Differential role of estrogen receptor isoforms in the cardiovascular system of young and senescent rats [Elektronische Ressource] / vorgelegt von Virginija Jazbutyte

Differential role of estrogen receptor isoforms in the
cardiovascular system of young and senescent rats








Dissertation zur Erlangung des
naturwissenschaftlichen Doktorgrades
der Bayerischen Julius- Maximilians- Universität Würzburg




vorgelegt von
Virginija Jazbutyte, MSc
aus Skuodas, Litauen



Würzburg, 2007



Eingereicht am:


Mitglieder der Promotionskommission:


Vorsitzender: Prof. Dr. M. Müller

Gutachter: Prof. Dr. rer. nat. K. Scheller
Gutachter: Priv. Doz. Dr. med. O. Ritter



Betreuer: Dr. med. Theo Pelzer



Tag des Promotionskolloquiums:

Doktorurkunde ausgehändigt am:









The study was performed under supervision of Dr. med. Theo Pelzer in the laboratory
of Molecular Cardiology, Medical Clinic and Policlinic I (Director Prof. Dr. G. Ertl) at
Julius- Maximilians University in Würzburg, Germany. My PhD studies were
supported by the German Academic Exchange Service (“DAAD”,
Promotionstipendium) and by the University Clinics and Policlinics in Würzburg
(Ausbildungsstipendium).








Declaration

Hereby, I declare that the submitted dissertation was completed by myself and no
others and that I have not used any sources or materials others than those enclosed.
Moreover, I declare that the dissertation has not been submitted further in this form or
any other form and has not been used for obtaining any other equivalent qualification
in any other organization. Additionally, I have not applied or will attempt to apply for
any other degree or qualification in relation to this work.






Würzburg, 11.05.2007



(Virginija Jazbutyte, MSc)







To my family






































Content

Content

Summary……………………………………………………………………………...... 4
Zusammenfassung……………………………………………………….................. 6
1. Introduction......................................................................................8
1.1. Estrogen receptor structure and biology.................... 9
1.2. Estrogen receptor function and regulation..................................................... 11
1.3. Estrogen receptor protein- protein interactions.............................. 14
1.4. Estrogen receptors in the heart.................................................... 16
1.5. Natural and synthetic ER ligands................................................. 17
1.6. The role of aging in estrogen substitution efficacy and estrogen
20
metabolism............................................................................................................
1.7. Cardiac hypertrophy....................................................................................... 22
1.8. Spontaneously hypertensive rats (SHR) as a model of cardiac
hyperhypertrophy.................................................................................................. 22
1.9. Myosin heavy chain........................................................................................ 23
2. Aims of the study............................................................................................ 25
3. Materials and Methods................................26
3.1. Study design.................................................................................................. 26
3.2. Animal model and treatment...................................... 26
3.3. Morphometric analysis................................................................. 27
3.4. Hemodynamic analysis.............................................. 27
3.5. Determination of serum hormone levels........................................................ 28
3.6. Molecular analysis.......................................................................................... 28
3.6.1. Myosin heavy chain electrophoresis......................................... 28
3.6.2. Generation of rabbit polyclonal antibodies against estrogen
receptor beta......................................................................................................... 29
3.6.3. Protein expression analysis by Western blotting....................... 30
3.6.4. Immunofluorescent staining........................................................................ 31
3.7. Construction of a bait vector containing rat estrogen receptor
32 alpha ligand binding domain (rER α_LBD).............................................................
3.7.1. Preparation of total rat heart RNA............................................................... 32
1Content
3.7.2. Amplification of rat ER αLBD........................................................................ 32
3.7.3. Ligation and transformation of One- shot competent bacterial cells........... 33
3.7.4. Construction of bait vector containing ER α_LBD insert.............................. 34
3.8. ER α ligand binding domain protein- protein interaction partners
36 in the heart............................................................................................................
3.8.1. Neonatal human heart cDNA library screening for ER α
36 protein- protein interaction partners......................................................................
3.8.2. Cloning of ER αLBD protein- protein interaction partners
candidate genes.................................................................................................... 38
3.8.3. Generation of recombinant human 17 β- hydroxysteroid
39 dehydrogenase type 10 protein.............................................................................
3.8.4. Generation of recombinant vectors for mammalian expression
40 of rat ER α_LBD.....................................................................................................
3.8.5. Mitochondria preparation for 17 βHSD10 enzymatic assay and
41 Western blot analysis............................................................................................
3.8.6. 17 βHSD10 enzymatic assay....................................................... 41
3.8.7. Co- immunoprecipitation of ER αLBD and His_17 βHSD10......................... 42
3.9. Statistics......................................................................................................... 43
4. Results........................................................................................... 44
4.1. Hormone influence on morphological parameters of SHR rats...................... 44
4.2. Hemodynamic measurements....................................................................... 48
4.3. Estrogen, its precursors and metabolites in the plasma.
Quantitative analysis............................................................................................. 51
4.4. Myosin heavy chain (MHC) expression in the SHR rat hearts....................... 55
4.5. Cardiac estrogen receptor alpha and beta protein expression
57 in young and senescent SHR................................................................................
4.6. Protein expression levels of SERCA 2, phospholamban
60 and phosphorylated phospholamban....................................................................
4.7. Neonatal Heart cDNA library screening for protein- protein interaction
63 partners of rat ER α ligand binding domain (LBD) (Bacterial Two Hybrid system)
4.8. Localization of 17 β- hydroxysteroid dehydrogenase 10 in the heart.............. 66
4.9. ER αLBD and 17 β- HSD protein- protein interaction
70
(GST pull- down assay).........................................................................................
2Content
4.10. 17 β- hydroxysteroid dehydrogenase 10 enzymatic assay........................... 71
4.11. 17 β- HSD10 activity studies in estrogen receptor deficient mouse heart..... 76
4.12. Identification and characterization of BLCAP 10 in the heart....................... 78
5. Discussion....................................................................................................... 82
5.1. Influence of hormone replacement on morphological parameters................. 83
5.2. Influence of estrogens on cardiac function..................................................... 85
5.3. Sex hormone profiles................................................................... 86
5.4. Cardiac myosin heavy chain expression...................................... 88
5.5. Protein expression analysis........................................................................... 89
5.6. Protein- protein interaction partners of ERα in the heart................................ 91
6. Outlook............................................................................................................. 95
7. Acknowledgements......................................