Dosage-sensitive RBFOX2 autoregulation promotes cardiomyocyte differentiation through transcriptome maturation

Haploinsufficiency of the RNA splicing regulator, RBFOX2 , is linked to congenital heart disease (CHD), yet its pathogenic mechanisms remain unclear. Here, we demonstrate that RBFOX2 is essential for progressing cardiomyocyte (CM) differentiation by shifting exon usage profiles to more mature states in sarcomere, cytoskeletal, and focal adhesion genes, including ALPHA-ACTININ-2 (ACTN2). This maturation program is initiated by critical levels of RBFOX2 that facilitate self-splicing at mutually exclusive exons encoding early and late isoforms with distinct functional roles. In heterozygous CMs, self-splicing is disrupted, which skews isoform ratios and generates a dominant-negative caused by exon co-inclusion. Finally, we demonstrate that overexpression of correctly spliced ACTN2 rescues heterozygous, but not null, phenotypes by restoring contractility, which triggers a mechanosensing feedback loop involving upregulation of RBFOX2 from the wildtype allele and transcriptome maturation. Our data suggest that decreased RBFOX2 dosage and self-splicing impairs CM differentiation, contributing to CHD pathogenesis and heart failure susceptibility.