In:
Circulation Research, Ovid Technologies (Wolters Kluwer Health), Vol. 129, No. Suppl_1 ( 2021-09-03)
Abstract:
Transcriptional changes in cardiomyocytes drive heart failure progression, however, precise control over endogenous gene expression remains challenging. The expression of Krueppel-like factor 15 ( KLF15 ), an evolutionary conserved nuclear and cardiomyocyte specific inhibitor of WNT/CTNNB1 signalling in the heart, is lost upon cardiac remodelling, and accompanied by aberrantly active WNT/CTNNB1 resulting in heart failure progression. We investigated KLF15 expression dynamics employing CRISPR/Cas9-based tools in mouse cardiomyocytes in vivo and in human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CM) under the hypothesis that re-establishment of KLF15 levels in myocardial stress conditions prevents heart failure progression. Using a mouse model expressing enzymatically inactive Cas9 (dCas9) fused to transcriptional activators (VPR) under Myh6 -promoter control, we activated Klf15 in a murine pressure overload model by transverse aortic constriction. Delivery of Klf15 gRNAs targeted to the Klf15 promoter region via AAV9 induced Klf15 expression sufficiently to re-normalize Klf15 expression to transcript levels comparable to sham surgery hearts. This was accompanied by reduced decrease of fractional shortening as well as reduced cardiomyocyte hypertrophy in stressed Klf15 re-activated hearts compared to non-trageted (NT) gRNA hearts (n=3-8 per group, echo data from 4 and 8 weeks post-surgery). We achieved titratable KLF15 activation in dCas9VPR transgenic hiPSC-CM by selection of single and multiple gRNAs (n=3-4 replicates) and used these cells to generate human engineered myocardium by combining hiPSC-CM and fibroblasts which we subjected to isometric contractions in order to induce mechanical stress, which resulted in KLF15 expressional decrease in line with our in vivo data. This transcriptional loss was rescued in CRISPR/dCas9VPR hiPSC-CM targeted to the KLF15 locus compared to controls (n=6-9/2/4 tissues per group/casting sessions/differentiations). Additionally, TGFB1 induced cardiomyocyte stress resulted in decreased KLF15 expression levels in 2D hiPSC-CM cultures which were rescued by dCas9VPR- KLF15 targeting (n=3 experiments). In conclusion, we report controllable gene activity by CRISPR/dCas9VPR to restore the loss of KLF15 in stressed mouse and human cardiomyocytes. We furthermore evaluate the potential to gain full control over gene dose titratability with these models to validate and define novel therapeutic targets for the prevention of heart failure progression.
Type of Medium:
Online Resource
ISSN:
0009-7330
,
1524-4571
DOI:
10.1161/res.129.suppl_1.MP217
Language:
English
Publisher:
Ovid Technologies (Wolters Kluwer Health)
Publication Date:
2021
detail.hit.zdb_id:
1467838-X
