310 Chapter 12 (DCC/FAM155Ahigh CMs) (Supplementary Figure 5E). The CM2 cluster is highlighted by the presence of classical cardiac genes such as sarcomeric protein TNNT2 and the Ca2+ handling protein Ryanodine receptor 2 (RYR2 ) (TNNT2/RYR2high CMs). While the expression levels of cardiac markers troponin T (TNNT2) and myosin heavy chain 7 (MYH7) did not reveal an overall significant difference between conditions (Supplementary Figure 5F), PLN-R14del spheroids contained more cells in the CM2 and fibroblast-like single cells, cluster 3, were detected in 7.51% of PLN-R14del cells compared to the control (1.87%) confirming the increased fibroblast number after culturing PLN-R14del cells (Supplementary Figure 5D). Cluster 3 is highlighted by fibroblast matrix proteins Fibronectin 1 (FN1) and type I collagen (COL1A1) (Supplementary Figure 5G-H). Gene set enrichment analysis of the top 100 pathways revealed differences in specific pathways that were previously described to be affected by the PLN-R14del mutation (Figure 4D). Multi-omics integration13 and mitochondrial functional assays12 have shown an impaired mitochondrial function and decreased fatty acid oxidation metabolism in PLNR14del hiPSC-CMs. Here, we showcase 4 pathways involved in mitochondrial function (Figure 4D, yellow bars), such as fatty acid oxidation, biogenesis, oxidative phosphorylation, and triglyceride catabolism, that were all significantly decreased in the PLN-R14del single cells. Moreover, previous single-cell RNA sequencing revealed the induction of the unfolded protein response (UPR) pathway (Figure 4D, red bars) in PLN R14del compared with isogenic control hiPSC-CMs.13 Similarly, we found autophagy, UPR activation, and aggrephagy pathways all significantly increased in the PLN-R14del single cells, confirming our previous findings.13 A decreased force in engineered heart tissues has revealed impaired contractility in PLNR14del CMs13,14, which was confirmed by our pathway analysis of muscle contraction and dilated and hypertrophic cardiomyopathies (DCM/HMC) related protein expressions in PLNR14del single cells (Figure 4D, blue bars). Furthermore, increased arrhythmic Ca2+ cycling16 and decreased decay time and amplitude37 revealed a critical role of abnormal Ca2+ handling in the pathogenesis of PLN-R14del. We observed a significant decrease in the cardiac conduction, Arrhythmogenic Right Ventricular Cardiomyopathy (ARVC), and myogenesis pathways, whereas the Ion homeostasis was decreased in PLN-R14del single cells although not significantly (Figure 4D, purple bars). Most PLN-R14del hearts develop significant cardiac fibrosis, mainly in the posterolateral left ventricle wall. Interestingly, to the best of our knowledge, we show for the first time that pathways involved in fibrosis are affected in in vitro cardiac spheroids by our single-cell sequencing approach (Figure 4D, orange bars). Specifically, we found significantly different expressed pathways in the extracellular matrix (laminin/ECM) and pathways involved in EIF and PI3K/AKT in PLN-R14del single cells, indicating a profibrotic response in spheroids harboring the PLN-R14del mutation.
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