323 Modeling and Rescue of PLN-R14del Cardiomyopathy Phenotype in Human iPSC-Derived Cardiac Spheroids 12 the latter is regulated by PLN is not certain, but it could be related to paracrine signaling to nonmyocytes such as fibroblasts, possibly via cardiomyocyte-specific expression of cytokine (as reviewed elsewhere).70 Moreover, profibrotic factors could modulate the endoplasmic reticulum Ca2+ release and Ca2+ influx into fibroblasts, which could result in an increased cytosolic Ca2+ concentration, thereby initiating fibrosis-promoting proliferation/differentiation of fibroblasts.71 In a disease state, CFs undergo activation to a myofibroblast phenotype, characterized by increased proliferation, ECM production, and decreased contractility.72 Interestingly, geneset enrichment analysis revealed the top 1 and 4 elevated genesets to be Netrin-1 signalling. Netrin-1 is a laminin-like protein, which is involved in embryonic development and is upregulated in cancer-associated fibroblasts.38 Despite the role of Netrin-1 in the heart has not been fully elucidated, this protein is highly expressed in muscle tissue.73 A study in neonatal rat cardiomyocytes revealed that the downregulation of Netrin-1 was found to significantly alleviate the degrees of myocardial hypertrophy, fibrosis, apoptosis, and heart failure.74,75 Future studies are required to investigate the role of netrin-1 in the PLNR14del cardiomyopathy. Transient receptor potential (TRP) channels play an important role in the differentiation of fibroblasts to myofibroblasts, ECM remodelling, and the fibrogenesis cascade and have emerged as the most important ion channels that mediate Ca2+ signals in cardiac fibroblasts.76–78 TRP mechanosensitive Ca2+-permeable channels 3 and 4 (TRPM3/4) were significantly upregulated in PLN-R14del hCSs, hinting towards a potential role of TRP in the onset of fibroblast activation in PLN-R14del CMs. Further unbiased and spatiotemporal RNA sequencing and proteomics attempts could identify key regulators of cytosolic Ca2+ overload-mediated cardiac dysfunction, fibroblast activation, and fibrogenesis. It would be of great interest to further study all of the disease-specific traits of the PLN-R14del disease in an earlier stage versus the disease state. Knowing when and why these pathophysiological traits derail would be the key to unravelling the optimal time window and most suitable therapeutic intervention. Our hCSs model largely mimics the cardiomyopathy characteristics seen in the PLN-R14del disease: mitochondrial dysfunction, UPR activation, PLN aggregation, reduced contractility, and impaired Ca2+ handling, which therefore could be used as a high-throughput screening tool to identify novel therapeutic targets. Additionally, our hCSs model showed the important role of activated CFs in PLN-R14del, indicating the importance of fibrosis for the pathogenesis of ACM/DCM and potential therapeutic interventions. Importantly, the identification of clinically relevant fibroblast activation may inform us of disease pathology and hint at other potential therapeutic opportunities. In the last years, three clinical trials using AAV-mediated gene replacement have been designed targeting heart failure (reviewed elsewhere79). We note that to date, only a fairly limited number of reports have employed organoids to study AAV gene therapy (summarized here80). In this study, we show, for the first time, the feasibility of using a cardiac spheroids platform to test AAV-delivered gene augmentation as a therapeutic strategy for genetic
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