20 Chapter 1 Chapter 9, we started by using transcriptomics of hiPSC-CMs and compared the differentially expressed pathways in PLN-R14del vs isogenic control. Here, we found the unfolded protein response as a compensatory mechanism of the PLN-R14del disease. We will show how this mechanism, directly and indirectly, suggests a mechanistic link between protein toxicity and PLN aggregate formation in the PLN-R14del-induced pathophysiology. Furthermore, we explored the therapeutic potential of activating the UPR with a small molecule activator, BiP (Chapter 9). In Chapters 8 and 9, we made use of only one severely affected PLN-R14del patient. To study the molecular mechanism of the PLN-R14del of various patients, we generated hiPSC lines derived from six patients carrying the pathogenic PLN-R14del variant and two non-carrier family members. (Chapter 10) In order to investigate these hiPSC-CMs of many PLN-R14del patients, we present a scalable, high-throughput screening-compatible workflow for the generation, maintenance, and optical analysis of cardiac spheroids in a 96- well-format (Chapter 11). Additionally, these small cardiac spheroids can be cryopreserved, allowing researchers to create next-generation living biobanks. Lastly, we use the spheroid model to study the PLN-R14del disease, screening hiPSC-CMs generated from 6 hiPSC lines. Here, we found the calcium handling parameters such as decay time, rise time, calcium transient duration, and peak value (amplitude) to be reduced in PLN-R14del spheroids derived from three individual patients (Chapter 12). Lastly, translating these findings back to clinical care, we will investigate the potential improvement of an AAV-mediated I-1c gene augmentation therapy on the PLN-R14del disease. Findings from the trial should be able to serve as pivotal evidence for therapy potentials and guidelines. GENERAL DISCUSSION AND SUMMARY As concluding considerations, we will put all the aforementioned findings into contemporary and future perspective, provide recommendations for future research, and provide an outlook for the future of hiPSC-CMs in disease modeling and therapeutic screening (Chapter 13). The thesis ends with a summary of the previous chapters (Chapter 14). Table 2. Overview of the general introduction and related thesis chapters on the specific introduction sections. Introduction section Related thesis chapter number 1.1 Clinical relevance Chapter 7 1.2 PLN-R14del cardiomyopathy Chapters 7-10 & 12 1.3 Lessons learned from cardiogenesis Chapter 2 1.4 Cardiomyocyte generation and expansion Chapters 3-5 1.5 Cardiomyocyte maturation Chapters 6, 8, 9, 12 1.6 hiPSC-CM models Chapters 3-6 & 8-12 1.7 High-throughput integrative disease modeling and drug screenings Chapters 11,12
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