The Study Of SIRT3 Attenuates Cardiac Fibrosis By Inhibiting Myofibroblasts Transdifferentiation

BackgroundsMyocardial fibrosis emerges in the late course of chronic hypertension.The main feature of cardiac fibrosis is the imbalance between secretion and degradation of extracellular matrix,which leads to massive collagen accumulation and reduced myocardial compliance.The diastolic function is impaired at first,and lower systolic function in combined as the diseases progress,so cardiac fibrosis plays an important role in the pathogenesis and development of heart failure.In addition,the alteration of myocardial structure provides anatomic basis for the occurrence of malignant arrhythmias,which can lead to sudden death.The production of collagen increases while the degradation decreases,so myocardial fibrosis occurs.The elevated generation of collagen fibers is significantly associated with the activation of myofibroblasts.The myofibroblasts do not exist in normal cardiac tissues,they are transdifferentiated by a variety of cell types in conditions such as myocardial infarction or pressure overload,the sign of its differentiation is α-smooth muscle actin(α-SMA)expression.Studies have shown that the transdifferentiation of cardiac fibroblasts into myofibroblasts plays a leading role in the development of myocardial fibrosis.Factors such as inflammatory mediators,cytokines and mechanical tension can stimulate the transdifferentiation of myofibroblasts which harbors stronger secretion ability,they secret more collagen fibers to promote ventricular remodeling.SIRT3 is an NAD+-dependent mitochondrial deacetylase,it is widely expressed in heart,liver,kidney,brown adipose tissue,and it’s involved in cellular senescence,differentiation,apoptosis and energy metabolism process.In recent years,it has been gradually discovered that SIRT3 has been implicated in many biological processes to deacetylate some non-histone proteins,which has attracted more and more attention from researchers.Studies have shown that compared with wild type mice,SIRT3 knockout mice have spontaneous myocardial hypertrophy,fibrosis and contraction dysfunction with age.They develop more severe myocardial hypertrophy and fibrosis under pressure overload.The possible mechanism is that SIRT3 can deacetylate transcription factor FOXO3a,thus increasing the its expression,while the latter further promote the expression MnSOD and IDH2 to reduce oxidative stress damage,to delay the onset of myocardial hypertrophy.However,the relationship between SIRT3 and cardiac fibroblasts transdifferentiation and the underlying mechanism are still unknown.Nuclear factor of activated T cells(NFAT)is a transcription factor with multiple regulating functions,its mainly activated by calcium-calcineurin signaling pathways to regulate gene expression.It regulates the expression of a-SMA in smooth muscle cells.Studies have shown that TRPC6(syndecan-4)-Calcineurin-NFAT pathway can promote the transdifferentiation of cardiac fibroblasts and play an important role in the development of myocardial fibrosis.The NFAT family includes NFAT1(NFATc2),NFATc or NFATcl,NFAT3(NFATc4)and NFAT4(NFAT3).NFATc4 plays a role in the differentiation of fibroblasts,but the effect of NFATc2 is not ambiguous,and the NFATc2 knockout mice have less ventricular fibrosis than the wild-type mice under pressure overload.In a study related to pulmonary hypertension,SIRT3 deficiency can activate STAT3 to promote its nuclear translocation and thus affect its ranscriptional activity,and the expression and activation of NFATc2 is regulated by STAT3 in smooth muscle cells in the lungs.STAT3 is an important member of the STAT family and is pivotal in the JAK-STAT signaling pathway,which plays an important role in the development of the tumor.Of all STAT members,STAT3 had the most intimate relationship with the cardiovascular system,and previous studies showed that STAT3 had a tendency to worsen myocardial fibrosis.As a transcription factor shuttle between mitochondria and the nucleus,STAT3 have specific acetylating sites.The role of STAT3 in the development of myocardial fibrosis deserves further discussion,whether can it be deacetylated by SIRT3 to affect its activity,how about its influence on the downstream NFATc2?To sum up,this study intends to explore the role of SIRT3 in angiotensin Ⅱ-induced myocardial fibrosis and the involved signaling pathways both in vivo and in vitro.Hope our study could bring new ideas for the prevention and treatment of myocardial fibrosis,to provide laboratory basis to design new drugs targeting SIRT3.Objectives1.To investigate whether SIRT3 knockout mice develop more serious myocardial fibrosis than normal wild-type ones under the stimulus of angiotensin Ⅱin vivo2.To study whether SIRT3 plays a role in the transdifferentiation of fibroblasts into myofibroblasts in vitro3.To explore the possible molecular mechanisms of SIRT3 in inhibiting myofibroblasts transdifferentiation,and to clarify the role of the STAT3-NFAT2 signaling pathway.Methods1.Animal modelWe chose 8-week old male WT and SIRT3-/-mice to divide into four groups as follows:WT,SIRT3-/-WT+Ang Ⅱ and SIRT3-/-+Ang Ⅱ,15 mice in each group.AngⅡ(Sigma Aldrich,USA,dissolved in sterile ddH20)was subcutaneously infused at the rate of 2000ng/kg/min for 28 days by implanting of Osmotic Minipumps.2.Cardiac fibroblasts isolation and cultureCardiac fibroblasts were isolated from the hearts of 1-3-day-old WT and SIRT3-/-mice.The cells were then maintained in DMEM containing 10%FBS and penicillin-streptomycin in a 5%C02 humidified incubator at 37℃.3.Cell transfection and treatmentWT and SIRT3-/-cardiac fibroblasts were treated with Ang Ⅱ(10-7mol/L)for 48 h.We transfected SIRT3 lentivirus into WT fibroblasts,to observe the transdifferentiation of myofibroblasts.We transfected si-STAT3 into SIRT3-/-cardiac fibroblasts to study the expression of downstream NFATc2 and a-SMA.4.Western blot analysisProteins were harvested from cell lysates and mouse hearts.The PVDF membranes were blocked in 5%non-fat milk for 2 hours.Then,the blocked membranes were incubated overnight with the primary antibodies at 4℃.The membranes were incubated with the rabbit or mouse secondary antibodies for 1 hour at room temperature next day.The protein bands were visualized with enhanced chemiluminescence AI600.5.Histology and immunohistochemistryHearts were fixed in 4%paraformaldehyde,embedded in paraffin,and sectioned at 5-μm intervals.Hematoxylin and eosin,Masson’s Trichrome and Picrosirius red staining were performed using standard procedures.For immunohistochemistry,briefly,the sections were incubated with primary antibodies overnight at 4℃.Then after being washed with phosphate-buffered saline,the slides were incubated with secondary antibodies at 37℃ for 1h.6.ImmunofluorescenceCardiac Fibroblasts were fixed in 4%paraformaldehyde for 25 min and then perforated with 0.5%Triton X-100 for 20 min.After being blocked in 1%bovine serum albumin for 30min,the cells were then incubated with primary antibodies overnight at 4℃.After that,secondary antibodies were added.Nuclei were stained with DAPI for 5 min.The images were acquired by a laser scanning confocal microscope.7.Statistical analysisAll the results were obtained from at least three independent experiments.Statistical analysis was performed using GraphPad 6.0 Prism.Data are presented as the mean ± standard deviation.Student’s t-test was used to assess between-group differencesand the results were considered statistically significant at P<0.05.Results1.SIRT3 deficiency aggravated AngⅡ-induced murine interstitial fibrosisTo investigate the role of SIRT3 in cardiac fibrosis,we subjected both the wild-type(WT)and SIRT3-knockout(SIRT3-/-)mice to chronic Ang Ⅱ infusion for four weeks.The immunoblot analysis showed that SIRT3 levels decreased by 21%after AngⅡ infusion compared to their WT controls(P<0.05).HE staining showed more cardiomyocytes loss and expanded cross-sections area in the hearts of mice subjected to AngⅡ,especially the SIRT3-/-group.Masson’s trichrome and Picrosirius red staining indicated that the fibrosis area fraction in SIRT3-/-mice was higher compared to WT mice at baseline and increased further after chronic AngⅡ infusion(P<0.05).The AngⅡ-treated SIRT3-/-mice exhibited most collagen accumulation showed by immunohistochemical staining of collagen Ⅰ and collagen Ⅲ(P<0.05),and its confirmed by western blot.Altogether,these results indicated that SIRT3 might be involved in preventing collagen deposition and cardiac fibrosis.2.SIRT3-/-fibroblasts transdifferentiated into myofibroblasts spontaneouslyWe observed more a-smooth muscle actin(a-SMA),the marker of well-differentiated myofibroblasts,expressed in the SIRT3-/-mice.To further investigate the role of SIRT3 in fibroblasts transdifferentiation,we isolated primary neonatal fibroblasts from WT and SIRT3-/-mice.Immunoblots and immunofluorescence analysis indicated that SIRT3-/-fibroblasts exhibited more a-SMA,collagen I and fibronection expression(P<0.05),which suggested fibroblasts transdifferentiate into myofibroblasts in a spontaneous way.Then we infected fibroblasts from WT neonatal mice with LV.SIRT3,and the expression of a-SMA and collagen I decreased as SIRT3 expressed highly(P<0.05).In summary,our data demonstrated that SIRT3 plays a vital role in inhibiting fibroblast transdifferentiation.3.SIRT3-/-fibroblasts acquired more evident phenotype conversion with AngⅡstimulationWe treated WT and SIRT3-/-fibroblasts with AngⅡ.SIRT3-/-fibroblasts with AngⅡ stimulation manifested maximum a-SMA expression and concomitant elevation of collagen,fibronectin(P<0.05).The immunoblot analysis of SIRT3-/-murine hearts and myofibroblasts showed higher TGF-β compared to their controls(P<0.05).TGF-β plays critical role in fibrogensis by promoting fibroblasts transdifferentiation.It suggested that it existed positive feedback between TGF-βstimulation and fibroblast transdifferentiation with SIRT3 deficiency.4.SIRT3 deacetylated and inhibited the activation of STAT3Though these results demonstrated that SIRT3 could regulate fibroblasts transdifferention,the underlying mechanism remains unclear.It noticed that there was higher ratio of p-STAT3/STAT3 in SIRT3-/-myofibroblasts,implying more STAT3 activation compared to their WT controls(P<0.05).And SIRT3 overexpression reversed this phenomenon.We proposed the hypothesis that SIRT3 could deacetylate STAT3 to inhibit its activity.We found SIRT3 deficiency was associated with elevated acetylation level of STAT3 by immunoprecipitation.(P<0.05).Co-immunoprecipitation displayed that there exists interaction between SIRT3 and STAT3.It’s concluded that SIRT3 could deacetylate STAT3 and inhibit its activity.5.NFATc2 expression and nuclear translocation were increased in SIRT3-/-myofibroblastsFurthermore,we observed increased NFATc2 level in SIRT3-/-myofibroblasts and decreased NFATc2 level in LV.SIRT3 myofibroblasts,compared to the wild type controls(P<0.05).And SIRT3 deficiency promoted nuclear translocation of transcription factor NFATc2 in fibroblasts.Following the infection of si-STAT3 in SIRT3-/-fibroblasts,the NFATc2 expression was reduced markedly as well as a-SMA(P<0.05).These results indicated STAT3-NFATc2 pathway was involved in the myofibroblasts transdifferentiation.Conclusions1.SIRT3 knockout mice develop more serious myocardial fibrosis than normal wild-type ones under the stimulus of angiotensin Ⅱ2.SIRT3 inhibits the transdifferentiation of fibroblasts into myofibroblasts to attenuate cardiac fibrosis3.The molecular mechanisms of SIRT3 in inhibiting myofibroblasts transdifferentiation are partially relevant to the STAT3-NFAT2 signaling pathway.BackgroundsWith the popularity of high-calorie diets and sedentary lifestyles,obesity has become a worrying global health problem.Epidemiological investigation has found that the increasing incidence of obesity is closely related to heart failure.In 1973,Smith and Willius first identified the link between obesity and ventricular function.Studies done by Hubert have shown that obesity can affect ventricular function,and that obesity is a major risk factor for heart failure.Recent studies have shown there are abnormal cardiac structure and function even in mild to moderate obesity,therefore,obesity cardiomyopathy should be defined as that cardiomyopathy which can’t be explained by diabetes,high blood pressure,coronary artery disease or other reasonable causes in obese individuals.It varies from asymptomatic left ventricular dysfunction to symptomatic dilated cardiomyopathy.The main cause of obesity cardiomyopathy has not been identified yet.It is now clear that hemodynamic abnormalities(high hemodynamic states)cause increased ventricular load,which in turn leads to left ventricular hypertrophy and ventricular dilatation.The main histologic feature is the diffuse increase in the number of hypertrophied myocyte and apoptosis.Researches have found significant fibrosis in both obese animal models and clinical studies,and such fibrosis is often accompanied by tissue degeneration and inflammation in animal models.Silent information adjustment factor(Sirtuins)is a kind of histone deacetylase dependent on NAD +,mainly expressed metabolic organs such as liver,kidney and fat,and they regulate a variety of important cellular functions.SIRT3 is the only subtype that has been found to be related to prolonged human lifespan.Exercise and caloric restriction can improve the expression of SIRT3.SIRT3 is widely found in mitochondria and nucleus and plays an important role in the oxidation of fatty acids and the maintenance of cellular ATP levels.Our research group has been working on the protection of SIRT3 in the cardiovascular system.In our previous studies,we found that SIRT3 gene knockout mice showed more severe myocardial hypertrophy and cardiomyocyte energy metabolism disorder than wild-type mice under pressure overload.It may be related to decreased activity of LCAD.In previous studies on obesity,it was found that SIRT3 knockout may worsen cardiac function by reducing the myocardial angiogenesis of myocardial tissue.Obesity itself is in a state of chronic inflammation,whether SIRT3 could affect the cardiac structure and function by influencing the expression and release of inflammatory cytokines has not been reported,and it is worth further research.In adipose tissue of obese mice induced by high-fat diet,the expression of monocyte chemotactic protein-1(MCP-1)mRNA was significantly increased,accompanied by a large concentration of macrophages.In the study conducted in vitro cultured renal proximal tubule cells,SIRT3 was shown to be negatively correlated with mRNA of MCP-1,that is,the expression of MCP-1 after interfering with SIRT3 was increased.However,the role of MCP-1 in the heart of high-fat diet-induced obese mice is not clear,whether it affects the infiltration of inflammatory factor in heart alone,and whether its associated with SIRT3 and their joint action on cardiac remodeling in obese mice has not been reported.Other inflammatory factors such as IL-6,TGF-β,TNF-α play an important role in the development of atherosclerosis,and whether they are highly expressed in the obesity model induced by high-fat diet remain unknown.Their relationship with SIRT3,and the effects on cardiac structure and cardiac function needs further research.Based on the above theories and research foundation,we designed and carried out the current experiment.In this study,we gave the male wild-type and SIRT3 knockout mice a normal diet or a high-fat diet for 16 weeks,and the heart phenotype was first studied after successful modeling.We mainly concentrated on cardiac inflammation and fibrosis related to cardiac remodeling in further research.Finally,we explored the pathophysiological process the signaling pathways involved.It is hoped that our research data can provide relevant laboratory evidence to provide a certain basis for future clinical development of related drugs for obesity-related cardiomyopathy.Objectives1.To clarify the effect of SIRT3 on the phenotypic change and cardiac function of obese cardiomyopathy.2.To explore the mechanism of SIRT3 affecting the phenotype of obese cardiomyopathy,with emphasis on the release of inflammatory factors and changes in myocardial fibrosis.3.To illustrate that SIRT3 can reduce the expression of MCP-1 in myocardial tissue and infiltration of macrophages by inhibiting ROS-NF-κB signaling pathway,ultimately reducing the occurrence of myocardial inflammation and myocardial fibrosis,thus improving myocardial remodeling and cardiac function.Methods1.Animal model constructionWe selected 8-week old male WT and SIRT3-/-mice to divide into four groups as follows:WT+CD,SIRT3-/-+CD,WT+HFD and SIRT3-/-+HFD,15 mice in each group.The chow diet and high-fat diet(60%kcal,D12492;(New Brunswick,NJ,USA)were respectively fed for 16 weeks to induce obesity models.2.EchocardiographyAll the mice were tested by ultrasound with Vevo770 by the special technical personnel who did not know about the project before taking the material.The isoflurane inhalation of 1%was used for anesthesia in mice,then M-mode,2 dimension,pulsed wave doppler(PW)and tissue doppler imaging(TDI)were applied respectively to record all the parameters,such as ventricular septal thickness(IVST),left ventricular posterior wall thickness(LVPWT),left ventricular contraction,end-diastolic diameter and volume(LVESV LVESD,LVEDD,LVEDV),left ventricular ejection fraction(LVEF),E/A,E ’/A,etc.3.Histopathological staining and histochemical analysisAfter the hearts of each group were taken,the tissue was soaked in 4%paraformaldehyde,and the P2 process was performed to dehydrate it.Then the hearts were buried in paraffin for reserve.Paraffin sections with a thickness of about 5um were used for the subsequent histopathological staining.For immunohistochemical analysis:the selected slice were incubated with primary antibodies after antigen repair overnight at 4℃,then incubated with the secondary antibodies at 37 ℃ for 1 h the next day.Then we observed microscopically by using horseradish peroxide enzymatic enhancement until tan particles appeared.4.Western blot analysisThe total protein of the animal heart tissue was collected respectively.After electrophoresis,the protein was transferred to PVDF membrane using the method of wet transfer,then the membranes were blocked in 5%non-fat milk for 2 hours at room temperature.The incubation with specific antibodies continued on the shaking bed at 4 ℃ for the night.The next day,the corresponding rabbit or rat was incubated at room temperature for 1 hour,and the blots was fully washed and then illuminated with an automatic luminescence device.5.Real-time quantitative PCR.RNA was extracted from each group of heart tissues by Trizol method,and its concentration and purity were detected respectively,and the reverse transcription was done to get cDNA under appropriate conditions,and the expression of each target gene was detected by SYBR green method.6.DHE dyeingFreshly drawn mouse hearts were immediately frozen with OCT compounds and quickly made into a frozen section with thickness of 5um.The slices were first soaked in PBS for 5 minutes,then incubated for 15 minutes in a dark room with a DHE solution of 0.5mmol.After being washed fully with PBS solution,the image was photographed with a fluorescence microscope as soon as possible.7.Statistical analysisAll the results were obtained from at least three independent experiments.Statistical analysis was performed using GraphPad 6.0 Prism.Data are presented as the mean ± standard deviation.Student’s t-test was used to assess between-group differencesand the results were considered statistically significant at P<0.05.Results1.SIRT3-/-mice acquired more weight gain and worse cardiac diastolic dysfunction after high-fat dietTo study the role of SIRT3 in cardiac remodeling in obesity-related cardiomyopathy,we placed both wild-type(WT)and SIRT3-knockout(SIRT3-/-)mice on a high-fat diet for 16 weeks.We found more weight gain as well as increased heart weight/body weight ratio in HFD feeding SIRT3-/-mice than WT controls(P<0.05).The echocardiography showed left ventricle hypertrophy and diastolic dysfunction induced by high-fat diet,especially in SIRT3-/-mice(P<0.05).We evaluated the diastolic function by measuring E/A ratio.The systolic function remains unaltered2.SIRT3 deficiency deteriorated cardiac fibrosis in HFD-fed miceFirstly,we measured SIRT3 protein levels in all the murine hearts.SIRT3 expression decreased by 19%in WT controls after HFD feeding(P<0.05).Masson’s trichrome indicated that the fibrosis area fraction in SIRT3-/-mice was higher than that in WT mice at baseline and increased further after HFD feeding(P<0.05).The HFD-fed SIRT3-1-mice exhibited most collagen accumulation proven by immunohistochemical staining of collagen I and collagen III.And we confirmed this finding by western blot(P<0.05).Altogether,these results revealed that SIRT3 might be involved in preventing collagen deposition and cardiac fibrosis in obesity-related cardiac remodeling.3.SIRT3-/-mice fed on HFD showed the maximum inflammatory cytokine expressionsWe measured and compared the levels of inflammatory cytokines including IL-6,TGF-β,TNF-α,IL-10 in murine hearts by western blot.The results showed more pro-inflammatory cytokines expression in HFD-fed mice,especially in SIRT3-/-mice(P<0.05).On the contrary,the anti-inflammatory factor IL-10 decreased most significantly in SIRT3-/-mice fed on HFD among all the groups(P<0.05).We confirmed this discovery by PCR.The PCR analysis revealed the same trend as immunoblot.4.Maximum MCP-1 expression and macrophage infiltration in HFD-fed SIRT3-/-miceTo further illuminate the underlying relation between cardiac fibrosis and inflammation in obesity-related cardiac remodeling,we carried out more experiments.Both immunohistochemical staining and western blot showed the highest MCP-1 expression in SIRT3-/-mice placed on HFD(P<0.05).HE staining showed more cardiomyocytes loss and inflammatory cells accumulation in the hearts of HFD-fed mice,especially the SIRT3-/-group(P<0.05).Further immunohistochemistry verified the inflammatory cells as macrophage marked by MOMA-2 staining.5.We observed most ROS generation and activated NF-κB in SIRT3-/-mice fed on HFDAs MCP-1 increased greatly in SIRT3-/-mice after HFD feeding,we determined to explore its upstream molecules.So we test NF-κB activation by measuring p-NF-κB as well as nuclear translocation of NF-κB in all the groups.DHE staining was performed to evaluate the amount of ROS in cardiac tissue.The results showed more ROS generation and activated NF-κB after HFD feeding in mice,especially in SIRT2-/-+HFD group(P<0.05).Conclusions1.The obese SIRT3-/-mice had more obvious cardiac remodeling and decreased diastolic function.2.SIRT3 may improve myocardial remodeling and cardiac function by reducing the release of inflammatory factors and improving myocardial fibrosis.3.SIRT3 may reduce the expression of MCP-1 and the infiltration of macrophages in myocardial tissue by inhibiting the activation of ROS-NF-κB signaling pathway,thus eventually reducing the occurrence of myocardial inflammation and myocardial fibrosis.