| BackgroundLeft ventricular hypertrophy (LVH) is an adaptive response to pressure or volume stress, mutations of sarcomeric proteins or loss of contractile mass from prior infarction, which is accompanied by many forms of heart disease, including hypertension, valvular disease and ischemic disease. In these types of cardiac pathology, pressure overload-induced concentric hypertrophy is believed to have a compensatory function by enhancing contractile strength, diminishing wall stress and oxygen consumption. However, ventricular hypertrophy is also associated with significantly increased risk of heart failure and malignant arrhythmia. Thus, it is important to suppress hypertrophy without provoking circulatory insufficiency.To accomplish this goal, it is critical to elucidate the mechanisms underlying the maladaptive features of hypertrophy. Of the many maladaptive features of hypertrophy, energy metabolism, which reverses the oxidation of long-chain fatty acid to increased glucose utilization, makes a large difference in the process of hypertrophy. On the one hand, it decreases oxygen consumption per mole of ATP generated, which could help reduce cellular reactive oxygen species (ROS), and on the other hand, maladaptive features exist, including increased lipid accumulation in the heart stemming from chronically impaired oxidation of fatty acid, lactic acid accumulation, and diminished maximal ATP generation from glycolysis. Van et al. raised the concept of myocardial metabolic remodeling at 2004, namely by changes in cardiac energy metabolism pathways, mitochondrial dysfunction and high-energy phosphate metabolic abnormalities which all happens during the process of myocardial chronic pressure-overload and substrate supply shift. However, the molecular mechanism through which lipid accumulates in the heart during hypertrophy remains unknown.The sirtuins are a class of NAD+-dependent deacetylase comprising seven members. Shore et al. discovered the first sirtuin in 1984 in yeast, interest did not really take off until an effect on life span was noted. The nature of sirtuins as NAD+-dependent deacetylase was recognized in 2000 by Imai et al. Once their enzymatic actions in the cell started to be elucidated it was soon realized that their apparent ability to extend life span involved similar pathways as utilized by calorie restriction. Since all sirtuins are activated by NAD+, and the levels of intracellular NAD+are mainly decided by the state of energy metabolism in cells, the sirtuins are important in the transduction pathways emanating from energy sensingRecent studies have found that sirtuins are critical regulators of many cellular processes, including insulin secretion, the cell cycle, and apoptosis. It has been proven that sirtuins are novel therapeutics that inhibit metabolic disorders and combat associated diseases. Of the seven sirtuin analogues, SIRT3 is the only member whose increased expression has been linked to the longevity of humans. It has been proven that SIRT3 facilitates lipid, amino acid and carbohydrate metabolism. Moreover, recent studies have demonstrated that SIRT3 also exhibits protection effects on cardiomyocytes by reducing ROS production. SIRT3 has been proven to deacetylate and activate lots of mitochondrial key metabolic enzymes, such as acetyl CoA synthetase 2 (AceCS2), long-chain acyl CoA dehydrogenase (LCAD), succinate dehydrogenase and isocitrate dehydrogenase. Among which, LCAD is a key enzyme involved in fatty acid oxidation. However, the molecular basis of SIRT3-mediated protection on hypertrophy-related lipid accumulation remains unknown.Aims1.To explore the role of SIRT3 in mouse cardiac hypertrophy.2.To explore the role of SIRT3 in cardiac mitochondrial lipid metabolism.3.To explore the mechanism that overexpression of SIRT3 could attenuate lipid accumulation in cardiac mitochondria.Experiment materials and methods1. AnimalsThe animal experimental protocol complied with the Animal Management Rules of the Chinese Ministry of Health (Document No.55,2001) and was approved by the Animal Care and Use Committee of Shandong University. The SIRT3-KO mice were purchased from the Jackson Laboratories (USA).129 Wild-type mice at 6-7 weeks of age were purchased from the Department of Laboratory Animal Science of Peking University and served as controls (Beijing, China).2. Hypertrophic modelEight-week-old male mice (age-matched wild-type and SIRT3-KO mice) were subjected to constriction of the thoracic aorta. The animals were anesthetized using sodium pentobarbital, and the aorta was isolated from the adjacent tissue and banded between the carotid arteries over a 27-gauge needle, which was immediately removed. The animals subjected to sham surgery underwent an identical procedure with the exception of band placement. The animals were sacrificed six weeks after surgery, and their hearts were removed and analyzed for the development of cardiac hypertrophy and lipid analyses.3. ImmunohistochemistryHematoxylin and eosin staining and Masson’s trichrome staining were performed using standard procedures. The collagen volume fraction was quantified blindly using quantitative morphometry with an automated image analysis system (Image-Pro Plus).4. EchocardiographyThe animals were imaged in the left lateral decubitus position with a VisualSonics Vevo 770 machine using a 30 MHz high frequency transducer. Images were captured from M-mode, two-dimensional (2-D), pulse wave (PW) Doppler.5. Transmission electron microscopyThe embedded sections were stained using lead citrate and observed using a transmission electron microscope (TEM, H-7000FA, Hitachi, Tokyo, Japan, X15000).6. Triglyceride and cholesterol assaysThe triglyceride (TG) and cholesterol contents in the heart extracted by a chloroform/methanol (2:1) mixture were determined using the TG Assay Kit (Biovision Inc. Mountain View, CA, USA) and a Cholesterol/Cholesteryl Ester Quantitation Kit (Biovision Inc.).7. Isolated Langendorff perfusion and pa Imitate oxidation rateThe hearts were cannulated via the aorta and perfused in the Langendorff mode with KH buffer (gassed with 95% O2,5%CO2) at 37℃ and a constant perfusion pressure of 100 mmHg. After an initial 15-min stabilization period, the hearts were perfused with 250 ml of recirculating KH buffer containing 1 mM palmitate bound to 1.5% fatty acid-free bovine serum albumin (Sigma, USA) and 0.2 μCi ml-1 [9,10-3H] palmitate for 1 h. Aliquots of recirculating buffer were collected every 8 min during the perfusion protocol, and the palmitate oxidation rates were determined. Briefly, 3H2O was separated from 3H-palmitate in the buffer samples through chloroform: methanol Folch extraction, and the upper aqueous phase was assessed for radioactivity. The steady-state palmitate oxidation rates were calculated from the linear increase in 3H2O, which was expressed as μmol x gram of whole heart wet weight (gww-1) min-1.8. Cell culture, transfection and treatmentRat cardiomyocytes (H9C2) were obtained from American Type Culture Collection (ATCC) and maintained in a 5% CO2 humidified incubator at 37℃. For all of the virus transfection experiments, the viruses were used at a multiplicity of infection of 10. Rat cardiomyocytes (H9C2) were treated with PE (20μM) for 48 h to induce cardiomyocyte hypertrophy in vitro.9. Oil Red O staining and cholesterol and triglyceride measurements of cardiomyocytesThe lipid contents in cardiomyocytes were stained with Oil Red O. The cells were fixed in 10% formalin for 90 min. After washing thoroughly with distilled water, the cells were incubated with a working solution of Oil Red O for 3 h. The cholesterol and triglyceride contents in the cells were measured quantitatively through enzymatic colorimetric assays using kits purchased from Wako (Richmond, VA, USA) according to the manufacturer’s protocols. The concentrations of cellular proteins in these cells were measured using a protein assay kit obtained from Bio-Rad (Hercules, CA, USA).10. Western blotting analysisFrozen tissue and cell proteins were lysed by RIPA. And the extracted total proteins were separated by SDS-PAGE and transferred onto a PVDF membrane using a wet transfer apparatus (Bio-Rad, Hercules, CA, USA). The membranes were blocked with 5% non-fat milk, incubated overnight at 4℃ with the primary antibodies, and then incubated with the secondary antibodies labeled with horseradish peroxidase. The protein bands were visualized through enhanced chemiluminescence (Millipore). The protein levels were detected using an ImageQuant LAS4000 chemiluminescence reader (GE, USA).11. Statistical analysisData are presented as mean±standard error of the mean (SEM). Data analysis was performed with unpaired t-test or one-way analysis of variance (ANOVA) using SPSS18.0 software. P< 0.05 was considered as statistically significant.Results:1. SIRT3 was downregulated in the murine hypertrophic heart. We subjected the mice to TAC for six weeks. To ensure that the aortic binding was definite, we explored the velocity of the binding aorta one week after sham or TAC through echocardiology, and the velocity was significantly elevated after binding. The mice subjected to TAC produced nearly 20%,40%, and 60% cardiac hypertrophy, respectively, two, four, and six weeks after TAC, and we found that the short form of SIRT3 was downregulated after TAC compared with the sham controls, whereas the long form was slightly increased during mild hypertrophy but did not show obvious changes during severe hypertrophy. These results agreed with the findings of a previous study conducted by Sundaresan et al, indicating that only the short form of SIRT3 was consistently downregulated in mice subjected to TAC.2.SIRT3-KO mice had a propensity to develop heart failure in response to TAC.To further explore the role of SIRT3 in the development of cardiac hypertrophy, we subjected SIRT3-KO mice and their WT controls to TAC. In our experiments, we observed that the ratio of the heart weight to the body weight (HW/BW) in SIRT3-KO mice subjected to TAC was nearly 15% greater than that observed in the WT controls. Under sham conditions, the ratio was also higher in the SIRT3-KO mice. The histological examination of cardiac tissue sections from SIRT3-KO mice revealed signs of cellular necrosis in the hearts as shown by areas of cardiomyocyte loss or dropout, and higher levels of fibrosis were also observed in the SIRT3-KO mice compared with the WT controls under both sham and TAC conditions. Echocardiology was used to evaluate the cardiac function noninvasively, and the LV wall thickness of the SIRT3-KO mice was greater than that in the WT mice at baseline and was increased to significantly higher levels than those observed in the WT controls after TAC. In addition, the quantification of LV fractional shortening showed that the SIRT3-KO mice developed severe contractile dysfunction. These findings revealed that the SIRT3-KO mice had a propensity to develop impaired cardiac function and pathogenesis of heart failure, suggesting that SIRT3 may be required to block cardiac hypertrophy.3.SIRT3-KO mice displayed excessive lipid accumulation and decreased palmitate oxidation rates in the heart.Cardiac tissue sections from these mice were further examined by transmission electron microscopy, which showed marked changes in the mitochondrial morphology. It appeared that the SIRT3-KO mice did not exhibit any obvious cardiac phenotypes under normal conditions, and the transmission electron micrographs revealed an unusual pattern of lipid body formation in both types of mice after TAC. However, compared with the WT mice, the mitochondria from the transgenic mice became significantly swollen, the mitochondrial cristae were highly disturbed, and large areas of blebbing could be observed in the mitochondria. We then used a metabolomics approach to screen multiple metabolic pathways in the heart. The levels of triglyceride and cholesterol were increased in both WT and SIRT3-KO mice subjected to TAC for six weeks, and the level of triglyceride was even higher in the SIRT3-KO mice than the controls, which is suggestive of incomplete oxidation of fatty acid, verifying the transmission electron microscopy results. To directly assess fatty acid oxidation, the degree of ex vivo palmitate oxidation was measured in the hearts from WT and SIRT3-KO mice. In the WT mice, the palmitate oxidation rates were 30% lower in the hypertrophic hearts compared with the sham controls, and in the SIRT3-KO mice, the palmitate oxidation rates were significantly lower under both sham and TAC conditions. Collectively, these results revealed that SIRT3-KO mice subjected to hypertrophic stimuli displayed excessive lipid accumulation and decreased fatty acid oxidation rates in the heart.4.SIRT3 controlled the acetylation status of LCAD in vivo and in vitro.Because the enzymes involved in fatty acid oxidation are localized in the mitochondria, we intended to explore whether the short form of SIRT3 (28 kDa) participates in the regulation of cardiac fatty acid oxidation. The results showed obvious hyperacetylation of the mitochondrial proteins in SIRT3-KO mice, and in WT mice, a relative increase in the acetylation level of mitochondrial proteins was accompanied by a parallel decrease in the short form of SIRT3 (28 kDa). The endogenous mitochondrial proteins were immunoprecipitated with anti-acetyllysine antiserum and were analyzed by western blotting using an antibody specific to LCAD. This experiment demonstrated that LCAD was acetylated under normal conditions and became hyperacetylated during hypertrophy (Figure 4C), and when the same experiment was conducted using mitochondria extracted from SIRT3-KO mice, LCAD was profoundly hyperacetylated under both normal state and hypertrophy (Figure 4C). This result demonstrated that SIRT3 was necessary for LCAD deacetylation. To test the ability of SIRT3 to directly deacetylate LCAD, expression vectors encoding FLAG-tagged murine LCAD were cotransfected with an expression vector for SIRT3 or vehicle into cardiomyocytes. The acetylation levels for murine LCAD were measured by western blotting with an antibody for acetyllysine after immunoprecipitation with anti-FLAG antiserum. This experiment demonstrated that the overexpression of SIRT3 could deacetylate LCAD directly. Together, these observations showed that SIRT3 may be able to regulate fatty acid oxidation through the deacetylation of LCAD in the heart.5.Overexpression of SIRT3 attenuated lipid accumulation in vitro and blocked the cardiac hypertrophic response.We used phenylephrine (PE) (20 μM) or blank control (saline) to treat cardiomyocytes that were infected with Ad.SIRT3 or vehicle for 48 h. The overexpression of SIRT3 reduced the expression of a-SMA in cardiomyocytes. The PE-induced protein synthesis was measured by [3H]-leucine incorporation into the total cellular protein, which showed that the induction of protein synthesis was inhibited by the overexpression of SIRT3, and the analysis of the ANF and 3-MHC mRNA levels also demonstrated that the overexpression of SIRT3 could block the activation of fetal gene expression induced by PE stimuli. In addition, using an optical microscope, some droplets were observed in the cytosol of the cells in response to PE stimuli, and the cardiomyocytes of different groups were then stained with oil red, which could detect lipid accumulation in the cells. It was shown that the overexpression of SIRT3 attenuated lipid accumulation in response to PE stimuli. We also determined the intracellular accumulation of cholesterol and triglyceride in cardiomyocytes to confirm the apparent increase in the lipid content in response to PE stimuli. In the cells pretreated with Ad.SIRT3 infection compared with the controls, the cholesterol and triglyceride accumulation was reduced 40% and 50% respectively. These findings revealed that SIRT3 was able to protect cardiomyocytes from hypertrophic stimuli by attenuating lipid accumulation.Conclusions1.SIRT3 can improve mouse cardiac hypertrophy.2. SIRT3 can inhibit hypertrophy-related lipid accumulation in cardiac mitochondria.3.SIRT3 could regulate fatty acid oxidation by deacetylation of LCAD in mouse heart.BackgroundThe most extensive fibrotic remodeling of the ventricle is found associated with varieties of heart disease. Pressure overload, induced by hypertension or aortic stenosis, results in extensive cardiac fibrosis that is initially associated with increased stiffness and diastolic dysfunction; a persistent pressure load may eventually lead to ventricular dilation and combined diastolic and systolic heart failure. In animal models, pressure overload induces early hypertrophy, fibrosis, and diastolic dysfunction, followed by decompensation, dilative cardiomyopathy, and the development of systolic dysfunction.Disturbance of the tightly regulated balance between the synthetic and degradative aspects of collagen metabolism results in profound structural and functional abnormalities of the heart. Fibrosis disrupts the coordination of myocardial excitation-contraction coupling in both systole and diastole and may result in profound impairment of systolic and diastolic function. Increased deposition of interstitial collagen in the perimysial space is initially associated with a stiffer ventricle and diastolic dysfunction. However, active fibrotic remodeling of the cardiac interstitium is also associated with matrix degradation leading to the development of ventricular dilation and systolic failure. Disturbance of the collagen network in the fibrotic heart may cause systolic dysfunction through several distinct mechanisms. First, loss of fibrillar collagen may impair transduction of cardiomyocyte contraction into myocardial force development resulting in uncoordinated contraction of cardiomyocyte bundles. Second, interactions between endomysial components (such as laminin and collagen) and their receptors may play an important role in cardiomyocyte homeostasis. Finally, fibrosis may result in sliding displacement (slippage) of cardiomyocytes leading to a decrease in the number of muscular layers in the ventricular wall and subsequent left ventricular dilation. Beyond its profound effects on cardiac function, fibrotic ventricular remodeling also promotes arrhythmogenesis through impaired conduction and subsequent generation of re-entry circuits.Sirtuin3 is dependent on nicotinamide adenine dinucleotide (NAD) for its activity. SIRT3 is the only analogue whose increased expression has been found to be associated with extended lifespan of humans. SIRT3 level has been shown to be elevated by exercise and calorie restriction. Initial studies have shown that SIRT3 plays a major role in free fatty acid oxidation and maintenance of cellular ATP levels. In the heart SIRT3 has been found to block development of cardiac hypertrophy and protect cardiomyocytes from oxidative stress-mediated cell death. Both nuclear and mitochondrial substrates of SIRT3 have been identified, Expression of SIRT3 has also been implicated in the synthesis and maintenance of cellular ATP levels and could protect myocytes from genotoxic and oxidative stress mediated cell death.The desire to live longer and probably forever has long fascinated mankind. It seems our forefathers found a way to live longer and healthy by undergoing calorie restriction, a diet regimen that is considered to be impractical for modern society where food is surplus and time is scarce. Even though vaccination, antibiotics, better child care, and early disease-detection techniques in combination with modern drugs have helped us to increase our average lifespan, the quest to increase maximal lifespan still remains elusive. The major advances in ageing research that we have witnessed in the past two decades are the rediscovery of benefits of calorie restriction, and the delineation of the molecular mechanism involved in its protective effects. Many studies have proposed that the beneficial effect of calorie restriction is mediated through a set of genes collectively called sirtuins.Although CR also has beneficial effects in humans, such a diet is unlikely to be widely adopted and will pose significant risks to the frail, critically ill, and elderly. Therefore, the development of "CR mimetic" compounds has been the subject of focus to provide some of the benefits of CR without a reduction in calorie intake. The polyphenol resveratrol (RSV) is the first compound discovered able to mimic CR by stimulating SIRTs. In mice, long-term administration of RSV induces gene expression patterns that can be induced by CR and delays aging-related deterioration. Furthermore, RSV can inhibit myocardial hypertrophy and fibrosis in spontaneously hypertensive rats.Together, these studies imply an important role of SIRT3 in cardioprotection. Moreover, the positive health effects of CR and activation of SIRTs in aging-related diseases have provoked intense interest in the development of small-molecule activators of SIRTs. This study intended to build pressure load of myocardial hypertrophy model with TAC and explore the role of SIRT3 in cardiac fibrosis and differentiation of myofibroblasts, clarify its possible mechanism and signal pathway.Aimsl.To explore the role of SIRT3 in pressure overload-induced cardiac fibrosis.2.To examine the effect of resveratrol on expression of SIRT3 in the mouse heart.3.To explore the mechanism that activation of SIRT3 could improve cardiac fibrosis.Experiment materials and methods1. AnimalsThe animal experimental protocol was approved by the Animal Care and Use Committee of Shandong University and complied with the Animal Management Rules of the Chinese Ministry of Health (Document No.55,2001).129 Wild-type mice at 6-7 weeks of age were purchased from the Department of Laboratory Animal Science of Peking University and served as controls (Beijing, China). The SIRT3-KO mice were purchased from the Jackson Laboratories (USA).2. Dietary experimentsDetails of the methods used to house and feed the mice have been previously described. Control diet (CD)-fed groups were fed 86.4 kcal/wk of the precision pellet diet AIN-93M (BioServ, Frenchtown, NJ), and CR groups were fed 64.8 kcal/wk (25% CR) of the precision pellet diet AIN-93M. Mice were treated with RSV supplemented in drinking water daily, and mice were fed with RSV at 1.8 mg·kg-1·day-1.3. Hypertrophic modelEight-week-old male mice (age-matched wild-type and SIRT3-KO mice) were subjected to constriction of the thoracic aorta to establish mouse reactive cardiac fibrosis model. The animals were anesthetized using sodium pentobarbital, and the aorta was isolated from the adjacent tissue and banded between the carotid arteries over a 27-gauge needle, which was immediately removed. The animals subjected to sham surgery underwent an identical procedure with the exception of band placement. The animals were sacrificed eight weeks after surgery, and their hearts were removed and analyzed for the development of cardiac hypertrophy and lipid analyses.4. ImmunohistochemistryHematoxylin and eosin staining and Sirius red staining were performed using standard procedures. The collagen volume fraction was quantified blindly using quantitative morphometry with an automated image analysis system (Image-Pro Plus).5. EchocardiographyThe animals were anesthetized with isoflurane inhalation and imaged in the left lateral decubitus position with a VisualSonics Vevo 770 machine using a 30 MHz high frequency transducer. Images were captured from M-mode, two-dimensional (2-D), pulse wave (PW) Doppler.6. Cardiac fibroblast isolation, cultureCardiac fibroblasts were prepared from the hearts of adult male WT mice as previously described. Cells were maintained in DMEM (GIBCO) containing 10%FBS and penicillin-streptomycin (Invitrogen, Carlsbad, CA) in a 5%CO2 humidified incubator at 37℃.7. Cardiac fibroblast fibrotic treatment.As an in vitro model of fibroblast-to-myoblast differentiation, cardiac fibroblasts were treated with ANG II (1 u mol/1) for 48 h as previously described.8. Immunofluorescence.Cardiac fibroblasts were fixed for 15 min in 4% paraformaldehyde prepared in complete medium at 37℃ in an incubator. Briefly, cells were incubated with primary antibodies for α-SMA or Smad3 (1:200, diluted in 50% blocking buffer) overnight at 4℃ in a humidified chamber. All imaging analyses were performed in the digital confocal microscopy core facility.9. Western blotting analysisFrozen tissue and cell proteins were lysed by RIPA. And the extracted total proteins were separated by SDS-PAGE and transferred onto a PVDF membrane using a wet transfer apparatus (Bio-Rad, Hercules, CA, USA). The membranes were blocked with 5% non-fat milk, incubated overnight at 4℃ with the primary antibodies, and then incubated with the secondary antibodies labeled with horseradish peroxidase. The protein bands were visualized through enhanced chemiluminescence (Millipore). The protein levels were detected using an ImageQuant LAS4000 chemiluminescence reader (GE, USA).10. Real-time PCR analysisTotal RNA extracted with TRIzol reagent (Invitrogen) according to the manufacturer’s instructions, was reverse transcribed using a Transcriptor First Strand cDNA Synthesis Kit for RT-PCR following the indications of the manufacturer (Roche). Real-time PCR was conducted on a MyiQ Real-Time PCR System (Bio-Rad) using SYBR green Real-Time PCR (Roche) per the protocol11. Statistical analysisData are presented as mean±standard error of the mean (SEM). Data analysis was performed with unpaired t-test or one-way analysis of variance (ANOVA) using SPSS18.0 software. P< 0.05 was considered as statistically significant.Results:1.SIRT3 elevated the expression of SIRT3 in mouse heart.We subjected WT mice to CR diets and RSV-supplemented diets for 8 wk. Cardiac expression levels of SIRT3 in the CR group were twice those observed in the CD group (*p<0.05). Moreover, RSV (1.8 mg·kg-1·day-1) significantly increased SIRT3 expression in mouse hearts in a manner similar to CR. Next; we subjected WT and SIRT3 KO mice to sham operation or TAC (mice were fed with different diets throughout until euthanization). We observed that SIRT3 levels decreased by 50% in the WT+TAC group 2 wk after TAC. However, in the WT+RSV+TAC group the decrease of SIRT3 caused by TAC was overtly reserved by RSV.2. SIRT3 was required for RSV-mediated prevention of hypertrophy.TAC produced more severe hypertrophy in SIRT3 KO groups, as assessed by the heart weight-to-body weight ratio (73% in the SIRT3 KO+vehicle+TAC group and 32% in the WT+vehicle+TAC group, P<0.05). Moreover, RSV alleviated cardiac hypertrophy in the WT+RSV+TAC group, whereas in the SIRT3 KO+RSV+TAC group, RSV did not elicit similar protective effects. Expression levels of other hypertrophic markers, such as atrial natriuretic peptide and myosin heavy chain-7, were also significantly higher in the SIRT3 KO+vehicle+TAC group than in the WT+vehicle+TAC group, and RSV significantly reduced the expression of fetal genes in the WT+RSV+TAC group but not in the SIRT3 KO+RSV+TAC group. Collectively, these data indicate that SIRT3 was required for the RSV-mediated prevention of hypertrophy.3. Activation of SIRT3 by RSV prevented the loss of cardiac function during hypertrophy.We performed echocardiography to examine cardiac function. LV posterior wall thickness was significantly higher in the SIRT3 KO+vehicle+TAC group (1.45+0.07 mm, P<0.05 vs. the WT+vehicle+TAC group). LV fractional shortening decreased significantly after TAC, particularly in the SIRT3 KO+vehicle+TAC group, but LV ejection fraction did not exhibit obvious changes between sham and TAC groups. Diastolic dysfunction was obvious, as reflected by increased Aa wave velocity and a decreased Ea-to-Aa ratio. In the present study, the Doppler indexes all demonstrated that diastolic dysfunction existed in both WT and SIRT3 KO groups after TAC and was more severe in the SIRT3 KO+vehicle+TAC group, cardiac function appeared normal in the WT+RSV+TAC group, which suggested that LV function was preserved by RSV. In contrast, RSV treatment did not elicit any protective effects in the SIRT3 KO+RSV+TAC group. Together, these results indicate that the activation of SIRT3 by RSV was able to prevent the loss of cardiac function, particularly diastolic function in response to TAC.4. Activation of SIRT3 by RSV alleviated cardiac fibrosis in mice.Fibrosis is an integral feature of the remodeling characteristics of the failing heart. Total collagen fractions observed under polarized light (as determined by golden staining) were higher in both WT+vehicle+TAC and SIRT3 KO+vehicle+TAC groups than in their corresponding sham control groups, and SIRT3 KO mice developed cardiac fibrosis spontaneously, RSV treatment alleviated cardiac fibrosis in the WT+RSV+TAC group but not in the SIRT3 KO+RSV+TAC group, which indicated that SIRT3 was required for the RSV-mediated block of cardiac fibrosis. Both collagen type I and collagen type III increased after TAC in the mouse hearts. RSV treatment reduced collagen type I and collagen type III in the WT+RSV+TAC group, which was not observed in the SIRT3 KO+RSV+TAC group.5. Activation of SIRT3 by RSV reduced TGF-β, a-SMA, and TIMP-2 expression and inhibited TGF3-/Smad3 signaling in cardiac tissue.TGF-β, α-SMA, and TIMP-2, determined as markers of extracellular matrix synthesis and degradation, In the present study, both WT and SIRT3 KO mice exhibited significantly increases in the expression of TGF-β, α-SMA, and TIMP-2 after TAC. RSV treatment significantly decreased the expression of TGF-β, α-SMA, and TIMP-2 in the WT+RSV+TAC group but had no effect in the SIRT3 KO+RSV+TAC group. The TGF-(3/Smad3 pathway was activated in cardiac tissue in response to TAC, as shown by the high level of p-Smad3. TGF-β receptors (TR(31 and TRβ2), which can increase the phosphorylation of Smad3, were downregulated by RSV in the WT+RSV+TAC group, which indicated that RSV inhibited TGF-(3 signaling in a Smad3-dependent manner. However, this effect was not observed in the SIRT3 KO+RSV+TAC group. Thus, the activation of SIRT3 by RSV decreased TGF-β, α-SMA, and TIMP-2 expression and might inhibit cardiac fibrosis via TGF-(3/Smad3 signaling in mouse hearts.6. SIRT3 was activated by RSV in a manner similar to CR in cardiac fibroblasts and prevented cardiac fibroblast-to-myoblast transformation.We used different doses of RSV (0-100 u M) to treat cardiac fibroblasts grown in serum-containing medium for 24 h, and we found that cells treated with 80 u M RSV exhibited twofold higher levels of SIRT3 than did control cells. We used RSV (80 u M) to treat cardiac fibroblasts for 48 h, and cells treated with RSV (80 u M) for 18 h exhibited twofold higher levels of SIRT3 in vitro. To investigate the role of the activation of SIRT3 by RSV in fibroblast-to-myoblast transformation, we used ANG II to stimulate cardiac fibroblasts, as previously described. Immunofluorescence analyses revealed that α-SMA, a major marker of myoblasts, was strongly expressed by cardiac fibroblasts in response to AN6 II (Fig.6A). In contrast, the protein level of a-SMA was markedly suppressed by pretreatment with RSV; however, when SIRT3 was knocked down using SIRT3-specific siRNA, RSV failed to reduce a-SMA expression. Real-time PCR further confirmed that fibroblasts treated with ANG II exhibited significantly increased mRNA expression of a-SMA and TGF-P, whereas pretreatment with RSV attenuated these effects. However, in cardiac fibroblasts transfected with siRNA to SIRT3, the protective effect of RSV was not observed.7. Activation of SIRT3 by RSV blocked the TGF-β pathway in cardiac fibroblasts and inhibited transcription factor Smad3.In the present study, expression levels of TGF-β both in vivo and in vitro increased in response to TAC and ANG II. To investigate whether the antifibrosis effect of SIRT3 was the result of inhibition of the TGF-P pathway, we analyzed the TGF-β pathway in cardiac fibroblasts in response to ANG II. Our results revealed that RSV treatment decreased TRP1; TRP2, p-Smad3, and Smad3 levels in response to ANG II. Moreover, PAI-1, which is an important Smad3 target and controls the activities of plasmin-dependent matrix metalloproteinases, was also downregulated by the activation of SIRT3. Again, RSV treatment did not modify the expression of the TGF-β pathway when cells were transfected with siRNA to SIRT3. Taken together, these results suggest that the contribution of SIRT3 to antifibrosis effects was mediated through the TGF-β pathway. Next, w... |
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