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Vascular ageing is a key risk factor for cardiovascular diseases and is characterised by a continuous decline in endothelial cell function. Despite progress in recent years, the molecular mechanisms for this deterioration remain incompletely understood. Long non-coding RNAs (lncRNAs) are a heterogeneous class of RNAs that have been shown to regulate gene expression and protein function, however, little is known about their role in the ageing-associated dysregulation of endothelial cell (EC) function. In this study, we aimed to identify and functionally characterise a novel ageing-regulated lncRNA in ECs. Using RNA sequencing data of cardiac ECs derived from 12 weeks young and 20 months old mice, we identified Mirial as an ageing-induced lncRNA (1.32-fold, p=0.00005). Mirial is conserved between mice and humans and has no obvious coding potential. GapmeR-mediated silencing of MIRIAL in human umbilical vein ECs (HUVECs) decreased cell proliferation by 50%, migration by 24% (p=0.045) and basal angiogenic sprouting by 53% (p=0.0029), without affecting apoptosis or senescence. Additionally, silencing of MIRIAL increases mitochondrial mass (1.8-fold, p Taken together, MIRIAL is an ageing-induced lncRNA in endothelial cells acting as a key regulator of metabolic and cellular function. MIRIAL promotes cell proliferation, migration and basal angiogenic sprouting while decreasing mitochondrial function. We hypothesise that MIRIAL influences these cellular functions by affecting the p53 pathway and mitochondrial respiration through FoxO signalling. The results from the present study suggest that modulation of cellular MIRIAL expression may be a promising strategy to prevent or even reverse ageing-induced functional decline of ECs, both in vitro and in vivo. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Deutsche Forschungsgemeinschaft - Collaborative Research Centre (SFB) 834 - Project B9Deutsche Forschungsgemeinschaft - Collaborative Research Centre/Transregio (TRR) 267 - Project B4

Introduction Cardiac disease induces remodelling which can include fibrosis, cardiomyocyte hypertrophy, cardiac dilation, and finally can lead to heart failure. Age is one of the main risk factors of cardiovascular disease and induces heart remodelling, in particular, it has profound effects on the microcirculation. Pericytes are microvascular mural cells involved in the maintenance of stability and homeostasis of the vascular network. Although the phenotypes that arise from cardiac remodelling have been well studied in cardiomyocytes, endothelial cells and fibroblasts, the effect of aging on pericytes remain largely unknown. Purpose The purpose of this study is to characterise pericyte responses to cardiac ageing and disease in order to determine the therapeutic potential of these mural cells to reverse, or at least reduce, structural remodelling. Methods We have studied 12-week-old and 18-month-old mice. We have performed histological analysis and single-nucleus-RNA-sequencing (snRNAseq). For our in vitro experiments we have used primary human pericytes. Results Age affects the structure of the microcirculation in the heart. Pericyte coverage is reduced and capillary diameter is increased. Gene ontology analysis of differentially expressed genes in pericytes revealed an upregulation of genes related to filopodia and actin cytoskeleton, while a reduction of genes related to focal adhesion in the pericytes of the aged heart. Interestingly, we detected a downregulation of Regulator of G-protein signalling 5 (RGS5), a repressor of GPCRs signalling. RGS5 knockdown induces a contractile, pro-inflammatory and pro-fibrotic gene expression profile reducing pericytes proliferation and migration. Mechanistically, we show that RGS5 post-transcriptionally regulates PDGFRβ, a crucial tyrosine kinase receptor for pericyte-endothelial cell interaction. RGS5 knockdown reduces the expression of the receptor at the protein level, but not at the gene expression level and furthermore reduces the phosphorylation of AKT, a downstream signal of PDGFRβ activity. Furthermore, we have identified that T-Box Transcription Factor 20 (Tbx20), a cardiogenic transcription factor, is enriched in aged pericytes. Silencing and upregulation studies have revealed that Tbx20 is a repressor of PDGFRB, the gene that encodes for PDGFRβ, and that it controls pericyte adhesion. Conclusions Together, these observations have identified RGS5 and Tbx20 as crucial key players maintaining pericyte function in the aged heart. We propose that RGS5 and Tbx20 regulate pericyte function by controlling PDGFRβ signalling and cellular proliferation, adhesion and migration. Given the importance of pericytes in keeping vessel homeostasis, maintaining or recovering pericyte function in context of cardiac stress would be a potential approach to reduce the malignant effects of cardiac remodelling in the aged heart. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): SFB1366 (DFG)