353 General Discussion - hiPSC-CMs Disease Modelling and Future Perspectives 13 heart tissue. This RNA-seq data showed that in hiPSC-CMs, the cell cycle gene expression is inversely correlated to sarcomere gene expression and maturity of CMs. In Chapter 8, we used the metabolic maturation of patient-derived hiPSC-CMs, to provoke the molecular scenery of the cardiomyocytes from a PLN-R14del patient. To clarify the cell regulation on all levels, we combined the transcriptional regulation analysis in human primary tissue and validated this expression in a unique long-term (160 days) matured hiPSC-CM model. First, the multi-omics integration hinted us to disturbed mitochondrial function and (lipid) metabolism in PLNR14del hearts based on the changed histone acetylation levels of annotated gene promoters, predicted transcription factor binding motifs (TFBMs), and altered gene expression. Next, we demonstrated, that PLN-R14del hiPSC-CMs displayed a lower fatty acid oxidation (FAO) profile than the controls at both mRNA and functional levels, the suppression pattern remained consistent even though PLN-R14del hiPCS-CMs were given excessive amounts of FAs or glucose, indicating the profoundly impaired lipid metabolism. Additionally, to the best of our knowledge, we showed for the first time, the potential of bezafibrate in re-activating mitochondrial FAO and improving Ca2+ transients, which provides a novel strategic path for developing precision medicine for PLN-R14del patients, such as targeting FAO upstream regulators (i.e. PPARA). In a recent study, multi-omics analysis (plasma/cardiac tissue metabolomics, genome-wide RNA-seq, and proteomic studies) revealed profound metabolic abnormalities in human failing hearts in 87 explanted human hearts from 39 patients with end-stage HF compared with 48 non-failing donors.28 Substantial reductions in fatty acids and acylcarnitines were observed in these failing hearts, despite plasma elevations, suggesting the same defective import of FA into cardiomyocytes as we described in Chapter 8 and Chapter 12. As a result, the glucose levels were elevated in the end-stage HF hearts, similar to our glucose dependency of the PLN-R14del hiPSC-CMs. This study proved that starting with ChiP-sequencing and RNA-seq from failing hearts identify gene pattern alterations, which could be confirmed by the RNA-seq of hiPSC-CMs of heart failure patients in the early stage of the disease. Next, we showed that single-cell transcriptomic analysis of metabolically matured patientderived hiPSC-CMs revealed the induction of the unfolded protein response (UPR) pathway in PLN-R14del hiPSC-CMs as compared to isogenic control hiPSC-CMs (Chapter 9). Additionally, this disturbance of UPR expression was also detected in bulk RNA-sequencing of human hearts from PLN-R14del patients. Next, we used both 2D and 3D in vitro models to evaluate the impact of modulating disease-relevant pathways in PLN-R14del hiPSC-CMs. hiPSC-CM contractility analysis in both the 2D and 3D models recapitulated the contractile deficit associated with the disease in vitro, which could be restored by activating the UPR with a small molecule activator. Both studies (Chapter 8 and Chapter 9) provided insight into the transcriptional regulation of both human tissues and hiPSC-CMs of PLN-R14del patients to understand the molecular consequences of the pathogenic mutation and subsequent development of therapeutic interventions.
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