Abstract 12802: Intron-Mediated Enhancement of Titin ( TTN ) Regulates Sarcomere Formation and Function

Abstract

Background: Heterozygous truncating variants in the sarcomere protein titin (TTN) are the most common genetic cause of heart failure, a major cause of morbidity and mortality. This causality indicates that even two-fold changes in the amount of TTN can profoundly disturb cardiac physiology. Although a critical role of TTN in sarcomere formation and cardiomyocyte contractility is well established, the mechanisms regulating transcription of the TTN gene remain poorly understood. Methods: We performed bioinformatics analysis to identify a putative transcriptional enhancer of TTN . Next, we created biallelic deletion of the enhancer in human induced pluripotent stem cell derived cardiomyocytes and performed enhancer reporter assays both in vitro and in vivo to demonstrate necessity and sufficiency of the enhancer in TTN gene expression, respectively. Furthermore, we performed massive parallel reporter assay to define critical transcriptional factors of the TTN enhancer activity and analyzed whole genome sequencing (WGS) data of human patients with unexplained dilated cardiomyopathy (DCM). Results: We identified an intron mediated enhancer that promotes cardiac-specific TTN expression. Global deletion of this element downregulated TTN expression in cardiomyocytes and impaired sarcomere development, while transgenic expression promoted cardiac expression in mice. Using mutational scanning we defined key transcription factor binding sites, including NKX2-5 and MEF2 motifs that promote TTN expression in cardiomyocytes. Consistent with these functions, analyses of WGS data in 69 patients with unexplained DCM revealed one rare variant that disrupted the conserved MEF2 transcriptional factor binding motif. Conclusions: Discovery of a TTN enhancer advances our understanding of cardiomyocyte development, provides an opportunity to modulate TTN transcriptional activity, and ultimately develop therapeutic strategies to treat dilated cardiomyopathy caused by TTN haploinsufficiency.

Arrhythmias as Presentation of Genetic Cardiomyopathy

“…194 An intron-mediated TTN enhancer that promoted cardiac-specific TTN expression in similarly derived cardiomyocytes promoted normal TTN expression in mice. 195 Similarly, adenoviral mediated modulation of RNA splicing in induced pluripotent stem cells with an HCM-associated MYBPC3 mutation corrected the hypertrophic phenotype. 196 The first-in-human gene transfection with the LAMP2 gene has been reported for 3 patients with Danon disease, a HCM phenocopy.…”