Untersuchungen zur Funktion der humanen atrialen essentiellen leichten Myosinkette (ALC-1) in einem transgenen Rattenmodell

Untersuchungen zur Funktion der humanen atrialen essentiellen leichten Myosinkette (ALC-1) in einem transgenen RattenmodellAhmed Ihab Abdelaziz7273741101.1111.212131.2.114151.2.21617181.2.2.11920211.2.32223241.2.3.1251.2.3.22627281.2.3.3291.2.3.43031321.2.3.51.2.3.6331.3342352.1362.23738393403.13.2413.3423.43.5433.6443.7453.8463.93.9.1473.9.23.9.33.10483.114494.1504.25152534.354554.45657584.559604.6626364565666768697071ABBREVIATIONS7 REFERENCES757677787980818283 APPENDIX84Publications Curriculum Vitae858687 Acknowledgments 88 Erklärung89enTable of contentsHelpAus dem Institut für Physiologie der Medizinischen Fakultät der Charité-
Universitätsmedizin BerlinDISSERTATIONUntersuchungen zur Funktion der humanen atrialen essentiellen leichten Myosinkette (ALC-1) in einem transgenen RattenmodellZur Erlangung des akademischen Grades
Doctor medicinae (Dr.med.)vorgelegt der Medizinischen Fakultät der Charité –
Universitätsmedizin BerlinVon
Ahmed Ihab Abdelaziz aus Kairo, Ägypten
Prof. Dr. Joachim W. Dudenhausen
Prof. Dr. med Martin Paul Prof. Ingo Morano, Max-Delbrueck-Center for Molecular Medicine & Charite University Medicine, Berlin, Germany Prof. Guy Vassort, INSERM Montpellier France Prof. Anders Arner MD, PhD, Lund University Sweden Datum der Promotion: 20.09.2004

The blood in the right ventricle of the
heart must reach the left ventricle by way
of the lungs and not through a passage
connecting the ventricles.
[Ibn al-Nafis (d.1288/678). commentary on
the Canon of Medicine by Ibn Sina
(Avicenna)]

Dedicated to

My Father

for his uncompromising principles that guided my life

My Mother

for leading her children into intellectual pursuits

My WIFE

for her magnificent and infinite give and support

My Children

For making everything worthwhile

SUMMARY

Most patients with hypertrophic cardiomyopathy and congenital heart diseases express the atrial essential myosin light chains (ALC-1) in their ventricles, partially replacing the ventricular essential light chains (VLC-1). This VLC-1/ALC-1 isoform shift is correlated with an increase in cross-bridge cycling kinetics as measured using chemically skinned fibers from the hypertrophied ventricles of human hearts. To study the functional importance of hALC-1 in the whole intact perfused-heart, a transgenic rat model overexpressing hALC-1 (TGR/hALC-1) in the heart was generated. WKY rats were used as the respective genetically correct control strain.

Using hALC-1_HIST protein as a standard, the amount of transgenic protein expression was quantified by Immunoblot analysis of the left ventricular tissue extracts of the transgenic rats. Twelve-week-old TGR/hALC-1 expressed around 17μg hALC-1 per mg of whole SDS-soluble protein.

The transgene was localized in-between the Z-lines of the sarcomere by immunofluoresecnce microscopy. Furthermore, the proper integration of the transgene into the rat ventricular myosin was confirmed by purifying myosin from rat ventricular tissues.

Whole ventricular proteome analysis by 2D-PAGE, resolved approximately 3000 proteins spots in each TGR/hALC-1 and WKY animal. The whole protein expression patterns in both animal groups showed no differences with the exception of the transgenic hALC-1 protein spot.

The perfused heart contractility parameters were evaluated using the Langendorff preparation. Expression of hALC-1 was accompanied by statistically significant improvements (p<0.001) in all measured contractile parameters of the hearts of the TGR/hALC-1 compared to the age matched control (WKY) animals, represented by increases in the developed left ventricular pressure, maximal rate of pressure increase and the relaxation rate. Age dependent decrease in the transgene expression caused a decline of the improved contractility parameters to normal values.

These results demonstrate that the expression of the hALC-1 in the heart not only improved the function of in-vitro preparations, but also of the whole intact organ.

ZUSAMMENFASSUNG

Die meisten Patienten mit hypertropher Kardiomyopathie und kongenitalen Herzerkrankungen exprimieren die atriale essentielle leichte Myosinkette (ALC-1) im Ventrikel, wo sie teilweise die ventrikuläre essentielle leichte Myosinkette (VLC-1) ersetzt. Diese VLC-1/ALC-1 Isoformveränderung korrelierte mit einem Anstieg der Zykluskinetik der Myosin-Querbrücken in chemisch gehäuteten Herzfasern aus hypertrophierten Humanventrikeln. Um die funktionelle Bedeutung der ALC-1 im gesamten intakten Herzen zu untersuchen, habe ich in der vorliegenden Arbeit ein transgenes Rattenmodell charakterisiert, das die humane ALC-1 (hALC-1) im Herzen exprimiert (TGR/hALC-1). WKY-Ratten dienten als genetisch korrekter Kontrollstamm.

Mittels rekombinanter hALC-1 als Standard wurde die exprimierte hALC-1-Menge in SDS-Extrakten linker Ventrikel der TGR/hALC-1 im Western-Blot untersucht. 12 Wochen alte TGR/hALC-1 exprimierten etwa 17 µg hALC-1/mg SDS-Extrakt. Das exprimierte Transgen konnte in der Immunfluoreszenz zwischen den Z-Linien der Sarkomere lokalisiert werden. Die gerichtete Integrations des Transgens in das kardiale Myosinmolekül wurde zusätzlich noch in hochgereinigten Myosinpräparationen nachgewiesen.

Analyse des linksventrikulären Proteoms durch 2D-PAGE, das zur Identifikation von etwa 3000 Proteinen führte, zeigte vergleichbare Proteinmuster in WKY und TGR/hALC-1.

Die Untersuchungen der Kontraktilität des intakten isoliert perfundierten Herzen wurden mit Langendorff-Präparationen durchgeführt. Die Expression des hALC-1-Transgen führte zu statistisch signifikanten (p<0.001) Steigerungen aller gemessenen Kontraktilitätsparameter (linksventrikuläre Druckentwicklung, maximale Geschwindigkeit der Kraftentwicklung, maximale Erschlaffungsgeschwindigkeit). Die altersbedingte Abnahme der Transgenexpression führte zu einer Abnahme der verbesserten Kontraktilitätsparameter auf das Kontrollniveau der WKY-Tiere.

Diese Ergebnisse belegen, dass die Expression der hALC-1 im Herzen nicht nur die Funktion von in vitro-Präparaten, sondern auch die des gesamten intakten Organs zu steigern vermag.

Table of contents

1 INTRODUCTION
- 1.1 Cardiac Excitation-Contraction-Coupling
- 1.2 Cardiac myofilaments
  - 1.2.1 Sarcomeric proteins associated with cardiac myofilaments
  - 1.2.2 Thin Filament
    - 1.2.2.1 Thin filament regulation in myocardial contraction
  - 1.2.3 Myosin filament
    - 1.2.3.1 Myosin cross-bridge
    - 1.2.3.2 Cross-bridge cycle kinetics
    - 1.2.3.3 Myosin heavy chains (MHC)
    - 1.2.3.4 Myosin light chains (MLC)
    - 1.2.3.5 Regulation of ALC-1 expression in the heart
    - 1.2.3.6 Functions of ALC-1 in the heart
- 1.3 Aim of the study
2 MATERIALS
- 2.1 Equipments
- 2.2 Chemicals
3 METHODS
- 3.1 Transgene construction and generation of transgenic rats
- 3.2 Genotyping of the genomic DNA from the transgenic-rat tails.
- 3.3 Generation of the hALC-1 specific antibody
- 3.4 Recombinant human ALC-1
- 3.5 Myosin purification.
- 3.6 Composition analysis of purified myosin and human atrial tissue
- 3.7 Quantification of transgene expression.
- 3.8 Immunofluorescence microscopy.
- 3.9 Analysis of the ventricular proteome by 2D-PAGE.
  - 3.9.1 Preparation of the protein samples:
  - 3.9.2 2D electrophoresis:
  - 3.9.3 Silver staining, spot detection and mass spectrometry
- 3.10 Isolated perfused hearts (Langendorff method)
- 3.11 Statistical analysis
4 RESULTS
- 4.1 Generation and basic characterization of the transgenic rats
- 4.2 Quantification of the expressed hALC-1
- 4.3 MLC analysis
- 4.4 Immunofluorescence Microscopy
- 4.5 Comparative proteome analysis
- 4.6 Functional properties of the transgene
5 DISKUSSION
ABBREVIATIONS
REFERENCES
APPENDIX
- Publications
- Curriculum Vitae
- Acknowledgments
- Erklärung

Tables

Table 1 . Recipes for separating and stacking polyacrylamide gels, based on the buffer system of Laemmli.

Images

Figure 1 . Scheme of cardiac excitation-contraction-coupling events in a ventricular myocyte.
Figure 2 . Ultra-structure of the working myocardial cells.
Figure 3 . Major components of the cardiac sarcomere:
Figure 4 . Thin filaments:
Figure 5 . Regulation of thin filament in contraction
Figure 6 . Schematic representation for myosin molecule structure:
Figure 7 . Three-dimensional ribbon structure of Myosin S1
Figure 8 . Cross-Bridge cycle kinetics.
Figure 9 . Schematic representation of the construct used to generate the transgenic rats.b
Figure 10 . Genotyping of transgenic rats.
Figure 11 . Heart weight/body weight relationship (HW/BW).
Figure 12 . hALC-1 HIST Standard Curve .
Figure 13 . Quantification of the expressed hALC-1.
Figure 14 . MLC composition analysis.
Figure 15 . Immunofluorescence localization of the transgene-expression.
Figure 16 . Expression-patterns of the transgene.
Figure 17 . Analysis of the ventricular proteome by 2D-PAGE.
Figure 18 . Developed left ventricular pressure (LVP):
Figure 19 . Maximum rate of pressure increase (+dP/dt max) (contraction rate):
Figure 20 . Maximum rate of pressure decrease (-dP/dt max) (relaxation rate):
Figure 21 . Amino acid sequence comparison of the human, mouse and rat atrial essential myosin light chains (ALC-1):