Treatment with integrin {α_1tnv [alpha-v] inhibitor abolishes compensatory cardiac hypertrophy due to altered signal transduction and ECM gene expression [Elektronische Ressource] / vorgelegt von Rongxue Wu

Medizinischen Fakultät

ürde der zur Erlangung der Doktorw

der Bayerischen Julius -Maximilians -Universität zu Würzburg

von

Wu

vorgelegt

Rongxue

aus Kunming, China

Würzburg, Dezember 2006

Treatment with integrin Įv inhibitor abolishes compensatory cardiac

hypertrophy due to altered signal transduction and ECM gene expression

Aus der Medizinischen Klinik I

der Universität Würzburg

rg Ertl ofessor Dr. med. GeorDirektor : P

-

Inaugural Dissertation

Referent : Prof. Dr. med.Georg Ertl

Korreferent: Priv.-Doz. Dr. med. Oliver Ritter

.Frosch Dekan: Prof. Dr. M

Tag der mündlichen Prüfung: 17.01.2007

Die Promovendin ist Ärztin

Für meine geliebten Eltern

Content

......................................................................................... Introduction1

y in cardiac remodelling …………………………………………………………… Hypertroph1.1.

1.1.1. cardiac hypertrophy ……………………………………………………... Definition of

response versus mal-adaptation…………… Cardiac hypertrophy: compensatory 1.1.2.

cardiac hypertrophy ………………………………………………………Inhibition of 1.1.3.

cardiac hypertrophy …………………………… Alterations in Gene expression of 1.1.4.

Mechanisms and signal transduction pathways of cardiac hypertrophy…………………...1.2.

Stimuli inducing cardiac hypertrophy..………………...……..…………………......... 1.2.1.

Mechanical 1.2.1.1. ……….……………...... stress.………….……………………….

1.3.

1.4.

……………………….. and hormones……………………....1.2.1.2. Growth factors

….…… Signal transduction of stretch stimuli …………………..…………………….1.2.2.

1.2.2.1. The mitogen-activated protein kinase (MAPK) pathway ...…………….….

............ kinase (ERK) pathway…....................r-regulated extracellula The

NK) pathway……….……….…….. J The c-Jun N-terminal protein kinase (

The p38 MAPK Pathway……….……...………..…………………………….

1.2.2.2. The janus kinase/signal transducers and activators of transcription…...

1.2.2.3. Calcineurin-dependent pathway ……………..………………………………

pertrophy……………………………………………….. in cardiac hyof integrins Implication

1.3.1. cardiac hypertrophy ………………………….. Integrin structure and expression in

…………… myocardium……………………….Integrin-mediated signalling in the 1.3.2.

1.3.3. Function of Integrin Įv molecule in heart……………………………………………..

Extracellular matrix in cardiac hypertrophy and failure……………………………………

.............. Extracelluar matrix and cardiac fibrosis………………………………….....1.4.1.

………………………… Functions and modulation of different component of ECM 1.4.2.

nd their functions…………………….............. Different elements of cardiac ECM a1.4.3.

……………………………………………………………………….………… Collagen

……………………........………………………………………………….... Fibronectin

Vitronectin………………………………………………………………………………...

…………………………………………….………… SPARC…….. ……………………

1.5. Aim of this study …………………………………………………………………………………

1

1

1

2

3

4

6

5

5

5

6

7

7

9

10

10

11

12

12

14

16

17

17

18

19

19

19

20

21

22

………………………………………………........2. Method and materials

…... ………………………………...……2.1. Animals…………………….…………...……

......................…2.2. Experiments…………………………………………….………..……

2.2.1. Experimental design ………………………………………….………………....

….. sure overload-induced hypertrophy in mice …………………...……2.2.2. Pres

on of integrin alpha v ……………………………………….…... 2.2.3. In vivo inhibiti

2.2.4. Hemodynamic measurements………………………..……………....………..

sthoracic Echocardiogram (M-mode)………………………..………2.2.5. Tran…...

2.2.5.1.

2.2.5.2.

aphy……………………………………………………...Echocardiogr

Measurements………………………………………………………….

Ventricular mass calculation…………………………………………………… 2.2.6.

RNA isolation……. ………………...…………2.3. …. ………………………………..……

………………………………………..……….……….............cDNA Microarray ……2.4.

……………………………. idization…2.4.1. RNA preparation and Gene Chip hybr

tion Data Analysis……………………………………………… 2.4.2. Post-Hybridiza

methods……………………………...…………………………..…….……….. 2.5. Protein

on for Western.blot…………………………………..………… 2.5.1. Protein extracti

2.5.2. Western blotting ………………………………………………………………….

2.6. Material…………………………………………………………………………………...

……Antibodies……………….………………………………………………...… 2.7.1.

.. ..…rs and Solutions…………………………………………………...…2.7.2. Buffe

….. ………...……………………………………..…2.7.3. Chemicals ……… …….…

2.7.4. Software and websites………………………………...……… …….……….. ..

2.7.5. Lab devices…………………………………….……………………….………..

3. Results………………………………………………………………………………….in alpha v for early cardiac hypertrophy and survival……... Requirement of Integr3.1

3.1.1. ………………………………………..………......... ........………rtality.........Mo

….……………………........ rphometry..…………………………..…………3.1.2. Mo

sthoracic Echocardiography………………………………………………. 3.1.3. Tran

3.1.4. Effects of integrin alpha v inhibitor on hemodynamic measurements…..….

3.2. Integrin alpha v requirement for pressure overload -activated protein kinase

23

23

23

23

23

25

26

27

27

28

29

29

30

30

31

32

32

33

33

33

36

39

42

42

44

44

44

45

47

47

3.3.

activity during heart remodelling ………………………………………………..……..

3.2.1. Activation of Fak and Src in the mouse heart following 7 days of aortic

v inhibitorĮbanding was abrogated by treatment with integrin

Regulation of ERK 1/2 and p-38 expression in heart after AB 3.2.2.

3.2.3. ion following aortic banding v on ANF expressEffect of integrin alpha

ion and its modulation by integrin alpha v inhibitor following ECM gene express

…………………………………………………………………. aortic banding………

ium...….. …………………………… ession In myocard3.3.1. Integrin alpha v expr

3.3.2. Regulation of ECM gene expression upon pressure overload …………..

4. Discussion…………………………………………………………….…………...hy…………………………..……. c hypertropequired for cardiagrin alpha v is r4.1. Inte

ed in development 4.2. Integrin alpha v mediated signal transduction is involv

pertrophy upon pressy of hure overload……………………………………………….

M and integrin……………………..…………………………..…….. C4.3. Cross talk of E

5. Abstract 6. References

Appendi

ces……...………...……………………………………………………….…………... ……………………………………………………….…………... ................................scription of Gene-Array.............. Gene de...…………....…….

Abbreviations……………………………………………………………………………

Honours………………………………………………………………………………….

Acknowledgments

54

54

56

58

59

59

60

62

65

65

68

70

75

76

86

86

91

93

1. INTRODUCTION

in cardiac remodelling ypertrophHy1.1.

pertrophyrdiac hya1.1.1. Definition of c

alpertrophy is the cellular response to an increase in biomechanicCardiac hy

stress, such as arterial hypertension or alveolar heart disease. The essential cardiac

response to a fixed increase in hemodynamic load is an increase in cardiac mass.

al This frequently results in cardiac hypertrophy, which is induced by the mechanic

stress on the cardiomyocytes and the activation of neuroendocrine mechanisms,

mpathetic nervous in-aldosterone system and the sy the renin-angiotensparticularly

e defining fsystem. Thte size, e in cardiomyocyeatures of hypertrophy are an increas

ation of the sarcomere. The enhanced protein synthesis, and a higher organiz

e membrane yte growth involvcomplex molecular processes that lead to cardiomyoc

and transcription factors. The common final step of receptors, second messengers,

all these intracellular pathways whose variations are being ion, is gene express

pertrophy represents an independent risk revealed in increasing detail. Myocardial hy

as, and chemic heart disease, arrhythmifactor for cardiovascular events such as is

of heart failure. (Levy, predictor for the developmentsudden death and is a powerful

Garrison et al. 1990).

Figure 1.1. Artificial model of cardiacshows a normal structure of heart, The front heart wall is detached to reveal the chypertrophy. A: Ithambers
and valves inside. B: It blood pressure. The muscular wall ofshows the long-term effects of incr the left hear ventricle is considerably thickened and theeased heart activity due to high
rounded. art is visibly tip of the he

- 1 -

1.1.2. Cardiac hypertrophy: compensatory response versus maladaptation

itially beneficial since it augments the Development of cardiac hypertrophy is in

e units and reduces ventricular wall stress to normal levelsnumber of contractil

cepted that cardiac . It is generally acaccording to the law of Laplace (Basford 2002)

hypertrophy can be adaptive in some situations, s