Membranprotein-Komposition von Kardiofibroblasten in Normoxie und Hypoxie

Membrane protein composition of cardiac fibroblasts in normoxia and hypoxia

von Johannes Böttger
Dissertation
Datum der mündl. Prüfung:2019-10-24
Erschienen:2019-10-08
Betreuer:Dr. Marieke Claudia Wottawa
Gutachter:Prof. Dr. Dörthe Katschinski
Gutachter:Prof. Dr. Peter Schu
Zum Verlinken/Zitieren: http://dx.doi.org/10.53846/goediss-7661

 

 

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Englisch

Hypoxia is a key element in the pathophysiology of ischemic, inflammatory and malignant neoplastic diseases. It also plays an essential role in the intrauterine environment during embryonic development as well as in stem-cell niches. Oxygen tension within the blood or tissues are subject to rapid alterations. Therefore, cells must be able to adapt quickly to declines in oxygen supply to ensure cell survival and tissue function. The HIF (hypoxia-inducible factor) transcription factors are the master regulators of oxygen homeostasis in all mammalian cells. The myocardium is particularly dependent of a constant oxygen supply. Myocardial development, structure and function, as well as remodeling, are modulated by cardiac fibroblasts. The necessary interaction between a cell and its environment are mediated via the plasma membrane. Its composition as well as a multitude of biologic processes are controlled by cellular membrane trafficking. The aim of this study was to investigate the impact of short-term hypoxia on cellular membrane trafficking and the plasma membrane composition of cardiac fibroblasts. Total membrane turnover of primary cardiac fibroblasts was analyzed by confocal microscopy after application of the membrane marker FM® 1-43 (N-(3-triethylammoniumpropyl)-4-(4-(dibutylamino) styryl) pyridinium dibromide). Endocytotic membrane internalization was significantly increased after six hours of hypoxic incubation. In addition, intracellular vesicles increased in size. Similar results were observed in fibroblasts of different topographic origin. The effect was fully reversible 30 minutes after reoxygenation. We hypothesized these kinetic properties might be a hint towards a HIF-independent mechanism. Earlier investigations of specific membrane trafficking sub-categories and protein expression at the plasma membrane used heterogenous methodology and showed in parts conflicting evidence. So far, there has been no investigation of whole membrane turnover in primary cells. A SILAC-screen was performed in order to analyze the effects of this phenotype on the membrane protein composition of cardiac fibroblasts. Hypoxia led to an increased abundance of small, monomeric G proteins at the plasma membrane. Many of these proteins are involved in the regulation of membrane trafficking. These findings give insight into the adaptation of cellular processes in hypoxia. Further investigations of the precise kinetics and physiologic consequences of the hypoxia-induced alterations in membrane trafficking as well as their underlying molecular mechanisms might contribute to our understanding of hypoxia-associated cell function and pathology.
Keywords: hypoxia; membrane trafficking; silac
 
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