Stress induced lncRNA Lockd inhibits proliferation and angiogenesis of myocardial endothelial cells and promotes maladaptive cardiac remodelling during pressure overload

Myocardial endothelial cells play a pivotal role in modulating the cardiac stress response and influencing heart growth and function. While past research has predominantly focused on endothelial-derived proteins, recent evidence points to the involvement of long non-coding RNAs (lncRNAs) in these processes. This study sought to investigate the significance of the lncRNA Lockd in angiogenesis, endothelial cell function, and cardiac responses during cardiac pressure overload.

Through a systematic examination of differentially regulated lncRNAs in cardiac endothelial cells under pressure overload conditions, Lockd, an intergenic lncRNA downstream of Cdkn1b was identified. Lockd expression was notably upregulated in heart tissue during cardiac pressure overload induced by transverse aortic constriction (TAC). It peaked one-week post-TAC in cardiomyocytes and endothelial cells, but not in fibroblasts, gradually returning to basal levels thereafter. Interestingly, Lockd RNA expression was also significantly upregulated in failing vs. healthy human heart samples. Functional studies of Lockd in endothelial cells revealed that its overexpression suppressed angiogenesis in a sprouting assay, while its knockdown enhanced sprouting activity. Additionally, Lockd downregulation led to increased endothelial cell proliferation, whereas its overexpression reduced cell proliferation, as demonstrated by BrdU proliferation assay and phospho-histone-3 staining. RNA sequencing of Lockd knockdown endothelial cells unveiled reduced expression of genes associated with extracellular matrix organization and regulation of angiogenesis, while genes linked to cell cycle progression were upregulated. Analysis of single-cell sequencing data from isolated cardiac endothelial cells revealed Lockd's exclusive presence in a specific cluster associated with cell cycle and cell division. Protein-RNA pull-down assays demonstrated Lockd's binding to ribosomal proteins, possibly influencing the translation of selected proteins. Indeed, polysome fractionation revealed that Lockd has a strong affinity to the assembled 80S ribosome. Knockdown of Lockd significantly reduced the localisation of Lockd to the ribosome. Furthermore, chromatin RNA immunoprecipitation showed Lockd's association with specific chromatin regions, suggesting its potential direct involvement in gene expression regulation also at the level of transcription. To validate the functional relevance of Lockd in vivo, its upregulation post-TAC in mice was inhibited using antisense GapmeRs. This intervention significantly improved systolic cardiac function and reduced hypertrophy compared to control GapmeR-treated mice. Staining of heart tissue post-TAC demonstrated increased capillary density and decreased cardiomyocyte hypertrophy in mice with reduced Lockd expression upon Lockd GapmeR treatment. RNA sequencing of cardiac endothelial cells one-week post-TAC indicated substantial dysregulation of cell-cycle-related genes upon Lockd knockdown, emphasizing its role in cell cycle progression and cell proliferation in vivo.

In conclusion, these findings suggest that Lockd upregulation inhibits cell-cycle progression by regulating gene-expression at the transcriptional and post-transcriptional level, thereby counteracting endothelial proliferation and angiogenesis after TAC. Consequently, Lockd upregulation in heart tissue appears to be detrimental during cardiac pressure overload and cardiac remodelling.