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. 2024 Oct 17;24(12):1335–1347. doi: 10.1007/s12012-024-09932-8

Protective Role of (-)-Epicatechin on Trimethylamine-N-Oxide (TMAO)-Induced Cardiac Hypertrophy via SP1/SIRT1/SUMO1 Signaling Pathway

Siting Hong 1, Jing Lu 2,3, Jiaoyan Li 1,4, Yingchun Luo 5, Dongxue Liu 1, Yuanyuan Jin 1, Zeng Wang 1, Yibo Wang 1, Hao Zhang 6, Xin Zhang 1, Yang Li 1, Haoruo Zhang 1, Zengxiang Dong 2,3, Zhaojun Wang 1,✉, Lin Lv 7,✉, Zhaoguang Liang 1,✉
PMCID: PMC11564365  PMID: 39419946

Abstract

(-)-Epicatechin (EPI) is beneficial for cardiovascular health. Trimethylamine N-oxide (TMAO), a gut microbe-derived food metabolite, is strongly associated with the risk of cardiovascular diseases. However, the effects and underlying mechanisms of EPI on TMAO-induced cardiac hypertrophy remain unclear. This study aimed to determine whether EPI inhibits TMAO-induced cardiac hypertrophy. Plasma levels of TMAO in control participants and patients with cardiac hypertrophy were measured and analyzed. Male C57BL/6 mice were randomly divided into control group, TMAO group, EPI group and TMAO + EPI group. According to the groups assignments, mice received intraperitoneal (i.p.) injection of normal saline or i.p. injection of TMAO (150 mg/kg/day) for 14 days. The EPI group was given intragastric (i.g.) administration of EPI alone (1 mg/kg/day) for 21 days, and TMAO + EPI group received i.g. administration of EPI for 7 days before starting i.p. injection of TMAO, continuing until the end of the TMAO treatment. Histological analyses of the mice’s hearts was accessed by H&E and Masson staining. In vitro, H9c2 cells were induced to hypertrophy by TMAO (10 µM) for 24 h and were pre-treated with or without EPI (10 µM) for 1 h. Protein level of cardiac hypertrophy markers and Sp1/SIRT1/SUMO1 pathway were determined by western blot. The plasma level of TMAO was 2.66 ± 1.59 μmol/L in patients with cardiac hypertrophy and 0.62 ± 0.30 μmol/L in control participants. EPI attenuated TMAO-induced hypertrophy in H9c2 cells. In vivo, TMAO induced cardiac hypertrophy and impaired the cardiac function of mice. Pathological staining showed that TMAO induced cardiac hypertrophy and collagen deposition in mice. EPI treatment improved the cardiac function, inhibited the myocardial hypertrophy induced by TMAO. EPI significantly attenuated the TMAO-induced upregulation of ANP and BNP and the downregulation of SP1, SIRT1 and SUMO1 in vivo and in vitro. EPI may suppress TMAO-induced cardiac hypertrophy by activating the Sp1/SIRT1/SUMO1 signaling pathway.

Keywords: (-)-Epicatechin, Trimethylamine N-oxide, Cardiac hypertrophy, SP1/SIRT1/SUMO1

Introduction

Cardiac hypertrophy (CH) is a major consequence of heart overload and pathophysiological injury [1]. Typical features of cardiac hypertrophy include the reactivation of fetal cardiac genes, increased heart mass, and associated changes in the shape of the left ventricle. Initially, cardiac hypertrophy occurs as a compensatory mechanism. However, as hypertrophy progresses, it can lead to heart failure and arrhythmia, which are among the leading causes of sudden death [2–4]. Given the severe outcomes of cardiac hypertrophy, numerous studies have focused on identifying the underlying mechanisms and exploring potential therapeutic strategies for its treatment.

The gut microbiota plays a crucial role in the pathogenesis of cardiovascular disease [5–7]. Previous research has revealed that trimethylamine N-oxide (TMAO), a gut microbe-derived metabolite of dietary choline and other trimethylamine-containing nutrients, is strongly associated with the risk of cardiovascular disease [8]. In recent years, TMAO has been identified as a novel independent risk factor for major adverse cardiovascular events [9]. For instance, TMAO promotes vascular inflammation, induces atherosclerosis, and increases platelet hyperreactivity and thrombosis risk [10–12]. Furthermore, TMAO has been recognized as a significant factor in the development of heart failure [13, 14], directly promoting the progression of cardiac hypertrophy and fibrosis both in vitro and in vivo [15].

Epicatechin (EPI) is considered as a significant candidate responsible for the beneficial effects of flavanol-rich foods [16, 17]. Pharmacological studies have demonstrated that EPI protects against oxidative stress injury, myocardial ischemia–reperfusion injury, and permanent coronary occlusion [18–20]. EPI treatment also improves mitochondrial structure and enhances cardiac and/or skeletal muscle function in patients with heart failure [21]. These findings underscore the cardioprotective role of EPI in heart failure. However, it remains unclear whether EPI has a direct effect on TMAO-induced cardiac hypertrophy, and the mechanism underlying its antihypertrophic role has yet to be elucidated.

Specific protein 1 (SP1), a member of the transcription factors family, is involved in various cellular processes, including growth, differentiation, angiogenesis, apoptosis, cardiac hypertrophy, and tumorigenesis [22, 23]. SIRT1, an NAD-dependent histone deacetylase, belongs to the mammalian sirtuin family and regulates apoptosis, autophagy, inflammation, oxidative stress, and fibrosis [24–26]. Small ubiquitin-related modifier (SUMO), a post-transcriptional regulator, is implicated in transcriptional regulation, cell-cycle progression, and DNA repair [27]. Both SIRT1 and SUMO1 have been shown to participate in EPI-inhibited myofibroblast transformation [28]. Additionally, studies have indicated that SUMOylation of SIRT1 stabilizes protein levels and alleviates myocardial ischemia/reperfusion (MI/R) injury [29, 30].

Therefore, this study aimed to determine the role of EPI in TMAO-induced cardiac hypertrophy. Specifically, we evaluated the anti-hypertrophic effects of EPI and elucidated the underlying mechanisms involved in the regulation of the SP1/SIRT1/SUMO1 signaling pathway in cardiomyocyte hypertrophy both in vitro and in vivo.

Materials and Methods

Participants

The patients enrolled in this study were recruited from the First Affiliated Hospital of Harbin Medical University (Harbin, China) between May 2016 and August 2017. The inclusion criteria for the cardiac hypertrophy group were patients diagnosed with cardiac hypertrophy by a cardiologist. Patients were excluded from the study if they had chronic inflammation, active malignancy, or severe hepatic and renal dysfunction. The control group consisted of seven participants with no clinical history of cardiac hypertrophy. The study included two groups: control participants (n = 7) and patients with cardiac hypertrophy (n = 7). The clinical characteristics of the study population are summarized in Table 1.

Table 1.

Baseline characteristics of subjects stratified by CON and CH (cardiac hypertrophy)

Characteristic Control (n = 7) CH (n = 7) P value
Age (years) 60.1 ± 3.43 59.1 ± 13.1  > 0.05
Male (%) 3(42.9%) 3(42.9%)  > 0.05
Hypertension (%) 0(0.0%) 4(57.1%) 0.0180
DM (%) 1(14.3%) 1(14.3%)  > 0.05
Dyslipidemia (%) 1(14.3%) 3(42.9%)  > 0.05
LVEF (%) 67.3 ± 5.1 63.9 ± 4.6  > 0.05
LAD (mm) 32.7 ± 2.7 42.7 ± 3.6  < 0.0001
LVEDD (mm) 44.9 ± 4.7 47.0 ± 4.0  > 0.05
IVSd (mm) 9.6 ± 1.6 20.4 ± 3.5  < 0.0001
LVPWd (mm) 9.4 ± 1.3 11.5 ± 1.7 0.0200
Hemoglobin (g/L) 137.4 ± 15.3 148.1 ± 8.3  > 0.05
Blood glucose (mmol/L) 6.59 ± 4.26 5.06 ± 3.98  > 0.05
BUN (mmol/L) 5.5 ± 1.4 6.6 ± 1.5  > 0.05
Creatinine (μmol/L) 62.6 ± 15.8 71.0 ± 20.0  > 0.05
BNP (pg/mL) 25.7 ± 10.2 303.9 ± 263.7 0.0164
TC (mmol/L) 4.8 ± 0.9 4.4 ± 1.1  > 0.05
TG (mmol/L) 1.3 ± 1.4 1.7 ± 0.9  > 0.05
β-blockers (%) 0(0.0%) 5(71.4%) 0.0053
ACEI or ARB (%) 1(14.3%) 1(14.3%)  > 0.05
CCB (%) 0(0.0%) 2(28.6%)  > 0.05
hs-TnI (ng/mL) 0.006 ± 0.010 0.019 ± 0.009 0.0299
CKMB (ng/mL) 1.00 ± 0.57 1.56 ± 0.69  > 0.05
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Data are presented as mean ± standard deviation or proportions. DM diabetes mellitus; LVEF left ventricular ejection fraction; LAD left atrial diameter; LVEDD left ventricular end-diastolic dimension; IVSd interventricular septal thickness at end-diastole; LVPWd left ventricular posterior wall thickness in diastole; BUN blood urea nitrogen; BNP brain type natriuretic peptide; TC total cholesterol; TG triglyceride; ACEI angiotensin converting enzyme inhibitors; ARB angiotensin receptor blocker; CCB calcium channel blockers; hs-TnI high-sensitive troponin I; CKMB creatine kinase MB. P > 0.05 versus control

Ethical Approval of Studies and Informed Consent

All human studies were approved by the Institutional Ethics Committee of the First Affiliated Hospital of Harbin Medical University (No. IRB-AF/SC-08/05.0), and written informed consent was obtained from all patients.

Determination of Plasma TMAO Levels

Blood samples were collected using vacutainer tubes containing EDTA after at least 12 h of fasting. The samples were then centrifuged immediately at 765 g (centrifuge: Allegra 64R, Beckman Coulter, USA; rotor: Beckman F1010, USA) for 15 min at 4 ℃. The plasma samples were frozen at –80 ℃ until further analysis. The plasma TMAO concentration was measured using LC–MS/MS with 50 μM TMAO-d9 (TRC, Canada) as an internal standard, as described previously [31, 32].

Chemicals and Animal Care

( −)-EPI (purity ≥ 98%) and all other chemicals used in this study were purchased from Sigma-Aldrich (St. Louis, MO, USA). EPI or TMAO was dissolved in sterile water (pH 7.4). All animal experimental procedures were approved by the Laboratory Animals Ethics Committee of First Affiliated Hospital, Harbin Medical University and complied with the Guide for the Use and Care of Laboratory Animals published by the National Institutes of Health (NIH Publication No. 85-23, revised 1996).

Cell Culture

The H9c2 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin and streptomycin. The cells were incubated at 37 °C and 5% CO2 environment. The culture medium was replaced with fresh medium after 48 h. Before experiments, cells were cultured in non-serum DMEM for 12 h. To investigate whether TMAO could induce cardiac hypertrophy, H9c2 cells were treated with TMAO at concentrations ranging from 5 to 20 µmol/L. Subsequently, the cells were pretreated with 10 µM EPI or phosphate buffer saline (PBS) for 1 h and then stimulated with 10 µM TMAO (Sigma, St. Louis, MO, USA) for 24 h.

Measurement of Cell Surface Area

After treatment, the cells were fixed with 4% paraformaldehyde for 20 min and permeabilized with 0.1% Triton X-100 in PBS. This was followed by blocking with 3% bovine serum albumin (BSA) for 1 h at room temperature. The cardiomyocytes were then incubated with monoclonal antibody against sarcomeric α-actinin (TRITC Phalloidin) at 4 °C overnight. For nuclear staining, the sample was incubated with 4′,6-diamidino-2-phenylindole (DAPI, Sigma, St. Louis, MO, USA) at room temperature for 10 min. The stained cardiomyocytes were visualized and imaged at 200 × magnification using a Carl Zeiss Axio VertA1 microscope (Carl Zeiss Microimaging, Thornwood, NY, USA). Image Pro-Plus Image analysis software was used to analyse the proportions of fluorescent cells. The relative surface area was read with arbitrary units (the number of pixels) to evaluate hypertrophy and the cell surface area in control cells was expressed as 1.

Animal Experiments

8-week-old C57BL/6 mice were supplied by the Medical Experimental Animal Center of Harbin Medical University, China. The mice were housed under standard animal room conditions (temperature 25 ± 1 °C; humidity 50–60%) and allowed food and water ad libitum throughout the experiments. The animals were randomly distributed into the four groups: control (n = 10), TMAO stimulation (TMAO, n = 10), EPI treatment (EPI, n = 10), and TMAO stimulation with EPI pretreatment (TMAO + EPI, n = 10). According to the groups assignments (Fig. 1A), mice received either an intraperitoneal (i.p.) injection of normal saline or TMAO (150 mg/kg/day) for 14 days. The EPI group was given intragastric (i.g.) administration of EPI alone (1 mg/kg/day) for 21 days. The TMAO + EPI group received i.g. administration of EPI for 7 days prior to the start of of TMAO injections, and continued receiving EPI until the end of the TMAO treatment. After drug administration period, mice continued to receive food and water ad libitum. Their hearts were collected under anesthesia, and the heart weight/body weight (HW/BW) ratio was calculated. The heart samples were then frozen in liquid nitrogen and stored at –80 °C.

Fig. 1.

Fig. 1

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Plasma TMAO levels and TMAO-induced H9c2 cellular hypertrophy. A Schematic of the experimental protocol. B TMAO levels in control participants and patients with cardiac hypertrophy. n = 7. **P < 0.01 versus the control group. CH: cardiac hypertrophy. C, D Protein expression levels of ANP and BNP in H9c2 cells induced by different concentration gradients of TMAO, as determined by western blot analysis. Summarized data of cardiac ANP and BNP protein expression levels. β-actin was used as a protein loading control. Data are presented as mean ± SEM (n = 6 per group). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 versus the control group

Western Blot Analysis

Total protein was extracted from the cultured H9c2 cells and mouse hearts. Protein concentrations were determined using a BCA protein assay kit with BSA as the standard. Protein samples were separated using sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF transfer membrane (Millipore, Bedford, MA, USA). Membranes were blocked with 5% non-fat milk for 1 h at room temperature and then incubated overnight at 4 °C with the following primary antibodies: anti-SP1, anti-SIRT1, anti-SUMO1 (1:1000 dilution, Abcam, MA, USA), anti-ANP (1:500 dilution, ABclonal, Wuhan, China), anti-BNP (1:500 dilution, ABclonal, Wuhan, China), and anti-β-actin (1:1000 dilution, ZSGB-Biotech, Beijing, China). The next morning, the membranes were washed three times for 10 min in TBST and incubated with secondary antibody (1:2000–4000 dilution) at room temperature for 1 h. Finally, the western blot bands were visualized using a Universal Hood II chemiluminescence apparatus and quantified using Image Lab v2.0.1, software (LI-COR Biosciences, Lincoln, NE, USA) by measuring the band intensity (area × OD) in each group.

Echocardiographic Assessment

Echocardiographic examination of cardiac structure and function was performed using ultrasonic echocardiographic instruments (Visualsonic Vevo 1100, Canada). The following parameters were measured: left ventricular systolic internal diameter (LVIDs), left ventricular diastolic internal diameter (LVIDd), left ventricular posterior wall thickness in systole (LVPWs), left ventricular posterior wall thickness in diastole (LVPWd), left ventricular ejection fraction (LVEF), left ventricular fractional shortening (LVFS), interventricular septal thickness at end systole (IVSs), and interventricular septal thickness at end diastole (IVSd). All measurements were averaged over 6 consecutive cardiac cycles.

Histology

Heart tissue specimens were fixed with 4% paraformaldehyde and then embedded in paraffin. Cross-sectional slices along the minor axis were obtained using a microtome and stained with Mayer’s hematoxylin and eosin (H&E). The cell area was determined by measuring at least 100 cells per slide. To evaluate cardiac fibrosis, a Masson’s trichrome staining kit (Solarbio, Beijing, China) was performed. Both H&E and Masson-stained sections were imaged at 200 × magnification using a Carl Zeiss microscope (Carl Zeiss Microimaging, Thornwood, NY, USA). The cross-sectional areas of the cardiomyocytes and the percentage of collagen deposition were analyzed using Image-Pro Plus software (Media Cybernetics, Rockville, MD, USA).

Statistical Analysis

All figures and statistical analyses were performed using Prism (Graphpad) and SPSS 13.0 software. The data were presented as the means ± standard error of mean (SEM). Two independent Student’s t-tests and one-way ANOVA were used for difference comparisons. All statistical significance was two-tailed, with the significance level was set at 0.05. A value of P < 0.05 was considered to be statistically significant.

Results

Patients with Cardiac Hypertrophy Exhibit Elevated Plasma TMAO Levels, and TMAO Induces Cardiac Hypertrophy In Vitro

Baseline characteristics of the 7 control subjects and 7 patients with cardiac hypertrophy are displayed in Table 1, categorized by control (CON) and cardiac hypertrophy (CH) groups. There was no statistically significant difference in the age and sex distribution (P > 0.05). However, high-sensitive troponin I (hs-TnI) and brain type natriuretic peptide (BNP) levels were significantly higher in CH patients compared to controls (P < 0.05). Patients with cardiac hypertrophy exhibited higher levels of LAD (P < 0.0001), IVS (P < 0.0001), and LVPWd (P < 0.05) compared to the control group. No significant differences were found in hemoglobin, blood glucose, blood urea nitrogen (BUN), creatinine, total cholesterol (TC), triglyceride (TG), creatine kinase-MB (CK-MB) and left ventricular ejection fraction (LVEF%) between the groups (P > 0.05) (Table 1).

Notably, plasma level of TMAO was significantly higher in cardiac hypertrophy group (2.66 ± 1.59 μmol/L) compared to the control group (0.62 ± 0.30 μmol/L) (P < 0.01; Fig. 1B). These results an association between elevated TMAO levels and cardiac hypertrophy. Previous studies have also indicated that TMAO can induce cardiovascular diseases [10–12].

In vitro, H9c2 cells were treated with TMAO (5–20 µmol/L) as described in the Methods section. We observed that content of proteins such as atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) markedly increased (P < 0.05, P < 0.01, P < 0.001, P < 0.0001 vs. control group) in a concentration-dependent manner with increasing in TMAO levels (Fig. 1C–D).

EPI Inhibits TMAO-Induced Cardiomyocyte Hypertrophy In Vitro

To investigate the effects of EPI on cardiac hypertrophy, we examined the impact of 10 µM TMAO on markers of cardiac hypertrophy in H9c2 cells. Cardiomyocyte immunofluorescence revealed that TMAO significantly expanded the area of cardiomyocytes (P < 0.001), whereas EPI inhibited the effects (Fig. 2A–B). Furthermore, TMAO enhanced the expression of ANP and BNP (P < 0.05, P < 0.01 vs. control group) (Fig. 2C–D). In contrast, treatment with 10 µM EPI significantly attenuated the TMAO-induced expression of ANP and BNP (P < 0.05 vs. TMAO group). These results indicated that TMAO-induced hypertrophy in H9c2 cells can be mitigated by EPI in vitro.

Fig. 2.

Fig. 2

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Effect of EPI on cardiac hypertrophy induced by TMAO in vitro. A, B TMAO induced increase of the H9c2 cell area. Representative photographs of H9c2 cells and summarized data of cell area. Cells were identified (× 200) with sarcomeric ɑ-actinin antibody (red signal), and nuclei were stained with bisbenzamide (blue). The relative surface area was read with arbitrary units (the number of pixels) to evaluate hypertrophy and the cell surface area in control cells was expressed as 1. n = 5 per group. Scale bar = 50 µm. C, D Protein expression levels of ANP and BNP in H9c2 cells, as determined by western blot analysis. Summarized data of cardiac ANP and BNP protein expression. β-actin was used as the protein loading control. Data are presented as the mean ± SEM (n = 4 per group). *P < 0.05, **P < 0.01, ***P < 0.001 versus the control group. #P < 0.05, ####P < 0.0001 versus the TMAO group

EPI Plays an Antihypertrophic Role in TMAO-Induced Cardiac Hypertrophy In Vivo

To further investigate the antihypertrophic role of EPI in TMAO-induced cardiac hypertrophy, we examined the beneficial effects of EPI in vivo. Echocardiography was performed to monitor cardiac function in the mice (Fig. 3), measuring parameters such as LVEF, LVFS, LVIDs, LVIDd, LVPWs, LVPWd, IVSs, and IVSd. TMAO decreased LVEF and LVFS while increasing LVIDd and LVIDs compared to the control group, (P < 0.05, P < 0.01 vs. control group). These adverse effects were ameliorated by EPI treatment (P < 0.05, P < 0.01 vs. TMAO group), suggesting that intragastrically administered EPI inhibits the TMAO-induced decline in cardiac function in mice.

Fig. 3.

Fig. 3

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Effect of EPI on cardiac function in mice. A Representative cardiac echocardiography images of the indicated groups. B–I The following parameters were analyzed: LVEF left ventricular ejection fraction, LVFS left ventricular fractional shortening, LVIDs left ventricular systolic internal diameter, LVIDd left ventricular diastolic internal diameter, LVPWs Left ventricular posterior wall thickness in systole, LVPWd left ventricular posterior wall thickness in diastole, IVSs interventricular septal thickness at endsystole, IVSd interventricular septal thickness at end-diastole. Statistical analysis of LVEF, LVFS, LVIDs, LVIDd, LVPWs, LVPWd, IVSs, and IVSd. Data are shown as mean ± SEM (n = 6–7). *P < 0.05, **P < 0.01 versus the control group; #P < 0.05, ##P < 0.01 versus the TMAO group; ns, not significant (P > 0.05)

H&E staining of gross heart tissue (Fig. 4A and C) confirmed the protective effect of EPI against cardiac hypertrophy. Under a light microscope, the control group cardiomyocytes displayed compact-arranged fibers with no intercellular space. In contrast, the TMAO group cardiomyocytes exhibited hypertrophy with severely ruptured cardiac muscle fibers. TMAO-induced cardiomyocyte hypertrophy was significantly ameliorated by EPI treatment. Masson's trichrome staining showed increased collagen content in the heart tissues of the TMAO group compared to the control group, which was then significantly reduced by EPI treatment (Fig. 4B and D).

Fig. 4.

Fig. 4

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In vivo inhibition of TMAO-induced pathological cardiac remodeling by EPI. A, B Representative graph of H&E and Masson staining resuls. The magnification is × 200, scale bar = 100 µm. C Quantification of myocyte cross-sectional area. D Summarized data of cardiac fibrosis ratio. Data are shown as mean ± SEM (n = 6). **P < 0.01, ****P < 0.0001 versus the control group; ###P < 0.001, ####P < 0.0001 versus the TMAO group

As shown in Fig. 5A, the heart weight (HW) to body weight (BW) ratio increased in mice treated with TMAO (P < 0.0001 vs. control group), which was also successfully decreased by EPI pretreatment (P < 0.01 vs. TMAO group). EPI also significantly attenuated the TMAO-induced increase in the protein expression of ANP and BNP (P < 0.01 vs. TMAO group) (Fig. 5B–C). In summary, these results demonstrate that EPI plays an anti-hypertrophic role in TMAO-induced cardiac hypertrophy in vivo.

Fig. 5.

Fig. 5

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Amelioration of TMAO-induced cardiac hypertrophy in mice by EPI. A Statistical analysis of HW/BW index, HW heart weight, BW body weight. N = 8 per group. B, C Protein expression levels of ANP and BNP in mice heart as determined by western blot analysis. Summarized data of cardiac ANP and BNP protein expression. β-actin was used as a protein loading control. Data are presented as the mean ± SEM (n = 4–5 per group). **P < 0.01, ***P < 0.001, ****P < 0.0001 versus the control group. ##P < 0.01 versus the TMAO group

EPI Protects Cardiac Hypertrophy Induced by TMAO via the SP1/SIRT1/SUMO1 Signaling Pathway

To further investigate the mechanism by which EPI exerts its anti-cardiac hypertrophy effects, we studied the impact of EPI and TMAO on the SP1/SIRT1/SUMO1 signaling pathway. As shown in Fig. 6A–C, TMAO treatment led to a significant decrease in SP1, SIRT1, and SUMO1 protein expression in H9c2 cells (P < 0.05, P < 0.01 vs. control group). Conversely, EPI treatment counteracted this effect by significantly increasing the expression levels of SP1, SIRT1, and SUMO1 that were decreased by TMAO (P < 0.05, P < 0.01, vs. TMAO group). In addition, EPI alone increased the protein levels of SUMO1 in H9c2 cells (P < 0.001 vs. control group). Similarly, in vivo experiments showed that TMAO treatment caused a significant reduction in the protein expression of SP1, SIRT1, and SUMO1 in mouse heart tissue (P < 0.01, P < 0.001 vs. control group) (Fig. 6D–F). EPI treatment inhibited the TMAO-induced decrease in SP1, SIRT1, and SUMO1 expression in the mouse hearts (P < 0.05 vs. the TMAO group). Interestingly, EPI alone increased the protein levels of SP1 and SIRT1 in mouse heart tissue (P < 0.01, P < 0.0001 vs. control group). These results suggest that EPI can attenuate TMAO-induced cardiac hypertrophy through the activation of the SP1/SIRT1/SUMO1 signaling pathway.

Fig. 6.

Fig. 6

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Protein expression levels of SP1, SIRT1, and SUMO1 determined by western blot analysis. A–C Protein expression levels of SP1, SIRT1, and SUMO1 in H9c2 cells as determined by western blot analysis. n = 3–4 per group. D–F Protein expression levels of SP1, SIRT1, and SUMO1 in mice heart tissues as determined by western blot analysis. β-actin was used as the protein loading control. Data are presented as the mean ± SEM (n = 5–7 per group). * P < 0.05, **P < 0.01,***P < 0.001,****P < 0.0001  versus the control group; # P < 0.05, ##P < 0.01 versus the TMAO group; ns, not significant (P > 0.05)

Discussion

To the best of our knowledge, this is the first study to investigate the role of the SP1/SIRT1/SUMO1 signaling pathway in the cardioprotective effects of EPI in TMAO-induced cardiac hypertrophy. The major findings are as follows: (1) elevated plasma TMAO levels in cardiac hypertrophy patients; (2) EPI inhibits TMAO-induced hypertrophy in vivo and in vitro; and (3) EPI protects against cardiac hypertrophy via the SP1/SIRT1/SUMO1 signaling pathway.

Gut microbes play an important role in regulating human health and disease [33]. TMAO, a gut microbiota metabolite, is a key mediator in the development of cardiovascular diseases [34]. There is a well-established association between elevated blood TMAO levels and increased cardiovascular risk [10–12]. Our previous research found that TMAO is an independent predictor of coronary heart disease; additionally, TMAO levels are strongly associated with diabetes in Chinese coronary heart disease patients [32]. In this study, we found that the plasma level of TMAO was significantly higher in patients with cardiac hypertrophy (2.66 ± 1.59 μmol/L) compared to control participants (0.62 ± 0.30 μmol/L). Furthermore, TMAO-induced cell hypertrophy was attenuated by EPI treatment. Cardiac structure and function, which were compromised in the TMAO group, were significantly improved by EPI treatment. A study found that after 6 weeks of TAC treatment, TMAO levels were significantly elevated in SD rats [15]. Similarly, in cultured cardiomyocytes, TMAO stimulation induced cardiac hypertrophy, as evidenced by an increase in cardiomyocyte size and elevated expression of hypertrophic markers. Notably, TMAO also promotes the transformation of fibroblasts into myofibroblasts, along with their migration and collagen secretion, leading to cardiac dysfunction in mice through the activation of the NLRP3 inflammasome [35]. Recent evidence has shown that reducing circulating TMAO can prevent the progression of cardiac and renal dysfunction in a rat model of chronic cardiorenal syndrome [36]. Thus, these results indicated that TMAO can promote the progression of cardiac hypertrophy and fibrosis, leading to impaired cardiac function; however, the specific mechanisms underlying these effects require further investigation.

EPI is commonly found in numerous foods and drinks and has been observed to suppress cardiac hypertrophy and fibrosis induced by myocardial ischemia in vivo [37]. Given these cardioprotective properties, it is essential to explore the effects of EPI on TMAO-induced cardiac hypertrophy. EPI’s antioxidant effects have been demonstrated to inhibit cancer, diabetes mellitus, and insulin resistance [38–40]. Additionally, EPI has been shown to decrease the likelihood of developing cardiovascular disease by increasing flow-mediated dilation of the endothelium and decreasing platelet aggregation [41, 42]. Some studies have demonstrated that EPI alleviates cardiomyocyte hypertrophy in hypertensive rats [43, 44], which is consistent with the results of the present study. The protective effect of EPI against cardiomyocyte hypertrophy is thought to occur through the reduction in oxidative stress and improvement in mitochondrial structure and function [18, 19]. Previous studies have shown that in rat models of angiotensin II (Ang II) and abdominal aortic constriction, EPI can inhibit the activation of NF-κB, reduce collagen synthesis and fibronectin expression, thereby alleviating pressure overload-induced cardiac hypertrophy [45]. Moreover, research indicates that in vitro, EPI can activate AMP-activated protein kinase (AMPK) expression in phenylephrine-induced hypertrophy of H9c2 cardiomyocytes, reduce the mRNA expression of ANP and BNP, and decrease the surface area of hypertrophic H9c2 cells [46]. Similarly, our study found that EPI could significantly reduce the TMAO-induced expression of hypertrophic markers ANP and BNP.

EPI has been shown to prevent cardiac fibrosis by increasing protein expression of SP1 and regulating SUMO1-dependent modulation of SIRT1 [28]. SP1 a member of the the SP/KLF family, plays a critica in gene transcription and expression [47, 48]. Upregulated nuclear Sp1 levels are associated with proteasome-dependent degradation mechanism during the G1 phase of the cell cycle, leading to the proliferation of Sp1-responsive genes such as ODC and cyclin D1 [49]. SP1 can also enhance the expression of HDAC4 (histone deacetylase 4), a SUMO E3 ligase that stabilizes SIRT1 and promotes SUMO modification of the target proteins [29, 50]. SIRT1 is known for its protective effects against cardiovascular diseases [51]. Previous studies have shown that SP1 binds to the SIRT1 gene promoter, indicating that SIRT1 may play an essential role in the antihypertrophic effects of EPI via SP1 [23, 52]. Meanwhile, studies have found that the upregulation of Sp1 can block Ang II-induced cardiac hypertrophy and increase SIRT1 protein levels in vitro [23]. Additionally, SIRT1 in its SUMOylated form has been found to play a protective role during MI/R injury [30]. SIRT1 mitigates ischemia-induced endothelial dysfunction by regulating the expression of endothelial nitric oxide synthase (eNOS) and manganese superoxide dismutase (MnSOD), serving as a cardioprotective molecule [53]. Another study found that suppressing SIRT1 in patients with coronary artery disease (CAD) led to an increased affinity of monocytes for endothelial cells [54]. However, there is not yet sufficient data from studies to confirm whether the inhibition of SIRT1 in monocytes is elevated in CAD [55]. Our study demonstrated that the expression of SP1, SIRT1, and SUMO1 was reduced during TMAO-induced cardiac hypertrophy and that their protein levels were significantly upregulated by EPI treatment. The results revealed that EPI effectively alleviated TMAO-induced cardiac hypertrophy and increased the levels of SP1, SIRT1, SUMO1, which are key targets for the cardioprotective effects of EPI (Fig. 7).

Fig. 7.

Fig. 7

Open in a new tab

Proposed mechanism underlying TMAO-induced pathological cardiac hypertrophy

Our study has several limitations. Firstly, the relatively small group size may affect the generalizability of our findings. Additionally, the short duration of TMAO exposure in mice may not fully capture the long-term effects of TMAO-induced cardiac hypertrophy. Therefore, follow-up studies with larger sample sizes and extended durations of TMAO exposure are needed to validate our results. Future studies should also aim to confirm the changes in the RNA expression of the related target proteins and further elucidate the role of SP1/SIRT1/SUMO1 pathway in cardiac hypertrophy. Investigating the transcriptional regulation of these proteins will provide a more comprehensive understanding of how EPI modulates this signaling pathway. Furthermore, it is essential to explore whether different timings and modes of TMAO administration impact its effect on cardiac hypertrophy.

Conclusions

In conclusion, our findings demonstrate that EPI can prevent TMAO-induced cardiac hypertrophy by targeting the SP1/SIRT1/SUMO1 signaling pathway both in vivo and in vitro. These results suggest that EPI may represent a novel approach for the prevention and treatment of TMAO-induced cardiac hypertrophy in the future.

Acknowledgements

Schematic diagram was created using BioRender.

Author Contributions

SH, ZD, ZW, LL, and ZL contributed to the study conception and design. Material preparation, data collection, and analysis were performed by SH, JL, JL; YL, DL, YJ, ZW, YW, HZ, XZ, YL, HZ and LL. The first draft of the manuscript was written by SH. ZD, ZW, LL, and ZL made contributions to supervision and writing-review & editing. All authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

Funding

This study was supported by National Natural Science Foundation of China (Grant No. 82000297, 81870169, 81400250); Postdoctoral Research Fund of China (Grant No. 2018M631963); Support Project for Outstanding Young Talents of the First Affiliated Hospital of Harbin Medical University (Grant No. 2024YQ15) and Doctor Funds of the First Affiliated Hospital of Harbin Medical University (Grant No. 2019B09).

Data Availability

No datasets were generated or analysed during the current study.

Code Availability

Not applicable.

Declarations

Competing interests

The authors declare no competing interests.

Ethical Approval

The study was approved by the Institutional Ethics Committees of the First Affiliated Hospital of Harbin Medical University (No. IRB-AF/SC-08/05.0). The present study was conducted according to the “Guide for the Care and Use of Laboratory Animals” (NIH Publication No.85-23, revised in 1996). The animal experiments were approved by the Institutional Animal Care and Use Committee of First Affiliated Hospital, Harbin Medical University.

Consent to Participate

The study protocols and the procedures for handling human samples were approved by the Ethics Committee of the First Affiliated Hospital of Harbin Medical University (Harbin, China). All patients provided informed consent, and all the methods were carried out in accordance with the approved guidelines.

Consent for Publication

Not applicable.

Footnotes

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Contributor Information

Zhaojun Wang, Email: hyd2013@qq.com.

Lin Lv, Email: dicklin1992@163.com.

Zhaoguang Liang, Email: zhaoguangliangsupper@126.com.

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