221 Fatty Acid Oxidation in PLN R14del Cardiomyopathy 8 DISCUSSION In this study, we provide new information on changes in chromatin activity and global transcriptional regulation in human myocardium obtained from patients with PLN-R14del cardiomyopathy compared to other types of cardiomyopathy and healthy controls. Multiomics integration points to the inhibited mitochondrial function and (lipid) metabolism in PLN-R14del hearts based on the changed histone acetylation levels of annotated gene promoters, predicted TFBMs and altered gene expression. These datasets will serve as the basis for upcoming biomarker and novel therapeutic strategies. We particularly focused on the inhibited PPARA-mediated FAO in PLN-R14del hearts. PPARA is a key TF that regulates cardiac lipid metabolism to enhance FAO.29 It is predominantly located within the nucleus,30 however, external signals and pathways regulate the transport of PPARA from the nucleus to other subcellular compartments and mediate the biological functions of PPARA.31 We obtained the enriched TFBM of PPARA in differentially acetylated promoters between PLNR14del and controls. Although the histone acetylation of PPARA promoter and mRNA level of PPARA remained comparable between PLN-R14del and control hearts, we showed a significant decline of PPARA signal inside cardiomyocyte nuclei in PLN-R14del hearts. Taken together, the loss of nuclear PPARA in PLN-R14del cardiomyocytes suggested suppressed PPARA-mediated biological processes that take place inside nuclei, including substrate inflexibility promoting FFA uptake and lipid accumulation, and ensuing cardiovascular complications. The promoters of PPARA-regulated downstream targets in lipid metabolism, such as HADHA, HADHB, MLYCD, and PNPLA2,32,33,34 showed suppressed acetylation levels in PLN-R14del hearts. Furthermore, by comparing PLN-R14del hearts with non-PLN-R14del-related cardiomyopathies, we identified PLN-R14del-specific regions and many annotated genes in these regions also involved in lipid metabolism (e.g. AGPAT2, HADHA, HADHB, MLYCD, PLPP1, and PTGDS). Additionally, several of these metabolic genes also showed decreased mRNA levels in PLN-R14del versus control hearts, including HADHA and HADHB. In line with these omics-based data, we also observed accumulated lipid droplets and abnormal mitochondrial morphology in PLNR14del hearts. Combined, the impaired PPARA-regulated FAO could play a critical role in PLN-R14del cardiomyopathy. A recent study also showed that 18-33 days old PLN-R14del engineered heart tissues had impaired energy metabolism reflected at the protein level, including suppressed FAs metabolism and accumulation of lipid droplets.6 Here, we further explored and identified affected genes in the metabolic regulation in long-term cultured PLNR14del and control hiPSC-CMs (>110 days). We demonstrated for the first time that PLN-R14del hiPSC-CMs displayed a lower FAO profile than the controls at both mRNA and functional levels, and this suppression pattern remained consistent even though PLN-R14del hiPCSCMs were given excessive amounts of FAs or glucose, indicating the profoundly impaired lipid metabolism. FAO changes serve as an indicator of an early adapted or maladapted metabolic response.35 Normally, the majority of the energy demand of the heart comes from mitochondrial FAO, especially free circulating FAs.36 In contrast, diseased cardiomyocytes suffer from a decreased FAO and an increased intracellular lipid accumulation resulting in lipotoxicity and cell death.37,38 We also showed lipid accumulation and a decreased cell viability of PLN-
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