315 Modeling and Rescue of PLN-R14del Cardiomyopathy Phenotype in Human iPSC-Derived Cardiac Spheroids 12 AAV-mediated overexpression of I-1c rescues spheroid morphology and cardiac expression patterns and improves contractility in PLN-R14del spheroids Next, we asked whether gene augmentation with I-1c might alleviate the disease phenotype by restoring cardiac spheroid function. I-1c decreases protein phosphatase 1 (PP1) activity, thereby upstream regulating the SERCA2a/phospholamban complex, and is also closely linked to the β-adrenoceptor system.40 Upon activation of I-1c, PP1 is suppressed, thereby increasing SERCA2a activity and phospholamban phosphorylation which leads to improved basal cardiac performance.41 AAV.I-1c previously demonstrated clinically meaningful benefits in ischaemic heart failure pigs by improving left ventricle ejection fraction (LVEF), contractility, and decreased fibroblast activation42, whereafter AAV2/8.I-1c is currently in clinical trials43. In this gene augmentation approach, the constitutively active I-1c is used to restore Ca2+ An increased MOI rate demonstrated an exponential increase in expression of I-1c from 28 up to 34.000-fold in PLN-R14del transduced hiPSC-CMs over non-transduced cells (Supplementary Figure 6A). The percentage of functional contracting spheroids was similar in all conditions, suggesting that AAV.I-1c does not affect spheroid viability (Supplementary Figure 6B). Interestingly, AAV.I-1v improved Ca2+ handling in PLN-R14del hCSs by all used MOIs. An MOI of 10,000 was selected for the rest of the experiments based on physiological levels of I-1v and significant changes in Ca2+ handling (Supplementary Figure 6C). Seven-day-old healthy control and PLN-R14del spheroids were transduced with AAV.I-1c at an MOI of 10.000 (Figure 6A) and subsequently analyzed after 14 days for spheroid size. When PLN-R14del hCSs were generated in 6 wells, we again observed large spheroids with reduced Ca2+ flux activity (Figure 6B). After a single dose of AAV.I-1c treatment, we observed increased Ca2+ intensity and more regular peak widths in treated PLN-R14del hCSs (Figure 6C). AAV.I-1c reduced spheroid size in all 3 PLN-R4del patients hCSs (462 ± 55 μm versus 931 ± 200 μm in non-transduced PLNR14del hCS) (Figure 6D) while not affecting the size of healthy control spheroids (306 ± 50 μm versus 294 ± 62 μm in non-transduced control hCS) (Figure 6E). Moreover, to study the effect of AAV.I-1c on contractility, Engineered Heart Tissues (EHTs) were generated of all hiPSC lines as previously reported.34 The administration of AAV.I-1c for 7 days improved contraction in both the CTR spheroids and significantly in the PLN-R14del EHTs (Figure 6F). Secondly, gene expression after transduction of control and PLN-R14del spheroids with AAV.I-1c was evaluated by RT-PCR analysis. Interestingly, treatment of AAV.I1c decreased the expression of fibroblast activation genes such as ɑ-Smooth muscle actin (aSMA), Fibronectin 1 (FN1), and Pro-collagen 1 (COL1A1), and the proliferation marker Ki67 in PLN-R14del spheroids (Supplementary Figure 7A). The hypertrophy-responsive genes, such as atrial natriuretic peptide (NPPA) and Myosin Heavy chain 7 (MYH7), did not significantly reduce after AAV.I-1c treatment (Supplementary Figure 7B), whereas both plakophilin-2 (PKP2) and calsequestrin 2 (CASQ2) showed a non-significant trend towards higher levels (Supplementary Figure 7C). The thin myofilament genes Troponin I (TNNI3) and ɑ-actinin (ACTA1) did not significantly increase (Supplementary Figure 7D), however, both PLN and SERCA2A gene expression was
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