Senolytics rejuvenate the reparative activity of human cardiomyocytes and endothelial cells

Abstract

Abstract Background Senescent cells have emerged as bona fide drivers of ageing and age-related cardiovascular disease, with senescent cells accumulating in the aged heart and following damage/injury. We have shown that removal of senescent cells using senolytics can rejuvenate the regenerative capacity of the aged heart. Purpose We investigate the effects of cell senescence and action of the senolytics, Dasatinib (D) and Quercetin (Q), on human cardiomyocyte and endothelial cell viability, survival, proliferation, angiogenesis and senescence in vitro. Methods We developed a transwell insert co-culture stress-induced premature senescence model system in vitro. Doxorubicin was used to induce premature senescence to cardiac stromal progenitor cells or HUVECs, which were seeded on the bottom chamber. In separate well plates, untreated cells (non-senescent) were seeded on the bottom chamber and acted as control. Human iPSC-cardiomyocytes (iPSC-CMs) or HUVECs were seeded on the top chamber insert. Co-cultures were left for 7 days and then iPSC-CMs or HUVECs in the top chamber were analysed for viability, proliferation and markers of senescence, and the conditioned medium analysed for SASP factors. The cultures were then treated with D+Q (0.5μM D with 20μM Q) for 3 days to clear the senescent cells in the bottom chamber. Then 7 days later the iPSC-CMs or HUVECs in the top chamber were analysed for viability, proliferation and the markers of senescence, and conditioned medium analysed for SASP factors (total of 17 days). Results Co-culture of iPSC-CMs with senescent cells led to decreased iPSC-CM viability (64±11% vs. 100±3% control; p<0.05) and proliferation (5±2% vs. 11±1% control; p<0.05). There was no increased senescence in co-cultured iPSC-CMs. Treatment with senolytics D+Q resulted in rescue of viability (79±23% 17d co-culture+D+Q; 64±9% 17d co-culture; 100±4% 17d control) and proliferation (7±2% 17d co-culture+D+Q; 3±0.3% 17d co-culture; 8±0.3% 17d control). Similar effects were observed with HUVECs, which showed decreased viability (83±5% vs. 100±6% control; p<0.05) and proliferation (21±7% vs. 44±7% control; p<0.05) when co-cultured with senescent HUVECs. Treatment with senolytics ameliorated HUVEC viability (61±22% 17d co-culture+D+Q; 21±12% 17d co-culture; 100±5% 17d control), proliferation (30±5% 17d co-culture+D+Q; 22±4% 17d co-culture; 48±4% 17d control) and angiogenesis (Score relative to control: 1.1±0.5 D+Q conditioned media; 0.2±0.1 senescent cell conditioned media; 1.0±0.3 control media). Luminex analysis of the senescent cell conditioned media revealed upregulation of SASP factors, but the level of SASP factors were reduced with application of D+Q. Conclusion We show that D+Q senolytics have therapeutic potential in rejuvenating the reparative activity of cardiomyocytes and endothelial cells. These results open the path to further studies on using senolytic therapy in age-related cardiac deterioration and rejuvenation. Funding Acknowledgement Type of funding sources: Other. Main funding source(s): Heart Research UK