326 Chapter 12 In conclusion, the generation of patient-derived spheroids is simple, scalable, and rapid, and works robustly for a range of different pluripotent stem cell lines of both HF patients, isogenic controls, and healthy individuals. Cardiac spheroids represent a new preclinical model that mimics the sophisticated spatiotemporal processes of the PLN-R14del disease. Here, our findings support that this experimental approach can be broadly used to identify additional processes that contribute to pathogenic mutations in genetic cardiomyopathies and demonstrate the feasibility of using AAV-delivered I-1c as a promising therapeutic option that may represent a potential antiarrhythmic concept in cardiomyopathy patients. The evaluation of the efficacy of therapeutic options in a 3D model, such as hCSs, represents an intermediate step toward clinical application for these patients. Still, further studies with a large animal model harboring the PLN-R14del cardiomyopathy are required to test AAV doses and delivery before going to the first-in patient. STUDY LIMITATIONS HiPSC-CM presents a readily available human cardiomyocyte model that can be generated on demand in large quantities, making it a promising model for investigating electrophysiological abnormalities in patients with inherited cardiac arrhythmias. However, our data showed that the hCSs decreased in their Ca2+ handling and beating rate after 6 weeks of culturing (Supplementary Figure 2). This observation is in line with another study describing an apoptotic response in CM-only spheroids cultured for 8 weeks. Interestingly, spheroids generated from all four cardiac-cell types, in the ratio of CMs, Ecs, SMCs, and CFs (4:2:1:1) remained viable throughout the culture period, by presumably following the distribution trends found in human myocardium.69 Recently, several studies have investigated the role of a controlled combination of hiPSC-CMs, cardiac fibroblasts, and cardiac endothelial cells, which showed enhanced maturation and allowed the disease modeling of ACM and strikingly recapitulated features of the disease, indicating a multicell-type cause of genetic cardiomyopathies.19,70 This finding suggests the importance of physiological ratios of all cell types of the heart, to study the full cell-cell communication in vitro and could be also of value in this model. Another explanation for the reduced Ca2+ handling and beating rate after 6 weeks of culturing is an edge effect, which we observed possibly due to the evaporation of the cell culture medium, which plays a critical role in multi-well-based cellular assays because it can deteriorate assay performance compared to 6-wells cultured hCSs (data not shown). Another limitation of the model is that not every aspect of the disease mechanism can be mimicked with the current spheroid model (e.g., using immature hiPSC-CMs, contractile force, and the lack of inflammatory cells). Additionally, the mechanisms underlying the decreased Ca2+ handling are beyond the scope of the present study. Nevertheless, altered mRNA expression of the underlying Ca2+ regulators SERCA2A and PLN renders intrinsic di erences of its expression levels between PLN-R14del and healthy control hCSs likely. Further analysis
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