363 General Discussion - hiPSC-CMs Disease Modelling and Future Perspectives 13 Figure 2. Intracellular calcium cycling in PLN-R14del hiPSC-CMs compared to control. (A) Calcium transient and corresponding decay time modified from the study by Badone et al.76 (B) Calcium transient and corresponding decay time modified from Chapter 12. SERCA2a function is modulated by the binding of PLN. Over the last decade, more PLNbinding partners have been described, including HAX-1 and GM, the anchoring subunit of protein phosphatase 1 (PP1), as well as AKAP the anchoring subunit of protein kinase A (PKA), allowing the fine-tuning of the PLN binding/phosphorylation status and thus, SERCA activity.77 PP1 dephosphorylates PLN, causing PLN to inhibit SERCA2a’s Ca2+ pumping. Upon β-adrenergic stimulation, PKA activity increases, whereby PKA phosphorylates PLN, relieving SERCA2a inhibition and enhancing SR Ca2+ transport as well as cardiac relaxation.78 The β-AR mediated phosphorylation of PLN has been shown to occur mainly through the effects of PKA at serine 16, illustrating the importance of this mechanism for beta-adrenergic-induced cardiac relaxation.79 Importantly, multiple studies described that the deletion of arginine 14 in the PLN-R14del disease, disrupts its R-R-X-S motif.80,81 This protein motif is required for the phosphorylation by PKA, on only two amino acids higher, serine 16. The missense of arginine 14 could be associated with conformational changes in PLNs coil domain, which is believed to provide flexibility associated with PLN phosphorylation and impaired SERCA2a regulation.77,82 If the PKA-Ser16 phosphorylation is hampered, the release of PLN from SERCA2a could be delayed. Together, we can hypothesize that the reduced PLN phosphorylation by PKA-Ser16, could result in impaired SERCA2a activation, thereby reducing the SR Ca2+ transport (Figure 3). Previously, it was described that even if the PLNR14del can be phosphorylated by PKA, it will not reverse the inhibition of SERCA activity, leading to the super-inhibition of SERCA2a.83 Although SERCA2a super-inhibition by very strong PLN binding in the basal state has been described to be a suitable explanation for the clinical phenotype in PLN-R14del patients, after our observations, this hypothesis makes it difficult to agree with completely. The observed decreased decay time and contractile incompetence in mice and multiple human cardiomyocyte studies provide evidence of the delayed release of PLN-R14del from SERCA2a due to disturbed phosphorylation.76,84,85 Thus, our observation of decreased calcium parameters (such as amplitude and decay time) and decreased PLN and SERCA2a expression in PLN-R14del spheroids confirmed the decreased SERCA2a activity upon PKA phosphorylation in PLN-R14del CMs (Chapter 12). In failing hearts, increased PP1 activity, coupled with
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