325 Modeling and Rescue of PLN-R14del Cardiomyopathy Phenotype in Human iPSC-Derived Cardiac Spheroids 12 Figure 8. The schematic hypothesis of the PLN-R14del-mediated pathological mechanisms in the PLN-R14del cardiomyopathy. Purple; reduced contractility by potential Ca2+ overload and the observed downregulation of RNA-binding motif protein 20 (RBM20), cardiac troponin I-interacting kinase (TNNI3K), SPHK1 Interactor, AKAP Domain Containing (SPHKAP) and genesets muscle contraction and myogenesis. Red; Activation of the unfolded protein response pathway by misfolded PLN-R14del proteins and activation of (HSP90B1), (HSPA5), (UBC), (LC3), and the aggrephagy geneset. Upregulation of autophagy relates genes Sterol regulatory element-binding proteins (SREPBs) and Mechanistic target of rapamycin complex 1 (mTORC1) could also influence the lipid accumulation in PLN-R14del CMs. Yellow; Mitochondrial dysfunction by potential Ca2+ overload and downregulation of the mitochondrial genes Four And A Half LIM Domains 2 (FHL2) and Transmembrane Protein 71 (TMEM71). Geneset downregulation of triglyceride metabolism and upregulation of cholesterol biosynthesis could also increase lipid droplet accumulation due to reduced fatty acid metabolism. Blue; Reduced Ca2+ handling by PLN-R14del malfunction and reduced expression of Ca2+ channel/binding proteins sarcoplasmic/endoplasmic reticulum Ca-ATPase (SERCA), Connexin 43 (Cx43), Ca2+ Voltage-Gated Channel Subunit Alpha1 G (CACNA1G), Junctophilin 2 (JPH2) and Copine 5 (CPNE5), potentially leading to a toxic cytosolic Ca2+ overload. The AAV.I-1c therapy suppresses SR-associated PP1 activity, thereby increasing SERCA2a activity, and PLN phosphorylation, thereby enhancing Ca2+ handling and contractility. Orange; fibroblast activation by potential Ca2+ overload or increased mechanical stress by the upregulation of transient receptor potential Ca2+-permeable channels 3 and 4 (TRPM3/4) and downregulation of desmosomal gene Plakophilin 2 (PKP2). Laminin-like protein Netrin 1 (NTN1) and transcription factor Eukaryotic Translation Initiation Factor 3 Subunit A (EIF3A) could also play a role in the activation of fibroblasts. After fibroblast activation, fibroblast proliferation is increased as indicated by proliferation marker ki67 and the mature myofibroblasts express excessive extracellular matrix (ECM), ECM receptor Integrin Subunit Beta 3 (ITGB3), and α-smooth muscle actin (ɑ-SMA) stress fibers. Abbreviations: Ca2+; Calcium, I-1c; Inhibitor-1, ROS; Reactive Oxygen Species,. The illustration is created with Biorender.com.
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