215 Fatty Acid Oxidation in PLN R14del Cardiomyopathy 8 cultured PLN-R14del and healthy control hiPSC-CMs and confirmed these observations at the protein level by immunofluorescence staining (Fig.3B and Fig.3C). Therefore, we extended the culture time of hiPSC-CMs in the following experiments, which showed an overall improved maturation status but a more distinguishable metabolism-phenotype between PLN-R14del and control hiPSC-CMs. Since our data obtained from PLN-R14del hearts indicated a disrupted lipid metabolism, we cultured control and PLN-R14del hiPSC-CMs to further elucidate the metabolic activities in three culture media containing different amounts of glucose and lipids, which are the two main metabolic substrates for cardiomyocytes (Fig.3A). In total, we identified 952, 1,321, and 2,104 differentially expressed genes in PLN-R14del versus control hiPSC-CMs cultured in the maturation, the glucose-rich, and the lipid-rich medium, respectively (Table S7A-C). Notably, regardless of culture media, pathway enrichment analyses using downregulated genes in PLN-R14del versus control hiPSC-CMs consistently pointed towards metabolic activities, such as oxidative phosphorylation/GO:0006119 (Fig.3D, Fig.S8A-C, and Table S7D-F). However, it is important to note that the metabolic genes involved in the enriched biological processes (i.e., oxidative phosphorylation), which were shared among three conditions, were not the same (Fig.S8DF). Similarly, regardless of culture media, pathway enrichment analyses using upregulated genes in PLN-R14del versus control hiPSC-CMs consistently pointed towards fibrosis and (cardiovascular) development, which were in line with the results obtained from the ex vivo human cardiac tissues (Table S7D-F). Disturbed fatty acid oxidation (FAO) and metabolic flexibility in PLN-R14del hiPSCCMs Besides the downregulated transcriptional regulation of lipid metabolism in PLN-R14del cardiomyopathy, a significantly lower cellular metabolic activity/viability was also observed in PLN-R14del versus control hiPSC-CMs (Fig.4A). This suppression remained when excessive glucose or FAs were given to the cells (Fig.S7B-C). To further elucidate whether the obtained transcriptomic data could predict affected metabolism in PLN-R14del cardiomyopathy, we compared the FAO metabolism, a key metabolic program in cardiomyocytes (Fig.4B), by evaluating mitochondrial respiration via ETO-inhibited FAO and 2-DG-inhibited glycolysis using the Seahorse analysis (Fig.4C). In the maturation medium, which contains both glucose and FAs, we observed a comparable oxygen consumption rate (OCR) between PLN-R14del and control hiPSC-CMs at the baseline level and after ETO-inhibited FAO (Fig.4E), suggesting a similar FAO-dependence of both groups. However, after blocking glycolysis by 2-DG, we observed an increased OCR in control hiPSC-CMs, whereas the OCR of PLN-R14del hiPSCCMs continued to decline significantly, suggesting control hiPSC-CMs are less dependent on glycolysis and have more metabolic adaptive characteristics than PLN-R14del hiPSC-CMs. Similarly, in the glucose-rich medium, OCR was comparable between PLN-R14del and control hiPSC-CMs at the baseline level (Fig.4F). After blocking glycolysis, a significantly higher OCR was observed in control versus PLN-R14del hiPSC-CMs, once again suggesting better metabolic flexibility and substrate utilisation in control hiPSC-CMs. Notably, in the lipid-rich medium, the
RkJQdWJsaXNoZXIy MTk4NDMw