160 Chapter 7 at serine 16 and/or Ca2+/calmodulin-dependent protein kinase II (CaMKII) at the threonine 17 residue. As more Ca2+ is accumulated in the SR, a greater Ca2+ store is available for release in a subsequent beat, leading to enhanced contractile force.113 Production of pluripotent stem cells (hiPSC) from human adult somatic tissues provides the opportunity to generate large numbers of PLN-R14del patient-specific stem cells.89,94,107 The first direct evidence of PLN-R14del mutation affecting calcium cycling has been shown in these (hiPSC)-derived cardiomyocytes (iPSC-CMs) from a DCM patient carrying the mutation.46 This study revealed progressive dysregulation of Ca2+ cycling in PLN-R14del hiPSC-CMs, characterized by oscillations and extra peaks, a hallmark of an arrhythmic profile114,115, which is consistent with the increased arrhythmogenic vulnerability seen in patients with this mutation. Similarly, irregular Ca2+ transients were observed in a PLN-R14del knock-in hiPSC line generated by inserting the mutation in a control iPSC line via CRISPR/Cas9.61 Subsequently, a study from Cuello et al. showed prolonged late Ca2+ decay time and a Ca2+ concentration-dependent increase in irregular beating patterns in 2D PLN-R14del-CMs, but no observations of abnormal Ca2+ waveforms were reported. A deeper calcium transient analysis was performed by Badone et al., in the same PLN-R14del hiPSC line used by Cuello et al. where heterogeneous calcium transient profiles in the cell population of both wild-type and mutant lines were observed.85 However, the majority of mutant cells showed a typical profile characterized by fast rise and decay time as compared to wild-type, indicating increased SERACA2a activity, possibly due to a loss of PLN inhibitory function as a consequence of the mutation. These data are in contrast with the current described theory of a “super-inhibitory” effect of PLN-R14del and indicate that PLN-R14del mutation might have other, yet unknown, consequences. Interestingly, all studies using engineered heart tissues (EHTs) reported a reduced contractile force in EHTs generated from multiple PLN-R14del patient lines or healthy donor introduced R14del.51,71,72 EHTs contain more relevant physiological structures compared to 2D culturing techniques and can promote cell phenotype, metabolic activity, maturation, and functionality, leading to more reliable and more predictive models. The variation of PLNR14del calcium handling outcomes in 2D hiPSC-CMs might be explained by the inefficient model format which it would be interesting to study the calcium handling in 3D hiPSC-CMs models harboring the PLN-R14del disease. The first mouse models, overexpressing PLN-R14Del, revealed histopathologic abnormalities, super-inhibition of the Ca2+ affinity for SERCA2a, and premature death.1,35 Calcium kinetic and contractile parameters have been further investigated in isolated cardiomyocytes from a novel knock-in humanized PLN-R14del mouse model.76 The study reported depressed calcium cycling and contractile parameters, specifically in right ventricular (RV) myocytes from young mutant mice (12-13 weeks). This suggests that the arrhythmogenic phenotype associated with the PLN-R14del mutation may originate in the RV compartment during early stages. In the same mouse model, at 12 months of age, contractile parameters and calcium transients
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