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TMAO: how gut microbiota contributes to heart failure

  • Yixin Zhang
    Affiliations
    Beijing Anzhen Hospital, Capital Medical University, Key Laboratory of Remodeling-Related Cardiovascular Diseases, Ministry of Education, Beijing Collaborative Innovation Center for Cardiovascular Disorders, Beijing, China

    Beijing Institute of Heart, Lung and Blood Vessel Disease, Beijing, China
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  • Yuan Wang
    Affiliations
    Beijing Anzhen Hospital, Capital Medical University, Key Laboratory of Remodeling-Related Cardiovascular Diseases, Ministry of Education, Beijing Collaborative Innovation Center for Cardiovascular Disorders, Beijing, China

    Beijing Institute of Heart, Lung and Blood Vessel Disease, Beijing, China
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  • Bingbing Ke
    Affiliations
    Beijing Anzhen Hospital, Capital Medical University, Key Laboratory of Remodeling-Related Cardiovascular Diseases, Ministry of Education, Beijing Collaborative Innovation Center for Cardiovascular Disorders, Beijing, China

    Beijing Institute of Heart, Lung and Blood Vessel Disease, Beijing, China
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  • Jie Du
    Correspondence
    Reprint requests: Jie Du, Beijing Anzhen Hospital, No. 2 Anzhen Rd, Chaoyang District, Beijing 100029, China
    Affiliations
    Beijing Anzhen Hospital, Capital Medical University, Key Laboratory of Remodeling-Related Cardiovascular Diseases, Ministry of Education, Beijing Collaborative Innovation Center for Cardiovascular Disorders, Beijing, China

    Beijing Institute of Heart, Lung and Blood Vessel Disease, Beijing, China
    Search for articles by this author
Open AccessPublished:August 21, 2020DOI:https://doi.org/10.1016/j.trsl.2020.08.007
      An increasing amount of evidence reveals that the gut microbiota is involved in the pathogenesis and progression of various cardiovascular diseases. In patients with heart failure (HF), splanchnic hypoperfusion causes ischemia and intestinal edema, allowing bacterial translocation and bacterial metabolites to enter the blood circulation via an impaired intestinal barrier. This results in local and systemic inflammatory responses. Gut microbe-derived metabolites are implicated in the pathology of multiple diseases, including HF. These landmark findings suggest that gut microbiota influences the host's metabolic health, either directly or indirectly by producing several metabolites. In this review, we mainly discuss a newly identified gut microbiota-dependent metabolite, trimethylamine N-oxide (TMAO), which appears to participate in the pathologic processes of HF and can serve as an early warning marker to identify individuals who are at the risk of disease progression. We also discuss the potential of the gut–TMAO–HF axis as a new target for HF treatment and highlight the current controversies and potentially new and exciting directions for future research.

      Abbreviations:

      AHF (acute HF), BNP (brain natriuretic peptide), CHF (chronic HF), CVDs (cardiovascular diseases), DASH (Dietary to Stop Hypertension), DIM (3,3ʹ-diindolylmethane), DMA (dimethylamine), DMB (3,3-dimethyl-1-butanol), ER (endoplasmic reticulum), FMC (fluoromethylcholine), FMO (flavin-containing monooxygenase), FMT (fecal microbial transplantation), HF (heart failure), HFpEF (HF with preserved ejection fraction), HFrEF (HF with reduced ejection fraction), IL (interleukin), IMC (iodomethylcholine), LPS (lipopolysaccharide), LV (left ventricular), LVAD (left ventricular assist device), LVEF (LV ejection fraction), NF-κB (nuclear factor-κB), NLRP3 (nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing 3), NT-proBNP (N-terminal pro BNP), PKC (protein kinase C), RCT (randomized controlled trial), SCFA (short-chain fatty acid), SIRT3 (sirtuin 3), SOD2 (superoxide dismutase 2), TCA (tricarboxylic acid), TGF-β1 (transforming growth factor-β1), TMA (trimethylamine), TML (trimethyllysine), TMAO (trimethylamine N-oxide), TNF (tumor necrosis factor), VCAM (vascular cell adhesion molecule), γBB (γ-butyrobetaine)

      Introduction

      Heart failure (HF) is the end-stage of various types of cardiovascular diseases (CVDs) and is a common cause of disability and death. Despite recent advances in new drugs and therapeutic strategies, the overall prognosis for patients with HF remains poor, which is mainly reflected in a high rate of readmission and mortality.
      • Chioncel O
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      • et al.
      Clinical phenotypes and outcome of patients hospitalized for acute heart failure: the ESC Heart Failure Long-Term Registry.
      ,
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      • Laroche C
      • et al.
      In-hospital and 1-year mortality associated with diabetes in patients with acute heart failure: results from the ESC-HFA Heart Failure Long-Term Registry.
      The pathophysiological mechanisms of HF are very complicated, including abnormal hemodynamics, activation of neuroendocrine system, cardiac remodeling and inflammatory response, etc. The activation of the neuroendocrine pathways, consisted of renin-angiotensin-aldosterone system, sympathetic nervous system, and natriuretic peptide system, is traditionally believed to be the main cause for HF. It will lead to a series of pathologic myocardial remodeling processes such as myocardial hypertrophy, apoptosis, and extracellular matrix deposition and resultant fibrosis.
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      In search of new therapeutic targets and strategies for heart failure: recent advances in basic science.
      ,
      • Mudd JO
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      Tackling heart failure in the twenty-first century.
      Therefore, current treatment strategies are mainly based on neuroendocrine inhibition.
      • Ponikowski P
      • Voors AA
      • Anker SD
      • et al.
      2016 ESC guidelines for the diagnosis and treatment of acute and chronic heart failure.
      However, the mechanisms underlying HF development and progression are still being explored. To reduce the disease and economic burdens that are associated with HF, it is particularly important to clarify the molecular mechanisms through which HF develops, identify crucial mediators, and further explore new potential therapeutic targets.
      Recent evidence suggests the potential significance of the gut microbiota and its metabolites in mediating or modulating HF pathophysiology.
      • Tang WHW
      • Backhed F
      • Landmesser U
      • Hazen SL
      Intestinal microbiota in cardiovascular health and disease: JACC state-of-the-art review.
      ,
      • Tang WH
      • Kitai T
      • Hazen SL
      Gut microbiota in cardiovascular health and disease.
      The gut hypothesis of HF indicates that decreased cardiac output and alteration of systemic circulation will contribute to bowel hypoperfusion and mucosal ischemia. The impaired gut barrier, in turn, may increase gut permeability, facilitate the translocation of microorganisms, and allow the presence of microbial metabolites into the blood circulation, which ultimately leads to low-grade chronic inflammation in HF patients.
      • Chioncel O
      • Ambrosy AP.
      Trimethylamine N-oxide and risk of heart failure progression: marker or mediator of disease.
      In 2013, based on an untargeted metabolomic analysis, researchers first showed that trimethylamine-N-oxide (TMAO), a molecular metabolite that is derived from the gut microbiota, predicted an increased risk of cardiovascular events in 4007 stable cardiac patients undergoing elective coronary angiography.
      • Wang Z
      • Klipfell E
      • Bennett BJ
      • et al.
      Gut flora metabolism of phosphatidylcholine promotes cardiovascular disease.
      Gut microbiota plays an obligatory role in converting dietary choline into trimethylamine (TMA), which enters the circulatory system before being subsequently oxidized to TMAO in liver.
      • Hernandez D
      • Janmohamed A
      • Chandan P
      • Phillips IR
      • Shephard EA
      Organization and evolution of the flavin-containing monooxygenase genes of human and mouse: identification of novel gene and pseudogene clusters.
      Recently, TMAO has emerged as a significant mediator, demonstrating a close relationship between gut microbiota and multiple CVDs such as atherosclerosis, hypertension, diabetes, and myocardial infarction.
      • Ivashkin VT
      • Kashukh YA.
      Impact of L-carnitine and phosphatidylcholine containing products on the proatherogenic metabolite TMAO production and gut microbiome changes in patients with coronary artery disease.
      • Ge X
      • Zheng L
      • Zhuang R
      • et al.
      The gut microbial metabolite trimethylamine N-oxide and hypertension risk: a systematic review and dose-response meta-analysis.
      • Tang WH
      • Wang Z
      • Li XS
      • et al.
      Increased trimethylamine N-oxide portends high mortality risk independent of glycemic control in patients with type 2 diabetes mellitus.
      • Senthong V
      • Wang Z
      • Fan Y
      • Wu Y
      • Hazen SL
      • Tang WH
      Trimethylamine N-oxide and mortality risk in patients with peripheral artery disease.
      • Tan Y
      • Sheng Z
      • Zhou P
      • et al.
      Plasma trimethylamine N-oxide as a novel biomarker for plaque rupture in patients with ST-segment-elevation myocardial infarction.
      Remarkably, TMAO is also a powerful prognostic marker, participating in the progression of HF.
      • Tang WH
      • Wang Z
      • Fan Y
      • et al.
      Prognostic value of elevated levels of intestinal microbe-generated metabolite trimethylamine-N-oxide in patients with heart failure: refining the gut hypothesis.
      Subsequent preclinical experiments show that TMAO may directly affect the heart by inducing myocardial hypertrophy and fibrosis, endothelial cell and vascular inflammation, as well as cardiac mitochondrial dysfunction, thereby aggravating the progress of HF.
      • Li Z
      • Wu Z
      • Yan J
      • et al.
      Gut microbe-derived metabolite trimethylamine N-oxide induces cardiac hypertrophy and fibrosis.
      • Sun X
      • Jiao X
      • Ma Y
      • et al.
      Trimethylamine N-oxide induces inflammation and endothelial dysfunction in human umbilical vein endothelial cells via activating ROS-TXNIP-NLRP3 inflammasome.
      • Makrecka-Kuka M
      • Volska K
      • Antone U
      • et al.
      Trimethylamine N-oxide impairs pyruvate and fatty acid oxidation in cardiac mitochondria.
      Here, we review current knowledge about the gut–TMAO–HF axis, and consider its potential translational value as a new therapeutic target in HF.

      DISORDERED INTESTINAL METABOLISM IN HF

      HF is characterized by reduced cardiac output and insufficient blood supply to meet the body's demand. The gut is an endocrine organ that is significantly affected by this reduced blood supply. Ischemia and hyperemia caused by a decreased oxygen supply in the intestinal tract will lead to a series of metabolic disorders.

       Functional dysbiosis of the gut

      Generally, the gut contains a highly complex microbiota that plays a vital role in maintaining health by digesting nutrients, producing vitamins and hormones, interfering with pathogen colonization, and shaping healthy mucosal immunity.
      • Carbone S
      • Billingsley HE
      • Abbate A
      The Mediterranean diet to treat heart failure: a potentially powerful tool in the hands of providers.
      • Caesar R
      • Nygren H
      • Oresic M
      • Backhed F
      Interaction between dietary lipids and gut microbiota regulates hepatic cholesterol metabolism.
      • Spencer SP
      • Fragiadakis GK
      • Sonnenburg JL
      Pursuing human-relevant gut microbiota-immune interactions.
      • Buffie CG
      • Pamer EG
      Microbiota-mediated colonization resistance against intestinal pathogens.
      The gut is highly abundant in blood, accounting for approximately 40% of the total human blood. In HF, it is the first organ to undergo ischemia and the last to recover, during which the intestinal villi (and microvilli) are prone to functional anoxia.
      • Takala J.
      Determinants of splanchnic blood flow.
      In addition, visceral venous congestion caused by right HF may also lead to decreased blood flow to intestinal epithelial cells, resulting in cell hypoxia, anaerobic metabolism, and overexpression of the sodium/hydrogen exchanger 3, thereby increasing sodium transport and lowering the lumen pH.
      • Polsinelli VB
      • Marteau L
      • Shah SJ
      The role of splanchnic congestion and the intestinal microenvironment in the pathogenesis of advanced heart failure.
      All these factors eventually contribute to a shifting composition of the gut microbiota, which is mainly manifested by a reduction in Bacteroides and Bifidobacteria and an increase in Firmicutes and Proteobacteria.
      • Chen K
      • Zheng X
      • Feng M
      • Li D
      • Zhang H
      Gut microbiota-dependent metabolite trimethylamine N-oxide contributes to cardiac dysfunction in western diet-induced obese mice.
      Moreover, increased concentrations of enteropathogenic Candida such as Salmonella, Shigella, and Campylobacter were found in fecal samples from chronic HF (CHF) patients.
      • Pasini E
      • Aquilani R
      • Testa C
      • et al.
      Pathogenic gut flora in patients with chronic heart failure.
      Sandek et al
      • Sandek A
      • Bauditz J
      • Swidsinski A
      • et al.
      Altered intestinal function in patients with chronic heart failure.
      also observed excessive bacterial growth and adhesion in the intestinal mucosa of patients with HF.

       Intestinal barrier dysfunction triggers chronic inflammation

      HF has been considered as a chronic systemic inflammatory disease, manifested by a significant increase in the levels of various plasma pro-inflammatory cytokines. Unresolved inflammation is a major component of CVD, but its origin remains unclear.
      • Pullen AB
      • Jadapalli JK
      • Rhourri-Frih B
      • Halade GV
      Re-evaluating the causes and consequences of non-resolving inflammation in chronic cardiovascular disease.
      ,
      • Liljestrand JM
      • Paju S
      • Pietiainen M
      • et al.
      Immunologic burden links periodontitis to acute coronary syndrome.
      Recent evidence suggests that disordered gut microbiota and increased intestinal permeability may be triggers for chronic inflammation, leading to further impaired cardiac function.
      • Sandek A
      • Bauditz J
      • Swidsinski A
      • et al.
      Altered intestinal function in patients with chronic heart failure.
      ,
      • Cox AJ
      • West NP
      • Cripps AW
      Obesity, inflammation, and the gut microbiota.
      Structurally, systemic congestion in HF patients can cause intestinal wall edema, which results in increased intestinal permeability.
      • Sandek A
      • Bauditz J
      • Swidsinski A
      • et al.
      Altered intestinal function in patients with chronic heart failure.
      The disrupted intestinal epithelial barrier will lead to the entry of bacteria and bacterial products into the circulation.
      • Sandek A
      • Anker SD
      • von Haehling S
      The gut and intestinal bacteria in chronic heart failure.
      Patients with bacterial DNA in peripheral blood have significantly higher levels of inflammatory markers such as hypersensitive C-reactive protein and interleukin (IL)-6.
      • Wang F
      • Jiang H
      • Shi K
      • Ren Y
      • Zhang P
      • Cheng S
      Gut bacterial translocation is associated with microinflammation in end-stage renal disease patients.
      Endotoxin (lipopolysaccharide: LPS), an important toxic and immunogenic component of gram-negative bacteria, also enters the blood through the swollen intestinal wall and is a strong stimulator for activated pro-inflammatory cytokines in HF patients.
      • Anker SD
      • Egerer KR
      • Volk HD
      • Kox WJ
      • Poole-Wilson PA
      • Coats AJ
      Elevated soluble CD14 receptors and altered cytokines in chronic heart failure.
      ,
      • Munger MA
      • Johnson B
      • Amber IJ
      • Callahan KS
      • Gilbert EM
      Circulating concentrations of proinflammatory cytokines in mild or moderate heart failure secondary to ischemic or idiopathic dilated cardiomyopathy.
      LPS induces the production of various downstream inflammatory factors by acting directly on cardiomyocytes, cardiac fibroblasts, and macrophages through Toll-like receptor 4 pattern recognition receptors.
      • Lu YC
      • Yeh WC
      • Ohashi PS
      LPS/TLR4 signal transduction pathway.
      ,
      • Frangogiannis NG.
      The inflammatory response in myocardial injury, repair, and remodelling.
      Studies have found that serum levels of multiple cytokines such as IL-1, IL-6, and tumor necrosis factor (TNF) are elevated in HF patients and associated with severe clinical symptoms and worse survival rates.
      • Rauchhaus M
      • Doehner W
      • Francis DP
      • et al.
      Plasma cytokine parameters and mortality in patients with chronic heart failure.
      • Deswal A
      • Petersen NJ
      • Feldman AM
      • Young JB
      • White BG
      • Mann DL
      Cytokines and cytokine receptors in advanced heart failure: an analysis of the cytokine database from the Vesnarinone trial (VEST).
      • Conraads VM
      • Bosmans JM
      • Schuerwegh AJ
      • et al.
      Intracellular monocyte cytokine production and CD 14 expression are up-regulated in severe vs mild chronic heart failure.
      Meanwhile, these cytokines are also involved in the process of cardiomyocyte apoptosis, hypertrophy, and fibrosis.
      • Mann DL.
      Innate immunity and the failing heart: the cytokine hypothesis revisited.
      However, current attempts to treat the inflammatory response in HF patients by blocking cytokines have not achieved success.
      • Mann DL
      • McMurray JJ
      • Packer M
      • et al.
      Targeted anticytokine therapy in patients with chronic heart failure: results of the Randomized Etanercept Worldwide Evaluation (RENEWAL).
      ,
      • Torre-Amione G
      • Anker SD
      • Bourge RC
      • et al.
      Results of a non-specific immunomodulation therapy in chronic heart failure (ACCLAIM trial): a placebo-controlled randomised trial.
      Thus, more attention has been averted to the role of microbial metabolites in HF.

       Changes in microbial metabolites: focus on TMAO

      An increasing amount of evidence suggests that gut microbiota may have systemic effects on the host by generating bioactive metabolites, such as short-chain fatty acids, bile acids, and TMAO.
      • Tang WHW
      • Li DY
      • Hazen SL
      Dietary metabolism, the gut microbiome, and heart failure.
      These metabolites influence intestinal health and other physiological systems, particularly the circulatory system. Although most bacterial metabolites are healthy under normal conditions, harmful metabolites will increase when the gut microbiota's balance is disrupted, and this may be involved in HF-related cardiac pathologic processes. One of the metabolites, TMAO, was shown to be associated with the prognosis of patients with HF.

      TMAO BIOGENESIS AND METABOLISM

      Gut microbiota can transform various dietary nutrients into trimethylamine (TMA). Most of the TMA enters the circulatory system and is subsequently oxidized to TMAO by hepatic flavin-containing monooxygenase (FMO). The excess directly decomposes into dimethylamine (DMA) or methane.
      • Bennett BJ
      • de Aguiar Vallim TQ
      • Wang Z
      • et al.
      Trimethylamine-N-oxide, a metabolite associated with atherosclerosis, exhibits complex genetic and dietary regulation.
      The host FMO family contains 5 functional enzymes, and FMO3 is the key rate-limiting enzyme that is involved in the transformation of TMA to TMAO, with the highest conversion efficiency.
      • Hernandez D
      • Janmohamed A
      • Chandan P
      • Phillips IR
      • Shephard EA
      Organization and evolution of the flavin-containing monooxygenase genes of human and mouse: identification of novel gene and pseudogene clusters.
      FMO3 gene mutation contributes to less TMA oxidization, and excessively accumulated TMA is excreted in the urine and sweat and by respiration, producing a strong “fishy” odor.
      • Messenger J
      • Clark S
      • Massick S
      • Bechtel M
      A review of trimethylaminuria: (fish odor syndrome).
      Previous studies revealed that TMAO could accumulate in the heart, kidney, or other tissues, participating in various biological processes, such as activating platelet aggregation, increasing foam cell formation, inducing inflammatory responses, and decreasing reverse cholesterol transport.
      • Tang WHW
      • Backhed F
      • Landmesser U
      • Hazen SL
      Intestinal microbiota in cardiovascular health and disease: JACC state-of-the-art review.
      ,
      • Wang Z
      • Klipfell E
      • Bennett BJ
      • et al.
      Gut flora metabolism of phosphatidylcholine promotes cardiovascular disease.
      ,
      • Zhu W
      • Buffa JA
      • Wang Z
      • et al.
      Flavin monooxygenase 3, the host hepatic enzyme in the metaorganismal trimethylamine N-oxide-generating pathway, modulates platelet responsiveness and thrombosis risk.
      In most cases, the majority of TMAO is eliminated by the kidney, and the rest will be reduced to TMA by the enzyme TMAO reductase in the gut.
      • Dai Q
      • Zhang H
      • Liu Y
      Trimethylamine-N-oxide and cardiovascular events in chronic kidney disease.
      ,
      • Pascal MC
      • Burini JF
      • Chippaux M
      Regulation of the trimethylamine N-oxide (TMAO) reductase in Escherichia coli: analysis of tor::Mud1 operon fusion.
      Red meat, eggs, fish, and dairy products are abundant in TMA nutritional precursors, such as choline, betaine, L-carnitine, crotonobetaine, trimethyllysine, γ-butyrobetaine, phosphatidylcholine, glycerophosphocholine, and trimethylamine-N-oxide.
      • Organ CL
      • Otsuka H
      • Bhushan S
      • et al.
      Choline diet and its gut microbe-derived metabolite, trimethylamine N-oxide, exacerbate pressure overload-induced heart failure.
      ,
      • Wang Z
      • Zhao Y.
      Gut microbiota derived metabolites in cardiovascular health and disease.
      These precursors can be converted to TMA via specific intestinal microbial enzymes. To date, 4 different microbial enzyme systems have been identified: choline-TMA lyase (cutC/D),
      • Craciun S
      • Marks JA
      • Balskus EP
      Characterization of choline trimethylamine-lyase expands the chemistry of glycyl radical enzymes.
      carnitine monooxygenase (cntA/B),
      • Zhu Y
      • Jameson E
      • Crosatti M
      • et al.
      Carnitine metabolism to trimethylamine by an unusual Rieske-type oxygenase from human microbiota.
      betaine reductase,
      • Andreesen JR.
      Glycine metabolism in anaerobes.
      and TMAO reductase.
      • Pascal MC
      • Burini JF
      • Chippaux M
      Regulation of the trimethylamine N-oxide (TMAO) reductase in Escherichia coli: analysis of tor::Mud1 operon fusion.
      In addition, yeaW/X, which is homologous to cntA/B and can utilize a variety of substrates, also promotes TMA synthesis (Fig 1).
      • Koeth RA
      • Levison BS
      • Culley MK
      • et al.
      gamma-Butyrobetaine is a proatherogenic intermediate in gut microbial metabolism of L-carnitine to TMAO.
      Fig 1
      Fig 1TMAO biogenesis and metabolism. Dietary nutrients (ie, choline) are biotransformed into TMA by TMA lyases (ie, cutC/D) in the gut. Most TMA enters the circulatory system and is further oxidized into TMAO by FMO3 in the liver, and the excess is decomposed into DMA and methane. Circulating TMAO can activate platelet hyper-reactivity, increase foam cell formation, induce inflammatory responses, and decrease reverse cholesterol transport. These effects contribute to the progression of atherosclerosis, heart failure, or chronic kidney disease. Finally, TMAO is mainly eliminated by the kidney, and the rest will be reduced to TMA by the TMAO reductase.
      The change in gut microbiota composition that is caused by HF can alter circulating TMAO levels.
      • Koeth RA
      • Wang Z
      • Levison BS
      • et al.
      Intestinal microbiota metabolism of L-carnitine, a nutrient in red meat, promotes atherosclerosis.
      Researchers have discovered 9 human intestinal strains that are capable of producing TMA, such as Firmicutes and Proteobacteria.
      • Romano KA
      • Vivas EI
      • Amador-Noguez D
      • Rey FE
      Intestinal microbiota composition modulates choline bioavailability from diet and accumulation of the proatherogenic metabolite trimethylamine-N-oxide.
      This is consistent with findings of an increased proportion of these gut bacterial strains in HF patients, which suggests that changes in gut microbiota may affect TMAO levels by regulating intestinal TMA synthesis.
      It is worth noting that genetic factors also play an important role in the production of intestinal metabolites. Studies have revealed that host genetic directly affects the composition of the gut microbiome and regulates immune pathways and metabolic phenotypes.
      • Jacobs J
      • Braun J.
      Host genes and their effect on the intestinal microbiome garden.
      ,
      • Hall AB
      • Tolonen AC
      • Xavier RJ
      Human genetic variation and the gut microbiome in disease.
      Goodrich et al
      • Goodrich JK
      • Waters JL
      • Poole AC
      • et al.
      Human genetics shape the gut microbiome.
      found that the susceptibility of developing diseases such as obesity may be partially due to genetics which can alter the gut microbiota. Knights et al
      • Knights D
      • Silverberg MS
      • Weersma RK
      • et al.
      Complex host genetics influence the microbiome in inflammatory bowel disease.
      successfully identified 48 polymorphisms related to inflammatory bowel disease and suggested a complex link between host-genetics and microbial composition of this population. In a comprehensive analysis of genome-wide host–microbiota associations, genetic factors accounted for approximately 10% of the gut microbiome variation.
      • Wang J
      • Thingholm LB
      • Skieceviciene J
      • et al.
      Genome-wide association analysis identifies variation in vitamin D receptor and other host factors influencing the gut microbiota.
      Therefore, based on the important role genetics plays in the composition of intestinal flora, it is presumable that in addition to environmental, dietary and disease factors, genetics also plays a role in the production of TMAO.

      PATHOLOGICAL MECHANISMS OF TMAO IN HF

      Several experimental studies have demonstrated that TMAO directly or indirectly affects the heart and exacerbates the procession of HF (TableI).
      Table IMain pathophysiological mechanisms of TMAO in heart failure: directly and indirectly
      Authors (year)Species/cellsMain findingsRef
      Direct effect
      Li et al (2018)RatsTMAO promoted myocardial hypertrophy and fibrosis via Smad3 signaling pathway.
      • Li Z
      • Wu Z
      • Yan J
      • et al.
      Gut microbe-derived metabolite trimethylamine N-oxide induces cardiac hypertrophy and fibrosis.
      Wang et al (2020)Mice3,3-dimethyl-1-butanol attenuated cardiac remodeling by reducing plasma TMAO levels, which negatively regulated the TGF-β1/Smad3 signaling and p65 NF-κB signaling pathways.
      • Wang G
      • Kong B
      • Shuai W
      • Fu H
      • Jiang X
      • Huang H
      3,3-Dimethyl-1-butanol attenuates cardiac remodeling in pressure-overload-induced heart failure mice.
      Organ et al (2016)MiceMice fed TMAO or choline showed pathological LV dilation, reduced LV ejection fraction, increased circulating BNP levels, pulmonary edema and myocardial fibrosis.
      • Organ CL
      • Otsuka H
      • Bhushan S
      • et al.
      Choline diet and its gut microbe-derived metabolite, trimethylamine N-oxide, exacerbate pressure overload-induced heart failure.
      Seldin et al (2016)MiceTMAO promoted recruitment of activated leukocytes to endothelial cells.

      Activation of NF-κB signaling was necessary for TMAO to induce inflammatory gene expression.
      • Seldin MM
      • Meng Y
      • Qi H
      • et al.
      Trimethylamine N-oxide promotes vascular inflammation through signaling of mitogen-activated protein kinase and nuclear factor-kappa B.
      Sun et al (2016)Human umbilical vein endothelial cellsTMAO induced inflammation and endothelial dysfunction via activating ROS-TXNIP-NLRP3 inflammasome.
      • Sun X
      • Jiao X
      • Ma Y
      • et al.
      Trimethylamine N-oxide induces inflammation and endothelial dysfunction in human umbilical vein endothelial cells via activating ROS-TXNIP-NLRP3 inflammasome.
      Chen et al (2017)Human umbilical vein endothelial cells and MiceTMAO promoted vascular inflammation by activating the NLRP3 inflammasome, which was mediated through inhibition of the SIRT3-SOD2–mitochondrial ROS signaling pathway.
      • Chen ML
      • Zhu XH
      • Ran L
      • Lang HD
      • Yi L
      • Mi MT
      Trimethylamine-N-oxide induces vascular inflammation by activating the NLRP3 inflammasome through the SIRT3-SOD2-mtROS signaling pathway.
      Ma et al (2017)Human umbilical vein endothelial cellsEffect of TMAO on endothelial dysfunction was partly attributable to activation of PKC/NF-κB, leading to elevated expression of VCAM-1 and monocyte adhesion.
      • Ma G
      • Pan B
      • Chen Y
      • et al.
      Trimethylamine N-oxide in atherogenesis: impairing endothelial self-repair capacity and enhancing monocyte adhesion.
      Makrecka-Kuka et al (2017)MiceTMAO concentration impaired pyruvate and fatty acid oxidation in cardiac mitochondria.
      • Makrecka-Kuka M
      • Volska K
      • Antone U
      • et al.
      Trimethylamine N-oxide impairs pyruvate and fatty acid oxidation in cardiac mitochondria.
      Savi et al (2018)RatsTMAO had a direct negative impact on cardiomyocyte contractile function and intracellular calcium handling.
      • Savi M
      • Bocchi L
      • Bresciani L
      • et al.
      Trimethylamine-N-oxide (TMAO)-induced impairment of cardiomyocyte function and the protective role of urolithin B-glucuronide.
      Indirect effect
      Tang et al (2015)MiceDietary TMAO promoted renal fibrosis and dysfunction.
      • Tan Y
      • Sheng Z
      • Zhou P
      • et al.
      Plasma trimethylamine N-oxide as a novel biomarker for plaque rupture in patients with ST-segment-elevation myocardial infarction.
      Zhu et al (2016)MiceTMAO directly increased platelet hyperreactivity and thrombosis formation.
      • Tang WH
      • Wang Z
      • Kennedy DJ
      • et al.
      Gut microbiota-dependent trimethylamine N-oxide (TMAO) pathway contributes to both development of renal insufficiency and mortality risk in chronic kidney disease.
      Wang et al (2011)MiceDietary supplementation of mice with TMAO promoted upregulation of multiple macrophage scavenger receptors linked to atherosclerosis.
      • Wang Z
      • Klipfell E
      • Bennett BJ
      • et al.
      Gut flora metabolism of phosphatidylcholine promotes cardiovascular disease.
      Abbreviations: LV, left ventricular; NF-κB, nuclear factor-κB; NLRP3, nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing 3; PKC, protein kinase C; ROS, reactive oxygen species; SOD2, superoxide dismutase 2; SIRT3, sirtuin 3; TAC, transverse aortic constriction; TGF-β1, transforming growth factor-β1; TMAO, trimethylamine N-oxide; TXNIP, thioredoxin-interacting protein; VCAM, vascular cell adhesion molecule.

       Direct effect

       TMAO affects myocardial hypertrophy and fibrosis

      In transverse aortic constriction-induced rats, a well-established model of pressure-overloaded HF, the circulating TMAO levels were significantly higher compared with sham-operated groups.
      • Organ CL
      • Otsuka H
      • Bhushan S
      • et al.
      Choline diet and its gut microbe-derived metabolite, trimethylamine N-oxide, exacerbate pressure overload-induced heart failure.
      Li et al
      • Li Z
      • Wu Z
      • Yan J
      • et al.
      Gut microbe-derived metabolite trimethylamine N-oxide induces cardiac hypertrophy and fibrosis.
      showed that in in vivo and in vitro studies, TMAO promoted myocardial hypertrophy and fibrosis via Smad3 signaling pathway. This promotion was blocked by a specific Smad3 inhibitor, SIS3. Subsequently, an inhibitor of TMA synthesis, 3,3-dimethyl-1-butanol (DMB) was found to be capable of preventing myocardial hypertrophy and fibrosis via regulating transforming growth factor-β1 (TGF-β1)/Smad3 and p65 nuclear factor-κB (NF-κB) signaling pathways,
      • Wang G
      • Kong B
      • Shuai W
      • Fu H
      • Jiang X
      • Huang H
      3,3-Dimethyl-1-butanol attenuates cardiac remodeling in pressure-overload-induced heart failure mice.
      which also confirmed the role of TMAO in ventricular remodeling. Additionally, mice fed TMAO or choline showed worse pulmonary edema, left ventricular (LV) dilation, cardiac fibrosis, and elevated circulating brain natriuretic peptide (BNP) levels compared with control groups.
      • Organ CL
      • Otsuka H
      • Bhushan S
      • et al.
      Choline diet and its gut microbe-derived metabolite, trimethylamine N-oxide, exacerbate pressure overload-induced heart failure.
      A similar result was obtained in another study, in which TMAO aggravated myocardial interstitial and perivascular fibrosis, as well as damaged cardiac compliance and function.
      • Chen K
      • Zheng X
      • Feng M
      • Li D
      • Zhang H
      Gut microbiota-dependent metabolite trimethylamine N-oxide contributes to cardiac dysfunction in western diet-induced obese mice.

       TMAO induces an inflammatory response

      In mice that were fed choline, TMAO directly activated inflammatory pathways such as NF-κB signaling, leading to inflammation in vascular smooth muscle cells.
      • Seldin MM
      • Meng Y
      • Qi H
      • et al.
      Trimethylamine N-oxide promotes vascular inflammation through signaling of mitogen-activated protein kinase and nuclear factor-kappa B.
      Moreover, it can promote the mitochondrial reactive oxygen species (mtROS) accumulation by inhibiting sirtuin 3 (SIRT3) expression and superoxide dismutase 2 (SOD2) activity, which will further activate nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing 3 (NLRP3) inflammasomes. When activated, the NLRP3 inflammasomes generate IL-1β and IL-18, eventually leading to endothelial cell inflammation.
      • Sun X
      • Jiao X
      • Ma Y
      • et al.
      Trimethylamine N-oxide induces inflammation and endothelial dysfunction in human umbilical vein endothelial cells via activating ROS-TXNIP-NLRP3 inflammasome.
      ,
      • Chen ML
      • Zhu XH
      • Ran L
      • Lang HD
      • Yi L
      • Mi MT
      Trimethylamine-N-oxide induces vascular inflammation by activating the NLRP3 inflammasome through the SIRT3-SOD2-mtROS signaling pathway.
      Additionally, Ma et al
      • Ma G
      • Pan B
      • Chen Y
      • et al.
      Trimethylamine N-oxide in atherogenesis: impairing endothelial self-repair capacity and enhancing monocyte adhesion.
      demonstrated that TMAO upregulated vascular cell adhesion molecule (VCAM)-1 expression by activating protein kinase C (PKC)/NF-κB, which directly resulted in endothelial dysfunction that was characterized by reduced self‐healing capacity and increased monocyte adhesion. In a study to investigate whether inhibiting TMAO prevented myocardial inflammation, Zhang et al
      • Zhang H
      • Meng J
      • Yu H
      Trimethylamine N-oxide supplementation abolishes the cardioprotective effects of voluntary exercise in mice fed a western diet.
      discovered that TMAO might induce myocardial inflammation by elevating TNF-α levels and decreasing IL-10 levels, thereby reversing the cardioprotective effect of exercise on myocardial fibrosis.

       TMAO exacerbates mitochondrial dysfunction

      Makrecka-Kuka et al
      • Makrecka-Kuka M
      • Volska K
      • Antone U
      • et al.
      Trimethylamine N-oxide impairs pyruvate and fatty acid oxidation in cardiac mitochondria.
      found that after feeding mice 120 mg/kg of TMAO for 8 weeks, the increased plasma TMAO affected cardiac energy metabolism and mitochondrial function by influencing the pyruvate and fatty acid oxidation, which finally led to the ventricular remodeling and HF development. Subsequently, Savi et al
      • Savi M
      • Bocchi L
      • Bresciani L
      • et al.
      Trimethylamine-N-oxide (TMAO)-induced impairment of cardiomyocyte function and the protective role of urolithin B-glucuronide.
      indicated for the first time that TMAO affected contractile function and intracellular calcium processing in cardiomyocytes, which could be attributed to reduce energy production because of TMAO-induced mitochondrial dysfunction (Fig 2).
      Fig 2
      Fig 2TMAO causes direct damage to the heart. Gut microbiota dysbiosis has been shown to occur with increased circulating TMAO levels. In cardiomyocytes, TMAO activates TGF-β1/Smad3 and p65 NF-κB signaling pathways and decreases energy metabolism and mitochondrial function by influencing the pyruvate and fatty acid oxidation, which are involved in the tricarboxylic acid (TCA) cycle. It also negatively affects the myocardial contractile function and intracellular calcium processing. In endothelial cells, TMAO induces IL-1β and IL-18 release via inhibiting SIRT3–SOD2 pathway and activating NLRP3 inflammasomes. It also promotes endothelial dysfunction by activating PKC/NF-κB/VCAM-1 pathway. Taken together, these mechanisms eventually contribute to the progression of HF.

       Indirect effect

      In addition to cardiac damage, the role of renal function in HF deterioration should not be ignored. Because the kidney plays a vital role in the process of excreting TMAO, impaired renal function is closely associated with elevated plasma TMAO levels.
      • Roberts AB
      • Gu X
      • Buffa JA
      • et al.
      Development of a gut microbe-targeted nonlethal therapeutic to inhibit thrombosis potential.
      Data from the Framingham heart study showed that increased TMAO directly contributed to renal interstitial fibrosis and dysfunction, promoting sodium and water retention.
      • Tang WH
      • Wang Z
      • Kennedy DJ
      • et al.
      Gut microbiota-dependent trimethylamine N-oxide (TMAO) pathway contributes to both development of renal insufficiency and mortality risk in chronic kidney disease.
      All these pathophysiologic mechanisms could interact in a vicious cycle that further aggravates the procession of HF.
      For etiology, myocardial ischemia or infarction is the primary cause of HF. Studies have indicated that TMAO significantly induces platelet hyper-reactivity and increases the risk of thrombosis,
      • Zhu W
      • Gregory JC
      • Org E
      • et al.
      Gut microbial metabolite TMAO enhances platelet hyperreactivity and thrombosis risk.
      which can potentially lead to tissue infarction. Wang et al
      • Wang Z
      • Klipfell E
      • Bennett BJ
      • et al.
      Gut flora metabolism of phosphatidylcholine promotes cardiovascular disease.
      also found that TMAO promoted foam cell formation and aggravated atherosclerosis. All the above pathologic processes are involved in the occurrence of myocardial ischemia or infarction, thus exacerbating the progression of ischemic HF.

      TMAO AS A PROGNOSTIC MARKER OF HF

      TMAO was shown to be a promising cardiovascular risk marker, representing an independent tool for predicting adverse events in patients with HF.

       AHF and CHF

      Suzuki et al
      • Suzuki T
      • Heaney LM
      • Bhandari SS
      • Jones DJ
      • Ng LL
      Trimethylamine N-oxide and prognosis in acute heart failure.
      first assessed the role of TMAO in acute HF (AHF) and found that circulating TMAO was a marker for predicting death and death/HF within 1 year. However, after adjusting for renal function parameters, TMAO lost the ability of independent prediction, possibly because of the significant associations between TMAO and renal function parameters (urea and estimated glomerular filtration rate). Combining TMAO with the current clinical risk algorithm improved the risk stratification of in-hospital mortality, and adding the N-terminal pro BNP (NT-proBNP) to this model further enhanced the prediction efficiency of death/HF within 1 year. Moreover, this study also identified that patients with increased levels of both markers (TMAO and NT-proBNP) had the highest risk of death/HF. Recently, after investigating about whether the associations between TMAO levels and HF outcomes were influenced by ethnicity, this team found that only elevated TMAO levels in Caucasian patients showed increased association with adverse outcomes, but not in non-Caucasian patients.
      • Yazaki Y
      • Aizawa K
      • Israr MZ
      • et al.
      Ethnic differences in association of outcomes with trimethylamine N-oxide in acute heart failure patients.
      However, clinical data on TMAO in AHF are very limited, and validation in a larger, multicenter study with a broader cohort is needed to gain additional information and enhance our understanding of the clinical application of TMAO in evaluating the postadmission outcomes in AHF patients.
      Studies of TMAO in CHF are more in depth. In a study of 720 stable patients with CHF, the association between elevated TMAO levels and cardiovascular events was identified for the first time. Compared with age- and sex-matched subjects without HF, CHF patients had significantly higher TMAO levels that were associated with a 3.4-fold increased mortality risk. Even following adjustment for traditional risk factors and cardiorenal indexes, the elevated TMAO levels still could predict an increased risk of 5-year mortality.
      • Tang WH
      • Wang Z
      • Fan Y
      • et al.
      Prognostic value of elevated levels of intestinal microbe-generated metabolite trimethylamine-N-oxide in patients with heart failure: refining the gut hypothesis.
      Another study observed that in addition to TMAO, choline and betaine both seemed to participate in the aggravation of LV diastolic dysfunction; however, only elevated TMAO levels showed an adverse prognostic value after adjusting for cardiopulmonary parameters.
      • Tang WH
      • Wang Z
      • Shrestha K
      • et al.
      Intestinal microbiota-dependent phosphatidylcholine metabolites, diastolic dysfunction, and adverse clinical outcomes in chronic systolic heart failure.
      A similar finding was subsequently reported in a Norwegian cohort, where circulating TMAO levels in CHF patients were associated with the New York Heart Association classification, ischemic etiology, mortality, and survival rate after heart transplantation.
      • Troseid M
      • Ueland T
      • Hov JR
      • et al.
      Microbiota-dependent metabolite trimethylamine-N-oxide is associated with disease severity and survival of patients with chronic heart failure.
      The BIOSTAT-CHF (biological research on the personalized treatment of CHF) study first investigated the response of TMAO levels to treatments and discovered that the current guideline-based medications for CHF might not affect circulating TMAO levels. Patients who had low TMAO levels at baseline or follow-up showed higher survival; however, continuous elevated TMAO levels before and after treatment were related to a higher mortality rate.
      • Suzuki T
      • Yazaki Y
      • Voors AA
      • et al.
      Association with outcomes and response to treatment of trimethylamine N-oxide in heart failure: results from BIOSTAT-CHF.
      This cohort was a multicenter study involving 11 countries in Europe, and another study based on it explored the impact of geographical factors on TMAO levels. It was found that even after adjusting for confounding, TMAO levels differed by region and their relationships with HF outcomes were also different.
      • Yazaki Y
      • Salzano A
      • Nelson CP
      • et al.
      Geographical location affects the levels and association of trimethylamine N-oxide with heart failure mortality in BIOSTAT-CHF: a post-hoc analysis.

       HFrEF and HFpEF

      HF subtypes are classified as HF with preserved ejection fraction (HFpEF) and HF with reduced ejection fraction (HFrEF) based on the LV ejection fraction (LVEF). Their pathophysiological mechanisms and prognosis are quite different. To explore the role of TMAO in both HF types, a clinical study of 823 patients found that elevated TMAO levels predicted cardiovascular events in HFrEF patients, but not in HFpEF patients.
      • Schuett K
      • Kleber ME
      • Scharnagl H
      • et al.
      Trimethylamine-N-oxide and heart failure with reduced versus preserved ejection fraction.
      Conversely, in another study focusing on HFpEF, TMAO levels contributed to risk stratification of HFpEF patients, especially when BNP levels were not high. Moreover, the author suggested that the combination of BNP and TMAO concentrations will provide more valuable prognostic information to HFpEF patients.
      • Salzano A
      • Israr MZ
      • Yazaki Y
      • et al.
      Combined use of trimethylamine N-oxide with BNP for risk stratification in heart failure with preserved ejection fraction: findings from the DIAMONDHFpEF study.
      Because the results of the 2 studies are opposite, further investigations are required to elucidate the specific value of TMAO in HFpEF patients.

       Advanced HF

      Patients who have been operated heart transplant may represent a subset of the population with advanced HF. In a double-blind, multicenter, open-label study, Troseid et al
      • Troseid M
      • Mayerhofer CCK
      • Broch K
      • et al.
      The carnitine-butyrobetaine-TMAO pathway after cardiac transplant: Impact on cardiac allograft vasculopathy and acute rejection.
      evaluated circulating levels of γ-butyrobetaine (γBB), TMAO, carnitine, and trimethyllysine (TML) in patients who received heart transplantation and found that the baseline levels of TMAO, carnitine and TML in heart transplant recipients were higher than controls. During the next 3 years of follow-up, plasma TMAO levels were negatively correlated with renal function; increased γBB and TML levels were associated with changes in total atheroma volume within 3 years; increased γBB and carnitine levels from baseline to 1 year were associated with high incidence of acute rejection within 1 year of transplantation. Recently, a cross-sectional study explored changes in gut microbiota and inflammatory environment in advanced HF patients after left ventricular assist device (LVAD) or heart transplantation, finding that the diversity of gut microbiota decreased, while the endotoxemia and systemic inflammation increased. This result indicates that the restoration of hemodynamics is only partially responsible for improving the overall state of HF and attention to the new type of intestinal dysfunction-inflammation partnership is necessary.
      • Madan S
      • Mehra MR.
      The heart-gut microbiome axis in advanced heart failure.
      ,
      • Yuzefpolskaya M
      • Bohn B
      • Nasiri M
      • et al.
      Gut microbiota, endotoxemia, inflammation, and oxidative stress in patients with heart failure, left ventricular assist device, and transplant.

      GUT–TMAO–HF AXIS AS A POTENTIAL THERAPEUTIC TARGET FOR HF

      Emerging evidence from various groups and clinical observations showed the relationship among gut microbiota disorder, circulating TMAO level, and HF susceptibility, suggesting that the gut–TMAO–HF axis is a new and attractive therapeutic target for HF treatment (TableII).
      Table IIRepresentative research on targeting gut–TMAO–HF axis for the treatment of heart failure
      TreatmentInterventionPatients/modelMain findingsRef
      DietMediterranean dietAHFAdherence to the Mediterranean diet was associated with decreased rate of AHF rehospitalization during the next yearCarbone et al (2018)
      • Carbone S
      • Billingsley HE
      • Abbate A
      The Mediterranean diet to treat heart failure: a potentially powerful tool in the hands of providers.
      NoneHFHigher dietary approaches to stop hypertension diet scores were associated with lower mortality in women with HF.Levitan et al (2013)
      • Levitan EB
      • Lewis CE
      • Tinker LF
      • et al.
      Mediterranean and DASH diet scores and mortality in women with heart failure: the Women's Health Initiative.
      NoneHealthy volunteers; Vegetarians; Vegans; 0mnivoresLow adherence to the Mediterranean diet corresponded to an increase in urinary TMAO levels;

      High-level consumption of plant foodstuffs was associated with beneficial microbiome-related metabolomic.
      De Filippis et al (2016),
      • De Filippis F
      • Pellegrini N
      • Vannini L
      • et al.
      High-level adherence to a Mediterranean diet beneficially impacts the gut microbiota and associated metabolome.
      ProbioticsLactobacillus rhamnosus GR-1RatsRats administered GR-1 exhibited a significant attenuation of left ventricular hypertrophy and improved hemodynamic parameters.Gan et al (2014)
      • Gan XT
      • Ettinger G
      • Huang CX
      • et al.
      Probiotic administration attenuates myocardial hypertrophy and heart failure after myocardial infarction in the rat.
      Lactobacillus plantarum 299vMiceSupplementing Antibiotic-treated mice with a Lactobacillus probiotic prior to MI yielded cardioprotective effects and shifted the balance of SCFAs towards propionate.Tang et al (2019)
      • Tang TWH
      • Chen HC
      • Chen CY
      • et al.
      Loss of gut microbiota alters immune system composition and cripples postinfarction cardiac repair.
      Saccharomyces boulardiiCHF patientsCHF patients treated with S. boulardii for 3-months presented reduced inflammatory biomarkers and improved cardiovascular function.Costanza et al (2015)
      • Costanza AC
      • Moscavitch SD
      • Faria Neto HC
      • Mesquita ET
      Probiotic therapy with Saccharomyces boulardii for heart failure patients: a randomized, double-blind, placebo-controlled pilot trial.
      PrebioticsInulin-propionate ester/inulinAdults with overweight and obesitySupplementing the diet with inulin-propionate ester/inulin improved glucose homeostasis and reduced inflammatory markers in humans.Chambers et al (2019)
      • Chambers ES
      • Byrne CS
      • Morrison DJ
      • et al.
      Dietary supplementation with inulin-propionate ester or inulin improves insulin sensitivity in adults with overweight and obesity with distinct effects on the gut microbiota, plasma metabolome and systemic inflammatory responses: a randomised cross-over trial.
      TMA-lyase inhibitorsFMC/IMCMiceFMC and IMC sustainably suppressed host TMAO levels without observed toxicity.Roberts et al (2018)
      • Roberts AB
      • Gu X
      • Buffa JA
      • et al.
      Development of a gut microbe-targeted nonlethal therapeutic to inhibit thrombosis potential.
      DMBMiceDMB attenuated pressure overload-induced cardiac remodeling by reducing plasma TMAO levelsWang et al (2020)
      • Wang G
      • Kong B
      • Shuai W
      • Fu H
      • Jiang X
      • Huang H
      3,3-Dimethyl-1-butanol attenuates cardiac remodeling in pressure-overload-induced heart failure mice.
      Natural phytochemicalsResveratrolMiceResveratrol attenuated atherosclerosis by decreasing TMAO levels and increasing hepatic bile acid neosynthesis via gut microbiota remodeling.Chen et al (2016)
      • Chen ML
      • Yi L
      • Zhang Y
      • et al.
      Resveratrol attenuates trimethylamine-N-oxide (TMAO)-induced atherosclerosis by regulating TMAO synthesis and bile acid metabolism via remodeling of the gut microbiota.
      AllicinMiceDietary allicin might be capable of protecting the host from producing TMAO when carnitine was consumed through its impact on gut microbiota.Wu et al (2015)
      • Wu WK PS
      • Ho CT
      • Kuo CH
      • Wu MS
      • Sheen LY
      Dietary allicin reduces transformation of L-carnitine to TMAO through impact on gut microbiota.
      FMTIntestinal microbiota from lean donorsPatients with metabolic syndromeSix weeks after infusion of microbiota from lean donors, insulin sensitivity of recipients increased.Vrieze et al (2012)
      • Vrieze A
      • Van Nood E
      • Holleman F
      • et al.
      Transfer of intestinal microbiota from lean donors increases insulin sensitivity in individuals with metabolic syndrome.
      AntibioticsPolymyxin B/tobramycin regimenCHF patientsPolymyxin B/tobramycin significantly reduced fecal endotoxin load and improved peripheral endothelial function.Conraads et al (2004)
      • Conraads VM
      • Jorens PG
      • De Clerck LS
      • et al.
      Selective intestinal decontamination in advanced chronic heart failure: a pilot trial.
      Metronidazole/ciprofloxacinHealthy adultPlasma TMAO levels were markedly suppressed after administration of antibiotics and then reappeared after withdrawal of antibiotics.Tang et al (2013)
      • Tang WH
      • Wang Z
      • Levison BS
      • et al.
      Intestinal microbial metabolism of phosphatidylcholine and cardiovascular risk.
      Abbreviations: AHF, acute heart failure; CHF, chronic heart failure; DMB, 3-dimethyl-1-butanol; FMC, fluoromethylcholine; FMT, fecal microbial transplantation; HF, heart failure; IMC, iodomethylcholine; MI, myocardial infarction; SCFA, short-chain fatty acid; TMAO, trimethylamine N-oxide.

       Dietary intervention

      Plasma TMAO levels are closely related to diet, and how food impacts upon the risk of CVDs has long been a question of interest in the scientific community. Because the gut microbiome composition fluctuates considerably throughout the life cycle,
      • Voreades N
      • Kozil A
      • Weir TL
      Diet and the development of the human intestinal microbiome.
      it is hoped to be modified by dietary interventions, which will cause rapid and substantial changes in particular nutrients.
      • David LA
      • Maurice CF
      • Carmody RN
      • et al.
      Diet rapidly and reproducibly alters the human gut microbiome.
      The western diet, which is rich in saturated fats, animal protein, and sugars, has been shown to contribute to gut microbiota dysbiosis, upregulated plasma TMAO levels, and an increased risk of CVDs.
      • Koeth RA
      • Wang Z
      • Levison BS
      • et al.
      Intestinal microbiota metabolism of L-carnitine, a nutrient in red meat, promotes atherosclerosis.
      Moreover, overconsumption of eggs and fish also will lead to significantly higher TMAO levels in plasma and urine.
      • Wang Z
      • Bergeron N
      • Levison BS
      • et al.
      Impact of chronic dietary red meat, white meat, or non-meat protein on trimethylamine N-oxide metabolism and renal excretion in healthy men and women.
      • Miller CA
      • Corbin KD
      • da Costa KA
      • et al.
      Effect of egg ingestion on trimethylamine-N-oxide production in humans: a randomized, controlled, dose-response study.
      • Svensson BG
      • Akesson B
      • Nilsson A
      • Paulsson K
      Urinary excretion of methylamines in men with varying intake of fish from the Baltic Sea.
      However, the Mediterranean diet, which is characterized by a high intake of fruits and vegetables, nuts, whole grains, and limited quantities of meat, eggs, and sugar, is likely to promote optimal gut microbiota status and significantly reduce the incidence of HF.
      • Carbone S
      • Billingsley HE
      • Abbate A
      The Mediterranean diet to treat heart failure: a potentially powerful tool in the hands of providers.
      A systematic review and meta-analysis of randomized controlled trials (RCTs) showed that the Mediterranean diet reduced the incidence of HF by 70%.
      • Liyanage T
      • Ninomiya T
      • Wang A
      • et al.
      Effects of the Mediterranean Diet on cardiovascular outcomes—a systematic review and meta-analysis.
      Similarly, a retrospective cohort study of 3215 postmenopausal women showed a tendency for the Mediterranean diet to be associated with reduced HF mortality, though not to a statistically significant degree. Nevertheless, Dietary to Stop Hypertension (DASH) is deeply associated with lower HF mortality rate.
      • Levitan EB
      • Lewis CE
      • Tinker LF
      • et al.
      Mediterranean and DASH diet scores and mortality in women with heart failure: the Women's Health Initiative.
      Two other analyses suggested that both DASH and Mediterranean diet might lead to reduced HF morbidity and possibly contribute to secondary prevention.
      • Sanches Machado d'Almeida K
      • Ronchi Spillere S
      • Zuchinali P
      • Correa Souza G
      Mediterranean diet and other dietary patterns in primary prevention of heart failure and changes in cardiac function markers: a systematic review.
      ,
      • Dos Reis Padilha G
      • Sanches Machado d'Almeida K
      • Ronchi Spillere S
      • Correa Souza G
      Dietary patterns in secondary prevention of heart failure: a systematic review.
      In addition, a high-fiber diet has been reported to prevent the development of HF and effectively improve cardiac remodeling.
      • Marques FZ
      • Nelson E
      • Chu PY
      • et al.
      High-fiber diet and acetate supplementation change the gut microbiota and prevent the development of hypertension and heart failure in hypertensive mice.
      Kerley et al
      • Kerley CP.
      A review of plant-based diets to prevent and treat heart failure.
      found that for HF patients, a plant-based diet rich in fruits, vegetables, beans and whole wheat might be beneficial; the role of nuts, dairy products, and poultry is controversial; yet red or processed meat, eggs, and refined carbohydrates seem to be harmful.
      In a subsequent postmortem cross-sectional analysis, participants who were on the Mediterranean diet showed lower urinary TMAO levels.
      • De Filippis F
      • Pellegrini N
      • Vannini L
      • et al.
      High-level adherence to a Mediterranean diet beneficially impacts the gut microbiota and associated metabolome.
      Other studies supported this result, showing that reducing red meat
      • Wang Z
      • Bergeron N
      • Levison BS
      • et al.
      Impact of chronic dietary red meat, white meat, or non-meat protein on trimethylamine N-oxide metabolism and renal excretion in healthy men and women.
      and fat intake
      • Erickson ML
      • Malin SK
      • Wang Z
      • Brown JM
      • Hazen SL
      • Kirwan JP
      Effects of lifestyle intervention on plasma trimethylamine N-oxide in obese adults.
      and increasing fiber consumption
      • Sandek A
      • Bauditz J
      • Swidsinski A
      • et al.
      Altered intestinal function in patients with chronic heart failure.
      could lower plasma TMAO levels and alleviate ventricular remodeling. Recently,
      • Organ CL
      • Li Z
      • Sharp 3rd, TE
      • et al.
      Nonlethal inhibition of gut microbial trimethylamine N-oxide production improves cardiac function and remodeling in a murine model of heart failure.
      discovered that dietary withdrawal of TMAO improved HF remodeling and cardiac function, and the use of microbial choline TMA lyase inhibitor alleviated choline diet-induced cardiac insufficiency, which suggested that strategies to reduce circulating TMAO levels might counteract the negative effects of dietary choline and TMAO on HF.
      Overall, a healthy diet is the most cost-effective way to prevent and treat HF by positively affecting the gut microbiota. A promising treatment in the future is to conduct tailored monitoring of TMAO and to provide dietary advice based on individuals and their circumstances.

       Probiotics, prebiotics, and archaea

      Probiotics are live microorganisms that are beneficial to gut health.
      • Fuller R.
      Probiotics.
      They play an essential role in altering intestinal microbiota composition, maintaining host intestinal homeostasis, and improving human health. There is an increasing amount of evidence that probiotics may be involved in regulating myocardial remodeling in HF patients. For example, Lactobacillus plantarum exerts cardioprotective effects on rats during ischemia–reperfusion injury by reducing the LV infarction area and improving recovery of cardiac function.
      • Lam V
      • Su J
      • Koprowski S
      • et al.
      Intestinal microbiota determine severity of myocardial infarction in rats.
      Similar results were also reported in some other Lactobacillus species, such as Lactobacillus rhamnosus GR-1. In HF rats administered GR-1, the LV hypertrophy significantly decreased, and a significant improvement of systolic and diastolic hemodynamic parameters could be observed. Experiments showed that these effects were achieved by reducing serum leptin and increasing tissue taurine levels.
      • Gan XT
      • Ettinger G
      • Huang CX
      • et al.
      Probiotic administration attenuates myocardial hypertrophy and heart failure after myocardial infarction in the rat.
      Tang et al
      • Tang TWH
      • Chen HC
      • Chen CY
      • et al.
      Loss of gut microbiota alters immune system composition and cripples postinfarction cardiac repair.
      recently linked gut microbiota to cardiac function after myocardial infarction. In a mouse model of left anterior descending branch ligation, oral poorly absorbed antibiotics inhibited gut microbiota and significantly increased the rate of ventricular rupture and death. However, supplementing antibiotic-treated mice with probiotic Goodbelly (containing Lactobacillus plantarum 299v and Bifidobacterium lactis Bi-07) prior to myocardial infarction exerted a cardioprotective effect by shifting the host short-chain fatty acid (SCFA) balance to propionic acid. Additionally, recent studies showed that probiotics reduced TMAO levels, suggesting that their protective effects on the heart may be partly achieved by reducing circulatory TMAO.
      • Qiu L
      • Yang D
      • Tao X
      • Yu J
      • Xiong H
      • Wei H
      Enterobacter aerogenes ZDY01 attenuates choline-induced trimethylamine N-oxide levels by remodeling gut microbiota in mice.
      ,
      • Qiu L
      • Tao X
      • Xiong H
      • Yu J
      • Wei H
      Lactobacillus plantarum ZDY04 exhibits a strain-specific property of lowering TMAO via the modulation of gut microbiota in mice.
      To verify whether this finding can be applied clinically, Costanza et al conducted a 3-month probiotic treatment trial in systolic chronic HF patients. Compared with the placebo groups, they observed significantly decreased inflammatory markers and increased cardiac systolic function in the experimental groups that were treated with Saccharomyces boulardii.
      • Costanza AC
      • Moscavitch SD
      • Faria Neto HC
      • Mesquita ET
      Probiotic therapy with Saccharomyces boulardii for heart failure patients: a randomized, double-blind, placebo-controlled pilot trial.
      Preliminary results from another ongoing RCT showed that brady yeast can increase LVEF by 5% in patients with systolic HF, and the final results are expected to be achieved in 2020.
      • Mayerhofer CCK
      • Awoyemi AO
      • Moscavitch SD
      • et al.
      Design of the GutHeart-targeting gut microbiota to treat heart failure-trial: a phase II, randomized clinical trial.
      In conclusion, although studies of probiotics in HF patients remain scarce, this treatment strategy has a great prospect.
      Another strategy for regulating gut homeostasis is the administration of prebiotics, which are nondigestible carbohydrates that may modulate the microbiota activity
      • Gibson GR
      • Roberfroid MB.
      Dietary modulation of the human colonic microbiota: introducing the concept of prebiotics.
      and provide health benefits to the host. In obese mice, Gibson et al
      • Everard A
      • Lazarevic V
      • Derrien M
      • et al.
      Responses of gut microbiota and glucose and lipid metabolism to prebiotics in genetic obese and diet-induced leptin-resistant mice.
      discovered the ability of prebiotics to improve intestinal permeability, reduce metabolic endotoxemia, and decrease inflammation response. Moreover, Bifidobacterium animalis subsp. lactis LKM512, which is a type of prebiotics, was observed to decrease intestinal TMA levels in healthy volunteers.
      • Matsumoto M KY
      • Shimomura Y
      • Naito Y
      Bifidobacterium animalis subsp. lactis LKM512 reduces levels of intestinal trimethylamine produced by intestinal microbiota in healthy volunteers: a double-blind, placebo- controlled study.
      Inulin, a well-recognized prebiotic to promote the growth of beneficial bacteria, can improve diversity and function of gut microbiota to modulate the side effects of antibiotics.
      • Johnson LP
      • Walton GE
      • Psichas A
      • Frost GS
      • Gibson GR
      • Barraclough TG
      Prebiotics modulate the effects of antibiotics on gut microbial diversity and functioning in vitro.
      It is known that inulin can stimulate the production of SCFA.
      • Sarbini SR
      • Kolida S
      • Naeye T
      • et al.
      In vitro fermentation of linear and alpha-1,2-branched dextrans by the human fecal microbiota.
      In a RCT study of obese patients, dietary inulin or inulin-propionate supplementation increased the release of SCFA propionate from the colon, which therefore improved insulin sensitivity and reduced systemic inflammatory markers.
      • Chambers ES
      • Byrne CS
      • Morrison DJ
      • et al.
      Dietary supplementation with inulin-propionate ester or inulin improves insulin sensitivity in adults with overweight and obesity with distinct effects on the gut microbiota, plasma metabolome and systemic inflammatory responses: a randomised cross-over trial.
      Similarly, inulin-enriched diet is able to promote weight loss in obese patients and its effect is related to the characteristics of intestinal flora.
      • Hiel S
      • Gianfrancesco MA
      • Rodriguez J
      • et al.
      Link between gut microbiota and health outcomes in inulin-treated obese patients: lessons from the Food4Gut multicenter randomized placebo-controlled trial.
      Several studies have also found the role of prebiotics in lowering blood pressure,
      • Marques FZ
      • Nelson E
      • Chu PY
      • et al.
      High-fiber diet and acetate supplementation change the gut microbiota and prevent the development of hypertension and heart failure in hypertensive mice.
      preventing low-density lipoprotein oxidation,
      • Fava F
      • Lovegrove JA
      • Gitau R
      • Jackson KG
      • Tuohy KM
      The gut microbiota and lipid metabolism: implications for human health and coronary heart disease.
      and improving visceral obesity.
      • Anhe FF
      • Roy D
      • Pilon G
      • et al.
      A polyphenol-rich cranberry extract protects from diet-induced obesity, insulin resistance and intestinal inflammation in association with increased Akkermansia spp. population in the gut microbiota of mice.
      Although the current scientific evidence does not support the use of prebiotics to treat HF patients or reduce plasma TMAO levels, it can still be used in further trials for CVD.
      Archaea are a group of prokaryotic microorganisms that live in extreme environments and morphologically resemble bacteria. Currently, nearly 400 archaeal genomes have been published. Studies found that archaea could reduce gut TMA by partially converting it into methane
      • Eme L
      • Doolittle W.
      Archaea.
      ,
      • Fennema D
      • Phillips IR
      • Shephard EA
      Trimethylamine and trimethylamine N-oxide, a flavin-containing monooxygenase 3 (FMO3)-mediated host-microbiome metabolic axis implicated in health and disease.
      ; however, it is unclear whether archaea can be developed into a therapeutic target in the future, and relevant basic and human studies have not been carried out.
      Taking these observations together, we conclude that although the current data on this section are limited, most of the findings support their possible beneficial role in HF and more research will be expected in the future.

       Microbial TMA-lyase inhibitors

      Hazen's team developed 2 potent inhibitors of cutC/D: fluoromethylcholine (FMC) and iodomethylcholine (IMC), which permanently inactivated cutC/D without affecting the viability of the symbiotic bacteria. In animal models, FMC and IMC significantly reduced the systemic TMAO levels, and reversed TMAO-induced platelet hyperactivity and thrombus formation.
      • Roberts AB
      • Gu X
      • Buffa JA
      • et al.
      Development of a gut microbe-targeted nonlethal therapeutic to inhibit thrombosis potential.
      Subsequently, in mice that were fed choline, DMB was shown to reduce circulating TMAO concentrations by inhibiting TMA formation.
      • Wang G
      • Kong B
      • Shuai W
      • Fu H
      • Jiang X
      • Huang H
      3,3-Dimethyl-1-butanol attenuates cardiac remodeling in pressure-overload-induced heart failure mice.
      DMB is a natural product that is present in extra-virgin olive oil, which is one of the main integral constituents of Mediterranean diet. Therefore, a mechanistic link may exist within the Mediterranean diet and a reduction in TMAO production.

       Natural phytochemicals

      Some chemicals from natural plants have also been shown to reduce plasma TMAO levels. For example, dietary allicin, an active antibacterial compound in garlic, can reduce TMAO formation by affecting the gut microbiota.
      • Wu WK PS
      • Ho CT
      • Kuo CH
      • Wu MS
      • Sheen LY
      Dietary allicin reduces transformation of L-carnitine to TMAO through impact on gut microbiota.
      Resveratrol, a polyphenolic plant antitoxin with an anti-inflammatory effect, has been reported to rescue TMAO-induced atherosclerosis by shifting the gut microbiota composition, reducing plasma TMAO levels, and accelerating liver bile acid synthesis.
      • Chen ML
      • Yi L
      • Zhang Y
      • et al.
      Resveratrol attenuates trimethylamine-N-oxide (TMAO)-induced atherosclerosis by regulating TMAO synthesis and bile acid metabolism via remodeling of the gut microbiota.
      As described above, a key regulator in the TMAO synthesis pathway is FMO3, which rapidly converts TMA to TMAO. Phytochemicals such as berberine, 3,3ʹ-diindolylmethane (DIM) and indole-3-carbinol have shown promise in inhibiting FMO3 activity and reducing TMAO production.
      • Shi Y
      • Hu J
      • Geng J
      • et al.
      Berberine treatment reduces atherosclerosis by mediating gut microbiota in ApoE-/- mice.
      ,
      • Chen S
      • Henderson A
      • Petriello MC
      • et al.
      Trimethylamine N-oxide binds and activates PERK to promote metabolic dysfunction.
      However, FMO3 inhibitors may lead to the occurrence of acute hepatitis and fishy syndrome,
      • Wang Z
      • Roberts AB
      • Buffa JA
      • et al.
      Non-lethal inhibition of gut microbial trimethylamine production for the treatment of atherosclerosis.
      so their adverse effects should be considered carefully before clinical application.

       Fecal microbial transplantation

      Fecal microbial transplantation (FMT) is another approach that can be applied in diseases that are linked to gut dysbiosis, which involves the transplantation of microbes from healthy individuals into the intestines of high-risk patients. Currently, FMT has been clinically shown to be effective for treating intestinal diseases, such as Clostridium difficile infection and inflammatory bowel disease.
      • Zellmer C
      • De Wolfe TJ
      • Van Hoof S
      • Blakney R
      • Safdar N
      Patient perspectives on fecal microbiota transplantation for Clostridium difficile infection.
      Some parenteral diseases are also being explored, such as autism,
      • Vendrik KEW
      • Ooijevaar RE
      • de Jong PRC
      • et al.
      Fecal microbiota transplantation in neurological disorders.
      obesity,
      • Guirro M
      • Costa A
      • Gual-Grau A
      • et al.
      Effects from diet-induced gut microbiota dysbiosis and obesity can be ameliorated by fecal microbiota transplantation: a multiomics approach.
      and type 2 diabetes.
      • Zhang PP
      • Li LL
      • Han X
      • et al.
      Fecal microbiota transplantation improves metabolism and gut microbiome composition in db/db mice.
      Vrieze et al
      • Vrieze A
      • Van Nood E
      • Holleman F
      • et al.
      Transfer of intestinal microbiota from lean donors increases insulin sensitivity in individuals with metabolic syndrome.
      performed FMT treatment on patients with metabolic syndrome, and the result indicated that patients receiving gut flora from leptin donor displayed improved insulin sensitivity and increased butyrate production. This finding was validated in another RCT, suggesting that FMT might be a potential treatment strategy for metabolism-related diseases.
      • Kootte RS
      • Levin E
      • Salojarvi J
      • et al.
      Improvement of insulin sensitivity after lean donor feces in metabolic syndrome is driven by baseline intestinal microbiota composition.
      In a double-blind RCT, patients with metabolic syndrome received FMT from vegan-donors; however, their ability to produce TMAO was not affected.
      • Smits LP
      • Kootte RS
      • Levin E
      • et al.
      Effect of vegan fecal microbiota transplantation on carnitine- and choline-derived trimethylamine-N-oxide production and vascular inflammation in patients with metabolic syndrome.
      Changes in plasma TMAO level depend on the specific microbiota with TMA lyases. Romano et al
      • Romano KA
      • Vivas EI
      • Amador-Noguez D
      • Rey FE
      Intestinal microbiota composition modulates choline bioavailability from diet and accumulation of the proatherogenic metabolite trimethylamine-N-oxide.
      identified nine intestinal strains that were capable of generating TMA, and they found that serum TMAO accumulated in mice that were transplanted with TMA-producing bacteria. Subsequent studies showed that transplantation of high TMA-producing microbes could transmit the potential to generate TMAO to the recipients.
      • Zhu W
      • Wang Z
      • Tang WHW
      • Hazen SL
      Gut Microbe-generated trimethylamine N-oxide from dietary choline is prothrombotic in subjects.
      ,
      • Gregory JC
      • Buffa JA
      • Org E
      • et al.
      Transmission of atherosclerosis susceptibility with gut microbial transplantation.
      To date, FMT has not been used in patients with HF, it is expected that TMAO can be reduced by transplanting low-yield TMAO intestinal flora, but no such clinical studies have been conducted.
      Although the initial research is encouraging, the current limitations of FMT still need to be addressed, namely the high risk for both infection and rejection.
      • Kelly CR
      • Kahn S
      • Kashyap P
      • et al.
      Update on fecal microbiota transplantation 2015: indications, methodologies, mechanisms, and outlook.
      Further work is required to test the clinical significance of FMT in the field of cardiometabolic disorders. In addition to feces, transplanting specific types of bacterial strains may be an alternative to FMT.
      • Wymore Brand M
      • Wannemuehler MJ
      • Phillips GJ
      • et al.
      The altered schaedler flora: continued applications of a defined murine microbial community.

       Antibiotic therapy

      Antibiotics can affect microbe-driven disease by altering the abundance or composition of the gut microbial community. For example, oral administration of vancomycin reduced the infarct area of the left ventricle and improved the cardiac function in a rat model of myocardial ischemia–reperfusion.
      • Lam V
      • Su J
      • Koprowski S
      • et al.
      Intestinal microbiota determine severity of myocardial infarction in rats.
      ,
      • Lam V
      • Su J
      • Hsu A
      • Gross GJ
      • Salzman NH
      • Baker JE
      Intestinal microbial metabolites are linked to severity of myocardial infarction in rats.
      Moreover, subjects on short-term, low-absorbance antibiotics showed inhibited TMAO synthesis.
      • Koeth RA
      • Wang Z
      • Levison BS
      • et al.
      Intestinal microbiota metabolism of L-carnitine, a nutrient in red meat, promotes atherosclerosis.
      ,
      • Tang WH
      • Wang Z
      • Levison BS
      • et al.
      Intestinal microbial metabolism of phosphatidylcholine and cardiovascular risk.
      However, whether the use of antibiotics has a protective effect in HF patients is controversial. Although the antibiotic polymyxin B can reduce the production of pro-inflammatory cytokines and improve the endothelial function of patients with HF,
      • Conraads VM
      • Jorens PG
      • De Clerck LS
      • et al.
      Selective intestinal decontamination in advanced chronic heart failure: a pilot trial.
      it is clinically limited because of the toxicity. In addition, using antibiotics to inhibit the excessive proliferation of harmful bacteria may also lead to the presence of drug-resistant microbiota in the gut.
      • Tang WH
      • Wang Z
      • Levison BS
      • et al.
      Intestinal microbial metabolism of phosphatidylcholine and cardiovascular risk.
      Therefore, clinicians must weigh the potential side effects of antibiotics carefully before using this medication, and further investigations are needed to assess whether the rational use of antibiotics in certain situations will have favorable effects on heart function and improve HF patient survival.
      In an ongoing randomized, open-label, controlled trial called GutHeart, which is investigating the potential relationship between gut microbiota and inflammation pathways in the cardiovascular system, 150 stable HFrEF patients were assigned, in a blinded manner, to rifaximin, brady yeast, or no treatment groups, and the primary endpoint is the LVEF that is measured by echocardiography after 3 months. This study aims to reveal whether targeting gut microbiota using antibiotics can be a new potential strategy to improve the survival rate of HF patients (Fig 3).
      • Mayerhofer CCK
      • Awoyemi AO
      • Moscavitch SD
      • et al.
      Design of the GutHeart-targeting gut microbiota to treat heart failure-trial: a phase II, randomized clinical trial.
      Fig 3
      Fig 3Potential therapeutic targets (green) in gut–TMAO–HF axis. Replacing the western diet with the Mediterranean diet is likely to promote optimal gut microbiota status and significantly reduce the incidence of HF. Targeting the intestinal flora, antibiotics, probiotics, prebiotics, and FMT have been shown to regulate gut disorders, as well as some natural phytochemicals such as allicin and resveratrol. DMB, FMC, and IMC, as inhibitors of TMA lyases, can reduce TMA biosynthesis. Archaea is expected to reduce TMA levels by metabolizing it into methane. Additionally, FMO3 inhibitors such as DIM, berberine, and indole-3-carbinol were shown to inhibit FMO3 activity and reduce TMAO production. Thus, the gut–TMAO–HF axis offers promising routes to address HF via these described mechanisms.

      CURRENT CONTROVERSIES

      Although elevated circulating TMAO is described as a risk factor for HF and is directly involved in the HF pathologic processes, some studies seem to contradict this conclusion, indicating that TMAO may be beneficial to the cardiovascular system.
      Tomasz et al
      • Tomasz H AD
      • Marta G
      • Marek K
      • et al.
      Chronic, low-dose TMAO treatment reduces diastolic dysfunction and heart fibrosis in hypertensive rats.
      found that chronic, low-dose TMAO significantly reduced plasma NT-proBNP and vasopressin concentrations, LV diastolic pressure, and myocardial fibrosis in hypertensive rats. Similarly, Collin et al
      • Collins HL
      • Drazul-Schrader D
      • Sulpizio AC
      • et al.
      L-Carnitine intake and high trimethylamine N-oxide plasma levels correlate with low aortic lesions in ApoE(-/-) transgenic mice expressing CETP.
      showed that in ApoE(−/−) mice that were fed a high-dose L-carnitine, elevated TMAO levels were negatively associated with the size of the aortic lesion without altering the cholesterol content, suggesting that TMAO might delay the formation of aortic lesions and have a protective effect against the occurrence of atherosclerosis. In clinical research of ischemic encephalopathy patients, Yin et al
      • Yin J
      • Liao SX
      • He Y
      • et al.
      Dysbiosis of gut microbiota with reduced trimethylamine-N-oxide level in patients with large-artery atherosclerotic stroke or transient ischemic attack.
      showed that CVDs were associated with significantly imbalanced gut microbiota and decreased plasma TMAO levels. However, those with asymptomatic atherosclerosis had no changes in either the gut microbiota or in TMAO levels.
      All the above preclinical and clinical studies seem to indicate that a modest increase in plasma TMAO levels appears to have no adverse effect on the circulatory system. However, the limitations of these studies cannot be ignored. In Collin et al’s study,
      • Collins HL
      • Drazul-Schrader D
      • Sulpizio AC
      • et al.
      L-Carnitine intake and high trimethylamine N-oxide plasma levels correlate with low aortic lesions in ApoE(-/-) transgenic mice expressing CETP.
      the circulating TMAO levels in the L-carnitine group were far less than the estimated levels in patients, and the relationship between TMAO level and aortic lesion size was not apparent. In Yin et al’s study,
      • Yin J
      • Liao SX
      • He Y
      • et al.
      Dysbiosis of gut microbiota with reduced trimethylamine-N-oxide level in patients with large-artery atherosclerotic stroke or transient ischemic attack.
      both the disease and the treatment might have significant effects on TMAO levels. For example, stroke patients need to control their dietary intake, which may reduce TMAO levels. Additionally, aspirin is a routine drug for stroke treatment, and it has been demonstrated to decrease plasma TMAO levels.
      • Wu WK
      • Sheen LY
      • Wu MS
      Response to the letter: identification of trimethylamine N-oxide (TMAO)-producer phenotype is interesting, but is it helpful?.
      Endoplasmic reticulum (ER) stress is involved in foam cell formation and progression of atherosclerosis.
      • Oh J
      • Riek AE
      • Weng S
      • et al.
      Endoplasmic reticulum stress controls M2 macrophage differentiation and foam cell formation.
      In rodent studies, TMAO acted as a small molecular chaperone that inhibited ER responses.
      • McCarty MF
      • DiNicolantonio JJ
      Trimethylamine-N-oxide and heart failure.
      However, in mice, TMAO was shown to promote atherosclerosis by activating the overexpression of CD36 and scavenger receptor A in macrophages.
      • Koeth RA
      • Wang Z
      • Levison BS
      • et al.
      Intestinal microbiota metabolism of L-carnitine, a nutrient in red meat, promotes atherosclerosis.
      A reasonable explanation for this paradox is that TMAO may have specific adverse effects on foam cells, which outweighs its benefits on ER stress inhibition.
      Additionally, TMAO is also an osmotic substance that plays a vital role in adapting cells to osmotic and hydrostatic pressure. For example, it was observed to stabilize the structure of peptide regions, thereby protecting the protein from damage caused by hydration.
      • Arakawa T
      • Timasheff SN.
      The stabilization of proteins by osmolytes.
      ,
      • Hu CY
      • Lynch GC
      • Kokubo H
      • Pettitt BM
      Trimethylamine N-oxide influence on the backbone of proteins: an oligoglycine model.
      An example is deep-sea fish, which can use TMAO to support protein stability from osmotic and hydrostatic pressure.
      • Seibel BA
      • Walsh PJ.
      Trimethylamine oxide accumulation in marine animals: relationship to acylglycerol storage.
      ,
      • Yancey PH.
      Organic osmolytes as compatible, metabolic and counteracting cytoprotectants in high osmolarity and other stresses.
      Therefore, TMAO seems to function as a protein-mate for stable protein structure. Considering the beneficial effects of TMAO in cells, a hypothesis proposes that the elevated TMAO levels are the result of a compensatory response to HF, similar to BNP. When overloaded with volume and pressure, the failing heart will release BNP to alleviate overload by increasing diuresis. Therefore, it is speculated that the increase in TMAO levels during HF is a result of its beneficial role in protecting proteins from the osmotic and hydrostatic pressure.
      Another argument for TMAO focuses on fish, especially deep-sea fish, which contain 1.7 g or more of TMAO per pound. A study showed that TMAO exposure from 1 pound of fish might be an order of magnitude more compared with 1 pound of red meat.
      • McCarty MF
      L-carnitine consumption, its metabolism by intestinal microbiota, and cardiovascular health.
      However, epidemiological investigations suggested that high fish consumption was significantly related to a reduction in CVD risk factors.
      • Mozaffarian D
      • Lemaitre RN
      • Kuller LH
      • et al.
      Cardiac benefits of fish consumption may depend on the type of fish meal consumed: the Cardiovascular Health Study.
      The plausible explanation is that omega-3 fatty acids that are contained in certain fish can protect the cardiovascular system, and the potential benefits outweigh the risks from TMAO.
      • Heydari B
      • Abdullah S
      • Shah R
      • et al.
      Omega-3 fatty acids effect on post-myocardial infarction ST2 levels for heart failure and myocardial fibrosis.
      ,
      • Kris-Etherton PM
      • Richter CK
      • Bowen KJ
      • et al.
      Recent clinical trials shed new light on the cardiovascular benefits of omega-3 fatty acids.
      Given the controversies of whether TMAO is beneficial or harmful to the organisms, some scholars believe that it is TMA, the precursor of TMAO, that has adverse biological effects on the circulatory system. Under physiological conditions, the plasma TMA level is 5–7 times higher compared with TMAO.
      • Jaworska K
      • Bielinska K
      • Gawrys-Kopczynska M
      • Ufnal M
      TMA (trimethylamine), but not its oxide TMAO (trimethylamine-oxide), exerts haemodynamic effects: implications for interpretation of cardiovascular actions of gut microbiome.
      As early as 1981, TMA was discovered to be a uremia toxin that had a cytotoxic effect.
      • Wills MR
      • Savory J.
      Biochemistry of renal failure.
      It was then shown to be elevated in patients with CVDs, and negatively correlated with eGFR.
      • Jaworska K
      • Hering D
      • Mosieniak G
      • et al.
      TMA, a forgotten uremic toxin, but not TMAO, is involved in cardiovascular pathology.
      Therefore, some scientists consider that it is TMA, rather than TMAO, that matters in patients with a higher cardiovascular risk, and future studies should assess both TMAO and TMA.

      FUTURE DIRECTIONS

      Several human clinical studies have indicated the profound relationship between gut microbiota composition or its metabolites and HF. However, these studies are typical correlation studies, and it is still challenging to determine whether changes in the gut microbiota are a cause or consequence of HF. Therefore, it is necessary to further establish a prospective research cohort, combining omics data, comprehensive clinical data, and dietary factors to link the gut microbiota composition with disease progression, thereby exploring whether there is a causative relationship between gut microbiota and HF.
      Generally, TMAO has 2 values in HF: (1) as a prognostic marker. TMAO is located in a new pathological pathway of HF and it represents the degree of intestinal dysbiosis. It is closely associated with the poor survival of HF patients, which helps clinicians to screen out high-risk groups and pay more attention to their follow-up monitoring as well as enhance HF-related treatments. However, the problem is that the existing clinical research subjects are mainly Caucasian patients and there is a lack of diversity. Therefore, more cohort studies enrolling patients of other races are required to show whether TMAO can be a widely used prognostic marker for HF; and (2) as a potential therapeutic target. Based on previous studies, reducing plasma TMAO levels by specific interventions is expected to reduce mortality in HF patients. However, the mechanisms of TMAO's participation in HF are not clear, and more research is needed to reveal the underlying pathologic process. In addition, there is no evidence showing that reducing plasma TMAO levels are beneficial, so prospective intervention studies to investigate whether lowering TMAO levels improves the prognosis of HF patients are urgently needed.
      Although gut microbial genome sequencing is increasingly becoming a part of research studies, there is still a long way to go before it can be used in clinical practice. Instead, measuring gut microbe-derived metabolites in blood or urine to guide targeted interventions will have a higher clinical translational value.

      CONCLUSIONS

      Over the past few years, multiple studies have firmly established a direct link between gut microbiota and CVDs. Now we are aware that TMAO, a metabolite produced by the gut microbiota, may provide novel insights into how the gut microbiota contributes to HF. These findings provide an excellent opportunity to develop interventions targeting the gut microbiota for treating HF, such as personalized dietary interventions, probiotics, prebiotics, and FMT. Phytochemicals targeting TMAO metabolism, such as DMB, are also expected to provide potential therapeutic value. However, a wide variety of metabolites can contribute to heart disease, and TMAO may represent the tip of the iceberg. In the future, regulating the composition of gut microbiota and targeting the gut–TMAO–HF axis are both likely to have a profound influence on HF patient survival.

      Acknowledgments

      This work was supported by the National Natural Science Foundation of China (Major Program, grant no. 81790622 ) and the Ministry of Science and Technology of China (Key Projects of Precision Medicine Program, grant no. 2017YFC0908400 ). Dr. Jie Du is the Fellow of the Collaborative Innovation Center for Cardiovascular Disease Translational Medicine, Nanjing Medical University; the Molecular Imaging Precision Medical Collaborative Innovation Center, Shanxi Medical University. All authors have read the journal's authorship agreement and policy on disclosure of potential conflicts of interest. The manuscript has been reviewed by and approved by all named authors.

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