364 Chapter 13 decreased cAMP-pathway signaling, contributes to a decreased cardiac function. The effect of the R14del on the dephosphorylation of PLN by PP1, remains still, unknown. Interestingly, we demonstrated that the AAV-mediated activation of inhibitor-1 (I-1c), an endogenous inhibitor of PP1, restored the PLN-R14del calcium handling abnormalities (Chapter 12). Figure 3. Overview of the calcium handling in healthy cardiomyocytes vs the suggested disturbed calcium handling in PLN-R14del cardiomyocytes. (A) Calcium regulation in healthy controls. A1) The Ca2+ transient begins when membrane depolarization opens L-type Ca2+ channels (LTCC). A2) The Ca2+ influx leads to a sharp rise in Ca2+ within the dyadic space, triggering the opening of ryanodine receptors (RyRs) and the release of Ca2+ from the sarcoplasmic reticulum (SR). A3) Ca2+ then binds to the myofilaments, triggering myocyte contraction. A4) The Ca2+ transient is terminated as RyRs close and Ca2+ is removed from the cytosol by the Sarco/endoplasmic reticulum Ca2+ ATPase 2a (SERCA2a). A5) Relaxation is mediated mainly by PKA phosphorylation of PLN and thereby the dissociation from SERCA2a, increasing the rate of calcium reuptake into the SR. (B) Suggested disturbed calcium handling in PLN-R14del cardiomyocytes. B4) Impaired phosphorylation of PLN by PKA results in increased cytosolic calcium leading to reduced contractility, UPR activation, mitochondrial dysfunction, and fibroblast activation. (C) Ca2+ transient time course in cardiac spheroids of isogenic control (blue) and derived from a PLNR14del patient hiPSC-CMs (red). Numbers around the peak height correspond to the calcium handling regulation order described in 3A and 3B. Created with BioRender.com. If SERCA2a does not function as well in removing Ca2+ from the sarcoplasm, this could lead to two consequences: 1) there is an increase in resting sarcoplasmic Ca2+ that contributes to reduced relaxation and diastolic dysfunction; and 2) there is less Ca2+ released from the sarcoplasmic reticulum during contraction, which means that the force of contraction is reduced. The decreased contractile function has been described in patients86, mice87, and hiPSC-CMs84 harboring the PLN-R14del mutation. In Chapter 9, we also observed a 50% force reduction in EHTs from PLN-R14del hiPSC-CMs. The rapid change in free Ca2+ levels in the
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