Mengmeng Huang

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.

SUMMARY: 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 patterns in sarcomere, cytoskeletal, and focal adhesion genes, including alpha-actinin-2 ( ACTN2 ). This maturation program is initiated by critical levels of RBFOX2 that facilitate autoregulatory splicing at mutually exclusive exons encoding early and late isoforms with distinct functional roles. In heterozygous CMs, autoregulation is disrupted, which skews isoform ratios and generates a dominant-negative product caused by exon co-inclusion. Finally, we demonstrate that overexpression of 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 autoregulation impair CM differentiation, contributing to CHD pathogenesis and heart failure susceptibility.

Aortic root aneurysm is a common cause of morbidity and mortality in Loeys-Dietz and Marfan syndromes, where perturbations in transforming growth factor beta (TGFβ) signaling play a causal or contributory role, respectively. Despite the advantages of cross-species disease modeling, animal models of aortic root aneurysm are largely restricted to genetically engineered mice. Here, we report that zebrafish devoid of the genes encoding latent-transforming growth factor beta-binding protein 1 and 3 (ltbp1 and ltbp3, respectively) develop rapid and severe aneurysm of the outflow tract (OFT), the aortic root equivalent. Similar to syndromic aneurysm tissue, the distended OFTs display evidence for paradoxical hyperactivated TGFβ signaling. RNA-sequencing revealed significant overlap between the molecular signatures of disease tissue from mutant zebrafish and a mouse model of Marfan syndrome. Moreover, chemical inhibition of TGFβ signaling in wild-type animals phenocopied mutants but chemical activation did not, demonstrating that TGFβ signaling is protective against aneurysm. Human relevance is supported by recent studies implicating genetic lesions in LTBP3 and, potentially, LTBP1 as heritable causes of aortic root aneurysm. Ultimately, our data demonstrate that zebrafish can now be leveraged to interrogate thoracic aneurysmal disease and identify novel lead compounds through small-molecule suppressor screens.This article has an associated First Person interview with the first author of the paper.