Background: The regenerative capacity of the failing heart is limited. We identified DLK1 as a developmentally strongly expressed gene that is downregulated in failing myocardium but upregulated in regenerative organs. In addition, loss of DLK1 has been associated with a fibrotic cardiomyopathy phenotype in mice.

Hypothesis: DLK1 expression can be enhanced by CRISPR activation in human cardiomyocytes and protects from a cardiomyopathy phenotype.

Methods and Results: First, we confirmed the downregulation of DLK1 expression in failing human heart samples from patients with dilated cardiomyopathy (44±14% reduction in expression compared to non-failing heart, n=3). To further investigate DLK1 regulation we treated human iPSC derived cardiomyocytes with pro-fibrotic TGFβ1 (5 ng/ml) and found a comparable 51±24% (n=4) decrease in DLK1 expression. To simulate heart failure in vitro, we generated Engineered Human Myocardium (EHM) from iPSC-derived cardiomyocytes and fibroblasts and treated them with L-noradrenaline (NA, 10 µM) and TGFβ1 (T, 5 ng/ml) for 7 days. Contractile dysfunction and fetal gene expression in NAT–treated EHM was associated with a decrease in DLK1 expression similar to in vivo conditions. To enhance and modify DLK1 expression, we used a transgenic CRISPRa iPSC cell line, which stably expresses a nuclease inactive (d)Cas variant recruiting transcriptional factors to regulatory gene loci. We designed gRNA directly aiming at the transcriptional start site of the DLK1 gene locus. Out of 7 tested gRNAs 2 significantly increased DLK1 expression in HEK cells. Lentiviral delivery of the most effective gRNA showed a transcriptional activation of DLK1 by 1.5±0.2 fold (n=8) in CRISPRa iPSC derived cardiomyocytes.

Conclusion: CRISPR activation is suitable to increase DLK1 expression in human cardiomyocytes. Further work is targeted at improving the efficiency and testing functional effects in the established in vitro heart failure model.