Trimethylamine N-oxide activates cardiac fibroblasts transformation through NLRP3 inflammasome activation with changes in mitochondrial dynamics and ER-mitochondrial contact under ER stress.

Trimethylamine N-oxide (TMAO), a gut microbiota-derived metabolite, has been linked to cardiovascular diseases. This study aimed to explore the role of TMAO in cardiac fibrosis by examining its effects on the NLRP3 inflammasome, endoplasmic reticulum stress (ERS), mitochondria-associated membranes (MAMs), and mitochondrial dynamics in cardiac fibroblasts (CFs), alongside clinical data from acute myocardial infarction (AMI) patients and unstable angina (UA) patients and AMI animal model data. Plasma TMAO levels were measured in AMI patients and healthy controls. In vitro, CFs were treated with TMAO to assess cellular activation and fibrosis markers. Western blot, immunofluorescence, and RNA sequencing identified key pathways and proteins related to ERS, NLRP3 inflammasome activation, and mitochondrial dynamics. In vivo, Masson's trichrome staining, Hematoxylin-Eosin (HE) staining and Immunohistochemical were used to evaluate the effects of TMAO on AMI mice. Plasma TMAO levels were significantly higher in the AMI group. TMAO promoted cardiac fibroblast activation and fibrosis by increasing α-SMA and Collagen I expression. It induced ERS, marked by elevated GRP78, p-PERK, and CHOP, and upregulated Sigma-1R, enhancing MAM formation. TMAO also altered mitochondrial dynamics via DRP1 phosphorylation and Mfn2 expression. RNA sequencing identified macrophage migration inhibitory factor (MIF) as a key mediator linking TMAO to NLRP3 inflammasome activation. TMAO exacerbates myocardial injury and fibrotic remodeling in AMI mice. TMAO exacerbates cardiac fibrosis via ERS and NLRP3 activation, with implications for mitochondrial dynamics and MAM formation. Elevated TMAO levels in AMI patients underscore its potential as a therapeutic target for ventricular remodeling fibrosis.