Antisense-mediated regulation of exon usage in the elastic spring region of Titin modulates sarcomere function

Alternative splicing of Titin (TTN) I-band exons produce protein isoforms with variable size and elasticity, but the mechanisms whereby TTN splice factors regulate exon usage and thereby determining cardiomyocyte passive stiffness and diastolic function, is not well understood. Non-coding RNA transcripts from the antisense strand of protein-coding genes have been shown to regulate alternative splicing of the sense gene. The TTN gene locus harbours >80 natural antisense transcripts (NATs) with unknown function in the human heart. The aim of this study was to determine if TTN antisense transcripts play a role in alternative splicing of TTN. RNA-sequencing and RNA in situ hybridization (ISH) of cardiac tissue from heart failure patients (HF), unused donor hearts and human iPS-derived cardiomyocytes (iPS-CMs) were used to determine the expression and localization of TTN NATs. Live cell imaging was used to analyze the effect of NATs on sarcomere properties. RNA ISH, immunofluorescence was performed in iPS-CMs to study the interaction between NATs, TTN mRNA and splice factor protein RBM20.We found that TTN-AS1-276 was the predominant TTN NAT in the human heart and that it was upregulated in HF. Knock down of TTN-AS1-276 in human iPS-CMs resulted in decreased interaction between the splicing factor RBM20 and TTN pre-mRNA, decreased TTN I-band exon skipping, and markedly lower expression of the less compliant TTN isoform N2B. The effect on TTN exon usage was independent of sense-antisense exon overlap and polymerase II elongation rate. Furthermore, knockdown resulted in longer sarcomeres with preserved alignment, improved fractional shortening and relaxation times. We demonstrate a role for TTN-AS1-276 in facilitating alternative splicing of TTN and regulating sarcomere properties. This transcript could constitute a target for improving cardiac passive stiffness and diastolic function in conditions such as heart failure with preserved ejection fraction.