19 General Introduction and Thesis Outline 1 PART I: CLUES FROM HEART DEVELOPMENT TOWARDS OPTIMIZED HIPSCCMS MODELS In the first part of this thesis, we describe the steps of cardiogenesis and the pathways involved (Chapter 2). This leads to the identification of particular mechanisms involved in cardiomyocyte proliferation and the strategies for cardiomyocyte production, which are also presented here. By this rationale, differentiation strategies that have proven to generate hiPSC-CMs effectively may be repurposed for the massive expansion of hiPSC-CMs. We describe that concomitant GSK-3β inhibition and removal of cell-cell contact inhibition via low cell density serial passaging resulted in a massive proliferative response of hiPSC-CMs (Chapter 3). With this knowledge, we invented a highly efficient method for the expansion and passaging of functional hiPSC-CMs that can routinely be cryopreserved and subsequently used as a stable cell source for downstream applications (Chapter 4). This method is put in a new perspective in Chapter 5, in which live imaging in a hiPSC-CM culture system is used to follow the sequence of sarcomere breakdown during the mitotic phases of CM cell division. Again, going one step deeper in understanding and utilizing the mechanism of action, we will describe the magnitude of Wnt activation in hiPSC-CMs, which results in increased efficiency of non-viral vector incorporation. These findings give an insight into the regulation of sarcomere homeostasis during mitotic cell phases and provide a tool for further molecular and engineering studies (Chapter 5). However, the expanded hiPSC-CMs physiological immaturity severely limits their utility as a model system and their adoption for drug discovery. Therefore, we avail ourselves of a maturation media designed to provide oxidative substrates adapted to the metabolic needs of hiPSC-CMs. Part I concludes with a model of cardiac ischemic damage in metabolically matured hiPSC-CMs and exemplarily evaluates the cardioprotective effect of the RIP1 kinase inhibitor necrostatin-1 (Chapter 6). PART II: MODELING THE PHOSHOLAMBAN R14DEL MUTATION USING PATIENT-SPECIFIC HIPSC-CMS In the second part of this thesis, we aim to provide the most complete approach to investigating the molecular mechanism behind the genetic cardiomyopathy caused by the deletion of arginine 14 in the phospholamban gene (PLN-R14del). First, we will use a systematic review describing studies conducted to investigate the PLN-R14del disease (Chapter 7). We describe the currently available observational evidence that suggests a possible molecular mechanism and the therapeutic strategies used to improve the disease phenotype. Hereafter, we combined the approach of transcriptional regulation analysis in human primary tissue and validation in a unique long-term (160 days) matured hiPSC-CM model. We demonstrate a dysregulated PPARA-mediated mitochondrial fatty acid oxidation (FAO) signaling in PLN-R14del hearts and hiPSC-CMs. By activating PPARA in PLN-R14del hiPSC-CMs using bezafibrate, we observed an improved mitochondrial structure and calcium handling function, further indicating the importance of FAO in the molecular mechanism behind the PLN-R14del disease (Chapter 8). In
RkJQdWJsaXNoZXIy MTk4NDMw