Cardiac T-Tubule Membranes - Nanostructure and Remodeling Mechanisms in Disease
von Eva Wagner
Datum der mündl. Prüfung:2012-12-10
Betreuer:Prof. Dr. Stephan E. Lehnart
Gutachter:PD Dr. Michael Hoppert
Gutachter:Prof. Dr. Stefan Jakobs
EnglischTransverse tubules (T-tubules, TTs) are continuous invaginations of the plasma membrane which form a complex network of excitable membranes inside mammalian ventricular cardiomyocytes. The TT network couples electrical with chemical signals in the relatively large cardiomyocytes and enables the synchronous rise of intracellular Ca2+ concentrations in response to membrane depolarization during systole. It is assumed that the functional coupling of voltage-dependent Ca2+ channels in the TT membranes and ryanodine receptor Ca2+ release channels in the junctional sarcoplasmic reticulum is essential to assure Ca2+ induced Ca2+ release. Previous studies reported changes of the TT structure in cardiomyocytes from diseased hearts. However, the methods used to visualize TTs were either diffraction limited (confocal microscopy) or restricted to fixed samples (electron microscopy). In this thesis, Stimulated Emission Depletion (STED) microscopy, a super-resolution technique, was used to quantitatively characterize TT membrane structures in living cardiomyocytes. Applying different image analysis strategies, the properties of individual TTs and the TT network in living healthy and diseased cardiomyocytes were quantified. Pathological TT changes were analyzed in two different mouse disease models, which were induced either by myocardial infarction (MI) or by transverse aortic constriction (TAC). Imaging and analysis of the TT nanostructure were complemented by functional measurements of intracellular Ca2+ transients and by the analysis of proteins which might play a role in TT remodeling. During heart failure (HF) development after MI, a progressive and heterogeneous enlargement of individual TT cross-sections was observed. In addition, length and complexity of the TT network progressively increased after MI. A significant increase of longitudinal TT elements was identified 4 weeks after MI representing an early time point of disease development. The observed TT remodeling was accompanied by differential expression changes of caveolin-3 and junctophilin-2. Furthermore, the differential spatial reorganization of TT elements correlated with a loss of intracellular Ca2+ release synchrony. Increased TT dimensions and proliferative TT network remodeling were also observed during HF development in the TAC model. These data suggest that TT remodeling after TAC may occur over a shorter period of time than TT remodeling after MI. This thesis introduces STED microscopy for imaging of intact TT membranes in living cardiomyocytes. Importantly, relatively early during HF development, individual TT elements and the cell-wide network properties are significantly altered through proliferative mechanisms. The data obtained here further suggest that TT remodeling during HF development might lead to excitation-contraction uncoupling, which can directly contribute to electrical and contractile dysfunction of diseased hearts.
Keywords: T-Tubule, heart failure, super-resolution imaging, STED microscopy