319 Modeling and Rescue of PLN-R14del Cardiomyopathy Phenotype in Human iPSC-Derived Cardiac Spheroids 12 DISCUSSION Various pathogenic genetic variants cause severe heart failure, including mutations in the coding region of the PLN gene. Specifically, the deletion of the arginine 14 codon in the PLN gene (PLN-R14del) is associated with prevalent ventricular arrhythmias, fibrosis, dilatation of the heart, and sudden cardiac death.9 The pathogenic signature of how the PLN-R14del contributes to the overall clinical presentation is limited, especially in in vitro models. This unknown pathogenic signature translates back into the current lack of disease-specific therapeutic screening strategies. Patient-derived hiPSC-CM models provide a platform for modelling various genetic cardiomyopathies in vitro to understand the underlying pathological mechanisms. However, the complexity of the human heart by the cellular composition and conduction characteristics presents a limiting factor to defining the total disease-specific phenotype in vitro. To overcome these challenges and explore further PLN-R14del phenotypes, we generated 3D human cardiac spheroids (hCSs) from healthy controls, an isogenic control, and three PLN-R14del patient lines. The hiPSC-CMs for hCSs were successfully generated in a highly reproducible way from 6 individual patients, which can be frozen for later usage without altering their functional properties.23 hCSs were formed from just 10,000 cells by self-aggregation in 96/384 wells plates and cultured for an optimal time of 21 days. In contrast to other 3D systems, including engineered heart tissues (EHT)44, our hCS model allows automated analysis of Ca2+ transients in a high-throughput fashion. Here, we showed, for the first time, that the optical 3D construct analysis in 384-well plates revealed decreased Ca2+ handling and Ca2+ peak amplitude in PLN-R14del hCSs (Figure 1). Interestingly, even after 7 days of culturing, a reduced decay time and CTD10 were observed, hinting toward the first functional pathological phenotype of this disease. However, the reduction in all Ca2+ handling parameters after 21 days, including rise time, decay time, and the Ca2+ transient duration, indicates an overall reduced Ca2+ flux in PLN-R14del hCSs, instead of a prolonged decay time as previously described. Indeed, the reduced Ca2+ handling parameters were all decreased, despite the increased beating rate in the PLN-R14del spheroids (Supplemetary Figure 2). Genetic analysis of the PLN-R14del hCSs revealed a decreased expression of Ca2+ channels JPH2, PLN, and CACNA1G and an increase in CPNE5. The gene CPNE5 is a poorly characterized Ca2+-dependent membrane protein45 and merits further attention. Taken together, an aberrant Ca2+ handling and amplitude due to the R14 deletion in PLN was observed in the hCSs. Recently, a decreased decay time was observed in 2D hiPSC-CMs of an unrelated PLN-R14del patient, which was described to be caused by an enhanced SERCA2a activity.37 Pharmacological SERCA2a activation by PST3093 did not alleviate the accelerated systolic and diastolic Ca2+ dynamics by the overserved enhanced SR function. Cardiac DWORF overexpression in homozygous PLN-R14del mice delayed cardiac fibrosis and heart failure. However, these mice displayed an already enhanced Ca2+ reuptake, wherefore the DWORF overexpression did not improve Ca2+ S/ER reuptake.46 The authors state that the PLN
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