HRASV12G overexpression as a driver of senescence in human cardiac fibroblasts

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Abstract Background Recent studies suggest that the senescence of cardiac fibroblasts (cFib) plays a critical role in cardiac aging and disease. In particular, the senescence of cardiac cells, especially cFib and other non-cardiomyocytes, has been implicated in the pathogenesis of doxorubicin (Dox)-induced cardiotoxicity. However, oncogene-induced senescence in primary human cFib has not yet been investigated. Purpose Here, we addressed the question of whether cFib also undergo senescence in response to oncogene-induced senescence, by overexpression of HRASV12G. Methods cFib were isolated from right atrial appendage specimens collected during cardiac surgery involving extracorporeal circulation. These cells were subjected to oncogene-induced senescence by overexpressing HRASV12G-eGFP, while cFib overexpressing eGFP alone were used as controls. The key steps of the overexpression method are summarized in the graphical abstract. Senescence was assessed by the typical markers of senescence such as staining for senescence-associated (SA) β-galactosidase, EdU incorporation, and RT-PCR analysis of CDKN1A expression. Results The main results are summarized in the graphical abstract. HRASV12G overexpression consistently induced senescence in cFib, as highlighted by significantly increased SA-β-gal activity and CDKN1A expression, and reduced EdU uptake compared to control. Interestingly, HRASV12G-overexpressing cFib exhibited a peculiar vacuolated morphological phenotype not previously described in the literature as being associated with senescence. Conclusions HRASV12G overexpression induces senescence in cFib, as demonstrated by the expression of typical senescence markers, and a unique vacuolated phenotype. Further analyses, including RNA-sequencing and transmission electron microscopy, are ongoing to characterize the phenotypic features and identify distinctive molecular signatures of HRASV12G-induced senescence in cFib.

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