324 Chapter 12 cardiomyopathies. Previous studies have demonstrated that the administration of a constitutively active I-1c delivered by this cardiotropic AAV2i8 (BNP116) virus in ischaemicinduced heart failure pigs improved LV cardiac function with an EF of 5.7%.20 Additionally, investigators reported that the delivery of I-1c was safe in three non-ischemic cardiomyopathy patients who exhibited LVEF improvements of 10.5%, 22%, and 6%, respectively, at 12 months of follow-up.81 We observed that a single dose of AAV.I-1c was sufficient to rescue PLN-R14del spheroids size, Ca2+ function, EHT contractility, and cardiac expression patterns (Figure 6). It is important to note that contractile function is strongly decreased in PLN-R14del myocytes and EHT constructs.13,14 Additionally, we observed an overall increase in all Ca2+ handling parameters after a single dose of AAV.I-1c treatment (Figure 7). However, we observed a prolonged decay time and CTD90 compared to non-transduced spheroids, indicating a decreased SR Ca2+ uptake. The increase in decay time might be caused by the overall increased total amount of Ca2+ as indicated by the increase in peak value. Additionally, we observed a 3 to 5-fold increase in SERCA2A expression compared to non-transduced control and PLNR14del hCSs respectively. Increased I-1c levels suppress SR-associated PP1 activity, thereby increasing SERCA2a activity, and PLN phosphorylation at both Ser16 and Thr17 and enhancing contractility both basally and after β-adrenergic stimulation.82 Indeed, the upregulation of I-1c increases SERCA2A activation and Ca2+ transient amplitude, possibly decreasing PP-1 activity and improving phospholamban phosphorylation. Lastly, I-1c gene augmentation has a positive effect on mRNA levels of proteins associated with fibrosis and Ca2+ handling function (Supplementary Figure 7). For instance, COL1A1 and FN1, which are known to be higher in activated myofobroblasts83, were significantly reduced after 14 days of AAV.I-1c therapy. AAV.I1c increased mRNA levels of Ca2+ handling proteins, confirming the observed improvement in cardiac function. While the restoration of the hCSs size, gene expression, contractility, and Ca2+ handling was evident in our study, further studies are required to define the link between the fibroblast activation, cardiomyocyte function, and the protective role of AAV.I-1c we observed. Our work on AAV2i8-based gene augmentation with the constitutively active I-1c complements animal studies with a human-specific system and evaluates the rescue of PLN-R14del phenotype, which may constitute a therapeutic strategy in failing hearts. We are aware that additional systematic testing of different AAV-mediated strategies such as WT PLN overexpression might lead to a more direct improvement, as previously demonstrated by AAV6-mediated overexpression of PLN, which induced downregulation of endogenous PLN-R14del transcripts and sufficient to improve CM function.16 Nevertheless, this is the first proof of concept of AAV-mediated gene augmentation in cardiac spheroids. Based on our results, we hypothesize the potential pathological mechanism underlying the PLN-R14del cardiomyopathy to be the toxic cytosolic Ca2+ overload due to the reduced Ca2+ handling and protein misfolding by PLN-R14del. Multiple pathological pathways emerge, from UPR activation, metabolic dysfunction, and reduced contractility to the activation of myofibroblasts (Figure 8).
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