Medizinische Universität Graz Austria/Österreich - Forschungsportal - Medical University of Graz
Gewählte Publikation:
Unterer, G.
Zytosolische Na+ Homöostase in KArdiomyozyten der Maus mit chronischer beta1-adrenerger Stimulation
Humanmedizin; [ Diplomarbeit/Master Thesis ] Graz Medical University; 2010. pp.68.
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- Autor*innen der Med Uni Graz:
- Betreuer*innen:
- Heinzel Frank
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- Abstract:
- Introduction: Mice with a cardiospecific overexpression of the beta1-receptor (beta1-TG mice) develop heart failure through chronic adrenergic stimulation. An increased cytosolic Ca2+-load of the cardiomyocytes was detected and was shown to be causally involved in the progression of the disease. However the causes of impaired Ca2+-handling are unsolved. Intracellular Na+-homeostasis plays an important role in the cardiac Ca2+-regulation through the sarcolemmal Na+-Ca2+ exchanger (NCX) and therefore may contribute to impaired Ca2+-handling. Thus we analyzed if a disturbed Na+-homeostasis of the beta1-TG mouse in the form of elevated intracellular Na+-levels ([Na+]i) is present in early stage heart failure. Methods: An epifluorescent microscopic setup for ratiometric measurements was modified for the fluorescent dye SBFI-AM (Na+-indicator). [Na+]i was controlled by exposing the cardiomyocytes to various solutions with defined [Na+] in the presence of the Na+-K+ ionophore Gramicidin. The fluorescence signal of single SBFI-loaded mouse cardiomyocytes was detected at excitation wavelengths of 340 and 380 nm and the 340/380 nm fluorescence ratio was calibrated to the known [Na+]i. With this method it was possible to isolate ventricular cardiomyocytes of young (age: 8-9 weeks) beta1-TG mice and wildtype (WT) mice of the same age and to ratiometrically determine their [Na+]i. Results: The ratiometric epifluorescent microscopic setup was successfully modified. SBFI was calibrated in vivo in the mouse cardiomyocytes. These measurements showed stable fluorescence values but also an unexpected dye-unrelated decay of the fluorescence signal right after the permeabilisation of the cell, consistent with an oxidation of NADH. Based on the collected data we developed an algorithm to compensate this decay. We discovered a trend toward elevated [Na+]i in cardiomyocytes of beta1-TG mice at rest as well as at 1 Hz and 3 Hz as compared to cardiomyocytes of the wildtype. Conclusion: For ratiometric measurements of [Na+]i using SBFI in mouse cardiomyocytes at excitation wavelengths in the UV-range the dye-unrelated decay of the fluorescence signal after cell permeabilisation must be considered. There is a trend toward cytosolic [Na+] elevation in young beta1-TG mice. This elevated [Na+]i in the early stage of myocardiac remodeling might contribute to impaired Ca2+-handling and therefore to the manifestation and progression of chronic heart failure.