Oncogenic HRASV12G as a strong driver of senescence in human cardiac stromal cells

Abstract Background Recent studies suggest that senescence plays a critical role in cardiovascular aging and disease. Oncogene-induced senescence is well described in non-cardiac cells, especially fibroblasts, but has not yet been investigated in cardiac cells. Purpose Here, we addressed the question of whether cardiac stromal cells (CStCs) undergo oncogene-induced senescence in response to overexpression of HRASV12G. Methods Human CStCs were isolated from right atrial appendage specimens collected during cardiac surgery with extracorporeal circulation. These cells were subjected to oncogene-induced senescence by overexpressing HRASV12G-eGFP, while CStCs overexpressing eGFP alone were used as controls. The key steps of the overexpression method are summarized in Graphical abstract. Senescence was assessed by evaluation of typical markers, such as staining for senescence-associated (SA) β-galactosidase (β-gal), EdU uptake, and RT-PCR analysis of CDKN1A expression. In addition, the transcriptomic profile and the secretory phenotype of HRASV12G- and eGFP-overexpressing CStCs were investigated by RNA sequencing and commercial antibody arrays. Results HRASV12G overexpression consistently induced senescence in CStCs, as highlighted by significantly increased SA-β-gal activity and CDKN1A expression (Figure 1D-B), and reduced EdU uptake compared to control (Figure 1C). HRASV12G-overexpressing CStCs also contained large numbers of variable-size vacuoles (Figure 1A). Transcriptomic analysis revealed upregulation of genes encoding the senescence-associated secretory phenotype (SASP), including pro-inflammatory factors, SERPINs, and matrix metalloproteinases, and activation of cholesterol biosynthesis pathways (Figure 1E). While some senescence-associated factors, such as GDF-15, were also enriched in the conditioned medium of HRASV12G-overexpressing CStCs, other, unconventional proteins were differentially expressed in HRASV12G- vs GFP-overexpressing cells, including CD14, Angiopoietin-2, TfR, C-Reactive Protein and Cripto-1 (Figure 1F). Conclusions Overexpression of the oncogene HRAS in CStCs induces typical markers of senescence, but also distinctive features that have not been reported after HRAS overexpression in stromal cells from other organs. The implications of this aberrant senescent phenotype need to be explored by future studies, in particular the role in – or against - cardiac tumorigenesis.For image description, please refer to the figure legend and surrounding text. For image description, please refer to the figure legend and surrounding text.