Trimethylamine N-oxide drives cardiac hypertrophy through H3K27ac-mediated enhancer activation of ketohexokinase.

Trimethylamine N-oxide (TMAO), a gut microbiota-derived metabolite, promotes cardiac hypertrophy, yet the molecular mechanisms linking microbial metabolism to cardiac gene expression remain incompletely defined. Ketohexokinase (KHK), the rate-limiting enzyme of fructose catabolism, is implicated in cardiac pathology, but its role in TMAO-induced hypertrophy is unknown. Here, we show that dietary choline supplementation elevates plasma TMAO and induces pathological cardiac hypertrophy in mice, effects significantly attenuated by the microbial inhibitor 3,3-dimethyl-1-butanol (DMB). RNA sequencing identified Khk as a top upregulated transcript in TMAO-exposed cardiomyocytes, confirmed at the protein level both in vitro and in vivo. Functionally, lentiviral-mediated Khk silencing mitigated hypertrophic growth, mitochondrial dysfunction, and oxidative stress, while pharmacological KHK inhibition with osthole ameliorated cardiac hypertrophy and preserved cardiac function. Mechanistically, TMAO selectively enriched histone H3 lysine 27 acetylation (H3K27ac) at a downstream enhancer of Khk, as revealed by CUT&Tag sequencing and qPCR. Pharmacological blockade of histone acetyltransferase activity with C646 abolished this enhancer activation and KHK upregulation. Targeted motif discovery within the Khk enhancer further identified conserved binding motifs for GATA family transcription factors, which are known to cooperate with p300 to modulate H3K27ac deposition and regulate cardiac hypertrophy genes, suggesting a potential mechanism by which this enhancer may be regulated. Collectively, these findings define a novel TMAO-H3K27ac-KHK axis linking gut microbial metabolism to cardiac remodeling, and identify KHK as a promising druggable target for therapeutic intervention in microbiota-associated heart disease.