387 General Summary 14 a straightforward procedure for whole-spheroids biobanking, providing the opportunity to create next-generation living biobanks. We conclude that this step-by-step protocol produces high-quality and functional CSs for an optimal cardiac disease modeling and drug screening platform. In Chapter 12, we investigated the molecular mechanism of PLN-R14del in 3D human cardiac spheroids (hCSs) from healthy controls, an isogenic control, and three PLN-R14del patient lines. We observed a striking pathological increase of 2.4-fold in PLN-R14del hCSs size. We confirmed the decreased contractility and calcium handling, mitochondrial function, lipid droplet accumulation, and ER/UPR stress, thereby confirming various human PLN-R14del cardiomyopathy features. Interestingly, single-cell sequencing analysis revealed additional pathological pathways involved in fibroblast activation and ECM secretion such as netrin-1 and transient receptor potential channels to be significantly different in the PLN-R14del spheroid cells, indicating the importance of fibrosis for the pathogenesis of ACM/DCM and potential therapeutic interventions. In this final thesis study, we revealed fibroblast activation, PLN aggregates, and a decreased Ca2+ handling in PLN-R14del spheroids, which have not been described in previous hiPSC-CMs studies. Lastly, we assessed the therapeutic potential of AAVbased I-1c gene therapy and showed improved calcium handling, contractility, and decreased fibroblast activation expression in PLN-R14del hCSs. We concluded that hCSs represent the pathological phenotypes of the PLN-R14del disease and that I-1c AAV2/8-based gene therapy can potentially be used for the treatment of genetic cardiomyopathy modeled in hiPSC-CMs. PART III: GENERAL DISCUSSION AND SUMMARY In Chapter 13, we put the aforementioned findings into methodological, mechanistic, and clinical perspectives. We use the insights from cardiogenesis to improve cardiomyocyte proliferation and maturation for therapeutic strategies to repair the damaged heart. Secondly, we discuss multiple examples of hiPSC-CM models for the understanding of the pathophysiological and molecular mechanisms of genetic cardiomyopathies. We address currently unanswered questions about which triggers underlie the onset of pathological features in PLN-R14del cardiomyopathy. Lastly, we discuss the usage of hiPSC-CMs to assess therapeutic options to cure genetic cardiomyopathies. We conclude that further improvement of the standardization, quality metrics, and algorithms, as well as clear guidelines for designing and executing any in vitro studies testing clinical therapeutic strategies, are required for the optimization of the choice of therapeutic intervention. Considering the output from more than five years of translational hiPSC-CM research summarized in this thesis, we conclude to have—hopefully—not only added data that will improve these ‘mini-hearts’ to predict, prevent and cure the burden of cardiomyopathies in people we try to help so hard - the patients.
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