Abstract Body: Background: Hypoplastic Left Heart Syndrome (HLHS) is a severe congenital heart defect that disrupts normal systemic circulation. Infants with HLHS are born with an underdeveloped left ventricle, a narrowed or absent ascending aorta, a malformed mitral valve, and defects such as a patent ductus arteriosus and atrial septal abnormalities. Because the left side of the heart cannot effectively pump blood to the body, survival depends on a series of complex surgeries to reroute circulation. Despite surgical intervention, individuals with HLHS face lifelong cardiovascular challenges and require ongoing medical care. Mutations in the RNA-binding protein RBFOX2 is strongly linked to HLHS. It is poorly understood how the loss of an RNA binding protein contributes to cardiovascular defects. To investigate this, we generated Rbfox2 conditional-knockout mouse model (Rbfox2-CKO) defects in formation of 4-chambered heart, outflow tract and yolk sac vasculature. Importantly, our Rbfox2 conditional knockout mouse model recapitulated several molecular and phenotypic features of HLHS.
Hypothesis: Based on our preliminary results, we hypothesize that RBFOX2 is important for cardiovascular development.
Method/Results: To determine the molecular drivers of cardiovascular defects in Rbfox2-CKO mouse model, we performed enhanced crosslinking immunoprecipitation (eCLIP) followed by RNA-sequencing to determine RNA targets of RBFOX2 in embryonic mouse hearts. Our eCLIP-seq data revealed genes involved in venous vs arterial specification as targets of RBFOX2. To further investigate this, we generated human iPSCs with HLHS patient mutations (RBFOX2^HLHS-hiPSCs) and differentiated them to venous vs arterial ECs similar to isogenic iPSC line. Our preliminary results revealed RBFOX2^HLHS-hiPSCs exhibited aberrant in alternative splicing and expression levels of genes required for artery and vein commitment.
Conclusions: Our preliminary results suggest that RBFOX2 loss disrupts RNA processing and expression levels of target genes required for EC specification. Our work will investigate the mechanistic insights into how RBFOX2 mediated RNA processing impacts endothelial cell identity.