These data reveal a pivotal role for the lncRNA H19 in controlling endothelial cell aging.
Long non-coding RNAs (lncRNAs) contribute to cardiac (patho)physiology. Aging is the major risk factor for cardiovascular disease with cardiomyocyte apoptosis as one underlying cause. Here, we report the identification of the aging-regulated lncRNA Sarrah (ENSMUST00000140003) that is anti-apoptotic in cardiomyocytes. Importantly, loss of SARRAH (OXCT1-AS1) in human engineered heart tissue results in impaired contractile force development. SARRAH directly binds to the promoters of genes downregulated after SARRAH silencing via RNA-DNA triple helix formation and cardiomyocytes lacking the triple helix forming domain of Sarrah show an increase in apoptosis. One of the direct SARRAH targets is NRF2, and restoration of NRF2 levels after SARRAH silencing partially rescues the reduction in cell viability. Overexpression of Sarrah in mice shows better recovery of cardiac contractile function after AMI compared to control mice. In summary, we identified the anti-apoptotic evolutionary conserved lncRNA Sarrah, which is downregulated by aging, as a regulator of cardiomyocyte survival.
Vascular ageing is a key risk factor for cardiovascular diseases and is characterised by a continuous decline in endothelial 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 from 12 weeks young and 20 months old mice, we identified Mirial as an ageing-induced lncRNA (1.32-fold, p=0.ehab724.33565). 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), while increasing VEGF-A-stimulated sprouting by 50% (p=0.0139) and not affecting apoptosis or senescence. Subcellular fractionation of HUVECs revealed that MIRIAL was predominantly associated with the chromatin (80%). Pathway analysis of RNA sequencing data showed an overrepresentation of upregulated p53 target genes upon MIRIAL knockdown in HUVECs which was validated using qRT-PCR (1.8–5.2-fold increased). Using siRNA against p53 we showed that this effect is fully dependent on the presence of p53. Moreover, p53 and its phosphorylated form (Ser15) were both increased (1.8-fold, p=0.01 and 2.9-fold, p=0.02) after MIRIAL silencing. Intriguingly, RNA immunoprecipitation revealed that MIRIAL physically interacts with p53 (3.75-fold enriched, p=0.0067). To further study the interactome of MIRIAL, we performed RNA pulldown assays followed by mass spectrometry analysis of bound proteins, which identified the ageing-associated prohibitin (PHB) 1 and 2 to potentially interact with MIRIAL. Similar to MIRIAL knockdown, siRNA-mediated PHB 1 or 2 silencing caused proliferative defects. Further, PHBs are known to physically interact with p53 and control mitochondrial metabolism, a key factor in cellular ageing. Interestingly, silencing of MIRIAL in HUVECs increased mitochondrial mass (1.8-fold, p=0.0008) and spare respiratory capacity (1.95-fold) with the latter being decreased in isolated aged murine ECs. Taken together, MIRIAL is an ageing-induced lncRNA in ECs acting as a key regulator of metabolic and cellular function. MIRIAL promotes cell proliferation, migration and basal angiogenic sprouting while decreasing mitochondrial function and VEGF-A-stimulated sprouting. We hypothesise that MIRIAL influences p53 signalling and mitochondrial respiration through PHB 1 and 2. The present study suggests that modulation of MIRIAL expression may be a promising strategy to prevent or even reverse ageing-induced functional decline of ECs. FUNDunding Acknowledgement Type of funding sources: Public grant(s) – EU funding. Main funding source(s): European Research Council (ERC) Starting Grant: Non-coding RNA in Vascular Ageing (NOVA)
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<0.01) and spare respiratory capacity (1.95-fold). Preliminary data from the hearts of Mirial knockout mice confirm the elevated mitochondrial mass after Mirial ablation (1.26-fold, p=0.05). In HUVECS, MIRIAL is mainly associated with the chromatin (80%), suggesting a role in the regulation of gene expression. Pathway analysis showed an overrepresentation of p53 target genes that were upregulated upon MIRIAL knockdown, which was validated using qRT-PCR (1.8–5.2-fold increases). Interestingly, this effect is fully dependent on the presence of p53. Moreover, p53 and phospho-p53 (Ser15) were both increased (1.8-fold, p=0.01 and 2.9-fold, p=0.02, respectively) after MIRIAL silencing. Pulldown of MIRIAL identified DDX5 and MRPL41 as direct p53 interactors and RNA immunoprecipitation revealed that MIRIAL physically interacts with p53 (3.75-fold enrichment, p<0.01). Gene set enrichment analysis of RNA sequencing data revealed that 10% of deregulated genes after MIRIAL knockdown have a binding site for Forkhead Box O (FoxO) transcription factors. In particular, FoxO1 is known as one of the key players in endothelial proliferation and regulation of angiogenesis as well as in mitochondrial biogenesis. 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
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