Abstract
Life-saving coronary artery bypass and vascular grafting procedures are associated with a deleterious ischaemia reperfusion injury (IRI). The carbon monoxide (CO) releasing molecule oCOm-21 has been shown to provide cardioprotection in cardiac IRI. CO is postulated to inhibit the nucleotide-binding oligomerisation domain (NOD), leucine-rich repeat (LRR)- containing protein (NLR) family pyrin domain containing 3 (NLRP3) inflammasome. NLRP3 and pro-interleukin (IL)-1β priming is triggered by sterile inflammatory signals, facilitating inflammasome assembly via NLRP3 and apoptosis-associated speck-like protein containing a caspase activation and recruitment domain (CARD) (ASC) interactions. Following assembly, the inflammasome activates caspase-1, which cleaves pro-IL-1β into IL-1β. This study aimed to examine the effect of oCOm-21 on NLRP3 in two models of cardiac IRI. 1; In ex vivo isolated non-hypertrophic and moderately hypertrophic CYP1A1-Ren2 rat hearts exposed to warm global ischaemia (30 minutes) and reperfusion (60 minutes); and 2; in an in vitro hypoxia and reoxygenation model of cardiac IRI in AC16 cardiomyocytes.
All statistical analyses were conducted using one-way or two-way ANOVA, with Bonferroni post-hoc analysis. The effects of oCOm-21 on NLRP3 in ex vivo studies involved immunofluorescent microscopy, Western blotting, and enzyme-linked immunosorbent assay (ELISA) techniques. Results revealed 3 μM oCOm-21 inhibited NLRP3 fluorescent expression in non-hypertrophic and hypertrophic rat hearts compared to vehicle-treated hearts (32.4% and 28.8% (P < 0.05) respectively) but not pro-IL-1β expression (P > 0.05). 3 μM oCOm-21 significantly reduced the Pearson’s Correlation Coefficient (r) of NLRP3 and ASC co- localisation in hypertrophic rat hearts compared to vehicle-treated hearts (0.57 ± 0.05 to 0.31 ± 0.04 respectively, P < 0.05) but not ASC oligomerisation. The expression of caspase-1 p20, ratio of active IL-1β/pro-IL-1β, and secreted IL-1β remained unchanged with oCOm-21 treatment (P > 0.05). The effects of oCOm-21 on NLRP3 in in vitro studies were investigated via cell viability and ELISA techniques. Both 3 μM oCOm-21 and 1 μM MCC950 (a validated NLRP3 inhibitor) increased viability in cells exposed to hypoxia and reoxygenation by 22.7% and 23.4% (P < 0.05) respectively, compared to normoxic controls. This recovery was associated with a significant reduction in IL-1β expression (levels undetectable, P < 0.05). These findings suggest that 3 μM oCOm-21 inhibits the priming and assembly of the NLRP3 inflammasome in ex vivo and in vitro cardiac IRI, and that inhibition of NLRP3 is a promising cardioprotective target for CO intervention therapies in cardiac IRI.