321 Modeling and Rescue of PLN-R14del Cardiomyopathy Phenotype in Human iPSC-Derived Cardiac Spheroids 12 the highly upregulated UPR pathway in 21-day-old hCSs and aggregates could indicate that the elevated UPR could trigger the accumulation of lipid droplets. For example, activating the UPR branch ATF6 is known to down-regulate PPARα causing a reduction in β‐oxidation and an accumulation of lipid droplets in mice.53 Similar to ATF6, sterol regulatory element‐ binding proteins (SREBPs) are ER‐resident proteins possessing transcription factor activity to activate a large number of genes involved in the synthesis of FA derivatives and cholesterol. The reactome of ‘gene expression by SREBP is significantly upregulated in the PLN-R14del CMs geneset analysis. Additionally, autophagy leads to the release of lipids from protein breakdown, of which a portion of autophagy-released lipids is immediately re-esterified to form triacylglycerol. This process is under the control of the master growth regulator, mTOR complex 1 (mTORC1)54, which is also significantly upregulated in the PLN-R14del CMs geneset. Contractility-associated genes that were affected by the PLN-R14del included sarcomere kinases and regulators TNNI3K, RBM20, and SPHKAP. TNNI3K has been described in atrial tachyarrhythmia and dilated cardiomyopathy55, the loss of RBM20 causes an abnormal intracellular Ca2+ handling, and arrhythmias56 and SPHKAP is a membrane-anchoring protein in the Z-bodies associated with heart failure57. Three of the top twenty genesets involved in cardiac contractility; myogenesis, muscle contraction, and HCM, were significantly downregulated in PLN-R14del CMs. We and others have shown a significant decrease in contractile force in EHTs.13,14 We have not analyzed contraction in our cardiac spheroids, although this model could improve high-throughput analysis of contractile parameters. In addition to the reduced Ca2+ handling and known PLN-R14del pathological metabolic and UPR expression patterns, we observed a striking 2.4-fold increase in PLN-R14del hCS size after 21 days of 3D culturing (Figure 2). This observed size increase was not related to the initial cell number, size, or purity of the CMs. Previously, a 3-fold size increase has been observed in vascularized cardiac spheroid after the addition of doxorubicin, a known inducer of myocardial fibrosis.58 We did observe increased expression of the proliferation marker ki67, collagen production, and in multiple genes such as α-SMA (ACTA2), and EIF3A, in PLN-R14del CMs of hCSs. EIF3A and ITGB3 are involved in fibrosis through via regulation of the TGF-b1/SMAD3 signaling pathway.59,60 Cardiac fibroblasts and the differentiation to myofibroblasts underlie the development of pathological cardiac fibrosis, leading to arrhythmias and heart failure. Matured myofibroblasts are characterised by increased α-smooth muscle actin (α-SMA) fiber expression, secretion of collagens, and changes in proliferation.61,62 Additionally, a controlled addition of hiPSC-CFs and hiPSC-CMs, creating various cell ratios, did not induce the hCSs size increase (Figure 3). Interestingly, the addition of >50% hiPSC-CM derived from a PLN-R14del background initiated this massive increase in size, suggesting that this phenotype is linked to the CM PLNR14del mutation. Importantly, the significant upregulation of all fibroblast genes is accompanied by the decrease of cardiomyocyte-specific genes. This confirms the reduced cells positive for the sarcomeric protein alpha-actinin that we observed by the immunofluorescent imaging. Currently, the underlying molecular mechanisms of cardiomyocytes causing fibroblast activation
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