HIF-1 regulates common bidirectional gene expression changes during hypoxia and iron deficiency in human induced pluripotent stem cell derived cardiomyocytes

Abstract

Abstract Background Heart failure regardless of cause is characterised by extensive transcriptional reprogramming and a switch to foetal developmental programmes via unknown mechanisms (1-3). An additional factor that adversely impacts heart failure patients is iron deficiency (ID)(4). ID is also associated with worse prognosis in acute coronary syndromes (5). However, the mechanisms by which ID interferes with clinical outcomes in heart disease remain incompletely understood. Purpose The purpose of this study was to investigate gene expression changes in human cardiomyocytes during hypoxia and ID, two important stressors in the context of ischaemia and heart failure. Methods In this study, we use human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) derived through a protocol that promotes left ventricle cardiomyocyte differentiation (6). Cells were harvested at different stages of differentiation (day 6 and day 32) and mature (day 32) hiPSC-CMs were treated with 30 μM deferoxamine to induce ID either under normoxic or hypoxic conditions with 1% O2. hiPSC contraction was assessed using musclemotion software and metabolism through use of a Seahorse extracellular flux analyser. RNA was isolated using standard isolation kits and RNA-seq (n=5) was performed. Data was analysed for gene expression by alignment with the human hg19 reference genome using the software STAR. A log2 fold change of 1 and P value of <0.0001 were considered significant. Results We show that both hypoxic and ID hiPSC-CMs demonstrate impaired contractility and reduced oxygen consumption, features of cardiomyocytes from failing hearts. Importantly, hiPSC-CMs exposed to hypoxia, ID or the combination of the two exhibit bidirectional gene expression changes with significant overlap (Figure 1). Focusing on the commonly affected gene set, it becomes evident that hypoxia & ID work via similar mechanisms involving activation of the HIF-1 pathway, that in turn orchestrates the metabolic switch to glycolysis and glutamine oxidation similar to foetal cells. Overall, hypoxia promotes a shift towards immature gene programmes, with 45% of dysregulated genes in hypoxia mimicking expression patterns of less mature cells. Interestingly changes in long non-coding RNA (lncRNA) profiles in hypoxia and ID mimic gene expression changes in these conditions (figure 2). Although restoring oxygen levels in hypoxic hiPSC-CMs rescues the hypoxic transcriptional signature, hiPSC-CMs previously exposed to hypoxia can use glycolytic pathways more efficiently upon further exposures to hypoxia. Conclusion These results shed light into the mechanisms ID impacts heart disease patients and are suggestive that HIF-1 is a key player in orchestrating the gene switch in ID and hypoxia.