Functional role of NFAT in ventricular cardiomyocytes of rat [Elektronische Ressource] / by Attia Anwar

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• Publié par: justus-liebig-universitat_giessen
• Publié le: 01 janvier 2006
• Nombre de lectures: 12
• Langue: English
• Poids de l'ouvrage: 1 Mo

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FUNCTIONAL ROLE OF NFAT IN
VENTRICULAR CARDIOMYOCYTES
OF RAT
ATTIA ANWAR
INAUGURAL DISSERTATION
submitted to the
Faculty of Medicine
in partial fulfillment of the requirements
for the PhD-Degree
édition scientifique
VVB LAUFERSWEILER VERLAG of the Faculties of Veterinary Medicine and Medicine
of the Justus Liebig University GiessenVVB LAUFERSWEILER VERLAG ISBN 3-8359-5091-6
STAUFENBERGRING 15 D-35396 GIESSEN
Tel: [phone] Fax: -5599890
[e-mail]
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édition scientifique
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Functional Role of NFAT in Ventricular
Cardiomyocytes of Rat



INAUGURAL DISSERTATION
submitted to the Faculty of Medicine
in partial fulfillment of the requirements
for the PhD-Degree
of the Faculties of Veterinary Medicine and Medicine
of the Justus Liebig University Giessen



by


Attia Anwar
of
Gujrat-Pakistan


Giessen 2006


From the Institute of Physiology of the Justus Liebig University Giessen

Director / Chairman: Prof. Dr. Dr. H. M. Piper
of the Faculty of Medicine of the Justus Liebig University Giessen













First Supervisor and Committee Member: Priv.-Doz. Dr. G. Euler
Second Supervisor and Committee Member: Prof. Dr. Axel Gödecke
Committee Members: Prof. Dr. Heinz-Jürgen Thiel (Chairman) and
Prof. Dr. Martin Diener



thDate of Doctoral Defense: October 09 , 2006








Dedicated to

My Parents

Who have always worked very hard for making the best
available education accessible to me. I thank them for always
believing in me and always being there for me. This thesis is truly a
fruit of their lifelong affection and prayers.

&

My Sweet Husband

Whose constant encouragement and somewhat prolonged
banishment together provided me incentive and opportunity to
complete the compilation of this thesis.





. Table of contents

Abbrevations 1
1. Introduction 4
1.1. Myocardial hypertrophy and heart failure
1.2. α-Adrenoceptor stimulation induces hypertrophy 4
1.2.1. Calcium-calcineurin-NFAT-AP1- signaling 7
1.3. Effect of β-adrenoceptor agonists in hypertrophy 8
1.4. Contractile function of cardiomyocytes 9
1.5. Cell culture model 13
1.6. Aims of the study 14
2. Materials 16
2.1. Chemicals 16
2.1.1. Inhibitors 18
2.2. Decoy oligonucleotides
2.3. Antibodies
2.4. RT-PCR reagents 18
2.4.1. Real-time RT-PCR Kit 19
2.4.2. Primers 19
2.5. Equipments
2.5.1. General objects of utility 19
2.5.2. Special objects of utility 20
2.5.2.1.Cell culture 20
2.5.2.2. Other instruments 20
2.5.3. System for the measurement of cell contraction parameters
2.5.4. Consumables 21
2.5.5. Software for analysis 21
3. Methods 22
3.1. Isolation of ventricular cardiomyocytes 22
3.1.1. Laboratory Animals 22

3.1.2. Preparation of isolated ventricular cardiomyocytes from
rat hearts 22
3.1.3. Procedure for the preparation of cardiomyocytes 23
3.2. Culturing of cardiomyocytes 24
3.2.1. Preincubation of culture plates 24
3.2.2. Plating of cardiomyocytes 24
3.2.3. Culturing of cardiomyocytes 25
3.2.4. Treatment of cardiomyocytes 25
3.3. Transformation of cardiomyocytes 25
3.3.1 Hybridization of decoy oligonuleotides 25
3.3.2. Transformation of cardiomyocytes with decoy-
oligonucleotides 26
3.4. Determination of hypertrophic growth 26
3.4.1. Determination of the rate of protein synthesis 26
3.4.2. Quantification of protein by Bradford Method 28
3.4.3. Determination of cell size 28
3.5. Determination of cell shortening 29
3.5.1. Sample preparation 29
3.5.2. Electrical stimulation of cardiomyocytes
3.5.3. Determination of parameters of cell contraction 30
3.5.4. Measurement of cell contraction 31
3.6. Western blot 32
3.6.1. Lysis of cells
3.6.2. SDS Polyacrylamide gel (7.5%) 32
3.6.2.1. Preparation of gel 34
3.6.3. Blotting of proteins 34
3.6.3.1. Preparation of blotting chamber and transfer of protein
to membrane 35
3.6.4. Incubation of membrane with antibodies
3.6.5. Antibody dilutions 36
3.6.6. Staining solution 37 3.7. RNA isolation 37
3.7.1. Harvesting of cells 37
3.7.2. RNA isolation 38
3.7.3. DNAse treatment of RNA 38
3.8. Reverse transcription-polymerase chain reaction (RT-PCR) 39
3.8.1. cDNA synthesis 39
3.8.2. Polymerase Chain Reaction (PCR) 40
3.8.3. Agarose gel electrophoresis 42
3.9. Real-time RT-PCR 43
3.10. Retardation assay 46
3.10.1. Fluorescence labeling of oligonucleotides 47
3.10.2. Nuclear extraction 47
3.10.3. Binding reaction of oligonucleotide with specific proteins 49
3.10.4. Retardation assay gel electrophoresis 49
4. Results 51
4.1. Analysis of NFAT involvement in hypertrophic growth of
ventricular cardiomyocytes of rat under stimulation with
α-adrenoceptor agonist phenylephrine (PE) 51
4.1.1. NFAT is activated under phenylephrine (PE) 51
4.1.2. NFAT is inhibited by decoy oligonucleotides 52
4.1.3. NFAT is not involved in hypertrophic growth induced by PE 54
4.2. Effect of PE on cell contraction 58
4.2.1. Cell shortening is reduced at low beating frequency (0.5 Hz) 58
4.2.2. Relaxation velocity under PE is enhanced at high beating
frequency at 2.0 Hz 60
4.2.3. PE induces SERCA2A expression 61
4.2.4. Signaling pathway of SERCA2A up-regulation 63
4.2.4.1. PKC is not involved in SERCA2A up-regulation 63
2+ 4.2.4.2. Ca / Calcineurin pathway is involved in SERCA2A
up-regulation 67
4.2.4.3. NFAT is involved in SERCA2A up-regulation 69