SIRT1 Activation by Resveratrol Alleviates Cardiac Dysfunction via Mitochondrial Regulation in Diabetic Cardiomyopathy Mice

Background Diabetic cardiomyopathy (DCM) is a major threat for diabetic patients. Silent information regulator 1 (SIRT1) has a regulatory effect on mitochondrial dynamics, which is associated with DCM pathological changes. Our study aims to investigate whether resveratrol, a SRIT1 activator, could exert a protective effect against DCM. Methods and Results Cardiac-specific SIRT1 knockout (SIRT1KO) mice were generated using Cre-loxP system. SIRT1KO mice displayed symptoms of DCM, including cardiac hypertrophy and dysfunction, insulin resistance, and abnormal glucose metabolism. DCM and SIRT1KO hearts showed impaired mitochondrial biogenesis and function, while SIRT1 activation by resveratrol reversed this in DCM mice. High glucose caused increased apoptosis, impaired mitochondrial biogenesis, and function in cardiomyocytes, which was alleviated by resveratrol. SIRT1 deletion by both SIRT1KO and shRNA abolished the beneficial effects of resveratrol. Furthermore, the function of SIRT1 is mediated via the deacetylation effect on peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), thus inducing increased expression of nuclear respiratory factor 1 (NRF-1), NRF-2, estrogen-related receptor-α (ERR-α), and mitochondrial transcription factor A (TFAM). Conclusions Cardiac deletion of SIRT1 caused phenotypes resembling DCM. Activation of SIRT1 by resveratrol ameliorated cardiac injuries in DCM through PGC-1α-mediated mitochondrial regulation. Collectively, SIRT1 may serve as a potential therapeutic target for DCM.


Introduction
Diabetes mellitus is an emerging threat to global human health. According to an official report, diabetes will affect approximately 400 million patients universally [1]. Diabetic cardiomyopathy (DCM) has been a major cause for increased morbidity and mortality in diabetic patients, contributing to over 50% diabetic death [2]. Epidemiological studies have revealed that diabetic people have a 2-to 5-fold increase of risk in developing heart failure compared with age-matched healthy subjects, indicating the necessity of DCM research [3,4]. DCM is characterized by left ventricular hypertrophy, fetal gene reactivation, and lipid accumulation in cardiac cells together leading to contractile dysfunction in myocardium [5,6]. The pathophysiology of DCM is complex and not clearly elucidated, including mitochondrial dysregulation, inflammation, disruption of intracellular transport of Ca 2+ , and myocardium fibrosis [7]. Elucidation of the mechanisms for DCM is essential for the development of effective treatment strategies. Of particular interest, mitochondrial dysfunction has recently been reported to be a major contributor to the development of DCM. Mitochondrial dysfunction occurs by several mechanisms, involving impaired cardiac insulin and glucose homeostasis, impaired cellular and altered cardiac substrate metabolism, oxidative stress, and mitochondrial uncoupling [8]. Additionally, abundant evidence indicated that impaired mitochondrial biogenesis contributed to cardiac dysfunction in diabetic hearts [9].
Emerging evidence manifested that mitochondrial alterations might be a central mediator for the pathologic process in DCM. Therefore, searching for appropriate therapeutic approaches targeting mitochondrial biology holds a promise for the management of DCM [10].
Sirtuin 1 (SIRT1) is one of the seven mammalian homologs (SIRT1-SIRT7) of yeast silent information regulator 2 (Sir2). SIRT1 is an NAD + -dependent protein deacetylase [11]. It played multiple roles in cells including longevity, apoptosis, DNA repair, inflammation, and mitochondrial regulation [12]. As a pivotal protein in cellular metabolism, the regulatory effect of SIRT1 on mitochondrial dynamics has gained much attention. Recently, several studies reported that SIRT1 may play a beneficial role in DCM [13,14], but the underlying mechanisms are not clearly elucidated.
In a previous study, 21 different molecules were identified as activators of SIRT1, of which resveratrol (2,3,4 ′ -trihydroxystilbene) gained most attention [15]. Resveratrol, found to be linked to the cardiovascular benefits of red wine, has been shown to significantly increase SIRT1 activity through allosteric interaction, resulting in the increase of SIRT1 affinity for both NAD + and the acetylated substrate [15,16]. Resveratrol is a potential candidate for the treatment of cardiovascular diseases (including atherosclerosis, hypertension, myocardial ischemia, and heart failure), owing to its protective antioxidant, anti-inflammatory, and anti-angiogenic properties [17,18]. In the majority of studies to date, resveratrol has been employed as an effective activator for SIRT1. In the work of Yu et al., Cote et al., and Liu et al., resveratrol was demonstrated to produce beneficial effects by enhancing the activation of SIRT1 [19][20][21][22].
To date, it is still unknown whether the regulatory effect of SIRT1 on mitochondrial dynamics could be beneficial in the pathological process of DCM. In the present study, we hypothesized that SIRT1 may exert a protective effect against the development of DCM through mitochondrial regulation. We applied DCM mouse model and in vitro high glucose (HG) cultured H9c2 cell model to investigate whether SIRT1 played an essential role in the development of DCM. To further confirm the crucial benefits of SIRT1, cardiac-specific SIRT1 knockout mice were generated and lentiviral vector targeting SIRT1 shRNA was synthesized. Besides, we investigated mitochondrial biogenesis and function indexes including mitochondrial DNA amount, ATP production, mitochondrial membrane potential, and mitochondrial morphological alterations. Finally, expressions of downstream proteins including PGC-1α were tested to determine the signaling pathway.

Methods
2.1. Ethics. All animal study procedures were performed in accordance with the Chinese National Institutes of Health. The experimental protocol was approved by the Fourth Military Medical University Committee on Animal Care.

2.2.
Generation of the Cardiac-Specific SIRT1 Knockout (SIRT1 KO ) Mice. Cardiac-specific SIRT1 knockout mice (SIRT1 KO ) were generated by crossbreeding SIRT1 flox5-6/ flox5-6 with Myh6-Cre + transgenic mice. SIRT1 flox5-6/flox5-6 129/FVB/Black/Swiss transgenic mice were generously presented by Professor Yongzhan Nie (State Key Laboratory of Cancer Biology and Xijing Hospital of Digestive Diseases, Xi'an, Shaanxi, China). Myh6-Cre C57BL/6a transgenic mice were purchased from the Jackson Lab (011038, the Jackson Laboratory, USA). Successful knockout of SIRT1 in myocardium was confirmed by PCR and Western Blot analysis. The PCR cycling conditions for SIRT1 were a primary denaturation at 94°C for 5 min, followed by 30 cycles of 45 s at 94°C, annealing temperature at 59°C for 45 s, and 72°C for 45 s, with a final extension of 5 min at 72°C. Primer sequences for PCR were as follows: Myh6-Cre: forward ATGACAGACAGATCCCTCCTATCTCC and reverse CTCATCACTCGTTGCATCATCGAC and floxed SIRT1 gene: forward GTGGAGGTCAGAAGATCAACC and reverse CACATCTTACACAGATCCAC.
2.3. Animal Grouping and Treatment. Mice were divided into six groups: control group (Con), diabetic cardiomyopathy group (DCM), DCM + resveratrol-treated group (DCM + RES), cardiac-specific SIRT1 knockout mouse group (SIRT1 KO ), SIRT1 KO + DCM group (SIRT1 KO + DCM), and SIRT1 KO + DCM + resveratrol-treated group (SIRT1 KO + DCM + R) (n = 10, each group). DCM mouse model was conducted as follows: eight-week-old mice were intraperitoneally injected with streptozotocin (STZ, Sigma, St. Louis, MO, USA) at the concentration of 150 mg/kg in citrate buffer (pH = 4.5) for seven consecutive days, while controlled mice received citrate buffer of the same volume. The blood glucose level was detected with a glucometer (Sannuo Biotech Ltd., Changsha, Hunan, China). In vivo experiments including echocardiography, PET/CT imaging, historical staining, and Western Blot were performed at least eight weeks after the establishment of a diabetic animal model. Mice with the fasting blood glucose level of higher than 350 mg/dL were considered as diabetic. RES-grouped mice were intraperitoneally treated with resveratrol of 25 mg/kg/d for five consecutive days. All animals had free access to water and food during the experiment. Animals were kept in plastic cages with well-ventilated stainless steel grid tops with a 12-hour light cycle (8am-8pm). The room temperature was maintained at 18-22°C.
2.6. TMRM Fluorescence Imaging. Mitochondrial membrane potential was measured using tetramethylrhodamine methyl ester (TMRM) fluorescence imaging with a MitoPT TMRM Assay Kit (ImmunoChemistry Technologies, LLC, Bloomington, USA) according to the manufacturer's instruction. Fluorescence images were visualized by a confocal microscope (Olympus FV 1000, Olympus, Tokyo, Japan) at 543 nm excitation and 580 nm emission. 2.9. Mitochondrial DNA Amount. Mitochondrial DNA (mtDNA) amount was determined by the ratio of mtDNA to nucleic DNA, which were measured by quantitative real-time PCR. Quantitive PCR was performed using the following primer sequences: mtDNA-specific PCR: forward CCGCAAGGGAAAGATGAAAGA and reverse TCGTTTG GTTTCGGGGTTTC and nuclear DNA-specific PCR: forward GCCAGCCTCTCCTGATGT and reverse GGGAAC ACAAAAGACCTCTTCTGG. The PCR amplification conditions were a primary denaturation at 94°C for 10 min, followed by 30 cycles of 1 min at 94°C, 1 min at 56°C, and 1 min at 72°C, with a final extension of 5 min at 72°C.

Western Blot Analysis.
Myocardium tissue was harvested for Western blot as described previously [23]. Cells of each group were harvested at appropriate time. Cells were washed three times with PBS and collected after ice-cold lysis buffer digestion. Protein lysates were separated on 10% SDS-PAGE gels and transferred onto nitrocellulose (NC) membrane. Membranes were blocked with 5% milk in 1 × TBS-Tween-20 buffer and incubated overnight at 4°C with primary antibodies. Then, membranes were washed in Tris-buffered saline with Tween, followed by incubation with the corresponding secondary antibodies at room temperature for 1 h. The blots were developed using an enhanced chemiluminescence kit (Millipore, Billerica, MA, USA) and visualized with UVP Bio-Imaging Systems. Blot densities were analyzed using ImageJ Software (National Institutes of Health, Bethesda, MD).

Transmission Electron Microscopy (TEM)
. TEM was performed to observe morphological mitochondrion changes in myocardium as previously described. Briefly, hearts were removed from anesthetized mice and washed with PBS solution. A specimen of the left ventricular myocardium was cut into ultrathin sections with a thickness of 60-64 nm. Sections were taken after fixation, stepwise alcohol dehydration, embedding, polymerization, sectioning, and staining. Images were observed with an electron microscope (JEM-2000EX TEM, JEOL Ltd., Tokyo, Japan). Random sections were taken and visualized by a blinded technician.

Statistical
Analysis. Data was expressed as mean ± standard deviation (SD). SPSS15.0 (SPSS Inc., USA) and Prism5.0 (GraphPad Software, USA) were used to perform the one-way analysis of variance (ANOVA) for evaluating the differences among different experimental groups. Pairwise multiple comparisons were used to identify the parameter differences between the two groups using the ANOVA-conjuncted Tukey test. Data was analyzed using parametric test assuming Gaussian distribution. p value < 0.05 was considered significant.

Resveratrol Alleviated Cardiac Dysfunction in DCM
Mouse Heart. Ventricular hypertrophy, myocardial fibrosis, and cardiac dysfunction are major characteristics of DCM hearts. As shown in Figures 1(a) and 1(b), myocardial hypertrophy in STZ-induced DCM mice was characterized by increased heart weight/tibia length (89.2 ± 2.86 versus 72.0 ± 5.73, p < 0 05, DCM versus Con group) and enhanced ventricle/body weight (g/kg, 3.16 ± 0.22 versus 2.47 ± 0.09, p < 0 05, DCM versus Con group). In addition, myocardial hypertrophy in DCM was also evidenced by increased expression of atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) (Figure 1(c)). Echocardiography results (Figure 1 Figure 1(e)). Additionally, masson trichrome staining demonstrated bits of fibrogenesis in the myocardium of the DCM group, and resveratrol markedly alleviated these changes in DCM mice (Figure 1(e) Figure 2(a)) were perceived as heterozygous (Heter) mice in which SIRT1 was partially expressed in myocardium.
Mice with genotype of SIRT1 flox−/− and Myh6-Cre + (same background, but normal SIRT1 expression, shown in the third column in Figure 2(a)) were used as control mice. Mice with genetype of SIRT1 flox+/+ and Myh6-Cre + (shown in the fourth column in Figure 2(a)) were considered as SIRT1 KO mice. Figure 2(b) shows that there was almost no SIRT1 mRNA in SIRT1 KO mouse cardiac tissue (0.53 ± 0.07 in the Heter group, and 0.03 ± 0.02 in the SIRT1 KO group). In addition, SIRT1 protein was also barely expressed in SIRT1 KO mouse myocardium (Figure 2(c)), indicating the successful knockout of SIRT1 in myocardium. As for the organ-specific knockout characteristics of Cre-loxP recombination system, SIRT1 was normally expressed in other organs such as the lungs, kidneys, and brain (shown in Supplementary Figures S1A and S1B available online at https://doi.org/10.1155/2017/4602715).  There was almost no SIRT1 mRNA in SIRT1 KO mouse cardiac tissue ( * p < 0 05 in Heter group, * * p < 0 01 in SIRT1 KO group). (c) SIRT1 protein was also barely expressed in SIRT1 KO mice myocardium ( * * p < 0 01 in SIRT1 KO group). (d) Increased heart weight/tibia length ratio in SIRT1 KO mouse myocardium and ventricle/heart weight in SIRT1 KO mouse myocardium ( * p < 0 05). (e) The mRNA expressions of atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) were significantly increased in SIRT1 KO mice ( * p < 0 05). (f ) SIRT1 KO mouse hearts displayed structural changes and fibrogenesis in the myocardium. (g) Cardiac function was impaired in SIRT1 KO mice as compared with WT and Heter mice ( * p < 0 05) (Figure 1(g)).
* p < 0 05 versus WT. WT: wild type; Heter: heterozygous. ventricular hypertrophy and cardiac dysfunction. Figure 2( (Figure 2(e)). Resembling DCM mice, SIRT1 KO mouse hearts also displayed structural changes, such as unbalanced cellular structures or aggregation of necrotic and inflammatory myocytes, and masson trichrome staining demonstrated bits of fibrogenesis in the myocardium of SIRT1 KO heart (Figure 2(f) (Figure 1(g)). These data suggest that cardiac-specific SIRT1 knockout of SIRT1 was sufficient to duplicate the phenotype of DCM, demonstrating that the existence of SIRT1 may exert a crucial role in cardiac function and structure modeling in diabetic hearts.

Myocardial Metabolic and Mitochondrial Alterations in
DCM and SIRT1 KO Mice. TEM images (Figure 3(b)) revealed morphological and mitochondrial impairment in both DCM and SIRT1 KO mice. Control heart showed normal tightly packed interfibrillar mitochondrion appearance. While in DCM and SIRT1 KO myocardium, interfibrillar mitochondria were observed less uniform and more fragmented, displaying swollen appearance with loss of discernable cristae. Excessive accumulation of glucose granules was also observed in DCM heart. Additionally, SIRT1 activation by resveratrol could alleviate these mitochondrial changes in DCM + RES mice (Figure 3(a)). Mitochondrial DNA (mtDNA) amount was an effective indicator for mitochondrial biogenesis. Figure 3(b) demonstrates that mtDNA amount was significantly decreased in the DCM, SIRT1 KO 18 F-FDG uptake in myocardium, which was significantly improved by resveratrol. Interestingly, SIRT1 KO also resulted in decreased 18 F-FDG uptake, but resveratrol did not ameliorate this in SIRT1 KO + DCM + R mice (Figure 3(d)).

SIRT1 Downregulation in H9c2 Cells by shRNA Lentiviral
Vector. GFP fluorescence images and flow cytometry results showed that H9c2 cells were optimally transfected at the MOI of 100 (Supplementary Figures S2A and S2B), which was the selected concentration in our later experiments. Western blot was done to reveal the effectiveness of SIRT1 downregulation in four different shRNA interfering sequences (Supplementary Figure S2C) Figure S2D).

Discussion
Diabetic cardiomyopathy (DCM) is one of the most important causes for morbidity and mortality in diabetic patients, characterized by diastolic dysfunction in early stages, proceeding decreased systolic function and eventual heart failure, which are independent of other cardiac diseases such as coronary heart disease or atherosclerosis [25]. The pathogenesis of DCM is not clearly illuminated, and existing treatment options are limited. Several studies considered mitochondria as a promising target for the management of DCM. Our present study demonstrated that cardiac-specific knockdown of SIRT1 is sufficient to cause phenotypes resembling DCM in mice heart and that SIRT1 played a beneficial role against the development of DCM. SIRT1 activation by resveratrol alleviated decreased cardiac function, impaired mitochondrial biogenesis, and function in DCM mice. Furthermore, SIRT1 improved mitochondrial dynamics through the deacetylation of PGC-1α and regulation of downstream proteins such as NRF-1, NRF-2, ERR-α, and TFAM. Clinically, diabetes mellitus (DM) is categorized into type 1 DM and type 2 DM [26,27]. Type 1 insulin-dependent DM accounts for about ten percent while type 2 DM is considered as the etiology of over 80 percent of all diabetics. Apart from a single state of hyperglycemia, type 2 diabetes is usually accompanied by obesity-induced insulin resistance and hyperinsulinaemia, which could have a nonnegligible effect on insulin signaling and lead to cardiac hypertrophy [28]. The use of type 1 diabetic model could effectively avoid these changes. In most animal studies of type 1 diabetes mellitus, diabetes is induced by the administration of pancreatic beta-cell toxin streptozotocin (STZ) [29]. In vivo researches in these rodent models have provided echocardiography evidence for systolic and diastolic dysfunction [30,31]. Some studies were also performed in isolated perfused hearts and revealed depressed cardiac function, according to a recent review by Severson [32]. Additionally, animal models provide the opportunity to conduct mechanistic studies for DCM. Several hypotheses have been proposed, including impaired calcium homeostasis, activation of the reninangiotensin system, increased oxidative stress, mitochondrial dysfunction, and altered substrate metabolism [29]. In our present study, we established type 1 diabetic model by STZ administration even though the fact is that type 2 diabetes is more popular than type 1 diabetes in humans. Cardiac hypertrophy and fibrosis are two essential characteristics of DCM that caused diabetic cardiac dysfunction. As shown in our Results, STZ-induced DCM model was accompanied by enhanced ventricle weight, increased ANP and BNP level, myocardial fibrosis, and impaired cardiac function.
Considering the multiple functions of SIRT1 in various cell types, generalized knockout of SIRT1 may exert a complicated effect on metabolism in the whole body, which would confuse the results in our study. Furthermore, high perinatal mortality was reported in generalized SIRT1-deficient mice [33], and whole body SIRT1 knockouts suffer from severe growth retardation and a number of developmental defects [34]. Thus, we used Cre-loxP recombination system, by which the desired gene modification can be restricted to certain cell types, to generate cardiac-specific SIRT1 knockout mice to avoid abovementioned conditions. [35]. As shown in our Western blot results, SIRT1 protein was specifically knocked out in heart tissue while normally expressed in other tissues such as lung, brain, and kidney. Additionally, specific SIRT1 deletion in myocardium did not affect the survival rate and whole body weight in SIRT1 KO mice, which indicated that SIRT1 deficiency did not affect growth and development in immature mice. A major innovative finding of our present study is that cardiac-specific knockdown of SIRT1 is sufficient to induce cardiac phenotypes resembling DCM in mice. Cardiac function in SIRT1 KO mice was markedly reduced as compared with that in WT mice, accompanied with cardiac hypertrophy and fibrosis, indicating the crucial role of SIRT1 in cardiac function. Moreover, interestingly, cardiac function reduction in DCM mice was accompanied by decreased SIRT1 expression, demonstrating that the impaired heart function was associated with SIRT1 deficiency.
Even though antidiabetic effects of resveratrol have been widely studied, the low bioavailability of resveratrol raises questions about whether the beneficial effects of oral resveratrol can act directly on diabetic myocardial tissue [22]. We show here that intraperitoneal injection of resveratrol reversed DCM-induced reduction in SIRT1 protein level while also enhancing cardiac function in DCM heart. However, due to the multifunctional properties of resveratrol, it could also exert beneficial effects against DCM via other mechanisms, such as antioxidant and anti-inflammatory effects [36]. A study by Guo et al. demonstrated that resveratrol attenuated high glucose-induced oxidative stress and cardiomyocyte apoptosis through the suppression of NADPH oxidase-derived ROS generation and the activation of antioxidant defenses via the regulation of AMPK pathway [37]. Additionally, Huang et al. reported that resveratrol prevented cardiac dysfunction in diabetes by relieving nitrosative and oxidative stress [38]. In a recent study by Bagul and his colleagues, it was revealed that resveratrol ameliorated cardiac oxidative stress in diabetes through deacetylation of NF-kB and histone 3 [39]. In this current study, cardiac-specific SIRT1 knockout mice provided the opportunity to directly assess the effects of resveratrol in animals lacking functional SIRT1. Using this model, we clearly demonstrate that the ability of resveratrol to improve cardiac function in DCM is, at least partially, dependent on SIRT1.
Currently, the underlying mechanisms for the physiopathologic progression of DCM remain exclusive. Abundant evidence has shown that DCM is associated with multiple factors including impaired myocardial insulin signaling and calcium homeostasis, mitochondrial dysfunction, endoplasmic reticulum stress, abnormal coronary microcirculation, and activation of the sympathetic nervous system or renin-angiotensin-aldosterone system. These changes lead to excessive oxidative stress, myocardial fibrosis, cardiac diastolic dysfunction, and eventually systolic heart failure [40]. DCM-associated myocardial apoptosis and fibrosis contributed to the loss of cardiac function [41]. In accordance with previous findings, we found enhanced apoptosis and fibrosis in diabetic hearts accompanied by a significant reduction in cardiac function of EF and FS. Interestingly, similar tendency was found in SIRT1 KO mice. SIRT1 activation by resveratrol markedly reversed these changes in DCM mice but not in DCM + SIRT1 KO mice. Our results manifested that SIRT1 played an essential role in myocardium, and downregulation of SIRT1 due to DCM or SIRT1 KO was associated with cardiac dysfunction and increase in myocardial apoptosis and fibrosis.
It is well recognized that in diabetic hearts, the use of glucose is decreased. As evidenced by our data, SIRT1 is essentially involved in the regulation of cardiac metabolism. Similar to DCM hearts, the glucose uptake is inhibited in SIRT1 low-expressed hearts. More importantly, we provided evidence that SIRT1 knockdown led to cardiac insulin resistance, as low expression of SIRT1 caused reduced IRS2 expression and impaired insulin signaling in both in vivo and in vitro models. Insulin resistance is associated with mitochondrial dysfunction due to reduced insulinstimulated mitochondrial activity [42].
Mitochondria are the center of cellular metabolism and, thus, are highly linked to impaired metabolism associated with DCM. Significant data indicates that mitochondrion alterations may play an essential role in the development of DCM [43]. Here, our results demonstrate that a normal heart showed regular tightly packed interfibrillar mitochondria while DCM and SIRT1 KO hearts displayed swollen mitochondria with loss of discernable cristae and accumulation of glucose granules [44]. It was also reported that STZtreated mice had significant changes to interfibrillar mitochondrial population, including reduced mitochondrial size, cardio-lipid content, and electron transport activity [45]. Apart from morphological mitochondrial abnormities, the reduction of mitochondrial DNA (mtDNA) amount both in in vivo and in vitro under HG and SIRT1 KO conditions suggested a dysregulation in mitochondrial biogenesis. We further investigate mitochondrial functional indexes including mitochondrial complex IV enzyme activity and ATP production. Myocardium has a high rate of ATP production, and turnover is required to maintain continuous mechanical work. Normal myocardium depends on abundant mitochondrial ATP supply to properly develop force. During DCM, however, myocardial energetic balance is disturbed, contributing to the systolic and diastolic dysfunction. However, it must be noted that even though we observed concurrent mitochondrial dysfunction and altered mitochondrial morphology in DCM myocardium, the causative nature between functional and morphological changes of mitochondria in DCM is unclear [46]. In a recent study by Ni et al., mitochondrial ATP synthase and insufficient ATP production are important mechanisms contributing to DCM [47]. Their results are partially in accordance with our present, but they focus on ATP synthase complex V. In our current study, we found decreased complex IV enzyme activity and reduced ATP generation in HG and SIRT1 KO cells. Resveratrol increased complex IV enzyme activity and ATP production under HG condition, but this effect was diminished when SIRT1 was deficient. Mitochondrial dysfunction and abnormal biogenesis are central upstream defect inflicted on the heart of DCM [48]; herein, we demonstrate the possibility of targeting mitochondrial energetics through the activation of SIRT1 for the management of DCM.
As a result of these findings above, there has been a surge of interest in understanding the molecular mechanism and targets of SIRT1's protective effect against DCM. Notably, one gene whose decreased expression is consistently implicated in diabetic mice is the peroxisome proliferatoractivated receptor γ coactivator (PGC-1α) [49][50][51]. PGC-1α, a transcriptional coactivator, plays a central role in the regulation of myocardial metabolism and mitochondrial biogenesis. Although PGC-1α was reported to be upregulated in diabetes, we found unchanged PGC-1α expression in DCM and SIRT1 KO hearts. However, acetylated level of PGC-1α was altered, contributing to functional PGC-1α changes. SIRT1 physically interacted with deacetylated PGC-1α, consequently increasing PGC-1α activity. As a consequence, functional deacetylated PGC-1α was decreased under the condition of DCM and SIRT1 KO , leading to decreased mRNA and protein expression of mitochondrion-related genes of NRF-1, NRF-2, ERR-α, and TFAM. NRF-1, NRF-2, ERR-α, and TFAM are four of the most essential mitochondrial regulatory genes. The regulation effect of NRF-1 and NRF-2 on nucleus-encoded mitochondrial transcription factors is essential to the control of mitochondrial biogenesis [52]. Functional PGC-1 by SIRT1 stimulated a powerful induction of NRF-1 and NRF-2 gene expression, consequently increasing the expression of proteins involved in oxidative phosphorylation, and thus played an important role in the regulation of mitochondrial biogenesis and function [53]. ERR-α responds to signals central to the regulation of mitochondrial biogenesis and function, such as upon exposure to cold, fasting, and exercise [54,55]. Previous studies have shown that ERR-α is required for the ability of exogenously expressed PGC-1α to induce mitochondrial biogenesis and respiration [56]. Of particular, TFAM is a main regulator for the mtDNA copy number and plays a critical role in the stability of mtDNA via formation of nucleoid structure. TFAM is a key factor for mtDNA maintenance, and the expression of human TFAM in a mouse increased the amount of mtDNA almost in parallel with the increase in the TFAM [57].
However, there are some limitations in our current study. Firstly, even through resveratrol is widely used as an activator for SIRT1, its agonist effect is not exclusive to SIRT1. Chen et al. reported the activation effect of resveratrol on SIRT3 and the consequential cardiac protective effect [58]. Although we used SIRT1 KO transgenic mice and lentiviral vector targeting SIRT1 shRNA to testify that the beneficial effect of resveratrol was dependent on SIRT1 activation, specific SIRT1 agonist is still needed to obtain more convincing results. Secondly, as SIRT1 can interact with a variety of mitochondrion-related proteins apart from PGC-1α, more molecular experiments are needed to investigate more about the signaling pathway.

Conclusion
In conclusion, we have demonstrated that the expression of SIRT1 was markedly reduced in DCM hearts. And we have found, for the first time, that cardiac-specific low expression of SIRT1 caused both compromised insulin signaling and mitochondrial dynamic abnormity, contributing to phenotypes resembling DCM in the mouse heart. Our results strongly support the conclusion that SIRT1 have a beneficial effect on cardiac dysfunction caused by DCM or HG through mitochondrial biogenesis and functional regulations. Furthermore, the protective role of SIRT1 on mitochondria is via the deacetylation effect on PGC-1α, thus inducing the increased expression of mitochondrial regulatory genes of NRF-1, NRF-2, ERR-α, and TFAM. Collectively, SIRT1 may serve as a potential therapeutic target for the management of DCM.

Conflicts of Interest
No potential conflicts of interest relevant to this article were reported.