202 Chapter 8 fetus-like energy management is insufficient to meet adult energy consumption needs and it will disturb the capacity of FAO-based ATP synthesis, leading to further starvation of the heart and disease progression.24 The impaired lipid metabolism could hereby contribute to the disease progression in PLN-R14del cardiomyocytes and warrant further investigation. Here, we explored the role of transcriptional regulation on disturbed (lipid) metabolism in PLNR14del cardiomyopathy. We showed that even after the removal of the dominant fibrofatty replacement in the subepicardial layer, the fingerprint of (lipid) metabolic dysregulation was still present in the remaining human myocardium. We further confirmed the disturbed (lipid) metabolism in human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) from PLN-R14del carriers. We also identified key transcription factors involved in the affected (lipid) metabolism, which can serve as promising targets for future therapeutic strategies. Furthermore, we examined the preference of energy sources and the metabolic flexibility in switching energy sources between PLN-R14del and wildtype hiPSC-CMs, including the effect of major energy substrate depletion. Finally, we employed two rescue methods, namely CRISPR/ Cas9-based gene correction and an FAO-modulating compound bezafibrate), to investigate the association of the PLN-R14del pathogenic variant with intracellular lipid accumulation, mitochondrial lipid metabolism and the Ca2+ handling properties. Collectively, our data on the negative effects of energy substrate dysregulation in PLN-R14del cardiomyopathy provide novel insights into the new therapeutic strategies and clinical practice (i.e. drug repurposing). METHODS Study design and samples. This study was approved by the Biobank Research Ethics Committee, University Medical Centre Utrecht, Utrecht, The Netherlands, under protocol number 12-387 (cardiac tissues), and 14-513 (skin biopsies). Written informed consent was obtained or in certain cases waived by the ethics committee when obtaining informed consent was not possible due to the death of the individual. Cardiac tissues: Heart samples collected at autopsy or transplantation were obtained from a homogeneous cohort of PLN-R14del variant positive patients (n=6). Four control hearts obtained from rejected organ donors (n=3) or autopsy (n=1) were used as a reference. To further elaborate on PLN-R14del-specific changes, hearts from patients with ischemic cardiomyopathy (n=4) and non-ischemic cardiomyopathy based on pathogenic variants in genes encoding sarcomeric proteins (n=6) were also included. An overview of cardiac tissues is presented in Table S1A. Cardiac tissues used for ChIP-seq and RNA-seq were obtained from regions halfway between the atrioventricular valves and the apex and were stored at -80°C. The epicardial region, where fibrofatty replacement profoundly occurred, was excluded in PLN-R14del hearts and the cardiomyocyte-rich myocardial region was included in this study. From each individual, an adjacent block of tissue from the same biopsy was used for ChIP-seq and was paraffin-embedded and stained with Masson’s trichrome (Fig.S8A). High-resolution
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