The prevalence of obesity has increased exponentially worldwide in the past 3 decades, resulting in high number of diabetes, cardiovascular diseases, and other metabolic disorders.1 Cardiovascular deaths account for most deaths associated with obesity,2 which are mainly mediated by 3 metabolic risk factors: blood pressure, cholesterol, and glucose.3 However, a systematic review reported that nearly half of cardiovascular diseases associated with obesity is not associated with these metabolic mediators.3 Adiposity leads to myocardial structural and functional changes. Cardiac inflammation and fibrosis play a significant role in the alterations of obesity-related cardiac remodeling.4,5 Oxidative stress and inflammation are implicated in obesity-related fibrosis,6 which leads to cardiac stiffness and subsequent dysfunction. Obesity causes a low-degree inflammatory state; however, the underlying molecular pathways are unknown. The link between cardiac inflammation and fibrosis in the context of obesity should be explored.
SIRT3 is a member of the conserved family of NAD+-dependent class III histone deacetylases. Among the 7 sirtuin analogues, SIRT3 is the only protein whose increased expression is associated with increased human life-span.7 SIRT3, the main mitochondrial deacetylase modulates diverse cellular processes by regulating multiple enzyme activations. For instance, SIRT3 accords cardiac protection against hypertensive stimuli through deacetylation of STAT3 and FOXO1.8,9 Furthermore, SIRT3 regulates ROS production by activating mitochondrial antioxidant enzymes such as superoxide dismutase 2 (SOD2), manganese superoxide dismutase (MnSOD), and catalase (CAT).10,11 As a precursor, nicotinamide riboside (NR) confers protection against high fat diet-induced metabolic abnormalities by increasing NAD+ levels and activating SIRT3.12 In addition, PIKfyve inhibition attenuates obesity-related cardiometabolic phenotype by reducing mitochondrial oxidative stress and apoptosis in a SIRT3-dependent manner.13 SIRT3-KO mice fed on a high-fat diet (HFD) showed increased obesity, hepatic inflammation, and fibrosis compared with wild-type controls.14 Despite the above reports, the specific role of SIRT3 in obesity-related cardiac remodeling is still obscure.
Monocyte chemoattractant protein–1 (MCP-1) mRNA levels increase in the adipose tissue of HFD-induced obese mice, and which results in the concomitant increase in macrophage accumulation.15 A study on cultured proximal tubular cells, showed that SIRT3 was negatively correlated to MCP-1 mRNA expression.16 However, the role of MCP-1 in HFD-fed mice heart is vague, its independent and the associated effect of SIRT3 on cardiac remodeling is unknown.
Based on the above theoretical basis, we designed this study to address these issues. In summary, male wild-type and SIRT3-knockout mice were fed on chow diet or HFD for 16 weeks. Furthermore, we studied SIRT3 effect on cardiac remodeling by exploring cardiac inflammation and fibrosis in the animal models. In addition, we conducted further investigations to explore the molecular mechanisms involved in the pathogenesis. The purpose of this study is to provide a basis for the development of novel therapeutic strategies to combat obesity-related cardiac remodeling.
MATERIALS AND METHODS
The animal protocols conformed to the Animal Management Rules of the Chinese Ministry of Health (Document no. 55, 2001) and the study was approved by the Animal Care and Use Committee of Shandong University. SIRT3-KO (129-SIRT3tm1.1Fwa/J) mice were purchased from the Jackson Laboratories. Wild-type (WT, 129S1/SvImJ) mice were obtained from the Department of Laboratory Animal Science of Peking University (Beijing, China) as controls. We made them reproduce for our experiments. All animals were maintained at the key Laboratory of Cardiovascular Remodeling and Function Research in Qilu Hospital of Shandong University. The 8-week old male WT and SIRT3-KO mice were chosen and randomly divided into 4 groups as WT+CD, SIRT3-KO+CD, WT+HFD and SIRT3-KO+HFD. The first 2 groups were fed on chow diet, and the last 2 groups were fed on a HFD [60% kcal, D12492; Research Diets (New Brunswick, NJ)] for 16 weeks to induce obesity model. All animals were given free access to water throughout the study, and housed under standard lighting (12:12 hours, day-night rhythm), temperature (20–22°C), and humidity (50%–60%) conditions.
Antibodies against SIRT3 (rabbit monoclonal) (D22A3) , p- NF-κB p65 (Ser 536) (rabbit monoclonal) (93H1) , NF-κB p65 (rabbit monoclonal) (D14E12) , and Histone H3 (rabbit monoclonal) (D1H2)  were obtained from Cell Signaling Technology (Boston, MA). Primary antibodies against collagen type I (rabbit polyclonal) [ab34710], collagen type III (rabbit polyclonal) [ab7778], TGF-β1 (rabbit polyclonal) [ab92486], MCP-1 (rabbit polyclonal) [ab7202], MOMA-2 (Rat monoclonal) [ab33451], TNF-α (rabbit polyclonal) [ab6671], IL-10 (rabbit polyclonal) [ab9969] were obtained from Abcam (London, United Kingdom). GAPDH (mouse monoclonal) [BM1623], IL-6 (rabbit polyclonal) [PB0060] were bought from Boster (Wuhan, China).
Echocardiography for Mice
Echocardiography was performed on mice anesthetized via continuously delivered gas inhalation of 1% isoflurane. Animals were examined in the left lateral decubitus position with a Visual Sonics Vevo 770 machine and a 30-MHz high-frequency transducer. Images were captured with M-mode, two-dimensional (2-D), pulse wave Doppler and tissue Doppler imaging. The operator was blind to the mice type.
Histology and Immunohistochemistry
After echocardiography, hearts were harvested from the study mice. The isolated hearts were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned at 5-μm intervals. Hematoxylin and eosin staining was performed following standard procedures, and Masson's trichrome was conducted to evaluate myocardial fibrosis. For immunohistochemistry, the sections were incubated overnight with primary antibodies against collagen I (10 µg/mL), collagen III (10 µg/mL), MCP-1 (10 µg/mL) at 4°C. Furthermore, the slides were washed with phosphate-buffered saline and incubated with secondary antibodies at 37°C for 1 h. The immunohistochemical staining for MOMA-2 was performed using frozen sections. The results were analyzed by an automated image analysis system (Image-Pro Plus, Version 7.0; Media Cybernetics, Silver Spring, MD).
Western Blot Analysis
Proteins were harvested from freshly dissected mouse hearts using cell lysis buffer. Tissue lysates were electrophoresed on 10% SDS/PAGE gels and the separated proteins were transferred onto PVDF membranes. The membranes were blocked with 5% nonfat milk for 2 hours. Furthermore, the membranes were incubated overnight with the primary antibodies against collagen I (0.2 µg/mL), collagen III (0.2 µg/mL), TGF-β1 (0.5 µg/mL), SIRT3 (0.5 µg/mL), MCP-1 (0.5 µg/mL), NF-κB (0.5 µg/mL), P-NF-κB (0.5 µg/mL), IL-6 (0.5 µg/mL), TNF-α (1 µg/mL), IL-10 (1 µg/mL), H3 (0.2 µg/mL), and GAPDH (0.5 µg/mL). The membranes were washed with Tris-buffered saline containing 0.1% Tween, and incubated with rabbit or mouse secondary antibodies (1:5000) for 1 h at room temperature. Protein bands were visualized with enhanced chemiluminescence (Millipore, MA) whereas protein levels were detected using an Image Quant LAS4000 chemiluminescence reader (GE, Fairfield, CT). Relative protein levels were quantified using Image J software.
Real-Time Polymerase Chain Reaction (PCR) Analysis
Total RNAs were purified from cardiac tissue using TRIzol. RNA was reverse-transcribed into cDNA using the Transcriptor First Strand cDNA synthesis kit. PCR amplifications were quantified using a MyiQ Real-Time PCR System (Bio-Rad, Hercules, CA) and the results were normalized using internal GAPDH control. The primers for PCR analysis were as follows: TNF-α, forward 5′-AGCCGATGGGTTGTACCTTG-3′ and reverse 5′-ATAGCAAATCGGCTGACGGT'; IL-6 forward5′-TCCAGTTGCCTTCTTGGGAC-3′ and reverse5′-AGTCTCCTCTCCGGACTTGT-3'; GAPDH forward 5′-CAAGATCATTGCTCCTCCTG-3′ and reverse 5′-TCATCGTACTCCTGCTTGCT-3'; IL-10 forward 5′-TAACTGCACCCACTTCCCAG-3′ and reverse 5′-TTGGCAACCCAAGTAACCCTTA-3'.
Dihydroethidium (DHE) Staining
Newly cut mouse hearts were frozen using O.C.T. compound, then frozen ventricular sections were prepared. The slides were dipped into PBS for 5 minutes and then incubated with 0.5 mM DHE for 15 minutes in the dark. Images were taken immediately using a laser scanning confocal microscope (LSM710; Carl Zeiss, Oberkochen, Baden-Wurttember, Germany). The relative density of DHE fluorescence was analyzed using image-analysis software (Image J, NIH).
All the results were obtained from at least 3 independent experiments. Statistical analysis was performed using GraphPad 6.0 Prism (version 5.00 for Windows, GraphPad Software). Data were presented as means ± SEM. Student's t test was used to assess between-group differences and multiple-group comparisons were performed with one-way analysis of variance analysis followed by Tukey post-hoc test when the F test indicated a significant effect. If the variances were not equal, then Tamhane T2's test was used instead. The results were considered statistically significant at P < 0.05.
SIRT3-KO Mice Showed High Weight Gain and Severe Cardiac Diastolic Dysfunction after High-Fat Diet
To investigate the role of SIRT3 in obesity-related cardiac remodeling, we fed both wild-type (WT) and SIRT3-knockout (SIRT3-KO) mice with a HFD for 16 weeks. As shown in Table 1, we observed higher weight gain and increased heart weight in HFD-fed SIRT3-KO mice compared with WT controls. Echocardiography showed left ventricle hypertrophy and diastolic dysfunction induced by HFD, especially in SIRT3-KO mice (Fig. 1A). Furthermore, we evaluated the diastolic function by measuring E/A and E'/A′ ratio (Fig. 1A). Notably, the systolic function was unaltered.
TABLE 1. -
Cardiac Parameters and Echocardiographic Analysis
||Body Weight (g)
||Heart Weight (mg)
||Heart Rate (Beats/min)
|WT + CD
||23.49 ± 0.66
||127.8 ± 6.3
||414.8 ± 1.7
||0.2135 ± 0.02
||0.8136 ± 0.02
||2.870 ± 0.05
||73.00 ± 0.71
||1.30 ± 0.03
||1.28 ± 0.02
|WT + HFD
||29.71 ± 0.50*
||162.1 ± 2.4*
||417.8 ± 0.8
||0.2760 ± 0.01*
||1.277 ± 0.02*
||2.974 ± 0.06
||71.60 ± 0.51
||0.93 ± 0.02*
||0.97 ± 0.03*
|SIRT3-KO + CD
||26.58 ± 0.45
||134.3 ± 4.8
||417.0 ± 0.9
||0.2240 ± 0.02
||0.8532 ± 0.02
||2.942 ± 0.04
||72.00 ± 0.55
||1.29 ± 0.04
||1.22 ± 0.03
|SIRT3-KO + HFD
||34.28 ± 0.76†
||196.0 ± 5.1†
||420.3 ± 1.5
||0.3033 ± 0.03†
||1.3840 ± 0.03†
||3.032 ± 0.03
||70.20 ± 0.58
||0.82 ± 0.03†
||0.84 ± 0.02†
Data are presented as means ± SEM from 3 independent experiments (n = 8, each).
*P < 0.05 versus WT+CD group.
†P < 0.05 versus WT+HFD group.
E/A, the peak flow rate ratio in early and late diastolic phase. E'/A′, the ratio of tissue motion velocity obtained from the LV posterior wall in early and late diastole; IVS, interventricular septum thickness; LVEDD, left ventricular end-diastolic diameter; LVEF, left ventricular ejection fraction; LVPW, left ventricle posterior wall thickness.
SIRT3 Deficiency Deteriorated Cardiac Fibrosis in HFD-Fed Mice
First, we measured SIRT3 protein levels in all the murine hearts. Immunoblot analysis confirmed loss of SIRT3 protein in SIRT3-KO mice, whereas SIRT3 expression levels decreased by 19% in WT controls after HFD feeding (Fig. 1D). Furthermore, we compared HFD effect on WT and SIRT3-KO mice. Masson's trichrome indicated that the fibrosis area fraction in SIRT3-KO mice was higher compared with WT mice at baseline and further increase was observed after HFD feeding (Fig. 2A). The HFD-fed SIRT3-KO mice exhibited the most collagen accumulation as shown by immunohistochemical staining of collagen I and collagen III (Figs. 2B–C). We confirmed this finding by western blot (Fig. 1E). These findings revealed that SIRT3 may be involved in preventing collagen deposition and cardiac fibrosis in obesity-related cardiac remodeling.
HFD-Fed SIRT3-KO Mice Showed High MCP-1 Expression Levels and Macrophage Infiltration
To further understand the underlying relation between cardiac fibrosis and inflammation in obesity-related cardiac remodeling, we conducted more experiments. Both immunohistochemical staining and western blot showed the high MCP-1 expression levels in SIRT3-KO mice placed on HFD compared with the other groups (Figs. 3A–C). HE staining showed more cardiomyocytes loss and inflammatory cells accumulation in the hearts of HFD-fed mice, especially the SIRT3-KO group (Fig. 3D). Further immunohistochemistry showed that inflammatory cells were macrophages marked by MOMA-2 staining (Fig. 3E).
SIRT3-KO Mice Fed on HFD Showed High Expression Levels of Inflammatory Cytokine
We measured and compared inflammatory cytokine levels including IL-6, TGF-β, TNF-α, and IL-10 in murine hearts by western blot (Fig. 4F). The results showed more pro-inflammatory cytokine expression in HFD-fed mice, with higher levels observed in SIRT3-KO mice. On the contrary, the anti-inflammatory factor IL-10 decreased significantly in HFD-fed SIRT3-KO mice. We confirmed this observation through PCR. PCR results agreed with immunoblot results.
HFD-Fed SIRT3-KO Mice Showed Increased ROS Generation and NF-κB Activation
As MCP-1 increased greatly in SIRT3-KO mice after HFD feeding, we sought to explore its upstream molecules. Therefore, we tested NF-κB activation by measuring p-NF-κB and nuclear translocation of NF-κB in all the groups (Fig. 4A–D). DHE staining was performed to evaluate the ROS levels in cardiac tissue. The results showed more ROS generation (Fig. 4G) and activated NF-κB after HFD feeding in mice, with higher levels observed in SIRT3-KO+HFD group.
The obesity prevalence in both developed and developing countries has been increasing steadily nowadays. The high morbidity of cardiovascular and other metabolic abnormalities associated with obesity has increased the economic and health burden in modern society. Previous epidemiology studies have reported the correlation between obesity and heart failure providing key information on the concept and pathogenesis of “obesity cardiomyopathy.”4,17 The myocardial changes in obesity-related cardiomyopathy include structural left ventricle hypertrophy and diastolic dysfunction. Cardiac inflammation and fibrosis are the main factors implicated in obesity-related cardiomyopathy pathophysiology.5 Cardiac fibrosis disrupts myocardium integrity and coordination and impairs both systolic and diastolic functions. Furthermore, it accelerates the progress of heart failure to decompensated stage. In addition, cardiac fibrosis provides substantial condition for malignant arrhythmia and subsequent sudden deaths.18
Previous studies on the link between obesity and inflammation mainly focused on adipose tissue and metabolic disorders such as diabetes and insulin resistance.19,20 Currently, there is limited research on the relationship between inflammation and cardiac remodeling in the context of obesity. The systemic inflammatory state could contribute to development of cardiovascular disease either by direct myocardial effects or influencing other components of the cardiovascular system. In this study, we observed high collagen accumulation levels and high expression levels of IL-6, TGF-β, and TNF-α in SIRT3-KO mice placed on HFD compared with other groups. IL-6 mediates myocardial fibrosis and diastolic dysfunction identical to hypertensive heart phenotype in rats.21 Notably, anti-TNF-α therapeutic approaches are ineffective in heart failure treatment,22 TNF-α inhibition attenuates adverse myocardial remodeling in a rat model of volume overload.23 TGF-β acts as the main fibrogenic mediator in cardiac fibrosis pathogenesis by increasing collagen deposition and promoting myofibroblast transdifferentiation.8 On the contrary, we observed decreased secretion of anti-inflammatory cytokine IL-10. In a myocardial ischemia/reperfusion model, the IL-10-knockout mice showed higher mortality rates correlated with increased neutrophil recruitment.24 IL-10 inhibits the expression of pro-inflammatory cytokines such as TNF-α,25 so the elevated TNF-α level observed in our study could be explained partially by the low levels of IL-10.
In SIRT3-KO mice fed on a HFD, we observed the highest MCP-1 expression levels, which is in accordance with a previous study conducted in obese rats.6 MCP-1, a main chemotactic factor is important in macrophage infiltration in adipose tissue of HFD-fed mice and hypertensive myocardial fibrosis.15,26 To investigate the role of macrophages in cardiac remodeling in our study, we conducted more experiments to confirm the infiltrated inflammatory cells as macrophage by immunohistochemistry staining. Thus, we speculate the positive causative relation between MCP-1 and macrophage accumulation in myocardium just like in adipose tissue. Macrophage is the main source of numerous inflammatory factors such as TGF-β and IL-6, thus it plays a vital role in the progression of cardiac fibrosis.26,27
In a former study conducted on proximal tubular cells, the investigators reported a negative correlation between SIRT3 and MCP-1. High MCP-1 expression levels were induced by activated NF-κB, while NF-κB activation was stimulated by its upstream MAPKs and ROS formation.16 Oxidative stress has been implicated in metabolic syndrome and numerous cardiovascular diseases.28 Although excessive ROS accumulation leads to oxidative damage, it participates in critical signaling pathways under physiological conditions.29 Heng et al showed that HFD feeding in mice induced increased ROS production in the murine hearts.30 In our animal models, we detected NF-κB activation by measuring its nuclear translocation as well as phosphorylated NF-κB (p-NF-κB) in myocardium. The results showed higher activation of NF-κB in mice after HFD, with the most activation observed in SIRT3-KO+HFD group. To further explain the pathway involved, we assessed the ROS levels on cardiac tissue by DHE staining. Notably, we observed the same trend as NF-κB activation with SIRT3-KO+HFD group displaying higher ROS production compared with the other groups. Therefore, we deduced that NF-κB activation is associated with elevated ROS production.
In conclusion, SIRT3 ablation deteriorates obesity-related cardiac remodeling by augmenting cardiac inflammation and fibrosis through ROS-NF-κB-MCP-1 signaling pathway.
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