Clin Res Cardiol (2021). 10.1007/s00392-021-01933-9

Hans-Jürgen-Bretschneider-Abstract-Preis:
Krueppel-like factor 15 transcriptional restoration with CRISPR gene activation prevents cardiomyocyte remodeling upon pressure overload
E. Schoger1, R. Kim1, C. Rocha1, M. Jassyk1, F. Bleckwedel1, S. Doroudgar2, O. J. Müller3, L. Cyganek4, W.-H. Zimmermann1, L. Zelarayán1
1Institut für Pharmakologie und Toxikologie, Universitätsmedizin Göttingen, Göttingen; 2Klinik für Innere Med. III, Kardiologie, Angiologie u. Pneumologie, Universitätsklinikum Heidelberg, Heidelberg; 3Klinik für Innere Medizin III, Schwerpunkt Kardiologie und Angiologie, Universitätsklinikum Schleswig-Holstein, Kiel; 4Herzzentrum Göttingen - Stem Cell Unit, Universitätsmedizin Göttingen, Göttingen;
Transcriptional disbalance drives cardiomyocyte hypertrophy and concomitant loss during cardiac remodeling resulting in progression to heart failure. We identified a physical interaction of the hypertrophic repressor, Krueppel-like factor 15 (KLF15), and CTNNB1 as a part of a multiprotein complex inhibiting WNT/CTNNB1 signaling in the adult heart. Expression of KLF15 is reduced in the diseased myocardium concomitant with a de-repression of WNT/CTNNB1 signaling associated with disease progression. We therefore hypothesized that transcriptional re-normalization of KLF15 prevents disease progression mediated in part via inhibition of WNT/CTNNB1 signaling in diseased cardiomyocytes. We used a mouse model for cardiomyocyte-specific endogenous gene activation with enzymatically inactive Cas9 (dCas9) fused to transcriptional activators (VPR) and targeted the Klf15 promoter with gRNAs delivered with AAV9 upon induced pressure overload. Non-targeted (NT) gRNAs served as control. Homogenous and robust expression of both dCas9VPR and gRNA components was confirmed by co-expressed fluorescent reporters. At baseline, Klf15 gRNA did not show functional differences compared to NT gRNA and saline injected mice. Upon pressure overload, we observed a progressive decline in fractional shortening in NT gRNA mice (-50% reduced fractional shortening) which was blunted in the Klf15 gRNA group (-15% reduced fractional shortening) 8-weeks post-surgery. This was accompanied by reduced organ hypertrophy and increased survival compared to control groups including sham operated. Importantly, we detected Klf15 transcript levels in Klf15 gRNA hearts comparable to sham control groups 16 weeks post-surgery (control groups: -25% loss of Klf15, Klf15 gRNA group: +10% increase compared to sham control groups, n = 3-8) overall indicating transcriptional restoration with concomitant blunting of a hypertrophic response in vivo. To translate these findings into human cardiomyocytes, we generated induced pluripotent stem cell derived cardiomyocytes expressing dCas9VPR and delivered KLF15 gRNAs via lentiviral particles. We embedded these cells in a collagen matrix together with fibroblasts to generate engineered human myocardium (EHM) and forced these tissues into isometric contractions to mimic a pressure overload stress in a dish. EHM cultured under isotonic contraction conditions served as control. We observed KLF15 transcriptional loss in NT-gRNA (control) EHM under stress conditions (-40% loss of KLF15) similar to the in vivo condition which was re-normalized to baseline conditions in KLF15 gRNA induced EHM (n = 6-9 from 2 independent experiments and 4 hiPSC-CM differentiations). In conclusion, we demonstrated unprecedented control over endogenous gene activity which will allow to reprogram cardiomyocytes to a homeostatic state targeting candidate genes such as Klf15.
https://dgk.org/kongress_programme/ht2021/BS951.htm