Gut microbiota production of trimethyl-5-aminovaleric acid reduces fatty acid oxidation and accelerates cardiac hypertrophy

Numerous studies found intestinal microbiota alterations which are thought to affect the development of various diseases through the production of gut-derived metabolites. However, the specific metabolites and their pathophysiological contribution to cardiac hypertrophy or heart failure progression still remain unclear. N,N,N-trimethyl-5-aminovaleric acid (TMAVA), derived from trimethyllysine through the gut microbiota, was elevated with gradually increased risk of cardiac mortality and transplantation in a prospective heart failure cohort (n = 1647). TMAVA treatment aggravated cardiac hypertrophy and dysfunction in high-fat diet-fed mice. Decreased fatty acid oxidation (FAO) is a hallmark of metabolic reprogramming in the diseased heart and contributes to impaired myocardial energetics and contractile dysfunction. Proteomics uncovered that TMAVA disturbed cardiac energy metabolism, leading to inhibition of FAO and myocardial lipid accumulation. TMAVA treatment altered mitochondrial ultrastructure, respiration and FAO and inhibited carnitine metabolism. Mice with γ-butyrobetaine hydroxylase (BBOX) deficiency displayed a similar cardiac hypertrophy phenotype, indicating that TMAVA functions through BBOX. Finally, exogenous carnitine supplementation reversed TMAVA induced cardiac hypertrophy. These data suggest that the gut microbiota-derived TMAVA is a key determinant for the development of cardiac hypertrophy through inhibition of carnitine synthesis and subsequent FAO.


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Sample size
Sample sizes were predetermined based on effect size, standard deviation, and significance level required to attain statistical significance of p<0.05 with a 90% probability on the basis of previous experiments using similar methodologies and were deemed sufficient to account for any biological/technical variability.
Data exclusions Outliers were excluded.

Replication
In vivo experiments were carried out on multiple independent cohorts of animals with similar results. Proteomic data were further followed up by alternative approaches, such as RT-qPCR.
Randomization For in vivo studies, mice in each genotype were randomly assigned to treatment groups.

Blinding
Investigators were not blinded during animal experiments but were blinded in human study.

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Wild animals
The study did not involve wild animals.

Field-collected samples
The study did not involve samples collected from the field.

Ethics oversight
All protocols for mouse experiments were approved by the Ethics Committee of Animal Research, Peking University Health Science Center and the Animal Care and Ethics Committee of Tongji Medical College of Huazhong University of Science and Technology.
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Human research participants
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Population characteristics
The learning cohort leading to the discovery of TMAVA was described previously in our recent publication[Zhao, M. et al. TMAVA, a metabolite of intestinal microbes, is increased in plasma from patients with liver steatosis, inhibits γ-butyrobetaine hydroxylase, and exacerbates fatty liver in mice. Gastroenterology 158, 2266Gastroenterology 158, -2281Gastroenterology 158, .e2227 (2020.]. TMAVA levels in patients with hypertension in that cohort were analyzed in this new study. The identification of hypertensive patients was based on a clearly documented medical history of hypertension with a systolic blood pressure≥140 mm Hg or a diastolic blood pressure≥90 mm Hg. The baseline characteristics of subjects in the learning cohort are presented in Supplementary Table 1 in the manuscript. The validation cohort, on the other hand, is a prospective population-based cohort study. At recruitment, 1647 participants with heart failure were enrolled between 2008-2017, corresponding to the longest 84-month follow-up visit (Supplementary Table 2 in the manuscript). Follow-up for mortality and specific outcome of adverse events were implemented by trained interviewers. The participants were tracked until the first occurrence of defined outcomes, including cardiac death and heart transplantation. All blood samples were collected at the fasting state, and stored at -80 °C immediately until analysis.

Recruitment
Inclusion and diagnosis of these patients were assessed by cardiologists from the Tongji Hospital Affiliated to Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China. Indications for the enrolment of HF and exclusion criteria have been reported previously[Huang, J. et al. ADRB2 polymorphism Arg16Gly modifies the natural outcome of heart failure and dictates therapeutic response to beta-blockers in patients with heart failure. Cell Discov. 4, 57 (2018).]. Briefly, subjects with clinically significant valvar heart disease, acute myocardial infarction or unstable angina within one month, as well as those with malignant tumor, were excluded from the study. Patients with severe coronary heart disease without complete revascularization therapy were not specifically excluded. Concomitantly, control subjects without significant cardiac disease consecutively screened with coronary angiogram and echocardiography were selected at random in the same hospital from October 2013 to March 2017.