CRISPR/Cas9 Activation of Delta-like non-canonical Notch ligand 1 (DLK1) to Protect from Contractile Dysfunction in Engineered Human Myocardium

by Niklas Bader
Doctoral thesis
Date of Examination:2025-06-12
Date of issue:2025-07-01
Advisor:Prof. Dr. Wolfram-Hubertus Zimmermann
Referee:Prof. Dr. Wolfram-Hubertus Zimmermann
Referee:Prof. Dr. Karl Toischer
Referee:Prof. Dr. Heidi Hahn
Sponsor:Deutsches Zentrum für Herz- und Kreislaufforschung
Persistent Address: http://dx.doi.org/10.53846/goediss-11321

 

 

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Abstract

English

Delta-like non-canonical Notch ligand 1 (DLK1) is a gene that is highly upregulated in all three germ layers during the development of the human body, but is almost absent in end-stage heart failure. This work confirmed the decreased DLK1 expression in a heart failure phenotype in vivo and in vitro. We mimicked neurohormonal stress in monolayer induced pluripotent stem cell-derived cardiomyocytes by supplementation of Transforming Growth Factor-β1 or L-noradrenaline for 3 days, demonstrating diminished DLK1 expression upon stress. To reactivate the DLK1 expression in vitro, we utilized a Clustered Regularly Interspaced Short Palindromic Repeats activation (CRISPRa). Lentiviral gene delivery of our tested guide ribonucleic acid E was utilized to transduce induced pluripotent stem cell-derived cardiomyocytes. To further validate the successful reactivation on the protein level, we applied the Western blot technique, demonstrating sufficient DLK1 overexpression. As we observed that DLK1 protein abundance in human serum samples is abolished in heart failure, we analyzed supernatants of monolayer induced pluripotent stem cell-derived cardiomyocytes after CRISPR mediated DLK1 enhancement. Concordantly, we saw increased DLK1 protein abundance in our samples. To further study functional effects mediated by cardiomyocyte specific DLK1 activation, we used our established Engineered Human Myocardium model. DLK1 activated Engineered Human Myocardium displayed functional differences in their contractile force generation compared to their respective controls. Furthermore, we demonstrated that increased preloading of Engineered Human Myocardium improved functional maturation. To recapitulate a heart failure phenotype in Engineered Human Myocardium, we applied neurohormonal stress for 1 week by daily supplementation of Transforming Growth Factor-β1 and L-noradrenaline. Engineered Human Myocardium with CRISPR enhanced DLK1 transcription is partially protected from NAT induced contractile dysfunction. We further elucidated transcriptomic changes and found evidence for enhanced mitotic activity. From our data, we conclude that DLK1 induces a pro-regenerative program, preventing contractile deterioration. In accordance with the recent literature, we observed an influence of DLK1 activation on Transforming Growth Factor-β1 signaling. An inhibitory role of DLK1 on Notch signaling was not detected. Perspectively, DLK1 is a promising target to rejuvenate the heart and ameliorate contractile function loss in heart failure.
Keywords: CRISPR; Cardiology; Heart failure
 
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