Role of predisposing genetics, telomere signaling and crosstalk of cardiac fibroblasts and cardiomyocytes in a pluripotent stem cell model of dilated cardiomyopathy
by Wiebke Maurer
Date of Examination:2022-09-26
Date of issue:2023-02-16
Advisor:Prof. Dr. Katrin Streckfuß-Bömeke
Referee:Prof. Dr. Katrin Streckfuß-Bömeke
Referee:Prof. Dr. Susanne Lutz
Referee:Prof. Dr. Bernd Wollnik
Referee:Prof. Dr. Ralf Dressel
Referee:PD Dr. Laura Zelarayan-Behrend
Referee:Prof. Dr. Rüdiger Behr
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EnglishDespite rapid scientific and medical advances, cardiovascular diseases represent a major threat to human health worldwide. Among these, cardiomyopathies are one of the leading causes of heart failure. Dilated cardiomyopathy (DCM) represents one of the most common causes of heart failure and is the main indication for heart transplantation worldwide. It is characterized by dilation of the left ventricle and systolic dysfunction, but no left ventricular wall thickening. The underlying molecular causes of most DCM cases remain unknown. Nevertheless, it was shown that up to 35% of all DCM cases have a family history and are associated with mutations in more than 50 gene loci. Thus, identifying the underlying molecular mechanisms contributing to DCM development is indispensable. Hence, this project aims to identify disease-causing genetic variants and unravel underlying molecular mechanisms contributing to the DCM pathophysiology. To establish a human in vitro DCM model of a four-member index family consisting of patients with severe DCM (the father and oldest daughter) and healthy controls (the mother and second daughter), patient-derived iPSC-cardiomyocytes were generated. They were differentiated into beating iPSC cardiomyocytes with high purity. Since whole exome sequencing (WES) and panel sequencing identified a new and disease-causing missense variant in the highly conserved domain 20 of the filamin C (FLNC) gene the CRISPR/Cas9-technology was used to generate isogenic control iPSC lines. We identified a FLNC dependent dysregulated sarcomeric structure in diseased iPSC-cardiomyocytes as the main disease phenotype, giving first evidence for the crucial role of the FLNC variant within the DCM pathophysiology. Based on this finding, we analyzed the role of the FLNC variant further on molecular and functional levels. No differences were found in FLNC expression or localization between diseased and control (iPSC-) cardiomyocytes, suggesting impairments of FLNC interactions with important binding partners. Both patients showed FLNC dependent contraction defects and increased stiffness in 3D-engineered heart muscle (EHM), composed of patient-specific iPSC- cardiomyocytes, fibroblasts, and collagen. This further demonstrates the crucial role of the FLNC variant within the (iPSC-) cardiomyocytes and highlights its important contribution to the DCM pathophysiology. Moreover, FLNC dependent compaction and contraction of diseased engineered connective tissue, composed of (iPSC-) cardiac fibroblasts and collagen, were decreased in the patients. With this, we could, to our knowledge, for the first time demonstrate a role of FLNC within cardiac fibroblasts in general and identify a decisive role for the identified FLNC variant in cardiac fibroblasts as an important player in the DCM pathophysiology. Engineered connective tissue of primary cardiac fibroblasts additionally displayed increased stiffness, pointing towards fibrotic processes, which is in line with the demonstrated increased fibrosis of the father`s explanted myocardium of the left ventricle. These results clearly identified the new FLNC missense variant as an underlying disease cause within the DCM family, primarily leading to a dysregulated sarcomeric structure, contraction defects, and increased stiffness. These are all DCM hallmarks which could be linked to the FLNC variant. Furthermore, FLNC dependent functional impairments in cardiac fibroblasts were identified, pointing to FLNC as a crucial player within the DCM pathophysiology.
Keywords: Dilated cardiomyopathy (DCM); Filamin C (FLNC); iPSC-cardiomyocytes; Cardiac fibroblasts; Sarcomere; 3D-engineered heart muscle (EHM); 3D-engineered connective tissue (ECT); CRISPR/Cas9