Feria A. Ladha

BACKGROUND: Haploinsufficiency of the RNA splicing regulator RBFOX2 has been linked to congenital heart disease, including hypoplastic left heart syndrome, yet its pathogenic mechanisms remain unresolved. METHODS: Human induced pluripotent stem cells heterozygous or null for RBFOX2 were created and their cardiomyocyte (CM) derivatives were characterized in monolayer culture and engineered heart tissues. In addition to cellular phenotyping, molecular profiling by RNA sequencing and enhanced crosslinking and immunoprecipitation sequencing was performed to detect changes in gene expression, exon usage, and RBFOX2 (RNA-binding fox-1 homolog 2) binding. These data sets were integrated to identify RBFOX2-bound RNAs with altered expression and splicing and to compare them with publicly available data sets from progressive CM differentiation time points. Rescue experiments were performed in induced pluripotent stem cell-derived CMs, engineered heart tissues, and zebrafish. RESULTS: We demonstrate that RBFOX2 is essential for CM differentiation by driving transcriptome maturation through gene expression- and alternative splicing-based isoform switching in transcripts primarily encoding sarcomere, cytoskeletal, and focal adhesion components, including the critical Z-disc factor ACTN2 (alpha-actinin-2). We further find that threshold levels of RBFOX2 facilitate autoregulatory splicing of mutually exclusive exons, generating early and late RBFOX2 isoforms with distinct functions. In heterozygous CMs, this threshold is not reached, resulting in isoform imbalance and production of an aberrant transcript, which further reduces functional RBFOX2 levels to well below 50% of wild-type. Finally, we demonstrate that ACTN2 overexpression rescues heterozygous, but not null, phenotypes, suggesting that ACTN2 activates an RBFOX2-dependent feedback loop that promotes transcriptome maturation. CONCLUSIONS: Our data demonstrate that reduced RBFOX2 dosage and impaired autoregulation stall CM differentiation by inhibiting transcriptome maturation, contributing to congenital heart disease pathogenesis and potentially increasing heart failure susceptibility following surgical intervention.