251 Unfolded Protein Response in PLN R14del Cardiomyopathy 9 Figure 2. Single-cell RNA sequencing of isogenic human induced pluripotent stem cell–derived cardiomyocytes (hiPSC-CMs) carrying the phospholamban (PLN) loss of arginine at position 14 (R14del) mutation. A and B, Unbiased identification of cell clusters using t-distributed stochastic neighbor embedding (tSNE)–based clustering of single-cell transcriptomes showing a 2-dimensional visualization with distinctly isolated cell subpopulations (n=5279 cells, healthy donor [phospholamban wild-type (PLN WT)]; n=3965 cells, healthy donor PLN R14del introduced [PLN R14del]). C and D, Subpopulations were classified according to canonical marker gene expression. E, Heat map display of 77 differentially expressed genes in the cardiomyocyte subpopulations. F, Gene set enrichment analysis pathway enrichment analysis. G, Comparison of unfolded protein response (UPR) hallmark differential gene expression between cardiomyocyte and noncardiomyocyte subpopulations in a healthy donor (HD) and R14del introduced hiPSC-CMs. H, Western blot expression analysis of UPR proteins from paired isogenic hiPSC-CM lines (n=3 batches). I, Assessment of the UPR activity in living hiPSC-CMs (PLN R14del and PLN WT) transduced with AAV-F-XBP1ΔDBD (3 batches, n=4–6 wells each). Statistical significance represented as differences between PLN WT and PLN R14del in either control or isoproterenol (iso) conditions. Data are presented as mean±SEM. *P<0.0005.
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