213 Fatty Acid Oxidation in PLN R14del Cardiomyopathy 8 which were annotated in PLN-R14del-specific hypoacetylated clusters were shown in Fig.1I. Combined, we detected differentially acetylated regions that distinguish PLN-R14del from other types of cardiomyopathies and they annotated metabolic-related genes were profoundly affected. Enriched metabolic pathways by differentially expressed genes in PLN-R14del hearts Besides differentially acetylated regions, we obtained 1,668 up- and 1,873 downregulated genes in PLN-R14del versus control hearts using RNA-seq (Fig.2A and Fig.2B, Table S5A). In line with the well-established suppression of SERCA2A/ATP2A2 at the protein level,6 we showed its suppression at the mRNA level for the first time. Additionally, metabolic genes, such as HADHA and HAHDB, which were annotated from PLN-R14del-specific hypoacetylated clusters, also showed significantly lower mRNA levels in PLN-R14del versus control hearts (Fig.2C). We further demonstrated decreased HADHA and SERCA2A/ATP2A2 protein levels in PLN-R14del versus the control heart by immunofluorescence staining (Fig.2D). Consistent with enriched biological processes and pathways by annotated genes from differentially acetylated regions, fibrosis, (cardiovascular) development, and chromatin assembly were enriched by upregulated genes (Table S5B) and metabolism (oxidative phosphorylation, ATP metabolic process, metabolic pathways, etc.) were enriched by downregulated genes (Fig.2E and Table S5C). Notably, among 200 TFs annotated from differentially acetylated regions, 39 TFs showed significantly altered mRNA levels in PLN-R14del versus control hearts, including 26 up- and 13 down-regulated TF-coding genes. Enriched protein-protein-interaction network by 39 TF-coding genes again suggests (negatively) affected metabolism (Fig.2F). Thus, we identified a panel of upstream TFs and downstream targets in metabolic processes, which were disrupted in PLN-R14del cardiomyopathy. Differentially expressed genes in PLN-R14del hiPSC-CMs suggest altered lipid metabolic pathways Monolayers of hiPSC-CMs have been used to study the molecular mechanisms underlying several major cardiomyopathies, including ion-related, structural, and metabolic cardiomyopathies. However, the physiological immaturity of hiPSC-CMs severely limits their utility as a prediction model for adolescent genetic myopathies. To improve the cardiac immaturity limitation, we cultured hiPSC-CMs for 160 days in a maturation media designed to provide oxidative substrates adapted to the metabolic needs27 (Table S6). These long-term cultured hiPSC-CMs showed well-developed structural and mitochondrial organization as stained by the sarcomeric and mitochondrial marker (Fig.S7A). Hereafter, we studied the transcriptome profiles in PLN-R14del and healthy control hiPSC-CMs (Fig.3A and Table S1B). First, we examined markers of cytoskeletal components (ACTN1, TNNT2, MYH7, and MYL2), ion channels (KCNA5 and KCNJ4), and mitochondrial components (ATP5F1A and HSP60), and lipid metabolism (PPARA, ACAT1, FABP3, and Nile red staining). In general, we observed an increase in mRNA levels of most markers from short- and long-term (25 and 160 days, respectively)
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