Choline Diet and Its Gut Microbe-Derived Metabolite, Trimethylamine N-Oxide, Exacerbate Pressure Overload-Induced Heart Failure

Abstract

Background: Trimethylamine N-oxide (TMAO), a gut microbe-dependent metabolite of dietary choline and other trimethylamine-containing nutrients, is both elevated in the circulation of patients having heart failure and heralds worse overall prognosis. In animal studies, dietary choline or TMAO significantly accelerates atherosclerotic lesion development in ApoE-deficient mice, and reduction in TMAO levels inhibits atherosclerosis development in the low-density lipoprotein receptor knockout mouse.

Methods and results: C57BL6/J mice were fed either a control diet, a diet containing choline (1.2%) or a diet containing TMAO (0.12%) starting 3 weeks before surgical transverse aortic constriction. Mice were studied for 12 weeks after transverse aortic constriction. Cardiac function and left ventricular structure were monitored at 3-week intervals using echocardiography. Twelve weeks post transverse aortic constriction, myocardial tissues were collected to evaluate cardiac and vascular fibrosis, and blood samples were evaluated for cardiac brain natriuretic peptide, choline, and TMAO levels. Pulmonary edema, cardiac enlargement, and left ventricular ejection fraction were significantly (P<0.05, each) worse in mice fed either TMAO- or choline-supplemented diets when compared with the control diet. In addition, myocardial fibrosis was also significantly greater (P<0.01, each) in the TMAO and choline groups relative to controls.

Conclusions: Heart failure severity is significantly enhanced in mice fed diets supplemented with either choline or the gut microbe-dependent metabolite TMAO. The present results suggest that additional studies are warranted examining whether gut microbiota and the dietary choline → TMAO pathway contribute to increased heart failure susceptibility.

Keywords: choline; fibrosis; left; metabolomics; pulmonary edema; ventricular dysfunction.

Figure 1

Experimental protocol for transverse aortic constriction (TAC) studies in mice fed either control diet, trimethyamine N-oxide (TMAO), or choline. Mice were fed experimental diets starting at 3 weeks prior to TAC surgery and left ventricular (LV) function and structure were evaluated via two-dimensional echocardiography at 3-week intervals following baseline echocardiography studies at 1 week prior to TAC surgery. Upon completion of the study, mice were sacrificed at 12 weeks for heart and lung weights, cardiac histology and pathology as well as molecular determinations.

Figure 2

Dietary trimethylamine N-oxide (TMAO) exacerbates cardiac dilation and dysfunction following transverse aortic constriction (TAC) in mice, as compared to a control diet. (A) Left ventricular (LV) end-systolic diameter (LVESD; in mm), (B) LV end-diastolic diameter (LVEDD; in mm), (C) Intraventricular septal wall diastolic diameter (IVSd; in mm) and (D) LV ejection fraction (%) from baseline to 12 weeks. Results are presented as mean ± SEM. A linear mixed effects model was used to evaluate the change of all parameters during the 12 week study period when compared to baseline. Two-sample Wilcoxon tests were used to compare between TMAO and control groups at different time points for 10 – 12 animals per group.

Figure 3

Dietary choline (1.2%) exacerbates cardiac dilation and dysfunction following transverse aortic constriction (TAC) in mice, as compared to a control diet. (A) Left ventricular (LV) end-systolic diameter (LVESD; in mm), (B) LV end-diastolic diameter (LVEDD; in mm), (C) Intraventricular septal wall diameter (IVSd; in mm) and (D) LV ejection fraction (%) from baseline to 12 weeks. A linear mixed effects model was used to evaluate the change of all parameters during the 12 week study period when compared to baseline. Results are presented as mean ± SEM. Two-sample Wilcoxon tests were used to compare between choline and control groups at different time points for 10 – 12 animals per group.

Figure 4

Dietary trimethylamine N-oxide (TMAO) and choline exacerbate cardiac enlargement and hypertrophy following transverse aortic constriction (TAC). (A) Ratio of heart weights to tibia length in TMAO vs. control. (B) Ratio of left atrial (LA) weights to tibia length in TMAO vs control. (C) Ratio of lung weights to tibia length in TMAO vs. control. (D) Ratio of heart weights to tibia length in choline vs. control. (E) Ratio of LA weights to tibia length in choline vs. control. (F), Ratio of lung weights to tibia length in choline vs. control. Results are presented as mean ± SEM. Two-sample Wilcoxon tests were used to compare between TMAO or Choline vs. control for 10 – 12 animals per group.

Figure 5

Increased circulating levels of brain natriuretic peptide (BNP) in mice following transverse aortic constriction (TAC) and the development of pressure overload induced heart failure. (A–C) Representative photomicrographs of control (TAC + control diet), trimethylamine n-oxide treated (TAC + TMAO diet), and choline treated (TAC + choline diet) hearts at 12 weeks following TAC. (D) Mean circulating BNP levels (ng/mL) in mice 12 weeks post TAC. Two-sample Wilcoxon tests were used for two group comparison for 10 – 12 animals per study group.

Figure 6

Dietary trimethylamine N-oxide (TMAO) and choline initiated at 3 weeks prior to transverse aortic constriction (TAC) worsens interstitial fibrosis. (A) Representative photomicrographs of Masson’s trichrome stained heart sections in Control, Choline and TMAO groups of mice illustrating intermuscular fibrosis at 12 weeks post TAC. (B) Representative photomicrographs of Picrosirius red stained heart sections in Control, Choline and TMAO mice illustrating intermuscular fibrosis at 12 weeks post TAC. (C) Cardiac fibrosis as a percent (%) of left ventricular area calculated from Picrosirius red stained heart. Scale bars on photomicrographs represent 500 micrometers. Results are presented as mean ± SEM. Two-sample Wilcoxon tests were used for two group comparison.for 6 – 7 animals per group.

Figure 7

Dietary trimethylamine N-oxide (TMAO) and choline exacerbate perivascular fibrosis in the setting of transverse aortic constriction (TAC). (A) Representative photomicrographs of Masson’s trichrome stained heart sections in Control, Choline and TMAO groups of mice illustrating perivascular fibrosis at 12 weeks post TAC. (B) Representative photomicrographs of Picrosirius red stained heart sections in Control, Choline and TMAO mice illustrating perivascular fibrosis at 12 weeks post TAC. (C) Cardiac fibrosis as a percent (%) of left ventricular area calculated from Picrosirius red stained heart tissue. Scale bars on photomicrographs represent 500 micrometers. Results are presented as mean ± SEM. Two-sample Wilcoxon tests were used for two group comparison.for 6–7 animals per group.