313 Modeling and Rescue of PLN-R14del Cardiomyopathy Phenotype in Human iPSC-Derived Cardiac Spheroids 12 PLN-R14del hCSs express an increased fibroblast activation pattern. Interestingly, the pathological mechanisms observed in PLN-R14del spheroids were observed in various stages of severity in spheroid size and sarcomeric marker alpha-actinin (ɑ-ACT) presence. In some PLN-R14del spheroids, one or multiple non-positive holes were observed, whereas some spheroids did reveal only a few nuclei with sarcomere expression (Figure 5A). However, all PLN-R14del spheroids revealed nuclei with no sarcomere proteins present (Figure 5A), and a significant reduction of ɑ-ACT area/nucleus (Figure 5B). Many nuclei in the PLN-R14del spheroids were found positive for the proliferation marker Ki67 and were observed in different locations, either in the middle or on the outer layers (Figure 5B), whereas the collagen production was mainly found in the outer layer of the spheroids (Figure 5E). Quantification of proliferation showed an increase in Ki67-positive nuclei (Figure 5D) and a significant increase in Pro-COL1A1 accumulation (Figure 5F). RT-qPCR analyses revealed that PLN-R14del hCSs displayed a significant increase in activated fibroblast genes (aSMA (ACTA2), COL1A1, FN1), proliferation gene ki67, fetal genes (NPPA/MYH7) and a significantly reduced expression of genes that regulate cardiomyocyte function e.g. CASQ2, ATP2A, PLN, PKP2, GJA1, TNNI3 and ACTA1 (Figure 5G). A heat map was generated using Euclidean distance showing hierarchical clustering of control and PLN-R14del hCSs, based on log2 fold expression of the genes described in Figure 3G (Figure 5H), confirming similar expression patterns between control hCSs and the three PLN-R14del patient hCSs. The reduction of cardiac markers and increase in proliferation and collagen expression is variable in each PLN-R14del spheroid but could be the result of CM depletion and replacing fibrosis also observed in the myocardium of PLN-R14del patients.
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