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

Celik, Selvi LU ; Hyrefelt, Ludvig ; Czuba, Tomasz LU ; Li, Yuan LU ; Assis, Juliana ; Martinez, Julia LU orcid ; Johansson, Markus ; André, Oscar LU ; Synnergren, Jane and Sandstedt, Joakim , et al. (2025) In Cardiovascular Research 121(4). p.629-642
Abstract

Aims 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. Methods and results RNA-sequencing and RNA in situ hybridization (ISH) of cardiac tissue... (More)

Aims 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. Methods and results RNA-sequencing and RNA in situ hybridization (ISH) of cardiac tissue from heart failure (HF) patients, 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 analyse the effect of NATs on sarcomere properties. RNA ISH and 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 up-regulated in HF. Knockdown of TTN-AS1-276 in human iPS-CMs resulted in decreased interaction between 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. Conclusions 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.

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organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
Non-coding RNA, Sarcomere function, Splicing, Titin
in
Cardiovascular Research
volume
121
issue
4
pages
14 pages
publisher
Oxford University Press
external identifiers
  • scopus:105004745931
  • pmid:40042822
ISSN
0008-6363
DOI
10.1093/cvr/cvaf037
language
English
LU publication?
yes
id
702909bc-fc4c-4917-8f9f-e1c3d2adb893
date added to LUP
2025-08-13 10:42:43
date last changed
2025-08-13 12:36:27
@article{702909bc-fc4c-4917-8f9f-e1c3d2adb893,
  abstract     = {{<p>Aims 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 &gt;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. Methods and results RNA-sequencing and RNA in situ hybridization (ISH) of cardiac tissue from heart failure (HF) patients, 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 analyse the effect of NATs on sarcomere properties. RNA ISH and 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 up-regulated in HF. Knockdown of TTN-AS1-276 in human iPS-CMs resulted in decreased interaction between 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. Conclusions 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.</p>}},
  author       = {{Celik, Selvi and Hyrefelt, Ludvig and Czuba, Tomasz and Li, Yuan and Assis, Juliana and Martinez, Julia and Johansson, Markus and André, Oscar and Synnergren, Jane and Sandstedt, Joakim and Nordenfelt, Pontus and Vukusic, Kristina and Smith, J. Gustav and Gidlöf, Olof}},
  issn         = {{0008-6363}},
  keywords     = {{Non-coding RNA; Sarcomere function; Splicing; Titin}},
  language     = {{eng}},
  month        = {{03}},
  number       = {{4}},
  pages        = {{629--642}},
  publisher    = {{Oxford University Press}},
  series       = {{Cardiovascular Research}},
  title        = {{Antisense-mediated regulation of exon usage in the elastic spring region of Titin modulates sarcomere function}},
  url          = {{http://dx.doi.org/10.1093/cvr/cvaf037}},
  doi          = {{10.1093/cvr/cvaf037}},
  volume       = {{121}},
  year         = {{2025}},
}