Hypoplastic left heart syndrome (HLHS) is an often-lethal congenital heart defect in which babies are born with malfunctioning or under developed left ventricles. Infants with HLHS undergo a series of open-heart surgeries to restore systemic circulation. Even after surgery, children can develop heart failure and other complications due to the overload on the right ventricle (RV) that serves as the single ventricle. RV failure is a common cause of death and heart transplantation in children with HLHS. Very little is known about the etiology and pathogenic mechanisms of HLHS. Recently, aberrant alternative splicing (AS) and mRNA levels have been identified in the RV of infants with HLHS. Molecular mechanisms responsible for transcriptome changes in HLHS RVs are widely unknown. Our data show that the majority of transcripts differentially expressed in HLHS patient RVs are targets of the RNA binding protein Rbfox2, a master regulator of alternative splicing. We find that these Rbfox2 target genes have important roles in macromolecular metabolism, muscle structure/integrity, transcription and cell cycle regulation. In HLHS patient RVs, Rbfox2 subcellular distribution is altered such that its function in RNA metabolism is impaired. Consistent with our findings on the role of Rbfox2 in HLHS pathogenesis, a human genetic study have identified de novo damaging mutations in Rbfox2 that significantly associated with HLHS phenotype. Based on these findings, our hypothesis is that dysregulation of Rbfox2 contributes to HLHS pathogenesis by modulating AS and mRNA networks critical for heart function. We will test this hypothesis by three specific aims using several experimental systems including cardiac specific Rbfox2 knockout mouse model and heart biopsy samples from HLHS patients with respective control heart samples. In the first aim, we will define mechanisms that disrupt Rbfox2 localization and function in HLHS patient RVs. We will investigate the consequences of newly identified Rbfox2 mutations associated with HLHS phenotype on Rbfox2 localization and function in RNA metabolism. Ultimately, our findings will provide new tools to restore Rbfox2 activity and correct global transcriptome changes observed in infants with HLHS. In the second aim, we will determine the mechanism by which Rbfox2 controls pathophysiologically relevant AS events altered in HLHS infant hearts. Discovery of functionally relevant AS events in HLHS patients will allow us to initiate future translational studies to fix AS defects using modified oligos that target Rbfox2 binding sites within pre-mRNAs mis-spliced in HLHS patients. In the last aim, we will elucidate how Rbfox2 regulates mRNA targets differentially expressed in HLHS patient hearts. Our findings will impact future studies to identify novel strategies to reestablish normal mRNA patterns. Results from these studies will identify a post-transcriptional network coordinately regulated by Rbfox2 that is disrupted in HLHS patients, and ultimately enable the development of innovative therapeutic strategies by restoring aberrant gene expression using modified oligos or by correcting Rbfox2 localization and activity.
Even though mutations in Rbfox2 gene are significantly associated with hypoplastic left heart syndrome phenotype, it is unclear how Rbfox2 contributes to hypoplastic left heart syndrome pathogenesis. In this project, we will directly test the effect of human mutations on Rbfox2 function in RNA metabolism and determine how Rbfox2 target genes aberrantly expressed in hypoplastic left heart syndrome patient hearts contribute to disease pathogenesis. Ultimately, our findings may provide novel and effective therapeutic options for infants with hypoplastic left heart syndrome by restoring Rbfox2 function.
|Belanger, KarryAnne; Nutter, Curtis A; Li, Jin et al. (2018) CELF1 contributes to aberrant alternative splicing patterns in the type 1 diabetic heart. Biochem Biophys Res Commun 503:3205-3211|
|Nutter, Curtis A; Kuyumcu-Martinez, Muge N (2018) Emerging roles of RNA-binding proteins in diabetes and their therapeutic potential in diabetic complications. Wiley Interdiscip Rev RNA 9:|
|Belanger, KarryAnne; Nutter, Curtis A; Li, Jin et al. (2018) A developmentally regulated spliced variant of PTBP1 is upregulated in type 1 diabetic hearts. Biochem Biophys Res Commun :|
|Nutter, Curtis A; Jaworski, Elizabeth; Verma, Sunil K et al. (2017) Developmentally regulated alternative splicing is perturbed in type 1 diabetic skeletal muscle. Muscle Nerve 56:744-749|