Gut microbe-derived metabolite trimethylamine N-oxide accelerates fibroblast-myofibroblast differentiation and induces cardiac fibrosis.

Trimethylamine N-oxide (TMAO), a gut microbe-derived metabolite of dietary choline and other trimethylamine-containing nutrients, has been associated with poor prognosis in coronary heart disease. However, the role and underlying mechanisms of TMAO in the cardiac fibrosis after myocardial infarction (MI) remains unclear. We used mouse MI models and primary cardiac fibroblasts cultures to study the role of TMAO in the heart and in cardiac fibroblasts. C57BL/6 mice were fed a control diet, high choline (1.2%) or/and DMB diet or a diet containing TMAO (0.12%) starting 3 weeks before MI. DMB, a structural analogue of choline, inhibited microbial TMA lyases and reduced the level of TMAO in mice. Cardiac function was measured 7 days after MI using echocardiography. One week post MI, myocardial tissues were collected to evaluate cardiac fibrosis, and blood samples were evaluated for TMAO levels. The expression of TGF-β receptor, P-Smad2, α-SMA or collagen I in myocardial tissues and fibroblasts were analyzed by western blot or immunocytochemistry. We demonstrated that cardiac function and cardiac fibrosis were significantly deteriorated in mice fed either TMAO or high choline diets compared with the control diet, and DMB reversed the cardiac function damage of high choline diet (p < .05). Cardiomyocyte necrosis, apoptosis and macrophage infiltration after MI was significantly increased after treatment with TMAO or high choline diets. The size and migration of fibroblasts were increased after TMAO treatment compared with non-treated fibroblasts in vitro. Furthermore, TMAO increased TGF-β receptor I expression, which promoted the phosphorylation of Smad2 and up-regulated the expression of α-SMA and collagen I. The ubiquitination of TGF-βRI was decreased in neonatal mouse fibroblasts after TMAO treatment. TMAO also inhibited the expression of smurf2. Inhibition of TGF-β1 receptor with the small molecule inhibitor SB431542 decreased TGF-β receptor I expression, reduced the phosphorylation of Smad2, down-regulated TMAO-induced α-SMA and collagen I expression in cardiac fibroblasts. Cardiac function and cardiac fibrosis were significantly exacerbated in mice fed diets supplemented with either choline or TMAO, probably through accelerating the transformation of fibroblasts into myofibroblasts, indicating activation of TGF-βRI/Smad2 pathway.