Linköping Studies in Science and Technology.

Dissertations No. 1330

Cardiovascular β-adrenergic

signaling

Maturation and programming effects of hypoxia in a

chicken model

Isa Lindgren

Department of Physics, Chemistry and Biology

Linköping University

Linköping 2010

Front cover illustrations

Top: Model of a G-protein coupled receptor

Bottom: 19 day-old broiler chicken embryo in ovo. Photo: Isa Lindgren

Copyright © Isa Lindgren 2010, unless otherwise stated

Lindgren, I

Cardiovascular β-adrenergic signaling - Maturation and programming effects of hypoxia in a

chicken model

Available from Linköping University Electronic Press

http://www.ep.liu.se/

ISBN: 978-91-7393-352-0

ISSN 0345-7524

Published articles and figures have been reprinted with the permission of the copyright holder.

Printed by LiU-Tryck, Linköping, Sweden 2010

During the course of the research underlying this thesis, Isa Lindgren was enrolled in Forum

Scientium, a multidisciplinary graduate school at Linköping University, Sweden.

Linköping Studies in Science and Technology. Dissertations No. 1330

“The heart, a center for attraction, Has domination of the whole;

Its bulging muscles are in action From early start till final toll”

From “The Avian Embryo” (1960)

by Alexis Romanoff

“The heart has its reasons which reason knows nothing about” Blaise Pascal

i

ABSTRACT

Despite the importance of β-adrenergic receptors (βARs) in cardiovascular disease, not

much is known about how prenatal hypoxia effects βAR signaling in the postnatal animal.

Thus, the aim of this thesis was to characterize the pre- and postnatal maturation of the

cardiovascular βARs and the effects of chronic prenatal hypoxia on βAR signaling in the

embryo and adult animal using the chicken as experimental model.

βARs belong to the seven-transmembrane receptor family of G-protein coupled

receptors and are crucial for cardiovascular development, growth and regulation. In the

cardiovascular system there are two dominant subtypes, β1AR and β2AR, whose main

ligands are the biogenic catecholamines epinephrine and norepinephrine. When

stimulated, βARs primarily couple to the stimulatory G-protein (Gs) that stimulates

adenylyl cyclase to convert ATP to cAMP. cAMP increases ino- and chronotropy of the

heart and causes relaxation of blood vessels. β2ARs also have the ability to switch to

inhibitory G-protein (Gi) signaling that decreases the cAMP production. To protect the

cardiovascular system from overstimulation, the βARs desensitize and downregulate in

the case of prolonged elevation of catecholamines. This blunts the cardiovascular

response and the mechanisms behind desensitization/downregulation, including the

β2AR switch to Gi signaling, are closely linked to cardiovascular disease and are of

immense importance in medical therapeutics.

Hypoxic stress releases catecholamines and thereby triggers βAR responses and

desensitization/downregulation mechanisms. Hypoxia quite commonly occurs in utero

and it is well known that prenatal insults, like malnutrition or hypoxia, are coupled to an

increased risk of developing adult cardiovascular disease. This is referred to as

developmental programming and constitutes an important and modern field of research.

In this thesis, I show that; 1) the developmental trajectory for organ growth,

especially the heart, is affected by hypoxia, 2) chronic prenatal hypoxia causes cardiac

embryonic βAR sensitization, but causes desensitization postnatally suggesting that

there is a hypoxia-induced “programming” effect on adult β-adrenoceptor function, 3) the

adult βAR desensitization following prenatal hypoxia is linked to a decrease in

β1AR/β2AR ratio, a decrease in cAMP following βAR stimulation with isoproterenol and

an increase in Gαs, 4) the chorioallantoic (CA) membrane arteries display hypoxic

vasoconstriction, but lack α-adrenergic reactivity and 5) hypotension of the chronically

hypoxic chicken embryo is linked to a potent βAR relaxation of the CA vasculature and

an increased βAR sensitivity of the systemic arteries with no changes in heart rate.

ii

In conclusion, chronic prenatal hypoxia causes growth restriction, re-allocation

and has programming effects on the βAR system in the adult. The latter indicates that

the βAR system is an important factor in studying hypoxic developmental programming

of adult cardiovascular disease.

iii

POPULÄRVETENSKAPLIG SAMMANFATTNING

Trots dagens kunskap om adrenalinreceptorer och deras djupa involvering i

hjärtkärlsjukdom känner vi inte till mycket om hur syrebrist (hypoxi) i fosterstadiet

påverkar adrenalinreceptorsignalleringen i den vuxna individens hjärtkärlsystem. Därför

är målet med denna avhandling att karaktärisera mognaden av adrenalinreceptorer i

hjärtkärlsystemet före och efter födseln, samt att undersöka effekten av kronisk

fosterhypoxi på adrenalinreceptorsignallering i både fostret och den vuxna individen.

Detta gör vi genom att använda oss av kyckling som modelldjur.

β-adrenoreceptorer (βARr) utgör en av grupperna av adrenalinreceptorer. Enkelt

uttryckt så är de proteiner insprängda i cellens membran med en del utanför cellen och

en del inuti. De plockar upp signaler på cellens utsida och översätter dem till signaler

inne i cellen genom att koppla till G-proteiner på cellens insida. De två dominerande

subtyperna, β1AR och β2AR, är centrala i hjärtkärlsystemets utveckling, tillväxt och

reglering och de stimuleras främst av adrenalin och noradrenalin. När adrenalin eller

noradrenalin fäster till receptorns yttre del skickas en signal in i cellerna som ökar

hjärtats slagfrekvens och kontraktionskraft, samt utvidgar blodkärlen. För att skydda

hjärtkärlsystemet från överstimulering (t.ex. vid långdragen stress med höga halter

adrenalin) finns det mekanismer för desensitisering och nedreglering av receptorerna.

Detta gör att hjärtat reagerar sämre på adrenalin/noradrenalinstimulering. Mekanismerna

bakom desensitisering/nedreglering är tätt länkade till hjärtkärlsjukdom och är därför

viktiga måltavlor för läkemedel.

Hypoxi är en sorts stress och utlöser därmed utsöndring av adrenalin och

noradrenalin och triggar både ökad hjärtkärlrespons och desensitiserings/nedreglerings-

mekanismer. Att foster utsätts för hypoxi genom tryck på navelsträngen eller nedsatt

funktion av moderkakan är inte helt ovanligt och det är väl känt att onormal stress under

fosterstadiet (t.ex. undernäring eller hypoxi) är kopplat till ökade risker att utveckla

hjärtkärlsjukdom i vuxen ålder och detta kallas för utvecklingsprogrammering. Effekterna

av hypoxi under utvecklingen utgör därför ett viktigt och modernt forskningsområde.

I denna avhandling visar jag att kronisk fosterhypoxi minskar födelsevikten och

ökar βAR-känsligheten i själva fostret, men minskar adrenalinkänsligheten i den vuxna

individen, något som återfinns hos hjärtsjuka personer. Dessa resultat visar på att

fosterhypoxi har en ”programmerande” effekt på vuxen βAR-funktion. Fosterhypoxi ökar

också halten av stimulerande G-protein, men ändrar β1AR/β2AR-proportionerna till fördel

för β2AR i det vuxna hjärtat. Dessa förändringar liknar också de observerade i

iv

hjärtkärlsjukdom och ger ytterligare en fingervisning om att fosterhypoxi leder

till ”programmering” av βAR-systemet. Utöver detta visar jag att choriallantois-

membranets (CAMs) blodkärl (motsvarande moderkakans blodkärl i däggdjur) helt

saknar klassisk adrenalin-inducerad sammandragning och reagerar istället uteslutande

på adrenalin genom utvidgning. Direkt hypoxi kring det isolerade kärlet orsakar däremot

sammandragning. Jag presenterar också bevis för att hjärtfrekvensen hos embryot inte

ändras med kronisk fosterhypoxi och att det observerade blodtrycksfallet hos hypoxiska

kycklingembryon med stor sannolikhet beror på βAR-utvidgningen av CAMs blodkärl.

Den viktigaste slutsatsen från vår studie är att förändringen av vuxen βAR-funktion

genom fosterhypoxi påminner om förändringarna vi ser i hjärtkärlsjukdom. Därmed

kopplas förändringar av βAR-systemet som en möjlig mekanism bakom utvecklings-

programmering av vuxen hjärtkärlsjukdom.

v

PAPERS INCLUDED IN THESIS

PAPER I Lindgren I, Altimiras J . Sensitivity of organ growth to chronically low oxygen levels during incubation in red junglefowl and domesticated chicken breeds. Submitted Student’s contribution: Isa Lindgren carried out the experiments, analyzed the results and contributed to the writing of the paper. PAPER II Lindgren I, Altimiras J. Chronic prenatal hypoxia sensitizes β-adrenoceptors in the embryonic heart but causes postnatal desensitization. Am J Physiol Regul Integr Comp Physiol 2009;297:R258-264. Student’s contribution: Isa Lindgren designed the study and formulated the questions together with Jordi Altimiras. IL executed all experiments, analyzed the data and wrote the paper with some help from Jordi Altimiras. PAPER III Lindgren I . Postnatal β-adrenergic desensitization caused by chronic prenatal hypoxia is linked to an increase in Gαs and decreased β1AR/β2AR ratio. Manuscript Student’s contribution: Isa Lindgren designed the study, carried out the experiments with the help from two students, analyzed the data and wrote the paper guided by Jordi Altimiras. PAPER IV Lindgren I, Zoer B, Altimiras J, Villamor E. Reactivity of chicken chorioallantoic arteries, avian homologue of human fetoplacental arteries. Submitted Student’s contribution: Isa Lindgren executed all experiments with the help of Bea Zoer. IL analyzed all data and contributed to the written paper equally together with the other authors. Experiments were mainly planned and supervised by Eduardo Villamor. PAPER V Lindgren I, Crossley D, Villamor E, Altimiras J. Hypotension in the chronic hypoxic chicken embryo origins in a potent β-adrenergic response of the chorioallantoic arteries and not bradycardia. Submitted Student’s contribution: Isa Lindgren executed all experiments except the cannulation heart rate and blood pressure measurements that were done by Dane Crossley. IL analyzed the data and wrote the paper under main supervision of Jordi Altimiras and partly Eduardo Villamor.

vi

vii

TABLE OF CONTENTS ABSTRACT ................................................................................................................. i

POPULÄRVETENSKAPLIG SAMMANFATTNING .................................................... ii

PAPERS INCLUDED IN THESIS ................................................................................ v

1 G-PROTEIN COUPLED RECEPTORS ....................................................................... 1

1.1 GPCR structure .................................................................................................... 1

1.2 G-proteins ............................................................................................................. 1

1.3 The GPCR signaling cascade ............................................................................. 2

1.4 β-adrenergic receptors ........................................................................................ 5

2 CARDIOVASCULAR β-ADRENOCEPTORS IN HEALTH

AND DISEASE ............................................................................................................ 7

2.1 βAR signaling in the cardiovascular system .................................................... 7

2.2 β2AR cross-signaling and compartmentation ................................................... 9

2.3 βARs in cardiovascular disease ......................................................................... 11

3 DEVELOPMENTAL PROGRAMMING ........................................................................ 13

3.1 Phenotypic plasticity and developmental “windows” ...................................... 13

3.2 Developmental Origins of Health and Disease .................................................. 14

3.3 The beginning - “Barker’s hypothesis” and

the “thrifty phenotype” ....................................................................................... 15

3.4 The “Predictive Adaptive Response” theory .................................................... 16

3.5 Criticism of PAR ................................................................................................... 18

3.6 My definition of developmental programming ................................................... 19

4 HYPOXIC CARDIOVASCULAR REGULATION AND THE ROLE OF β-ADRENERGIC

RECEPTORS .............................................................................................................. 21

4.1 Adult and fetal cardiovascular regulation during hypoxia................................ 21

4.2 Effect of hypoxia on the βAR system ................................................................ 24

viii

5 SUMMARY OF PAPERS ............................................................................................ 27

5.1 Summary Paper I ................................................................................................. 27

5.2 Summary Paper II ................................................................................................. 28

5.3 Summary Paper III ............................................................................................... 28

5.4 Summary Paper IV ............................................................................................... 29

5.5 Summary Paper V ................................................................................................ 30

6 DISCUSSION ............................................................................................................... 31

6.1 Comments on the experimental model .............................................................. 31

6.2 Developmental programming (Papers II and III) ............................................... 31

6.3 Growth and allocation (Paper I) .......................................................................... 32

6.4 Vascular reactivity and cardiovascular response to chronic hypoxia in the

chicken embryo (Papers IV and V) ........................................................................... 33

6.5 Conclusions ......................................................................................................... 34

ACKNOWLEDGEMENTS ...................................................................................................... 37

REFERENCES ....................................................................................................................... 41

1

1 G-PROTEIN COUPLED RECEPTORS

The key to regulate cell function globally is cell-to-cell communication. This relies on one

cell releasing the transmitter substance and another being specialized for receiving it.

Depending on the nature of the ligand, the signal transduction can take two main

avenues; if the ligand is lipophilic and able to diffuse over the cell membrane, it can

reach cytosolic or nuclear receptors inside the cell. If it is hydrophilic, it could have an

active transporter across the membrane, but more likely it will have its receptor on the

cell surface, i.e. a membrane receptor. Membrane receptors are divided into three main

classes; 1) Ligand gated ion-channels, 2) Enzyme-linked receptors and 3) G-protein

coupled receptors (GPCRs). The GPCR family is the largest of the three (52) and

encompasses over 1000 known receptors (43).

1.1 GPCR structure

The ubiquitous GPCR secondary and tertiary structure is dominated by seven

transmembrane (7TM) α-helices, connected by extra- and intracellular loops, and

arranged in a bundle to create a center core (Fig.1). Clearly, the 7TM regions are

hydrophobic since they span the cell membrane, but the core they create between them

is more or less hydrophilic and is important for ligand binding and the subsequent

conformational change that the binding of the ligand causes (52). TM helix 3 is

suggested to be the main ligand binding site, while TM helix 5 and 6 are involved in

initiating the intracellular signaling cascade (47). The whole structure is built up by a

single amino acid chain with an extracellular N-terminal and an intracellular C-terminal.

While the α-helices are quite homologous, the terminals and loops are subjected to the

largest variations between different GPCRs, thus being involved in giving the receptor its

unique identity and ligand specificity (43, 52). The variations in the intracellular loops and

C-terminus determine G-protein specificity (99) and are the regions of the receptor that

are exposed to intracellular kinases and are therefore important phosphorylation sites.

1.2 G-proteins

The proteins that gave GPCRs their name and are responsible for the first step in the

intracellular signaling cascade are called Guanine nucleotide-binding proteins, or just G-

proteins, and are, just like the GPCRs, highly conserved through evolution. They are

trimeric proteins belonging to the GTPase enzyme group and act as ON/OFF switches

2

regulating cell processes as disparate as sensory perception and cell differentiation (95).

The three subunits that make up a G-protein are named with greek letters as α, β and γ,

with the β and γ being tightly bound to form a βγ complex that is dissociated from the α

subunit (Gα) at the time of activation (70). As hinted by the name, the G-proteins bind

guanine nucleotides and are activated when GTP binds and inactivated by the hydrolysis

of GTP to GDP due to the intrinsic enzymatic activity of the protein (55). The hydrolysis

of GTP is irreversible and to activate the G-protein again, GDP has to be replaced with

GTP by the help of guanine nucleotide-exchange factors, or GEFs (95). The binding site

for the guanine nucleotides are located in the Gα and because of its enzymatic activity it

was long thought that it was only the Gα that was involved in transmitting the signal. We

now know that the βγ complex has its own signaling pathways and that it evokes other

types of responses than the α-mediated signal (63, 85).

The G-protein is mostly referred to by its α-subunit and four different subfamilies of

Gα proteins are known; Gαs (stimulatory), Gαi (inhibitory), Gαq and Gα12 (70). As previously

mentioned, all Gα subunits bind guanine nucleotides to regulate their activity. This is not

to be confused with that Gαs and Gαi are both acting in the pathway where the second

messenger cyclic adenosine monophosphate (cAMP) is generated through conversion

of ATP by adenylyl cyclase (AC). As their names indicate, Gαs stimulates the cAMP

production, while Gαi inhibits it. Gαq acts via phospholipase C to generate the second

messengers IP3 and DAG, while Gα12 couple to GEFs to remodel and regulate the

cellular cytoskeleton (63). The Gαq and Gα12 pathways are however beyond the scope of

this thesis and from hereon only Gαs and Gαi will be considered.

1.3 The GPCR signaling cascade

A ligand can have many different effects on a GPCR depending on what kind of ligand

and what type of GPCR. One thing that all ligand-bound GPCRs do have in common is

that the 7TM regions of the receptor will change their conformation upon agonist

stimulation (70). The conformation of the agonist-bound receptor aids the translocation

of a G-protein to the cell membrane. Once the G-protein/GPCR interaction is established,

the receptor acts as a GEF to exchange the bound GDP to a GTP in the Gα subunit,

thereby activating the Gα, changing its conformation and causing the dissociation of the

βγ complex (63) (Fig. 1). The enzymatic activity of the Gα then hydrolyses GTP to GDP

and the Gα thereby regains its affinity for the βγ complex and re-associates and the

3

Figure 1. Illustration of the classical G αs-mediated GPCR signaling pathway. Agonist

stimulation of the GPCR leads to a conformation change that allows intracellular interaction with

the trimeric G-protein. The interaction leads to an exchange of GDP to GTP in the Gαs subunit,

making it active and dissociating the G-protein into a free Gαs and a βγ complex. The βγ complex

have disparate pathways for signaling and goes on further to give a cell response, while the

activated Gαs stimulates adenylyl cyclase (AC) to convert ATP cAMP. The cAMP in turn acts like

the second messenger and activates kinases like G-protein coupled receptor kinase (GRK) and

protein kinase A (PKA). The activated kinases phosphorylate different targets to give a cell

response or to desensitize the receptor to further signaling.

effector signal is turned off. Thus, it has been proposed that the duration of the receptor

signal is dependent on the lifetime of the GTP-bound Gα (63).

As previously mentioned, the activated Gαs stimulates AC to catalyze the

conversion of ATP to cAMP. The second messenger cAMP has many targets in the cell,

but in the classical cAMP-mediated pathway the most important proteins it

phosphorylates (and thereby activates) are protein kinase A (PKA) and G-protein

AC

Gas

ß?

cAMP ATP

DissociationGas

ß?

GDP

GTP

PKA(Active)

GRK(Active)

GPCR phosphorylation

CELL RESPONSE

Agonist

AC

Gas

ß?

ß?

cAMP ATP

DissociationGas

ß?

GDP

Gas

ß?

ß?

GDP

GTP

PKA(Active)

GRK(Active)

GPCR phosphorylation

CELL RESPONSE

Agonist

4

coupled receptor kinases (GRKs) (Fig. 2). Once activated, PKA also has many

phosphorylation targets that eventually lead to either a cellular response or an

interruption of the signal, while GRKs are specifically phosphorylating GPCRs to cease

the signal. The phosphorylation decouples the G-protein from its receptor, serving as a

negative-feedback loop (70). The action of making the receptor irresponsive to further

stimulation on the level of G-protein coupling, despite the presence of agonist, is known

as desensitization and is one of many means for fine-tuning and regulation of the signal

and protecting the cell from overstimulation (78). Being quite promiscuous, the activated

PKA may phosphorylate other GPCRs not involved in the initiation of the signal, thus

desensitizing other pathways not related to the one where the PKA activation originated.

This is called heterologous desensitization. Conversely, the GPCR-specific

desensitization carried out by GRKs is called homologous desensitization and mostly

regulates the same pathway it was activated by (70).

Following the phosphorylation-mediated uncoupling of the G-protein from its

GPCR, the fate of the receptor is to go down one of many routes, most of them

intracellular after the receptor has been sequestered from the cell surface. The receptor

can be sequestrated (or internalized) in different ways including clathrin-coated pits and

caveolae (56, 78). The internalization process is in most cases closely related to an

association of the receptor with a group of proteins called β-arrestins. β-arrestins interact

with the phosphorylated parts of the receptor and further block the site for G-protein

binding. In addition to the direct, steric desensitizing action, β-arrestin is involved in

receptor trafficking via clathrin-coated vesicles (70) (Fig. 2). Recently, β-arrestin has also

been demonstrated to be involved in independent signaling through the mitogen-

activated protein kinase (MAPK) pathway by acting as a scaffold for additional proteins

and also in recruiting phosphodiesterases to the cell membrane to speed up the

degradation of cAMP (56). Mediated by β-arrestin or not, once the receptor is in an

intracellular endosome it can either be de-phosphorylated (and thus resensitized) by

phosphatases and brought back to the cell surface for further signaling, or it can simply

be degraded (70) (Fig. 2). Through the MAPK pathway, the protein expression of the

receptor can be regulated and together with endosomal degradation of the receptor

protein, this longer-term regulation of receptor availability is commonly referred to as

receptor downregulation.

5

Figure 2. GPCR trafficking. The kinases activated in the GPCR signaling pathway (e.g. GRK)

phosphorylate the intracellular regions of the receptor that are involved in G-protein coupling. The

receptor phosphorylation per se leads to desensitization, but additional desensitizing steps are

possible. This figure depicts how β-arrestin (βArr) associates with the phosphorylated part of the

receptor, further blocking any G-protein interaction. Additionally, the β-arrestin is involved in

recruiting the receptor into clathrin-coated invaginations, leading to sequestration of the receptor

from the cell surface by endocytosis. Once in the endosome, the receptor can either be de-

phosphorylated by phosphatase (P-ase) and returned to the surface for further signaling, or

degraded and thus permanently removed from the signaling cycle.

1.4 β-adrenergic receptors

βARs are a group of GPCRs whose ligands are biogenic amines (primarily the

catecholamines epinephrine and norepinephrine) and there is a plethora of studies of

their structure, signaling pathways and functional regulation. Together with the closely

related α-adrenergic receptor group, they are present in most tissues of the body and

ßArr

Agonist

GRK(Active)

P P Phosphorylation

ßArr

PP

Clathrin-coated

vesicle

Endosome a) Degradation

b) De-phosphorylation

P-aseP P

Receptor recycling

ßArr

Agonist

GRK(Active)

P PP P Phosphorylation

ßArr

PP

PP

Clathrin-coated

vesicle

Endosome a) Degradation

b) De-phosphorylation

P-aseP PP P

Receptor recycling

6

are some of the most studied GPCRs to this date. In fact, the β2AR subtype was one of

the first GPCRs to be crystallized to enable structure determination of the intact receptor,

something that is hard to obtain in GPCRs because of their structural instability in vitro

(74).

There are three known subtypes of βAR receptors; β1, β2 and β3 (48, 89). Although

they can be found throughout the body, β1ARs are found in abundance in the heart,

while β2ARs are classically identified in for example blood vessels and airway smooth

muscle (48). β3ARs are predominantly found in adipose tissue and because their role in

the cardiovascular system is not yet fully understod, it will not be discussed further in this

thesis.

All βARs signal mainly through Gαs. However, β2ARs also have the ability to signal

through Gαi , something that will be discussed in the next chapter. βARs follow the

classical GPCR/Gαs/cAMP pathway and thereby activate both PKA and GRKs. More

specifically, βARs activate GRK2 that is also called β-adrenoceptor kinase 1 (βARK1) due

to its specificity to βARs (89). The targets of the activated PKA include proteins like

phospholamban (PLB) or receptor operated calcium channels (ROCCs) that will result in

an increase in intracellular Ca2+ (89). The mechanisms and cellular response to βAR

stimulation are however different in different cell types and will be discussed further in

the next chapter.

7

2 CARDIOVASCULAR β-ADRENOCEPTORS IN

HEALTH AND DISEASE

Although βARs are widespread throughout our bodies and essential in many

physiological processes, they are dominant in the cardiovascular system. Their

significance throughout the lifespan of an organism is demonstrated all the way from

their early embryonic presence when knocking out the βARs is lethal (72), to their

regulation of adult cardiovascular function and involvement in cardiovascular disease

and senescence (84, 96).

2.1 βAR signaling in the cardiovascular system

As previously mentioned, βARs are GPCRs that signal through Gαs, resulting in an

increase of cytosolic cAMP and subsequent activation of kinases like PKA. This same

pathway increases contractility in cardiac muscle and decreases contractility (causes

relaxation) in vascular smooth muscle.

In the heart, one possible target for the activated PKA are the L-type calcium

channels. “L” stands for “Long-lasting” and is based on the fact that the channel is open

for a relatively long time. The long refractory time is one of the features contributing to

the unique characteristics of the heart muscle – the action potentials last longer and

prevent arrhythmias and re-entry of action potentials to enable the cyclic synchronization

of all cardiac cells (90). When PKA phosphorylates the L-type calcium channel, the

channel conductance is not increased, but the mean opening time is, meaning that the

probability for more channels to be open at the same time increases and thereby also

increases the inward calcium current (90). The increased intracellular Ca2+ triggers a

calcium-induced calcium release from ryanodine receptors (RyRs) in the sarcoplasmic

reticulum (SR) and the muscle contracts (53). At the same time, PKA can also

phosphorylate RyRs directly to increase their opening probability (58) (Fig.3). Another

primary target for PKA is PLB in the SR membrane. In its unphosphorylated state, PLB

inhibits the sarco/endoplasmatic reticulum calcium-ATPase (SERCA), a “pump” in the

SR membrane that is responsible for the reuptake of Ca2+. When phosphorylated, the

inhibitory action of PLB on SERCA is lifted and Ca2+ can be pumped back into the SR

(58). This means that, when βARs are stimulated, not only the rate of contraction is

increased, but also the rate of relaxation (lusitropy).

8

In vascular smooth muscle, the relaxation in response to βAR stimulation is

mediated primarily by β2ARs and ascribed to the PKA phosphorylation of PLB and the

subsequent sequestration of Ca2+ from the cytosol by SERCA (84). PKA can also

phosphorylate plasma membrane ion channels to cause an efflux of Ca2+ out of the

cytosol. Ultimately, the decrease of cytosolic Ca2+ is what is causing the βAR-mediated

relaxation in vascular smooth muscle.

Ca2+

Ca2+

PKA(Active)

CONTRACTION

Phosphorylation

L-type Ca 2+ channel

Ca2 +

Ca2+

Ca2+

Ca2 +

Ca2+

P

Channel opening

Ca2+

Ca2+

Ca2+Ca2+

Ca2+

Ca2+

Ca2+

Ca2 +

RyRCa2+

Ca2+-induced Ca 2+ release

SR

P

Ca2+

Ca2+

PKA(Active)

CONTRACTION

Phosphorylation

L-type Ca 2+ channel

Ca2 +

Ca2+

Ca2+

Ca2 +

Ca2+

PP

Channel opening

Ca2+

Ca2+

Ca2+Ca2+

Ca2+

Ca2+

Ca2+

Ca2 +

RyRCa2+

Ca2+-induced Ca 2+ release

SR

P

Figure 3. Effect of βAR activation in cardiomyocytes. PKA activated via the βAR/cAMP pathway

phosphorylates the L-type calcium channel and increases its opening probability. Intracellular

calcium (Ca2+) increases and when sensed by the ryanodine receptor (RyR) in the sarcoplasmic

reticulum (SR) membrane, additional Ca2+ is rapidly released into the cytosol and leads to

contraction of the cardiomyocytes. Activated PKA can also directly phosphorylate RyR to cause

Ca2+ release.

9

2.2 β2AR cross-signaling and compartmentation

The Gαs/cAMP signaling pathway is the classical example of βAR signaling. However,

the β2AR has the ability to cross-signal with the Gαi, inhibiting AC, resulting in a

decreased cAMP production (42, 44, 100). The switching between stimulatory and

inhibitory G-protein signaling takes place at higher concentrations of catecholamines. It

is mediated by PKA phosphorylation of the β2AR, which in turn decreases the affinity of

the receptor to Gαs and increases the affinity for Gαi (22). It is also demonstrated that the

βγ complex from pertussis toxin-sensitive G-proteins (i.e. Gi proteins) evokes a separate

signal through the MAPK pathway, further dampening the effect from the initial cAMP

pathway (22). The β2AR G-protein switching is thought of as a protective mechanism of

the βAR system and is shown to be antiapoptotic in the heart (104). This is further

supported by studies of βARs in heart failure, where β1ARs are downregulated to a

larger extent than β2ARs (16, 25). This naturally connects to the fact that decreased

levels of cAMP will lead to decreased activation of kinases such as GRKs and PKA,

consequently leading to a decrease in receptor phosphorylation and subsequent

downregulation. Attenuated relaxant effects on arteries through β2AR-induced Gαi

coupling has been documented (8) and in Papers IV and V of this thesis, Gαi switching of

the β2AR is a probable explanation for our observations in concentration-response

curves to the synthetic βAR agonist isoproterenol in chorioallantoic arteries from the

chicken embryo; at higher concentrations the agonist turned the response from relaxing

to contracting, in some cases contracting the vessel even more than the initial pre-

contraction (Fig. 4, Paper IV). This is further supported by the lack of αAR-mediated

contraction response in the studied vessels (Papers IV and V) and the isoproterenol

stimulation therefore has to be β2AR mediated.

10

Figure 4. Original tracing of the response of a chicken chorioallantoic artery to the β-

adrenoceptor (βAR) agonist isoproterenol (Iso) obtained by wire myography. The βAR-induced

relaxation of blood vessels is primarily mediated by β2ARs signaling through Gαs. However,

β2ARs have the tendency to switch from Gαs to Gαi signaling at higher concentrations of agonist,

which leads to contraction. The trace shows a clear contraction in response to 10-6 M Iso and

higher (concentrations shown as logarithmic values in figure). The vessel was precontracted by

depolarization with 62.5 mM KCl. Modified from Paper IV.

The difference in β2AR signaling compared to β1ARs is also ascribed to the spatial

localization of the receptors in the cell. It has been demonstrated that the cAMP

produced by β2AR stimulation fails to elicit phosphorylation of for example PLB and thus

failing to increase lusitropy (53, 102) and despite the higher cAMP production from β2AR

stimulation compared to β1ARs it is uncoupled from inotropic effects in cardiac cells (58).

Additionally, intact caveolae have been shown to be a requirement for β2AR-mediated

regulation of calcium channels, but nor for β1ARs (7). This growing body of knowledge

has lead to the hypothesis that the β2ARs are compartmentalized in lipid rafts, caveolae

or other spatially defined structures in the cell membrane (58, 85) and a seminal paper

was recently published, showing that β2ARs in the healthy heart are confined to deep

transverse T-tubules, while β1ARs are diffusely spread throughout the cell membrane

(67).

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2.3 βARs in cardiovascular disease

Physiological stress is well known to alter βAR signaling since stress is related to the

release of catecholamines that will stimulate βARs and, depending on the duration of the

stress, eventually lead to receptor desensitization and downregulation (83). Stress- or

non-stress induced, heart failure (HF) is connected to an increase in sympathetic activity

(25, 78). The sympathetic nerve endings are the source of the elevated levels of

norepinephrine that can be measured in HF patients (19). The downregulation of βARs

seen in HF due to the sympathetic hyperactivity decreases the contractile response of

the cardiac muscle and the suboptimal cardiac performance thereby fails to meet the

circulatory demands from the body. As an early intervention method, βAR agonist were

used to try to increase cardiac performance, but it showed to be detrimental when it only

reinforced the negative effects from the initial sympathetic overstimulation (77). Contra-

intuitively, βAR blocking has instead been shown to have long-term positive effects in HF

patients and is now an accepted treatment for HF patients (58, 78).

The damages on the heart from βAR overstimulation are proposed to be mainly

through β1AR Gαs signaling and include increased apoptosis, arrhythmia, cardiac

remodeling and hypertrophy (64, 77, 101). In HF, the β1ARs are selectively

downregulated, while the β2AR population is more or less retained (16, 25). Interestingly,

β2AR signaling has been shown to ameliorate HF and have antiapoptotic effects (16, 77,

101) and treating cardiomyocytes with isoproterenol will induce apoptosis in cells

expressing only β1ARs, but not in cells expressing only β2ARs (103) (Fig. 5).

Figure 5. Micrographs of TUNEL stained mouse cardiomyocytes treated with 1 µM isoproterenol

(ISO). Cells expressing only β1ARs are apoptotic (arrows indicating apoptotic condensed nuclei),

while cells expressing only β2ARs are not. Reprinted with permission from Zhu et al., 2001.

12

The protective, antiapoptotic effect of β2ARs is related to the cross-signaling of β2ARs

with Gαi proteins (101). Gαi proteins are up-regulated in HF and although the increase in

Gαi has been proposed to contribute to the decreased inotropy in the failing heart (73), it

might also be a protective measure from the heart to prevent further damage. The long-

term positive effect of βAR blockers has been suggested to be because of the block of

the detrimental β1ARs and promoting antiapoptotic and reverse remodeling effects

mediated by Gαi and β2AR agonists could therefore be a possible treatment for HF due

to the stimulation of protective pathways through Gαi (16).

13

3 DEVELOPMENTAL PROGRAMMING

Things that are dear or interesting to us tend to assimilate many names describing

virtually the same phenomenon – something that is certainly true in our field of work. I

have chosen to call the concept under which I have carried out most of the work

comprising this thesis “developmental programming”, while others have referred to it as

“developmental origins of health and disease” (DOHaD), “fetal programming” or

“predictive adaptive response” (PAR). As confusing as it may seem, these theories and

concepts are of common origin and merely differ in nuance or complexity levels; they all

assume that the embryo and fetus possesses an inherent plasticity and that, due to this

plasticity, changes in the pre- or neonatal environment can trigger physiological and

functional changes that will echo into adulthood.

3.1 Phenotypic plasticity and developmental “windows”

Phenotypic plasticity is defined as the ability of one set genotype to produce different

phenotypes due to changes in the environment and can be seen as the interaction

between genotype and the environment (71). Although quite a simple definition, it is not

as trivial deciding if the plastic phenotype is adaptive or not. Normally, a plastic

phenotype is considered adaptive if it gives a higher fitness in the new environment than

other phenotypes would, but a lower fitness in another environment (75). However, the

fitness associated to the phenotypic change is relative since it also comes with a cost; if

the environment is fluctuating, investing in the “new” phenotype is risky and if the

environment changes back, the fitness received from the initial phenotypic change may

now cause a decrease in fitness. Thus, for a plastic phenotype to be adaptive, i.e.

increasing fitness, the environment needs to be relatively stable. There are also more

direct costs involved in plasticity, such as maintenance of the sensory and regulatory

mechanisms and genetic costs (i.e. when plasticity genes are linked to genes reducing

fitness) (24). So, even if the plastic phenotype per se is beneficial in the new

environment, all of the costs must be taken into account to decide if the phenotype is

truly adaptive or not.

There are many examples of phenotypic plasticity in juvenile or adult animals, such

as crabs increasing their claw size when their prey get thicker shells or Daphnia

developing thick neck spines in the presence of midge larvae that preys on them (23). In

most mammals, however, the greatest plasticity exists during early life before cell

14

differentiation is completed, i.e. it is a developmental plasticity (34). Because cells

eventually do differentiate and differentiation in most cell types is irreversible, the

phenotypic changes induced during the plastic period will eventually also be fixed and

irreversible, which is of uttermost importance if the changes result in a maladaptive

phenotype that will eventually lead to disease – it cannot be reverted. This, however,

also means that it is important when during development an insult takes place. Different

organs have different proliferation periods and different physiological functions have

different timings for onset. Therefore, if the insult takes place during a period of growth,

the outcome can be expected to be less severe than if it takes place during a

proliferation period. These periods of different sensitivity are referred to as critical

windows of development and are different between organs and functions depending on

their maturation at the time of the insult (86).

3.2 Developmental Origins of Health and Disease

Before going into detail about the development of the different views and theories of fetal

events influencing adult health and function, it is in place to shortly review the concept of

Developmental Origins of Health and Disease (DOHaD). DOHaD is quite a modern

concept elaborated from the precedent FOAD (Fetal Origins of Adult Disease) and can

be seen as the “umbrella concept” under which all theories described below are

gathered. The modernization of the concept from FOAD to DOHaD was necessary as

the rapidly growing body of knowledge made it clear that not only fetal events can have

an effect on later disease risks, but events throughout the plastic phase of the organism

(32). Additionally, it was necessary to emphasize health in the concept, both since fetal

events are not restricted to negative, but can also have positive, effects on the postnatal

animal and because prevention and health promotion is an important part of medical

research (32). Nowadays, there is an international society of DOHaD that was set up to

“…promote research into the fetal and developmental origins of disease and involves

scientists from many backgrounds.” (1) and the Journal of Developmental Origins of

Health and Disease that “…publishes leading research in the field of developmental

origins of health and disease (DOHaD), focusing on how the environment during early

animal and human development, and interactions between environmental and genetic

factors, influence health in later life and risk of disease.” (2). Together they represent the

importance and rapid growth of this field of research.

15

3.3 The beginning - “Barker’s hypothesis” and the “thrifty phenotype”

Although DOHaD might represent the modern view of early life effects on adult health,

all big ideas have their antecedents. In 1977, the Norwegian scientist Forsdahl published

a paper where he noted that coronary heart disease mortality in a cohort of men over 40

was correlated with the infant mortality from the same region at the time the men were

born (29). About 10 years later, Barker and colleagues awoke a world interest around

the same subject when they published a series of now highly cited papers that founded a

new area of research (11, 12). Similar to Forsdahl in 1977, they managed to provide

epidemiological evidence for the relationship between infant mortality in 1921-25 and

coronary heart disease mortality in 1968-78 based on prosperity and nutritional status of

the British regions studied (11). Following the initial study, they further showed a

correlation between low birth weight and increased risk of ischemic heart disease in

adulthood (12). From these important studies arose what we now know as “the Barker’s

hypothesis”, something that Barker himself defines as when: “…undernutrition in utero

permanently changes the body’s structure, function and metabolism in ways that lead to

(…) disease in later life” (10).

Since the introduction of the hypothesis almost 30 years ago, countless studies

have verified the theory, firmly establishing that intrauterine growth restriction is

correlated to many adult diseases, not only cardiac disease, but also diabetes type 2,

obesity and hypertension (27, 76). However, it is a hypothesis that encompasses only

the relationship between low birth weight and adult disease and so, after having

prevailed for a long time, the Barker’s hypothesis has been suggested to have served its

purpose and that, although it will always be the base for the whole research field, it is

time to move on and use the findings to investigate the actual mechanisms behind the

observed phenomenon to be able to assess new targets for intervention and thereby

prevention (68).

In 1992, Barker and colleagues presented another refined and extended

hypothesis based on the previous “Barker’s hypothesis” that they called “the thrifty

phenotype” (39). The new hypothesis was already narrowing the old hypothesis down to

a more defined field of interest, namely pancreatic development and type-2 diabetes. In

their review (39), they link poor intrauterine growth to changes in glucose-insulin

metabolism leading to insulin-resistance. Since then, many more epidemiological studies

in humans have presented evidence of the relation between impaired fetal growth due to

16

malnutrition and metabolic syndrome and the body of empirical data from animal models

is vast as reviewed by Bertram and Hanson (14).

Another important addition to the hypothesis was that of uterine conditions giving

a “hint” of what is to be expected in postnatal life, meaning that if nutrition is sparse, the

organism can interpret the low nutritional level as a signal to adapt its metabolism to low

food conditions and thereby be prepared for the life conditions after birth. If the postnatal

diet is instead rich, the adult would suffer a higher risk of developing type-2 diabetes

when it faces the high metabolic load (92).

Although the thrifty phenotype hypothesis started off focusing on metabolic disease, it

has later been established that it is a general model that can be applied in evaluating the

effect of intrauterine experience on all organ systems (69, 92).

3.4 The “Predictive Adaptive Response” theory

As mentioned in the introduction to this chapter, the different theories around the role of

the developmental environment on future health are very similar, but they do, however,

emphasize quite different aspects. Building on the “thrifty phenotype” hypothesis,

Bateson et al. suggested that the early organism receives information while in the

prenatal environment about the quality of the postnatal environment, much like a

“weather forecast” (13, 94). The adaptations induced by the “sensing” of the environment

to come was not seen as ideal, but more making the most of a bad situation (92).

The “Predictive Adaptive Response” theory (PAR) was coined by Gluckman and

Hanson in the beginning of the 21st century and takes the “forecasting” hypothesis even

further (33, 34). PAR emphasizes the importance of the fetal/young animals’ prediction

of the future environment and they partly define PARs as being “…induced by

environmental factors acting in early life, most often in pre-embryonic, embryonic or fetal

life, not as an immediate physiological adaptation, but as a predictive response in

expectation of some future environment” (34). In other words, this suggests that the

altered growth trajectories and physiological changes induced in the fetus by an

environmental challenge are not just to help the fetus cope with the immediate challenge

and survive until birth, but more importantly, describe the challenge (eg. nutrient

availability) as a “cue” from the mother, preparing the fetus of what is to come. If the

challenge is true, i.e. the postnatal nutritional status matches the one experienced by the

fetus, the animal is presumably well adapted and the “prediction” is correct, thus there is

a fitness benefit. However, if the experienced nutritional status is low and the postnatal is

17

adequate (e.g. in the case of placental malfunction), there is a nutritional mismatch, i.e.

the prediction is faulty and the maladaptation may lead to adult disease (34). To put it

very simply, one can say that PAR is really a form of developmental plasticity, but with a

predictive power (36).

Additionally, the PAR theory tries to distinguish the relevance of severity of the

fetal/neonatal challenge; it is only a PAR if the challenge works within the scope of the

organism’s plasticity (34). If it is too severe, the fetus will not be able to cope and the

challenge will thus be disruptive. On that scale, between the PAR-inducing and the

disruptive intensity of the challenge, is a gray area where the organism is forced to

“cope” with the environment, a “coping” that might encompass simply homeostatic

changes or more drastic changes necessary to ensure survival that might leave traces

that will affect the fitness of the adult individual (37) (Fig.6).

PARs or not, this is an important issue when interpreting empirical data; are we

measuring what we can call “PAR mismatch” or “programming effects” or are we

measuring disruptive damage? There is no easy answer to that question, but it is one

that should be kept in mind when analyzing data. In addition to the initial focus mainly on

nutritional shortage, Gluckman et al. (37) extended the theory to also include nutritional

excess as a potential cue that would either be disruptive or cause a mismatch (Fig.6).

18

Deficitdisruption

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Figure 6. Suggested relationship between early life substrate availability (e.g. nutrients, oxygen

etc.) and adult fitness according to the “predictive adaptive response” (PAR) hypothesis. PARs

are induced within the “scope” of plasticity. Intense challenges forces the organism to “cope” with

the challenge through remodeling/adjusting to assure survival, but may cause a disadvantage in

adulthood. Extremely high or low substrate availability causes disruption, e.g. malformation or

growth retardation. Modified from Gluckman, Hanson, Spencer and Bateson, 2005.

3.5 Criticism of PAR

Members of the group which formulated the PAR theory are very active members of the

DOHaD research community and have published many papers and books in the field,

firmly establishing PAR as a valid theory (32-37, 40, 50). However, being one of the

biggest theories in the field, it has not passed uncriticized.

Altimiras and Milberg (2005) question the term “predictive adaptive” by stressing

the need for a stimulus to receive a response, pointing out that adaptation can only be

based on factors currently present and cannot be based on those in the future, i.e. they

cannot be “predictive” (4). Wells (2007) goes further in his criticism to say that the PARs

concept is flawed for several reasons; firstly, he questions the validity of the “prediction”

in a long-lived species like humans (93, 94). Unlike a prediction in a species with short

19

generation time, a correct prediction of the postnatal life can be hard to make in a long-

lived species since the probability that the environment will change is large. Secondly,

he claims that PAR overlooks the fact that mammalian fetuses are robustly protected

from external perturbations due to the mother’s own buffering capacity, and will thus

never get correct information about the external world, only cues that have already been

modified and translated by the mother (92, 93). This goes hand in hand with that the

mother’s fitness is maximized if her offspring is strong and wants to protect her fetus

from disturbances. Wells emphasizes that, because of the maternal-offspring fitness

trade-off during pregnancy and the implications that this conflict has for maternal fitness

especially, the trajectory of the fetus is controlled by the status of the mother and

therefore not “predictive”, but rather backward-looking (93). This is further supported by

Kuzawa (54) that suggests that growth responses to short-term environmental

fluctuations are more about adopting the maternal phenotype and thereby adjust, not to

small periodical changes, but to a longer history of fluctuations that has influenced the

previous generations. This supposedly “smoothes out” fluctuation peaks, or the “noise”

of seasonality and enables the fetus to actually adjust according to longer term trends

(54). Lastly, Wells argues that the structure of the group in social animals, like humans,

will have an impact on the nutritional status of different mothers in the group, even

though the physical environment is the same. Social hierarchy will make a female of

lower social status receive less food. To maintain high fitness, it is therefore best for a

lower ranked mother to have a small progeny since it is likely that the offspring also will

be low in rank and receive less food (93).

3.6 My definition of developmental programming

In this jungle of expressions and definitions of the DOHaD phenomenon, I have chosen

to talk about “developmental programming” to describe my work. Although the full term

“developmental programming” is not as clearly defined as the theories presented above,

Lucas (1992) defined “programming” as: “…programming occurs when an early stimulus

or insult, operating at a critical or sensitive period, results in a permanent or long-term

change in the structure or function of the organism” (59). Barker and colleagues

sometimes use the expression “fetal programming” (9, 38), but for the same reason

presented for the change from FOAD to DOHaD, I have chosen to use the term

“developmental programming” to encompass both pre- and postnatal growth and not just

in utero events.

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21

4 HYPOXIC CARDIOVASCULAR REGULATION AND THE ROLE

OF β-ADRENERGIC RECEPTORS

When a vertebrate is faced with oxygen deficit caused by for example physical activity or

altitude, there are several mechanisms that will set in to regulate cardiovascular function

and retain homeostasis. These mechanisms are different between species and have

different onsets during development.

4.1 Adult and fetal cardiovascular regulation during hypoxia

The cardiovascular response to hypoxia in adult mammals is not uniform, since it is

complicated by ventilatory actions that affect the heart rate (HR) and blood pressure

(BP). When faced with hypoxaemia1, or low blood oxygen, chemoceptors in the carotid

bodies (located close to the bifurcation of the carotid artery in the neck) sense the low

pO2. This triggers an efferent reflex response that includes both increased vagal activity

that potentially could lower heart rate and increased sympathetic tone that could

increase blood pressure (62). However, the chemoreflex primarily affects the pulmonary

ventilation and the cardiovascular response is thereby subjected to secondary ventilatory

effects; vagal activity is suppressed by the activity of inspiratory neurons and pulmonary

stretch receptors and the outcome of HR and BP may therefore be different depending

on the respiratory response (49). In spontaneously breathing adult animals, hypoxia

leads to hyperventilation that can cause tachycardia or bradycardia combined with either

vasoconstriction or vasodilatation depending on the animal model (62). When pulmonary

ventilation is controlled, however, tachycardic and vasodilatory responses are

completely abolished and unmask bradycardia and vasoconstriction as the “true”

chemoreflex-induced response of the cardiovascular system (49).

In one of the most common fetal hypoxia models, the fetal sheep, the chemoreflex

is present relatively early (51) and the magnitude is dependent on age together with the

depth and duration of the hypoxic insult (91). Hypoxia has no effect on fetal breathing

movements in the sheep fetus, which is not unexpected since fetal breathing movements

have no role in gaseous exchange (31). Without the ventilatory effects, the

cardiovascular response to hypoxia in the near-term sheep fetus is therefore similar to

1 Note that hypoxaemia is used when talking about low partial pressure of oxygen in the blood in particular, while hypoxia is used as a wider concept, mostly referring to the oxygen level of the environment the organism is in. Hypoxaemia is often a result of hypoxia, but in this thesis I use hypoxia indifferently.

22

the one in the adult with controlled ventilation; hypoxia induces an initial bradycardia and

increased peripheral vascular resistance mediated by carotid chemoreceptors (31) (Fig.

7). The increase in systemic blood pressure is instrumental in the redistribution of

cardiac output (CO) to crucial organs like the heart, brain and adrenals on behalf of

organs such as the kidneys and liver (28, 66). If the hypoxic episode is prolonged, the

release of catecholamines will oppose the vagal tone and eventually restore HR (28).

In the model used for this thesis, the chicken embryo, the cardiac response to

hypoxia differs from that of the fetal sheep. As shown by microsphere injections, the

redistribution of CO in response to hypoxia is present also in the chicken (66), but both

the vagal and sympathetic limb of the reflex are non-functional, suggesting that

cardiovascular control relies mainly on humoral factors such as epinephrine (20). The

acutely hypoxic chicken embryo is bradycardic, possibly due to direct effects of low

oxygen on the cardiac muscle (21), and hypotensive. In Paper V we investigate the

effect of chronic hypoxia on BP and HR, something that will be further discussed in

Chapter 6.

23

Figure 7. Cardiovascular response to hypoxia in the near-term sheep fetus. Intact fetuses (solid

lines) experience hypoxic bradycardia (top) and an increase in peripheral vascular resistance

(bottom), while carotid denervated fetuses (dashed lines) do not, demonstrating that the initial

hypoxic response in the late fetal sheep are due to a functional chemoreflex mediated through the

carotid chemoreceptors. Modified from Giussani et al. 1993.

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4.2 Effect of hypoxia on the βAR system

Hypoxia is an environmental stress. The physiological response to stress is linked to the

release of catecholamines and elevated levels of catecholamines are indeed

documented in both the hypoxic adult and fetus (17, 18, 21, 60, 65). However, elevated

catecholamines and hypoxia have different effects on adult and embryonic βARs.

Hypoxia or infusion of βAR agonists in the intact adult elicit the classical βAR

regulation pathway of desensitization and downregulation with a concomitant loss of

surface receptors in both the cardiac and vascular tissue (5, 41, 98). However, the

hypoxia-induced loss of βAR-mediated cAMP production in adult hearts can be restored

by normoxic recovery (45).

In the fetus and neonate, βAR regulation differs from the adult. In contrast to the

adult desensitization, Auman et al. (2001, 2002) have shown that infusion of βAR

agonists do not desensitize the βARs, but rather sensitizes them. We found similar

results in response to chronic hypoxia in Paper II where we show that, despite the loss

of surface receptors, there is an increase in sensitivity of the system. The fetal and

neonatal βAR sensitization has been linked to a higher concentration of β2ARs in the

fetal/neonatal heart (6) and the fact that β2ARs 1) are resistant to downregulation and

have a protective effect on the heart, 2) do not induce cardiomyocytes enlargement

(hypertrophy) like β1ARs do (the increased volume-to-area ratio directly means

decreased sensitivity) and 3) have, in opposite to in the adult, contractile effects in the

neonatal heart (101). Although we found that there is an increase of Gαi in the chronically

hypoxic chicken embryo heart (Unpublished data, Isa Lindgren 2010), the contractile

effect of β2ARs is thought to be because of the lack of Gαi coupling in the fetal heart and

that Gαi coupling is aquired with maturation (101). The increased βAR sensitivity has also

been linked to an increase in Gαs/AC coupling and altered expression of AC subtypes

promoting a more active isoform of AC (79).

The effects of hypoxia on βARs described above are implicitly related to the

increased release of catecholamines based on that the observations are made in intact

animals where catecholamines have been shown to increase with hypoxia (see

beginning of this section). This is also supported by similar results from studies where

catecholamines have been infused. However, in addition to the secondary hypoxic effect

on βARs through increased levels of catecholamines, hypoxia per se has been shown to

have an effect on βARs (61). Marsh and Sweeney (1989) incubated cultured embryonic

chicken cardiomyocytes myocytes in hypoxia induced a >50% decrease of cell surface

25

βARs that was restored to control values with re-oxygenation (61). This is interesting

since these cardiomyocytes are isolated and thereby not subjected to catecholamine

stimulation of adrenomedullary or sympathetic origin. There is, however, evidence of

intrinsic cardiac adrenergic cells (ICA cells) in the fetal heart releasing epinephrine and

norepinephrine that can affect the pacing rate of spontaneously beating cardiomyocyte

cultures (46). Whether hypoxia stimulates ICA cell catecholamine release or hypoxia per

se is the reason for the hypoxic βAR downregulation is not yet known, but it is an

interesting question that warrants further investigation.

26

27

5 SUMMARY OF PAPERS

The first foundation for our future life and health is laid during fetal development and

understanding developmental processes and what influences them is therefore of great

interest and importance. In this thesis I have looked at not only the cardiovascular β-

adrenergic signaling system, but also how it is altered by chronic prenatal hypoxia

connected to the progression of growth and development throughout fetal life. The β-

adrenoceptors are of particular interest, since they are crucial for cardiovascular

development, function and regulation and also since hypoxia trigger the release of their

main ligands epinephrine and norepinephrine.

5.1 Paper I

Lindgren I, Altimiras J . Sensitivity of organ growth to chronically low oxygen levels

during incubation in red junglefowl and domesticated chicken breeds. Submitted

Chicken domestication has resulted in the appearance of numerous phenotypic traits

connected to animal productivity (egg laying or meat production). This paper

demonstrates that some of these traits are already present during embryonic

development and this makes some breeds more susceptible to environmental

manipulation such as oxygen shortage. In the paper we use a modeling approach to

illustrate the effects of hypoxia and developmental age on organ growth, built on organ

mass data from different developmental stages. In particular, we show that the

developmental trajectory for cardiac growth is affected by hypoxia. Because βARs are

closely related to cardiac development and growth (72), this observation alone prompted

the follow-up work on cardiac function in papers II and III. The paper is also an

interesting contribution to the discussion about developmental plasticity, allocation

theory and the evolutionary significance of plasticity/developmental programming.

28

5.2 Paper II

Lindgren I, Altimiras J. Chronic prenatal hypoxia sensitizes β-adrenoceptors in the

embryonic heart but causes postnatal desensitization. Am J Physiol Regul Integr Comp

Physiol 2009;297:R258-264.

Prenatal oxygen deficit (hypoxia) is correlated with low birth weight, which is in turn

correlated with increased risk of adult morbidity like diabetes and cardiovascular disease.

This phenomenon is referred to as developmental programming and in this light we

describe how chronic prenatal hypoxia affects βAR density and sensitivity in both

embryonic and juvenile broiler chicken hearts, since β-adrenoceptors are crucial for the

embryonic development and adult function of the heart. Broiler eggs were incubated in

normoxia or hypoxia (14% O2) throughout development and one batch from each

treatment was also hatched and raised to 14 or 35 days of age. Based on [3H]CPG-

12177 binding in ventricular tissue slices and concentration-response curves to

epinephrine in electrically paced muscle strip preparations, we found that chronic

prenatal hypoxia increases the pEC50 (i.e. sensitivity) of epinephrine-stimulated cardiac

strips in the 19 day embryo, despite a significant decrease of receptor density. At the

same time, we found that the prenatal hypoxia did not affect receptor density in the 35

day old juvenile chickens, but caused βARs desensitization. 14 day old chickens were

not affected. This supports that prenatal hypoxia has a “programming” effect on adult β-

adrenoceptor function that may be an indication of early symptoms of heart failure.

5.3 Paper III

Lindgren I . Postnatal β-adrenergic desensitization caused by chronic prenatal hypoxia

is linked to an increase in Gαs and decreased β1AR/β2AR ratio. Manuscript

This paper is the continuation of paper II and is an attempt to explain the β-adrenergic

desensitization seen in the 35 day juvenile chickens suffering from chronic prenatal

hypoxia. Others have suggested that altered receptor sensitivity could depend on a

decrease in β1AR/β2AR subtype ratio (since β2ARs do not significantly contribute to

contraction in the adult heart) and Gαs/Gαi protein (more Gαi could potentially increase the

probability of β2AR switching, thus inhibiting cAMP production) (6, 79, 101). We

investigated the β1AR/β2AR subtype ratio using selective [3H]CPG-12177 binding in

29

ventricular tissue slices by blocking β1ARs (CGP-20712A) or β2ARs (ICI-118,551).

Gαs/Gαi protein expression levels were determined by Western blot on cardiac whole-

tissue preparations and cAMP produced in response to βAR stimulation with

isoproterenol was measured with an immunological assay. The significantly lower cAMP

level in prenatally hypoxic hearts compared to the controls supported our previous

observations in Paper II of βAR desensitization in 5 week chickens prenatally exposed to

hypoxia. We also found that there is indeed a decrease in β1AR/β2AR ratio. Surprisingly,

Gαs increased in prenatally hypoxic hearts and not Gαi as hypothesized. When again

considering the lower cAMP accumulation in response to βAR stimulation however, the

increased Gαs seems to be inactive and might not be recruited to the cell membrane,

something that is necessary for taking part in the signaling. Based on that the 8 month-

old chicken exposed to prenatal hypoxia display cardiomyopathy, we speculate that what

we see in our 5 week chickens is a phenotype of early cardiac failure.

5.4 Paper IV

Lindgren I, Zoer B, Altimiras J, Villamor E. Reactivity of chicken chorioallantoic

arteries, avian homologue of human fetoplacental arteries. Submitted

The chorioallantoic (CA) membrane is the avian homologue to the mammalian placenta,

but nothing is yet known about the vasoreactivity of this vascular bed. Characterizing the

vascular reactivity of the CA arteries throughout development is essential to fully

understand the hemodynamics of the embryo since a large proportion of vascular

volume is contained within the extraembryonic circulation. In this paper we used wire

myography techniques to perform concentration-response curves in isolated CA arteries

to contracting (KCl [4.75-125mM], the thromboxane A2 mimetic U46619 [1nM-1µM],

endothelin-1 [ET-1; 1nM-100nM] and α1 adrenoceptor agonist phenylephrine [Phe;

10nM-100mM]) and relaxing agents (acetylcholine [ACh; 10nM-100mM], the nitric oxide

donor sodium nitroprusside [SNP; 10nM-100mM], the β-adrenergic agonist isoproterenol

[Iso; 1nM -1mM] and the adenylate cyclase activator forskolin [Forsk; 1nM-10mM]). In

the relaxation experiments the vessels were pre-contracted with either 62.5 mM KCl or

U46619. We also carried out experiments of acute hypoxia on the vessels and our

results show that CA arteries share many of the characteristics of placental arteries, but

also that they do not react to Phe (indicating lack of αAR receptors), that they contract,

rather than relax, to ACh and that they display vasoconstriction to acute hypoxia. In

30

addition to supplying us with information to better understand the hemodynamic

regulation of the chicken embryo, the obtained results suggest that it is an interesting

model artery for studying the mechanisms of hypoxic vasoconstriction.

5.5 Paper V

Lindgren I, Crossley D, Villamor E, Altimiras J. Hypotension in the chronic hypoxic

chicken embryo origins in a potent β-adrenergic response of the chorioallantoic arteries

and not bradycardia. Submitted

Despite the increasing importance of the embryonic chicken as a model to study

development, cardiovascular responses to hypoxia and developmental programming of

adult disease, the hypoxic hemodynamic response and regulation is still not fully

understood. In this paper we try to answer the question if the chronic hypoxic chicken

embryo is hypotensive like the acutely hypoxic embryo (21) and if this possible

hypotension is due to chorioallantoic (CA) artery β-adrenoceptor-mediated relaxation

and/or bradycardia. We measured heart rate both acutely by cannulation on day 19 of

incubation and continuously throughout development by a non-invasive impedance

method. Additionally, blood pressure was measured in the cannulated embryos.

Concentration-response curves to isoproterenol and forskolin in normoxic and hypoxic

CA and femoral arteries were obtained by wire myography of isolated vessels. We found

that heart rate was not changed in the chronic hypoxic chicken embryo and that CA

arteries were not affected by hypoxia, but displayed a potent β-adrenergic relaxation

together with an increased βAR sensitivity of the hypoxic femoral vessels. This suggests

that the CA vascular bed, together with an increased vasorelaxation of the systemic

arteries, is an important contributor to the hypoxic hypotension response in the chicken

embryo, while heart rate is not involved.

31

6 DISCUSSION

6.1 Comments on the experimental model

The chicken model used in my studies is an increasingly used model to study hypoxic

effects on prenatal growth and development (30, 57, 81, 97, 105), much because it

enables manipulation without confounding maternal effects. The chicken genome is

sequenced (88), which adds additional value to the model.

Commercially, the broiler chicken grows to 2.6 kg in 6 weeks after hatching. The

rapid growth of especially muscle mass puts a heavy load on the cardiovascular system

to supply the tissues with oxygen and nutrients. This predisposes the broilers to develop

cardiovascular disease already as juveniles and because we know that cardiovascular

problems can occur already before adulthood2, 5 week animals are suitable for studies

of fetal programming of cardiovascular disease. However, rearing the birds in line with

commercial breeding is not suitable for research animals and thus our birds did not grow

quite as fast as indicated above.

Although lower levels of oxygen can be successfully used in experiments of acute

or intermittent hypoxia, the level of hypoxia in my chronic experiments is between 15-

14% O2. The choice of oxygen concentration is based on pilot experiments where I

found that, in a chronic setting, 12% O2 killed all embryos after day 11 of incubation with

a peak mortality at Hamilton-Hamburger stage 25-26 (corresponding to day 5).

6.2 Developmental programming (Papers II and III)

If you boil down the information presented so far in this thesis, you end up with quite a

simple trail of events: Developmental hypoxia leads to fetal growth restriction, fetal

growth restriction leads to increased risk of adult cardiovascular disease and adult

cardiovascular disease is closely related to changes in the βAR system. Despite the

seemingly obvious connection between low birth weight - adult cardiovascular disease -

βARs, there is not much known about possible programming effects of prenatal hypoxia

on the adult βAR system. In one of the few studies on programming effects of βAR

function, Rohlicek et al. (2005) show that 10 days of hypoxia with subsequent normoxic

recovery in the neonatal rat does not change the βAR density in the adult heart, but

decreases cAMP production, likely due to changes in AC levels. Because of the evident

lack of data on fetal programming effects of hypoxia on the βAR system and its

2 Adulthood defined as after sexual maturity, approximately 20 weeks of age in the chicken.

32

importance in cardiovascular disease, one of the most important aims with this thesis

was to investigate whether prenatal hypoxia has a programming effect on adult βAR

function or not. I show in Paper II that it does; chronic prenatal hypoxia in the chicken

increases βAR sensitivity in the embryo, but has no effect in the 2 week old postnatal

animal. However, in the 5 week old chicken, βAR sensitivity to epinephrine is decreased.

It is important to consider all three of these stages when discussing whether this is

programming or not; the fact that the 2 week old chicken is unaffected and that the

desensitizing effect occurs later in life is key. In Paper III we look for possible

mechanisms behind this desensitization and find that the β1AR/β2AR ratio is decreased,

resembling what is seen in adult individuals suffering from cardiovascular disease,

suggesting that my 5 week old chickens might be in the early stage of developing heart

failure. This is further supported by a study from Tintu et al. (2009) showing how 8

month-old chickens prenatally exposed to hypoxia display left ventricular dilatation

cardiomyopathy and cardiac remodeling (87). Surprisingly, I also found an increase in

Gαs, the opposite to the hypothesized increase in Gαi. However, a mere increase of Gαs

does not necessarily mean that the expressed proteins are available for signaling. The

protein increase could be in the unavailable cytosolic fraction, or the coupling of the βAR

to the Gαs could be affected through elevated receptor phosphorylation due to an

increase in GRKs or an increase in β-arrestin availability to block the receptors (58).

Additionally, even if Gαi do not increase, it has been shown that the functional activity of

Gαi is increased (26), which would contribute to the decreased βAR response. My

suggestions for further studies is the expression and activity of AC isoforms, cell

fractionation of G-proteins, Gαs functional activity and the activity and abundance of GRK

and β-arrestin in the prenatally hypoxic heart. My ultimate conclusion from Paper II and

III is however, that prenatal hypoxia does have a programming effect on adult βAR

function.

6.3 Growth and allocation (Paper I)

Another important part of this thesis is dealing with the effect of hypoxia on growth and

resource allocation – a foundation for discussing the growth restriction factor of

developmental programming. It is established that the organism has a basal energy

budget for maintaining minimal functionality and all energy available on top of that is

allocated to growth, reproduction, fat storage and physical activity (54). Consequently, if

energy availability is on the verge to what is required for maintaining basal function,

33

there will be a re-allocation of energy to save the most vital functions and organs to

secure survival. In the case of prenatal hypoxia where oxygen is the limiting metabolic

factor, the re-allocation from growth to preserving vital functions is illustrated by the re-

distribution of cardiac output away from the carcass towards the heart, brain and

adrenals (66). This contributes to the well-established fetal growth reduction and sparing

of the before mentioned organs. I show in Paper I that the sparing is different in different

strains of chickens. The heart and the liver are spared in the early broiler embryo, but

stunted in the late embryo. Considering that about 50% of the embryonic growth takes

place in the last five days of incubation (80) and that it is the period when the oxygen

demand reaches its peak, hypoxia has a greater impact on growth in the end of

incubation. This is emphasized by the fact that the loss of embryonic mass in response

to 14% O2 is absent at day 7, 10% at day 11 and around 30% at day (unpublished data,

Isa Lindgren, 2010). It is possible that my level of hypoxia is not affecting the early

embryo because its oxygen demand is low, but that it is detrimental in the late embryo

because of its higher metabolic rate. Referring back to Fig. 6 in Chapter 3, the milder

hypoxic insult in the early embryo could be speculated to be within the “coping” section

of the graph, making the best of the situation and thus being able to re-allocate

resources to spare the heart. Later on in development when the intensity of the hypoxia

is increased due to higher oxygen demand, it might be so severe that it pushes the

response into the “disruptive” zone, leading to the recess of all organ growth. A future

perspective to organ growth and resource allocation in the chicken is to decrease the

severity of the hypoxia (tentatively by inducing intermittent hypoxia) to investigate

whether sparing of the heart can be kept until hatching. Broberg et al. (2003) show that a

10-day period of anemia in the fetal sheep actually increases the contractile function of

the adult heart in response to hypoxia (15), something that would be very interesting to

attempt in the chicken due to its rapid postnatal growth and subsequent tissue hypoxia.

6.4 Vascular reactivity and cardiovascular response to chronic hypoxia in

the chicken embryo (Papers IV and V)

Despite the popularity of the chicken embryo as a model to study development and

hypoxic response (30, 57, 81, 97, 105), the understanding of something as basal as the

cardiovascular regulation in response to chronic hypoxia is still incomplete.

34

Crossley et al. (2003) hypothesizes that the hypotension seen in the acutely

hypoxic chicken is due to βAR dilatation of the CAM. Because the basic vasoreactivity of

the CAM has never been assessed, I started by characterizing the contractile and

relaxant responses of the CAM (Paper IV). With the integrative cardiovascular response

to chronic hypoxia in mind, my most important findings were that the CA arteries did not

contract to phenylephrine (indicating lack of αARs), but relaxed to both βAR stimulation

by isoproterenol and AC stimulation by forskolin. I also found that CA arteries share

many of its relaxing and contracting responses with placental vessels to common

vasoactive drugs such as endothelin-1 and sodium nitroprusside. A quite significant

exception, however, was a substantial contraction to acetylcholine in K+ pre-contracted

vessels. CA arteries, like placental, also display hypoxic vasoconstriction indicating an

oxygen sensing capability. For future studies it would be interesting and important to

define the hypoxic vasoconstriction of the CA arteries and the impact it has on the

hemodynamics of the chicken in hypoxia. This is not, however, within the scope of this

thesis.

The characterization of CA arteries in Paper IV was important per se, since it is a

large vascular bed with previously unknown vasoreactivity. It was also the necessary

foundation for Paper V where I investigated the BP and HR response to chronic hypoxia.

The baroreflex is not well developed in the chicken embryo (3) and in the absence of a

baroreflex, a drop in HR is positively correlated to BP, i.e. low HR leads to hypotension. I

showed in Paper V, however, that HR is not lower in chronically hypoxic embryos, and

thus not contributing to the hypotension. Instead, I showed that CA arteries display a

potent βAR relaxation that is not altered with hypoxia and that femoral (systemic)

arteries βAR relaxation was increased with hypoxia. Thus, what is likely causing the

hypotension in the hypoxic chicken embryo is βAR stimulation by increased levels of

catecholamines.

The results from Papers IV and V are important not only to elucidate new aspects

of fetal cardiovascular regulation, but also to understand mechanistics behind βAR

regulation. Tissue-specific βAR regulation has been described before (5) and it is

apparent that βARs in the CAM and the femoral vessels are differently regulated under

identical conditions. An interesting dimension to this discussion is the role of innervation;

Ruijtenbeek et al. (2000) has demonstrated hyperinnervation of the femoral artery in

hypoxic chicken embryos (82), while I have shown that the CAM is not innervated (Paper

IV). The sympathetic innervation in the chicken embryo, although present, has been

35

suggested not to be functional until very late in incubation (21), but might be involved in

the disparate regulation of systemic vs. extra-embryonic vascular βARs.

6.5 Conclusions

With this thesis I have further defined and refined the prenatally hypoxic chicken model

and used it to show that chronic prenatal hypoxia causes growth restriction, re-allocation

and that it has programming effects on the βAR system in the adult. The latter indicates

that the βAR system is an important factor in studying hypoxic developmental

programming of adult cardiovascular disease.

36

37

ACKNOWLEDGEMENTS

I would like to dedicate my first thank you to my supervisor Jordi. Not just because it is

expected from you to thank your supervisor first when writing your acknowledgements,

but because it is from the very depth of my heart when I say that I am so incredible

grateful to have had you as my supervisor. You saw something in me (I am still not sure

I understand what, because you asked me at my interview how I would compensate for

not being so book smart or exceptionally bright…) and gave me the opportunity of a life

time when you took me on as you PhD student. There is so much I have to say, but at

least let me thank you for your never ending commitment, your stamina, drive and

enthusiasm, your contagious interest in science and teaching, your wit, your cheeriness,

your know-how, your straightforwardness, your way of always fighting for what you

believe in and for rubbing all of the above off on me. Thanks for teaching me that a

physiology lab is just as much a work-shop and that all physiologists need to know how

to solder. Thanks for all the fun and laughs, but also for being an ******* sometimes, only

so that I could realize later that you were only being honest and teaching me reality,

independence and trusting my own ways – it took me sometime to realize that (even if

you see him as your science hero) sometimes your boss does not know everything and

you might just be right yourself! At last, thank you for just being the crazy, happy

Spaniard that makes coming into work everyday a treat and adventure! It is with a heavy

heart and with the deepest respect for you as a person and a researcher that I leave

your (our!) lab, but you promised me you would be my life-long friend and mentor and I

will hold you to it! Thanks also to your wonderful family for putting up with the both of us

and for all the great times we have had together.

Secondly, I would like to thank the other Spanish keystone of my PhD - my co-

supervisor Eduardo Villamor. Thank you for taking me in to your lab in Maastricht and

teaching me so much about vascular physiology in only three months. 40% of my papers

in this thesis are the products of my work in your lab and that is not even covering all the

data we produced – one cannot call that anything but a productive collaboration! Thank

you for always being so enthusiastic and encouraging, it was a true pleasure working in

you lab and it certainly helped me get my new job! Thanks also to the rest of my

colleagues at the University hospital of Maastricht for making my stay so much fun.

I am also very grateful to Carl-Gunnar Runemark and colleagues at Lantmännen

SweHatch in Väderstad. Thanks for being so helpful and accommodating throughout the

years we have collected the eggs for our research at your hatching facility.

38

Thanks to all my wonderful colleagues throughout my years at Biology, you all

contributed to that it has always been interesting, rewarding and fun to come in to work.

Special thanks to Johan B for all the good ol’ years sharing an office, to the loyal Friday

beer crew for making Friday afternoons half an hour shorter and to Magnus E and Ida G

(the two people in Biology except me who know what working for Jordi is like!) for always

being there when I need to vent and for putting an extra smile on my face, preferably

over lunch ☺. Thanks also to all the students that have passed through the lab for the

effort that you made, but mostly for teaching me a whole lot about myself, forcing me to

be a “boss”.

Thanks to my chemistry posse – Daniel K, Roz, Alma, Ina and the others – not

only for your support and being good friends through my last year of my PhD, but for

making my life with one foot in integrative physiology and one foot in biochem sooo

much easier with all your know-how! You are all stars and super-brains ☺.

A big thank you to Stefan Klintsröm, director of Forum Scientium, and all of the

Forum students for enriching my time as a PhD student. Through Forum I have not only

made many friends and learned about new places, ideas and techniques, but also

gained important knowledge about the part of science you cannot learn from books –

communicating science, research politics and life after your PhD.

Science is a lot of fun, but sometimes you need to take your mind off things, and

what better way to do it than with friends from back-in-the-days – thank you my

wonderful, sassy, honest, loving and caring Matfors-girls of “fnittergänget” - Lisa, Martina,

Backlund and Bengtsson - for putting things in perspective and loving me for who I am.

Thanks to my fellow PhD girls outside LiTH, Dana, Kattis, Anneli and Sanna for being

such a source of inspiration (and red wine!). Thanks also to the rest of my Linköping-

gang through the years (no one mentioned, no one forgotten - there are too many of you,

but you know who you are!) for putting up with me constantly yapping about my work.

You have kept my feet on the ground and made me feel that I am not alone when things

have been tough!

Now to the most important people behind the scenes – my wonderful, wonderful

family. To my extended family through David and Miras – I am so happy that you (and

your families) are part of ours and I can not explain how great it feels having you

interested in my progress and cheering me on. Thank you Fredriks family for always

being so wonderful, interested and encouraging. You make me feel like a superstar!

Thank you to the two best sisters anyone could ever have – Kim and Moa. Your bare

39

presence means so much to me and it does not matter how stressed I might be – with

you guys I am relaxed, because I know I do not have to accomplish anything or perform

around you, just be me. Love you dearly. An extra thanks to Kim and David for making

me and Moa aunties to the most wonderful little Albert and Betty on the planet!

A mere thank you does not seem enough when it comes to my fantastic parents,

but it will have to do; thank you for giving me the best childhood one can ever ask for

and a rock solid foundation of always feeling loved and safe. I only dare throwing myself

into almost anything because I know I always have you backing me up. I am very lucky

to have you as my parents and I love you so much. An extra thanks to mom for always

taking the time to answer my questions about plants and animals and thus founding my

interest for biology from the time I was a toddler. Deep inside I am sure we all knew that

I was going to be a scientist one day…

My last thank you is directed to the most important person in my life – Fredrik. I

guess it is kind of weird or cliché to say thank you for loving me, but - thank you for

loving me so unconditionally! Thanks for believing in me, for making everyday

(EVERYDAY!) sunny and fun, for being so truly good-hearted, for listening to my

looooong harangues about work, thanks for making my friends and family love you and

thanks for always making me feel like I am invincible! And people, listen to this – thank

you for (voluntarily!) taking care of aaaall the cleaning, planning, laundry, cooking and

shopping for the last THREE MONTHS of my PhD! What a man! YEAH! Moving to the

US will be a piece of cake with you by my side and I am so excited about all the fun we

have to look forward to together! And whatever career path you may choose – I will

support you wholeheartedly, just like you have supported me. I love you.

Ps. Oh, and a big thanks to me – keep doing what you are doing Isa, you kick ass! ☺

Isa ”Bäst-i-världen-och-så-jävulskt-snygg-och.smart” Lindgren, August 2010

40

41

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