386 Chapter 14 have been reported about PLN-R14del cardiomyopathy. We described the impact that the discovery of PLN-R14del had on fuelling insights into the molecular mechanisms and the potential therapeutic approaches that have been tested in both in-vitro and in-vivo models. In Chapter 8, we identified the disturbance in the mitochondrial function and (lipid) metabolism in histone acetylation activities and the transcriptome regulation in PLN-R14del hearts and PLN-R14del hiPSC-CMs. Functional assessment of the mitochondria revealed reduced metabolic flexibility and increased glucose utilization, and morphological assessment revealed large lipid droplet accumulation and decreased cell viability in PLN-R14del hiPSCCMs. We assessed the therapeutic potential of a PPARA agonist (bezafibrate) and showed improved calcium handling parameters and increased mitochondrial trifunctional protein expression in PLN-R14del hiPSC-CMs. We concluded that restoration of the impaired FAO, lipid accumulation, and Ca2+ handling shed light on future therapeutic strategies for PLNR14del patients. In Chapter 9, we identified the activation of the Unfolded Protein Response (UPR) in hiPSC-CMs and hearts of PLN-R14del patients by (single-cell) RNA sequencing. The UPR is a cellular stress response and is activated in response to an accumulation of unfolded or misfolded proteins. In hiPSC-CMs, the PLN-R14del mutation activates the UPR transcriptional program and sensitizes R14del hiPSC-CMs to adrenergic stress. We conformed to the previously described aggregation of PLN proteins in the myocardium of PLN-R14del patients and observed an increased presence of UPR regulators. Secondly, we found that activation of UPR in PLN-R14del hiPSC-CMs is protective because molecular inhibition of each of the 3 UPR sensors, IRE1, PERK, and ATF6, exacerbated the contractile dysfunction. Lastly, we assessed the therapeutic potential of activating the UPR with a small molecule activator, BiP (binding immunoglobulin protein) inducer X, and showed a dose-dependent amelioration of the contractility deficit in PLN-R14del hiPSC-CMs. We concluded that the UPR exerts a protective effect and increasing the UPR activity could be therapeutically beneficial in PLN R14del cardiomyopathy. In Chapter 10, we generated 8 hiPSC lines, from 6 individuals carrying the PLN-R14del mutation, and 2 healthy proband family members. We showed efficient reprogramming by the characterization of pluripotency and healthy karyotyping of all hiPSC lines. These hiPSC lines increase the hiPSC sample size in order to improve the probability of identifying specific disease modeling phenotypes and predicting cardiac toxicity and therapeutic drug responses. In Chapter 11, we developed a high-throughput screening (HTS)-compatible workflow with easy scalability for the generation, maintenance, and optical analysis of human cardiac spheroids (hCSs). We observed a highly homogeneous morphology, size, maturation, functional calcium handling, and contractile activity in CSs. We automated the entire workflow from CSs generation to functional analysis, to enhance intra- and inter-batch reproducibility as demonstrated via HTS- imaging and -calcium handling analysis. In addition, we described
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