Abstract
Mutations and inadequate methylation profiles of CITED2 are associated with human congenital heart disease (CHD). In mouse, Cited2 is necessary for embryogenesis, particularly for heart development, and its depletion in embryonic stem cells (ESC) impairs cardiac differentiation. We have now determined that Cited2 depletion in ESC affects the expression of transcription factors and cardiopoietic genes involved in early mesoderm and cardiac specification. Interestingly, the supplementation of the secretome prepared from ESC overexpressing CITED2, during the onset of differentiation, rescued the cardiogenic defects of Cited2-depleted ESC. In addition, we demonstrate that the proteins WNT5A and WNT11 held the potential for rescue. We also validated the zebrafish as a model to investigate cited2 function during development. Indeed, the microinjection of morpholinos targeting cited2 transcripts caused developmental defects recapitulating those of mice knockout models, including the increased propensity for cardiac defects and severe death rate. Importantly, the co-injection of anti-cited2 morpholinos with either CITED2 or WNT5A and WNT11 recombinant proteins corrected the developmental defects of Cited2-morphants. This study argues that defects caused by the dysfunction of Cited2 at early stages of development, including heart anomalies, may be remediable by supplementation of exogenous molecules, offering the opportunity to develop novel therapeutic strategies aiming to prevent CHD.
Highlights
Despite clinical improvements and efforts to sensitize the population about heart disease risk factors and habits for prevention, the global burden of cardiovascular disease has increased worldwide[1]
Delayed mesoderm commitment of Cited2-depleted cells To investigate the mechanisms underlying the cardiogenic defects of Cited2-depleted embryonic stem cells (ESC), we compared gene expression profiles at day 4 (D4) of differentiation of cells derived from C2fl/fl[Cre] ESC treated at the onset of differentiation for 48 h either with 4-hydroxytamoxifen (4HT) to deplete Cited[2] or with ethanol, as previously described[22]
The functional enrichment analysis for these differentially expressed genes (DEGs) using Enrichr[30,31], indicated that many upregulated genes at D4 of differentiation are related to cardiovascular development, and epithelial-to-mesenchymal transition (EMT) which is an early event of ESC commitment to mesoderm (Fig. S1)
Summary
Despite clinical improvements and efforts to sensitize the population about heart disease risk factors and habits for prevention, the global burden of cardiovascular disease has increased worldwide[1]. Congenital heart disease (CHD) affects nearly 1% of new-borns and results from structural and functional cardiac anomalies occurring. Ventricular (VSD) and atrial (ASD) septal defects, transposition of the great arteries and the tetralogy of Fallot (TOF) are the most frequent heart anomalies associated with CITED2 variants in humans. Most of CITED2 variants identified in patients with CHD, only marginally affect the capacity of CITED2 to repress HIF-1α transcriptional activity and/or to co-activate TFAP2C transcription factor ex vivo[4,5,6,7,8,9,10]. CITED2 variants identified within the 5ʹ and 3ʹ-untranslated regions associated with ASD, VSD, and TOF4, may result in abnormal stability, processing, localization or translation of the CITED2 transcripts, and decrease
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