250 Chapter 9 Next, we explored the transcriptional changes associated with the R14del mutation. An advantage of single-cell over bulk RNA sequencing is that it affords greater statistical power to resolve differential expression because it focuses within cell-identity clusters rather than across the broader heterogeneous population in which cell type–specific information remains obscure. On the basis of the t-distributed stochastic neighbour embedding clustering and canonical marker expression, we restricted the analysis to the cardiomyocyte subclusters 1 to 4 and 6 to 7. Seventy-seven genes (Wilcoxon ranksum test) were differentially expressed in PLN R14del hiPSCCMs compared with the isogenic control hiPSC-CMs (Figure 2E). Gene enrichment analysis revealed genes associated with pathways related to ER stress and UPR signaling pathway (Figure 2F). Further bioinformatics analysis showed that several hallmark genes related to UPR pathway were activated in PLN R14del hiPSC-CMs, including gene members of the protein disulfide isomerase (PDIA3 and PDIA4), heat shock protein 90 (HSP90A1 and HSP90B1), and HSPA5 (also known as BiP [binding immunoglobulin protein]) compared with isogenic PLN WT controls. Comparison of the gene expression profiles of the cell cluster identified as fibroblasts showed no significant differences in the expression of the UPR hallmark genes between the PLN R14del and PLN WT cells (Figure 2G). We further corroborated the activation of UPR markers in the hiPSC-CMs at the protein level using Western blot analysis (Figure 2H). Finally, we monitored the activity of the UPR pathway in living hiPSC-CMs using an XBP1-splicing reporter construct7 (Figure 2I) and observed a significant increase in UPR activity in patient PLN R14del cells. Because adrenergic drive activates UPR response in cardiomyocytes,8,9 we also measured the UPR reporter activity on isoproterenol stimulation. Under adrenergic stress conditions (72 hours of treatment with 1 µmol/L), we noticed an increase in UPR signaling in PLN WT cells but an exaggerated (4-fold higher) response in hiPSC-CMs containing the mutation. Taken together, these data suggest that the PLN R14del mutation activates the UPR transcriptional program and sensitizes R14del hiPSC-CMs to adrenergic stress. Histopathologic Characterization of the UPR in PLN-R14del Patient Myocardium The accumulation of protein aggregates is often associated with dysfunctional UPR responses, for instance, in neurodegenerative diseases.10 Protein aggregation has been noted as a histopathologic characteristic of end-stage PLN R14del DCM hearts but not in hearts representative of other forms of DCM.11–13 Therefore, we examined human myocardial samples of PLN R14del hearts for evidence of UPR involvement. We also analyzed myocardial tissue from desmosomal arrhythmogenic right ventricular cardiomyopathy and ischemic cardiomyopathy hearts (Figure III and Table V in the Data Supplement). Consistent with previous studies,13 PLN immunolabeling revealed perinuclear aggregations in PLN R14del tissues (7±2%), but these aggregations were absent in arrhythmogenic right ventricular cardiomyopathy, ischemic cardiomyopathy, and control hearts (Figure 3A–3D and 3M). BiP is an essential regulator of the UPR that monitors ER stress, and its expression is markedly increased in the presence of unfolded proteins in the ER.14,15 Accordingly, we observed the highest BiP levels in PLN R14del
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