The Role Of SIRT3 Signaling Pathway In Mediating The Protective Effects Of Melatonin On Cardiovascular Diseases

Background:Melatonin(Mel)is an endogenous hormone secreted mainly by the pineal gland and its biological activities include sedation,hypnotic effect,blood pressure-regulation,anti-oxidation,anti-inflammation,anti-apoptosis and so on.In recent years,a large number of basic and clinical researches have shown that melatonin can effectively ameliorate various cardiovascular diseases.In addition,considering that melatonin is cheap and safe,it represents a promising drug to treat cardiovascular diseases.Convicing evidence have suggested that the cardioprotective effects of melatonin are mainly due to its ability to regulate mitochondrial function,but the underlying mechanisms remain elusive,which largely limits its clinical application.SIRT3 is a NAD+-dependent deacetylase mainly distributed in mitochondria.It closely participates in regulating mitochondrial pathophysiological processes such as oxidative stress,energy metabolism,apoptosis and mitochondrial biogenesis via deacetylating mitochondrial proteins.More importantly,a growing number of studies have shown that SIRT3 is a potential intervention target for various cardiovascular diseases such as myocardial ischemia reperfusion(MIR)injury,myocardial infarction,diabetic cardiomyopathy(DCM),chemotherapy drug-induced cardiotoxicity and heart failure,which reflects that it has become a hot spot in the field of cardiovascular protection.However,whether SIRT3 mediates the cardioprotection of melatonin has not bee n reported.In addition,further studies are needed to clarify how melatonin re gulates SIRT3 signaling pathway in cardiovascular diseases.Objective:The aims of this study were to investigate the protective effects of melatonin on MIR injury and cardiac remodeling in type 2 diabetes mellitus by in vivo and in vitro experiments,and to clarify the following key issues:1)Whether the SIRT3 signaling pathway mediates the protective effects of melatonin against MIR injury;2)Whether the SIRT3 signaling pathway mediates the protective effects of melatonin against cardiac remodeling in T2DM;3)To elucidate the upstream and downstream signaling pathways of SIRT3 in mediating the cardioprotective effects of melatonin.Methods:1)Construction of MIR-injured mouse model: Mouse MIR injury model was established by transient ligation(30 minutes)of coronary artery left anterior descending artery;a single dose of melatonin(20 mg/kg,i.p.)was administrated 10 minutes before reperfusion.The protein expression of molecules and the parameters of oxidative stress were measured 3 hours after reperfusion.The heart function,myocardial infract size and myocardial apoptotic ratio were detected 24 hours after reperfusion.2)Construction of type 2 diabetes mellitus(T2DM)mouse model: The T2 DM mouse model was established by a single intraperitoneal injection of str eptozotocin(STZ,100 mg/kg)after 3 months of high-fat diet(HFD).Five days after STZ injection,the 3 h-fasting blood glucose of mice in each group was detected,and the mice with the blood glucose level ≥ 13.88 mmol/L were considered to be T2 DM mice.T2 DM mice were then treated with HFD and melatonin(20 mg/kg/day,via drinking water)for 4 months.3)Echocardiography: The left ventricular ejection fraction(LVEF)and left ventricular fractional shortening(LVFS)were measured using the Vevo 2100 ultrasound imaging system for small animal research.4)Hemodynamic testing: The in vivo hemodynamic parameters in each group were measured using the Millar catheter.The indices measured or calculated included the left ventricular end-diastolic pressure(LVEDP),the maximal slope of the systolic pressure increment(+d P/dt)and the maximal slope of the diastolic pressure decrement(-d P/dt).5)Determination of myocardial infarct size and serum lactate dehydrogenase(LDH)activity after ischemia reperfusion: The myocardial infarct size of each group was detected using TTC/Evans blue double staining technique.The blue-stained portion indicates the nonischemic,normal region;the red-stained portion indicates the ischemic/reperfused but not infarcted region;and the non-stained portion indicates the infarcted region.The infarct size was expressed as the infarct area/the ischemic area×100%.In addition,serum LDH activity of mice in each group was measured using a LDH activity detection kit.6)Determination of oxidative stress: The reactive oxygen species(ROS)production in cardiac tissue was detected by DHE staining.The ROS production in H9c2 cells and cardiac fibroblasts were detected by DCFH-DA staining and Mito-Tracker/Mito-SOX double staining techniques,respectively.The malonaldehyde(MDA)content,superoxide dismutase(SOD)activity and glutathione peroxidase(GSH-Px)activity were detected by commercially available kits.The protein expression of NADPH oxidase 2(NOX2),nuclear factor(erythroid-derived 2)-like 2(Nrf2)and NAD(P)H quinone dehydrogenase 1(NQO1)were detected by Western blotting.7)Determination of apoptosis: The apoptotic ratio was detected by TUNEL/DAPI staining,and was expressed as TUNEL-positive nuclei/total nuclei×100%.In addition,the protein expression of B-cell lymphoma-2(Bcl-2),Bcl-2-associated X protein(Bax),mitochondrial Bax(Mito-Bax),Caspase-3,cleaved Caspase-3 and cytoplasmic cytochrome C(Cyt-Cyto C)were detected by Western blotting.8)Determination of inflammatory response: The macrophage infiltration in the heart tissue was detected by immunofluorescence staining of CD68(a marker of macrophage).The protein expression of tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β)and interleukin-6(IL-6)were detected by Western blotti ng.9)Determination of cardiac hypertrophy: The heart weight/tibial length(HW/TL)ratio was calculated by measuring the heart weight and tibial length;the mea n cross-sectional area of the left ventricular myocytes was detected by HE staining.10)Determination of cardiac fibrosis: The perivascular and interstitial collagen deposition were detected by Masson and Sirius red staining.The expression of collagen I and collagen III in heart tissue were detected by immumohistochemical staining.The protein expression of transforming growth factor β1(TGF-β1),connective tissue growth factor(CTGF),p-Smad3 and Smad3 were detected by Western blotting.11)Cell viability: The cell viability of H9c2 cells and cardiac fibroblasts were detected using the CCK-8 kit.12)Fibroblasts to myofibroblasts transformation: Fibroblasts to myofibroblasts transformation was detected by measuring the expression of α-smooth muscle actin(α-SMA)using cellular immunofluorescence staining and Western blotting.Results:1)Melatonin effectively ameliorated MIR injury in mice.Compared with the Sha m group,the LVEF and LVFS were significantly decreased,while the myocardial infarct size and serum LDH activity were significantly increased in the IR+V group.Compared with the IR+V group,melatonin significantly increased LVEF and LVFS,reduced myocardial infarct size,and decreased serum LDH activity following MIR injury.In addition,compared with the Sham group,the expression and activity of SIRT3 were significantly decreased,while the acetylation of SOD2 was significantly increased in the IR+V group.Compared with the IR+V group,melatonin up-regulated the expression and activity of SIRT3 and decreased the acetylation of SOD2.2)Melatonin attenuated oxidative stress and myocardial apoptosis induced by MIR in mice.Compared with the Sham group,the expression of gp91 pho x and the content of MDA were significantly increased,while the activities of SOD and GSH-Px,and the expression of Nrf2 and NQO1 were significantly decreased in the IR+V group,suggesting that MIR induced oxidative stress in heart tissues.In addition,compared with Sham group,the apoptotic ratio and the expression of Cyt-Cyto C,total Bax,Mito-Bax,Caspase-3 and cleaved Caspase-3 were significantly increased,while the expression of Bcl-2 was significantly decreased in the IR+V group,suggesting that the myocardial apoptosis is activated after MIR.Melatonin largely reversed the above changes caused by MIR,indicating that melatonin could inhibit oxidative stre ss and myocardial apoptosis induced by MIR.3)SIRT3/SOD2 signaling pathway mediated the protective effects of melatonin against MIR injury in mice.Firstly,compared to the IR+Mel group,LVEF and LVFS were significantly reduced,and myocardial infarct size a nd serum LDH activity were significantly increased in the IR+Mel+3-TYP(SIRT3 selective inhibitor)group.Secondly,compared with the IR+Mel group,the expression of gp91 phox and the content of MDA were significantly increased,while the activities of SOD and GSH-Px and the expression of NQO1 were significantly decreased in the IR+Mel+3-TYP group.In addition,compared with the IR+Mel group,the apoptotic ratio and the expression of Cyt-Cyto C,total Bax,Mito-Bax,Caspase-3 and cleaved Caspase-3 were signi ficantly increased,while the expression of Bcl-2 was significantly decreased in the IR+Mel+3-TYP group.4)Melatonin ameliorated ischemia reperfusion injury in H9c2 cells through activating the SIRT3/SOD2 signaling pathway.In order to determine whether melatonin directly acted on cardiomyocytes and further elucidate the underlying mechanisms of melatonin against MIR injury,the H9c2 cells were treated with an ischemic buffer for1 h and then treated with serum-free DMEM for 4 h to simulate ischemia reperfusion(SIR)injury.The results showed that the cell viability of H9c2 cells was significantly reduced after SIR.Melatonin(50,100 and 200 μM)showed little effect on the cell viability of normal H9c2 cells,however,melatonin increased the cell viability o f SIR-treated H9c2 cells in a dose-dependent manner.The pro-survival effect was most obvious with 100 μM melatonin.Compared with the SIR group,melatonin treatment significantly inhibited SIR-induced oxidative stress and cardiomyocyte apoptosis.Consistent with the in vivo results,melatonin activated the SIRT3/SOD2 signaling pathway in H9c2 cells following SIR treatment.Inhibiting the SIRT3 expression using SIRT3 si RNA largely attenuated the protective effects of melatonin mentioned above.5)Melatonin activated SIRT3/SOD2 signaling pathway in heart tissue after ischemia-reperfusion in a silent information regulator 1(SIRT1)-dependent manner.Notably,the expression levels of SIRT1 and SIRT3 in heart tissue were both significantly decreased after MIR,and melatonin could simultaneously up-regulate the expression of SIRT1 and SIRT3.Inhibiting the SIRT1 signaling pathway using EX527 or SIRT1 si RNA decreased the expression and activity of SIRT3.Inhibiting the SIRT3 signaling pathway using 3-TYP or SIRT3 si RNA showed little effect on the expression and activity of SIRT1.These data indicate that SIRT1 acts as the upstrea m signal of SIRT3 during MIR injury.6)The AMPK-SIRT3 signaling pathway in the heart tissue of T2 DM mice is significantly impaired,while melato nin preserved the AMPK-SIRT3 signaling pathway.To elucidate the role of the AMPK-SIRT3 signaling pathway in melatonin’s cardioprotective effects against cardiac remodeling in diabetic cardiomyopathy,we used Compound C(CC)and 3-TYP to selectively inhibit AMPK and SIRT3,respectively.Compound C treatment significantly inhibited AMPK phosphorylation and decreased SIRT3 expression and activity;while 3-TYP or SIRT3 si RNA inhibited SIRT3 signaling pathway,and showed little effect on AMPK phosphorylation.T hese data indicate that AMPK acts as the upstream signal of SIRT3.7)Melatonin improved cardiac hypertrophy and cardiac systolic and diastolic dysfunction induced by T2 DM through activating the AMPK-SIRT3 signaling pathway.Compared with the Control group,the HW/TL ratio,the mea n cross-sectional area of the left ventricle and LVEDP were significantly increased,while LVEF,LVFS,+d P/dt and-d P/dt were significantly decreased in the DM group.Melatonin significantly reversed the above changes caused by T2 DM.Inhibiting the AMPK-SIRT3 signaling pathway largely attenuated the above-mentioned protective effects of melatonin against cardiac hypertrophy and cardiac dysfunction.8)Melatonin inhibited cardiac fibrosis induced by T2 DM through activating the AMPK-SIRT3 signaling pathway.Compared with the Control group,the amount of perivascular and interstitial collagen deposition,the expression of Collagen I,Collagen III,TGF-β1,CTGF and α-SMA and the phosphorylation of Smad3 were significantly increased in the DM group.Melatonin significantly reversed the above changes caused by T2 DM.Inhibiting the AMPK-SIRT3 signaling pathway largely attenuated the above-mentioned protective effects of melatonin against cardiac fibrosis.9)Melatonin inhibited myocardial inflammation and oxidative stress induced by T2 DM through activating the AMPK-SIRT3 signaling pathway.Compared with the Control group,the expression of TNF-α,IL-6 and IL-1β and macrophage infiltration were significantly increased in the DM group.In addition,compared with the Control group,the ROS production,MDA content and NOX2 expression were significantly increased,while the SOD activity and GSH-Px activity were significantly decreased in the DM group.Melatonin significantly reversed the above changes caus ed by T2 DM.Inhibiting the AMPK-SIRT3 signaling pathway largely attenuated the anti-inflammatory and anti-oxidative effects of melatonin mentioned above.10)Melatonin inhibited high glucose-induced cardiac fibroblasts proliferation,fibroblast-to-myofibroblast transformation,and synthesis of fibrosis-associated proteins through activating the AMPK-SIRT3 signaling pathway.Compared with the NG group,the AMPK-SIRT3 signaling pathway was significantly impaired in the HG group,while melatonin preserved the AMPK-SIRT3 signaling pathway in high glucose-treated cardiac fibroblasts.In addition,compared with the NG group,the cell viability,the expression of Collagen I,Collagen III,TGF-β1,CTGF and α-SMA,and the phosphorylation of Smad3 were significantly increased in the HG group.Melatonin significantly reversed the above changes caused by high glucose.Inhibiting the AMPK and SIRT3 signals using Compound C and SIRT3 si RNA largely attenuated the anti-fibrotic effects of melatonin mentioned above.11)Melatonin inhibited high glucose-induced oxidative stress in cardiac fibroblasts through activating the AMPK-SIRT3 signaling pathway.Compared with the NG group,the ROS production,MDA content and NOX2 expression were significantly increased,while the SOD activity and GSH-Px activity were significantly decreased in the HG group.Melatonin alleviated the oxidative stress in high glucose-treated cardiac fibroblasts,while inhibiting the AMPK-SIRT3 signaling pathway largely attenuated the anti-oxidative effects of melatonin.Conclusions:1)We first demonstrated that melatonin ameliorates MIR injury by inhibiting myocardial mitochondrial oxidative stress damage and the activation of apoptotic pathways via activating the SIRT3 signaling pathway,and that melatonin activates t he SIRT3 signaling pathway in cardiac tissue in a SIRT1-dependent manner;2)We first demonstrated that melatonin alleviates cardiac remodeling in type 2 diabetic cardiomyopathy by inhibiting cardiac oxidative stress and inflammatory response via activating the SIRT3 signaling pathway,and that melatonin activates the SIRT3 signaling pathway in type 2 diabetic heart mainly in an AMPK-dependent manner.In addition,melatonin inhibits high glucose-induced cardiac fibroblasts proliferation and fibroblast-to-myofibroblast transformation by inhibiting oxidative stress via an AMPK-SIRT3-dependent manner;3)Through the in vivo and in vitro experiments,we confirmed that melatonin represents an effective strategy for relieving MIR injury and cardiac remodeling in type 2 diabetic cardiomyopathy,elucidated the underlying mechanisms of melatonin in detail,and provided a new theoretical basis for the clinical promotion and application of melatonin in cardiovascular diseases.