Background: The human heart displays limited functional regeneration after an injury. During the progression from a compensated hypertrophy into heart failure expression of Delta-like non-canonical Notch ligand (DLK1) decreases substantially and is almost absent in end stage heart failure. DLK1 is highly expressed during development and in tissues, which inherit the capacity for functional regeneration.

Hypothesis: DLK1 expression can be restored in “diseased” Engineered Human Myocardium (EHM) and ameliorates contractile dysfunction.
Methods and Results: First, we observed that patients with heart failure and reduced EF displayed decreased DLK1 serum levels in comparison to the non-failing control cohort (n=8). Furthermore, a correlation (R=0.798) of DLK1 serum concentration and left-ventricular ejection fraction was found. In addition, expression of DLK1 in adult human heart biopsies is reduced by 44±14% in patients suffering from dilated cardiomyopathy (n=3) or aortic stenosis (n=14). To enhance DLK1 expression in a human heart model, we utilized a human iPSC line, which stably expresses an enzymatically inactive dCas9 to activate gene expression (CRISPR activation). Seven guide RNAs were established and validated in HEK293T to navigate the dCas9 activation complex to the transcriptional start site of DLK1. The most efficient gRNA (29±5 fold increase, n=4) was delivered to CRISPR activation cardiomyocytes by a lentivirus. Engineered Human Myocardium (EHM) was generated with DLK1 activated or non-target gRNA control cardiomyocytes and cardiac fibroblasts and subjected to a neurohormonal stress protocol by treatment with L-noradrenaline (NA, 10 µM) and TGFβ1 (T, 5 ng/ml) for 7 days. EHM exhibited a loss of DLK1 expression upon simulated heart failure resembling the in vivo situation, which was restored via CRISPR mediated DLK1 activation. DLK1 activated EHM exhibited a significantly reduced loss of contractile function in comparison to their respective controls under simulated neurohormonal stress.
Conclusion: CRISPR mediated DLK1 activation promotes a rescue of contractile function in EHM subjected to simulated heart failure. Ongoing work elucidates the molecular mechanisms which contribute to the improved phenotype.