Regulation of sarcomere formation and function in the healthy heart requires a titin intronic enhancer
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
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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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 5

Predicted MEF2- and NKX2-5–binding motifs within E1 are critical for its transcriptional activity in vivo.

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Predicted MEF2- and NKX2-5–binding motifs within E1 are critical for its...
(A) Schematic of the TTN enhancer constructs used for transgenic mouse reporter assay. (B) Transgenic mouse enhancer assay. Addition of human E1 sequences to the minimal Shh promoter was sufficient to induce transgene (β-gal) expression in a cardiac-specific manner, which was visualized as blue on a whole-mount staining of E11.5 mouse embryos. Mutation of the predicted MEF2- and NKX2-5–binding sites within E1 abrogated its expression. Expression pattern of the reporter gene was consistent among 5–8 transgenic mouse embryos within each construct group tested. Photos of representative embryos are shown in this figure. Scale bar: 200 μm. (C) A rare noncoding variant in E1 identified in a patient with DCM. Among 69 LV samples from patients with DCM studied by WGS, 1 DCM sample had a rare (MAF <2.00 × 10–5) variant within a highly conserved residue in a predicted MEF2-binding motif in E1 (chr2:178,806,843T>C; red box). Modified from the UCSC genome browser (31) and the Integrative Genomics Viewer (60). (D) TTN expression based on single nucleus RNA-Seq analysis. Each dot represents a single cell. The P value was calculated by Wilcoxon rank-sum test and adjusted for multiple testing. WT: 5 replicates from 3 differentiations; MEF2_2_T>C: 4 replicates from 2 differentiations. (E) Flow cytometric analysis of TTN-GFP hiPSC-CMs harboring a biallelic MEF2_2T>C mutation. Composite data are from 4 independent CM differentiations.