Trimethylamine N-oxide drives bioprosthetic heart valve calcification via macrophage pyroptosis in juvenile rats

Abstract

Bioprosthetic heart valves (BHVs) constructed from bovine pericardium (BP) are widely used in valve replacement due to their favorable biocompatibility. However, early structural degeneration, particularly in younger recipients, remains a critical challenge, primarily driven by calcification. Trimethylamine N-oxide (TMAO), a gut microbiota-derived metabolite elevated by high-choline diets, has been implicated in vascular and valvular calcification, but its role in BHV deterioration remains unclear. This study aimed to evaluate the effects of TMAO on BP calcification. Three-week-old Sprague-Dawley rats were assigned to six diet groups: Normal Chow Diet (NCD), High Choline Diet (HCD), High Fat Diet (HFD), combined High Fat and Choline Diet (HFD+HCD), HFD with TMAO supplementation (HFD+TMAO), and HFD with both HCD and the TMAO inhibitor 3,3-dimethyl-1-butanol (DMB). BHVs made of BP were implanted subcutaneously, and after 8 weeks, we assessed calcium deposition, osteogenic markers, plasma metabolites, inflammatory cytokines, inflammatory cell proportions, and macrophage pyroptosis using techniques such as colorimetry, immunohistochemistry, ELISA, flow cytometry, and immunofluorescence. In vitro, RAW264.7 macrophages were exposed to TMAO, and pyroptosis was assessed by Western blotting, ELISA, and electron microscopy. Results indicated that HCD and HFD significantly increased BP calcification, osteogenic marker expression, and inflammatory responses in BHVs. The HFD+HCD and HFD+TMAO groups exhibited pronounced calcific and inflammatory effects, which were reduced by DMB. In vitro, TMAO induced macrophage pyroptosis, contributing to calcification. These findings suggest that TMAO promotes BP calcification through pyroptosis-driven inflammation, and that targeting TMAO via dietary or microbial modulation may offer a promising strategy to improve BHV durability, particularly in young patients.