Upregulation of the Absent-in-melanoma 2 Inflammasome Correlates with Markers of DNA Damage and Cell Cycle Dysregulation in Peripheral Arterial Disease

Introduction: We showed that myoblasts (Mb) in peripheral arterial disease (PAD) differentiate poorly and have evidence of inflammasome activation. Absent-in-melanoma 2 (AIM2), an inflammasome and mediator of pyroptosis, binds damaged DNA which also triggers the G2/M DNA damage checkpoint including CDC2 and its regulator, polo-like kinase 1 (PLK1). Dysregulated checkpoints can cause faulty replication, but a link between checkpoint function and AIM2 is not known. We hypothesize that AIM2 expression in PAD Mb correlates with increased DNA damage and a dysregulated G2/M DNA damage checkpoint. Methods: Cells were harvested from ischemic and perfused muscle obtained during amputations for PAD. Mb (Pax7-/MyoD+) were isolated using sequential plating and cell sorting. Mb from healthy donors (PAD -) were purchased (Cook MyoSite®). AIM2, γH2AX (DNA damage marker), PLK1 and CDC2 were measured with immunofluorescence (5 images/dish). Mb were treated with the PLK1-inhibitor BI2536 (10nM; 1h) to assess checkpoint responsiveness. ANOVA with post-hoc analysis confirmed statistical significance (α=0.05). Results: γH2AX expression was elevated in all PAD groups compared to PAD (-) Mb (N=3-4/PAD group). While PLK1 expression was similar across groups, CDC2 was lower in all ischemic PAD groups relative to perfused PAD Mb. BI2536 attenuated CDC2 expression in perfused PAD Mb (p = 0.006) while Mb in ischemic PAD remained unresponsive. AIM2 expression was significantly higher in ischemic Mb compared to perfused and PAD (-) Mb, correlating with high γH2AX and low CDC2. Results are summarized in Figure 1. Conclusion: Here, we show that in PAD Mb, AIM2 correlates with DNA damage, attenuated DNA damage checkpoint protein expression and diminished CDC2 responsiveness to BI2536. Thus, AIM2 may promote pyroptosis in the setting of defective DNA damage mechanisms that would otherwise prompt apoptosis. Whether these pathways are reversed by revascularization is a topic of further study.