Regulation of sarcomere formation and function in the healthy heart requires a titin intronic enhancer
Yuri Kim, … , J.G. Seidman, Christine E. Seidman
Yuri Kim, … , J.G. Seidman, Christine E. Seidman
Published December 17, 2024
Citation Information: J Clin Invest. 2025;135(4):e183353. https://doi.org/10.1172/JCI183353.
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Research Article Cardiology Genetics

Regulation of sarcomere formation and function in the healthy heart requires a titin intronic enhancer

Abstract

Heterozygous truncating variants in the sarcomere protein titin (TTN) are the most common genetic cause of heart failure. To understand mechanisms that regulate abundant cardiomyocyte (CM) TTN expression, we characterized highly conserved intron 1 sequences that exhibited dynamic changes in chromatin accessibility during differentiation of human CMs from induced pluripotent stem cells (hiPSC-CMs). Homozygous deletion of these sequences in mice caused embryonic lethality, whereas heterozygous mice showed an allele-specific reduction in Ttn expression. A 296 bp fragment of this element, denoted E1, was sufficient to drive expression of a reporter gene in hiPSC-CMs. Deletion of E1 downregulated TTN expression, impaired sarcomerogenesis, and decreased contractility in hiPSC-CMs. Site-directed mutagenesis of predicted binding sites of NK2 homeobox 5 (NKX2-5) and myocyte enhancer factor 2 (MEF2) within E1 abolished its transcriptional activity. In embryonic mice expressing E1 reporter gene constructs, we validated in vivo cardiac-specific activity of E1 and the requirement for NKX2-5– and MEF2-binding sequences. Moreover, isogenic hiPSC-CMs containing a rare E1 variant in the predicted MEF2-binding motif that was identified in a patient with unexplained dilated cardiomyopathy (DCM) showed reduced TTN expression. Together, these discoveries define an essential, functional enhancer that regulates TTN expression. Manipulation of this element may advance therapeutic strategies to treat DCM caused by TTN haploinsufficiency.

Authors

Yuri Kim, Seong Won Kim, David Saul, Meraj Neyazi, Manuel Schmid, Hiroko Wakimoto, Neil Slaven, Joshua H. Lee, Olivia Layton, Lauren K. Wasson, Justin H. Letendre, Feng Xiao, Jourdan K. Ewoldt, Konstantinos Gkatzis, Peter Sommer, Bénédicte Gobert, Nicolas Wiest-Daesslé, Quentin McAfee, Nandita Singhal, Mingyue Lun, Joshua M. Gorham, Zolt Arany, Arun Sharma, Christopher N. Toepfer, Gavin Y. Oudit, William T. Pu, Diane E. Dickel, Len A. Pennacchio, Axel Visel, Christopher S. Chen, J.G. Seidman, Christine E. Seidman

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Figure 2

TTN regulatory elements are sufficient to drive gene expression in CMs and necessary for TTN expression.

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TTN regulatory elements are sufficient to drive gene expression in CMs a...
(A) GFP reporter gene assay in hiPSC-CMs. TTN regulatory elements E0 and E1 when inserted into a GFP expression vector with minimal promoter were able to induce GFP expression in hiPSC-CMs at differentiation day 30. In contrast, neither E2 nor E3 was able to activate GFP expression in hiPSC-CMs. Scale bar: 100 μm. (B) Quantification of GFP intensity by flow cytometric analysis. GFP intensity of hiPSC-CMs, which were transduced with lentivirus containing each TTN regulatory element, minimal promoter, and the GFP reporter gene, is shown. (–) indicates lentivirus containing minimal promoter and the GFP reporter gene. Data represent the mean ± SD. **P < 0.01 and ***P < 0.001, by unpaired, 2-tailed t test with Welch’s correction. TTN expression in hiPSC-CMs upon deletion of the TTN regulatory element E0 (C) and E1 (D). Multiplexed single nuclei RNA-Seq analysis was performed using hiPSC-CMs at differentiation day 30 from 2 and 4 independent differentiations, respectively. ****P < 0.0001, by unpaired, 2-tailed t test with Welch’s correction. Transcript level of full-length (E) and Cronos (F) isoforms of TTN. qPCR was performed in hiPSC-CMs at differentiation day 30. n = 3 per genotype, each set (WT, E1del/+, and E1del/del) from independent differentiations. TTN expression values normalized to WT are shown. P values were calculated using an unpaired, 2-tailed t test with Welch’s correction. (G) Total protein analysis of hiPSC-CMs using SDS-agarose gel stained with Coomassie blue. n = 3 per genotype, each set (WT, E1del/+, and E1del/del) from independent differentiations. Cell lysates were collected on differentiation day 30. Quantification of (H) N2BA and N2B and (I) T2 and Cronos TTN isoforms relative to WT. MHC was used for normalization. P values were calculated using an unpaired, 2-tailed t test with Welch’s correction.