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Activation of Silent Information Regulator 6 Signaling Attenuates Myocardial Fibrosis by Reducing TGFβ1-Smad2/3 Signaling in a Type 2 Diabetic Animal Model

Yu, Liming1,∗; Wang, Jian2; Dong, Xue3; Hu, Yue1; Luo, Linyu1; Xue, Xiaodong1; Wang, Yang1

Editor(s): Xu, Tianyu; Fu, Xiaoxia

Author Information
doi: 10.1097/CD9.0000000000000031
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  • Exogenous activation of silent information regulator 6 (SIRT6) signaling protected against diabetes-induced myocardial fibrosis through AMP-activated protein kinase (AMPK) signaling.
  • SIRT6-AMPK signaling regulated the progression of cardiac remodeling in diabetic animals by suppressing the TGFβ1-Smad2/3 signaling pathway.


  • This study provides experimental data and a theoretical basis for the management of diabetes-induced myocardial fibrosis.
  • SIRT6 may serve as a novel clinical target for treating diabetes-related cardiovascular diseases.


As a well-established contributor to cardiovascular diseases, diabetes can result in specific myocardial dysfunction in the absence of hypertension and ischemic events.[1] This was initially characterized by Rubler et al[2] as diabetic cardiomyopathy. Indeed, diabetes-related cardiovascular diseases have been recognized as major causes of death in diabetic individuals. It has been accepted that long-term diabetes can lead to ventricular hypertrophic remodeling, tissue fibrosis, myocardial metabolic defection, and eventually, heart failure.[1,3] Moreover, diabetic conditions can also blunt the beneficial effects of multiple cardioprotective agents.[4] Despite advances in recent years, there is still a lack of effective clinical interventions.

Silent information regulator 2 family (SIRTs) contains evolutionarily conserved class III histone deacetylase enzymes that play important roles in regulating redox status and senescence.[5–7] Among the 7 members (SIRT1-7) that have been identified so far, SIRT6 is mainly distributed in the nucleus and has been demonstrated to show beneficial effects against cardiovascular diseases.[8] Growing evidence indicates that SIRT6 activation exerts protective effects against cardiac ischemic disease,[9] ventricular hypertrophic remodeling,[10] and heart failure.[10] However, in the case of diabetes, whether the direct regulation of myocardial SIRT6 signaling affects cardiac performance and its underlying mechanism in this disease remain largely unknown.

AMP-activated protein kinase (AMPK) signaling significantly contributes to maintaining cellular metabolic balance.[11] Previously, Cui et al[12] demonstrated that suppression of SIRT6-related pathways in muscle tissue could reduce expression of the genes involved in mitochondrial oxidative phosphorylation through AMPK inhibition. Additionally, several studies have demonstrated that upregulation of AMPK pathways protects the heart against diabetic cardiomyopathy and preserves cardiac performance.[13] More importantly, Wang et al[14] revealed that upregulation of cardiac SIRT6 exerted a positive effect on myocardial AMPK signaling and eventually protected against myocardial ischemia/reperfusion (MI/R) injury. Recent evidence also suggests that the SIRT6-AMPK pathway inhibits mitochondrial abnormalities in podocytes or biliary epithelial cells.[15,16] In view of these critical findings, we hypothesized that in diabetic conditions, SIRT6 upregulation might retards diabetes-induced ventricular remodeling through the AMPK pathway.

In this study, our data demonstrate that SIRT6 signaling was inhibited and TGFβ1-Smad2/3-induced myocardial fibrosis exacerbated in a type 2 diabetic rat model. Exogenous activation of SIRT6 signaling protects against diabetes-induced myocardial fibrosis through AMPK signaling. Intracellular SIRT6 might therefore be a promising therapeutic target to protect the heart in type 2 diabetic individuals against myocardial fibrosis.

Materials and methods


Sprague Dawley (SD) rats (HUAFUKANG Bioscience Co., Ltd, Beijing, China) were randomly assigned to the following groups (n = 10, respectively): non-diabetic group, diabetic group, diabetic group injected with empty adenoviral vectors (Ad-EV group) and adenoviral vectors expressing SIRT6 (Ad-SIRT6 group). The animal experiments in this study were approved by the General Hospital of Northern Theatre Command Animal Care Committee (2017011032) and conformed to the Principles of Laboratory Animal Care published by the US National Institutes of Health.

Diabetes induction

Diabetes induction was established as described in our previous study.[17] SD rats in diabetic group were fed a high-fat diet (Research Diets, New Jersey, USA) for 28 days and then administered streptozotocin (prepared in citrate buffer, 40 mg/kg, intraperitoneal; Sigma-Aldrich, Missouri, USA). After injection, the animals were continuously fed a high-fat diet, and those with fasting plasma glucose ≥11.1 mmol/L were defined as type 2 diabetic subjects.

Rats in non-diabetic group were fed a normal chow diet.

Experimental design

Rats in non-diabetic and diabetic groups were kept for another 8 weeks. Ventricular proteins were extracted and analyzed. Additionally, members in the diabetic group (8 weeks after the establishment of diabetes) were allocated to the Ad-EV or Ad-SIRT6 group. The experimental animals were fed a high-fat diet and kept for another 4 weeks before being sacrificed. Tissue samples were collected and analyzed.

Adenovirus injection

Ad-EV and Ad-SIRT6 were constructed by Hanbio Co., Ltd (Shanghai, China). For adenovirus injection, the rats were anesthetized, and the heart was exposed by thoracotomy surgery. The adenoviruses were administered to ventricle tissue (approximately 5 × 1010 genomes/rat) using a special microsyringe (Hamilton Co., Reno, Nevada, USA).


Animal myocardial performance was assessed as described previously using a high-resolution Doppler ultrasound imaging system (VINNO, Suzhou, China).[18] Five SD rats in each of non-diabetic group, diabetic group, Ad-EV group and Ad-SIRT6 group were included in echocardiographic assessment.

Masson's trichrome staining

Cardiac remodeling was assessed by Masson's trichrome staining (Baso Company, China), as described in previous work.[19] Five SD rats in each of non-diabetic group, diabetic group, Ad-EV group and Ad-SIRT6 group underwent histochemical Staining.

Western blotting

Western blotting was performed as previously described.[17] Primary antibodies for SIRT6, AMPKα, and p-AMPKα (Thr172) were obtained from Cell Signaling Technology (Massachusetts, USA). The remaining antibodies used in this study were obtained from Santa Cruz Biotechnology (California, USA). The blots were scanned and analyzed using the Tanon Analysis System (Tanon Technology, Shanghai, China). Five SD rats in each of non-diabetic group, diabetic group, Ad-EV group and Ad-SIRT6 group were included in western blotting measurement.


Data are presented as means ± standard error of mean (SEM) and analyzed using Student's t test (GraphPad Software, California, USA). Statistical significance was set at P values <0.05.


Myocardial fibrosis after 8 weeks of diabetic induction

As shown in Figure 1, the diabetic myocardium exhibited impaired cardiac performance and increased myocardial fibrosis.

Figure 1:
Myocardial functional measurement and histological assessment of cardiac fibrosis. The interstitial collagen deposition markedly increased in diabetic group (n = 5) compared with non-diabetic group (n = 5). (A) Representative M-mode echocardiographic images; (B) LVEF and LVFS; (C) Masson's trichrome staining images (scale bar = 100 μm); (D) Quantitative analysis of fibrotic area. Each value represents the mean ± SEM of 5 independent experiments. P < 0.01 vs. non-diabetic group. LVEF: Left ventricular ejection fraction; LVFS: Left ventricular fractional shortening; SEM: Standard error of mean.

Myocardial SIRT6-AMPK signaling and TGFβ1-Smad2/3 signaling during the progression of cardiomyopathy

As shown in Figure 2A, the diabetic group showed significantly reduced SIRT6 expression compared to the non-diabetic group. The level of AMPK phosphorylation at Thr172 was reduced in diabetic subjects, implying that changes in the SIRT6 and AMPK pathways might affect the progression of myocardial dysfunction.

Figure 2:
Western blot analysis of myocardial SIRT6-AMPK signaling and TGFβ1-Smad2/3 signaling. Diabetic group showed significantly reduced SIRT6-AMPK signaling and enhanced TGFβ1-Smad2/3 signaling. (A) Western blot and associated quantitative analysis of myocardial SIRT6, p-AMPKα (Thr172) and AMPKα expressions; (B) Western blot and associated quantitative analysis of myocardial TGFβ1 and Smad2/3 expressions. Each value represents the mean ± SEM of 5 independent experiments. P < 0.01 vs. non-diabetic group. AMPK: AMP-activated protein kinase; SEM: Standard error of mean; SIRT6: Silent information regulator 6.

Diabetes induced a significant increase in TGFβ1 and Smad2/3 protein levels [Figure 2B]. We investigated the potential effects of SIRT6 on the progression of diabetes. As shown in Figure 3A and B, SIRT6 overexpression markedly increased SIRT6 protein levels. Meanwhile, AMPK signaling was also markedly enhanced, as evidenced by the increased phosphorylation of AMPK at Thr172 [Figure 3C], indicating that AMPK serves as a potential effector of myocardial SIRT6 signaling in diabetic conditions.

Figure 3:
Western blot analysis of myocardial SIRT6-AMPK signaling. Ad-SIRT6 group showed significantly enhanced SIRT6-AMPK signaling. (A) Representative western blot images; (B) SIRT6 expression; (C) p-AMPKα phorsphorylation level. Each value represents the mean ± SEM of 5 independent experiments. P < 0.01 vs. Ad-EV group. Ad-EV: Empty adenoviral vectors; AMPK: AMP-activated protein kinase; SEM: Standard error of mean; SIRT6: Silent information regulator 6.

Importantly, we further demonstrated that SIRT6 activation improved myocardial function and reduced myocardial fibrosis, as evidenced by increased left ventricular ejection fraction and left ventricular fractional shortening and decreased blue-stained area in Figure 4A–D. Furthermore, myocardial TGFβ1-Smad2/3 was also suppressed by SIRT6 overexpression, indicating that SIRT6 exerted an inhibitory role against fibrotic remodeling during the progression of diabetes by reducing the TGFβ1-Smad2/3 signaling pathway [Figure 4E–G].

Figure 4:
Myocardial fibrosis evaluation and western blot analysis of myocardial TGFβ1-Smad2/3 signaling. The interstitial collagen deposition markedly reduced in Ad-SIRT6 group compared with Ad-EV group. Ad-SIRT6 group showed significantly decreased TGFβ1-Smad2/3 signaling. (A) Representative M-mode echocardiographic images; (B) LVEF and LVFS; (C) Masson's trichrome staining images (scale bar = 100 μm); (D) Quantitative analysis of fibrotic area; (E) Representative western blot images; (F) TGFβ1 expression; (G) Smad2/3 expression. Each value represents the mean ± SEM of 5 independent experiments. P < 0.05 vs. Ad-EV group. P < 0.01 vs. Ad-EV group. Ad-EV: Empty adenoviral vectors; AMPK: AMP-activated protein kinase; LVEF: Left ventricular ejection fraction; LVFS: Left ventricular fractional shortening; SEM: Standard error of mean; SIRT6: Silent information regulator 6.


Our experimental results demonstrate that diabetes markedly downregulated SIRT6 expression and exacerbated TGFβ1-Smad2/3-induced myocardial fibrosis. Exogenous activation of SIRT6 signaling protected against diabetes-induced myocardial fibrosis through AMPK signaling. Intracellular SIRT6 might be a promising cardioprotective target against fibrosis in type 2 diabetic individuals.

Previous studies have indicated that long-term diabetes can lead to heart failure. Myocyte hypertrophy, necrosis, programmed cell death, and myocardial fibrosis all participate in this process,[20] although the mechanisms are not fully understood. Myocardial fibrotic remodeling is one of the major causes of diabetes-related myocardial dysfunction. In diabetic conditions, myocardial fibroblasts exhibit significantly enhanced proliferation and extracellular matrix markedly accumulated in cardiac tissue, which causes significantly reduced heart function.[4,21] To date, many studies have shown that TGFβ1 serves as a pivotal contributor to cardiac remodeling and plays a key role in the pathogenesis of tissue fibrotic changes.[22] It has been demonstrated that in diabetic subjects, cardiac collagen levels are positively correlated with TGFβ1 levels, and TGFβ1 mediates the development of myocardial fibroses.[22,23] A study by Suematsu et al[24] showed that streptozotocin induced an increase in myocardial TGFβ1 in diabetic mice, which led to myocardial fibrosis accompanied by cardiac dysfunction. Interestingly, the involvement of the Smad pathway in the pathogenesis of cardiac remodeling has also been well established.[25] Targeting TGFβ1-Smad2/3 signaling has proved to be a key therapeutic approach to defend against myocardial fibrosis.[26] In the present study, we found that, with the development of diabetes, myocardial TGFβ1-Smad2/3 signaling was markedly activated. These results coincide with previous reports demonstrating that TGFβ1-Smad2/3 signaling serves as a key contributor to myocardial fibrosis.

Recently, SIRT6 has attracted much attention for its modulatory role in human telomeres and genome stabilization,[27] longevity, DNA repair,[28] and glucose metabolism.[29] A study by Sundaresan et al[30] showed that SIRT6 deletion led to severe myocardial remodeling and tissue fibrosis in mice, while activation of SIRT6 signaling markedly inhibited ventricular dysfunction. Additionally, Kanwal et al[31] reported that SIRT3 and SIRT6 regulate each other's activity and attenuate diabetic cardiomyopathy. In this study, our data show that diabetes significantly reduced myocardial SIRT6 expression, indicating that inactivation of SIRT6 signaling probably participates in the development of diabetes-related myocardial dysfunction. Thus, we further investigated AMPK signaling, since interplay between these 2 proteins has been well documented.

In this study, our results showed that overexpression of cardiac SIRT6 induced significantly higher AMPK phosphorylation levels, indicating that the cardioprotective effect elicited by SIRT6 against myocardial fibrosis was likely through AMPK signaling. It is worth noting that the potential modulatory effect of AMPK on myocardial TGFβ1-Smad2/3 signaling has been reported. Recently, Yu et al[32] showed that Ginsenoside Re improved myocardial infarction-induced ventricular dysfunction and mitigated myocardial tissue remodeling by modulating the AMPK-TGFβ1-Smad2/3 signaling pathways. Another study by Ge et al[33] indicated that the adenosine derivative IMM-H007 could serve as an effective activator of AMPK and could reduce TGFβ1 expression during fibrosis. Therefore, we investigated the levels of TGFβ1-Smad2/3 signaling. We found that SIRT6 activation markedly inhibited the expression of TGFβ1 and Smad2/3 and reduced myocardial fibrosis. Based on a previous report, we demonstrated that SIRT6-AMPK signaling ameliorates diabetes-induced cardiac fibrosis by inhibiting the TGFβ1-Smad2/3 signaling pathway in the diabetic state.

Taken together, we demonstrated by a series of in vivo experiments that SIRT6-AMPK signaling regulated the progression of cardiac remodeling in diabetes mellitus animals by suppressing the TGFβ1-Smad2/3 signaling pathway. These results provide experimental data and a theoretical basis for the management of diabetes-induced myocardial fibrosis. SIRT6 may serve as a novel clinical target for treating diabetes-related cardiovascular diseases.


This study was supported by grants from the National Natural Science Foundation of China (81700264), Postdoctoral Science Foundation Grant of China (2018M643839), and the Natural Science Foundation of Liaoning Province (2020-MS-036).

Author Contributions

Jian Wang and Xue Dong participated in the writing of the paper; Liming Yu, Jian Wang, Xue Dong, and Xiaodong Xue participated in the performance of the research; Yue Hu, Lin-Yu Luo, and Yang Wang participated in data analysis.

Conflicts of Interest



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AMP-activated protein kinase; Diabetes; Fibrosis; Silent information regulator 6

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