Background: Myocardial endothelial cells (ECs) are able to modulate the cardiac stress response during health and disease and thereby exert a major impact on heart growth and function. While so far mainly endothelial derived proteins were studied, growing evidence suggests that long non-coding RNAs (lncRNAs) can also contribute to these effects.

Aim of the study was to investigate how the lncRNA Lockd contributes to angiogenesis, endothelial cell survival and fibrotic/mesenchymal gene expression during cardiac pressure overload.

Methods and Results: In a screen for differentially regulated lncRNAs in cardiac endothelial cells during pressure overload, we identified the so far largely unstudied intergenic lncRNA downstream of Cdkn1b, Lockd. It is significantly upregulated in heart tissue during cardiac pressure overload induced by transverse aortic constriction (TAC). We found that Lockd expression peaks in cardiac tissue one week after TAC operation (approx. 11-fold increase vs. Sham, p<0.05, n=5), and gradually declines back to basal levels, indicating that Lockd is induced during the compensatory phase of cardiac remodelling.

Separation of cardiac cells with MACS/Langendorff one week after TAC and Sham revealed that Lockd upregulation occurs in cardiomyocytes and endothelial cells, but not in fibroblasts.

Lockd expression in cardiac ECs also peaked one week after TAC surgery (approx. 6-fold increase compared to Sham, p<0.005, n=6) and then gradually fell back to basal values.

We next investigated the functional impact of Lockd on endothelial cells in vitro. Adenoviral overexpression of Lockd in ECs reduced angiogenesis in a sprouting assay. In contrast, down-regulation of Lockd by siRNA by 90% triggered a stronger sprouting activity. Furthermore, down-regulation of Lockd triggered increased EC proliferation, while its overexpression reduced cell proliferation, as demonstrated by BrdU proliferation assay.

RNA sequencing in siRNA control versus siLockd treated endothelial cells showed that Lockd downregulation entailed reduced expression of genes related to extracellular matrix organization (Fn1, Col4a3, Col4a4) and to blood vessel development (Mtor, Pdgfb, Ccn2). On the other hand, it triggered increased expression of genes associated with cell cycle progression (Cdc25b, Ccnd3, Uhmk1). ChIRP-MS pulldown experiment, which is a technique for studying endogenous ribonucleoprotein complexes, showed that Lockd specifically binds to Khdrbs1, a protein involved in cell cycle progression.

Targeted inhibition of Lockd upregulation after TAC with antisense GapmeRs in mice in vivo strongly improved systolic cardiac function and reduced hypertrophy in comparison to Control GapmeR treated mice. Histological and RNA sequencing studies from whole hearts and isolated ECs from these mice are ongoing and will be presented.

Conclusion: Lockd expression increases in cardiac ECs in the early phase of induced pressure overload. As suggested by our results, Lockd upregulation inhibits cell cycle progression, thereby counteracting angiogenesis, and promoting mesenchymal gene expression in endothelial cells. Therefore, Lockd upregulation in heart tissue might be detrimental during cardiac pressure overload. Indeed, downregulation of Lockd in mice in vivo during TAC ameliorated cardiac dysfunction and fibrosis. These findings identify Lockd as a new potential therapeutic target in heart failure.