3,3-Dimethyl-1-butanol attenuates cardiac remodeling in pressure-overload-induced heart failure mice

Journal Pre-proof 3,3-dimethyl-1-butanol attenuates cardiac remodeling in pressure overload-induced heart failure mice

Guangji Wang, Bin Kong, Wei Shuai, Hui Fu, Xiaobo Jiang, He Huang PII:

S0955-2863(19)30592-3

DOI:

https://doi.org/10.1016/j.jnutbio.2020.108341

Reference:

JNB 108341

To appear in:

The Journal of Nutritional Biochemistry

Received date:

9 June 2019

Revised date:

23 December 2019

Accepted date:

30 December 2019

Please cite this article as: G. Wang, B. Kong, W. Shuai, et al., 3,3-dimethyl-1-butanol attenuates cardiac remodeling in pressure overload-induced heart failure mice, The Journal of Nutritional Biochemistry(2020), https://doi.org/10.1016/j.jnutbio.2020.108341

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© 2020 Published by Elsevier.

Journal Pre-proof

3,3-dimethyl-1-butanol attenuates cardiac remodeling in pressure overload-induced heart failure mice Guangji Wanga,b,c,1, Bin Konga,b,c,1, Wei Shuaia,b,c, Hui Fua,b,c, Xiaobo Jianga,b,c, He Huanga,b,c,*

Jiefang Road, Wuhan 430060, Hubei Province, PR China .

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a Department of Cardiology, Renmin Hospital of Wuhan University, Wuhan, Hubei, China b Cardiovascular Research Institute, Wuhan University, Wuhan, Hubei, China c Hubei Key Laboratory of Cardiology, Wuhan, Hubei, China *Correspondence to: Department of Cardiology, Renmin Hospital of Wuhan University, Cardiovascular Research Institute of Wuhan University, Hubei Key Laboratory of Cardiology, 238

Abstract

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E-mail address: [email protected] (H. Huang). 1 Guangji Wang and Bin Kong contributed equally to this work

Trimethylamine N-oxide (TMAO) is closely related to cardiovascular diseases particularly

heart

failure

(HF).

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(CVD),

Recent

studies

shows

that

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3,3-dimethyl-1-butanol (DMB) can reduce plasma TMAO levels. However, the role of DMB in overload-induced HF is not well-understood. In this research study, we explored the effects and the underlying mechanisms of DMB in overload-induced HF.

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Aortic banding (AB) surgery was performed in C57BL6/J mice to induce HF and a subset group of mice underwent a sham operation. After surgery, the mice were fed

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with a normal diet and given water supplemented with or without 1% DMB for 6

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weeks. Cardiac function, plasma TMAO level, cardiac hypertrophy and fibrosis, expression of inflammatory, electrophysiological studies and signaling pathway were analyzed at the 6th week after AB surgery. DMB reduced TMAO levels in overload-induced heart failure mice. Adverse cardiac structural remodeling, such as cardiac hypertrophy, fibrosis and inflammation were elevated in overload-induced HF mice. Susceptibility to ventricular arrhythmia also significantly increased in overload-induced HF mice. However, these changes were prevented by DMB treatment. DMB attenuated all of these changes by reducing plasma TMAO levels, hence negatively inhibiting the p65 NF-κB signaling pathway and TGF-β1/Smad3 signaling pathway. DMB plays an important role in attenuating the development of cardiac structural remodeling and electrical remodeling in overload-induced HF mice. This may be attributed to the p65 NF-κB signaling pathway and TGF-β1/Smad3 signaling pathway inhibition. Keywords: Trimethylamine N-oxide; 3,3-dimethyl-1-butanol; structural remodeling;

Journal Pre-proof electrical remodeling 1. Introduction There is high global incidences and mortality rates of cardiovascular diseases (CVD) [1]. Recent studies have revealed that the gut microbiota has a metabolic potential with significant impact on the incidence and development of CVD [1,2]. Gut microbe metabolism of some dietary nutrients (L-carnitine, choline and betaine) produces trimethylamine (TMA) [3-6], which is easily absorbed and oxidized to trimethylamine N-oxide (TMAO) by Flavin monooxygenase 3 (FMO 3) in the liver [7]. Clinical studies have proved that elevated plasma TMAO levels are associated

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with cardiac adverse effects, such as acute coronary syndrome, atrial fibrillation,

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stroke, heart failure (HF), and even death [8-12]. Furthermore, animal studies confirmed that TMAO promotes cardiac hypertrophy [13], atherosclerosis [14], atrial

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fibrillation [15], heart failure and death [16]. Therefore, regulating the level of TMAO

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in circulating blood may be a new treatment of CVD.

Previous studies have demonstrated that antibiotics and berberine can reduce the

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synthesis of TMAO [5,13,17,18]. However, attention needs to be paid on the potential adverse effects of antibiotics and berberine, such as antibiotics may lead dysbacteriosis, bacterial drug-resistance and berberine may induce liver tissue damage

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[18]. 3-dimethyl-1-butanol (DMB), a structural analogue of choline, was detected in

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some balsamic vinegars, red wines, and in some cold-pressed extra virgin olive oils and grape seed oils [19]. Notably, DMB can significantly reduce the level of TMAO

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in mice fed a high choline or carnitine diet, inhibit the formation of atherosclerosis, and have no toxic and side effects [19]. Similarly, DMB can reduce TMAO levels in obese [20], kidney disease (CKD) [21], reduced uterine perfusion pressure (RUPP) [22] and aged individuals [23]. To date, there are no studies that have explored the function of DMB in HF. Therefore, herein, we investigated the effects of DMB in HF condition after aortic banding (AB). 2. Methods 2.1. Experimental animals C57BL6/J male mice (8-10 weeks old) were obtained from Laboratory Animal Center of Renmin Hospital of Wuhan University. The experiment protocol followed the guidelines of the National Care and Use of Laboratory Animals and was authorized by the Animal Ethics Committee of Renmin Hospital, Institute of Health, Wuhan University. All mice were controlled on light cycles (12 h light and 12 h dark),

Journal Pre-proof temperature (20-25 ℃) , humidity (50±5%). 2.2. Study protocol The mice underwent AB surgery to induce HF as previously described [24]. After surgery, the mice were fed with a normal diet [25] and given water containing or without 1% DMB (Sigma, St.Louis, Mo., USA) for 6 weeks. It has been shown that this dose of DMB can effectively inhibit TMA formation and decrease plasma TMAO level in other animal disease models [19-23]. The experimental animals were divided into three groups: sham group, AB group, and AB+DMB group. Echocardiography and hemodynamic were used to measure the cardiac function at the 6 th week after the

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AB operation. Blood samples were collected by removing eyeball after isoflurane

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anesthesia. After blood sampling, the mice were euthanized. The heart, lung and tibia were rapidly isolated. Heart weight (HW), body weight (BW), lung weight (LW),

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tibiae length (TL) were measured in the three groups. The hearts were divided into two parts. One part of the left ventricle (LV) was fixed in 4% paraformaldehyde for

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histomorphology examination, the other part was stored in – 80 °C refrigerator for

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molecular biology examination. Electrophysiological studies were done using isolated perfused hearts at the 6th week after the AB operation. 2.3. Echocardiography and hemodynamic assessment

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The cardiac function of mice was measured as previously described [26,27].

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Briefly, mice were anaesthetized with an isoflurane (1.5% isoflurane in 98% O2) inhalation [26]. Mylab30CV (ESAOTE) ultrasound system was used to measure the

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interventricular septum diameter in diastole (IVSd), interventricular septum diameter in systole (IVSs), left ventricular posterior wall diameter in diastole (LVPWd), left ventricular posterior wall diameter in systole (LVPWs), left ventricular end-diastolic diameter (LVEDD), left ventricular end-systolic diameter (LVESD), left ventricular fractional shortening (LVFS) and left ventricular ejection fraction (LVEF). After echocardiography examination and mice were awake for 1 hour. Thereafter, the mice were anesthetized by continuous isoflurane inhalation and hemodynamic assessment was done. [27]. A microtip catheter transducer was then pushed into the LV through the right carotid artery. Subsequently, the ARIA pressure-volume conductance system was used to record left ventricular end-systolic pressure (LVESP), left ventricular maximum and minimum rates of pressure rise (+dP/dT and -dP/dT ). The data were calculated with LabChart 7 software. 2.4. Histological analysis

Journal Pre-proof The heart was embedded in paraffin and cut into 5-um thick sections. The cross-sectional area of LV cardiomyocytes were measured via Haematoxylin-eosin (H&E) staining. The degree of interstitial and perivascular fibrosis was evaluated using Picrosirius red (PSR) staining and Masson trichrome staining [16]. The degree of myocardial fibrosis was evaluated based on the ratio of fibrotic tissue area to the total myocardial area. The data were measured with Image Pro-Plus software. 2.5. Quantification of plasma levels of TMAO The plasma levels of TMAO were measured by liquid chromatography tandem mass spectrometry [16]. Briefly, the impurities were removed by protein precipitation

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method, and the same amount of internal standard (TMAO-D9) was added to the

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sample. The atomic or molecular ions in the sample to be measured were transformed into charged particles by electrospray ion source (ESI). Separated according to the

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mass charge ratio of the ions, spectral peaks were plotted, and the intensities of

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different ion peaks were measured. The exact concentration of TMAO was calculated from the ratio of peak area of TMAO to TMAO-D9 in the samples measured.

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2.6. Real-time polymerase chain reaction (RT-PCR) analysis TRIzol reagent was used to extract the total RNA from the tissues. The mRvioNA levels (ANP, BNP, β-MHC, collagen Iα, collagen III and CTGF) of the

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hearts from the different groups were measured using RT-PCR. The mRNA levels

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were calculated using the relative standard curve method and the calculated mRNA levels were normalized by GAPDH mRNA level. Data were analyzed using the

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previous method [13].

2.7. Western blot analysis

Firstly, proteins were extracted from the frozen LV tissues. Bicinchoninic Acid (BC-A) protein concentration assay kit was used to determine the sample protein concentration. Sodium dodecylsulphate (SDS)-polyacrylamide gel electrophoresis (PAGE) was performed to separate samples and were then transferred to polyvinylidene difluoride membranes. The membranes were blocked with 5% non-fat dry milk for 60 min and then incubated overnight at 4 °C with primary antibodies, i.e. TNF-α, IL-1β, IL-6, phospho-Smad3, Smad3, TGF-β1, phospho-P65, P65 and β-actin. All above used antibodies were obtained from Abcam (Cambridge, MA, USA) and Cell Signaling Technology (Beverly, MA, USA). Thereafter, TBST was performed by washing the membranes three times, and then incubating with secondary antibodies for 30 minutes at room temperature. The Alpha Ease FC software was used to

Journal Pre-proof measure the optical density value of the Protein strip [27]. 2.8. Surface ECG recording and analysis This method was used as previously described [27]. Animals were lightly anesthetized using pentobarbital sodium. Subsequently, surface electrode was placed subcutaneously to record body surface using lead electrocardiogram (lead II) for 15-30 min. The data was analyzed with LabChart 7 software. 2.9. Monophasic action potential recording and ventricular tachycardia inducibility Langendorff-perfused hearts and HEPES-buffered Tyrode’s solution were prepared as previously described [26,27]. A pair of electrodes was used to stimulate

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the right ventricular basal surface. Before the stimulation, all isolated hearts were

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perfused for 20 min to recover a regular spontaneous rhythm. Setting the pacing cycle length (PCL) at 150 ms, recorded the monophasic action potential (MAP) and

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calculated the 90% action potential duration (APD90). APD90 was defined as 90%

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repolarization time of MAP at 150 ms PCL. The electric alternans threshold was measured by graded incremental stimulation. When the difference of APD90 was

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more than 5% or the amplitude difference of APD90 was ≥ 10% for at least 10 times, it was considered that an electric alternans had occurred. The longest PLC induced electric alternans was regarded as the electric alternans threshold. A 2s burst (pulse

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duration for 2 ms) was used to induce ventricular arrhythmia (VA). The data was

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analyzed with LabChart 7 software. 2.10. Statistical analysis

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SPSS 20.0 and GraphPad Prism 7 software were used to analyze data. All data measurement were summarized by mean ± SD and then evaluated by one-way ANOVA, followed by Tukey’s post hoc test or Tamhane’s T2 post hoc test. Categorical data were presented as rate (%) and applied for Chi-square test or Fisher exact test. The difference was regarded as statistical significance with P < 0.05. 3. Results 3.1. DMB improved cardiac function in HF mice Six weeks after AB operation, the mice in the AB group had increased IVSd, IVSs, LVPWd, LVPWs, LVEDD, LVESD, LVESP and decreased LVFS, LVEF, +dP/dT and -dP/dT than Sham group. DMB treatment significantly improved LVEF (50.44±2.46%

vs

45.89±4.73%,

P

<

0.05),

+dP/dt

(7944.44±360.94

vs

6911.11±527.84 mmHg/s, P < 0.05) and −dP/dt (6611.11±236.88 vs 6200.00±212.13 mmHg/s, P < 0.05) compared with the AB group (Table 1). These data indicate that if

Journal Pre-proof mice underwent a AB surgery and developed HF, DMB can improve cardiac functions in the mice. 3.2. DMB reduced plasma TMAO levels in HF mice The plasma TMAO levels was markedly increased in the AB group compared with the Sham group (25.5±3.6 vs 13.5±1.1 ng/ml, P < 0.05), but upon DMB treatment, plasma TMAO levels decreased in the AB group (18.2±1.2 vs 25.5±3.6 ng/ml, P < 0.05) (Fig. 1). 3.3. DMB attenuated cardiac hypertrophy and fibrosis in HF mice To further evaluate the effect of DMB on the cardiac structural remodeling,

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Haematoxylin-eosin (H&E) staining, Picrosirius red (PSR) staining and Masson

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Trichrome staining were performed to assess cardiac hypertrophy and fibrosis at the 6th week after AB surgery. H&E staining demonstrated that the cross-sectional area of

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LV cardiomyocytes was larger in AB group than Sham group (315.9±10.4 vs

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202.9±14.7 um2 l, P < 0.05), DMB treatment significantly reduced the increase of the cross-sectional area of LV cardiomyocytes compared to AB group (284.3±6.5 vs

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315.9±10.4 um2, P < 0.05) (Fig. 2A-B). Mice in the AB group gained more significant increase in the ratios of HW/BW (9.2±0.2 vs 5.1±0.1 mg/g, P < 0.05), HW/TL (14.9±1.6 vs 8.3±1.0 mg/mm, P < 0.05) and LW/TL (14.4±2.2 vs 7.6±1.3 mg/mm, P

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< 0.05) than Sham group, DMB treatment significantly inhibited the increase in the

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ratios of HW/BW (8.5±0.5 vs 9.2±0.2 mg/g, P < 0.05), HW/TL (12.2±1.4 vs 14.9±1.6 mg/mm, P < 0.05) and LW/TL (10.5±0.6 vs 14.4±2.2 mg/mm, P < 0.05) compared

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with the AB group (Fig. 2C-E). Furthermore, compared to Sham group, the mRNA levels of cardiac hypertrophy markers (ANP, BNP and β-MHC) were higher in the AB group (P < 0.05), and DMB treatment significantly inhibited the increase in ANP, BNP and β-MHC mRNA expression compared with the AB group (P < 0.05) (Fig. 2F-H). All these results suggest that DMB treatment prevented AB-induced increase in cardiac hypertrophy (Fig. 2). PSR and Masson Trichrome staining demonstrated that area of LV interstitial fibrosis was more serious in AB group than that in Sham group (8.8±1.1% vs 2.5±0.7%, P < 0.05), DMB treatment significantly reduced the area of LV interstitial fibrosis compared with the AB group (6.2±0.5% vs 8.8±1.1%, P < 0.05) (Fig. 3A-B). The data of Masson Trichrome staining is presented in Fig. 3B. Using the same method of tissue staining and analysis, we observed similar results in the perivascular sections of heart tissues collected 6 weeks after AB surgery, DMB treatment significantly reduced the area of LV perivascular fibrosis compared with the

Journal Pre-proof AB group (5.8±0.8% vs 11.4±1.5%, P < 0.05) (Fig. 3C-D). Furthermore, the mRNA levels of myocardial fibrosis marker (collagen Iα, collagen III and CTGF) were increased in AB group than Sham group (P < 0.05) (Fig. 3E-G), and DMB treatment significantly inhibited the mRNA expression of collagen Iα, collagen III and CTGF compared with the AB group (P < 0.05). All these findings proved that AB-induced increase in cardiac fibrosis was attenuated by treatment with DMB (Fig. 3). 3.4. DMB attenuated expression of inflammatory cytokines after AB surgery Previous researches have confirmed that elevated TMAO promotes the expression of inflammatory cytokines [15,20] and DMB can reverse this trend [20].

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However, it is not clear whether DMB can attenuate the expression of inflammatory

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cytokines in mice after AB surgery. As indicated in Fig. 4A-D, the protein levels of TNF-α, IL-6 and IL-1β clearly increased in the AB group than in Sham group (P <

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0.05). However, DMB treatment attenuated these expressions in mice heart after AB

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surgery (P < 0.05).

3.5. DMB attenuated electrical remodeling after AB surgery

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Surface ECG (lead II) data revealed that QRS intervals prolonged in AB+DMB group compared to Sham group (14.3±2.5 vs 11.1±1.5 ms, P < 0.05) and QTc intervals were prolonged in AB group compared to Sham group (55.8±1.4 vs 48.4±2.9

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ms, P < 0.05). The QRS intervals and QTc intervals had no significant changes

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between AB group and AB+DMB group (Fig. 5A-B). There were no statistical (Fig. 5A-B).

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significant differences detected in PR intervals and RR intervals among the 3 groups The APD90 (83.6±1.4 vs 61.0±1.2 ms, P < 0.05) and threshold of electric alternans (88±2.2 vs 69.0±2.3 ms, P < 0.05) was markedly prolonged in AB group than in Sham group, but DMB treatment significantly suppressed the increase of APD90 (67.6±0.7 vs 83.6±1.4 ms, P < 0.05) and threshold of electric alternans (79±2.6 vs 88±2.2 ms, P < 0.05) compared with the AB group (Fig. 5C-F). As shown in Fig. 5G-H, the VA inducibility rate from burst stimuli was significantly higher in AB group compared to Sham group (63.6% vs 10%, P < 0.05). However, the VA inducibility rate was lower in AB+DMB group than AB group (16.7% vs 63.6%, P < 0.05). 3.6. DMB modulated TGF-β1/Smad3 signaling pathways in mice with HF after AB surgery Previous studies have shown that TMAO promotes cardiac hypertrophy and

Journal Pre-proof fibrosis

by activating TGF-β1/Smad3 signaling pathway [13].

Thus,

the

TGF-β1/Smad3 signaling pathway was investigated to test whether DMB modulated this signaling pathway to attenuate cardiac hypertrophy and fibrosis. Western blot analysis showed that the protein levels of TGF-β1 and p-Smad3/Smad3 were increased in AB group than in Sham group (P < 0.05), but were decreased in AB+DMB group than in AB group (P < 0.05) (Fig. 6A-C). Altogether, these results demonstrate that DMB can reduce cardiac hypertrophy and fibrosis in HF via TGF-β1/Smad3 signaling pathways. 3.7. DMB modulated p65 NF-κB signaling pathway in mice with HF after AB surgery

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According to previous studies, TMAO can significantly activate the p65 NF-κB

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pathway [15,28], which has been demonstrated to play an important role in regulating inflammation [29]. However, it is not clear whether DMB modulates p65 NF-κB

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signaling pathway to attenuate expression of inflammatory after AB surgery. As

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shown in Fig. 6D-E, the protein levels of p-p65 NF-κB was clearly increased in the AB group than the Sham group (P < 0.05), but it was decreased upon DMB treatment

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(P < 0.05). This data suggests that DMB attenuated the expression of inflammatory cytokines by inhibiting p65 NF-κB signaling pathway. 4. Discussion

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In this study, the following findings were obtained: (1) the level of plasma

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TMAO was significantly increased in the overload-induced HF, and treatment with DMB reduced the TMAO levels; (2) the cardiac remodeling from HF was attenuated

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by treatment with DMB; (3) DMB attenuates cardiac structural and electrical remodeling at least partially by regulating TGF-β1/Smad3 signaling pathway and p65 NF-κB signaling pathway. These findings indicate that DMB has an important impact on reducing LV structural remodeling and electrical remodeling after AB surgery. TMAO, a gut microbiota dependent metabolite, was closely related with the development and severity of cardiovascular diseases [8-12], including HF [11,12]. Elevated TMAO levels can predict increased risk of hospitalization for patients with HF [30]. In addition, the level of TMAO was not only elevated in chronic HF, but also in acute HF. A total of 972 plasma samples results revealed that TMAO levels were significantly elevated in patients with acute HF. This was related to poor prognosis at 1 year and was a univariate predictor of death and death/HF [31]. Moreover, a recent study proved that supplemental TMAO directly promoted the development of HF [16]. In addition, TMAO was reported to facilitate the progression of atrial fibrillation [15].

Journal Pre-proof DMB is a natural plant extract, as an inhibitor of TMA formation, DMB has showed many beneficial pharmacological efficacy with no side effects in multiple fields [19-22]. In consistent with these results, we revealed that plasma TMAO levels were significantly elevated in the overload-induced HF mice and treatment with DMB inhibited plasma TMAO levels elevation. More importantly, we found that DMB attenuated cardiac structural remodeling and electrical remodeling in HF mice model. HF is closely related to cardiac hypertrophy and fibrosis. Increased cardiac hypertrophy and fibrosis may play an influential role in mice cardiac dysfunction after AB surgery [24,32,33]. Furthermore, TMAO can enhance cardiac hypertrophy,

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fibrosis and worsen cardiac function [13,16]. Similar to the previous studies,

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measurement values of heart function suggested that DMB significantly improved cardiac function (Table1). It was also evident from our study that cardiac hypertrophy

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and fibrosis was increased in HF mice and treatment with DMB attenuated cardiac

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hypertrophy and fibrosis (Fig.2, Fig.3). It is interesting to note that previous studies have shown that dietary TMAO can enhance renal fibrosis [34] and treatment with

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DMB could reverse renal fibrosis [21].

As described previously, elevated TMAO can cause inflammation [35]. Recent studies have found that DMB could decrease the level of inflammatory cytokines

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[19-23]. Consistent with previous findings, our study found significant increase in

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expression of inflammatory cytokines (TNF-α, IL-6 and IL-1β) in the heart of HF mice. However, upon treatment with DMB, the expression of the above inflammatory

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cytokines reduced (Fig.4).

Recent studies have show that increased plasma TMAO is associated to the formation of atrial fibrillation and TMAO can facilitate the progression of atrial fibrillation via directly activating the atrial autonomic ganglion plexus (GP) [9, 15]. TMAO can also activate the cardiac sympathetic nervous system (CSNS) and aggravate ischemia-induced VA [36]. Besides, TMAO can enhance cardiac fibrosis and inflammation [13, 35], earlier studies have shown that myocardial fibrosis and inflammation is important in the pathogenesis of VA [32, 37]. However, the relationship between DMB and VA is not well-understood. We previously reported that the APD90, threshold of electric alternans, VA inducibility were increased in overload-induced heart failure [32]. These results are consistent to the ones in our present study. More importantly, this study confirms that DMB shortens APD90, decreases the threshold of electric alternans and VA inducibility rate in mice HF

Journal Pre-proof model (Fig.5). The mechanism of DMB decreased VA inducibility rate may due to that DMB attenuated cardiac fibrosis and inflammation by reducing the level of plasma TMAO. On the other hand, DMB may inhibition of CSNS to shortens APD90, decreases the threshold of electric alternans and VA inducibility rate by reducing level of plasma TMAO. Previous studies have shown that TMAO promotes cardiac hypertrophy and fibrosis by activating TGF-β1/Smad3 signaling pathway [13]. TMAO was also reported to significantly activate the p65 NF-κB pathway [15, 28], which has been demonstrated to play an important role in regulating inflammation and atrial

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fibrillation [15, 29]. DMB is an inhibitor of TMA formation. Whether DMB exerts

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cardioprotective effect on overload-induced HF via TGF-β1/Smad3 signaling pathway and p65 NF-κB signaling pathway as well? Thus, the protein level of TGF-β1,

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p-Smad3, Smad3, p-p65 and p65 was investigated to test whether DMB modulated

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TGF-β1/Smad3 signaling pathway and p65 NF-κB signaling pathway to attenuate cardiac remodeling in pressure overload-induced HF mice. The result of western blot

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demonstrated that the protein levels of TGF-β1, p-Smad3/Smad3, p-p65/p65 were increased in AB group compared to Sham group. DMB reversed the increase of these protein expression in AB group (Fig.6), indicating that DMB attenuates cardiac

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NF-κB signaling pathway.

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structural and electrical remodeling via TGF-β1/Smad3 signaling pathway and p65 Conclusion

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In conclusion, this study reveals that DMB attenuates pressure overload-induced cardiac remodeling by reducing plasma TMAO levels, hence negatively regulating the TGF-β1/Smad3 signaling and p65 NF-κB signaling pathways. Sources of funding

This study was supported by the National Natural Science Foundation of China(No.81570306 and No.81570459) and National Key R&D Program of China (2017YFC1700500). Disclosures None.

References [1] Tang WH, Kitai T, Hazen SL. Gut Microbiota in Cardiovascular Health and Disease. Circulation research. 2017;120:1183-96. [2] Verbrugge FH, Dupont M, Steels P, Grieten L, Malbrain M, Tang WH, et al. Abdominal contributions to cardiorenal dysfunction in congestive heart failure. Journal of the American College of Cardiology. 2013;62:485-95.

Journal Pre-proof [3] Wang Z, Klipfell E, Bennett BJ, Koeth R, Levison BS, Dugar B, et al. Gut flora metabolism of phosphatidylcholine promotes cardiovascular disease. Nature. 2011;472:57-63. [4] Koeth RA, Levison BS, Culley MK, Buffa JA, Wang Z, Gregory JC, et al. gamma-Butyrobetaine is a proatherogenic intermediate in gut microbial metabolism of L-carnitine to TMAO. Cell metabolism. 2014;20:799-812. [5] Koeth RA, Wang Z, Levison BS, Buffa JA, Org E, Sheehy BT, et al. Intestinal microbiota metabolism of L-carnitine, a nutrient in red meat, promotes atherosclerosis. Nature medicine. 2013;19:576-85. [6] Boutagy NE, Neilson AP, Osterberg KL, Smithson AT, Englund TR, Davy BM, et al. Probiotic supplementation and trimethylamine-N-oxide production following a high-fat diet. Obesity (Silver Spring, Md). 2015;23:2357-63. [7] Bennett BJ, de Aguiar Vallim TQ, Wang Z, Shih DM, Meng Y, Gregory J, et al. Trimethylamine-N-oxide, a metabolite associated with atherosclerosis, exhibits complex genetic and dietary regulation. Cell metabolism. 2013;17:49-60.

f

[8] Li XS, Obeid S, Klingenberg R, Gencer B, Mach F, Raber L, et al. Gut microbiota-dependent trimethylamine

oo

N-oxide in acute coronary syndromes: a prognostic marker for incident cardiovascular events beyond traditional risk factors. European heart journal. 2017;38:814-24.

pr

[9] Svingen GFT, Zuo H, Ueland PM, Seifert R, Loland KH, Pedersen ER, et al. Increased plasma trimethylamine-N-oxide is associated with incident atrial fibrillation. International journal of cardiology. 2018;267:100-6.

e-

[10] Haghikia A, Li XS, Liman TG, Bledau N, Schmidt D, Zimmermann F, et al. Gut Microbiota-Dependent Trimethylamine N-Oxide Predicts Risk of Cardiovascular Events in Patients With Stroke and Is Related to

Pr

Proinflammatory Monocytes. Arteriosclerosis, thrombosis, and vascular biology. 2018;38:2225-35. [11] Troseid M, Ueland T, Hov JR, Svardal A, Gregersen I, Dahl CP, et al. Microbiota-dependent metabolite trimethylamine-N-oxide is associated with disease severity and survival of patients with chronic heart failure.

al

Journal of internal medicine. 2015;277:717-26.

[12] Tang WH, Wang Z, Fan Y, Levison B, Hazen JE, Donahue LM, et al. Prognostic value of elevated levels of

rn

intestinal microbe-generated metabolite trimethylamine-N-oxide in patients with heart failure: refining the gut hypothesis. Journal of the American College of Cardiology. 2014;64:1908-14.

Jo u

[13] Li Z, Wu Z, Yan J. Gut microbe-derived metabolite trimethylamine N-oxide induces cardiac hypertrophy and fibrosis. Laboratory investigation; a journal of technical methods and pathology. 2019;99:346-57. [14] Geng J, Yang C, Wang B, Zhang X, Hu T, Gu Y, et al. Trimethylamine N-oxide promotes atherosclerosis via CD36-dependent MAPK/JNK pathway. Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie. 2018;97:941-7.

[15] Yu L, Meng G, Huang B, Zhou X, Stavrakis S, Wang M, et al. A potential relationship between gut microbes and atrial fibrillation: Trimethylamine N-oxide, a gut microbe-derived metabolite, facilitates the progression of atrial fibrillation. International journal of cardiology. 2018;255:92-8. [16] Organ CL, Otsuka H, Bhushan S, Wang Z, Bradley J, Trivedi R, et al. Choline Diet and Its Gut Microbe-Derived Metabolite, Trimethylamine N-Oxide, Exacerbate Pressure Overload-Induced Heart Failure. Circulation Heart failure. 2016;9:e002314. [17] Chen ML, Yi L, Zhang Y, Zhou X, Ran L, Yang J, et al. Resveratrol Attenuates Trimethylamine-N-Oxide (TMAO)-Induced Atherosclerosis by Regulating TMAO Synthesis and Bile Acid Metabolism via Remodeling of the Gut Microbiota. mBio. 2016;7:e02210-15. [18] Shi Y, Hu J, Geng J, Hu T, Wang B, Yan W, et al. Berberine treatment reduces atherosclerosis by mediating gut microbiota in apoE-/- mice. Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie. 2018;107:1556-63.

Journal Pre-proof [19] Wang Z, Roberts AB, Buffa JA, Levison BS, Zhu W, Org E, et al. Non-lethal Inhibition of Gut Microbial Trimethylamine Production for the Treatment of Atherosclerosis. Cell. 2015;163:1585-95. [20] Chen K, Zheng X, Feng M, Li D, Zhang H. Gut Microbiota-Dependent Metabolite Trimethylamine N-Oxide Contributes to Cardiac Dysfunction in Western Diet-Induced Obese Mice. Frontiers in physiology. 2017;8:139. [21] Li T, Gua C, Wu B, Chen Y. Increased circulating trimethylamine N-oxide contributes to endothelial dysfunction in a rat model of chronic kidney disease. Biochemical and biophysical research communications. 2018;495:2071-7. [22] Chen H, Li J, Li N, Liu H, Tang J. Increased circulating trimethylamine N-oxide plays a contributory role in the development of endothelial dysfunction and hypertension in the RUPP rat model of preeclampsia. Hypertension in pregnancy. 2019;38:96-104. [23] Li T, Chen Y, Gua C, Li X. Elevated Circulating Trimethylamine N-Oxide Levels Contribute to Endothelial Dysfunction in Aged Rats through Vascular Inflammation and Oxidative Stress. Frontiers in physiology.

f

2017;8:350.

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[24] Li H, He C, Feng J, Zhang Y, Tang Q, Bian Z et al. Regulator of G protein signaling 5 protects against cardiac hypertrophy and fibrosis during biomechanical stress of pressure overload. Proc Natl Acad Sci USA

pr

2010;107:13818–13823.

[25] Wang J, Li D, Wang P, Hu X, Chen F. Ginger prevents obesity through regulation of energy metabolism and activation of browning in high-fat diet-induced obese mice. The Journal of nutritional biochemistry.

e-

2019;70:105-15.

[26] Shuai W, Kong B, Fu H, Shen C, Jiang X, Huang H. MD1 Deficiency Promotes Inflammatory Atrial

Pr

Remodelling Induced by High-Fat Diets. The Canadian journal of cardiology. 2019;35:208-16. [27] Jiang X, Kong B, Shuai W, Shen C, Yang F, Fu H, et al. Loss of MD1 exacerbates myocardial ischemia/reperfusion injury and susceptibility to ventricular arrhythmia. European journal of pharmacology.

al

2019;844:79-86.

[28] Seldin MM, Meng Y, Qi H, Zhu W, Wang Z, Hazen SL, et al. Trimethylamine N-Oxide Promotes Vascular

rn

Inflammation Through Signaling of Mitogen-Activated Protein Kinase and Nuclear Factor-kappaB. Journal of the American Heart Association. 2016;5.

Jo u

[29] Capiralla H, Vingtdeux V, Zhao H, Sankowski R, Al-Abed Y, Davies P, et al. Resveratrol mitigates lipopolysaccharide- and Abeta-mediated microglial inflammation by inhibiting the TLR4/NF-kappaB/STAT signaling cascade. Journal of neurochemistry. 2012;120:461-72. [30] Lever M, George PM, Slow S, Bellamy D, Young JM, Ho M, et al. Betaine and Trimethylamine-N-Oxide as Predictors of Cardiovascular Outcomes Show Different Patterns in Diabetes Mellitus: An Observational Study. PloS one. 2014;9:e114969. [31] Suzuki T, Heaney LMBS, Jones DJ, Ng LL. Trimethylamine N-oxide and prognosis in acute heart failure. Heart. 2016;102:841-8. [32] Peng J, Liu Y, Xiong X, Huang C, Mei Y, Wang Z, et al. Loss of MD1 exacerbates pressure overload-induced left ventricular structural and electrical remodelling. Scientific reports. 2017;7:5116. [33] Xiong X, Liu Y, Mei Y, Peng J, Wang Z, Kong B, et al. Novel Protective Role of Myeloid Differentiation 1 in Pathological Cardiac Remodelling. Scientific reports. 2017;7:41857. [34] Tang WH, Wang Z, Kennedy DJ, Wu Y, Buffa JA, Agatisa-Boyle B, et al. Gut microbiota-dependent trimethylamine N-oxide (TMAO) pathway contributes to both development of renal insufficiency and mortality risk in chronic kidney disease. Circulation research. 2015;116:448-55.

Journal Pre-proof [35] Sun X, Jiao X, Ma Y, Liu Y, Zhang L, He Y, et al. Trimethylamine N-oxide induces inflammation and endothelial dysfunction in human umbilical vein endothelial cells via activating ROS-TXNIP-NLRP3 inflammasome. Biochemical and biophysical research communications. 2016;481:63-70. [36] Meng G, Zhou X, Wang M, Zhou L, Wang Z, Wang M, et al. Gut microbe-derived metabolite trimethylamine N-oxide activates the cardiac autonomic nervous system and facilitates ischemia-induced ventricular arrhythmia via two different pathways. EBioMedicine. 2019;44:656-64 [37] Esposito CT, Varahan S, Jeyaraj D, Lu Y, Stambler BS. Spironolactone improves the arrhythmogenic substrate in heart failure by preventing ventricular electrical activation delays associated with myocardial interstitial fibrosis and inflammation. Journal of cardiovascular electrophysiology. 2013;24:806-12

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Fig.1. Effects of DMB on TMAO levels. n=6.*P< 0.05 vs Sham group, #P< 0.05 vs

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AB group.

Fig.2. Effects of DMB on cardiac hypertrophy. (A) Representative images of Gross

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hearts and Haematoxylin-eosin (HE) staining at 6 weeks after sham or AB surgery (n = 7-8). (B) Quantitative analysis of the cell sectional areas (n = 100 + cells). (C-E)

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Ratio of Heart weight (HW)/body weight (BW), HW/tibiae length (TL), and lung weight (LW)/TL (n=9). (F-H) Levels of the hypertrophic markers ANP, BNP and

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β-MHC mRNA in the 3 groups (n=4).*P< 0.05 vs Sham group, #P< 0.05 vs AB group.

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Fig.3. Effects of DMB on cardiac fibrosis. (A) Representative images of picrosirius red (PSR) and Masson staining, illustrating interstitial fibrosis in 6 weeks after sham

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or AB surgery (n = 7-8). (B) Quantitative analysis of interstitial fibrosis calculated

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from Masson-stained heart. (C) Representative images of picrosirius red (PSR) and Masson trichrome staining, illustrating perivascular fibrosis in 6 weeks after sham or AB surgery (n = 7-8). (D) Quantitative analysis of perivascular fibrosis calculated from Masson-stained heart. (F-H) Levels of mRNA of the fibrosis markers collagen Iα, collagen III and connective tissue growth factor (CTGF) in the 3 groups (n=4). *P< 0.05 vs Sham group, #P< 0.05 vs AB group. Fig.4. Effects of DMB on the expression of TNF-α, IL-6 and IL-1β. (A-D) Representative examples and relative expression of TNF-α, IL-6 and IL-1β across different groups (n=4).*P< 0.05 vs Sham group, #P< 0.05 vs AB group. Fig.5. Effects of DMB on electrical remodeling. (A, B) Representative images of ECG and quantitative analysis of Surface electrocardiograph parameters in the 3 groups (n=6-9). (C, D) Representative images of the monophasic action potential (MAP) recordings at a pacing cycle length (PCL) of 150 ms and quantitative analysis of 90% action potential duration (APD90) in the 3 groups (n=6-8). (E, F)

Journal Pre-proof Representative images of the monophasic action potential (MAP) recordings of action potential duration (APD) alternans and quantitative analysis of threshold interval for action potential duration (APD) alternans in the 3 groups (n=6-8). (G, H) Representative images of the monophasic action potential (MAP) recordings after burst pacing and quantitative analysis of ventricular arrhythmia (VA) inducibility in the 3 groups (n=10-12). *P< 0.05 vs Sham group, #P< 0.05 vs AB group. Fig.6. Effects of DMB on TGF-β1/Smad3 signaling pathways and p65 NF-κB signaling pathway. Representative images (A, D) of Western blot (B, C, E) and their corresponding statistical analysis (n=4).*P< 0.05 vs Sham group, #P< 0.05 vs AB

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Table 1 Echocardiographic and haemodynamic measurements in three groups Sham

AB

AB+DMB

IVSd, mm

0.78±0.02

0.81±0.02*

0.80±0.02*

IVSs, mm

1.13±0.41

1.22±0.32*

1.19±0.32*

LVPWd, mm

0.77±0.01

0.83±0.02*

0.81±0.02*

LVPWs, mm

1.13±0.04

1.27±0.03*

1.24±0.02*

LVEDD, mm

4.06±0.25

5.30±0.60*

4.83±0.42*

LVESD, mm

2.24±0.26

4.29±0.58*

3.84±0.43*

LVFS, %

45.13±4.07

19.21±2.71*

20.6±2.18*

LVEF, %

81.22±4.02

45.89±4.73*

50.44±2.46*#

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Echocardiography

110.22±2.54

142.56±1.88*

144.56±1.13*

+dP/dt, mmHg/s

10044.44±815.64

6911.11±527.84*

7944.44±360.94*#

6200.00±212.13*

6611.11±236.88*#

−dP/dt, mmHg/s

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LVESP, mmHg

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Hemodynamics

8055.56±591.84

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n=9 for each group. Data are expressed as mean±SD. IVSd, interventricular septum diameter in diastole; IVSs, interventricular septum diameter in systole; LVPWd, left ventricular posterior wall diameter in diastole; LVPWs, left ventricular posterior wall diameter in systole; LVEDD, left ventricular end-diastolicdiameter; LVESD, left ventricular end-systolic diameter; LVFS, left ventricular fractional shortening; LVEF, left ventricularEjection fraction; LVESP, left ventricular end-systolic pressure; +dP/dt, left ventricular maximum rates of pressure rise; −dP/dt, left ventricular minimum rates of pressure rise *P<0.05 VS Sham group ; #P<0.05 VS AB group.

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Figure 4

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