Establishment of a mouse model of TMAO‐induced cardiac injury and application of MICT intervention

Abstract

Background

This study aims to explore the establishment of an animal model of cardiac injury induced by trimethylamine‐N‐oxide (TMAO), a metabolite secreted by gut microorganisms, and to investigate its application in moderate‐intensity continuous training (MICT) intervention.

Methods

C57BL6/J mice were randomly divided into four groups: normal mice (Nor, n = 15); mice administered TMAO (TMAO, n = 15); mice undergoing (Nor+MICT, n = 15); mice undergoing (MICT) and administered TMAO (TMAO+MICT, n = 15). Mice in the TMAO and TMAO+MICT groups received daily gavage of high‐dose TMAO for 8 weeks, whereas those in the Nor+MICT and TMAO+MICT groups underwent MICT for 8 weeks (60 min per session, 5 days per week, at 50% maximal running capacity). Cardiac function was evaluated using ultrasound, myocardial histology was examined using hematoxylin and eosin (HE) staining, and nuclear magnetic resonance (NMR)‐based metabolomics was employed for multivariate statistical and metabolic pathway analyses.

Results

Relative to the Nor group, TMAO‐treated mice exhibited significant weight loss, elevated heart rate, and reduced ejection fraction and left ventricular fractional shortening, indicating cardiac impairment. Importantly, the TMAO+MICT group demonstrated significant improvements in these parameters compared to the TMAO group, alongside distinct alterations in myocardial metabolic profiles. TMAO altered five metabolic pathways relative to controls, whereas MICT induced significant changes in three pathways in TMAO‐treated mice.

Conclusion

Eight weeks of high‐dose TMAO administration induced significant cardiac dysfunction in mice, which was effectively mitigated by MICT intervention. Consequently, this animal model serves as a valuable tool for investigating the mechanisms underlying the impact of MICT on cardiovascular diseases.

This study aims to explore the establishment of an animal model of cardiac injury induced by trimethylamine‐N‐oxide (TMAO), a metabolite secreted by gut microorganisms, and to investigate its application in moderate‐intensity continuous training (MICT) intervention. C57BL6/J mice were randomly divided into four groups: normal mice (Nor, n = 15); mice administered TMAO (TMAO, n = 15); mice undergoing (Nor+MICT, n = 15); mice undergoing MICT and administered TMAO (TMAO+MICT, n = 15). Eight weeks of high‐dose TMAO gavage (800 mg/kg/day) induced significant cardiac dysfunction, evidenced by reduced ejection fraction (EF), left ventricular fractional shortening (LVFS), elevated heart rate (HR), and histopathological myocardial damage. MICT intervention (60 min/day, 5 days/week at 50% maximal running capacity) reversed these effects, restoring cardiac function and aerobic capacity. Nuclear magnetic resonance (NMR)‐based metabolomics revealed distinct myocardial metabolic perturbations. The analytical outcomes revealed that TMAO induction led to substantial modifications in five distinct metabolic pathways: phenylalanine, tyrosine, and tryptophan biosynthesis; phenylalanine metabolism; starch and sucrose metabolism; glycerolipid metabolism; nicotinate and nicotinamide metabolism. Compared to the TMAO group, the TMAO+MICT group exhibited significant alterations in three metabolic pathways, including glycerolipid metabolism, purine metabolism, and TCA cycle. This model demonstrates TMAO's cardiotoxicity and validates MICT as a nonpharmacological strategy to mitigate gut microbiota‐mediated cardiac injury. Consequently, this animal model serves as a valuable tool for investigating the mechanisms underlying the impact of MICT on cardiovascular diseases.

1. INTRODUCTION

Cardiovascular disease (CVD), characterized by its high incidence and mortality rates, poses a significant threat to global health. Statistics reveal that, annually, CVDs claim up to 15 million lives globally, with as many as 2.6 million of those fatalities occurring in China, and remain the leading cause of death and morbidity in developed countries. ¹ Given the intricate and multifactorial nature of CVD, current therapeutic medications and medical interventions have not yet yielded substantial improvements in reducing the incidence and mortality rates of these conditions. Consequently, there is an urgent and critical need to actively explore and develop more effective strategies for the prevention and treatment of CVDs to enhance their control and management.

Exercise is widely acknowledged as a potent nonpharmacological treatment and prevention tool. There is evidence that exercise can enhance metabolic risk factors for the heart, bolster the functionality of the cardiovascular system, decrease the incidence of cardiac arrhythmias, and ameliorate vascular responses during episodes of coronary ischemia and reperfusion. ² , ³ , ⁴ , ⁵ Specifically, moderate‐intensity continuous training (MICT) has demonstrated efficacy in the prevention of numerous chronic diseases. Research has shown that aerobic exercise can help normalize myocardial metabolism by increasing physical strength and improving myocardial function and antioxidant capacity. ⁶ Swimming can improve left ventricular conformation and attenuate the symptoms of heart failure in hypertensive rats induced by a high‐salt diet. ⁷ In these studies, animal models, especially mouse models, as their greater homology with humans compared to other species, have served as a good and effective tool. Because of wild‐type mice are resistant to the developing pathological changes, apolipoprotein E‐deficient mice (ApoE^−/− mice) are the most widely used in current mouse models of atherosclerosis. But, atherosclerotic lesions in ApoE^−/− mice arise due to the dietary induction of pro‐atherosclerotic lipoproteins, and the implications of these outcomes warrant careful consideration. ⁸ Furthermore, a multitude of factors, including genetics, diet, and environment, play crucial roles in the intricate field of cardiovascular pathophysiology; it is nearly impossible to correlate specific diseases with a singular experimental model. Consequently, we need more animal models of cardiac injury for studies related to CVD.

Recent studies have uncovered a significant influence of metabolites secreted by gut microorganisms on the development of CVD. ⁹ , ¹⁰ , ¹¹ Of particular concern is trimethylamine‐N‐oxide (TMAO), a metabolite generated in the action of intestinal microbiota and hepatic flavin monooxygenase, which is widely considered to be closely related to the prognosis of CVD and plays a key role in atherosclerosis. ¹² , ¹³ Employing metabolomics approach, researchers found a distinct dose‐dependent association between circulating TMAO levels and the risk of CVD, marking the first instance that TMAO has been explicitly correlated with cardiovascular risk. ¹⁴ Multiple studies have shown that elevated plasma TMAO levels significantly increase the risk of stroke, ¹⁵ coronary heart disease, ¹⁶ and overall CVD. ¹⁷

In animal experimental models, high doses of TMAO have been demonstrated to directly induce atherosclerosis and thrombosis, ¹⁷ , ¹⁸ , ¹⁹ with the underlying mechanisms still requiring further clarification. A study found that TMAO raises intracellular Ca²⁺, upregulates the hypertrophic markers ANP and MYH7, and downregulates SERCA2a, indicating its potential to induce cardiac hypertrophy. ²⁰ In primary cultures of both rat and human VSMC, TMAO evoked a dose‐dependent rise in PRMT5 and VCAM‐1 at 100–1000 μmol/L and a time‐dependent induction of both proteins after exposure to 600 μmol/L TMAO. ²¹ In a large, multiethnic cohort with repeated TMAO measurements, those in the highest quintile showed a 32% greater risk of incident atherosclerotic CVD compared to the lowest. ²² Nonetheless, it has also been evidenced that the perfusion with 1 mmol/L TMAO has no effect on isolated cardiac function, and the administration of TMAO at a dose of 120 mg/kg in the drinking water for 14 weeks did not affect cardiac function in rats. ²³

Therefore, there is a burgeoning consensus that TMAO consumption is correlated with the incidence of CVD, and that its impact on the body may be either detrimental or beneficial, is intricately linked to both the quantity and the temporal pattern of consumption. The heart, as a highly metabolically active organ, has its functionality closely intertwined with the cardiac metabolic state. ²⁴ The cardioprotective role of exercise is widely acknowledged, and there is a consensus that it mitigates myocardial damage by influencing TMAO, a metabolite produced by gut microorganisms. However, the precise mechanisms through which exercise affects TMAO require further investigation. The objective of this study was to develop a straightforward, reproducible, and low‐mortality model of cardiac injury in wild‐type mice induced by TMAO, which is closely associated with cardiovascular health, to investigate the effects of exercise intervention. By applying this model to MICT intervention experiments, we aimed to lay a novel scientific foundation for cardioprotection and the prevention of related diseases.

2. MATERIALS AND METHODS

2.1. Animal

C57BL/6J male mice (SPF, 11 weeks) were purchased from Beijing Huafukang Biotechnology Co., Ltd. (Beijing, China) and accommodated in the animal laboratory of Beijing Sport University, where they were maintained in a controlled environment with a temperature of 22–24℃ and a relative humidity of 55%–70%. The mice were group‐housed in rearing cages, with four mice per cage, under a 12:12‐h light–dark cycle to simulate natural day–night conditions, and provided with ad libitum access to food and water. The Experimental Animal Center of Beijing Sports University (Beijing, China) granted approval for all experimental protocols (2021127A), ensuring the study's adherence to ethical standards and the welfare of the animals involved. The mice were randomly assigned to four experimental groups, with 15 mice per group. The groups were as follows: the Nor group, in which mice were neither subjected to exercise nor treated with TMAO (n = 15); the TMAO group, in which mice were gavaged with TMAO without exercise (n = 15); the Nor+MICT group, in which mice were given saline in conjunction with MICT (n = 15); and the TMAO+MICT group, in which mice were gavaged with TMAO while undergoing MICT (n = 15).

2.2. TMAO administration

TMAO, produced by Sigma‐Aldrich, USA, is cataloged under item number 317594‐5G and is presented as a light‐yellow powder. In our experimental protocol, the dosage of TMAO administered via gavage was set at 800 mg/kg, calibrated according to the individual body weight of each mouse. The TMAO was thoroughly dissolved in saline solution, with each mouse receiving a daily dose of 0.3 mL of this solution. The control groups were received an equivalent volume of saline solution. The gavage procedure was conducted daily at 9:00 a.m., followed by the exercise regimen at 4:00 p.m., establishing a consistent routine for all subjects involved in the study.

2.3. Maximal running capacity test protocol

The maximal running capacity (MRC) test protocol was adapted from the research of Martinez‐Huenchullan et al. ²⁵ and it commenced with a 10‐min warm‐up period, beginning at a speed of 6 m/min. The treadmill velocity was then incremented by 3 m/min every 3 min, continuing until the mice reached exhaustion. Exhaustion was determined when the mice could no longer reach the end of the treadmill track after they received more than five gentle tail prods with a stimulation rod, indicating that their maximal running capacity had been achieved.

2.4. Training protocol

After a week of acclimatization feeding, the mice underwent a week of acclimatization exercise training, comprising a total of five sessions based on the following regimen: they exercised for 10 min at a speed of 6 m/min on day 1, and this speed was increased by 1 m/min each subsequent day while the duration remained unchanged. After the adaptation exercise, the mice were given a 2‐day respite before they underwent the MRC test. Subsequently, the Nor+MICT and TMAO+MICT groups engaged in an 8‐week MICT program. This program was structured to include a 5‐min warm‐up, 50 min of exercise at 50% of their MRC intensity, and a final 5‐min cool‐down period, summing up to a 60‐min session. The training protocol was curated based on existing literature ²⁶ , ²⁷ , ²⁸ and refined using data from preliminary trials. The MRC test was conducted once every 2 weeks to facilitate the adjustment of the MICT intensity.

2.5. Sample collection

Cardiac tissue was collected 24 h following the final exercise session. The mice were initially anesthetized with isoflurane gas, after which their cardiac function was meticulously evaluated. After the assessment, the mice were euthanized, and the heart tissue was carefully extracted. The tissue was then meticulously washed with saline to eliminate any remaining blood, and the weight of the heart was precisely documented.

2.6. Cardiac testing

2.6.1. Ultrasound examination

Mice designated for ultrasound assessment were gently anesthetized using isoflurane gas. Following this, the chest hair was meticulously removed using depilatory agents. The mice were carefully positioned on the operating table with their abdomens facing up, and their limbs were gently secured with medical tape to maintain proper extension. Ultrasound gel was applied to the chest area, and the portable cardiac ultrasound device, VIVID I, was then activated to measure cardiac function. The transducer was immersed in the gel, angled at 10°–30° to the sternum, and initially positioned to obtain long‐axis views of the heart. With precision, the transducer was moved to capture M‐mode images. Throughout the procedure, heart rate (HR), ejection fraction (EF), left ventricular end‐systolic diameter (LVIDs), left ventricular end‐diastolic diameter (LVIDd), and left ventricular fractional shortening (LVFS) were diligently recorded. Each parameter was measured at least thrice to ensure reliability, with the mean values being documented for further statistical analysis.

2.6.2. HE staining

After the procurement of myocardial tissue, it was initially immersed in a 4% paraformaldehyde solution for a 24‐h fixation period. The tissue was then subjected to a graded ethanol dehydration process prior to being embedded in paraffin. Using a microtome, we meticulously prepared 5‐μm sections and affixed them to glass slides. The slides were subsequently baked in a slide warmer at 70℃ for 10 min for pretreatment. Deparaffinization was carried out using xylene in two 10‐min intervals, ensuring the removal of paraffin. The slides were then transferred to a graded ethanol series to eliminate any remaining xylene traces, followed by a thorough rinse in distilled water. The sections were stained with hematoxylin for approximately 10–30 min, followed by three water rinses and a brief treatment with 1% hydrochloric acid ethanol solution for controlled destaining. After another set of three washes, the sections were stained using 0.5% eosin solution and finally sealed with neutral gum.

2.6.3. Measurement of cardiomyocyte cross‐sectional area

Typically, three to five fields of view are randomly selected for each mouse. The following criteria are to be observed when selecting cells: (i) the cell cross‐section is approximately circular; (ii) a complete and centered circular/ovoid cell nucleus is clearly visible. The cell membrane boundary is clearly delineated. The freehand selection function using ImageJ should be used to delineate the outer periphery of the cell membrane, and the measure function will directly provide the area (μm²). The number of cells measured for each mouse ranged from 30 to 50, with the mean value being calculated as the cellular surface area (CSA) of the respective mouse. The CSA measurement was conducted on six mice per group.

2.7. NMR‐based metabolomic analysis methods

To detect differences in metabolic patterns between groups and analyze the perturbed metabolic pathways, we conducted NMR‐based metabolomic analyses. The sample preparation process was as follows: First, the water‐soluble metabolites from mouse cardiac tissue were extracted. Approximately 50 mg of cardiac tissue was cut, and CH₃OH, CHCl₃, and dd H₂O were added in proportion. The mixture was homogenized, centrifuged, dried under nitrogen, and lyophilized. NMR buffer was then added to prepare the samples for NMR. Subsequently, spectroscopic data were acquired using a Bruker Avance III 850 MHz NMR spectrometer equipped with a TCI cryoprobe (Bruker Bio Spin, Rheinstetten, Germany). The spectra were preprocessed and normalized, and metabolites were identified using Chenomx NMR Suite software and the Human Metabolome Database (HMDB, http://www.hmdb.ca/). The specific methods of NMR‐based metabolomics have been explained in the Supporting Information.

2.7.1. Multivariate statistical analysis

Multivariate statistical analysis of the normalized NMR spectrum data was performed using SIMCA‐P software (version 14.1.0, MKS Umetrics, Umea AB, Sweden). Initially, a Pareto scaling was applied to the data to enhance the weighting of metabolites present at lower levels. Subsequently, a supervised partial least squares discriminant analysis (PLS‐DA) was performed, with the aim of identifying and reinforcing trends between sample groups. To ascertain the stability and reliability of the PLS‐DA model, a cross‐validation process involving 200 iterations of response permutation test (RPT) was implemented.

2.7.2. Metabolic pathway analysis

To uncover metabolic pathways that exhibit significant changes, metabolic pathway analysis was performed using the MetaboAnalyst 5.0 web server (https://www.metaboanalyst.ca) provided by McGill university in Montreal, Canada. This analysis integrated metabolite set enrichment analysis and pathway topology analysis (PTA) to provide a holistic view of the metabolic shifts. The metabolite enrichment analysis was dedicated to pinpointing differential metabolites with biological significance across groups, assessed against stringent statistical criteria, with a p‐value threshold of <0.05 to denote statistical significance. Furthermore, PTA of the relative intermediate approach was also utilized to determine the pathway impact value (PIV). By integrating the outcomes of these analyses, p < 0.05 (−log(p) > 1.3) and PIV > 0.1 were identified as characteristic metabolic pathways capable of inducing significant metabolic changes.

2.8. Statistical analysis

Data analysis and processing of the experimental results were performed using GraphPad Prism 8.3.0 software (La Jolla, CA, USA). Variance within the data was assessed using one‐way analysis of variance (ANOVA). For groups that exhibited significant differences, subsequent pairwise comparisons were conducted using unpaired t‐tests to further clarify the underlying reasons for these disparities. The threshold for determining statistical significance was set at p < 0.05.

3. RESULTS

3.1. Body weight fluctuations of mice in the Nor and TMAO groups

Prior to gavage, the average body weights of mice in the Nor and TMAO groups were 23.45 ± 0.72 and 23.47 ± 0.95 g, respectively (Table 1), with no significant difference between the two groups. After 8 weeks of gavage, the TMAO group exhibited lower than the Nor group, with respective values of 24.99 ± 1.02 and 26.42 ± 1.32 g, and this difference was found to be statistically significant (p < 0.01).

TABLE 1.

Body weight fluctuations in the Nor and TMAO groups of mice (g, mean ± SD).

Time (weeks)	Groups	Body weight (g)	95% CI
		Mean ± SD	Lower bound	Upper bound
Preintervention	Nor	23.45 ± 0.72	23.05	23.85
	TMAO	23.47 ± 0.95	22.94	23.99
First week	Nor	22.73 ± 1.41	21.95	23.52
	TMAO	22.58 ± 0.98	22.04	23.12
Second week	Nor	23.60 ± 1.06	23.01	24.19
	TMAO	23.69 ± 1.12	23.06	24.31
Third week	Nor	24.08 ± 1.02	23.52	24.64
	TMAO	23.87 ± 1.43	23.08	24.66
Fourth week	Nor	24.89 ± 1.03	24.32	25.47
	TMAO	24.34 ± 1.32	23.61	25.07
Fifth week	Nor	25.56 ± 1.15	24.93	26.19
	TMAO	24.79 ± 1.21	24.13	25.46
Sixth week	Nor	25.79 ± 1.29	25.08	26.51
	TMAO	24.97 ± 1.22	24.29	25.64
Seventh week	Nor	25.95 ± 1.40	25.17	26.72
	TMAO	25.07 ± 1.35	24.33	25.82
Eighth week	Nor	26.42 ± 1.32	25.58	27.26
	TMAO	24.99 ± 1.02**	24.38	25.61

Note: Statistical significances: **p < 0.01.

Abbreviations: CI, confidence interval; SD, standard deviation.

3.2. HE staining of myocardial tissue from mice in the Nor and TMAO groups

Under microscopic examination, the nucleus of cardiomyocytes appeared bluish‐brown, and the cytoplasm exhibited a pinkish‐red hue. Comparative analysis revealed that myocardial tissue from the TMAO group exhibited subtle indications of damage when contrasted with the tissue from the Nor group (Figure 1). Specifically, after TMAO treatment, the cross‐sectional area of mouse cardiomyocytes was significantly larger than that of the Nor group (Figure 1B).

FIGURE 1.

Hematoxylin and eosin (HE) staining of myocardial tissue from mice (A) and cross‐sectional area of cardiomyocytes (B) in the Nor and TMAO groups. Scale bar: 700 μm Statistical significance: ****p < 0.0001.

3.3. Heart weights of mice in the Nor and TMAO groups

The heart weights of mice in the Nor and TMAO groups were recorded as 108.45 ± 5.52 and 106.31 ± 6.36 mg, respectively, as detailed in Table S1. Although the mean heart weight in the TMAO group was slightly less than that of the Nor group, statistical analysis revealed no significant difference between the two groups (Figure S1, p > 0.05).

3.4. Ultrasound assessments in mice from the Nor and TMAO groups

HR in the Nor and TMAO groups was recorded at 721.43 ± 54.31 and 858.97 ± 83.70 bpm, respectively, as detailed in Table S2. Statistical analysis revealed significant differences between the two groups. As depicted in Figure 2, there was a marked increase in relative HR (p < 0.01) and notable decreases in both EF and LVFS (p < 0.01, p < 0.05) in the TMAO group compared to the Nor group. These findings suggest a detrimental impact on cardiac function due to long‐term, high‐dose TMAO consumption.

FIGURE 2.

Cardiac ultrasound assessments of heart rate (HR), ejection fraction (EF), and left ventricular fractional shortening (LVFS) in mice from the Nor and TMAO groups. Statistical significance: *p < 0.05, **p < 0.01, ***p < 0.001.

3.5. Assessment of exercise endurance in mice from the Nor+MICT and TMAO+MICT groups

Exercise endurance in Nor+MICT and TMAO+MICT groups was assessed at multiple time points: before gavage and after gavage for 2, 4, 6, and 8 weeks. Prior to gavage, the mean exercise endurance for Nor+MICT and TMAO+MICT groups was 55.93 ± 8.84 and 60.18 ± 11.64 min, respectively. The t‐test indicated no significant difference between the two groups (Table S3; Figure S2). Except for the 2‐week mark, a decline was observed in the TMAO+MICT group at 4, 6, and 8 weeks compared to the Nor+MICT group (Figure 3). Notably, after 8 weeks of gavage, a significant difference emerged between the two groups (Figure 3, p < 0.001), suggesting that 8 weeks of TMAO administration reduced the aerobic capacity of the mice.

FIGURE 3.

Exercise endurance in the Nor+MICT and TMAO+MICT groups of mice at multiple time points: Second week, fourth week, sixth week, and eighth week. Statistical significance: ns, p > 0.05, *p < 0.05, ***p < 0.001.

3.6. Implementation of MICT intervention in an 8‐week TMAO‐induced cardiac injury mouse model

We endeavored to utilize this model in the intervention study involving MICT to elucidate the mechanisms through which MICT may mitigate cardiac damage in mice. The study aims to establish not only the successful establishment of an 8‐week TMAO‐induced cardiac injury model but also to demonstrate the efficacy of MICT in reducing the severity of cardiac injury.

3.6.1. MICT intervention alleviates cardiac dysfunction in an 8‐week TMAO‐induced mouse model of cardiac injury

Relative to the TMAO group, the TMAO+MICT group exhibited a markedly significant reduction in HR, LVIDd, and LVIDs (Figure 4A,D,E; Table S4; p < 0.05), along with a notable increase in both EF and LVFS (Figure 4B,C; Table S4; p < 0.05). These findings suggest that an 8‐week TMAO challenge compromised cardiac function in mice. However, MICT substantially ameliorated the extent of cardiac dysfunction. This indicates that the model is apt for investigating the effects of MICT intervention on impaired cardiac function.

FIGURE 4.

Ultrasound assessments in mice from three groups. (A) Heart rate (HR). (B) Ejection fraction (EF). (C) Left ventricular fractional shortening (LVFS). (D) Left ventricular end‐diastolic diameter (LVIDd). (E) Left ventricular end‐systolic diameter (LVIDs). Statistical significance: ns, p > 0.05, *p < 0.05, **p < 0.01.

3.6.2. MICT intervention modifies the metabolic patterns in an 8‐week TMAO‐induced mouse model of cardiac injury

Multivariate statistical analysis

To determine whether MICT intervention would alter the metabolic patterns in the myocardium of mice induced by 8 weeks of TMAO, we established a supervised PLS‐DA model among the Nor, TMAO, and TMAO+MICT groups. The results indicated that there were distinct metabolic profiles among these three groups of mice (Figure 5A), and the metabolic profile of the TMAO group was significantly different from that of the Nor group at t[1] (Figure 5B), whereas the metabolic profiles of the TMAO+MICT group were significantly different from those of the TMAO group at t[1] and t[2] (Figure 5C). The cross‐validation plots (Figure 5D–F) revealed that the blue regression line representing Q2 intersected the y‐axis at a negative value. Notably, at X = 1, the R2 and Q2 values were significantly elevated compared to the remaining data points in the pairwise group comparisons, with the exception of the Nor versus TMAO comparison. This absence of overfitting in the PLS‐DA model underscores its reliability and accuracy. These results indicate that TMAO induces metabolic disruptions within myocardial tissues, whereas MICT partially ameliorates these metabolic imbalances, facilitating a return to metabolic equilibrium in the cardiac muscle.

FIGURE 5.

Multivariate analysis of identified metabolites in ¹H‐NMR spectra of aqueous extracts derived from the hearts in three groups of mice. (A–C) Partial least squares discriminant analysis (PLS‐DA) score plots. (D–F) Permutation validation of the relative PLS‐DA model.

Metabolic pathway analysis

To discern the alterations in myocardial metabolic pathways prompted by MICT intervention in mice subjected to 8 weeks of TMAO exposure, a comprehensive metabolic pathway analysis was executed. For the analysis, significantly altered metabolic pathways were identified on the basis of changes in metabolite concentration levels, as determined by p < 0.05 and PIV > 0.1. The analytical outcomes revealed that TMAO induction led to substantial modifications in five distinct metabolic pathways (Figure 6A): (a) phenylalanine, tyrosine, and tryptophan biosynthesis; (b) phenylalanine metabolism; (c) starch and sucrose metabolism; (d) glycerolipid metabolism, and (e) nicotinate and nicotinamide metabolism. Compared to the TMAO group, the TMAO+MICT group exhibited significant alterations in three metabolic pathways (Figure 6B), including: glycerolipid metabolism, purine metabolism, and TCA cycle.

FIGURE 6.

Significantly altered metabolic pathways identified from the pairwise comparisons between the three groups of the hearts of mice. (A) TMAO group versus Nor group. (B) TMAO+MICT group versus TMAO group. (a: Phenylalanine, tyrosine, and tryptophan biosynthesis; b: Phenylalanine metabolism; c: Starch and sucrose metabolism; d: Glycerolipid metabolism; e: Nicotinate and nicotinamide metabolism; f: Purine metabolism; g: TCA cycle).

These results suggest that 8 weeks of TMAO administration induces metabolic disturbances in mouse myocardial tissue, which can be alleviated by MICT by modulating multiple metabolic pathways. This approach allows for the modeling of myocardial injury and can be used for MICT intervention studies.

4. DISCUSSION

Over the past few decades, the development of animal models of CVD has played a crucial role in elucidating the pathogenesis and correlates of the disease, as well as in exploring possible therapeutic strategies. Throughout the experiments, researchers continued to improve the models to better replicate the conditions of the disease in question. Currently, among the mouse models of atherosclerosis, aortic aneurysms (AAs), and hypertension, low‐density lipoprotein receptor‐deficient mice (LDLR^−/− mice) and apolipoprotein E‐deficient mice (ApoE^−/− mice) are the most commonly used. ¹ , ²⁹ There are three primary induction methods for modeling CVD: surgical intervention, ³⁰ , ³¹ drug‐induced disease, ³² , ³³ and electrical techniques. ³⁴ Each model possesses its own characteristics, offering researchers tailored tools to meet their specific requirements. ³⁵

In this study, we proposed the hypothesis that the chronic administration of high doses of TMAO could effectively establish a model of cardiac injury in mice, with the aim of applying this model to investigate the efficacy of MICT interventions. Our study found that the administration of TMAO at a dose of 800 mg/kg per day with gavage for 8 weeks resulted in a significant decrease in body weight (Table 1), slight signs of myocardial tissue damage (Figure 1), a significant increase in relative HR (Figure 2; p < 0.01), a significant decrease in EF and LVFS (Figure 2, p < 0.01 and p < 0.05), and a significant reduction in exercise duration in C57BL/6J mice (Figure 3), indicating that the heart function of mice is impaired and their aerobic capacity is reduced. Gut microorganisms and their metabolites are pivotal in the regulation of various metabolic processes in the body, exerting a profound influence on overall health status. ³⁶ , ³⁷ Seafood, red meats, shellfish, and eggs into your daily diet provide a bountiful supply of phosphatidylcholine (PC), a nutrient that is pivotal for various bodily functions. This PC is subsequently metabolized by the gut microbiota into TMAO. Emerging research has implicated TMAO as a significant risk factor associated with the development of CVD, cancers, and other chronic diseases. ¹⁰ , ¹³ Additionally, a study suggested that TMAO might exacerbate cognitive impairments in APP/PS1 mice, ³⁸ which could lead to kidney damage. ¹² Acute high‐dose TMAO injection increased thrombus formation in a mouse model of carotid artery injury. ³⁹

The administration of TMAO at a plasma concentration of 86 μmol/L to LDLR^−/− mice has been observed to trigger heightened levels of inflammatory biomarkers. This treatment also prompts the activation of MAPK, ERK, and NF‐κB pathways. ⁴⁰ In a study, rats exhibited cardiac hypertrophy and fibrosis following intraperitoneal injection of TMAO, and this effect was significantly observed after injection of antibiotics that inhibited TMAO synthesis. ⁴¹ However, recent studies on rodents have reported a lack of evidence linking TMAO to the pathogenesis of CVD even at high concentrations. Research has demonstrated that 2‐week infusion of TMAO significantly elevated blood levels of TMAO from 0.6 to 60 μmol/L, which is a 100‐fold increase. Despite this substantial increase, it was observed that there were no discernible toxic effects on the rats. ⁴² Nevertheless, evidence has emerged, indicating that the exogenous supplementation of TMAO can mitigate the advancement of heart failure. Prolonged administration of a low dose of TMAO has been demonstrated to attenuate the incidence of heart failure and myocardial fibrosis in a model of spontaneously hypertensive rats. Gawrys et al. ⁴³ concluded that low doses of TMAO exert a pronounced diuretic effect, which alleviates the volume load in rats and significantly contributes to the enhancement of cardiac function.

In light of these two starkly contrasting conclusions, it has been hypothesized that TMAO could serve as a feedback mechanism against disease progression, acting more as a biomarker of disease rather than a causative agent. ⁴⁴ , ⁴⁵ Additionally, many studies reporting that TMAO induces atherosclerosis have been conducted in ApoE^−/− mice. ⁸ In contrast, TMAO has been shown to exert a protective effect against the development of atherosclerosis in the E‐Leiden/CETP mouse model. ⁴⁶ A study has suggested that the discrepancies observed in research on the role of TMAO may stem from three factors: the dose of exposure, the route of administration, and the activity of FMO3—the enzyme responsible for converting TMA into TMAO. ⁴⁷

Acute 40‐μmol/L TMAO recovered insulin production, insulin granule formation, and insulin secretion by upregulating the IRE1α unfolded protein response to GLT‐induced endoplasmic reticulum (ER) and oxidative stress. In contrast, β‐cells exposed to higher concentrations of 80 and 160 μmol/L exhibited impaired mitochondrial viability and reduced glucose‐stimulated insulin secretion (GSIS). ⁴⁸ Organ et al. ⁴⁹ found a marked reduction in LVEF, enlargement of the heart, and increased myocardial fibrosis in mice fed a diet containing excess chow or TMAO for 12 weeks compared to mice maintained on a standard diet. A study showed that chronic exposure to 0.12% TMAO for 16 weeks suppresses the demethylase FTO and RNA‐binding protein IGF2BP2, thereby increasing NLRP3 m6A modification and inflammasome activation in microglia, which amplifies neuroinflammation and exacerbates ischemic stroke injury. Concurrently, it was demonstrated that a 3‐week regimen of 0.12% TMAO supplemented with the chow diet did not exert any discernible influence on cardiac parameters. ⁵⁰

Our study demonstrated that after a 2‐week period, the TMAO+MICT group exhibited a significantly extended exercise duration compared to the Nor+MICT group (Figure 3). However, the administration of TMAO via gavage at a dosage of 800 mg/kg per day for 8 weeks impaired cardiac function in mice. Subsequently, we used the 8‐week TMAO‐induced cardiac injury model to investigate the effects of MICT intervention. The results showed that MICT intervention alleviated cardiac function in mice (Figure 4). Furthermore, a multivariate statistical analysis of the metabolic profiles indicated that the TMAO+MICT group displayed markedly distinct metabolic patterns from the TMAO group (Figure 5C,F). This suggests that using this model to study MICT results in a positive intervention, which is consistent with most of the trends of MICT, and it can be used to explore the mechanism of MICT.

The present study showed that TMAO induction significantly altered five metabolic pathways: (a) phenylalanine, tyrosine, and tryptophan biosynthesis; (b) phenylalanine metabolism; (c) starch and sucrose metabolism; (d) glycerolipid metabolism; and (e) nicotinate and nicotinamide metabolism (Figure 6A). These essential amino acids play a crucial role in the tricarboxylic acid (TCA) cycle, either directly or indirectly contributing to the generation of energy, ⁵¹ and these amino acids are also indispensable for protein synthesis. A study has shown that MICT activates the AMPK–PGC‐1α axis, enhancing mitochondrial biogenesis and fatty‐acid oxidation efficiency to improve myocardial energy supply. ⁵² Previous studies have also shown that phenylacetylglutamine, a metabolite of phenylalanine, is associated with the development of CVD. ⁵³ , ⁵⁴ Furthermore, tyrosine is an indispensable precursor in the biosynthesis of catecholamines, which are pivotal in the regulation of cardiovascular function. Specifically, catecholamines, such as adrenaline and norepinephrine, are intimately linked to the progression of cardiac hypertrophy. ⁵⁵ Consequently, it is reasonable to deduce that metabolic perturbations involving phenylalanine and tyrosine are notably evident in TMAO‐induced mouse model. These metabolic imbalances could potentially precipitate pathological conditions, including cardiac dysfunction and hypertrophy, by impacting several critical facets of the cardiovascular system, for instance, the synthesis and secretion of catecholamines. Study has demonstrated that 4 weeks of MICT accelerates cardiac blood flow and enhances the shear stress along the endocardial surface, triggering neuroendocrine changes—such as decreased angiotensin II and catecholamines—that ultimately increase myocardial protein content; ⁵⁶ these observations are consistent with our findings.

The current study also revealed that MICT had a pronounced beneficial impact on myocardial metabolism, influencing three key metabolic pathways: (d) glycerolipid metabolism, (f) purine metabolism, and (g) TCA cycle (Figure 6B). Previous research has demonstrated that glycine serves as a protective agent against myocardial injury triggered by a variety of factors, including chemotherapeutic agents, hypoxia, and endotoxins. The metabolism of glycine, along with serine and threonine, is pivotal not only for modulating the immune system's response but also exerts a substantial influence on the functionality of various other organs. ⁵⁷ , ⁵⁸ Therefore, we postulate that MICT may mitigate TMAO‐induced myocardial injury by bolstering immune system function. Purine metabolism plays a critical role in significantly elevating ATP levels, improving myocardial energy metabolism and cardiac diastolic function. This metabolic process is particularly beneficial for patients with heart failure, as it contributes to a reduction in morbidity and mortality rates. ⁵⁹ The application of allopurinol as an inhibitor of xanthine oxidase in the treatment of CVD has garnered extensive attention within the medical community. Research has demonstrated that allopurinol contributes positively to the amelioration of cardiac function and the enhancement of exercise tolerance in both hyperuricemic and non‐hyperuricemic chronic heart failure patients. ⁶⁰ , ⁶¹ Collectively, these data indicate that MICT may alleviate the myocardial injury induced by high‐dose TMAO and restore energy homeostasis by activating these metabolic pathways and augmenting purine synthesis, thereby preserving myocardial metabolic health and functional integrity, findings consistent with previous investigations.

5. CONCLUSIONS

We have developed an animal model of cardiac injury precipitated by TMAO, a metabolite produced by the gut microbiota, with the intention of utilizing it in the context of an MICT intervention study. Our findings reveal that the administration of TMAO via gavage at a concentration of 800 mg/kg over an 8‐week period led to a reduction in body weight of C57BL/6J mice, along with myocardial tissue damage, diminished cardiac function, and a shortened exercise duration. The establishment of this injury model has paved the way for its application in MICT intervention studies, where it has been demonstrated to partially ameliorate the cardiac impairments caused by TMAO. NMR metabolomics analysis has unveiled significant differences in the myocardial metabolic profiles among the three groups, indicating that multiple metabolic pathways have been disrupted. These results align with previous studies, further validating that 8 weeks of high‐dose TMAO administration via gavage leads to impaired cardiac function. Moreover, this cardiac impairment could be substantially mitigated by MICT intervention. Thus, the cardiac injury animal model we have established may serve as a valuable tool for investigating the mechanisms by which MICT can exert its positive influence on cardiovascular health.

AUTHOR CONTRIBUTIONS

Zhongping Xie: Conceptualization; data curation; investigation; methodology; software; validation; writing – original draft. Hong Zou: Conceptualization; data curation; investigation; software; writing – original draft. Lijing Gong: Data curation; formal analysis; funding acquisition; investigation; resources; software; visualization. Minghui Lin: Formal analysis; visualization; writing – review and editing. Caihua Huang: Conceptualization; funding acquisition; methodology; project administration; resources; supervision; writing – review and editing.

FUNDING INFORMATION

This work was supported financially by grants from the National Natural Science Foundation of China (No. 32271496) and China Fundamental Research Funds for the Central Universities (Beijing Sport University File No. 2024TZJK001).

CONFLICT OF INTEREST STATEMENT

The authors declare no conflicts of interest.

ETHICS STATEMENT

The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Sports Science Experiment Ethics Committee of Beijing Sport University (2021127A).

Supporting information

Table S1.

Xie Z, Zou H, Gong L, Lin M, Huang C. Establishment of a mouse model of TMAO‐induced cardiac injury and application of MICT intervention. Anim Models Exp Med. 2026;9:142‐153. doi: 10.1002/ame2.70125

DATA AVAILABILITY STATEMENT

The data are contained within the article or Supporting Information.