Dokument: Arretierung embryonaler Rattenkardiomyozyten induziert den Titin-Turnover unter Beteilung intrazellulärer Systeme der Protein-Qualitätskontrolle
| Titel: | Arretierung embryonaler Rattenkardiomyozyten induziert den Titin-Turnover unter Beteilung intrazellulärer Systeme der Protein-Qualitätskontrolle | |||||||
| Weiterer Titel: | Arrest of embryonic rat cardiomyocytes induces titin turnover involving intracellular protein quality control systems | |||||||
| URL für Lesezeichen: | https://docserv.uni-duesseldorf.de/servlets/DocumentServlet?id=69854 | |||||||
| URN (NBN): | urn:nbn:de:hbz:061-20250616-082016-7 | |||||||
| Kollektion: | Dissertationen | |||||||
| Sprache: | Deutsch | |||||||
| Dokumententyp: | Wissenschaftliche Abschlussarbeiten » Dissertation | |||||||
| Medientyp: | Text | |||||||
| Autor: | Weber, Sina [Autor] | |||||||
| Dateien: |
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| Beitragende: | Prof. Dr. Martina Krüger [Gutachter] PD Dr. Doreen Floß [Gutachter] Kötter, Sebastian [Beitragender] | |||||||
| Stichwörter: | Titin, Proteinqualitätskontrolle, Proteasom, Ubiquitin, Autophagie, Myokard, Sarkomer | |||||||
| Dewey Dezimal-Klassifikation: | 600 Technik, Medizin, angewandte Wissenschaften » 610 Medizin und Gesundheit | |||||||
| Beschreibungen: | Das Muskelprotein Titin ist mit einer Größe von 3-3,7 MDa das größte bekannte Protein. Lokalisiert ist es innerhalb der kleinsten kontraktilen Einheit der quergestreiften Herz- und Skelettmuskulatur, dem Sarkomer. Die zwei im Herzen vorkommenden Isoformen sind die längere und elastischere Isoform N2BA und die kürzere und steifere Isoform N2B. Während der embryonalen Entwicklung wird zunächst eine embryonale N2BA-Isoform exprimiert, welche postnatal durch adulte N2BA- und N2B-Isoformen ersetzt wird. Der Abbau von Titin stellt aufgrund seiner festen Verankerung im Sarkomer und der Größe des Proteins eine komplexe Aufgabe für die Zelle dar. Die an diesem Prozess beteiligten molekularen Mechanismen sind noch nicht vollständig verstanden und waren daher Gegenstand dieser Arbeit. In vorherigen Arbeiten konnte gezeigt werden, dass den intrazellulären Abbausystemen, das Ubiquitin-Proteasom-System (UPS) und die Autophagie, hierbei eine entscheidende Rolle zukommt, weshalb wir deren Beteiligung am Abbau Titins untersuchten. Indem wir spontan kontrahierende embryonale Rattenkardiomyozyten durch Kultivierung Ansätzen mit KCL,- und Blebbistatin-versetztem Nährmedien mechanisch arretierten, sollte ein durch Nicht-Nutzung beschleunigter Abbau des kontraktilen Apparats erreicht werden. So konnten wir zeigen, dass das Titin-Degradationsprodukt in arretierten Zellen vermehrt anfällt und das Titin bevorzugt gegenüber anderen Sarkomerproteinen abgebaut wird. Wir fertigten biochemische Analysen verschiedener Autophagie- und Proteasomenmarker-, sowie Marker beteiligter Signaltransduktionswege an und analysierten den Ubiquitinierungsstatus Titins über eine Kultivierungsdauer von 15 Tagen. Die Arretierung der Zellen ging mit vermehrter Ubiquitinierung Titins und mit einem beschleunigten Isoformen-switch einher. Es konnte eine Verringerung der Aktivität proteasomaler Enzyme gemessen werden, während es zu einem Anstieg eines proteasomalen Markerproteins kam. Da wir einen sequenziellen Abbau der verschiedenen Titindomänen vermuteten, analysierten wir die Strukturveränderung der Zellen, indem wir Immunfluoreszenzfärbungen anfertigten. Hier zeigte sich kein klares Bild einer sequenziellen Degradation. Zusammenfassend konnten wir nachweisen, dass durch die Arretierung der kontraktilen Zellen der Titinturnover induziert werden kann und es zu einem beschleunigten Isoformen-switch kommt. Hierdurch erlangen die Zellen vorzeitig ihre adulte Isoformenkomposition. Es kommt weiter zu einem Anstieg ubiquitinierten Titins, was auf eine Beteiligung der intrazellulären Abbausystemen hinweist.With its size of 3-3,7 MDa, the muscle protein Titin is the largest known protein in humans. It is localised within the smallest contractile unit of the striated heart- and skeletal muscle, the sarcomere in heart muscle tissue. Titin is expressed as two major isoforms- the longer and more compliant isoform N2BA and the shorter and stiffer isoform N2B. During embryonic development a embryonic N2BA isoform is expressed, which is replaced in the perinatal period by adult N2BA- and N2B-isoforms. Due to its massive size and its tight incorporation within the sarcomere Titin degradation is a complex task for the cell and the molecular mechanisms involved in titin turnover are not fully understood. In this work we aimed to investigate the Titin degradation especially regarding the impact of systems of protein quality control. Previous research could already show that the two important systems of protein quality control, the Ubiquitin-Proteasom-System (UPS) and the Autophagy, play central roles in this process. By cultivation of spontaneously contracting embryonic rat cardiomyocytes in KCL- and Blebbistatin-containing media, we achieved the mechanical arrest of the cells. Cardiomyocyte arrest accelerated the degradation of the contractile machinery of the muscle cells, due to the resulting non-usage of the contractile machinery. That way we could show a higher production of the Titin degradation product T2 in arrested cells and a preferred degradation of Titin over other sarcomere proteins. We also investigated different marker proteins for the protein quality control systems autophagy and UPS, along with proteins important for signal transduction. Furthermore, we analysed the ubiquitination levels of Titin, using Western blot analysis. We performed our analysis over a cultivation duration of 15 days and found higher ubiquitination levels of Titin and an accelerated embryonic isoform-switch. Furthermore, we aimed to measure the activity of the proteasome to find out more about the role of the UPS within the degradation process of Titin. For the observed period, we could observe decreased proteasomal enzyme activity in arrested cells, while we could show elevated levels of a proteasomal marker protein. Assuming a sequential degradation of the different Titin domains, we analysed structural alterations in arrested cells, using immunofluorescent staining and microscopy. Here we could not show such a sequential degradation. Summarizing our results, we could show that mechanical arrest, either caused by treating the cells with KCL or Blebbistatin, induces Titin turnover and the Titin isoform-switch. An increase of ubiquitinated titin indicates the involvement of autophagy and the UPS. | |||||||
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| Lizenz: |  Dieses Werk ist lizenziert unter einer Creative Commons Namensnennung 4.0 International Lizenz | |||||||
| Bezug: | 04/2021-06/2025 | |||||||
| Fachbereich / Einrichtung: | Medizinische Fakultät | |||||||
| Dokument erstellt am: | 16.06.2025 | |||||||
| Dateien geändert am: | 16.06.2025 | |||||||
| Promotionsantrag am: | 11.02.2025 | |||||||
| Datum der Promotion: | 10.06.2025 |
