211 Fatty Acid Oxidation in PLN R14del Cardiomyopathy 8 Genes annotated to differentially acetylated regions in PLN-R14del cardiac tissues are enriched in metabolic pathways To identify genes potentially regulated by the differentially acetylated regions, we focused on regions in close vicinity to promoters and annotated genes 5,000 bases up- and downstream from the transcription start site of a gene as used previously (Fig.1B).25,26 Out of 968 hyperacetylated regions, 295 genes were identified in close vicinity to 251 hyperacetylated regions, and out of 1,149 hypoacetylated regions, 568 genes were identified in the close vicinity to 462 hypoacetylated regions (Fig.1C, Table S2B and 2C). To examine which biological processes and pathways are affected, we performed gene set enrichment analysis using genes annotated to differentially acetylated regions. We observed that hyperacetylation-related genes were mostly involved in fibrosis, (cardiovascular) development, and chromatin assembly (Table S2D and Fig.S2), while hypoacetylation-related genes were related to metabolism (Fig.1D and Table S2E). The transcription factor binding motifs (TFBMs) overrepresented in hypoacetylated regions are enriched in metabolic pathways To identify possible upstream acting transcription factors (TFs), which regulate genes involved in the pathogenesis of the disease, we studied the overrepresentation of TFBMs in differentially acetylated regions. By using the DNA sequences of all differentially acetylated regions in PLN-R14del versus control hearts (Fig.1B), we detected enrichment in 202 TFBMs and annotated them to 200 TF-encoding genes (Table S3A). Consistently, several of the most enriched biological processes annotated to TFs pointed towards altered metabolism, such as adipogenesis and mitochondrial structure (Table S3B and Fig.S3). Notably, PPARA, a major regulator of cardiomyocyte lipid metabolism, particularly FAO, was also annotated from enriched motifs together with other interacting TFs (Fig.1E). Therefore, we further investigated the localization of PPARA in cardiac tissues by immunofluorescence staining and observed a significant decrease in the nuclear PPARA signal of PLN-R14del cardiomyocytes versus the controls, whereas the PPARA signal in non-myocyte cells remained comparable between PLN-R14del and control hearts (Fig.1F). Hypoacetylated regions associated with metabolic pathways specific for PLNR14del cardiomyopathy as compared to other cardiomyopathies Besides non-failing control hearts, we also compared PLN-R14del hearts with other cardiomyopathies, including ischemic cardiomyopathy (n=4) and non-ischemic dilated cardiomyopathy (sarcomeric group, n=6, Fig.1G). K-mean clustering analysis revealed four PLN-R14del-specific clusters when compared to other cardiomyopathy groups and the controls (Fig.1H, Fig.S4, and Table S4A). Genes located in the vicinity of these PLN-R14delspecific clusters were again highly enriched in metabolic signalling (Table S4B and 4C, and Fig.S5). Examples of metabolic genes, including HADHA/HADHB, SLC25A20, PDK2, and CPT1B,
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