298 Chapter 12 ABSTRACT A deleterious mutation of the arginine 14 codon in the phospholamban (PLN) gene (PLNR14del)) in dilated cardiomyopathy patients is associated with severe ventricular arrhythmias, contractile dysfunction, heart failure, and sudden cardiac death. Using induced pluripotent stem cells (hiPSCs) generated from three symptomatic patients with the PLN-R14del mutation, we established a 3D cardiac spheroid (hCS) in vitro model that displayed an increase in spheroid size, nuclei number, and decreased CM number upon culturing, as compared to isogenic and healthy control lines. Single-cell RNA sequencing revealed patient-like pathological phenotypes, among others, mitochondrial dysfunction, increased fibrosis, affected unfolded protein response, and reduced calcium (Ca2+) handling and contraction in PLN-R14del patient-derived cardiac spheroids. For the first time, we further revealed fibroblast activation, decreased expression of cardiac-specific genes, and decreased Ca2+ handling in PLN-R14del 3D spheroids, thereby confirming various human PLN-R14del cardiomyopathy features. Adeno-associated virus (AAV)-mediated overexpression of constitutively active inhibitor-1 (I-1c) partially rescued the disease phenotype of the PLN-R14del hCSs, by restoring spheroid size, cardiac gene expression levels, preventing fibroblast activation and improving contractility and Ca2+ handling functionality. This study provides evidence for (1) the feasibility of generating 3D hCS for high-throughput screenings to model the PLN-R14del phenotype in vitro and (2) efficient gene augmentation in hCSs as a potential therapeutic strategy for genetic cardiomyopathies.
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