365 General Discussion - hiPSC-CMs Disease Modelling and Future Perspectives 13 cytoplasm is essential for the proper initiation of muscle contraction and relaxation. Therefore, the chronic accumulation of Ca2+ in the cytosol due to the reduced SERCA2a activation can affect the contractile function in PLN-R14del. Additionally, upon high cytosolic Ca2+ levels, the protein folding capacity of the endoplasmic reticulum (ER) is reduced, which leads to the accumulation and aggregation of unfolded proteins, resulting in ER stress. As a result, this ER stress leads to the activation of three ER-resident transmembrane proteins of the unfolded protein response (UPR). The UPR can greatly impact the folding capacity and can eventually induce ER stress-mediated apoptosis. In the myocardium of PLN-R14del patients, we observed the aggregation of PLN proteins, similarly, we observed the increased presence of UPR regulators in both PLN-R14del hearts and the metabolically matured hiPSC-CMs (Chapter 9). So, the observed ER stress by the Ca2+ imbalance could be described as another pathological result of the PLN-R14del. Moreover, mitochondria regulate Ca2+ dynamic oscillation due to their high Ca2+-buffering capacity.88 Mitochondrial Ca2+ overload in the PLN-R14del CMs could therefore, increase mitochondrial Ca2+ uptake, which in turn leads to altered metabolism and increased production of ROS.89 In Chapter 8, we studied the decline of fatty acid oxidation (FAO) metabolism transcription in both PLN-R14del hearts and hiPSC-CMs. Here, we found, for the first time, that PLN-R14del hiPSC-CMs displayed a lower FAO profile than the controls at both mRNA and functional levels. Additionally, we observed many PLN-R14del hiPSC-CMs filled with large lipid droplets, indicating the profoundly impaired lipid utilization metabolism. Recently, De Bortoli et al. revealed similar a lower overall density of sodium current and a higher intracellular lipid accumulation in ACM patient-derived hiPSC-CMs compared to asymptomatic hiPSC-CMs.90 Therefore, it is tempting to speculate that the Ca2+ overload in PLN-R14del CMs could induce mitochondrial dysfunction, which could lead to a reduction in the FAO metabolism. However, the precise pathophysiological effect of the overload on mitochondrial Ca2+ transport machinery needs further exploration. Lastly, cardiac fibrosis has been found in histological examination of heart tissues from PLNR14del patients with end-stage disease.91,92 Interestingly, a specific predominance of fibrosis in the inferolateral wall of the left ventricle in PLN-R14del carriers was observed, together with a preserved LV systolic function.93 This finding could suggest that cardiac fibrosis is an early feature of PLN-R14del cardiomyopathy before LV function decreases. In fact, recent data show that total collagen turnover correlates weakly to moderately with clinical parameters in PLN-R14del patients.94 In Chapter 12, we described for the first time, the upregulation of multiple markers known for myofibroblast activation in an in vitro PLN-R14del model by single-cell sequencing. Interestingly, we found that the cardiac spheroids massively increased in size over time, together with the loss of calcium-handling activity. Moreover, in Chapter 12, we combined hiPSC-derived cardiac fibroblasts and CMs in various ratios and observed the increase of PLN-R14del spheroid size when combining 50% or more hiPSC-CMs in the spheroids. This preliminary experiment suggests that the increase in size is caused by the PLN-
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