312 Chapter 12 the SPLIT-seq dataset and corresponding immunofluorescent stainings. * P< 0.05, ** P<0.01, **** P<0.0001 vs. CTR group by Student’s unpaired T-test. Scale bars indicate 50 uM and the yellow box indicates the zoomed-in location of the PLN-R14del spheroid (right picture). Abbreviations; CTR; Control hCSs (1CiCTR, 273iCTR, C31iCTR), R14del; PLN-R14del hCSs (D4iR14del, 6BiR14del, 10BiCTR), FHL2; Four And A Half LIM Domains 2, MT-ND4L; Mitochondrially Encoded NADH Ubiquinone Oxidoreductase Core Subunit 4L, HSP90B1; Heat Shock Protein 90 Beta Family Member 1, BIP/HSPA5; Immunoglobulin heavy-chain-binding protein, LC3; Microtubule-associated protein 1A/1B-light chain 3, TNNI3K; Troponin I-interacting kinase 3, RBM20; RNA binding motif protein 20, JPH2; Junctophilin, PLN; Phospholamban, ACTA2; Actin Alpha 2 and EIF3A; Eukaryotic Translation Initiation Factor 3 Subunit A. Next, we investigated genes involved in the pathological mechanisms of the 5 pathways described in the PLN-R14del disease; mitochondrial function, UPR, contractility, Ca2+ handling, and fibrosis. The top 20 of differentially expressed genes revealed the expression of genes involved in all 5 pathways described in the PLN-R14del disease, such as Polyubiquitin-C (UPR) but also Golgi and ER protein modification, which could also be involved in protein toxicity (GOLGA1, ICMT) (Supplementary Table 2). The top 20 differentially expressed genesets revealed mainly the reduced contraction genes. PLN-R14del cells also expressed upregulated Netrin-1 genesets, which are involved in embryonic development and are upregulated in cancer-associated fibroblasts.38 Additionally, Gli protein expression was elevated in PLNR14del, which is known to be involved in cell fate determination, proliferation, and patterning in many cell types and most organs during embryo development39 (Supplementary Table 3). We additionally selected 2 genes of the SPLiTSseq dataset in each PLN-R14del pathway and performed immunofluorescent imaging to visualize pathological protein patterns within the PLN-R14del spheroids. We observed mitochondrial genes Four And A Half LIM Domains 2 (FHL2) and Mitochondrially Encoded NADH Ubiquinone Oxidoreductase Core Subunit 4L (MT-ND4L) to be significantly decreased and the Nile red staining revealed visually increased lipid droplet size in PLN-R14del spheroid cells as compared to control (Figure 4E). The expression levels of the UPR genes; Heat Shock Protein 90 Beta Family Member 1 (HSP90B1) and Immunoglobulin heavy-chain-binding protein (BIP or HSPA5) were also significantly upregulated, and autophagy was confirmed with an increased protein imaging of LC3 (Figure 4C). The contractility genes; Troponin I-interacting kinase 3 (TNNI3K) and RNA binding motif protein 20 (RBM20) and the immunofluorescent staining of cardiac sarcomere protein alpha-actinin, which showed a significant reduction in the gene expression and a visually reduced presence of sarcomere positive cells in the PLN-R14del spheroids (Figure 4E). Next, the Ca2+ handling gene Junctophilin (JPH2) was significantly decreased, indicating the decreased junctions in the PLN-R14del spheroid cells. However, the PLN expression levels were not significantly decreased, although we could observe, for the first time, PLN protein aggregates in the PLN-R14del spheroids (Figure 4D). Lastly, we analysed the fibroblast genes; Actin Alpha 2 (ACTA2) and Eukaryotic Translation Initiation Factor 3 Subunit A (EIF3A) and the ECM protein vimentin were increased in PLN-R14del spheroid cells as compared to control (Figure 4E). The increased lipid droplets, fibrotic ECM accumulation, elevated autophagy, PLN aggregates, and reduced sarcomere-positive cells prove the spheroid capability to mimic the PLN-R14del pathological mechanisms in vitro.
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