Trimethylamine-N-oxide promotes fibrotic activation of quiescent valvular interstitial cells via endoplasmic reticulum stress

Calcific aortic valve disease currently lacks effective treatments beyond aortic valve replacement, largely due to an incomplete understanding of its pathogenesis. Emerging evidence suggests that the gut microbiome influences cardiovascular health through the production of metabolites derived from dietary components. Among these metabolites, trimethylamine-N-oxide (TMAO) has been identified as a potential causal factor for several cardiovascular conditions. However, its specific role in the development of aortic valve disease remains poorly understood. This study sought to investigate the impact of TMAO on valvular interstitial cells (VICs), the most abundant cell type in the aortic valve. Here, we demonstrate that TMAO activates aortic VICs towards a myofibroblastic phenotype through the induction of endoplasmic reticulum stress. Using a novel in vitro protocol to generate quiescent VICs, we found that TMAO induces the upregulation of the myofibroblastic proteins α-smooth muscle actin and transgelin in a sex-independent manner. These quiescent VICs were more sensitive to TMAO than conventionally cultured VICs. Treatment with TMAO also elevated extracellular matrix production and oxidative stress, phenotypic hallmarks of an activated profibrotic state. Finally, inhibition of the endoplasmic reticulum stress kinase prior to TMAO treatment blocked all effects of this metabolite on VIC phenotype. These findings suggest that TMAO contributes to the early stages of valve disease by promoting VIC activation and extracellular matrix production through endoplasmic reticulum stress mechanisms. Understanding the role of TMAO and other gut-derived metabolites in the pathogenesis of valve disease could inform the development of novel preventive or therapeutic strategies to modify or delay disease progression. Furthermore, these insights underscore the importance of host-microbiome interactions for cardiovascular health and highlight the potential for targeted dietary interventions to mitigate disease risk.