The E3 ubiquitin ligase MARCH9 alleviates pyroptosis by regulating NLPR3 ubiquitination following myocardial ischemia reperfusion

Abstract NLRP3 inflammasome activation-induced pyroptosis has emerged as a key mediator of pathological myocardial ischemia-reperfusion (MI/R) injury. Nevertheless, the underlying molecular mechanisms remain poorly understood. In this study, we aimed to investigate the regulatory mechanisms underlying NLRP3 inflammasome activation and its mediation of pyroptotic cell death following MI/R. The current research examined the impact of MARCH9 on NLRP3 inflammasome-induced pyroptosis both in vitro following hypoxia/reoxygenation (H/R) and in vivo using a MI/R mouse model. Wild-type and MARCH9-Tg mice underwent analysis through TTC, Evan's blue dye, and immunohistochemistry staining. H9c2 cells were transfected with si-MARCH9, while HEK293T cells received transfections of MARCH9 and NLRP3 overexpression plasmid, along with their respective mutants. Western blot analysis was utilized to quantify levels of MARCH9, NLRP3, Caspase-1, GSDMD, and GSDMD-N. Protein interactions were assessed via immunoprecipitation, and protein colocalization within cells was observed using a confocal microscope. In this study, our data suggest that MARCH9 plays a key role in the regulation of the NLRP3 inflammasome activation and pyroptosis during myocardial I/R injury. We also examine the function of MARCH9 as an E3 ubiquitin ligase specifically targeting NLRP3. The interaction between MARCH9 and NLRP3 results in the promotion of K48-linked polyubiquitination of NLRP3, ultimately leading to its proteasomal degradation and subsequent inhibition of NLRP3 activation. These findings provide novel insights into the involvement of MARCH9 in the regulation of NLRP3 inflammasome-induced pyroptotic cell death.