Subzelluläre Mechanismen der Dysferlin-vermittelten Tubulusmembranbiogenese bei kardialer Hypertrophie
Subcellular mechanisms of Dysferlin-mediated tubular membrane biogenesis in cardiac hypertrophy
by Carolin Fleischhacker
Date of Examination:2025-08-21
Date of issue:2025-07-31
Advisor:Prof. Dr. Stephan E. Lehnart
Referee:Prof. Dr. Stephan E. Lehnart
Referee:Prof. Dr. Niels Voigt
Sponsor:Deutsche Gesellschaft für Kardiologie (DGK)
Sponsor:Deutsches Zentrum für Herz-Kreislauf-Forschung (DZHK)
Sponsor:Studienstiftung des Deutschen Volkes
Persistent Address:
http://dx.doi.org/10.53846/goediss-11414
Files in this item
Name:Dissertation FINAL eDiss C. Fleischhacker.pdf
Size:2.98Mb
Format:PDF
These files are not accessible until the state exam has been completed.
Abstract
English
Dysferlin is a 238 kDa calcium-binding transmembrane protein out of the ferlin family which is expressed in striated muscle cells. Dysferlin-gene mutations are associated with skeletal muscle disorders known as Dysferlinopathies. In skeletal muscle cells, Dysferlin is involved in calcium-dependent membrane repair. Further, its involvement in tubule membrane biogenesis of the transverse axial tubule (TAT) network, whose reorganization is associated with limitations in myocyte function, is also being discussed. Dysferlinopathie-patients also develop a mild form of dilated cardiomyopathy. In the heart, Dysferlin is most strongly expressed in the ventricle, and its expression increases significantly in the context of pressure overload induced hypertrophic remodeling processes. However, the mechanisms of Dysferlin-mediated maladaptive tubule reorganization in pressure overload induced hypertrophy and the associated development of heart failure are not yet fully understood. The aim of this dissertation is to investigate in more detail Dysferlin-mediated subcellular mechanisms of cell membrane repair and tubule membrane biogenesis in cardiac hypertrophy. For experiments, a Dysferlin knockout mouse line was used. A general characterization revealed no impairment of the mice in terms of overall survival, but a gender-specific analysis showed increased mortality in knockout male mice. However, histological examinations failed to reveal any changes in cell morphology or fibrosis as a cause of increased mortality. Transverse aortic constriction (TAC) surgery was performed to induce cardiac pressure overload, and myocardial infarction (MI) surgery was performed to induce an anterior wall infarction. However, post-TAC, the Dysferlin-deficient males did not show increased mortality, which can be explained by a protective aspect of reduced maladaptive remodeling due to Dysferlin deficiency. Both, echocardiographic measurements and analyses of single ventricular cardiomyocytes (VM) showed a reduction in pressure overload induced cardiac hypertrophy in Dysferlin deficiency. In wildtype mice, Dysferlin expression is already increased up to 143.2 ± 9.857% one week post-TAC. Using stimulated emission depletion nanoscopy, more accurate localization analyses of Dysferlin and possible interacting proteins could be performed. Here, it was shown that Dysferlin is localized along the transverse axial tubule (TAT) network and is particularly close to proteins of the calcium release units (CRU). Dysferlin may stabilize the CRU, thereby preventing arrhythmogenic potentials that contribute to the development of heart failure. Post-TAC, Dysferlin is increasingly localized along newly formed axial tubules in hypertrophied wildtype VM. Our results show for the first time that Dysferlin is involved in axial tubule biogenesis, thus suggesting that Dysferlin plays an important role in hypertrophic remodeling undergoing pressure overload. To ensure rapid cell membrane repair, it seems reasonable to assume that Dysferlin is stored intracellularly, from where, for example, calcium-triggered Dysferlin can be recruited for membrane repair. To investigate this hypothesis, this dissertation quantified sarcolemmal-expressed proteins, which we were able to establish for the first time in living VM. Using a membrane-impermeable crosslinker, proteins with extracellular lysines were linked to complexes with a higher molecular weight and thus quantitatively determined. Our experiments showed Dysferlin expression on the cell membrane of 22.14 ± 0.6156% under baseline conditions. Provoked by hypertrophic remodeling, Dysferlin expression on the cell membrane increased significantly, suggesting storage of Dysferlin within the membrane of intracellular vesicles. Dysferlin is recruited to the cell membrane surface, where it may work together with intracellular membrane reservoirs to repair and remodel the cellular membrane. In the context of myocardial infarction events, initial studies have detected an increase in Dysferlin expression, particularly along the infarct scar, but also globally in the left ventricular myocardium. Analogous to the role of Dysferlin in pressure overload induced remodeling, we suspect that Dysferlin is also involved in the stabilization and reorganization of surviving, postmitotic VM. Dysferlin may protect VM from mechanical overload and the resulting cell death. STED studies also showed a colocalization of Dysferlin and CRU proteins in ischemic events. More detailed studies of Dysferlin in myocardial infarction events need to be focussed in future experiments. This dissertation demonstrated the relevant role of Dysferlin in sarcolemmal reorganization and the repair mechanisms of VM, particularly in the context of pressure-induced cardiac hypertrophy. Cumulative membrane damage and maladaptive remodeling influence the cell membrane structure and action potential conduction, leading to the development of heart failure. A more detailed understanding of these processes can help prevent the development of heart failure before cardiac decompensation, which is associated with enormous limitations in the quality of life of patients, occurs. Therapeutic goals could be (1) to stabilize the connections between the tubule and sarcoplasmic reticulum for adequate electromechanical stimulus conduction, (2) promoting the regeneration of the TAT membrane in heart failure, and (3) controlling the remodeling of the TAT membrane and the growth of the VM in pressure overload induced cardiac hypertrophy. However, further studies are necessary for clinical implementation, particularly with regard to the activation and regulation of Dysferlin.
Keywords: Dysferlin; cardiac hypertrophy; tubular membrane remodeling; heart failure
Schlagwörter: Dysferlin; Herzinsuffizienz; Tubulusmembranbiogenese; kardiale Hypertrophie
