362 Chapter 13 metabolic, and contractile pathogenesis. Here, an integrated screening approach that combines the HT analysis with first a physiological function screening, whereafter the same cells are subsequently followed by a secondary screening for the protein/gene expression that is affected by the disease would be optimal. However, to fulfill this promise, it must be understood that HTS platforms require more than the simple linear extension of a single cell line approach by adding additional cell lines. Thus, future HTS research should focus on a standardized approach for all the different aspects before the screening, as almost every study now uses only a few hiPSC lines, variable differentiation protocols (Chapter 2, Figure 3), and different maturation stages of hiPSC-CMs (Table 1). Moreover, without the proper maturation of hiPSC-CMs, there is a potential risk of uncovering biology relevant to a fetal rather than an adult disease manifestation. Taken together, the HTS of hiPSC-CM models is more complicated than the present in vitro testing approaches, and optimization is, in many ways a study itself, demanding its unique protocols, robotics, standardization, and automated data analysis. Given the above, it can be assumed that hiPSC-CM models could eventually be included in regulatory documents as the first CTiD screen before drug evaluation in patients. Next, the combination of the clinical findings with the CTiD data with machine learning algorithms could help the clinical choices of care by cardiologists. Moreover, eventually, the HTS platform holds the promise of reducing the need for in vivo studies, although the secondary effects in pharmacodynamic, tissue interaction, and metabolite activity remain challenging to study in in vitro. Studying PLN-R14del hiPSC-CMs reveals multiple pathological mechanisms The PLN-R14del mutation causes ventricular arrhythmias, decreased contractility, protein aggregation/toxicity, mitochondrial dysfunction, fibrosis, and inflammation. However, identifying the primary effect leading to this phenotype is crucial to determining ‘at-risk’ patients, which is important for disease prediction and prevention. We were, to our knowledge, the first to report the altered Ca2+ transient parameters in PLN-R14del spheroids, corresponding to the decreased cardiac function (Chapter 12). The SERCA2a/PLN complex plays a pivotal role in mediating intracellular calcium homeostasis and aberrant calcium handling by the decreased SERCA2a activity is a consistent finding in heart failure. As described in Chapter 7, data converging to argue for PLN p.Arg14del may cause the ‘super-inhibition’ of SERCA2a, due to increased PLN-R14del/SERCA2a binding.75 The decreased activity of SERCA2a by PLN-R14del is expected to cause prolonged calcium reuptake by SERCA2a, therefore increasing the decay time. However, calcium analysis data from others (Figure 2A)76 and our calcium data (Figure 2B, Chapter 12) displayed a very similar pattern of decreased decay time in hiPSC-CMs. These observations raised the question of ‘’which alternative mechanisms could connect PLN-R14del defects in Ca2+ handling with low-force development?’’
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