CRISPR-mediated Activation of DLK1 to Induce a Regenerative Pattern In Engineered Human Myocardium

Background: The human heart, known for its limited potential to recover from injury, could benefit from the reactivation of genes, which have the potential to induce a regenerative state. Through bioinformatic analysis of transcriptome data from patients with aortic stenosis and preserved or impaired left ventricular pump function, we identified Delta-like non-canonical Notch ligand-1 (DLK1) as a gene that could play a crucial role in the transition to congestive heart failure. This imprinted gene, highly active during embryo-fetal development, re-emerges in regenerative organs post-injury. Our study takes a unique and novel approach by exploring the potential of enhancing DLK1 expression in engineered human myocardium (EHM) to induce a regenerative response.

 

Methods and Results: We found that expression of DLK1 in the adult human heart is reduced by 44±14% in patients suffering from dilated cardiomyopathy (n=3) or aortic stenosis. Additionally, we analyzed muscle biopsies from non-failing (n=14) and failing (n=12) human hearts via RNA sequencing, which showed a reduced expression of DLK1 in heart failure. Concordantly, soluble DLK1 was reduced in patients' serum suffering from heart failure and correlated to left ventricular ejection fraction. To test if DLK1 restoration prevents contractile dysfunction, we used a CRISPRa transgenic iPSC cell line, which stably expresses an enzymatic inactive dCas9 complex to increase and modulate DLK1 expression in vitro. We tested the functional effects of DLK1 activation in an Engineered Human Myocardium (EHM) model of simulated heart failure by supplementation of L-noradrenaline (NA, 10 µM) and TGFβ1 (T, 5 ng/ml) for 7 days. Similar to the in vivo situation, DLK1 expression decreased in EHM under neurohormonal stress. Cardiomyocyte-specific DLK1 activation (DLK1a) in EHM made with primary cardiac fibroblasts in a collagen hydrogel enhanced global DLK1 expression levels to 12±5-fold (n=5) of non-target (NT) control in simulated heart failure. While DLK1a EHM showed comparable baseline function, contractile dysfunction by simulated heart failure was attenuated in DLK1a EHM (Force of contraction: -13±5 % vs -45±5 % in DLK1a vs NT EHM, n=8, p<0.05). RNA sequencing identified a regenerative transcriptome pattern in DLK1a EHM with differential expression of cell motility, extracellular matrix, cell cycle, and metabolism-related processes.

 

Conclusion: Our study's findings on the reactivation of the developmental gene DLK1 not only induce a regenerative state but also hold the potential to protect from heart failure.