366 Chapter 13 R14del CMs, rather than the initial amount of cardiac fibroblasts in the spheroids. Recently, cardiac spheroids treated with ISO/hypoxia/TGFβ1 to induce cardiac injury revealed the downregulation of several calcium handling, ion channels, fatty acid metabolism-related genes, and the upregulation of ECM and fibroblast activation genes.95 Nonetheless, the exact pathophysiological mechanism and trigger behind the fibroblast activation in PLN-R14del cardiomyopathy remains to be investigated. The chicken or the egg? What is the first trigger of the PLN-R14del phenotype? In patients, the first described subclinical PLN-R14del disease alteration before the onset of heart failure symptoms is the post-systolic shortening in the left ventricular apex, which is detected by echocardiographic deformation imaging.96 Accordingly, it was hypothesized by these authors that the dysregulation of intracellular calcium handling in PLN-R14del mutation carriers could lead to altered myocardial mechanical behavior observed in these pre-symptomatic mutation carriers.97 In this thesis, we used hiPSC-CMs and described the impaired SERCA2a activation, reduced contractile force, the activation of ER stress, fibroblast activation, and the reduced FAO metabolism in the PLN-R14del disease. However, one big question remains; What is the first trigger of the PLN-R14del phenotype? Meaning; Which came first, the chicken or the egg? Understanding the underlying pathophysiology of the PLN-R14del disease would improve insights into a patient's disease course and would create the opportunity for conceiving treatment strategies. After so many intense years of studying the PLN-R14del phenotype in vitro, the onset of the observed PLN-R14del hiPSC-CMs phenotypes described in this thesis differs. First, on day 30 (spheroids week 1), impaired calcium handling is observed (Chapter 12). Next, on day 42 post differentiation, we described the reduced contractility and UPR activation (Chapter 9). The fibroblast activation seems to occur after 2 weeks of spheroids culturing, also around differentiation day 42 (Chapter 12). Later, between days 60 and 180, lipid droplet accumulation in hiPSC-CMs is observed (Chapter 8). However, both contractility and the metabolic phenotype have been described in earlier 2D and 3D models (day ~30).84 Together, in this thesis we successfully recapitulate the diverse phenotype, highlighting the utility of hiPSC-CMs in modeling more complex cardiac diseases. Overall, a reduced SERCA2a activity has been described to increase cytosolic Ca2+ levels, resulting in mitochondrial and ER stress, ventricular arrhythmias, reduced contractility, and eventually heart failure.98,99 After combining all our data described in this thesis, we would like to propose the following onset of the PLN-R14del phenotype hypothesis (Figure 4). 1) First, The mutation of arginine 14 causes a conformation change, resulting in the reduced PLN phosphorylation by PKA-Ser16 and thereby delayed release of PLN from SERCA2a, 2) The impaired SERCA2a activation, reduces the SR Ca2+ transport, leading to Ca2+ overload.
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