320 Chapter 12 aggregation could trigger cardiomyocyte dysfunction, leading to cardiomyocyte cell loss and fibrosis, which finally culminates in severe cardiac dysfunction and death. Given the recently described increased enhanced Ca2+ reuptake in both PLN-R14del 2D hiPSC-CMs and mice, the interpretation of Ca2+ handling and potential interventions may need to be re‐evaluated. We used the single-cell SPLiT-seq technique, which represents a new approach to determining gene expression variability at the single-cell level. Here, we used comprehensive transcriptome analysis of 386 (PLN-R14del) and 374 (healthy control) single cells from cardiac spheroids to classify them in an unbiased manner, independent of any prior knowledge of cell subtypes (Figure 4). To study the late stage of the PLN-R14del cardiomyocytes, we removed very large (fibrotic) spheroids and used smaller spheroids after 3 weeks of culturing from all 6 donors. Our day 75 3D spheroid cells cluster similarly to a previously described expression pattern in 2D day 90 CMs25, indicating the enhanced maturity of the spheroid cells upon sequencing. We classified the spheroid cells into three groups; CM1 (DCC/FAM155Ahigh CMs), CM2 (TNNT2/ RYR2high CMs) and fibro (FN1/COL1A1high) The CM1 group contained cells positive for alphaactinin but was also distinguished from the CM2 group by the abundance of the genes DCC and FAM155A, which are involved in organogenesis/cardiomyocyte survival47 and sodium leak channel48. Interestingly, even though we removed very large spheroids, the fibro group was more present in the PLN-R14del cells, indicating the increased fibroblast presence in the PLN-R14del spheroids despite the initial smaller size. When comparing GO-term pathway analysis, and the top 20 genes and genesets, we observed significant differences in pathways that were previously described in the PLN-R14del pathophysiology and pathology. The mitochondrial genes FHL2 and TMEM71 were downregulated, which were also previously described to be downregulated in hypertrophic cardiomyocytes.49,50 In the top 20 genesets, triglyceride metabolism and cholesterol biosynthesis were found to be significantly expressed in PLN-R14del compared to the control. Both triglycerides and cholesterol have been found to accumulate in lipid droplets.51 Lipid droplets have been observed in the PLN-R14del spheroids (Figure 4E), showcasing the detrimental outcome of mitochondrial dysfunction in the PLNR14del disease. The impairment of the ER/mitochondria compartment and fatty acid oxidation has been previously described14,15, which was confirmed in PLN-R14del cardiac spheroids. The top differentially expressed gene we found is Ubiquitin C (UBC), a polyubiquitin precursor activated by proteotoxic stress to degrade misfolded proteins. Other highly expressed genes include HSP90B1 and HSPA5, both members of the UPR. The geneset ‘aggrephagy’ was also significantly increased in the PLN-R14del CMs from the cardiac spheroids and was confirmed by the presence of autophagy protein LC3. The activation of the UPR has also previously been observed as the top significant pathway in day 42-old 2D hiPSC-CMs by single-cell sequencing.13 Our model revealed both lipid droplets and additonally PLN accumulation, which has not been observed in vitro before. Hence, the mechanism connecting impaired lipid droplet biogenesis to UPR activation remains unclear. Recent findings indicate that the UPR can be directly activated by altering the lipid composition of the ER.52 In contrast,
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