Functional analysis of TTN uORFs reveals context-dependent translational regulation

Summary

Dilated cardiomyopathy is a condition where the heart becomes enlarged and weaker, which can make it harder to pump blood properly. One important cause is a change in a gene called TTN, which helps make a protein called titin. People with some TTN changes make too little titin.

In this study, researchers looked at two small sections of TTN called uORFs. These sections can act like brakes, reducing how much protein a cell makes. In simple laboratory tests, removing the uORFs increased protein production.

The researchers then tested the same idea in heart cells made from human stem cells. They used CRISPR to remove the uORFs from the natural TTN gene. Surprisingly, this did not increase titin levels, either in healthy cells or cells carrying a TTN disease-causing change.

The study shows that a genetic change that works in a simple laboratory test may not work in real cells. The findings suggest that targeting TTN uORFs is unlikely to be a useful treatment for this form of heart disease.

Keywords

Dilated cardiomyopathy, TTN, titin, genetic disease, CRISPR-Cas9, gene regulation, uORFs, antisense oligonucleotides, cardiomyocytes, stem cells, gene therapy

Abstract

Familial dilated cardiomyopathy (DCM) is a common condition with a high clinical burden, and no therapies that target the underlying genetic mechanisms. The leading genetic cause of DCM is heterozygous truncating variants in TTN, which are thought to drive disease through haploinsufficiency, where titin is reduced. This raises the possibility of treating disease through therapeutic upregulation of endogenous TTN expression. Upstream open reading frames (uORFs) are regulatory elements that repress translation and have been targeted to upregulate gene expression in a range of genes. Two uORFs in the TTN 5' untranslated region have been shown to repress expression in luciferase reporter assays, suggesting potential for therapeutic targeting. Here, we investigated the role of TTN uORFs in reporter systems and in the endogenous locus in a human-induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) model. While disruption of uORFs increased expression in luciferase reporter gene assays, including in hiPSC-CMs, CRISPR-Cas9-mediated disruption of uORFs at the endogenous TTN locus in hiPSC-CMs did not increase titin protein expression. These findings indicate that uORF-mediated repression of TTN is context-dependent and not recapitulated in a disease-relevant model. This highlights the complex regulation of TTN expression and the importance of validating regulatory elements in their endogenous context.

Keywords: MT: RNA/DNA editing; TTN; cardiomyopathy; dilated cardiomyopathy; genetic therapy; titin; uORF; upstream open reading frame.