From Gut to Heart: Targeting Trimethylamine N-Oxide as a Novel Strategy in Heart Failure Management

Inflammation is closely linked to HF [14]. Higher concentrations of TMAO, such as 600 μmol/L, have been shown to enhance the expression of inflammatory genes and activate proinflammatory cytokines, thus amplifying inflammatory responses [54]. Boini et al. [55] experimentally demonstrated that TMAO stimulated the secretion of inflammatory cytokines, including interleukin (IL)-1β, by activating the nucleotide-binding oligomerization domain-like receptor containing pyrin domain 3 (NLRP3) inflammasome, leading to endothelial inflammation. TMAO also inhibited the deacetylation of superoxide dismutase 2 (SOD2) by suppressing Sirtuin 3 (SIRT3), thereby reducing SOD2 activity. This process promoted mitochondrial reactive oxygen species (mtROS) generation, leading to elevated overall reactive oxygen species (ROS) levels [56,57]. This increased thioredoxin-interacting protein (TXNIP) levels, subsequently activating the NLRP3 inflammasome [57]. Such activation elicited a dose- and time-dependent release of IL-1β and IL-18 [58], culminating in an inflammatory response.

Moreover, TMAO may influence HF by modulating inflammatory pathways. Zhang and colleagues [59] reported that TMAO could possibly exacerbate myocardial inflammation in mice by elevating TNF (tumor necrosis factor)-α and reducing IL-10 levels. Additionally, their study showed that TMAO supplementation (120 mg/kg) in drinking water may counteract the cardioprotective benefits of voluntary exercise [59].

Abnormal energy metabolism plays a pivotal role in the development of HF. In HF, due to the increased dependence of cardiac metabolism on glucose, the primary energy source shifts from fatty acid (FA) oxidation to glucose metabolism [60]. Additionally, hypoxia reduces oxidative phosphorylation (OXPHOS) and enhances glycolysis [61]. These metabolic alterations contribute to an insufficient energy supply, thus worsening the condition of HF.

Elevated TMAO levels are strongly linked to impaired cardiac energy metabolism. HFD-induced TMAO accumulation inhibited mitochondrial activity and consequently compromised energy metabolism [62]. Saaoud et al. [54] revealed that 600 μmol/L TMAO initiated a metabolic switch from OXPHOS to glycolysis. This shift occurred by upregulating mitochondrial regulators such as perilipin 4 (PLIN4), overlapping activity with m-AAA protease 1 (OMA1), and oxoglutarate dehydrogenase L (OGDHL), ultimately disrupting energy homeostasis. Brown adipose tissue (BAT) enhances thermogenesis and supports cardiac function [63,64], but hypoxia induces brown adipocyte apoptosis, resulting in BAT dysfunction [28]. In physiological conditions, BAT absorbs and metabolizes circulating choline, potentially to maintain its cellular membrane integrity. In a murine model of HF, BAT dysfunction led to choline accumulation, which may be partially converted to TMAO via the choline-TMA-TMAO pathway; this increased TMAO synthesis exacerbated HF [28]. This study further revealed that TMAO significantly attenuated creatine phosphate (CP) and adenosine triphosphate (ATP) concentrations in cardiac tissue by suppressing mitochondrial complex IV activity, further impairing cardiac energy metabolism. This led to a vicious cycle of BAT dysfunction and cardiac dysfunction ultimately [28].

In addition, impaired calcium ion (Ca²⁺) handling in myocardial cells increases the energy demand of the failing heart [65], compromising both systolic and diastolic function and worsening HF. TMAO disrupts Ca²⁺ regulation in cardiac cells. In endothelial cells, 100 μmol/L TMAO suppressed purinergic response-induced increase in Ca²⁺ influx and prolongation of Ca²⁺ signaling, indicating loss of Ca²⁺ homeostasis [66]. High glucose and high fat could also induce TMAO production, possibly predisposing individuals to Ca²⁺ overload [67]. Esposito and coworkers [68] proved that 100 μmol/L TMAO transiently suppressed contractile recovery of myocardial cells by elevating intracellular Ca²⁺ currents.

Oxidative stress contributes to the pathophysiological HF via multiple mechanisms. Studies have demonstrated that TMAO can mediate oxidative stress. TMAO concentrations were remarkably higher in preeclampsia patients. Transplanting fecal microbiota from these patients into antibiotic-treated mice resulted in pronounced oxidative stress in the recipients [69]. In addition, TMAO can provoke oxidative stress by distinct pathways. A study involving mice and healthy human subjects revealed that TMAO promoted oxidative stress by increasing superoxide levels and impairing endothelial nitric oxide synthase (eNOS) activity [46]. Chen et al. [57] confirmed that TMAO, at concentrations of 150, 300, 600, and 900 μmol/L, augmented ROS generation, particularly mtROS, through modulating the SIRT3-SOD2-mtROS signaling cascade, thus fostering oxidative stress. Moreover, TMAO induced oxidative stress through activation of NADPH oxidase 2 (NOX2) [70].

Elevated TMAO levels have been demonstrated to initiate HF through oxidative stress. Recent research revealed that TMAO induced oxidative stress by downregulating piezo type mechanosensitive ion channel component 1 (PIEZO1), thereby exacerbating cardiac diastolic dysfunction in mice suffering from HFpEF [25]. Therefore, decreasing TMAO concentrations and alleviating oxidative stress appear to be a viable therapeutic approach for HF.

Myocardial remodeling stands as a significant mechanism underlying the occurrence and progression of HF. Its primary features consist of pathological cardiomyocyte hypertrophy and myocardial fibrosis. TMAO directly influences this process. Research in mice indicated that a Western diet (WD) or TMAO supplementation increased TMAO levels, promoted myocardial fibrosis, and impaired cardiac function [59]. Studies demonstrated that TMAO stimulated myocardial remodeling in Sprague-Dawley rats and triggered hypertrophic responses in rat cardiomyocytes, enlarging cell size while upregulating hypertrophy markers such as atrial natriuretic peptide (ANP) and β-myosin heavy chain (β-MHC). This effect might involve activation of the transforming growth factor (TGF)-β1/Smad3 signaling pathway [71]. Another study indicated that transverse aortic constriction-induced HF in mice correlated with elevated TMAO levels, exacerbated myocardial hypertrophy and fibrosis, and compromised cardiac function. The mechanisms could be associated with activation of the TGF-β1/Smad3 and p65 nuclear factor (NF)-κB signaling pathways [27].

TMAO aggravates HF by affecting adverse cardiac remodeling. Organ et al. [72] discovered that diets containing choline (1.2%) or TMAO (0.12%) markedly increased circulating TMAO and brain natriuretic peptide (BNP) levels in HF mice. Concurrently, left ventricular (LV) fractional shortening and ejection fraction were decreased, accompanied by exacerbated LV remodeling, intensified myocardial fibrosis, and deteriorated cardiac dysfunction, ultimately accelerating HF progression. Subsequent experiments showed that discontinuing TMAO intake or inhibiting TMA lyase effectively lowered the HF marker BNP levels, suppressed numerous profibrotic gene expressions, and ultimately mitigated ventricular remodeling and cardiac dysfunction [73].

TMAO indirectly contributes to the emergence and progression of HF by mediating several mechanisms such as endothelial dysfunction [46], renal insufficiency [74], elevated blood pressure [75], enhanced platelet activity and thrombosis [34], and lipid metabolism disorders [9,31].

Current evidence indicates that dietary intervention could effectively lessen circulating TMAO levels, probably aiding in the prevention and treatment of HF [21]. As noted, consuming foods abundant in TMA precursors—including choline, L-carnitine, and betaine—or high-fat foods increases TMAO concentrations in humans. In animal studies, a choline-rich diet significantly increased TMAO and BNP levels, worsened ventricular remodeling, impaired cardiac function, and further aggravated HF [72]. Mo et al. [32] demonstrated that an HFD elevated plasma TMAO concentrations in naturally aging rats. In healthy rats, prolonged HFD intake triggered low-grade systemic inflammation and diminished beneficial SCFA levels, while long-term fructose consumption heightened oxidative stress, elevated blood pressure, and expanded Enterobacter and Escherichia coli populations [98]. The WD, distinguished by high amounts of TMA precursors, fats, and sugars [9,47,62], is strongly associated with increased TMAO concentrations [52]. Consuming WD alters gut microbiota composition, diminishes its diversity, and consequently promotes TMAO production, rising blood TMAO concentrations, even in healthy people [99]. The WD has been proven to elevate circulating TMAO levels, thus inducing cardiac fibrosis and systemic inflammation. These pathological processes can subsequently cause cardiac dysfunction, heightening the risk of developing AS, coronary artery diseases (CADs), and HF [9,21,47,62]. Furthermore, animal experiments demonstrated that a high-salt diet may reduce the ratio of SCFAs to TMAO, lower Lactobacillus abundance, and exacerbate immune and inflammatory responses [100]. Additionally, insufficient dietary fiber could worsen gut microbiota dysbiosis [101]. Hence, it is advisable to steer clear of overindulging in foods rich in TMA precursors, fats, sugars, or salts [99], while augmenting dietary fiber intake.

Studies have shown that consuming red meat raises TMAO concentrations. Decreasing consumption of choline- or L-carnitine-rich foods such as red meat can hinder TMAO synthesis, ultimately reducing HF incidence [7,52]. A plant-based diet primarily consists of fruits, vegetables, seeds, nuts, legumes, whole grains, and herbal plants. It may adhere strictly to a vegetarian diet or incorporate moderate quantities of animal-derived foods like fish, seafood, dairy products, and eggs [102]. This dietary pattern has been demonstrated to modulate gut microbiota, augment the generation of advantageous metabolites such as SCFAs, and diminish TMAO levels [103,104,105]. The Mediterranean diet (MD), a well-established healthy pattern, emphasizes ample fruits, vegetables, nuts, and whole grains, along with moderate meat, eggs, sugar, olive oil, and wine [93]. It is low in saturated FAs, salt, and phosphates, but rich in unsaturated FAs, antioxidants (such as polyphenols, vitamins, and flavonoids), nitrates, and dietary fiber [9,37,67]. Healthy diets like the MD could modulate gut microbiota composition and its metabolites, influencing the occurrence and progression of HF. Studies have indicated that MD mitigates oxidative stress and inflammation while enhancing antioxidant capacity and endothelial function by augmenting the gut microbial diversity and abundance, raising SCFA production, and reducing TMAO concentrations. These effects collectively improve cardiac performance and lower CVD risk [9,106,107]. Adherence to the MD has been linked to decreased HF incidence and mortality [108], and it has exhibited additional benefits such as restoring gut microbial balance and lessening TMAO concentrations [109]. Estruch and colleagues [110] demonstrated that MD consumption, especially when enriched with extra-virgin olive oil, effectively attenuated TMAO levels, thus diminishing major cardiovascular events such as MI and stroke. Kaluza et al. [111] uncovered that certain foods, including vegetables, fruits, and nuts, lessened HF risk in individuals with smoking addictions by modifying gut microbiota, suppressing oxidative stress, and inhibiting cell death. Merques and coworkers [112] revealed that a high-fiber diet regulated gut microbiota, lowered blood pressure, and ameliorated cardiac fibrosis and LV hypertrophy, thereby contributing to the prevention of HF. Furthermore, obese patients following a low-calorie diet may experience decreased TMAO concentrations [113,114]. In summary, healthy eating serves as a practical and economical way to manage HF, mainly by modulating gut microbiota and their metabolites [93].

Tailored exercise training can enhance exercise capacity and quality of life in patients with HF, improve survival rates, and lower the likelihood of HF-related hospitalization [115,116]. Additionally, physical activities have been shown to modulate gut microbiota [117], leading to lower TMAO levels. For example, sprint training decreased serum and urinary TMAO concentrations in male participants [118,119]. Physical activity also notably reduced plasma TMAO levels in a study of 16 obese adults [113]. Zhang et al. [120] discovered that voluntary wheel running in mice alleviated gut microbiota dysbiosis and decreased serum levels of TMAO and its precursors, TMA and betaine.

Physical activities confer beneficial effects on the heart by downregulating TMAO concentrations. After being fed a WD, mice that engaged in voluntary exercise demonstrated reduced plasma TMAO levels, diminished myocardial inflammation and fibrosis, and enhanced cardiac function compared with sedentary controls [59]. Brandao and colleagues [121] found that a combined exercise regimen—incorporating both strength and aerobic training—decreased TMAO levels, thereby mitigating cardiovascular risk and strengthening physical function in obese women.

Probiotics, prebiotics, and synbiotics serve as prevalent therapeutic avenues for preserving gut microbiota homeostasis [122]. Probiotics, live microorganisms capable of adjusting the quantity and composition of gut microbiota, confer health benefits [35]. Prebiotics consist of non-digestible substrates selectively utilized by beneficial microorganisms in the host to promote their growth and/or activity, ultimately improving host health [122,123]. Synbiotics combine live microorganisms with substrates selectively metabolized by host microbiota, effectively integrating the benefits of both probiotics and prebiotics to foster host well-being. The synergistic and complementary effects of live microorganisms and substrates allow synbiotics to effectually influence microbiota composition and immune function [124].

Research has shown that probiotics, prebiotics, or synbiotics can remodel the gut microbiota in animals, thereby reducing gut-derived TMA synthesis, inhibiting or blocking the TMA/TMAO pathway, and ultimately decreasing TMAO levels [7]. Probiotics, including Lactobacillus plantarum ZDY04, Bifidobacterium breve Bb4, Bifidobacterium longum BL1 and BL7, and Lacticaseibacillus rhamnosus L34, may decrease TMAO concentrations by adjusting gut microbiota and restricting TMA generation [44,125,126]. Wang et al. [125] discovered that Bifidobacterium lessened the abundance of TMA-producing Ruminococcaceae UCG-009 and UCG-010, thereby lowering plasma TMAO concentrations. Among lactobacilli strains with robust adherence capability, Lactobacillus amylovorus LAM1345 and Lactiplantibacillus plantarum LP1145 individually reduced serum TMAO in choline-fed mice. Whereas, a multistrain formula (MF) containing these two strains plus Limosilactobacillus fermentum LF33 exhibited the most pronounced reduction, likely due to the additive and synergistic actions in MF [127]. Furthermore, following a 12-week synbiotic intervention decreased serum TMAO and improved metabolic profiles in patients with dyslipidemia [128].

Probiotics, prebiotics, and synbiotics hold potential in treating CVDs, like HF, via various pathophysiological mechanisms [129,130]. The gut microbiota of HF patients showed reduced probiotic abundance and elevated harmful bacteria [131]. Several probiotics, such as Lactobacillus plantarum ZDY04 [44], Lactobacillus rhamnosus GG strain [132], and Bifidobacterium lactis Probio-M8 [133], reduced TMAO concentrations, aid in CVD management, and diminish adverse cardiovascular events, indicating TMAO inhibition contributed to their cardioprotective effects. Sánchez-Quintero et al. [134] transplanted gut microbiota from patients with IHD into mice, elevating TMAO concentrations in recipients. Prebiotic essential oils from parsley (Petroselinum crispum) and rosemary (Rosmarinus officinalis) lessened plasma TMAO levels by reshaping gut microbiota in these mice. Similarly, thyme (Thymus vulgaris) and oregano (Origanum vulgare) essential oils functioned as effective prebiotics and produced comparable effects in gnotobiotic mice colonized with gut microbiota from CAD patients [135]. Synbiotics also reduced NT-proBNP levels and ameliorated the inflammatory status in patients with CHF [130]. As a result, probiotics, prebiotics, synbiotics, and their derivatives may offer safe and effective strategies for HF prevention and treatment through mechanisms including modulating gut microbiota, reducing TMAO levels, and maintaining host intestinal homeostasis [93].

Archaea, ancient single-celled prokaryotes inhabiting extreme environments and morphologically similar to bacteria [136], are also present in the human gastrointestinal tract. Certain archaea partially convert gut-derived TMA into methane, reducing TMA content [137]. Ramezani and coworkers [138] found that intestinal colonization with Methanobrevibacter smithii, a methanogenic archae, reduced plasma TMAO in ApoE^−/− mice, consequently attenuating AS progression. Although current research on archaea-based therapies for CVDs is still limited, this approach appears highly promising.

As previously mentioned, choline TMA lyase—which metabolizes choline into TMA—and FMOs—which oxidize TMA to produce TMAO—play critical parts in the TMAO synthesis and thus represent crucial targets for impeding TMAO production. Current methods for reducing TMAO generation typically focus on inhibiting TMA lyase or FMOs employing pharmacological inhibitors or genetic modification.

Choline TMA lyase (CutC), encoded by adjacent genes within a gene cluster, is a glycosyl radical enzyme. Both CutC and its activating protein CutD are critical for TMA biosynthesis in the gut microbiota [139]. 3,3-dimethyl-1-butanol (DMB), a choline analog, is the most widely studied inhibitor of this enzyme. It is naturally present in vinegar, extra virgin olive oil, wine, and grape seed oil; notably, extra virgin olive oil and wine are components of the MD [93,140]. Hence, DMB may partly account for the TMAO-lowering effects associated with the MD. Wang and colleagues [140] first showed in vitro and in vivo that DMB can partially inhibit TMA lyase activity in different microorganisms, blocking TMA formation from various substrates, including choline and carnitine, ultimately decreasing plasma TMAO concentrations. Additionally, DMB is non-toxic and non-lethal. Nevertheless, it does not inhibit TMA generation from certain substrates such as γ-butyrobetaine, nor does it affect TMA oxidation by FMO3 [140]. Based on DMB, the Hazen team developed a new generation of TMA lyase inhibitors: fluoromethylcholine (FMC) and iodomethylcholine (IMC). FMC and IMC irreversibly impede CutC/D, thereby effectively and continuously attenuating host TMA and TMAO levels. Furthermore, they exhibit poor absorption by the host, leave commensal bacteria unaffected, demonstrate limited systemic exposure, and are non-toxic and associated with a reduced risk of side effects [85]. Meanwhile, FMO inhibitors lower plasma TMAO by blocking hepatic TMA oxidation [141]. Methimazole, a high-affinity substrate of FMO3, acts as a competitive inhibitor, whereas indoles are non-substrate competitive FMO3 inhibitors. Both have been shown to suppress FMO3 activity and decrease TMAO concentrations [28,142]. However, the impact of methimazole on the thyroid cannot be overlooked. Therefore, inhibiting important TMAO-producing enzymes, specifically TMA lyase and FMO3, offers a plausible approach to lessening TMAO levels and ultimately managing CVDs like HF.

TMA lyase inhibitors have been extensively employed in animal models to hinder TMAO generation and ameliorate cardiovascular impairments. Studies have shown that DMB may attenuate inflammation, cardiomyocyte hypertrophy, and fibrosis, and improve cardiac function in mice with compromised hearts or HF, mainly through TMAO reduction. Furthermore, chronic DMB treatment exhibits no overt toxicity [25,27]. In mice fed WDs, DMB ameliorated excessive TMAO levels, vascular dysfunction, reduced exercise tolerance, and frailty. It also alleviated cardiac inflammation and fibrosis, consequently improving cardiac function and ultimately diminishing CVDs likelihood [143]. IMC, another TMA lyase inhibitor, reduced TMAO and BNP levels in HF mice, downregulated profibrotic gene expression, and mitigated TMAO-induced cardiac remodeling and dysfunction. Importantly, IMC also proved pharmacological safety in relevant tests [73]. FMC and IMC have demonstrated notable curative effects in lowering plasma TMAO levels and suppressing choline-triggered platelet hyperreactivity and thrombosis, without increasing bleeding risk [85]. Although DMB, IMC, and FMC have shown preliminary efficacy and safety in HF models, clinical data remain scarce. Moreover, Yoshida et al. [28] reported that either administering the FMO inhibitor methimazole or depleting the FMO2 gene lessened TMAO levels in HF mice, alleviating cardiac fibrosis and dysfunction. Antisense oligonucleotide-targeted inhibition [84] or knockout [144] of the FMO3 gene effectively lowered TMAO levels and subsequently mitigated TMAO-induced platelet hyperreactivity and thrombosis in mice. These results suggest that pharmacological or genetic inhibition of FMOs to decrease TMAO synthesis may offer a therapeutic avenue for HF. Nevertheless, clinical translation constitutes challenges, considering that FMOs participate in the oxidative metabolism of diverse drugs and chemicals in the body besides TMAO. Inhibition or deficiency of FMOs can cause hepatotoxicity and lead to liver diseases [145]. Additionally, loss of FMO3 function in humans may result in harmful TMA buildup, triggering side effects including TMAU [48]. Therefore, potential adverse effects must be thoroughly evaluated before FMO inhibitors are applied clinically.

In conclusion, targeted inhibition of TMAO-producing enzymes represents a promising tactic for addressing HF, whereas significant gaps remain between experimental studies and clinical application, warranting further comprehensive investigation [35].

Phytomedicines can modulate gut microbiota and their metabolites, reduce TMAO concentrations, and exert certain preventive and therapeutic effects on CVDs such as HF [176]. Phytomedicines, distinguished by multi-component, multi-target, and multi-pathway attributes, could alleviate drug resistance via compensatory mechanisms and reduce adverse drug reactions to some extent [177]. They thus provide underlying new strategies for inhibiting TMAO production and treating CVDs.

Research indicated that berberine (BBR), a bioactive compound derived from Rhizoma coptidis, showed efficacy in managing HF [178]. BBR reduced TMAO generation in animal models and AS patients by remodeling gut microbiota, downregulating TMA-producing enzymes and FMO3 [179], and suppressing the choline-TMA-TMAO pathway [180], thereby curbing TMAO-induced AS. Importantly, BBR exhibited no hepatorenal toxicity [179]. The findings suggest that BBR possesses significant research and development potential. Resveratrol (RES), a natural extract from plants like Polygonum cuspidatum, lowered TMAO levels by decreasing TMA generation via remodeling gut microbiota [181], and was shown to alleviate HF in mice [182]. Garlic (Allium sativum), renowned for its antibacterial properties, contains allicin, which has been proven effective in addressing CVDs, including HF [183]. In vitro and in vivo studies confirmed that allicin and fresh garlic juice rich in allicin suppressed the TMAO synthesis through gut microbiota modulation [184]. Recent investigation has revealed that aged garlic oligosaccharides mitigate AS in ApoE^−/− mice fed a high-fat and high-cholesterol diet by regulating gut microbiota and decreasing TMAO levels [185]. Hawthorn (Crataegus) and its extracts also exhibited therapeutic potential in HF [186,187]. He et al. [92] reported that hawthorn fruit extract lowered TMAO concentrations in mice dose-dependently, mediated by anti-inflammatory and antioxidant properties. Gypenosides (GYP), the primary constituent of Gynostemma pentaphyllum, lessened plasma TMAO levels by reshaping gut microbiota and inhibiting TMA lyase activity [188]. Moreover, GYP exerted cardioprotective effects and enhanced cardiac function by promoting mitochondrial autophagy, ultimately ameliorating HF [189]. Ji and coworkers [190] discovered that enemas with rhubarb (Rhei Radix et Rhizoma) modulated gut microbiota, decreasing serum TMAO and TMA concentrations and downregulating inflammatory markers in rats. Additional evidence indicated that the main constituents of rhubarb, emodin [191,192] and rhein [193,194], ameliorated pathological cardiac hypertrophy. These findings indicate that rhubarb and its extracts have promise as an innovative therapeutic agent for lowering TMAO concentrations and aiding in managing HF. Lycium barbarum polysaccharides (LBPs), important ingredients of Fructus lycii, could modulate gut microbiota, reduce intestinal permeability, downregulate inflammatory cytokines, significantly lessen serum TMAO levels, and subsequently improve LV function in mice [195]. Puerarin (PU), derived from Puerariae lobatae Radix, enhanced cardiac function in rats suffering from adriamycin-induced HF [196]. A systematic review and meta-analysis showed that combining PU injection with conventional drug therapy was safer and more effective than conventional drug therapy alone in acute heart failure [197]. Recent investigation has demonstrated that PU attenuates TMAO concentrations by altering gut microbiota composition and hindering TMA synthesis, thus exerting anti-AS effects [198]. Polymethoxyflavones (PMFs) from citrus (Citrus sinensis L.) peel decreased TMAO levels by inhibiting CutC/D and FMO3 activity, ultimately reducing CVD risk [199,200]. In addition, 5-Demethylnobiletin, a natural PMF, ameliorated isoproterenol-induced cardiac injury in mice [201].

In summary, TMAO stands out as a potential novel therapeutic target for HF, as illustrated in Figure 3.