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Abstract

Heart muscle cells (cardiomyocytes) have to fulfill a demanding task. They have to ensure continuous and regular heartbeat and maintain their integrity despite undergoing constant mechanical stress during muscle contraction. To accomplish this, they feature a characteristic membrane architecture: the Transverse-Axial Tubular System (TATS), a network of membrane invaginations – the Transverse Tubules (TT) – which allows the fast translation of an electrical stimulus into a mechanical response. Further, specialized membrane protein complexes ensure membrane flexibility and stability during cycles of contraction and relaxation. Structural alterations of the TATS and the membrane protein complexes are linked to cardiac pathologies.

This thesis presents the subdiffraction image based investigations of the cardiac membrane architecture and membrane nanodomains using STimulated Emission Depletion (STED) microscopy of mouse ventricular cardiomyocytes (VM). Experimental and analytical methods comprising single- and multicolor, one- and two-photon-excitation STED microscopy are developed and applied. Special focus is laid on the three-dimensional (3D) TATS topology, on the membrane lipids Cholesterol (Chol) and Ganglioside GM1 (GM1), and on the membrane associated proteins Caveolin-3 (Cav-3) and Dystrophin (Dyst).

Novel fluorescent Chol analogs are characterized and established as a class of membrane labels with superior properties for STED microscopy of living VM. These dye compounds allow the visualization of the TATS with an unprecedented lateral resolution of below 35 nm and can be used for both membrane bulk staining and labeling of nanoscopic membrane compartments.

Using a custom-built two-photon-excitation-STED (2P-Exc-STED) microscope, the new Chol dyes enable the acquisition of 3D subdiffraction images of the TATS of living VM. These 3D images reveal that TT bud from Chol rich membrane domains and that these Chol rich domains can also form shallow membrane invaginations which are hypothesized to be caveolae.

The signal patterns of the caveolae-associated protein Cav-3 and of Chol are comparatively investigated and their similarities quantitatively evaluated. The dramatic effect of membrane Chol depletion on the nanoscopic Cav-3 signal distribution is assessed. The correlation between the Cav-3 and Chol membrane patterns is further supported by two-color STED microscopy of VM labeled for Cav-3 and GM1, and Chol and GM1.

Finally, the spatial association between Cav-3 and the cytoskeletal protein Dyst is studied in detail. For this, two- and three-color STED imaging protocols and image analysis procedures are developed. To determine the molecular orientation of the Dyst protein with respect to Cav-3 and with respect to the cardiac membrane, a multicolor “intra-protein” labeling protocol is developed that is based on immunofluorescence staining using different primary antibodies that target specific epitopes along the Dyst protein.

A cardiac membrane nanodomain model summarizing the presented observations and findings is derived, validated, and discussed in detail.