecutive production of heat
and is responsible for thermogenesis in hibernating animals (Krauss et al.
2005). It disperses the mitochondrial proton (H+) gradient generated by the
respiratory chain, producing heat instead of ATP (Cannon and Nedergaard
1985; Klaus et al. 1991). It has been shown that the uncoupling activity of UCP1
is increased by FFA (Rial et al. 1983; Strieleman et al. 1985; Cunningham et al.
1986; Skulachev 1991) and long chain fatty acyl CoA esters (Strieleman and
Shrago 1985; Katiyar and Shrago 1991), and decreased by purine nucleotide
di- or tri- phosphates (Heaton et al. 1978; Rial et al. 1983).
UCP2 and UCP3 were isolated and cloned recently as homologues of
UCP1 and have wider tissue distribution than UCP1 (Barazzoni and Nair 2001).
UCP2 and UCP3 showed high homology of 59% and 57%, respectively, by
matching the sequence of amino acids with UCP1, whereas the homology of
UCP4 and UCP5 appeared to be much less related to UCP1 (Borecky et al.
2001; Ledesma et al. 2002). UCP4 and UCP5 are present only in brain.
Therefore, they were named Òbrain-specific mitochondrial carrier proteinsÓ
(BMCP) (Yu et al. 2000). UCP2 is expressed in most tissues and cell types,
including β-cells of the pancreas and the heart (Van Der Lee et al. 2000;
Barazzoni and Nair 2001; Blanc et al. 2003), whereas UCP3 is predominantly
expressed in skeletal muscle, myocardium and brown adipose tissue (Vidal-
10

Introduction

Puig et al. 1997). While the expression of UCP2 and UCP3 in different tissues
was shown mainly on the mRNA level, the protein expression is not always
identical (Sivitz et al. 1999; Pecqueur et al. 2001). For instance UCP2 mRNA
was detected in myocardial muscle, skeletal muscle and BAT, but UCP2 protein
was not detected in these tissues (Pecqueur et al. 2001; Krauss et al. 2005).
Overexpressing UCP2 in yeast led to increased uncoupling of oxidative
phosphorylation, decreased membrane potential and decelerated growth
(Fleury et al. 1997; Gimeno et al. 1997). Similarly, overexpression of UCP3 led
to increased uncoupling of ATP production (Clapham et al. 2000). Hence, UCPs
may lead to insufficient amounts of cardiac energy and play an important role in
the development of heart failure.
1.4. The impact of TGF-β1, Ang II and β-adrenergic system on
sUCP Currently, little is known about the association between cardiac UCPs,
growth factors such as TGF-β1 and neuroendocrine systems such as the RAAS
and β-adrenergic system. However, some studies point to correlations in other
cell types and organ systems. For example, TGF-β1 concentration- and time-
dependently induces the expression of UCP mRNA in BAT. It was postulated
that TGF-β1 has physiological importance as an endocrine factor regulating the
differentiation of BAT (Teruel et al. 1995). Therefore, with respect to
cardiomyocytes, where UCPs are expressed, TGF-β1 could affect energy
metabolism and therefore represent an unknown regulatory mechanism in
myocardial hypertrophy and function. This hypothesis would explain the results
of preliminary studies on TGF-β1-TG mice, which showed a lower contractile
reserve upon dobutamine stimulation, than the WT (C57BL/6) controls
(Rosenkranz et al. 2003). It is assumed that upregulation of UCPs through TGF-
β1 leads to reduction of ATP-production in the mitochondria, resulting in
insufficient energy production and myocardial dysfunction. This hypothesis is
supported by the fact that the contractile response to dobutamine in TGF-β1-TG
11

Introduction

mice was restored by treatment with a neutralizing antibody (sTGF-β1-Fc)
(Rosenkranz et al. 2003).
Based on the established connection between Ang II and TGF-β1, it is
expected that UCPs are also induced by Ang II. Indeed, it has been shown that
the intracerebroventricular infusion of Ang II in rats led to a significant
upregulation of UCP1 mRNA expression in BAT (Porter et al. 2003). Likewise, it
has been shown that the upregulation of UCP2 mRNA in a model of aortic
regurgitation (chronic volume overload) was suppressed by the ACE inhibitor
perindopril, which was associated with preservation of myocardial function
(Murakami et al. 2002). Furthermore, the reduction of postinfarction cardiac
remodeling and improvement of contractility by bisoprolol and captopril in rats
correlated with an improvement of cardiac energy metabolism (Hugel et al.
. 1999)In summary, the above mentioned studies suggest important connections
between the UCPs, the RAS and TGF-β1, which may be relevant for cardiac
energy metabolism and myocardial function.
1.5. Hypothesis and main goal
Despite the importance of heart failure, studies and extensive data known
to date, signal transduction pathways, which play a role in the development of
cardiac hypertrophy and myocardial dysfunction, have not been fully
characterized. The main goal of the project was to investigate the role and
regulation of UCPs and mitochondrial energy metabolism within the scope of
TGF-β1-induced myocardial hypertrophy and failure. This proposed mechanism
could be important for the progression of cardiac hypertrophy to failure. The
inhibition of TGF-β1-induced UCPs could play an important clinical role in the
development of novel therapeutic interventions. Therefore, the purpose of this
dissertation was to address the following questions:

12

Introduction

1. Does regulation of UCPs via TGF-β1 take place in isolated
cardiomyocytes?
2. Does TGF-β1 regulate UCPs in isolated mitochondria from heart and
skeletal muscle in vivo?
3. Does the inhibition of UCPs affect cardiac function in vitro and in vivo?
Hence, we wanted to detect whether the amounts of UCPs in isolated
cardiomyocytes and mitochondria from TGF-β1-TG mice hearts differ from the
amounts of UCPs in WT (C57BL/6) mice. Next, we intended to analyze whether
upregulation of UCPs leads to deterioration of myocardial function in vitro and in
vivo. In this respect, we postulate the following hypothesis: various stimuli
increase the concentration of TGF-β1, leading to upregulation of mitochondrial
UCPs, which then presumably decrease the synthesis of ATP. The latter may
potentially lead to the development of myocardial dysfunction in the
hypertrophied heart.

13

Introduction

Different stimuli
(pressure overload, Ang II, etc.)
β-TGF1 CPU Mitochondrial energy metabolism
ATP Hypertrophy Heart
Failure

Different stimuli
(pressure overload, Ang II, etc.)
TGF-β1
CPU Mitochondrial energy metabolism
ATP Hypertrophy Heart
Failure
Figure 5: Schematic highlight of the demonstrated hypothesis.
Different stimuli, generated through mechanical overload or Ang II lead to increased
concentration of TGF-β1. TGF-β1 upregulates the UCPs: an increased expression of UCPs
leads to decreased production of ATP. This may represent a so far unknown mechanism for the
development of heart failure and the transition from compensated myocardial hypertrophy to
failure.

14

Materials and Methods

2. Materials and Methods
2.1. Materials
Chemicals and compounds 2.1.1. 10 mM dATP, dGTP, dCTP and dTTP
Acrylamid
AllStars Negative Control siRNA
6-Aminohexane acid
Ammoniumpersulfate
Aprotinin
Ascorbic acid
Bisacrylamid (N,N`-methilen-bis-acrylamid)
Bradford BioRad-protein-assay
β-mercaptoethanol
Bovine serum albumine
Bovine serume albumine for Bradford protein-assay
Bromophenol blue
Calcium chloride
0.1 M dithiothreitol
Dry milk, low-fat milk powder
ECL-detection-reagent
Ethylene diamine tetracetic acid
Ethanol
5x first strand buffer
Genipin
Glucose
15

Invitrogen
amgSi Qiagen amgSi arvSegSi am amgSi amgSi Rad-oBi amgSi amgSiFermentas
amgSi rckeMInvitrogen
Hochwald
Amersham
amgSi RothInvitrogen
koaW amgSi

Materials and Methods

Glycerol
Glycin
Halt protease inhibitor cocktail
High molecular weight marker
HiPerFect Transfection Reagent
Hyperfilm MP
Leupeptin
Low molecular weight marker
Magnesium chloride
Mastermix for RT-TaqMan¨ PCR
Methanol
Mitochondria isolation kit
Mm/Hs_MAPK1 control siRNA
M-MLV-Reverse transcriptase
One-way-Pellet-Pistill for 1.5 ml microtubes
Pellet-Pistill homogenizer
PageRulerTM prestained protein ladder
Pipettes
Phenylmethylsulphonyl fluoride
Polyoxyethylene (20) sorbitan monolaurate (TWEEN 20)
Potassium chloride
Potassium hydrogen phosphate
Ponceau S solution
Proteinase K
(-)-Propanol
PVDF Membrane
Random primers for cDNA-Synthesis
Recombinant human TGF-β1
RNase-free DNase set
RNase-free water
RNase-OUT
RNAlater RNA stabilization reagent
RNeasy Mini Kit for tissues
siRNA against UCP2 and UCP3-genes
16

rckeM rckeM rceePi amgSi QiagenAmersham
amgSi amgSi rckeMApplied Biosystems
Roth rceePi QiagenInvitrogen
Fisher Scientific
Fisher Scientific
Fermentas
amgSiamgSi amgSi rckeM rckMe Roth Qiagen amgSi Rad-oBiInvitrogen
PromoKine
Qiagen RothInvitrogen
Qiagen Qiagen Qiagen

Materials and Methods

rckeM Roth rckeM rckeM arvSe amgSi RothSi amgAmbion

Sodium acid Merck
Sodium chloride Roth
Sodium hydrogen carbonate Merck
di-Sodium hydrogen phosphate Merck
Sodium Dodecyl Sulfate Serva
N,N,N',N'-Tetramethylethylenediamine Sigma
Tris-(hydroxymethyl)-aminomethan Roth
Triton X-100 Sigma
Yeast-RNA Ambion
2.1.2. Antibodies
Anti-cytochrome c oxidase complex IV (COX-I) Invitrogen
Anti-mitochondrial UCP3 Affinity Bioreagents
Anti-mitochondrial UCP2 Santa Cruz
Anti-mitochondrial UCP2 Alpha Diagnostics
Anti-mouse-IgG (whole molecule), peroxidase conjugate Sigma
Anti-rabbit-IgG (whole molecule), peroxidase conjugate Sigma
Anti-goat-IgG (whole molecule), peroxidase conjugate Sigma
Anti-SERCA2 Affinity Bioreagents
Anti-Calsequestrin Affinity Bioreagents
Anti-p38-MAPK Sigma
Anti-Ras-GAP Dr. A. Kazlauskas,
Harvard Medical
School, USA
Anti-ANT Dr. M. Huntgeburth,
Harvard Medical
School, USA

Anti-ANT

17

Materials and Methods

2.1.3. Primers
UCP-3 mouse forward-primer (5«-TGC TGA GAT GGT GAC CTA CGA-3«)
UCP-3 mouse reverse-primer (5«-CCA AAG GCA GAG ACA AAG TGA-3«)
UCP-3 mouse TaqMan probe (5«-AAG TTG TCA GTA AAC AGG TGA GAC
TCC AGC AA-3«)
UCP-2 mouse forward-primer (5«-TCA TCA AAG ATA CTC TCC TGA AAG C-
«)3UCP-2 mouse reverse-primer (5«-TGA CGG TGG TGC AGA AGC-3«)
UCP-2 mouse TaqMan probe (5«-TGA CAG ACG ACC TCC CTT GCC ACT-3«)
β-actin mouse forward-primer (5«-CTG CCT GAC GGC CAA GTC-3«)
β-actin mouse reverse-primer (5«-CAA GAA GGA AGG CTG GAA AAG A-3«)
β-actin mouse TaqMan probe (5«-CAC TAT TGG CAA CGA GCG GTT CCG-3«)
2.1.4. TGF-β1-transgenic mice
To examine the role of TGF-β1 in the myocardium, experiments were
performed in TGF-β1 overexpressing mice. These were developed and kindly
provided by Dr. N. Sanderson and Dr. S.S. Thorgeirrson (National Cancer
Institute, NIH, Bethesda, MD/USA) (Sanderson et al. 1995). In brief, the 4.7-kb
transgene was composed of the murine albumine promoter and enhancer,
linked to a porcine TGF-β1 construct and the 3Õ region of the human growth
hormone gene, which contains a polyadenylation signal. The TGF-β1 cDNA
encodes cysteine/serine substitutions at amino acid residues 223 and 225,
resulting in preferential secretion of mature TGF-β1 (Samuel et al. 1992).
In the presented work we used mice from strain 25, which show the
highest transgene expression of TGF-β1 in the liver. The fact that all transgenic
mice were males indicates that the modified gene is located on the Y
chromosome.

18

Materials and Methods

All investigations were performed in 8-week old male Alb/TGF-β1 (Cys223,
225Ser) transgenic mice and WT (C57BL/6) mice according to the National
Institutes of Health Guide for Care and Use of Laboratory Animals and
Institutional Animal Care and Use Guidelines. All animals were anesthetized
before the examinations. The mice were painlessly sacrificed and weighed.
Subsequently, the heart and skeletal muscle (M. quadriceps) of mice were
extracted, weighed and used for further analysis, such as preparation of tissue
homogenates, isolation of mitochondria, isolation of cardiomyocytes and RNA
isolation. Heart lysates and isolated mitochondria were used for further Western
blot analysis. Contractility was measured by using isolated cardiomyocytes.
Isolated RNA was used for further reverse-transcriptase cDNA first-strand
synthesis, which was used in Real-Time quantitative RT-PCR (TaqMan¨ PCR)
with primers and TaqMan probes.
2.1.5. Treatment of TGF-β1-transgenic mice
The mice were treated from week three (immediately after weaning) to
week eight with either metoprolol (350 mg/kgBW/d) or telmisartan (10
mg/kgBW/d), each supplied with the drinking water, or by intraperitoneal
application of soluble TGF-β receptor-Fc (sR-Fc; 1 mg/kgBW every other day)
as indicated. The latter compound was previously shown to act as a potent
TGF-β antagonist (Lee et al., 2001).
2.1.6. Human heart tissue
Left ventricular tissue was obtained from explanted hearts of patients
undergoing heart transplant. In DCM hearts, LVEF was <40%. Non-failing
controls represent hearts that could not be implanted for whatever reason.
19

Materials and Methods

2.2. Methods
2.2.1. Cardiomyocyte cultures
Isolation of cardiomyocytes and measurement of contractility were
performed in cooperation with the department of Physiology, University Clinic of
Giessen, Germany (Klaus-Dieter Schlter). Ventricular cardiomyocytes were
isolated from male Wistar Kyoto rats and male Alb/TGF-β1 (Cys223, 225Ser)
transgenic or WT (C57BL/6) mice as previously described (Zhang et al. 2004).
Briefly, the animals were injected with heparin (5,000 IU/kg, i.p.) 20 min prior to
the experimental protocol, anesthetized and then sacrificed by cervical
dislocation. The heart was excised and the aorta was cannulated rapidly. The
cannulated heart was mounted on a Langendorff perfusion apparatus with
constant flow, where perfusion pressure was monitored. The initial perfusion
pressure was maintained at 40 mmHg by regulating the flow rate. The heart
was digested by 0.05% crude collagenase I at 37¡C and the enzymatic
digestion was terminated immediately when the perfusion pressure was lowered
to 28 mmHg. The heart was then cut off the cannula, and the atria and aorta
were dissected. The ventricular tissue was chopped and the single myocytes
were dispersed gently by a wide-tipped pipette. The viability of freshly isolated
cardiomyocytes was more than 70%. The cardiomyocytes were kept in Joklik's
minimum essential medium containing 1% BSA and 10 mmol/L BDM, then
extracellular calcium was restored step-wise to a final concentration of 1.25
mmol/L. The viability of cardiomyocytes reduced to 40-50% after 2 h standing.
More than 90% of rod-shaped cardiomyocytes were quiescent and had visible
cross striations and sharp edges.
20

20

Materials and Methods

2.2.2. Measurement of cardiomyocyte contractility
The impact of TGF-β1 overexpression on the contractile function of
cardiomyocytes was evaluated by measuring the cell shortening of isolated
cardiomyocytes under electrical stimulation (Rosenkranz et al. 1997). Cardiac
myocytes were suspended in a thermostatically controlled Perspex chamber of
400 µl volume and superfused at 0.6 ml/min with a modified TyrodeÕs solution
containing in mmol/l: 119.6 NaCl, 5.36 KCl, 1.0 CaCl2, 1.05 MgCl2, 22.6
NaHCO3, 0.42 NaHPO4, 5.046 glucose, 0.283 ascorbic acid and 0.05 EDTA.
The medium was oxygenated with carbogen (95% O2, 5% CO2) and
temperature was maintained at 32±0.5¡C. The cells were electrically field-
stimulated with platinum electrodes at 0.5 Hz with 5-ms pulses by an electronic
stimulator (Stim2; Scientific Instruments, Heidelberg, Germany). Contraction
amplitudes were measured using a phase-contrast microscope (Diaphot 300,
Nikon, Tokyo, Japan) connected to a one-dimensional camera (ZK4, Scientific
Instruments, Heidelberg, Germany), which detects the edges of the cell by light-
dark contrasts in the microscope image. These signals were digitalized on an
oscilloscope (HM 205-3, Hameg Instruments, Kowloon, Hong Kong) and cell
images were analyzed by a computer using the MUCELL program (Scientific
Instruments, Heidelberg, Germany), which measured the diastolic length of the
myocyte and cell shortening, synchronized to electrical stimulation. The twitch
amplitude was calculated as the maximal shortening length subtracted from the
resting length. In order to evaluate contractile function precisely, twitch
amplitudes were normalized by dividing each by the resting length (Sakai et al.
1992) 21

21

Materials and Methods

2.2.3. Inhibition of UCPs with siRNA and genipin and measurement of
cardiomyocyte contractility.
Silencing of UCP-gene was performed using siRNA against UCP2, UCP3
and both UCP2 and UCP3-genes according to the reverse-transfection protocol
for 6-well plates from Qiagen. Shortly before transfection, 1.5-6x105 neonatal
cardiomyocytes per well were seeded in 2.3 ml of culture medium. The
cardiomyocytes were incubated at 37¡C until transfection. 150 ng siRNA
(corresponding to 6 µl from 2 µM stock solution) were diluted in 400 µl of culture
medium without serum, so that the final concentration of 5 nM was reached. 12
µl of HiPerFect Transfection Reagent were added to the diluted siRNA and
mixed by vortexing. The samples were incubated at room temperature (15-
25¡C) for 5-10 min to allow the formation of transfection complexes. The
complexes were added drop-wise onto the cells and the plate was gently
swirled to ensure uniform distribution of the transfection complexes. The
cardiomyocytes were incubated for 24 hours under normal growth conditions for
gene silencing. Mm/Hs_MAPK1 Control siRNA was used as positive control and
AllStars Negative Control siRNA was used as negative control. Mock-
transferred cells were used as mock transfection control, which go through the
transfection process without addition of siRNA (cells are treated with
transfection reagent only).
An alternative approach to inhibit UCPs in isolated cardiomyocytes was
also performed, as cardiomyocytes were preincubated with herbal substance
genipin. In the presence of genipin, the contractile response of cardiomyocytes
to isoprenaline was measured. The measurements of cardiomyocyte
contractility were performed as described above.
22

22

Materials and Methods

2.2.4. Preparation of whole heart lysates
Mouse hearts from WT (C57BL/6) and TGF-β1-TG mice were excised and
frozen as previously described. Left ventricular tissue was homogenized by
incubation in extraction buffer (10 mM cacodylicacid, 150 mM NaCl, 1 µM
ZnCl2, 20 mM CaCl2, 1.5 mM NaN3, 0.01% Triton-X100, pH 5.0) for 12 h at 4¡C
and subsequent centrifugation for 10 min at 1.200 x g.
For further processing, heart tissues were slowly thawed on ice and
homogenized with a Glass-Teflon-Potter (900U/min) in 1 ml of homogenization
buffer (0.1 mol/l Tris, 1 mmol/l EDTA, 1 mmol/l PMSF, 50 µl aprotinin 1 mg/ml,
50 µl leupeptin 1 mg/ml) per 100 mg tissue at 4¡C, homogenized 5 times for 5
sec in an ultrasound bath and incubated on ice for 15 min. The homogenates
were centrifuged for 20 min at 4¡C and 10.000 x g (Beckmann, Rotor JA 20).
The supernatants were aliquoted, stored at -80¡C and used for further Western
blot analysis. Protein concentrations were determined using the Bradford
Protein-Assay (Bradford 1976).
2.2.5. Isolation of mitochondria
The isolation of mitochondria was performed according to the Mitochondria
Isolation Kit protocol (Pierce Biotechnology). After extraction of the tissues
(heart and skeletal muscle) they were washed twice in 2 ml phosphate buffered
saline (PBS) [137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.76 mM
KH2PO4], cut into small pieces, put into a microcentrifuge tube with 800 µl
BSA/Reagent A solution [4 mg/ml] + 1:100 EDTA free Halt Protease Inhibitor
and homogenized using Dounce Homogenization. Subsequently, the
homogenates were mixed with Reagent C solution + 1:100 EDTA free Halt
Protease Inhibitor and centrifuged for 10 min at 4¡C at 700 x g. The supernatant
was then decanted into a new 2 ml microcentrifuge tube and centrifuged for 15
min at 4¡C at 3.000 x g. The supernatant (cytosol fraction) was frozen at -20¡C
23

Materials and Methods

for further analysis. The pellet was washed with 500 µl washing buffer (1:1
Reagent C solution and bidistilled water) and centrifuged for 5 min at 4¡C at
12.000 x g. The pellet, which contains only mitochondria, was harvested in
bromophenol blue-free probe buffer (2.0 ml glycerol, 2.0 ml 10% SDS, 2.5 ml
stacking gel buffer [pH 6.8: 6.06 g Tris, 4.0 ml 10% SDS, ad 100 ml aqua dest.],
500 µl β-mercaptoethanol ad 10 ml aqua dest.) and gently mixed by pipetting.
The final protein concentration was measured using the Bradford BioRad-
Protein-Assay (Bradford 1976). The mitochondria were kept at -20¡C and were
subsequently used for Western blot analysis.
2.2.6. Determination of protein concentrations
Protein concentrations were determined according to the Bradford method
using the Bradford BioRad-Protein-Assay. This is based on a colorimetric
method, using the Coomassie Brilliant Blue G-250. In approximately 2 minutes
the binding of the dye to the protein takes place and the complex remains
dispersed for approximately 1 hour (Bradford 1976). The absorbance was
measured at 595 nm using a spectral photometer (Mithras LB 940, Berthold
Technologies) with support of MikroWin 2000 software (Mikrotek Laborsysteme
GmbH). The calibration curve was created based on the BSA-standard curve
with a concentration range between 1 and 20 mg/ml.
2.2.7. Western blotting
After the preparation of lysates and isolation of mitochondria, the samples
were mixed with bromophenol blue-containing probe buffer (2.0 ml glycerol, 2.0
ml 10% SDS, 0.025 mg bromophenol blue, 2.5 ml stacking gel buffer [pH 6.8:
6.06 g Tris, 4.0 ml 10% SDS ad 100 ml aqua dest.], 500 µl β-mercaptoethanol
24

Materials and Methods

ad 10 ml aqua dest.), so that the final concentration was 20 µg. The probes
were then heated for 3 min at 95¡C, briefly centrifuged and used for Western
blotting. Equal amounts of protein (20 µg) were loaded onto a 12.5% SDS-
polyacrylamide gel. The separation of the proteins was performed by sodium
dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) through the
separation gel (4x separation gel buffer: 1.5 M Tris/HCl pH 8.8; 0.8% (w/v)
SDS), after the proteins ran through a stacking gel (4x stacking gel buffer: 0.5 M
Tris/HCl pH 6.8; 0.8% (w/v) SDS). The polymerization of the gels was reached
by adding 0.04% ammonium persulfate (APS) [(NH4)2SO8] as a trigger of
radical formation and 0.2% N,N,N',N'-Tetramethylethylenediamine (TEMED).
The running buffer contained 25 mM Tris/HCl, 192 mM glycin and 0.1% SDS.
The SDS-PAGE ran at constant amperage of 15 mA/Gel at room temperature.
PageRulerTM Prestained Protein Ladder was used as a marker for determining
protein size. Proteins were then transferred to a PVDF membrane (with pore
size 0.2 µm), as previously described, using a semi-dry blotting method (Towbin
et al. 1979). The membrane was activated in 99.8% methanol for 1 minute, after
which it was soaked in AP2-buffer (300 mM Tris pH 10.4, 20% methanol) for 1
min. The transfer to the membrane was performed using the following layers: 3
pieces of Watman 3MM paper with a size of the separation gel, soaked for 1
min in AP1-buffer (25 mM Tris/HCl pH 10.4, 20% methanol); 2 pieces of
Watman 3MM paper, soaked for 1 min in AP2-buffer (300 mM Tris pH 10.4,
20% methanol); the activated PVDF-membrane, soaked for 1 min in AP2-buffer;
stacking gel, soaked for 1 min in CP-buffer (25 mM Tris/HCl pH 9.4; 40 mM 6-
amino hexane acid; 20% methanol). The ÕÕsandwichÕÕ was built, the bubbles
between the layers were carefully removed and the transfer of proteins onto the
membrane was performed using a blotting chamber for 1 hour at 100 mA/gel.
The membrane was blocked in 5% nonfat dry milk (5% in Tris buffered saline
(TBS) [0.05 M Tris base, 0.9% NaCl, pH 7.6]), after which it was washed for 5
min with TBS and incubated with primary antibodies overnight. The membrane
was then washed 3 times for 15 min with TBS-Tween 20 (0.05 M Tris base,
0.9% NaCl, 0.05% Tween 20, pH 7.6). After incubation with horseradish
polyclonal secondary antibodies for one hour the membrane was washed 3
times with TBS-Tween 20 and one time with TBS for 15 min, incubated in
western-blot-detection-reagent ECL for 1 min and the protein amount was
25

Materials and Methods

detected by enhanced chemiluminescence using Hyperfilm. The interpretation
of the bands took place using densitometry with Image Quant ÒPersonal
DensitometerÓ (Molecular Dynamics, Krefeld).
2.2.8. Isolation of total RNA
The isolation of RNA was performed using RNeasy Mini Kit for tissues and
cells, according to the protocol from Qiagen. After the extraction, heart tissue
(~30 µg of heart) was stabilized in RNAlater RNA stabilization reagent and kept
at 4¡C. The tissue or isolated cardiomyocytes were then put in 300 µl RTL
buffer and homogenized using Pellet-Pistill homogenizer and One-way-Pellet-
Pistill, after which 590 µl of RNase-free DEPC water were added to the
homogenate. 10 µl of proteinase K solution were added to the sample, which
was incubated at 55¡C for 10 min. After a centrifugation at 9.300 x g at room
temperature for 3 min, the supernatant was carefully transferred into a new 1.5
ml microcentrifuge tube. 0.5 volumes of 96-100% ethanol were added to the
cleared lysate and carefully mixed by pipetting. 700 µl of the sample were
pipetted into an RNeasy Mini Spin Column and centrifuged for 15 sec at 9.300 x
g. The same step was repeated with the rest of the sample. 350 µl of buffer
RW1 were pipetted into the RNeasy Mini Spin Column and centrifuged for 15
sec at 9.300 x g to wash. A mixture of 10 µl DNase I solution and 70 µl buffer
RDD was pipetted directly onto the RNeasy silica-gel membrane of RNeasy
Mini Spin Column and left for 15 min at room temperature. 350 µl of buffer RW1
were pipetted into the RNeasy Mini Spin Column and centrifuged for 15 sec at
9.300 x g. The RNeasy Mini Spin Column was transferred into a new 2 ml
collection tube, after which 500 µl of buffer RPE were pipetted onto the RNeasy
Mini Spin Column and centrifuged for 15 sec at 9.300 x g to wash the column.
Another 500 µl of buffer RPE were added to the RNeasy Mini Spin Column and
centrifuged for 2 min at 9.300 x g to dry the RNeasy silica-gel membrane. The
RNeasy Mini Spin Column was centrifuged for 1 min at full speed to ensure that
no ethanol is carried over during elution. The RNeasy Mini Spin Column was
26

Materials and Methods

transferred to a new 1.5 ml collection tube, 50 µl of RNase-free water were
pipetted directly onto the RNeasy silica-gel membrane and centrifuged for 1 min
at 9.300 x g to elute. The final concentration of RNA was measured using a
spectrophotometer and kept at -80¡C, until RNA was further used for First-
Strand cDNA Synthesis.
2.2.9. Reverse Transcriptase first-strand cDNA synthesis
Reverse Transcriptase (RT) cDNA first-strand synthesis was performed
according to the protocol from Invitrogen Life Sciences. The following
components were mixed together in a nuclease-free microcentrifuge tube: 150
ng random primers, 2 µg RNA, 1 µl 10mM dNTP-Mix (10 mM each dATP,
dGTP, dCTP and dTTP at neutral pH), RNase-free water to 12 µl. The mixture
was then heated at 65¡C for 5 min and was briefly centrifuged. The following
components were then added to the mixture: 4 µl 5x First-Strand Buffer (FSB),
2 µl 0.1 M DTT, and 1 µl RNase-OUT. The contents were carefully mixed and
incubated at 37¡C for 2 min. 1 µl (200 units) of M-MLV-Reverse Transcriptase
was added to the mixture and carefully mixed. The contents were then
incubated at 25¡C for 10 min, at 37¡C for 50 min and at 70¡C for 15 min. The
cDNA samples were stored at -20¡C and were subsequently used as a template
for amplification in Real-Time quantitative RT-PCR (TaqMan¨ PCR).
2.2.10. TaqMan¨ Real-Time quantitative RT-PCR
Real-Time TaqMan¨ RT-PCR was performed with an ABI-PRISM 7500
sequence detector system (Applied Biosystems) using the PCR Core Reagent
Kit (Applied Biosystems) and analyzed using the comparative threshold cycle
(Ct) method. A total volume of 25 µl of earlier synthesized cDNA samples from
27

Materials and Methods

TGF-β1-TG and WT (C57BL/6) male mice (n=9) were transferred into a 96-well
plate with the following primers and probes, labeled with carboxyfluorescein
dye: UCP-3: 5«-TGC TGA GAT GGT GAC CTA CGA-3« (forward-primer), 5«-
CCA AAG GCA GAG ACA AAG TGA-3« (reverse-primer), 5«-AAG TTG TCA
GTA AAC AGG TGA GAC TCC AGC AA-3« (probe); UCP-2: 5«-TCATCA AAG
ATA CTC TCC TGA AAG C-3« (forward-primer), 5«-TGA CGG TGG TGC AGA
AGC-3« (reverse-primer), 5«-TGA CAG ACG ACC TCC CTT GCC ACT-3«
(probe). The following conditions were used: 2 minutes at 50¡C and 10 minutes
at 94¡C, followed by a total of 40 cycles of two temperature cycles (15 seconds
at 95¡C and 1 minute at 60¡C). Each sample was analyzed in triplicate and
normalized to values for 18S mRNA.
2.2.11. Dobutamine stress echocardiography
The in vivo analysis of contractile function under resting conditions and
upon catecholamine stimulation was performed in cooperation with the
Department of Cardiology, University Clinic Bonn, Germany (Klaus Tiemann) in
male WT (C57BL/6) and treated or untreated TGF-β1-TG mice, using
dobutamine stress echocardiography (DSE). Images were obtained by using an
HDI-5000 ultrasound device (Philips Medical Systems, Bothell, WA, USA)
equipped with a linear array transducer (15 MHz) as described (Tiemann et al.
2003). Parasternal short and long axis views were obtained in two-dimensional
B-mode. At least 20 cardiac cycles were obtained for each view, and each
imaging plane was acquired three times to assess reproducibility. One-
dimensional M-mode imaging was performed two-dimensionally guided in the
parasternal short axis view at the level of the papillary muscle. Parasternal short
axis views were divided into six segments and long axis views were divided into
seven segments (Cerqueira et al. 2002). The endocardial borders were
manually traced on the innermost endocardial edge, while the epicardial
borders were defined by tracing along the first bright pixel adjacent to
myocardial tissue (Collins et al. 2001). Left ventricular mass (LVM) and left
28

Materials and Methods

ventricular ejection fraction (LVEF) were assessed as previously described
(Tiemann et al. 2003). The resistive index (RI) was calculated as 1 Ð
enddiastolic velocity/systolic velocity. After light anesthesia, the animals were
injected with dobutamine in various concentrations (10, 20, 40 µg/kgKGBW/min)
after microscopical cannulation of the tail vein. This procedure corresponds to
the recommended protocol of the American Society of Echocardiography that is
used in humans (Armstrong et al. 1998). Through echocardiographic standard
procedure the effect of TGF-β1 on cardiac function under exposure conditions
was examined in vivo on TGF-β1-TG and WT (C57BL/6) mice. 2D- and m-mode
registrations were recorded at each level of dobutamine. The measurements
were repeated after an intraperitoneal injection of genipin (100 mg/kg body
weight) 1 hour prior to the experiment.
2.2.12. Statistics
All results are presented as mean values ± SEM. Statistical significance
(p) was acquired using either student t-test for unrelated random samples or
ANOVA followed by Student-Neumann-Keuls test. A P-Value <0.05 was defined
as statistically significant. The results were presented with the help of Graph
Pad Prism software (Graph Pad Software Incorporated, San Diego, USA).

29

Results

3. Results
3.1. Regulation of the expression of UCPs in ventricular
cardiomyocytes under the influence of TGF-β1
To investigate whether TGF-β1 regulates the expression of UCPs in
cardiomyocytes, the expression of UCP2 and UCP3 was measured on the
mRNA-level in rat ventricular cardiomyocytes that had been pre-stimulated for
24 hours with 10 ng/ml of recombinant human TGF-β1. As demonstrated in
figure 6, stimulation with TGF-β1 for 24 hours induced the expression of the
UCP2 and UCP3 genes in cardiomyocytes as compared with unstimulated
cells. UCP2 mRNA increased approximately 9-fold, whereas UCP3 even
showed a 44-fold increase.

Figure 6: Expression of UCP2 and UCP3 mRNA in isolated ventricular cardiomyocytes of
the rat.
Quantitative Real-Time PCR (qPCR) of UCP2 mRNA (A) and UCP3 mRNA (B) in ventricular
cardiomyocytes, isolated from Wistar Kyoto rats, stimulated with recombinant human TGF-β1
versus non-stimulated cells. The demonstrated values correspond to mean values ± SEM (n=4).
*P<0.05 versus controls.
30

Results

3.2. Regulation of the expression of UCPs in mouse heart and
skeletal muscle under the influence of TGF-β1
To assess the expression of UCPs in mouse heart tissue, the mRNA and
protein-levels of UCP2 and UCP3 were measured using Real-Time RT-PCR
(TaqMan¨-PCR) and Western blotting. Figure 7 depicts significantly higher
expression of UCP2 and UCP3 mRNA within the hearts of TGF-β1-TG mice as
compared to WT (C57BL/6) control mice. Interestingly, chronic treatment with
the β-adrenoceptor blocker metoprolol as well as with the TGF-β1 antagonist
sR-Fc prevented the upregulation of UCP2 and UCP3 mRNA, whereas
treatment with the angiotensin AT1-receptor blocker telmisartan had no effect on
UCP expression in TGF-β1-TG mice.

Figure 7: Expression of UCP2 and UCP3 mRNA in heart tissue from WT (C57BL/6) and
TGF-β1-TG mice.
Quhearant ttiitsastivue e fRreomal -TiWmT e( PC5C7RB L(/q6P),C RTG) Fof- βU-CTGP2 mmicReN, Aa n(Ad ) aTGnFd- Uβ-CPTG3 mmiRceN, A w(Bhi)c hin hvaevnet rbiceuelanr
11treated with metoprolol, telmisartan or TGF-β1-antibody (TGFβR-Fc). The levels of UCP2 and
UCP3 mRNA were normalized for the level of 18S mRNA. The demonstrated values correspond
to mean values ± SEM (n=4-9). *P<0.05 versus WT.
31

31

Results

In order to answer the question, whether TGF-β1 regulates the expression
of UCP3 on the protein level, the specificity of a commercially available anti-
UCP3-antibody (Affinity BioReagents) was tested in Western blotting analysis
on mitochondrial extracts from the hearts of WT (C57BL/6) and UCP3-Knockout
mice (UCP3-KO) (kindly provided by Dr. Michael Huntgeburth, Harvard Medical
School, Boston/USA). As shown in Figure 8A, the antibody detects a protein
band at the expected protein level size (~32 kDa), whereas there was no signal
at the expected size in samples from UCP3-KO mice, indicating that the
antibody detected the correct protein band of UCP3.
The protein expression of UCP3 was measured in isolated mitochondria
from WT (C57BL/6) and TGF-β1-TG mice by Western blotting analysis using the
above antibody. The expression of UCP3 in heart mitochondria under the
influence of TGF-β1 is increased as compared to WT (C57BL/6) mice.
Densitometric analysis shows an approximately 2.3-fold increase of UCP3
expression in mitochondria isolated from TGF-β1-TG mouse hearts (P=0.008)
(Fig. 8B). Anti-cytochrome c oxidase complex IV (COX-I) antibodies in a
concentration of 0.25 µg/ml were used as a loading control, and anti-
mitochondrial UCP3 antibodies in a concentration of 1 µg/ml were used for
detecting mitochondrial UCP3.
In order to confirm the regulating function of TGF-β1 on the expression of
UCP3, protein expression was measured in other muscular tissue. To this end,
it could be demonstrated that the expression of UCP3 is also upregulated by
TGF-β1 in isolated mitochondria from skeletal muscle of musculus quadriceps
(Fig. 8D, E). In summary, these data demonstrate that the expression of UCP3
in mitochondria, isolated from either heart or skeletal muscle, is upregulated by
TGF-β1, in vivo.

32

Results

Figure 8: Protein expression of UCP3 in heart and skeletal muscle.
A) Representative immunoblots for verification of the specificity of the used antibody.
Mitochondria were isolated from the hearts of WT (C57BL/6) and UCP3-deficient (UCP3-KO)
mice. UCP3-KO serves as control for the specificity.
B) Representative immunoblots for the regulation of UCP3-protein in isolated mitochondria from
hearts of WT (C57BL/6) and TGF-β1-TG mice. Upper band: immunoblotting against cytochrome
c oxidase complex IV (COX-I) to control constant loading of proteins onto the gels. Bottom
band: immunoblotting against UCP3 in isolated heart mitochondria from WT (C57BL/6) and
TGF-β1-TG mice.
C) Densitometric analysis of UCP3 immunoblots from heart mitochondria (n=12). *P<0.01
versus WT control mice.
D) Representative immunoblots for regulation of UCP3-protein in isolated mitochondria from
skeletal muscle (musculus quadriceps) of WT (C57BL/6) mice and TGF-β1-TG mice. Upper
band: immunoblotting against cytochrome c oxidase complex IV (COX-I) to control constant
loading of proteins onto the gels. Bottom band: immunoblotting against UCP3 in isolated
skeletal muscle mitochondria from WT (C57BL/6) and TGF-β1-TG mice.
E) Densitometric analysis of UCP3 immunoblots from skeletal muscle mitochondria (n=14).
*P<0.01 versus WT (C57BL/6) control mice.

33

Results

The regulation of UCP2 was investigated in isolated heart mitochondria
from WT (C57BL/6) and TGF-β1-TG mice, using numerous antibodies. Despite
the intensive efforts, neither the expression of UCP2-protein, nor its induction by
TGF-β1 could be demonstrated (Fig. 9). Anti-cytochrome c oxidase complex IV
(COX-I) antibodies in a concentration of 0.25 µg/ml were used as a control and
anti-mitochondrial UCP2 antibodies from different companies in various
concentrations were used for detecting mitochondrial UCP2. The failure to
detect UCP2 protein in the heart is consistent with other investigations
(Pecqueur et al. 2001; Krauss et al. 2005)

FiRepgurrees e9n: taPtrivote eiimn mexunproeblsosti ondepi ocf tsU tChPe2 r ieng uthlaeti ohen oarft. U CP2-protein in isolated mitochondria from
the hearts of WT (C57BL/6) and TGF-β-TG mice. Upper band: immunoblotting against
cytochrome c oxydase complex IV (COX-I) to1 show the accurate isolation of mitochondria and to
inco isntorolal tecod nhsteaarntt lmoitaodcinhgo nodfr piar fortoeimn s WoTn to(C t5h7eB Lg/e6l)s. Band ottToGmF -βband-TG: i mmimcuen. oblotting against UCP3
1 3.3. Effect of UCP-inhibition on contractile function in vitro
Inhibition of UCP in vitro was examined on isolated cardiomyocytes from
WT (C57BL/6) and TGF-β1-TG mice by measuring their contractile response to
β-adrenergic stimulation with isoprenaline, with and without prestimulation with
herbal agent genipin, which was previously shown to act as a potent UCP
inhibitor (Zhang et al. 2006). First, the contractility of cardiomyocytes was
34

Results

measured on non-genipin-treated cardiomyocytes after stimulation with
isoprenaline, and then the same procedure was repeated with cardiomyocytes,
which had been pretreated with genipin (Fig. 10). While the contractile response
of isolated cardiac myocytes to isoprenaline was diminished in cells from
untreated TGF-β1-TG mice, pre-treatment with genipin (5 µM) reversed this
effect, so that cells from TGF-β1-TG mice responded better to isoprenaline than
cells from WT (C57BL/6) mice. These data indicate that the expression and
activity of UCPs are critically involved in the determination of the contractile
response to catecholamines in the hearts from TGF-β1-TG mice. This also
suggests that the improvement of inotropic reserve in TGF-β1-TG mice by
chronic β-blocker treatment as previously shown (Rosenkranz 2004) may at
least be partly explained by downregulation of the increased UCP levels in the
hearts from TGF-β1-TG mice.
Figure 10: Contractility of mouse cardiomyocytes.
Contractility of cardiomyocytes, isolated from WT (C57BL/6) and TGF-β1-TG mice. The
contractile response to isoprenaline was measured without and with pretreatment with 5 µM
genipin (n=4). *P<0.05 versus control.
In a second approach, it was attempted to repeat the experiments by
silencing UCP-genes with small interfering RNA (siRNA) before measuring the
35

Results

contractility of cardiomyocytes. Unfortunately, these experiments did not give us
valuable results in respect of contractility of cardiomyocytes, as transfection with
siRNA had significant impact on the viability of mouse cardiomyocytes, so that
contractility could not be reliably measured.
3.4. TGF-β1 does not regulate the expression of other
myocardial proteins such as SERCA2, p38-MAPK and
TAN To further support our hypothesis that the role of TGF-β1 in the attenuation
of cardiac function takes place through regulation of UCPs and not through
other pathways, we also assessed the effect of TGF-β1 overexpression on other
myocardial proteins such as sarco/endoplasmatic reticulum Ca2+-ATPase
(SERCA2), mitochondrial protein adenine nucleotide translocator (ANT), also
known as ADT/ATP translocator, and p38-MAPK. Whole heart lysates from WT
(C57BL/6) and TGF-β1-TG mice were prepared and the protein levels of
SERCA2 and p38-MAPK were determined by Western blot analysis.
Calsequestrin was used as a loading control for myocardial protein. Figure 11A-
C indicates that the increased concentration of TGF-β1 does not affect the
expression of SERCA2 and p38-MAPK. To evaluate the impact of increased
TGF-β1 levels on ANT expression, mitochondria were isolated from hearts that
were extracted from WT (C57BL/6) and TGF-β1-TG mice. In these experiments,
COX-I served as a loading control. Figure 11D-E demonstrates that
mitochondrial ANT expression was not altered in TGF-β1-TG mice.

36

Results

Figure 11: Protein expression of SERCA2, p38-MAPK and ANT in the hearts of WT
(C57BL/6) and TGF-β1-TG mice.
A) Representative immunoblots for regulation of SERCA2 and p38-MAPK in whole lysates from
the hearts of WT (C57BL/6) and TGF-β1-TG mice. Upper band: immunoblotting against
calsequestrin to control constant loading of proteins onto the gels. Middle band: immunoblotting
against SERCA2. Bottom band: immunoblotting against p38-MAPK.
B) Densitometric analysis of SERCA2 immunoblots from heart lysates of WT (C57BL/6) and
TGF-β1-TG mice (n=3).
C) Densitometric analysis of p38-MAPK immunoblots from heart lysates of WT (C57BL/6) and
TGF-β1-TG mice (n=3).
D)and ReTGprFe-sβe-ntTGativ mei ciem. mUpunpeoblr otsband: for irmmegulatunoblion ottofi ng ANagaiT inns th eCaOrt X-mIi ttooc choonntrdorila c oofn stWTan t l(Co5a7diBnLg/ o6f)
1proteins onto the gels. Bottom band: immunoblotting against ANT.
E) Densitometric analysis of ANT immunoblots from heart mitochondria of WT (C57BL/6) and
TGF-β1-TG mice (n=3).

37

Results

3.5. Effect of UCP inhibition on cardiac function in vivo
The effect of UCP inhibition on cardiac function was also examined in vivo
in WT (C57BL/6) and TGF-β1-TG mice, using dobutamine stress
echocardiography (DSE). Previous studies had shown that TGF-β1-TG mice
displayed a significantly decreased contractile reserve upon stimulation with the
β1-adrenoceptor agonist dobutamine as compared to WT (C57BL/6) mice
(Rosenkranz 2004). At peak stress (dobutamine 40µg/kg/min), the relative
increase of LVEF was 16±5% in TGF-β1-TG mice vs. 44±5% in control WT
(C57BL/6) mice (Rosenkranz 2004).
In order to assess whether UCPs are involved with the reduced inotropic
reserve in TGF-β1-TG mice, genipin was applied in TGF-β1-TG or WT
(C57BL/6) mice that underwent DSE. After intraperitoneal injection of genipin
(100 mg/kg BW) 2 hours prior to measurements, the contractile response to
dobutamine during DSE in TGF-β1-TG mice was restored and comparable to
the response of WT (C57BL/6) mice (relative increase of LVEF 42±4 vs.
44±5%; Fig. 6E) (Fig. 12), indicating that UCP activity is crucially involved in the
diminished contractile reserve of TGF-β1-TG mice.

38

Results

WT

TGF-β1-TG

WT TGF-β1-TG
Figure 12: Effect of acute UCP-inhibition on cardiac function in vivo.
Shown is the increase of left ventricular ejection fraction (EF) during dobutamine stress
echocardiography (DSE) after infusion of dobutamine (40 µg/kg/min) in WT (C57BL/6) and
TGF-β1-TG mice, as well as the effect of pretreatment with genipin in TGF-β1-TG mice (n=5).
Inhibition of uncoupling proteins by genipin (100 mg/kgBW) restored the contractile response to
dobutamine (40 µg/kg/min) in TGF-β1-TG mice. *P<0.05 versus baseline; #P<0.05 versus WT,
¤P<0.05 versus dobutamine alone in TGF-β1-TG mice.
3.6. TGF-β1 and UCP3 expression in human heart failure
To investigate whether this mechanism may also be relevant in humans,
we measured TGF-β1 and UCP3 expression in non-failing hearts and in
myocardium from patients with dilatative cardiomyopathy (DCM) who had either
been treated or not been treated with metoprolol. In a limited number of human
samples that was available to us, we show a trend towards increased TGF-β1
expression in DCM hearts regardless of β-blocker treatment (Fig. 13A).
Interestingly, there was a clear trend towards an increased expression of UCP3
in DCM hearts of patients who have not received metoprolol as compared to
non-failing myocardium. This trend did not reach statistical significance, which
39

Results

may be explained by the limited number of samples. In contrast to the
expression levels of TGF-β1, there was a lower UCP3 expression in DCM
hearts from metoprolol-treated patients as compared to those who had not
received metoprolol (Fig. 13B). Hence, increased expression of myocardial
UCPs and its downregulation by β-adrenoceptor blockade is found at least in
some patients with heart failure.

Figure 13: TGF-β1 (A) and UCP3 (B) expression in human heart.
Myocardial samples were obtained from non-failing myocardium (NF; n=3), and from DCM
hearts of patients who had not received β-blocker treatment (DCM; n=5) or patients who were
treated with metoprolol (DCM-METO; n=3). Data represent means ± SEM. *P<0,05 vs. DCM.

40

Discussion

4. Discussion
In the presented study, the impact of elevated myocardial TGF-β1 levels
on the development of cardiac hypertrophy and contractile dysfunction was
investigated. The main aim of the project was to investigate the mechanism, by
which TGF-β1 leads to the development of myocardial hypertrophy and cardiac
dysfunction. More specifically, the hypothesis was tested that diminished
contractile reserve in TGF-β1-TG mice may be due to the affection of energy
metabolism in cardiac myocytes by TGF-β1. We thus aimed to demonstrate a
link between TGF-β1 and mitochondrial UCPs in the heart.
One of the key findings was the evidence that TGF-β1 overexpression led
to upregulation of mitochondrial UCP2 and UCP3 both on the RNA and protein
levels. It was demonstrated that TGF-β1 upregulates UCP2 and UCP3 both in
vitro and in vivo. TGF-β1-TG mice, which have significantly hypertrophied heart
muscle and harbor reduced contractile response to β-adrenoceptor agonists,
demonstrated significantly higher expression of UCP2 on the RNA-level and
UCP3 on both the RNA and protein-levels. This observation was shown to be
highly relevant for the regulation of myocardial energy metabolism and thus
contractile function, as the inhibition of UCPs with herbal substance genipin,
representing a potent UCP-inhibitor (Sakaida et al. 2003), was able to restore
the contractile reserve in TGF-β1-TG mice.
Finally, it was found that cardiac TGF-β1 and UCP expressions were
elevated in heart failure in humans, and UCP Ð but not TGF-β1 Ð was
downregulated by β-blocker treatment.

41

Discussion

4.1. Regulation of mitochondrial UCPs through TGF-β1 and its
role in the regulation of energy metabolism
Numerous publications demonstrated an important role for TGF-β1 in the
development of myocardial hypertrophy, which is mainly due to induction of
proliferation of fibroblasts, collagen deposition, fibronectin synthesis, but also to
hypertrophy of cardiac myocytes. (Li et al. 1998; Pauschinger et al. 1999;
Rosenkranz 2004). Numerous studies have indicated that Ang II-induced
hypertrophy of cardiac myocytes depends on TGF-β1 (Rosenkranz 2004). For
instance, Schultz et al. proofed that the hypertrophic cardiomyocyte growth
induced by Ang II, is mediated by TGF-β1 in vivo (Schultz et al. 2002). Along
with that, it was shown by other experimental approaches that TGF-β1 is
required for Ang II-induced cardiomyocyte hypertrophy as it acts downstream of
the AT1 receptor (Rosenkranz 2004).
Although various morphological cardiac alterations that are induced by
TGF-β1 have been described in numerous studies (Brand and Schneider 1995;
Rosenkranz 2004; Bujak and Frangogiannis 2007), only little information is
known about the functional consequences of increased TGF-β1 activity in the
heart. It was previously shown that overexpression of TGF-β1 in transgenic mice
leads to an increased density of myocardial β-adrenoceptor and to
downregulation of negative regulators such as Giα and βARK-1 (Rosenkranz et
al. 2002). While the induction of a hypertrophic responsiveness to β-adrenergic
stimulation in TGF-β1-TG mice and its reversal by chronic β-adrenoceptor
blockade appears as a logical consequence of increased β-adrenergic
signaling, it is difficult to understand why the inotropic response to β-
adrenoceptor stimulation is opposedly affected.
The upregulation of cardiac mitochondrial UCPs by TGF-β1 provides a
molecular explanation for this finding. While TGF-β1 signaling has previously not
been linked to cardiac energy metabolism, a connection between TGF-β1 and
UCPs was shown in other systems. For instance, Teruel et al. showed a time-
and dose-dependent induction of UCP mRNA expression in rat fetal BAT
(Teruel et al. 1995). Recently, UCP2 was shown to be upregulated in an aortic
regurgitation model of heart failure, and mitochondrial uncoupling and an
42

Discussion

upregulation of UCP3 were demonstrated in viable myocardium of chronically
infarcted, failing rat hearts (Noma et al. 2001; Murray et al. 2008). Despite the
fact that those results havenÕt been related to TGF-β1, the upregulation of
mitochondrial UCPs correlates with an increased expression of TGF-β1 in
chronic myocardial infarction and heart failure, as shown in several animal
studies as well as in human heart (Rosenkranz 2004; Bujak and Frangogiannis
. 2007)There is a connection between increased UCP levels, derangement of
myocardial energetics and cardiac dysfunction. The contractile function of the
heart is dependent on a sufficient energy supply that has to be continuously
adapted to the energy demand. The required energy is brought by constant re-
synthesis of ATP by oxidative phosphorylation in the mitochondria. In spite of
this, cardiac energy metabolism involves three components: substrate
utilization, oxidative phosphorylation, and ATP transfer and utilization
(Neubauer 2007). A significant step in the latter component is the transfer of a
phosphoryl group from ATP to the high-energy phosphate compound,
phosphocreatine (PCr), by creatine kinase. The creatine kinase reaction
equilibrium favors ATP synthesis over PCr synthesis by a factor of ~100. Thus,
when the energy demand outstrips the energy supply, PCr levels decline,
whereas ATP levels remain unchanged, and free ADP levels rise. A reduction in
the ATP transfer capacity through creatine kinase leads to an insufficient
transport of high-energy phosphate bonds from the mitochondria to the
myofibrils, resulting in contractile dysfunction (Neubauer 2007). UCPs are
believed to play a critical role in this context, as they uncouple oxygen
consumption from ATP synthesis and thus impair myocardial energy
metabolism.
Interestingly, the functional importance of UCPs was apparent only under
the stimulation of β-adrenoceptors. Therefore, the energy supply appeared
adequate under resting conditions, but insufficient under high work-load
conditions. These could be due to that the fact, that when the hypertrophied
heart undergoes β-adrenergic stimulation, causing high workload conditions, the
PCr/ATP ratio decreases while the level of free ADP increases. The latter
correlates with an upregulation of mitochondrial UCPs.

43

Discussion

Our data show that TGF-β1 upregulates the expression of mitochondrial
UCP2 and UCP3 mRNA in cardiac myocytes, and that overexpression of TGF-
β1 in TGF-β1-TG mice is associated with increased levels of UCP3 protein both
in the heart and in skeletal muscle, which leads to a mediation of proton leak
and therefore to possible uncoupling of ATP-synthesis.
Despite the usage of multiple antibodies, it was not possible to prove the
regulation of UCP2 on the protein-level. Although these results are
unsatisfactory, they are consistent with the results from different other
publications. Pecqueur et al. (Pecqueur et al. 2001) and Krauss et al. (Krauss et
al. 2005) have reported in their studies, during which the specificity of UCP2-
antibody was examined in mitochondria isolated from UCP2-deficient mice
(UCP2-KO), that it was not possible to verify UCP2-protein in various tissues,
such as heart, skeletal muscle and brown adipose tissue. These results are in
line with our data. However, controversial debates in the literature still take
place. If despite the positive detection of UCP2 mRNA, UCP2-protein is being
expressed at all or if the lack of evidence is limited by quality of available
antibodies - still remains to be elucidated. The detection of UCP2-protein raises
doubts, since UCP2 has a very short half-life of merely 20-30 minutes (Rousset
et al. 2007). Hence, UCP2-protein could have been almost totally catabolized or
decreased during the preparation of lysates, isolation of mitochondria and the
determination of protein levels, and may therefore not be recognizable by
Western blot analysis.
4.2. Inhibition of UCP activity in vivo
A promising result of the present work is the finding that cardiac function,
and in particular contractile reserve, was improved after inhibition of UCP-
activity. Our in vivo examinations indicated that the cardiac response to
dobutamine was improved upon inhibition of UCPs with herbal substance
genipin. These results are in agreement with some of our previous studies,
demonstrating that reduced cardiac contractility in TGF-β1-TG mice under
44

Discussion

stimulation with the sympathomimetic agent dobutamine could be fully
recovered after administration of the β-blocker metoprolol or a selective TGF-β1
antagonist (sTGFβR-Fc). In contrast, this was not the case after the
administration of AT1-antagonist telmisartan (Rosenkranz 2004). Similar results
were also obtained in isolated cardiomyocytes, where the contractility of
cardiomyocytes was measured in non-genipin-treated cardiomyocytes with and
without stimulation with isoprenaline. Isoprenaline-induced increase of
contractility in cardiomyocytes, isolated from TGF-β1-TG mice, was clearly
lower, than in cardiomyocytes from WT (C57BL/6) mice. This effect was
reversible through previous treatment of cardiomyocytes with genipin, which
acts as a potent inhibitior of UCPs.
4.3. Inhibition of UCP-mediated proton leak by a herbal
substance genipin
Inchin-ko-to is an herbal medicine, which has been used for ages in
Chinese and Japanese medicine as an anti-inflammatory, antipyretic, choleretic
and diuretic agent against liver diseases (Sakaida et al. 2003). Different studies
have suggested beneficial effects in liver fibrosis, cholestatic liver disease
(Sakaida et al. 2003; Arai et al. 2004; Shoda et al. 2004), and liver cirrhosis
(Inao et al. 2004). The substance consists of three herbal components, known
as Inchin-ko (Artemisiae capillary spica), San-shishi (Gardeniae fructus) and
Dai-ou (Rhei rhizoma) (Kitano et al. 2006). The main bioactive component of
Inchin-ko is geniposide, which is converted to genipin in the digestive tract by
bacterial enzymes and is subsequently absorbed into the blood (Yim et al.
2004). In a recent study on UCP2-Knockout mice, Zhang et al. have shown that
a synthesized authentic sample of genipin inhibits superoxide- and HNE-
activated UCP2-mediated mitochondrial proton leak. It also increases
mitochondrial membrane potential and ATP levels, closes KATP channels and
stimulates insulin secretion in a UCP2-dependent manner (Zhang et al. 2006).

45

Discussion

Our results showed that the inhibition of UCPs by genipin restored the
inotropic reserve in isolated cardiac myocytes as well as in vivo. Hence, it may
be implicated that the upregulation of UCPs is critically involved in the
diminished contractile β-adrenergic response in TGF-β1-TG mice.
4.4. UCPs in human heart
Although the data obtained in human myocardium are based on a small
number of samples and therefore have to be interpreted with caution, they
indicate that the above mechanisms may be relevant in humans. Myocardial
UCP3 levels were elevated in hearts from DCM patients not receiving β-blocker
treatment, while this was not the case in patients receiving metoprolol. These
data are consistent with recent reports, which indicate that energy deficiency in
heart failure is associated with increased cardiac mitochondrial UCP
expression, and/or activity in humans. Murray and co-workers reported that
UCP2 and UCP3 were upregulated in atrial appendage samples of patients with
ischemia-associated cardiomyopathy (Murray et al. 2004). Likewise, increased
UCP activity was found in patients with obesity-related diabetic cardiomyopathy
(Boudina et al. 2007).
4.5. Conclusions
Our results indicate that the pathogenesis of cardiac hypertrophy and
dysfunction involves an axis involving Ang II, TGF-β1, mitochondrial UCPs and
the β-adrenergic system. More specifically, TGF-β1 induces the cardiac
expression of UCP2 and UCP3, both of which are involved in cardiac energy
metabolism. Upregulation of UCPs causes abrogation of the contractile

46

Discussion

response to β-adrenergic stimulation. Thus, inhibition of UCP

provide a novel approach to affect contractile function of the heart.

47

activity

am y

Summary

5. Summary
5.1. English summary
Cardiac hypertrophy is an independent risk factor for cardiac morbidity and
mortality. Multiple mechanisms are involved in the development of myocardial
hypertrophy, such as activation of the RAS and the β-adrenergic system. In
addition, local mediators such as TGF-β1 play a significant role. Ang II-induced
upregulation of TGF-β1 particularly takes place during the transition from stable
hypertrophy to heart failure. It has been postulated that the reduction of cardiac
energy plays an important role in the development of heart failure, and that
mitochondrial energy metabolism, which is regulated through mitochondrial
UCPs, is affected in the failing heart. However, a connection between cardiac
TGF-β1 and UCPs has not been characterized thus far. We hypothesized that
TGF-β1 affects energy metabolism of the heart by regulating mitochondrial
UCPs and therefore plays an important role for progressive contractile
dysfunction in the hypertrophied heart.
The primary goal of this dissertation was to unravel the regulation and
functional role of mitochondrial UCPs and their importance for cardiac energy
metabolism within the scope of TGF-β1-induced myocardial hypertrophy and
failure. Our results indicate that TGF-β1 upregulates mitochondrial UCP2 and
UCP3 in cardiac myocytes in vitro, and that cardiac hypertrophy in TGF-β1-
overexpressing mice is associated with increased UCP expression in vivo.
Chronic β-adrenoceptor blockade and antagonism of TGF-β1 by sTGFβR-Fc
reversed the upregulation of UCPs in TGF-β1-TG mice, whereas treatment with
the AT1-receptor antagonist telmisartan had no effect. The functional
importance of UCPs was evaluated by the use of genipin, representing a potent
UCP inhibitor. While the contractile response to β-adrenergic stimulation was
diminished in TGF-β1-TG mice in vitro as assessed by the contractility of
isolated cardiac myocytes and in vivo as assessed by dobutamine stress
echocardiography, pretreatment with genipin was able to fully salvage the
48

Summary

reduced contractile reserve in TGF-β1-TG mice. Finally, cardiac TGF-β1 and
UCPs expression were elevated in heart failure in humans, and UCPs Ð but not
TGF-β1 Ð was downregulated by chronic β-adrenoceptor blocker treatment.
It is concluded that TGF-β1 affects cardiac contractility by regulating
mitochondrial UCPs, and that inhibition of UCP activity may represent a novel
approach to improve contractile function of the heart.
5.2. Deutsche Zusammenfassung
Die Herzhypertrophie ist ein unabhngiger Risikofaktor fr die kardiale
Morbiditt und Mortalitt. Viele Mechanismen wie z.B. die Aktivierung des
Renin-Angiotensin-Systems (RAS) und des β-adrenergen Systems sind in die
Entwicklung der Herzhypertrophie miteinbezogen. Zudem haben lokale
Mediatoren wie Transforming Growth Factor-β1 (TGF-β1) eine signifikante
Bedeutung. Eine myokardiale Heraufregulation von TGF-β1 findet besonders
whrend des bergangs von der stabilen Herzhypertrophie zur Herzinsuffizienz
statt. Es wird postuliert, dass Vernderungen des kardialen
Energiemetabolismus, welcher durch mitochondriale Uncoupling-Proteine
(UCPs) reguliert wird, eine wichtige Rolle in der Entwicklung einer
Herzinsuffizienz spielen. Eine Verbindung zwischen kardialem TGF-β1 und
mitochondrialen UCPs wurde bislang nicht beschrieben. Wir stellten die
Hypothese auf, dass TGF-β1 den Energiemetabolismus des Herzens
beeinflusst, indem es mitochondriale UCPs reguliert und deshalb eine wichtige
Bedeutung fr die fortschreitende kontraktile Funktionsstrung im
hypertrophierten Herzen hat.
Das Hauptziel dieser Dissertation war es daher, die Regulation und
funktionelle Bedeutung von mitochondrialen UCPs sowie deren Einfluss auf den
kardialen Energiemetabolismus im Rahmen einer TGF-β1-verursachten
Herzhypertrophie zu erforschen. Unsere Ergebnisse zeigen, dass TGF-β1 in
vitro mitochondriales UCP2 und UCP3 in Kardiomyozyten heraufreguliert, und
dass die Herzhypertrophie in Musen, welche vermehrt TGF-β1 exprimieren, mit
49

Summary

einer erhhten UCP-Expression in vivo assoziiert ist. Chronische β-
Adrenozeptorblockade und Antagonisierung von TGF-β1 durch sTGFβR-Fc
hoben die Heraufregulation von UCPs in TGF-β1-transgenen (TGF-β1-TG)
Musen auf, whrend die Behandlung mit dem Angiotensin-Typ-1-
Rezeptorantagonisten Telmisartan keinen Effekt hatte. Die funktionelle
Bedeutung von UCPs wurde unter Anwendung von Genipin evaluiert, welches
einen wirksamen UCP-Inhibitor darstellt. Whrend die kontraktile Antwort auf
eine β-adrenerge Stimulation in TGF-β1-TG Musen vermindert war (kontraktile
Antwort von isolierten Kardiomyozyten auf Isoprenalin in vitro und von Wild-Typ
oder TGF-β1-TG Musen whrend Dobutamin-Stress-Echokardiographie in
vivo), war es durch die Vorbehandlung mit Genipin mglich, die verminderte
kontraktile Reserve in TGF-β1-TG Musen wiederherzustellen. In humanem
Myokard war die Expression von kardialem TGF-β1 und UCPs bei
Herzinsuffizienz erhht. Die Expression von UCPs - jedoch nicht von TGF-β1 Ð
wurde durch die chronische Behandlung mit einem β-Adrenozeptorblocker
weitgehend normalisiert. Zusammengefasst beeinflusst TGF-β1 die Kontraktilitt
des Herzens, indem es die mitochondrialen UCPs reguliert und somit zu einer
Dysfunktion des mitochondrialen Energiemetabolismus fhrt. Die Hemmung
von UCP-Aktivitt stellt mglicherweise einen neuen Ansatz zur Verbesserung
der kontraktilen Funktion des Herzens dar.

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