In:
Circulation Research, Ovid Technologies (Wolters Kluwer Health), Vol. 125, No. Suppl_1 ( 2019-08-02)
Abstract:
Dilated cardiomyopathy (DCM) is a leading cause for heart failure and is associated with a rate of mortality of 20% within 5 years of diagnosis. The most common genetic causes for DCM are mutations of the sarcomere protein titin (encoded by TTN ), which occurs in 10-20% of DCM cases. Dominant DCM mutations truncate titin (TTNtv) and result in haploinsufficiency. Thus, strategies to increase the expression of the wild type TTN allele could attenuate damaging effects of TTNtv. Utilizing bioinformatic tools, we identified a putative enhancer for TTN in its intron 1. We deleted a 658 bp region from intron 1 which encompasses the region of interest in human induced pluripotent stem cells (hiPSCs) using CRISPR/Cas9 genome editing to validate its function. Utilizing RNA sequencing and qPCR of RNA harvested from hiPSC-derived cardiomyocytes (hiPSC-CMs), we demonstrated that a homozygous deletion in this region leads to a drop in TTN expression compared to the wild type (WT) control (0.344-fold change, p 〈 0.001). To further characterize this region, we subdivided it into three parts which we called E1 (296 bp), E2 (206 bp), and E3 (139 bp). E1 includes a highly conserved region and a region of open chromatin as identified by the Assay for Transposase-Accessible Chromatin Sequencing (ATAC-Seq) performed on hiPSC-CMs. A homozygous E1 deletion resulted in a decreased TTN expression of 0.63-fold compared to the WT control (p 〈 0.001) when performing RNA sequencing on hiPSC-CMs. Both homozygous E2 and E3 deletions resulted in an increased TTN expression (1.56-fold change, p 〈 0.001; 1.19 fold change, p 〈 0.001). Utilizing a published sarcomere tracking platform, SarcTrack, to investigate hiPSC-CM physiology, we saw a decreased contractility of 6.6% in hiPSC-CMs carrying a homozygous E1 deletion compared to 10.1% in the WT control (p 〈 0.001). Cells carrying homozygous E2 or E3 deletions were hypercontractile (13.8%, p 〈 0.001; 13.7%, p 〈 0.001). Given our results, we hypothesize that TTN expression depends on the E1 region. If confirmed, we expect that increasing the activity of this enhancer using small molecules may provide a novel therapeutic target for DCM caused by TTNtv.
Type of Medium:
Online Resource
ISSN:
0009-7330
,
1524-4571
DOI:
10.1161/res.125.suppl_1.104
Language:
English
Publisher:
Ovid Technologies (Wolters Kluwer Health)
Publication Date:
2019
detail.hit.zdb_id:
1467838-X
