Ionizing radiation elicits senescence and a pro-inflammatory phenotype in human cardiac stromal cells

Background

Ionizing radiation (IR) can induce senescence in cardiac cells, which may contribute to the pathogenesis of cardiac dysfunction caused by radiation therapy and associated cancer treatments. While radiation-induced senescence of cardiomyocytes and endothelial cells has already been characterized, the effects of IR on cardiac stromal cells (CStCs) are yet to be studied.

Purpose

We thoroughly investigated IR-induced and – as positive control - replicative senescence (Rep) in primary human CStCs (hCStCs). We further assessed whether hCStCs acquired a pro-inflammatory senescence-associated secretory phenotype (SASP) upon irradiation and whether they propagated senescence to recipient cells (defined as secondary senescence).

Methods

hCStCs were isolated from right atrial appendages, collected during cardiac surgery with extracorporeal circulation. These cells were exposed to IR (5 Gray, Gy) and analyzed at 2 days and 14 days to assess both acute and chronic response to irradiation. Additionally, hCStCs were passaged until Rep was reached (P12-P14).

Senescence was assessed by staining for senescence-associated β-galactosidase (SA-β-gal), EdU uptake and by RT-PCR analysis for LMNB1 expression. Gene and protein levels of key pro-inflammatory cytokines were evaluated by RT-PCR and multi-antibody array of conditioned medium (CM), respectively.

Recipient hCStCs and murine neonatal ventricular cardiomyocytes (mNVCMs) were incubated with the CM of senescent hCStCs to determine whether they would undergo secondary senescence (graphical abstract).

Results

IR and prolonged culturing consistently induced senescence in hCStCs, highlighted by reduced EdU uptake (Fig 1A), increased SA-β-gal activity (Fig 1B), and LMNB1 downregulation (Fig 1C).

Senescent hCStCs also displayed upregulation of IL-6 and IL-8 at 14 days (Fig 1D). Multi-antibody arrays of CM collected from irradiated senescent hCStCs at 14 days confirmed the secretion of IL-8 (Figure 1E). Following treatment of recipient hCStCs and mNVCMs with CM from IR-primed senescent hCStCs, EdU uptake, SA-β-gal activity, and LMNB1 expression were not different from the control cells, suggesting that primary senescence initiated by IR is not transmitted to other cardiac cells in a paracrine way.

Conclusion

IR elicits senescence in hCStCs, which entails the release of pro-inflammatory cytokines. The pro-inflammatory phenotype of senescent hCStCs may have direct negative effects and may precipitate or compound the toxicity of other oncological drugs, especially acting on the immune system. Additional experiments, including RNA sequencing, are ongoing to further explore the implications of IR-triggered senescence in hCStCs.